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Journal of 

Medicinal Plants Research 

Volume 6 Number 5 9 February, 2012 

ISSN 1996-0875

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Elazıg Directorate of National Education 

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Information Center, 

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Lagos State University, Ojo, Lagos, 

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Omdurman Islamic University, Botany Department, 

Sudan. 

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Division of plant Biology, 

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Veterinary Medicine, 

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Lorestan University, 

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Inonu University Faculty of Medicine, 

Turkey. 

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Department of Crop and Environmental Protection, 

Ladoke Akintola University of Technology, 

Ogbomoso 

Nigeria. 

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Cairo 

Egypt.

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Journal of Medicinal Plants Research 

Table of Content: Volume 6 Number 5 9 February, 2012 

International Journal of Medicine and Medical Sciences 

Sciences 

Review 

ARTICLES 

Monograph of Tribulus terrestris 641 

Ghazala Shaheen, Irshad Ahmad, Khan Usmanghani Naveed Akhter, 

Mukhtiar Ahmad, Sabira Sultana and M. Akram 

Research Articles 

The effects of thyme (Thymus vulgaris) extract supplementation in 

drinking water on iron metabolism in broiler chickens 645 

Rahim Abdulkarimi and Mohsen Daneshyar 

Effects of borage extract in rat skin wound healing model, 

histopathological study 651 

Mohammad Reza Farahpour and Amir Hossein Mavaddati 

Evaluation of antioxidant activities of Withania somnifera leaves 

growing in natural habitats of North-west Himalaya, India 657 

R. K. Sharma, S. S. Samant, P. Sharma and S. Devi 

Phytochemical screening, antioxidant and antimutagenic activities of 

selected Thai edible plant extracts 662 

M. Phadungkit, T. Somdee and K. Kangsadalampai 

Ethnobotany and antimicrobial activity of medicinal plants of 

Bakhtiari Zagross mountains, Iran 675 

A. Ghasemi Pirbalouti, F. Malekpoor and B. Hamedi 

Proposing a methodology in preparation of olive orchards map as an 

important medicinal plant in Iran by remote sensing (RS) and 

geographical information system (GIS) 680 

Ali Mohammadi Torkashvand and Alireza Eslami


Sciences 

ARTICLES 

Traditional medicinal plants research in Egypt: Studies of antioxidant 

and anticancer activities 689 

Ahmed M. Aboul-Enein, Faten Abu El-Ela, Emad A. Shalaby 

and Hany A. El-Shemy 

Preliminary phytochemical screening and ethnomedicinal uses 

of Teucrium stocksianum from Malakand Division 704 

Gul Rahim, Rahmatullah Qureshi, Muhammad Gulfraz, M. Arshad 

and Sahib Rahim 

Antioxidant and antiapoptotic properties of chlorogenic acid on 

human umbilical vein endothelial cells 708 

Xing Wu, Song Lin and Xiaotang Zhang 

Elastase, tyrosinase and lipoxygenase inhibition and antioxidant activity 

of an aqueous extract from Epilobium angustifolium L. leaves 716 

Hulya Celik Onar, Ayse Yusufoglu, Gulen Turker and Refiye Yanardag 

Identification of superior varieties of tea (Camellia sinensis (l.) o. kuntze) 

in the selected UPASI germplasm using biomarkers 727 

S. Ramkumar, P. Sureshkumar, A. K. A. Mandal, K. Rajaram 

and P. Mohankumar 

Development of a sebum control cream from a local desert plant 

Capparis decidua 744 

Shahiq uz Zaman, Naveed Akhtar, Barkat Ali Khan, Tariq Mahmood, 

Akhtar Rasul, Arshad Mahmood, Muhammad Naeem Aamir and Atif Ali 

An ethnobotanical survey of medicinal plants used by indigenous 

people in Zangelanlo district, Northeast Iran 749 

Mohammad Sadegh Amiri, Parham Jabbarzadeh and Mahdi Akhondi


ences 

ARTICLES 

Reproductive behaviour and breeding system of wild and cultivated 

types of Withania somnifera (L.) Dunal 754 

Bilal Ahmad Mir, Sushma Koul, Arun Kuar, Shushant Sharma, 

Maharaj K. Kaul and Amarjit S. Soodan 

Effect of sodium tanshinon IIA silate on heart function of children 

with myocarditis 763 

Huang Weizhe, Zhang Dongtao, Zhang Ge and Li Tong 

Nutritional, phytochemical potential and pharmacological evaluation 

of Nigella Sativa (Kalonji) and Trachyspermum Ammi (Ajwain) 768 

Shabnam Javed, Ahmad Ali Shahid, Muhammad Saleem Haider, 

Aysha Umeera, Rauf Ahmad and Sobia Mushtaq 

Systematic anatomy and elemental dispersive spectrophotometer 

analysis of genus Pennisetum from Pakistan 776 

Shabnum Shaheen, Farah Khan, Rana Abrar Hussain, Riffat Sidique, 

Hanan Mukhtar, Zaryab Khalid, Amina Younis, Mushtaq Ahmad 

and Muhammad Zafar 

Nutritional assessment of a traditional local vegetable 

(Brassica oleracea var. acephala) 784 

Mariga I. K., Lutendo Mativha and Maposa D. 

Effect of salicylic acid application on biochemical changes in ginger 

(Zingiber officinale Roscoe) 790 

Ali Ghasemzadeh and Hawa Z. E. Jaafar 

Antidiabetic effect of Gynura procumbens leaves extracts involve 

modulation of hepatic carbohydrate metabolism in 

streptozotocin-induced diabetic rats 796 

Hui-Wen Lee, Pusparanee Hakim, Amir Rabu and Halimah Abdullah Sani

S 


ARTICLES 

Hypaconitine protects H9c2 cells from oxidative stress-induced apoptosis 813 

Zhi-Hui Li, Wanhong Xu, Guo-Hui Li, Ling Shen, Min-Jie Mao, 

Zhi-Gang Wu, Xue-Ting Shao and Kun Fang 

Antimicrobial activity and chemical composition of essential oils of 

chamomile from Neyshabur, Iran 820 

Motavalizadehkakhky Alireza 

Determination of saikosaponin, phenolic and podophyllotoxin contents 

of five endemic Bupleurum root extracts and their effects on 

MCF-7 cells 825 

Gökhan Kars, Meltem Demirel Kars, Mehtap Akin, Hatice Taner 

Saraçoğlu and Ufuk Gündüz 

Effects of astragaloside IV on L-type calcium channel currents in 

adult rat ventricular myocytes 833 

Shiqi Lu, Guolin Chen, Yiming Zhao and Wanping Sun 

Internal transcribed spacer-based identification of Bupleurum 

species used as sources of medicinal herbs 841 

Young Hwa Kim, Eung Soo Kim, Byoung Seob Ko, Md. Romij Uddin, 

Seung Eun Oh, Go Ya Choi, Seong Wook Chae, Hye Won Lee, 

Je Hyun Lee, Ju Young Park and Mi Young Lee 

Effect of catalpol on doxorubicin-induced cytotoxicity in H9c2 cells 849 

Xing Wu and Yanbin Mao 

Optimization of light quality for production of alkaloid and polysaccharide 855 

Fonge B. A., Egbe E. A., Fongod A. G. N., Focho D. A., Tchetcha D. J., 

Nkembi L. and Tacham W. N.

S 


ARTICLES 

Discrimination of Zhishi from different species using rapid-resolution 

liquid chromatography-diode array detection/ultraviolet 

(RRLC-DAD/UV) coupled with multivariate statistical analysis 866 

Zhenli Liu, Yuanyan Liu, Chun Wang, Zhiqian Song, Qinglin Zha, 

Cheng Lu, Chao Wang and Aiping Lu 

Isolation, chemical characterization and in vitro antioxidant 

activities of polysaccharides from Aconitum coreanum 876 

Bin Li, Xian-Jun Meng and Li-Wei Sun 

Preparative separation of hyperoside of seeds extract of 

Saposhnikovia divaricata by high performance 

counter-current chromatography 884 

Li Li, Yuge Gui, Jing Wang, Huirong Zhang, Xiaofei Zong 

and Chun Ming Liu 

Research update: Lectin enriched fractions of herb and dry extract 

of Urtica dioica L. 888 

Savickiene Nijole, Baniulis Danas, Bendokas Vidmantas, Balciunaite 

Gabriele, Draksiene Gailute, Peciura Rimantas and Serniene Loreta 

A clinical study on the effects and mechanism of Xuebijing injection 

in the treatment of traumatic intracranial hematoma 893 

Yong Guo, Kuipo Yan, Jiasheng Fang, Jinfang Liu, Mingyu Zhang 

and Jun Wu 

Cardiospermum grandiflorum leaf extract potentiates amoxicillin 

activity on Staphylococcus aureus 901 

Petra O. Nnamani, Franklin C. Kenechukwu and Wilfred N. Oguamanam 

The first Stolbur Phytoplasma occurrence on two St. John's Worth species 

(Hypericum perforatum L. and Hypericum barbatum L.) in Serbia 906 

Snezana Pavlovic, Dragana Josic, Mira Starovic, Sasa Stojanovic, Goran Aleksic, 

Vera Stojsin and Dragoje Radanovic

S 


ARTICLES 

Insecticidal activity of the essential oil of Lonicera japonica flower 

buds and its main constituent compounds against two grain 

storage insects 912 

Hai Yan Zhou, Na Na Zhao, Shu Shan Du, Kai Yang, Cheng Fang Wang, 

Zhi Long Liu and Yan Jiang Qiao 

Research on herbal combinations of traditional Chinese medicine 

for chronic gastritis based on network biology 918 

Peng Lu, Qingqiong Deng, Chenghe Shi, Yibao Gao, Jianqiang Yi, 

Mingquan Zhou, Yiping Yang and Yuhao Zhao 

GC-MS analysis of volatile oils from Bupleurum chinense DC. f. 

vanheurckii (Muell.-Arg.) Shan et Y.Li 926 

LIU Ze-kun and Chen-Haixia 

Isolation of cinnamic acid derivatives from the root of Rheum 

tanguticum Maxim.ex Balf. and its significance 929 

Liangliang Gao, Xudong Xu, Haijiang Nan, Junshan Yang, Guangli Sun, 

Haifeng Wu and Mingliang Zhong

Journal of Medicinal Plants Research Vol. 6(5), pp. 641-644, 9 February, 2012 

Available online at http://www.academicjournals.org/JMPR 

DOI: 10.5897/JMPR11.014 

ISSN 1996-0875 ©2012 Academic Journals 

Review 

Monograph of Tribulus terrestris 

Ghazala Shaheen 1 *, Irshad Ahmad 2 , Khan Usmanghani 3 Naveed Akhter 1 , Mukhtiar Ahmad 1 , 

Sabira Sultana 1 and M. Akram 3 

University College of Conventional Medicine, Faculty of Pharmacy and Alternative Medicine, The Islamia University 

Bahawalpur, Pakistan. 

Accepted 19 December, 2011 

Tribulus terrestris has long been used as a tonic and aphrodisiac, and a diuretic in Unani system of 

medicine. The diuretic effect was attributed to the presence of potassium salts in high concentration. 

So many studies have been done on pharmacological activities of T. terrestris. The major constituents 

of these plants are steroidal saponins namely: terrestrosins A, B, C, D and E, desgalactotigonis, Fgitonis, 

desglucolanatigoneis, gitnin etc. The biological activity exhibited by saponins include: 

pisicidal, antimicrobial, molluscicidal, haemolytic, antiviral, cytotoxic, antihepatotoxic, spermicidal, 

insecticidal, antioedematous, antiulcer analgesic, immunomodulatory, and sedative effects. 

Key words: Tribulus terrestris, steroidal saponins, aphrodisiac, diuretic. 

INTRODUCTION 

Tribulus terrestris is a flowering plant of the 

family Zygophyllaceae. It is native to warm temperate and 

tropical regions of the Old World in Southern Europe, 

Southern Asia, throughout Africa, and Australia 

(http://en.wikipedia.org/wiki/Tribulus_terrestris). 

Family: Zygophyllaceae 

Genus: Tribulus 

Species: Terrestris Linn. 

Botanical synonym: Tribulus languinosus Linn. 

English: Land-caltrops, Puncture-vine 

Part used: Dried spiny fruit 

Botanical description 

T. terrestris is an annual or perennial, prostrate herb with 

many slender, spreading branches and silky-villous 

young parts (Matthew et al., 1983). Leaves are abruptly 

simple, pinnate and opposite. Leaflets almost sessile, 

rounded or oblique at the base, mucronate at the apex, 

flowers bright yellow, solitary, pseudo axillary or leaf 

opposed. Fruits are 5 angled or winged spinous 

tuberculate woody schizocarp, separating into five cocci, 

each coccus having two long, stiff, sharp divaricate 

*Corresponding author. E-mail: ghazala.shaheen@iub.edu.pk. 

spines towards the distal half and two shorter ones 

nearer the base, seeds one or more in each coccus 

(Matthew et al., 1983). 

Geographical distribution 

The plant grows wild throughout India, the shrub thrives 

in well irrigated black soil upto attitudes of 3000 m. 

(Matthew et al., 1983) (Figure 1). 

Traditional uses 

The roots and fruits are sweet, cooling, diuretic, 

aphrodisiac, emollient, appetiser, digestive, anthelmintic, 

expectorant, anodyne, anti-inflammatory, alterant, 

laxative, cardiotonic, styptic, lithotriptic and tonic. They 

are useful in strangury, dysuria, vitiated conditions of vata 

and pitta, renal and vesical calculi, anorexia, dyspepsia, 

helminthiasis, spermatorrhoea, anaemina, scabies, 

ophthalmia, ulocace and general weakness. The leaves 

are astringent, diuretic, aphrodisiac, depurative, 

anthelmenthic and tonic. They are useful in gonorrhoea, 

inflammation, menorrhagia, strangury, leprosy, skin 

diseases, verminosis and general weakness. The seeds 

are astringent, strengthening and are useful in epistaxis, 

haemorrhages and ulcerative stomatitis. The ash of the 

whole plant is good for external application in rheumatic-

642 J. Med. Plants Res. 

Figure 2. Diosgenin. 

Figure 3. Ruscogenin. 

Figure 1. Tribulus terrestris. 

arthritis. The diuretic properties of the plant are due to the 

large quantities of the nitrates present as well as the 

essential oil which occurs in the seeds (Nadkarni et al., 

1993). 

Phytochemistry 

The major constituents of this plants are steroidal 

saponins 1 namely terrestrosins A, B, C, D and E, 

desgalactotigonis, F-gitonis, desglucolanatigoneis, gitnin 

etc., which on hydrolysis yield jdiosgenis, hecogenis and 

neotigogenin etc. (Zafar et al., 1989). There are other 

minor constituents like alkaloids (uncharacterised) (Yan 

et al., 1996) common phyto sterols namely, �-sitosterol, 

stigmasterol, a cinnamic amide derivative - terrestiamide 

and 7-methylhydroisdamone (Mahato et al., 1978). 

Active constituents 

The fruit of T. terrestris contain of glycosides, especially 

saponin glycosides. The saponins on hydrolysis yields 

diosgenin, ruscogenin, gitogenin, three flavone 

glycosides etc. (Figures 2, 3 and 4). 

PHARMACOLOGY 

Antiurolithiatic activity 

In a preliminary study the diuretic effect of T. terrestris 

and Hygrophila spinosa water extracts in albino rats was 

evaluated. The diuretic effect was attributed to the 

presence of potassium salts in high concentration 

(Kumari and Iyer, 1967). The diuretic action with minimal 

side effect of T. terrestris in albino rats was confirmed

Figure 4. Gitogenin. 

(Singh et al., 1991). Further studies were conducted to 

evaluate the therapeutic use of T. terrestris in various 

urinary disorders including urolithiasis. The ethanol 

extract was tested for activity against artificially induced 

urolithiasis in albino rats. The extract was administered 

orally at 25, 50 and 100 mg/kg daily for 4 months. It 

exhibited dose dependent antiurolithiatic activity and 

almost completely inhibited stone formation. Other 

biochemical parameters in urine and serum which were 

altered during the process of stone formation were 

normalized by the plant extract in a dose dependent 

manner (Anand et al., 1994). The effect of an aqueous 

extract of T. terrestris administered orally at a dose of 5 

g/kg body weight was studied in rats with induced 

hyperoxaluria (Intraperitoneal injection of 4 –OH proline 

at a dose of 2.5 g/kg body weight for three successive 

days) and maintained by sodium glycolate, twenty four 

hour urine was collected and analyzed for creatinine and 

oxalate. The oxalate excretion reversed to normal from 

1.97 + 0.314 to 0.144 + 0.004 mg/mg creatinine (P


T. terrestris has the action of dilating coronary artery and 

improving coronary circulation and thus has better effects 

on improving ECG of mycocardial ischemia. No adverse 

reaction on blood system, hepatic and renal functions 

were noticed (Bowen et al., 1990). 

Safety profile 

The ethanolic (95%) extract was tested in rats 

intraperitoneally and the LD50 was found to be 56.4 

mg/kg (Dhar et al., 1968). The maximum tolerated dose 

in mouse was 100 g/kg, the extract used ethanol and 

water (1:1) intraperitoneally (Chakraborty and Neogi, 

1978). 

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of certain fractions of Tribulus terrestris fruits against experimentally 

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Tribulus terrestris '. Ind. J. Pharm. Sci., 40: 50-52. 

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of Hygrophila spinosa and Tribulus terrestris" Ind. J. Med. Res., 

55(7): 714-716. 

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plants for diosgenis "J. Ind. Chem. (India), 50(1): 49-50. 

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Published by The Rapinat Herbarium St.Joseph’s College- 

Tiruchirapalli, p. 185. 

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1230. 

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terrestris Linn (Chota Gokharu)”. Ind. Drugs, 22(6): 332 -333. 

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66. 

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“Therapeutic response of Tribulus terrestris (Gokhru) aqueous extract 

on hyperoxaluria in male adult rats”. Phytother. Res., 7: 116-119. 

Singh RG, Singh RP, Usha KP, Shukla KP, Singh P (1991). 

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terrestris Linn.) on albino rats", J. Res. Educ. Ind. Med., 10(1): 19-21. 

Yan W, Ohtani K, Kasai R, Yamasaki K (1996). " Steroidal saponin from 

fruits of Tribulus terrestris”. Phytochemistry, 42(5): 1417-1422. 

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knowledge". Ind. Drugs, 27(3): 148-158. 

http://en.wikipedia.org/wiki/Tribulus_terrestris



DOI: 10.5897/JMPR11.090 


Full Length Research Paper 

The effects of thyme (Thymus vulgaris) extract 

supplementation in drinking water on iron metabolism 

in broiler chickens 

Rahim Abdulkarimi 1 and Mohsen Daneshyar 2 * 

1 Islamic Azad University, Boukan Branch, Boukan, Iran. 

2 Department of Animal Science, Faculty of Agriculture, Urmia University, Urmia, Iran. 

Accepted 23 March, 2011 

The effects of 0 (ZT), 0.2 (LT), 0.4 (MT) and 0.6% (HT) thyme extract in drinking water on the amounts of 

blood iron, hemoglobin (Hb), red blood cell (RBC), hematocrit (Hct) and total iron binding capacity 

(TIBC) on days 21 and 42 broilers were investigated. No significant differences were observed between 

the treatments for the amounts of blood Hb, RBC and Hct, but blood iron concentration of birds that 

received the thyme extract was lower as compared to that of control birds (ZT) on both ages (P


bioavailable. The absorption of this form is not affected 

by other factors presented in foods. Non heme iron is the 

other form of iron present in vegetable, cereals and dairy 

product (Saltzman and Russell, 1998). Adversely with 

heme iron, this form have a low absorption and markedly 

affected by gastro intestinal acidity, tannins, polyphenols, 

phytates, calcium and phosphate (Kaltwasser et al., 

1998; Hurrell et al., 1999; Davidsson et al., 2001; Cook et 

al., 1991). The presence of absorption inhibitors such as 

phytic acid or polyphenol compounds in the plant foods is 

a major cause of iron deficiency (Fairweather and Hurrell, 

1996). Polyphenol compounds are widely present in the 

human diets as components of fruits, vegetable, spices, 

cereals, tea, coffee, red wine, cacao and different herb 

teas (Hurrell et al., 1999). The phenolic compounds are 

released from the foods and beverage during digestion 

process and can complex with iron and making it 

unavailable for absorption (Hurrell et al., 1999). 

Negative effects of phenolic compounds in black tea 

(Thankschan et al., 2008), grape seed extract (King et al., 

2008), spinach and aubergine (Gillooly et al., 1983) on 

iron absorption has earlier been reported. However no 

information is published regarding the thyme plant or its 

extract effects on iron absorption. Thymus vulgaris is a 

perennial medicinal herb in the Lamiaceae family and 

cultivated throughout the world for culinary, cosmetic and 

medical purposes. This species has special activities 

such as antispasmodic, expectorant, antiseptic, 

antimicrobial and antioxidant (Abudarwish et al., 2009; 

Hertrampf, 2001). Herbal thyme contains about 1 to 2.5% 

essential oils (Franz et al., 2005) such as thymol (44.4 to 

58%), carvacrol (2.4 to 4.2%) and γ-terpipen (6.9 to 

18.9%) that have been identified as the phenolic 

compounds (Sengul et al., 2008). 

Since thyme extract does have essential oil, tannins, 

glycosides, saponins and other components (Escop, 

2003) and inhibitory effects of these components were 

indicated previously, it was hypothesized that thyme 

extract may changes the iron metabolism. So thyme 

extract supplementation in drinking water effects were 

evaluated on the amounts of blood iron, Hemoglobin 

(Hb), red blood cell (RBC), Hematocrit (Hct) and total iron 

binding capacity (TIBC) in broiler chickens. According to 

previous suggestions of Conway et al. (2006), plasma 

iron concentration was used as an indicator of iron 

absorption in recent experiment. These authors provided 

some beneficial information regarding the use of serum 

iron concentration in individuals as a good estimate of 

relative bioavailability and iron absorption in foods or 

meals. 

MATERIALS AND METHODS 

One hundred and sixty one-day-old broiler chicks (Ross 308) were 

provided from a local hatchery, weighed on arrival and randomly 

divided between 16 pens (1×1 m) of 10 birds each and each four 

were assigned to each treatment. Water and feed were provided for 

ad libitum consumption. All the chickens were fed the same starter 

(from day one to 21 of age) and grower (from day 22 to 42 of age) 

diets in pellet form (Table 1) but received 0.0% (ZT), 0.2% (LT), 

0.4% (MT) and 0.6% (HT) T vulgaris alcoholic extract (having 0.06 

thymol and pH=5) in drinking water from day one to day 42 of age. 

T. vulgaris alcoholic extract was prepared using the standard 

maceration method of Zhang et al. (2005). Vegetative parts of the 

shade dried T. vulgaris full bloom stage were crushed and soaked 

in ethanol 80% in 1:5 ratios (w/v) for 72 h on a shaker. The extract 

strained and its thymol content pH were determined by TLC method 

(Thin Layer Chromatography) and a pH meter instrument (HACH, 

HQ40D, USA), respectively. 

At days 21 and 42 of age, two birds per pen (eight per dietary 

treatment) were randomly selected and killed by decapitation to 

obtain the blood samples. Blood in microcapillary tubes were used 

for blood hematocrit (Hct) measurements after centrifugation (5000 

rpm) for 7 min. Blood samples were collected in anticoagulant tubes 

(citrate sodium 3.6% solution). A commercial kit (Zist-Shimi 

Company, Iran) was used for hemoglobin determination. In this 

method, ferrous ions of hemoglobin were oxidized to the ferric state 

by potassium ferricyanide to form hemiglobin (methemoglobin). 

Hemiglobin reacts with cyanide to form hemiglobincyanide 

(cyanmethemoglobin) that can be measured spectrophotometry. 

The amounts of blood iron and TIBC were determined 

colorimetrically with a commercial kit (Zist-Shimi Company, Iran) 

using a spectrophotometer (Unico 2100, Japon). Red blood cells 

concentration was determined by a hemocytometer manually. The 

data were subjected to one way analyses of variance using SAS 

statistical package, version 9.1 (SAS Institute, Cary, NC, USA) and 

analyzed based on a completely randomized design using the 

General Linear Model procedure. Turkey–Kramer Multiple 

Comparison Test at significance level of 0.05 was used to compare 

the mean values. In addition, regression models (linear and 

quadratic) were performed to show the changes in the amounts of 

blood iron and TIBC by thyme extract supplementation at days 21 

and 42 of the experiment. 

RESULTS 

The amounts of blood iron, Hb, and RBC, Hct and TIBC 

of the birds in different treatments are presented in Table 

2. There was no significant differences between the 

treatments for the amounts of blood Hb, and RBC and 

Hct at days 21 and 42 of age (P>0.05) but blood iron 

concentration of all the thyme extract received birds was 

lower as compared to that of control birds (ZT). Blood 

iron concentration of LT and MT birds was lower than that 

of ZT birds but higher than that of HT birds (P

Table 1. Composition of experimental diets. 

Abdulkarimi and Mohsen Daneshyar 647 

Ingredients (%) Starter (0-21 d) Grower (21-42 d) 

Corn 54.87 61.78 

Soybean meal (44% protein) 36.72 26.36 

Fish meal 1.31 4.50 

Vegetable oil 3.00 4.00 

Limestone 1.15 1.05 

Dicalcium phosphate 1.94 1.49 

Vitamin and mineral premix 1 0.50 0.50 

Salt 0.30 0.30 

DL-methionine 0.21 0.02 

Total 100.00 100.00 

Calculated analysis 

ME (kcal/kg) 2937 3100 

CP (%) 21.44 19.37 

Calcium (%) 1.05 1.00 

A. Phosphorus (%) 0.51 0.50 

Sodium (%) 0.16 0.14 

Arginine (%) 1.41 1.23 

Methionine + Cystine (%) 0.91 0.69 

Lysine (%) 1.20 1.10 

Tryptophan (%) 0.31 0.26 

1 provide per kilogram of diet: retinol, 15000 IU; cholecalciferol,8000 IU; vitamin K3, 3 mg; B12, 15 µg; niacin, 32 mg; choline, 840 mg; 

biotin, 40 µg; thiamine, 4 mg; riboflavin, 6.6 mg; pyridoxine, 5 mg; folic acid, 1 mg; zinc, 80 mg; manganese, 100 mg; selenium, 200 

mg; iron, 80 mg; magnesium, 12; copper, 10 mg; calcium, 15 mg; iodeine,1 mg. 

Table 2. blood Hematocrit (Hct) and total iron binding capacity (TIBC) percents, and Hemoglobin (Hb), Red blood cell (RBC), iron 

concentrations of broiler chickens 1 received free thyme extract water (ZT) or 0.2 (LT), 0.4 (MT) and 0.6% (HT) of thyme extract in 

drinking water. 

Treatment Hct (%) Hb (g/dl) 

RBC 

(× 10 6 cells/mm3) 

iron (mg/dl) TIBC (%) 

d 21 d 42 d 21 d 42 d 21 d 42 d 21 d 42 d 21 d 42 

ZT 26.75 28.5 8.9 9.5 2.97 3.17 15.88 a 16.88 a 41.63 b 35.50 c 

LT 27.75 25.75 9.24 8.58 3.21 2.86 12.88 b 15.00 b 46.50 a 41.38 ab 

MT 26.88 26.38 8.96 8.79 2.99 2.93 13 b 14.63 b 47.13 a 40.88 b 

HT 26 26.13 8.67 8.71 2.89 2.9 11.13 c 12.62 c 48.13 a 42.75 a 

P value 0.48 0.33 0.48 0.33 0.14 0.33 0.0001 0.0001 0.0001 0.0001 

SEM 0.38 0.57 0.13 0.19 0.05 0.06 0.3 0.34 0.51 0.54 

a-b Means with no common superscript letter in each columns differ significantly (P


Blood iron (mg/dl) 

18 

16 

14 

12 

10 

8 

6 

4 

2 

0 

day 21 day 42 

0 0.2 0.4 0.6 

Thyme extract (%) 

Figure 1. The relationship between the level (%) of Thymus vulgaris supplementation in drinking 

water and the blood iron concentration at day 21(quadratic regression, y = 13.6 - 1.68x + 0.281x 2 , 

P

used as a model to study the cystic fibrosis (Craig- 

Schmidt et al., 1986) and proposed to study the cause of 

Kashin-Beck disease (Cook, 2000) in human. 

No effects of thyme extract on the amounts of blood 

Hct and Hb and RBC were observed in present study. 

Consistently results have been published regarding the 

nonexistence effects of phenolic compounds on these 

indices. No association was found between the black tea 

consumption and either of blood hemoglobin or ferritin 

concentrations in adult South African (Hogenkamp et al., 

2008). Siegenberg et al. (1991) did not indicate any 

changes of blood hemoglobin concentration in human 

subjects that fed from a bread meal containing different 

proportions of phytate-containing maize bran (14 to 58 

mg) or from a bread meal with increasing doses of tannic 

acid (12 to 833 mg) in his attempt to explain the inhibitory 

effects of polyphenols and phytates on iron absorption by 

ascorbic acid,. In other consistent results, Wren et al 

(2002) observed no changes of blood hemoglobin, RBC 

or MCV in Dawley rats fed 0, 0.5, 1.0, or 2.0% grape 

seed extract for a period of 90 days but show decreased 

serum iron levels in rats fed the high-dose extract. 

As expected thyme extract supplementation decreased 

the blood iron concentration in broiler chickens. There is 

no information regarding the inhibitory effects of thyme on 

iron absorption but in a contradictory research, no effects 

of red (high polyphenol contain) and white (low 

polyphenol contain) beans (Pha seolus vulgaris L) was 

indicated on iron bioavailibilities in pigs (Tako et al., 

2009). However, the inhibited absorption of iron by 

phenolic compounds from other plants or their extracts is 

in agreement with the results of our experiment. Drinking 

of 200 ml tea (prepared from 5 g dry tea) by individuals 

inhibited the iron absorption from solutions of FeCl3 and 

FeSO4, bread, a meal of rice with potato and onion soup. 

The authors connected this inhibition effects to the tannin 

contents of tea and the formation of insoluble iron-tannin 

complex in the intestinal lumen (Disler et al., 1975). 

Hurnell et al. (1999) observed the inhibited iron 

absorption by tea from a bread meal by 50-90% 

depending on the kind of the tea and the concentration of 

polyphenols in the brewed tea. 

Thankschan et al. (2008) investigated the effects of 

drinking tea (1 or 2 cup black tea) on iron absorption in 

iron deficient and iron adequate foods with rice meal in 

women and observed an inhibited iron absorption by a 

similar amount in both groups and mentioned the low 

bioavailability of iron from plant-based diet containing 

mineral absorption inhibitors such as polyphenols and 

phytates as the reason. Moreover a negative correlation 

was observed between the amount of spinach and 

aubergine (rich in polyphenols) with iron absorption in 

men (Gillooly et al., 1983). As same as tea, spinach, 

aubergine and coffee, thyme extract have polyphenolic 

components such as essential oils (Escop, 2003). Thymol 

and carvacrol phenols are the main constituent volatile 

oils of thyme extract (Massada, 1976). Phenolic 

Abdulkarimi and Mohsen Daneshyar 649 

compounds influence the iron absorption by complexing 

iron in the intestinal lumen. The functional group of 

polyphenol compounds is an aromatic ring structure with 

one or more hydroxyl groups and combines with iron and 

causes destruction of iron absorption (Harborne, 1986). 

Moreover, phenolic compounds could inhibit the iron 

absorption by lowering the intestinal permeability 

(Harborne, 1986). King et al. (2008) examined the 

influence of the dietary polyphenols epigallocatechin-3gallate 

(EGCG) and grape seed extract (GSE) on 

transepithelial iron transport in CaCo -2 intestinal cells and 

reported the inhibited non heme iron absorption by 

polyphenol compounds from apical iron import in 

intestinal cells. Besides thyme contains the components 

such as tannins, glycosides and saponins (Escop, 2003) 

that affect the iron absorption. It was suggested that 

saponins may interfere with iron metabolism either by 

forming complexes with the dietary iron thereby 

unavailable for absorption or by producing changes in 

mucosal function with long-term consumption thus 

reducing the efficiency nutrients absorption (Southon et 

al., 1988a, b). Siddiqi (1994) reported the reduced iron 

absorption by Cicer arietinum consumption (rich in 

saponins) and the insoluble iron-saponin complex 

formation at the stomach pH. Thyme extract saponins 

and its low pH may be other reason of low iron absorption 

in our study. We used a T. vulgaris extract with low pH 

(pH = 5) that possibly has caused the greater decrease in 

gastrointestinal pH and consequently the easier formation 

of complex between saponin and iron in the intestinal 

lumen. 

An opposite relations for blood TIBC and iron with the 

thyme extract supplementation were found in our 

experiment. This shows that thyme supplementation 

decreases the blood iron concentration and increases the 

blood iron requirements for maximum saturation of blood 

transferring since TIBC is the blood capacity to bind iron 

with transferrin or the amount of iron needed to 100% 

saturation of transferrin. Blood TIBC often increases in 

iron deficiency and decreases in choronic inflammatory 

disorders and hematochromatosis (Tietz, 1999). In the 

same way, Furugouri (1972) observed higher blood TIBC 

at 10 and 20 days of age in the pigs with anemia and 

unsupplemented ferrous fumarate diets than in pigs 

receiving ferrous fumarate. 

In conclusion, supplementation of thyme extract in 

drinking water decreases the blood iron concentration in 

broiler chickens and possibly in human. Inhibited 

absorption of iron by phenolic compounds or the 

saponins in thyme extract is the possible reason. 

ACKNOWLEDGEMENTS 

We thank Farhad Farhanpajhoh (Central Laboratory of 

Veterinary, Faculty of Veterinary, Urmia University, Iran) 

for helping during the laboratory works of the experiment.


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DOI: 10.5897/JMPR11.111 



Effects of borage extract in rat skin wound healing 

model, histopathological study 

Mohammad Reza Farahpour 1 * and Amir Hossein Mavaddati 2 

1 Department of Veterinary Surgery, Islamic Azad University, Urmia Branch, Urmia-Iran. 

2 DVM, Department of Veterinary Surgery, Islamic Azad University, Urmia Branch, Urmia-Iran. 

Accepted 23 March, 2011 

Nowadays, borage flowers and leaves are used medicinally for wound healing because of plant 

constituents, gamma-linolenic acid, alpha linolenic acid and delta-6-fatty acid. The aim of the present 

study was to evaluate the ability of borage extract ointment wound healing process. Under surgical 

anaesthesia, excisional wounds were made on back of 30 rats by punching. Rats were divided into 3 

groups of borage 1.5% ointment, eucerin-vaseline, and control. All the rats were treated with topical 

ointments daily for 21 days. Histopathological examination was performed on the 3rd, 7th, 14th, and 

21st days and the wound healing was assessed. The results showed that wound size of the test groups 

were reduced early as compared to control group. The significant results of histopathological 

evaluation were obtained with borage, when compared to the other groups as well as to the control and 

the placebo. Present study demonstrated that borage extract was capable of promoting wound healing 

process. 

Key words: Borage, herbal extract, histopathological study, rat, wound healing. 

INTRODUCTION 

Nowadays, the promotion of wound healing in some 

diseases and chronic disorders with the aid of herbal 

extracts is more challenging than ever before. That is 

why new compounds prepared for this purpose have 

been widely accepted. Borage (Echium amaenum) is a 

large hairy annual herb that is a member of Boraginaceae 

family. It grows in most of Europe, in the Mediterranean 

region, and also in northern parts of Iran. The flowers are 

bright blue and star-shaped and the fruit consists of four 

brownish-black nutlets. Borage flourishes in ordinary soil 

and may be propagated by division of rootstocks and by 

cuttings of shoots in sandy soil in a cold frame in summer 

and autumn or from seeds sown in good light soil from 

mid of March to May. The flowers and the leaves of 

borage are used medicinally in France as an antifebrile, 

antidepressive, for the treatment of stress and of 

circulatory heart diseases, for pulmonary complaints, as a 

poultice for inflammatory swellings (Kapoor and 

*Corresponding author. E-mail: mrf78s@gmail.com. Tel: 

+98(441)4373676. Fax: +98(441)3460980. 

Klimaszewski, 1999), as a diuretic (due to potassium 

nitrate), as a laxative, emollient and demulcent (due to 

the mucilage), and as a possible protective factor against 

cancer (Gonzalez et al., 1993). The plant constituents 

have been isolated by different investigators; they include 

gamma-linolenic acid (GLA), alphalinolenic acid (ALA), 

delta 6 fatty acid denaturase, delta 8 sphingolipid 

desaturase (Coupland, 2008), pyrrolizidine alkaloids, 

mucilage, resin, potassium nitrate, and calcium salt 

combined with mineral acids. Therefore, it can improve 

wound healing process. 

Normal healing response begins immediately after 

tissue injuring. Platelets, the cells present in the highest 

numbers shortly after a wound occurs, release a number 

of things into the blood, including ECM proteins and 

cytokines, including growth factors. Growth factors 

stimulate cells to speed their rate of division. Platelets 

also release other proinflammatory factors like serotonin, 

bradykinin, prostaglandins, prostacyclins, thromboxane, 

and histamine, which serve a number of purposes, 

including to increase cell proliferation and migration to the 

area and to cause blood vessels to become dilated and 

porous. Following homeostasis, the neutrophils then


enter the wound site and begin the critical task of 

phagocytosis to remove foreign materials, bacteria and 

damaged tissue. As part of this inflammatory phase, the 

macrophages appear and continue the process of 

phagocytosis as well as releasing more PDGF and TGF . 

Once the wound site is cleaned out, fibroblasts migrate in 

to begin the proliferative phase and deposit new 

extracellular matrix. The new collagen matrix then 

becomes cross-linked and organized during the final 

remodeling phase. In order to this efficient and highly 

controlled repair process, numerous cell-signaling events 

are required (Robert and Melissa, 2004). Many biological 

dressings and indigenous medicines have been reported 

to possess wound healing properties. However, none of 

these has been completely effective and free of side 

effects. Many herbal drugs can be effective in wound 

healing acceleration. 

The wound healing activities of plants have since been 

explored from ancient times. With the development of 

scientific research methods, the significant successes 

reported have led to investigation into medicinal plants 

with a view to confirming these acclaimed properties. 

Findings have been shown that different parts of plants 

contain some active components that are antimicrobial 

and nutritive in function that could have benefits on 

wound healing process. Borage is a hairy annual herb 

commonly known as ‘borage. It has been known for its 

mood elevating properties. The plant is reputed as 

antispasmodic, antihypertensive, antipyretic, aphrodisiac, 

demulcent, diuretic and is also considered useful to treat 

asthma, bronchitis, cramps, diarrhea, palpitations and 

kidney ailments (Duke et al., 2002). Decoction of the 

plant is used as nerve and cardiac tonic and a home 

remedy for blood purification. 

Phytochemical studies reveal the presence of tannins, 

resins, ascorbic acid, beta-carotene, niacin, riboflavin, 

thiamine, silicic acid, choline arabinose, unsaturated 

pyrrolizidines alkaloids including amabiline, lycopsamine 

and supinidine, polyphenolics including phenolic acid, 

vanillic acid, p-coumaric acid, p-hydroxy benzoic acid, 

gentisic acid, caffeic acid, rosmarinic acid and 

chlorogenic acid, scopoletin and flavonoids (Gudej and 

Tomczyk, 1996). 

Borage oil has been reported to lower serum 

cholesterol, phospholipids and triglyceride levels (Gu et 

al., 1998) and increases the levels of omega-6 

polyunsaturated fatty acids in the plasma, liver, aorta and 

renal artery tissues. Dietary use of borage oil exhibited 

immuno-modulatory (Harbige et al., 2000) and blood 

pressure lowering effects in normal and spontaneously 

hypertensive rats through unknown mechanism. It 

exhibited kidney protective potential through angiotensin 

II receptor blockade (Engler et al., 1998), cytotoxic and 

free radical scavenging activities (Bandoniene and 

Murkovic, 2002; Lin et al., 2002). Despite the fact that 

borage has been used traditionally, it has not been widely 

studied to justify its use in abdominal colic, diarrhea, 

asthma and hypertension. In this investigation, we report 

the presence of Ca +2 antagonist-like constituents, which 

provide the pharmacological basis for the use of borage 

in hyperactive gastrointestinal, respiratory and 

cardiovascular disorders. As a dietary supplement, 

borage oil (BO) has been reported to exert clinical 

efficacy in a variety of skin diseases and in the 

suppression of proliferation and inflammation in the skin 

(Chung et al., 2002; Mork-Hansen et al., 1983). The 

major constituent of BO underlying this clinical efficacy is 

believed to be oxidative metabolites of essential fatty 

acids (EFA), gama-linolenic acid (GLA) prostaglandin E1 

(PGE1), and 15-hydroxyeicosatrienoic acid 15HETrE), 

which have been reported to exert antiproliferative and 

anti-inflammatory effects in vitro (Chung et al., 2002; 

Miller et al., 1988). Fatty acids belong to a class of 

compounds formed by a long hydrocarbon chain and a 

terminal carboxylic group. They have three main 

functions: They are structural components of biological 

membranes, they act as precursors of intracellular 

messengers and they are oxidized to produce adenosine 

triphosphate (ATP) (Hatanaka and Curi, 2007). 

From the beginning of the 1970s, there have been 

studies on the effects of fatty acids on the immune 

response. Such compounds interfere with various events 

of the inflammatory process, such as vascular 

contraction, chemotaxis, adhesion, diapedesis, activation 

and cell death, where the majority of these occur via 

arachidonic acids such as prostaglandins, leukotrienes, 

thromboxanes and lipoxins (Hatanaka and Curi, 2007). 

Polyunsaturated fatty acids (PUFAs) should be pointed 

out among the various fatty acids present in the plasma 

and in leukocytes. Besides their structural function, they 

can modulate cell to cell interactions and intracellular 

signaling. Thus, the alteration of the composition of fatty 

acids of phospholipids in the cell membrana can modify 

fluidity and change the binding of cytokines to their 

receptors (Cardoso et al., 2004). Linoleic acid is an 

essential fatty acid of 18 carbons. Through a desaturation 

process, it gives rise to arachidonic acid (20 carbons), a 

precursor of prostaglandins, leukotrienes, thromboxane 

and lipoxins, which in turn act as mediators of platelet 

function and of inflammatory, vascular, motor and 

sensory processes, among others (Hatanaka and Curi, 

2007; Ortonne and Clévy, 1994). It has been observed 

that linoleic acid is capable of inhibiting the growth of 

Staphylococcus aureus by inhibiting protein synthesis of 

cell wall, nucleic acids and cell membrane during division. 

Linoleic acid has also been shown to participate in cell 

proliferation and inflammatory process, where in the latter 

it plays a role as a mediator of leukocyte function having 

chemotactic and stimulatory effects on neutrophils (Moch 

et al., 1990). 

In present study, number of leukocytes and other 

parameters that show the repair development such as 

congestion, neovascularization, leucocytes (neutrophils, 

lymphocytes and etc), fibroblast level, stratum corneum,

epithelium thickness, collagen mass, collagen maturation, 

granulation tissue level and fibrin level. 

Grading was according following criteria: For example 

in study region, if lymphocytes are more than 90% of total 

counted cells, the grade is four, between 60 to 90% the 

grade is three, between 40 to 60% the grade is two, 

between 10 to 40% the grade is one and at last less than 

10% or none, the grade is zero. 


Plant material and preparation of the extract 

The borage found in Iran is Echium amoenum, which is different 

from the borage grown in Europe, Borago officialis L. 

(Boraginaceae). Dried borage flowers were collected from 

Hamedan province, in northwestern Iran in mid August. Cold 

aqueous extract (pH 5.8) of dried E. amaenum flowers (5%, w/v) 

was used in all the experiments. Dried flowers (15 g) were steeped 

for 6 h at 4°C in 300 ml distilled water, with consta nt stirring. The 

material was centrifuged and the supernatant was filter-sterilized 

and then freeze-dried. 

Experimental animals 

Male Wister rats (150 to 200 g) of 2 to 3 months were used as 

experimental animals. The animals were housed in standard 

environmental conditions of temperature (22 ± 3°C), hum idity (60 ± 

5%), and a 12 h light/dark cycle. During experimental time, rats 

were given standard pellet diet (Pastor Institute, Iran) and water ad 

libitum. 

Surgical procedures 

After anaesthesia induction with xylizaine 2% and ketamine 10% 

(I.M. 60 mg/kg) rats were fixed in ventral posture on surgery table. 

Then the dorsal area from scapula to ilium were scrubbed and 

prepared to surgery. Two circle shapes, full thickness surgical 

wounds with 7 mm diameters in both side of the backbone, 1 cm 

away from backbone and 5 cm away from each other were made 

with biopsy punch with 7 mm. With this excisional wounding 

method, epidermis, dermis, hypodermis and Panniculus Carnosus 

layers were removed completely (Luisa and DiPietro, 2003). 

Treatment 

After made of surgical wound, all rats randomly were colored with 

none toxic color and divided to three groups. In group 1, ointment 

with 1.5% borage extract was administered. Group 2 as control did 

not receive any administration and group 3 as placebo were 

administrated with eucerin and Vaseline. All rats were followed 21 

days. Daily observation was performed and any wound fluid or any 

evidence of infection or other abnormalities were noted. 

Histopathological study 

The healing tissues samples obtained during days 3, 7, 14 and 21, 

from all four groups of animals and were processed for histological 

study. The samples fixed in formalin and installed on slides, stained 

with Hematoxylin and Eosin and were reviewed under light 

microscope. Recorded factors were eschar, inflammatory cells, 

Farahpour and Mavaddati 653 

kind of inflammatory cells, angiogenesis, fibroplasia, epithelial 

growth, hyperemia, collagen density fibroblast, fibrin, thickness of 

corneal layer and Fibroblastic aggregation. 

Statistical analysis 

The relative wound area was statistically analyzed using one-way 

ANOVA by the program SPSS 16 and comparison of the means of 

the wound areas at different days evaluated by Turkey test at P < 

0.05 level. 

RESULTS 

Histopathological results 

The extract of borage showed significant wound healing 

activity when topically administered in rats. The wound 

area measurement showed the wound size of the test 

groups were reduced early as compared with control 

group. The best histopathological evaluation were 

obtained from borage in comparison with other groups as 

well as compared with control and the placebo (Table 1). 

3 rd day of 1.5% borage ointments 

After evaluation of samples with 1.5% ointment 

administration, it is observed that eschar formation was 

low but volume of inflammatory cells in tissue section was 

high. Neutrophil was the main of these cells. The 

angiogenesis was mild and the primary stage of 

fibroplasia was clear. There was no growth in epithelial 

cells region. Also, there was a low amount of tissue 

collagen and fibroblastic concentration. But there was a 

high volume of fibrin in area (Table 1). 

7 th day of 1.5% borage ointments 

In day 7 eschar formation was low but there is medium 

amount of inflammatory cells in tissue section. The most 

of these cells were lymphocytes. The angiogenesis and 

fibroplasia was low. Also, there was a mild epithelial cell 

growth. Fibrin level was low but collagen level and 

fibroblastic concentration was medium. The thickness if 

corneum layer and fibroblastic maturation was not the 

same as day 3 (Table 1). 

14 th day of 1.5% borage ointments 

In day 14, there was no eschar and levels of 

inflammatory cells were reduced. The majority of these 

cells were lymphocytes and angiogenesis is low. But 

fibroplasia and epithelial growth was increased. Collagen 

levels were low and fibroblastic concentration was 

increased. There was little amount of fibrin. The thickness


Table 1. effect of topical administration of borage oil extract on wound healing during days 3, 7, 14 and 21. 

Studied factors 

groups 

Eschar 

Inflammatory 

cells infiltration 

Kind of 

inflammatory 

cells 

Angiogenesis Fibroplasia 

Epithelial 

growth 

Hyperemia 

Collagen 

density 

Fibroblast Fibrin 

Thickness of 

corneal layer 

3 rd day.1.5% 1+ 3+ Neutrophil 2+ +1 0 1+ 1+ 1+ 3+ 0 0 

7 th day.1.5% 1+ 2+ Lymphocyte 1+ 2+ 1+ 1+ 2+ 2+ 1+ 0 0 

14 th day.1.5% 0 1+ Lymphocyte +1 3+ 3+ 1+ 2+ 3+ 0 1+ 1+ 

21 st day.1.5% 0 1+ Lymphocyte 0 3+ 3+ 0 3+ 3+ 0 2+ 2+ 

3 rd day control group 2+ 1+ Neutrophil +2 1+ 0 1+ 0 1+ 3+ 0 0 

7 th day control group 2+ 3+ Neutrophil +2 2+ 1+ 1+ 2+ 1+ 3+ 0 0 

14 th day control group 0 1+ Lymphocyte +1 2+ 2+ 0 3+ 2+ 0 1+ 1+ 

21 st day control group 0 1+ Lymphocyte +1 2+ 2+ 0 3+ 1+ 0 1+ 2+ 

3 rd day Placebo group 2+ 3+ Neutrophil +2 1+ 0 2+ 1+ 1+ 2+ 0 0 

7 th day Placebo group 3+ 3+ Lymphocyte +3 1+ 1+ 2+ 1+ 2+ 2+ 0 0 

14 th day Placebo group 0 2+ Lymphocyte +2 2+ 2+ 1+ 2+ 2+ 0 1+ 1+ 

21 st day Placebo group 0 1+ Lymphocyte +1 2+ 3+ 0 3+ 2+ 0 1+ 2+ 

increased if corneum layer and fibroblastic 

maturation was increased (Table 1). 

21 st day of 1.5% borage ointments 

In day 21, eschar was same with scar in day 14, 

but levels of inflammatory cells were reduced. The 

majority of these cells were lymphocytes. 

Because of wound constriction, there is no 

angiogenesis and fibroblastic concentration. Fibrin 

level was very low. The thickness corneum layer 

and fibroblastic maturation was medium but 

overall it was better than control group (Table 1). 

DISCUSSION 

In this study, the process of tissue repair of 

experimentally induced cutaneous wounds in rats 

was evaluated in vivo based on the 

histopathological over time. Wound healing 

involves a complex and coordinated number of 

events which include inflammation, cell 

proliferation, and contraction of the wound and 

tissue remodeling. Thus, in this study, the effect of 

borage oil was analyzed, applied topically, on rat 

skin wound considering these results comparing 

to findings from earlier studies that demonstrated 

the efficacy of these agents in accelerating wound 

healing. 

Borage oil extract contains therapeutics that 

gamma-3, 6 fatty acids are one of the most 

effective and most knowing of them and most of 

borage oil extract therapeutic effects are referred 

to mentioned components. Research has sought 

to define benefits to wound healing of specific lipid 

types. The gamma-3 fatty acids, which exhibit 

anti-inflammatory properties by inhibiting the 

production of eicosanoids and other mediators, 

such as platelet-activating factor, interleukin-1, 

and tumor necrosis factor alpha (Albina et al., 

Fibroblastic 

aggregation 

1993; Endres et al., 1989; Simopoulos, 1991; 

Kremer et al., 1987) are among the most widely 

investigated. 

The true benefit of gamma-3 fatty acids, 

therefore, may be in their immune modulation of 

the host rather than in improved wound healing 

per second. Studies of healing burns in humans 

and guinea pigs, however, have demonstrated 

improved immune function, improved survival, and 

reduced infectious complications after the 

administration of a diet rich in gamma-3 fatty acids 

to this specific subset of injured patients 

(Gottschlich et al., 1990; Alexander et al., 1986). 

Up to the 3rd day, subjective observation 

indicated slow retraction of the wound in the 

Control group, with hyperemia and scab 

formation. In the Test group, hyperemia and 

discrete formation of scab on the edges of the 

lesion were observed. Hyperemia, edema and 

fibrin were seen in the placebo group. 

In another study in which the effects of

polyunsaturated fatty acids on the healing of cutaneous 

wounds were evaluated, a macroscopic difference in 

lesion repair was demonstrated in the first 48 h after the 

surgical procedure (Cardoso et al., 2004). The greater 

infiltration of polymorphonuclear cells and macrophages 

in the first three days can correspond to the phase of 

exudation and inflammation in the wounds treated with 

triglycerides as demonstrated in a study conducted in 

dogs (De Nardi et al., 2004). 

In the same period in the present study, there were no 

signs of exudates by macroscopic observation. From the 

3rd to the 7th day, there was progressive repair of the 

wound area, which indicated development of granulation 

tissue in concordance with findings in the literature 

(Mandelbaum et al., 2003). The macroscopic appearance 

showed the wound area with the presence of fibrin and 

scab in the Reference group, complete scab formation in 

the Control group, and discrete hyperemia in the Test 

group. 

From the 7th to 14th day, a greater rate of repair 

occurred in the Reference group. The test preparation 

showed evident granulation tissue and greater tissue 

contraction around the edges of the wound, which had 

become irregular. In the Control group, gaps in the scab 

were seen on the 7th day, along with irregular edges due 

to contraction of the wound, besides the presence of 

granulation tissue. The Reference group remained with 

the largest wound area and with irregular edges, which 

was also found in a study using linoleic acid (Marques et 

al., 2004). The treatments in the three groups contributed 

to the almost complete closing of the lesions, suggesting 

that growth factors were probably responsible for the 

hyperplasia of the epithelium as reported in the literature 

(Mandelbaum et al., 2003). 

In this period, there was agreement with a study that 

considered cytokines as being important mediators of 

neoangiogenesis, fibroplasia and maturation, which are 

released by cells such as platelets, neutrophils, 

macrophages, lymphocytes, mast cells and fibroblasts, 

making it easy to understand the importance of 

chemotactic properties of the test preparation in the 

repair of the lesions (De Nardi et al., 2004). 

A study that examined wound healing in sheep showed 

that linoleic acid constituted a powerful pro-inflammatory 

mediator, being essential for the regulation of the 

biochemical events that precede fibroplasia in addition to 

stimulating growth factors and neovascularization15. It is 

possible that in this study, the presence of linoleic acid in 

the test preparation contributed to a similar event. A 

study carried out with polyunsaturated fatty acids showed 

a tendency for the wound area to diminish in the first ten 

days of treatment, and demonstrated overall that PUFAs 

may play an important role in wound healing (Cardoso et 

al., 2004). 

On the 14th day, clinical observation showed a smaller 

wound area characterized by the proximity of the edges 

(contraction) with irregular outlines and better presence of 

granulation tissue, like that seen in the study with 

Farahpour and Mavaddati 655 

linoleic acid in sheep (Marques et al., 2004). Proliferation 

(fibroplasia and matrix formation) was demonstrated by 

scholars in the past as being extremely important in the 

formation granulation tissue. This depends on fibroblasts 

which produce elastin, fibronectin, glucosaminoglycans 

and proteases (Mandelbaum et al., 2003). The repair of 

wounds by secondary union showed that contraction 

could have been responsible for the reduction in wound 

area in the three groups; in concordance with a study in 

which contraction was shown to be able to reduce the 

surface of the cutaneous defect. 

In conclusion, present study demonstrated that borage 

oil extract was capable of promoting wound healing 

activity. Due to pharmacological evidence and 

histological observations borage oil extract accelerate 

wound healing process. 

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DOI: 10.5897/JMPR11.257 



Evaluation of antioxidant activities of Withania 

somnifera leaves growing in natural habitats of 

North-west Himalaya, India 

R. K. Sharma*, S. S. Samant, P. Sharma and S. Devi 

Pant Institute of Himalayan Environment and Development, Himachal Unit, Mohal-Kullu-175 126, 

Himachal Pradesh, India. 


Evaluation of antioxidant properties of medicinal plants from Indian Himalayan region has been very 

rarely carried out. Withania somnifera L. is one of the commercially available and most preferred 

medicinal plants in the Himalayan region due to its aphrodisiac property and potential to cure various 

diseases. The review of literature has indicated that the antioxidant activities of W. somnifera have not 

been carried out for so long. Therefore, in the present study an attempt was made to evaluate the 

antioxidant properties of W. somnifera collected from two different habitats that is, forest and roadside 

at Kullu, north-west Himalaya. The total phenolic and flavonoid contents and DPPH (1, 1-Diphenyl-2pycrylhydrazyl) 

scavenging potential of leaves extract of W. somnifera varied significantly between the 

habitat (p


exhaust emissions are oxides of nitrogen (NOx), oxides 

of carbon, oxides of sulphur (SOx), carbon particles, 

heavy metals, water vapour and hydrocarbons including 

aldehydes, single and poly aromatic hydrocarbons, 

alcohols, olefins, alkylnitriles besides a number of 

secondary pollutants such as ozone, etc., causing 

serious environmental and health impacts. W. somnifera 

L. commonly known as Ashwagandha has been a 

commercially viable medicinal shrub in Ayurvedic and 

Indigenous Systems of Medicines for many centuries in 

India (Jaleel et al., 2008). It is also known as the Indian 

Ginseng due to its ability of curing a large number of 

diseases (Samant et al., 2008). The free radical 

scavenging potential of the W. somnifera from different 

habitats of the Himalaya has not been reported yet. 

Therefore, an attempt has been made to evaluate the 

antioxidant potential of the methanol extracts of the 

leaves of W. somnifera collected from two different 

habitats that is, forest and roadside at Kullu valley of the 

Himachal Pradesh, India. 


Chemicals were purchased from Sigma and Merck Chemical 

Company India. All the chemicals and reagents were of the 

analytical grade. 

Sampling and analysis 

Five mature plants, each of W. somnifera having heights of 60 cm 

were collected in March 2010 from the roadside habitat of NH-21 at 

a distance of 50 m both sides of the road and from forest habitat, 

300 to 500 m away from the NH-21. The numbers of vehicles 

running on NH-21 were at Kullu averaged from 2,900 per day 

during 6 am to 6 pm (Kuniyal et al., 2007). The samples were 

washed with running tap water and air dried. The leaves were 

separated and used for chemical analysis. For extraction, 1g of leaf 

was weighed and crushed in 10 ml of 80% methanol using a mortar 

and pestle, and were kept at 0°C for 24 h. Finally, the supernatant 

was taken and final volume was maintained to 10 ml by adding 80% 

methanol and stored in refrigerator for further analyses. The 

amounts of total phenolics in methanol extracts of leaf samples 

were determined by the modified method of Wolfe et al. (2003). An 

aliquot (1ml) of the extract was mixed with 1ml of Folin-Ciocalteu 

Phenol Reagent (previously diluted with double distilled water in 1:1 

v/v) and 2 ml of 2% of sodium carbonate. The final volume was 

maintained to 10 ml by double distilled water and whole mixture 

was then heated at 80°C for 30 min or till the blue color appeared. 

The absorbance of blue color solution after cooling at room 

temperature was determined using Spectrophotometer (Ultraspec 

2100 Pro) at 650 nm. The content of the total phenolics was 

expressed in mg tannic acid g -1 fresh leaf. A standard curve was 

also prepared using different concentrations of the tannic acid. 

Total flavonoids contents in the methanol extracts of the leaf 

samples was quantified using the modified method of Ordon-Ez et 

al. (2006) based on the formation of complex flavonoid-aluminum 

and absorbance of yellow color was determined using 

Spectrophotometer (Ultraspec 2100 Pro) at 420 nm. An amount of 

1 ml of aliquot was mixed to 1 ml of 2% ethanolic aluminium 

chloride (AlCl3) vigorously. The reaction mixture was left for 1 h at 

room temperature. Total flavonoid contents were calculated as mg 

quercetin g -1 fresh leaf using standard curve prepared from different 

concentrations of quercetin. 

The effect of methanol extracts of leaf samples on scavenging 

DPPH radical was estimated using the method of Liyana-Pathirama 

and Shahidi (2005). A solution of 0.135 mM DPPH in methanol was 

prepared and 5 ml of the solution was mixed vigorously with 1ml of 

leaf extract in methanol. This mixture was left for 30 min in dark at 

room temperature. The absorbance of mixture was measured using 

Spectrophotometer (Ultraspec 2100 Pro) at 517 nm. The ascorbic 

acid or butylated hydroxyl toluene (BHT) solutions were used as 

reference solution. The DPPH free radical scavenging activity was 

calculated using following equation. 

DPPH radical scavenging activity (%) = (O.DC– O.DS) × 100 / O.DC 

Where O. DC is the absorbance of DPPH radical + methanol; O. DS 

is the absorbance of DPPH radical + leaf extract/standard. 


Means and standard error for the each site were analysed. The 

significance differences between the sites were analysed using 

Student’s T-test at probability levels (0.05, 0.01). All statistical 

analyses were performed by using SPSS software, version 12. 

RESULTS AND DISCUSSION 

The contents of the total phenolics and flavonoids, and 

DPPH scavenging potential of the methanol extracts of 

fresh leaves of W. somnifera L. are presented in Table 1 

and Figure 1, respectively. The results showed that 

methanol extracts of leaves of W. somnifera L. collected 

from the roadsides had significantly higher contents of 

total phenolics and flavonoids (100.10±0.90 and 92.88 ± 

1.12 mg g -1 fresh leaf, respectively) as compared to forest 

(Table 1). On the other hand, DPPH radical scavenging 

potential was found significantly higher (p

Sharma et al. 659 

Table 1. Total phenolics, flavonoid contents and DPPH inhibition potential of methanol extracts of the fresh leaves 

of W. somnifera collected from roadside and forest of north-west Himalaya. 

Parameter 

Methanol extracts 

Forest site Roadside 

Total phenolics (µg tannic acid. g -1 ) 74.27 ± 0.76 100.10** ± 0.90 

Total flavonoids (µg quercetin. g -1 ) 75.12 ± 0.89 92.88* ± 1.12 

DPPH inhibition (%) 81.63 ± 0.56 54.25* ± 3.44 

Values are means ± S.E. of five replicates. The significant differences between polluted and non-polluted sites were 

analyzed using the student’s t-test. Level of significance: **p≤0.01, *p≤0.05. 

Figure 1. DPPH radical scavenging activity of methanol extracts of the leaves of W. somnifera 

collected from roadside and forest habitats of north-west Himalaya. 

ozone and heavy metals in plants (Tiwari et al., 2010; 

Sharma et al., 2010). Mir et al. (2009) have reported 

increased levels of total flavonoids and phenolic content 

in selected medicinal plants growing on roadsides. 

Vehicular emissions have reduced the chlorophyll and 

protein content and reduced leaf areas in roadside plants 

(Wagh et al., 2006). Photosynthetic pigments content 

decreased progressively as a stress increased. 

Decreased in chlorophyll level under a stress condition 

may be due to reduction in pigment biosynthesis or 

enzymatic chlorophyll degradation and slight reduction in 

carotenoides may be due to their protective role against 

ROS. 

Vehicular emission can lead to oxidative stress and 

causing significant decrease to photosynthetic system. 

Similarly protein content is also reduced under vehicular 

emission which is present in the form of enzyme in 

plants. These enzymes provide additional defense 

against oxidative stress and keep the metabolic activities. 

Under stress conditions, enzyme activities increase in the 

plants. The continued exposure of soil to vehicular 

pollution may get rich in toxic chemicals such Cd, Pb, 

etc., which may be ascribed to increased levels of total 

phenolics and flavonoids in roadside plants. The free 

radical scavenging potential of medicinal plants mainly 

depends on its active ingredients. The phenolic 

compounds have antioxidant activity due to their redox 

properties, which play an important role in adsorbing and 

neutralizing free radicals, quenching singlet and triplet 

oxygen, or decomposing peroxides. Total phenolics are 

also effective free radical scavengers and have 

antioxidative property. The positive correlations between


Table 2. DPPH scavenging capacities of methanol extracts of the fresh leaves of W. somnifera L. collected from 

roadside and forest of north-west Himalaya. 

Extract Forest Roadside BHT Ascorbic acid 

IC50 (μg . ml -1 ) 206.77 224.96 60.87 27.47 

antioxidant potential and phenolic compounds of the 

extracts of Tanacetum sp. have also been described 

(Tepe and Sokmen, 2007). Plants containing flavonoids 

have also been reported to have strong antioxidant 

properties. It was estimated that vehicles account for 

70% of CO, 50% of HC, 30-40% of NOX , 30% of SPM 

and 10% of SO2 of the total pollution load in the major 

metros of India, of which two thirds are contributed by two 

wheelers alone (CPCB, 2010). 

DPPH, a chemical compound has a proton free radical 

with a characteristic absorption which decreased 

significantly due to exposure to reagents/solutions 

containing proton radical scavengers (Yamaguchi et al., 

1998). According to Baumann et al. (1979) and Chen and 

Ho (1995) DPPH radical scavenging by the natural or 

synthetic antioxidants is attributed to their hydrogen 

donating ability. The dose response curve for inhibition 

(%) of DPPH radicals by the tested samples at a range of 

0.02 to 0.10 mg ml -1 are presented in Figure 1. The 

present study observed that a concentration of 0.1 mg ml - 

1 , the scavenging activity of the methanol extracts of the 

leaves of all the tested plants from the roadsides reached 

to 23.73%, while at the same concentration, that of 

leaves from forest was 27.10%. The DPPH radical 

scavenging abilities of the leaves extracts from of the 

tested plants were found further very less than those of 

BHT (60.07%) and ascorbic acid (82.38%). In addition, 

the IC50 values (the concentration that inhibits radical 

formation by 50%) of different tested samples are given 

in Table 2. Among the tested samples, the IC50 value was 

found maximum for the plant extracts from the polluted 

site followed by non-polluted site, BHT and ascorbic acid 

(Table 2). The present study reported for the first time 

that the methanol extracts of W. somnifera of Himalayan 

origin from the roadsides have higher antioxidant 

contents, but a lower (51%) free radical scavenging 

potential as compared to a forest. Thus, based on the 

present study it can be suggested that the medicinal 

plants for commercial purpose could not be grown near 

the national highways as their medicinal properties get 

reduced due to their continuous exposure to vehicular 

emissions. 


Authors are thankful to the Director, G.B. Pant Institute of 

Himalayan Environment and Development, Kosi- 

Katarmal, Almora, Uttarakhand, India for providing 

necessary facilities and encouragements. 

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DOI: 10.5897/JMPR11-517 



Phytochemical screening, antioxidant and 

antimutagenic activities of selected Thai edible plant 

extracts 

M. Phadungkit 1 *, T. Somdee 2 and K. Kangsadalampai 3 

1 Faculty of Pharmacy, Mahasarakham University, Kantarawichai, Maha Sarakham, 44150, Thailand. 

2 Faculty of Public Health, Mahasarakham University, Kantarawichai, Maha Sarakham, 44150, Thailand. 

3 Institute of Nutrition, Mahidol University, Salaya, Nakhon Pathom, 73170, Thailand. 


Antioxidant and antimutagenic activities of plant extracts possess great potential as functional foods 

for cancer prevention. The aims of the current study were to evaluate antioxidant and antimutagenic 

activities and to study the chemical constituents of five Thai edible plant extracts. Antioxidant activity 

was expressed as the ability of each extract to scavenge the free radicals 1,1-diphenyl-2-picrylhydrazyl 

(DPPH). Antimutagenic activity was evaluated with the Ames test using Salmonella typhimurium strains 

TA 98, and TA 100. The results showed that Oroxylum indicum (Linn.) Kurg. and Tiliacora triandra Diels. 

extracts had high antioxidant activity with the EC50 values at concentrations of 12.69 ± 1.02 and 14.51 ± 

0.67 �g/ml, respectively, whereas Basella alba Linn. extract had the strongest antimutagenicity with 

both strains of S. typhimurium with percentage of inhibition values ranging from 54.06 ± 1.52 to 86.08 ± 

2.78 %. In the present study, flavonoids and phenolic compounds from the herbal extracts are proposed 

to be antioxidant and antimutagenic agents, respectively. The apparent antioxidant and antimutagenic 

activities of Thai edible plants further suggests their potential usefulness in cancer prevention. 

Key words: Phytochemical screening, antioxidant, antimutagenic. 

INTRODUCTION 

Several studies provide strong evidence that vegetables, 

fruits and phytochemicals protect against some cancers 

(Lako et al., 2007; Geoffrey et al., 2010). Many 

antioxidative agents and antimutagens have been 

identified as anticarcinogens (Ames, 1983). Therefore, 

the regular intake of plants possessing antimutagenic or 

antioxidative agents can reduce genotoxic effects of 

mutagenic and carcinogenic factors (Ikken et al., 1999). 

Thai people consume many plants as part of their diet. 

Therefore, it is of interest to know whether traditional 

edible plants have antioxidant and antimutagenic 

activities. If they did then they could be useful in dietary 

supplement development with the herbal extracts as 

agents for cancer prevention. The aims of this 

investigation were to test the antioxidant and anti 

*Corresponding author. E-mail: phadang_p@hotmail.com. Tel: 

664-37-54360. 

mutagenic activities and to screen the chemical 

constituents of selected Thai edible plant extracts. 

EXPERIMENTAL METHOD 

Plant materials 

Oroxylum indicum (Linn.) Kurg (fruits), Tiliacora triandra Diels. 

(leaves), Morinda citrifolia Linn. (leaves), Basella alba Linn.(entire 

plant) and Sauropus androgyrus (L.) Merrill (leaves) obtained from 

Maha Sarakham province and identified by one of the authors (Dr. 

Phadungkit). Voucher specimens have been deposited in the 

Herbarium at the Faculty of Pharmacy, Mahasarakham University, 

Thailand. Each plant was extracted with 95% ethanol by the 

maceration method. The macerates were evaporated to dryness in 

a rotary evaporator and each crude herbal extract was kept at 4°C. 

Phytochemical screening 

The solutions of each plant extract were freshly prepared from the 

crude extract described previously. They were analyzed for the

Table 1. Phytochemical screening of the herbal extracts. 

Sciencetific name Alkaloids 

Condensed 

tannins 

Phenolic 

compounds 

Phadungkit et al. 663 

Triterpenes Steroids Flavonoids Saponins Antraquinones 

Oroxylum indicum + - + - + + - - 

Tiliacora triandra - + - + - + + - 

Morinda citrifolia - - + - + + - - 

Basella alba - - + - + - - - 

Sauropus androgyrus - - + - + - - - 

Key: - absent; + present. 

presence of alkaloids, condensed tannins, phenolics, triterpenes, 

steroids, saponins and anthraquinones according to the methods 

described by Trease and Evans (1989). 

Antioxidant activity assay 

Antioxidant activity of each sample was determined based on its 

ability to react with the stable 1,1-diphenyl-2-picryl hydrazyl (DPPH) 

free radical (Yamasaki et al., 1994). An aliquot (750 �l) of the 

extract (50 to 1000 �g/ml in absolute ethanol) was added to 750 �l 

of 152 �M DPPH in absolute ethanol. After incubation at room 

temperature for 20 min, the absorbance of each solution was 

determined at 520 nm. Percentage of inhibition and the 

concentration of sample required for 50% scavenging of the DPPH 

free radical (EC50) were determined. Ascorbic acid was used as the 

reference standard. 

Mutagenicity activity 

Salmonella typhimurium test strains TA 98 and TA 100 provided by 

Dr. Wannee Kusamran (National Cancer Institute, Ministry of Public 

Health, Thailand) were used throughout this study. The test strains 

were manipulated as suggested by Maron and Ames (1983). An 

overnight culture of bacteria was prepared by inoculating 10 mL of 

Oxoid nutrient broth No. 2 from a frozen stock culture (after thawing 

at room temperature) and incubating at 37°C. This was used for the 

mutagenesis assay. 1-Aminopyrene (1-AP) treated with nitrite in 

acid solution was used as the positive mutagen (Kangsadalampai 

et al., 1996). The pre-incubation method suggested by Yahagi et al. 

(1975) was used to determine the mutagenicity of the positive 

standard and each sample in the Ames test throughout this study. 

Antimutagenicity activity 

The antimutagenic effect of the plant extracts on the mutagenicity of 

nitrite treated 1-AP was studied. Briefly, 0.1 ml quantities of DMSO 

containing 3.75 to 30 mg of each crude extract was mixed with 0.7 

ml volumes of sodium phosphate buffer (0.2 M, pH 7.4) and 0.1 ml 

of overnight culture of S. typhimurium. Then, 0.1 ml quantities of 

DMSO were prepared both with and without 0.1 μg of the positive 

standard mutagen. 

The entire mixtures were then separately pre-incubated in 20 mL 

of Oxoid nutrient broth No. 2 at 37°C for 20 min before 2.0 ml agar 

containing NaCl (5.0 g/l), L-histidine (0.025 mM), biotin (0.025 mM) 

and agar (6.0 g/l) was added. The mixture was then poured onto a 

minimal glucose agar plate. The histidine revertant colonies were 

counted after incubation at 37°C for 48 h. Each sample was 

assayed using triplicate plates. The inhibitory effect of each of the 

herbal extracts on the mutagenicity of the standard direct mutagen 

was determined as a percentage of inhibition as described 

subsequently: 

Percentage of inhibition = (A-B)/(A-C) x 100 

Where A is the number of revertants per plate induced by the 

positive mutagen; B is the number of revertants per plate induced 

by the positive mutagen in the presence of each extract; and C is 

the number of spontaneous revertants per plate. The inhibition of 

each herbal extract was considered according to Calomme et al. 

(1996) as being strong, moderate or weak when the value was 

higher than 60, 40 to 60 or 20 to 40%, respectively. The values of 

less than 20% were considered negligible. 



The screening for phytochemical constituents showed the 

presence of some bioactive compounds in the herbal 

extracts (Table 1). These results indicated phenolic 

compounds and steroids in the herbal extracts. However, 

anthraquinones were not found in any herbal extract. It is 

well understood that plants are a major source of 

phenolic compounds, which are synthesized as 

secondary metabolites during normal development in 

response to stress conditions, such as wounding and UV 

radiation among others (Stahl and Sies, 2003). Plants 

may contain simple phenolics, phenolic acids, coumarins, 

flavonoids and stilbene (Naczk and Shahidi, 2006). The 

information found in this study suggests that phenolic 

compounds are the major component in the herbal 

extracts except those of T. triandra. 

Antioxidant activity assay 

The antioxidant activity of the herbal extracts is 

summarized in Table 2. All herbal extracts possessed 

lower antioxidant activity than that of the standard 

ascorbic acid in terms of EC50 values. The two strongest 

antioxidant scavengers were the herbal extracts of O. 

indicum and T. triandra. Their EC50 values were 12.69 ± 

1.02 and 14.51 ± 0.67 and �g/ml for O. indicum and T. 

triandra, respectively. Furthermore, most reports 

indicated that the protective effect against oxidative


Table 2. DPPH scavenging activity of the herbal extracts. 

Family name Sciencetific name 50% DPPH scavenging activity (EC50 µg/ml) 

Bignoniaceae Oroxylum indicum 12.69 ± 1.02 

Menispermaceae Tiliacora triandra 14.51 ± 0.67 

Rubiaceae Morinda citrifolia 36.27 ± 1.08 

Basellaceae Basella alba 102.99 ± 4.37 

Euphorbiaceae Sauropus androgyrus 179.11 ± 15.11 

Ascorbic acid 5.61 ± 0.37 

damage of any samples or compounds was attributable 

to phenolic compounds (Robbins, 2003). In addition, 

Tenpe et al. (2009) concluded that the O. indicum leaf 

extract was a good antioxidant and expressed its 

hepatoprotective activity. This activity was probably due 

to the presence of polar phenolic compound such as 

flavonoids, tannins etc. According to the result shown in 

Table 2, herbal extracts, which had strong antioxidant 

activity, had phenolic and flavonoid contents. Most of the 

antioxidant substances in plants are phenolic compounds 

and phenolic substances serve as oxidation terminators 

by scavenging radicals to form resonance stabilized 

radicals (Rice-Evans et al., 1997) and the finding of 

flavonoids in the present investigation may suggest its 

antioxidant role previously reported by Cook and 

Samman (1996). The high antioxidant activity of the 

extract of O. indicum was similar to the result of Kalaivani 

and Lazar (2009). 

Mutagenicity activity 

The concentrations of herbal extracts used to evaluate 

the mutagenic activity were neither toxic nor mutagenic, 

since the numbers of revertant colonies were not higher 

than that of the negative control. The range of revertant 

colonies of most samples was 10 to 22 for TA 98 and 96 

to 168 for TA 100 which were less than the spontaneous 

revertants. Yen et al. (2001) evaluated the mutagenic 

effect of new leafy vegetables in Taiwan and found that 

B. alba extract was not mutagenic on TA 98 or TA 100 

cultures either with or without the activating system. The 

range of herbal extracts on revertant colonies was similar 

to those reported previously by several authors (Maron 

and Ames, 1983; Gonzalez de Mejia et al., 1999; 

Cardador-Martํ nez et al., 2002). 

Antimutagenicity activity 

The main product of nitrite treated 1-aminopyrene that did 

not require metabolic activation before expressing its 

mutagenicity was determined to be 1-nitropyrene (Kato et 

al., 1991). The percentage of inhibition on strains TA 98 

and TA 100 revertants had values ranging from 77 to 86 

and 54 to 63, respectively (Table 3) indicating that 

Basella alba extract (3.75 to 30 mg/plate) was strongly 

antimutagenic against nitrite treated 1-aminopyrene. Yen 

et al. (2001). reported that the extract from B. alba was a 

moderate inhibitor of 2-amino-3-methyl-imidazo [4,5f]quinoline 

(IQ) in S. typhimurium TA 98 and TA 100. 

The high antimutagenic activity of B. alba extracts may 

be due to the presence of phenolic compounds as 

suggested by Jayaprakasha et al. (2007). The correlation 

of antimutagenicity of natural compounds of plant origin 

was positive with polarity of flavonols; also phenolic 

compounds were classified as potent antimutagens 

(Edenharder et al., 1997). Phenolic compounds 

presented in plants were considered to be responsible for 

their antimutagenic activity (Romina and Luis, 2006). 

Rocha et al. (2007) also suggested that phenolic 

compounds present in the acetone extracts from beans 

were potent antimutagens against the direct mutagen 1nitropyrene 

in S. typhimurium TA 98. 

B. alba extract expressed high antimutagenic activity. 

The possible mechanisms may be classified as follows. 

Firstly, B. alba extracts might include the blocking of the 

mutagen transfer into the cytosol by phenolic binding or 

insertion into the transporters of the outer membrane of 

the cell (Hour et al., 1999). Elvira et al. (1999) suggested 

that phenolic compounds could interact directly and nonenzymatically 

with the proximate and/or ultimate 

mutagen; or form a complex with known mutagens e.g. 

Benzo[a]pyrene (B[a]P), thereby reducing the 

bioavailability of mutagen. Secondly, B. alba extracts 

modified the permeability for mutagens across bacterial 

membranes. Edenharder and Tang, (1997) reported that 

1-nitropyrene were in general more effectively 

antagonized by potent antimutagenic flavonoids. They 

suggested that different mechanism of antimutagenesis 

were at work. Antimutagenic flavonoids might modulate 

the mutagenic response of the nitroarenes, which have 1nitropyrene 

as a member, tested by modification of the 

permeability of bacterial membranes. 

Antimutagens and/or anticarcinogens are found in all 

categories of foods, but mainly in fruits and vegetables 

(Nakamura et al., 1998). Five herbal extracts in the 

present study, especially from the B. alba, that are 

generally consumed as vegetables or herbs showed 

antimutagenic activity in the Ames test. These data

Table 3. Antimutagenic activity of the herbal extracts. 

Family name Scientific name Concentration (mg/plate) 

Bignoniaceae Oroxylum indicum 

Menispermaceae Tiliacora triandra 

Rubiaceae Morinda citrifolia 

Basellaceae Basella alba 

Euphorbiaceae Sauropus androgyrus 

further indicated that B. alba may also be a healthpromoting 

food. 

Conclusion 

The present study indicates that the herbal extracts of O. 

indicum and T. triandra, which posses high antioxidant 

activity, and the herbal extracts of B. alba, which posses 

potent antimutagenic activity, have high potential to be 

further developed as functional foods for cancer 

prevention. Increased consumption of these plants would 

seem to be of great benefit to most consumers since 

theses plants are cheap and plentiful. Phenolic 

compounds are suggested to be responsible for the 

antioxidant and antimutagenic activity exhibited in this 

study. Thus total phenolic content may be used to predict 

the ability of the extracts to scavenge free radicals and to 

decrease the mutagenicity induced by environmental 

toxicants. 

ACKNOWLEDGEMENT 

The authors would like to thank The Thailand Research 

Fund for financial support. 

Phadungkit et al. 665 

%Inhibition 

TA 98 TA 100 

3.75 75.43 ± 3.83 -13.16 ± 2.16 

7.5 84.38 ± 1.77 -14.43 ± 3.89 

15 89.91 ± 2.07 21.39 ± 1.03 

30 97.95 ± 3.76 35.06 ± 1.53 

3.75 50.09 ± 2.56 43.26 ± 3.46 

7.5 51.37 ± 5.73 47.74 ± 1.08 

15 47.65 ± 4.10 48.63 ± 1.77 

30 44.84 ± 8.98 58.84 ± 10.81 

3.75 37.33 ± 2.13 27.63 ± 1.19 

7.5 8.09 ± 1.94 3.75 ± 0.75 

15 51.52 ± 0.11 57.59 ± 10.33 

30 74.30 ± 15.57 78.49 ± 3.10 

3.75 77.48 ± 1.41 54.06 ± 1.52 

7.5 82.71± 5.33 59.77 ± 2.64 

15 83.84 ± 2.28 63.02 ± 6.83 

30 86.08 ± 2.78 63.11 ± 2.54 

3.75 83.55 ± 13.92 14.49 ± 1.48 

7.5 88.72 ± 16.07 42.52 ± 2.09 

15 89.25 ± 4.01 43.64 ± 2.83 

30 94.42 ± 5.79 44.58 ± 6.25 

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DOI: 10.5897/JMPR11.930 



Ethnobotany and antimicrobial activity of medicinal 

plants of Bakhtiari Zagross mountains, Iran 

A. Ghasemi Pirbalouti*, F. Malekpoor and B. Hamedi 

Shahrekord Branch, Islamic Azad University, Department of Medicinal Plants, Researches Centre of Medicinal Plants 

and Ethno-veterinary, P. O. Box: 166,Shahrekord, Iran. 


The major aims of this study was to determine ethnobotany and antimicrobial activity of sixteen 

medicinal, aromatic and nutraceutical plants from high altitude of Bakhtiari Zagross mountains, which 

are Iranian medicinal plants. Antimicrobial activities of extract of sixteen Iranian folklore plants 

including, Heracleum lasiopetalum Boiss. (Apiaceae), Satureja bachtiarica Bunge. (Lamiacae), Thymus 

daenensis Celak. (Lamiacae), Ziziphora teniur L. (Lamiacae), Echiophora platyloba L. (Apiaceae), 

Dracocephalum multicaule Benth. (Lamiacae), Kelussia odoratissima Mozff. (Apiaceae), Mentha 

longifolia Hudson. (Lamiacae), Achillea kellalensis Boiss. (Asteraceae), Stachys lavandulifolia Vahl. 

(Lamiacae), Hypericum scabrum L. (Hypericaceae), Quercus brantii Lindley. (Fagaceae), Myrtus 

communis L. (Myrtaceae), Pistachia atlantica Desf. (Anacardiaceae), Arnebia euchroma (Royle.) 

Johnston. (Boraginaceae) and Salvia hydrangea DC. (Lamiacae) were investigated against strains of 

Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Escherichia 

coli O157:H7, Yersinia enterocolitica, Bacillus cereus, Listeria monocytogenes, Campylobacter coli, 

Campylobacter jejuni and Candida albicans by agar disc diffusion and serial dilution assays. The 

results of the study reveal that some of species play an important role in primary healthcare system of 

these tribal communities. Some of the medicinal plants showed relatively high antimicrobial activity 

against all the tested bacteria and fungi. In conclusion, it can be said that the extract and essential oil of 

some of the medicinal plants from high altitude of Bakhtiari Zagross Mountains could be used as 

natural antibacterial agents in the food preservation and human health. 

Key words: Medicinal plants, ethnobotany, antimicrobial activity, high altitude, Zagross mountains. 

INTRODUCTION 

Plant substances play a major role in primary health care 

as therapeutic remedies in many developing countries 

until today (Zakaria, 1991). Medicinal herbs contain 

physiologically active principles that over the years have 

been exploited in traditional medicine for the treatment of 

various ailments as they contain anti-microbial properties 

(Kelmanson et al., 2000; Srinivasan et al., 2001). 

The antifungal and antibacterial activity exhibited by 

extract and essential oil of medicinal plants has been 

demonstrated by several researchers (Aktug and 

Karapinar, 1986 ; Arora and Kaur, 1999 ; Delgado et al., 

2004 ; El-Khateib and Abd El-Rahman, 1987 ; Nasar- 

Abbas and Kadir Halkman, 2004 ; Ozcan and Erkmen, 

*Corresponding author. E-mail: ghasemi@iaushk.ac.ir. 

2001 ; Fazeli et al., 2007 ), but unfortunately, there are few 

data related to the antimicrobial activity of extracts 

obtained from different medicinal plants in Chaharmahal 

va Bakhtiari (Iran). Numerous Iranian folklore herbs for 

example: Heracleum lasiopetalum Boiss., Satureja 

bachtiarica Bunge., Thymus daenensis Celak., Ziziphora 

teniur L., Echiophora platyloba L., Dracocephalum 

multicaule Benth., Kelussia odoratissima Mozff., Mentha 

longifolia Hudson., Achillea kellalensis Boiss., Stachys 

lavandulifolia Vahl., Hypericum scabrum L., Quercus 

branti Lindley., Myrtus communis L., Pistachia atlantica 

Desf., Arnebia euchroma (Royle.) Johnston. and Salvia 

hydrangea DC. have been utilized as traditional 

medicines by the indigenous people of Chaharmahal va 

Bakhtiari, Southwest Iran (Ghasemi Pirbalouti, 2009 ). 

Hence, it is necessary to establish the scientific basis for 

the therapeutic actions of traditional plant medicines


as these may serve as the source for the development of 

more effective drugs. In this study, it was aimed to 

determine antimicrobial activity of ethanol extract and 

essential oils of 16 plant species which are Iranian 

endemic plants. 


An ethnobotanical survey was conducted in Chaharmahal va 

Bakhtiari Province, South-West of Iran. The study area is located 

between latitude 31º 10´ to 32º 45´ N and longitude 49º 29´ to 52 º 

34´ E. It occupies an area of 10893 km². The elevation range is 

between 1009 m in the south to 4250 m in the west districts. The 

survey was conducted by interviewing traditional healers in each 

locality using the local language. Each interview followed a semistructured 

questionnaire designed to obtain the following 

information: Scientific and local plant names; habit; plant parts 

used; uses/ailments treated. The information was collected from 55 

persons (20 men and 35 women) in 15 villages, mostly villages of 

the southern and western parts of the area. The plants were 

collected from mountain areas of Zagross, Chaharmahal va 

Bakhtiari district, during 2008 to 2010. Their identity was confirmed 

and voucher specimens were deposited at the Researches Centre 

of Medicinal and Aromatic Plants, Islamic Azad University, 

Shahrekord Branch, Iran. Dried plant material were powered (200g) 

and subjected to hydro-distillation (2000 ml distillated water) for 4 h 

using a Clevenger- type apparatus according to the method 

recommended in British pharmacopoeia (British Pharmacopoeia, 

1988). 

Air-dried and powdered Leaves and flowers (100 g) were 

macerated at room temperature with 1 L of ethanol: water (80:20) 

for 24 h. The extractions continued two times and then were 

concentrated in a rotary evaporator under reduced pressure (Model 

Zirbus 302®, Italy). The extract samples were stored in universal 

bottles and refrigerated at 4°C prior to use. 

The antimicrobial activity of 16 Iranian medicinal plants was 

determined against Staphylococcus aureus, Escherichia coli, 

Pseudomonas aeruginosa, Klebsiella pneumonia, Escherichia coli 

O 157 : H 7 , Yersinia enterocolitica, Bacillus cereus, Listeria 

monocytogenes, Campylobacter coli, Campylobacter jejuni and 

Candida albicans . This microorganism was obtained from 

Department microbiology of Islamic Azad University, Shahrekord 

Branch, Iran. A final bacterial suspension containing 10 7 CFU/ml 

was made from the flask broth culture. Subsequent dilutions were 

made from the above suspension, which were then used in tests. 

The disc diffusion method of Iennette (1985) was used with some 

modification to determinate rate of inhibition growth of bacteria and 

fungi by plant extract and essential oil. BHI agar (Merck, Germany) 

was used to prepare the culture medium and autoclaved at 121°C 

for 15 min. Briefly, plates (8-cm diameter) were prepared with 10 ml 

agar inoculated with 1 ml bacterial suspension. Sterile paper discs 

(6 mm in diameter) were impregnated with 60 µl of dilutions of 

known extract concentrations (100 µg/disc) and incubated at 35°C 

for 72 h. The extracts were dissolved in dimethyl sulfoxid (DMSO, 

15 µl) before the test for antimicrobial activity. Antimicrobial growth 

inhibition was determined as the diameter of the inhibition zones 

around the discs (mm). The growth inhibition diameter was an 

average of three measurements, taken at three different directions. 

All tests were performed in triplicate. 

RESULTS 

The results of the ethnobotanical survey are presented in 

Table 1. The Growth inhibition value of extract and 

essential oil on bacteria and fungi strains are shown in 

Table 2. Most of the extracts and essential oils showed 

relatively antimicrobial activity against the tested bacteria 

and fungi with the diameter of inhibition zone ranging 

between 7 and 30 mm. Antimicrobial activities of extract 

and essential oil of plants varied related to the test 

organism. There were significant differences in the 

antibacterial activities of plant extract and essential oils . 

Among the plants tested essential oil of Satureja 

bachtiarica and Thymus daenensis showed the best 

antimicrobial activity. 

Subsequent experiments were conducted to determine 

minimal inhibitory concentration and minimal bactericidal 

and fungicidal concentration of all selected plant extracts 

and essential oils. The MIC values for active extract and 

essential oil ranging between 0.039 and 10 mg/ml. The 

most active of the concentrations was 10 mg/ml 

concentration inhibiting completely the growth of bacteria 

and fungi. The results obtained appeared to confirm the 

antibacterial potential of the plants investigated. 

DISCUSSION 

Antimicrobial activities of extract and essential oil of 

plants varied related to the test organisms. The results 

showed that bacteria (E. coli, L. monocytogenes and B. 

cereus) were more sensitive than bacteria (K. 

pneumonia, E. coli O 157 : H 7 , Y. enterocolitica, C. coli, 

and C. jejuni ). In this experimental, E. coli was the most 

sensitive, while E. coli O 157 : H 7 was the most resistant. 

Shan et al. (2007) reported that Gram-positive bacteria 

(L. monocytogenes, S. aureus and B. cereus) were 

generally more sensitive to the tested extracts than 

Gram-negative (E. coli and Salmonella anatum). 

A possible explanation for these observations may lie in 

the significant differences in the outer layers of Gramnegative 

and Gram-positive bacteria. Gram-negative 

bacteria possess an outer membrane and a unique 

periplasmic space not found in Gram-positive bacteria 

(Duffy and Power, 2001). The resistance of Gramnegative 

bacteria towards antibacterial substances is 

related to the hydrophilic surface of their outer membrane 

which is rich in lipopolysaccharide molecules, presenting 

a barrier to the penetration of numerous antibiotic 

molecules and is also associated with the enzymes in the 

periplasmic space, which are capable of breaking down 

the molecules introduced from outside (Nikaido, 1994; 

Gao et al., 1999). Gram-positive bacteria do not have 

such an outer membrane and cell wall structure. 

Antibacterial substances can easily destroy the bacterial 

cell wall and cytoplasmic membrane and result in a 

leakage of the cytoplasm and its coagulation (Kalemba 

and Kunicka, 2003). 

Some studies claim that the phenolic compounds 

present in spices and herbs might also play a major role

Table 1. Ethnobotany of Medicinal plants from high altitude of Bakhtiari Zagross mountains, Iran. 

Scientific name Family name Local name Habit* Parts used Uses/ailments treated 

Achillea kellalensis Boiss. and Hausskn. Asteraceae Golberenjaz H flowers Wound, carminative, indigestion 

Arnebia euchroma (Royle.) Johnston. Boraginaceae Sorya H rhizome, root Burn wound, anti-eczema, antimicrobial, anti- inflammatory 

Pirbalouti et al. 677 

Dracocephalum multicaule Montbr and Auch. Lamiaceae Zarrin giah, Zeravi H leaves, flowers Sedative, analgesia, inflammatory, anti-bacterial, anti-septic, foot pain 

Echinophora platyloba DC. Apiaceae Khosharizeh S aerial plant Anti fungal, spice and culinary 

Heracleum lasiopetalum Boiss Apiaceae Goolpar, Kereson H fruit Anti-septic, spice and condiment 

Hypericum scabrum L. Hypericaceae Golraye dayhimi H flowers Green tea, sedative, headache, analgesic 

Kelussia odoratissima Mozaff. Apiaceae Kelus H leaves Edible as vegetable, flavoring, indigestion, rheumatism, sedative 

Mentha longifolia (L.) Hudson. Lamiaceae Pooneh, Pineh H leaves, flowers Edible as vegetable, flavoring, indigestion, cough 

Myrtus communis L. Myrtaceae Mort T leaves Skin discords, digestive discords, astringent, good hair condition, bronchodilatotor 

Pistachia atlanta Desf. Anacardiaceae Baneh, T resin, fruit Indigestion, tonic, toothache, astringent 

Quercus branti Lindley. Fagaceae Balout T fruit Anti-ulcer, indigestion 

Salvia hydrangea DC. Lamiaceae Gool ouroneh H leaves, flowers Cough, emollient, sore throat, antibacterial 

Satureja bachtiarica Bung. Lamiaceae Marzeh Koohi H leaves, flowers Edible as vegetable, flavoring, indigestion, cough, anti-bacterial 

Thymus daenensis Celak. Lamiaceae Oushon H leaves, flowers Green tea, spice, culinary, cough, anti-bacterial, carminative 

Stachys lavandulifolia Vahl. Lamiaceae Lolopashmak, Chaye Koohi H leaves, flowers Green tea, anti-bacterial, skin diseases, menorrhagia 

Ziziphora tenuior L. Lamiaceae Kakouti H leaves, flowers Green tea, spice, culinary, anti-bacterial, carminative, anti-asthmatic 

*Habit: T: Tree, H: Herb. 

in their antimicrobial effects (Hara-Kudo et 

al., 2004). There has been no large scale 

systematic investigation of the relationship 

between bacterial inhibition and total 

phenolic content of spices and herbs. 

Previous studies (Shan et al., 2005) showed 

that a highly positive linear relationship exists 

between antioxidant activity and total 

phenolic content in some spices and herbs. 

Many herb and spice extracts for example T. 

daenensis and S. bachtiarica contained 

high levels of phenolics and exhibited 

antibacterial activity against food-borne 

pathogens. Previous studies (Rasooli et al., 

2006) on the antimicrobial activity of the 

essential oils of some Thymus spp., most of 

them possessing large quantities of phenolic 

monoterpenes, have shown activity against 

viruses, bacteria, food-derived microbial 

strains and fungi. Pervious works (Ghasemi 

Pirbalouti et al., 2009a) showed that 

essential oils of T. daenensis and Thymbra 

spicata (Elam ecotype) flowers exhibited 

antibacterial activities against L. 

monocytogenes from chicken meat. In a 

previous study, the minimum inhibitory 

concentration (MIC > 50 % growth inhabitation) against 

L. monocytogenes for T. daenensis and T. 

spicata were 0.700 and 1.7 mg/ml, 

respectively. Fazeli et al. (2007) studied 

antimicrobial effects of two medicinal plants 

(R. coriaria L. and Z. multiflora Boiss.) used 

in Iranian traditional medicine were


Table 2. Antibacterial tests of the investigated plants in agar diffusion assay (100 µg/disc). 

Plant species Extraction E.c S.a P.a K.p E.c O 157 :H7 B.c L.m Y.e C.c C.j C.a 

Achillea kellalensis Boiss. and Hausskn. 

Arnebia euchroma (Royle.) Johnston. 

Ethanol extract 21 11 10 9 13 

Essential oil 18 17 12 13 

Ethanol extract 7 13 14 13 

Aqueous extract 10 12 16 12 

Dracocephalum multicaule Montbr and Auch. Ethanol extract 22 12 11 23 17 

Echiophora platyloba L. Ethanol extract 12 9 10 16 12 12 

Heracleum lasiopetalum Boiss 

Hypericum scabrum L. 

Kelussia odoratissima Mozaff. 

Mentha longifolia (L.) Hudson. 

Myrtus communis L. 


Essential oil 17 12 15 14 9 23 


Aqueous extract 18 11 9 11 

Ethanol extract 10 12 10 9 20 13 10 13 

Essential oil 16 9 16 17 9 14 



Ethanol extract 19 12 10 


Pistachia atlanta Desf. Ethanol extract 22 27 12 12 

Quercus branti Lindley. Ethanol extract 

Salvia hydrangea DC. 

Satureja bachtiarica Bung. 

Thymus daenensis Celak. 

Ethanol extract 16 13 11 



Essential oil 23 21 22 14 17 14 15 16 

Ethanol extract 16 8 16 14 12 14 13 11 

Essential oil 18 22 17 19 7 25 16 15 19 

Stachys lavandulifolia Vahl. Ethanol extract 12 13 14 

Ziziphora tenuior L. Ethanol extract 18 11 8 10 

E.c: Escherichia coli ; P.a: Pseudomonas aeruginosa; S.a: Staphylococcus aureu; K.b: Klebsiella pneumoniae ; E.c O 157 :H7: Escherichia coli O 157 :H7 ; Y.e: Yersinia enterocolitica ; B.c: 

Bacillus cereus ; L.m: Listeria monocytogenes ; C.c: Campylobacter coli ; C.j: Campylobacter jejuni; C.a: Candida albicans.

investigated against some pathogenic food-borne 

bacteria. The minimum inhibitory concentrations of R. 

coriaria and Z. multiflora were determined against several 

strains of Gram-positive and Gram-negative bacteria. 

They have reported that B. cereus was found to be the 

most sensitive bacteria to R. coriaria showing the MIC of 

0.05%., while S. aureus and Proteus vulgaris ranked next 

with 0.10% followed by Shigella flexneri, E. coli and 

Salmonella typhi with MIC of 0.20%. 

According to a report (Rasooli et al., 2006) extract and 

essential oils of Thymus erioealyx and Thymus porlock 

inhibited the growth of L. monocytogenes. The essential 

oil and extract of some aromatic plants (for example mint 

family, Lamiaceae) with a higher percentage of cavracrol 

and thymol have a higher efficacy against strain bacterial 

(Rasooli et al., 2006). 

The result of a study (Ghasemi Pirbalouti et al., 2009b) 

showed that extract and essential oil of S. bachtiarica, 

Scrophularia deserti and Zizyphus spina-christi inhibited 

the growth of C. albicans. Ghasemi Pirbalouti et al. 

(2009a) reported that the essential oils of T. daenensis 

and T. spicata (MIC > 50 % = 0.63 µl ml -1 and MLC > 99.9 % = 

22 µl ml -1 ) and ethanol extract of Mentha longifolia 

showed higher of inhibition against the Saprolegnia 

parasitica than the other extracts. Kokoska et al. (2002) 

reported that the ethanol extract of Salvia officinalis had 

strong antimicrobial activity against 

B. cereus, E. coli, and S. aureus. 

According to a report (Rasooli et al., 2006) extract and 

essential oils of T. erioealyx and T. porlock inhibited the 

growth of L. monocytogenes. The essential oil and 

extract of some aromatic plants (for example mint family, 

Lamiaceae) with a higher percentage of cavracrol and 

thymol have a higher efficacy against strain bacterial 

(Rasooli et al., 2006). 

Conclusions 

It can be said that the extract and essential oil of some of 

the medicinal plants from high altitude of Bakhtiari 

Zagross Mountains could be used as natural antibacterial 

agents in the food preservation and human health. 

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DOI: 10.5897/JMPR11-938 



Proposing a methodology in preparation of olive 

orchards map as an important medicinal plant in Iran 

by remote sensing (RS) and geographical information 

system (GIS) 

Ali Mohammadi Torkashvand* and Alireza Eslami 

Rasht Branch, Islamic Azad University, Rasht, Iran. 


Present study focuses on identification and mapping of olive in the part of Roodbar region, Guilan, Iran 

by IRS images and GIS. Two methods were evaluated to separate olive orchards spectrum reflex from 

the other surface covers. At the first method, upper and lower limit of digital number of olive orchards 

were determined by the addition and subtraction of standard deviation from the mean in each band and 

initial classified map of olive orchards was prepared in every band. The final map of olive orchards was 

prepared from crossing three initial maps of olive orchards. At the second method, olive orchards map 

was prepared by four models include: Box classifier, maximum likelihood, minimum distance and 

minimum Mahalanobis distance. Methods accuracy was evaluated from crossing the map of training 

points (pixel) with olive orchards map. The results indicated that in classification of less-condensed 

olive orchards, because of spectrum wave interference of olive green canopy and the soil zone between 

the canopy cover, the interference of digital number of low-condensed olive is observed with the other 

vegetation cover and bare lands. There was this issue even for wave interference of low-condensed 

olive with urban and residential regions. There is the interference of spectrum reflexes between the 

agriculture land and olive. This issue also exists for different methods of supervised classification. 

Key words: Spectrum reflex, olive, training points, map. 

INTRODUCTION 

Olea earapae is a medicinal plant that grows as wild and 

cultivated in north and west of Iran. Oil, olive leaves and 

bark have a medicinal value. This plant can be planted in 

areas prone to developing cultivation. In addition to 

financial benefits for the residents of these areas, it 

prevent of deforestation and soil erosion. The use of 

medicinal plants without an estimation of cultivated areas 

tends to lack of precise programming. The most 

important principle to exploit of forests and pastures is 

continuous production (Eslami, 2008). 

The information of digital imagery acquired by Remote 

Sensing technology can be used for mapping, monitoring 

and assessing the properties of the environmental and 

*Corresponding author e-mail: m.torkashvand54@yahoo.com, 

orkashvand@iaurasht.ac.ir. 

terrestrial features. Land use and land cover is found as 

significant information in an area that may assist 

managers and decision-makers to take drastic measures. 

One of the most important land uses is agriculture lands 

as well as orchards, which may play an important role in 

providing human food. Land-use changes in farming 

areas that derive from agricultural practices or 

overexploitation of water resources in many semiarid 

regions of the world have led to designing urgent 

strategies of water management towards sustainable 

development (Hugget, 1993; Brandt and Thornes, 1996). 

As human demands increase, the sustainability of land 

use is in question. Better land management involves 

identifying land-use changes, understanding current landuse 

patterns or features, and assessing economic and 

ecological benefits and costs that arise from land-use 

practices, as well as finding the best alternatives for each 

area (Wu et al., 2001). Remotely sensed data frequently

Figure 1. Satellite image of study region (band 2 of IRS satellite). 

are used to map land surface cover for use in a variety of 

resource assessment, land management, and modeling 

applications. Mapping from coarse spatial resolution 

images and with multispectral instruments necessarily 

has focused on land cover and broad vegetation types 

(Loveland, 2000) rather than discrimination of vegetation 

at a species level. 

Crop yield forecast in an area usually requires crop 

area estimation, which is mainly concerned by some 

relating organizations. Satellite data along with remote 

sensing technique may be employed as a useful and 

effective tool to estimate crop area. Recent developments 

of image processing technique as well as availability of 

high resolution satellite imageries avail to use this 

technique as a quick and low cost method in compare to 

conventional methods for crop area estimation while it is 

obvious that due to limited period of growing season in 

case of some crops it is very tedious and difficult task to 

estimate crop area using time consuming conventional 

methods. 

Many studies have been conducted concerning the use 

of satellite data for different crop type area estimation 

throughout the world. Olive area estimation has been 

carried out in a few olive-growing countries as Turkey, 

Spain and Portugal. Ediz (2004) used Land sat 7 and IRS 

data to survey olive, pistachio and vineyard in Gazin-Tab 

area Turkey. He used image supervised classification 

technique to estimate olive plantation area. Teresa and 

Granado (2004) has compared land sat images with 

aerial photo to map olive gardens in Portugal. Ramos et 

al. (2007) quantified the eventual land movement and the 

Torkashvand and Eslami 681 

subsequent displacement of olive trees produced by 

continuous tillage erosion. They analyzed these 

movements on a property of olive orchards located on 

variable sloping land. Berni et al. (2009) applied models 

based on canopy temperature estimated from high 

resolution airborne imagery to calculate tree canopy 

conductance (Gc) and the crop water stress index 

(CWSI) of heterogeneous olive orchards. 

In this research, surveying of olive orchards was 

investigated by using IRS Satellite images in the region 

including some parts of Roudbar, Manjil, Loushan and 

Abbar, Guilan, Iran. 


The study area is located between eastern longitudes of 48�55�48� 

and 49�52�54�; and northern latitudes of 36�31�19� and 36�59�57� 

that the region area is 459 km 2 . Administrative boundary of the 

study area includes Roodbar Township along southern part of 

Guilan province, Iran. Figure 1 shows studying region on band 2 of 

IRS satellite. From the physiographic point of view, lands are gently 

sloping foothills, mountainous lands and upper terraces. Lithic units 

are sandstone, limestone, marl, conglomerate, andesite and diorite. 

During recent years, government policy with increasing demand 

has promoted the area of this crop and development of olive 

processing factories in this region. Different image processing 

techniques are usually available to highlight a certain land use. In 

present research, two techniques were employed to highlight olive 

orchards from other land covers which are going to be described in 

following: 1. By spectral reflectance stochastic (DN) of different land 

covers and slicing and 2. By different methods of supervised 

classification. 

IRS images of July 2006 were used to map olive farming area


Table 1. Some properties of IRS bands. 

Band No. Standard Deviation Median Mean 

1 13.3 28 19.58 

2 51.33 99 67.3 

3 47.99 98 67.7 

Figure 2. The final map of olive orchards prepared from crossing three initial maps of olive orchards in their spectrum bands. 

and software ILWIS 3.3 Academic was used for processing data. 

Some properties of IRS bands are observed in Table 1. 

Topographic maps of 1:25000 prepared by National Cartographic 

Center and 23 ground control points were used for geo-referencing 

of images. 

Global positioning system (GPS) was also employed for training 

area selection in the field. Field views (248 points) were done to 

determine accurate positions of land covers including: 1. Olive, 2. 

Hard wood forest, 3. Soft wood forest, 4. Cultivation lands (paddy), 

5. Bare lands, 6. Non olive-plant covers, 7. Water area and 8. 

Urban regions. 

Spectral reflectance stochastic 

A point map of training and auxiliary point of different land covers 

was prepared to overlay on a sample set of color composite (Bands 

1, 2 and 3). The mean and standard deviation of training and 

auxiliary pixels of olive orchards was calculated. Upper and lower 

limits of digital number of olive orchards class pixels determined 

with the addition and subtraction of standard deviation from the 

mean in each band and initial classified map of olive orchards 

prepared in each band with them (Equation 1). 

X B , B , B ) � 2S. 

d 

( 1 2 3 

Olive orchards map was prepared by slicing of these limits in every 

band, separately. The final map of olive orchards prepared from 

crossing three initial maps of olive orchards in there spectrum 

bands that Figure 2 indicates this map. The method accuracy was 

evaluated from crossing the map of training points (pixel) with olive 

orchards map. 

Supervised classification 

Taking into consideration training and auxiliary points of different 

land covers, the supervised classification of IRS images was done 

in four methods: 1. Box Classifier, 2. Maximum likelihood, 3. 

Minimum distance and 4. Minimum Mahalanobis distance. They 

were selected as the most common supervised per-pixel 

classification useful for obtaining thematic maps from multispectral 

satellite data (Richards and Jia, 2006). Supervised algorithm were 

tested by changing bias and threshold values in order to select the 

best boundaries in the spectral space beyond that a pixel has such 

(1)


Table 2. Indicates mean, standard deviation and upper/lower limits of training pixels spectrum reflexes-Olive in order to image slicing in 

Bands 1, 2 and 3. 

Band number 

X * 

S.d 

* * 

X � 2S. 

d 

X � 2S. 

d 

1 2 3 1 2 3 1 2 3 1 2 3 

24.1 64.6 102.3 0.8 6.7 6.2 22.5 51.3 90.0 25.7 77.9 114.7 

*Digital numbers mean of training points in olive orchards class. **Digital numbers standard deviation of training points in olive orchards class. 

Table 3. Indicated the crossing result of training points map with Olive map. According to results, more than 60% of training points in 

classified Olive map recognized as olive class. Spectrum reflexes interference of agricultural and paddle lands with Olive had been 

found, so that, 17.7% of training pixels of agricultural lands in the classified map had olive class. 

Nt* Nt-o** Nt-o/Nt*** 

Olive 2016 1416 70.23 

Hard wood forest 10884 97 0.89 

Soft wood forest 208 - - 

Non olive-plant covers 293 270 92.15 

Bare lands 38734 - - 

Cultivation Lands (paddy) 1213 215 17.72 

Water area 8597 - - 

Urban 1134 - - 

*Total numbers of training points. **Numbers of olive class-pixels after crossing classified map of olive with training points map. ***Olive classpixels/total 

pixels ratio (%). 

a low probability of inclusion in a given class that it is excluded from 

that class. 

In the supervised classification methods, we differentiated dense 

olive orchards of its low dense orchards. Finally, the olive orchards 

map has been crossed by training point map to calculate the 

accuracy of method. The classification accuracy was assessed on 

the entire study area by estimating the overall (A), producer's (PA) 

and user's (UA) accuracies and Cohen's Kappa coefficient (K) 

(Congalton and Green, 1999) derived from the error matrix that is 

the core of accuracy assessment of a classified map (Foody, 2002; 

Liu et al., 2007). 

1 

A � n 

(2) 

ii 

N 

r 

� 

i�1 

n PA / � (3) 

ii icol n 

UA � n / 

(4) 

K � N 

ii nirow 

r 

r 

2 

�nii �� 

nicolnirow 

/ N �� 

i�1 

i�1 

i�1 

r 

n 

icol 

n 

irow 

Where nii is the number of pixels correctly classified in a category; 

N is the total number of pixels in the confusion matrix; r is the 

number of rows; and nicol and nirow are the column (reference data) 

and row (predicted classes) total, respectively. The overall accuracy 

incorporates the major diagonal and gives the crude percentage of 

pixels correctly allocated. PA and UA detail the omission and 

commission errors, respectively. PA shows what percentage of a 

category on the ground is correctly classified by the analyst, and 

(5) 

can define a measure of pixels omitted from its reference class 

(omission error). Likewise, commission error can be estimated from 

UA that, highlighting the interest in quantifying the correspondence 

between the two maps by users, implies a residual percentage of 

pixels of a category that do not “truly” belong to the reference class, 

but are committed to other ground-truth classes (commission error). 

K includes off-diagonal elements also taking into account the 

commission and omission errors. K, by including also information 

on these errors, represents a more realistic and reliable indication 

about the probability that a pixel classified on the map actually 

represents that category on the ground. 

RESULTS 

Table 3 indicates the user’s accuracy in various methods 

of surface cover classifications. Producer accuracy of 

different methods for preparing the classified map of 

surface covers is showing in Table 4. It must be consider 

that user’s accuracy is the main criterion to method 

accuracy in preparing the map of olive. As it can see, 

user’s accuracy in box classifier with 0.73 threshold of 

standard deviation is 41.77% and user’s accuracy 

reduced sharply in classification of olive, so that, in the 

threshold of 2.73 and 3.73 standard deviation, user’s 

accuracy to classify olive is 2.67 and 46%, respectively. 

Therefore, it seems that even in low threshold, this 

method is not enjoying of suitable accuracy to prepare 

olive orchards map. It must be considered that in 

maximum likelihood method, the change of threshold


Table 4. The Users Accuracy in different methods of supervised classification. 

Classification method kind 

of lands cover 

Box classifier 

Maximum 

likelihood 

Minimum distance 

to mean 

Minimum Mahalanobis 

distance 

Standard deviation Thresholds 10, 50 

Search radius (m) Search radius (m) 

0.73 1.73 2.73 3.73 and 100% 

1 50 and 10 100 1 10, 50 and 100 

Olive 41.7 12.0 2.6 0.04 52.5 66.6 54.9 57.1 66.4 65.5 

Low dense olive 62.5 36.6 8.0 3.3 75.5 0 51.6 63.4 53.5 55.8 

Hard wood forest 99.2 98.2 95.2 69.0 97.6 97.9 97.2 73.2 99.2 97.4 

Soft wood forest 100 100 99.5 100 98.0 100 100 100 100 97.4 

Non olive-plant covers 65.7 84.2 82.9 55.3 41.1 0 44.9 42.1 39.0 31.8 

Bare lands 53.0 24.5 9.5 4.4 56.3 66.6 56.7 48.4 62.1 48.1 

Cultivation Lands (paddy) 95.8 89.7 93.5 98.0 89.4 100 85.1 64.8 96.9 73.8 

Water area 100 100 99.9 99.7 99.6 100 99.9 99.7 100 99.5 

Urban 79.1 88.5 86.2 66.6 70.4 0 55.3 65.1 51.4 61.4 

Table 5. The producer accuracy in different methods of supervised classification. 

Classification method 

kind of lands cover 


Maximum 

likelihood 


to mean 


distance 

Standard deviation Thresholds 10, 

Search radius (m) Search Radius (m) 

0.73 1.73 2.73 3.73 50 and 100% 1 50 and 10 100 1 10, 50 and 100 

Olive 80.7 75.1 9.4 0.18 79.3 66.6 76.9 74.0 75.9 71.7 

Low Dense Olive 65.4 51.7 34.2 42.8 54.9 0 60.0 61.1 62.2 38.2 

Hard wood forest 100 100 99.9 100 99.9 100 99.2 98.1 100 99.9 

Soft wood forest 100 89.1 78.7 38.8 67.3 100 68.6 8.9 98.3 78.4 

Non olive-plant covers 22.6 14.7 11.4 1.7 15.1 0 19.1 17.7 22.6 16.0 

Bare lands 99.0 99.2 98.3 96.2 98.9 100 98.3 98.7 87.0 97.4 

Cultivation Lands (paddy) 91.0 63.0 42.2 14.6 64.1 66.6 69.0 13.3 87.0 75.9 

Water area 100 100 99.9 99.7 99.8 100 100 99.8 100 99.9 

Urban 2.9 2.2 1.9 1.6 3.2 0 2.2 97.2 2.3 2.3 

had no effect on map accuracy and user’s 

accuracy in classification of olive orchards was 

52.5%. The most important point is that box 

classifier accuracy in all standard deviations in 

classification of low condensed olive had been 

more than the classification of condensed olive. 

With regard to the results of Table 5, it is found 

that user’s accuracy in maximum likelihood 

method in classification of condensed olive in 

various thresholds of likelihood percent was 

52.5%, but this accuracy with the mentioned 

method in classification of low condensed olive

Table 6. The classified area of different lands covers as compared with basin area in different methods of supervised classification. 


kind of lands cover 


Maximum 

likelihood 


to mean 



distance 

Standard deviation Thresholds 10, 


0.73 1.73 2.73 3.73 50 and 100% 

1 50 and 10 100 1 10, 50 and 100 

Olive 1440.8 5151.9 6990.7 6862.6 14200.5 14.9 5887.3 19723.5 1886.5 19068.7 

Low dense olive 4638.7 13049.8 5497.8 1285.6 20762.7 4.8 6019.1 17535.5 2685.1 19949.2 

Hard wood forest 13092.8 61861.9 73849.0 63834.5 79799.9 205.1 26352.9 47334.4 20468.3 78455.6 

Soft wood forest 29.1 505.3 1452.5 4950.6 2133.4 1.1 1753.9 27916.7 43.2 1061.9 

Non olive-plant covers 2694.0 17547.3 45333.7 126150.4 17661.6 9.2 5239.8 10610.3 1403.8 11689.1 

Bare lands 66145.9 32328.9 10712.3 4015.6 125761.8 206.0 78781.2 11523.7 80549.0 174194.5 

Cultivation Lands (paddy) 1178.3 9364.2 20576.0 39799.3 12338.5 12.8 7879.4 44607.8 1663.8 6870.6 

Water area 553.5 2606.8 2933.6 3095.3 3153.1 10.7 2357.0 3272.0 765.5 3102.7 

Urban 65549.0 254627.5 282128.5 208299.6 183187.4 0.0 78997.4 172134.1 41138.8 144606.5 

Total Area (ha) 155322.1 397043.6 449474.1 458293.7 458999.5 464.6 213268.0 354658.0 150604.0 458999.5 

Unknown area of Basin (ha) 303677.1 61955.9 9525.4 705.8 - 458534.9 245731.5 104341.5 308395.5 - 

Classified area ratio to total area of basin (%) 33.8 86.5 97.9 99.8 100 0.1 46.5 77.3 32.8 100 

orchards is more than 75%. Table 6 indicates the 

classified area of different lands covers as 

compared with basin area in different methods of 

supervised classification. It is correct that in box 

classifier, user’s accuracy is reduced in 

classification of low condensed and condensed 

olive as increasing the threshold, but it must be 

considered that in 0.73 threshold, just 33.8% of 

area had been classified and 67.2% of area had 

been considered as unknown cells. As increasing 

the threshold, the percent of classified area will be 

increase, so the accuracy will be reduced. In 

maximum likelihood method where user’s 

accuracy in classification of condensed olive in 

little, total area of the basin had been classified. 

Therefore, maximum likelihood method had been 

given more suitable response in classified area 

and user’s accuracy. 

In minimum distance method, the most users’ 

accuracy in classification of olive obtained in 1 m 

search radius, although in 100 m search radius, 

user’s accuracy had been had more than 10 m 

search radius. In one m search radius, only 0.1% 

of area had been classified. In this method with 1 

m radius, no cells located in low condensed olive 

category. Also, as increasing the amount of 

search radius to 50 m, it is not found a difference 

in olive accuracy with 10 search radius. 

In minimum Mahalanobis distance method, in 1 

m radius, user’s accuracy in classification of 

condensed olive orchards is 66.47% and in 

classification of low condensed olive is 53.5%. 

The increase of search radius in this method to 

10, 50 and 100 m had been caused to little reduce 

of user’s accuracy in classification of condensed 

olive orchards and to increase of classification 

accuracy of low condensed olive orchards. 

Table 7 indicates the overall accuracy and 

Kappa coefficient in various methods of surface 

cover classifications. The largest overall accuracy 

and Kappa coefficient is related to minimum 

distance method. Overall accuracy and Kappa 

coefficient have decreased with increasing 

standard deviation or search radius in box 

classifier or minimum distance and minimum 

mahalanobis distance methods. 

DISCUSSION 

Separating olive orchards helping spectrum reflex 

statistics caused to separate these orchards from 

forest zones, well. Of course it must consider that, 

in each of spectrum bands, there is wave 

interference of some of surface covers, so 

considering 3 spectrum bands with each other, 

caused to reduce the interference of reflexes and 

to increase the possible to separate olive. The 

results indicated that there would be possible to 

separate olive spectrum reflexes from broad


Table 7. Overall accuracy and Kappa coefficient of different lands covers classification in different methods of supervised classification. 

Maximum 

likelihood 


to mean 


distance 



Standard deviation Thresholds 10, 50 


0.73 1.73 2.73 3.73 and 100% 

1 10 and 50 100 1 10, 50 and 100 

Overall accuracy 61.8 48.8 39.4 29.9 70.4 87.9 69.8 59.4 71.3 65.0 

Kappa coefficient 59.4 39.2 32.4 24.6 58.6 82.8 58.3 46.4 54.8 53.0 

leaf forest (hard wood forest), conifer forest (soft 

wood forest), urban and residential regions, bare 

lands, ranges and water zones, but because of 

intensive spectrum reflexes interference, it was 

not possible to separate the olive from non- olive 

crops including the other orchards, woods and 

parks green spaces. Shrimali et al. (2001) in the 

classification of land use/land cover derived an 

overall accuracy of 83%, that some classes such 

as agriculture (terraced), agriculture (fallow), and 

water body had a classification accuracy of more 

than 90%. The classification of forest into three 

classes (dense, moderate and open mixed forest) 

was 85.5% accurate. The reason for the 

percentage of error was signature overlapping of 

fallow areas with grass cover and overlapping of 

grass and moderate (dense) forests with open 

mixed forests. Accuracy could be further improved 

if the quantification of standing bio-mass in the 

forest area could be done in the training sets. In 

this study, there is overlapping of olive orchards 

with other vegetation, while there was no such 

problem for dense forest. 

In supervised classification of surface area, the 

number of training points, dispersion of training 

points, classification method, defined threshold, 

the number and kind of surface covers classes 

are influencing on map accuracy from 

classification. 

The aim of current study was to separate the 

olive orchards regions from the other surface 

area, so as the condensed of olive canopy cover 

impact on spectrum reflections, olive orchards 

have been considered in two condensed and low 

condensed categories. When there is a low 

condensed olive orchard, it is natural that in one 

pixel, spectrum reflections is influencing on olive 

green canopy cover and soil zone of the lands 

between olive trees. 

In various regions, the type of surface 

phenomenon impact on map accuracy from 

classification, intensively. For example, separating 

of water zones in IRS 3-bands images from 

surface phenomenon maybe possible, simply 

which in turn have its own certain condition , so, 

when the issue of separation one vegetation from 

the other vegetation is consider the possible of 

separating is most difficult . In this research, olive 

consider as one class and the other vegetation 

including orchards, woodlands , garden and etc 

had been considered in another class by the title 

of non-olive vegetation. Also, the vegetation of 

broad-leaf and conifer are considered in a 

separate class. 

To identify a suitable method to prepare surface 

area map, some indexes must be consider: 1. 

User’s accuracy 2. Overall accuracy 3. Kappa 

coefficient and 4. The capacity of method in 

classification various vegetation and classified 

area. 

Minimum distance method was not a suitable 

method to prepare olive map. In this method, 

when search radius rate was 1 m, only 0.1% of 

area classified as identified pixels or in other 

words, nearly all area of unknown domain 

classified. In this method also in higher search 

radius, more part of area classified as unknown 

area. As it could find, spectrum reflections 

interference of olive and non-olive vegetation 

cause to hesitate in using minimum distance 

method. When the spectrum classes are close to 

each other, this classification method is not so 

good (Alavi panah, 2004). 

Out of minimum distance methods, classification 

method with 100 m search radius because of 

77.3% cover area and 64.37% user’s accuracy in 

classification of olive orchards was relatively 

better than the other rate of search radius in this 

method. It must be consider that the overall 

accuracy of this method was about 60%, but 

Kappa coefficient was less than 50 and 46.4%, so 

as the goal is to prepare olive orchards map, the 

main judge criteria is user’s accuracy in 

classification of olive, since it consider the Kappa 

coefficient, correct classified pixels and error 

pixels of all surface vegetation classes. Cuneo et 

al (2009) provided a map of African olive 

distribution was produced from the image analysis 

and checked for accuracy at 337 random 

locations using ground observation and

comparison with existing vegetation maps. Results 

indicated that a total area of 1907 ha of dense African 

olive infestation was identified, with an omission error of 

7.5% and a commission error of 5.4%. 

Minimum distance method was not a suitable method 

to prepare olive map. In this method, when search radius 

rate was 1 m, only 0.1% of area classified as identified 

pixels or in other words, nearly all area of unknown 

domain classified. In this method also in higher search 

radius, more part of area classified as unknown area. As 

it could find, spectrum reflections interference of olive and 

Sepulcre-Canto (2009) monitored a total of 1076 olive 

orchards in area in southern Spain, gathering the field 

location, field area, tree density, and whether the field 

was drip irrigated or rainfed by. An approach based on a 

cumulative index using temperature and the normalized 

difference vegetation index (NDVI) information for the 6year 

ASTER time-series was capable of detecting 

differences between irrigated and rainfed open-canopy 

orchards, obtaining 80% success on field-to-field 

assessments. The method considered that irrigated 

orchards with equal vegetation cover would yield lower 

temperature and NDVI than rainfed orchards; an overall 

accuracy of 75% and a kappa (kappa) of 0.34 was 

obtained with a supervised classification method using 

visible, near infrared and temperature information for the 

6-year ASTER imagery series. 

Maximum likelihood method in all likelihood percent 

thresholds could classify 100% of domain area but in box 

classifier, it was only in 2.73 standard deviation threshold 

that nearly whole area was classified, so they were not 

correct method in olive orchards classification. Unal et al. 

(2004) used maximum likelihood method to classify 

cultivated land and separation of pistachio garden and 

orchard from the other vegetation in Gaziantep province 

of Turkish. Also, Muschen et al. (2001) tried to separate 

agricultural area from non-agricultural area using 

controlled classification of integrated images of TM5 with 

ORS IC PAN land sat and ERS 2 radar by this method 

(maximum likelihood). Maybe, minimum mahalanobis 

distance is the best method in user’s accuracy classified 

area of the domain. In 10 and 50 m thresholds, this 

method had 65.57% and 55.87% classified user’s 

accuracy in the classification of condensed and low 

condensed olive, respectively, which in the whole, its 

user’s accuracy in olive orchards classification was 

60.21% which was the most user’s accuracy in the 

classification of olive. 

Overall accuracy indicates the efficiency of a method in 

classification of various surface covers, but it is possible 

in an overall accuracy that user’s accuracy be less in 

classification of olive. For example, in maximum 

likelihood method, overall accuracy and user’s accuracy 

in classification of olive was 60.4 and 64.6%, 

respectively, but in the minimum Mahalanobis distance 

method with 10 m or more radius, overall accuracy was 

less than the maximum likelihood method (65.0%), but it 


enjoyed the higher user’s accuracy in classification of 

olive (60.2%). Therefore, as the goal is to classify olive, 

user’s accuracy is enjoying from the most importance in 

classification of this olive orchards. Ahadnejad (2003) in a 

research concluded that PCA analysis is the most 

effective method to increase discrimination factor among 

different classes. Color composites of PCA1 PCA2, 

PCA3, consisting the majority of information were used 

for training area selection. He employed maximum 

likelihood classifier to highlight olive farming area that 

olive area estimated around 3843 ha. Mohammadi and 

Nikkami (2008) compared the accuracy of different 

methods including satellite imagery and data layers 

integration and concluded that differentiating 

photomorphic units in satellite imagery makes more 

uniform units for using as working units in erosion feature 

studies. 

Since spectrum waves interference of various surface 

cover cause to raise error in classification and true pixels 

of a certain class locating in a difference class of surface 

cover, so Kappa coefficient is enjoying certain importance 

because with regarding the whole of pixels correct 

classified pixels, user’s accuracy and producer accuracy 

is the more reliable coefficient comparing to overall 

accuracy. In classification of condensed olive orchards, 

as there is high error in classification of olive, non- olive 

vegetation and agricultural lands, so the increase of 

kappa coefficient indicating less error and more capacity 

of this method in classification of surface covers and 

olive. 

In classification of less-condensed olive orchards, 

because of spectrum wave interference of olive green 

canopy cover and the soil zone between the canopy 

cover, the interference of digital number of lowcondensed 

olive observed not only with the other 

vegetation cover but also with bare lands. There was this 

issue even for wave interference of low-condensed olive 

with urban and residential regions as some part of olive 

located in urban and residential regions and one pixel 

digital number can be an average of reradiating wave of 

olive canopy cover and urban and residential region. So, 

some true pixels of low-condensed olive had been 

classified as residential region or vice versa. 

Conclusion 

To compare various classification methods to spectrum 

reflections statistic classification indicate that the 

classification based on spectrum reflections statistic while 

having accuracy same as the best image supervised 

classification, enjoying more simplicity. In this method, 

because of image classification, only focusing on olive 

spectrum reflexes statistic, the likelihood olive regions 

had been separated and preparing the maps is done with 

regard to goal, that is, olive and the other surface covers 

are not consider.


As a whole, it seems that, if preparing the map of olive 

orchards is doing with the help of spectrum reflexes 

statistic in the regions with olive and non-olive vegetation, 

the separated area must indicated under the title of 

mixed olive land and non-olive vegetations. Also, it must 

be consider that the commune spectrum reflexes found 

between the agriculture land and olive. This issue is true 

to supervised classification with box classifier, maximum 

likelihood, minimum distance and minimum Mahalanobiss 

distance. 

With regard to this issue that olive always has green 

canopy cover in Mediterranean climate, it is suggested 

that the possible to prepare olive map study using 

satellite picture in winter season. 

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DOI: 10.5897/JMPR11.968 



Traditional medicinal plants research in Egypt: 

Studies of antioxidant and anticancer activities 

Ahmed M. Aboul-Enein 1 , Faten Abu El-Ela 1 , Emad A. Shalaby 1 and Hany A. El-Shemy 1,2 * 

1 Biochemistry Department, Faculty of Agriculture, Cairo University, 12613 Giza, Egypt. 

2 Faculty of Agriculture Research Park (FARP) and Biochemistry Department, Faculty of Agriculture, Cairo University, 

12613 Giza, Egypt. 

Accepted 10 October, 2011 

Plants have played a significant role in the treatment of cancer and infectious diseases for the last four 

decades. Natural products have been rediscovered as important tools for drug development despite 

advances in combinatorial chemistry. Egyptian flora, the most diverse in the world, has become an 

interesting spot to prospect for new chemical leads or hits due to its species diversity. Screening 

programs have been established in Egypt as a strategy to identify potentially active substances. High 

throughput screening techniques allow for the analysis of large numbers of extracts in a relatively short 

period of time and can be considered one of the most efficient ways of finding new leads from natural 

products. In our study, 23 wild plants were extracted by ethanol and water in addition to 24 ethanolic 

and aqueous extracts from spices and herbs and tested in vitro as anticancer agents. The trypan blue 

technique was used for the anticancer activity against Ehrlich Ascites Carcinoma Cells (EACC) while 

SRB technique was used against HepG2 cells. The antioxidant activity of the 90 plant extracts was 

determined by 2, 2 diphenyl-1-picrylhydrazyl (DPPH) assay. Results showed that both of ethanolic and 

water extracts of some plant possessed high cytotoxic and antioxidant activities and inhibited the cell 

growth of cancer cells. On the other hand, some ethanolic extract gave cytotoxic and antioxidant 

activities more than aqueous extract but other aqueous extracts possessed the opposite trend. We 

believe that the flora of Egypt can be a valuable source of plants rich in, cytotoxic compounds and 

antioxidant agents. 

Key words: Anticancer, antioxidant, polar extracts, Egyptian flora. 

INTRODUCTION 

Scientists are interested in investigating medicinal plants 

which are commonly used by public and derived from 

folklore or anecdotal information (Helton, 1996; Mail et 

al., 1989). In this concern, El-Shemy et al. (2007) 

reported that the medical use of herbs is deeply rooted in 

*Corresponding author. E-mail: helshemy@hotmail.com. Tel: 

+2-(0)2-3774-2600. Fax: +2-(0)2-3571-7355. 

Abbreviations: ROS, reactive oxygen species; O-2, superoxide 

radical; OH, hydroxyl radical; ROO . , hydroperoxyl radical; RNS, 

nitrogen species; . ONOO-, peroxynitrite; NO . , nitric oxide; DNA, 

deoxyribonucleic acid; EACC, Ehrlich Ascites Carcinoma Cells; 

DPPH, 2, 2 diphenyl-1-picrylhydrazyl; NCI, National Cancer 

Institue; HepG-2, human hepatocellular cancer cell line; CNE2, 

carcinoma cell line; MGC-803, gastric cancer cell line. 

human history and folklore and incorporated into the 

historical medicine of virtually all human cultures. He 

described the history of Ginseng and Garlic as two 

famous plants widely used –till now- in traditional 

medicine and proved to have many active constituents. 

Some famous selected examples used to represent the 

importance of those plants based on human observation, 

trial and error, religious advices and from various 

generations’ accumulated experiences, which should 

never neglected or classified as unscientifically based 

treatment. The medicinal plants derived from folklore are 

huge hence; Zingiber officinale (Amara et al., 2008) and 

Nigella sativa (Ferrigni et al., 1982; Ferrigni and 

Mchaughlin, 1984) were taken in consideration as 

extracts containing antitumor compounds. 

Herbal or ‘botanical’ medicines, recorded in developing 

countries with ancient civilizations, such as Egypt and


China, provide an abundant Pharmacopoeia of products 

that have been prescribed for many diseases over many 

centuries. The natural products underlying traditional 

medicines have received increased scientific attention 

lately (Han et al., 2002; Vickers, 2002). Since there are 

national and indigenous rights over plant derived 

resources, basic scientific investigations based on 

medicinal plants and indigenous medical systems have 

increased in developing countries (Han et al., 2002; El 

Shemy et al., 2007). In addition, Egypt includes wide 

areas of desert and tropical regions which encourage the 

growing of wild plants resistant for those hard conditions. 

Therefore, these plants might contain different secondary 

metabolites with high biological value which can be used 

for treatment of different diseases including cancer. 

The Mediterranean region, despite its location in a 

temperate zone far from the diversity hotspots 

popularized by the media, it is one of the areas with the 

greatest diversity on the planet and thus it is considered 

that it should be maintained as a conservation sanctuary 

(Myers et al., 2000). About 10% of the world’s higher 

plants can be found in this area, which represents only 

1.6% of the Earth’s surface (Médail and Qu´ezel, 1999). 

Around 25,000 species are found in the region. 

Cancer is a general term applied to malignant diseases 

characterized by rapid and uncontrolled abnormal cells 

formation which may mass together to form a growth or 

proliferate throughout the body and it may progress until 

it causes death. Medicinal plants are the most exclusive 

source of life saving drugs for the majority of the world’s 

population. Medicinal herbs have been widely used for 

treatment of diseases in traditional way for several 

generations. An interaction between traditional medicine 

and modern biotechnological tools is to be established 

towards new drug development. The interference 

between cell biology, in vitro assays and structural 

chemistry will be the best way forward to obtain valuable 

leads. There is considerable scientific evidence to 

suggest that nutritive and non nutritive plant-based 

dietary factors can inhibit the process of carcinogenesis 

effectively. 

Cancer chemoprevention involves pharmacologic 

intervention with synthetic or naturally occurring 

chemicals to prevent, inhibit or reverse carcinogenesis or 

prevent the development of invasive cancer. Out of an 

estimated 250,000 higher plants, less than 1% has been 

screened pharmacologically (El-Shemy et al., 2007.). In 

recent years, focus on plant research has increased all 

over the world. 

Antioxidants are a group of substances that are useful 

for fighting cancer and other processes that potentially 

lead to diseases such as atherosclerosis, Alzheimer's, 

Parkinson's, diabetes and heart disease (Valko et al., 

2007). Unlike cytotoxic agents that damage tumor cells, 

antioxidants act by preventing the onset of cancer during 

carcinogenesis, and they are generally beneficial to cells. 

Oxidants such as reactive oxygen species (ROS) that 

include the superoxide radical (O - 

2), hydroxyl radical 

(OH), hydroperoxyl radical (ROO . ) and nitrogen species 

(RNS) such as peroxynitrite ( . ONOO-) and nitric oxide 

(NO . ) damage macromolecules, including proteins, lipids, 

enzymes and deoxyribonucleic acid (DNA) (Sies, 1993). 

To combat these radicals, living organisms produce 

enzymes (for example, catalase, superoxide dismutase 

and peroxidase) or rely on nonenzymatic molecules, such 

as glutathione, cysteine, ascorbic acid, flavonoids and 

vitamin K for protection (Sies, 1993). 

The aim of this study is to evaluate the anticancer and 

antioxidant capacity of traditionally and fresh medicinal 

plants collected from different regions in Egypt. 


Collection of plants materials 

Wild plants materials used in this investigation were collected from 

El-Alaemeen-Coastel area, Sedi Abd El-Rahman, Egypt, during 

February – March, 2010. Taxonomy section for the wild plants was 

preceded by Prof. Dr. Sherif S. El-Khanagrs, ARC, CAIM Herbarium 

of Museum, Dokki, Egypt. The plant samples (Whole plant) was air 

dried then grounded to a powder using mechanical mortar. Also ten 

fresh samples were tested for anticancer and antioxidant activity. 

The fresh samples were purchased from local commercial shops 

which include: Capsicum annuum, Solanum lycopersicum, Daucus 

carota, Psidium guajav, Citrus limon (L.) Burm, Ficus carica, 

Phoenix dactylifera, Vitis vinifera except Eichhornia compressa 

which was collected from Nile River (Tables 1 to 3). 

Preparation of extracts 

Different crude extracts of wild and traditional plants as well as, 

fresh plants were prepared according to Ferrigni et al. (1982). To 

obtain the aqueous and ethanolic extracts samples were cleaned 

and air dried (except fresh samples) then grinded to be ready for 

testing. The specific plant samples (roots, leaves, fruits and bulbs) 

were extracted with water and ethanol as follows: 5 g of air dried 

sample were grinded in the presence of about 30 ml cold distilled 

water and filtered then centrifuged at 3000 rpm for 10 min. The 

supernatant was separated solvents removed under reduced 

pressure. Each extract was then transferred to an 10 ml weighed, 

small and clean glass vial and the crude extract weight was 

determined. All the extracted materials were preserved at -20°C 

until analysis of biological activities. 

Viability of Ehrlich Ascites Carcinoma Cells (EACC) using 

trypan blue-exclusion technique 

A line of Ehrlich Ascities Carcinoma from National Cancer Institute 

(NCI) Cairo, Egypt has been used. The tumor line is maintained in 

female Swiss albino mice by weekly intraperitoneal (ip) 

transplantation of 2.5 × 10 6 cells. The cells were taken from tumor 

transplanted animals after ≈ 7 days of transplantation. The cells 

were centrifuged at 1000 rpm for 5 min, washed with saline then the 

needed number of cells was prepared by suspending the cells in 

the appropriate volume of saline according to the tests used. 

Transplantation in animals for cell line, the appropriate volume of 

ascities can be used directly. 

The viability percentages of tumor cells after incubation with 

aqueous or ethanolic extract as well as saline as control were

Aboul-Enein et al. 691 

Table 1. Scientific name, Family, plant part, Arabic name and plant picture of wild plants (Collected from El-Alameen , Sidi Abd El-Rahman 

and Marsa Matrouh regions at winter season 2010). 

Scientific name Family Plant part tested Arabic name Plant picture 

Atriplex sp Chenopodiaceae Whole plant فطقل 

Euphorbia 

paralias L. 

Cakile maritime 

Scop. 

Panax 

quinquefolius 

Rosmarinus 

officinalis 

Zygophyllum 

album 

Euphorbiaceae Whole plant ةنٌبل 

Cruciferae Whole plant لٌكاك 

Araliaceae Seeds جنسنج 

Lamiaceae 

Whole plant نابل ًصح 

Zygophyllaceae Whole plant لابلب


Table 1. Contd. 

Asparagus stipularis Liliaceae Whole plant سٌجاربسا 

Kochia indica Chenopodiaceae Whole plant اٌكوك 

Retama raetam (Forssk) 

Webb 

Leguminosae Whole plant مترلا 

Olea europaea L. Oleaceae Whole plant ىبرولاا نوتٌزلا 

Pituranthos tortusous Umbelliferae Whole plant حازق 

Limoniastrum monopetalum 

(L.) Boiss. 

Plumaginaceae Whole plant ةتٌز


Cistanche phelypaea (L.) Orobanchaceae Whole plant نوند 

Moricandia nitens Cruciferae Whole plant يدناكروم 

Zygophulum simplex L. 

Arum palaestinum 

Anabasis artiaulata (Forssk.) 

Moq. 

Thymelaea hirsute (L.) 

Endl. 

Zygophyllaceae 

Araceae 

Whole plant طٌرطرلا 

Leaves فول 

Chenopodiaceae Whole plant مرجعلا 

Thymelaeaceae Whole plant نانتملا 

Aboul-Enein et al. 693



Astragalus pinosus Leguminosae Whole plant سلجارتسا 

Asphodelus microcarpus Liliaceae Whole plant لصنعلا 

Solanum nigrum Solanaceae Whole plant بٌدلا بنع 

Lotas polyphylles 

Beta vulgaris 

Leguminosae Whole plant 

ساتول 

Chenopodiaceae Whole plant ركسلا رجنب

Table 2. Scientific name, Family, plant part, Arabic name and plant picture of Spices and herbes. 

Scientific name 

Camellia sinensis 

Cinnamomum verum. 

Punica granatum 

Glycyrrhiza glabra 

Capsicum annuum 

Family 

Theaceae 

Lauraceae 

Punicaceaea 

Galegeae 

Solanaceae 

Plant part 

tested 

Leaves 

Bark 

Fruit 

Seeds 

Fruit 

Arabic name 

ضر 

ةفرق 

نامر 

خا ياش 

سوسقرع 

رمحا لفلف 

Plant picture 

Aboul-Enein et al. 695



Ocimum basilicum 

Zingiber officinale 

Curcuma longa 

Cassia italca 

Lamiaceae 

Zingiberaceae 

Zingiberaceae 

Leguminosae 

Seeds 

Rhizome 

Rhizome 

Leaves 

measured by the modified cytotoxic trypan blue-exclusion technique 

of Bennett et al. (1976). Two ml of media containing EACC (2 ×10 4 

cells) were transferred into a set of tubes each, then 100 μg/ml from 

different extract were added into the appropriate tube as well as 

saline. The tubes were incubated at 37°C for 2 h then centrifuged at 

1000 rpm for 5 min and the separated cells were suspended in 2 ml 

saline. For each examined materials (and control), a new clean, dry 

small test tube was used and 10 μl of cell suspension, 80 μl saline 

and 10 μl trypan blue (0.4%) were added and mixed, then the 

number of living cells (non stained) was calculated using a 

homocytometer slide by microscope (Nikon, TMS). 

Viability of HepG-2 cells using SRB assay 

Human hepatocellular cancer cell line (HepG-2), was obtained from 

the Vaccera (Giza, Egypt). Cells were maintained in RPMI-1640 

supplemented with 100 µg/ml streptomycin, 100 units/ml penicillin 

and 10% heat-inactivated fetal bovine serum in a humidified, 5% 

(v/v) CO2 atmosphere at 37ºC. The cytotoxicity of fresh crude 

ناحٌر 

لٌبجنز 

مكرك 

ًكمانس 

extract was tested against HepG-2 cells by SRB assay as 

previously described (Skehan et al., 1990). Exponentially growing 

cells were collected using 0.25% Trypsin-EDTA and plated in 96well 

plates at 1000-2000 cells/well. Cells were exposed to each test 

compound for 72 h and subsequently fixed with TCA (10%) for 1 h 

at 4ºC. After several washings, cells were exposed to 0.4% SRB 

solution for 10 min in dark place and subsequently washed with 1% 

glacial acetic acid. After drying overnight, Tris-HCl was used to 

dissolve the SRB-stained cells and color intensity was measured at 

540 nm. 

DPPH method 

The 2, 2 diphenyl-1-picrylhydrazyl (DPPH) test was carried out as 

described by Burits and Bucar (2000). One ml of plant extract (100 

μg/ml) was mixed with 1ml DPPH reagent (0.002% (w/v)/methanol 

solution). After an incubation for 30 min in the dark at room 

temperature, the absorbance was measured at 517 nm Butylated 

hydroxyl toluene (100 μg/ml) was used as positive control.

Table 3. Scientific name, Family, plant part, Arabic name and plant picture of fresh vegetables and fruits. 




Daucus carota 

Psidium guajava L 

Solanum lycopersicum 

Family 

Solanaceae 

Solanaceae 

Umbelliferae 

Myrtaceae 

Solanaceae 

Plant part tested 

Whole plant 

Whole plant 

Whole plant 

Leaves 

Bark 

Arabic name 

رمحا لفلف 

يمور لفلف 

رزج 

ةفاوج 

مطامط 


Plant picture



Citrus limon (L.) Burm 

Vitis vinifera 

Ficus carica 

Phoenix dactylifera 

Eichhornia azurea 

Rutaceae 

Vitaceae 

Moracea 

Palmaceae 


Pontederiaceae 

Fruit 

Whole plant 

Whole plant 

Whole plant 

Fruit 

Trypan blue assay was used for the evaluation of 

نومٌل 

بنع 

نٌت 

حلب 

لٌنلا درو 

anticancer activity of ethanolic and aqueous extracts 

against EACC. The anticancer activities of 35 plant 

extracts showed that 17 ethanolic and 18 aqueous

Table 4. Anticancer and Antioxidant activity of different extracts from wild plants. 



Anticancer activity 

Water Ethanolic 

Antioxidant activity 

Ethanolic Water 

Atriplex Sp. 100 49 70.8 50.5 

Euphorbia paralias L. 3.3 2.4 81.1 51.8 

Cakile maritime Scop. 89.7 90.78 56.3 55.6 

Panax quinquefolius 64 2.55 11.7 56 

Zygophulum album L.F 61.13 32.86 80.3 64.8 

Asparagus Stipularis) 13 5.2 72.7 70.9 

Kochia indica wight 2.88 1.6 50.4 72.4 

Retama raetam (Forssk) Webb 2.6 1.4 80.2 78.1 

Olea europaea L. 0 7.98 50.5 81.1 

Pituranthos tortusous 11.21 14.28 58.4 81.4 

Limoniastrum monopetalum (L.) Boiss 52.9 3.8 85.6 82 

Cistanche phelypaea (L.) 37 100 50.7 85.6 

Moricandia nitens 89.19 51 89.8 85.6 

Zygophulum simplex L. 61.13 32.86 85.7 44.1 

Arum palaestinum 97.29 19.44 12.7 43.1 

Anabasis artiaulata (Forssk.) Moq 25 10 40.8 42.7 

Thymelaea hirsute (L.) Endl. 54 18 78.6 35.3 

Astragalus pinosus. 100 15.83 28.4 19.5 

Asphodelus microcarpus salzm 9.09 1.94 60.3 49.5 

Solanum nigrum 100 89.74 85.7 55.6 

Lotas polyphylles 7.15 7.9 27 27 

Beta vulgaris 64 6.99 41.1 30.3 

extracts gave anticancer activity more than 70% (Tables 

4 and 5). 

The maximal inhibition (100%) was observed in the 

ethanolic extracts obtained from Solanum nigrum, 

Atriplex sp. and Astragalus spinosus followed by Arum 

palaestinum (97.29%) as in Tables 4 and 5. While 

ethanolic extract obtained from Cakile maritime, N. sativa 

and Z. officinale possessed anticancer activity of 89.7, 81 

and 80%, respectively. It was observed that the ethanolic 

extract obtained from Ocimum basilicum, Cassia italca, 

Panax quinquefoliu and Zygophulum simplex were in the 

third category which gave anticancer activity more than 

60% (77.21, 66, 64.1 and 61%, respectively), while 

Thymelaea hirsute and Limoniastrum monopetalum 

showed anticancer activity more than 50% (54 and 53%). 

On the other hand, twenty two ethanolic extracts gave 

weak anticancer activity (0.0 to 47.83%) (Tables 4 and 5). 

In the water extracts tested, data also showed that the 

maximal cancer inhibition was observed by Cistanche 

phelypaea, Solenostemma argel, C. italca and Cakile 

maritime extracts (100, 95, 92 and 90.78%, respectively). 

Extracts from, S. nigrum, camellia sinensis and 

Glycyrrhiza glabra showed anticancer activity more than 

80% (89.7, 86.4 and 81%, respectively). It was clear from 

the results that both aqueous and ethanolic extracts of S. 

nigrum, C. maritime and O. basilicum possessed high 

anticancer activity such inhibited completely cell growth 

of EACC at the 100 μg/ml concentration. 

However, some other aqueous extracts provided high 

anticancer activity more than ethanolic extract such as 

Arum palaestinum, N. sativa, P. quinquefolius, Z. 

simplex, T. hirsute and L. monopetalu. 

These results were in agreement with the results 

obtained by Nawab et al. (2011) who reported that 

exposure of aqueous extract of S. nigrum (due to 

steroidal glycosides and glycoprotein) exerted an 

inhibitory effect on cell growth and colony formation of the 

prostate, breast and colorectal cells. In addition, Li et al. 

(2008) found that aqueous extract of S. nigrum inhibits 

growth of cervical carcinoma (U14). Extract of C. sinensis 

inhibited three tumor cell lines (HeLa cell line, poorly 

differentiated nasopharyngeal carcinoma cell line (CNE2) 

and gastric cancer cell line (MGC-803) due to inhibition of 

DNA topoisomerase II (Bingfen et al., 1994). G. glabra 

extract used to treat chronic hepatitis, other viruses, 

various types of ulcers and this extract is composed of 

triterpenes saponins, flavonoids, polysaccharides, pectin, 

simple sugars, amino acids, mineral salts, and various 

other substances (Saxena, 2005). 

In other side, Verotta and El-Sebakhy (2001) revealed 

that, Astragalus species were used in Chinese traditional 

medicine as antiperspirant, antihypertensive, antidiabetic,


Table 5. Anticancer and antioxidant of spices and herbs. 


Anticancer activity 

Water Ethanol 

Antioxidant activity 

Ethanol Water 

Rosmarinus oficinalis 80.04 61 38.4 65.1 

Camellia sinensis 85 86.4 85.4 70.6 

Cockatiel 9.76 22.88 56.7 71.4 

Punica granatum 6.08 4 85.7 75.8 

Glycyrrhiza glabra 36 81 47.4 84.1 

Capsicum annuum 24.35 68.63 57.3 25 

Ocimum basilicum 77.21 76.29 72.3 9.8 

Zingiber officinale 47.83 4.93 55.9 35.5 

Curcuma longa 39.42 72.4 6.4 43.4 

Cassia italca 89.7 90.78 55.4 30.7 

Nigella sativa 81 2.54 8.4 8.8 

Solenostemma argel 24.66 95 41.3 7 

Parviflora 7.83 1.55 42.7 40.3 

diuretic and tonic. The pharmacologically active 

constituents of Astragalus were classified to two different 

types, polysaccharides and saponins and the most 

interesting pharmacological properties were 

hepatoprotective, immunostimulant and antiviral activity. 

Husein et al. (2011) reported that highest cytotoxicity 

for ethanolic extracts of A. palaestinum against breast 

cancer. Moreover, the anticancer activity of N. sativa may 

be attributed to the quinone constituents of the seed 

(Mahfouz and El-Dakhakhny, 1960). The anticancer 

activity of the methanol extract of Cassia italica against 

two types of cancer cell lines, (Hep-G2, Hela cell lines) 

was also detected. On the other side, anticancer activity 

of ten fresh samples against HepG2 was evaluated and 

the observed data showed that five aqueous extracts 

possessed high anticancer activity more than 90% (Table 

4). Several studies have also demonstrated anti-cancer 

or anti-mutagenic effect of the chilli (C. annuum) extracts. 

Carotenoids present in chilli extracts were found to have 

a synergistic anti-mutagenic and in vitro anti-tumourpromoting 

activity (de Mejia et al., 1998; Maoka et al., 

2001). D. carota has anticancer constituent, epilaserine, 

in its lipophilic fraction (Jing et al., 2008). Therefore, Sato 

et al. (2010) suggested that a combination bark leaf and 

root extract inhibited growth of B16 melanoma cells. 

Epidemiological studies have suggested that an 

inverse association exists between consumption of 

vegetables and fruits and the risk of human cancers at 

many sites (Riboli and Norat, 2003). Phenolic 

compounds, including flavonoids are especially promising 

candidates for cancer prevention. Much information is 

available on the reported inhibitory effects of specific 

plant phenolic compounds and extracts on mutagenesis 

and carcinogenesis (Myers et al., 2000). The potential 

ability of polyphenol combinations to prevent cancer 

progression has not been adequately studied. Scientists 

have suggested that it appears extremely unlikely that 

any one substance is responsible for all of the 

associations seen between plant foods and cancer 

prevention because of the great variety of dietary 

phenolics, including flavonoids, and the many types of 

potential mechanisms reported (Birt et al., 2001; El- 

Shemy et al., 2007). 

These results were in agreement with that of our 

previous work (Nassr-Allah et al., 2009) which showed 

that the hot water and ethanolic extracts of S. arghel and 

hot water extracts of Colocasia antiquorum may have an 

immuno-modulatory potential via stimulating antiproliferation 

of tumor cells. However, hot water and ethanolic 

extracts of S. arghel and hot water extracts of C. 

antiquorum significantly inhibited the growth of AML, ALL 

and EACC cells in vitro and in-vivo. This appears to 

involve apoptosis-induced cell loss; a lowering in the 

proliferation rate of AML cells. The immuno-modulatory 

components were associated with the content of 

phenolics, including flavonoids. 

Further, the total phenolic contents correlated 

significantly (P

Table 6. Anticancer and Antioxidant activity of fresh samples (Vegetables and fruits). 

Scientific name Moisture content % 

Anticancer activity Antioxidant activity 


Water ethanolic Ethanolic extract Water extract 

Capsicum annuum 89.57 92.36 91.32 90.3 81.8 

Capsicum annuum 89 77.2 74.7 86 73.2 

Daucus carota 90.7 92.09 93.25 85.6 81.5 

Psidium guajava L 97.07 91.13 92.09 97.3 88.4 

Solanum lycopersicum 93.9 81.3 82.8 82.8 82.6 

Citrus limon (L.) Burm 82.9 86.7 91.3 91.3 70.4 

Vitis vinifera 85.08 82.6 89.2 90.5 85 

Ficus carica 91.25 82.6 83.4 84 80.5 

Phoenix dactylifera 72.67 88.6 79.3 83.4 77 

Eichhornia azurea 91.3 90.5 54.1 50.5 

activity more than 50% while the other samples were less 

(Table 3). It was observed that the aqueous extracts 

obtained from Retama raetam, Punica granatum, Kochia 

indica, Cinnamomum verum and Asparagus stipularis 

came in the third category which gave more than 70% 

antioxidant activity (78.1, 75.8, 72.4, 71.1, 70.9 and 

70.6%, respectively). The results also showed that seven 

aqueous extract obtained from Zygophyllum album, 

Rosmarinus oficinalis, P. quinquefolius, C. maritime, S. 

nigrum, Euphorbia paralias and Atriplex sp. showed 

moderate antioxidant activity (50.8 to 64.8%). The other 

plant extracts had only weak antioxidant activity ranged 7 

to 49.5% (Tables 4 and 5). 

From the 35 ethanolic plant extracts tested Moricandia 

nitens, Z. simplex, P. granatum, Limoniastrum 

monopetalum, Zygophulum album, E. paralias, and 

Retama raetam gave the maximum antioxidant activity 

(80 to 89%). Also from table (3) it was observed that four 

ethanolic plant extracts from T. hirsute, A. stipularis, O. 

basilicum and Atriplex sp. gave antioxidant activity more 

than 70% (78.6, 72.7, 72.3 and 70.8%, respectively). 

Finally, the data showed that both aqueous and ethanol 

extracts of some plants possessed high antioxidant 

activity at the 100 μg/ml concentration such as M. nitens, 

L. monopetalum and R. raetam. However, aqueous 

extract of other plants gave high antioxidant activity more 

than ethanolic extract such as R. raetam, O. europaea 

and Pituranthos tortuosus. While, ethanolic extracts of 

some plants possess high antioxidant activity more than 

aqueous extract such as Atriplex sp., S. nigrum and Z. 

album (Tables 4 and 5). Furthermore, data’s in Table 6 

showed the antioxidant activity of ten fresh samples. It 

was observed that ethanolic extract from P. guajava 

possessed high antioxidant activity (97.3%), other three 

extract showed antioxidant activity more than 90% which 

include C. limon, V. vinifera and C. annuum (91.3, 90.3 

and 90.5%, respectively). Five ethanolic extracts gave 

antioxidant activity more than 80% (C. annuum, D. 

carota, F. carica, P. dactylifera and S. lycopersicum). 

Different extracts from the tested plants have been 

previously evaluated for their antioxidant activity and 

results agreed with the findings that reported by 

Hassimotto et al. (2005) for C. phelypaea, P. guajava and 

M. nitens which gave the most active antioxidant against 

DPPH. M. nitens belongs to Cruciferae which has 

antioxidant activity as a result of glucosinolates as well as 

possessing a high content of flavonoids, vitamins and 

mineral nutrients (Moreno et al., 2006). The antioxidant 

activity of G. glabra extract has been reported also by 

Saxena (2005). In investigation by Trabelsi et al. (2010) 

extract from L. monopetalum exhibits antidysenteric 

properties against infectious diseases and antioxidant 

properties as a result of phenolic compound in these 

plant. P. tortuosus has antioxidant activity due to 

presence of flavonial glycosides, steroids and 

furanocoumarins (Abdel and Hafez, 1995). O. europaea 

showed antioxidant activity (Benavente-Garcيa et al., 

1999; Cلrcel et al., 2010) while C. limon besides its 

antioxidant activity (Hoyle and Santos, 2010), anticancer 

activity has been found (Silalahi, 2002). Aqueous extracts 

from Z. album and V. vinifera showed good results in 

scavenging DPPH and hydroxyl radicals (Khafagi et al., 

2001). Antidiabetic activity of V. vinifera was reported 

(Şendogdu and Asalan, 2006). As the extracts are better 

soluble in aqueous media than are the synthetic 

antioxidants, they offer a promising alternative as food 

ingredients with antioxidant activity. 

Our preliminary studies showed a good relationship 

between antioxidant efficacy of plant extracts and 

anticancer potency. All of the extracts which gave high 

anticancer potency have high antioxidant activity while 

the opposite trend is not. In this concern, cancer is a 

multistage process defined by at least three stages: 

initiation, promotion, and progression (Ames and Gold, 

1992; Guyton and Kensler, 1993; Schulte-Hermann et al., 

1990). Oxidative stress interacts with all three stages of 

this process. During the initiation stage, ROS may 

produce DNA damage by introducing gene mutations and 

structural alterations into the DNA. In the promotion 

stage, ROS can contribute to abnormal gene expression,


blockage of cell-to-cell communication, and modification 

of second-messenger systems, thus resulting in an 

increase in cell proliferation or a decrease in apoptosis of 

the initiated cell population. Finally, oxidative stress may 

also participate in the progression stage of the cancer 

process by adding further DNA alterations to the initiated 

cell population (Klaunig et al., 1998). 

The encouraging results obtained from this screening 

work represent an important step towards the effective 

isolation, characterization of the active principles in these 

plants and to understand the mechanism of cytotoxic of 

these compounds. We also working plan to carry more 

biological activities including the in vivo studies thus; 

these plants could be as a source for new lead structures 

in drug design to combat cancer and natural antioxidants. 

Conclusion 

It has become clear that in Egypt many plants and herbs 

might provide effective anti cancer therapeutics. Such 

extracts should be more widely used in developing 

countries for prevention and treatment of dangerous 

diseases like cancer. The extracts should be considered 

as good sources for drug discovery. 


This work was fully supported by a grant from the 

Science and Technology Development Fund (STDFproject 

ID:312), Cairo, Egypt. 

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DOI: 10.5897/JMPR11.1004 



Preliminary phytochemical screening and 

ethnomedicinal uses of Teucrium stocksianum from 

Malakand Division 

Gul Rahim 1 , Rahmatullah Qureshi 1 *, Muhammad Gulfraz 1 , M. Arshad 1 and Sahib Rahim 2 

1 Department of Botany, Pir Mehr Ali Shah Arid Agriculture University, Murree Road, Rawalpindi, Pakistan. 

2 Department of environmental Sciences, International Islamic University, Islamabad, Pakistan. 

Accepted 22 November, 2011 

The extracts of Teucrium stocksianum were screened for secondary metabolites by using methanol, 

chloroform and n-hexane solvents. The methanolic extracts gave positive results for alkaloids, tannins, 

flavonoids, saponins, steroid, reducing sugar, terpenoid, anthraquinone, phlobatannin and glycoside. In 

the case of chloroform, alkaloids, tannins, reducing sugar, saponins, flavonoids, terpenoid were 

present and anthraquinone, glycoside, phlobatannin, steroid were found to be absent, while n- hexane 

extracts contained tannins, reducing sugar, flavonoids and failed to detect alkaloids, anthraquinone, 

glycoside, phlobatannin and terpenoid. This paper highlights the significance of plant in traditional 

medicine and distribution of various chemical constituents with respect to Malakand Division, Pakistan. 

Key words: Teucrium stocksianum, secondary metabolites, traditional medicine, phytochemical constituents. 

INTRODUCTION 

For the existence of life on the earth, plants played very 

importance role from the time immemorial. Human being 

is directly relayed on plants to fulfill their daily life 

requirement. They are used as food, fuel, ornamentals, 

flavor and medicine. Throughout the world higher plants 

are being employed to treat various infectious diseases. 

They provide number of natural products including 

medicaments to fight against diseases. 

To maintain quality of drug, there is need to evaluate 

active constituents of raw material to ascertain their 

therapeutic effects. It enables the pharmacist to prescribe 

numerical values and ensure uniformity of standards 

(Amen, 1996). Medical plant can be defined as any part 

of the plant which contains substances that can be used 

for therapeutic purposes and its precursor for the 

synthesis of useful drugs. They contain nutrients and 

phytochemicals that can heal ailments of the body 

(Trease and Evans, 1978). Plant material that has cellular 

*Corresponding author. E-mail: rahmatullahq@yahoo.com, 

rahmatullahq@uaar.edu.pk. 

structure is referred as organized drug in pharmacy, 

whereas, non-cellular structure as unorganized or a 

cellular drug (Akinpelu and Onakoya, 2006). 

Medicinal and aromatic plants (MAP) are a large group 

of economically important plants that provide raw 

materials for pharmaceuticals, perfumery, flavor and 

cosmetic industries. Teucrium stocksianum Boiss. 

(Lamiaceae) locally known as Speer botay is a perennial, 

woody, aromatic herb which is native to the mountainous 

regions of the United Arab Emirates (UAE), northern 

Oman (Western 1989; Nadaf et al., 2003), Pakistan 

(Ahmad et al., 2002) and Iran (Mojab et al., 2003). This 

plant attains a height of 10 to 25 cm having densely 

packed stem and grey-green leaves. The leaves and 

young shoots of T. stocksianum are commonly used for 

the preparation of traditional medicines to treat several 

ailments including diabetes, gastro-intestinal ailments 

and inflammatory conditions (Radhakrishnan et al., 

2001). In addition to the gastro protective effect, 

decoction of T. stocksianum has been used for the 

treatment of diabetes and burning feet syndrome 

(Barkatullah and Hussain, 2009). 

Secondary metabolites are chemicals produced

through secondary reactions resulting into primary 

carbohydrates, amino acids and lipids (Ting, 1982). Their 

direct function in plant metabolism is not well recognized 

till date. However, their role in ecosystems (Dey and 

Harborne, 1997), particularly in plant herbivore interaction 

(Feeny, 1976; Swain, 1979) and chemotaxonomy (Gibbs, 

1974) has been well known. Plants contain secondary 

metabolites such as alkaloids, saponins and tannins are 

generally avoided by grazing animals and leaf feeding 

insects. Their presence in plants and intake at high level 

reduces the nutrient utilization, feed efficiency, animal 

productivity and in some cases may cause death (Makkar 

and Goodchild, 1996). 

The extract of T. stocksianum has been found to have 

anti-ulcerogenic and cytoprotective properties when 

applied on experimentally-induced gastric lesions (Islam 

et al., 2002). In addition, the plant extract has also been 

analyzed for analgesic and anti-inflammatory activities, 

which supports the traditional use of the plant in the 

treatment of painful inflammatory conditions 

(Radhakrishnan et al., 2001). This plant is also used as a 

blood purifier, a remedy for the treatment of hypertension 

and epilepsy (Ahmad et al., 2002) and sore throat (Iqbal 

and Hamayun, 2004). 

The people of Malakand division have good knowledge 

about this plant for its medicinal use. Based on this 

indigenous knowledge of T. stocksianum, present study 

was carried out to analyze phytochemicals in order to 

seek scientific logic for their use in folk medicine. 


Collection and identification of plant materials 

The whole plant (leaves, stem, root, and floral head) of T. 

stocksianum was collected in the month of May to June 2011 from 

the different areas of Talash (Dir lower), Malakand Division, Khyber 

Pakhtun Khwa (KPK), Pakistan. One set of specimens (No. 2214) 

was made using conventional method of pressing, drying and 

mounting on standard herbarium sheet. The plant was described 

and identified by Dr. Rahmatullah Qureshi in Taxonomy Lab. by 

employing Flora of Pakistan (Hedge, 1990). The identified 

specimen was deposited in the department of Botany, Pir Mehr Ali 

Shah Arid Agriculture University, Rawalpindi for further record. 

Preparation of the extracts 

The plant material was washed thoroughly 2 to 3 times with running 

water and once with sterile distilled water immediately after 

collection to remove dirt prior to the drying process and shade 

dried. These were then finely powdered (80 mesh) using a 

laboratory grinding mill and stored in refrigerator. The powdered 

material was soaked in methanol, chloroform and n-hexane (1:10) 

by shaken for 24 h at 37°C. The extracts were then f iltered using 

Whatman filter paper No. 1. The filtrates of methanol, chloroform 

and n-hexane were combined and concentrated under reduced 

pressure at 40°C using rotary evaporator. Blackish extra cts of 

methanol, chloroform and n-hexane were obtained for 

phytochemical screening. 


Rahim et al. 705 

Chemical tests were carried out in methanol, chloroform and nhexane 

extracts of T. stocksianum using standard procedures to 

identify the constituents as described by Sofowora (1993), Trease 

and Evans (1978) and Harborne (1973). 

Test for alkaloids 

About 0.2 g of the extracts was warmed with 2% H2SO4 for two 

minutes. The filtrate was added with few drops of Dragendorff's 

reagent (solution of potassium bismuth iodide). Presence of orange 

red precipitate indicated as positiveness for alkaloids. 

Test for tannins 

Little amount of extract was mixed with water and heated on water 

bath. The mixture was filtered and ferric chloride was added to the 

filtrate. The dark green solution indicated the presence of tannins. 

Test for anthraquinones 

About 0.5 g extract was boiled with 10% HCl for few minutes in 

water bath. The filtrate was allowed to cool and equal volume of 

chloroform was added. 

It was added with few drops of 10% NH and the mixture was 

heated. The appearance of rose-pink color indicated the presence 

of anthraquinone. 

Test for glycosides 

The extract was hydrolyzed adding with HCl and neutralized with 

NaOH solution. Afterward few drops of Fehling’s solution A and B 

were added. The occurrence of red precipitate showed the 

presence of glycosides. 

Test for reducing sugars 

The extract was shaken with distilled water, filtered and boiled with 

drops of Fehling’s solution A and B for minutes. Showing of an 

orange red precipitate indicated the occurrence of reducing sugars. 

Test for saponins 

About 0.2 g of the extract was shaken with 5 ml of distilled water 

and then heated to boil. Frothing (appearance of creamy miss of 

small bubbles) shows the presence of saponins. 

Test for flavonoids 

The 0.2 g extract was dissolved in diluted NaOH and HCl was 

added. A yellow solution that turned colorless within few minutes 

indicated the presence of flavonoids. 

Test for phlobatanins 

The 0.5 g extract was dissolved in distilled water and filtered. It was 

boiled with 2% HCl solution. The appearance of red precipitation 

revealed the presence of phlobatanins.


Test for steroids 

Table 1. Phytochemical screening of T. stocksianum using different solvents. 

Phytochemicals 

Solvent 

Chloroform Methanol n-hexane 

Alkaloids +ve +ve -ve 

Tannin +ve +ve +ve 

Saponins +ve +ve +ve 

Anthraquinone -ve +ve -ve 

Steroid -ve +ve -ve 

Phlobatannin -ve +ve -ve 

Terpenoid +ve +ve -ve 

Flavonoids +ve +ve +ve 

Glycoside -ve +ve -ve 

Reducing sugar +ve +ve +ve 

2 ml acetic anhydride was added to 0.5 g plant extract along with 2 

ml of H2SO4. The change of color from violet to blue or green 

indicated the presence of steroids. 

Test for terpenoids (Salkowski test) 

The 0.2 g plant extract was mixed with 2 ml chloroform and 3 ml 

concentrated H2SO4. The formation of reddish brown color to the 

interface was formed that indicated positive sign for terpenoids. 

Ethnobotanical use 

Indigenous communities of Malakand Division were interviewed to 

get important recipes and anecdotal uses of T. stocksianum 

following the methodology of Qureshi and Bhatti (2008). 

RESULTS 

The results of phytochemical screening of T. stocksianum 

by using methanol, chloroform and n-hexane solvents are 

summarized in Table 1. The methanolic extracts 

exhibited positive for the presence of all screened 

phytochemicals such as alkaloids, tannins, flavonoids, 

saponins, steroid, reducing sugar, terpenoid, 

anthraquinone, phlobatannin and glycoside. The chloroform 

extracts detected alkaloids, tannins, reducing sugar, 

saponins, flavonoids, terpenoid except anthraquinone, 

glycoside, phlobatannin, steroid. Similarly, n- hexane 

extracts traced tannins, reducing sugar, flavonoids and 

failed to detect alkaloids, anthraquinone, glycoside, 

phlobatannin and terpenoid. 

Ethnobotanical use 

The people of Malakand Division have good knowledge 

about this plant for its medicinal use. Ethnobotanically, 

the juice of T. stocksianum is given for the treatment of 

jaundice and as blood purifier as well as cooling agent. 

The decoction of plant is also prescribed to treat chronic 

fever. Leaves soaked in water overnight and the juice is 

given before breakfast to diarrhea and abdominal pain. 

Young leaves are boiled in water and obtained juice is 

used for curing cough. 

DISCUSSION 

The present research work reveals that there are various 

important phytochemicals such as alkaloids, tannin, 

saponins, anthraquinone, steroid, phlobatannin, 

terpenoid, flavonoids, glycoside and reducing sugar were 

present in T. stocksianum (Table 1). Besides, tannin, 

saponins, flavonoids and reducing sugar were uniformly 

detected by all solvents (Figure 1). The chemical 

constituents extracted by various solvents were in the 

order of methanol> chloroform> n-hexane. 

It is an established fact that secondary metabolites 

present in plants derive therapeutic activities (Rabe, 

2000). Particularly, alkaloids are reported the most 

important therapeutic significant substance (Njoku and 

Akumefula, 2007). The present study stratified that 

alkaloids were detected by methanol and chloroform 

solvents that may signify for further investigation from 

these solvent base extracts. Tannins and flavonoids are 

also reported to possess biological activities which are 

used for the prevention and management of many 

diseases (James et al., 2007). Our investigation from T. 

stocksianum reveals that tannins and flavonoids are 

traced by all solvents and link logically with ethnomedicinal 

use of this plant in traditional system of 

medicine. The medicinal properties of the plant could be 

attributed to the presence of one or more of the detected 

plant natural products. These findings give credibility to 

the traditional medicinal relevance of the plant as 

remedies for abdominal pain, chronic fever, diarrhea, 

blood purification, jaundice and cough. However, the 

pharmacological actions of the plant cannot be

3 

2.5 

2 

1.5 

1 

0.5 

0 

Figure 1. Phytochemical analysis of Teucrium stocksianum using various solvents. 

ascertained by the result of the phytochemical analysis 

only. Results of this investigation offer a scientific 

foundation for carrying out in vitro and in vivo activities. In 

conclusion, isolation and purification of the phytochemical 

followed by a detailed study might result in identification 

lead compound for curing various diseases. 

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DOI: 10.5897/JMPR11-1044 



Antioxidant and antiapoptotic properties of 

chlorogenic acid on human umbilical vein endothelial 

cells 

Xing Wu*, Song Lin and Xiaotang Zhang 

Department of Internal Medicine, the Sixth Hospital of Shenyang, Liaoning 110006, China. 


Chlorogenic acid (CGA) is one of the phenols isolated from Lonicera japonica Thumb. This study was 

designed to investigate whether CGA prevents human umbilical vein endothelial cells (HUVECs) from 

hydrogen peroxide (H2O2)–induced damage. The cell viability was examined by methylthiazolyl 

tetrazolium (MTT) assay. Malondialdehyde (MDA) level was measured using the thiobarbituric acid 

method. Reactive oxygen species (ROS) levels were determined using the 2',7'-dichlorofluorescindiacetate 

(DCFH-DA) assay. The antioxidant enzymes activities, including glutathione peroxidase (GPx) 

and superoxide dismutase (SOD) were examined photometrically. Total antioxidant capacity (T-AOC) 

was measured using the ferric reducing activity of plasma (FRAP) assay. Apoptotic cells were detected 

by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end 

labeling (TUNEL), annexin-V binding and by assessment of caspase-3. Protein and mRNA expression of 

Bax and Bcl-2 were determined by western blot and reverse transcription-polymerase chain reaction 

(RT-PCR). In the process of H2O2-induced damage, the expression of Bcl-2 was decreased and the 

expression of Bax was increased. The antioxidant enzymes activities and T-AOC levels were decreased. 

The content of MDA and the level of ROS were increased. Pretreatment with CGA significantly reduced 

oxidative stress by increased T-AOC levels and antioxidant enzymes activities, decreased ROS levels 

and lipid peroxidation. CGA significantly reduced apoptosis by up-regulation the expression of Bcl-2 

and down-regulation the expression of Bax. Taken together, these results suggest that CGA exhibits 

antioxidant and antiapoptotic properties to prevent H2O2-induced cytotoxicity in HUVECs. 

Key words: Chlorogenic acid, human umbilical vein endothelial cells (HUVECs), hydrogen peroxide, oxidative 

stress, apoptosis. 

INTRODUCTION 

Oxidative stress is caused by the imbalance between 

production of pro-oxidants and the antioxidant defenses. 

Reactive oxygen species (ROS) are natural byproducts of 

cellular metabolism. They may injure cells by causing 

membrane lipid peroxidation and DNA strand breaks 

(Apel and Hirt, 2004) and consequently lead to apoptosis 

or death (Aoki et al., 2001). Cellular defense systems 

against this free-radical consist of antioxidant enzymes 

*Corresponding author. E-mail: xingwu1997@sina.com. Tel: 

+86-024-23387410. Fax: +86-024-23388643. 

such as glutathione peroxidase (GPx) and superoxide 

dismutase (SOD). They can scavenge ROS and inhibit 

lipid peroxidation, thereby suppressing the resulting 

injury. 

Endothelial cells layer inside blood vessels and play a 

pivotal role in maintaining physiological function of the 

cardiovascular system. The loss of endothelial cells by 

apoptosis is likely to enhance atherosclerotic lesion 

formation (Verma et al., 2003) and leads to cardiovascular 

disease (Gorlach et al., 2000; Herkert et al., 2002). 

It is very important to inhibit endothelial cells apoptosis 

to prevent the development of atherosclerosis. Apoptosis 

is a highly regulated program of cell death and plays an

important role in the pathogenesis in a variety of 

cardiovascular diseases (Lee and Gustafsson, 2009, Aoki 

et al., 2001). Many genes are associated with the 

regulation of apoptosis. The Bcl-2 family members are 

involved in the regulation of apoptosis via the 

mitochondrial pathway (Fiers et al., 1999). Bcl-2 induces 

cell survival, while Bax promotes cell death (Bruckheimer 

et al., 1998). 

Chlorogenic acid (CGA) is an effective component 

extracted from Lonicera japonica Thumb, it has many 

pharmacological actions. In recent studies, CGA has 

been found to have potent cytoprotective effects (Xu et 

al., 2010; Huang et al., 2008). However, there have been 

no studies on whether CGA is able to protect the 

endothelial cells against oxidative stress-induced 

damage. In the present study, we examined for the first 

time whether CGA protects endothelial cells against 

H2O2-induced damage and tried to find out the potential 

mechanism. 


Reagents 

Human umbilical vein endothelial cells (HUVECs) were purchased 

from the American Type Culture Collection (ATCC, Shanghai, 

China). CGA was purchased from National Institute for the Control 

of Pharmaceutical and Biological Products (Beijing, China). 

Dulbecco′s modified Eagle′s medium (DMEM) and fetal bovine 

serum (FBS) were purchased from GIBCO BRL Life Technologies 

(Grand Island, USA). MTT and dimethyl sulfoxide (DMSO) were 

purchased from Sigma-Aldrich Corporation (St. Louis, USA). 

Annexin V and Propidium Iodide (PI) were purchased from Jingmei 

Biotechnology (Shenzhen, China). 

Primary antibodies, horseradish peroxidase (HRP) conjugated 

secondary antibodies, 3,3'-diaminobenzidine (DAB) kit and TUNEL 

apoptosis detection kit were purchased from Boster Biological 

Technology (Wuhan, China). Trizol and RNA PCR kit were 

purchased from TaKaRa Biotechnology Co. (Dalian, China). 

RevertAid H Minus M-MuLV reverse transcriptase was purchased 

from Biometra (Goettingen, Germany). Enhanced 

chemiluminescence kit was purchased from Amersham 

(Buckinghamshire, UK). Assay kits for antioxidant enzyme activities, 

total antioxidant capacity (T-AOC) assay kit, MDA test kit and 

dichlorofluorescein diacetate (DCFH-DA) detection kit were 

purchased from Beyotime Institute of Biotechnology (Jiangshu, 

China). All other chemicals were of analytical grade, and purchased 

from Beijing Chemical Factory (Beijing, China). 

Cell culture 

The HUVECs were maintained in DMEM supplemented with 10% 

FBS, 100 μg/ml of penicillin and 100 μg/ml of streptomycin. Cells 

were incubated in a humidified atmosphere of 5% CO2 at 37°C. 

Grouping and intervention 

There were five groups in this study: (1) Control group. Cells were 

untreated. (2) H2O2 group. To induce oxidative stress, cells were 

cultured with the medium containing 100 μΜ of H2O2 for 24 h. (3, 4, 

Wu et al. 709 

5) CGA group. Cells were treated with CGA at a dosage of 0.1, 1 

and 10 μg/ml for 24 h and then exposed to 100 μM H2O2 for 24 h. 

MTT assay 

Cell viability was assessed using MTT assay according to the 

manufacturer's protocol. Briefly, at the end of each treatment 

period, 20 µl of MTT was added into each well and the microplate 

was incubated for 4 h at 37°C. The medium with MTT was removed 

and 150 µl DMSO was added to each well. The plate was shaken 

for 10 minutes. The absorbance was measured at 490 nm. Wells 

without cells were used as blanks. Results were expressed as a 

percentage of control. 

TUNEL assay 

The apoptotic cells were detected using a TUNEL apoptosis 

detection kit following manufacturer's protocol. Briefly, after being 

fixed with 4% paraformaldehyde, the cells were incubated with 

0.3% hydrogen peroxide for 10 min. After being washed thrice with 

distilled water, the cells were incubated in the TUNEL reaction 

mixture containing both terminal deoxynucleotidyl transferase and 

biotin-11-deoxyuridine triphosphate at 37°C for 2 h. The cells were 

washed in PBS and marked using DAB as a chromogenic 

substrate. Cells were rinsed in water and then observed via a light 

microscopy (Olympus, Shimadzu, Japan). The nuclei of apoptotic 

cells were stained brown. 

Annexin V-FITC/PI assay 

HUVECs were collected and washed with PBS three times. Cells 

were resuspended in 100 μl binding buffer (10 mM HEPES/NaOH, 

140 mM NaCl, 2.5 mM CaCl2, pH 7.4), and 5 µL of Annexin-V FITC 

and 10 µL of PI were added. After incubation for 15 min at 25°C in 

the dark, 400 μl binding buffer was added. Flow cytometry 

(FACScalibur, Becton-Dickinson, USA) was used to analyze these 

cells and AnnexinV-FITC positive cells were counted as apoptotic 

cells (Rayner et al., 2006). 

Intracellular ROS and Lipid peroxidation 

The levels of intracellular ROS were measured using DCFH-DA 

detection kit. Briefly, cells were first washed with phosphate 

buffered saline (PBS) three times and then incubated for 30 min at 

37°C with the same solution containing 40 μM of DCFH-DA. The 

absorbance was read at 525 nm. 

Lipid peroxidation level was determined by measuring the 

production of MDA using the thiobarbituric acid method. The assay 

is based on the reaction of MDA with thiobarbituric acid (TBA); 

forming a pink chromogen compound (MDA-TBA adduct) whose 

absorbance was measured at 535 nm. 

FRAP assay 

The T-AOC of HUVECs were evaluated by using FRAP assay as 

described previously (Benzie and Strain, 1996). Each sample (5 μl) 

was added to 180 μl of FRAP reagent. After the mixture stood at 

37°C for 4 min, the absorbance at 593 nm was determined against 

blank. Fresh aqueous solutions of ferrous sulphate (FeSO4) were 

used for calibration, and the standard curve was obtained using 

FeSO4 concentration (0.15-1.5 mM). The results were plotted 

against the standard curves for each of them. The values for FRAP 

were expressed as μmol ferrous iron equivalents per mg protein.


Enzyme activity assay 

Table 1. Preventive effects of CGA on cell viability against H2O2-induced damage determined by MTT assay. 

Group Cell viability (%) 

Control 100 

H2O2 

54.9 ± 6.3** 

H2O2 + CGA (0.1 μg/ml) 73.3 ± 4.8 ## 

H2O2 + CGA (1 μg/ml) 81.3 ± 4.3 ## 

H2O2 + CGA (10 μg/ml) 89.0 ± 2.7 ## 

The data from 5 independent experiments were expressed as means ± S.D. **P < 0.01 compared to control group. # P 

< 0.01 compared to H2O2 group. 

The total SOD activity was assayed by inhibition of nitroblue 

tetrazolium reduction by xanthine oxidase/xanthine system using a 

total superoxide dismutase assay kit according to the 

manufacturer’s protocol. The absorbance was read at 560 nm. The 

activities of SOD were expressed as units per mg protein. One unit 

is defined as the amount of enzyme needed to exhibit 50% 

dismutation of the superoxide radical. 

The GPx activity was assayed by quantifying the rate of oxidation 

of the reduced glutathione to the GSSG by H2O2 using a cellular 

glutathione peroxidase assay kit according to the manufacturer's 

protocol. The absorbance was read at 340 nm. The activities of 

GPx were expressed as units per mg protein. One unit of GPx 

activity was defined as the amount required to oxidize 1 μmol 

NADPH to NADP + in 1 min at 25°C, pH 8.0. 

Western blot assay 

HUVECs were collected, washed with PBS three times, and then 

resuspended in 100 μl lysis buffer (pH=7.5, 50 mM Tris-HCl, 150 

mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% 

sodium dodecyl sulfate and 1 mM phenylmethylsulfonyl fluoride) for 

30 min on ice. After centrifugation, the extracted protein 

concentration was determined using the Bradford assay. The 

proteins (50 μg), added in the same volume of sample buffer, were 

first subjected to 10% sodium dodecyl sulfate polyacrylamide gels 

and then transferred to nitrocellulose membranes. The membranes 

were incubated for 4 h with skimmed milk in TBST buffer (pH 7.5, 

10 mM Tris–HCl, 150mM NaCl and 0.1% (v/v) Tween-20). 

Subsequently, the membranes were probed with specific primary 

antibodies (anti cleaved caspase-3, Bcl-2, Bax, and β-actin 

antibodies) at 4°C overnight, followed by incubation with 

corresponding horseradish peroxidase-coupled secondary 

antibodies for 1 h at room temperature. Then the blots were 

detected using enhanced chemiluminescence technique. 

Semiquantitative real-time RT-PCR assay 

Total RNA was extracted using Trizol reagent following the 

manufacturer’s instructions. RNA concentrations were quantified by 

spectrophotometry (UV300, Hampshire, England). cDNA was 

synthesized with Revert Aid H Minus M-muLV reverse 

transcriptase. Real-time RT-PCR was performed using an ABI 7700 

Prism sequence detection system and TaqMan � probes (Applied 

Biosystems, Foster City, CA). The total reaction volume was 20 μl: 

2 μl cDNA, 10 μl SYBR � Premix Ex Taq � , 0.4 μl of each primer (10 

μM) and 7.2 μl ultrapure water. Cycler conditions were as follows: 

Taq activation at 95°C for 30 s, amplification at 40 cycles of 95°C 

for 5 s and final extension at 60°C for 30 s. Glyceraldehyde-3- 

phosphate dehydrogenase (GAPDH) was used as an internal 

control. The primer sequences were: Bax forward: 5′-TGC TTC 

AGG GTT TCA TCC A-3′, reverse: 5′-GAC ACT CGC TCA GCT 

TCT TG-3′; Bcl-2 forward: 5′-GGG AGA ACA GGG TAC GAT AA-3′, 

reverse: 5′-GCT GGG AGG AGA AGA TGC-3′ ; GAPDH forward: 5′- 

GGA TTT GGT CGT ATT GGG-3′, reverse: 5′-TCG CTC CTG GAA 

GAT GG-3′. 


All data are presented as mean ± standard deviation (S.D.). SPSS 

software version 13.0 (SPSS Inc., Chicago, USA) was used for data 

analysis. Statistical comparison within groups was carried out with 

one way ANOVA. The accepted level of significance was p

Figure 1. Apoptosis was evaluated by TUNEL method. TUNEL staining was performed after HUVECs were 

pretreated with CGA (10 μg/ml) for 24 h and then exposed to 100 μM of H2O2 for 24 h. The nuclei of apoptotic cells 

were stained brown (×400). (A) Control group (B) H2O2 group (C) CGA (10 μg/ml) group. 

Wu et al. 711 

Figure 2. Annexin V-FITC/PI assay for apoptosis rate. HUVECs were pretreated with different concentrations of CGA 

(0.1, 1, 10 μg/ml) for 24 h and then exposed to 100 μM H2O2 for 24 h. Cells were collected and the apoptosis rates 

were determined by flow cytometry using Annexin V-FITC/PI double labeling. (A) Control group; (B) H2O2 group; (C) 0.1 

μg/ml CGA group; (D) 1 μg/ml CGA group; (E) 10 μg/ml CGA group; (F) Histogram showing quantitative data. Values 

are means ± S.D. from three independent experiments. **P


Relative express of cleaved caspase-3 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

0.0 

H2O2 (100 µM) 

CGA (µg/ml) 

Figure 3. The level of cleaved caspase-3 was analyzed by western blot. β-Actin was 

used as the internal control. Cleaved caspase-3 bands were measured using optical 

densitometry, and the data were normalized to a control sample. Values are means ± 

S.D. from three independent experiments. **P < 0.01 compared to control group. # P < 

0.05 and ## P < 0.01 compared to H2O2 group. 

Inhibition of ROS generation and lipid peroxidation 

by CGA 

Compared with control, the intracellular ROS manifested 

as fluorescence intensity increased remarkably in 

HUVECs exposed to H2O2. The fluorescence intensity 

decreased remarkably by pretreatment with CGA (0.1, 1 

or 10 μg/ml) in a dose dependent manner (Table 2). 

In comparison with the control group, the content of 

MDA increased remarkably in the H2O2 group. CGA (0.1, 

1 or 10 μg/ml) significantly reduced lipid peroxidation in a 

dose dependent manner (Table 2). 

Effects of CGA on T-AOC, GPx and SOD 

Compared with control, notable reductions in T-AOC, 

GPx and SOD levels were observed after exposure to 

H2O2 for 24 hours. Pretreatment with CGA (0.1, 1 or 10 

μg/ml) significantly increased the levels of T-AOC, GPx 

and SOD in a dose dependent manner (Table 3). 

Adjustment of the expression of Bcl-2 and Bax by 

CGA 

The mRNA and protein expression levels of Bcl-2 were 

reduced in H2O2 group compared to control group, and 

were significantly increased in CGA groups (0.1, 1, 10 

μg/ml) compared to the H2O2 group in a dose dependent 

manner (Figure 4A and Table 4). The mRNA and protein 

expression levels of Bax were increased in H2O2 group 

compared to control group and were significantly 

decreased in CGA groups (0.1, 1, 10 μg/ml) compared to 

the H2O2 group in a dose dependent manner (Figure 4B 

and Table 4).

DISCUSSION 

Table 2. The effects of CGA on the levels of MDA and the intracellular ROS in HUVECs. Cells were pretreated with 

different concentrations of CGA (0.1, 1, 10 μg/ml) for 24 h and then exposed to 100 μM H2O2 for 24 h. 

Group ROS MDA (nmol/ mg protein) 

Control 36.8 ± 4.7 1.41 ± 0.22 

H2O2 101.6 ± 10.7** 3.74 ± 0.31** 

H2O2 + CGA (0.1 μg/ml) 84.0 ± 6.2 ## 2.85 ± 0.14 ## 

H2O2 + CGA (1 μg/ml) 66.5 ± 3.6 ## 2.01 ± 0.18 ## 

H2O2 + CGA (10 μg/ml) 41.9 ± 5.8 ## 1.52 ± 0.11 ## 

The data from 5 independent experiments were expressed as means ± S.D. **P


Relative express of Bcl-2 protein 

1.4 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 

H2O2 (100 µM) 

CGA (µg/ml) 

Relative express of Bax protein 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 

H2O2 (100 µM) 

CGA (µg/ml) 

Figure 4. The levels of Bax and Bcl-2 were analyzed by western blot. β-Actin was used as the internal control. The 

protein bands were measured using optical densitometry, and the data were normalized to a control sample. Values 

are means ± S.D. from three independent experiments. *P

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DOI: 10.5897/JMPR11.1127 



Elastase, tyrosinase and lipoxygenase inhibition and 

antioxidant activity of an aqueous extract from 

Epilobium angustifolium L. leaves 

Hulya Celik Onar 1 , Ayse Yusufoglu 1 , Gulen Turker 2 and Refiye Yanardag 3 * 

1 Faculty of Engineering, Department of Chemistry, Organic Division, Istanbul University, Avcilar-Istanbul, 34320, Turkey. 

2 Faculty of Science and Arts, Department of Chemistry, Canakkale Onsekiz Mart University, Canakkale, Turkey. 

3 Department of Chemistry, Biochemistry Division, Faculty of Engineering, Istanbul University, Avcilar-Istanbul, 34320, 

Turkey. 

Accepted 22 September, 2011 

Herbs have been utilized to treat acute and chronic disorders since thousand years. Epilobium 

angustifolium L. (Onagraceae) is used as herbal and digestive plant all over the world. The Epilobium 

extracts are reported to have analgesic, anti-microbial, antimotility, antiproliferative, antiinflammatory, 

antitumor and antiandrogenic activities. In the present study, the antioxidant activity and elastase, 

tyrosinase, lipoxygenase inhibitor capacity of E. angustifolium L. were examined. Total phenolic and 

total flavonoid content, reducing power, superoxide anion radical scavenging, hydroxyl radical 

scavenging, 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging, 2,2'-azino-bis(3ethylbenzothiazoline-6-sulfonic 

acid) (ABTS) radical scavenging activities were used to evaluate the 

antioxidant activities. The results were compared with natural and synthetic antioxidants. The aqueous 

extract of E. angustifolium exhibited strong elastase (EC50= 42.72 � 2.38 �g/ml), tyrosinase (EC50= 33.03 

� 3.71 �g/ml) and lipoxygenase inhibitory activities (EC50= 0.57 � 0.06 �g/ml). The extract also showed 

good antioxidant activity in all antioxidant tests. According to these results, E. angustifolium may be 

considered as an important source for pharmaceutical, cosmetic and food manufactures due to its 

elastase, tyrosinase, lipoxygenase inhibitory activities and antioxidant activities. 

Key words: Epilobium angustifolium L., antielastase, antityrosinase, antioxidant, activity, lipoxygenase, 

inhibition. 

INTRODUCTION 

Skin is a fundamentally important organ of the body, 

protecting it from damage caused by direct contact with 

outside environment. Ultraviolet light (UV) leads to 

alterations in the composition of the skin, including the 

accumulation of elastic fibres, collagen reduction and 

degenaration and deposition of glycosaminoglycans 

(Moon et al., 2010). Oxidative stress derived from UV in 

sunlight induces different hazardous effects in the skin, 

including sunburn, photo-aging and DNA mutagenesis. 

Elastin an important structural protein of extracellular 

*Corresponding author. E-mail: refiyeyanardag@yahoo.com. 

Tel: +90 212 4737037. Fax: +90 212 4737180. 

matrix, is the major component of the elastic fibres, which 

provide resilience and elasticity to many tissues including 

skin, lungs, ligaments and arterial walls (Wiedow et al., 

1990). Elastase is the only enzyme capable of deprading 

elastin, an insoluble elastic fibrous protein of animal 

connective tissue (Antonicelli et al., 2007). Elastase is 

known to cause rheumatoid arthritis, pulmonary 

emphysema, and other chronic inflammatory diseases by 

the protein degradation of human tissues. It also 

degrades elastin, which is closely related to the elasticity 

and restoration of skin, and induces wrinkles and the lack 

of elasticity. Elastase activity increased significantly with 

age and results in reduced skin elasticitic properties, 

aging and sagging (Robert, 2001). Inhibition of the 

elastase activity could be employed as a useful method

to protect against skin aging (Wiedow et al., 1990). 

Tyrosinase is an enzyme of the undesirable browning of 

fruits and vegetables, and is involved in the natural 

development of skin, hair and eye coloring (Roh et al., 

2004) and in the initial step of melanin synthesis. Overactivity 

of this enzyme leads to overproduction of melanin 

leading to hyper-pigmentation of the skin. Hyperpigmentation 

can also be caused by excessive exposure 

to UV light, drug reaction and also occurs during ageing. 

The accumulation of an excessive level of epidermal 

pigmentation can cause some dermatological disorders 

associated with freckles, melasma, ephelide and senile 

lentigines (Khan et al., 2006). Tyrosinase is linked to 

Parkinson disease and other neurodegenaritive diseases 

in mammals (Xu et al., 1998: Asanuma et al., 2003). In 

addition, tyrosinase has been reported to be involved in 

the molting process of insects (Shiino et al., 2001) and it 

may be characterized as a potential target site for the 

control of insects pests. Therefore, the control of 

tyrosinase is important in relation to browning control of 

vegetables and fruits, and potent tyrosinase inhibitors 

have also gained increasing importance in medicinal and 

cosmetic products in relation to hyperpigmentation. Some 

commercially available tyrosinase inhibitors used as 

chemical and fungal derived skin-lightening agents have 

been proven to have chronic, cytotoxic and mutagenic 

effects on humans (Wang et al., 2006). Therefore, it is 

still necessary to search tyrosinase inhibitors with potent 

activities and lower side effects. 

Lipooxygenases (LOX) are a family of iron–contaning 

enzymes that catalyse the dioxygenation of 

polyunsaturated fatty acids in lipids. Lipooxygenases 

have recently become of interest, as they are considered 

as the key enzymes in the biosynthesis of leukotrienes 

that have been postulated to play an important role in the 

pathophysiology of several inflammatory and allergic 

diseases. They also play a significant role in cancer, cell 

growth, metastasis, cell survival and induction of tumor 

necrosis factor (TNF) (Khan et al., 2007). Increased 

expression of cyclooxygenase (2) and specific LOX 

enzymes have been observed in several epithelial 

cancers such as breast, renal, pancreatic and prostate 

cancers. Inhibitors of lipooxygenases have attracted 

attention initially as potential agents for the treatment of 

inflammatory and allergic diseases, but their therapeutic 

potential has been expanded to certain types of cancer 

and cardiovascular disease (Werz and Steinhilber, 2006). 

Free radical species and reactive oxygen species (ROS) 

may cause oxidative damage. Antioxidants can react with 

ROS and quench free radicals giving rise to restriction of 

radical chain propagation, eventually preventing tissue 

damage. Many antioxidants based on drug formulations 

are used for the prevention and treatment of complex 

diseases like stroke, cancer, atherosclerosis, and 

Alzheimer’s disease. Oxidation of lipids, which is the 

main cause of quality deterioration in many food systems, 

may lead to off-flavors and formation of toxic compounds, 

and may lower the quality and nutritional value of foods. 

Onar et al. 717 

Lipid oxidation is also associated with aging, membrane 

damage, heart disease and cancer (Ramarathnam et al., 

1995). Natural antioxidant properties from plant species 

have medicinal functions. Natural antioxidant constituents 

are the most important compounds in cosmetic and food 

industries because of their capacities to decrease the 

free radical mediated degradations of tissues and cells in 

human organism (Jin et al., 2004). Consequently, there is 

a requeriment to explore new natural sources of 

antioxidants to replace synthetic antioxidants. 

The genus Epilobium is widely distributed all over the 

world and consist of over 200 species, among which the 

most known are E. angustifolium, E. hirsutum and E. 

parviflorum, perennial herbs generally named Willow 

herb, with reference to the willow-like nature of their 

leaves (Battinelli et al., 2001). Various members of the 

genus Epilobium have been used in folk medicine 

internally for prostate disease (Melchior, 1972), menstrual 

and gastrointestinal disorders (Hiermann et al., 1986), 

rectal bleeding and externally as antiphlogistic (Štajner et 

al., 2007). In Russia, due to a sweet and pleasant taste, it 

is usually consumed as tea for the treatment of stomach 

ulceration, gastritis and sleeping disorders (Štajner et al., 

2006). The leaves and young shoot tips can be used in 

salads, soups or cooked as a vegetable (Štajner et al., 

2007). 

Phytochemical screenings of Epilobium species have 

revealed the presence of steroids, triterpenes, fatty acids, 

phenolic acids, macrocyclic tannins, and flavonoids in the 

aerial parts (Barakat et al., 1997). The antioxidant activity 

and phenolic compounds, including other Epilobium 

subspecies have been studied, but only limited studies 

have been reported on the antioxidant activity of E. 

angustifolium L. aqueous extracts (Štajner et al., 2007). 

In this study, we have investigated the elastase, 

tyrosinase, lipoxygenase inhibitory activities of the 

aqueous extract from E. angustifolium L. for the first time 

as well as its antioxidant activity. The total phenolic and 

flavonoid contents were also determined to find out the 

relationship between free radical scavenging assays. 


Plant material 

E. angustifolium leaves were collected in September from 

Canakkale in Turkey and identified by Prof. Dr. Kerim Alpinar from 

the Faculty of Pharmacy, Istanbul University. Voucher specimens 

are deposited in the Herbarium of the Faculty of Pharmacy, Istanbul 

University (Herbarium code number, ISTE 83909). The leaves were 

washed with deionized water and dried for 5 to 7 days in the shade 

at room temperature. The dried leaves were manually ground to a 

fine powder. 

Preparation of extract 

Ground dried leaves powder (8 g) was extracted with boiled distilled 

water (200 ml) for 15 min while stirring, in order to prepare a 4%


(w/v) infusion. The extract was filtered and evaporated to dryness 

under reduced pressure at 40°C in a rotary evaporator, then 

weighed to determine the total extractable compounds (EC). The 

crude extract was transferred to vials and kept at -20°C and 

dissolved in water for the assessment of antioxidant activity. 

Elastase inhibitory activity 

Elastase activity was examined by using N-succinyl-Ala-Ala-Ala-pnitroanilide 

(STANA) as a substrate and by the measuring the 

release of p-nitroaniline at 410 nm 

(James et al., 1996). The reaction was carried out in a 200 mM Tris- 

HCl buffer (pH 8.0) containing 5 mM N-suc-(Ala)3 p-nitroanilide and 

20 μg/ml elastase. 50 μl of elastase inhibitor solution was mixed 

with the elastase and incubated for 15 min at 37°C prior to the 

addition of the STANA substrate. Then 0.9 ml of 50 mM Tris-HCl 

buffer (pH 7.8) and 20 µl 50 mM STANA were added and incubated 

for 40 min at 37°C. The change in absorbance was measured at 

410 nm. The percent inhibition of elastase was calculated as 

follows: 

Inhibition (%) = (1- B/A) × 100 

Where A is the enzyme activity without inhibitor and B is the activity 

in the presence of inhibitor. The EC50 defined as the concentration 

of plant extract required to inhibit elastase activity by 50% was 

determined. 

Tyrosinase inhibitor activity 

The tyrosinase inhibitior activity of the sample was estimated 

according to the method of Vanni et al. (1990). Briefly, test reaction 

mixtures were prepared by adding 10 μl tyrosinase to 10 μl plant 

extract and then adding 20 μl 1.5 mM L-tyrosine and 110 μl of 0.1 M 

sodium phosphate buffer (pH 6.5). The resulting mixture (150 μl) 

was incubated for 10 min at 37°C and absorption at 490 nm was 

measured. The percent inhibition of tyrosinase activity was 

calculated as follows: 

Inhibition (%) = (1 – B/A) × 100 


in presence of inhibitor. The EC50 defined as the concentration of 

plant extract required to inhibit tyrosinase activity by 50%, was 

determined. 

Lipoxygenase inhibitor activity 

Lipoxygenase inhibitor activity was determined by slightly modifiying 

the spectrophotometric method developed by Tappel (1962). A 

mixture of 0.8 ml of 100 mM phospate buffer (pH 8), 0.1 ml (U/ml) 

lipoxygenase and 50 μl plant extract was incubated for 10 min at 

25°C. 

The reaction was then initiated by addition of 50 μl 20 μmM 

linoleic acid. This resulting solution mixed well and incubated and 

room temperature. After 6 min, the change of absorbance at 234 

nm was read to measure conjugated diene produced. The percent 

inhibition of lipoxygenase activity was calculated as: 

Inhibition (%) = (1 – B/A) ×100 


in presence of inhibitor. 

The EC50 defined as the concentration of plant extract required to 

inhibit lipoxygenase activity by 50%, was determined. 

Determination of total phenolic contents 

Total phenolics in E. angustifolium extract were determined with 

Folin-Ciocalteau reagent, according to the method of Slinkard and 

Singleton (1977) with some modifications. Briefly, 0.1 ml of the E. 

angustifolium extract (1000 to 1500 µg/ml) was transferred into a 

test tube and the volume made up to 4.6 ml with distilled water. 

After addition of 0.1 ml Folin-Ciocalteau (previously diluted 3-fold 

with distilled water) and 0.3 ml 2% Na2CO3 solution, the tube was 

vortexed and then allowed to stand for 2 h with intermittent shaking. 

The absorbance was measured at 760 nm in a spectrophotometer. 

The amount of total phenolic compounds in the E. angustifolium 

extract was calculated as mg of pyrocatechol equivalent from the 

calibration curve and as mg pyrocatechol equivalents per mg of 

extract. 

Determination of total flavonoid contents 

Total flavonoid content was determined by using a method 

described by Sakanaka et al. (2005) using catechin as a standard 

flavonoid compound. Briefly, 0.25 ml of the extract (1000 to 3000 

μg/ml) or (+)-catechin standard solution (20 to 100 μg/ml) was 

mixed with 1.25 ml of distilled water in a test tube, followed by the 

addition of 75 μL of a 5% sodium nitrite solution. After 6 min, 150 μL 

of a 10% aluminium chloride solution was added and the mixture 

was allowed to stand for a further 5 min before 0.5 ml of M NaOH 

was added. The mixture was brought to 2.5 ml with distilled water 

and mixed well. The absorbance was measured immediately at 510 

nm using a spectrophotometer. The results were expressed as mg 

of (+)-catechin equivalents per mg of extract. 

Reducing power 

The reducing power of the E. angustifolium extract was measured 

according to the method of Oyaizu (1986). Various concentrations 

of extracts (20 to 100 �g) in 1 ml of distilled water were mixed with 

2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml potassium 

ferricyanide (1%, w/v), and the mixture was incubated at 50°C for 

30 min. 

Afterwards, 2.5 ml of trichloroacetic acid (10%, w/v) was added 

and the mixture was centrifuged at 3000 rpm for 10 min. Finally, 2.5 

ml of upper-layer solution was mixed with 2.5 ml distilled water and 

0.5 ml FeCl3 (0.1 %, w/v), and the absorbance was measured at 

700 nm. �-Tocopherol, butylated hydroxyanisole (BHA) and 

butylated hydroxytoluene (BHT) were used as standard 

antioxidants. 

Superoxide anion radical scavenging activity 

Superoxide anion scavenging activity of water extract of E. 

angustifolium was determined according to the method of Liu et al., 

(1997). Superoxide radicals were generated in a non-enzymatic 

phenazine methosulphate (PMS)-NADH system by oxidation of 

NADH and assayed by the reduction of nitroblue tetrazolium (NBT). 

The reaction mixture consisted of 3 ml of Tris-HCl (16 mM, pH 8), 1 

ml of NADH (78 �M) and 1 ml of NBT (50 �M) and 1 ml of diluted 

samples. 

The reaction was initated by adding of 1 ml of phenazine 

methosulphate (PMS) (10 �M) to the mixture. The tubes were 

incubated at 25°C for 5 min and the absorbance at 560 nm was 

recorded against blank samples in a spectrophotometer. A lower 

absorbance of the reaction mixture indicated a higher superoxide 

anion scavenging activity. 

The percentage inhibition of superoxide anion generation was 

calculated using the following formula:

Superoxide anion radical scavenging activity (%) = (A0 - A1 / A0) x 

100 

A0 is the absorbance of the control. A1 is the absorbance of the 

sample. 

Hydroxyl radical scavenging activity 

The effect of E. angustifolium extract on hydroxyl radicals was 

assayed by using the deoxyribose method (Chung et al., 1997). 

The reaction mixture contained 0.45 ml of 0.2 M sodium phosphate 

buffer (pH 7.4), 0.15 ml of 10 mM 2-deoxyribose, 0.15 ml of 10 mM 

FeSO4-EDTA, 0.15 ml of 10 mM hydrogen peroxide, 0.525 ml of 

distilled water and 0.075 ml of extract in a tube. The reaction was 

started by the addition of hydrogen peroxide. After incubation at 

37�C for 4 h, the reaction was stopped by adding 0.75 ml of 2.8% 

TCA and 0.75 ml 1.0% of thiobarbituric acid. The mixture was 

boiled for 10 min, cooled in an ice bath and then measured at 520 

nm. Hydroxyl radical scavenging activity was calculated in the 

following equation: 

Hydroxyl radical scavenging activity (%)= (A0 - A1 / A0) x 100 

A0 is the absorbance of the control and A1 is the absorbance of 

sample. 

DPPH radical scavenging activity 

The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging 

activity of the E. angustifolium extract was measured according to 

the procedure described by Brand-Williams et al. (1995). Dilution 

series (0.25 to 1 mg/ml) were prepared for each aqueous extract in 

methanol, 0.1 ml of each dilution was added to 3.9 ml of a 6x10 -5 M 

methanolic solution of DPPH followed by vortexing. The mixture 

was shaken vigorously and allowed to stand in the dark at room 

temperature for 30 min. The decrease in absorbance of the 

resulting solution was then measured spectrophotometrically at 517 

nm against methanol. The DPPH radical scavenging activity was 

calculated using the following equation: 

DPPH radical scavenging activity (%) = (A0 - A1 / A0) x 100 

A0 is the absorbance of the control. A1 is the absorbance of the 

sample 

ABTS radical scavenging activity 

The ABTS �+ scavenging activity of the E. angutsifolium extract was 

measured according to the procedure described by Arnao et al. 

(2001). The stock solutions included 7.4 mM ABTS �+ solution and 

2.6 mM potassium persulfate solution. The working solution was 

prepared by mixing the two stock solutions in equal quantities and 

allowing them to react for 12 h at room temperature in the dark. The 

solution was then diluted by mixing 1 ml ABTS �+ solution with 60 ml 

methanol to obtain an absorbance of 1.1 ± 0.02 units at 734 nm 

using the spectrophotometer. Fresh ABTS �+ solution was prepared 

for each assay. 

E. angustifolium extracts (150 μL) were allowed to react with 

2850 μL of the ABTS �+ solution for 2 h in a dark condition. Then the 

absorbance was taken at 734 nm using a spectrophotometer. The 

ABTS �+ scavenging activity was calculated using the following 

equation: 

ABTS radical scavenging activity (%) = (A0 - A1 / A0) × 100 


A0 is the absorbance of the control and A1 is the absorbance of the 

sample. 


Results were expressed as mean ± standart deviation (SD) of 

triplicate analyses. The correlation coefficient (r 2 ) between the 

parameters tested was established by regression analysis. 


Elastase inhibitory activity 

Elastase is known to cause rheumatoid arthritis, 

pulmonary emphysema, and other chronic inflammotary 

diseases by the protein degradation of human tissues. It 

also degrades elastin, which is closely related to the 

elasticity and restoration of skin, and induces wrinkles 

(An et al., 2005). Recently, there is an increased demand 

for natural substances such as green plant products have 

been in increased demand in the global market as new 

agents for cosmeceutical purposes (Aburjai and Natsheh, 

2003). In the present study, E. angustifolium was 

investigated for potential effectiveness as antiaging agent 

for the ingredients of cosmetic preparations. The 

inhibition effect of elastase activity is shown in Table 1. 

We have found that all concentrations exerted inhibitory 

effects on elastase in a dose-dependent manner. The 

inhibition was increased with increasing extract 

concentration. The inhibition percentage of E. 

angustifolium extract at 100 µg/ml concentration was 

82.19 ± 2.38%. EC50 values were found 42.72 ± 2.38 

µg/ml. A high correlation was observed between 

antielastase activity and phenolic compounds (r 2 = 

0.9982). Kang et al. (1994) reported that the inhibition of 

the elastase activity of ginseng was about 90% at 0.14 

mg/ml. The present results indicate that the E. 

angustifolium has a better activity than the other sources 

reported (An et al., 2005). Terpenoids, flavonoids, 

phenolic compounds have been shown to possess 

antielastase activity (Lee et al., 2001). This high 

antielastase activity is attributed to the presence of 

natural antioxidants such as flavonoids, terpenoids and 

phenolic compounds in E. angustifolium. Therefore, it can 

be concluded that E. angustifolium may have a potential 

role on skin care in the cosmetics for its enzyme inhibition 

activity. 

Tyrosinase inhibitor activity 

Tyrosinase inhibitors are chemical agents capable of 

reducing enzymatic reactions, such as food browning and 

melanisation of human skin. Therefore, these inhibitors 

have good commercial potential in food and cosmetic 

industries (Lim et al., 2009). Tyrosinase inhibitor activity 

of E. Angustifolium is reported the first time in this study


Table 1. Elastase, tyrosinase, and lipoxygenase inhibitor activities of Epilobium angustifolium L. extracts. 

Concentration 

(μg/ml) 

Elastase activity Tyrosinase activity Lipoxygenase activity 

Inhibition 

(%) 

EC50 

(μg/ml) 

Concentration 

(μg/ml) 

Inhibition 

(%) 

EC50 

(μg/ml) 

Concentration 

(μg/ml) 

Inhibition 

(%) 

10 11.03 ± 1.03 

0.1 4.9 ± 0.70 

0.1 21.81 ± 15.12 

25 

50 

42.72 ± 2.38 

72.16 ± 2.88 

42.72 ± 2.38 

1 

5 

12.07 ± 1.53 

13.89 ± 0.13 

33.03 ± 3.71 

0.2 

0.5 

31.25 ± 1.76 

46.58 ± 0.46 

100 82.19 ± 2.38 10 15.67 ± 5.19 

Values are means ± SD. 

and the results are shown in Table 1. It was found 

that the E. angustifolium inhibited the tyrosinase 

enzyme activity. The inhibition was increased with 

increasing extract concentration (Table 1). A high 

antityrosinase (15.67± 5.19%) was seen in 10 

µg/ml (Table 1). EC50 values were found 33.03 � 

3.71 µg/ml. The tyrosinase activity was correlated 

with phenolic compounds (r 2 = 0.9942). It was 

evident that E. angustifolium extract had a 

stronger effect on tyrosinase inhibition compared 

to various plant extracts in literature (Moon et al., 

2010). 

Jo et al. (2011) have reported that Magnolia 

species showed low tyrosinase inhibition with an 

average 8.63% inhibition of 1000 µg/ml (IC50 = 

10555.55 µg/ml). Various naturally occurring anti 

melanogenesis reagents contain a phenolic 

structure (Kim, 2007). Many tyrosinase inhibitors 

are polyphenol derivatives of flavonoids (Briganti 

et al., 2003; Seo et al., 2003). Structural 

differences in phenolic compounds found in E. 

Angustifolium may also play a critical role in 

occurrence of tyrosinase inhibition. Nerya et al. 

(2004) stated that the most important factor in 

efficacy of the chalcones against tyrosinase is the 

location of the hydroxyl groups on aromatic ring. 

The mechanism of tyrosinase inhibition activity is 

an important factor for skin whitening in cosmetic 

industry and for food browning inhibition in food 

industry (An et al., 2005; Vamos–Vigyazo, 1981). 

Lipoxygenase inhibitory activity 

The effect of the aqueous extract of E. 

angustifolium on lipoxygenase activity is shown in 

Table 1. Lipoxygenase inhibitory activity of E. 

angustifolium was found to be increased dose 

dependently. The results were expressed as EC50 

values, calculated from the regression equations 

preperad from the concentration of the samples. 

A high lipoxygenase inhibition (46.58 ± 0.46%) 

was seen in 0.5 µg/ml extract concentration 

(Table 1). EC50 values were found 0.57 ± 0.06 

µg/ml. Kiss et al. (2011) have demonstrated that 

Epilobium species inhibited the activity of 

lipoxoygenase with IC50 = 16 to 28 µg/ml. Our 

result (0.57 µg/ml) showed that E. angustifolium 

water extract had higher lipooxygenase inhibitor 

activity. 

A good relationship between polyphenol 

contents and lipoxygenase inhibitor activity has 

been reported by Aquila et al. (2009). This good 

lipoxygenase inhibitor activity is attributed to the 

presence of natural antioxidants such as phenolic 

compounds in E. angustifolium. This plant is used 

by people against prostate cancer in Turkey. In 

this study, it is found that the water extract of 

EC50 

(μg/ml) 

0.57 ± 0.06 

E. angustifolium has a good lipoxygenase inhibitor 

activity, therefore the usage of this plant by people 

against prostate cancer is scientifically confirmed. 

Total phenolic and flavonoid contents 

Phenols are very important plant constituents as 

they are good scavengers due to their hydroxyl 

groups. Phenolic compounds and flavonoids have 

been shown to be responsible for the antioxidant 

activity of many plants (Geetha et al., 2005). 

These antioxidants also possess diverse 

biological activities such as antiinflammatory, 

antiatherosclerotic and anticarcinogenic which 

may be related to their antioxidant activities 

(Chung et al., 1997). It was determined that there 

was 42.10 ± 10.76 μg pyrocatechol equivalent of 

phenolic compounds in 1 mg of the E. 

angustifolium extract. A high correlation was 

observed between the total phenolic content and 

hydroxyl radical scavenging activity (r 2 = 0.8577), 

DPPH radical scavenging activity (r 2 = 0.9285), 

and superoxide radical scavenging activity (r 2 = 

0.9999) of the extract. These data are in 

accordance with others, which have shown that a 

high total phenolic content increases antioxidant 

activity, and that there is a linear correlation 

between phenolic content and antioxidant activity

Absorbance (700 nm) 

Figure 1. Reducing power of the water extract from Epilobium angustifolium L. BHT and tocopherol 

were used as reference antioxidants. Values are means ± SD (n=3). 

(Holasova et al., 2002). These results indicate that the 

high antioxidant activity of E. angustifolium extract may 

be associated with its total phenolic content. Polyphenolic 

compounds like flavonoids have been labelled as “high 

level”natural antioxidants based on their abilities to 

scavenge free radicals and active oxygen species (Birt et 

al., 2001). They contain conjugated ring structures and 

hydroxyl groups having the potentional function as 

antioxidants in vitro or cell free systems by scavenging 

superoxide anion, singlet oxygen, lipid peroxyradicals, 

and stabilizing free radicals involved in oxidative 

processes through hydrogenation or complexing with 

oxidizing species (Klahorst, 2002). 

The concentration of flavonoids was 22.76 ± 1.34 μg 

catechin equivalent of phenolic compounds in 1 mg of the 

water extract. Phytochemical screening of Epilobium 

species have revealed the presence of phenolic acid, 

macrocyclic tannins, and flavonoids (in particular 

myricitrin, isomyricitrin, quercitrin, and quercetin-3-O- �- 

D- glucuronide) in the aerial parts of the plant (Barakat et 

al.,1997). 

Reducing power 

0.45 

0.4 

0.35 

0.3 

0.25 

0.2 

0.15 

0.1 

0.05 

0 

Fe 3+ reduction is often used as an indicator of electron- 

donating activity, which is an important mechanism of 


phenolic antioxidant action, and can be strongly 

correlated with other antioxidant properties (Dorman et 

al., 2003). In the reducing power assay the presence of 

reductants (antioxidants) in the samples could result in 

the reduction of the Fe 3+ / ferricyanide complex to its 

ferrous form. Amount of Fe 2+ complex can then be 

monitored by measuring the formation of Perl’s Prussian 

blue at 700 nm. The reducing capacity of a compound 

may serve as a significant indicator of its potential 

antioxidant activity. 

Figure 1 shows the dose-response curves for the 

reducing powers of the extract from E. angustifolium and 

standards. The reducing power of water extract was 

nearly equal to that of BHT. Results of the reducing 

power indicate that the reducing capacity of E. 

angustifolium was higher when compared to standard 

tocopherol, indicating that it is an excellent natural 

antioxidant. 

Reducing power of extract and standards decreased in 

order of water extract� BHT>tocopherol. These results 

revealed that the extract of E. angustifolium was electron 

donor and also could react with free radicals, converting 

them to more stable products. The outcome of the 

reducing reaction is to terminate the radical chain 

reactions that may otherwise be very damaging. This 

good antioxidant activity is attributed to the presence of 

natural antioxidants such as phenolic compounds in


E. angustifolium. 

Figure 2. Superoxide radical scavenging activity of the water extract from Epilobium 

angustifolium L. Trolox was used as reference antioxidant. Values are means ± SD (n=3). 

Superoxide anion radical scavenging activity 

Superoxide anion is a free radical created from the 

normal process of energy generation in the human body. 

Superoxide anion is toxic to cells and tissues and can act 

as precursor to other ROS (Korychka-Dahl and 

Richardson, 1978). These species are produced by a 

number of enzyme systems. The radicals may also play 

an important role during the peroxidation of unsaturated 

fatty acids and other potential susceptible substances 

(Sakanaka et al., 2005; Halliwell and Gutteridge, 1984). It 

has also been reported that antioxidant properties of 

some flavonoids are effective mainly via scavenging of 

superoxide anion radicals (Yen and Duh, 1994). 

In a biological system, its toxic role can be eliminated 

by superoxide dismutase (SOD) (Chung et al., 2005). 

Figure 2 shows the dose response curves of superoxidescavenging 

activities of the extracts from E. angustifolium 

by the PMS-NAOH superoxide-generating system. Water 

extract at 100 mg/ml exhibited 39.32 � 0.02% superoxide 

radical scavenging activity. On the other hand at the 

same concentration, Trolox exhibited (74.36 ± 0.02%) 

activity. These values were lower than that of the same 

dose of Trolox (74.36 � 0.02%). The superoxide radical 

scavenging activity of those samples were as follows: 

Trolox>water extract. EC50 values of E. angustifolium 

extract and Trolox were found to be 127.15 � 0.08 and 

7.41 � 0.008 mg/ml, respectively. When compared to 

Trolox, the superoxide scavenging activity of the extract 

was found to be low. This could be due to the presence 

of reactive concentration of bioactive constituents and 

mixture of other nutritients in the extract. The superoxide 

radical scavenging activity in the water extract was 

correlated with phenolic compounds (r 2 = 0.9999). These 

results indicated that the radical scavenging capacity of 

the water extract might be mostly related to its 

concentration of phenolic hydroxyl group. 


The hydroxyl radical is an extremely reactive free radical 

formed in biological systems and has been implicated as 

a highly damaging species in free radical pathology, 

capable of damaging almost every molecules found in 

living cells (Hochestein and Atallah, 1988). This radical 

has the capacity to join nucleotides in DNA and cause 

strand breakage which contributes to carcinogenesis, 

mutagenesis and cytotoxicity. This reactive hydroxyl 

radical can cause oxidative damage to DNA, lipids and 

proteins (Shukla et al., 2009). Hydroxyl radical 

scavenging capacity of an extract is directly related to its 

antioxidant activity (Babu et al., 2001). Figure 3 shows 

the dose response curves of radical scavenging activities 

of the extracts and reference antioxidant (ascorbic acid) 

on the hydroxy radicals. Water extract and ascorbic acid 

scavenging hydroxyl radicals by 8.89 � 0.83 and 5.56 �


Figure 3. Hydroxy radical scavenging activity of the water extract from Epilobium angustifolium L. Ascorbic acid was used 

as reference antioxidant. Values are means ± SD (n=3). 

0.18% at 20 mg/ml, and 15.60 � 1.32 and 13.84 � 0.12% 

at 100 mg/ml, respectively. These values were 

significantly higher than the value of the positive control 

ascorbic acid. The scavenging abilities on hydroxyl 

radicals were in descending order: water>ascorbic acid. 

A higher hydroxyl radical scavenging activity is 

associated with a lower EC50 value. EC50 values for 

extracts, and ascorbic acid on hydroxyl radical 

scavenging activity were found as 2.64 ± 0.22, and 2.34 ± 

0.02 mg/ml, respectively. A high correlation was 

observed between hydroxyl radical scavenging activity 

and phenolic compounds (r 2 = 0.8577). E. angustifolium 

extract was also capable of reducing DNA damage at all 

concentrations used. All results showed antioxidant 

activity in dose dependent manner. These results 

indicated that water extracts from E. angustifolium are 

effective scavengers for hydroxyl free radicals. The high 

value of hydroxyl radical scavenging activity of this plant 

might be the reason why it is widely used in traditional 

medicine for prostate cancer. 


DPPH has been widely used to evaluate the free radical 

scavenging effectiveness of various antioxidant 

substance (Ozcelik et al., 2006). A chain in lipophilic 

radicals was initiated by the lipid autoxidation. DPPH is a 

stable free radical at room temperature and accepts an 

electron or hydrogen radical to become a stable 

diamagnetic molecule. Antioxidants react with DPPH, 

reducing a number of DPPH molecules equal to the 

number of their available hydroxyl groups (Xu et al., 

2005). Figure 4 shows, the dose response curves of 

DPPH radical scavenging activity of the extracts from E. 

angustifolium. The extract was capable of scavenging 

DPPH radicals in a concentration-dependent manner. 

The scavenging activity of E. angustifolium extract, 

butylated hydroxyanisole (BHA), butylated 

hydroxytoluene (BHT) and ascorbic acid on DPPH 

radicals increased between 5 to 10 mg/ml and were 

90.68 ± 0.45, 94.19 ± 0.29, 85.84 ± 0.61 and 93.50 ± 

0.23% at a concentration of 10 mg/ml, respectively. 

Water extract, BHA and BHT showed similar DPPH 

radical scavenging activities, while ascorbic acid was a 

considerably less effective DPPH radical scavenger. The 

DPPH radical scavenging activity was correlated with 

phenolic compounds (r 2 = 0.9286). These results 

indicated that the radical scavenging capacity of the 

water extract might be mostly related to concentration of 

phenolic hydroxyl groups. EC50 values for ascorbic acid, 

water extract, BHA and BHT on DPPH radical 

scavenging activity were found as 2.59 ± 0.01, 2.70 ± 

0.09, 2.94 ± 0.03 and 3.47 ± 0.07 mg/ml. Štajner et al. 

(2007) has reported that Willow herb’s showed a 

relatively high DPPH radical scavenging effect


Figure 4. DPPH radical scavenging activity of the water extract from Epilobium angustifolium L. BHA, BHT and 

ascorbic acid were used as reference antioxidants. Values are means ± SD (n=3). 

(95.57 ± 6.57%). This value is smilar to the our findings.. 

ABTS radical scavenging activity 

The ABTS �+ scavenging activity is also one of the most 

commonly used methods to evaluate the antioxidant 

activity (Zhang et al., 2009). The ABTS �+ scavenging 

activity of water extract compared to Trolox are shown in 

Figure 5. They increased with increasing concentration 

reaching 96.10 � 0.12% and these values were 

comparable to those of the positive control Trolox 96.49 � 

0.03% at a concentration of 0.5 mg /ml. EC50 values for 

E. angustifolium extract and Trolox on ABTS �+ 

scavenging activity were found as 0.052 � 0.003 and 

0.051 � 0.004 mg/ml, respectively. A high correlation was 

observed between ABTS �+ scavenging activity and 

phenolic compounds (r 2 = 0.6546). 

Conclusions 

The results of the present study are indicating that water 

extract of E. angustifolium L. exhibits good antioxidant, 

antielastase, antityrosinase and lipoxygenase inhibitor 

activities. We have demonstrated that the aqueous 

extract of E. angustifolium L. contained high level of 

flavonoid and phenolic compounds. Phenolic compounds 

and flavonoids present in the plant kingdom are mainly 

resonsible for the antioxidant and enzyme inhibitor 

activity. Therefore, E. angustifolium L. could be used as a 

source of natural antioxidant and antielastase, 

antityrosinase, and lipoxygenase inhibitor in 

pharmaceutical, cosmetic and food industries. The high 

value of antioxidant and lipoxygenase inhibitor activity of 

this plant obtained in this study may partly support and 

explain the use of its extracts in folk medicine for prostate 

disease treatment. 

The water extract of E. angustifolium L. could be 

beneficial for conventional herbalism and used as a 

natural remedy in the future. In conclusion, E. 

angustifolium L. extract may be considered as a main 

lipoxygenase, tyrosinase and elastase inhibitor and free 

radical scavenger. Therefore, this plant may be 

appreciated as a medicinal food mainly at skin, prostate, 

Parkinson and other diseases. 


The authors would like to thank Prof. Dr. Kerim Alpinar 

(Istanbul University, Faculty of Pharmacy) for the 

identification of the plant. 

Abbreviations: ABTS, 2,2'-azino-bis(3ethylbenzothiazoline-6-sulfonic 

acid) diammonium salt; 

BHA, butylated hydroxyanisole; BHT, butylated 

hydroxytoluene; DPPH, 2,2-diphenyl-1-picryl-hydrazyl; 

DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); EC50, efficiency 

concentration; EC, extractable compounds; ferrozine, 3- 

(2-pyridyl)-5,6-bis(4-phenyl-sulfonic acid)-1,2,4-triazine; 

LOX, lipoxygenase; NADH, reduced nicotinamide 

adenine dinucleotide; NADPH2, reduced nicotinamide 

adenine dinucleotide phosphate; NBT, nitroblue 

tetrazolium; PMS, phenazine methosulphate;

Figure 5. ABTS radical scavenging activity of the water extract from Epilobium angustifolium L. Trolox was 

used as reference antioxidant. Values are means ± SD (n=3). 

ROS, reactive oxygen species; STANA, N-succinyl-Ala- 

Ala-Ala-p-nitroanilide; TBA, tiobarbituric acid; TCA, 

trichloroacetic acid; Trolox, 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic 

acid; TNF, tumor 

necrosis factor; UV, ultraviolet light. 

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DOI: 10.5897/JMPR11.1243 

ISSN 1992 -1950 ©2012 Academic Journals 


Identification of superior varieties of tea (Camellia 

sinensis (l.) o. kuntze) in the selected UPASI germplasm 

using biomarkers 

S. Ramkumar 1 , P. Sureshkumar 2 *, A. K. A. Mandal 3 , K. Rajaram 2 and P. Mohankumar 1 

1 Plant Physiology and Biotechnology Division, UPASI TRF, UPASI Tea, Research Institute, Nirar Dam BPO, Valparai 

642 127, Coimbatore District, Tamilnadu, India. 

2 Department of Biotechnology, Anna University of Technology, Tamil nadu, Triuchirappalli- 620 024, India. 

3 School of Bioscience and Technology, Vellore Institute of Technology, Vellore, Tamilnadu, India. 


Biomarkers are used as a vital tool in cultivar improvement programme for woody perennial tree crops 

such as (Camellia sinensis (L.) O. Kuntze). Commercially important fifteen accessions were selected 

and investigated for total polyphenol oxidase (PPO) activity (U/mg of protein), based on the PPO activity 

range, the accessions were further separated into three groups, namely high (P/11/10, UPASI-16, UPASI- 

18, UPASI-14, I/30/17), moderate (UPASI-3, UPASI-17, UPASI-13, UPASI-1, UPASI-21) and low (I/30/9, 

p/11/15, I/30/30,MGL-16, MGL- 8) PPO activity. To study the pattern of genetic diversity, random 

amplified polymorphic DNA (RAPD) analysis was performed using twenty decamer primers. The RAPD 

amplification results revealed that genetic similarity (GS) among the accessions tested ranged 0.64 to 

0.918 with an average of 0.28%. RAPD dendogram showed three distinct clusters of high, moderate and 

low using un-weighed pair-group method for arithmetic averages analysis (UPGMA) method. This 

genetic diversity studies on tea showed effectively for the initial assessment of partitioning the intra 

specific level of genetic variation correlated to the total PPO enzyme activity. 

Key words: Camellia sinensis, random amplified polymorphic DNA (RAPD), polyphenol oxidase, genetic 

diversity. 

INTRODUCTION 

Tea (Camellia sinensis (L) O. Kuntze), beverage is made 

from tender leaves of the tea plant. India is the largest 

producer of tea (an important economic crop). Polyphenol 

oxidase (PPO) is an important enzyme in tea plants, 

especially for tea quality. High PPO activity is necessary 

for the enzymatic oxidation during process of the black 

tea manufacture and it should be deactivated promptly in 

the green tea making process. Polyphenol oxidase (EC1. 

*Corresponding author. E-mail: sureshbiotech2003@yahoo.co.in. 

Abbreviations: GS, Genetic similarity; PPO, polyphenol 

oxidase; RAPD, random amplified polymorphic DNA; UPASI, 

United Planters Association of South India; UPGMA, unweighed 

pair-group method for arithmetic averages analysis. 

10.3.2) is also known as phenol oxidase, tyrosinase, odiphenol 

oxidase, catechol oxidase, phenolase, and 

chlorogenic acid oxidase. These enzymes in higher 

plants oxidize a great variety of monophenolic and odiphenol 

compounds and catalyze two types of reactions 

(Yelena et al., 1996). First reaction involves the 

hydroxylation of a monophenol to give a diphenol and 

second involves the removal of hydrogen’s from diphenol 

to give quinone (Robertson and bendall, 1983). During 

the enzymatic oxidation PPO enzyme leads to catechin 

for production of black tea pigments, viz; theflavins (TF) 

and thearubigis (TR). This polyphenol oxidase enzyme 

provides an important role in plant metabolism; provide 

some defense against predators by their astringency. 

This enzyme mostly found in higher plants like apple, 

peach, mushroom tobacco, coffee and tea (whitaker,


Table 1. Basic information regarding the tea origin and descriptive characters of prominent south Indian tea cultivars used 

in the study. 

S/N Clone Varital type Source of the material 

1 P/11/10 Assam Paraley estate selection, valparai 

2 UPASI-16 Assam (B/6/182) Brooklands estate, the nilgiris. 

3 UPASI- 18 Cambod (B/6/57) Brooklands estate, the nilgiris. 

4 UPASI -14 Cambod S/6/99 (Singara) Singara estate, the nilgiris. 

5 I/30/17 Nil Iyerpadai estate selection, valparai 

6 UPASI-3 Assam B/5/63 (Sundaram) Brooklands estate, the nilgiris. 

7 UPASI-17 Cambod B/6/203 (Swarna) Brooklands estate, the nilgiris. 

8 UPASI-13 Assam (B/6/137) Brooklands estate, the nilgiris. 

9 UPASI-21 Assam (B/4/198) Brooklands estate, the nilgiris 

10 UPASI-1 Assam (B/4/141 (Ever green)) Brooklands estate, the nilgiris. 

11 I/30/9 Assam Iyerpadai estate selection, valparai 

12 P/11/15 Assam Paraley estate selection, valparai 

13 I/30/30 Assam Iyerpadai estate selection, valparai 

14 MGL – 8 China Murugaley estate selection, valparai 

15 MGL - 16 China Murugaley estate selection, valparai 

1994). Genetic diversity assessments at inter and intraspecies 

levels are important basis for collection, 

conservation, evaluation, and utilization of tea 

germplasm. Some traditional methods, such as morphological 

characteristics, chemical components, esterase, 

isozymes, and karyotype, have been employed to 

describe the phylogenetic relationships among tea plants. 

Molecular markers are valuable tools in the 

characterization and evaluation of genetic diversity within 

and between species and populations. 

In tea different molecular markers have been used by 

different workers to study the genetic diversity. In a preliminary 

study, Tanuka et al. (1995) attempted to detect 

variations among Korean, Japanese, Chinese, Indian and 

Vietnamese tea using 10-mer and 12-mer primers. They 

concluded that China, Korean tea has undergone some 

genetic diversification and Japanese tea showed a close 

similarity with the Chinese tea. Recently, random 

amplified polymorphic DNA (RAPD) have also been used 

for the investigation of genetic relationship (Wacheria et 

al., 1997), identification of parentage (Tanaka et al., 

2001), genetic diversity (Kaundun et al., 2000; Mondal, 

2000; Chen et al., 2005) and genetic mapping (Hackett et 

al., 2000) of tea plants (C. Sinensis). Genetic variability of 

in vitro raised tea plants were investigated by Mondal 

and Chand (2002), who reported that while both Assam 

and China have specific band, Japanese tea are more 

closer to Chinese tea than others and same of the tea 

varieties from Vietnam are the hybrids of Assam and 

China. Lai et al. (2001) have studied the genetic 

relationship of 37 tea samples that comprised 21 clones 

of china, 3 clones of Assam, and 6 individual samples of 

native Taiwanese wild tea by using RAPD and ISSR 

markers. The evaluation of genetic diversity in tea is a 

prerequisite for screening superior variety of tea. Hence, 

the present work investigates to identify the superior 

varieties of tea among the 15 commercially available tea 

accessions (UPASI germplasm) by using biomarkers like 

biochemical (total PPO activity) and molecular markers 

(RAPD). 



The 15 tea accessions (UPASI germplasm) were collected from 

Valparai (UPASI) and the Nilgiris estates for the present study 

(Table 1). 

Preparation of crude polyphenol oxidase (PPO) enzyme 

The soluble and bound component of the PPO crude enzyme was 

extracted using acetone from 25 g of crop shoots by homogenizing 

the tissue of the shoots using chilled acetone (−20°C) with acid 

washed sand powder using a pre chilled pestle and mortar. The 

homogenate was filtered through Whatmann No.1 filter paper. The 

retentate was washed free of phenolics by passing through chilled 

acetone then with cold aqueous acetone (80:20 acetone/water, v/v) 

and finally washed with acetone. The previous homogenate slurry 

was filtered through Whatmann No.1 paper filter. The powder was 

then stored in evacuated desiccators to allow complete drying. The 

dried white powder called as an acetone powder, is used for 

preparation of the enzyme extracts. All the enzyme preparation and 

its activity were done at 25°C. 

Enzyme preparation 

The soluble component of the enzymes was extracted with 5 g

acetone powder by gentle grinding in a pestle and mortar with 

distilled water (1:10 w/v) and incubated 10 min followed by 

centrifugation at 4000 rpm for 10 min. The supernatant obtained 

was soluble enzyme. Subsequently, the residue was extracted by 

regrinding with 5 mL of 0.2 M sodium sulphate (Na2SO4) solution 

and incubated at 30 min followed by centrifugation for 10 min at 

10000 g. The supernatant obtained was ionically bound enzyme. 

PPO activity was calculated using formula according to previously 

published protocol by Singh and Ravindranath et al. (1990). 

Polyphenol oxidase (PPO) enzyme assay 

Polyphenol oxidase activity was determined using UV- 

Spectrophotometer (Ultra spec 2100 pro, GE Healthcare 

Biosciences Ltd). The principle is based on the initial increase in the 

rate of the absorbance at 380 nm. Firstly, Mix 1.5 ml of 0.1M 

sodium phosphate buffer (pH 5.6) with 100 µl of 10 mM of catechin 

as substrate in a 3 ml cuvette, then extracted 100 µl of crude 

enzyme was added in 1.0 cm path length cuvette and the 

absorbance was recorded immediately 30 s at 3 min interval at 

25°C. The instrument was tarred using the same mixture without an 

enzyme. Triplicate measurement was recorded for each assay. One 

unit of PPO enzyme activity was defined as that amount which 

caused a rate of change of 0.001 absorption unit’s min -1 at 380 nm. 

Overall enzyme activity was expressed as U mg/protein. 

Protein estimation 

Protein content was determined according to the coomassie blue 

binding method of Bradford (1976). 


All extractions and determinations were conducted 3 times at least. 

Data were expressed as means ± standard error of the mean of the 

mean of three independent experiments carried out in duplicate. A 

one way ANOVA with Duncan’s test was employed to evaluate the 

significance of results. A probability (p) value �0.05 was considered 

significant (Gomez and Gomez, 1976). 

Random amplified polymorphic DNA (RAPD) Analysis 

DNA isolation from tea leaves 

Young and fresh leaves were collected from the 15 accessions with 

similar age and uniformly pruned at 26" (about 60 cm) above 

ground level were selected and subjected for RAPD analysis. 

Leaves were washed thoroughly in sterile distilled water. Equal 

quantity (100 mg) of leaf tissue was weighed and used for DNA 

isolation. DNA was extracted from the young tea leaves using the 

CTAB method with some modifications. Young leaf tissues (0.5 g) 

were ground in liquid nitrogen and mixed with 10 ml of CTAB buffer 

and incubated at 65°C for one hour. Samples were extracted with 

equal volume of chloroform/isoamyl alcohol (24:1 v/v) and the 

aqueous phase was mixed with 2/3 volume of chilled isopropanol. 

Precipitated DNA was collected by centrifugation and washed with 

70% ethanol. DNA was air dried and re-suspended in 1 ml of sterile 

distilled water. Later it was treated with RNase A (1 mg/ml) for two 

hour at 37°C and purified using 500 ml of equilibrated phenol and 

750 ml of chloroform/isoamyl alcohol (24:1 v/v). The purified DNA 

was re-precipitated from the aqueous phase using chilled ethanol, 

air-dried and re-suspended in sterile water. The high molecular 

Ramkumar et al. 729 

weight DNA was checked for quality and quantity using agarose gel 

(0.8%) electrophoresis. Observed gel under in a UV light 

transilluminator. 

List of primers used Polymerase chain reaction (PCR) 

amplification 

Fifteen decamer random primers (from OPERON - Qiagen 

Company, U.S.A.) were used for all PCR reactions. List of primers 

used and their sequences are presented in Table 2. 

Polymerase chain reaction (PCR) and gel electrophoresis 

Polymerase chain reaction was carried out in peltier thermal cycler 

(PTC-200, MJ Research, Inc., and U.S.A) using ten decamer 

random primers (Table 2). Each 25 μl reaction mixture contained 1 

unit of Taq DNA polymerase, 0.2 mM each dNTPs, 1X PCR buffer, 

3 mM MgCl2 (Bangalore Genei Pvt. Ltd., India), 10 pmole of primer 

(OPERON-Qiagen Company, U.S.A.) and approximately 50 ng of 

template genomic DNA. PCR conditions were as follows: initial 

denaturation at 94°C for 4 min, followed by 45 cycles of 

denaturation at 94°C for 45 s, annealing at 36°C for 60 s and 

extension at 72°C for 120 s followed by final extension at 72°C for 

10 min. The amplified products were separated on 2% agarose gel 

using 1X TBE buffer followed by staining in ethidium bromide 

solution (1 μg/ml) and documentation was carried out by placing of 

the stained gel on UV-Transilluminator. The reproducibility of the 

amplification products was checked thrice for each polymorphic 

primer. Bands were scored from photographs. 

Scoring and analysis of data 

Bands were scored as present (1) or absent (0) in all the samples. 

All the DNA samples were repeated at least twice and only 

reproducible bands were scored. Molecular weight of each band 

was estimated using 1 kb DNA ladder (Fermentas Life science, 

Germany) as a standard. Similarity coefficient matrix was 

constructed by calculating Jaccard’s similarity coefficient values for 

each pair wise comparison between samples (Jaccard, 1908). A 

dendogram was generated (using average linkage procedure) 

from this matrix following un-weighed pair-group method for 

arithmetic averages analysis (UPGMA) method using NTSYS 2.1 

(Rohlf, 2002). 

RESULTS 

Polyphenol oxidase (PPO) activity 

Polyphenol oxidase (PPO) enzyme activity of fifteen 

accessions (UPASI germplasm), eight from UPASI 

released clones and seven accessions from Paraley, 

Iyerpadai and Murugaley estates selections were assayed. 

Activity of PPO as bound and soluble form and total 

activity has been shown in Table 3. 

UPASI released clones showed higher PPO activity 

compared to selections from other estates (Paraley and 

Murugaley estate, Valparai). The fifteen accessions were 

divided into three groups (high, moderate and low) based 

on the total PPO (soluble and bound) enzyme activity, 

each groups having five clones each. The high group


Table 2. List of primers along with their sequence used in the present study. 

(700 to 1300 U/mg of protein), namely P/11/10, UPASI- 

16, UPASI-18, UPASI-14, I/30/17, moderate (300 to 500 

U/mg of protein), namely UPASI-1, 3, 13, 17 and 21, low 

(50 to 90 U/mg of protein) namely, MGL-8, 16, I/30/9, 

I/30/30 and P/11/15, respectively (Table 3). 

Our results showed out of 15 accessions screened for 

total PPO activity, UPASI-18 (Cambod Type) showed that 

highest activity (1233.57 U/mg of protein) followed by 

UPASI - 16 (Assam type; 1151.29 U/mg of protein) and 

P/11/10 (Paraley estate; 1085.14 U/mg of protein). 

Random amplified polymorphic DNA (RAPD) analysis 

The amplification profiles of total genomic DNA of the 

fifteen accessions, which were grouped into high, 

moderate and low based on their PPO activity, were 

tested for genetic diversity studies. Out of the twenty 

primers tested only 14 provided good amplifications 

(Figures 2 to 7, 9, 10, 13, 14, 16 to 18 and 20) primers 

which detected good polymorphisms remaining six 

primers (Figures 8, 11, 12, 15, 19 and 21) had showed 

low reproducibility in all accessions. A total of six hundred 

and eighty one amplicons were obtained with 100 to 1500 

bp in size and nearly 70% were monomorphic. The 

number of bands ranged 30 to 35 per primer. 

Similarity coefficiency between the accessions was 

derived by Nei’s correlation. The pair wise (Nei and Li, 

1979) genetic distance co-efficient values for fifteen 

S/N Primer Sequences5’ 

1 OPA 02 TGCCGAGCTG 

2 OPA 03 AGTCAGCCAC 

3 OPA 05 AGGGGTCTTG 

4 OPA 17 GACCGCTTGT 

5 OPB 03 CATCCCCCTG 

6 OPB 07 GGTGACGCAG 

7 OPB 10 CTGCTGGGAC 

8 OPB 18 CCACAGCAGT 

9 OPB 20 GGACCCTTAC 

10 OPD 02 GGACCCAACC 

11 OPD 04 TCTGGTGAGG 

12 OPD 07 TTGGCACGGG 

13 OPD 13 GGGGTGACGA 

14 OPD 19 CTGGGGACTT 

15 OPK 02 GTCTCCGCAA 

16 OPK03 CCAGCTTAGG 

17 OPK 07 AGCGAGCAAG 

18 OPK 11 AATGCCCCAG 

19 OPK 13 GGTTGTACCC 

20 OPK 17 CCCAGCTGTG 

accessions ranged 0.64 to 0.918 indicating the diversity 

(Table 4). The UPGMA analysis of the scored data and 

the Jaccard’s similarity co-efficient values were used for 

clustering to develop the dendogram. The cluster 

analysis indicated that fifteen different accessions belong 

to (C. sinensis (L.) O. Kuntze) formed two major clusters 

based on similarity index (Figure 1). First major clusters 

were further divided into two minor sub clusters. The first 

minor sub clusters contained five accessions that is, 

UPASI-18, UPASI-16 and UPASI-14 (similarity indics 

0.86) grouped with P/11/10 and I/30/17. The second 

minor sub cluster contained five accessions, four 

accessions like UPASI-13 UPASI-17 UPASI-13 and 

UPASI-21 are grouped together with similarity index of 

0.91 and this was grouped with UPASI -1. 

The second minor cluster was divided into two minor 

sub cluster, the first contained I/30/9 and MGL-16 with 

similarity index of 0.81 grouped with second minor sub 

cluster. The second minor group contained three 

accessions, two of them I/30/30 and MGL-8 (with 

similarity index 0.85) grouped with P/11/15. 

DISCUSSION 

The genetic diversity among the germplasm resource has 

been demonstrated using morphological character was 

described by Sealy (1958), Chang (1984) and Ming 

(1992). Chen et al. (2000) reported that Thea

Table 3. Summary of results of tea PPO polyphenol oxidase. 


Sample Soluble PPO ( a ) (U/mg of protein) Bound PPO ( a ) (U/mg of protein) Total PPO ( a ) (U/mg of protein) Groups (PPO activity range) 

P/11/10 12.68 ± 2.68 1072.46 ± 3.68 1085.14 ± 1.37 c 

UPASI -16 103.35 ± 4.59 1047.94 ± 5.97 1151.29 ± 3.41 

High 

b 

UPASI - 18 190.82 ± 4.32 1042.75 ± 3.04 1233.57 ± 4.56 a 

UPASI - 14 84.97 ± 1.80 744.08 ± 5.43 829.05 ± 2.23 d 

I/30/17 7.97 ± 0.93 781.18 ± 1.28 789.15 ± 2.02 e 

UPASI - 3 12.78 ± 1.43 432.89 ± 3.81 445.68 ± 2.84 f 

UPASI - 17 8.97 ± 0.04 369.51 ± 5.79 378.48 ± 4.0 ih 

UPASI - 13 13.35 ± 0.55 387.24 ± 1.76 400.59 ± 1.76 h 

UPASI - 1 3.53 ± 0.83 391.30 ± 2.13 394.82 ± 1.03 i 

UPASI - 21 113.38 ± 0.23 315.84 ± 0.54 434.99 ± 1.28 g 

I/30/9 4.36 ± 1.21 79.76 ± 0.58 84.12 ± 0.57 jk 

P/11/15 5.21 ± 0.80 38.75 ± 2.60 43.96 ± 3.14 l 

1/30/30 8.86 ± 0.43 54.53 ± 1.07 63.39 ±1.03 k 

MGL-16 1.67 ± 0.17 88.62 ± 2.18 90.29 ± 3.08 jk 

MGL- 8 3.02 ± 0.22 88.04 ± 1.47 91.06 ± 1.35 j 

Enzyme Specific activity ( a ) - One unit of enzyme defined as the amount which caused a rate of change of 0.001/OD units min -1 at 380nm using catechin as substrate. *Means followed by the 

same letter are not significantly different at P



A14 0.667 0.691 0.697 0.673 0.673 0.684 0.681 0.698 0.662 0.686 0.797 0.811 0.846 1 

A15 0.647 0.711 0.676 0.673 0.667 0.709 0.725 0.723 0.726 0.757 0.801 0.791 0.0.779 0.804 1 

Figure 1. DNA based genetic relationship elaborated through the dendogram of 15 tea clones (Camellia 

sinensis (L.) O. Kuntze) constructed through bivariate data matrix generated using 20 RAPD primers. 

Figure 2. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. Kuntze) using OPB7 primer.

Figure 3. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. Kuntze) using OPA 2 primer. 

Figure 4. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. Kuntze) using OPA 3 

primer. 

Ramkumar et al. 733


Figure 5. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. Kuntze) using OPA 5 primer. 

Figure 6. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. Kuntze) using OPA 17 primer.

Figure 7. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. Kuntze) using OPB10 primer. 

Figure 8. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. Kuntze) using OPB18 primer. 

Ramkumar et al. 735


Figure 9. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. Kuntze) using OPB 20 primer. 

Figure 10. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. Kuntze) using OPB3 primer.

Figure 11. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. Kuntze) using OPD2 primer. 

Figure 12. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. Kuntze) using OPD4 primer. 

Ramkumar et al. 737


Figure 13. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. Kuntze) using 

OPD7 primer. 

Figure 14. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. Kuntze) 

using OPD13 primer.

Figure 15. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. Kuntze) using OPK 2 primer. 

Figure 16. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. Kuntze) using OPK 3 primer. 

Ramkumar et al. 739


Figure 17. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) 

O. Kuntze) using OPK7 primer. 

Figure 18. RAPD profile of 15 accessions of tea (Camellia 

sinensis (L.) O. Kuntze) using OPK13 primer.

Figure 19. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. Kuntze) using OPK 11 

primer. 

Figure 20. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. 

Kuntze) using OPK 17 primer. 

Ramkumar et al. 741


Figure 21. RAPD profile of 15 accessions of tea (Camellia sinensis (L.) O. Kuntze) using OPD 19 primer. 

Showed wide morphological variations in tree height, tree 

habit, leaf, size and shape of flower and fruit characters. 

The biochemical characterization also plays an important 

role in the diversity like, biochemical compositions (Du et 

al., 1990), esterase isozymes (Lu et al., 1992) and 

polyphenol oxidase (Singh and Ravindranath, 1990). 

The karyotype (Liang et al., 1994) was used to 

discriminate tea plants and their wild allied species. 

However, it was found that all were not reproducible 

because of different growing environments, developing 

stages, seasons and even experimental conditions. 

Hence, the biochemical like enzyme activity related to 

RAPD analysis can be used to identify the high quality 

tea varieties. Molecular markers like RAPD discriminate 

germplasm at intra and inter specific levels. Conner and 

Wood (2001) described DNA fingerprinting analysis also 

provide a good method for the intra specific level of 

germplasm. 

The biochemical marker (PPO activity) results showed 

three different range of activity (high, moderate and low), 

each containing 5 accessions. In order to correlate the 

biochemical marker study with molecular marker, all the 

15 accessions were analysed (RAPD) using 20 decamer 

primers. The primers were selected based on their ability 

to produce maximum number of bands, reproducibility 

and the ability to produce polymorphism. Among the 20 

RAPD primers were used, fourteen primers showed high 

level of polymorphism. The OPD13, OPB20 and OPB10 

primers were capable of producing high level of 

polymorphism. This could be explained by the capability 

of individual primers to amplify the less conserved and 

highly repeated regions of the genomic DNA. There is 

high possibility for the amplified fragments to contain 

repeated sequences. 

In cluster analysis based on the dendogram, the 

shared fragments divided into three groups, high, 

moderate and low. Among the two major clusters based 

on similarity index, first major clusters were further 

divided into two minor sub clusters. The first minor sub 

clusters contained five accessions (high), the second 

minor sub cluster contained five accessions (moderate) 

and the second minor (low). 

The UPASI 18 (“Cambod”) was found to be 0.28% 

different from the other accessions. This shows that there 

is considerable variability among the accessions selected 

and can be further utilized for crop improvement. 

In this analysis of genetic diversity within the population 

showed most variable in “Cambod” types of tea plants. 

However, other accessions were collected from the same 

geographic region (Brooklands Estate) exhibited high 

level of similarities, presumably because of selection 

being made on the same natural populations as reported 

(Wachira et al., 1995). The genomic diversity between 

the Assam and China clones showed wide variations in

their geographical origin. Mondal et al., (2002) described 

that an average, 57% within and 43% between 

population’s variability and the Chinese clones as more 

genetically diverse than Assam clones. 

Conclusion 

The present investigation concludes that, results of 

biochemical markers studies correlated well in RAPD 

analysis. The pattern of clusters segregated very well in 

the groups (high, moderate and low) which related to the 

total PPO activity of the selected UPASI germplasm. 

This is the first report showing the identifications of 

superior clones performed by using total PPO and RAPD 

analysis. 


The authors thank UPASI TRF, UPASI Tea Research 

Institute, Nirar Dam BPO, Valparai 642 127, Coimbatore 

District, Tamilnadu, India for providing financial 

assistances. 

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Mondal TK (2002). Assessment of genetic diversity of tea (Camellia 

sinensis (L.) O Kuntze) by inter-simple sequence repeat polymerase 

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DOI: 10.5897/JMPR11.1259 



Development of a sebum control cream from a local 

desert plant Capparis decidua 

Shahiq uz Zaman *1 , Naveed Akhtar 1 , Barkat Ali Khan 1 , Tariq Mahmood 1 , Akhtar Rasul 1 , 

Arshad Mahmood 2 , Muhammad Naeem Aamir 3 and Atif Ali 1 

1 Department of Pharmacy, Faculty of Pharmacy and Alternative Medicine, the Islamia University of Bahawalpur, 

Pakistan. 

2 Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology Abbottabad, Pakistan. 

3 University of Veterinary and Animal Sciences (UVAS), Lahore, Pakistan. 


The aim of this study was to develop a stable cream from a local desert plant capable of producing 

Antisebum effects. Two creams (emulsions) were prepared, both of which were of w/o type. One was 

the formulation in which 5% extract obtained from the plant Capparis decidua was added during the 

preparation of the cream and the other was the base or control in which the extract was not added while 

other ingredients were the same as that of the formulation. Thirteen healthy male volunteers were 

selected and their initial sebum readings of both the cheeks were noted with the help of Sebumeter. The 

volunteers were given both the creams and asked to apply the creams on the face daily two times, the 

base on the right side and the formulation on the left side. The readings were taken every fifteen days 

for a period of three months. At the end of the study period, it was found that the formulation 

significantly decreased the sebum values on the left side. On the other hand, an increase in the sebum 

values was observed on the right side where the base was applied, although the increase was not 

significant statistically. The results showed that the cream prepared from the local desert plant C. 

decidua had the ability to produce Antisebum effects in the human volunteers. 

Key words: Antisebum, extract, Capparis decidua, Sebumeter, Sebum. 

INTRODUCTION 

Capparis decidua which is also known as Capparis 

aphylla is a desert plant (Mishra et al., 2007; 

Sarathchandiran et al., 2007) (Figure 1). It is very 

commonly found in the dry regions of Pakistan, India, 

Tropical Africa and Egypt (Neelkamal, 2009a). In 

Pakistan, this plant is abundantly present in the Cholistan 

desert (Mohammad et al., 2008). C. decidua plant 

belongs to the family Capparidaceae (Govind et al., 

2009) and is a densely branching shrub having scanty, 

small caducous leaves (Ravi et al., 2010). A large 

number of biological compounds, alkaloids, phenols, 

sterols or glycosides (Mishra et al., 2007; Rajni and 

Rajbala, 2010), terpenoids as well as some fatty acids 

have been reported in the plant C. decidua (Neelkamal, 

*Corresponding author. E-mail: shahiq75@yahoo.com. Tel: 

00923333854488. Fax: 0092629255243 

2009a; Baby and Jini, 2011). The presence of these 

constituents has resulted in the use of this plant in 

asthma, gout, rheumatism, ulcer, ear infection, as 

hypoglycemic and antidiabetic agent, in lowering 

oxidative stress in diabetes, as anthelmentic, as 

constipative, as purgative and diuretic as well as in some 

skin disorders (Mishra et al., 2007; Ravi et al., 2010; 

Dangi and Mishra, 2010; Yamini and Ranjana, 2001). 

The extract of this plant has also been reported to 

cause reductrion in plasma triglycerides, total lipids and 

phospholipids, because of which it is also used as a 

hypocholesterolemic (Dheeraj and Ranjay, 2011; Yamini 

and Ranjana, 2001; Rajni and Rajbala, 2010). High 

contents of isothiocyanate glucoside, glucocapparin, 

stachydrine, n-triacontane, β-carotene and β-sitosterol 

have been revealed in the Phytochemical screening of 

the plant, while other constituents found include ntriacontanol, 

n-pentacosane and phthalic acid (Pradeep 

et al., 2011).

Figure 1. The plant Capparis decidua. 

Sebum is produced by the sebaceous glands and it is the 

main component of skin surface lipids. It has several 

functions, among which one of the most important is the 

controlling of the moisture balance in the stratum 

corneum. The production of sebum by sebaceous glands 

is controlled by the androgens (Robert and Shirley, 

2008). 

Excessively, oily facial skin is due to overactive 

sebaceous glands. In such case the skin becomes 

greasy and shiny. It develops large pores, feels 

unpleasant, and may also become a serious cosmetic 

problem. Moreover, such type of skin has much more 

chances to develop acne and seborrheic dermatitis 

(Hristo, 2007). Increased sebum secretion is one of the 

most important cause of Acne which is the most common 

skin disease of the people of age between 11 and 30 

years (Naveed et al., 2010). Therefore, it is very 

important to control the excessive oiliness of the skin 

(Hristo, 2007). 

The aim of this study was to develop a cream from the 

extract of a natural plant that could control the excessive 

sebum production. The common products available to 

control the problems associated with excessive sebum 

production generally contain benzoyl peroxide which 

usually causes irritation, swelling or redness and 

moreover people also get allergic to it (Carol and Jeffrey, 

2007). Other treatments like antibiotics and steriods carry 

Zaman et al. 745 

the risk of severe side effects. The cream prepared from 

the extract of a plant can therefore be of great benefit and 

may solve all these problems. 


C. decidua plant was collected from the Cholistan desert near 

Bahawalpur, Abil ® EM90 was purchased from Franken Chemicals 

Germany, Paraffin oil from Merck Germany; Methanol from BDH 

England, Distilled water was prepared in the Pharmaceutical Labs 

of Department of Pharmacy, The Islamia University of Bahawalpur, 

Pakistan. The Lemon oil was purchased from the local market. 

Identification of Plant 

The identification of the plant (C. decidua) was performed at the 

Cholistan Institute of Desert Studies (CIDS), The Islamia University 

of Bahawalpur, Pakistan. The specimen was deposited in the 

Herbarium of the Islamia University Bahawalpur. 

Instruments 

The instruments used in the experimental work were; centrifuge 

machine (Hettich EBA 20, Germany), incubator (Sanyo MIR-153, 

Japan), Incubator (Sanyo MIR-162, Japan), conductivity-meter 

(WTW COND-197i, Germany), sebumeter MPA 5 (Courage + 

Khazaka,Germany), digital humidity meter (TES Electronic Corp, 

Taiwan), electrical balance (Precisa BJ-210, Switzerland),


homogenizer (Euro-Star, IKAD 230, Germany), pH-meter (WTW 

pH-197i, Germany), refrigerator (Dawlance, Pakistan) and rotary 

evaporator (Eyela, Co. Ltd., Japan). 

Preparation of the formulation 

C. decidua plant was cut into small pieces, 200 g of the plant 

material was weighed on an electrical balance and macerated with 

one litre of analytical grade methanol in a container. The container 

was carefully sealed and kept at room temperature for 72 h. The 

container was shaken for 10 to 15 min after every 12 h. The 

macerated plant material was then filtered through multiple layers of 

muslin cloth. The filtered extract was then filtered through Whatman 

No.1 filter paper to obtain a particle free extract. The filtered extract 

was then evaporated by using the rotary evaporator under reduced 

pressure at 40°C temperature. The evaporation was continued until 

the extract was reduced to one third of its original volume. The 

extract was collected in a container and stored in a refrigerator at 

4ºC. 

The formulations selected for this study were W/O emulsions 

(creams) which were prepared by adding the aqueous phase into 

the oily phase with continuous agitation (Barkat et al., 2010). The 

oily phase contained 16% of paraffin oil and 3.5% of ABIL - EM 90, 

which was used as the emulsifier. The aqueous phase was 

comprised of distilled water quantity sufficient to make the volume 

100%. Both the oily and the aqueous phases were heated up to 75 

± 5°C, and 5% extract of C. decidua was added in the aqueous 

phase. The aqueous phase was then added drop by drop to the oily 

phase. The Stirring was continuously done at 2000 rpm by the 

homogenizer for about 15 min until all of the aqueous phase had 

been added to the oily phase. 

A few drops of lemon oil were added during stirring in order to 

give good fragrance to the formulation. When whole of the aqueous 

phase had been added, the speed of the homogenizer was reduced 

and it was operated at a speed of 1000 rpm for 5 min, afterwards 

the speed of the homogenizer was further reduced and it was 

operated at 500 rpm for 5 min so that complete homogenization 

may occur and emulsion cools down to the room temerature. The 

Base or control was prepared by the same method, the only 

difference was that the 5% C. decidua extract was not added to it 

and its volume was replaced by the distilled water. 

Study design 

Thirteen healthy volunteers were selected for this study. The age of 

the volunteers was between 20 to 35 years. All the selected 

volunteers were male. The volunteers were examined by a skin 

specialist for any serious skin disease or damage especially on 

cheeks and forearms. A volunteer protocol was provided to each of 

the volunteer before the study. The protocol stated the terms and 

conditions of the study. All the volunteers had to sign the consent 

form to agree with the terms and conditions of the testing 

individually. The contents of the formulations were kept secret from 

the volunteers. The readings were taken at 25±1ºC temperature 

and 40±2% relative humidity conditions. Before the start of the 

study the patch test was performed on the forearms of the 

volunteers for 48 h. Such test is done to check if any volunteer feels 

the irritation, itch or redness on the skin from the application of the 

formulation. 

The volunteers who report such problems are removed from the 

studies. In this study, none of the volunteers reported such problem 

after the 48 h patch test. Therefore, the zero hour Sebum readings 

of the volunteers were taken from the cheeks of the volunteers and 

noted. Each volunteer was then given two creams, a formulation 

having the extract of the plant and a base or control, without the 

extract. The volunteers were instructed about the proper use of the 

creams. Each volunteer was told to come to the laboratory every 

second week for the measurements of skin sebum production upto 

the end of the study period, which was three months. 

Ethical standards 

The approval of this study was taken from the Board of the 

Advanced Study and Research (BASR), the Islamia University, 

Bahawalpur and the Institutional Ethical Committee, Faculty of 

Pharmacy and Alternative medicine, The Islamia University, 

Bahawalpur. 

Mathematical and statistical analysis 

The sebum values of the right and left cheek of the volunteers were 

calculated at zero hour, 2 nd week, 4 th week, 6 th week, 8 th week, 10 th 

week and 12 th week. The data obtained was then analyzed by the 

SPSS 17.0 on the computer by using the two-way ANOVA for 

variation between different time intervals and the paired sample ttest 

for the variation between the two formulations. The level of 

significance was 0.5 %. 

RESULTS 

In vitro evaluation of creams 

The stability of both the creams, base and the formulation 

was evaluated by keeping them at four different storage 

conditions which were 4, 25, 40 and 40°C + 75% RH for 

eight weeks (2 months). Both the centrifugation test and 

storage at accelerated conditions of temperature are very 

important parameters for detecting the stability of creams. 

No phase separation was observed during the study 

period of two months. The liquefaction was not observed 

at 4 and 25°C through out the study period of 2 months, 

while slight liquefaction was observed at 40 and 40°C + 

75% RH at the 6 th and 8 th week in case of the base and 

only 8 th week in case of the formulation. 

In vivo evaluation of the creams on skin sebum 

In this study an increase in the sebum values occurred on 

the right cheeks where the base was applied. There were 

variations seen but generally an increase in sebum 

occurred by the base as shown in the Table 1. The 

ANOVA test showed that the increase was not significant 

(p = 0.171) with respect to time. The increase was less 

than 2.5% upto the 6 th week but it was more than 5% on 

8 th , 10 th and 12 th week (Figure 2). In case of formulation, 

a continuous decrease in the values of the sebum was 

observed throughout the study period of three months on 

the left cheeks as shown in the Table 1. The application 

of ANOVA test showed that the decrease in the values of 

sebum was highly significant (p = 0.000) with respect to 

time. The decrease in sebum values was less than 5% 

upto the 4 th week but it became more than 12% after 6 th 

week and onwards. The heighest decrease was seen at 

the end of the study period which was 18% (Figure 2).

Table 1. Mean of the Sebum values of the volunteers after the application of base and formulation. 

Zaman et al. 747 

Skin Sebum (Mean ± SEM) 

Time 0 Hour 2 nd week 4 th week 6 th week 8 th week 10 th week 12 th week 

Base 132.23±8.84 131.77±9.11 134.69±9.49 134.00±8.16 137.85±8.20 138.46±8.73 138.15±8.61 

Formulation 136.00±9.78 133.92±10.48 130.15±9.98 120.08±9.68 114.85±8.31 112.31±8.55 111.62±8.29 

Percentage change in Sebum values 

10 

Figure 2: Percentage of change in the Sebum values of volunteers after the application 

of Base and Formulation. 

DISCUSSION 

8 

6 

4 

2 

0 

-2 

-4 

-6 

-8 

-10 

-12 

-14 

-16 

-18 

-20 

0.03 

-1.99 

2.28 2.20 

-4.68 

Base Formulation 

-12.23 

5.53 5.45 

2nd week 4th week 6th week 8th week 10th week 12th week 

Time 

-15.46 

-17.52 

5.04 

-18.00 

Figure 2. Percentage of change in the Sebum values of volunteers after the application of Base and 

Formulation. 

The stability characteristrics are of prime importance for 

any kind of formulation and if a formulation does not meet 

the stability standards it becomes unfit for use. In case of 

creams, the elevated temperature can cause change in 

viscosity because of which phase separation or 

liquefaction may occur. As Abil®EM90 was used in this 

formulation which is a lipophilic surfactant and is more 

stable at elevated temperature (Raymond et al., 2009), 

therefore the creams showed good stability 

characteristics. The liquefaction was not observed at 

4 and 25°C through out the study period of 2 months, 

while slight liquefaction was observed at 40 and 40°C + 

75% RH at the 6 th and 8 th week in case of the base and 

only 8 th week in case of the formulation. 

Sebum is an oily substance which is produced by the 

sebaceous glands in the skin. These are tiny glands 

mostly located on the scalp, face and around the anus. 

The production of sebum is partly controlled by male sex 

hormones. Sebum which is composed of fat and wax 

lubricates the skin and protects it from becoming soggy 

when wet, as well as from becoming cracked when 

exposed to hot, dry temperatures. Sebum also helps


protect the skin from bacteria and fungi, but oversecretion 

of sebum causes Seborrhea (oily skin and scalp) and 

may lead to seborrheic dermatitis or acne. Although, the 

exact cause of this excess production is not fully 

understood but it is observed that male sex hormones 

(androgen hormones) do play a role in its excess 

production. This is the reason that the problem of 

Seborrhea is very common in adolescent boys and men 

(Carol and Jeffrey, 2007). 

This study showed an increase in the sebum values on 

the right cheeks where the base was applied, although it 

was not significant (Figure 2). The increase in sebum 

values may be due to the oily nature of the cream as 

Paraffin oil was used in the formulation (Naveed et al., 

2011). In case of formulation a regular decrease was 

observed in the sebum values throughout the study 

period of three months (Table 1) and the ANOVA test 

showed that the decrease was highly significant. It has 

been reported that the androgens stimulate the sebum 

production. The enzyme 5α-reductase is the enzyme that 

metabolizes the testosterone into its more potent form the 

dihydrotestosterone in the skin, which causes the 

enlargement of sebaceous glands and increased 

secretion of sebum. It is supposed that the application of 

inhibitors of 5α-reductase type 1 or dual inhibitor may be 

effecctive in lowering the sebum level. Many botanical 

compounds are thought to inhibit 5α-reductase, like Saw 

palmetto extract, essential fatty acids (γ-linolenic acid,αlinolenic 

acid, linoleic, and oleic acids), and phytosterols 

(β-sitosterol) (Hristo, 2007). 

It has been reported that C. decidua contains βsitosterol 

(Mishra et al., 2007; Neelkamal, 2009b; 

Satyanarayana et al., 2008), fatty acids as well as 

thioglucosides which release the isothiocyanates or 

Mustard oils (Dheeraj and Ranjay, 2011). These 

constituents have the ability to inhibit the enyme 5αreductase, 

which may inhibit the excesive sebum 

secretion (Hristro, 2007). As these constituents are 

present in C. Decidua, so they may be responsible for the 

decrease of sebum values in case of formulation. 

Conclusions 

This study shows that a cream from the extract of a local 

desert plant can be formulated; which has the ability to 

control the excessive sebum secretion and therefore can 

be used by people having oily and those who have 

developed acne or seborrhea due to excessive sebum 

secretion. The main advantage of this cream lies in the 

fact that it is prepared from the extract of plant and so it is 

free of the adverse effects of strong antibiotics, steroids 

or products containing strong chemicals. 

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of Hippophae rhamnoides fruit extract. J. Pharm. Res., 3(6): 

1342-1344. 

Carol T, Jeffrey SD (2007). The Encyclopedia of Skin and Skin 

Disorders. Third edition. Facts On File, Inc. New York, USA, 6: 

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10(1): 146-155. 

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DOI: 10.5897/JMPR11.1280 



An ethnobotanical survey of medicinal plants used by 

indigenous people in Zangelanlo district, Northeast Iran 

Mohammad Sadegh Amiri*, Parham Jabbarzadeh and Mahdi Akhondi 

Department of Biology, Payame Noor University, 19395-3697 Tehran, Iran. 


A survey was conducted to document the ethnobotanical potential of Zangelanlo district during 2009 to 

2010. The study was mainly focused on gathering information on traditional uses of plants from local 

peoples. Local inhabitants are extremely knowledgeable about the utilization of indigenous flora of the 

study area. The main uses of the herbal drugs were as febrifuge, anthelmintic and in digestive 

problems, jaundice, respiratory ailments, urinary diseases, skin diseases and diabetes. In this present 

investigation, 52 plants species belonging to 48 genera and 26 families were included. The major plant 

families which contributed in folk herbs included Lamiaceae (9 spp.), Asteraceae (8 spp.), Apiaceae (4 

spp.) and Fabaceae (3 spp.). For each species, botanical name, vernacular name, used plant part(s) and 

medicinal uses are provided. The results of this survey indicated that the studied area is rich in 

medicinal plants to treat a wide spectrum of human ailments. Therefore, this work will also contribute 

for the search of new drugs and treatments. 

Key words: Ethnobotany, medicinal plants, Lamiaceae, Asteraceae, Apiaceae, Fabaceae, Zangelanlo, Iran. 

INTRODUCTION 

Herbal remedies are the oldest form of health care known 

by mankind. Prior to the development of modern 

medicine, traditional medicine systems that have evolved 

over the centuries among various communities, were still 

maintained as a great traditional knowledge basis in 

herbal medicines (Mukherjee and Wahil, 2006). This 

knowledge has been passed on orally from generation to 

generation without any written document (Perumal Samy 

and Ignacimuthu, 2000) and is still retained by various 

indigenous groups around the world. During the last few 

decades there has been an increasing in the study of 

medicinal plants and their traditional use in different parts 

of the world (Lev, 2006). Today according to the World 

Health Organization reports, as many as 80% of the 

world's people depend on traditional medicine for their 

*Corresponding author. E-mail: M.S._Amiri@pnu.ac.ir. Tel: 

00985825230015. Fax: +985825229291. 

primary health care needs. There are considerable 

economic benefits in the development of indigenous 

medicines and in the use of medicinal spices for the 

treatment of various diseases (Azaizeh et al., 2003). 

Ethnobotanical survey has been found to be one of the 

reliable approaches to drug discovery (Fabricant and 

Farnsworth, 2001). Several active compounds have been 

discovered from plants on the basis of ethnobotanical 

information and used directly as patented drugs (Carney 

et al., 1999). The extent of the knowledge of traditional 

medicine practice based on medicinal plants should be 

documented through botanical surveys. Botanical 

collection and documentation of the associated 

ethnobotanical knowledge should be carried out before 

such rich heritages are lost due to various anthropogenic 

and other natural causes. In addition, the conservation of 

ethnobotanical knowledge as part of living cultural 

knowledge and practice between communities and the 

environment is essential for biodiversity conservation 

(Martin, 1995). Some of this species are


Handelia trichophylla (Schrenk) Heimerl is very rare in 

this area and is known only from one locality and 

consider being critically endangered (Amiri and Joharchi, 

2010). The main focus of the present study is to ascertain 

the detailed information on the use of plants and their 

therapeutic practices among indigenous people of 

Zangelanlo district, Khorassan province, Iran. 


The district of Zangelanlo is situated in the Northeast of Iran 

between 37°13� to 37°27� north latitude and 59° 8� to59° 35� east 

longitude belonging to the Irano-Turanien Plant Geography Region. 

The present data were collected on a field research carried out as a 

part of floristic and ethnobotanical studies during 2009 to 2010 

(Amiri and Jabbarzadeh, 2010). A questionnaire was administered 

to the local people, through face to face interviews. About 8 villages 

around the area were visited and surveyed where interviews of 

about 27 local informants especially women were made. The 

questionnaire was administered only to people who had knowledge 

of medicinal plants. At the end of each interview, the participants 

were asked to show the researchers these wild plants in the field. 

Then, specimens of these plants were collected and identified. 

Specimens were identified by using various Floras (Rechinger, 

1963, 2005; Assadi et al., 1988, 2008; Davis, 1965, 1985; Komarov, 

1934, 1958) and consulting different herbal literature (Amin, 1991; 

Ghorbani, 2005; Hooper, 1937; Zargari, 1989, 1992). Data 

tabulated were plant name, botanical family, local name, used parts 

and plant uses (Table 1). The family plant names were listed in 

alphabetic order. 


During the survey, 52 herbs belonging to 48 genera and 

26 plant families showed to present medicinal uses 

(Table 1). The families which contributed with species 

included as folk medicines were: Lamiaceae (9 spp.), 

Asteraceae (8 spp.), Apiaceae (4 spp.), Brassicaceae, 

Fabaceae and Malvaceae (3 spp.), Plantaginaceae and 

Scrophulariaceae (2 spp. each), while the remaining 18 

families had only one medicinal species. Different parts of 

medicinal plant species were used by indigenous people 

of this area as medicine. For curing ailments, the use of 

aerial plant parts was higher (85%) than the underground 

parts (15%). Among the aerial parts, leaf was used in 

majority of cases (28 species). Leaves were used the 

most, constituting (25%) of the total uses. This was 

followed by fruits (19%), flowers (17%), roots (15%), 

seeds (12%), whole plants (8%), barks (2%) and gums 

(2%). Maximum use of leaves medicinal purpose 

indicates either these plants are easily availability or they 

may have strong medicinal properties. The main 

remedies used for treating the ailments in the studied 

area were: febrifuge, anthelmintic, digestive problems, 

jaundice, respiratory ailments, urinary diseases, skin 

diseases and diabetes. 

Based on Table 1 high numbers of medicinal plant 

species were used for the treatment of skin diseases 

(23%) and indigestion (21%). Common health ailments in 

the studied area were skin problems such as wounds, 

cuts, dermal allergies and a larger number of remedies 

were used to treat these ailments. Common medicinal 

plants such as Althaea officinalis L., Falcaria vulgaris 

Bernh., Fumaria vaillantii Loisel., Handelia trichophylla 

Heimerl, Plantago major L. and Scutellaria luteo-coerulea 

Bornm and Sint. Ex Bornm were used for skin diseases. 

Most of the reported preparations in the area were drawn 

from a single plant, mixtures were rarely used. It was 

observed that 70% among women and 30% among men 

were knowledgeable about plants. It was noted that elder 

people had more knowledge about the folk uses of 

medicinal plants than younger generation. The local 

inhabitants use 52 species of plants for treating various 

ailments. Most species had multi uses. The plants were 

mostly used in their crude form. This data offers basic 

information to the pharmaceutical industry for further 

research in the treatment and control of ailments. 

Conclusion 

The data collected shows that these medicinal plants 

have been used to cure 43 types of ailments. Generally, 

the people of the studied area still have a strong belief in 

the efficacy and success of the herbal medicine. The 

results of the present study provide evidence that 

medicinal plants continue to play an important role in the 

healthcare system of this tribal community. The medicinal 

-botanical survey revealed that the people from this area 

have significant herbal drugs knowledge but as the 

people are in progressive exposure to modernization, 

their knowledge of traditional uses of plants may be lost 

in due course. So it is important to study and record the 

uses of plants by different tribes and sub-tribes for future 

studies. Ethnobotanical studies enable knowledge 

transference of plant-based treatments to the future 

generations. Further pharmacological studies are 

required before encouraging the use of herbal drugs from 

Zangelanlo district in the northeast Iran. Finally, to 

conclude, this research article will attract the attention of 

ethno botanists, phytochemists and pharmacologists for 

further critical investigation of medicinal plants present in 

the districts of Zangelanlo, Iran. 

ACKNOWLEDGMENTS 

Authors thank the traditional healers of the studied plants 

area for sharing their valuable ethnobotanical knowledge.

Table 1. Medicinal plants used to cure various ailments in Zangelanlo district, Iran. 

No. Family Scientific name Local name Part used Uses 

Amiri et al. 751 

1 Apiaceae Bunium persicum Gharah zireh Fruit Indigestion, flavoring and carminative 

2 Apiaceae Falcaria vulgaris Ghaz Yaghi Leaves Treat wounds 

3 Apiaceae Ferula gumosa Ghasni Root and gum Stomach problems, appetiser, emmenagogue and anthelmintic 

4 Apiaceae Ferula latisecta Sasekoma Leaves Indigestion and anthelmintic 

5 Asteraceae Achillea Wilhelmsii Gulechaghgher Flower Wounds, cutting, antihemorrhoids and indigestion 

6 Asteraceae Centaurea behen Bikh-e-Sefid Root Aphrodisiac 

7 Asteraceae Cichorium intybus Kasni Whole plant Jaundice, febrifuge, blood cleanser and cooling 

9 Asteraceae Handelia trichophylla Sarahgool Aerial parts Respiratory ailments, skin problems, kidney stone and anti-hemorrhage 

10 Asteraceae Scorzonera pusilla Tolli Root Stomach problems 

11 Asteraceae Silybum marianum Gharamdolagh Aerial parts Kidney stone 

12 Asteraceae Tripleurospermum disciforme Babuneh Flower Sore throat, febrifuge and to treat cold 

13 Beberidaceae Berberis integrrima ZelK Fruit and root Analgesic and depurative 

14 Brassicaceae Alyssum campestre Ghodumeh Leaves Laxative and anti-tussive 

15 Brassicaceae Descurainia sophia Khakshir Seed Constipation, Anti-thirst cooling and depurative 

16 Brassicaceae Eruca sativa Mando Aerial parts General weakness and sedative 

17 Capparidaceae Capparis spinosa Kalir Fruit Indigestion and anthelmintic, 

18 Caryophylaceae Acanthophyllum speciosum Chubak Root Herpes and washing 

19 Chenopodiaceae Chenopodium botrys Therekh Whole plant Antidiarrhea, indigestion, carminative, anthelmintic and respiratory 

disorders 

20 Eleagnaceae Eleagnus angustifolius Igdeh Fruit Antidiarrhea and rheumatism 

21 Fabaceae Alhagi persarum Dovatikan Aerial parts Antidiarrhea, diuretic and kidney stone 

22 Fabaceae Colutea buhsei Gadook Fruit and leaves Toothache 

23 Fabaceae Glycyrrhiza glabra Soosook Root Stomach problems, general weakness and mouth ulcers 

24 Fumariaceae Fumaria vaillantii Shatara Aerial parts Jaundice, indigestion, antiacid, treatment of dermal allergies and depurative 

25 Hypericaceae Hypericum scabrum Chayoti Flower Anti-hemorrhage body heat and analgesic 

26 Juglandaceae Juglans regia Gouz Fruit and leaves Antidiarrhea and hair color 

27 Lamiaceae Hymenocrater elegans Shonook Flower and leaves Respiratory ailments, carminative, hypnotic and nerve tonic 

28 Lamiaceae Lallemantia royleana Balengoo Seed Anti-thirst , constipation, sore throat and cough 

29 Lamiaceae Nepeta bracteata Zufa Aerial parts Carminative and treatment of cold 

30 Lamiaceae Perovskia abrotanoides Hoosh Whole plant Respiratory ailments, sedative, dysentery, rheumatism and toothache



31 Lamiaceae Scutellaria luteo-coerulea Geylan Aerial parts Indigestion, carminative and skin problems 

32 Lamiaceae Stachys lavandulifolia Toklijeh Flower Treatment of colic, treatment of cold and nerve tonic 

33 Lamiaceae Teucrium polium Bayramnokhodi Aerial parts Diabetes and stomach problems 

34 Lamiaceae Ziziphora clinopodioides Annokh Aerial parts Respiratory ailments, carminative, indigestion, anthelmintic and dysentery 

35 Lamiaceae Ziziphora teniuor Kakuti Aerial parts 

36 Liliaceae Eremurus olgae Cherish Root and leaves Edible as vegetable, sticking and dermal infection 

37 Malvaceae Malva sylvestris Panirak Flower and fruit Febrifuge, respiratory ailments, depurative and mouth ulcers 

38 Malvaceae Malva neglecta Hmamchoorieh Flower and fruit Sore throat and anti-tussive 

39 Malvaceae Althea officinalis Charm Giah Root Wounds, broken bone, swellings due to wounds and snake bites 

40 Nitrariaceae Peganum harmala Ispand Seed Diabetes and antiseptic 

41 Plantaginaceae Plantago major Kalghooreh Seed and leaves Treatment of dermal allergies, treatment of cold, toothache blood-cleaning 

and laxative 

42 Plantaginaceae Plantago psyllium Gharenyarg Seed Gastric ulcer, mouth ulcers and antitussive 

43 Polygonaceae Rheum ribes Rivis Fruit and petiole Diuretic, blood-cleaning and jaundice 

44 Portulacaceae Portulaca oleracea Khorfeh Seed and leaves Indigestion, depurative, edible as vegetable and anti-thirst 

45 Pteridaceae Adiantum capillus-veneris Paresiavash Aerial parts Febrifuge, sore throat, antitussive and mouth ulcers 

46 Salicaceae Salix alba Sooyet Leaves and bark Sedative, cooling and fever 

47 Scrophulariaceae Scrophularia striata Mokhaleseh Whole plant Dysentery, treatment of colic, carminative and rheumatism 

48 Scrophulariaceae Verbascum songaricum Doch Leaves Anti-hemorrhage, antidiarrhea and indigestion 

49 Solanaceae Solanum nigrum Sabok angur Fruit Analgesic and expectorant 

50 Urticaceae Urtica dioica Gazgazoonak Aerial parts Indigestion and anemia 

51 Violaceae Viola odorata Banafsheh Flower Fever of children and treatment of cold 

52 Zygophyllaceae Tribulus terrestris Gornook Fruit and leaves Increases sexual vigour, Kidney stone and anthelmintic 

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of the Asteraceae-Anthemideae for the flora of Iran. The 

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Watershed, Khorassan, Iran. J. Taxonomy and 

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(eds.). (1988-2008). Flora of Iran. Vols. 1-60. Research 

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Azaizeh H, Fulder S, Khalil K, Said O (2003). Ethnomedicinal 

knowledge of local Arab practitioners in the Middle East 

Region. Fitoterapia. 74: 98-108. 

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Hsu VL, Moswa JL (1999). Maprouneacin, a new daphnane 

diterpenoid with potent antihyperglycemic activity from

Maprounea africana. J. Nat. Prod., 62: 345-347. 

Davis OH (ed.) (1965-1988). Flora of Turkey. Vols. 1-10. Edinburgh 

University Press, Edinburgh. 

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traditional medicine for drug discovery. Environ. Health Perspectives 

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region of Turkmen Sahra, north of Iran (Part1): General results. J. 


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Botanical Institute of Science the U.S.S.R. Leningrad. Translated by 

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Zargari A (1989-1992). Medicinal Plants. Tehran University 

Publication, Tehran, 1-5: 4678.



DOI: 10.5897/JMPR11.1303 



Reproductive behaviour and breeding system of wild 

and cultivated types of Withania somnifera (L.) Dunal 

Bilal Ahmad Mir 1,2 *, Sushma Koul 1 , Arun Kuar 1 , Shushant Sharma 3 , Maharaj K. Kaul 1 and 

Amarjit S. Soodan 3 

1 Biodiversity and Applied Botany Division, Indian Institute of Integrative Medicine (CSIR), Jammu 180001, India. 

2 Department of Botany, University of Kashmir, Hazratbal, Srinagar-160001, India. 

3 Botanical and Environmental sciences Department, Guru Nanak Dev University, Amritsar, India. 


The Indian germplasm of Withania somnifera (L.) Dunal shows remarkable genetic variability both in the 

cultivated and the wild populations. The utilization of this variability through conventional breeding 

requires a clear understanding of its reproductive biology and breeding system. It is an amphimictic 

species practicing open pollination. However, a proximal placement of the stigma and the anther and a 

synchrony between the receptivity and dehiscence of anthers strongly predispose the species to self 

pollination and selfing. However open pollination results in equally high percentage of fruit and seed 

set as on controlled selfing indicating that the species shows facultative autogamy. This raises the 

probability of genetic improvement through hybridization. The somatic complements revealed a diploid 

number of 2n=48 in all the accessions tested. The absence of karyomorphological differences indicated 

that numerical and structural changes do not have a role in controlling the genetic variability of the 

species. Experimental crosses between the cultivated and the wild accessions produced viable seeds. 

A significantly higher fruit set and seed germ inability in crosses involving the cultivated types as the 

seed parent point to the existence of maternal effect. 

Key words: Withania somnifera, floral biology, autogamy, geitonogamy, open pollination, ovule ratio and 

reproductive effort. 

INTRODUCTION 

Withania somnifera (L.) Dunal (Ashwagandha, 

Solanaceae), popularly known as Indian ginseng and 

Winter cherry, is one of the most reputed medicinal herbs 

that forms an essential constituent of over 100 traditional 

medicine formulations (Kaileh et al., 2007). The well 

described pharmacological activities of the plant include 

physiological and metabolic restoration, anti-arthritic, antiaging, 

anti-cancer, anti-bacterial, cognitive function 

improvement in geriatric states and recovery from 

neurodegenerative disorders (Owais et al., 2005; Lal et 

al., 2006; Misra et al., 2008; Mirjalili et al., 2009; Koduru 

et al., 2010; Ven Murthy et al., 2010). In vitro and in vivo 

pharmacological investigations have elucidated the 

*Corresponding author. E-mail: meerbilal82@gmail.com. 

Tel:91-191-2569000-10, 09018808438 Ext. 228. 

leaves and roots (Sangwan et al., 2004; Mirjalili et al., 

association of these activities to withanolides present in 

2009). The species grows wild in several states of India 

viz., Madhya Pradesh, Uttar Pradesh, Andhra Pradesh, 

Gujarat, Maharashtra, Rajasthan and Punjab extending 

to the mountainous regions of Himachal Pradesh and 

Jammu up to an elevation of 1500 m (Singh and Kumar, 

1998). In view of increasing demand in 

phytopharmaceutical industries, an annual morphotype 

has been in cultivation in Madhya Pradesh, Rajasthan 

and Andhra Pradesh (Kothari et al., 2003). The cultivated 

plants differ from the wild ones not only in their 

therapeutic properties but also in morphological 

characters of roots, stems, leaves, flowers, seeds and 

fruits. For this reason, Kaul (1957) suggested a new 

species Withania ashwagandha for the cultivated plants 

as distinct from Withania somnifera for the wild ones. 

In recent literature, Kumar et al. (2007) reported that

wild and cultivated populations of Ashwagandha differ not 

only in morphology but also in their chemical architecture 

and suggested that the wild and the cultivated 

populations be recognized as separate chemotypes. 

Recently our group reported the differences in molecular 

make up of the wild and the cultivated populations (Mir et 

al., 2010; Kumar et al., 2011). Joshi et al. (2010) also 

confirmed the distinct nature of wild and cultivated plants 

on a number of morphological, bio-chemical and 

physiological traits. Moreover, protocols of breeding 

require a thorough understanding of the reproductive 

behavior and crossability of the morphotypes. In view of 

paucity of such data on the Indian chemotypes, the 

present investigations were conducted to fill this gap of 

information. Our work is a sequel to integrated 

investigations on the Indian germplasm of W. somnifera 

under the New Millennium Indian Technology Leadership 

Initiative (NMITLI) programme. We took leads from the 

initial work of Kaul et al. (2005) who studied some of the 

aspects of the reproductive biology of W. somnifera. 

Reference to the work on the related species Withania 

aristata by Anderson et al. (2006) has also proved 

helpful. 



Data were collected from populations of twenty three wild and 

cultivated accessions of W. somnifera maintained in the 

experimental plots of Indian Institute of Integrative Medicine, 

Jammu, India. For comparison, two elite accessions (AGB002 and 

AGB025) were taken as representative of the wild and cultivated 

accessions, respectively. The site of study is located at 32 0 44' N, 

75 0 55' E. It is 400 m asl with the annual temperature ranging 

between 5 and 45°C, and annual rainfall up to 110 cm. Studies 

were carried out during the peak flowering period (April to June) for 

three consecutive years (2007 to 2009). 

Floral biology 

Individuals in both the populations (wild and cultivated) bore perfect 

flowers. Morphology and morphometry was done from mature 

flowers. Flowering phenology was studied on day to day basis from 

twenty plants marked in each population at the pre-anthesis and 

post-anthesis phases. 

Pollen/ovule measurement 

Anthers collected from twenty fully mature flowers at pre-anthesis 

stage were stored in 1% glycine. The anthers were chopped, spun 

on vortex mixer and pollen grains were counted with a 

haemotocytometer (Osankoyo, 1999). Pollen size was recorded 

with the help of a set of ocular and stage micrometers. Pollen 

output per flower was calculated by the estimated number of pollen 

grains per anther times the number of anthers (5) per flower. Ovule 

count per ovary was made by clearing fresh pistils with 1 N NaOH, 

staining with 1% aceto-carmine and gentle squashing. Ovules were 

counted under a stereo-microscope. Pollen-ovule ratio was 

obtained by dividing the pollen number by the number of ovules per 

flower (Cruden, 1977). Pollen viability was estimated by 3, 5- 

Mir et al. 755 

triphenyltetrazolium chloride (TTC 2,) (Stanley et al., 1974) and 

Fluorescein Diacetate (Heslop-Harrison and Heslop-Harrison, 

1970) tests. For ultra-structure of the pollen surface, pollen was 

acetolyzed and mounted on glass plate following the method of 

Caccavari and Dome (2000). Thereafter, plates were prepared for 

electro-micrographs under a Scanning Electron Microscope (JEM- 

JEOL-100 CX II EM with ASID) at 40 KV. 

Pollination 

Stigma receptivity was checked by the method of Shivana and 

Rangaswamy (1992). Frequency of flower visitors was recorded at 

regular intervals of time during the peak flowering period between 

9.30 am and 5.00 pm for three consecutive days. 

Reproductive effort 

Dry biomass was estimated from ten plants each from wild 

(AGB002) and cultivated (AGB025) populations. The plants were 

disjointed into leaves, roots, flowers and fruits. Leaf area was 

measured by leaf area meter and seed weight with a single pan 

electronic balance. Foliar, floral and reproductive shoot parts were 

oven dried at 60°C to a constant weight. Reproductive effort (RE) 

was estimated by three indices. 

GW 

RE1 = (Wolff, 1999) 

GW � LB 

FB 

RE2 = (Dunn and Sharitz, 1991) 

LB � GW 

SW 

RE3 = (Primack and Antonovics, 1982) 

LA 

Where, GW, generative weight (Floral biomass plant -1 + 

Reproductive shoot biomass plant -1 ); LB, leaf biomass plant -1 ; FB, 

floral biomass plant -1 ; SW, seed weight (total) plant -1 ; and LA = leaf 

area plant -1 . 

Breeding experiments 

Controlled crosses were carried out to test the wild and cultivated 

populations for autogamy, geitnogamy and xenogamy. Hand 

pollinations were conducted during 0900 to 1100 h during April to 

May for three consecutive years (2007 to 2009). 

Autogamy 

In a sample, twenty plants flowers were emasculated before anther 

dehiscence and pollinated during peak stigmatic receptivity with 

pollen from previous day flowers and bagged. Self-pollination 

(passive autogamy) was tested by bagging intact flowers. 

Geitonogamy 

Hand pollination of 155 flowers was conducted in twenty plants 

each of the wild and the cultivated population with pollen from other


flowers of the same plant. 

Xenogamy 

Here again, twenty plants each from wild and cultivated populations 

were involved in experiments to test for xenogamy. An 

inflorescence from each of the selected plants was marked and 

open flowers were removed. Flower buds ready to open were 

carefully emasculated avoiding damage to the pistil. For hand 

pollination, mature pollen from the male parent were put on 

receptive stigma of the emasculated flowers (n=300) with the help 

of a paint brush and bagged. After time for fruit set elapsed, bags 

were opened and percentage fruit set and number of seeds/fruit 

was recorded. At fruit maturity, seeds from different crossing 

experiments were dried and put in Petri plates at 25°C in controlled 

conditions for germination; percentage germination was recorded 

after 30 days. 

Somatic complements 

Seeds from the parent populations and those raised by 

hybridization were germinated in the dark at 27°C on moist filter 

paper in Petri-dishes. Root tips ~ 0.5 cm long were excised pretreated 

in a saturated solution of p-dichlorobenzene (PDB) for 4 h 

and rinsed thoroughly 5 to 6 times with double distilled water. Then 

they were fixed in acetic: ethanol (1:3) mixture for 24 h and stored 

at 4°C in 70% ethanol. For chromosomal studies, fixed root tips 

were washed thoroughly in distilled water to remove the traces of 

ethanol. Root tips were then hydrolyzed in 5N HCl for 30 min at 

room temperature, rinsed thoroughly in distilled water and kept in 

leucobasic fuschin for 45 min in dark conditions and squashed in 

1% acetocarmine. Some of the cells showed well spread 

metaphase complements. 

Chemical profiling 

The leaf and root samples for each accession were collected from 

twenty plants at the same stage of development. They were dried at 

45 to 50°C in oven for 48 to 50 h. Dried and powdered roots and 

leaves were percolated separately four times with ethanol: water 

(1:1) at retention time (RT). Extracts were centrifuged and 

concentrated to 1/8 th of the original volume under reduced pressure 

at 50±5°C. The concentrated extract was thoroughly extracted with 

chloroform. Thereafter, chloroform was distilled off under reduced 

pressure to yield a residue. Qualitative analysis of leaf and root 

residue was carried out for two withanolides (withaferin A and 

withanolide A) by High-performance liquid chromatography (HPLC) 

method according to the procedure of Khajuria et al. (2004). 

RESULTS 

Plant habit and morphology 

Plants of the wild population (AGB002) were perennial 

shrubs, 120 to 130 cm tall, having sharply acute, slightly 

haired membranous leaves with entire margins. The leaf 

blade was 7.0 to 8.5 cm × 4.5 to 5.5 cm in size. Six to 

seven shoots arose from the crown. Flowers were borne 

in clusters of 6 to 10 at nodes in the axils of leaves. The 

flowers were pentamerous, actinomorphic and perfect 

with a hypogynous ovary. Androecium composed of five 

epipetalous stamens. The ovary was bilocular with axile 

placentation and an exserted pistil. The fruiting calyx was 

globular and very faintly ribbed. The berries were red and 

the seeds oily to touch. Biochemically, withaferin A was 

present in significantly higher concentration in the leaves 

(0.423 to 0.679 g 100 g -1 ) than in the roots (0.003 to 

0.010 g 100 g -1 ). The amount of withanolide A ranged 

from 0.001 to 0.003 g 100g -1 in the leaves and 0.100 to 

0.300 g 100g -1 in the roots. The third withanolide, namely 

withanone also varied from 0.008 to 0.170 g 100 g -1 in 

leaves and 0.011 to 0.017 g 100 g -1 in the roots. Plants of 

cultivated population yield the Ashwagandha of 

commerce. Plants were much shorter than the wild types 

(rarely more than 40 cm tall) having small ovate, sub 

acute, stellately pubescent and subcoriacious leaves with 

inconspicuous veins and undulate margin. There were no 

differences from the wild types either in the size or 

structure of the essential and the accessory whorls 

(Table 1). However, the fruiting calyx was a little more 

elongated, and prominently ribbed, enclosed 

yellow/orange berries with non-oily and dry seeds (Figure 

1). The species exhibits perpetual flowering with a peak 

period in April to June. Withanolide A and withanone 

were absent in the leaves but withaferin A showed a 

higher range (0.850 to 1.255 g 100 g -1 ). In the roots, all 

the three marker compounds were present in lower 

quantities. 

Floral biology and breeding system 

The opening of petal lobes at the tip marked the 

beginning of anthesis which occurs between 0090 to 

1100 h. At pre-anthesis phase, the carpel was longer 

than stamens but post-anthesis stamens increased in 

length exceeding the height of the carpels at the time of 

anther dehiscence. The filaments were inserted into 

stapets or stirrups which were fused with the corolla tube. 

In both the populations the stapet (filament base) was 

adpressed to the ovary and a narrow groove between the 

stapets and the ovary allowed nectar to ooze upwards 

from the nectary located at the base of the ovary. After 

anthesis, the petals lasted for about three days after 

which the corolla, which was pale greenish at anthesis 

turned brown and withered. By the next day, the petals 

dropped. Anthers dehisced longitudinally. On the inner 

side, they were in touch with stigma causing sufficient 

pollen deposition on it. Pollen grains were yellow, smooth 

walled, slightly sticky, uniform in size and nearly as long 

as broad both in the wild and cultivated types (Table 1). 

Stigmas were papillose and greenish and had a wet 

surface at the receptive stage. In both the populations 

pollen grains were found to be trizonocolporate with a 

scabrate-granulate exine pattern. The non-viable pollen 

grains were deformed (Figure 2). In both the

Table 1. Comparison of essential traits in the floral biology of wild and cultivated populations of W. somnifera. 

Character (Flower size) Wild population Cultivated population 

(Length × Breadth) mm 

Before antesis 5.2±0.015×5.3±0.027(75) * 5.03±0.022×4.50±0.022 

After anthesis 5.34±0.020×5.3±0.026(75) 5.33±0.018×5.23±0.020 

Pistil length (mm) 

Before anthesis 3.87±0.011 3.69±0.006 

After anthesis 4.52±0.011 3.76±0.006 

Length of stamen before 

Anthesis (mm) 3.09±0.06 (75) 3.48±0.13 (75) 

Length of stamen after 

Anthesis (mm) 3.76±0.05 (75) 3.75±0.09 (75) 

Pollen anther -1 5800±175.7 (20) * 7650±201.5 (20) 

Pollen flower -1 29000 38250 

Pollen viability 

TTC test 66.37±1.84 (2100) 62.4±1.13 (2320) 

FDA test 60.50±0.85 (2100) 50.06±7.57(2320) 

Pollen shape Spherical to sub-spherical Slightly triangular 

Pollen size (µm) 28.0±0.07 27.3±0.07 

Stigma type Pappilose wet type Pappilose wet type 

No. of ovules flower -1 35.5±1.85 (20) 31.5±1.20 (33) 

Pollen ovule ratio flower -1 817:1 1214:1 

* Mean ± Standard Error; Values in parenthesis represent number of observations. 

representative accessions (AGB002 and AGB025), 

berries took 30 to 40 days to mature. Both the wild and 

the cultivated types as also their synthetic hybrids had a 

somatic number (2n) = 48. They did not show any 

karyomorphological differences (Figure 2 A to B). 

Essential data on the reproductive morphometry and 

pollen and ovule output are summarized in Table 1. 

Experimental crosses 

Manual pollination of flowers with self pollen resulted in 

82.5± 2.36 and 76.80±1.68% fruit set in wild and 

cultivated populations, respectively (Table 2). In the wild 

population, of the total fruit set, 83.0± 2.89% developed 

mature seeds and out of the total mature seeds 

60.3±4.57% showed germination. However in the 

cultivated plants, seed set and germination percentage 

were 74.5±1.28 and 58.3±4.41, respectively (Table 2). 

Percentage fruit and seed set as also percentage 

germination on geitnogamy were found to be in the same 

range as for autogamy and open pollination (Table 2). 

Artificial crossing between plants of wild and cultivated 

populations led to very low fruit set percentage 4.04±3.21 


(for W♀ X C♂) and 8.3±2.71 (for C♀ X W♂) (Table 2). 

The seeds obtained as a result of crossing showed a 

germination percentage of 10.8±4.93 in direct (W♀ X C♂) 

and 20.3±5.15 in reciprocal (C♀ X W♂) crosses, 

respectively. In crossing experiments higher figures for 

fruit set, seed set as well as germination percentage were 

obtained when wild plant was the source of pollen. 

Reproductive effort 

Dry biomass of foliar and floral parts exhibited striking 

differences between the two populations. Total leaf 

biomass and floral biomass per plant averaged to 21.35 

and 13.40 g, respectively in the wild as compared to 

only3.51 and 11.01 g in the cultivated population (Table 

2). The RE1 was a little lower in wild population (81.26%) 

as compared to the cultivated (93.31%) types. However 

the second estimator of RE2 revealed much lower figures 

(11.75%) for wild population as compared to the 

cultivated (24.52%). And the third estimator of RE3 yielded 

comparable figures in the cultivated (9.94 mg cm -2 ) and the 

wild (8.98 mg cm -2 ) populations (Table 3).


Figure 1. Comparative morphology of wild; A to E and cultivated populations; F to K of W. somnifera: Habit; A, F, Flower at anthesis; B, G, 

L.S. of flower at anthesis; C, H, Inflated calyx; D, J, Mature berry; E, K and (I) Fruit of cultivated type exposed to show attachment. 

Insect visits 

Both the populations bore scentless and unattractive light 

green flowers. Nevertheless, they were visited by Apis 

dorsata, Apis florae, butterflies and some other insect 

species. Insect visits commenced early in the morning 

with a peak activity from 0800 to 1200 h. 

DISCUSSION 

Flowering plants possess a wide array of contrivances 

controlling breeding system (Eckert and Barrett, 1994; 

Gituru et al., 2002). Breeding systems range from 

obligate selfing to obligate out-crossing and are 

controlled by various types of self compatible and self


Figure 2. Mitotic chromosome complements of wild; A and cultivated; B plants; pollen viability by TTC; C and FDA; D 

tests (arrows indicate non-viable pollen); scanning electron micrographs of non-viable; E and viable pollen grains in 

anterior; F and posterior; G views. 

incompatible systems which are often accompanied by 

peculiarities in floral architecture (Darlington, 1958; Lewis 

and John, 1963; Bawa et al., 1985; Ramirez and Brito, 

1990; Talavera et al., 1993; Lalonde and Roitberg, 1994). 

Even though a lot of work on reproductive biology and 

breeding system of medicinal plants has been reported, 

work on sex expression and reproductive behaviour of W. 

somnifera is scanty. The present studies indicate 

autogamous nature of the species. Apart from a 

monoecious type of sex expression, there are other 

indicators of autogamy in the material under 

investigation. Equal length of stamens and carpels, close 

proximity of anthers and stigma and synchrony between 

stigma receptivity and anther dehiscence strongly 

predispose this species to self-pollination and autogamy. 

A similar mechanism of self pollination caused by 

reflexing of stigma to contact its own anthers has been 

reported in Petrocoptis viscose, an endemic herb of the 

North-West Iberian Peninsula (Navarro and Guitian, 

2002).


Table 2. Pollination type, percentage fruit set and seed germination in wild and cultivated populations of W. somnifera. 

S/N Pollination Population Fruit set (%) Seed set/berry (%) Germination (%) 

1 Autogamy 

2 Geitnogamy 

3 Controlled crossing (Xenogamy) 

4 Open pollination (Control) 

* Mean ± Standard Error; (n) = number of flowers scored; W-wild, C-cultivated. 

In addition to proximal placement of stigmas and the 

anthers, high pollen load both in the wild (817) and the 

cultivated (1214) per ovule checks competition from 

foreign pollen and greatly reduces the possibility of cross 

pollination. Dichogamy at the floral level is an important 

mechanism controlling out-crossing in a large number 

(more than 75%) of bisexual angiospermic species 

(Bertin and Newmann, 1993; Griffin et al., 2000). 

However in our material, perfect synchrony in the male 

and the female phases favour self pollination. Also 

unattractive flowers of the species reduce the possible 

contribution of insect visitors in pollination and predispose 

the species to self pollination. This is a frequent 

feature reported in other self pollinating plants as well 

(Primack, 1985). Despite the existence of several 

pointers to the predominance of autogamy, crossing 

experiments provided the clinching evidence on the 

breeding system of the species. Most significantly, a high 

percentage of fruit and seed set in controlled self 

pollination in both wild and cultivated accessions 

confirmed their self compatible nature. Cross pollination 

after emasculation resulted in very low fruit set indicating 

partial fixation of autogamy in the species (Table 1). Kaul 

et al. (2005) also reported self compatibility in some other 

accessions of the species. That W. somnifera is 

autogamous both at the level of the flower and the 

individual was indicated by our experimental work. Both 

the cultivated and the wild accessions showed nearly as 

much percentage seed set on geitonogamy as on 

autogamy (Table 2). 

The difference in fruit set on autogamy and open 

pollination was also found to be insignificant providing 

strong evidence that the species is cross compatible at 

the population level and raises the possibility of interpopulational 

crossability between the wild and the 

cultivated types. However, fruit set, seed set and seed 

germinability of the hybrid seed by controlled crosses 

Wild 82.5±2.36* (n=400) 83.0±2.89 60.3±4.57 

Cultivated (n=273) 76.8±1.68 74.5±1.28 58.3±4.41 

Wild 70.8±4.37 (n=300) 81.8±3.16 55.3±1.41 

Cultivated 60.5±3.27 (n=203) 60.5±4.91 53.2±2.23 

♀W×♂C 4.04±3.21 (n=312) 38.5±4.03 10.8±4.93 

♀C×♂W 8.3±2.71 (n=173) 40.3±3.37 20.3±5.15 

Wild 87.3±2.28 (n=365) 87.3±3.08 58.10±1.60 

Cultivated 79.40±1.50 (n=215) 85.5±1018 63.0±2.68 

between the wild and the cultivated types was lower than 

that obtained on autogamy and open pollination by 

several orders of magnitude (Table 2). However figures 

for these parameters of crossability were significantly 

higher when the cultivated type were used as the seed 

parent indicating some kind of a maternal effect. 

Comparatively lower fruit and seed set in the interpopulation 

crosses points to genetic divergence between 

cultivated and wild accessions and lend support to the 

proposal that cultivated types be put into a separate 

species W. ashwagandha (Kaul, 1957). All along the 

period of cultivation, the cultivated types seem to have 

accumulated genetic differences from the wild; autogamy 

seems to provide a means for this fixation and 

consequent divergence. 

Resource allocation constitutes an important aspect of 

the life cycle of plants (Primack and Antonomvics, 1982; 

Dunn and Sharitz, 1991). Higher reproductive effort as 

estimated by three indices (RE1, RE2 and RE3) in the 

cultivated type is another result of domestication (Table 

3). Similar differences were reported in several species of 

the plantain genus Plantago. Cultivated species, 

Plantago ovata invests more on reproductive effort as 

compared to its wild allies- Plantago major and Plantago 

lanceolata (Sharma and Kaul, 1995). Despite genetic 

divergence, crossability of the types provides a channel 

for genetic exchange and improvement. Even earlier, 

crosses involving different chemotypes have been 

attempted for genetic improvement of the species. The 

biochemical profiles in the hybrids may be similar to one 

of the two parents, intermediate between them and even 

show novel constituents that are lacking in both the 

parents (Kirson et al., 1977). For example, interchemotypic 

hybrids between Israel chemotype III and 

Indian chemotype I produced an appreciable amount of 

withanolide D which was absent in both the parental 

types (Kirson et al., 1977; Singh and Kumar, 1998).

Table 3. Biomass allocation and reproductive effort in wild and cultivated populations 


Character Wild population Cultivated population 

No. of leaves plant -1 152.50±16.01* 70.25±10.28 

Dry biomass leaf -1 (g) 0.14±0.004 0.05±0.005 

Dry leaf biomass plant -1 (g) 21.35 3.51 

Floral biomass plant -1 (g) 13.40±2.31 11.01±2.24 

Rep. Shoot biomass plant -1 (g) 79.21±6.24 30.38±2.26 

Generative weight (g) 92.61 41.39 

Reproductive effort (RE1) 81.26% 93.31% 

Reproductive effort (RE2) 11.75% 24.52% 

Total leaf area plant -1 (cm 2 ) 2671.80 635.76 

Total seed weight plant -1 (g) 24.00±3.12 6.32±0.62 

Reproductive effort (RE3) mgcm -2 (Seed wt. /unit leaf area) 8.98 mg/cm 2 9.94 mgcm -2 

* Mean ± Standard Error. 

Chemotypic profiles of some other inter chemotypic 

hybrids in W. somnifera have also been worked out 

(Lavie et al., 1975; Nittala and Lavie, 1981; Eastwood et 

al., 1980). 

Conclusion 

W. somnifera is an open pollinated species. However, 

floral architecture in both the cultivated and the wild 

populations creates condition for selfing. The divergence 

seen between the cultivated and the wild accessions 

could probably have resulted from genetic fixation 

through selfing. However formation of fertile hybrids in 

interpopulation crosses indicates that the genetic barriers 

are partial. Synchrony in the flowering periods of the wild 

and cultivated accessions, monoecious sex expression 

and an amenability to emasculation and crossing further 

enhance the possibility of genetic improvement of this 

amphimictic species through hybridization. 


Authors are grateful to the Director, Indian Institute of 

Integrative Medicine (IIIM), Jammu for constant 

inspiration and encouragement. Authors are also thankful 

to P. R. Sharma for electron microscopy. 

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DOI: 10.5897/JMPR11.1309 



Effect of sodium tanshinon IIA silate on heart function 

of children with myocarditis 

Huang Weizhe 1 , Zhang Dongtao 2 , Zhang Ge 3 and Li Tong 4 * 

1 Department of Cardiac Pulmonary Surgery, The First Affiliated Hospital of Shantou University Medical College, 

Shantou, PR China. 

2 Department of Cardiovascular Medicine, The First Affiliated Hospital of Shantou University Medical College, Shantou, 

PR China. 

3 Departments of Orthopaedics and Traumatology, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, PR 

China. 

4 Department of Paediatrics, The First Affiliated Hospital of Shantou University Medical College, Shantou, PR China. 


This study intends to evaluate the effects of sodium tanshinon IIA silate on cardiac function of children 

with myocarditis. 69 children were randomly divided into myocarditis untreatment and sodium 

tanshinon IIA silate treatment groups. Another 24 healthy children served as normal control. Our 

experimental results demonstrated that the sodium tanshinon IIA silate treatment can significantly 

improve heart function in children with myocarditis. 

Key words: Sodium tanshinon IIA silate, myocarditis, children, troponin I. 

INTRODUCTION 

The topic of myocarditis is fraught with controversy, 

ranging from the mode of diagnosis to the optimal means 

of therapy. There is a broad range of presentation, 

ranging from virtually no symptoms to sudden cardiac 

death. So far, however, investigators have mostly focused 

on assessing either acute (Friedrich et al., 1998) or 

chronic (De Cobelli et al., 2006) myocarditis or have 

attempted to monitor only 1 of these injuries, for example, 

tissue necrosis (Mahrholdt et al., 2004). Danshen, the dry 

root and rhizome of Salvia miltiorrhiza Bge (Labiatae), 

*Corresponding author. E-mail: shantfygz@yahoo.com.cn. 

Abbreviations: HRV, Heart rate variability; LVEF, left 

ventricular ejection fraction; LVFS, left ventricular fraction 

shortening; VU-AMS, vrije universiteit ambulatory monitoring 

system; ECG, electrocardiogram; SDNN, standard deviations of 

all normal-to-normal intervals; RMSSD, root mean squares of 

the successive differences; CKMB, creatine kinase MB; SPSS, 

statistically package for social sciences; LF, low frequency; HF, 

high frequency; cTnI, cardiac troponin I; LVED, left ventricle 

end-diastolic diameter; WSC, water-soluble compounds; LDQ, 

lipophilic diterpenoid quinines; CK, Creatine kinase; FS, 

fractional shortening; EDD, end-diastolic dimension; ESD, endsystolic 

dimension; LVES, left ventricle end-systolic diameter. 

was widely used in therapeutic remedies in China and 

other countries (Wu et al., 2004). Many clinical studies 

indicated that Danshen and its preparations could treat 

coronary artery diseases, myocardial infarction, liver 

malfunction, etc (Wu et al., 2004; Bi et al., 2008; Oh et 

al., 2006; Lee et al., 2008). Tanshinon IIA is isolated from 

S. miltiorrhiza and one of the main ingredients of 

Danshen for cardioprotective effects (Zhou et al, 2005). 

Tanshinone IIA was shown to exert beneficial effects on 

cardio-vascular system with minimal reported side effects 

(Wu et al., 1993; Lin et al., 2006; Fu et al., 2007; Yang et 

al., 2009; Shan et al., 2009). This study is designed to 

evaluate the effect of sodium tanshinon IIA silate on heart 

function of children with myocarditis. 


Subject and experiment design 

A retrospective study of 69 patients (age 9 and 14 years) with 

myocarditis was carried out in the Department of our hospital 

between 2010 and 2011. Myocarditis was diagnosed by attending 

cardiologists clinical symptoms compatible with myocarditis were 

observed along with at least one of the following: elevated troponin 

I (>0.1 ng/ml); cardiomegaly (cardiothoracic ratio, >0.5) on the chest 

radiograph, or impaired heart contractility on echocardiography 

(ejection fraction


Table 1. Effect of sodium tanshinon IIA silate treatment on HRV time domain index’s in myocarditis children patients. 

Group SDNN (t/ms) SDANN (t/ms) SDNN index (t/ms) rMSSD (t/ms) PNN50 (%) 

I 148.4±17.8 131.6±11.9 74.5±12.6 53.7±10.8 27.1±4.7 

II 109.6±13.7 ** 117.8±12.8 * 40.2±5.8 ** 42.1±15.2 * 17.6±4.8 * 

III 145.6±19.7 ## 128.6±11.5 # 69.8±13.6 ## 50.4±7.3 # 25.2±7.8 # 

* P

9 

8 

7 

6 

5 

4 

3 

2 

1 

0 

TP (In[ms2]) LF (In[ms2]) HF (In[ms2]) LF/HF 

** * 

## # 

** 

I II III 

Group 

* 

Weizhe et al. 765 

Figure 1. Effect of sodium tanshinon IIA silate treatment on HRV frequency domain indexs in myocarditis children patients; * 

P


Table 4. Effect of sodium tanshinon IIA silate treatment on heart size and function in myocarditis children patients. 

Group LVED (l/mm) LAED (l/mm) LVEF (%) LVFS (%) 

I 37.95±3.99 22.57±1.87 71.43±9.07 42.06±6.06 

II 38.99±4.37 33.63±2.47 ** 60.74±10.76 * 38.13±4.38 

III 38.73±4.91 21.06±4.05 ## 70.53±8.68 # 41.96±5.52 

* P

whether the pain is due to a heart attack. It may also be 

ordered in a person with a high CK to determine whether 

damage is to the heart or other muscles. Increased CK- 

MB can usually be detected in heart attack patients about 

3 to 4 h after onset of chest pain (Kanemitsu and Okigaki, 

1994; Kanemitsu and Okigaki, 1992). Cardiac troponin is 

the standard for diagnosis or exclusion of acute 

myocardial infarction. Recent guidelines recommend a 

cutoff value at the upper 99th percentile (99 th %) of values 

for a reference population of healthy individuals and 

assay imprecision 10% at this cutoff (Leonardi et al., 

2008). 

Measurements of cTnI are used in the diagnosis and 

treatment of myocardial infarction and as an aid in the 

risk stratification of patients with acute coronary 

syndromes with respect to their relative risk of mortality 

(Song et al., 2011). Our present work showed that sodium 

tanshinon II asilate can increase CKMB and cTnI in 

myocarditis children patients. Fractional shortening (FS) 

is the fraction of any diastolic dimension that is lost in 

systole. When referring to endocardial luminal distances, 

it is End-diastolic dimension (EDD) minus End-systolic 

dimension (ESD) divided by EDD (times 100 when 

measured in percentage) (Costa et al., 2003). LVED is 

the most important measurement. It is measured at end 

diastole, on the frame after mitral closure (Haasler et al., 

1984). It normally corresponds to the largest cardiac 

dimension. Left ventricle end-systolic diameter (LVES) is 

measured at end systole, on the frame preceding mitral 

valve opening. It corresponds to the smallest cardiac 

dimension (Sambola et al., 2008). In our present study, 

LAED is reduced and LVEF and LVFS are increased in 

medicine-treated children group. 

Conclusion 

Our work suggests that sodium tanshinon II asilate is 

useful for therapy of myocarditis children patients. 

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DOI: 10.5897/JMPR11.1341 



Nutritional, phytochemical potential and 

pharmacological evaluation of Nigella Sativa (Kalonji) 

and Trachyspermum Ammi (Ajwain) 

Shabnam Javed 1 *, Ahmad Ali Shahid 1 , Muhammad Saleem Haider 1 , Aysha Umeera 1 , 

Rauf Ahmad 2 and Sobia Mushtaq 1 

1 Institute of Agricultural Sciences, University of the Punjab, Quaid-e- Azam Campus, Lahore-54590 Lahore. Pakistan. 

2 Pakistan Council of Scientific and Industrial Research, Lahore, Pakistan. 


Nigella sativa and Trachyspermum ammi were screened for phytochemical constituents and nutritional 

analysis. Tests for flavonoids, tannins, steroids were positive in both spices except saponins. The 

nutrition analysis indicated higher energy value (459.29 and 314.55%), carbohydrates (39.04 and 

47.54%), protein content (24.05 and 20.23%), fat content (21.67 and 4.83%), moisture content (5.4 and 

11.6%), fibre content (5.5 and 4.3%) and ash content (4.34 and 11.5%) in N. sativa and T. ammi seeds, 

respectively. Aqueous, methanolic, ethanolic, n-Hexane extracts of N. sativa and T. ammi and also 

essential oil of T. ammi were subjected to in vitro antifungal and antibacterial assay. Micro well dilution 

assay was adopted against three human pathogenic fungal strains Aspergillus flavus, Aspergillus 

niger, Candida albicans and four bacterial isolates Escherichia coli, Staphylococcus sp., Pseudomonas 

syringae and Bacillus subtilis. Among all the four extracts of N. sativa tested, methanolic extract 

showed maximum inhibitory potential against all the test fungi and bacteria. In case of T. ammi extracts 

and essential oil, all the aqueous, organic extracts especially ethanol, n-hexane and essential oil 

depicted marked antimicrobial potential against all pathogens. Anti bacterial activity of n-Hexane 

extract was maximum for all bacteria (4.76 cm) P. syringica, (4.03 cm) B. subtilis, (3.73 cm) E. coli and 

(4.76 cm) S. sp. Bactericidal action of different fractions of T. ammi decrease in the order n-Hexane 

extract>essential oil>ethanol extract>methanol extract>aqueous extract. 

Key words: Nigella sativa, Trachyspermum ammi, phytochemical analysis, nutrition value, 

antifungal/antibacterial. 

INTRODUCTION 

Spices have valued for their medicinal, flavoring and 

aromatic qualities for centuries, the synthetic products of 

the modern age suppressed their importance. In the 

present time herbal products are considered as safe 

alternatives of synthetic drugs that are regarded as 

unsafe to human and environment. However in the recent 

past increasing research evidence is getting 

accumulated, which clearly indicate the positive role of 

spices and plant extracts for health care. The use of plant 

drugs is increased in many of the developing countries 

because modern lives saving drugs are beyond the reach 

*Corresponding author. E- mail: shabnamjaved@yahoo.com. 

of three quarters of the third world’s population. Many of 

such developing countries spend 40 to 50% of their total 

wealth on drugs and health care reference. As a part of 

the strategy to reduce the financial burden on developing 

countries, it is obvious that an increased use of plant 

drugs will be followed in the future. Nigella sativa (Klonji) 

is a valuable spice, having distinctive aroma and taste; its 

seeds have been used in pickles, bread recepies and 

savoury dishes. N. sativa is regarded as a valuable 

remedy for various ailments, the seeds, oil and extracts 

have played an important role over the years in ancient 

Islamic system of herbal medicine. Bukhari reported that 

Holy Prophet Muhammad (peace be upon him) told” 

There exists, in the black grains, health care of all the 

diseases, except death (Gheznavi, 1988). The dried

seeds are used as astringent, bitter, diuretic, 

emmemagogue; stimulant and anthelmintic. Its decoction 

is useful in paralysis, jaundice, intermittent fever, 

dyspepsia, piles and skin diseases (Paarakh, 2010; Ali et 

al., 2003; Gilani et al., 2001; Hosseinzadeh et al., 2007). 

N. sativa seeds are reported to contain numerous 

chemical compounds. Active ingredients are nigellone, 

beta-sisterol, and thymoquinone. Other compounds 

include 2-methyl-4-isopropyl-p-quinones, anthraquinones, 

saponins, glycosoides, melanthin, essential oil, fixed oil, 

tannins, protein, mucilage resins and glucose, etc 

(Saeed, 1969; Ikram and Hussain, 1978; Ghaznavi, 1988; 

Akhtar, 1988). Thymoquinone improves respiration, 

decrease serum levels of glucose, cholesterol and reduce 

blood pressure (ElTahir et al., 1993; Al-Shabanah et al., 

1998; ElTahir et al., 1993). It has antioxidant, analgesic 

and anti-inflammatory effects (Ali and Blunden, 2003; Al- 

Gharably et al., 1997; Nagi et al., 1999). N. sativa has 

shown anti yeast activity against C. albicans, when used 

at 400 µg/disc or acid-ethyl alcohols at 0.20 ml/disc on 

agar plate. The dried seeds of N. sativa exhibited 

bactericidal activity against Pseudomonas aeruginosa 

(Hanafy and Hatem, 1991). Its hexane extract, water 

extract, ether extract and extract in 95% ethyl alcohol 

give weak activity against Streptococcus pyogenes, S. 

aureus and Streptococcus viridians (Morsi, 2000). Ajwain, 

Trachyspermum ammi (L.) used as a spice is a traditional 

herb widely used for curing various diseases in both 

humans and animals. Most utilizing part of Ajwain is its 

caraway like fruit, which is popular in cooking recipes, 

snacks, savory pastries and as spice. Aroma chemicals 

present in Ajwain; inhibit other undesirable changes in 

food affecting its nutritional quality, texture and flavor. 

Decoction of Ajwain seeds is used for treatments of 

abdominal discomfort, diarrhea, cough and stomach 

troubles (Anikumar et al., 2009). 

Fruit of Ajwain is reported to have antiseptic, antifungal/ 

antibacterial and antithelminitic effects (Morsi, 2000). In 

Ajwain, the major phenolic compound Thymol is present 

and has been reported to be an antispasmodic, germicide 

and antifungal agent (Nagalakshmi et al., 2000). In the 

essential oil of T. ammi, the principle active constituents 

of the oil are phenols, mainly thymol (35 to 60%) and 

some carvacrol (Tsimidou and Boskou, 1994). Both the 

phenols Thymol and carvacrol are responsible for the 

antiseptic, antitussive and expectorant properties (Treas 

and Evans, 2002). Thymol also has antiseptic activity and 

carvacrol possesses antifungal properties (Menphini et 

al., 1995). The essential oil and chloroform, n- Hexane, 

ethyl acetate and methanol extracts of T. ammi were 

found to contain remarkable antibacterial effects against 

food borne and spoilage bacteria, B. subtilis, Salmonella 

typhimurium, P. aeruginosa, Enterobactor aerogens and 

Staphylococcus aureus. The scanning electron 

microscopic studies also investigated the inhibitory effect 

of essential oil on the morphology of B. subtilis at the 

minimum inhibitory concentration (MIC) (Paul et al., 

Javed et al. 769 

2011). The objective of the present study is to evaluate 

the phytochemicals and nutritional value of Klongi and 

Ajwain to be used as a natural food additive and to 

compare the antimicrobial effects of their different 

fractions for medicinal use. 


Collection and identification of medicinal plant seeds 

The seeds of N. sativa (Klonji) and T. ammi (Ajwain) were 

purchased from local market of Lahore, during January 2011. The 

dried plant seeds were grinded using pestle and mortar and packed 

in polythene bags and placed in a dried place for further 

extractions. The plant species were identified by The Flora of 

Pakistan (Nasir and Ali, 1978). The voucher specimen 

IAGS.HHC.633 and IAGS. HHC.634 were given to N. sativa (Klonji) 

and T. ammi (Ajwain), respectively and deposited in the Herbarium 

of Institute of Agricultural Sciences, IAGS, University of the Punjab 

(PU) Lahore, Pakistan. 

Extract preparation 

Soxhlet extraction assembly was used for this purpose. Each of 50 

g dried and powdered seeds were mixed with 250 ml distilled water, 

methanol, ethanol and n-Hexane successively and continuous 

extraction was done for 5 to 6 h. After that extracts were filtered, 

aqueous extracts were dried by freeze drying technique, the 

organic extracts were dried in a rotary evaporator at reduced 

pressure. T. ammi (Ajwain) seeds were submitted to hydro 

distillation for 3 h. The extracted essential oil thus obtained was 

dried over anhydrous sodium sulphate and left overnight to remove 

traces of moisture. Finally powdered extracts and essential oil was 

weighed and transferred in clean and dried vial, then stored in 

refrigerator at 4°C until use. 


Chemical tests were carried out to evaluate the presence of the 

phytochemicals Alkaloids, Flavonoids, Saponins, Tannins and 

Sterols in the selected spices; using standard procedures described 

by Sofowora (1993); Trease and Evans (1989). 

Nutrition analysis 

The proximate analysis (Carbohydrate, Fats, Protein, Moisture and 

Ash) of N. sativa and T. ammi was determined by using the 

Association of Official Analytical Chemists (AOAC) methods 1990. 

Carbohydrate value was determined by difference (100– (moisture 

+ ash + protein + fat)). The nitrogen content was determined by 

kjeldahl method and multiplied to factor 6.25 to find the protein 

content. The weight difference method was used to find moisture 

and ash content. Soxhlet apparatus was used and solvent 

extraction method used for fat content determination, n-Hexane 

was used as solvent. Nutrient contents were valued in percentage. 

Investigation of antimicrobial activity 

Micro well dilution assay 

Micro well dilution methods are used to examine antifungal activity 

of the spices fractions against growth of three pathogenic fungi and


Table 1. Percentage yield of different fractions of N. sativa (Klonji) and T. ammi (Ajwain). 

Table 2. Qualitative analysis of the phytochemicals of seeds of N. sativa and T. ammi. 

Spice Alkaloids Tannins Saponins Flavanoids Sterols 

N. sativa + +++ _ ++ +++ 

T. ammi + ++++ _ ++++ ++++ 

Presence of constituent = +; Absence of constituent = -. 

antibacterial activity for four bacteria, respectively (Okeke et al., 

2001). Each fungal and bacterial concentration was made 

containing 10 CFU/ml. Stock solution was prepared by dissolving 

the powdered extracts and essential oil 0.4 g/ml of dimethyl 

sulfoxide (DMSO) solution. 60 µl of each fungal and bacterial 

suspension was spread on MEA/NA with L shaped glass spreader. 

Single well (8 mm in diameter) was made at the center of the plate 

using sterilized cork borer. About 70 µl of the aqueous, methanol, 

ethanol, n-Hexane extract and essential oil of Ajwain was poured 

into the well with the help of micropipette (10 to 100 µl) and then left 

on a clean place. Controls of DMSO and flucanozone were 

prepared for antifungal activity and streptomycin sulphate for 

antibacterial activity. The inoculated plates were incubated at 27°C 

for 72 h for fungal and bacterial isolates. Antimicrobial activity was 

estimated by measuring the zone of inhibition against the test 

microorganisms in comparison to control. Studies were performed 

in triplicate and mean value was calculated. 


Klonji that is, N. sativa and Ajwain T. ammi are frequently 

used spices and an active part of certain medicines and 

reported to treat different human ailments Paarakh et al., 

2010; Bonjar, 2004). 

Percentage yield 

Extract fraction 

Percentage yield of different extracts of N. sativa showed 

maximum amount of n-hexane extract 9.6% and 

minimum of ethanol extract 3.9%. In contrast T. ammi 

yielded highest concentration of ethanol extract 4.94% 

and lowest by aqueous extract 3.5% (Table 1). 

Phytochemical investigation 

All the test phytochemicals alkaloids, tannins, flavonoids 

% Yield 

Klonji Ajwain 

Aqueous 5 3.5 

Methanol 4.4 3.7 

Ethanol 3.9 4.94 

n-Hexane 9.6 2.92 

Essential oil - 4 

and sterols were detected in N. sativa and T. ammi seeds 

except saponins. In Klonji, tannins and sterols were 

detected in considerable amounts >75% (Table 2). 

Flavonoids and alkaloids were present in decreasing 

order 50 and 25%, respectively. Tannins, flavonoids and 

sterols were found maximum while alkaloids were 

minimum 25%. The presence of these phytochemicals 

has contributed to medicinal value of spices. Flavonoids 

have been reported to have antibacterial, antineoplastic 

activity, anti inflammatory, anti allergic and antiviral 

activity (Alan and Miller, 1996). Steroids are of much 

importance in pharmacy as sex hormones (Okwu, 2001). 

Alkaloids play significant role for fitness of survival to 

plant species. These have insecticidal properties and are 

an integral part of certain medicines and recreational 

drugs (Roger and Wink, 1998). 

Proximate analysis 

The nutrition analysis (carbohydrate, fats, fibre, protein, 

moisture, energy and ash) of N. sativa and T. ammi were 

determined by using AOAC (1990) methods. The (Klonji) 

N. sativa seeds possess high energy content viz. 

459.29%. The decreasing order of different food contents 

are given in order of carbohydrates> protein> fats which 

are in the range of 39.04 to 21.67%. Moisture, fibre and 

ash contents were found in the same range of 5.5 to 

4.34%, fibre content being maximum and ash contents 

were minimum Table 3. These findings are in line with 

that of Sultan et al. (2009), which reported 6.46% 

moisture, ash 4.20%, protein 22.80%, fiber 6.03% and fat 

31.16% Table 3. Similarly, (Ajwain) T. ammi seeds 

possess higher energy value that is, 314.55%. Also, 

Ajwain is rich source of carbohydrates 47.54%. Protein, 

moisture, ash and fibre contents were evaluated in the

Table 3. Proximate analysis of dried seeds of N. sativa and T. ammi. 

Nutrient 

Percentage ±S.D 

Nigella sativa Trachyspermum ammi 

Energy 459.29%±0.05 314.55±0.89 

Carbohydrates 39.04%±0.21 47.54±1.05 

Fat 21.67±1.55 4.83±0.16 

Protein 24.05±1.76 20.23±1.79 

Moisture 5.4±0.15 11.6±0.77 

Fiber 5.5±0.15 4.3±0.1 

Ash 4.34±0.73 11.5±0 

range of 20.23 to 4.3%, proteins being highest and fiber 

was the least (Table 3); very less fat content that is, 

4.83% were detected (Anonymous, 2003). In contrast 

depicted different results from present proximate analysis 

of Ajwain where carbohydrates 24.6%, protein 17.1%, fat 

21.1% and moisture 7.4% are studied. From the present 

nutritional analysis, one can deduce that, these spices 

when added in human diet supply the body with 

carbohydrates, proteins and energy. The presence of 

higher energy content, carbohydrates, proteins and less 

fat contents contribute to nutritive value of selected 

spices and thus can be rich sources of useful foods. 

Antimicrobial activity 

In this study antifungal and antibacterial effects of 

aqueous, methanol, ethanol and n-Hexane extracts of N. 

sativa and T. ammi seeds oil were investigated. In the 

present study, polar solvent (water), solvents of 

intermediate polarity (like methanol and ethanol) and 

solvent of low polarity (hexane) were used to extract plant 

secondary metabolites that differ in polarity and structure 

and thus different solvent extracts showed distinct 

biological properties. During extraction process, solvents 

diffuse into the solid plant material and solubilise 

compounds of similar polarity (Green, 2004). The polarity 

of solvent affects quantity and composition of secondary 

metabolite of an extract. Secondary metabolites having 

antimicrobial activity are phenols, quinones, flavones, 

limonoids, essential oils, alkaloids, saponins, glycosides, 

flavonoids, tannins and coumarins, etc. Traditional 

healers primarily use water for extract preparation from 

plant extracts but organic solvents have been found to 

give more consistent antimicrobial activity compared to 

water extracts (Parekh et al., 2005). Most other bioactive 

compounds including phenols are generally soluble in 

polar solvents such as methanol. Many bioactive 

components are not water soluble and thus organic 

solvent extracts of plants have been found to be more 

effective (Parekh et al., 2006). Methanol, ethanol and 

water are the most commonly used solvents for 

antimicrobial investigations. (Bisignino et al., 1999; 


Lourens et al., 2004; Salie et al., 1996; Rojas et al., 2006; 

Parekh et al., 2005). Micro well dilution assay was 

adopted against three pathogenic fungal strains A. flavus, 

A. niger, C. albicans and four bacterial strains P. 

syringica, B. subtilis, E. coli and Staphylococcus sp. 

The results pertaining to the antifungal potential of the 

Klonji were given in (Table 4). Among different extracts of 

Klonji screened, the methanol extract showed highest 

antifungal potential against all the test fungi with 3.95, 

1.95 and 2.3 cm zone of inhibition, even marked 

significant from the flucanonozone where 0.5 and 0.66 

cm inhibition zone was recorded, in case of A. flavus and 

A. niger. The largest zone of inhibition (3.95 cm) was 

obtained with A. flavus and least (1.95 cm) with A. niger. 

Maximum bactericidal effect (2.43 cm) was observed with 

B. subtilis and minimum (2.36 cm) with P. syringica. 

Ethanol and n-hexane extracts of N. sativa were 

ineffective against all fungal pathogens except n-hexane 

extract in case of A. flavus (0.03 cm). Followed by 

methanol extract, aqueous extract showed marked 

antifungal activity where 1.0; 1.5 and 1.63 cm inhibition 

zone were recorded. Ethanol extract depicted no 

antifungal potential against all the test fungi. N-Hexane 

extracts showed slight antifungal potential (0.03 cm 

inhibition zone) against A. flavus where no inhibition zone 

was observed in case of A. niger and C. albicans. Similar 

to the antifungal results of N. sativa extracts, showed 

maximum inhibition zones were recorded by methanol 

extract, 2.36 cm P. syringica, 2.43 cm (B. subtilis), 2.7 

cm (E. coli) and 2.86 cm (Staphylococcus sp.) (Table 5). 

Streptomycin sulphate was taken as positive control. 

Minimum antibacterial activity was exhibited by n-Hexane 

extract where no inhibition zone was recorded in case of 

B. subtilis. Aqueous extract depicted marked bactericidal 

activity against all tested bacteria maximum for P. 

syringica (2.06 cm) and minimum for Staphylococcus sp. 

(2.1 cm). Similar is the case with ethanol extract showing 

significant decrease in bacterial growth. Mashhadian and 

Rakhshandeh (2005) investigated the antibacterial and 

antifungal effects of the aqueous, chloroform and 

methanol extracts of the Klonji seeds against pathogenic 

C. albicans, coagulase-positive S. aureus and P. 

aeroginosa isolated from wound, blood, urine and critical


Table 4. Effect of N. sativa on fungal isolates. 

Extract 

Mean diameter of zone of inhibition 

Aspergillus flavus Aspergillus niger Candida albicians 

Aqueous 1.5 1.5 1.63 

Methanol 3.95 1.95 2.3 

Ethanol 0 0 0 

N-hexane 0.1 0 0 

Flucanazone 1.5 2 3.3 

Control 0 1.05 1.55 

Table 5. Effect of N. sativa on bacterial isolates. 

Extract 

Pseudomonas syringica 


Bacillus subtilis Escherichia coil Staphylococcus sp 

Aqueous 2.06 1.9 1.83 2.1 

Methanol 2.36 2.43 2.7 2.86 

Ethanol 1.16 2.26 0.1 1.63 

N-hexane 0.1 0 0.1 0.1 

Str. sulphate 2.1 1.43 2.53 2.4 

Control 0 1.2 1.06 0 

success factors (CSF), compared with standard drugs 

cloxacillin, clotrimazole and gentamicin. 

Our antimicrobial results of N. sativa are in agreement 

with these previous findings, where the methanol extract 

of N. sativa seeds had shown the best antimicrobial 

activity using agar dilution, cylinder plate and disk 

diffusion methods. On contrary to present findings, in the 

previous study, aqueous extract did not show any 

antimicrobial activity against any test pathogen. Masood 

et al. (2008) investigated the antibacterial activity of 

aqueous infusions and decoctions of klonji (N. sativa) 

against 188 bacterial isolates of Gram-positive and 

Gram-negative belonging to 11 different genera isolated 

from oral cavity, using disc diffusion method. The 

aqueous decoctions of klonji showed (51%) antibacterial 

potential of the tested microorganisms. Preliminary 

clinical trials have investigated and reported the 

traditional therapeutic use of Klonji seeds for the 

treatment of diarrhoea, hypertension, asthma, diabetes, 

cough, bronchitis, inflammation, headache, eczema, 

influenza and dental caries (Ali et al., 2003; Gilani et al., 

2001). The chemical constituents of seeds of kalonji have 

above one hundred and thymoquinone is regarded as 

active ingredient, responsible for antibacterial activity. 

Other ingredients include dithymoquinone, nigellone, 

thymohydroquinone, ascorbic acid (vitamin C), tocopherol 

(vitamin E), linoeic acid, oleic acid, carvacrol, lipase, tanethole 

and 4-terpineol, etc (Ali et al., 2003). In case of 

T. ammi extracts and essential oil, all the aqueous, 

organic extracts especially ethanol, n-hexane and 

essential oil significantly masked fungal growth (Table 6). 

Fungitoxicity of n-hexane extract was maximum in case 

of A. flavus where inhibition zone 3.2 cm was recorded 

followed by essential oil (2.9 cm),ethanol extract (2.4 

cm),methanol extract (1.3 cm) and aqueous extract (1.2 

cm), respectively. In case of A. niger, mycelia growth was 

suppressed by Ajwain ethanol extract (3.6 cm) and 

minimum by aqueous extract (1.6 cm). Essential oil of 

Ajwain proved to possess strong antifungal toxicity 

against all the three fungal strains being highest in C. 

albicans (3.66 cm), after that ethanol extract (3.53 cm), n- 

Hexane extract (3.16 cm), methanol extract (1.65c m)and 

aqueous extract (1.45 cm). Present antibacterial study of 

T. ammi is in agreement with its antifungal study as all 

the aqueous, organic extracts and essential oil 

represented marked inhibition against all bacteria under 

observation. n-hexane extract significantly inhibited 

bacterial growth (4.76 cm) P. syringica, (4.03 cm) B. 

subtilis, (3.73 cm) E. coli and (4.76 cm) Staphylococcus 

sp. Bactericidal action of different fractions of Ajwain 

decrease in the order of n-Hexane extract>essential 

oil>ethanol extract>methanol extract> and aqueous 

extract (Table 7). Amongst the different extracts of N. 

sativa investigated, methanol extract showed the most 

remarkable activity. This might have resulted from 

solubility of the active constituents in methanol and water 

also. The investigated plant N. sativa did not show strong 

antimicrobial activity of its ethanol and n-hexane extracts 

as compared to methanol extract, negative results do not 

mean absence of bioactive constituents. Active

Table 6. Effect of Trachyspermum ammi on fungal isolates. 


Extract 

Aspergillus flavus 


Aspergillus niger Candida albicians 

Aqueous 1.2 1.6 1.45 

Methanol 1.3 1.35 1.65 

Ethanol 2.4 3.6 3.53 

N-Hexane 3.2 2.96 3.16 

Essential oil 2.9 2.4 3.66 

Flucanazone 1.5 1.55 3.3 

Control 0 1.1 1.55 

Table 7. Effect of Trachyspermum ammi on bacterial isolates. 

Extract 

Pseudomonas syringica 


Bacillus subtilis Escherichia coil Staphylococcus sp. 

Aqueous 1.9 0.9 0.8 2.2 

Methanol 2.23 2.03 1.43 2.03 

Ethanol 2.33 2.36 2.1 2.03 

n-Hexane 4.76 4.03 3.73 4.76 

Essential oil 2.83 3.33 3.21 3.53 

Str. sulphate 2.1 1.7 2.56 2.23 

Control 0 1.2 1.1 0 

compounds may be in insufficient quantities to show 

activity with the applied concentration (Taylor et al., 

2001). In case of T. ammi, organic extracts mainly 

essential oil and n-hexane showed significant 

antimicrobial activity. Aqueous extract depicted least 

activity; this might have resulted from the lack of solubility 

of the active constituents in water. The ajwain essential 

oil and acetone extract exhibited a broad spectrum of 

fungitoxicity against all tested fungi; A. niger, A. flavus, 

Aspergillus oryzae, Aspergillus ochraceus, Fusarium 

monoliforme, Fusarium graminearum, Pencillium citrium, 

Pencillium viridicatum, Pencillium madriti and Curvularia 

lunata. However, acetone extract was found less effective 

than the essential oil. Gas chromatography (GC) and Gas 

chromatograph and mass spectrometer (GC-MS) 

analysis of ajwain identified 26 components from 

essential oil and 18 components from acetone extract, 

Thymol (39.1%) was found as a major component in 

both, along with p-cymene, ç-terpinene, â-pinene, 

terpinene-4-ol and carvacrol 0.3% (Singh et al., 2004). 

The essential oil of Ajwain has proved as potential 

nematicide against the pinewood nematode. The 

nematicidal activity of ajwain oil was mainly attributed to 

the activity of thymol, major component of this oil and 

carvacrol (Park et al., 2007; Choi et al., 2007). Fungi 

toxicity of essential oil T. ammi has shown against 

Epidermophyton floccsum, Microsporum canis and 

Trichophyton mentagrophytes at 900 ppm concentration. 

Thymol has been identified as the fungitoxic chemical in 

Ajwain essential oil (Singh et al., 1986). The essential oil 

also extracted antibacterial activity against S. aureus, E. 

coli, Salmonella typhi, Shigella dysenteriae and Vibrio 

cholera (Syed et al., 1986; Anonymous, 1995; Mayaud et 

al., 2008. 

Moreover, seed extract at 1:20 dilution exhibited 

fungicidal action against Rhizoctonia solani (Ansari, 

1995). Different aqueous and organic extracts T. ammi 

extracts prepared exhibited variable activity against E. 

coli, P. aeruginosa, S. typhi and S. aureus justifying its 

use to cure for various gastrointestinal disorders (Kaur 

and Arora, 2009; Ahmad et al., 1998; Patel et al., 2008). 

Methanol extracts of T. ammi significantly in vitro 

inhibited hepatitis C virus (HCV) protease at a 

concentration of 100 µg/ml (Hussein et al., 2000). 

Secondary metabolites mechanisms of antimicrobial 

action are not fully understood but many investigations 

have been conducted. Single compound may not be 

responsible for the bioactivity but rather a combination of 

compounds interacting in an additive or synergistic 

manner. Gram-positive and negative bacteria showed 

differential sensitivity to plant extracts, might be due to 

the morphological differences. An outer phospholipidic 

membrane carrying structural lipopolysaccharide is 

present in Gram-negative bacteria making the cell wall 

impermeable to organic solutes, while porins make a 

barrier to the aqueous solutes. The Gram-positive


bacteria in this regard should be more sensitive since 

they lack this outer layer (Arias et al., 2004). Flavonoids 

action mechanism might be through cytoplasmic 

membrane, deoxyribonucleic acid (DNA) gyrase inhibition 

and β-hydroxyacyl-acyl carrier protein dehydratase 

activities (Cushnie and Lamb, 2005; Zhang et al., 2008). 

The cell morphology can be changed by isoflavone 

genistein through formation of filamentous cells and 

inhibiting the synthesis of DNA and ribonucleic acid 

(RNA) of Vibrio harveyi (Ulanowska et al., 2006). 

Terpenes abundantly present in essential oil promote 

membrane disruption, coumarins cause cell respiration 

reduction and tannins bind to polysaccharides or 

enzymes promoting inactivation and effect microorganism 

membrane (Ya, 1988; Cowan, 1999). Essential oils being 

lipophilic in nature make it permeable to cellular 

membrane (Bakkali et al., 2008). 

Conclusion 

From neutraceutical point of view, selected spices Klonji 

and Ajwain have served as important constituents of 

human diet supplying the body with sufficient amount of 

proteins, carbohydrates and energy. The presence of 

biologically active compounds also contributes to its 

nutritive value and thus proved to be potential sources of 

useful foods. The presence of phytochemicals justifies 

the biological properties of spices. The study supports the 

antibmicrobial potential of kalonji/ajwain especially N. 

sativa methanolic extract and T. ammi organic extracts 

and essential oil as a potent antibmicrobial agent. 

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DOI: 10.5897/JMPR11.1377 



Systematic anatomy and elemental dispersive 

spectrophotometer analysis of genus Pennisetum from 

Pakistan 

Shabnum Shaheen 1 , Mushtaq Ahmad 2,3 , Farah Khan 1 , Rana Abrar Hussain 2 , Zaryab Khalid 1 , 

Amina Younis 1 and Muhammad Zafar 2,3 

1 Department of Botany, Lahore College for Women University, Lahore Pakistan. 

2 Science and Technology University of Education Lahore, Pakistan. 

3 Department of Plant Sciences, Quaid-i-Azam University Islamabad Pakistan. 


Microscopic examination of five species of genus Pennisetum was carried out from Pakistan. The 

investigation of the species was based on the evidence from the epidermal leaf anatomy (LM and 

scanning electron microscopy (SEM) and elemental dispersive spectrophotometer analysis. Leaf 

epidermal anatomy of the selected species showed variation in size and shape of stomatal cells, micro 

and macro hairs, trichomes, silica bodies, hooks, papillae and long cells. Leaf epidermal anatomy 

proved a significant tool for the identification and classification in systematics. Energy dispersive 

spectrometer (EDS) emerged as a new taxonomic tool in the classification of the selected species and 

was done first time for the genus Pennisetum. 

Key words: Systematic anatomy, energy dispersive spectrometer (EDS), Pennisetum. 

INTRODUCTION 

The genus Pennisetum belongs to the grasses, which are 

a large, diverse and successful group of family Poaceae 

of monocotyledonous plants. There are about 10,000 

species that are categorized as grasses by taxonomists 

who traditionally follow embryonic, architectural and 

anatomical characters (Clayton and Renvoize, 2006). 

The Poaceae is generally divided into 5 (or 6) sub 

families: Poaideae, Panicoideae, Bambusoideae, 

Chloridoideae, Arundinoideae and sometimes 

Oryzoideae, a subtype of Bambusoideae. The genus 

Pennisetum belongs to the subfamily Panicoideae. 

Hubbard (1948) and Bor (1960) placed the genus 

Pennisetum in the tribe Paniceae, close to the genus 

Cenchrus. The generic name Pennisetum has been 

derived from two Latin words, Penna and Seta, meaning 

feather and bristles that is, feathery bristles (Brunken, 

2008). The genus includes about 140 species distributed 

in the world (Chaudhary, 1989). Pennisetum is an 

economically important genus as it comprises many grain 

*Corresponding author. E-mail: shabnumflora@hotmail.com. 

crops such as Pennisetum americanum and some 

species are also used as common fodder such as 

Pennisetum orientale. Some species are of medicinal 

imporatance such as Pennisetum divisum (Sujatha et al., 

1989). The Pennistum is also considered as a great 

source of millets. Millets are generally considered as 

minor crops and they are also used as both for forage 

and grains and when used as a grain they are considered 

as a cereal (Brunken, 2008). Despite the economic 

importance and worldwide distribution of the genus 

Pennisetum, the members of this genus have received 

relatively little attention from systematics. 

As the previously published account of this genus 

provides only the traditional scheme of systematics so 

has posed many taxonomic confusions and problems. 

These traditional taxonomic methods are based on gross 

morphology. Also the use of anatomical characters 

through light microscopy in taxonomy became a routine 

procedure. So the purpose of present study was to 

produce a classification system drawing upon evidence 

from not only the morpho-anatomical characters through 

light microscopy but also through scanning electron 

microscopy in conjugation with the elemental dispersive

Figure 1. Prickles and silica bodies of P. americanum. 

spectrophotometer, as the leaf epidermal anatomy is now 

considered as one of the most important tools in 

understanding the identification, classification and 

relationships of various species, genera and families of 

the angiosperms. In the family Poaceae the epidermal 

characters attained a high degree of differentiation and 

specialization. As early Prat in 1932 to 1936 used 

anatomical features in the classification of grasses and 

believed that the anatomical features were reflecting their 

true relationships more exactly than any other characters. 

So the aim of the present study was to explore the 

morpho-anatomical characters of the five species of the 

genus Pennisetum trough LM, SEM and EDS detector. 

EDS technique is used first time in systematics as a new 

tool for the identification and classification of the genus 

Pennisetum. 


The Pennisetum species listed were used in the present 

investigation to gain knowledge about the morpho-anatomical 

characterization. (1) Pennisetum americanum (2) Pennisetum 

divisum (3) Pennisetum lanatum (4) Pennisetum orientale (5) 

Pennisetum flaccidum. The fresh as well as the herbarium 

specimens of all the five species of the genus Pennisetum were 

used for the anatomical study. The peelings were made by scraping 

the epidermis with a sharp blade following the method described by 

the Cotton (1974) and Clark (1960) but with a little modification 

(Shaheen et al., 2010). The preparations of abaxial and adaxial 

epidermis of leaf samples were then observed under light 

microscope. For SEM the abaxial and adaxial epidermis of leaves 

Shaheen et al. 777 

were placed on the stubs and after gold coating put into SEM for 

getting digital images. The method was followed by the Terrel and 

Wergin (1979) and Hilu and Wright (1984) who concluded that SEM 

has proven to be extremely useful for examining the structure of 

leaf epidermis in extent. Also the SEM/EDS analysis of abaxial and 

adaxial epidermis of leaf samples was carried out. The quantitative 

analysis of phytoliths were taken, especially the mass percentage 

of Silicon was calculated in order to make a comparison between 

the different taxa of genus Pennisetum. Then the microphotographs 

of leaf samples through LM, SEM and “mapping” of the composition 

of the elements in the form of graphs were taken by using 

SEM/EDS analysis. 


P. americanum (L.) Leeke 

The distinctive epidermal anatomical features of P. 

americanum are presented in Figure 1 showing dumbbell 

shaped silica bodies and long cells are present 

between the veins with thick sinuous walls and beak 

shaped with sharp pointed end prickles are abundantly 

present between the veins shown in Table 1 whereas 

EDS analysis is presented in Figure 2 and shows that the 

mass percentage of Silicon is 20.63. 

P. divisum (Gmel.) Henr 


divisum are presented in Figure 3 having cross-shaped


Table 1. Distinguishing summary of leaf epidermal anatomy among the species of genus Pennisetum. 

Characteristics P. americanum P. divisum P. flaccidum P. lanatum P. orientale 

Short cells 

Silica bodies 

Macro-hairs 

Micro-hairs 

Prickles 

Hooks 

Stomata 

Long cells 

Counts 

15000 

13500 

12000 

10500 

9000 

7500 

6000 

4500 

3000 

1500 

0 

In rows of 3 to 4 

cells 

In rows of 2 to 3 

cells 

Dumb-bell shaped Cross-shaped and 

nodular shaped 

None seen Between the veins, 

30 to 40 �m 

Between the veins, 

6 to 8 �m 


beak shaped, 6 

to10 �m 

Between the veins, 5 

to 6 �m 

Between the veins 

and also over the 

veins, beak shaped, 

knife shaped, 6 to 10 

�m 

In rows of 2 or 

more cells 

Cross-shaped to 

dumb bell shaped 


more cells 

Cross-shaped to 

dumb bell shaped 


more cells 

Dumb-bell shaped 

None seen None seen None seen 

None seen Between the veins, 

9 �m 



veins, beak 

shaped, knife 

shaped, 4 to 7 �m 



veins, beak 

shaped, knife 



5 to 6 �m 



veins, beak 

shaped, knife 


None seen Present, 2 to 3 �m Present, 3 �m None seen Present, 2 �m 

With 5 to 6 rows, 

low dome-shaped 

subsidiary cells, 5 

to 6 �m 

Thick sinuous walls, 

1 to 30 �m 

pen.ame 

CKa OKa 

NaKa 

SiKa 


almost circular 

shaped subsidiary 

cells, to 6 �m 

Slightly sinuous 

walls, 25 to 30 �m 

ClKa ClKb 

CaKa 

CaKb 




to 8 �m 

Thick sinuous 



almost circular 

shaped subsidiary 

cells, 3 to 4 �m 

Thick sinuous 





to 6 �m 

Thick sinuous 


0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 

Figure 2. EDS analysis of P. americanum. 

AuMz 

AuMa 

AuMr 

keV 

AuLl 

AuLa

Counts 

Figure 3. Long cells, prickles and silica bodies of P. divisum. 

13500 

12000 

10500 

9000 

7500 

6000 

4500 

3000 

1500 

0 

Pen.dib 

CKa OKa 

SiKa 

ClKa ClKb 

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 

Figure 4. EDS analysis of P. divisum. 

and nodular shaped silica bodies. Micro-hairs are rarely 

present between the veins and prickles are abundantly 

present between the veins and also over the veins which 

are beak shaped with much broader base and knife 

shaped (Table 1). EDS analysis is presented in Figure 4 

and it shows that the mass percentage of Silicon is 12.45 

and shows the lowest value in genus Pennisetum. 

AuMz 

AuMa 

AuMr 

keV 

P. flaccidum Griseb 

AuLl 



flaccidum are presented in Figure 5, short-cells are over 

the veins, in a rows of 2 or more cells, each containing a 

closely paired silica body and cross-shaped to dumb-bell 

shaped silica bodies are present. Stomata are present 

AuLa


Figure 5. SEM Silica body of P. flaccidum. 

Counts 

16500 

15000 

13500 

12000 

10500 

9000 

7500 

6000 

4500 

3000 

1500 

0 

pen.flac 

CKa OKa 

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 

Figure 6. EDS Analysis of P. flaccidum. 

NaKa 

AuMz 

SiKa 

AuMa 

AuMr 

ClKa ClKb 

with 2 to 3 rows in intercostal zones, often tending to be 

with low dome-shaped subsidiary cells (Table 1). EDS 

analysis is presented in Figure 6 and it shows that the 

mass percentage of Silicon is 22.86 and shows the 

highest value in genus Pennisetum. 

keV 

P. lanatum Klotzsch 


lnatum are presented in Figure 7. Silica bodies are crossshaped 

to dumb-bell shaped and long cells are present 

AuLl 

AuLa

Counts 

Figure 7. Silica bodies and prickles of P. lanatum. 

13500 

12000 

10500 

9000 

7500 

6000 

4500 

3000 

1500 

0 

pen.lan 

CKa OKa 

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 

Figure 8. EDS Analysis of P. lanatum. 

NaKa 

SiKa 

ClKa ClKb 

between the veins with thick sinuous walls (Table 1) and 

EDS analysis is presented in Figure 8 and it shows the 

elemental composition of phytoliths. The mass 

percentage of Silicon is 16.21 and confirms the siliceous 

nature of phytoliths. 

AuMz 

AuMa 

AuMr 

keV 

P. orientale L. C. Rich 

AuLl 



orientale are presented in Figure 9. Prickles are present 

between the veins and also over the veins, beak shaped 

AuLa


Counts 

Figure 9. Long cells, stomata and prickles of P. orientale. 

16500 

15000 

13500 

12000 

10500 

9000 

7500 

6000 

4500 

3000 

1500 

0 

pen.ori 

CKa OKa 

NaKa 

AuMz 

SiKa 

ILesc AuMa 

ClKa 

ClKb 

ILl 

ILa 

CaKa 

CaKb 

ILb 

ILb2 

ILr ILr2, 

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 

Figure 10. EDS Analysis of P. orientale. 

with much broader base and knife shaped (Table 1). EDS 

analysis is presented in Figure 10 and it shows that the 

mass percentage of Silicon is 15.04, which play a vital 

role in the composition of silica bodies. A lot of variation 

was observed in the morpho-anatomical characters of 

AuMr 

keV 

different species of genus Pennisetum. Leaf epidermal 

anatomy was found taxonomically useful, which helps at 

the species level and showed that in all the five species 

of the genus leaf is bifacial. In P. americanum, dumb-bell 

shaped silica bodies are found (Figure 1) whereas 

AuLl 

AuLa

P. divisum comprises of cross-shaped and nodular 

shaped silica bodies (Figure 3). As indicated in the 

present research, features of the leaf epidermal anatomy 

are useful at specific level. P. americanum and P. 

divisum can be distinguished on the basis of the shape of 

silica bodies and number of rows of short cells. In P. 

americanum, short-cells are arranged predominantly in 

rows of 3 to 4 cells in the costal regions whereas in 

majority of the other taxa, the short cells are arranged in 

rows of two or more cells. Tateoka et al. (2009) said that 

epidermal characters are very useful for the 

differentiation of the different taxa at higher level. 

Macro-hairs are absent in majority of the species 

except P. divisum, which comprises of macro-hairs in the 

intercostal regions, on the adaxial surface only. Microhairs 

are present in majority of the species except P. 

flaccidum. In all the species, micro-hairs are found in the 

intercostal regions. P. divisum and P. orientale comprise 

micro-hairs in both abaxial and adaxial surfaces whereas 

in P. lanatum only abaxial surface and in P. americanum 

only adaxial surface possess micro-hairs (Table 1). Terrel 

and Wergin (1979) and Tateoka (2008) reported that the 

micro-hairs of grasses are characteristic bicellular 

trichomes, commonly found on the leaves, but also 

occurring elsewhere on the plant (for example, lemmas, 

paleas and lodicules). Watson et al. (2005) observed that 

micro-hairs are lacking in the subfamily Pooideae, but 

almost universally present in the other subfamilies. 

Prickles are found in all the species in genus 

Pennisetum. Beak-shaped and knife-shaped prickles are 

present in both the costal and intercostal regions in 

majority of the taxa (Figures 3, 4 and 9) except P. 

americanum, which possesses only beak-shaped prickles 

in the intercostal regions (Table 1). No hook cells are 

found in the P. americanum and P. lanatum whereas the 

other species comprise the hook cells. 

In general, the costal and intercostal zones showed the 

presence of long and short cells. According to Metcalfe 

(1960), the shape and outline of the long cells is variable, 

presenting from shorter elements with non-sinuous walls 

to long elements with sinuous thickened walls. Besides, 

the above-mentioned, author stated that the different 

kinds of long cells have been used in the solution of 

taxonomic problems, although he also pointed out that 

this character should not be used isolatedly, since 

intermediate forms can be used. The long cells in 

majority of the taxa comprise thick sinuous walls (Figure 

1) except P. divisum, which possesses slightly sinuous 

walls (Figure 3). No papillae are seen in all the species of 

genus Pennisetum. Esau (2007) discussed that the 

silicon is found in the epidermis of mature grass leaves in 

the form of discrete particles (silica bodies) produced in 

the specialized silica cells. A great variation is observed 

in the mass percentage of silicon in this genus. P. 

divisum shows the lowest value as 12.45% (Figure 4) 


whereas the P. flaccidum shows the highest value as 

23.58% (Figure 6). Dahlgren and Clifford (2002) 

concluded that the surface sculpture, shape, size and 

distribution pattern of silica bodies on grass epidermis are 

enormously variable from one species to another, and 

this variation has always been considered of great 

taxonomic value. 

REFERENCES 

Bor NL (1960). Grasses of Burma, Ceylon, India and Pakistan, London 

(Permamon Press), P. 767. 

Brunken JN (2008). Morphometric variation and the classification of 

Pennisetum section B revivalvula (Gramineae) in tropical Africa. Bot. 

J. Linnean Soc., 79(1): 51-64. 

Chaudhary SA (1989). Grasses of Saudi Arabia. Saudi national 

herbarium, Ministry of agriculture and water. Kingdom of Saudi 

Arabia, pp. 381-393. 

Clark J (1960). Preparation of leaf epidermis for topographic study. 

Stain. Technol., 35: 35-39. 

Clayton WD, Renvoize SA (2006). Genera Graminum: Grasses of the 

World. Kew Bulletin Additional Series XIII, Royal Botanical Gardens 

Kew, Her Majesty's Stationery Office, London. 

Cotton R (1974). Cytotaxonomy of the genus Vulpia. Ph. D. Thesis, 

Univ. Manchester, USA. 

Dahlgren RMT, Clifford HT (2002). The Monocotyedons: A Comparative 

Study. Academic Press, London, P. 379. 

Esau K (2007). Anatomy of seed plants. Second edition, New York, 

John Wiley and Sons, P. 550. 

Hilu KW, Wright K (1984). Systematics of Graminae: A cluster Analysis 

Study. Taxon, 33(1): 9-36. 

Hubbard CE (1948). In J. Hutchison, British Flowering Plants P.R. 

Gawthorn Ltd. VIII, P. 374. 

Metcalfe CR (1960). Anatomy of monocotykedons 1: Gramineae Jodrell 

L. B. Royal Botanic Gardens Kew. Oxford University Press. 

Prat, H (1932). The epidermis of grasses, el systematic anatomical 

study. Thesis, Paris. 

Prat H (1936). Revued 'agrostology towards a natural classification of 

the grasses. Bull. Soc. Bot. France, 107: 32-79. 

Shaheen S, Khan MA, Ahmad M, Sultana S (2010). A monograph on 

tribe Paniceae from Pakistan. Taxonomic studies. VDM Publishing 

House Ltd. Germany, pp. 28-30. 

Sujatha DM, Manga V, Murty MV (1989). Meiotic studies in some 

species of Pennisetum L. Rich. (Poaceae). Cytologia, pp. 461-652. 

Tateoka T (2008). Chromosome numbers of the genus Calamagrostis in 

Japan. Bot. Mag. Tokyo, 89: 99-114. 

Tateoka T, Inoue S, Kawano S (2009). Notes on some grasses. IX. 

Systematic significance of bicellular microhairs of leaf epidermis. Bot. 

Gazette (Chicago, Ill.), 121: 80-91. 

Terrel EE, Wergin WP (1979). Scanning electron microscopy and 

energy dispersive X ray analysis of leaf epidermis in Zizania 

(Gramineae). Scan. Electr. Microsc., 3: 81-88. 

Watson L, Clifford HT, Dalliwitz MJ (2005). The classification of 

Poaceae: Subfamilies and Subtribes. Aust. J. Bot., 33: 433-484.



DOI: 10.5897/JMPR11.1426 



Nutritional assessment of a traditional local vegetable 

(Brassica oleracea var. acephala) 

Mariga I. K. 1 *, Lutendo Mativha 2 and Maposa D. 3 

1 School of Agricultural and Environmental Sciences, University of Limpopo, P. Bag X1106, Sovenga 0727, South Africa. 

2 Limpopo Agro-Food Technology Station, P. Bag X 1106, Sovenga 0727, South Africa. 

3 School of Mathematical and Computational Sciences, University of Limpopo, P. Bag X1106, Sovenga 0727, 

South Africa. 


The leaf cabbage (Brassica oleracea var. acephala), is a traditional local vegetable widely grown in rural 

and urban areas and consumed mostly by the poor in southern and eastern Africa. It is easy to 

propagate and is highly productive throughout the year. Three lines of leaf cabbage were evaluated for 

their nutritional value against a commonly grown exotic vegetable, Swiss chard (Beta vulgaris var. 

cicla). Swiss chard was superior to the leaf cabbage lines in protein, total ash, vitamin A, sodium and 

iron content. On the other hand, the leaf cabbage lines had significantly higher quantities of fibre, 

carbohydrate, fat, calcium, vitamin C and energy than Swiss chard. The leaf cabbage types tested have 

many good nutritional attributes that justify their genetic improvement through breeding in aspects 

such as protein, vitamin A and iron content. 

Key words: Brassica oleracea, nutritional assessment, Swiss chard, traditional vegetable. 

INTRODUCTION 

Indigenous leafy vegetables play an important role in the 

tradition and food culture of African households and 

some are also used for medicinal purposes (Eifediyi et 

al., 2008). Traditional vegetables offer variety in family 

diets and help to ensure household food security (Luchen 

and Mingochi, 1995). Indigenous leafy vegetables are 

known as sources of many nutrients, vitamins, 

antioxidants, minerals and important proteins (Akula et 

al., 2007). The most commonly consumed exotic leaf 

vegetables in southern Africa include various types of 

kale (for example, Rape and Choumollier) and Swiss 

chard (Beta vulgaris var. cicla), which is often wrongly 

referred to as spinach (Spinacia oleracea), and various 

types of Cabbage (Brassica oleracea var. capitata). 

There is also one traditional vegetable: Brassica 

oleraceae var. acephala (leaf cabbage) that is now widely 

grown on both commercial and household scales in the 

*Corresponding author. E-mail: Irvine.Mariga@ul.ac.za. Tel: 

0027152682203. 

southern African sub-region. This kale has several local 

names, for example, ‘Rugare’(comfort) or Covo in 

Zimbabwe, ‘muRhodesia’ in northern part of South Africa 

and ‘Sukuma wiki’(push out the week) in eastern Africa. 

The Vavhenda farmers of Limpopo province refer to B. 

oleracea var. acephala as ‘muRhodesia’ implying that the 

lines of the vegetable found in their area were introduced 

from Zimbabwe (previously Rhodesia). B. oleracea var. 

acephala is also called ‘walking stick cabbage’ due to the 

tall woody stalk it produces as it grows upwards for many 

months. 

The vegetatively propagated types of leaf cabbage 

commonly grown in Zimbabwe were described by Mvere 

and van der Werff (2004) as Rugare and Viscose. 

Rugare is vegetatively propagated, rarely by seed except 

only at high altitudes, its plants are 2 to 3 m tall, offers 

repeated leaf pickings, has long life, has pale blue-green 

curly leaves but clones with different leaf colour exist. 

Viscose is a selection from Rugare which has improved 

hardiness in the field and segregates into different types, 

has darker green leaves and is more pronouncedly curled 

than Rugare. There are several clones in between

Rugare and Viscose. Rugare leaves are consumed 

mostly as a relish taken in with stiff porridge made from 

maize (Zea mays) flour. It is considered to be delicious. 

Several methods are used to prepare the vegetable in 

southern Africa. The tender fresh leaves of this vegetable 

are sliced and commonly boiled for a short period 

(remaining green), salted and a little cooking oil is added. 

Rugare prepared in this manner can be consumed with 

beef stew, or other types of meat, together with either 

maize stiff porridge or rice. Rugare leaves can also be 

eaten with stiff porridge made from sorghum (Sorghum 

bicolor), pearl millet (Pennisetum glaucum), and cassava 

(Manihot esculenta) or even boiled potatoes (Solanum 

tuberosum). In East Africa, B. oleracea var. acephala can 

also be eaten together with “chapatti”. Where the 

vegetable serves as the sole relish, its taste is often 

enhanced by addition of tomatoes and onions, and 

sometimes pepper. Some people add animal fat chunks 

and boil these with the vegetable to add a meaty taste to 

the vegetable. 

Another common way of preparing this vegetable is to 

add the vegetable to a pot with boiling meat and leave 

the vegetable to get cooked as the cooked meat is left to 

simmer. The sliced fresh leaves are sometimes boiled, 

salted and then a sauce made from smooth peanut butter 

is added. Cut leaves of ‘Rugare’ can also be salted and 

boiled briefly before they are sun-dried for later use in the 

dry season when availability of fresh vegetables becomes 

limited due to lack of irrigation water or when vegetable 

plant growth is adversely affected by high insect pest 

pressure during the summer months. Rugare is 

vegetatively propagated through plant apical cuttings or 

side suckers that develop from the leaf axil buds. These 

root readily as long as the soil is moist and the weather 

conditions are not very hot. The plant grows vigorously 

upwards for many months, and the leaves are plucked as 

it grows. With proper fertilization and watering, a plant 

can produce 3 to 4 harvestable leaves per week. As the 

plant reaches around 1.5 m height, growth vigour 

declines as seen in smaller leaves and longer frequency 

of harvest. At that stage, the plants can be re-established 

by planting apical cuttings and suckers. Most varieties of 

‘Rugare’ continuously develop suckers from axil buds 

even after the leaf has been harvested and also many 

shoots at the base and lower nodes. These suckers need 

to be periodically removed, unless required for 

propagation, so that leaf growth is not slowed down. 

If suckers are not disturbed, stem branches can be 

formed. Some growers leave some of the suckers to 

develop and these tend to keep the branched plants 

shorter with smaller leaves. Most vigorous growth is in 

the dry season as ‘Rugare’ suffers severe insect and 

disease damage in the rainy season. A wide range of 

varieties of ‘Rugare’ are grown. They differ in leaf 

structure and texture, duration to flowering and vigour of 

growth. The leaves differ considerably in taste and 

Mariga et al. 785 

texture but most indigenes prefer ‘Rugare’ to Swiss chard 

or cabbage. Rugare is widely grown in Zimbabwe and in 

eastern Africa, in both rural and urban areas. It is the 

commonest vegetable in small backyard gardens, 

particularly in urban areas. The crop has recently been 

grown on a commercial scale in rural areas of Zimbabwe 

and peri-urban smallholdings in the northern part of 

South Africa. Foeken and Owuor (2008) reported that 

nearly every household of the low-income dwellers of 

Nakuru, Kenya, survived on B. oleracea var. acephala. 

The respondents in their survey commended the 

vegetable for its ease to grow and that it can sustain 

longer than other vegetable crops. Because recognition 

of traditional and indigenous vegetables is recent, very 

little research has been conducted on them. Whilst it is 

generally accepted that B. oleracea var. acephala is 

delicious and nutritious, the nutritional profiles of local 

lines are not known. A number of Rugare lines were 

therefore selected for evaluation of their nutritional status. 


Fully grown fresh leaves of three lines of ‘Rugare’, differentiated on 

the basis of leaf characteristics, were plucked for laboratory 

analysis of nutritional value. For the purpose of this study, the three 

types were named (i) ‘purple petiole’, (ii) ‘viscose’ and (iii) ‘plain’. 

Swiss chard leaves were included as the standard. The leaves of 

viscose are dark green, have a curly surface and are soft; the 

petiole of purple petiole has a light purple tinge on the inside and 

the leaves are light green, smaller than those of Viscose and have 

a tough texture, while the leaves of the Plain type are green and 

soft. Both purple petiole and plain have leaves with smooth 

surfaces. 

Nutritional analyses 

Nutritional analysis of the three Rugare lines and Swiss chard was 

done at the Limpopo Agro-food Technology Station laboratory of 

the University of Limpopo, Mankweng. The fresh leaf samples were 

oven dried at 50°C overnight. They were ground to pass through a 

40-mesh sieve and stored in air-tight containers under refrigerated 

temperature for further use. Moisture, ash, crude protein, fat and 

dietary fibre were analyzed by the methods described in AOAC 

(1990). All the analyses were carried out on dry weight basis and 

expressed per 100 g of edible portion. Moisture content was 

determined using the drying oven method, by drying a 

representative 5 g sample in an oven at 105°C for 2 h. Ash content 

was determined by the incineration of a sample (4 g) in a muffle 

furnace at 600°C for 6 h until the ash turned white. Crude protein 

was estimated by the Kjeldahl method. Total protein was calculated 

by multiplying the evaluated nitrogen percentage by 6.25. Fat was 

determined by petroleum ether extraction in a Soxhlet apparatus. 

Dietary fibre was analyzed by an enzymatic gravimetric method 

using the Tecator Fibertec E System. 

Carbohydrates (g/100 g) were estimated by using a difference 

method described by FAO (1985), by subtracting the sum of the 

percentage of protein, moisture, fat and ash from 100. Mineral 

content (calcium, iron, sodium and potassium) was determined in 

the samples that were digested in a microwave digester. Two


replicates of each sample (approximately 0.5 g) from each of the 

homogenized plant specimens were weighed into Teflon vessels 

after which 3 ml concentrated nitric acid and 1 ml concentrated 

hydrogen peroxide were added. Each vessel was closed with its 

Teflon lid. Vessels were positioned on the rotor and were secured 

by placing a circular safety band around them. The digested 

contents from the vessels were transferred into 50 ml flasks and the 

volume was made up using double deionized water. Concentrations 

were determined with an inductively coupled plasma (ICP) Perkin– 

Elmer spectrometer. Samples of respective mineral solutions were 

quantified against standard solutions of known concentration that 

were analyzed concurrently (Perkin–Elmer, 1996). 

RESULTS 

The nutritional profiles of the four vegetables analyzed 

are given in Tables 1 to 3. Nutritional composition of 

selected exotic leaf vegetables is given in Table 4. 

Comparison of the moisture content means at 95% 

confidence interval indicates that plain had more moisture 

(p

Table 1. Proximate analysis of three varieties of Rugare and Swiss chard on a dry weight basis. 


Vegetable type Moisture content% Protein% CHO% Fat% Fibre% Ash% 

Swiss chard 8.21 a (0.02) 32.25 b (1.36) 33.32 a (2.19) 2.41 a (0.37) 6.16 a (0.12) 17.62 b (0.31) 

Plain 8.67 b (0.03) 20.71 a (0.10) 35.86 a (0.05) 3.71 ab (0.40) 11.78 d (0.06) 19.24 b (0.36) 

Viscose 8.37 ab (0.10) 21.76 a (0.26) 45.35 b (1.28) 3.89 ab (0.52) 7.18 b (0.23) 13.44 a (0.62) 

Purple petiole 8.47 ab (0.09) 21.25 a (0.47) 45.08 b (1.42) 4.29 b (0.39) 8.24 c (0.32) 12.65 a (0.78) 

P value 0.016* 0.000*** 0.002** 0.043* 0.000*** 0.001** 

LSD(0.05) 0.32 3.31 6.28 1.84 0.91 2.39 

CV% 0.89 3.21 3.66 11.92 2.53 3.53 

*, ** and *** = significantly different at P< 0.05, 0.01 and 0.001, respectively. CHO = carbohydrate; CV = coefficient of variation; Means are followed by 

standard deviation in brackets; Means in the same column, with same letters are not significantly different at 5% level of significance. 

Table 2. Mineral nutrient and vitamin content in leaves of three lines of ’Rugare’ and Swiss chard on a dry weight basis. 

Vegetable type Potassium % 

Calcium 

% 

Mineral nutrient Vitamins 

Sodium 

mg/kg 

Iron 

mg/kg 

Vit. A 

mg/100 g 

Vit. C 

mg/100 g 

Swiss chard 2.96 a 0.09) 0.78 a (0.01) 42977.5 c (3.53) 288.4 c (3.61) 2220 c (5.65) 57.9 a (3.11) 

Plain 2.55 a (0.21) 5.91 b (0.03) 1313.5 b (16.26) 180.9 b (4.04) 251.0 b (2.82) 218.2 b (5.80) 

Viscose 3.83 a (0.05) 3.62 b (0.21) 1327.5 b (7.43) 178.5 b (0.89) - - 

Purple petiole 3.03 a (0.68) 5.76 b (0.09) 1154.5 b (7.77) 161.6 a (1.14) 161.5 a (12.02) 234.2 b (7.92) 

P value 0.095 ns 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 

LSD(0.05) 1.56 0.49 43.03 12.04 33.74 18.16 

CV% 11.75 2.82 0.86 1.38 0.89 2.48 

*, ** and *** = significantly different at P


Table 4. Nutritional composition of exotic leaf vegetables per 100 g fresh weight. 

Vegetable Energy (kcal) Moisture (g) Protein (g) Fat (g) Fibre (g) 

Spinach 26 91 3.2 0.3 4.3 

Cabbage 24 92 1.3 0.2 5.4 

Lettuce 18 94 1.3 0.3 3.5 

Swiss chard 25 91 2.4 0.3 4.6 

Cauliflower 27 97 2.7 0.2 5.2 

Kale 53 83 6.0 0.8 9.0 

Source: Akula et al. (2007). 

reduction of risk of chronic diseases such as heart 

disease, certain forms of cancer and cataracts, and is 

essential to the activity of many enzymes (Insel et al., 

2004). Vitamin C also helps to protect the body against 

cancer and other degenerative diseases like arthritis and 

Type 1 diabetes mellitus (Eifediyi et al., 2008). All the 

three Rugare lines were superior to Swiss chard 

regarding calcium content. This is important because 

some of the poor people may not readily access other 

foods providing calcium such as milk, yoghurt and 

cheese. Hence adequate consumption of these 

vegetables may meaningfully contribute to their body 

requirements for calcium. Mineral calcium is required for 

strong bones, teeth, hair, nails and plays an important 

role in muscle contractions and relaxation, blood clotting, 

synoptic transmission and many cellular functions, such 

as production of energy and the maintenance of the 

immune function (Clicks, 2007). Swiss chard had 

comparable levels of potassium to that of the three 

Rugare lines. Potassium and magnesium are responsible 

for reducing blood pressure (Eifediyi et al., 2008). 

Swiss chard was superior to the Rugare lines in terms 

of iron content. This perhaps is another characteristic that 

needs improvement in the Rugare lines as iron is 

important in the body. Iron is needed for haemoglobin 

formation. Vegetables with significant amounts of iron are 

recommended for patients suffering from anaemic 

convalescence. Iron in leaf cabbage is available in an 

easily digestible form (Mvere and van der Werff, 2004). 

Swiss chard was much more superior to Rugare lines in 

terms of sodium content but this is not critical since most 

diets provide adequate salt intake and sodium in the salt 

form is associated with high blood pressure if taken in 

large amounts. Only plain had higher ash content than 

Swiss chard while viscose and purple petiole had 

significantly lower ash content than Swiss chard. This 

implies that the latter two have considerably less 

inorganic minerals than Swiss chard, which is an aspect 

that could be improved through breeding. The nutritional 

value of Brassica vegetables has medicinal implications. 

Leaf cabbage contains high levels of glucosinolates, 

which form compounds with antioxidant and anticancer 

activities during preparation (Mvere and van der Werff, 

2004). Mvere and van der Werff (2004) provided United 

States Department of Agriculture (USDA) data on 

nutritional composition of leaf cabbage per 100 g of raw 

leaf as: water 84.5 g, energy 209 kJ (50 kcal), protein 3.3 

g, fat 0.7 g, carbohydrate 10.0 g, total dietary fibre 2.0 g, 

Ca 135 mg, P 56 mg, Fe 1.7 mg, Mg 34 mg, Zn 0.44 mg, 

vitamin A 15,376 IU, thiamin 0.11 mg, riboflavin 0.13 mg, 

niacin 1.0 mg, folate 29 ug and ascorbic acid 120 mg. 

This nutritional profile suggests need for a more 

complete analysis of these Rugare lines for effective 

selection on nutritional attributes. Brassica vegetables 

are highly regarded for their nutritional value as they 

provide high amounts of vitamin C and soluble fibre and 

also contain multiple nutrients with potent anti-cancer 

properties. It has been recently discovered that 3, 3’- 

Diindolylmethane in Brassica vegetables is a potent 

modulator of the innate immune response system with 

potent anti-viral, anti-bacterial and anti-cancer activity 

(Wikipedia, 2009). The findings from this study generally 

show that Rugare, a traditional vegetable, has 

comparable nutritional value to commonly used exotic 

vegetables such as Swiss chard, spinach, lettuce and 

cabbage. Afoloyan and Jimoh (2008) and Kala et al. 

(2008) reported a similar situation for indigenous leafy 

vegetables. 

The nutritional (and medicinal) attributes of Rugare and 

its wide adoption in Eastern and Southern Africa warrants 

that researchers, particularly plant breeders, should pay 

more attention to this vegetable. They could help develop 

or identify a few cultivars of known nutritional profiles, 

having improved some of the existing lines on identified 

nutritional deficiencies. The need for more breeding and 

research work on cultural practices was also stressed by 

Mvere and van der Werff (2004). Foeken and Awuor 

(2008) cite many reports that indicate the importance of 

urban agriculture to the poor as it contributes to 

household food and nutritional security. Cultivation of 

Rugare does not require large pieces of land as it is 

highly productive. It is thus well suited to urban areas 

where residents in high density surbubs only have small 

pieces of their yards for gardening. Improvement in the 

nutritional value of lines of B. oleracea var. acephala can 

go a long way in improving the nutrition and health of the

poor in southern and eastern Africa. 

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29-31 August 1995, ICRAF-HQ, Nairobi, Kenya. 

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DOI: 10.5897/JMPR11.1459 



Effect of salicylic acid application on biochemical 

changes in ginger (Zingiber officinale Roscoe) 

Ali Ghasemzadeh 1 * and Hawa Z. E. Jaafar 2 

1 Department of Agronomy, Science and Research Branch, Islamic Azad University, Tehran, Iran. 

2 Department of Crop Science, Faculty of Agriculture, University Putra Malaysia, 43400 University Putra Malaysia (UPM) 

Serdang, Selangor, Malaysia. 


Salicylic acid (SA) belonging to plant phenolics group is found in some plant species and is capable of 

enhancing plant growth and yield. Effects of SA application (10 −3 and 10 −5 M) on synthesis of total 

soluble carbohydrate (TSC), total flavonoids (TF) and total phenolics (TP) were studied out in two 

ginger varieties (Halia Bentong and Halia Bara) under greenhouse conditions. In treated plants as the 

level of SA increased (from 10 −5 M to 10 −3 M) the production of TF increased while synthesis of TP 

decreased. SA induced production of TSC content in both varieties. Halia Bara exhibited a higher 

content of TSC (7.98 mg/g dry weight) compared to Halia Bentong (7.59 mg/g dry weight) when sprayed 

with low concentration (10 −5 M) of SA. The result of high performance liquid chromatography (HPLC) 

analysis showed that concentration of the some majority flavonoids (quercetin, catechin and 

kaempferol) decreased significantly in plants when treated with different concentration of SA. 

Accordingly, high concentrations of these flavonoids were found in control plants. Furthermore, SA 

application stimulated synthesis of phenolic acids (cinnamic acid, vanillic acid, ferulic acid and gallic 

acid) in both varieties. These increases might be due to an increase in TSC content. The results implied 

that SA could be used for improving biochemical synthesis in young ginger. 

Key words: Salicylic acid, ginger, total soluble carbohydrate, total phenolics, total flavonoids. 

INTRODUCTION 

Salicylic acid (SA) has been identified as one of the 

important phenolic compound in plants and also reported 

as allelopathic chemical (Chandra et al., 2007; Einhelling, 

1986). The results of previous studies showed that 

production of soluble carbohydrates, sugars and 

secondary metabolites enhanced in plants exposed to 

SA. Shraiy and Hegazi (2005) reported positive effects of 

SA application were correlated with significant increase in 

total soluble proteins, phenol, total soluble carbohydrates 

and sugars in pea (Pisum Sativum L.) seeds. Flavonoids 

belong to a large family of polyphenolic components 

synthesized by plants (Ghasemzadeh et al., 2010). High 

contents of natural phenolic acids and flavonoids are found 

in green tea, fruits and vegetables, while some amounts of 

phenolics exist in red wine and coffee (Yao, 2004). Free 

radicals and single oxygen are recognized as major factors 

causing various chronic diseases such as cancer, 

*Corresponding author. E-mail: upmali@yahoo.com. 

diabetes, etc. The uptake of high levels of antioxidant 

supplements can reduce the risk of these diseases. 

Phenolic compounds have been implicated as natural 

antioxidants that may reduce oxidative damage to the 

human body. In addition phenolic acids and flavonoids are 

antioxidants with health benefits such as anti-inflammatory 

and antitumor effect (Ghasemzadeh and Jaafar, 2011; 

Chun et al., 2003). Recent work by Sung-jin et al. (2008) 

has shown that some flavonoid components in green tea 

are effective in inhibiting cancer or induce mechanisms 

that may kill cancer cells and inhibit tumor invasion. The 

shikimic acid pathway participates in the biosynthesis of 

most plant phenolics (Conn, 1986). In addition, Soluble 

carbohydrates are basic compounds required to produce 

phenolic component in the shikimic acid pathway. The 

shikimic acid pathway is able to convert simple 

carbohydrate precursors derived from glycolysis and the 

pentose phosphate pathway to the aromatic amino acids 

(Bryant et al., 1983). It was also noted that increase in 

phenolic concentration relates to the balance between 

carbohydrate source-sink, such that the greater the

source:sink ratio, the greater the concentration of 

phenolic compounds (Shui-Yuan et al., 2009). Amin et al. 

(2007) found that salicylic acid regulates sugar contents 

(translocation from source to sink) and causes a 

significant increase in total soluble sugars. The plant 

hormones, salicylic acid (SA), ethylene and jasmonic acid 

(JA) have essential roles in mediating pathogen 

responses in plants (Navarro et al., 2008). Alonso- 

Ramirez et al. (2009) reported that giberellic acid 

stimulated in Arabidopsis may have an important role in 

SA biosynthesis and action, and that some of the 

physiological effects of this hormone may be mediated by 

SA. For instance, it has been proven that SA has an 

important role in heat and water stress responses (Singh 

and Usha, 2003; Clarke et al., 2004) or in the improved 

germination of Arabidopsis thaliana seeds under salt 

stress conditions (Rajjou et al., 2006). 

The objective of this study was to examine the effect of 

foliar application of salicylic acid on synthesis of primary 

(total soluble carbohydrate) and secondary (flavonoids 

and phenolic acids) metabolites in two varieties of 

Malaysian ginger (Zingiber officinale) namely Halia 

Bentong and Halia Bara. 

EXPERIMENTAL 

Plant material and maintenance 

Rhizomes of ginger varieties, Halia Bentong and Halia Bara (Z. 

officinale), were sprouted for two weeks in 10 cm diameter pots 

which were filled with peat. They were then transferred to 

polyethylene bags, which were filled with soilless mixture media 

including burnt rice husk and coco peat (ratio 1:1). The plants were 

grown in glasshouse at the glasshouse complex of University Putra 

Malaysia (UPM). The seedlings were raised in specially constructed 

growth houses receiving 12 h photoperiod and average 

photosynthetic photon flux density of 310 μmol/m 2 /s. Day and night 

temperatures were recorded at 30 ± 1.0 and 20 ± 1.5°C, respectively, 

and relative humidity at about 70 to 80%. When the ginger 

seedlings were at the second leaf stage, they were sprayed with 

two concentrations (10 –3 and 10 –5 M) of salicylic acid solution (SA; 

2-hydroxybenzoic acid + 100 μl dimethyl sulfoxide + 0.02% 

Polyoxyethylenesorbitan monolaurate, Tween 20, Sigma Chemicals; 

pH 6.5). The control plants were sprayed with same solution but 

without SA. Plants were sprayed once on the leaves early in the 

morning and every week until one month. 

Extract preparation for total flavonoids and total phenolics 

Leaves were dried (Freeze dry) to constant weights and powdered 

using mortar. Powdered sample (1 g) extracted using methanol (50 

ml). The solutions were shaken for 1 h at room temperature using 

an orbital shaker. Extracts were filtered under suction and stored at 

−20°C for further use. 

Determination of total soluble carbohydrate 

A few drops of ethanol (80%) were added onto 0.1 g of freeze 

Ghasemzadeh and Jaafar 791 

dried samples. Then 25 ml aqueous ethanol was added and mixed 

with shaking. Solutions were centrifuged at 5000 rpm. About 1 ml of 

supernatant was placed into test tubes and 10 ml of anthrone 

solution (0.15%) were added and finally the samples were heated. 

Tubes cooled down to room temperature. Absorption of the 

samples was recorded at 625 nm using a spectrophotometer (U- 

2001, Hitachi Instruments Inc., Tokyo, Japan) (Sivaci, 2006). Total 

soluble carbohydrate concentrations of the samples were 

calculated using the calibration curve drawn for glucose standard 

solutions. The soluble carbohydrate in the sample was expressed 

as mg glucose/g dry sample. 

Determination of total phenolic content 

The total phenolic content was determined using the method of Kim 

et al. (2003). Briefly, 1 ml of extract was added to deionized water 

(10 ml) and Folin–Ciocalteu phenol reagents 10% (1.0 ml). After 5 

min, 20% sodium carbonate solution (2.0 ml) was added to the 

mixture. Solution was kept in total darkness and after that the 

absorbance was measured at 750 nm using a spectrophotometer 

(U-2001, Hitachi Instruments Inc., Tokyo, Japan). 

Determination of total flavonoid Contrnt 

The TF were measured following a previously reported 

spectrophotometric method (Bushra et al. 2009). Briefly, extracts of 

each plant material (1 ml) were diluted with 4 ml water in a 10 ml 

volumetric flask. Initially, 5% NaNO2 solution (0.3 ml) was added to 

each volumetric flask; at 5 min, AlCl3 added total content of flask’s 

10%; and at 6 min, 1.0 M NaOH (2 ml) was added. Absorbance of 

the reaction mixture was read at 430 nm. 

High performance liquid chromatography (HPLC) 

Analysis of flavonoids composition by HPLC 

Reversed-phase HPLC was used to assay flavonoid compositions. 

The Agilent HPLC system used consist of a Model 1100 pump 

equipped with a multi-solvent delivery system and an L-7400 

ultraviolet (UV) detector. The column was an Agilent C18 (5 µm, 4.6 

× 250 mm). The mobile phase composed of: (A) 2% acetic acid 

(CH3COOH) and (B) 0.5% acetic acid-100% acetonitrile (CH3CN), 

(50:50 v/v), and gradient elution was performed as follows: 0 min, 

95:5; 10 min, 90:10; 40 min, 60:40, 55 min, 45:55; 60 min, 20:80; 

and 65 min, 0:100. The mobile phase was filtered under vacuum 

through a 0.45 µm membrane filter before use. The flow rate was 1 

ml/min and UV absorbance was measured at 280 to 365 nm. The 

operating temperature was maintained at room temperature. 

Identification of the flavonoids was achieved by comparison with 

retention times of standards, UV spectra and calculation of UV 

absorbance ratios after co-injection of samples and standards 

(Wang et al., 2007). Commercial standards were purchased from 

Sigma–Aldrich (St Louis, MO, USA). 

Analysis of phenolics acids composition by HPLC 

An Agilent HPLC system consisting of a Model 1100 pump 

equipped with a multi-solvent delivery system and a L-7400 

ultraviolet (UV) detector was used. The column was an Agilent C18 

(5 µm, 4.6 × 250 mm). The mobile phase was composed of (A) 

phosphoric acid (aqueous) and (B) acetonitrile and gradient elution,


Table 1. Effect of SA application (10 −3 and 10 −5 M) on total soluble carbohydrate (TSC), total phenolic (TP) and total flavonoids (TF) 

content in ginger (Zingiber officinale) varieties. 

Characteristics 

Halia Bentong Halia Bara 

Control SA 10 −5 SA 10 −3 Control SA 10 −5 SA 10 −3 

TSC 5.95 ± 0.46 b 7.59 ± 0.69 ab 7.41 ± 0.69 ab 6.3 ± 0.97 ab 7.98 ± 0.97 a 7.72 ± 1.32 a 

TF 9.3 ± 0.88 ab 7.98 ± 0.76 b 8.21 ± 0.92 b 10.87 ± 1.04 a 8.97 ± 0.78 b 9.35 ± 0.28 ab 

TP 39.6 ± 2.91 c 49.5 ± 0.72 ab 46.9 ± 3.01 ab 44.06 ± 3.85 bc 53.23 ± 5.4 a 50.1 ± 2.78 ab 

All analyses are the mean of triplicate measurements ± standard deviation; All of results expressed in mg/g dry weight; Means not sharing a 

common single letter were significantly different at P ≤ 0.05. 

was performed as follows: 0 min, 85:15; 12 min, 75:25; 20 min 

75:25; 22 min, 85:15 and 30 min, 85:15. The mobile phase was 

filtered under vacuum through a 0.45 µm membrane filter before 

use. The flow rate and injection volume were 1 ml/min and 20 μl. 

UV absorbance was measured at 220 to 365 nm. The operating 

temperature was maintained at room temperature. Identification of 

the phenolic acids was achieved by comparison with retention times 

of standards, UV spectra and calculation of UV absorbance ratios 

after co-injection of samples and standards (Standard Operating 

Protocol, 2001). Commercial standards were purchased from 

Sigma-Aldrich. 


The experimental results were expressed as mean ± standard 

deviation of three replicates. Data were analyzed using analysis of 

variance by Statistical Analysis System (SAS, system 9.0, 2002). 

Mean separation test between treatments was performed using 

Duncan multiple range test and P-value of < 0.05 was regarded as 

significant. 


Effect of foliar application of salicylic acid on total 

soluble carbohydrate (TSC), total flavonoids (TF) and 

total phenolics (TP) 

SA treatments significantly (P ≤ 0.05) affected TSC 

content (Table 1). Treated varieties by SA showed higher 

TSC content in comparison to control plants. Halia Bara 

had a higher content of TSC (7.98 mg/g dry weight) 

compared to Halia Bentong (7.59 mg/g dry weight) when 

the varieties were sprayed with 10 −5 M SA. This 

concentration of SA enhanced production of TSC at 

about 27.5% in Halia Bentong and 26.6% in Halia Bara. 

SA treatment might also be assumed to inhibit 

polysaccharide-hydrolyzing enzyme system on one hand 

and accelerate the incorporation of soluble sugars into 

polysaccharides. In this regard, soluble sugar content 

was also increased in tomato plants in relation to salt 

stress (Maria et al., 2000). Our assumption could be 

supported by the result that SA increased polysaccharide 

level that is related to soluble sugars (Jeyakumar et al., 

2008). In this context, Sharma and Lakhvir (1998) 

postulated that foliar spray of SA to ray plants resulted in 

decreasing their soluble sugar level. Khodary (2004) 

pointed out that SA application increased TSC content in 

maize. These results are in agreement with Amin et al. 

(2007) who reported that salicylic acid regulates sugar 

contents (translocation from source to sink) and causes a 

significant increase in total soluble sugars. 

From Table 1, it is apparent that TF and TP 

accumulation and partitioning in the plant were 

significantly affected by the SA application (P ≤ 0.05). 

High content of TF was observed in control plants (Halia 

Bentong: 9.3 mg/g dry weight; Halia Bara 10.87 mg/g dry 

weight). In treated plants, TF content increased from 7.98 

to 8.21 mg/g dry weight in Halia Bentong and from 8.97 

to 9.35 mg/g dry weight in Halia Bara with increasing in 

SA concentration from 10-5 to 10-3M. Conversely to TF, 

high content of TP was observed in treated plants (Halia 

Bentong: 49.5 mg/g dry weight; Halia Bara 53.23 mg/g 

dry weight) compared to control plants. However, with 

decreased SA concentration from 10-3 to10-5M, this 

resulted in the highest values in TP and lowest value in 

TF. In addition, TP content decreased from 49.5 to 46.9 

mg/g dry weight in Halia Bentong and from 53.23 to 50.1 

mg/g dry weight in Halia Bara when treated with higher 

concentration of SA (10-3M). Our results suggest the 

ability of SA application to alter or modify both the 

concentration and profiling of flavonoids and phenolic 

acids in ginger. According to our result, the increase in 

TP content might be due to increase in TSC production in 

the leaves. A positive and significant correlation between 

soluble carbohydrate and total phenolics was observed in 

previous studies (Ibrahim and Jaafar, 2011; 

Ghasemzadeh et al., 2010). 

Isolation of flavonoid and phenolic compounds using 

HPLC 

High performance liquid chromatography (HPLC) 

analysis of some flavonoids and phenolic acids are 

present in Table 2. Malaysian ginger especially Halia 

Bara contained considerably (P ≤ 0.05) high amount of 

quercetin (1.13 mg/g dry weight), catechin (0.553 mg/g 

dry weight) and kaempferol (0.06 mg/g dry weight) in 

control plants. According to the data obtained from Table 

2, the concentration of the majority flavonoids(quercetin, 

catechin and kaempferol) decreased significantly


Table 2. High performance liquid chromatography analysis of some flavonoid and phenolic constituents of SA-treated ginger (Zingiber 

officinale) varieties. 

Compounds 

Halia Bentong Halia Bara 

Control SA 10 −5 SA 10 −3 Control SA 10 −5 SA 10 −3 

Quercetin 0.893 ± 0.03 bc 0.736 ± 0.09 c 0.79 ± 0.06 c 1.13 ± 0.12 a 0.883 ± 0.07 bc 0.993 ± 0.07 ab 

Catechin 0.384 ± 0.049 c 0.276 ± 0.08 d 0.305 ± 0.09 d 0.553 ± 0.06 a 0.45 ± 0.06 b 0.55 ± 0.04 a 

Kaempferol 0.04 ± 0.009 b 0.038 ± 0.009 b 0.029 ± 0.018 b 0.06 ± 0.001 a 0.048 ± 0.004 ab 0.044 ± 0.006 ab 

Gallic acid 0.227 ± 0.049 a 0.29 ± 0.1 a 0.259 ± 0.045 a 0.259 ± 0.033 a 0.303 ± 0.097 a 0.304 ± 0.02 a 

Vanillic acid nd 0.037 ± 0.017 bc 0.028 ± 0.03 bc 0.077 ± 0.011 b 0.139 ± 0.046 a 0.134 ± 0.01 a 

Ferulic acid 0.117 ± 0.02 ab 0.173 ± 0.055 ab 0.158 ± 0.042 ab 0.102 ± 0.042 b 0.193 ± 0.03 a 0.182 ± 0.017 a 

Tannic acid 0.429 ± 0.049 a 0.332 ± 0.041 a 0.376 ± 0.122 a 0.354 ± 0.141 a 0.281 ± 0.05 a 0.269 ± 0.053 a 

Cinnamic acid nd 0.193 ± 0.045 b 0.184 ± 0.08 b nd 0.242 ± 0.046 a 0.231 ± 0.014 a 

All analyses are the mean of triplicate measurements ± standard deviation; All of results expressed in mg/g dry weight; Means not sharing a common 

single letter were significantly different at P ≤ 0.05.; n.d: not detected. 

in plants when treated with different concentration of SA. 

Conversely, amount of phenolic acids (gallic acid, 

cinnamic acid, ferulic acid and vanillic acid) increased 

significantly in both varieties when treated with different 

concentration of SA and only tannic acid did not show the 

highest content in this treatment. Furthermore, in both 

varieties of ginger, amount of vanillic acid, ferulic acid 

and cinnamic acid were observed to be higher at low 

concentration (10-5 M) of SA. 

Among phenolics acids, tannic acid and gallic acid had 

more content in both varieties followed by ferulic and 

vanillic acids. The interesting finding was that the 

application of SA in both varieties induced synthesis of 

vanillic acid and cinnamic acid. These compounds were 

not detected from the control plants. According to HPLC 

analysis results, it could be concluded that application of 

SA induce synthesis of phenolic acids and conversely 

inhibit flavonoids synthesis in ginger. 

The production of phenolic compounds is catalyzed by 

phenylalanine ammonia-lyase (PAL), (Figure 1). The 

results of our study are consistent with other studies and 

suggest that high content of some phenolic components 

such as cinamic acid can inhibit flavonoid biosynthesis 

with inhibition of PAL enzyme activity (Shui-Yuan et al., 

2009). In this study, cinnamic acid was not detected in 

the control plants where instead high content of 

flavonoids was registered; but foliar application of SA 

induced synthesis of cinnamic acid in both varieties and 

following that, amount of flavonoids decreased in these 

plants. At higher SA concentration (10 −3 M), the level of 

soluble phenolics in ginger leaves decreased and this 

reduction was already reported by Dixon and Paiva 

(1995). PAL is a key gateway enzyme in the secondary 

metabolic pathway leading to the synthesis of phenolic 

compounds (Keski-Saari, 2005). 

SA inhibits the activity of PAL, a key enzyme in the 

synthesis of phenolic compounds (Nicholson and 

Hammerschmidt, 1992) and stimulates activity of 

chalcone synthase (CS) a key enzyme in the synthesis 

of flavonoids. In this study at low concentrations (10 −5 M), 

SA stimulated the accumulation of soluble phenolic 

compounds in ginger leaves. It has been suggested that 

SA inhibits catalase activity leading to increased levels of 

H2O2 (Chen et al., 1993), which in turn induces PAL gene 

expression (Desikan et al., 1998) and synthesis of 

phenolic compounds (Dorey et al., 1997; Dihazi et al., 

2003). In addition, at low concentration of SA, PAL 

catalyses the deamination of L-phenylalanine, and the 

product, trans-cinnamate, is converted in plants to 

various phenylpropanoid compounds such as chlorogenic 

acid, lignin monomers and flavonoids (Dihazi et al., 

2003). This improvement in plant secondary metabolites 

might be due to increased total soluble content 

Conclusions 

Our assumption could be supported by the results that 

SA increased polysaccharide level related to soluble 

sugars. Furthermore, treatment with SA greatly increased 

synthesis of phenolic acids in ginger leaves whereas 

flavonoids synthesis decreased in both varieties. The 

results indicate that increasing in TP might be contributed 

by the increase in TSC content. Accordingly, synthesis of 

some phenolic acids like as vanillic acid and cinnamic 

acid induced by SA application whereas these 

compounds were not detected from control plants. 

The results of our study are consistent with other 

studies and suggest that high content of some phenolic 

components such as cinamic acid can inhibit flavonoid 

biosynthesis with inhibition of phenylalanine ammonia 

lyase (PAL) enzyme activity. 


The authors are grateful to the University Putra Malaysia 

(UPM) for supporting this work.


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DOI: 10.5897/JMPR11.1466 



Antidiabetic effect of Gynura procumbens leaves 

extracts involve modulation of hepatic carbohydrate 

metabolism in streptozotocin-induced diabetic rats 

Hui-Wen Lee*, Pusparanee Hakim, Amir Rabu and Halimah Abdullah Sani 

School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 

Bangi, Selangor, Malaysia. 


Gynura procumbens is an annual evergreen shrub found in Southeast Asia, especially Malaysia, 

Thailand and Indonesia, which is of considerable medicinal value. The present study aims to investigate 

the antidiabetic properties of G. procumbens leaves aqueous and ethanolic extracts and its possible 

underlying antihyperglycemic mechanisms of action involving liver carbohydrate metabolism in 

streptozotocin-induced diabetic rats. Experimental diabetes was induced via single intravenous 

injection of streptozotocin (55 mg/kg of body weight). G. procumbens leaves aqueous and ethanolic 

extracts were administered orally at 3 different doses (50, 100 and 150 mg/kg of body weight) for 42 

consecutive days. Administration of G. procumbens leaves aqueous and ethanolic extracts significantly 

(P

in mortality rate around the world (Altan, 2003; The 

Expert Committee on the Diagnosis and Classification of 

Diabetes Mellitus, 2003). 

Many diabetic treatments have been developed in the 

past decades with the same underlying goal to maintain 

adequate blood glucose concentration. To date, there are 

six classes of oral antidiabetic agents which have been 

used in the treatment of diabetes mellitus, such as 

sulfonylureas and meglitinides, biguanides, 

thiazolidinediones, α-glucosidase inhibitors and dipeptidyl 

peptidase-4 inhibitors. Each of these drug classes works 

on different mechanism of action, including stimulation of 

insulin secretion, reduction of hepatic gluconeogenesis, 

increasing peripheral insulin sensitivity, delaying intestinal 

digestion and absorption of carbohydrate, and increasing 

endogenous levels of incretin hormones that act in the 

presence of glucose to stimulate insulin secretion, 

respectively (Donner, 2006; Jain and Saraf, 2010; 

Klonoff, 2010). Unfortunately, these agents could 

produce various side effects, such as hypoglycemia, 

weight gain, lactic acidosis, vitamin B12 deficiency, 

edema, bone fractures, gastrointestinal problems and 

pancreatitis (Chiasson et al., 2002; Kar and Holt, 2008; 

Kirpichnikov et al., 2002; Klonoff, 2010; Loke et al., 2009; 

Nesto et al., 2003; Wulffele et al., 2003). 

Gynura procumbens (Lour.) Merr (family Compositae), 

also known locally as “Sambung Nyawa”, is found easily 

in Southeast Asia, especially Malaysia, Thailand and 

Indonesia. Traditionally, G. procumbens leaves have 

been known to possess high medicinal values and was 

used to treat illnesses such as eruptive fevers, rash, 

kidney diseases, migraines, constipation, hypertension, 

cancer and diabetes mellitus (Perry, 1980). Recently, 

pharmacological studies have indicated that G. 

procumbens has anti-inflammatory (Iskander et al., 

2002), anti-herpes simplex virus (Jiratchariyakul et al., 

2000; Nawawi et al., 1999), anti-hypertensive (Hoe et al., 

2007; Kim et al., 2006), anti-hyperlipidemic (Zhang and 

Tan, 2000), anti-sterility (Halimah et al., 2008; 

Pusparanee et al., 2008), and anti-oxidative capabilities 

(Akowuah et al., 2009; Puangpronpitag et al., 2010). 

Studies have also shown that n-butanol fraction of G. 

procumbens leaves methanolic extract possessed blood 

glucose lowering activity and the possible active chemical 

constituents from this plant had been isolated and 

identified to be flavonoids, saponins, tannins and 

terpenoids (Akowuah et al., 2001, 2002). Although it is 

known that G. procumbens contains anti-diabetic 

properties, however little information on the mechanism 

of action involved in the antidiabetic activity of G. 

procumbens is available. Moreover, most of the 

antidiabetic studies on G. procumbens were conducted 

on methanolic and aqueous extracts, but little is known 

on the antidiabetic effect of ethanolic extract of G. 

procumbens leaves. Therefore, the aim of the present 

study is to evaluate the antihyperglycemic properties of 

Lee et al. 797 

aqueous and ethanolic extracts of G. procumbens leaves 

in vivo and to investigate its possible underlying 

antidiabetic mechanism involving hepatic carbohydrate 

metabolism. 



Leaves of Gynura procumbens (Lour.) Merr. (Compositae) were 

collected and identified from the Green House Facility of Faculty of 

Science and Technology, Universiti Kebangsaan Malaysia, Bangi, 

Selangor, Malaysia. 

Preparation of Gynura procumbens extracts 

The leaves of G. procumbens were extracted in two different 

methods to produce crude aqueous and ethanolic extracts. 

Aqueous extraction was performed according to the method of 

Peungvicha et al. (1998). Fresh leaves of G. procumbens were 

washed, weighed and dried in oven (Sheldon Manufacturing, Inc., 

USA) at 45°C for 3 days. Upon drying, dried leaves were blended 

into powder form, then 20 g of dried leaves powder was mixed with 

400 ml of distilled water (ratio of 1:20) and heated in water bath 

(50°C) for 3 h with stirring of the extract at every 20 min interval. 

The extract was then cooled and centrifuged at 3000 rpm for 10 min 

(Kubota Corp., Japan). Supernatant obtained was separated and 

the pellet was subjected to another round of centrifugation at 3000 

rpm for 10 min. Both of the supernatants were combined and 

freeze-dried (LabConco Corporation, USA) to yield brown powder 

of G. procumbens crude aqueous extract. 

Ethanolic extract of G. procumbens leaves was prepared 

according to the method of Zhang and Tan (2000). Fresh leaves of 

G. procumbens (1 kg) were washed, blended and mixed with 95% 

ethanol (1.5 L) for 7 days at room temperature. The extract was 

then filtered and centrifuged at 3000 rpm for 20 min. Supernatant 

obtained was concentrated using rotary evaporator at 40°C (BUCHI 

Rotavapor R-200, BUCHI Labortechnik AG, Switzerland) and 

freeze-dried (LabConco Corporation, USA) to yield yellowish dark 

green powder of G. procumbens crude ethanolic extract. The 

extracts were stored at -20°C until needed in the experiment. 

Experimental animals 

Male Sprague Dawley rats aged 6 weeks (weighed 200 to 280 g) 

were obtained from the Animal House Facility, Faculty of Science 

and Technology, Universiti Kebangsaan Malaysia. The animals 

were acclimatized to laboratory conditions for 14 days prior to the 

experiments. Five rats were housed per polycarbonate cage and 

maintained on a standard laboratory rat chow diet (Barastoc, 

Australia) with water ad-libitum. The rats were maintained at 

ambient room temperature (25 ± 2°C) under a light/dark cycle of 12 

h. The experiment was approved by Universiti Kebangsaan 

Malaysia Animal Ethics Committee (UKMAEC Approval Number: 

FST/SBB/2010/HALIMAH/24-AUGUST/322-SEPTEMBER-2010- 

NOVEMBER-2011). All procedures in this study were performed 

according to the guidelines stated by UKMAEC. 

Induction of diabetes 

Diabetes was induced via intravenous injection of streptozotocin


(Calbiochem EMD Biosciences, Inc., USA) (55 mg/kg body weight 

freshly dissolved in cold sodium citrate buffer 0.1 M, pH 4.5 and 

kept in ice prior to use) to overnight fasted rats (16 h). Diabetes was 

identified by polyuria, polydipsia and high fasting blood glucose 

level 7 days after injection of streptozotocin. Rats with fasting blood 

glucose level exceeding 13 mmol/l were considered to be diabetic 

and were used in the experiment (Ravi et al., 2004). 

Experimental design 

A total of 81 rats (36 normal and 45 diabetic rats) were used. The 

normal and diabetic rats were divided randomly into five groups 

containing 4 rats per normal group (n = 4) and 5 rats per diabetic 

group (n = 5). Group 1 consisted of control animals treated with 

vehicle (distilled water, 1 ml/rat); Group 2 animals were treated with 

50, 100 or 150 mg/kg body weight (b.w.) of G. procumbens leaves 

aqueous extract; Group 3 animals were treated with 50, 100 or 150 

mg/kg b.w. of G. procumbens leaves ethanolic extract; Group 4 

animals were treated with 5 mg/kg b.w. of glibenclamide; and 

Group 5 animals were treated with 500 mg/kg b.w. of metformin. 

Both glibenclamide- and metformin-treated groups served as 

positive controls in this experiment. Gynura procumbens extracts, 

glibenclamide and metformin were suspended in distilled water and 

administered daily (1 ml/rat) via oral gavage for 42 consecutive 

days to the experimental animals. Fasting blood glucose level and 

body weight were measured prior to (day 0) and after treatment 

(day 43). At the end of 42 days treatment (day 43), rats were fasted 

overnight (16 h) and sacrificed via diethyl ether inhalation to obtain 

aortal blood and liver samples for analysis. Aortal blood samples 

were used to measure hemoglobin concentration and then 

processed immediately to obtain plasma for determination of 

plasma insulin concentration and packed erythrocyte for HbA1c 

assay. Liver organs were frozen in liquid nitrogen and stored at - 

20°C. These tissues were used for the assays of glycogen content, 

hexokinase, phosphofructokinase and fructose-1,6-bisphosphatase 

activities. 

Determination of fasting blood glucose level 

Blood was obtained from tail vein of rats fasted overnight (16 h) and 

fasting blood glucose concentration was measured on day 0 (before 

the start of treatment) and day 43 (end of treatment) using 

AccuChek® Performa glucometer (Roche Diagnostics GmbH, 

Germany). 

Preparation and determination of plasma insulin concentration 

Aortal blood samples (~1 ml) were collected into Vacuette EDTA 

tubes and centrifuged at 3000 rpm for 10 min at 4°C. The plasma 

obtained was separated into polypropylene microtubes and stored 

at -20°C until analysis. Insulin concentration in the plasma samples 

were assayed by enzyme-linked immunosorbent assay (ELISA) 

using the Ultra Sensitive Rat Insulin ELISA kit purchased from 

Crystal Chem, Inc. USA (Webster et al., 1990). 

Determination of HbA1c and hemoglobin concentrations 

Packed erythrocytes obtained from the preparation of aortal blood 

samples (~1 ml) collected into Vacuette EDTA tubes were used for 

determination of HbA1c concentration performed according to the 

method of Sudhakar Nayak and Pattabiraman (1981). The packed 

erythrocytes were washed with 2 ml 0.9% NaCl, through repeated 

centrifugation (3000 rpm, 10 min, 4°C) and washing for four times. 

Then, 2 ml of distilled water and 0.5 ml of carbon tetrachloride 

(CCl4) were added to the packed erythrocytes and incubated 

overnight at 4°C. The mixture was centrifuged at 3000 rpm for 15 

min at 4°C to obtain hemolysate. Aliquot of hemolysate (0.2 ml) was 

made up to 2 ml with distilled water, followed by the addition of 4 ml 

of 1 M oxalic acid in 2 N HCl. The mixture was heated in boiling 

water bath (100°C) for 4 h and cooled before the addition of 2 ml 

40% TCA. After adding 40% TCA, the hydrolysate was centrifuged 

at 3000 rpm for 10 min. Aliquot of hydrolysate (1 ml) was added 

with 0.05 ml 80% phenol, followed by 3 ml 95% H2SO4 and 

incubated at room temperature for 30 min before the measurement 

of absorbance. Absorbance was read at 480 nm wavelength using 

spectrophotometer (Hitachi U-1800 Spectrophotometer, Japan). 

Hemoglobin concentration in the blood samples were measured 

using Reflotron® Hemoglobin Strips (Roche Diagnostics GmbH, 

Mannheim, Germany). Glucose (200 µg/ml) was used as standard 

and HbA1c level was expressed as milligram of glucose per gram of 

hemoglobin. 

Determination of liver glycogen content 

Glycogen content was measured in the rat liver samples according 

to the method of Carroll et al. (1956). The frozen liver (50 mg) was 

hydrolysed with 2 ml 30% KOH for 15 min in boiling water bath 

(100°C). Liver hydrolysate was cooled and added with 2.4 ml 95% 

ethanol. The mixture was incubated overnight at 4°C and then 

centrifuged at 3000 rpm for 15 min. Supernatant was discarded and 

the tubes were allowed to drain in inverted position for 10 min to 

obtain glycogen pellet. Then, the glycogen pellet was dissolved in 1 

ml of distilled water via vigorous shaking, followed by the addition of 

5 ml of anthrone reagent (0.05% anthrone, 1% thiourea, 72% (v/v) 

H2SO4) with the tubes placed in cold water to prevent overheating. 

After cooling, the reaction mixture was heated in boiling water bath 

(100°C) for 15 min, and then cooled under running tap water. 

Absorbance was then read at 620 nm wavelength using 


Glucose (0.25 mg) was used as standard and glycogen content 

was expressed as milligram of glycogen per gram of wet liver 

tissue. 

Hexokinase activity assay 

Activity of liver hexokinase was assayed according to the method of 

Salas et al. (1963). The frozen liver was minced and homogenized 

with 2 volumes of ice-cold 0.1 M Tris-HCl, pH 7.4. Homogenates 

were immediately centrifuged at 20,000 × g for 30 min at 4°C 

(Eppendorf Centrifuge 5810R, Germany). The supernatant was 

then used for the measurement of hexokinase activity by the 

coupled enzyme assay procedure in a reaction mixture containing, 

in total volume of 2 ml, 50 mM Tris (pH 7.4), 0.2 IU glucose-6phosphate 

dehydrogenase, 0.25 mM β-NADP, 5 mM ATP, 5 mM 

MgCl2, 5 mM β-mercaptoethanol, 0.5 mM glucose and 25 µl of fresh 

homogenate. Control assay without ATP and glucose by replacing 

with distilled water was carried out in parallel. Reaction mixture was 

incubated at room temperature (21 to 25°C) for 5 min prior to 

assay. Reaction was started with the addition of liver homogenate 

into each assay and absorbance was read at 340 nm wavelength at 

every 1 min interval for 10 min using spectrophotometer (Hitachi U- 

1800 Spectrophotometer, Japan). Hexokinase activity was 

calculated by subtracting the activity with the activity of control. 

Activity was expressed in micromoles of glucose phosphorylated 

per minute per ml enzyme at room temperature (21 to 25°C).

Phosphofructokinase activity assay 

Phosphofructokinase activity in liver was assayed according to the 

method of Sakakibara and Uyeda (1983). The frozen liver was 

minced and homogenized with 3 volumes of ice-cold 0.1 M Tris-HCl 

(pH 7.4). Homogenates were immediately subjected to 

centrifugation at high speed (20,000 × g) for 20 min at 4°C 

(Eppendorf Centrifuge 5810R, Germany). Supernatant obtained 

was used for the measurement of phosphofructokinase activity by 

the coupled enzyme assay procedure. Reaction mixture was 

prepared, in total volume of 1 ml, containing 50 mM Tris-HCl (pH 

8.0), 1 mM fructose-6-phosphate, 1 mM ATP, 2 mM MgCl2, 0.16 

mM NADH, 2.5 mM dithiothreitol (DTT), 1 mM EDTA, 5 mM 

(NH4)2SO4, 0.4 IU aldolase, 2.4 IU triosephosphate isomerase, 0.4 

IU α-glycerophosphate dehydrogenase and 25 µl of fresh liver 

homogenate. Control assay was carried out in parallel and 

contained all the chemicals, except fructose-6-phosphate and ATP 

which was replaced by distilled water. Reaction mixture was 

incubated for 5 min at room temperature (21 to 25°C) prior to 


into each assay and absorbance was read at 340 nm wavelength at 

every 1 min interval for 10 min using spectrophotometer (Hitachi U- 

1800 Spectrophotometer, Japan). Phosphofructokinase activity was 

calculated by subtracting the activity with the activity of control. 

Activity was expressed in micromoles of fructose-1,6-bisphosphate 

produced per minute per ml enzyme at room temperature (21 to 

25°C). 

Fructose-1,6-bisphosphatase activity assay 

The method of Riou et al. (1977) was adopted for the measurement 

of fructose-1,6-bisphosphatase activity in the liver samples. The 

frozen liver was minced and homogenized with 4 volumes of icecold 

0.1 M Tris-HCl (pH 7.4). Homogenates were immediately 

centrifuged at 20,000 × g for 30 min at 4°C using Eppendorf 

Centrifuge 5810R (Germany). Supernatant obtained was subjected 

to coupled enzyme assay for the measurement of fructose-1,6bisphosphatase 

activity. Reaction mixture was prepared, in total 

volume of 1 ml, containing 100 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 

2 mM (NH4)2SO4, 2.5 mM β-mercaptoethanol, 0.05 mM EDTA, 0.2 

mM β-NADP, 3 IU glucose-6-phosphate dehydrogenase, 3 IU 

phosphoglucose isomerase, 70 µM fructose-1,6-bisphosphate and 

10 µl of fresh liver homogenate. Control assay containing all the 

chemicals, except fructose-1,6-bisphosphate was carried out in 

parallel. Reaction mixture was incubated for 5 min at 30°C prior to 


and fructose-1,6-bisphosphate into each assay. Absorbance was 

read at 340 nm wavelength at every 1 min interval for 10 min using 


Fructose-1,6-bisphosphatase activity was calculated by subtracting 

the activity with that of control. Activity was expressed in 

micromoles of fructose-1,6-bisphosphate hydrolyzed per minute per 

ml enzyme at 30°C. 

Protein determination 

Protein content of liver homogenate samples were determined 

according to the method of Bradford (1976) using bovine serum 

albumin as standard. 


Results were expressed as mean ± standard error of mean 


(S.E.M.). Statistical analysis was performed by paired T test using 

Minitab version 15. Differences with P


Table 1. Effect of Gynura procumbens extracts on body and liver weights of normal and diabetic rats. 

Test models Groups Dose (mg/kg) 

Normal 

Diabetic 

Day 0 

(Before treatment) 

Body weight (g) Liver weight (g) 

Day 43 

(End of treatment) 

Day 43 

(End of treatment) 

Control - 231.76 ± 5.3 346.83 ± 17.4 12.88 ± 0.79 

Glibenclamide 5 313.66 ± 14.2 399.66 ± 10.8 14.43 ± 0.61 

Metformin 500 284.81 ± 11.8 372.80 ± 12.1 13.57 ± 0.41 

Aqueous extract 50 207.06 ± 7.5 339.84 ± 7.7 11.57 ± 0.22 

Aqueous extract 100 278.61 ± 7.9 365.16 ± 14.6 13.85 ± 0.64 

Aqueous extract 150 222.70 ± 5.6 308.98 ± 9.3* 9.87 ± 0.72* 

Ethanolic extract 50 253.18 ± 2.8 389.96 ± 10.4 12.52 ± 0.63 



Control - 265.40 ± 13.7 197.84 ± 10.6 (-25.5) 9.55 ± 0.56 

Glibenclamide 5 214.79 ± 8.5 218.55 ± 19.7 (1.8) 10.50 ± 0.73 

Metformin 500 246.55 ± 16.7 229.23 ± 24.4 (-7.0) 11.31 ± 0.71 

Aqueous extract 50 208.64 ± 18.6 219.33 ± 14.5 (5.0) 9.60 ± 0.59 

Aqueous extract 100 226.18 ± 27.2 208.85 ± 20.0 (-7.7) 11.20 ± 1.11 

Aqueous extract 150 229.63 ± 15.8 198.14 ± 11.8 (-13.7) 10.13 ± 0.57 

Ethanolic extract 50 242.97 ± 15.6 210.87 ± 20.1 (-13.2) 9.12 ± 0.62 



Values are given as mean ± S.E.M. for five rats in each diabetic group (n=5) and four rats in each normal group (n=4). Treated groups were 

compared with controls at corresponding time-interval. Values in brackets show percentage change at the end of treatment (Day 43) as compared 

to before treatment (Day 0) for each corresponding group. Values are statistically significant at *P

Table 2. Effect of Gynura procumbens extracts on fasting blood glucose of normal and diabetic rats. 


Normal 

Diabetic 

Fasting blood glucose (mmol/l) 


Day 0 (Before treatment) Day 43 (End of treatment) 

Control - 4.75 ± 0.20 4.53 ± 0.05 

Glibenclamide 5 4.18 ± 0.33 3.93 ± 0.17* 

Metformin 500 4.35 ± 0.13 4.48 ± 0.10 

Aqueous extract 50 4.30 ± 0.25 4.23 ± 0.13 



Ethanolic extract 50 4.55 ± 0.13 4.55 ± 0.24 



Control - 18.76 ± 1.13 21.02 ± 2.00 

Glibenclamide 5 22.24 ± 0.60 18.68 ± 2.08 

Metformin 500 18.42 ± 0.70 12.38 ± 2.75 (41.4)* 

Aqueous extract 50 16.36 ± 1.14 15.84 ± 0.70 (24.6)* 

Aqueous extract 100 18.26 ± 2.00 16.92 ± 1.98 (19.5)* 

Aqueous extract 150 18.16 ± 1.59 17.36 ± 1.02 (17.4) 

Ethanolic extract 50 19.06 ± 0.88 10.72 ± 1.46 (49.0)* 

Ethanolic extract 100 18.74 ± 0.75 9.08 ± 1.95 (56.8)* 

Ethanolic extract 150 17.88 ± 0.51 9.32 ± 1.15 (55.7)** 

Values are given as mean ± S.E.M. for five rats in each diabetic group (n=5) and four rats in each normal group (n=4). Treated groups were 

compared with controls at corresponding time-interval and values in brackets show percentage change as compared to control. Values are 

statistically significant at *P


Control Aqueous Ethanolic Glibenclamide Metformin 

extract extract (5 mg/kg) (500 mg/kg) 

Normal group 



Diabetic group 

Figure 1. Effect of Gynura procumbens aqueous and ethanolic extracts on HbA1c level of normal (A) and diabetic rats (B). Results are 

expressed as mean ± S.E.M. with five rats in each diabetic group (n=5) and four rats in each normal group (n=4). Treated groups were 

compared with controls at corresponding time-interval. Values are statistically significant at *P

Table 3. Effect of Gynura procumbens extracts on plasma insulin concentration of normal and diabetic rats. 


Normal 

Diabetic 

Plasma insulin (ng/ml) 

Day 43 (End of treatment) 

Control - 1.476 ± 0.144 

Glibenclamide 5 1.841 ± 0.102 

Metformin 500 1.475 ± 0.217 

Aqueous extract 50 1.304 ± 0.199 



Ethanolic extract 50 1.338 ± 0.191 



Control - 0.145 ± 0.016 

Glibenclamide 5 0.189 ± 0.008* 

Metformin 500 0.148 ± 0.022 








Values are given as mean ± S.E.M. for five rats in each diabetic group (n=5) and four rats in each normal group (n=4). Treated groups 

were compared with controls at corresponding time-interval. Values are statistically significant at *P






Normal 

Normal 

group 

group 


Control Aqueous 

extract 

Ethanolic 

extract 

Glibenclamide 

(5 mg/kg) (500 

Metformin 

mg/kg) 


Diabetic 

Diabetic 

group 

group 

Figure 2. Effect of Gynura procumbens aqueous and ethanolic extracts on liver glycogen content of normal (A) and 

diabetic rats (B). Results are expressed as mean ± S.E.M. with five rats in each diabetic group (n=5) and four rats in 

each normal group (n=4). Treated groups were compared with controls at corresponding time-interval. Values are 


Hexokinase specific activity 

(x 10 -3 U/mg) 


(x 10 -3 U/mg) 


(x 10 -3 U/mg) 


(x 10 -3 U/mg) 

Control Aqueous Ethanolic Gilbenclamide Metformin 

Control Aqueous extract Ethanolic extract Gilbenclamide (5 mg/kg) Metformin (500 mg/kg) 


Normal group 

Normal group 

Control 

Control 

Aqueous 

Aqueous 

Ethanolic 

Ethanolic 

Gilbenclamide 

Gilbenclamide 

Metformin 

Metformin 



Diabetic 

Diabetic 

group 

group 


Figure 3. Effect of Gynura procumbens aqueous and ethanolic extracts on liver hexokinase specific activity of normal (A) 

and diabetic rats (B). Results are expressed as mean ± S.E.M. with five rats in each diabetic group (n=5) and four rats in 

each normal group (n=4). Treated groups were compared with controls at corresponding time-interval. Values are 



Phosphofructokinase specific activity 

(x 10 -3 U/mg) 

B 

B 


(x 10 -3 U/mg) 


(x 10 -3 U/mg) 


(x 10 -3 U/mg) 


(x 10 -3 U/mg) 


(x 10 -3 U/mg) 

1.4 

1.4 

1.2 

1.2 

1.0 

1.0 

0.8 

0.8 

0.6 

0.6 

0.4 

0.4 

0.2 

0.2 

0.0 

0.0 




Normal group 

Normal group 






Figure 4. Effect of Gynura procumbens aqueous and ethanolic extracts on liver phosphofructokinase specific activity of normal 

(A) and diabetic rats (B). Results are expressed as mean ± S.E.M. with five rats in each diabetic group (n=5) and four rats in 

each normal group (n=4). Treated groups were compared with controls at corresponding time-interval. Values are statistically 

significant at *P


(x 10 -3 U/mg) 


(x 10 -3 U/mg) 

DISCUSSION 





Normal 

Normal 

group 

group 





Diabetic 

Diabetic 

group 

group 

Figure 5. Effect of Gynura procumbens aqueous and ethanolic extracts on liver fructose-1,6-bisphosphatase 

specific activity of normal (A) and diabetic rats (B). Results are expressed as mean ± S.E.M. with five rats in each 

diabetic group (n=5) and four rats in each normal group (n=4). Treated groups were compared with controls at 

corresponding time-interval. Values are statistically significant at *P


selective destructive action of STZ on pancreatic β cells 

renders it a good diabetogenic agent and STZ-induced 

hyperglycemia in rodents is considered to be a good 

preliminary screening model for the study of antidiabetic 

agents (Singh et al., 2001). 

The limitations of currently available oral pharmacological 

agents for control of blood glucose have stimulated 

research on novel antidiabetic agents with different 

mechanism of action. Plant-derived antidiabetic agents 

are gaining popularity around the world for its effective 

antihyperglycemic activity and minimal side effects, such 

as resveratrol found in grapes (Palsamy and 

Subramanian, 2008), catechin from Cassia fistula (Daisy 

et al., 2010), myricetin from Abelmoschus moschatus (Liu 

et al., 2006) and berberine from Cortidis rhizoma (Chen 

et al., 2010). There are a few scientific reports relating on 

the antidiabetic potential of various extracts from G. 

procumbens demonstrated that G. procumbens extracts 

could normalize blood glucose level in diabetic animals 

(Akowuah et al., 2001, 2002; Zurina et al., 2008, 2010). 

This study was conducted to evaluate the antihyperglycemic 

effect of aqueous and ethanolic extracts of G. 

procumbens leaves in normal and STZ-induced diabetic 

rats by investigating the effect of these extracts on 

glucose profile, activity of hepatic enzymes such as 

hexokinase, phosphofructokinase and fructose-1,6bisphosphatase, 

and liver glycogen content, as well as 

the ability of these extracts to stimulate insulin secretion. 

One of the parameters to consider the amelioration of 

diabetic state is to ascertain the effect of treatment on the 

body weight (Al-Attar and Zari, 2010). In diabetes 

mellitus, deranged glucagon-mediated regulation of cyclic 

AMP formation in insulin deficiency leads to accelerated 

proteolysis (Rajasekaran et al., 2005). Since structural 

and tissue proteins contribute to 30 to 40% of total body 

weight, the excessive breakdown of tissue proteins due 

to diminished insulin response as well as the 

unavailability of carbohydrate for energy metabolism in 

diabetes mellitus results in decreased body weight 

(Gireesh et al., 2009; Palsamy and Subramanian, 2008; 

Zurina et al., 2010). Normalization of carbohydrate, 

protein and fat metabolism would alleviate the diabetic 

symptom of body weight loss; therefore body weight 

holds one of the key in evaluating the effectiveness of an 

antidiabetic treatment (Al-Attar and Zari, 2010). In the 

present study, treatment on diabetic rats with G. 

procumbens extracts showed decrease in body weight 

loss, which indicates the prevention of muscle tissue 

damage and protein wasting that is due to hyperglycemic 

condition, suggesting the potential of G. procumbens 

extracts in ameliorating diabetic state in STZ-induced 

diabetic rats. In addition, administration of G. 

procumbens extracts to normal rats did not produce 

significant changes in the body weight of normal rats, 

except 150 mg/kg b.w. G. procumbens aqueous extract- 

treated normal rats which showed significant reduction in 

body weight gain. Body weight is a sensitive indicator that 

reflects the state of health of experimental animals and 

decrease in body weight correlates with defects in body 

metabolism that is due to toxicity (Heywood, 1983). The 

reduced body weight gain which was only seen in 150 

mg/kg b.w. aqueous extract-treated normal rats might be 

due to the presence of antagonistic substances at higher 

doses of the aqueous extract. 

Present study also demonstrated that G. procumbens 

extracts lowered fasting blood glucose in diabetic rats, in 

which the ethanolic extract of G. procumbens leaves 

displayed higher extent of diabetic state amelioration than 

aqueous extract. The data presented in Table 2 displayed 

that fasting blood glucose was significantly lower for 100 

mg/kg b.w. G. procumbens ethanolic extract-treated 

diabetic rats than 50 and 150 mg/kg when compared with 

diabetic control. This suggested that the antihyperglycemic 

activity of G. procumbens occurs in a doseindependent 

manner. The decrease in antihyperglycemic 

activity at higher doses could be due to reduced or no 

effect of the components present in the extracts at higher 

doses and/or the presence of other antagonistic 

components in the extract. Since the extracts did not 

produce any hypoglycemic effect in normal rats, therefore 

the extracts may be considered to have good 

antihyperglycemic active principles without causing any 

hypoglycemic effect (Fatima et al., 2010). Furthermore, 

the blood glucose lowering effect of G. procumbens 

leaves ethanolic extract is comparable to that of 

metformin, showing the potential of G. procumbens 

ethanolic extract as plant-derived antidiabetic agent. 

Due to the diabetogenic nature of STZ, STZ-induced 

diabetic rats have diminished insulin secretion (Sefi et al., 

2011). As seen in this study, oral administration of G. 

procumbens extracts did not stimulate insulin secretion 

as demonstrated by the low levels of plasma insulin 

concentrations in the diabetic rats after 42 days of 

treatment. There were also no changes in the plasma 

insulin concentration of normal rats administered with G. 

procumbens extracts. The results obtained in this study is 

consistent with those reported by Zurina et al. (2008, 

2010), whereby oral administration of G. procumbens 

water extract did not improve the viability of pancreatic β 

cell and did not induce regeneration of the remaining 

partially destroyed pancreatic β cell of diabetic rats. 

According to Zurina et al. (2010), immunochemical 

staining for insulin of the pancreas of G. procumbens 

water extract-treated rats demonstrated minimal 

immunochemical staining for insulin, showing no 

activation of the β cells of pancreas. These findings 

together with the results obtained in our current study 

suggested that Gynura procumbens extracts do not 

possess insulinotropic activity. 

Increased non-enzymatic glycosylation is one of the 

possible mechanism linking hyperglycemia and vascular 

complications of diabetes. Prolonged hyperglycemia

promotes non-enzymatic glycosylation of enzymes and 

proteins, forming advanced glycation end-products 

(AGEs). One of the AGEs most commonly produced 

upon chronic hyperglycemia is glycosylated hemoglobin 

(HbA1c), which resulted from the reaction between 

excess glucose in the blood with hemoglobin (Al-Yassin 

and Ibrahim, 1981). Therefore, the concentration of 

glucose in the blood shows positive correlation with the 

level of HbA1c. Insulin exhibits anabolic effect on protein 

metabolism in which it stimulates protein synthesis and 

retards protein degradation (Devlin, 2006). However, 

diminished insulin response in diabetes mellitus retards 

protein synthesis and stimulates protein degradation, and 

thus decreasing the synthesis of hemoglobin. In the 

present study, STZ induction caused an increment in the 

level of HbA1c in the diabetic rats which is consistent with 

other studies (Kesari et al., 2007; Rajasekaran et al., 

2005). Oral administration of G. procumbens aqueous 

and ethanolic extracts have shown significant reduction 

on the HbA1c level of the STZ-diabetic rats which was 

due to improved glucose metabolism as well as 

increased hemoglobin synthesis (Rajasekaran et al., 

2005). These observations suggested the ability of G. 

procumbens extract in improving glycemic index of 

diabetic rats and the reduction of HbA1c level by Gynura 

procumbens extract further confirmed its ability in 

prevention of oxidative damage resulted from protein 

glycosylation reactions during diabetic condition, and thus 

reducing the risk of diabetic complication pathogenesis 

(Palsamy and Subramanian, 2008). 

The liver, which accounts for approximately 80% of 

endogenous glucose production, is an important organ 

that plays a pivotal role in the homeostasis of blood 

glucose and is primarily responsible for increased fasting 

blood glucose in diabetes mellitus (Xing et al., 2009). 

Glycogen is the primary intracellular storable form of 

glucose and its levels in various tissues directly reflect 

insulin activity as insulin promotes intracellular glycogen 

deposition by stimulating glycogen synthase and 

inhibiting glycogen phosphorylase. Since STZ selectively 

destroys β-cells of pancreas, markedly decreasing insulin 

biosynthesis and its concentration in the circulation, it is 

rational that glycogen levels in tissues especially liver and 

skeletal muscle decrease as they depend on insulin for 

glucose influx (Kalaiarasi and Pugalendi, 2009). Our 

findings that STZ-induced diabetic rats showed 

diminished glycogen storage in the liver are consistent 

with those of others (Adisakwattana et al., 2005; Bavarva 

and Narasimhacharya, 2008; Singh and Kakkar, 2009). 

Results of the present study demonstrated slight 

increment of liver glycogen content in STZ-diabetic rats 

after G. procumbens ethanolic extract administration for 

42 days, showing minimal effect of G. procumbens on 

liver glycogen of diabetic rats. In addition, our results also 

revealed that the antihyperglycemic effect of G. 

procumbens do not involve stimulation of insulin 


secretion by the pancreatic β-cells. This implies that the 

antihyperglycemic activity of G. procumbens does not 

mainly occur via improvement in glycogen metabolism, 

which is consistent with the results obtained on liver 

weight of diabetic rats as glycogen deposition in liver 

accounts up to 60% of liver weight in diabetic animals 

(Anderson, 1974). However, it is suggested that the slight 

improvement in liver glycogen storage observed plays a 

minor role in the improvement of blood glucose by G. 

procumbens. According to Daisy et al. (2010), catechin 

isolated from methanolic extract of C. fistula does not 

possess insulinotropic activity, but this flavan compound 

exerts antihyperglycemic effect by promoting glycogen 

storage in liver and muscle tissues of STZ-induced 

diabetic rats after 45 days of administration. 

The authors suggested that catechin possess 

insulinomimetic effect by acting as a molecule which 

mimics insulin in stimulating insulin-dependent 

processes, such as activation of glycogen synthase, and 

thus promoting glycogen synthesis in liver and muscle of 

diabetic rats. The lower glycogen content observed in the 

liver of G. procumbens aqueous and ethanolic extracttreated 

normal rats at doses of 50 and 150 mg/kg b.w. 

might be due to the presence of different types of active 

principles with a diverse range of biochemical activities 

which suppressed glycogen synthesis in the liver of 

normal rats. However, the suppression of glycogen 

storage in these normal rats did not significantly affect the 

fasting blood glucose and HbA1c levels of these normal 

rats, suggesting that the changes observed on glycogen 

storage in normal rats do not pose negative impact and 

does not affect blood glucose homeostasis in these rats. 

Besides glycogen metabolism, the liver is also an 

essential organ in glycolysis and gluconeogenesis for the 

regulation of blood glucose homeostasis. A partial or total 

deficiency of insulin causes derangement in carbohydrate 

metabolism that decreases the activity and expression of 

a few carbohydrate metabolic key enzymes such as 

hexokinase and phosphofructokinase, resulting in 

impaired peripheral glucose utilization and augmented 

hepatic glucose production contributing to hyperglycemia 

(Kalaiarasi and Pugalendi, 2009). In the present study, 

the decrease in activities of hepatic hexokinase and 

phosphofructokinase in STZ-induced diabetic rats which 

might be due to insulin deficiency is consistent with other 

studies on hexokinase (Kondeti et al., 2010; Rajasekaran 

et al. 2005) and phosphofructokinase (Adisakwattana et 

al., 2005). Chronic administration of G. procumbens 

extract to the diabetic rats markedly raised the activity of 

these two glycolytic enzymes, suggesting that 

antidiabetic action of G. procumbens is the result of 

increased glucose utilization by the liver. The lower 

hepatic glycolytic enzymes activity observed in G. 

procumbens aqueous extract-treated normal rats but not 

in ethanolic extract-treated normal rats might be due to 

the presence of antagonistic substances present in the


aqueous extract which suppressed the activity of 

glycolytic enzymes in the liver of normal rats. However, 

there were no significant changes observed on the 

fasting blood glucose of these normal rats, suggesting 

that these changes in activity of hepatic glycolytic 

enzymes in normal rats do not pose negative effect on 

the rats and does not affect blood glucose homeostasis in 

these rats. 

Insulin integrates hepatic carbohydrate metabolism by 

increasing the biosynthesis of enzymes of glycolysis and 

by inhibiting gluconeogenesis. Therefore, insulin 

deficiency that occurs in diabetes mellitus causes 

activation and expression of gluconeogenic enzymes 

(Kalaiarasi and Pugalendi, 2009). Activity of 

gluconeogenic enzyme, fructose-1,6-bisphosphatase in 

diabetic rats was significantly reduced by the 

administration of G. procumbens extract when compared 

with diabetic control. The reduction by G. procumbens 

ethanolic extract, especially 100 and 150 mg/kg b.w., was 

also comparable to that of metformin. The antidiabetic 

mechanism of metformin has been known to act mainly 

by inhibiting hepatic gluconeogenesis, resulting in 

decreased endogenous glucose production, and also by 

promoting peripheral glucose uptake via glucose 

transporters (Kim et al., 2008; Kirpichnikov et al., 2002). 

Thus, it is suggested that the antidiabetic effect of G. 

procumbens involves inhibition of hepatic 

gluconeogenesis. 

G. procumbens extracts did not show insulinotropic 

activity in this study, therefore the amelioration of diabetic 

state observed which occurs via stimulation of hepatic 

glucose utilization and inhibition of gluconeogenesis 

could be due to the presence of active principles in the 

extracts that possess insulinomimetic activity at the 

cellular level. Preliminary phytochemical analysis on G. 

procumbens methanolic extract conducted by Akowuah 

et al. (2001, 2002) led to the isolation of flavonol and 

flavonol glycosides, including rutin, quercetin, 

kaempferol, as well as their glycosides quercetin-3-Orhamnosyl(1-6)glucoside, 

quercetin-3-O-rhamnosyl(1- 

6)galactoside, kaempferol-3-O-glucoside and kaempferol- 

3-O-rhamnosyl(1-6)glucoside. High performance thin 

layer chromatography (HPTLC) analysis performed by 

Rosidah et al. (2009) and Zurina et al. (2008, 2010) 

revealed that methanolic and water extract of G. 

procumbens leaves contains 0.76 and 2.65% of 

kaempferol-3-O-rutinoside and astragalin, respectively. 

These flavonoids and their glycosides have been found to 

be responsible for blood glucose lowering activity in 

experimental animals (Chattopadhay, 1999; Zhang et al., 

2010) and might be responsible for promoting glucose 

uptake by muscle tissues in streptozotocin-induced 

diabetic rats (Zurina et al., 2010). Treatment of diabetic 

rats with ethanolic extract of G. procumbens seemed to 

exert better antihyperglycemic activity than aqueous 

extract as seen in improvement on glucose and glycogen 

metabolism in the diabetic rats. Ethanolic extract of G. 

procumbens had been previously reported to contain 

higher amount of polyphenols compared to aqueous and 

methanolic extracts (Puangpronpitag et al., 2010). 

Therefore, it is suggested that the antihyperglycemic 

activity in the ethanolic extract of G. procumbens could 

possibly due to the presence of higher amount of 

flavonoids and their glycosides which possess 

insulinomimetic activity. The antihyperglycemic effect of 

the G. procumbens ethanolic extract is comparable to 

that of metformin, suggesting its potential as an 

antidiabetic agent. 

Conclusion 

In conclusion, our findings demonstrated that the 

antidiabetic mechanism of G. procumbens extract is 

extra-pancreatic, which involves stimulation of glycolysis 

and inhibition of gluconeogenesis in the liver, leading to 

increased hepatic glucose utilization and decreased 

hepatic endogenous glucose production. The antidiabetic 

effect of G. procumbens extract may be due to the 

insulinomimetic ability of flavonoids present in the extract. 

Ethanolic extract of G. procumbens leaves showed 

promising antidiabetic effect and is comparable to 

metformin, suggesting its high potential to be developed 

as a plant-derived antidiabetic agent. 


This research was financially funded by the Ministry of 

Higher Education Malaysia through Fundamental 

Research 2007 (Grant No. UKM-ST-01-FRGS0051- 

2006). Facilities and help provided by the Animal House 

as well as the laboratory facilities in School of 

Biosciences and Biotechnology, Faculty of Science and 

Technology, Universiti Kebangsaan Malaysia are 

gratefully acknowledged. 

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DOI: 10.5897/JMPR11.1467 



Hypaconitine protects H9c2 cells from oxidative stressinduced 

apoptosis 

Zhi-Hui Li 1 , Wanhong Xu 2 , Guo-Hui Li 1 , Ling Shen 3 , Min-Jie Mao 1 , Zhi-Gang Wu 3 , Xue-Ting 

Shao 3 and Kun Fang 1 * 

1 Department of Intensive Care Unit, Hangzhou Red Cross Hospital, Zhejiang Chinese Medical University, China. 

2 Hangzhou High Throughput Drug Screening Center, ACEA Bio, Hangzhou, Zhejiang, China. 

3 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, Zhejiang 

University School of Medicine, China. 


The aim of the present study was to investigate the protective effect of hypaconitine on apoptosis 

induced by H2O2 and the underlying molecular mechanism in cardiac myocytes. First, cardiac myocytes 

were pretreated with different concentrations (0, 62.5, 125, and 250 ng/ml) of hypaconitine before 

exposure to 100 µM H2O2. Cell viability, apoptosis, and activation of caspase-3 and -9, p38 mitogenactivated 

protein kinases (MAPK), and nuclear factor κB (NF-κB) p65 protein were examined. In our 

study, H2O2 treatment resulted in a dose-dependent increase in the number of apoptotic cells. In 

addition, caspase-3 and -9, total and phorspho-p38 MAPK and phorspho-NF-κB p65, measured by 

western blot, were markedly activated by H2O2 treatment and, apoptosis induced by H2O2 was 

significantly reduced by pretreatment with hypaconitine in a dose-dependent manner. Similarly, the 

activation of caspase-3 and -9, phorspho-p38 MAPK, and phorspho-NF-κB p65 was blocked by 

hypaconitine; the strongest effect was observed at 250 ng/ml. In conclusion, in this study, we first 

demonstrated that hypaconitine protects cardiac myocytes from apoptosis triggered by H2O2 in a dosedependent 

manner ranging from 62.5 to 250 ng/ml. In addition, our results, at least partially, showed 

that hypaconitine inhibited cell apoptosis via blocking the activation of 3 important signaling pathways, 

MAPK pathway, NF-κB pathway, and caspase pathway, mediated by H2O2. 

Key words: Oxidative stress, heart failure, H9c2, apoptosis, hypaconitine. 

INTRODUCTION 

Heart failure is a leading cause of morbidity and mortality 

worldwide, and is accompanied by progressive left 

ventricular remodeling characterized by hypertrophy of 

the myocytes, impaired vascularization in the heart, 

abnormal extracellular matrix composition (fibrosis), and 

elevated cardiac myocytes cell death (Eguchi et al., 

2008). In heart failure after pressure-overload-induced 

cardiac hypertrophy, apoptosis of cardiac myocytes is 

proposed to be a critical point in the transition between 

compensatory hypertrophy and heart failure (Yamaguchi 

et al., 2003). Much attention has therefore focused on the 

*Corresponding author. E-mail: fangkun2010@hotmail.com. 

role of apoptosis and/or necrosis of contractile cardiac 

myocytes in the development of cardiac pathologies 

(Clerk et al., 2007). 

Oxidative stress is considered a major apoptotic 

stimulus in many cardiovascular diseases, and reactive 

oxygen species (ROS) can, in fact, trigger myocyte 

apoptosis by upregulating proapoptotic genes, which are 

inhibited by antioxidants (Xu and Wang, 2008). In vitro 

and in vivo studies have demonstrated that generation of 

reactive oxygen species (ROS) may activate necrosis, 

apoptosis, even hypertrophy in cardiac myocytes (Sheng 

et al., 2007). Furthermore, caspase, nuclear factor κB 

(NF-κB), and p38 mitogen-activated protein kinases 

(MAPK) signaling pathways are suggested to be involved 

in cardiac myocytes apoptosis (Zhuang et al., 2007;


Wang et al., 1998). Convincing data has been 

accumulated in the treatment of oxidative stress-induced 

cell injury by using natural products or plant extracts. For 

example, the investigations by Tuo et al. (2004) have 

demonstrated that onychin could exhibit a protective 

effect against lysophosphotidylcholine-mediated 

cardiovascular injury by preserving endotheliumdependent 

relaxation of rabbit aortic rings. Hypaconitine 

is a constituent of the aconite root, a traditional Chinese 

medicine that has been frequently prescribed to relieve 

muscular pain. In addition, it was concluded that 

hypaconitine, not aconitine, was the main constituent 

responsible for the action of the aconite root (Kimura et 

al., 1998; Muroi et al., 1990). Because higher doses of 

hypaconitine exhibit neuromuscular blocking effects that 

help relieve muscular pain, many studies focused their 

attention on its nerve toxicity (Kimura et al., 1998; Muroi 

et al., 1990), and few studies have focused on its 

beneficial effect on cardiac myocytes and the underlying 

mechanism. 

In the present report, we first investigated whether 

hypaconitine was involved in the protection of cardiac 

myocytes from H2O2-induced apoptosis. To explore the 

molecular mechanisms involved in the antiapoptotic 

effect of aconitine, the present study further examined 

how hypaconitine modulated the activity of MAP kinases 

and NF-κB p65 and the expression of cleaved caspase-3 

or -9 involved in oxidant-triggered apoptosis. 


All cell culture medium components were purchased from Invitrogen 

Life Technologies unless otherwise noted. H2O2 was purchased 

from Sigma (St. Louis, MO, USA) and prepared immediately in 

phosphate buffered saline (PBS) at 10, 25, 50, 100, 200, or 400 µM 

before use. Hypaconitine (Lot number 6798–9403, Beijing Drug 

Administration, Beijing, China, purity≥98%) was dissolved in 

Dulbecco’s modified Eagle’s medium (DMEM) at 2000 ng/ml. It was 

diluted to 62.5, 125, 250, 500, 1000, and 2000 ng/ml, respectively 

as final concentrations. Annexin V/FITC Kit was purchased from 

Bender MedSystems GmbH (Vienna, Austria). Antibodies used in 

the western blot analysis were rabbit anti-active caspase-3 and 

caspase-9 polyclonal antibodies (Chemicon, CA, USA) recognizing 

only the cleaved large subunit (17 kDa of caspase-3 and 37 kDa of 

caspase-9) and rabbit polyclonal antibody against β-actin was 

purchased from Santa Cruz Biotechnology, Inc., USA (43 kDa ). 

Phospho-p38 MAPK (38 kDa), total p38 MAPK (38 kDa) and 

phospho-NF-κB p65 (65 kDa, recognizing phosphorylation at the 

Ser536 position) were from Cell Signal (Beverly, MA, USA). 

Cell culture 

H9c2 cells, a clonal line of cardiac myocytes derived from 

embryonic rat heart tissue, were purchased from the American Type 

Culture Collection (ATCC, Manassas, VA, USA). Following the 

protocol provided by ATCC, the cells were cultured in complete 

medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) 

supplemented with 10% fetal bovine serum (FBS), 4 mM Lglutamine 

adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L 

glucose, and 1% (v/v) penicillin and streptomycin. The cultures 

were maintained at 37°C in a 5% CO2 humidified atmosphere. 

Cell viability assay (MTS) 

Cell viability was quantified on the basis of metabolic activity with 

the MTS assay (Promega, Madison, WI) according to the 

manufacturer’s protocol. Cardiac myocytes were seeded at a 

density of 5 × 10 4 /well in a 96-well plate in DMEM with 5% FBS. 

The cultures were incubated for 3 h at 37°C in serum-free medium 

containing 20 μl/well of the MTS tetrazolium compound. The 

absorbance of formazan products was photometrically measured at 

490 nm with a microplate reader. The cell viability was expressed 

as absorbance (OD). 

Flow cytometry analysis 

Recovery of the cells was monitored by examining the levels of 

apoptosis. Annexin V binding and propidium iodine (PI) staining 

was determined by flow cytometry. The cells were washed with icecold 

PBS and double stained with FITC-coupled Annexin V protein 

and PI for 20 min. Flow cytometry was performed with a 488-nm 

laser coupled to a cell sorter (FacsCalibur; BD Biosciences, San 

Jose, CA). Cells stained with both PI and Annexin V were 

considered necrotic and cells stained only with Annexin V were 

considered apoptotic. 

Western blot analysis 

The expression of caspase-3, caspase-9, phospho-p38 MAPK, total 

p38 MAPK, and phosphor-NF-κB p65 was detected by western blot. 

Cells were washed with PBS (4°C) and harvested under nondenaturing 

conditions by incubation (4°C/5 minutes) with lysis buffer 

as described above. Western blot was performed as described 

above. The immuno-blot was incubated for 16 h with monoclonal 

anti-β-actin antibody, rabbit anti-cleaved caspase-3 and -9, p38 

MAPK, antiphospho-p38 MAPK antibody, and rabbit antiphospho- 

NF-κB p65 antibody in PBS-Tween. The membrane was washed 

and incubated for 2 h at room temperature with a peroxidaselabeled 

goat anti-rabbit or anti-mouse immunoglobulin. After further 

washing, the proteins were detected by ECL chemiluminescence 

(from Amersham International, England). For the semi-quantitative 

determination of protein expression, western blotting images of 

some experiments were scanned on a flatbed scanner and the 

density of the bands were quantitated using ImageQuant software 

(Molecular Dynamic, Sunnyvale, CA). Densitometry results were 

reported as percentages of medium control after normalization with 

the average arbitrary integrated values of the β-actin signal. 


The results were expressed as the mean ± SD. For multiple 

comparisons, data were subjected to one-way ANOVA followed by 

Fisher’s multiple comparison test. p < 0.05 was considered 

statistically significant. 

RESULTS 

Effects of H2O2 on apoptosis of cardiac myocytes 

A number of studies have shown that H2O2, as an

Annexin V/PI (%) 

Concentration of H2O2 (μM) 

Figure 1. H2O2-induced apoptosis in a dose-dependent manner in H9c2 cells. 

example of oxidative stress, induces apoptosis of cardiac 

myocyte in vitro (Aoki et al., 2002). Moreover, cell 

apoptosis measurement determined the optimum 

concentration of H2O2 that induces apoptosis in our cell 

model. In the initial study, H9c2 cells were exposed to 

increasing concentrations (0~400 µM) of H2O2 for 24 h. 

As shown in Figure 1, the percentage of apoptotic cells 

was 1.51% in the absence of H2O2, and steadily 

increased to 13.26% when the cells were exposed to 25 

µM; 94.2% of cells were apoptotic when treated with 400 

µM. At 100 µM, the percentage of apoptotic cells was 

43.72%. 

Effect of hypaconitine on the viability of cardiac 

myocytes 

In the initial experiments, 100 µM H2O2 was the optimal 

concentration. Next, we wanted to identify the safe 

dosage of hypaconitine in cardiac myocytes. The 

cytotoxic effects of hypaconitine were evaluated by the 

MTS assay 24 h after the treatment of H9c2 cells with 

concentrations of hypaconitine ranging from 0 to 2000 

ng/ml. Although no cytotoxicity was evident at 

concentrations below 250 ng/ml, there was a significant 

(p = 0.00) dose-dependent decrease in cell number at 

concentrations above 250 ng/ml; 2000 ng/ml resulted in 

more than 50% loss in cell viability compared to the basal 

Li et al. 815 

level (Figure 2). In the end, we used hypaconitine in our 

following experiments at concentrations of 62.5, 125, and 

250 ng/ml. 

Hypaconitine blocked H9c2 cells apoptosis and the 

decrease in viability induced by H2O2 

To assess the effect of hypaconitine on H2O2-induced 

decreased viability and apoptosis, cells were randomly 

divided into 6 study groups and then incubated for 24 h 

(Table 1). In this part, the exposure of H9c2 cells to 100 

µM H2O2 for 24 h resulted in an obvious decrease in cell 

viability as measured by MTS, p=0.000 vs group1 (Figure 

3). When the cells were pretreated with hypaconitine 

(0~250 ng/ml) for 2 h, the cell damage was significantly 

attenuated in a concentration-dependent manner (p = 

0.000 every group vs the group2) and reached the best 

protective effect at 250 ng/ml (Figure 3). In other words, 

pretreatment with hypaconitine (0~250 ng/ml) dosedependently 

inhibited H2O2-induced H9c2 cell injury. 

When H9c2 cell apoptosis was quantified by flow 

cytometry, the control group had 2.54± 0.23% of cells 

were in the early phase of apoptosis (Annexin V-positive 

and PI-negative) (Figure. 4). An increase in the number of 

apoptotic cells (45.98± 4.94%) was observed in the H2O2 

alone-treated group with a lower number of living cells. 

Pretreatment with hypaconitine (62.5, 125, 250 ng/ml) for


Absorbance at 490 nm 

(OD) 

Figure 2. Confirmation of the safe dose of hypaconitine in H9c2 cells. 

Table 1. Groups in different culture conditions in this study. 

Condition/group 1 2 3 4 5 6 

H2O2 (µM) 0 100 100 100 100 0 

Hypaconitine (ng/ml) 0 0 62.5 125 250 250 

Absorbance at 490 nm (OD) 

Figure 3. Cytoprotective effect of hypaconitine on decreased H2O2-induced cell viability.

Figure 4. Inhibition of H2O2-triggered apoptosis by hypaconitine. 

2 h decreased the apoptotic rate to 32.07± 3.92, 

20.72±3.69, and 7.85±0.91%, respectively (Figure 4). 

Moreover, administration of hypaconitine (250 ng/ml) 

alone caused not only no alteration in cell viability but 

also no changes in the percentage of living and apoptotic 

cells compared to normal control values. 

Mechanism of hypaconitine defending against 

oxidative injury of H9c2 induced by H2O2 

Cell fate in response to severe stress will depend on the 

balance between antiapoptotic and proapoptotic factors 

(Cieslak and Lazou, 2007). Our foregoing data showed 

that hypaconitine pretreatment enhanced the H2O2induced 

suppressed viability and decreased apoptosis. 

To further confirm that hypaconitine possesses 

antiapoptotic properties in cardiac myocytes under 

oxidative stress and to explore its signaling pathways, 

cleaved caspase-3 and -9, and phosphorylation of p38 

MAPK and NF-κB p65 were examined by western 

Li et al. 817 

blotting. Figure 5A to C show that H2O2 stimulation 

triggered the activation of the above signaling molecules, 

as assessed by their protein levels using specific 

antibodies. These data indicated that apoptosis 

increasing was induced by H2O2 in a dose-dependent 

manner and it followed the activity increasing of the 

caspase-, p38 MAPK-, and NF-κB-dependent pathway in 

H9c2 cells. However, the expression of caspase-9 and -3 

decreased significantly when hypaconitine was used 

before treatment with H2O2 (Figure 5A), which showed 

that hypaconitine could protect against oxidative injury, at 

least partially, via reducing cleavage of caspase-3 and -9. 

In agreement with the data of caspase-3 and -9, protein 

expression studies showed that hypaconitine reduced the 

enhancement of phospho-NF-κB p65 protein levels 

(Figure 5C) and the phospho-p38/p38 MAPK ratio (Figure 

5B) induced by H2O2 in cardiac myocytes in a dosedependent 

manner. In addition, hypaconitine treatment 

alone did not influence these protein levels (Figure 5A to 

C). These data suggest that hypaconitine will also exert 

its antiapoptotic effect by decreasing phospho-NF-κB p65


Figure 5. Effect of hypaconitine on H2O2 induced early 

intracellular signaling events. 

protein levels and the phospho-p38/p38 MAPK ratio in 

oxidatively stressed cardiac myocytes. 

DISCUSSION 

Heart failure is a common end-stage event resulting from 

various cardiovascular diseases, and it is now well 

established that apoptosis of cardiac myocytes is an 

important component of cardiac remodeling, ultimately 

leading to heart failure (Eguchi et al., 2008). Oxidative 

stress has been shown to trigger apoptosis of cardiac 

myocytes in myocardial infarction, ischemia-reperfusion 

injury, cardiomyopathy, atherosclerosis, and heart failure 

(Wang et al., 2005; Haunstetter and Izumo, 1998). This 

study was undertaken to examine the effect of 

hypaconitine on H2O2-induced apoptosis of H9c2 cells 

and the possible molecular mechanisms involved. 

It is widely accepted that high concentrations of H2O2 or 

high levels of oxidative stress promote cardiac myocyte 

death, but some groups have reported that lower, 

nontoxic concentrations of H2O2 promote cytoprotection 

or growth (Clerk et al., 2007). Similarly, in our study, 

when cardiac myocytes were exposed to increasing 

concentrations of H2O2 ranging from 25 to 400 µM for 24 

h, the percentage of apoptotic cells (assessed by annexin 

V binding and PI staining) was significantly increased 

from 13.26 to 94.2% in a dose-dependent manner. As 

shown in Figure 1, H2O2 at 10 µM had no effect on cell 

survival (1.87% apoptotic cells vs. 1.51% H2O2-untreated 

cells), and we did not observe any cell-growth promoting 

effect. Maybe, this effect will be noticed at a 

concentration below 10 µM. H2O2 used at a concentration 

of 100 µM in this study, which has been reported to be in 

the range that mainly induces cell apoptosis but little 

necrosis (Xu and Wang, 2008), caused 43.72% apoptosis 

of cardiac myocytes in our study. Thus, in our 

subsequent study, 100 µM was chosen as the optimal 

concentration. 

Traditionally, hypaconitine from Fuzi is decocted with 

other herbal medicines, such as Panax ginseng, and is 

generally used in heart failure and other kinds of heart 

diseases (Luo et al., 2008). In the present study 

demonstrated, for the first time, a direct antiapoptotic 

effect of hypaconitine in cultured H9c2 cells. In H2O2induced 

cell injury, when H9c2 cells were preincubated 

with hypaconitine for 2 h, the safe concentration of 62.5- 

250 ng/ml of hypaconitine considerably boosted cell 

viability determined by the MTS assay. Meanwhile, 

hypaconitine significantly reduced the apoptotic rate of 

H9c2 cells as demonstrated in Figures 3 and 4. This 

effect became even more apparent when the 

concentration of hypaconitine increased from 62.5 to 250 

ng/ml, which caused significant reduction of cell death 

compared with the cells treated with H2O2 only. 

Furthermore, we showed that the 3 important signaling 

pathways involving p38 MAPK, NF-κB, and caspase 

activated by H2O2 were inhibited by hypaconitine in 

oxidatively stressed cardiac myocytes. Functionally, 

caspase-3 is a critical effector caspase of the apoptotic 

process and caspase-9 is an initiator of caspase-3 in the 

mitochondria-dependent pathway (Jiang et al., 2005). In 

our present study, H2O2 treatment significantly activated 

caspase-9 and caspase-3 in H9c2 cells. However, 

western blot analysis also showed that hypaconitine 

pretreatment remarkably suppressed proteolytic 

activation of caspase-3 and caspase-9 (Figure 5A). 

Further, oxidants can trigger the activation of multiple 

signaling pathways including the phosphorylation 

cascades leading to the activation of MAPKs, NF-κB 

(Wang et al., 1998) and the expression enhancing of Bax 

(Wu and Hu, 2011). Previous studies have shown that 

p38 MAPK is a downstream effector of the H2O2-induced 

apoptotic process (Tuo et al., 2004). Several groups 

observed increased p38 MAPK activity with apoptosis in 

stressed H9c2 cells, neonatal cardiac myocytes, adult

myocytes, and reperfused hearts. These results suggest 

that p38 MAPK may play a critical role in the 

development of cardiac apoptosis (He et al., 1999; 

Hreniuk et al., 2001; Yue et al., 2000). Consistent with 

these results, in our study, robust phospho-p38 MAPK 

was activated by H2O2 in H9c2 cells (Figure 5B). Among 

the potential MAPK-regulated transcription factors, NF-κB 

is known to be activated in response to oxidative stress 

(Wang et al., 1998). Although most published reports 

have provided evidence suggesting that NF-κB is 

antiapoptotic, whether this transcription factor acts as a 

proapoptotic or antiapoptotic factor may depend on the 

cell type and the activating pathway (Wang et al., 1998). 

It was therefore of interest to determine the effect of NFκB 

on the cellular response to H2O2 in our model. In 

keeping with our previous data, treatment with H2O2 also 

resulted in the remarkable activation of phospho-NF-κB 

p65 in our cells as shown in Figure 5C and, both the 

activation of phospho-p38 MAPK and phospho-NF-κB 

p65 were significantly suppressed by hypaconitine 

pretreatment in a dosage-dependent manner. The level 

of phospho-p38 MAPK and phospho-NF-κB p65 returned 

almost completely to the normal level when the 

concentration of hypaconitine was 250 ng/ml (Figure 5B 

and C). Further, hypaconitine treatment alone did not 

affect the protein level of cleaved caspase-3 and -9, the 

ratio of phospho-p38/p38 MAPK, and the protein level of 

phospho-NF-κB p65 (Figure 5A to C). 

In conclusion, the results of our study demonstrated 

that hypaconitine protects cardiac myocytes in a dosedependent 

manner against H2O2-mediated cytotoxicity 

and apoptosis. We clearly showed that hypaconitine 

inhibited apoptotic cell death in part mediated via 

activation of 3 important signaling pathways, MAPK 

pathway, NF-κB pathway, and caspase pathway, which 

are triggered by H2O2. Thus, hypaconitine may be an 

effective drug for protection cardiac myocytes from 

apoptosis in treating heart failure. 


This work was supported by grants from the Traditional 

Chinese Medicine Foundation (NO. 2008CA047 and 

NO.2006Y007), and the Medical and Health Science 

Foundation (No. 2006B033) of Health Bureau of 

Hangzhou, China. 

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DOI: 10.5897/JMPR11.1468 



Antimicrobial activity and chemical composition of 

essential oils of chamomile from Neyshabur, Iran 

Motavalizadehkakhky Alireza 

Department of Chemistry, Islamic Azad University, Neyshabur Branch, Neyshabur, Iran. 

E-mail: amotavalizadeh@yahoo.com. Tel: +989153510342 or +985516611720. 


Matricaria chamomilla L. or German chamomile is an annual plant of the composite family Asteraceae. 

In this study, M. chamomilla L. were collected from Neyshabur, Iran. Chemical constituent of essential 

oils of M. chamomilla L. were determined. Aerial parts (200 gr) were subjected to hydrodistillation in a 

Clevenger – type apparatus until there was no significant increase in the volume of the oil collected (2.5 

h). The yield of the blue oil was 0.9% (w/w). The essential oil was analyzed by GC and GC/MS. 

Identification of the components was based on GC retention indices computer matching with Wiley GC- 

MS library, and by comparison of the fragmentation patterns of the mass spectra with those reported in 

the literature (Adams, 2004). 47 components were identified constituting more than 83.1% of the oil. p- 

Cymene-8-ol(0.7%), Azulene(0.4%), p-Cymene (1.1%), 1,8-Cineole (2.1%), Artemisia Alcohol (0.2%), β- 

Elemene (0.9%), cis-β-farnesene (0.9%), trans-β-farnesene (5.2%), Borneol (0.8%), γ-Cadinene (0.4%), 

Spathulenol (9.4%), γ-Eudesmol (1.5%), α-Bisabolol oxide B(7.0%), α-Bisabolol oxide A (21.5%), α- 

Bisabolol (5.0%), Chamazulene (4.2%) and Germacrene D (0.8%) were major components in M. 

chamomilla L. oil. The oil was tested against seven strains of bacteria (Gram-positive and Gramnegative). 

The oil showed mild to significant antimicrobial activity associated mainly with Gram-positive 

and Gram-Negative bacteria. 

Key words: Matricaria chamomilla L., essential oil, antimicrobial activity, α-Bisabolol oxide A, α-Bisabolol oxide 

B. 

INTRODUCTION 

During recent years, there has been a growing increase 

in substances exhibiting antimicrobial properties that are 

supplied to human and animal organisms as food 

components or as specific pharmaceutics (Azuma et al., 

1995). Plant essential oil and extracts are the sources of 

naturally occurring antioxidants. Essential oils exhibit 

some antimicrobial properties (Ozer et al., 2007). 

Matricaria chamomilla L. or German chamomile also 

spelled camomile is an annual plant of the composite 

family Asteraceae. It usually grows near populated areas 

all over Europe and temperate Asia. Because the seeds 

need open soil to survive, it often grows near roads, 

around landing and in cultivated fields as a weed (Plant 

database USDA, 2008; Integrated toxonomic information 

system). 

M. chamomilla L. is an annual plant approximately 50 

cm, with drawn up stem, oarswoman. The branched stem 

is erect and smooth and grows to a height of 15 to 60 cm. 

The long and narrow leaves are bipinnate or tripinnate. 

The flowers are borne in paniculate capitula. The white 

ray florets are furnished with a ligule, while the disc 

florets are yellow. The hollow receptacle is swollen and 

lacks scales. This property distinguished German 

Chamomile from, Corn chamomile (Anthemis arvensis), 

which has a receptacle with scales. The flowers have a 

strong, aromatic smell and bloom in early to mid summer 

medically. German chamomile is used to treat sore 

stomach, irritable bowel syndrome, and as a gentle sleep 

aid. It is also used as a mild laxative and is antiinflammatory 

and bactericidal (Fatouma et al., 2011; 

Shivananda et al., 2007; Owlia, 2007). It can be taken as 

a herbal tea, which should be steeped for ten to fifteen 

minutes while covered to avoid evaporation of the volatile 

oils. The marc should be pressed because of the 

formation of a new active principle inside the cells, which 

can then be released by rupturing the cell walls, though 

this substance only forms very close to boiling point. For 

a sore stomach, some recommend taking a cup every

morning without food for two to three months. It is also 

used as a mouthwash against oral mucositis. It has 

acaricidal properties against certain mites, such as 

Psoroptes cuniculi. One of the active ingredients of the 

essential oil from German chamomile is the terpene 

bisabolol. Other active ingredients include farnesene, 

chamazulene, flavonoids (including apigenin, quercetin, 

patuletin and luteolin) and coumarin (McKay and 

Blumberg, 2006). Chamomile (M. chamomilla), a popular 

herb valued for centuries as a traditional medicine, has 

been used to treat various human ailments; however, its 

anticancer activity is unknown (Janmejai K, et al. 2007). 

Antifungal activity of M. chamomilla flower essential oil 

collected from Karaj, Iran, has been investigated against 

Aspergillus niger and identified as having 21 components 

in the plant oil (92.88%w/w), include bisabolol oxide A 

(2.19%) and chamazulene (2.18%) (Tolouee et al., 2010). 

Antimicrobial and antioxidant activities of essential oil and 

methanol extracted of M. chamomilla L. collected from 

Djibouti were investigated (Fatouma et al., 2011). 

Chemical composition of volatile essential oil from a 

chamomile sample cultivated in Botanical Garden of 

School of pharmacy in Isfahan, Iran, was studied. The oil 

plant represented 86.21%w/w of total oil and 63 

components were characterized. Bisabolol oxide A 

(25.01%) and bisabolol oxide B (9.43%) were the major 

constituents of the oil (Shams Ardakani MR et al., 2006). 

Inhibitory effect of essential oils against herpes simplex 

virus type 2 was investigated (Koch et al., 2008). In a 

clinical study chamomile solution or a 1% topical 

hydrocortisone ointment was used in the management of 

peristomal skin lesions in colostomy patients (Charousaei 

et al., 2011). Anti-allergic activity of German chamomile 

(Matricaria recutita L.) in mast cell mediated allergy 

model was studied (Chandrashekhar et al., 2011). 

Antihyperglycemic and antioxidative potential of M. 

chamomilla L. in streptozotocin-induced diabetic rats was 

investigated (Mustafa et al., 2008). Antiproliferative and 

apoptotic effects of chamomile extract in various human 

cancer cells were studied (Janmejai et al., 2007). 



Essential oil of M. chamomilla collected from Neyshabur, Khorasan- 

Razavi Province, Iran, in 2010 was obtained by hydrodistillation of 

the aerial parts. Voucher specimens of the plant have been 

deposited in the herbarium. 

Isolation of the essential oil 

Aerial parts (200 gr) of M. chamomilla were subjected to 

hydrodistilation using a Clevenger-type apparatus for 2.5 h. After 

decanting, the obtained essential oil was dried over anhydrous 

Alireza 821 

sodium sulfate and, after filtration, stored in refrigerator at -4°C until 

tested and analyzed. 

Gas chromatography and GC-MS 

GC analysis was performed on a shimadzu 15 A gas 

chromatography equipped with split/splitless injector (250°C) and a 

flame ionization detector (250°C). Nitrogen was used as carrier gas 

(1 ml/min) and the capillary column used was DB-5 (50 m × 0.2 

mm, film thickness 0.32 µm). The column temperature was kept at 

60°C for 3 min and then heated to 220°C with a 5°C / min rate and 

kept constant at 250°C for 5 min. Relative percentage amounts 

were calculated from peak area using a Shimadzu C-R4A 

chromatopac without the use of correction factors. 

GC-MS analysis was performed using a Hewlett – Packard 5973 

with a HP-5MS column (30 m × 0.25 mm, film thickness 0.25 µM). 

The column temperature was kept at 60°C for three mi nutes and 

programmed to 220°C at a rate of 5°C / min and kept co nstant at 

220°C for five minutes. The flow rate of Helium as c arrier gas (1 

ml/min). MS were taken at 70 eV. 

Identification of the constituents of each oil was made by 

comparison of their mass spectra and retention indices (RRI) with 

those given in the literature and those authentic samples (Adams, 

2004). Relative percentages of components were obtained from the 

peak area percent reports of volatiles from GC/MS data in Table 1. 

Antimicrobial assay of the oil 

In vitro antibacterial assay of the oil carried out according to disc 

agar diffusion method (Jirovets et al., 1999; Ajai et al., 2004). 

Antibacterial activity of the oil was tested against Gram-positive 

bacterial strains such as Bacillus cereus (MTCC430), Bacillus 

subtilis (MTCC441), Staphylococcus aureus subsp. aureus 

(MTCC2940) and Gram-negative bacterial strains; Klebsiella 

pneumonia (MTCC109), Escherichia coli (MTCC443), Proteus 

vulgaris (MTCC426) and Salmunella typhi (MTCC733) were grown 

in nutrient broth for 24 h (pH 7.2 to 7.4) and were used as 

inoculums. The Mueller-Hinton agar medium were poured into the 

plates to uniform depth of mm and allowed to solidify. Then the 

microbial suspentions were streaked over the surface of media 

using a sterile cotton swab to ensure the confluent growth of the 

organism. Aliquots of 10 µl of the oil at 1:2 dilutions in dimethyl 

sulfoxide (DMSO) were impregnated on Whatman No. 1 filter paper 

discs of 6 mm diameter. These discs were aseptically applied to the 

surface of the agar plates at well-spaced intervals. The plates were 

incubated at 37°C for 24 h and observed inhibition z ones including 

the diameter of the discs were measured. Control discs 

impregnated with 10 µl of the solvent DMSO and streptomycin (10 

µl /disc), reference for bacteria were used alongside the test discs 

in each experiment. The results are presented in Table 2. 


The volatile oil obtained in 0.90% w/w yield from 200 gr 

air dried aerial parts of M. chamomilla was blue color. 47 

components identified (out of 57 existing components) in 

oil. Among them monoterpenes were 3.7%, oxygenated 

monoterpene were 4.3%, sesquiterpenes were 11.6%, 

oxygenated sesquiterpenes were 58.8% and other 

components were 4.6%. p-Cymene-8-ol (0.7%), 

Azulene(0.4%), p-Cymene (1.1%), 1,8 - Cineole(2.1%), 

Artemisia Alcohol (0.2%), β-Elemene (0.9%),


Table 1. Chemical composition (%) of essential oil of aerial parts Matricaria chamomilla L. 

Compounds Aerial parts (%) RRI 

α-Pinene 0.1 958 

para-Cymene 1.1 1024 

(Z)-β-ocimene 0.2 1033 

1, 8-cineole 2.1 1034 

(E)-β-ocimene 0.1 1042 

Limonene 0.2 1034 

γ-Terpinene 0.3 1052 

Cis-sabinene hydrate 0.2 1062 

Artemisia alcohol 0.2 1062 

n-Nonanal 0.1 1075 

Camphor 0.1 1099 

Linalool 0.1 1100 

Cis-Chrysanthenol 0.1 1114 

Borneol 0.8 1117 

4-Terpineol 0.1 1171 

Para-Cymene-8-ol 0.7 1182 

Nonanoic acid 0.3 1201 

Azulene 0.4 1218 

Daucene 0.5 1253 

Cis-β-farnesene 0.9 1321 

Trans-β-farnesene 5.2 1331 

δ-Elemene 0.1 1331 

α-Muurolene 0.8 1355 

Cis-α-Bisabolene 0.3 1360 

β-Bisabolene 0.2 1364 

γ-Cadinene 0.4 1366 

α-Copaene 0.2 1368 

δ-Cadinene 0.2 1372 

Trans-γ-Bisabolene 0.1 1376 

β-Elemene 0.9 1386 

α-Cadinene 0.9 1386 

β-Caryophyllene 0.3 1410 

Spathulenol 9.4 1419 

γ-Eudesmol 1.5 1462 

β-Selinene 0.5 1475 

α-Bisabolol oxide B 7.0 1476 

β-Bisabolol 0.1 1482 

γ-Elemene 0.7 1431 

Germacrene-D 0.8 1471 

α-Bisabolene oxide A 10.0 1493 

α-Bisabolol 5.0 1499 

Chamazulene 4.2 1524 

α-Bisabolol oxide A 21.5 1557 

Caryophyllene oxide 0.7 1573 

β-Bisabolenal 0.8 1578 

Juniperol 0.9 1591 

α-Bisabolol acetate 1.8 1602


Grouped components 

Number of constituents 57 

Number of identified constituents 47 

Percentage identified 82 

Percentage of Monoterpene hydrocarbons 3.7 

Percentage of oxygen-containing monoterpenes 4.3 

Percentage of sesquiterpene hydrocarbons 11.6 

Percentage of oxygen-containing sesquiterpenes 58.8 

Percentage of others 4.6 

Percentage of total 83.1 

Table 2. Antibacterial activity of the aerial parts oil of Matricaria chamomilla L. 

Tested bacteria 

Gram-positive bacteria 

Zone of inhibition (mm) 

MTCC No. 

Oil in DMSO (1:2) Streptomycin 1 mg/ml 

Bacillus cereus 430 11.5 7.5 

Bacillus subtillis 441 8 11 

Staphylococcus aureus subsp. Aureus 2940 13.5 12.5 

Gram-negative bacteria 

Escherichia coli 443 8 13 

Klebsiella pneumoniae 109 9 9.5 

Proteus vulgaris 426 11.5 10 

Salmunella typhi 733 8.5 9 

cis-β-farnesene (0.9%), trans-β-farnesene (5.2%), 

Borneol (0.8%), γ-Cadinene (0.4%), Spathulenol (9.4%), 

γ-Eudesmol(1.5%), α-Bisabolol oxide B (7.0%), α- 

Bisabolol oxide A (21.5%), α-Bisabolol (5.0%), 

Chamazulene (4.2%), and Germacrene D(0.8%) were 

some major components in M. chamomilla L. oil. The oil 

in DMSO (1:2 dilutions) showed 153% inhibition against 

B. cereus, 73% inhibition against B. subtilis and 108% 

inhibition against S. aureus subsp. aureus as compared 

to the standard, streptomycin at 10 µl/disc. The oil 

showed comparable activity against Gram-negative 

bacteria: 95% against K. pneumonia, 62% against E. coli, 

115% against P. vulgaris and 94% against S. typhi (Table 

2). 


The author is thankful to Dr. Rustaiyan, Dr. Masoudi, Dr. 

Mehrzad, Dr. Taheri, and (MSc. Student) Zohreh 

Ebrahimi. 

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DOI: 10.5897/JMPR11.1471 



Determination of saikosaponin, phenolic and 

podophyllotoxin contents of five endemic Bupleurum 

root extracts and their effects on MCF-7 cells 

Gökhan Kars 1 *, Meltem Demirel Kars 2,3 , Mehtap Akin 1 , Hatice Taner Saraçoğlu 1 and 

Ufuk Gündüz 4 

1 Department of Biology, Faculty of Science, Selçuk University, 42075 Konya, Turkey. 

2 Sarayönü Vocational High School, Selçuk University, 42430 Konya, Turkey. 

3 Advanced Research and Application Center, Selçuk University, 42075 Konya, Turkey. 

4 Department of Biological Sciences, Middle East Technical University, 06531 Ankara, Turkey. 


Bupleurum species are among the plants used in Chinese medicine for phytotherapy. Various 

compounds obtained from plants have been found to exhibit anticancer activity. In this study, phenolic 

compounds, saikosaponins and podohyllotoxin contents of total root extracts from endemic Bupleurum 

species (Bupleurum sulphureum, Bupleurum lycaonicum, Bupleurum turcicum, Bupleurum heldreichii, 

Bupleurum pauciradiatum) were determined by high-performance liquid chromatography (HPLC). Total 

phenol content and free radical scavenging activities of total extracts were also identified. Finally, the 

effects of plant root extracts on viability of P-gp overexpressing paclitaxel resistant (MCF-7/Pac) and 

vincristine resistant (MCF-7/Vinc) MCF-7 mammary carcinoma cell lines and their parental line (MCF- 

7/S) were evaluated by cytotoxicity tests. Results showed that saikosaponin A, saikosaponin D and 

isoquercitrin contents of the root extracts were higher compared to podophyllotoxin, catechin and 

quercetin. While, B. lycaonicum root extract has about 1.5 fold more total phenol content with respect 

to others, B. turcium root extract has the highest free radical scavenging activity. According to 

cytotoxicity tests, B. turcicum and B. pauciradiatum root extracts were more toxic to the MCF-7/Pac cell 

line than the root extracts of other three species. In addition, B. heldrechii extract has been found to be 

the most toxic to the MCF-7/Vinc cell line among the others. 

Key words: Bupleurum species, plant phenolics, saikosaponin, podophyllotoxin, high-performance liquid 

chromatography (HPLC), MCF-7. 

INTRODUCTION 

Bupleurum species is among the plants used in 

traditional Chinese medicine (Yano et al., 1994) and 

major bioactive compounds have been found to be 

saikosaponins, phenolics, volatile oils and less polar 

substances (Zhang et al., 2010). Bupleurum 

scorzonerifolium is known as anti-inflammatory and antihepatotoxic 

plant and it was shown that saikosaponins 

found in plant root had anti-inflammatory and anticarcinogenic 

activities (Mahato et al., 1988; Hsu et al., 

*Corresponding author. E-mail: gkars2004@yahoo.com. Tel: 

+90 3322232771. 

2004). In addition, total root extract of B. scorzonerifolium 

has been proven to decrease the telomerase activity in 

lung carcinoma cell line by activating apoptosis (Cheng et 

al., 2003). Plant chemicals (phytochemicals) like 

polyphenols, organosulfurs, carotenoids, saponins, 

phytosterols, alkaloids, terpenes and more than thirty 

thousand types are the potential active ingredients 

exerting their effects on variety of the cells. Quercetin, 

rutin, isoquercitrin, catechin, robinin, tannin are examples 

for plant phenolic compounds which have antioxidant and 

chemopreventive activities (Cai et al., 2004). Saponins 

are glycosides with aglycan and glycan parts (Sparg et 

al., 2004). Saponins have been recently reported to 

possess anticancer activities by making complex with


Table 1. Specific locations of endemic Bupleurum species collected in Konya, Turkey. 

Species Location 

Bupleurum sulphureum Beyşehir yolu 

Bupleurum lycaonicum Beyşehir yolu 

Bupleurum heldreichii Karapinar 

Bupleurum turcicum Tuz gölü 

Bupleurum pauciradiatum Karaman-Başkişla 

cholesterols in cell membrane and producing pores that 

induce apoptosis (Sezgin and Artik, 2010). Plant lignans 

which are pinoresinol, steganacin and podophyllotoxin 

(PPT) are also polyphenolic compounds (Konuklugil, 

1994). PPT and its derivatives like etoposide and 

teniposide display a wide selection in medical 

applications such as purgative, vesicant, antirheumatic, 

antiviral, and antitumor agents. Their anticancer activities 

have been heavily under study and they were used in 

various chemotherapies, including lung cancer, 

lymphomas, and genital tumors (Gordaliza et al., 2004). 

In addition to antiproliferative effects, many natural 

compounds obtained from plants and their modified forms 

have been investigated in terms of their drug resistance 

reversal activity (Ugocsai et al., 2005; Engi et al., 2006; 

Molnar et al., 2004). Multiple drug resistance (MDR) 

acquired against chemotherapy by cancer cells causes 

most of the chemotherapeutic agents not to show 

expected impact on the patients and leads to the 

progression of the disease. Therefore, a natural 

substance which has low toxicity may potentially be used 

as a drug resistance reversal agent. In this study, five 

endemic Bupleurum species have been collected at 

different locations in Konya (Turkey) between May to 

August and their root extracts have been investigated in 

terms of their antioxidant activities, total phenolic 

contents and antiproliferative effects on MCF-7 cells. 

Phenolic compounds, saikosaponins and podophyllotoxin 

contents were further determined by high-performance 

liquid chromatography (HPLC) in detail. MCF-7 cell line is 

a model cell line for breast cancer. Paclitaxel (MCF- 

7/Pac) and vincristine (MCF-7/Vinc) resistant cell lines 

which were previously developed from sensitive MCF-7 

cells (MCF-7/S) were used in this study (Kars et al., 

2011b; İşeri et al., 2010). The resistant cell lines 

overexpress MDR1 gene and developed various 

resistance mechanisms as previously reported (Kars et 

al., 2011a). Not only the total root extracts but also the 

compounds which were found in high amount in the 

extracts of five endemic Bupleurum species have been 

tested on viability of sensitive and resistant MCF-7 cells. 


Plants and preparation of extracts 

Endemic Bupleurum sulphureum, Bupleurum lycaonicum, 

Bupleurum turcicum, Bupleurum heldreichii, Bupleurum 

pauciradiatum were collected from Central Anatolia, Konya 

province in Turkey between May and August (voucher specimens 

are deposited at the Herbarium in Selçuk University, Turkey: HP 

1001-1005 KNYA). 

Specific locations from where endemic Bupleurum species were 

collected are given in Table 1. The roots of plants were separated 

from the main plant bodies. Plant roots were grinded in a blender as 

powder form. 10 g of root powder from each species was extracted 

in 60 ml 70% ethanol by ultrasonication (Transsonic digitals) at 

100% power, 25 to 37°C, for 60 min. In order to protect heat-labile 

substances in the plant roots, ultrasonication has been chosen as 

an extraction method as it is done at relatively low temperatures. 

The remaining plant material was separated from the extract by 

filtration (Watman No. 1). The solvent of each extract was 

evaporated by rotary evaporator (Heidolph Laborota 4000), at room 

temperature for 40 to 45 min. The extracts were collected by 

distilled water to the vials and were frozen overnight at -20°C. 

Finally they were freeze dried in lyophilizer (Edwards Modulyo 4K 

Freezer), at -45°C. Dried extracts were stored at 8 to 10°C, in dark 

vials until use. 

Total phenol content determination 

The Folin-Ciocalteu method was used to assay total phenolics in 

the extracts (Lowry et al., 1951). A sample of 20 μl was mixed with 

6.5 ml of deionised water. 0.5 ml of non-diluted Folin’s Reagent 

(Sigma) and 3 ml of 10% anhydrous sodium carbonate solution 

were then added to the mixture. The mixture was kept in shaking 

incubator for 30 min at 40°C for color development. The 

absorbance was measured at 765 nm. Results were reported as 

milligram of gallic acid equivalents per gram of extract (mg GAE/g 

extract). Assay was repeated for two times independently. 

DPPH antioxidant activity determination 

2,2-diphenyl-1-picrylhydrazyl (DPPH) is a stable highly colored free 

radical that can abstract labile hydrogen atoms from phenolic 

antioxidants with concomitant formation of a colorless hydrazine 

(DPPH-H) (Diouf et al., 2009). Extracts from each species were 

dissolved in methanol as 10 mg/ml concentration. The samples 

were diluted in range of 10 to 0.078 mg/ml. Several dilutions of 

Butylated hydroxytoluene (BHT, Sigma) and L-Ascorbic acid (L- 

AsA, Sigma) with high free radical scavenging activity were used as 

positive controls (200 to 1.56 µg/ml). 3 ml of DPPH solution (20 

mg/L) was added to the sample solution and vortexed vigorously for 

30 s. The mixture was kept at room temperature in dark for 30 min 

before the absorbance at 517 nm was measured. The scavenging 

activity was determined by comparing the absorbance with that of 

the blank (100%) containing only DPPH solution and solvent. The 

total free radical scavenging activity of each extract was expressed 

as the concentration of extract that reduce 50% of DPPH (IC50). The 

Assay was repeated for two times independently.

HPLC analysis 

Table 2. Total phenolic contents of the root extracts in terms of gallic acid equivalents. 

Extract source mg GAE/g extract ± SD 

B. sulphureum 34.68 ± 0.02 

B. lycaonicum 61.48 ± 0.01 

B. turcicum 34.48 ± 0.00 

B. heldrechii 31.48 ± 0.00 

B. pauciradiatum 33.71 ± 0.00 

SD: Standard deviation (p < 0.05). 

HPLC analysis was performed using Varian Prostar HPLC system 

with pursuit 5u C18 (150 × 4.6 mm) column and PDA detector. The 

following conditions were applied during separation of 

saikosaponins, phenolics and podophyllotoxin in the extracts. 

Saikosaponin A and saikosaponin D (Sigma) were used as 

standards and they were detected and separated in the samples at 

25°C, with PDA detector at 203 nm, in acetonitril: water mobile 

phase. The flow rate was 0.8 ml/min and flow condition was at t = 0 

(30:70), t = 10 min (30:70), t = 18 min (40:60), t = 28 min (45:55), t 

= 35 min (45:55), t = 49 min (30:70). Cathechin, quercetin and 

isoquercitrin (Sigma) were used as standards for phenolic 

compounds. Standards and samples were detected and separated 

at 25°C, with PDA detector at 280, 360 and 360 nm for cathechin, 

quercetin, isoquercitrin respectively. Mobile phase was methanol: 

2.5% formic acid in water, flow rate was 1 ml/min, flow condition 

was t = 0 (0:100), t = 7 min (0:100), t = 42 min (20:80), t = 57 min 

(60:40), t = 58 min (0:100), t = 63 min (0:100). Podophyllotoxin 

(Sigma) was used as standard and the samples were detected and 

separated at 25°C, with PDA detector at 285 nm, in 

((methanol:acetonitril):water) mobile phase. The flow rate was 0.8 

ml/min and flow condition was at t = 0 ((20:30):50), t = 15 min 

((20:30):50. 

Cell lines 

In order to test the antiproliferative effect of plant root extracts, the 

sensitive and drug resistant MCF-7 cell lines have been used. The 

features and growth conditions of the parental cell line (MCF-7/S) 

and sublines resistant to 400 nM paclitaxel (MCF-7/Pac) and 120 

nM vincristine (MCF-7/Vinc) were described previously (Kars et al., 

2006; Kars et al., 2008). Not only the total root extracts but also the 

compounds which were found in high amount in extracts have been 

tested on aforementioned cell lines. 

Cytotoxicity assay 

The effects of the total extracts and compounds on the proliferation 

of sensitive and resistant MCF-7 cell lines were evaluated by 

means of the Cell Proliferation Kit (Biological Industries) in 96 well 

flat bottomed microtiter plates (Eskiocak et al., 2008). Briefly, first 

well is filled with 150 µl and all the wells except the cell control 

column (second) were filled with 100 µl medium. 200 µl of 

concentrated anticancer agent (prepared in medium) was added in 

to the third column and the compound was diluted horizontally by 

taking 100 µl portion of extract solution (3 mg/ml) or compound (100 

µM) from the third column and putting in to the next column. Finally, 

the cells were seeded in to 96-well microtiter plates 

(5×10 3 cells/well) and incubated for 72 h in medium containing 

horizontal dilutions of compound (except for medium control wells). 

Then, XTT reagent was applied to form a soluble dye. After 

Kars et al. 827 

incubation at 37°C for 4 h, the dissolution of formazan crystals that 

were produced by mitochondrial enzymes of the living cells 

occurred and then the optical density of chromogenic product was 

measured at 500 nm with a 96-well plate reader (BioTek microplate 

reader). The inhibition of cell proliferation and IC50 (inhibitory 

concentration 50) values were determined for each cell line. 

Statistics 

All the assays were repeated for two times independently. The 

results of assays were subjected to two-tailed t-test by using SPSS 

Software (SPSS Inc., Illinois, USA) to determine significant 

difference between means of groups (p < 0.05). The results were 

expressed as mean ± standard deviation. 

RESULTS 

Total phenol contents of plant extracts 

Natural phenols are very diverse group of compounds 

carrying one or more phenolic group, but some wellknown 

representatives of them could easily be identified 

in plant extracts by HPLC (Cai et al., 2004). In addition, 

total phenolics could be assayed by Folin-Ciocalteu 

method and expressed as milligram of gallic acid 

equivalents per gram of extract (mg GAE/g extract). Total 

phenolic contents of root extracts of B. sulphureum, B. 

lycaonicum, B. turcicum, B. heldrechii and B. 

pauciradiatum were found to be 35, 62, 35, 34 and 34 mg 

GAE/g root extracts respectively (Table 2). 

According to results, B. lycaonicum root extract has 

about 1.5 fold more total phenol content with respect to 

others (p < 0.05). 

DPPH antioxidant activity of plant extracts 

Free radical scavenging activities (FRSA) of the plant 

root extracts have been determined using DPPH which is 

a stable highly colored free radical. B. turcium root extract 

showed the highest free radical scavenging activity with 

57.37 µg/ml IC50 value (p


Table 3. Free radical scavenging activities (FRSA) of total extracts and known 

standards in terms of IC50. 

Extract source FRSA(IC50) (µg/ml) � SD 

B. sulphureum 403 ± 1.88 

B. lycaonicum 443 ± 2.76 

B. turcicum 57.37 ± 2.67 

B. heldrechii 184±2.19 

B. pauciradiatum 165 ± 1.82 

L-AsA 2.26 ± 0.17 

BHT 2.45 ± 0.30 

FRSA: Free radical scavenging activity; SD: standard deviation (p < 0.05); L-AsA: Lascorbic 

acid; BHT: butylated hydroxyltoluene. 

Table 4. HPLC results presenting the amounts of compounds in the root extracts. 

Compound mg/g extract ± SD 

Extract source Saikosaponin A Saikosaponin D Podophyllotoxin 

B. sulphureum ND ND ND 

B. lycaonicum 6.842 ± 0.146 1.735 ± 0.031 0.132 ± 0.003 

B. turcicum 12.990 ± 2.065 17.958 ± 0.308 ND 

B. heldrechii 3.608 ± 0.293 7.128 ± 0.400 0.496 ± 0.006 

B. pauciradiatum 8.200 ± 0.580 18.173 ± 0.223 ND 

ND: not detected; SD: standard deviation (p < 0.05). 

Table 5. HPLC results presenting the amounts of compounds in the root extracts. 

Compound mg/g extract ± SD 

Extract source Catechin Isoquercitrin Quercetin 

B. sulphureum 0.12 ± 0.03 5.78 ± 0.34 0.28 ± 0.03 

B. turcicum 0.11 ± 0.03 1.85 ± 0.11 0.21 ± 0.02 

B. lycaonicum ND 20.39 ± 1.23 0.51 ± 0.04 

B. heldrechii ND 2.27 ± 1.37 ND 

B. pauciradiatum ND 12.49 ± 0.75 0.39 ± 0.03 

ND: not detected; SD: standard deviation (p < 0.05). 

HPLC analysis of plant extracts 

Major well-known groups of phenolic and saponin 

compounds have been elucidated by HPLC and the 

results are listed in Tables 4 and 5. Saikosaponin A, 

saikosaponin D and isoquercitrin were detected in higher 

amount in the Bupleurum root extracts when compared to 

others. 

Antiproliferative activities of plant extracts and 

selected compounds 

Root extracts affected sensitive and drug resistant 

MCF-7 cell viabilities. According to the IC50 values, B. 

turcicum and B. pauciradiatum root extracts are more 

toxic to the MCF-7/Pac cell line than the other three plant 

extracts. Regarding MCF-7/Vinc cell line, B. heldrechii 

root extract was found to be the most toxic among the 

others. IC50 values are listed in Table 6. In addition to 

total extracts, the effects of saikosaponin A, saikosaponin 

D and isoquercitrin which were detected in higher 

amounts in the root extracts were also tested for 

cytotoxicity. IC50 values for saikosaponin A and 

saikosaponin D are presented in Figure 1. The highest 

application dose of isoquercitrin for XTT test was 1200 

µM however the compound did not exert any cytotoxicity 

and it was accounted as non-toxic with very high IC50

Table 6. Antiproliferative effects of root extracts on MCF-7 cell lines. 

Cell line Reagent/ Extract IC50 (mg/ml) � SD 

Paclitaxel 1.89 ×10 

MCF-7/S 

-3 ± 0.28 × 10 -3 

Vincristine 0.05 ± 0.00 



B. turcicum 0.22 ± 0.10 

B. heldrechii 0.79± 0.11 


MCF-7/Pac 

MCF-7/Vinc 

values for three cell lines. 

DISCUSSION 

SD: standard deviation (p < 0.05). 

Paclitaxel 0.27 ± 0.00 



B. turcicum 0.85 ± 0.00 



Vincristine 0.015 ± 0.003 



B. turcicum 3.47 ± 0.66 



Figure 1. Effects of saikosaponin A and saikosaponin D on the proliferation of cell 

lines. Error bars represent mean ± SD (p < 0.05). 

Cancer is a serious clinical problem and has a significant 

impact on the human health. Although, there are 


significant efforts developing new therapeutic strategies, 

the disease still affects millions of patients worldwide. 

Natural products including plants and microorganisms 

provide rich sources for anticancer drug discovery 

(Schwartsmann et al., 2002). Based on ancient and 

modern herbal medicine sources and Pharmacopoeia, 

there are many anticancer plants for the identification of


new sources for cancer therapy, and receive scientific 

attention recently (Bonham et al., 2002; Hu et al., 2002; 

Kao et al., 2001; Yano et al., 1994). For the purpose of 

finding the potential anticancer agents and/or MDR 

modulating agents, from natural sources, five endemic 

Bupleurum species have been collected from various 

locations in Turkey between May and August. And, 

ethanol extracts of the plant roots have been prepared 

using ultrasonication for the first time to test the 

cytotoxicity on the MCF-7/S, MCF-7/Pac and MCF-7/Vinc 

cell lines. We also clarified the major chemical contents 

of the root extracts in terms of phenolics, saikosaponins 

and podophyllotoxin amounts. 

The extracts were finally compared by their free radical 

scavenging activities. It was reported previously that 

ethanol extracted Chinese herbal mixture including 

Bupleurum species exerted FRSA about 430 µg/ml (Liu 

et al., 2005) which is very close to that of B. sulphureum 

and B. lycaonicum found in this study. However, B. 

turcicum has the highest FRSA activity with 57 µg/ml in 

our study. According to our findings, B. lycaonicum has 

the highest total phenolic content and also coherently it 

has the highest isoquercitrin amount among the other 

extracts. In a study done by Cai et al. (2004), it was 

reported that B. scorzonerifolium had 470 mg total 

phenolics/g extract, which is about 8 fold more than that 

of B. lycaonicum found in this study. 

When we consider the HPLC analysis results, the total 

extracts were found to contain saikosaponin A, 

saikosaponin D and isoquercitrin in considerable 

amounts. On the contrary, cathechin, quercetin and 

podophyllotoxin contents of the root extracts were not 

enough to be considered in cytotoxicity tests. In paralel to 

our findings saikosaponins, quercetin, rutin and 

isoquercitrin were found to be the major compounds of 

several Bupleurum species reported by different authors 

(Barrero et al., 2000; Cai et al., 2004). 

Antiproliferative effects of the total root extracts on drug 

resistant MCF-7 cell lines demontrate that the root 

extracts from the endemic Bupleurum species may be 

used in anticancer therapy strategies. According to the 

report of Cheng et al. (2005), acetone extracted B. 

scorzonerifolium root is about 10 fold more toxic to the 

different cancer cells including MCF-7 than the endemic 

species in our study. 

This may be due to the species differences or due to 

the extraction method. In addition to the total root 

extracts, we also tested the cytotoxicity of dominating 

molecules in the total extracts on the cell lines. 

Saikosaponin A and D exerted antiproliferative effects to 

some extend on MCF-7/S and MCF-7/Vinc that was 

about 1.5 to 2 fold more than that of on MCF-7/Pac. A 

study of Hsu et al. (2004) declares that saikosaponin D 

exerted antiprolifreative activity on A549 lung cancer cell 

line with IC50 value of 10 µM that is close to our findings 

with about 1 to 2 fold difference. When the 

antiproliferative effects of total extracts were compared to 

that of saikosaponin compounds, it is revealed that the 

saikosaponins are more cytotoxic to the cells when 

applied individually than root extracts which contain 

numerous compounds. Antiproliferative effects of 

saikosaponin A and D are dramatically high (50 to 150 

fold) on the cells than root extracts. 

Therefore, it can be deduced that saikosaponin A and 

saikosaponin D are important active ingredients of the 

endemic Bupleurum root extracts. Saponins make 

complex with cholesterol and produce pores on cell 

membrane and may induce apoptosis (Li et al., 2005). 

Saponins have amphipathic nature that may cause the 

absorption of macromolecules and polar drugs. It was 

reported that triterpen saponin obtained from Phytolacca 

americana reversed MDR in resistant ovarian cancer 

cells (Wang et al., 2008). In addition, Li et al. (2005) 

presented that saikosaponin A and D affected membrane 

fluidity. Most of the MDR reversing agents alter 

membrane fluidity and increase membrane permeability 

(Drori et al., 1995; Callaghan et al., 1993). The changes 

in the lipid membrane may modify the functional 

conformation of the P-gp. Alterations in the physical 

state of plasma membrane lipids can influence a 

number of important carrier-mediated processes and 

they appear to be important factors modulating efflux 

pump systems (Shin et al., 2006). Therefore, based on 

these facts, saikosaponins obtained from the endemic 

Bupleurum roots may be potential agents for MDR 

reversal. 

According to results presented here, isoquercitrin was 

not cytotoxic to the cell lines. This may be due to 

chemical characteristics or physiological effects of the 

compound in the cells (Table 7). The root extracts were 

found to contain considerable amount of phenolic 

compound isoquercitrin. Plant phenolics are known to 

have antioxidant activities and chemopreventive features 

(Cai et al., 2004). Phenolic compounds produce 

phenolates by hydroxyl groups of proteins in 

physiological conditions. Then they make hydrogen 

bonds with electronegative atoms of peptides or ionic 

bonds with basic aminoacids. These interactions may 

alter the three dimensional structures or activities of 

proteins. Manthey and Guthrie (2002) reported that P-gp 

may be inhibited due to such interactions by phenolic 

compounds. 

To conclude, these novel findings indicate that root 

extracts of the endemic Bupleurum species contain 

valuable compounds like saponins and phenolics. In 

addition, they have both free radical scavenging and 

antiproliferative activities. As further studies in the light of 

these results, MDR reversal activities of saikosaponin A, 

saikosaponin D and isoquercitrin will be evaluated in Pgp 

overexpressing MCF-7 cells by fluorescent methods. 

Their interactions with anticancer agents will also be 

determined by combination drug therapy in-vitro. 

Accordingly, we will suggest endemic B. turcicum, B. 

sulphureum, B. lycaonicum, B. pauciradiatum and

Table 7. Chemical formula and structure of agents found high amount in root extracts. 

http://www.chemblink.com, http://www.chemicalbook.com. 

Agents Chemical structure Chemical formula 

Saikosaponin A 

Saikosaponin D 

Isoquercitrin 

B. heldrechii for the use in anticancer therapy and/or as 

MDR reversal agents as natural sources in future. 


This study was supported by TÜBİTAK with the project 

number 110T552 and by the Selçuk University Research 

Fund with the project number 10401041. Dr. Özlem 

Darcansoy İşeri is also gratefully acknowledged for her 

contributions. 

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DOI: 10.5897/JMPR11.1482 



Effects of astragaloside IV on L-type calcium channel 

currents in adult rat ventricular myocytes 

Shiqi Lu 1 *, Guolin Chen 1 , Yiming Zhao 1 and Wanping Sun 2 

1 Department of Emergency, The First Affiliated Hospital of Soochow University, Suzhou 215006, China. 

2 Department of Pharmacology, College of Pharmaceutical Science, Soochow University, Suzhou 215123, China. 


Astragaloside IV (AGS-IV), a traditional Chinese herb, has been found to have potent cardioprotective 

effects. However, the relevant mechanisms still remain unclear. In the present study, using standard 

whole-cell patch clamp recording technique, we identified a novel functional role of AGS-IV in regulating 

L-type calcium channel currents (L-currents) in adult rat ventricular myocytes. Our results showed that 

AGS-IV exerted inhibitory effects on L-currents of the single adult rat ventricular myocytes. This 

L-current inhibitory action of AGS-IV was concentration-dependent. The current density at +10 mV was 

reduced by about 48.7% after exposure of the cells to AGS-IV (1 µM), from the control value of 13.7±1.7 to 

7.6±1.2 pA/pF. AGS-IV did not markedly affect the activation of L-currents but significantly shifted the 

inactivation curve to the left. Pretreatment of cells with H89, a protein kinase A (PKA) inhibitor, 

completely abolished AGS-IV-induced L-current inhibition. Similar results were obtained by another PKA 

inhibitor PKI 5-24. Taken together, these results suggested that AGS-IV inhibited L-currents via a 

PKA-dependent pathway in rat ventricular myocytes, which could contribute to its cardioprotective 

effects. 

Key words: Astragaloside IV, L-type calcium channels, ventricular myocytes, patch clamp. 

INTRODUCTION 

Astragalus membranaceus, a traditional Chinese herb, 

has been reported to have a range of pharmacological 

effects. Astragaloside IV (AGS-IV; 3-O-β-D- 

xylopyranosyl-6-O-β-D-glucopyranosyl-cycloastragenol, 

as the main active ingredient of Astragalus 

membranaceus, is a small molecular weight (MW 784) 

saponin. Current clinical and laboratory-based research 

has focused on the effects of AGS-IV with regards to 

anti-inflammation (Zhang et al., 2003), immunomodulation 

(Wang et al., 2002), ischemia injury protection (Zhang et 

al., 2006; Luo et al., 2004), and cardioprotection (Luo et 

al., 1995) etc. 

In cardiovascular system, AGS-IV produces protection 

against cardiac arrhythmias in animal and clinical trials. It 

exerts the protective action against hypoxia, and reduces 

the level of lactate dehydrogenase (LDH) and superoxide 

*Corresponding author. E-mail: lushiqi2004@126.com. 

dismutase (SOD) during myocardial infarction (Luo et al., 

1995). However, the direct evidence for the action 

mechanism of AGS-IV on the cardiomyocytes is still 

lacking. It is well established that cardiac ischemia/ 

reperfusion damage is associated with calcium overload 

((Stamm and del Nido, 2004) therefore, these 

cardioprotective effects of AGS-IV suggest that it may 

reduce cardiac intracellular calcium, which is highly 

relevant to the state of L-type calcium channels in 

cardiovascular system. However, to date, the detailed 

mechanisms remain still unclear. More research on the 

cellular mechanisms of AGS-IV will not only contribute to 

the understanding of the efficacies of astragalus 

membranaceus for clinical treatment, but would also lead 

to the development of potential therapeutic strategies for 

the treatment of relative diseases. 

In the present study, using the whole-cell patch clamp 

technique we first demonstrated that AGS-IV played a 

novel role in modulating L-type calcium channels in adult 

rat ventricular myocytes. Based on pharmacological


manipulation of AGS-IV-induced L-current inhibition, we 

propose that this response is coupled to a 

PKA-dependent pathway and could contribute to the 

cardioprotective effects induced by AGS-IV. 


Reagents and solutions 

All drugs were obtained from Sigma (USA), unless otherwise 

indicated. Astragaloside IV (JC Biotech, NJ) was prepared in 

dimethyl sulfoxide (DMSO). The concentration of DMSO in the bath 

solution is expected to be less than 0.01%, and had no functional 

effects on L-type calcium channels. The composition of the external 

solution for L-type calcium channel current recording was composed 

of (in mM): TEA-Cl 140, BaCl2 5, MgCl2 0.5, glucose 5.5, CsCl 5, 

and HEPES 10, pH 7.35, with TEA-OH. The pipette solution 

contained (in mM): CsCl 110, EGTA 10, ATP-Mg 4, GTP-Na 0.3, 

HEPES 25, Tris-phosphocreatine 10, pH 7.3 with CsOH, 290 mOsm. 

The Tyrode’s solution was composed of the following (in mM): NaCl 

150, KCl 5.4, MgCl 2.0, HEPES 10, glucose 11. pH was adjusted 

with NaOH to 7.4. The KB solution was composed of the following 

(in mM): KOH 70, KCl 40, L-glutamic acid 50, taurine 20, KH2PO4 20, 

MgCl2 3, HEPES 10, EGTA 0.5, glucose 10 (pH 7.35 to 7.40 with 

KOH). 

Isolation of single cardiac myocytes 

The animal use protocol was reviewed and approved by the Animal 

Care and Use Committee of Soochow University. Ventricle 

myocytes were dissociated enzymatically by a modified method 

described previously (Tao et al., 2004). Briefly, Sprague-Dawley rats 

(male, 200 to 300 g) were injected intraperitoneally with 1000 IU 

heparin and euthanized in a CO2 chamber. The heart was quickly 

removed and transferred to an ice-cold Tyrode’s solution. The aorta 

was cannulated and the heart was mounted on a Langendorff 

apparatus. The heart was perfused with prewarmed (at 37°C) and 

oxygenated Tyrode solution containing Protease Type XIV (Sigma) 

Collagenase Type I (Sigma C0130) for 10 min until the heart was 

flaccid. The ventricles were dissected out, cut into small pieces, and 

gently stirred in the Tyrode’s solution. Isolated cells were filtered and 

maintained in an oxygenated KB solution on ice. Only the cells with 

a rod shape and clear cross striation were used for experiments. 

The whole-cell patch clamp 

Voltage-clamp experiments for recording cardiac myocytes were 

performed at room temperature (20 to 22ºC) as described previously 

(Tao et al., 2009; Zhang et al., 2010; Wang et al., 2011). Electrodes 

were pulled from borosilicate glass microcapillary tubes (World 

Precision Instruments). They had resistances from 2 to 3 MΩ when 

filled with internal solution. We made recordings using a MultiClamp 

700B amplifier (Molecular Devices) and controlled voltage 

commands and digitization of membrane currents using a Digidata 

1440A interfaced with Clampex 10.2 of the pClamp software 

package (Molecular Devices), running on a personal computer. 

Currents were low-pass filtered at 2 to 5 kHz. Series resistance (Rs) 

and capacitance (Cm) values were taken directly from readings of 

the amplifier after electronic subtraction of the capacitive transients. 

Series resistance was compensated to the maximum extent 

possible (at least 70%). Current traces were corrected for linear 

capacitive leak with online P/6 trace subtraction. The steady-state 

inactivation curves were obtained from experiments by stepping 

from a holding potential of − 70 mV to a 30-ms normalizing pulse to 

0 mV followed by a family of 5-s-long prepulses from − 100 to 20 mV. 

A 100-ms test-pulse to 0 mV was recorded finally. Each test-pulse 

was normalized to the maximal current amplitude of the normalizing 

pulse. 

Data analysis 

All data are expressed as mean±S.E.M., and GraphPad Prism 

software was used for electrophysiological data plotting. Student’s 

t-tests or one-way ANOVA were used to compare the different 

values, and were considered significant at P

Current 

Control Current 

Control 

Lu et al. 835 

Figure 1. Characterization of L-type calcium channel currents in adult rat ventricular myocytes. A-B, Exemplary traces and 

pooled data showed the effects of Ni 2+ (100 μM, n=7, (A) or nifedipine (10 μM, n=6, (B) on barium currents elicited by a 

depolarizing step pulse from the holding potential of -70 mV to 0 mV. Current with 5 mM barium as a charge carrier were 

elicited by a 500-ms long depolarization step pulse. ***p


Peak current (nA) 

Current density (Pa/pF) 

Figure 2. AGS-IV dose dependently inhibited L-currents. A-C, Time course (A), exemplary traces (B) and 

pooled data (C) of L-type calcium channel currents (L-currents) recorded under the control conditions, during 

exposure to 1 µM AGS-IV, and washout. D, Dose-response curve for the inhibitory effects of AGS-IV on 

L-currents. The line represents the best fit of the data points to the sigmoidal Hill equation (see Methods and 

Materials). Number of cells tested at each concentration of AGS-IV is indicated in brackets. 

the presence of AGS-IV at 1 µM. These results suggested 

that AGS-IV did not affect the voltage-dependent 

activation. 

AGS-IV leftward shifted steady-state inactivation 

curve 

We further investigated whether the electrophysiological 

properties of L-type calcium channels were affected by 

AGS-IV. Steady-state activation and inactivation 

potentials of L-type calcium channels were then 

investigated (Figure 4A and B). We did not observe a 

significant shift in the hyperpolarized direction of the 

activation potential (V1/2 from -11.9±0.7 to -12.1±1.2 mV, 

Inhibition % by 0.1 �M ghrelin 

and k value from 6.5±0.8 to 6.3±0.7) (Figure 4A). 

However, AGS-IV at 1 μM leftward shifted the 

steady-state inactivation potentials of L-type Ca 2+ 

channels by -11 mV (V1/2 from -21.7±1.9 to -32.5±1.7 mV, 

and k value from -9.3±0.3 to -11.6±0.8) (Figure 4B). 

These results suggested that the reduced L-currents 

observed upon application of AGS-IV could be due to 

more channels remaining in the inactivated state after 

activation. 

Protein kinase A is involved in the AGS-IV-induced 

L-current inhibition 

We next investigated the detailed mechanism underlying

Figure 3. Effects of AGS-IV on current-voltage (I-V) curve. A-B, Exemplary traces (A) and 

pooled data (B) showed the effects of 1 µM AGS-IV on I-V curve. I-V curves were obtained 

from a holding potential of -70 mV, 500 ms depolarizing pulses to different membrane 

potentials (10 mV increments from -40 mV to +60 mV). 

Figure 4. AGS-IV hyperpolarized shifted steady-state inactivation curve. A, The steady-state 

activation of L-type calcium channels is not altered by 1 µM AGS-IV application. Tail currents were 

elicited by repolarization to -70 mV after 200 ms test pulses from -70 to 0 mV in increments of 10 

mV. B, AGS-IV shifted steady-state inactivation curve of L-type calcium channels to the 

hyperpolarizing direction. The steady-state inactivation curves were obtained from experiments 

by stepping from a holding potential of − 70 mV to a 30-ms normalizing pulse to 0 mV followed by 

a family of 5-s-long prepulses from − 100 to 20 mV. A 100-ms test-pulse to 0 mV was recorded 

finally. Each test-pulse was normalized to the maximal current amplitude of the normalizing pulse. 

AGS-IV-induced L-current inhibition. To determine 

whether protein kinase A (PKA) was involved in 

AGS-IV-mediated L-current inhibition, we pre-incubated 

the cells with a PKA inhibitor, H89 (1 μM). Our results 

showed that pretreatment of cells with H89 abolished the 

AGS-IV-induced L-current inhibition in adult rat ventricular 

Lu et al. 837 

myocytes (inhibition%=2.7±1.1, Figure 5A and B). In 

contrast, H85 (1 μM), a structurally related but inactive 

analogue, had no effect on AGS-IV-induced L-current 

effects (not shown). To further confirm the PKA-mediated 

L-current inhibition, we dialyzed the cells with a pipette 

solution containing another PKA inhibitor, PKI 5-24. Our


Figure 5. PKA was involved in AGS-IV induced L-current inhibition. A-D, Exemplary current traces and 

pooled data showed the effects of AGS-IV (1 µM) on L-currents in the presence of H89 (1 µM, A and B) or 

PKI 5-24 (1 µM, C and D). 

results showed that intracellular application of PKI 5 to 24 

(1 μM) to the recording pipette solution blocked 

AGS-IV-induced L-current inhibition (Figure 5C and D). 

DISCUSSION 

Peak current (pA) 

Peak current (pA) 

AGS-IV, the main functional ingredient of the Chinese 

herb Astragalus membranaceus, has many 

pharmacological functions. Although AGS-IV has a 

cardioprotective effect in the model of ischemic injury (Luo 

et al., 1995), there is almost no immediate evidence to 

demonstrate the cellular mechanism of AGS-IV on the 

protective effect of cardiomyocytes. In the present study, 

we first demonstrated that AGS-IV played a novel role in 

modulating L-type calcium channels in adult rat ventricular 

myocytes. Based on pharmacological manipulation of 

AGS-IV-induced L-current inhibition, we propose that this 

response is coupled to a PKA-dependent pathway and 

could contribute to the cardiac protective effects. 

To elicit calcium currents, 500-ms-long pulses were 

applied from a holding potential of -70 to 0 mV under 

conditions where calcium was the only charge carrier for 

inward current (Heubach et al., 2000). This calcium 

current was completely blocked by 1 µM nifedipine, a 

specific L-type calcium channel blocker, indicating that it 

was not contaminated by K + , Na + , and T-type calcium 

currents (Tao et al., 2004). Furthermore, Na + free solution

was used to eliminate the possibility of contamination by 

Na + current. This strategy has the additional virtue of 

disabling Na + -Ca 2+ exchange. Rundown of ionic currents 

is always a concern in whole-cell voltage-clamp recording 

(Tao et al., 2004). We minimized time-dependent changes 

in L-currents by using high resistance pipettes filled with 

Mg-ATP 4 µM and beginning the experiments within 5 min 

after membrane rupture (Belles et al.,1998). 

AGS-IV exerted some influence on the inactivation 

kinetics of L-type calcium channels. It shifted the 

inactivation curve of calcium currents to the left. These 

data strongly suggested that AGS-IV exerted its inhibitory 

effect via stabilizing the inactivation state of L-type 

calcium channels. Binding to the inactivated state is an 

important feature of the AGS-IV block, since strong 

inactivation at physiological resting potentials of L-type 

calcium channels could contribute to tissue selectivity. For 

example, dihydropyridines (DHP) that bind preferentially 

to the inactivated state of L-type calcium channels are 

useful as anti-hypertensive drugs by acting on vascular 

smooth muscle while having little effect on the heart 

(Triggle, 1992). 

There are several examples of L-current modulation by 

protein kinase C (PKC) activation. In mouse hippocampal 

neurons, activation of PKC inhibits L-currents while an 

inactive analogue has no effect (Zhang et al., 2010). By 

contrast, PKC-induced L-current increase was also 

described in rat and in chick ventricular myocytes (Bray 

and Mynlieff, 2011). In the present study, we found that 

the inhibitory effects of AGS-IV on L-currents were PKA, 

but not PKC-dependent. Our data showed that 

pretreatment of ventricular myocytes with H89 could 

completely abolish the inhibition of AGS-IV on L-currents. 

Our present results were supported by previous studies 

that L-current inhibition by CB1 cannabinoid receptor 

activation in GT1-7 hypothalamic neurons was prevented 

by application of PKA inhibitors (Hoddah et al., 2009). 

Similarly, Cav1.2 L-current inhibition by integrin receptor 

activation was blocked by addition of PKA inhibitor H89 

(Gui et al., 2006). In contrast, some previous workers 

have reported that T-type Ca 2+ currents were mildly 

affected by PKA (Collis et al., 2007). On the contrary, 

L-currents recorded from isolated cardiomyocytes from 

adult canine left ventricles, were shown to be increased 

by the cAMP-PKA pathway (Xiao et al., 2011). Together, 

these results suggest that the PKA regulatory effects on 

L-currents can be variable in different tissues expressing 

different L-type calcium channel subtypes (Cav1.2 or 

Cav1.3). 

In conclusion, this is the first report to 

electrophysiologically detail the inhibitory effects of 

AGS-IV on L-type calcium currents in adult rat ventricular 

myocytes. Our results provide ionic evidence of a possible 

link between the cardiacprotective effect of AGS-IV and 

calcium channels. Since L-type calcium channel inhibitors 

possess a high therapeutic potential in treating conditions 

like myocardial ischemia and hypertension (Tao et al., 

2004), AGS-IV may also be a potential therapeutic 

Lu et al. 839 

traditional Chinese Medicine agent under some 

pathological conditions, such as ischemia, hypoxia and 

myocardial infarction, in which calcium-overload plays an 

important role. 


This work was supported by the Youth Foundation of 

Soochow University (Q3134826). 

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IV protects against ischemic brain injury in a murine model of 

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pathway. Cell. Signal., 21(9): 1436-1443. 

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on T, B lymphocyte proliferation and peritoneal macrophage 

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peptide-1 enhances cardiac L-type Ca 2+ currents via activation of 

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Cell. Signal, 22: 1660-1668.



DOI: 10.5897/JMPR11.1484 



Internal transcribed spacer-based identification of 

Bupleurum species used as sources of medicinal herbs 

Young Hwa Kim 1 , Eung Soo Kim 2 , Byoung Seob Ko 1 , Md. Romij Uddin 3 , Seung Eun Oh 4 , 

Go Ya Choi 1 , Seong Wook Chae 1 , Hye Won Lee 1 , Je Hyun Lee 5 , Ju Young Park 6 and 

Mi Young Lee 1 * 

1 Aging Research Center, Korea Institute of Oriental Medicine, 1672 Yuseongdae-ro, Yuseong-gu, Daejeon 305-811, 

Korea. 

2 National Forensic Service, Seoul, Korea. 

3 Department of Crop Science, Chungnam National University, Daejeon, Korea. 

4 Department of Biological Sciences, Konkuk University, Seoul, Korea. 

5 Department of Oriental Medicine, Dongguk University, Gyeongju, Korea. 

6 Korea Food and Drug Administration, Cheongwon, Korea. 


Bupleuri Radix is a medicinal herb that is widely used in Asia, and several Bupleurum species are used 

as a source of active ingredients for traditional Chinese medicines. Among these species, Bupleurum 

longiradiatum Turcz., is toxic and is not used as a medicinal herb. Because B. longiradiatum is 

morphologically similar to other Bupleurum species, it is important to distinguish between them. In 

other to address this problem, we developed a polymerase chain reaction (PCR) method based on 

internal transcribed spacer (ITS) nucleotide sequences to discriminate B. longiradiatum from 

Bupleurum falcatum L. and Bupleurum chinense DC., which are the most frequently used medicinal 

Bupleurum species. The discriminatory power of the method was verified by gel and capillary 

electrophoresis. Three PCR primers specific to B. longiradiatum were developed for use in this assay to 

enable us reveal the four B. chinense haplotypes. 

Key words: Bupleurum longiradiatum, medicinal herb, molecular identification, internal transcribed spacer. 

INTRODUCTION 

Bupleuri Radix is a medicinal herb that is used 

extensively in Korea, China and Japan. It possesses 

numerous pharmacological activities, including 

antipyretic, analgesic, immuno-modulatory, antiinflammatory, 

antiallergic, anticlotting, antiatherosclerotic, 

and antitussive effects (Park et al., 2000; Kim and Park, 

2001). Its major constituents include triterpenoid 

glycosides (saikosaponins), essential oils and 

polysaccharides. The saikosaponins are the principal 

bioactive components (Hsu et al., 2000; Chiang et al., 

2003) and saikosaponin-a and saikosaponin-d are the 

dominant derivatives (Tian et al., 2009). Bupleuri Radix is 

*Corresponding author. E-mail: mylee@kiom.re.kr. Tel: 

+82428689504. Fax: +82428689301. 

known differently in Korea, China and Japan. The 

Japanese pharmacopoeia states that Bupleuri Radix is 

derived from Bupleurum falcatum L. (Apiaceae) (Ministry 

of Health, Labour and Welfare, 2006). The Korean Herbal 

Pharmacopoeia recognizes B. falcatum and B. chinense 

DC as Bupleuri Radix (Korea Food and Drug 

Administration, 2007). The Chinese Pharmacopoeia 

Commission confirms B. chinense and B. 

scorzonerifolium Willd. as Bupleuri Radix (Chinese 

Pharmacopoeia Commission, 2010).Thirty-six Bupleurum 

species and varieties are commonly used as Bupleuri 

Radix in various areas (Pan, 1996; Yang et al., 2007). 

Bupleurotoxin and acetylbupleurotoxin bestow toxic 

properties upon B. longiradiatum Turcz (You et al., 2002). 

Therefore, China prohibits the use of B. longiradiatum as 

a herbal drug source. B. longiradiatum Turcz. can be 

confused with more than 20 other Bupleurum species


Table 1. Bupleurum plant materials. 

Scientific name Collection location Collection date Voucher no Figure 2 Lane# 

Jinan and Korea 2002.5 G039005 1 

Sancheong and Korea 2006.9 G039022 2 

B. falcatum L. 

Pohang and Korea 2006.10 G039023 3 

Suwon and Korea 2007.7 G039029 4 

Toyama and Japan 2003.12 G039062 5 

B. chinense DC. 

B. longiradiatum Turcz. 

used as Bupleuri Radix because of similarities in root 

shapes (Yang et al., 2007; Huang et al., 2009). 

Processing of medicinal plants leads to changes in their 

morphology and chemical constituents (Hon et al., 2003; 

Yang et al., 2007). 

Various methods of discriminating medicinal herbs by 

using genetic tools facilitate accurate identification, 

despite such changes. For example, the nucleotide 

sequence of the ITS has been used successfully in 

studies aimed to define phylogenetic relationships among 

species and related genera (Wang et al., 2007; Lee et al., 

2008; Lin et al., 2008; Ryuk et al., 2010; Sun et al., 

2010). Up to date, more publications have revealed that 

ITS2 can serve as a novel universal barcode for the 

identification of a broader range of medicinal plant 

species and genera (Chen et al., 2010; Yao et al., 2010), 

but no method was reported to accurately identify 

medicinal plant of a species. ITS sequence analysis has 

also been used to identify Bupleurum species in China, 

(Yang et al., 2007; Xie et al., 2009), in a Korean study of 

Bupleurum species (including a native species) 

phylogeny (Moon et al., 2009), and for rapid 

discrimination of 3 Bupleurum species with a sequencespecific 

oligonucleotide probe (SSOP) array (Lin et al., 

2008). These studies identified species by simply 

comparing their ITS nucleotide sequences, but more 

simpler and effective methods for discrimination of 

Bupleurum species are necessary. Diagnostic analysis of 

PCR amplification products is typically achieved with gel 

electrophoresis (GE). Because capillary electrophoresis 

(CE) can improve resolution compared to GE, this 

technique more accurately discriminates PCR products. 

CE has been proven to be very useful for identification of 

Inner Mongolia and China 2004.8 G039012 6 

Jeonju and Korea 2006.6 G039019 7 

Sichuan and China 2007.6 G039026 8 

Qinghai and China 2007.7 G039027 9 

Gansu and China 2004.4 G039063 10 



Pyeongchang and Korea 2006.8 G039047 13 

Seongnam and Korea 2007.6 G039048 14 

Seongnam and Korea 2007.6 G039053 15 

soil bacteria (King et al., 2005), detection of genetically 

modified organisms during food production (Kim et al., 

2005), and plant nitrogen-glycan estimation using highthroughput 

quantitative techniques (Lee et al., 2009). In 

this study, we developed a reliable deoxyribonucleic acid 

(DNA) markers based on the ITS sequence to 

discriminate B. longiradiatum from B. falcatum and B. 

chinense and consummated the phylogenetic analysis of 

Bupleurum genus. The use of specific primers allowed us 

to unambiguously differentiate the PCR products of B. 

longiradiatum from those of other Bupleurum species by 

GE and CE, we first identify the most frequently used 

medicinal Bupleurum species by ITS sequences and 

pave way for further phylogenetic and/or evolutionary 

studies on B. longiradiatum and other Bupleurum 

species. 



The plant materials were collected from various locations in Korea, 

China and Japan. Prof. Je Hyun Lee of the Dongguk University 

identified the samples on the basis of their morphologies. The 

voucher samples were deposited with the Korea Institute of Oriental 

Medicine (Table 1). Photographs of representative dried samples 

are shown in Figure 1. 

DNA extraction 

Plant materials were frozen with liquid nitrogen and milled into a 

fine powder. Total cellular DNA was extracted from the powder 

using a Nucleospin ® PlantII kit (MACHEREY-NAGEL GmbH). 

Genomic DNA concentrations were measured using a model

Figure 1. Photographs of representative dried samples of three Bupleurum species. 

ND-1000 spectrophotometer (NanoDrop). 

PCR analysis 

PCR reactions were performed in 25 µl volumes containing 20 ng 

genomic DNA, 2× PCR Pre-Mix (Solgent), 10 pmol primers ITS1 

and ITS4 (White et al., 1990), and distilled deionized water. 

Reaction conditions were 95°C for 4 min; 35 cycles at 95°C for 30 

s, 55°C for 40 s, 72°C for 1 min and 72°C for 5 min, using a DNA 

Engine ® apparatus (Bio-Rad). The desired product migrated as an 

approximately 700- bp band through a 1.5% (w/v) agarose gel, 

which was visualized by LoadingSTAR (Dynebio) staining under 

ultraviolet illumination. PCR products were subsequently purified 

using a NucleoSpin ® Extract II Kit (MACHEREY-NAGEL GmbH). 

Nucleotide sequencing and design of specific primers 

PCR products were subcloned using a pGEM-T Easy Vector 

System (Promega), was used to transform Escherichia coli HIT TM - 

DH5α and red blood cells (RBC). Plasmid DNAs were isolated 

using a Plasmid Mini-prep Kit (Solgent). The nucleotide sequences 

of the cloned PCR product were determined using 5 samples 

isolate from different colonies by Solgent Company Limited. The 

forward and reverse strands of all samples were sequenced. 

BioEdit version 7.0.5 (Hall, 1999) was used for multiple sequence 

alignments. Based on Bupleurum species ITS nucleotide 

sequences, a 20-mer BL-F primer (forward primer), 34-mer BL-R 

primer (reverse primer), and a 20-mer B-I-F primer (forward primer) 

were designed to discriminate B. longiradiatum, from B. falcatum, 

and B. chinense. The ITS4 primer was also used as a reverse 

primer. Primer locations (boxed) and their sequences are presented 

in Figure 2 and Table 2, respectively. 

Specific PCR and capillary electrophoresis 

PCR amplifications to be analyzed by GE were performed in 

reactions using 20 µl volume containing 10× buffer (750 mM Tris- 

HCl pH 8.8, 200 mM (NH4) 2SO4, 0.1% (v/v) Tween ® 20), 200 μM 

deoxynucleotide triphosphates (dNTPs), 2 mM MgCl2, 5 ng 

genomic DNA, 10 pmol each primer (Table 2), and 1 U 

Thermoprime Taq DNA polymerase (Thermo Fisher Scientific Inc.). 

Kim et al. 843 

PCR conditions were 94°C for 7 min; 35 cycles at 94°C for 30 s, 52 

for 30 s and 72°C for 1 min and 72°C for 7 min. PCR was 

conducted using a DNA Engine ® apparatus (Bio-Rad). Amplified 

products separated in a 2% (w/v) agarose gel, were stained with 

LoadingSTAR (Dynebio), and were analyzed using a U: Genius 

(Syngene). PCR amplifications analyzed by CE were performed in 

25 µl volumes containing 1 ng genomic DNA, 2.5 µl 10× reaction 

buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl2, 0.01% 

(w/v) gelatin) (Applied Biosystems), 0.2 mM dNTP mixture, 10 pmol 

each primer, and 1.25 U AmpliTaq Gold Ⓡ Polymerase (Applied 

Biosystems). The 5′-end of each reverse primer was fluorescently 

labeled with JOE (6-carboxy-4�, 5�-dichloro-2�, 7�-dimethoxyfluorescein) 

or FAM (9-carboxy fluorescein) (Table 2). PCR 

conditions were 95°C for 12 min; 30 cycles at 94°C for 30 s, 51°C 

for 30 s, and 72°C for 30 s; and 72°C for 7 min using a DNA 

Engine Ⓡ (Bio-Rad). The fluorescent PCR products were mixed with 

14.7 µl Hi-Di Formamide (Applied Biosystems) and 0.3 µl 

Genescan-500 ROX size standards (Applied Biosystems), 

processed by CE using an ABI PRISM Ⓡ 310 genetic analyzer 

(Applied Biosystems), were viewed using GeneScan 3.1 software 

(Applied Biosystems). 


The sequences (655 bp) of all B. falcatum samples were 

identical and were represented as ITS1 exon (225 bp), 

5.8S exon (163 bp), and ITS2 exon (267 bp) (Figure 2). 

The sequence was identical to that of B. falcatum (Gen 

Bank accession number, AJ131344). Nucleotide 

sequencing of B. chinense revealed 4 haplotypes 

designated hap1, 2, 3, and 4, based on the differences at 

8 positions (Table 3). The hap4 nucleotide sequence is 

657 bp length because of the insertion of G at positions 

206 and 207 bp, which is absent in other haplotypes 

(hap1, 2, and 3). The B. chinense ITS1 hap1, 2, and 3 

exons each are 225 bp length, and that of hap4, 227 bp 

length. The 5.8S and ITS2 exons are 163 and 267 bp 

length, respectively (Figure 2). Sequences of the 4 

haplotypes have been deposited in GenBank as


B. falcatum (AJ131344) 

B. chinense -1 (FJ561306) 




B. longiradiatum (FJ561305) 











B. chinense -4 FJ561309 


























Figure 2. Nucleotide sequence comparisons of Bupleurum species’ DNA amplified using ITS1/ITS4 primers. Bold boxes indicate sequences used to design specific primers for B. 

longiradiatum Turcz.

Table 2. PCR primers for ITS amplification and identification of B. longiradiatum. 

Kim et al. 845 

Primer sequence (5′→3′) Fluorophore Reference 

ITS1 

Amplification of ITS region 

TCC GTA GGT GAA CCT GCG G White et al. (1990) 

ITS4 TCC TCC GCT TAT TGA TAT GC White et al. (1990) 

B. longiradiatum-specific primers 

BL-F TAG GCC GGG GGT GCC TAG TT 

BL-R CAA TTT CCG TCC CCC CGA ACA GCA ACG ACT CGA T JOE (green) 

B-I-F GTC GTG CGG CTG GTT TAA AA 

ITS4 TCC TCC GCT TAT TGA TAT GC FAM (blue) White et al. (1990) 

Table 3. B. chinense sequence haplotypes. 

Haplotype 

44 51 70 

Position (bp) 

119 206 207 428 615 Accession no 

B. chinense hap1 A T T A - - G A FJ561306 

B. chinense hap2 A A C G - - G G FJ561307 

B. chinense hap3 A A T G - - G G FJ561308 

B. chinense hap4 T A T G G G T G FJ561309 

Table 4. Bupleurum species sequence characterization. 

Species Accession no Code 

BF 

Sequence identity (%) 

BC-1 BC-2 BC-3 BC-4 BL 

% GC 

B. falcatum AJ131344 BF - 58.63 

B. chinense hap1 FJ561306 BC-1 98 - 58.32 

B. chinense hap2 FJ561307 BC-2 98 99 - 58.78 

B. chinense hap3 FJ561308 BC-3 99 99 99 - 58.63 

B. chinense hap4 FJ561309 BC-4 98 99 99 99 - 58.60 

B. longiradiatum FJ561305 BL 99 97 97 98 97 - 58.08 

B. chinense hap1 (FJ561306), B. chinense hap2 

(FJ561307), B. chinense hap3 (FJ561308), and B. 

chinense hap4 (FJ561309). All B. longiradiatum ITS 

nucleotide sequences were identical. The B. 

longiradiatum sequence differed at 5 positions when 

compared with that of B. longiradiatum (accession 

number, AY551291), and was, therefore, deposited in 

GenBank as a new B. longiradiatum nucleotide sequence 

(accession number, FJ561305). The hap1, 2, 3, and 4 

sequences were found to be 98 to 99% and 99% 

identical, for B. falcatum versus B. chinense and for B. 

falcatum versus B. longiradiatum, respectively. Thus, B. 

falcatum’s sequence was more closely related to that of 

B. longiradiatum than B. chinense. The lowest sequence 

identity (97%) determined was between B. chinense hap1 

and B. longiradiatum. B. longiradiatum’s (58.08%) and B. 

chinense’s (58.78%) hap2 GC contents were the lowest 

and the highest, respectively (Table 4). 

Comparison of ITS nucleotide sequences revealed 2 

positions (199 and 200 bp) at which all 3 species differed 

were B. falcatum, single base deletion; B. chinense, 2 

G’s, and B. longiradiatum, 2 A’s. Within these sites, 9 B. 

longiradiatum nucleotides differed from those of B. 

falcatum and B. chinense sequences (Table 5). The 

nucleotide sequences within the chloroplast rbcL region 

were also confirmed and displayed less variability than 

those of the ITS. Therefore, they were deemed 

inappropriate for identifying Bupleurum species. 

Furthermore, the highly variable ITS region is more 

effective for distinguishing Bupleurum species. We then 

designed BL-F, BL-R, and B-I-F primers based on the 9 

B. longiradiatum specific stretches to discriminate B. 

longiradiatum from B. falcatum and B. chinense (Table 2). 

PCR reactions employing 15 samples were listed in Table 

1, in combination with these primers, generated B. 

longiradiatum-specific bands, 204 and 351 bp length 

(Figure 3, Lanes 11 to 15) and unique B. falcatum (206 

bp, Figure 3, Lanes 1 to 5) and B. chinense (Figure 3,


Table 5. Comparison of Bupleurum sequences. 

Species 

82 109 110 114 

Position (bp) 

185 199 200 354 392 396 421 

B. falcatum T G C T T - - C T T G 

B. chinense C G C C C G G C A A G 

B. longiradiatum T - T T T A A T A T T 

Species 451 493 495 528 530 549 571 580 582 606 

B. falcatum G T T G A C C C G T 

B. chinense G T A T G A T A T T 

B. longiradiatum T C T G A C T C G C 

Figure 3. PCR Products Generated with B. longiradiatum-specific Primers. Lanes 1 to 15, Bupleurum species listed in Table 1. M = 100 

bp DNA size standard ladder. 

Lanes 6 to 10) bands. By using fluorescently labeled B. 

longiradiatum-specific primers (Table 2), we compared 

CE and GE. Reactions using templates of the DNA of B. 

falcatum and B. chinense DNAs as listed in Table 1 

yielded specific 206 bp products, in contrast to the 2 

specific B. longiradiatum 204 and 351 bp peaks (Figure 

4). 

The amplified products were similar in size as 

determined by both CE and GE analyses, confirming the 

suitability of the B. longiradiatum-specific primers for 

differentiating Bupleurum species. Five Bupleurum 

species growing naturally in Korea were classified into 3 

groups based on their external morphologies and number 

of somatic chromosomes (Ahn et al., 2008). B. falcatum 

and B. longiradiatum could further be discriminated from 

each other by microscopic analysis of their external root 

morphologies (Yang et al., 2004). However, because the 

external appearance or constituents of medicinal plants 

are influenced by various factors such as growth 

environment, harvesting time, and process flow (Yang et 

al., 2007; Hon et al., 2003; Tristezza et al., 2009), it is 

necessary to objectively and unambiguously differentiate 

Bupleurum species based on the molecular genetic 

information. Accordingly, to determine whether B. 

longiradiatum could be molecularly differentiated from B. 

falcatum and B. chinense, primers were developed for 

routine GE. The results demonstrated that an ITS-PCR 

method using these primers is useful for rapid and 

efficient discrimination of Bupleurum species. Although 

GE is relatively simple to perform, the technique is not 

easy to determine the exact size of PCR products or 

resolve similar sized bands. However, incorporating an 

internal size marker in CE can virtually eliminate the 

influence of external parameters, thereby permitting 

highly reproducible and accurate size determination of 

PCR products (Tristezza et al., 2009). 

Even a single-base pair difference can be detected by 

CE-based genotyping of Plasmodium falciparum 

(Liljander et al., 2009). Recently He et al. (2011) reported 

that Angelica anomala and Angelica dahurica are closely 

related and nested in this phylogeny tree. A. dahurica 

shown the high similarity with A. anomala than any other 

species. Therefore, we sought to develop a GE-based 

method capable of identifying B. longiradiatum swiftly, 

accurately, and economically and to enhance PCR 

accuracy based on CE using fluorescently labeled 

compounds. Further studies should aim to simultaneously 

identify multiple herbal plants by using CE and primers 

labeled with dyes that fluoresce at different wavelengths. 

In conclusion, ITS nucleotide sequence variation can 

be used to distinguish between B. falcatum, B. chinense, 

and B. longiradiatum. Furthermore, B. longiradiatum can

Figure 4. Capillary Electrophoresis Analysis of B. longiradiatum-specific PCR products; A B. falcatum; B B. chinense, and C B. longiradiatum. 

Kim et al. 847


be unambiguously identified using specific primers. GE 

and CE verified the specificity of this method, which may 

also prove to be useful in identifying other medicinal 

herbs that will not be easy to distinguish on the basis of 

their external appearance. 


This research was supported by a grant 

(08172KFDA264) from the Korea Food and Drug 

Administration and the Korea Institute of Oriental 

Medicine (K11101). 

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DOI: 10.5897/JMPR11.1493 



Effect of catalpol on doxorubicin-induced cytotoxicity 

in H9c2 cells 

Xing Wu* and Yanbin Mao 

Department of Internal Medicine, The Sixth Hospital of Shenyang, Liaoning 110006, China. 


The aim of the present study was to investigate the protective effect of catalpol, an effective component 

of Rehmannia glutinosa Libosch, against doxorubicin (DOX)-induced cytotoxicity in H9c2 cells and to 

find out its potential mechanisms. The cell viability was assayed by MTT assay. Apoptotic cells were 

evaluated by Hoechst 33258 staining. The lactate dehydrogenase (LDH) leakage, the malondialdehyde 

(MDA) activity and intracellular of reactive oxygen species (ROS) levels were examined. The activity of 

antioxidant enzymes, including catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase 

(SOD) were measured. Furthermore, we evaluated the effect of catalpol on the antitumor activity of DOX 

in HeLa cervical carcinoma cells with MTT assay. The results of MTT assay and measurement of LDH 

release showed that catalpol significantly reduced DOX-induced damage. Catalpol reduced intracellular 

ROS, decreased the concentration of MDA and increased the activities of antioxidant enzymes in DOXtreated 

H9c2 cells. Importantly, the MTT analysis showed that the addition of catalpol did not interfere 

with the antitumor activity of DOX in HeLa cells. Taken together, our findings indicate that catalpol 

could exert the cardioprotective effects against DOX-induced toxicity without affecting its antitumor 

activity of this anthracycline. 

Key words: Catalpol, doxorubicin, H9c2 cells, reactive oxygen species. 

INTRODUCTION 

Doxorubicin (DOX), an effective anticancer drug, is used 

to treat a variety of malignacies, such as leukemias, 

Hodgkin and non-Hodgkin lymphoma, and solid tumors 

(Young et al., 1981; Hortobagyi, 1997). Its clinical use is 

limited because of its dose-dependent cardiotoxicity. The 

mechanisms of DOX-induced cardiotoxicity are not 

completely understood, but a number of reports have 

indicated that the mechanism involves the generation of 

reactive oxygen species (ROS) (Takemura and Fujiwara, 

2007). ROS can damage all components of the 

cardiomyocyte, including lipid, DNA and protein, and 

consequently result in the apoptosis or death of cell 

(Arola et al., 2000; Menna et al., 2007; Kumar et al., 

2001; Wu et al., 2002). Antioxidant enzymes, such as 

catalase (CAT), glutathione peroxidase (GPx) and 

superoxide dismutase (SOD), are an important 

antioxidant defense in cells. They are the specific oxygen 

*Corresponding author. E-mail: xingwu1997@sina.com. Tel: 

+86-024-23387410. Fax: +86-024-23388643. 

free radical scavengers and can protect the cell against 

ROS-induced damage (Khalili et al., 2011). Catalpol is an 

effective component extracted from Rehmannia glutinosa 

Libosch, it has many pharmacological actions, and such 

as antioxidant activity (Zhang et al., 2009) and antiapoptotic 

function (Jiang et al., 2004) in neuronal cells. 

However, the effects of catalpol on DOX-induced 

cardiac damage have not been described. In the present 

study, we examined for the first time whether catalpol 

protects cardiomyocytes against DOX–induced 

cytotoxicity and tried to find out the potential mechanism. 


HUVECs and HeLa cells were obtained from Shanghai Institute of 

Cell Biology (Shanghai, China). Catalpol was purchased from 

National Institute for the Control of Pharmaceutical and Biological 

Products (Beijing, China). Dulbecco′s modified Eagle′s medium 

(DMEM) and fetal bovine serum (FBS) were purchased from 

GIBCO BRL Life Technologies (Grand Island, USA). MTT and 

dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich 

Corporation (St. Louis, USA). Assay kits for antioxidant enzyme


activities, MDA test kit and dichlorofluorescein diacetate (DCFH- 

DA) detection kit were obtained from Beyotime Institute of 

Biotechnology (Jiangshu, China). LDH-cytotoxic test kit was 

purchased from Jiancheng Bioengineering Institute (Nanjing, 

China). All other chemicals were of analytical grade and obtained 

from Beijing Chemical Factory (Beijing, China). 

Cell culture 

The H9c2 and HeLa cells were cultured in DMEM supplemented 

with 10% fetal bovine serum, 100 μg/ml of penicillin and 100 μg/ml 

of streptomycin. Cells were incubated in a humidified atmosphere of 

5% CO2 at 37°C. 

Experimental groups 

There were five groups in this study: (1) Control group (cells were 

untreated). (2) DOX group (cells were cultured with the medium 

containing 4 µmol/L of DOX for 3 h). (3, 4 and 5) Catalpol group 

(cells were treated with catalpol at the concentration of 0.1, 1 and 

10 μg/ml for 24 h and then exposed to 4 µmol/L of DOX for 3 h). 

MTT assay 

H9c2 cells were incubation with different concentrations of catalpol 

(0.1, 1, 10, 100 and 1000 μg/ml) for 24 h or 10 μg/ml catalpol for 

different amounts of time (3, 6, 12, 24 and 48 h) or with different 

concentrations of DOX (1, 2, 4, 8 and 16 μmol/L) for 3 h or 4 μmol/L 

DOX for different amounts of time (1, 3, 6, 12 and 24 h). Cell 

viability was assayed by using a MTT assay. At the end of each 

treatment period, 20 µl of MTT was added to each well and the 

microplate was incubated at 37°C for 4 h. The medium with MTT 

was removed and 150 µl DMSO was added to each well. The 

absorbance was measured at 490 nm. Wells without cells were 

used as blanks. 

Results were expressed as a percentage of control. HeLa cells 

were used to investigate the interference of catalpol on the 

antitumor activity of DOX. HeLa cells were incubation with10 μg/ml 

catalpol for 24 h or 4 μmol/L DOX for 3 h or pretreatment with10 

μg/ml catalpol 24 h then exposed to 4 μmol/L DOX 3 h. Cell viability 

was assayed by using a MTT assay. 

Cytotoxicity assay 

The LDH leakage was detected as described by the manufacturers 

of the LDH-cytotoxic test kit. Briefly, 20 μl of culture medium was 

incubated with nicotinamide adenine dinucleotide and 2 mM 

pyruvate at 37°C for 15 min. Then 0.25 ml of 2, 4- 

Dinitrophenylhydrazone was added and mixed, and then the 

reaction was stopped by the addition of 2.5 ml of 0.4 M NaOH. The 

absorbance was read at 440 nm. 

Hoechst 33258 staining 

Cells were harvested and washed with phosphate buffered saline 

(PBS) twice and fixed in 4% formaldehyde at 4°C for 10 min. 

Afterward, the cells were stained with Hoechst 33258 for 5 min in 

dark at 37°C. Nuclear morphology observed under a fluorescent 

microscope (Nikon, Yokohama, Japan). Apoptotic cells were 

identified by condensed, brightly stained nuclei. Cells from five 

random microscopic fields at 400 magnification were analyzed, and 

the numbers of apoptotic cells were expressed as percentages of 

the total cells. 

Measurement of ROS and lipid peroxidation 

The generation of ROS was determined using DCFH-DA detection 

kit according to the manufacturer's protocol. Briefly, cells were 

washed with PBS three times and then incubated with 20 μM 

DCFH-DA at 37°C for 30 min. The absorbance was read at 525 nm. 

The MDA was determined by the thiobarbituric acid method using 

MDA test kit according to the manufacturer's protocol. Briefly, 

sample was collected and incubated with 200 µl of TBA reagent at 

100°C for 15 min. After cooling to room temperature in an ice batch 

for 5 min, the cells were concentrated by centrifugation at 1000 g 

for 10 min. 200 µl supernatant was transferred to a 96 well 

microplate and the absorbance of the colored product was read at 

532 nm. Values of MDA level were expressed as nanomoles per 

milligrams of protein. 

Enzymatic activity 

The total SOD (T-SOD) activity was determined by the xanthine 

oxidase method using total superoxide dismutase assay kit 

according to the manufacturer's protocol. Briefly, sample was 

collected and incubated with xanthine-xanthine oxidase and 

nitroblue tetrazolium (NBT) at 37°C for 20 min. The absorbance 

was read at 550 nm. The SOD activity was expressed as units per 

milligrams of protein. One unit is defined as the amount of enzyme 

needed to exhibit 50% dismutation of the superoxide radical. 

The GPx activity was determined by cellular glutathione 

peroxidase assay kit according to the manufacturer's protocol. 

Briefly, sample was collected and incubated with 5 ul of the 10 mM 

NADPH and 0.4 µl of glutathione reductase. The reaction was 

initiated by the addition of 4 µl of the 15 mM tert-Butyl 

hydroperoxide solution. The absorbance was read at 340 nm. The 

GPx activity was expressed as units per milligram of protein. One 

unit of GPx activity was defined as the amount required to oxidize 1 

μmol NADPH to NADP + in 1 min at 25°C, pH 8.0. 

The CAT activity was determined using CAT assay kit according 

to the manufacturer's protocol. Briefly, sample was collected and 

incubated with 10 ul of the 250 mM hydrogen peroxide at 25°C for 5 

min. The reaction was terminated by the addition of potassium 

hydroxide. After incubation with the chromogenic agent at 25°C for 

15 min, the absorbance was read at 520 nm. The CAT activity was 

expressed as units per milligram of protein. One unit of CAT activity 

was defined as the amount of CAT required to decompose 1 μmol 

H2O2 in 1 min at 25°C, pH 7.0. 


Data are expressed as mean ± standard deviation (S.D). Statistical 

comparison within groups was carried out with one way ANOVA, 

and the Student-Neuman-Keuls test was used to analyze between 

two groups. Values of P < 0.05 are considered statistically 

significant. 

RESULTS 

Effect of catalpol and DOX on the survival of H9c2 

cells 

Treatment with different concentrations of catalpol (0.1, 1 

and 10 μg/ml) for 24 h increased the viability of H9c2 

cells in a dose-dependent manner. Cell viability was 

106.9 ± 3.0%, 114.0 ± 5.6% and 137.0 ± 9.8%, 

respectively. The protective effect of catalpol was

inhibited and cell viability reduced at a concentration of 

1000 μg/ml (Figure 1A). Treatment with 10 μg/ml catalpol 

for up to 48 h increased the viability of H9c2 cells in a 

time-dependent manner. Cell viability was 109.9 ± 4.8%, 

120.6 ± 4.6% and 128.5 ± 6.5%, 135.4 ± 5.5%, 143.1 ± 

6.8%, respectively (Figure 1B). Treatment with different 

concentrations of DOX (1, 2, 4, 8 and 16 μmol/L) for 3 h 

resulted in dose-dependent decreases in cell viability. 

Cell viability was 95.8 ± 3.4%, 82.2 ± 5.5%, 54.9 ± 6.3%, 

42.9 ± 4.1% and 28.8 ± 3.2%, respectively (Figure 1C). 

Treatment with 4 µmol/L DOX for different amounts of 

time (1, 3, 6, 12 and 24 h) resulted in time-dependent 

decreases in cell viability. Cell viability was 75.8 ± 3.4%, 

54.9 ± 6.3%, 44.8 ± 1.8%, 33.5 ± 3.9% and 25.6 ± 3.4% 

respectively (Figure 1D). The viability was 54.9 ± 6.3% 

associated with treatment 4 μmol/L DOX for 3 h and this 

concentration was used in subsequent experiments. 

Catalpol did not interfere with the antitumor activity 

of DOX 

After exposure to 4 µmol/L DOX for 3 h, the viability of 

HeLa cells significantly reduced compared with the 

control group (Table 1). Cell viability did not show any 

significant differences after pretreatment with both 

catalpol and DOX compared with the DOX group. 

Moreover, when pretreatment with catalpol alone, cell 

viability did not decreased significantly compared with 

control group. 

Catalpol increases the viability of H9c2 cells exposed 

to DOX in a dose-dependent manner 

H9c2 cells were pretreated with different concentrations 

of catalpol (0.1, 1 and 10 μg/ml) for 24 h and then 

exposed to DOX for 3 h. Cell viability was assayed by 

MTT (Figure 1E). The survival rate of H9c2 cells was 

decreased after exposed to 4 μmol/L of DOX for 3 h 

compared to control group. Catalpol increased the 

viability of H9c2 cells in a dose-dependent manner at 

concentration of 0.1 to 10 μg/ml. As shown in Table 3, 

LDH concentrations in medium from control group cells 

were minimal. Treatment with 4 μmol/L of DOX for 3 h led 

to marked increases in LDH levels. Pre-treatment with 

catalpol (0.1, 1 or 10 μg/ml) attenuated LDH activity 

significantly in a dose dependent manner. 

Catalpol prevents H9c2 cells from DOX–induced 

apoptosis 

Apoptosis was detected using Hoechst 33258 staining. 

Compared with control group, the apoptotic rate was 

significantly increased in DOX group. Catalpol (0.1, 1 or 

10 μg/ml) significantly reduced the apoptotic rate in a 

dose dependent manner (Table 2). 

Wu and Mao 851 

Catalpol reduced the levels of ROS and lipid 

peroxidation 

As shown in Table 3, the level of intracellular ROS was 

measured with fluorescence probe DCFH-DA. Compared 

with control, the intracellular ROS manifested as 

fluorescence intensity increased significantly in H9c2 

cells exposed to DOX. The fluorescence intensity 

decreased significantly by pretreatment with catalpol (0.1, 

1 or 10 μg/ml) in a dose dependent manner. 

As shown in Table 3, the content of MDA increased 

significantly in the DOX group compared with the control 

group. Catalpol (0.1, 1 or 10 μg/ml) significantly reduced 

lipid peroxidation in a dose dependent manner. 

Catalpol increased the activity of antioxidant 

enzymes 

Compared with control, notable reductions in CAT, GPx 

and T-SOD activities were observed after exposure to 4 

µmol/L DOX for 3 h. Pretreatment with catalpol (0.1, 1 or 

10 μg/ml) significantly elevated the activities of CAT, GPx 

and T-SOD in a dose dependent manner (Table 4). 

DISCUSSION 

A lot of traditional plants have been shown to have 

cardioprotective effects in experimental studies (Shao, 

2011; Xing and Jian, 2011; Ritter et al., 2010). Catalpol, 

listed in the 2010 edition of Pharmacopoeia of the 

People’s Republic of China, is an iridoid glucoside 

isolated from the root of R. glutinosa Libosch and used 

for the quality evaluation of R. glutinosa. Catalpol has 

many pharmacological actions, such as anti-brain 

ischemia, anti-senile dementia, promoting neuroremodeling 

and reducing capillary permeability (Zhu et 

al., 2009). However, there have been no studies at all on 

whether catalpol is able to protect the cardiomyocytes 

from DOX-induced damage. In this study, we examined 

the protective effect of catalpol on the H9c2 cells 

exposed to DOX by MTT assay, LDH release and 

Hoechst 33258 staining. The major finding of the present 

study is that pretreatment with catalpol (0.1, 1 and 10 

μg/ml) for 24 h markedly reduces the decrease in viability 

of H9c2 cells exposed to DOX. Catalpol dosedependently 

inhibited apoptosis and decreased 

cytotoxicity in H9c2 cells exposed to DOX. Moreover, 

catalpol did not interfere with the antitumor activity of 

DOX in HeLa cells by MTT test. DOX is a widely used 

antitumour agent; however, its clinical use is limited 

because of its dose-dependent cardiotoxicity. The 

production of ROS is considered to be the main


Figure 1. The viability of DOX and catalpol in H9c2 cells. It was measured using MTT assay. (A) The viability in H9c2 cells with different concentrations of catalpol (0.1, 1, 

10, 100 and 1000 μg/ml) for 24 h. (B) The viability in H9c2 cells with 10 μg/ml catalpol for different amounts of time (3, 6, 12, 24 and 48 h). (C) Cell viability in H9c2 cells with 

different concentrations (1, 2, 4, 8 and16 μmol/L) for 3 h. (D) The viability in H9c2 cells with 4 μmol/L DOX for different amounts of time (1, 3, 6, 12 and 24 h). (E) The 

viability in H9c2 cells with 4 μmol/L DOX for 3 h after pretreatment with different concentrations of catalpol (0.1, 1, 10 μg/ml) for 24 h. The data represent means ± S.D from 

5 independent experiments. ★P

Table 1. HeLa cells were used to investigate the interference of catalpol on the antitumor activity 

of DOX. Cell viability was assayed by using a MTT assay. 

Group Cell viability (%) 

Control 100 

Catalpol (10 μg/ml) 97.6 ± 2.4 

DOX 29.5 ± 2.9 ▲ 

DOX + catalpol (10 μg/ml) 28.6 ± 3.1 ▲ 

The data from 5 independent experiments were expressed as means ± S.D. ▲ P


Table 4. The effects of catalpol on the activities of antioxidant enzymes in H9c2 cells. 

Group CAT (U/mg protein) Gpx (U/mg protein) T-SOD (U/mg protein) 

Control 15.33 ± 2.75 20.14 ± 1.96 8.41 ±1.15 

DOX 7.50 ± 1.31 ▲ 

13.99 ± 2.30 ▲ 

3.85 ± 0.78 ▲ 

DOX + catalpol (0.1 μg/ml) 13.43 ± 1.31 ★ 20.62 ± 1.46 ★ 8.29 ± 1.19 ★ 

DOX + catalpol (1 μg/ml) 17.97± 1.18 ★ 27.41 ± 1.36 ★ 12.82 ± 2.52 ★ 

H2O + catalpol (10 μg/ml) 23.88 ± 3.06 ★ 34.41 ± 2.84 ★ 19.56 ± 1.87 ★ 

All values are means ± S.D. of three replicates. ▲ P



DOI: 10.5897/JMPR11.1494 



Ethnobotany survey and uses of plants in the Lewoh- 

Lebang communities in the Lebialem highlands, South 

West Region, Cameroon 

Fonge B. A. 1 *, Egbe E. A. 1 , Fongod A. G. N. 1 , Focho D. A. 2 , Tchetcha D. J. 1 , Nkembi L. 3 

and Tacham W. N. 2 

1 Department of Plant and Animal Sciences, University of Buea, P. O. Box 63 Buea, Cameroon. 

2 Department of Plant Biology, University of Dschang, P. O. Box 67 Dschang, Cameroon. 

3 Environment and Rural Development Foundation, (ERuDeF), Buea, Cameroon. 


Ethnobotanical investigations were conducted in four Lewoh-Lebang villages (Attuleh, Leleng, Mbindia 

and Nyitebong) in Lebialem Division, Southwest Region of Cameroon to identify the different plants 

used in traditional pharmacopoeia for the treatment of human diseases and also to find out other uses 

of plants in this community. Ethnobotanical information was collected randomly using semistructured 

questionnaires and open-ended discussion with male, female and traditional healers, using different 

age groups. A total of 108 respondents were interviewed and (56%) were male with age ≥55 years. Thirty 

(30) medicinal plants belonging to 21 families were identified and documented. Guarea thompsonii, 

Schefflera hierniana and Cyclomorpha solmsii are endemic/vulnerable species. 21 diseases were cured 

using 30 species with rheumatism being the most frequent ailment and the bark was the most 

frequently used plant part. 13 species were used as fuel wood, five for fencing, eleven as timber and 

fifteen for cultural activities. Vernonia conferta (25 citations), Psychotria strictistipula (21 citations), 

Psychotria penducularis (22 citations) and Coffea sp. (20 citations) (all shrubs) were species used as 

fuel wood mostly harvested by women. It is very important to conserve these plant species in order to 

improve on the traditional health care practices in this community. 

Key words: Ethnobotanical, Lewoh-Lebang, traditional pharmacopoeia, medicinal plant. 

INTRODUCTION 

Ethnobotany is an area of human ecology that defines 

the interface between people and their forests and offers 

clues needed for rural development based on sustainable 

yields of forest products (Focho et al., 2009a). Traditional 

societies in Africa and elsewhere have always used 

plants to promote healing (Idu et al., 2005; Bussmann, 

2006; Teferi et al., 2009) and about 80% of the world’s 

population depends on the use of traditional medicine for 

health care (WHO, 1993). In some African countries up to 

95% of traditional medicinal preparations are of plant 

origin (Abebe, 1986). In Cameroon, the traditional 

medicine is still unorganized making its integration in the 

health system ineffective (Nkongmeneck et al., 2007). 

*Corresponding author. E-mail: ambofonge@yahoo.com. 

The conceptual strategy of health envisage the 

organization of folk medicine for which, a strategic plan 

has been worked out in Cameroon to provide the main 

trends for the development and its integration 

(Anonymous, 2006). Plants are the basis for the 

development of modern drugs and medicinal plants have 

been used for many years in daily life to treat diseases all 

over the world (Ates and Erdogrul, 2003; Adewusi and 

Afolayan, 2010). Uses of the plant may include food, 

medicine, shelter, hunting, clothing and even for religious 

occasions. Plants are also important in ceremonies such 

as birth, marriage and death in some cultures (Nichter, 

1992). Traditional and indigenous knowledge of plants is 

rapidly eroding (Teferi et al., 2009). 

This is exacerbated by rapid habitat destruction, 

agriculture expansion and over-exploitation of plants 

used in health care and other purposes putting most


species under threat of extinction (Focho et al., 2009b). 

With loss of biodiversity and negative effects of 

mainstream culture, the traditional/folk medicinal 

knowledge of many ethnic groups in Africa and 

elsewhere is facing critical depletion (Nichter, 1992; Cox, 

1993; Zhang, 2000; Pei, 2001; Yanchun et al., 2009). 

Loss of traditional knowledge of plants and culture which 

is the same as the disappearance of biodiversity is not a 

reversible process (Huai and Pei, 2009). Deforestation 

process is a serious threat to biodiversity conservation in 

the tropics. If no action is taken to remedy the trend, a 

considerable number of plant and animal species are 

likely to become extinct even before they are known to 

science (Zapfack et al., 2001). According to Idu et al. 

(2011) younger respondents are not as resourceful in 

traditional knowledge compared with the elderly or middle 

aged respondents. This gap portends a present threat to 

the successful transfer of indigenous knowledge from the 

older to younger generation as the latter appear to 

demonstrate increasing apathy towards acquiring such 

vital knowledge. 

It accentuates the urgent need for proper 

documentation. It is important to take urgent action 

especially in developing African countries to ensure 

sustainable exploitation of plant resources so that these 

resources will continue to be available for posterity. 

Ethnobotanical investigations carried out in Cameroon 

have covered many parts of the country but left out 

certain regions despite their richness in plant diversity 

(Adjanohoum et al., 1996; Mbolo, 2002). An example of 

such a region is the Lewoh-Lebang area in the Lebialem 

Division. Lebialem division found in the South West 

region of Cameroon is characterized by a hilly 

topography with a rich diversity of flora and fauna. This 

mountain ecosystem has been under serious pressure 

from the local people. This ecosystem is a centre of high 

endemism for many taxa (plants, amphibians, mammals 

and birds) whose destruction could lead to their 

extinction. The region also holds some of the globally 

threatened and endemic species such as the critically 

endangered cross river gorilla, chimpanzee, flying 

squirrel, endangered Bannerman’s Turaco and Banded 

Wattle-eye, vulnerable Red-headed Picathartes (Nkembi, 

2004). The study area is part of the Bamboutous 

mountain range which is a stronghold of montane 

biodiversity. These ecosystems around the Bamboutos 

Mountain continue to provide valuable goods and 

services to local people in the region and is an important 

watershed lodging the tributaries of Manyu River that 

drain into the cross river. 

Forest destruction is causing the local extinction of 

globally threatened biodiversity (plants and mammals, 

etc), watershed destruction and degradation of livelihood 

systems, property and lives. Due to precedent geological 

and geographical history of these mountain areas and 

coupled with the high annual rainfall (2000 to 3000 mm) 

and humidity, these areas are perpetually having 

landslides (Ayonghe and Ntasin, 2008; Zogning et al., 

2007). Most of the landslides are caused by 

anthropogenic activities of the communities around the 

mountain. The action of the local people, have lead to 

untold suffering including homelessness loss of human 

lives, properties, forest land, substantial loss of 

biodiversity, habitats, loss of income sources leading to 

extreme levels of poverty (Ayonghe and Ntasin, 2008; 

Che et al., 2011). There is also loss of cultural values and 

serious degradation of habitats. The Nweh people (tribe 

in the study site) practice slash and burn agriculture with 

the bimodal annual farming cycle which is entirely 

dependent on the rain fall patterns resulting to frequent 

landslides. As in the case with other communities in 

Cameroon, the importance of plants as medicine cannot 

be over emphasized. 

Traditional medicine is even preferred to modern 

medicine, as they are less expensive and often regarded 

as being more effective (Abebe,1986; Komaromi, 2009). 

Women make the greatest use of wild plants for 

medicinal purposes as well as for food, fibers, utensils, 

cosmetics and ornamentals, whereas men use the wood 

for crafting, construction of house and bridges within 

community (Ochoa et al., 1998; Kappelle et al., 2000). In 

the study area, there is no nearby Health Centre/hospital 

as the nearest health centre is about 50 km making the 

local population to rely more on their forest for first aid 

and treatment. The area is a humid savanna and the 

forest in the study area is in patches. There are 

constraints in healthcare, fuel wood for cooking and wood 

for construction and craft work. This study therefore 

brings to light the plants used in traditional health care, 

fuel wood, fencing and timber for construction in the 

Lewoh- Lebang community in Cameroon. 


Study area 

Lebialem is located in the North Eastern part of the Southwest 

Region of Cameroon (Latitudes 5°38’N and 5°43’N and between 

Longitude 9°58’ E and 10°27’ E). Lewoh-Lebang is located between 

latitudes 5°45’ and 5°47’ N and longitudes 9°91’E and 9°94’ E and 

at altitudes ranging from 1456 to 1835 m (Figure 1). The climate of 

this region is similar to that of the Cameroon mountain range which 

is characterized by high winds and low sunshine. The average daily 

temperature varies very much with seasons but ranges from 17 to 

32°C (Nkembi, 2004). The mean annual rainfall 2000 was 3000 

mm. The main vegetation type is grassland with patches of 

montane and sub-montane forest. The forest is dominated by 

Chytranthus gilletii, Gambeya africana and Schefflera barteri 

(Focho et al., 2009a). 

Collection of information 

Ethnobotanical information was collected in four villages in Lewoh- 

Lebang (Nyitebong, Mbindia, Attuleh and Lelang) using methods 

adapted from Jovel et al. (1996) and Karehed and Odulg (1997), 

consisting of open-ended conversations and semi-structured

Figure 1. Study area. 

Fonge et al. 857


Table 1. Percentage of respondents in Nyitebong, Mbindia, Attuleh and Leleng according to their age groups. 

Age group (Years) Number of respondents Percentage of respondents (%) 

Youths (15 to 29) 11 10 

Adults (30 to 54) 44 41 

Elderly (55 and above) 53 49 

Table 2. Percentage of respondents in Nyitebong, Mbindia, Attuleh and Leleng according to their genders. 

Gender Number of respondents Percentage of respondents (%) 

Male 61 56 

Female 47 44 

questionnaires. Selection of respondents was done randomly in the 

villages following age groups. On the indigenous knowledge of the 

medicinal plants youth, women, men and traditional healers in the 

local community were randomly selected. After seeking their 

consent, the respondents were interviewed using semi-structure 

questionnaires and open-ended conversations. The number of men 

respondents was 61 while that of the female was 47. The data 

collected included local names of the plants, uses, modes of 

preparation and administration of herbal drugs and the plant parts 

used. Informants often accompanied the investigators to the field to 

collect plant material. In cases of illiterate informants, photographs 

and fresh plant specimens from the field were presented to them 

and questionnaires were filled from their responses. 

The working language was mostly the dialect spoken in the 

community (Nweh) and the authors faced no language problems 

since two of them were natives of the area. Feul wood collection 

was done by women using baskets. Fuel wood and timber for 

construction of houses and for carvings are not collected in sacred 

forests. Field trips were made from October 2010 to May 2011 

during which specimens were collected and pressed for 

identification for plants that could not be identified in situ. Standard 

methods were used in plant material collection, drying, mounting, 

preparation and preservation. Plants were identified by their 

vernacular names and later validated at the Limbe Botanic Garden 

Herbarium (SCA) and the Cameroon National Herbarium (YA). 

Voucher specimens were deposited in the University of Buea 

teaching Herbarium and Limbe Botanic Garden Herbarium. 

Data analysis 

The data on the plant species uses, vernacular names and disease 

treated were entered into excel worksheets and percentage and 

frequencies calculated. The data on plants used were summarized 

as proposed by Cook (1995) 

RESULTS 

Table 1 presents the percentages of respondents in 

terms of their ages. It shows that the respondents were 

mostly in the age group of 55 years and above (49%) 

while the youths were the least represented. Table 2 

gives the percentages of respondents in terms of their 

gender, indicating that most of the respondents were men 

(56%) and 44% were female. Table 3 shows the scientific 

name/family, local names, plant parts used, diseases 

treated, mode of preparation and administration of 

medicine and the frequency of citation for the different 

medicinal plants. A total of 30 medicinal plants belonging 

to 21 families were documented. Asteraceae (3 species) 

was the most commonly used plant family in the area of 

study followed by Apocynaceae, Rutaceae, Annonaceae, 

Liliaceae, Euphorbiaceae, Fabaceae and Sterculiaceae 

all having 2 species. The other most utilized medicinal 

plants were Psidium guajava (31citations) and Aloe vera 

(27citations). In this rural community, child birth is of 

great importance and gynecological problems are treated 

with a variety of plants. These include Zanthoxylum gilleti, 

Vernonia amygdalina, Eremomastax speciosa and Aloe 

vera. 

In Table 4, plant species are regrouped according to 

their use in the management different ailments. Some 

plants such as E. speciosa, (Acanthaceae) and Rauvolfia 

vomitoria Afzel. (Apocynaceae) are used to treat about 3 

different ailments. The plants were used to treat ailments 

ranging from common fatigue to complex pathological 

disorders relating to gastro-intestinal diseases, 

respiratory ailments, problems of the urinary system as 

well as infertility. Thirteen (13) major conditions were 

prevalent in the area (Table 4). Rheumatism had the 

highest number of herbal plants used for its treatment (6), 

followed by the central nervous system (5), the digestive 

system (4) and the respiratory system (4). The main 

methods of preparation of remedies were decoctions, 

concoctions and infusions while the mode of 

administration was oral for internal infections and topical 

for skin diseases. Figure 2 illustrates the percentages of 

plant parts used. 

The barks of plants (42%) were the most popular plant 

part used in the various herbal preparations and followed 

by the leaves (36%) the latex (5%), whole plant (5%), 

seeds (3%), roots (3%) and fruits (3%) were the least 

used. Table 5, 6 and 7 give the plant species used as fuel 

wood, fencing and timber, respectively. The main supply

Table 3. Documentation of ethnomedicine, treatment and plant uses by the people of Lewoh-Lebang. 

No Species and family names Local names Parts used Diseases 

treated/uses 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

Tabernaemontana crassa 

Benth; (Apocynaceae) 

Kigelia africana (Lam.) 

Benth. (Bignoniaceae) 

Cylicomorpha solmsii (Urb.) 

Urb. (Caricaceae) 

Santeria balsamifera Oliv. 

(Burseraceae) 

Zanthoxylum gilletii (De 

Wild.) P.G. Waterman 

(Rutaceae) 

Zanthoxylum buesgenii 

Engl. (Rutaceae) 

Xylopia africana (Benth.) 

Oliv.(Annonaceae) 

Schumanniophyton 

magnificum (K.Schum.) 

Harms (Rubiaceae) 

Harungana 

madagascariensis Lam.ex 

Poir (Clusiaceae) 

Vernonia conferta Benth 

(Asteraceae) 

Vernonia amygdalina Del. 

Cent. (Asteraceae) 

Euphorbia desmindi Keay 

and Milne-Redhead 

(Euphorbiaceae) 

Etong 

Bark, 

leaves 

Preparation and 

administration 

Fonge et al. 859 

Frequency 

Malaria Infusion is taken orally 10 

Ledongne Bark Chest complaints Decoction is taken orally 7 

Ledongne Leaves Rheumatism Infusion is taken orally 5 

Ledongne Bark 

Chest pains of 

children 

Evong Bark Toothache 

Mbem 

Seeds, 

bark 

Mbem Roots 

Decoction is taken orally 12 

Decoction is used as 

mouth wash 

rheumatism Decoction is taken orally 7 

Loss of libido, 

Low sperm count 


Ndeun Bark Rheumatism Decoction is taken orally 5 

Sekoh 

Etoueh 

Bark, 

leaves 

Bark, 

leaves 

Afounouh Leaves 

Thyphoid fever Decoction is taken orally 19 

Yellow fever Decoction is taken orally 9 

Stomach ache/ 

cramps 

Bekantsu Leaves Menstrual cramps 

Maceration of leaves is 

taken orally 

Maceration of leaves is 

taken orally 

Aboueh Bark Rheumatism Decoction is taken orally 5 

Euphorbia desmindi Aboueh Latex Appetizer 

Guarea cf glomerulata Harm 

(Meliaceae) 

Rauvolfia vomitoria Afzel. 

(Apocynaceae) 

Quat Bark 

Etong-mbin 

Bark, 

leaves 

Rheumatism, 

general fatigue 

Malaria, stomach 

aches, gastritis 

Mixture of latex with egg 

is taken orally 

2 

11 

13 

Decotion is taken orally 6 


3



15 

16 

17 

18 

19 

20 

21 

22 

23 

24 

25 

26 

27 

28 

Piptadeniastrum africanum 

(Hook.f) Brenan (Fabaceae) 

Polyscias fulva 

(Hiern) Harms (Araliaceae) 

Pycnanthus angolensis 

(Welw.) Warb. 

(Myristicaceae) 

Isolona maitlandii Keay 

(Annonaceae) 

Dichaetanthera Africana 

(Hook.f.) Jacq.-Fel. 

(Melastomataceae) 

Macaranga monandra Mull. 

Arg. (Euphorbiaceae) 

Psidium guajava Linn. 

(Myrtaceae) 

Hibiscus sabdariffa Linn. 

(Malvaceae) 

Eremomastax speciosa 

(Hochst.) Cufod. 

(Acanthaceae) 

Senna alata (Linn.) Roxb. 

(Fabaceae) 

Ageratum conyzoides Linn. 

(Asteraceae) 

Colocassia esculenta (L.) 

Schoot (Araceae) 

Aloe vera (L.) Burm 

(Liliaceae) 

Aloe barbadense Mill. 

(Liliaceae) 

Cola accuminata (Vent.) 

Schott and Endt. 

(Sterculiaceae) 

Epheck Bark General fatigue Decoction is taken orally 12 

Keukeu Bark Frontal headache Decoction is taken orally 14 

keukeu Latex Clean the eyes 

Ndeung-mbin Bark 

Ngnekeugnang Leaves, 

bark 

Rheumatism, Chest 

pain 

Coughs, Chest 

complaints, Fatigue 

Atchach Bark Tooth ache 

Guava Leaves Malaria and fever 

Hibiscus Leaves Stomach ache 

Mbanfen Leaves 

Venkouh 

Irregular 

menstruation, 

infertility in women 

Stomach cramps 

Leaves Rashes, Filariasis 

Whole 

plants 

Akouh Stem Sores 

Akockdem Leaves 

Akockdem Leaves 

Headache, tiredness 

Menstrual cramps 

Stomach ache 

fire burns, 

constipation, 

stomach aches, 

gastritis 

Ledze Fruit Abscesses 

Small quantity is put 

inside each eye ad 

covered, and then the 

clot is removed 

Decotion is taken orally 12 

Decoction is taken orally 

sweetened with honey 

Decoction is used as 

mouth wash 

Concoction with leaves 

of Sena alata and roots 

of Carica papaya is taken 

orally 

Macerate the leaves and 

take it orally and as a 

purgative 

Infusion of two plants (E. 

speciosa, Aloe vera) is 

taken orally. Also used 

as anal wash 


apply topically 

Infusion of whole plant is 

taken regularly 

Grate the stem and apply 

on the area 


take it orally 

Mix gel with juice of 

Ageratum conyzoides 

and apply topically. 

Maceration is taken 

orally 

Grinding the fruit and 

apply topically 

3 

22 

8 

31 

9 

37 

13 

15 

4 

35 

27 

6


29 

30 

Stellaria media (L.) Vills 

(Caryophyllaceae) 

Kalanchoe crenata 

(Andr.) Haw. 

(Crassulaceae) 

Ntouh Whole plant Eye pain 

Louh Leaves Ear pain 

Table 4. Diseases and plants used to manage them. 

Rap the plant in Colocassia 

leaf and worm it, squeeze 

the plant and drop the liquid 

into the eyes 

Squeeze the leaves and 

drop the liquid in the ears 

Ailment Plant used (numbers as in Table 3) 

Digestive system 12, 14, 21, 27 

Central nervous system 13, 15, 16, 17, 24 

Respiratory system 2, 3, 17, 18 

Sores 25 

Skin diseases 23, 25, 27 

Ear 30 

Female sterility/gynaecology 11, 22, 26 

Male infertilitylity/sexual dysfunction 6 

Eye 16, 29 

Dental/mouth 4, 19 

Malaria/Thyphoid fever/yellow fever 1,8,9,14,20 

Inflammation/abscesses 28 

Rheumatism 2, 5, 7, 12, 13, 17 

of fuel wood came from V. conferta (25 citations), P. 

strictistipula (21 citations), P. penducularis (22 citations) 

and Coffea sp. (20 citations). These species are shrubs 

making it easy for the women to harvest firewood for 

household needs. Women collect fuel wood in baskets 

and carry it on their backs at least once a day from the 

nearest forest. Fuel wood and timber for construction of 

houses and for carvings are not collected in sacred 

forests. 

Table 8 shows that 15 species of plants were used in 

cultural activities mostly for protection against accident 

and witchcraft. Table 9 gives the plant species used for 

other purposes such as fish poisoning and craft works. 

DISCUSSION 

The high percentage of the respondent with respect to 

ethnomedicine was greater than 55 years. This is 

because they are custodians of traditional knowledge and 

they know the uses of plants especially medicinal plants 

than the younger generation, who have moved to 

neighbouring towns since the area is prone to landslides. 

This result is similar to that of Idu et al. (2011). According 

to Zogning et al. (2007) the area was declared by the 


government as a region unfit for human habitats after the 

2003 landslides which resulted to destruction of 

biodiversity, life and properties in the area. This has 

brought fear that indigenous ethnobotanical knowledge 

would or shall rapidly disappear from this region because 

of an increasing western lifestyle among the people and 

also the exodus. Other authors have also reported the 

gradual disappearance of indigenous knowledge in other 

parts of the world as a result of changes in lifestyle. For 

example, among the Wabane people in Lebialem Division 

of Cameroon (Focho et al., 2009a), the Igede people of 

(Igoli et al., 2005), Waluguru people in East Uluguru 

Mountains in Tanzania (Mahonge et al., 2006) and 

among the people of District Attock (Pungals) of Northen 

Pakistan (Ahmad et al., 2007). 

The study revealed the use of barks as the most used 

plant part followed by leaves in this area. Barks are 

known to accumulate alkaloids, tannins and inulins which 

are active components of most herbal preparations 

(Okoegwale and Omefezi, 2001). Leaves have also been 

reported to be the most commonly used plant parts in 

Wabane (Lebialem) and in other parts of Africa (Focho et 

al., 2009a; Mahonge et al., 2006). In the study the main 

methods of preparation were decoctions, concoctions 

and infusions while the mode of administration was oral 

7 

6


Figure 2. Percentages of parts of plants used as medicine. 

Table 5. Plant species used as fuel wood. 

No Species and family names Local names Frequency 

1 Lecomtedoxa klaineana (Pierre ex Engl.) Dubard (Sapotaceae) Mbeghembin 13 

2 Strombosia scheffleri Engl.(Olacaceae) Cokembin 3 

3 Coffea sp. (Rubiaceae) Cofembin 20 

4 Pentadesma butyracea Sabine (Clusiaceae) Thieup 8 

5 Ficus mucuso Welw. Ex. Ficalho (Moraceae) Ndar 11 

6 Trilepisium madagaseriense DC. (Moraceae) Leboh 5 

7 Psychotria strictistipula Schnell. (Rubiaceae) Ndelebi 21 

8 Dacryodes klaineana (Pierre) H.J. Lam (Burseraceae) Ntsoh 14 

9 Santeria balsamifera Oliv. (Burseraceae) Evong 8 

10 Carapa grandifolia Harms (Meliaceae) Leven 11 

11 Beilschmiedia sp_2 (Lauraceae) Mbeh 15 

12 Psychotria penducularis (Salisb.) Steyerm. (Rubiaceae) Ndelente 22 

13 Gambeya Africana G. Don (Sapotaceae) Segnei 2 

14 Vernonia conferta (Asteraceae) Afounouh 25 

Table 6. Plant species used for fencing. 

No Species and family names Local names Frequency 

1 Dracaena arborea (Willd.) Link (Dracaenaceae) Aquahe 28 

2 Schefflera hierniana (Seem.) Harms (Araliaceae) Tenkwu 39 

3 Psychotria strictistipula Schnell. (Rubiaceae) Ndelebi 15 

4 Draceana mannii Bak. (Dracaenaceae) Nkeng 22 

5 Ficus mucuso Welw. Ex. Ficalho (Moraceae) Ndar 13

Table 7. Plant species used as timber. 

No Species and family names Local names 

1 Lecomtedoxa klaineana(Pierre ex Engl.) Dubard (Sapotaceae) Mbeghembin 

2 Carapa grandifolia Harms (Meliaceae) Leven 

3 Memecylon afzelii G. Don (Melastomataceae) Chanye 

4 Euphorbia desmindi Keay and Milne-Redhead (Euphorbiaceae) Aboueh 

5 Macaranga monandra Mull.Arg. (Euphorbiaceae) Alegreb 

6 Trichoscypha patens (Oliv.) Engl. (Anacardiaceae) Ndeghebang 

7 Piptadeniastrum africanum (Hook.f) Brenan (Fabaceae) Epheck 

8 Beilschmiedia sp_2 (Lauraceae) Mbeh 

9 Trilepisuim madagaseriense DC. (Moraceae) Leboh 

10 Gambeya africana G. Don (Sapotaceae) Segnei 

11 Guarea cf thompsonii Sprague and Hutch. (Meliaceae) Findwat 

for internal infections and topical for skin diseases and 

this type of finding was also reported by other authors 

(Focho et al., 2009 a,b; Nkongmeneck et al., 2007; Okoli 

et al., 2007). The study revealed that 30 plant species 

belonging to 21 families and 27 genera were medicinal 

plants of this area. Similar results have been reported by 

(Focho et al., 2009a). The high prevalence of digestive 

ailments may be due to the lack of portable water in the 

area resulting in a high occurrence of water borne 

diseases. E. speciosa (37 citations) was used in the 

treatment of many disease conditions including irregular 

menstruation, infertility in women and stomach cramps. 

Many plants were used either singly or in combination 

with other medicinal plants. According to Igoli et al. 

(2005), the joint use of multiple medicinal plants could be 

due to synergistic or additive effects of constituents. 

Many of the plant species in the area have been reported 

elsewhere in Africa although they are often used for 

treatment of ailments different from those in the study 

(Okoli et al., 2007). In the study area, Cylicomorpha 

solmsii which is an endemic/vulnerable species was used 

in the treatment of children chest pain and this could lead 

to the disappearance of this important species in the area 

due to continous harvesting. Other vulnerable species 

used in the area were S. hierniana used in fencing and 

Guarea thompsonii used as Timber. Fence plants play an 

important role in local communities in preventing wild and 

domestic animals from entering crop fields. In Nepal, 

plant species having spines, thorns and branches are 

mostly preferred for fencing (Bhattarai et al., 2006). Some 

of these plants are grown around the field permanently as 

a life fence whereas some are harvested and temporarily 

placed as a barrier. 

In the study area both the life fence and the barrier 

systems were used. According to Bhattarai et al. (2006) 

this fence plants are also used as wind break and erosion 

control. Fences in Lebialem are both homogenous as 

well as heterogonous but the heterogonous type of 

fencing is commonly seen surrounding crop fields and 

compounds (households). Five species in the study area 


were used for fencing including Dracaena arborea, 

Dracaena mannii, S. hierniana, P. strictistipula and Ficus 

mucuso. Focho et al. (2009b) have reported other uses of 

these plants in other parts of Cameroon. For example, 

Schefflera abyssinica, Schefflera mannii, and Carapa 

grandifolia are used for wood carving, construction and 

honey production in the Fundong area of Cameroon. 

Also, D. mannii and Dracaena aborea are used for 

building bridges and for traditional dances. Fuel wood in 

Lebialem is used for cooking and heating of houses. 

Fourteen species of plants were used as fuel wood. Fuel 

wood gathering was mostly done by females. This is in 

contrast with the findings of Kappelle et al. (2000) and 

Ochoa et al. (1998) who observed that fuel wood 

collection was mostly done by male. 

Conclusion 

This research has shown that Lewoh- Lebang 

communities in Cameroon are rich and diversified in 

medicinal plant species. The elderly people in the area of 

study are the custodians of indigenous knowledge on 

medicinal plants while there was rural urban exodus by 

the younger generation. The collection of information 

about natural flora, classification, management and use 

of plants by the people holds importance among the 

ethnobotanists. The local people and researchers face 

the challenging task of not only documenting knowledge 

on plants, but also applying the results of their studies to 

biodiversity conservation and community development. 

The population has to be educated on propagation and 

conservation of plants as it is used in primary health care, 

timber, energy source as well as in the protection of their 

fragile ecosystem. 


The authors gratefully acknowledge the collaboration of


Table 8. Plant species used in cultural activities. 

No Species and family names Local names Parts used Uses 

1 

Schumanniophyton magnificum (K. Schum.) 

Harms (Rubiaceae) 

Sekoh Leaves Protection against accident 

2 

Harungana madagascariensis Lam.ex Poir 

(Clusiaceae) 

Etoueh Leaves Protection against witchcraft 

3 Maesa lanceolata Mez (Myrsinaceae) Teshi Leaves Protection against witchcraft 

4 Dracaena mannii Bak. (Dracaenaceae) Nkeng Branches Traditional dance 

5 Kigelia africana (Lam.) Benth. (Bignoniaceae) Ledongne Stem 

6 Dracaena arborea (Willd.) Link (Dracaenaceae) Aquahe Branches Traditional dance 

7 

Euphorbia desmindi Keay and Milne-Redhead 

(Euphorbiaceae) 

Used to fabricate traditional torches 

used during hunting 

Aboueh Leaves Protection against witchcraft 

8 Guarea cf glomerulata Harm (Meliaceae) Quat Leaves Protection against accident 

9 Vernonia sp. (Asteraceae) Bakeulouh Leaves 

10 

Psychotria penducularis (Salisb.) Steyerm. 

(Rubiaceae) 

Ndelente Peduncle 

11 Santeria balsamifera Oliv. (Burseraceae) Evong Resin 

Protection against accident and 

witchcraft 

Protection against accident and 

witchcraft 

the coagulated resin is burned to drive 

away witchcraft 

12 Kigelia africana (Lam.) Benth. (Bignoniaceae) Gueve Leaves Protection against accident 

13 Vernonia sp.(Asteraceae) Tabang Leaves Protection against witchcraft 

14 Cestrum nocturnum Linn. (Solanaceae) 

Queen of the 

night 

Whole plant 

15 Kigelia africana (Lam.) Benth. (Bignoniaceae) Gueve Branch Fetish 

Table 9. Plant species used for other purposes. 

Plant the tree in compounds to drive 

away witchcraft. 

No Species and family names Local names Parts used uses 

1 Euphorbia desmindi Keay and Milne-Redhead (Euphobiaceae) Aboueh Stem Craft 

2 Gambeya africana G. Don (Sapotaceae) Seignei Stem Craft 

3 Zanthoxylum gilletii (De Wild.) P.G. Waterman (Rutaceae) Mbem Stem Carving 

4 Cola accuminata (Vent.) Schott and Endt. (Sterculiaceae) Ledze Fruits N.T.F. P 

5 Cola heterophylla (P.Beauv.) Schott and Endl (Sterculiaceae) Ngalle Fruits N.T.F.P, used as gums 

6 Guarea cf glomerulata Harm (Meliaceae) Quat Bark Fish poisoning

the villagers in the Lewoh-Lebang villages in this study as 

well as the Limbe Botanic Garden and Botanists of the 

Cameroon National Herbarium for their help in validating 

the identities of specimens. Also the financial support of 

the non governmental organization (NGO), Environment 

and Rural Development Foundation (ERUDEF) is 

gratefully acknowledged. 

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DOI: 10.5897/JMPR11.1504 



Discrimination of Zhishi from different species using 

rapid-resolution liquid chromatography-diode array 

detection/ultraviolet (RRLC-DAD/UV) coupled with 

multivariate statistical analysis 

Zhenli Liu #1 , Yuanyan Liu #2 , Chun Wang 1 , Zhiqian Song 1 , Qinglin Zha 3 , Cheng Lu 3 , Chao 

Wang 1 and Aiping Lu 3 * 

1 Institution of Basic Theory, China Academy of Chinese Medical Sciences, Beijing, China. 

2 School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China. 

3 Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Beijing, China. 


Zhishi is one of the most commonly used traditional Chinese medicinal plants. Many species of Citrus 

plants, such as Citrus aurantium L. and its cultivars, Citrus sinensis Osbeck and its cultivars, and 

Citrus junos Sieb. ex Tanaka, can be used as Zhishi. However, Zhishi from different species exhibited 

differences in their therapeutic effects. In addition, it was very difficult to distinguish the species due to 

their similar morphologies. Flavanone compounds are the main bioactive constituents of Zhishi; in this 

study, flavanones were used as chemotaxonomic markers to distinguish among different Citrus 

samples. The contents of four flavanones (narirutin, naringin, hesperidin and neohesperidin) of 77 

Zhishi samples from three Citrus species were determined using rapid resolution liquid 

chromatography-diode array detection/UV (RRLC-DAD/UV) and multivariate statistical analysis. 

Principal component analysis (PCA) of the quantitative data showed a clear separation of compositions 

among the three species of Zhishi samples. Furthermore, the established differential mode was 

subsequently applied to identify the origin of unknown commercial samples. 

Key words: Zhishi, Citrus species, flavanone, rapid resolution liquid chromatography (RRLC), multivariate 

statistical analysis. 

INTRODUCTION 

Citrus plants are commonly used to derive medicine 

and/or food, and they contain a host of active flavonoids 

that contribute to health. For instance, the major species 

of Citrus that produce immature fruit used as Zhishi are 

Citrus aurantium L. and its cultivars, Citrus sinensis 

Osbeck and its cultivars, and Citrus junos Sieb. ex 

Tanaka (Xie, 1991; Cai et al., 1999). Zhishi (immature 

*Corresponding author. E-mail: lap64067611@126.com. Tel: 

+86 10 64067611. Fax: +86 10 64013896. 

# These authors contributed equally to this work. 

Fructus aurantii) is one of the earliest and most 

commonly used medicinal herbs in Traditional Chinese 

Medicine (TCM). The earliest record of Zhishi appeared 

in the Shen Nong Ben Cao Jing (A.D. 102-200). Zhishi is 

used to treat digestive disturbances (Chinese 

Pharmacopoeia, 2010). The herbs from different 

medicinal species and cultivation locations are widely 

used in clinical applications. As reported in the literature 

(Hao et al., 2002; Li et al., 1996), the effect of different 

species of Zhishi on enhancing intestinal transit speed 

are clearly different due to differences in the types and 

quantities of the chemical substances they contain. 

However, the morphologies of different species of 

commercial Zhishi are too similar to distinguish visually.

Therefore, the development of a reliable analytical 

method for species differentiation among Citrus herbs is 

of great significance for ensuring the safety and 

effectiveness of clinical treatment. Flavonoids are very 

common and widespread secondary plant metabolites. 

They show a wide range of biological and physiological 

activities and usually serve as chemotaxonomic marker 

compounds (Wang et al., 2007; Zhou et al., 2006; Maria 

et al., 2007). Moreover, flavonoids, especially the 

flavanones contained in Zhishi, have been recognized as 

the main bioactive constituents. Flavanones in Citrus 

species, including narirutin, naringin, hesperidin and 

neohesperidin (Hu et al., 1994), are among the most 

prominent cancer-preventing agents (Albach et al., 1969; 

Castillo et al., 1992; Jourdan et al., 1985; Kawaii et al., 

1999) and exhibit relatively high abundances. According 

to our previous research (Zhang et al., 2007) and the 

literature (Qin et al., 2009; Wu et al., 2009; Chuang et al., 

2007), the flavonoid constituents of Citrus species vary 

according to the species and the cultivation locations of 

the Citrus plants. This variation in flavonoid constituents 

may influence the therapeutic effect of different 

medicines that are produced using different plants. For 

these reasons, the aims of this work were to develop a 

reliable analytical method using flavanones as marker 

compounds and to establish a multivariate statistical 

analysis technique that can be utilized to identify species 

of Citrus plants such as C. aurantium L. and its cultivars, 

C. sinensis Osbeck and its cultivars, and C. junos Sieb. 

ex Tanaka. 

Many studies have focused on the differentiation of 

Zhishi by chemical compounds detection or fingerprints 

(Chuang et al., 2007; Kawaii et al., 1999; Maria et al., 

2007; Nogata et al., 2006; Qin et al, 2009; Wang et al., 

2007; Zhang et al., 2007). However, in fingerprint test, 

more compounds are needed to be verified, and in some 

cases, reference standards were unavailable. Also the 

determinations of fingerprints are time-consuming. In this 

study, four flavanone compounds (narirutin, naringin, 

hesperidin and neohesperidin) were used to be quantified 

in 77 samples of Zhishi, which were classified as three 

different Citrus species by RRLC-DAD/UV. Furthermore, 

this convenient and reliable analytical method coupled 

with multivariate statistical analysis was utilized to 

establish an effective differential mode for the different 

species of Zhishi samples. Finally, the application of this 

established analytical method to unknown samples 

enabled the classification of ten commercial Zhishi 

samples as three different Citrus species. 


Chemicals and materials 

Methanol and acetonitrile were purchased from Fisher Scientific 

(Pittsburgh, PA, USA). The other reagents were from Beijing 

Chemical Inc. (Beijing, China). The naringin and hesperidin 

standards were purchased from the National Institute for the 

Liu et al. 867 

Control of Pharmaceuticals and Biological Products (Beijing, 

China). The narirutin and neohesperidin standards were obtained 

from Sigma-Aldrich (Tokyo, Japan). 

The 77 authentic Zhishi samples were collected from four local 

provinces of China: Jiangxi, Sichuan, Zhejiang and Guizhou. They 

all were identified as genuine samples of C. aurantium L. and its 

cultivars (CA), C. sinensis Osbeck and its cultivars (CS), and 

C. junos Sieb. ex Tanaka (CJ), respectively, by Professors Ge Fei, 

Yan Zhuyun, Zhang Yungui, Xu Jianguo and Ke Fuzhi. The dried 

voucher specimens (marked as CA-1∼CA-32, CS-1~CS-33 and CJ- 

1~CJ-12) were deposited at the Institute of Basic Theory, China 

Academy of Chinese Medical Sciences, Beijing, P. R. China. The 

ten commercial Zhishi samples (marked as CM-1~CM-10) were 

purchased from drug stores in different provinces of China. 

RRLC-DAD/UV analysis 

The data were obtained using an Agilent 1200 Series RRLC with 

DAD. The analytical conditions for recording chromatograms of the 

marker compounds in Zhishi were as follows: A Zobax Extend-C18 

column (4.6 × 50 mm; 1.8 µm; Agilent Technologies) was used. The 

mobile phase was a mixture of methanol-2% HAc (30: 70, v/v) at a 

flow rate of 1.0 mL/min, and the column temperature was set to 

25°C. The detection wavelength was set to 285 nm and the run 

time of chromatography was 8 min. 

Sample preparation 

Each pulverized dried sample (0.1 g) was refluxed with methanol 

(25 mL) for 30 min at 80°C. Then the supernatant solution was 

filtered through a 0.2 µm Millipore filter for the RRLC analysis. Each 

sample was prepared in triplicate. The injection volume was 2 µL. 

The calibration curves, precisions, and recoveries for the analyses 

were examined and validated. The limits of detection (Signal/Noise 

= 3) under the present conditions were 0.35 ~ 0.48 ng. 


The RRLC-DAD/UV data for different species of Zhishi samples 

were analyzed to identify potential discriminant variables. A 

quantitative data set, which consisted of values taken from the 

RRLC analyses of the 77 samples, was used for the multivariate 

analysis. Multivariate statistical analyses, including unsupervised 

principal component analysis (PCA) and supervised partial least 

squares (PLS), were performed using the SAS 9.1.3 statistical 

package (order no. 195557). PCA was used to observe the natural 

interrelationship among the chemical components for each of the 

three Citrus species, which could be applied to differentiate the 

three species according to the differentially expressed components. 

Furthermore, differential mode was carried out through the use of a 

more sophisticated PLS. The critical p value for all analyses in this 

study was set to 0.05. 


Characteristics of the collected samples 

For all 77 Zhishi samples, the original plant, local name, 

location of collection, year and growing environments are 

listed in Table 1. All of the samples were collected from 

the main Zhishi-producing provinces of China during a 

three-year period. According to previous results (Zhou et


Table 1. The origins of the 77 Zhishi samples collected. 

No. Original plant Local name Location and time of collection Growing environment 

Citrus aurantium L. 

CA-1 C. aurantium cv Xiucheng Xiucheng Xingan, Jiangxi; 2007 Plain (N 27° E 115°; Alt.20~30 m) 

CA-2 C. aurantium cv Xiucheng Xiucheng Qingjiang, Jiangxi; 2007 Hillsides (N 27° E 114°; Alt.100~200 m) 

CA-3∼CA-10 C. aurantium cv Xiucheng Xiucheng Xingan, Jiangxi; 2008 Hillsides (N 27° E 115°; Alt.50~60 m) 

CA-11∼CA-21 C. aurantium cv Xiucheng Xiucheng Xingan, Jiangxi; 2009 Hillsides (N 27° E 115°; Alt.50~60 m) 

CA-22 C. aurantium cv Jizicheng Jizicheng Zhangshu, Jiangxi; 2009 Plain (N 27° E 115°; Alt.20~30 m) 

CA-23∼CA-24 C. aurantium × P. trifoliata Citrange Yuanjiang, Hunan; 2007 Plain (N 28° E 112°; Alt.30~40 m) 

CA-25∼CA-27 C. aurantium L. Sour orange Jiangjin, Sichuan; 2008 Field margins (N 29° E 106°; Alt.200~230 m) 

CA-28 C. aurantium L. Sour orange Jiangjin, Sichuan; 2007 Hillsides (N 29° E 106°; Alt.231 m) 

CA-29 C. aurantium L. Jiangjin sour orange Jiangjin, Sichuan; 2009 Hillsides (N 29° E 106°; Alt.231 m) 

CA-30∼CA-31 C. aurantium cv Daidai Daidai Jiangjin, Sichuan; 2009 Hillsides (N 29° E 106°; Alt.229 m) 

CA-32 C. aurantium cv Morocco sour orange Morocco sour orange Huangyan, Zhejiang; 2009 Hillsides (N 28° E 121°; Alt.45 m) 

Citurs junos Sieb.ex Tanaka 

CJ-1∼CJ-4 C. junos Sieb. ex Tanaka Xiangcheng Xingan, Jiangxi; 2008 Hillsides (N 27° E 115°; Alt.50~60 m) 

CJ-5∼CJ-6 C. junos Sieb. ex Tanaka Tuanye Xiangcheng Jiangjin, Sichuan; 2009 Hillsides (N 29° E 106°; Alt.220 m) 

CJ-7∼CJ-12 C. junos Sieb. ex Tanaka Xiangcheng Xingan, Jiangxi; 2009 Hillsides (N 27° E 115°; Alt.50~60 m) 

Citrus sinensis Osbeck 

CS-1 C. sinensis Osbeck Sweet orange Jiangjin, Sichuan; 2007 Field margins (N 29° E 106°; Alt.200~230 m) 

CS-2 C. sinensis Osbeck Sweet orange Fengjie, Sichuan; 2008 Field margins (N 29° E 106°; Alt.200~230 m) 

CS-3∼CS-4 C. sinensis Osbeck Sweet orange Jiangjin, Sichua; 2008 Field margins (N 29° E 106°; Alt.200~230 m) 

CS-5 C. sinensis Osbeck cv Jin Cheng Jin Cheng Qinglong, Guizhou; 2008 Hillsides (N 25° E 105°; Alt.1200~1300 m) 

CS-6 C. sinensis Osbeck cv Navel orange Navel orange Qinglong, Guizhou; 2008 Hillsides (N 25° E 105°; Alt.1200~1300 m) 

CS-7 C. sinensis Osbeck cv Blood orange Blood orange Qinglong, Guizhou; 2008 Hillsides (N 25° E 105°; Alt.1200~1300 m) 

CS-8 C. sinensis Osbeck cv Valencia Valencia Qinglong, Guizhou; 2008 Hillsides (N 25° E 105°; Alt.1200~1300 m) 

CS-9 C. sinensis Osbeck cv Blood orange Blood orange Jiangjin, Sichuan; 2009 Hillsides (N 29° E 106°; Alt.238 m) 

CS-10 C. sinensis Osbeck cv Valencia Valencia Jiangjin, Sichuan; 2009 Hillsides (N 29° E 106°; Alt.238 m) 

CS-11 C. sinensis Osbeck cv Navel orange Navel orange Jiangjin, Sichuan; 2009 Hillsides (N 29° E 106°; Alt.238 m) 

CS-12 C. sinensis Osbeck cv Peng an 100 Peng an 100 Jiangjin, Sichuan; 2009 Hillsides (N 29° E 106°; Alt.238 m) 

CS-13 C. sinensis Osbeck cv Li Cheng Li Cheng Jiangjin, Sichuan; 2009 Hillsides (N 29° E 106°; Alt.238 m) 

CS-14 C. sinensis Osbeck cv Hong 6-6 Hong 6-6 Jiangjin, Sichuan; 2009 Hillsides (N 29° E 106°; Alt.238 m) 

CS-15 C. sinensis Osbeck cv Feng Chan Ji Cheng Feng Chan Ji Cheng Jiangjin, Sichuan; 2009 Hillsides (N 29° E 106°; Alt.238 m) 

CS-16 C. sinensis Osbeck cv Da Zhou Zao Shu Da Zhou Zao Shu Jiangjin, Sichuan; 2009 Hillsides (N 29° E 106°; Alt.239 m) 

CS-17 C. sinensis Osbeck cv Chang Ye Cheng Chang Ye Cheng Jiangjin, Sichuan; 2009 Hillsides (N 29° E 106°; Alt.238 m) 

CS-18 C. sinensis Osbeck cv Yin Zao Cheng Yin Zao Cheng Huangyan, Zhejiang; 2009 Hillsides (N 28° E 121°; Alt.40~60 m)



CS-19 C. sinensis Osbeck cv Omishima navel orange Omishima navel orange Huangyan, Zhejiang; 2009 Hillsides (N 28° E 121°; Alt.40~60 m) 

CS-20 C. sinensis Osbeck cv Delta Valencia Delta Valencia Huangyan, Zhejiang; 2009 Hillsides (N 28° E 121°; Alt.40~60 m) 

CS-21 C. sinensis Osbeck cv Liu Ben Cheng Liu Ben Cheng Huangyan, Zhejiang; 2009 Plain (N 28° E 121°; Alt.7 m) 

CS-22 C. sinensis Osbeck cv Early Gold sweet orange Early Gold sweet orange Huangyan, Zhejiang; 2009 Hillsides (N 28° E 121°; Alt.40~60 m) 

CS-23 C. sinensis Osbeck cv Washington Sanguine Washington Sanguine Huangyan, Zhejiang; 2009 Hillsides (N 28° E 121°; Alt.40~60 m) 

CS-24 C. sinensis Osbeck cv Olinda Valencia Olinda Valencia Huangyan, Zhejiang; 2009 Hillsides (N 28° E 121°; Alt.40~60 m) 

CS-25 C. sinensis Osbeck cv Seike navel orange Seike navel orange Huangyan, Zhejiang; 2009 Hillsides (N 28° E 121°; Alt.40~60 m) 

CS-26 C. sinensis Osbeck cv Feng Chan Ji Cheng Feng Chan Ji Cheng Huangyan, Zhejiang; 2009 Hillsides (N 28° E 121°; Alt.40~60 m) 

CS-27 C. sinensis Osbeck cv Hamlin Hamlin Huangyan, Zhejiang; 2009 Plain (N 28° E 121°; Alt.7 m) 

CS-28 C. sinensis Osbeck cv Jin Cheng Jin Cheng Huangyan, Zhejiang; 2009 Hillsides (N 28° E 121°; Alt.40~60 m) 

CS-29 C. sinensis Osbeck cv Navel orange Navel orange Huangyan, Zhejiang; 2009 Hillsides (N 28° E 121°; Alt.40~60 m) 

CS-30 C. sinensis Osbeck cv Midknight Valencia Midknight Valencia Huangyan, Zhejiang; 2009 Hillsides (N 28° E 121°; Alt.40~60 m) 

CS-31 C. sinensis Osbeck cv Hong Jiang Cheng Hong Jiang Cheng Huangyan, Zhejiang; 2009 Hillsides (N 28° E 121°; Alt.40~60 m) 

CS-32 C. sinensis Osbeck cv Ming Liu Cheng Ming Liu Cheng Huangyan, Zhejiang; 2009 Hillsides (N 28° E 121°; Alt.40~60 m) 

CS-33 C. sinensis Osbeck cv Fukumoto navel orange Fukumoto navel orange Huangyan, Zhejiang; 2009 Hillsides (N 28° E 121°; Alt.40~60 m) 

al., 2006; Maria et al., 2007), the concentrations of 

naringin and neohesperidin in the fruits of C. 

aurantium are at a maximum during the 

logarithmic phase of growth,gradually decreasing 

until the organs reach maximum development. 

Thus, in this study, the diameters of collected 

Zhishi samples were limited to 0.5 cm∼2.5 cm, 

which is also consistent with the requirements of 

the Chinese Pharmacopoeia (Chinese 

Pharmacopoeia, 2010). Thirty-two samples (CA- 

1~CA-32) were identified as C. aurantium and its 

five cultivars (C. aurantium cv Xiucheng, C. 

aurantium cv Jizicheng, C. aurantium × P. 

trifoliata, C. aurantium cv Daidai, and C. 

aurantium cv Morocco sour orange); 12 samples 

(CJ-1~CJ-12) were identified as C. junos; and 33 

samples (CS-1~CS-33) were identified as C. 

sinensis and its cultivars. The cultivars of C. 

sinensis are abundant in China, and 12 kinds of 

C. sinensis and its cultivars were collected in this 

study, as shown in Table 1. 

Method validation 

Calibration curves were prepared by plotting the 

peak area of marker compounds against the 

corresponding concentrations. Good linear 

relationships (R 2 = 0.9998 for naringin, and 

0.9999 for narirutin, hesperidin and 

neohesperidin) are demonstrated over a range of 

4∼50 µg/mL. Limits of detection (LOD) and 

quantification (LOQ) under the present 

chromatographic conditions were determined on 

the basis of response and slope of each 

regression equation at a signal to noise ratio of 3 

and 10, respectively. 

The LOD for the four marker compounds ranged 

from 0.04 to 0.25 µg/mL, and the corresponding 

LOQ ranged from 0.11 to 0.69 µg/mL. The 

accuracy of the analytical method was evaluated 

using the recovery test. The mean recoveries are 

from 99.9 to 102.1% with RSD less than 2.01% for 

the four reference compounds. 

The precision of the assay was determined by 

repeatability. The RSD of retention time and peak 

area ranged from 0.34 to 1.91% and 1.14 to 

1.96%, respectively. 

RRLC-DAD/UV analyses of the three different 

species of Zhishi samples 

In the 32 samples of CA, all of the four reference 

standards could be identified. The concentrations 

of narirutin, naringin, hesperidin and 

neohesperidin in this species of Zhishi samples 

are shown in Table 2. 

The concentrations of narirutin and hesperidin 

ranged from 0.41 to 1.12% and 0.41 to 1.96%,


Table 2. The concentrations of the four flavanones investigated in the 77 Zhishi samples from three species of Citrus plants. 

No. 

Narirutin 

Content RSD (%) 

Naringin 


Hesperidin 


Neohesperidin 


Total (%) Naringin/Narirutin 

CA-1 0.92 1.3 10.94 1.2 0.79 0.25 10.16 0.45 22.81 11.84 

CA-2 0.61 0.64 7.23 1.1 1.65 0.33 17.10 0.74 26.59 11.83 

CA-3 0.59 1.1 7.01 2.4 1.26 0.57 14.80 2.0 23.66 11.84 

CA-4 0.51 0.17 8.14 0.25 1.96 0.58 12.70 1.9 23.31 15.96 

CA-5 0.58 0.28 6.21 0.55 1.32 0.48 17.00 0.91 25.11 10.67 

CA-6 0.80 1.6 11.90 0.74 0.96 1.1 18.40 0.97 32.06 14.86 

CA-7 0.88 2.1 11.20 0.56 1.18 2.5 17.50 0.47 30.76 12.68 

CA-8 0.81 0.89 10.10 0.24 1.72 0.25 11.80 0.24 24.43 12.41 

CA-9 0.61 0.74 7.94 2.1 0.75 1.5 15.30 0.57 24.60 12.97 

CA-10 0.41 0.62 6.05 0.36 0.66 0.25 12.17 0.36 19.30 14.63 

CA-11 0.90 1.3 13.57 0.69 0.96 0.48 12.18 0.17 27.61 15.04 

CA-12 0.48 0.14 9.74 0.61 0.54 0.51 12.13 0.28 22.90 20.09 

CA-13 0.83 0.27 9.76 0.25 1.36 0.33 20.21 1.7 32.15 11.82 

CA-14 0.64 1.2 12.52 0.25 0.54 0.69 16.03 2.1 29.74 19.43 

CA-15 0.64 2.9 10.35 1.2 0.41 0.94 8.38 1.0 19.78 16.27 

CA-16 0.66 1.2 10.81 2.1 0.38 0.28 9.21 0.36 21.06 16.35 

CA-17 0.57 1.0 10.10 2.2 0.48 0.47 6.54 0.60 17.69 17.64 

CA-18 0.44 0.64 9.69 1.2 0.43 0.77 8.95 0.14 19.51 21.88 

CA-19 0.62 1.1 10.06 1.0 0.72 0.25 12.52 0.51 23.92 16.24 

CA-20 0.46 2.1 8.69 2.5 0.41 0.62 8.96 0.58 18.52 18.83 

CA-21 0.55 0.33 11.08 0.25 0.59 0.24 10.32 0.34 22.54 20.07 

CA-22 0.66 1.8 9.23 0.54 0.89 0.28 16.52 0.21 27.30 13.91 

CA-23 0.59 0.21 8.24 1.2 0.98 1.5 20.20 0.11 30.01 13.94 

CA-24 0.87 1.9 12.70 0.58 1.76 0.66 9.48 0.28 24.81 14.55 

CA-25 0.64 0.36 9.47 1.2 1.18 0.48 24.30 0.10 35.59 14.77 

CA-26 0.55 0.48 9.06 0.52 1.21 0.41 27.25 0.97 38.07 16.41 

CA-27 0.53 1.7 8.57 0.28 1.42 0.36 25.15 0.24 35.67 16.14 

CA-28 0.59 1.5 11.50 1.2 0.52 0.58 19.10 0.28 31.42 19.62 

CA-29 0.76 1.9 11.40 0.25 1.50 0.47 24.41 0.46 38.07 15.07 

CA-30 1.01 0.24 13.35 2.2 0.99 0.54 22.68 0.97 38.04 13.23 

CA-31 0.91 0.18 12.14 0.25 1.39 1.5 27.30 0.77 41.74 13.31 

CA-32 1.12 0.25 14.63 1.25 1.23 0.65 23.12 0.45 40.10 13.08 

CJ-1 13.20 1.6 0.15 0.25 12.40 1.5 0.12 0.62 25.88 0.01 

CJ-2 14.80 1.7 0.38 1.2 12.10 1.8 0.50 0.24 27.79 0.03 

CJ-3 12.30 2.3 0.19 0.25 10.90 0.36 0.17 0.48 23.56 0.02


CJ-4 13.30 0.69 0.14 1.5 13.20 0.61 0.13 0.15 26.77 0.01 

CJ-5 6.99 0.22 5.32 0.36 7.51 0.47 6.63 0.25 26.45 0.76 

CJ-6 4.85 0.55 2.94 0.96 4.99 1.3 3.10 0.87 15.90 0.61 

CJ-7 16.12 0.14 0.27 0.58 9.46 1.2 0.11 0.14 25.96 0.02 

CJ-8 15.66 0.14 0.14 2.1 8.77 2.5 0.09 0.16 24.67 0.01 

CJ-9 16.23 0.36 1.66 0.25 12.15 1.4 2.15 0.61 32.19 0.10 

CJ-10 15.67 1.5 0.31 0.24 13.50 1.6 0.21 0.89 29.69 0.02 

CJ-11 14.65 1.7 0.24 0.15 11.52 0.54 0.33 0.69 26.74 0.02 

CJ-12 11.08 1.9 0.17 0.25 10.14 0.44 0.27 0.41 21.66 0.02 

CS-1 0.74 0.66 ND — 21.40 0.36 ND — 22.14 — 

CS-2 0.76 1.5 ND — 21.90 0.48 ND — 22.66 — 

CS-3 1.58 0.24 ND — 21.70 0.15 ND — 23.28 — 

CS-4 1.23 0.67 ND — 24.30 0.67 ND — 25.53 — 

CS-5 0.92 0.36 ND — 27.60 0.19 ND — 28.52 — 

CS-6 1.76 0.14 ND — 19.70 0.97 ND — 21.46 — 

CS-7 0.56 0.25 ND — 26.00 0.14 ND — 26.56 — 

CS-8 0.34 0.14 ND — 27.30 1.6 ND — 27.64 — 

CS-9 1.45 0.69 ND — 25.22 0.39 ND — 26.67 — 

CS-10 2.25 0.98 ND — 19.55 0.75 ND — 21.80 — 

CS-11 3.50 0.87 ND — 17.92 0.89 ND — 21.42 — 

CS-12 1.12 1.87 ND — 18.70 0.14 ND — 19.83 — 

CS-13 1.32 2.0 ND — 18.35 1.7 ND — 19.66 — 

CS-14 2.06 2.1 ND — 17.93 1.3 ND — 19.99 — 

CS-15 2.99 1.7 ND — 17.56 0.65 ND — 20.55 — 

CS-16 1.90 0.36 ND — 16.54 0.61 ND — 18.44 — 

CS-17 1.20 0.54 ND — 29.99 0.41 ND — 31.19 — 

CS-18 1.31 0.48 ND — 17.44 0.39 ND — 18.75 — 

CS-19 0.71 1.1 ND — 8.68 0.91 ND — 9.38 — 

CS-20 0.43 0.94 ND — 23.89 0.14 ND — 24.32 — 

CS-21 0.40 0.22 ND — 16.46 1.9 ND — 16.86 — 

CS-22 0.99 0.32 ND — 19.47 2.4 ND — 20.47 — 

CS-23 0.16 0.14 ND — 9.04 1.6 ND — 9.21 — 

CS-24 1.15 1.5 ND — 25.32 0.24 ND — 26.47 — 

CS-25 2.28 2.1 ND — 10.54 1.6 ND — 12.82 — 

CS-26 1.86 2.6 ND — 10.48 0.27 ND — 12.34 — 

CS-27 1.12 1.6 ND — 23.15 1.6 ND — 24.28 — 

CS-28 0.78 0.29 ND — 18.72 2.4 ND — 19.50 — 

Liu et al. 871



CS-29 1.61 0.25 ND — 15.64 1.4 ND — 17.25 — 

CS-30 0.31 0.14 ND — 13.84 2.0 ND — 14.16 — 

CS-31 0.45 0.36 ND — 21.70 0.68 ND — 22.15 — 

CS-32 0.59 0.14 ND — 25.62 1.1 ND — 26.20 — 

CS-33 2.05 0.58 ND — 23.27 2.8 ND — 25.32 — 

ND = not detectable. 

respectively; the levels of naringin and 

neohesperidin were found to be much higher than 

the other two standards, ranging from 6.05 to 

14.62% and 6.54 to 27.3%, respectively. The total 

amount of the four compounds was 18.52 to 

41.74%. 

Compared to the CA samples, the 12 samples 

of CJ exhibited some salient differences. The 

narirutin and hesperidin concentrations were 

higher in the CJ samples, with concentrations 

ranging from 4.86 to 16.23% and 4.99 to 13.50%, 

respectively. However, the concentrations of 

naringin and neohesperidin were much lower in 

the CJ samples, 

In the 33 samples of CS, only narirutin and 

hesperidin were detectable, while naringin and 

neohesperidin could barely be detected (Kawaii et 

al., 1999; Rouseff et al., 1987). The concentrations 

of narirutin and hesperidin ranged from 0.16 

to 3.5% and 9.04 to 17.92%, respectively. The 

total amount of these two compounds was 9.21 to 

31.19%. 

These findings indicated that different kinds of 

flavanones occurred at various concentrations in 

the different Citrus species of Zhishi samples. 

Samples from the same species cultivated in 

different locations did not show differences with 

regard to the types and content of the four 

reference standards. Furthermore, a comparison 

of the relative concentrations of naringin to 

narirutin (naringin/narirutin) among the three 

Citrus species suggested that the ratio was much 

higher in the CA samples than in the CJ and CS 

samples, which was consistent with the reported 

literature (Hosoda et al., 1989; Nogata et al., 

2006). Though there were some differences of the 

four compounds in different species of Zhishi, it is 

hard to make clear differentiation among different 

species of Zhishi. Therefore, the variation among 

the different Citrus species was further 

investigated in this study using the multivariate 

statistical analysis technique PCA. 

Principal component analysis 

To differentiate the CA, CJ and CS samples, an 

unsupervised pattern recognition method (PCA) 

was performed. A two-component PCA score plot 

of RRLC-DAD/UV data was utilized to depict the 

general variation of the marker flavanones among 

the three Citrus species of Zhishi samples. The 

clear separation of the three different species was 

observed in the PCA scores plot, where each 

coordinate represented a sample (Figure 1). 

A two-component PCA model cumulatively 

accounted for 93% of variation. The PCA scores 

plot in Figure 1 could be divided readily into three 

distinct clusters (CA, CJ and CS), indicating that 

the concentrations and distribution of flavanones 

varied significantly in the different Citrus species. 

Each sample was represented as a point in a 

scores plot (CA = ★, CJ = ▲ and CS = ◆). The CJ 

samples and the others were clearly separated by 

the principal component 1 (PC1), whereas the CA 

and CS samples were clearly separated by the 

principal component 2 (PC2). This statistical 

method can be used as a powerful tool for the 

authentication of Citrus species of Zhishi samples. 

Furthermore, the differential mode produced by 

the SAS 9.1.3 software was established through 

the use of PLS to identify the species of unknown 

commercial Zhishi samples. 

Identifying the species of unknown 

commercial Zhishi samples 

Ten commercially available Zhishi samples (CM- 

1~CM-10) were purchased from drug stores in 

different provinces of China. It is very difficult to 

distinguish the Citrus species merely by 

examining their morphologies because they are 

extremely similar; therefore, the present study 

quantified all four flavanones (narirutin, naringin, 

hesperidin and neohesperidin) in the commercial 

samples using the method described previously, 

and the data are listed in Table 3. The quantitative 

data were analyzed using the established differential

PrinC 2 

Figure 1. PCA scores plot (PC1 vs. PC2) of the three Citrus species of Zhishi samples: 32 

samples from C. aurantium L. and its cultivars, 12 samples from C. junos Sieb. ex Tanaka and 33 

samples from C. sinensis Osbeck and its cultivars. 

Table 3. The concentrations of the four flavanones investigated in the ten commercial Zhishi samples. 

No. Location of purchase 

Narirutin 

(%) 

Naringin 

(%) 

Hesperidin 

(%) 

Neohesperidin 

(%) 


Identified as 

species 

CM-1 Guiyang, Guizhou province 2.42 ND 11.65 ND CS 

CM-2 Yantai, Shandong province 2.50 ND 10.25 ND CS 

CM-3 Dalian, Liaoning province 2.50 ND 13.62 ND CS 

CM-4 Beijing 1.49 ND 11.13 ND CS 

CM-5 Beijing 0.59 ND 6.30 ND CJ 

CM-6 Chongqing 0.97 ND 8.99 ND CS 

CM-7 Jiangjin, Sichuan province 1.27 12.05 1.31 12.68 CA 

CM-8 Nanchang, Jiangxi province 0.61 7.23 1.65 17.12 CA 

CM-9 Nanjing, Jiangsu province 0.69 11.54 0.92 16.05 CA 

CM-10 Zhengzhou, Henan province 0.37 ND 11.36 ND CS 

ND = not detectable. 

mode, and CM-1~CM-4, CM-6 and CM-10 were identified 

as C. sinensis and its cultivars. CM-5 was identified as C. 

junos, and CM-7~CM-9 was identified as C. aurantium 

and its cultivars, as shown in Figure 2. 

In this study, a convenient and reliable analytical method 

coupled with multivariate statistical analysis was used to 

evaluate and distinguish the following three species of 

Zhishi samples: C. aurantium and its cultivars, C. 

sinensis and its cultivars, and C. junos. Four active 

flavanones (narirutin, naringin, hesperidin and neohesperidin)


Residual 

Figure 2. Identification of the ten unknown commercial samples through the use of PLS. (CA = ★, CJ = ▲ 

and CS = ◆). 

were used as marker compounds, which were quantified 

using RRLC-DAD/UV, and the corresponding data were 

analyzed using PCA analysis. Using the methods 

described in the forgoing, 77 Zhishi samples were clearly 

identified as three Citrus species. 

Though we did not know whether we can use less 

marker compounds for the differentiation of different 

species of Zhishi, the established differential mode by 

using the four important marker compounds could be 

applied to identify unknown commercial samples. Hence, 

this work is of great importance for the evaluation and 

authentication of Zhishi samples, which is ultimately of 

great significance for ensuring the safety and 

effectiveness of its clinical use. 


This study was financially supported by the National 

Science Foundation of China (Project No. 30772726, No. 

30825047 and No. 81001623). 

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DOI: 10.5897/JMPR11.1519 



Isolation, chemical characterization and in vitro 

antioxidant activities of polysaccharides from 

Aconitum coreanum 

Bin Li 1 *, Xian-Jun Meng 1 and Li-Wei Sun 2 

1 College of Food Science, Shenyang Agricultural University, Shenyang 110866, China. 

2 College of Basic Medical, Beihua University, Jilin City 132013, China. 


The water-soluble crude polysaccharide Aconitum coreanum polysaccharides (ACPS) was extracted 

from A. coreanum and purified by Diethylaminoethyl cellulose and Sepharose CL-6B chromatography, 

giving three polysaccharide fractions coded as ACPSA-1, ACPSB-2 and ACPSB-3. Their chemical and 

physical characteristics of polysaccharide fractions, and antioxidant capacity, including scavenging 

activity against DPPH radicals, superoxide and hydroxyl radicals, and chelating ability, were valuated in 

this paper. Experiment results showed that ACPS-2 and ACPS-3 exhibited significantly antioxidant 

activity at a concentration-dependent manner. The polysaccharide fractions can be developed as new 

antioxidant agents. 

Key words: Aconitum coreanum, polysaccharide, purification, characterization, antioxidant activity. 

INTRODUCTION 

The recent abundant evidences suggest that reactive 

oxygen species (ROS), such as superoxide anion, 

hydrogen peroxide and hydroxyl radical, involve in the 

pathogenesis of various disorders and diseases (Niki, 

2010). According to the free-radical theory (Lin and Beal, 

2003; Muller et al., 2007), the disruption of the delicate 

balance between generation of reactive oxygen species 

and antioxidant scavenging systems could lead to a shift 

to an oxidative cellular milieu, and eventually lead to 

serious health problems such as diabetes and 

Alzheimer’s disease. So, much more attention has 

attracted to develop and utilize effective and natural 

antioxidants in the maintenance of human health and 

prevention and retardation the progress of many chronic 

diseases induced by free radical (Getoff, 2007). 

Current researches on free radicals have confirmed that 

traditional Chinese medicine rich in antioxidants play an 

essential role in the prevention of cardiovascular 

diseases, cancers, neurodegenerative diseases, 

inflammation and other free radical induced problems 

*Corresponding author. E-mail: libinsyau@163.com. Tel: + 

86-24-88488277. Fax: + 86-24-88488277. 

(Liao et al., 2008; Wu et al., 2004), and search for novel 

type of antioxidants from traditional Chinese medicine 

(TCM) has achieved considerable attention (Kirby and 

Schmidt, 1997). Polysaccharides as important natural 

products from traditional Chinese medicine exhibit 

significant antioxidant activities, which protect cells 

against the damaging effects of reactive oxygen species, 

prevention of the chronic and degenerative diseases 

(Chen and Yan, 2005; Kardošová and Machová, 2006; 

Song et al., 2010). 

The genus Aconitum is well known as poisonous and 

medicinal plants, which comprises ca. 400 species, and 

more than a half of them are growing in China. The most 

important variety of Aconitum genus, Aconitum coreanum 

(Lèvl.) Rapaics was distributed in Liaoning, Jilin, 

Neimenggu and Heilongjiang Province of north China. Its 

roots had been used as one of the most centuries-old 

traditional Chinese medicines documented in ancient 

Chinese medicinal literature. Supposedly originated from 

China, A. coreanum was discovered to be an antidote for 

poisonous herbs by a great herbalist, Shen Nong, about 

4700 years ago. 

To date, A. coreanum has been used to treat various 

kinds of disorders such as cardialgia, facial distortion, 

epilepsia, migraine headache, vertigo, tetanus, infantile

convulsion and rheumatic arthralgia (Alessandra et al., 

2003; Liou et al., 2005). 

However, there have been seldom reports on free 

radical scavenging activities of polysaccharide fractions 

from A. coreanum. In order to fully develop the wild 

resources and extend the potential use of A. coreanum in 

antioxidant biomedicine, the present study was carried out 

to investigate antioxidant activities of polysaccharide 

fractions from Aconitum coreanum with three in vitro 

antioxidant models, including 

1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging 

activity, superoxide radical scavenging activity, hydroxyl 

radical scavenging activity and ferrous ion-chelating 

activity, as well as their chemical and physical 

characteristics. 

EXPERIMENTAL 

Materials and chemicals 

Aconitum coreanum was purchased from a local medicine market, 

and identified according to the identification standard of 

Pharmacopeia of the People’s Republic of China. Phenazine 

methosulfate (PMS), nicotinamide adenine dinucleotide (NADH), 

nitroblue tetrazolium (NBT), safranine, ferroizine, T-series dextrans, 

Diethylaminoethyl cellulose, 1,1-diphenyl-2-picrylhydrazyl (DPPH) 

and standard sugars were purchased from Sigma Chemical Co. (St. 

Louis, MO, USA). Sepharose CL-6B was purchased from 

Amersham Pharmacia Co. (Sweden). Trifluoroacetic acid (TFA), 

EDTA-Na2, sodium hydroxide, hydroxylamine, inositol, acetic 

anhydride, pyridine, methanol and acetic acid were from Beijing 

Chemicals and Reagents Co. (Beijing, China). All other chemical 

reagents used were analytical grade. 

Isolation and purification of polysaccharide fractions 

After the roots of A. coreanum were cleaned, they were ground in an 

electric mill, and the powder was extracted with hot water at pH 7.0 

for three times, 2 h for each time. The whole extract was filtered, 

centrifuged and concentrated, and then precipitated with 3 volumes 

of ethanol at 4°C overnight. The crude polysaccharide precipitate 

was collected by centrifugation, deproteinated by a combination of 

proteinase and Sevag method (Staub, 1965), and then obtained 

crude A. coreanum polysaccharides (cACPS). 

The cACPS was dissolved in distilled water, and then loaded onto 

DEAE–cellulose column, eluted successively with distilled water and 

0.5 M NaCl. Fractions were collected, and monitored with the 

phenol–sulfuric acid method. The two main fractions (ACPSA and 

ACPSB) was collected, dialyzed, lyophilized, and were further 

fractioned on a Sepharose CL-6B column, eluted with 0.15 M NaCl 

to yield three main fractions, codes as ACPSA-1, ACPSB-2 and 

ACPSB-3. All the fractions were collected, dialyzed and lyophilized. 

Molecular weight determination 

Molecular weights of the different polysaccharide fractions were 

determined by high performance liquid chromatography (HPLC). 

The samples of polysaccharide fractions were dissolved in distilled 

water, applied to Agilent HPLC system (Agilent Technologies, USA) 

equipped with a TSK-GEL G3000 PWXL column, eluted with 0.1 

mol/L Na2SO4 solutions and detected by a RID-10A Refractive Index 

Li et al. 877 

Detector. Dextran standards with different molecular weights 

(T-2000, T-70, T-40, T-20, and T-10) were to calibrated the column 

and establish a standard curve. 

Monosaccharide composition analysis 

Polysaccharide fractions were hydrolyzed and acetylated according 

to Lehrfeld (1985). Simply, the samples were hydrolyzed with TFA 

and then hydrolyzed product was reduced with KBH4, followed by 

neutralization with acetic acid. After adding myo-inositol and 

Na2CO3, the residue was concentrated. The reduced products were 

added with pyridine–propylamine, and acetylated with 

pyridine–acetic anhydride. The acetylated products were analyzed 

by gas chromatography (GC), and identified and estimated with 

myo-inositol as the internal standard. GC was performed on a 

Agilent 6890 instrument (Agilent Technologies, USA) equipped with 

HP-5 capillary column (30 m × 0.32 mm × 0.2 μm) and 

flame-ionization detector (FID) and temperatures programmed from 

120 to 250°C at a rate of 8°C /min. 

Measurement of carbohydrate and protein contents 

Total carbohydrate contents of the polysaccharide fractions were 

determined by phenol-sulfuric acid colorimetric method (Dubois et 

al., 1956). Total uronic acid contents were measured by 

m-hydroxydiphenyl method (Filisetti-Cozzi and Carpita, 1991). 

Protein contents were quantified according to the Bradford’s method 

(Bradford, 1976). 

DPPH free radical scavenging activity 

Radical scavenging activity against the stable radical 

1,1-diphenyl-2-picrylhydrazyl (DPPH) was measured by the method 

of Fan et al. (2009) with a minor modification. Samples were 

dissolved in distilled water at 0 (control), 0.5, 1, 2, 4, and 8 mg/ml. 

One milliliter samples were mixed with 2 ml of freshly prepared 

DPPH (0.1 mM) in 50% ethanol. The mixture was incubated at 25°C 

for 30 min in the dark, and then the absorbance was measured at 

517 nm. The experiment was carried out in triplicate and averaged. 

The scavenging activity of DPPH radicals was calculated by the 

following formula: 

Scavenging effect (%) = (1 - Asample/Acontrol) × 100%. 

Superoxide radical scavenging assay 

The scavenging effects of polysaccharide fractions on superoxide 

radicals were assayed by the method of photoreduction of NBT (Shu 

and Lung, 2008) with some modifications. Reaction mixtures in a 

final volume of 3 ml contained the following reagents at final 

concentration: 60 μM phenazine methosulfate (PMS), 468 μM 

nicotinamide adenine dinucleotide (NADH), 150 mM nitroblue 

tetrazolium (NBT), and various concentrations of samples. The 

mixture reacted at 20ºC for 10 min and then the absorbance was 

measured at 560 nm. Each value was expressed by the mean of 

triplicate measurements with standard deviation. The capability of 

scavenging the superoxide radical was calculated using the 


Scavenging effect (%) = (1−Asample/Acontrol) ×100%



The hydroxyl radical assay was measured by the method of Fenton 

reaction (Xia et al., 2005) with a minor modification. Briefly, Samples 

were dissolved in distilled water at 0 (control), 0.5, 1, 2, 4, and 8 

mg/ml. The reaction mixture contained 1 ml of safranine (0.36 mM), 

0.5 ml of EDTA-Fe (2 mM), 1.5 ml of H2O2 (3.0%) and 1 ml samples 

of varying concentrations. After incubation at room temperature for 

20 min, the absorbance of the mixture was measured at 520 nm. 

Hydroxyl radicals gave a crimson colour, so the absorbance change 

of the reaction mixture indicated the scavenging ability for hydroxyl 

radicals. The hydroxyl radical-scavenging activity was expressed as: 

Scavenging effect (%) = (1 - Asample/Acontrol) × 100%. 

Metal chelating assay 

The ferrous ion chelating ability of all different fractions was 

investigated according to the method of Lin et al. (2009). Briefly, 

samples were dissolved in distilled water at 0 (control), 0.5, 1, 2, 4, 

and 8 mg/ml. The reaction mixture contained 0.1 ml FeCl2 (2 mM), 

0.4 ml ferroizine (5 mM) and 1 ml samples of varying concentrations. 

After shaken well and incubated for 10 min at room temperature, the 

absorbance of the mixture was measured at 562 nm. The ability of 

different fractions to chelate ferrous ion was calculated using the 


Chelating ability (%) = (1−Asample/Acontrol) ×100% 


Isolation and purification of polysaccharides 

The yield of the crude polysaccharide extracted from A. 

coreanum was 0.7%. After deproteinated by a 

combination of proteinase and Sevag method, the crude 

polysaccharide sample (cACPS) was loaded onto the 

DEAE-cellulose column (Figure 1), de-ionized water was 

used to elute the unbound component (ACPSA); and the 

retained components (ACPSB) was eluted with 0.5 M 

NaCl. Then ACPSA and ACPSB were loaded onto 

Sephacryl S-300 column, respectively, eluted with 0.15 M 

NaCl, and three main fractions (ACPSA-1, ACPSB-2 and 

ACPSB-3) were separated for further analysis of 

physicochemical properties and in vitro antioxidant 

activities. 

Physicochemical properties and chemical 

compositions 

The total sugar, protein, uronic acid contents, molecular 

weight and monosaccharides composition of the 

polysaccharide fractions are summarized in Table 1. The 

polysaccharide fractions ACPSB-2 and ACPSB-3 had 

higher total carbohydrate content (95.1% in ACPSB-2 and 

96.5% in ACPSB-3) than ACPSA-1 (91.4%). According to 

Bradford method using bovine serum albumin (BSA) as 

standard, the protein contents of ACPSA-1, ACPSB-2 and 

ACPSB-3 were 7.5, 2.7 and 2.4%, respectively. 

Furthermore, the uronic acid content was measured by 

m-hydroxydiphenyl method, and the results showed that 

the uronic acid contents in ACPSB-2 and ACPSB-3 were 

19.2 and 28.7%, respectively, not detected in ACPSA-1. 

The average molecular weights of ACPSA-1, ACPSB-2 

and ACPSB-3 calculated by HPLC, according to the 

calibration curve with standard dextrans, were 74.5, 68,4 

and 22,5 kDa, respectively. 

Moreover, composition analysis of polysaccharide is an 

important procedure to control quality standard and give 

basic information of the polysaccharide fractions. 

According to GC analysis, ACPSA-1 was composed of 

arabinose, xylose, galactose and glucose with molar 

ratios of 23:12:10:41. ACPSB-2 and ACPSB-3 were both 

composed of five monosaccharides: arabinose, xylose, 

galactose, glucose and galacturonic acid with molar ratios 

of 15:8:10:43:16 and 11:14:10:32:21, respectively. 

Glucose was the predominant monosaccharide in all the 

fractions. 


The principle of scavenging the stable DPPH radical 

model, which is widely used to evaluate antioxidant 

activities, is based on the reduction of the stable DPPH 

solution (purple) in the presence of a hydrogen donating 

antioxidant, leading to the formation of non-radical form 

DPPH-H (yellow). 

Figure 2 demonstrated DPPH scavenging activity 

caused by polysaccharide fractions at different 

concentrations. ACPSB-2 and ACPSB-3 both exhibited 

significant radical scavenging activities. When the 

concentration was more than 2 mg/ml, DPPH scavenging 

activity of ACPSB-2 and ACPSB-3 was significantly 

higher (P < 0.01) than that of ACPSA-1. The scavenging 

activity increased steadily at the concentration rang of 0.5 

to 8 mg/ml. The DPPH radical scavenging rate of 

ACPSA-1, ACPSB-2 and ACPSB-3 reached 52.4, 77.5 

and 70.2% at 8 mg/ml, respectively. There was no 

significant difference in scavenging activity between 

ACPSB-2 and ACPSB-3 at the concentration range of 0.5 

to 8 mg/ml (P >0.05). The concentration required to inhibit 

50% radical scavenging effect (IC50) was determined from 

the results of a series of concentrations mentioned above. 

A lower IC50 value corresponds to a greater scavenging 

activity. The IC50 values of ACPSA-1, ACPSB-2 and 

ACPSB-3 were 6.6, 2.8, and 3.3 mg/ml, respectively, 

revealing that the fraction ACPSB-2 possessed the 

highest DPPH radical scavenging activity. 

Superoxide radical scavenging activity 

Superoxide anions are a precursor to active free radicals, 

which is normally formed first in cellular oxidation 

reactions. Although, it is not highly reactive, it can produce

a 


1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

ACPSA 

De-ionized water 

ACPSB 

0.0 

0.0 

0 10 20 30 40 50 60 70 80 90 100 

Fraction number 


c 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

NaCl (0.5M) 

490 nm 

280 nm 

1.4 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 



b 

ACPSB-2 

0.0 

0.0 

0 10 20 30 40 50 60 70 80 90 100 


1.0 

0.8 

0.6 

0.4 

0.2 

ACPSA-1 

Li et al. 879 

490 nm 

280 nm 

0.0 

0.0 

0 10 20 30 40 50 60 70 80 90 100 


ACPSB-3 

490 nm 

280 nm 

Figure 1. Polysaccharide fractionations isolated from Aconitum coreanum with DEAE-cellulose (a) and Sepharose CL-6B (b, c). Crude 

polysaccharide (cACPS) was loaded onto the DEAE-cellulose column, de-ionized water was used to elute the unbound component 

(ACPSA); and the retained components (ACPSB) was eluted with 0.5 M NaCl. Then ACPSA and ACPSB were loaded onto Sepharose 

CL-6B column, respectively, eluted with 0.15 M NaCl, giving three main fractions (ACPSA-1, ACPSB-2 and ACPSB-3). 

other ROS such as hydrogen peroxide, hydroxyl radical, 

and singlet oxygen. Furthermore, superoxide anion 

radical and its derivatives can cause damage in lipids, 

proteins, and DNA. Therefore, it is of great important to 

scavenge superoxide anion radical (Xie et al., 2008). 

As shown in Figure 3, superoxide scavenging activities 

of three fractions increased significantly (P < 0.01) with 

increasing concentrations from 0.5 to 8.0 mg/ml, and the 

superoxide radical scavenging rate of ACPSA-1, 

ACPSB-2 and ACPSB-3 at 8.0 mg/ml was 32.5, 47.1 and 

78.5, respectively. The IC50 values of ACPSA-1, 

ACPSB-2 and ACPSB-3 were 11.5, 8.6, and 3.0 mg/ml, 

respectively. Scavenging activity of ACPSB-3 was 

significantly higher (P < 0.01) than that of ACPSA-1 and 

ACPSB-2. 

1.0 

0.8 

0.6 

0.4 

0.2 



Hydroxyl radicals are considered to be the most reactive 

oxygen radicals. The Fenton reaction, that is, Fe 2+ + H2O2 

→·OH + OH - + Fe 3+ , is a standard method used to 

determine the hydroxyl radicals scavenging capacity of 

antioxidant compound. 

The hydroxyl radicals scavenging abilities of 

polysaccharide fractions were shown in Figure 4. 

ACPSA-1, ACPSB-2 and ACPSB-3 exhibited distinct 

scavenging ability, ACPSB-2 and ACPSB-3 against 

hydroxyl radical were better than that of ACPSA-1. 

Furthermore, the scavenging ability increased with the 

increasing concentrations from 0.5 to 8.0 mg/ml. At 8.0 

mg/ml, the scavenging rate of ACPSA-1, ACPSB-2 and 

0.9 

0.8 

0.7 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

Absorbance at 280 nm


Table 1. Molecular weight and composition of different polysaccharide fractions isolated from Aconitum coreanum. 

ACPSA-1 ACPSB-2 ACPSB-3 

Molecular weight 74,500 68,400 22,500 

Total sugar (%) 91.4 95.1 96.5 

Protein (%) 7.5 2.7 2.4 

Uronic acid (%) nd a 19.2 28.7 

Sugar components (mol%) 

Arabinose 23 15 11 

Xylose 12 8 14 

Galactose 10 10 10 

Glucose 41 43 32 

Galacturonic acid nd 16 21 

a nd: not detect. 

Scavenging rate (%) 

80% 

60% 

40% 

20% 

0% 

ACPS-A1 

ACPS-B2 

ACPS-B3 

0 1 2 3 4 5 6 7 8 

Concentration (mg/ml) 

Figure 2. Scavenging rate of different polysaccharide fractions isolated from Aconitum 

coreanum against DPPH radical. Results were presented as means ± S.D. (n=3). 

ACPSB-3 against hydroxyl radical was 29.4, 55.3 and 

68.7%, respectively. The IC50 values of ACPSA-1, 

ACPSB-2 and ACPSB-3 were 16.2, 5.2 and 3.5 mg/ml, 

respectively. These results showed that ACPSB-2 and 

ACPSB-3 had a moderate hydroxyl radical scavenging 

activity. 

Ferrous ion-chelating effect of polysaccharide 

fractions 

Iron can stimulate lipid peroxidation and also accelerate 

peroxidation by degradation lipid hydroperoxides into 

peroxyl and alkoxyl radicals. Metal chelating capacity was 

significant since it reduced the concentration of the 

catalyzing transition metal in lipid peroxidation. Ferrous 

ions (Fe 2+ ) can catalyze and induce superoxide anion to 

form more harmful hydroxyl radicals. Ferrozine can form 

complexes with Fe 2+ quantitatively, and the complex 

formation is disrupted with the result that the red color of 

the complex is decreased in the presence of chelating 

agents. Therefore, measurement of color reduction allows 

estimation of the chelating activity (Yuan et al., 2006). 

The chelating ability of the samples are shown in Figure 

5, all of the polysaccharide fractions ACPSB-2 and 

ACPSB-3 had an excellent chelating ability, while 

ACPSA-1 showed a moderate chelating ability. The 

chelating ability increased significantly (P < 0.01) with 

increasing concentrations from 0.5 mg/ml to 8.0 mg/ml, 

and the chelating ability of ACPSA-1, ACPSB-2 and 

ACPSB-3 at 8.0 mg/ml was 49.5, 75.3 and 68.7%, 

respectively, suggesting that they could capture ferrous



80% 

60% 

40% 

20% 

0% 

ACPS-A1 

ACPS-B2 

ACPS-B3 

0 1 2 3 4 5 6 7 8 


Figure 3. Scavenging rate of different polysaccharide fractions isolated from Aconitum coreanum against 

superoxide radical. Results were presented as means ±S.D. (n=3). 

80% 

60% 

40% 

20% 

0% 

ACPS-A1 

ACPS-B2 

ACPS-B3 

0 1 2 3 4 5 6 7 8 


Figure 4. Scavenging rate of different polysaccharide fractions isolated from Aconitum coreanum 

against hydroxyl radical. Results were presented as means ± S.D. (n=3). 

Li et al. 881


ion before ferrozine. 

Conclusions 

Chelating effect (%) i 

100% 

80% 

60% 

40% 

20% 

0% 

ACPS-A1 

ACPS-B2 

ACPS-B3 

0 1 2 3 4 5 6 7 8 


Figure 5. Ferrous ion-chelating ability of different polysaccharide fractions isolated from 

Aconitum coreanum. Results were presented as means ±S.D (n=3). 

Highly reactive free radicals and reactive oxygen species 

(ROS) commonly exist in the biological systems of live 

organs, which can oxidize nucleic acids, proteins, lipids or 

DNA inducing many kinds of degenerative diseases. 

Natural antioxidant compounds, such as phenolic acids, 

polyphenols, flavonoids and polysaccharides can 

maintain human healthy, prevent and retard the 

progresses of many free radical-induced chronic 

diseases. In this paper, three polysaccharide fractions 

(ACPSA-1, ACPSB-2 and ACPSB-3) from the roots of A. 

coreanum were obtained by DEAE-cellulose and 

Sepharose CL-6B chromatography. Several in vitro 

models were applied to evaluate the antioxidant potential 

of A. coreanum polysaccharides. Experiment results 

showed that ACPSB-2 and ACPSB-3 exhibited significant 

antioxidant activity in a concentration-dependent manner. 

Further structural analysis of A. coreanum 

polysaccharides will be important for their application in 

medicinal fields. 


This work was financially supported by National Key 

Technology R&D Program (No. 2007BAI38B02) and 

National Natural Science Foundation of China 

(81041091), Foundation for Agro-scientific Research in 

the Public Interest, No.201103037and the Young 

Teachers of Shenyang Agricultural University (20101007). 

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Chem. Lett., 16: 1329-1330.

Journal of Medicinal Plants Research Vol. 6(5), pp. 884-887 9 February, 2012 


DOI: 10.5897/JMPR11.1528 



Preparative separation of hyperoside of seeds extract 

of Saposhnikovia divaricata by high performance 

counter-current chromatography 

Li Li, Yuge Gui, Jing Wang, Huirong Zhang, Xiaofei Zong and Chun Ming Liu* 

The Central Laboratory, Changchun Normal University, Changchun, 130032, China. 


Hyperoside was isolated and purified from the seeds of Saposhnikovia divaricata for the first time by 

high performance counter-current chromatography (HPCCC) using a solvent system of ethyl acetate-nbutanol-ethanol 

-water (1:1:0.1:2, v/v/v/v). By injecting ca. 500 mg of n-butanol extract of S. divaricata 

seeds for five times, a total of 232.7 mg of hyperoside was purified from 2.5 g of the n-butanol extract of 

S. divaricata seeds, at 96.3% purity as determined by high performance liquid chromatography (HPLC). 

The identification of the purified compound was achieved by congruent retention time, ultraviolet (UV) 

spectra and the data of high-performance liquid chromatography- electrospray ion source mass 

spectroscopy (LC-ESI-MS n ) in the positive mode with that of the authentic standard and literature 

reports. 

Key words: S. divaricata seeds, hyperoside, HPCCC. 

INTRODUCTION 

Saposhnikovia divaricata (Turcz.) Schischk is an 

important medicine in China, and the dried root has been 

used to treat headache, generalized aching, inflammatory 

symptoms and cancer (Okuyama et al., 2001; Xue et al., 

2000). The chromones are the major bioactive 

constituents in the root of S. divaricata (Dai et al., 2008; 

Jiang et al., 2006; 2007; Sasakiet al., 1982; Gui et al., 

2011). 

However, compared to the many reported methods on 

the dried root of S. divaricata (Okuyama et al., 2001; 

Jiang et al., 2007; Sasaki et al., 1982; Gui et al., 2011), 

effective methods for the isolation, purification and 

structural characterization of constituents in seeds of S. 

divaricata are scarce. HPCCC methods have become an 

effective alternative to the conventional chromatographic 

techniques for the separation of hyperoside from some 

plant extracts (Sheng et al., 2009; Xie et al., 2010). The 

present paper describes the successful preparative 

separation and purification of hyperoside from the seeds 

of S. divaricata for the first time using preparative 

*Corresponding author. E-mail: lilchem@163.com. Tel: 

+8643186168376. Fax: +8643186168875. 

HPCCC. 

EXPERIMENTAL 

Chemicals and reagents 

The seeds of S. divaricata were purchased from HeiLongJiang 

NongKe; hyperoside was purchased from National Institute for the 

Control of Pharmaceutical and Biological Products (Beijing China). 

All organic solvents used for HPCCC separation were of analytical 

grade and purchased from Beijing Chemicals (Beijing, China); 

Acetonitrile and acetic acid were HPLC grade and purchased from 

Fisher Scientific Company. Water was purified on a Milli-Q water 

purification system (Millipore, France). 

Sample preparation 

The seeds of S. divaricata (500 g) was milled to powder and 

extracted with 2000 ml of 80% aqueous ethanol for ten times at 

room temperature, each for 12 h. Each time, the extraction mixture 

was filtered and the combined filtrates were concentrated to 

dryness in vacuo at 40°C. The extract was re-dissolved in 500 ml of 

water and was then defatted five times each with 500 ml hexane. 

The water layer was then extracted successively five times each 

with 500 ml of n-butanol. The combined n-butanol layer was 

concentrated to dryness in vacuo at 40°C, which yielded 11.02 g. 

The n-butanol extract was stored at -20°C before HPCCC 

separation.

HPLC analysis 

Figure 1. HPLC profiles of the n-butanol extract of the seeds of S. divaricata (A), purified compound 1 (B) and 

UV spectra of purified compound. The related compound was identified as hyperoside. 

An agilent technology 1100 series HPLC system equipped with a 

quaternary pump, a degasser, thermostatic auto-sampler and a 

photodiode array detector (DAD) was used for the analysis of 

hyperoside in the n-butanol extract of S. divaricata seeds, the 

partition coefficient (K) and to monitor the fractions collected from 

the HPCCC separation. The HPLC method following the same 

procedures published earlier (Gui et al., 2011). Briefly, an Agilent 

Zorbax Extend C18 column (250 × 4.6 mm, 5 μm) was used. The 

binary mobile phase consisted of acetonitrile (solvent A) and water 

containing 0.5% acetic acid (solvent B). The flow-rate was kept 

constant at 0.5 ml/min for a total run time of 25 min. The system 

was run with a gradient program: 0 to18 min: 80% B to 40% B; 18 

to 20 min, 40% B to 80% B; and 20 to 25 min, 80% B to 80% B. 

Peaks of interest were monitored at 254 nm by a DAD detector. 

HPCCC separation 

Preparative HPCCC was carried out in a Spectrum HPCCC (DE, 

England). The apparatus has one coil of 132 ml for preparation. The 

HPCCC system was equipped with two check pumps (Smartline 

Pump 100, KNAUER, Germany), a 2500 sensitive UV detector 

(KNAUER, Germany). The rotation speed can be regulated with a 

speed controller in the range of 0 to 1600 rpm. Sample injection 

was accomplished through an injection valve with a 10-ml sample 

loop. A solvent system of ethyl acetate-n-butanol-ethanol-water 

(1:1:0.1:2, v/v/v/v) was selected for separating compound 1 in 

Figure 1. This system provided a K value of 1.16 for compound 1. 

The 132 ml coil was used and the apparatus was set in the 

reversed phase mode. The entire coiled column was first filled with 

the upper phase, which serves as the stationary phase. 

The temperature was held at 30°C. The rotation rate of the 

apparatus was set at 1600 rpm and the lower phase (mobile phase) 

was pumped into the column at a flow rate of 3 ml/min. When the 

hydrodynamic equilibrium was established, a sample (ca. 500 mg) 

dissolved in 6 ml of the mixture of ethyl acetate-n-butanol-ethanolwater 

(1:1:0.1:2, v/v/v/v) was loaded into the injection valve after the 

Li et al. 885 

system reached hydrodynamic equilibrium. Then time was recorded 

after the injection. The effluent from the outlet of the column was 

continuously monitored by a UV detector at 254 nm and collected 

into test tubes with a fraction collector set at 2 min for each tube. 

Fractions from the HPCCC that had the same single peak as 

determined by HPLC were combined and freeze-dried. The purity of 

the peak as determined by HPLC was 96.3% for compound 1. The 

purified compound was stored at -20°C before LC-ESI-MS n 

analyses. 

LC-ESI-MS for Identification 

A Thermo Scientific LCQ Fleet mass spectrometer was connected 

to Thermo Scientific Surveyor Liquid chromatography (LC) Plus 

system via electrospray ionization (ESI) interface (ThermoFisher, 

USA). The LC-ESI-MS method following the same procedures 

published earlier (Gui et al., 2011). Briefly, the operating parameters 

in the positive ion mode were as follows: the sheath gas and 

auxiliary flow rates were set at 30 and 5 (arbitrary unit), 

respectively. The capillary voltage was set at 34.98 V and its 

temperature was controlled at 350�C. The entrance lens voltage 

was fixed at -35 V and the multipole retention factor (RF) amplitude 

was set at 406 V. The ESI needle voltage was controlled at 4.5 kV. 

The multipole lens 1 offset was -15 V. The electron multiplier 

voltage was set at - 980 V for ion detection. 


The n-butanol extract of S. divaricata seeds and the 

fractions corresponding to compound 1 isolated by 

HPCCC were analyzed by HPLC and the results are 

given in Figure 1, a good separation was achieved within 

25 min. Peak 1 was separated and detected with 

retention times at 12.11 min. Tentative identification of 

this peak was achieved by congruent retention time, UV


Figure 2. The HPCCC chromatogram of the n-butanol extract of S. divaricata seeds. The coil volume was 132 ml; The 

temperature was held at 30°C; The rotation rate of the apparatus was set at 1600 rpm; The lower phase (mobile phase) was 

3 ml/min; Sample (ca. 500 mg) dissolved in 6 ml of the mixture of ethyl acetate-n-butanol-ethanol-water (1:1:0.1:2, v/v/v/v); 

UV detector at 254 nm. 

spectra with that of the authentic standard and the peak 1 

was identified as hyperoside. In our experiment, a solvent 

system containing ethyl acetate-n-butanol-ethanolwater 

(1:1:0.1:2, v/v/v/v) was selected to separate compound 1, 

which has been used in HPCCC to separate four 

chromones from S. divaricata root (Gui et al.. 2011). We 

found that this system was suitable for the separation of 

compound 1 from the n-butanol extract of S. divaricata 

seeds. The K value for compound 1 in this system was at 

1.16. When this solvent system was applied to the 

HPCCC separation with a sample load of ca.500 mg and 

flow rate at 3 ml/min, the compound of interest was 

separated within 130 min (Figure 2). 

A total of 232.7 mg of hyperoside was purified from 2.5 

g of the n-butanol extract of S. divaricata seeds, at 96.3% 

purity as determined by HPLC. To further investigate the 

structure of this peak, LC-ESI-MS n experiment was 

attempted. Under LC-ESI-MS conditions, compound 

related to the peak 1 in Figure 1 exhibited intense 

molecular ions [M+H] + at m/z 465 in the positive mode 

and low intensity dimer [M+Na] + ion at m/z 487, from 

which the molecular weight of peak 1 was confirmed to 

be 464, the same as that for hyperoside (Li et al., 2005; 

Chen et al., 2002). In the LC-ESI-MS 2 experiment, the ion 

at m/z 465 formed one major fragment ion at 303. The ion 

at m/z 303 was produced directly from the parent ion of 

m/z 465 due to the loss of a galactosyl moiety. By 

comparing the molecular weight information and LC-ESI- 

MS n data with that of the authentic standard and literature 

report (Li et al., 2005; Chen et al., 2002), the identities of 

compound 1 were confirmed as hyperoside. 


This work was supported by the National Natural Science 

Foundation of China (No. 30970299) and the Natural 

Science Foundation of Jilin Province of China (No. 

200905109, 20090936, 2009D206, 2011-187, 20102203 

and 2010D036). 

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separation of chromones in plant extract of Saposhnikovia 

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of four components in the chromone fraction of Saposhnikovia 

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Li et al. 887 

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233.



DOI: 10.5897/JMPR11.1529 



Research update: Lectin enriched fractions of herb and 

dry extract of Urtica dioica L. 

Savickiene Nijole 1 *,Baniulis Danas 2 , Bendokas Vidmantas 2 , Balciunaite Gabriele 1 , 

Draksiene Gailute 3 , Peciura Rimantas 3 and Serniene Loreta 4 

1 Department of Pharmacognosy, Medical Academy of Lithuanian University of Health Sciences, Lithuania. 

2 Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry, Lithuania. 

3 Department of Drug Technology and Pharmaceutical Management, Medical academy of Lithuanian University of 

Health Sciences, Lithuania. 

4 Department of Food Safety and Animal Hygiene, Veterinary Academy of Lithuanian University of 

Health Sciences, Lithuania. 


Urtica dioica L is a plant rich in flavonoids, carotenoids, caffeoylmalic acid and has an established 

medical value. Although content of mineral and organic substances of U. dioica L. herb is well 

characterized, presence of bioactive polypeptides is much less appreciated. Seeds and roots of nettle, 

have been established as a common source for isolation of lectins. Therefore data on the presence of 

lectins in herb of nettle is ambiguous. Lectin-enriched protein fractions were isolated from herb (fresh 

and dry) and dry extract of U. dioica L. by using homogenisation with fluid nitrogen, extraction in 0.01 

M phosphate-buffer saline (PBS), concentrating, salting and precipitation. The amount of protein was 

measured using photometric Bradford method. A proteomic analysis using 2D gel electrophoresis was 

performed for lectin – enriched protein fractions isolation and analysis. We estimated quantity of 

protein and lectins, assessed their blood cell agglutinating activity using tests employing rabbit 

erythrocytes. The highest concentration of protein and specific hemagglutination activity was observed 

for protein fractions isolated from fresh herb. The highest lectins content was presented in protein 

fractions isolated from the dry extract. 

Key words: Urtica dioica L., lectins, hemaglutination, electrophoresis. 

INTRODUCTION 

Plant lectins are a class of carbohydrate-binding 

nonimmune origin protein (Goldstein et al., 1980; Sharon, 

1989; Peumans et al., 2001; de Meija et al., 2003). In 

past few decades a lot of lectins were purified and 

identified. They have attracted great interest because of 

their various biological activities, such as cell 

agglutination, antiproliferative, antitumor, immunomodulatory, 

antifungal and antiviral (Broekaert et 

al.,1989; Wang et al.,1996; Does et al., 1999; Wong et 

al., 2003; Singh et al., 2004). Urtica dioica L. is widely 

used herb in medicine. Stinging nettle is well known for 

its diuretic, anti-inflammatory effects. It is also used for 

joint and muscle rheumatic disease, bile system disease 

*Corresponding author. E-mail. savickienenijole@takas.lt. 

and arthritis (Bauer et al., 2009). Roots of the nettle have 

been established as a common source for isolation of 

lectins. Stinging nettle rhizomes contain considerable 

amounts of a lectin which exhibits carbohydrate binding 

specificity for N-acetylglucosamine oligomers (Chapot et 

al., 1986). 

An unusual lectin has been isolated from stinging nettle 

(U. dioica L.) rhizomes. It is a small (8.5 kDa) monomeric 

protein with high contents of glycine, cysteine and 

tryptophan. The U. dioica agglutinin (UDA) is not blood 

group-specific and is specifically inhibited by Nacetylglucosamine 

oligomers. As compared to other plant 

lectins. UDA has a very low specific agglutination activity. 

Nevertheless, it induces HuIFN-y in human lymphocytes 

at concentrations comparable to those of other inducers 

(Peumans et al., 1984). Our research novelty is 

purificating experiments of lectins with well known herb of

nettle (U. dioica L.). Therefore data on the presence of 

lectins in the herb of stinging nettle is ambiguous. In this 

study, we prepared lectin-enriched protein fractions from 

extracts of fresh and dry herb and dry extract of U. dioica 

L. and estimated protein and lectin quantity. We 

assessed lectin blood cell agglutinating activity using 

tests employing rabbit erythrocytes. 


The dry herb (humidity – 11.8%) and dry extract of U. dioica L. herb 

(DER: (5-10:1), extraction solvent: water) was imported from 

Poland. The fresh herb (humidity – 85%) of stinging nettle was 

collected in Lithuania, Kaunas district, Cekiskes. 

Homogenisation 

40 g of plant material (for choice - dry herb, fresh herb or dry extract 

of U. dioica L.) was homogenized with fluid nitrogen in mortar. 

Extraction 

Homogenised material was extracted in 1:5 (w/w) 0.01 M 

phosphate-buffer saline (PBS), pH 7.2, with protheasis inhibitor and 

2% PVPP (Polyvinyl prolidone) for 2 to 16 h at 4 to 5°C. PBS 

consists of 10.9 g NaH2PO4 (anhydrous), 3.2 g NaCl, 90 g H2O in1 

liter. Protheasis inhibitorconsists of 5 mM (0.8 mg/ml) Benzamidyne 

5 mM (0.66 mg/ml) ε-amino-capronic acid. 

Concentrating and salting 

The extract was filtered through filter and centrifuged at 1500 rpm 

for 30 min at 4 to 5°C. The filtrate was purified followed by 40% 

saturation with ammonium sulfate (w/v) and constant stirring for 15 

min. Then the solution with precipitate was centrifugated again at 

15000 rpm for 30 min. The extract was then subjected to a “salting 

out” step with 40 to 80% ammonium sulfate (w/v) and stirred about 

24 h. Forthcomming precipitate (first protein fraction) was dissolved 

in the little amount of PBS solution and left at 4°C. The 80% 

saturated solution was centrifuged at 15000 rpm for 30 min.The 

filtrate was purified followed by 80 to 100% saturation with 

ammonium sulfateand stirred again for 24 h. Forthcomming 

precipitate (second protein fraction) was dissolved in the little 

amount of PBS solution and left at 4°C. The 100% saturated 

solution was centrifuged at 15000 rpm for 30 min. Final filtrate was 

eliminated and precipitate (third protein fraction) was dissolved in 

the little amount of PBS solution. The precipitate (of three fractions) 

was resuspended and dialyzed against three changes of PBS for 

48 to 72 h. The amount of ammonium sulfate was: 0 to 40%- 

233,38 g/L; 40 to 80% - 266,41 g/L, 80 to 100% -143,35 g.L/ at 4°C. 

Peptide solutions were concentrated using Centiprep YM-3 filters. 

Methods prepared according to Dhuna (2005, 2010), Tulasi (2002) 

and Gupta (1997). 

Protein precipitation 

Protein precipitation accomplished with trichloracetic acid (TCA), 

0.2 M TCA (0,4 g/2 ml) mixed with 2 ml of peptide solution. 

Coagulation of protein can be prevented at reduced temperature (0 

to 4°C). Peptide solution were kept on ice for 30 min to avoid 

protein denaturation. Solutions centrifugated at 20000 rpm 30 min 

Nijole et al. 889 

at 4°C supernatant was poured. TCA residual was washed with 

acetone (at temperature -20°C) and incubated on ice for 10 min. 

Protein solution was centrifugated at 2000 rpm for 10 min. Acetone 

was removed and washing repeated once again. Solution dissolved 

in sodium dodecyl sulfate (SDS) buffer and kept for 30 min at 60°C 

(Bell et al., 1983). 

Measurement of protein amount 

The amount of protein was measured using photometric Bradford 

method. Standarts were prepared in 0.5 ml test-tubes: 0.125; 0.25; 

0.5 and 1.0 mg/ml Bull Serume Albumine (BSA) solutions. 

Standarts were devided in to small volumes and kept at 20°C. 

Negative control was prepared in 0.5 ml test-tube–5 µl buffer. 

Examples were prepared in 0.5 ml test-tubes -5 µl protein extract. 5 

volumes of Roti-Quant (Carl Roth GmbH, Karlsruhe, Germany) 

reagent diluted with 4 volumes of deionisated water. 250 µl of 

Rothi-Quant reagent was poured on standarts, negative standarts 

and mixed. Solutions were kept for 5 to 30 min at 20°C. Light 

absorbtion was measured with Eppendorf BioPhotometerin 595 nm 

wavelength (Lowry, 1951). 

Gel electrophoresis 

Electrophoresis is widely used in trials for protein isolation and 

analysis. Most of protein trials are made in 2D polyacrylamide gel 

electrophoresis (PAGE) method. Protein are separated by their 

charge with isoelectric focusing method and fractionated by their 

size in polyacrylamide gel. Protein are visible like spots after 

staining. PAGE was carried at pH 8.8 using 12% (w/v) acrylamide 

slab gel with running conditions 12 mA per mini-gel for 2 h 

(Ausubel, 2003). Coomasie Blue G-250 (Fackelmayer, 1998) and 

silver nitrate (Møller et al., 2003) were used to visualize the protein 

bands. 

Preparation of erythrocytes 

Rabbit blood was used during experiments, got from Lithuanian 

Univercity of Health Sciences, Veterinary academy. 2% erythrocyte 

solution was prepared, where amount of erythrocytes was 5x10 9 ml. 

1 ml of rabbit blood mixed with 14 ml PBS solution. Erythrocytes 

were washed 4 times: 2% erythrocyte solution was centrifugated at 

800 rpm for 10 min at 4°C, the supernatant was eliminated, 

precipitate resuspended in 15 ml PBS solution and procedure with 

centrifugation was repeated. After washing procedure, erythrocytes 

were treated with 7.5 ml trypsin (Biochrom AG, L2103, 1310 USP 

U/mg) solution (1 mg/ml PBS), incubated for 1 h at 37°C. 

Trypsintreated erythrocytes were washed 4 times: it was provided 

centrifugation at 800 rpm for 10 min at 4°C, Supernatant was 

poured, precipitate resuspended with 15 ml of PBS solutions and 

centrifugated at 800 rpm for 10 minat 4°C. After washing procedure 

erythrocytes resuspended with 15 ml of PBS solution (Ove et al., 

2003). 

Hemagglutionation test 

50 µl 0.01 M PBS solution (7.0 mM Na2HPO4, 2.7 mM NaH2PO4, 

154.0 mM NaCl, pH 7.2) was added to U shaped microplate wells. 

To the first well was poured 50 µl protein extract and mixed. 50 µl of 

extract was moved to the next well. Dilution repeated in 1 to 15 

wells. 50 µl of sample from 15„th well was eliminated. To the 16„th 

well added 50 µl PBS solution, mixed and 50 µl eliminated. In each 

well 50 µl 2% trypsine-treated erythrocytes solution were added. 

Microplate was softly mixed and covered. Titre was measured on


Table 1. Characteristics of protein fractions from Urtica dioicaL. herb (fresh and dry) and dry extract. 

Source / isolation step 

Protein quantity 

mg /g weight 

Specific hemoagglutinating 

activity 1 

Lectin content, % 2 

Fresh herb 

Precipitate of 40% saturated AS 1.18 ± 0.0253 2.95 ± 0.0131 0.01 ± 0.0014 

Precipitate of 80% saturated AS 2.85 ± 0.0376 2.23 ± 0.0856 0.01 ± 0.0007 

Dry herb 

Precipitate of 40% saturated AS 0.09±0.0141 0.44±0.0468 0.05±0.0051 


Supernatant of 80% saturated AS 0.64±0.0481 1.7±0.0653 0.01±0.0064 

Dry extract 



Supernatant of 80% saturated AS 0.24±0.0181 0.06±0.0046 0.33±0.0045 

1- Specific hemagglutinating activity was defined as the ratio of the titer/ml and protein concentration (mg/ml); Titer per ml was defined 

as the reciprocal of the highest dilution giving visible agglutination of the rabbit erythrocytes. 2 - Lectin content is based on cell 

agglutinating activity as compared to specific hemagglutinating activity of lectin from S. tuberosum. Tables include representative 

results from at least three separate experiments. AS,ammonium sulphate. 

Figure 1. Gel electrophoresis of dry 

Urtica dioica L. herb, pH 8.8 using 

12% (w/v) acrylamide slab gel. Flow 

direction fom top (+) to the bottom (- 

), 12 mA per minigel, duaration 2 

h.Std - PageRuler TM Protein Ladder. 

Total amount of proteins in each well 

: Net1 – 0.3 µg in 40 % AS saturated 

precipitate; Net2 – 0.6125 µg in 80% 

AS saturated precipitate; Net3 – 1.25 

µg in 80 % AS saturated 

supernatant. Sample was stained 

with silver nitrate. 

the light table after 30 min of incubation at 22°C. Hemagglutination 

titre (per 1 ml of sample) is the maximum dilution in which total 

aglutination was observed. Specific activity of hemagglutination was 

calculated by dividing the value of titre from protein concentration in 

the sample. Specifical hemagglutination activity was counted by 

dividing hemagglutination titre meaning from protein concentration 

in example (Laija et al., 2010). 


Analysis of standart deviation of means were carried out on all data 

using statistical analysis system (SAS 76, SAS Institute Inc). 

RESULTS 

Protein concentration (mg/g), specific hemagglutinating 

activity and lectins content (%) of U. dioica L. herb (fresh 

and dry) and dry extract presented in Table 1. Protein 

quantity of fresh and dry herb increased in precipitate 

from 40 to 80% saturated ammonium sulfate (AS) 

samples. Meanwhile protein quantity in dry extract 

decreased in precipitate from 40 to 80% saturated AS 

samples. Fresh U. dioica L. herb protein extract 

contained relatively high level of hemagglutinating activity 

against trypsin-treated rabbit erythrocytes. Maximum of 

specific hemagglutinating activity of dry U. dioica L. herb 

was in 80% AS saturated precipitate. Maximum of 

specific hemagglutinating activity of dry U. Dioica L. 

extract reached in 80% saturated AS supernatant. 

sodium dodecyl sulfate polyacrylamide gel 

electrophoresis (SDS-PAGE) electrophoresis test was 

made with protein fractions of Urtica dioica L. herb dry 

(Figure 1). In protein precipitate fraction (40 % saturated 

AS) (Net1) was found particles sized 200 kDa, 120 kDA

Figure 2. Hemagglutination test results of fresh, dry U. dioica L. herb and dry extract lectin enriched fractions. 

Std - Soy bean lectin standart, 1 – fresh U. dioica L. herb 40 % AS saturated precipitate, 2 - fresh U. dioica L. 

herb 80 % AS saturated extract precipitate, 3 Dry U. dioica L. herb 40 % AS saturated precipitate. 4 – dry U. 

dioica L. 80 % AS saturated precipitate, 5 – Dry U.dioica L. 80 % AS saturated supernatant, 6 – Dry U. dioica 

L. extract 40 % AS saturated precipitate, 7 - Dry U. dioica L. extract 80 % AS saturated precipitate, 8 Dry U. 

dioica L. extract 80 % AS saturated supernatant. 

and 30 kDA. In protein precipitate fraction (80% saturated 

AS) (Net2) particles size was 120 kDA and 30 kDA. In 

protein supernatant fraction (80% saturated AS) (Net3) 

particles size was 120 kDA. According to literature lectin 

size is from 29 kDA to 34 kDa (Hänsel et al., 2007). 

Hemaglutination activity titre was measured by full 

agglutination of rabbit erythrocytes, diluted in serial way 

(ratio 1:1) lectin extracts. Std –Soy bean lectin standart 

(starting concentration (conc.) 0.5 mg/ml). Standart 

control agglutination founded in Std-a 0.5 mg/ml conc., 

Std-b 0.25 mg/ml conc. and Std-c 0.125 mg/ml conc. 

Hemagglutination was found in 1a, 1b, 1 c, 2a, 2b, 3a, 

4a, 5a, 6a, 6b, 7a, 7b and 8a wells, Lectin content in 

each column : 1 to 0.01, 2 to 0.01, 3 to 0.05, 4 to 0.1, 5 to 

0.01, 6 to 0.02, 7 to 0.02, 8 to 0.33% based on cell 

agglutinating activity as compared to specific 

hemagglutinating activity of lectin from Solanum 

tuberosum. (Figure 2). 

DISCUSSION 

Nijole et al. 891 

It was investigated several U. dioica L. rhizomes samples 

and was found the general lectin –UDA in previous 

experiments. UDA is actualy a mixture of up to 11 

different isolectins, which are monomeric proteins formed 

by 80 to 90 amino acids. With a molecular weight 

between 8300 and 9500 Dalton these lectins are among 

the smallest so far identified (Peumans et al.,1984).The 

quantitative results obtained by capillary electrophoresis 

(the total lectin content varied from 0 to 0.42% in the 

samples) were accurate. SDS-PAGE electrophoresis test 

showed that protein fractions include lectins particles. 

According to literature lectin size is from 29 kDA, to 34 

kDa (Hänsel et al., 2007). In this study protein fractions of 

U. dioica L. herb and dry extract were isolated. The total 

concentration of protein in herb and dry extract of U. 

dioica L. revealed that lectin-like glycoprotein enriched


fractions contained specific hemagglutination activity up 

to 1.3 and 3.0 mg protein/ml, respectively and protein 

concentration was 0.09 to 2.25 mg /g weight. We 

established particles sized of 200, 120 and 30 kDA. The 

finding of separated lectins requires more detailed 

investigation. 

Conclusions 

Presence of hemagglutination activity was observed for 

all of the protein fractions isolated by salting out with 

ammonium sulphate from fresh and dry herb of U. dioica 

L. and dry extract of stinging nettle. The highest 

concentration of protein and specific hemagglutination 

activity were observed for protein fractions isolated from 

fresh herb. Estimated lectins content in fresh U. dioica L. 

herb based on functional activity using lectin from S. 

tuberosum as a standard 0.01%, in dry U. dioica L. herb 

varied from 0.01 to 0.1% and in dry U. dioica L. extract 

varied from 0.02 to 0.33%. The highest lectins content 

was presented in protein fractions isolated from the dry 

extract. However, hemagglutination activity was not 

detected in 80% saturated ammonium sulphate fraction. 


Research was supported by a Grant (No.MIP–10180) of 

Lithuanian Foundation for Research and Studies for the 

Projects according initiative of scientists. 

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DOI: 10.5897/JMPR11.1542 



A clinical study on the effects and mechanism of 

Xuebijing injection in the treatment of traumatic 

intracranial hematoma 

Yong Guo 1 , Kuipo Yan 2,3,4 , Jiasheng Fang 1 *, Jinfang Liu 1 , Mingyu Zhang 1 and Jun Wu 1 

1 Department of Neurosurgery, Xiangya Hospital, Central South University, 410008 Changsha, China. 

2 Institute of Integrated Traditional Medicine and Western Medicine, Xiangya Hospital, Central South University, 410008 

Changsha, China. 

3 Key Unit of Traditional Chinese Medicine Gan of SATCM, Xiangya Hospital, Central South University, 410008 


4 Key Unit of the 11th Five-year Plan of SATCM in Cerebrosis, Xiangya Hospital, Central South University, 410008 



Xuebijing injection (XBJ) is one of Chinese materia medica standardized products which was extracted 

from Salvia miltiorrhiza Bge, Carthamus tinctorius L, Paeonia lactiflora Pall, Ligusticum chuanxiong 

Hort. and Angelica sinensis, has the actions of activating blood circulation and clear the meridians to 

melt away toxin factor. Our aim was to observe whether or not XBJ have neuroprotective effects on the 

patients with traumatic intracranial hematoma (TICH). If so, we would explain the mechanism involved. 

Forty patients with TICH were randomly assigned to trial group and a control group (20 patients per 

group). Routine medication was given to the patients of the two groups, and XBJ was administered 

intravenously to patients in the trial group additionally. The scores of GCS was recorded pre and posttreatment 

of the two groups, along with GOS after therapy. We also measured each patient’ volume of 

the intracranial hematoma and coagulation indexes pre- and post-treatment. In addition, the activities of 

antioxidative enzymes were also evaluated in the study. XBJ could promote glasgow coma scale (GCS) 

and glasgow outcome scale (GOS) after therapy for the trial group compared with the control group 

(p


(TICH) can expand or develop late after head injury 

(Sawauchi et al., 2001). Furthermore, hemorrhage 

expansion is an independent determinant of death and 

disability (Davis et al., 2006). Identification of risk factors, 

early diagnosis and proper treatment of such lesion are 

important for improving the prognosis of these patients. 

Recent studies (Wu et al., 2006; Lima et al., 2008) 

have reported that the pervasive action of oxidative 

stress on neuronal function and plasticity after traumatic 

brain injury (TBI) including TICH is becoming increasingly 

recognized. 

Physiologic regulation keeps the dynamic balance 

between oxidant and antioxidant. However, in the bodies 

of patients with TICH a series of free radical chain 

reactions were gravely aggravated, the dynamic balance 

between oxidation and antioxidation was seriously 

disrupted, and oxidative stress was clearly exacerbated 

(Bakey et al., 1986), which is closely related to many 

disorders, such as brain edema, increased intracranial 

pressure, cerebral capillary spasm, increased 

vasopermeability, hemorheological changes, and so on 

(Sun et al., 2009). These factors may further accelerate 

cerebral ischemia and hypoxia which is the therapy key 

of TICH (Tang et al., 2006). So, antioxidant, that is 

scavenging free radicals, is a good strategy in clinics (Wu 

et al., 2010). 

Computed tomography (CT) is central to acute TBI 

including TICH diagnostics, and millions of brain CT 

scans are conducted yearly worldwide (David et al., 

2010), which is helpful to improve the diagnosis rate of 

TICH, make clinical tracing, observe absorption of small 

and moderate-sized hematomas in nonsurgical 

treatment. Symptomatic treatments such as dehydration 

therapy to reduce cranial pressure, pain relief, and so on 

are often adopted in neurosurgery for those having small 

and moderate-sized, non-life-threatening hematomas 

(Sun et al., 2009). Given its prognostic significance, 

mostly, there is no satisfactory clinical results obtained (Li 

et al., 2003). 

However, studies have been completed to establish 

that TICH treated with additional Chinese materia medica 

in the early phase may result in satisfactory therapeutic 

effects with improved safety (Zhou et al., 2009; Wei et al., 

2004). Xuebijing injection (XBJ) is one of Chinese 

materia medica standardized products which was 

extracted from Salvia miltiorrhiza Bge, Carthamus 

tinctorius L., Paeonia lactiflora Pall, Ligusticum 

chuanxiong Hort. and Angelica sinensis, has the actions 

of activating blood circulation and clear the meridians to 

melt away toxin factor. 

Pharmacological studies show that XBJ can antagonize 

the disruptive effects of endotoxin (Zhang et al., 2006), 

regulate body immune functions (Dai et al., 2009), 

decrease the stress-induced organ or tissue damage (Li 

et al., 2006), and notably improve the survive rate of 

animals with systemic inflammatory response syndrome 

(SIRS) or multiple organ dysfunction syndrome (MODS) 

(Wang et al., 2006). Moreover, according to some clinical 

researches, early Xuebijing injection treatment showed 

favorable therapeutic effects and reliable safety on the 

patients with sepsis (Wang et al., 2007) or MODS (Zhang 

et al., 2002). Meanwhile, it may also reduce the whole 

body and local inflammatory reaction degree and exhibit 

protection of brain tissue (Yuan et al., 2009b). The Safflor 

yellow A is the main chemical component in XBJ, and 

studies reported that the Safflor yellow A can protected 

rat brain (Ye et al., 2008) and cardiomyocytes (Liu et al., 

2008) against I/R injury, exhibit neuroprotective effects 

after permanent middle cerebral artery occlusion in rats 

(Zhu et al., 2003) and antagonize binding of platelet 

activating factor to its receptor (Zang et al., 2002). XBJ 

has been applied extensively to the treatment of TICH 

and has showed significant therapeutic effect in our 

hospital. Therefore, the aim of the clinical study was to 

observe the short- and long-term therapeutic effects of 

XBJ in the treatment of acute TICH, and to explore its 

possible mechanisms. 


Selection of subject 

Over the period from January 2010 to September 2011 in Xiangya 

Hospital, Changsha, China. Patients 18 years of age or older with 

TICH documented by CT scan within 3 h after causes ranged from 

traffic accident injury to falling from higher places, to being hit were 

eligible for enrollment. Subjects were randomly assigned to the trial 

group (XBJ treatment, twenty cases) and the control group (twenty 

cases) upon admission to the hospital. The diagnostic criteria and 

exclusion criteria referred to the previous study (Sun et al., 2009), 

as follows: those who were admitted within 24 h after a cerebral 

injury confirmed by CT; with an intracranial hemorrhage volume 

between 10 and 40 ml; with no continuously enlarged hematoma 

after consecutive head CT examinations within 3 days, but not 

scheduled for surgery; aged between 16 and 65 years; and all with 

Glasgow coma score (GCS) greater than 8. The exclusion criteria 

were: severe cardio-/cerebral vascular diseases; liver or renal 

diseases; diabetes mellitus; other severe organ injuries; coagulation 

disorders; obviously enlarged hematoma confirmed by head CT 

and/or aggravated symptoms indicating a need for surgery. The 

study was approved by the institutional ethics committee. Informed 

consent was obtained from all patients or from their guardians prior 

to inclusion. 

The data were comparable between the two groups with no 

significant differences in gender, age, type, volume of intracranial 

hemorrhage in Table 1. 

TREATMENT 

Routine therapy (that is, dehydration therapy to reduce cranial 

pressure, pain relief, antinausea, antiinfection, neurotrophic activity, 

e t c.) anti-microbial and sputum elimination agents were given to 

patients of both the two groups. Xuebijing injection produced by 

Tianjin Hongri Pharmaceuticals Co. Ltd. was additionally used in 

the trial group, 50 ml of Xuebijing injection with 100 ml of normal 

saline administered through intravenous drip twice a day. The 

treatment course was 14 days for both the two groups.

Table 1. Subject demographic and baseline characteristics. 

Guo et al. 895 

Variable Trial Group (n = 20) Control Group (n = 20) 

Gender 

Age 

Type 

Volume of intracranial hemorrhage 

Glasgow coma score (GCS) and glasgow outcome score (GOS) 

GCS (Graham et al., 1974) and GOS were recorded pre-treatment 

and on the 14th day of the therapy. The intracranial hemorrhage 

volume and GCS was used to judge the short-term efficacies and 

GOS (Jennett et al., 1981): (1: death; 2: persistent vegetative state; 

3: severe disability; 4: moderate disability; 5: good recovery) was 

used to evaluate the long-term efficacies in the two groups. 3 

months post-treatment was set as the evaluation time (Sun et al., 

2009). 

Coagulation detection 

Venous blood samples (withdrawn pre-treatment and on the 14th 

day of the therapy) were anticoagulated by the addition of 4.5 ml of 

whole blood to 0.5 mL of 0.105 mol/L citrate. Plasma samples were 

double centrifuged at 3000 g × 10 min and frozen at -80°C until 

analysis at a later date. Prothrombin time (PT), PT international 

normalized ratio (INR), activated partial thromboplatin time (APTT), 

thrombin time (TT), fibrinogen (Fbg), D-dimer (DD) were detected 

according to the protocols by the technicians in Xiangya Hospital 

affiliated to Central South University. 

Intracranial hematoma volume determination 

Intracranial hematoma volume was determined in the following 

manner (Broderick et al., 1994): On the CT slice with the largest 

area of intracranial hematoma, the largest diameter (A) of the 

hematoma was measured by use of the centimeter scale on the CT 

film. The diameter of the hemorrhage perpendicular to the largest 

diameter represented the second diameter (B). The height of the 

hematoma was calculated by multiplying the number of slices 

involved by the slice thickness, providing the third diameter (C). The 

three diameters were multiplied and then divided by 2 (A×B×C/2) to 

obtain the volume of intracranial hematoma. Head CT scan were 

conducted as pre-treatment, on the 7th and 14th days posttreatment 

to confirm the absorption of intracranial hematoma. 

Biochemical estimation 

Fasting blood samples of each patient in the two groups was drawn 

Male 15 16 

Female 5 4 

Range (yr) 19-64 20-65 

Mean±SD (yr) 39.43±12.35 38.96±13.79 

Epidural hematoma 7 6 

Subdural hematoma 5 6 

TICH 5 6 

Multiple hematoma 3 2 

Range (ml) 15-36 14-35 

Mean±SD (ml) 28.12±7.96 27.46±8.07 

by medical officer between 6:00 to 8:00 a.m. pre-treatment and on 

the 14th day of the therapy. Serum was obtained by centrifugation 

at 1500 × g for 15 min of blood samples taken without 

anticoagulant. Serum was kept at -80°C until the biochemical 

estimation of different parameters. Malondialdehyde (MDA), 

superoxide dismutase (SOD) activity, catalase (CAT) activity, 

glutathione (GSH) estimation in the serum were performed by the 

method of Santanu et al. (2008) 

Efficacy assessment and monitoring of adverse effects 

The common adverse effects (that is, dizziness, cardiopalmus, 

fever, rash, etc.) of Chinese herbs were observed by a specifically 

appointed person according to the previous reports (Yang et al., 

2011). Blood routine, alanine transferase (ALT), aspartate 

transaminase (AST), blood urea nitrogen (BUN), creatinine, 

electrocardiogram were tested pre-treatment and on the 14th day of 

the therapy. 

All the tests were operated according to the protocols provided by 

Xiangya Hospital affiliated to Central South University. 


All the data in the experiment are expressed as mean ± standard 

deviation. All data were analyzed by SPSS 16.0 software. 

Comparisons between pre- and post- treatment was carried out 

using a paired t-test. An independent-samples t-test was used to 

compare the post-treatment data between the trial group and the 

control group, and a Chi-square test was taken for nonparametric 

data. The values of p< 0.05 were considered to be statistically 

significant. 

RESULTS 

Effect on GCS 

Figure 1 showed that the pre-treatment GCS (9.76±1.24 

and 9.83±1.31) of the two groups were comparable with 

no significant differences (p>0.05). The post-treatment 

score in trial group and control group was significantly


Figure 1. Comparison of GCS score in the control and trial groups (Score). ※ P>0.05, trial group vs. control 

group of pre-treatment; **P

nmol/mg protein 

nmol H202 decompose 

/min/mg protein 

Volume of intracranial hematoma (ml) 

Figure 2. Comparison of intracranial hematoma volume after 1 week and 2 weeks of 

treatment. **p


alanine transferase (ALT), aspartate transaminase (AST), 

blood urea nitrogen (BUN), creatinine, electrocardiogram 

tests were seen in all patients. Only one patient reported 

a mild fever which resolved without symptomatic therapy. 

Apart from this, no other obvious adverse reaction 

occurred. 

DISCUSSION 

TICH belongs to the category of „„blood stasis‟‟ in 

traditional Chinese medicine (TCM) (Ma et al., 2008). 

Traditional Chinese medicine (TCM) thought that “blood 

circulating out of vessels was known as blood stasis” and 

blood stasis is an important underlying pathology of many 

disease processes according to traditional Chinese 

medicine. Described in TCM theory as a slowing or 

pooling of the blood due to injury or disruption of heart Qi, 

it is often understood in biomedical terms in terms of 

hematological disorders such as hemorrhage, 

congestion, thrombosis, local ischemia (microclots) and 

tissue changes (Zhao et al., 2008). The strategy of 

activating blood circulation and resolving stasis has been 

extensively in clinical application of Chinese materia 

medica. Numerous studies reported that it can improve 

microcirculation, eliminate toxic products, and attenuate 

cerebral edema, thus promoting the absorption of 

intracranial hematoma and improving the functions of the 

nervous system (Ma et al., 2008). 

The Chinese material medica, such as S. miltiorrhiza 

Bge, C. tinctorius L, P. lactiflora Pall, L. chuanxiong Hort. 

and A. sinensis, which are consisted in XBJ, all have the 

function of activating blood circulation and resolving 

stasis in accordance with the principals of TCM. Safflor 

yellow A, the main component of XBJ, mainly has the 

same function according to TCM principals and modern 

pharmacological studies. Some research also reported 

XBJ protect brain (Wei et al., 2005), lung tissue (Qiu et 

al., 2009), pancreas (Sun et al., 2007) through antioxidative 

effect. Moreover, Safflor yellow A could protect 

HUVECs from hypoxia induced injuries by inhibiting cell 

apoptosis and cell cycle arrest (Ji et al., 2009), inhibit 

thrombosis and platelet aggregation (Tian et al., 2003). It 

provided neuroprotection against cerebral 

ischemia/reperfusion injury through its antioxidant action 

(Wei et al., 2005) and exhibited neuroprotective effects 

after permanent middle cerebral artery occlusion (MCAO) 

in rats (Zhu et al., 2003). But there was no report on the 

effects of XBY on TICH, and the mechanisms of its 

neuroprotective effect are remains poorly understood. 

In the study, we observed the clinical therapeutic 

efficacy of XBJ on the treatment of traumatic intracranial 

hematoma. Trial group treatment in combination with XBJ 

could obviously improve the prognosis (promote the 

scores of GCS in Figure 1 and GOS) and make 

intracranial hematomas absorbed (Figure 2), compared 

with control group treatment. As expected, XBJ exhibited 

neuroprotective effects on patients with TICH. Its 

mechanism of action may be achieved by lowering 

fibrinogen content, promoting fibrolysis, and improving 

microcirculation (Table 2). Except XBJ, some other 

studies (Yuan et al., 2009a; Chen et al., 2007; Deng et 

al., 2009) showed that traditional Chinese herbs could 

significantly improve clinical effect, improve nerve 

impairment, and accelerate hematoma absorption. 

Interestingly, Shuxuetong injection (Jin et al., 2008) and 

Danhong injection (Sun et al., 2009) (Chinese materia 

medica injection) also demonstrated the similar effects to 

XBJ on clinical neuroprotective effects just as reported in 

the study. However, the mechanism of oxidative stress 

was studied in our research, which was not seen in these 

reports. 

Oxidative stress is the result of an imbalance between 

the generation of reactive oxygen species (ROS) and the 

antioxidant system in favour of the former (Santanu et al., 

2008). So, intensity of oxidative stress is determined not 

only by the free radicals production but also by 

antioxidants (enzymatic and non-enzymatic) defense 

(Beltowski et al., 2000). Oxidative stress is increased in 

brain injury, playing significant roles in neuronal apoptosis 

(Yu et al., 2002). 

Malondialdehyde (MDA), the degradation product of the 

oxygen-derived free radicals and lipid oxidation, can 

interfere with the metabolism of protein, glucose, and 

nucleic acid, which results in the decrease in activity of 

enzyme, template dysfunction of nucleic acid, and injury 

of tissues and cells (Zheng et al., 2008). Antioxidant 

defense system protects our body from the deleterious 

effect of reactive oxygen metabolites. Catalase (CAT) act 

as preventive antioxidants and superoxide dismutase 

(SOD), a chain breaking antioxidant, play an important 

role in protection against the deleterious effect of lipid 

peroxidation (Dinkovo-Kostova, 2002). Depletion of GSH 

is one of the primary factors that permit lipid peroxidation 

(Konukoglu et al., 1998). Figure 3 showed that trial and 

control treatment both could reduce the serum level of 

MDA, increased the activities of SOD and the serum level 

of GSH. To our surprise, the indexes in the trial group 

was superior to that of the control group (p

and exert synergistic therapeutic effects. The study 

provides evidence for the first time to elucidate the 

integrated protective effects of XBJ for improve the 

prognosis of TICH mainly through anti-oxidation and anticoagulation. 

The studies on its more and precise 

mechanism of action are underway. Therefore, XBJ may 

be safely used as an effective and promising agent for 

both TICH and other complex diseases. 

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DOI: 10.5897/JMPR11.1544 



Cardiospermum grandiflorum leaf extract potentiates 

amoxicillin activity on Staphylococcus aureus 

Petra O. Nnamani, Franklin C. Kenechukwu* and Wilfred N. Oguamanam 

Drug Delivery Research Unit, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Nigeria, 

Nsukka 410001, Enugu State, Nigeria. 


Concurrent administration of orthodox and herbal antibacterial agents could help overcome the 

tendency with which the former is inactivated by bacterially-produced endogenous enzymes. This study 

was designed to evaluate the antimicrobial interaction between the ethanol extract of Cardiospermum 

grandiflorum leaf (CGL) which is used in a remote village in Nsukka, Nigeria as a bathing sponge for 

treatment of skin infections and amoxicillin (AMX), an extended-spectrum but penicillinase-susceptible 

penicillin. The antimicrobial interaction between these two agents was evaluated by modification of the 

checkerboard technique using Staphylococcus aureus (a penicillinase-producing Gram-positive 

bacterium)and Bacillus subtilis (a non penicillinase-producing Gram-positive bacterium) as the test 

organisms. The MIC of the ethanolic extract against S. aureus and B. subtilis was respectively, 25.0 ± 

0.1 and 50.0 ± 0.5 mg/ml while the MICs of amoxicillin were 0.05 ± 0.01 and 0.025 ± 0.002 mg/ml against 

B. subtilis and S. aureus, respectively. The effect of combination of the ethanol extract of C. 

grandiflorum leaf with amoxicillin was dependent on both the ratio of combination and the test 

organism employed for the evaluation. Overall, the combined antimicrobial effect was predominantly 

synergistic against S. aureus. 

Key words: Cardiospermum grandiflorum leaf, antibacterial interaction, checkerboard technique, Bacillus 

subtilis, Staphylococcus aureus, amoxicillin. 

INTRODUCTION 

Recently, scientific interest in medicinal plants have 

burgeoned due to the increased efficiency of plant 

derived drugs and less side effects of the latter compared 

to modern medicines, continuing emergence of drug 

resistant organisms and adaptations by microbial 

pathogens to commonly used antimicrobials (Nair and 

Chanda, 2006; Parek et al., 2006; Audu et al., 2004; 

Adeniyi et al., 2005; Akinpelu and Onakoya, 2006; Nkere 

and Iroegbu, 2005). Ballon vine (Cardiospermum 

*Corresponding author. E-mail: chimafrankduff@yahoo.com, 

frankline.kenechukwu@unn.edu.ng. Tel: +234 8038362638. 

Abbreviations: CGL, Cardiospermum grandiflorum leaf 

ethanolic extract; AMX, Amoxicillin; FICAMX, fractional inhibitory 

concentration of amoxicillin; FICCGL, fractional inhibitory 

concentration of C. grandiflorum leaf ethanolic extract; Syn, 

synergism; Add, additivity; Ind, indiffernce; Ant, antagonism. 

grandiflorum Fam. Sapindaceae), a vigorous, vine-like 

climber (twinner), pubescent or nearly glabrous annual or 

perennial plant has slender branches, ternately 

compound, membraneous, depressed, pyriform capsule 

leaves wrangled at the angles with black seeds having 

large white shaped aril (Aluka, 2008). Various parts of the 

plant such as the leaves, roots and seeds have been 

widely used in traditional medicines for curing various 

human ailments including treatment of arthritis, 

amenorrhea, lumbago, neuropathy and rheumatism, 

stiffness of limbs and snake bite, nervous disorders and 

piles, diarrhoea, diabetes, convulsion and bacterial 

infections (Aluka, 2008; Banso, 2007). 

Amoxicillin, a synthetic extended-spectrum penicillin is 

known to decrease the stability of the cell wall by 

inhibiting both the transpeptidase and the D-alanine 

carboxypeptidase enzymes (Chambers, 2004). 

Staphylococcus aureus is a normal flora of the skin and is 

also implicated in some opportunistic infections of the


Figure 1. Picture of C. grandiflorum leaf. 

skin (Nair and Chanda, 2006; Parek et al., 2006; Nkere 

and Iroegbu, 2005). Amoxicillin is inactivated by 

penicillinases including those produced by S. aureus 

(Chambers, 2004). 

The interest in the present study was spurred by our 

observation, over the years, that a large number of 

patients who had some skin infections (characterized by 

massive dry skin with somewhat large patches of 

spreading scaly non-itching skin) on several occasions in 

a remote village in Nsukka L.G.A. of Enugu State, Nigeria 

were successfully treated by traditional medicine 

practitioners with the fresh leaves of C. grandiflorum as 

bathing sponge. These patients resorted to traditional 

medicines after unsuccessful attempts to treat the skin 

infection with conventional antibiotics due to 

reoccurrence of the infection. To authenticate this 

folkloric use, the plant was taken to a botanist for proper 

identification, hence the conception of the work. 

In rational drug therapy, the concurrent administration 

of two or more antimicrobial agents is often essential and 

sometimes mandatory in order to achieve the desired 

therapeutic aim to treat specific infections and co-existing 

diseases as well as prevent the emergence of resistant 

micro-organisms (Nnamani et al., 2005). Drug interaction 

may result in synergistic, antagonistic, indifferent or 

additive effects (Esimone et al., 2002). It is highly 

expedient that the in vitro interaction of combination of 

antimicrobials be evaluated using suitable test 

microorganisms before such combinations are clinically 

used. Although a study has been carried out that 

established the antimicrobial activities of crude ethanol 

extract from leaf of C. grandiflorum (Banso, 2007), to the 

best of our knowledge there is paucity of information on 

the antibacterial properties of crude ethanol extract from 

leaf of C. grandiflorum in combination with conventional 

antibiotics. 

In this paper therefore, we report on the antibacterial 

interaction of crude ethanol extract of C. grandiflorum leaf 

with amoxicillin, an extended-spectrum but penicillinasesusceptible 

penicillin. 


Reagents 

Analytical grades of ethanol 99% (Fluka, Germany) and 

dimethylsulphoxide, DMSO (Merk, Germany) were used for 

extraction and dilution respectively of the C. grandiflorum leaf 

extract. Distilled water was collected from an all-glass still. Nutrient 

agar (Fluka, Germany) was used as medium for the study. 

Amoxicillin pure powder (Afrab-Chem. Ltd., Nigeria) was used as 

synthetic antibiotic. Laboratory isolates of S. aureus and Bacillus 

subtilis were obtained from stock cultures in the Pharmaceutical 

Microbiology laboratory, Department of Pharmaceutics, University 

of Nigeria, Nsukka. 

Collection and identification of plant material 

Fresh leaves of the C. grandiflorum were obtained in June, 2009 

from Nkalagu-Obukpa in the Nsukka locality (Figure 1). 

Authentication of the leaves was done by Mr. A. O. Ozioko of the 

Bioresources Development and Conservation Programme Center 

(BDCP), Nsukka, Enugu State, Nigeria and a voucher specimen 

(PC98132) is preserved in the Pharmacognosy Herbarium, 

University of Nigeria, Nsukka. 

Preparation of the Cardiospermum grandiflorum leaf ethanol 

extract 

The C. grandiflorum leaves were air dried under shade for two 

consecutive days and then pulverized using electric blender at the 

Soil Science Department of the University of Nigeria, Nsukka. 

Approximately 250 g of the fine powder was extracted with one litre 

of 99% ethanol by the cold maceration method for 24 h. The extract

was further filtered, allowed to evaporate to a semi-solid residue 

and stored at 25°C until required for use. 

Preparation of culture media 

The growth medium employed was nutrient agar and it was 

prepared using the methods specified in the Oxoid manual (Oxoid, 

England). The stock microbial cultures were maintained on nutrient 

agar slants at 4°C. In order to activate these cultures, subcultures 

were freshly prepared and incubated at 37°C for 18 to 24 h before 

use. Standard suspensions of each test microorganisms were 

made by transferring a colony from the subculture containing 

approximately 10 9 colony forming unit per ml (cfu/ml) of the 

organisms into 5 ml of sterile distilled water, and adjusting the 

volume to obtain a cell population of approximately 10 6 cfu/ml. A 

volume of 0.1 ml of such suspensions was used as inoculum in all 

the tests. 

Preliminary antimicrobial screening 

Preliminary antimicrobial screening of the C. grandiflorum leaf 

extract was carried out using the cup-plate agar diffusion method 

(Hassan et al., 2003). This method depends on the diffusion of 

antibiotics from holes on the surface of the microbial seeded agar. 

Molten nutrient agar (20 ml) was inoculated with 0.1 ml of S. aureus 

broth culture. It was mixed thoroughly, poured into sterile Petri 

dishes and rotated for even distribution of the organism. The agar 

plates were allowed to set and a sterile cork borer (8 mm diameter) 

was used to bore six holes in the seeded agar medium. Two drops 

of each of the two-fold dilution of the extract in DMSO (100, 50, 25, 

12.5, 6.25 µm, 3.125 mg/ml) was added into each labeled hole 

using a sterile pipette. 

The plates were allowed to stand at room temperature for 15 min 

to enable the samples to diffuse into the medium before incubating 

at 37°C for 24 h. The experiment was repeated for B. subtilis. Three 

replicate tests were performed in each case. Growth was examined 

after incubation and the diameter of each inhibition zone was 

measured and the average determined. A control experiment was 

also set up against each test organism using DMSO as a control 

diluent. The whole experiment was similarly repeated for 4 mg/ml of 

amoxicillin using sterile distilled water as the solvent for dilution. 

Determination of the minimum inhibitory concentration (MIC) 

The MIC of the C. grandiflorum leaf extract was obtained using the 

agar dilution technique (Ofokansi et al., 2008). A stock solution of 

the extract (100 mg/ml) was prepared by dissolving 200 mg of the 

extract in 2 ml of 50% DMSO (that is, one part of DMSO in one part 

of water). Then two-fold serial dilutions were made with sterile 

distilled water to obtain concentrations between 50 and 3.125 

mg/ml. A volume of each of the concentrations equal to 0.5 ml was 

transferred into an agar plate and made up to 20 ml with molten 

agar and then allowed to set. The surface of the agar was then 

dried and streaked with isolates. 

An over-night (24 h) broth culture was used for this experiment. 

The same procedure was repeated with amoxicillin but in this case 

a stock solution of 4 mg/ml was prepared and the final 

concentrations obtained in agar plates ranged from 0.4 to 0.025 

mg/ml. Control plate having 5 ml of 50% DMSO in 15 ml of molten 

agar was prepared for C. grandiflorum. The plates were then 

incubated at 37°C for 24 h. The MIC was taken to be the lowest 

concentration which showed no visible growth of each of the test 

isolate on the agar surface. The experiment in each case was 

carried out in three replicates. 

Nnamani et al. 903 

Evaluation of the interaction between Cardiospermum 

grandiflorum leaf extract and amoxicillin 

Stock solutions of C. grandiflorum leaf extract (100 mg/ml) and 

amoxicillin (0.1 mg/ml) were prepared for evaluation of their 

combined effect on S. aureus and B. subtilis. The two agents were 

mixed in varying ratios ranging from 0:10 to 10: 0 of C. grandiflorum 

leaf extract and amoxicillin in accordance with the continuous 

variation checkerboard technique (Esimone et al., 2002; Ofokansi 

et al., 2008). Each of the eleven combinations of these two 

antimicrobial agents was serially diluted (2-fold) in 3 ml of sterile 

water into eight places. Two millilitres each of the dilutions of the 

stock mixtures was seeded into 18 ml of moltenagar. After setting, 

the surface of the agar was then streaked with the test 

microorganisms. The streaked agar plates were then incubated at 

37°C for 24 h. The combined effect of the antimicrobials on the test 

microorganisms was determined and recorded from the fractional 

inhibitory concentration (FIC) index. The experiment was done in 

triplicate to ensure reproducibility of results. The FIC index was 

calculated as follows (Ofokansi et al., 2008; Esimone et al., 2002): 

where FICAMX is the fractional inhibitory concentration of amoxicillin 

and FICCGL is fractional inhibitory concentration of C. grandiflorum. 

RESULTS 

The MICs of the ethanol extract of C. grandiflorum leaf 

against B. subtilis and S. aureus was evaluated to be 

50.0 ± 0.5 and 25.0 ± 0.1 mg/ml, respectively while that 

of amoxicillin was calculated to be 0.025 ± 0.002 and 

0.05 ± 0.01 mg/ml against S. aureus and B. subtilis, 

respectively. The recorded MIC values were the mean of 

three replicate studies. Tables 1 and 2, respectively, 

show the results of the combined antimicrobial effect of 

the ethanol extract of C. grandiflorum leaf and amoxicillin 

against the test organisms. Table 1 shows the combined 

activity of ethanol extract of C. grandiflorum leaf and 

amoxicillin against S. aureus. Synergistic effects were 

recorded at AMX/CGL ratios of 7:3, 4:6, 3:7 and 2:8, 

additivity (8:2 and 6:4), indifference (1:9) and antagonism 

(9:1 and 5:5). In Table 2, synergism was recorded at 

AMX/CGL ratios of 9:1 and 7:3; antagonism (5:5 and 

1:9), and indifferent effect (8:2, 6:4, 4:6, 3:7 and 2:8) 

against B. subtilis. 

DISCUSSION 

It could be seen from the MIC results that whereas 

amoxicillin showed very high activities against the 

(1) 

(2) 

(3)


Table 1. The combined antibacterial effect of the ethanol extract of C. grandiflorum leaf and amoxicillin against S. aureus. 

Drug combination 

ratio (AMX:CGL) 

MIC of AMX 

(mg/ml) 

MIC of CGL 

(mg/ml) 

FIC of AMX FIC of CGL FIC index Effect 

10:0 0.0500 - - - - - 

9:1 0.0900 0.0100 1.80 0.20 2.00 Ant 

8:2 0.0400 0.0100 0.80 0.20 1.00 Add 

7:3 0.0175 0.0075 0.35 0.15 0.50 Syn 

6:4 0.0300 0.0200 0.60 0.40 1.00 Add 

5:5 0.0500 0.0500 1.00 1.00 2.00 Ant 

4:6 0.0100 0.0150 0.20 0.30 0.50 Syn 

3:7 0.0075 0.0175 0.15 0.35 0.50 Syn 

2:8 0.0075 0.0175 0.10 0.40 0.50 Syn 

1:9 0.0050 0.0450 0.45 0.90 1.35 Ind 

0:10 - 0.0500 - - - - 

Syn, Synergism; Ind, indifference; Add, additivity; Ant, antagonism; MIC of AMX and CGL evaluated from agar dilution method against 

S. aureus were 0.025 ± 0.002 and 25.0 ± 0.1 mg/ml, respectively. 

Table 2. The combined antibacterial effect of the ethanol extract of C. grandiflorum leaf and amoxicillin against B. Subtilis. 

Drug combination 

ratio (AMX:CGL) 

MIC of AMX 

(mg/ml) 

MIC of CGL 

(mg/ml) 

FIC of AMX FIC of CGL FIC Index Effect 

10:0 0.0500 - - - - - 

9:1 0.0225 0.0025 0.45 0.20 0.65 Syn 

8:2 0.0400 0.0100 0.80 0.80 1.6 Ind 

7:3 0.0175 0.0075 0.35 0.60 0.95 Syn 

6:4 0.0150 0.0100 0.30 0.80 1.1 Ind 

5:5 0.0500 0.0500 0.60 4.00 4.6 Ant 

4:6 0.0100 0.0150 0.20 0.12 1.4 Ind 

3:7 0.0075 0.0175 0.15 1.40 1.55 Ind 

2:8 0.0050 0.0200 0.10 1.60 1.7 Ind 

1:9 0.0050 0.0450 0.02 3.60 3.62 Ant 

0:10 - 0.0125 - - - - 

Syn, Synergism; Ind, indifference; Add, additivity; Ant, antagonism; MIC of AMX and CGL evaluated from agar dilution method 

against B. subtilis were 0.05±0.01 and 50.0 ± 0.5 mg/ml, respectively. 

Gram-positive organisms, S. aureus, B. subtilis 

(Chambers, 2004), and C. grandiflorum leaf extract 

showed only a marginal activity against the two 

organisms. The FIC index is interpreted as synergism if 

its value is less than 1.0 additivity if it is equal to 1.0, 

indifference if more than 1.0 but less than 2.0 and 

antagonism if more than 2.0 (Esimone et al., 2002; 

Ofokansi et al., 2008). It is clear from Tables 1 and 2 that, 

although the combined antimicrobial effect against S. 

aureus and B. subtilis did not show a regular pattern, 

synergism was recorded at certain AMX/CGL 

combinations. In summary, the combined antimicrobial 

effect of the interaction between C. grandiflorum and 

amoxicillin was predominantly synergistic against S. 

aureus. This could be seen to mean a potentiation of the 

effect of amoxicillin (a penicillinase-susceptible penicillin) 

against S. aureus (a penicillinase-producing bacterium 

which is also implicated in some opportunistic infections 

of the skin) in the presence of ethanol extract of C. 

grandiflorum leaf. A probable explanation of the 

enhanced activity of amoxicillin by C. grandiflorum leaf 

extract in combination is that the amoxicillin and the 

antimicrobial principles in ethanol extract of C. 

grandiflorum leaf may possibly have different mechanism 

of action or may be inhibiting two different steps in the 

same biosynthetic pathway of the organism resulting in 

an overall synergy at certain combinations. The 

mechanism of the synergy demonstrated with these 

agents is yet unknown. However, it has been noted that 

two antimicrobial agents may interact antagonistically if 

one is bacteriostatic and the other bactericidal (Nnamani 

et al., 2005). Amoxicillin is known to decrease the stability

of the cell wall by inhibiting both the transpeptidase and 

the D-alanine carboxypeptidase enzymes (Chambers, 

2004) and so acted synergistically with C. grandiflorum 

leaf extract. A more critical look at Tables 1 and 2 would 

reveal that the combined effect of the two antimicrobial 

agents is not only dependent on the ratio of combination 

but also on the type of the test microorganism empolyed 

as exemplified by S. aureus (a penicillinase-producing 

Gram-positive bacterium which is also implicated in some 

opportunistic infections of the skin) and B. subtilis (a non 

penicillinase-producing Gram-positive bacterium). Since 

the predominant interaction between AMX/CGL were 

positive (synergism and additivity as shown in Table 1), 

this justifies the folkloric use as a skin sponge in treating 

skin infections. 

Moreover, it has been established that C. grandiflorum 

leaf possessed good antimicrobial activity against S. 

aureus (Banso, 2007). The result of our investigation 

revealed that C. grandiflorum had marginal activity 

against S. aureus. The slight variation in the results of the 

antimicrobial studies could be related to variation in the 

strains of S. aureus employed in the studies. 

Furthermore, amoxicillin is inactivated by penicillinases 

including those produced by S. aureus (Chambers, 

2004). Consequently, infections caused by amoxicillin 

resistant isolates such as S. aureus could be treated with 

some amoxicillin/C. grandiflorum leaf extract 

combinations at certain C. grandiflorum leaf extract 

concentration as occurred in the 7:3, 4:6, 3:7 and 2:8 

ratios of AMX/CGL combination, bearing in mind the 

inefficiency of amoxicillin to Staphylococcal infections due 

to inactivation of the drug by some penicillinases 

produced by some strains of S. aureus. 

Conclusions 

The results from this investigation has provided a 

preliminary evidence of some kind of bacterial interaction 

between ethanol extract of C. grandiflorum leaf and 

amoxicillin against S. aureus and B. subtilis. There is an 

indication that combinations of amoxicillin and C. 

grandiflorum leaf extract may have some usefulness in 

chemotherapy of infections in which S. aureusis 

implicated. 

Conversely, the combined effect of the interaction 

against B. subtilis may not be highly significant. In 

Nsukka L.G.A. of Enugu State, Nigeria, where C. 

grandiflorum leaf is commonly used as a bathing sponge 

in the treatment of skin infections; the possible 

therapeutic implications of using the leaf concomitantly 

with amoxicillin cannot be overlooked. Further studies 

would seek to evaluate the efficacy of amoxicillin/C. 

Grandiflorum leaf ethanol extract combinations as topical 

agents for the treatment of skin infections caused by S. 

aureus in experimental animals. 

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traditionally used medicinal plants for potential antibacterial 

activity. Indian J. Pharm. Sci., 68(6): 832-834.



DOI: 10.5897/JMPR11.1584 



The first Stolbur Phytoplasma occurrence on two St. 

John's Worth species (Hypericum perforatum L. and 

Hypericum barbatum L.) in Serbia 

Snezana Pavlovic 1 *, Dragana Josic 2 , Mira Starovic 3 , Sasa Stojanovic 3 , Goran Aleksic 3 , Vera 

Stojsin 4 and Dragoje Radanovic 1 

1 Institute for Medicinal Plant Research “Dr. Josif Pancic”, Belgrade, Serbia. 

2 Institute for Soil Science, Belgrade, Serbia. 

3 Institute for Plant Protection and Environment, Belgrade, Serbia. 

4 Faculty of Agriculture, Novi Sad, Serbia. 


The symptoms indicating phytoplasma like leaf yellowing, reddening and early drying were observed 

on two St. John’s worth species (Hypericum perforatum L. and Hypericum barbatum L.) on infected 

fields (Pancevo, Indjija and Stara Pazova) in Serbia in 2008. Electron microscopy examination of the 

ultra-thin sections revealed the presence of numerous polymorphic phytoplasma-like bodies in the 

phloem tissue of leaf midribs and petioles. The phytoplasma etiology was confirmed by polymerase 

chain reaction (PCR) using 3 sets of primers (P1/P7, P1/16S-Sr and R16F2n/R16R2). Restriction 

fragment length polymorphism (RFLP) analysis of amplification products of 1.2 kb (obtained with 

R16F2n/R2 primer pair) in 51 from 60 symptomatic plants, indicated the presence of 16SrXII-A 

phytoplasma subgroup from all three affected localities. Sequence of R16F2n/R2 amplicon for 

representative phytoplasma H. perforatum L. isolate Hp22 was deposited in the GeneBank with 

accession number JQ033928. This is the first report of the natural occurrence of Stolbur phytoplasma in 

two cultivated St. John’s worth species in Serbia. 

Key words: Hypericum perforatum, Hypericum barbatum, phytoplasma diseases, 16SrXII-A subgroup. 

INTRODUCTION 

In the last few decades, the trends in increased use of 

herb in alternative medicinal purposes, have led to 

increased cultivation of medicinal plants such as 

Hypericum. In these new crops, unique diseases and 

pest problems are emerging. Some of these are rare or 

unknown in the wild, and have emerged by the use of 

agricultural systems (Scheffer, 1997). Hypericum 

(Hypericaceae) is one of the plants used traditionally in 

medicine for over 2000 years (Patocka, 2003) and one of 

the best – selling herbs of the past decade (Grunwald, 

1999). In order to preserve the natural population of St. 

John’s worth and due to a huge demand, Hypericum 

perforatum has been cultivated on as a crop since 1997 

*Corresponding author. E-mail: spavlovic@mocbilja.rs. Tel: 

+381113283251. Fax: +38111 3031649. 

in Serbia. 

Phytoplasmas are associated with serious plants 

disseases. They are plant-pathogenic bacteria of the 

class Mollicutes, have no cell wall and inhabit plant 

phloem of numerous plants. Cell sizes of phytoplasmas 

are 0.1 to 0.8 µm in diameter and genome size are the 

smallest among bacteria (Hren et al., 2009). Current 

classification of the phytoplasmas is based on the 

nucleotide sequence and restriction fragment length 

polymorphism (RFLP) of the 16S rRNA gene. 

Polymerase chain reaction (PCR) amplification of 16S 

rDNA of the phytoplasmas has significantly contributed to 

the identification and characterization of unidentified 

phytoplasmas (Montano et al., 2001; Harrison et al., 

2002). 

Diverse phytoplasma infections have been found on 

various cultivated medicinal plants: Galega oficinalis L., 

Digitalis lutea L., Hyssopus officinalis L., Parietaria

Pavlovic et al. 907 

Table 1. Presence of Stolbur Phytoplasma detected by nested PCR in the samples of H. perforatum and H. barbatum with symptoms 

in 3 localities in Serbia during 2010. 

Locality 

Pancevo 

Stara 

Pazova 

Indjija 

Symptomatic plants Asymptomatic plants 

Samples No. nested PCR positive/ analysed Samples No. nested PCR positive/ analysed 

HpP* 1-10 10/10 HpP 31-33 0/3 

HbP 1-10 9/10 HbP 31-33 0/3 

HpS 11-20 7/10 HpS 34-36 0/3 

HbS 11-20 8/10 HbS 34-36 0/3 

HpI 21-30 9/10 HpI 37-39 0/3 

HbI 21-30 8/10 HbI 37-39 0/3 

*Hp- H.perforatum; Hb- H.barbatum; P- Pancevo; S- Stara Pazova; I-Indjija. 

officinalis L., Tagetes patula L., Spartium junceum L., 

Vinca rosea (Lee et al., 2000), Marticaria perforata 

(Khadair et al., 1999), H. perforatum L. (Bruni et al., 

2005), Valeriana officinalis (Khadhair et al., 2008), 

Echinacea purpurea (Radisek et al., 2008, Pavlovic et al., 

2011), Plantago lanceolata L. (Credi et al., 2006, Franova 

and Simkova, 2009) and Plantago major (Josic et al., in 

press). 

During the observation of the St. John’s worth plants in 

the locality Pancevo, at the end of May 2008, the 

phytoplasma like symptoms were noted: yellowing, 

followed by reddening of leaves and proliferation. The 

following year, all plants developed a yellow-red color on 

all green part of the plants: stem and leaves. The 

diseased tissue showed necrosis, drying and death 

before flowering. The percentage of affected plants 

expressed as the reduction in yield was over 70 in some 

fields. This very high percentage of diseased plants 

brought into the question the commercial viability of the 

production. 


Electron microscopy 

Samples for the observation of phloematic areas by transmission 

electron microscopy were taken from the symptomatic plants during 

June 2009 and May 2010. Main veins and petioles from 

symptomatic and asymptomatic leaves were fixed in 5% 

glutaraldehyde in 0.1 M potassium phosphate buffer (pH 7.2) for at 

least for 2 days at 4°C and, subsequently, post-fixed in 2.0% 

osmium tetroxide in the same buffer. The specimens were 

dehydrated by an ethanol series. Ultra-thin sections were stained 

with uranyl acetate in 70% ethanol and examined by the 

transmission electron microscopy (TEM) (Hopkins et al., 1973). 

Disease symptoms and incidence 

Samples of H. perforatum and Hypericum barbatum showing 

symptoms of phytoplasma infection were collected in May 2010 

from three different localities in Serbia - Pancevo, Stara Pazova 

and Indjija (Table 1). The symptoms included leaf yellowing, 

premature leaf drying and, occasionally, desiccation of the whole 

plant. 

Disease incidence, expressed as a percentage of the area with 

symptomatic plants, was calculated approximately in all three 

localities during 2008 to 2010. 

Phytoplasma detection 

A total of 30 H. perforatum and 30 H. barbatum symptomatic leaf 

samples were collected. Eighteen asymptomatic plants (3 plants 

per locality for both species) were also collected as control 

samples. 

For detection of phytoplasmas, total DNA was extracted from the 

veins of symptomatic and asymptomatic leaves using the protocol 

of Angelini et al. (2001). Oligonucleotide universal primers P1/P7 

(Deng and Hiruki, 1991; Schneider et al., 1995) were used in the 

direct PCR assays to amplify a 1.8 kb fragment including the 16S 

rRNA gene, the 16S–23S spacer region and the 5’ end of the 23S 

rRNA gene. 

The second set of primers was P1/16S-Sr and amplicons of 1.5 

kb were expected in infected plants. For nested PCR, the 

R16F2n/R16R2 primers (Gundersen and Lee, 1996) were used to 

amplify a 1.2 kb fragment of the 16S rRNA gene included in the 1.8 

or 1.5 kb fragment. Amplifications were performed in 50 µl reaction 

mixture using Dream Taq Green master mix (Fermentas, Lithuania). 

PCR using primer pairs P1/P7 and P1/16S-Sr were performed for 

35-cycles: denaturation at 95°C for 60 s (2 min for first cycle), 

annealing for 30 s at 56°C and primer extension for 90 s at 72°C. 

For the nested PCR the same conditions were applied, except that 

annealing was done at 57°C. The PCR products were separated on 

1.2% agarose in TBE buffer, stained with ethidium bromide and 

visualized on UV transilluminator. 

Phytoplasma identification 

For the polymerase chain reaction-restriction fragment length 

polymorphism (PCR-RFLP) analysis, the samples that yielded 

amplicons with R16F2n/R16R2 primers were used for further 

characterization using AluI and TruI restriction enzymes 

(Fermentas, Lithuania) as recommended by the manufacturer. 

RFLP profiles were analyzed by electrophoresis trough 2.5% 

agarose with GeneRuler SM0331 marker (Fermentas, Lithuania) as 

the DNA marker size standard. The following phytoplasma isolates


Figure 1. H. perforatum (a) and H. barbatum (b) plants infected with Stolbur phytoplasma. 

Figure 2. Ultra-thin cross-section of H. perforatum main 

vein from the symptomatic leaves (bar = 300 nm). 

were used as references for PCR/RFLP analysis: STOLphytoplasma 

DNA, grapevine FD-C and AY (kindly provided by M. 

Martini) phytoplasmas. 

The PCR product of representative isolate Hp22 primed by 

R16F2n/R16R2 primers for partial 16S rRNA gene was subjected to 

the sequence analysis using facilities of IMGGI, Belgrade. 

RESULTS 

Disease symptoms and incidence 

During the three years of observation (2008, 2009 and 

2010) the first symptoms, consisting in diffuse yellowing 

and reddening of the leaves, proliferation and stunting, 

were observed from the beginning of May (Figure 1), 

increasing in severity with time. Symptomatic plants died 

within one or two months. The percentage of infected 

plants in first year of observation (2008) varied from 15 

(Indjija and Stara Pazova) to 20% (Pancevo); in the 

second year from 40 (Indjija and Stara Pazova) to 70% 

(Pancevo), and in the third year of observation, this 

percentage in locality Indjija and Stara Pazova were 

higher than 70 while in Pancevo was over 85%. 

Electron microscopy 

Thin section of the leaf main veins revealed the presence 

of typical phytoplasma-like bodies of 75 x 100 to 100 x 

300 nm in diameter (Figure 2). They were observed in 

mature and immature phloem sieve tubes. In the control 

(healthy plants) there were no phytoplasma like bodies 

present. 

Phytoplasma detection 

The PCR amplification from total DNA of symptomatic 

plants generated 1.8 kb (in 28 out of 40 samples) or 1.5 

kb (in 17 out of 20 samples) DNA fragments of the 16S 

ribosomal DNA, when universal primers P1/P7 and 

P1/16S-Sr (Figure 3a) were used, respectively. The PCR 

amplification of a 1.2 kb (Figure 3b) fragments were 

obtained in 51 samples with R16F2n/R2 primers (Table 

1). No PCR products were obtained from asymptomatic, 

healthy, control plants. 

Phytoplasma identification 

All R16F2n/R2 amplified products gave identical RFLP 

patterns corresponding to the profile of the Stolbur 

phytoplasma (subgroup 16SrXII-A) using AluI and TruI 

restriction enzymes. RFLP patterns of representative 

isolates were shown in Figure 4. Partial 16S rRNA gene

M 1 2 3 4 5 6 7 8 9 10 11 12 13 M 

1.5 kb 

a b 

M 1 2 3 4 5 6 7 8 9 10 11 12 M 


1.2 kb 

Figure 3. PCR amplification of 16S ribosomal DNA using (a) P1/16S-Sr primers: lane 1. HpI37; lane 2-7: 

HpI22; HpI24; HpI26; HpI27; HpI28; HpI30; lane 8-12: HbI21; HbI22; HbI23; HbI25; HbI27; HbI29; lane 13. 

HbI 38; (b) R16F2n/R2 primers: line 1-6. HpP1; HpP7; HpP33; HpS36; HpS14; HpS19; lane 7-12. HbP1; 

HbP4; HbP7; HbS11; HbS15; HbS36; M-Marker: GeneRuler DNA Ladder mix SM0331 (Fermentas, 

Lithuania). 

1 2 3 M 4 5 6 7 8 9 10 11 M 

500 bp 

Figure 4. RFLP analysis of the 1.2-kb PCR product (R16F2n/ R2) digested by AluI (lane 1-6): lane 1. 

control 16SrXII group (STOL); lane 2. control AY phytoplasma; lane 3. control FD-C phytoplasma; 

Marker; lane 1-3: HpS19; HpI22; HbP4; and TruI (lane 7-11): lane 7-8: HpI22; HbP4; lane 9. control 

FD-C phytoplasma; lane 10. control AY phytoplasma; lane 11. control 16SrXII group (STOL); Marker: 

GeneRuler DNA Ladder mix SM0331 (Fermentas, Lithuania). 

and the 16S–23S spacer region sequence of 

representative isolate Hp22 was deposited in the 

GenBank database under accession No. JQ033928. 

DISCUSSION 

The ultra-thin transverse or longitudinal sections of the 

deseased tissues clearly show the presence of typical 

phytoplasma – like bodies of 75 to 300 nm in diameter, 

which corresponds to the known dimensions already 

cited in literature (Franova et al., 2004; Irti et al., 2008; 

Hren et al., 2009). 

According to the results of the RFLP analysis, all 

symptomatic H. perforatum and H. barbatum plants 

showed the same pattern as STOL reference isolate. 

The analysis of partial 16S rRNA gene sequence 

(JQ033928) showed high level of similarities with 16S 

ribosomal RNA gene partial sequences of Bulgarian 

'Rubus fruticosus' phytoplasma Stolbur-Rubus-Bg 

deposited by Bobev and De Jonghe (2011) (under 

accession number JF293091.1), Italian 'Bois noir'


phytoplasma strain CH-1 from periwinkle (HQ589193.1) 

deposited by Kube et al. (2010) and Russian Stolbur-Rus 

phytoplasma strain Rus93 (GU004375.1) deposited by 

Lee et al. (2010). 

Phytoplasmas of the Stolbur group (subgroup 16SrXII- 

A) infect a wide range of vegetable crops. In Serbia, 

Stolbur is a long known pathogen of pepper plant 

(Martinovic and Bjegovic, 1950) and bindweed 

Convolvulus arvensis (Aleksic et al., 1969). The high 

incidence of Stolbur occurred in past decade, mostly 

infecting grapevine (Duduk et al., 2004; Kuzmanovic et 

al., 2008; Josic et al., 2010), corn (Duduk and Bertaccini, 

2006), peach (Duduk et al., 2008) and weed species 

such as Cirsium arvense (Rancic et al., 2005). Recently, 

Stolbur disease was detected in important medicinal 

plants - purple coneflower (Pavlovic et al., 2011) and 

plantain (Josic et al., in press). 

The presence of pathogens has a significant influence 

on the composition of the secondary metabolites in 

plants. A study of Bruni et al. (2005) revealed that H. 

perforatum plants infected with an ash yellows 

phytoplasma (ribosomal group 16SrVII) produced lower 

amounts of essential oil (0.11%) than healthy plants 

(0.75%), and that a higher sesquiterpene and aliphatics 

ratio was observed in the infected plants. 

The chemical variation in plants affected by 

phytoplasmas may decrease their therapeutic efficacy 

and commercial value. The first detection of Stolbur 

phytoplasma in H. perforatum and H. barbatum plants in 

the three locations in Serbia (Pancevo, Stara Pazova and 

Indjija) indicates the need for further investigations in the 

way in which phytopatological conditions affect the quality 

of secondary metabolites in medicinal plants and 

commercial crops. 


We thank Dr Marta Martini (Università di Udine, Italy) for 

kindly providing the AY phytoplasma. This research was 

supported by the Ministry of Education and Science of 

the Repbulic of Serbia, through Projects TR-31018 and 

III46007. 

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DOI: 10.5897/JMPR11.1592 



Insecticidal activity of the essential oil of Lonicera 

japonica flower buds and its main constituent 

compounds against two grain storage insects 

Hai Yan Zhou 1 , Na Na Zhao 2 , Shu Shan Du 3 , Kai Yang 2 , Cheng Fang Wang 3 , Zhi Long Liu 2 and 

Yan Jiang Qiao 1 * 

1 Beijing University of Chinese Medicine, 11 Beisanhuan East Road, Chaoyang District, Beijing 100102, China. 

2 Department of Entomology, China Agricultural University, 2 Yuanmingyuan West Road, Haidian District, Beijing 100193, 

China. 

3 State Key Laboratory of Earth Surface Processes and Resource Ecology, Beijing Normal University, Beijing 100875, 

China. 


The aim of this research was to determine acute toxicity of the essential oil of Lonicera japonica Thunb. 

(Caprifoliaceae) flower buds against the booklouse (Liposcelis bostrychophila Badonnel) and the maize 

weevils (Sitophilus zeamais Motschulsky). Essential oil of L. japonica flower buds was obtained by 

hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS). A total of 25 

components of the essential oil were identified. The principal compounds in the essential oil were 

estragole (80.17%) and linalool (6.05%). The essential oil exhibited strong contact toxicity against S. 

zeamais and L. bostrychophila with LD50 values of 21.54 µg/adult and 64.04 µg/cm 2 , respectively. The 

constituent compounds, estragole (LD50 = 49.95 µg/cm 2 ) and linalool (LD50 = 172.54 µg/cm 2 ) also 

possessed contact toxicity against L. bostrychophila. L. japonica essential oil and its constituent 

compounds (estragole and linalool) exhibited fumigant toxicity against S. zeamais with LC50 values of 

13.36, 14.10 and 10.46 mg/L, respectively. The essential oil of L. japonica (LC50 = 0.20 mg/L) and its 

constituent compounds, estragole (LC50 = 0.16 mg/L) and linalool (LC50 = 0.41 mg/L) possessed fumigant 

toxicity against L. bostrychophila. The results indicated that the essential oil of L. japonica and its 

constituent compounds showed potential in terms of contact and fumigant toxicity against grain 

storage insects. 

Key words: Lonicera japonica, Liposcelis bostrychophila, Sitophilus zeamais, contact toxicity, fumigant, 

essential oil composition, estragole, linalool. 

INTRODUCTION 

The maize weevil (Sitophilus zeamais Motschulsky) is 

one of the major pests of stored grains and grain 

products in the tropics and subtropics (Liu and Ho, 1999). 

Infestations not only cause significant losses due to the 

consumption of grains; they also result in elevated 

temperature and moisture conditions that lead to an 

accelerated growth of molds, including toxigenic species 

(Magan et al., 2003). The booklouse (Liposcelis 

*Corresponding author. E-mail: zhilongliu@cau.edu.cn; 

yjqiao@263.net. Tel: +86-10-62732800. Fax:+86-10-62732800 

bostrychophila Badonnel) is frequently found in storedproduct 

grains, often in extremely high numbers, in 

amylaceous products (Nayak et al., 2005). Currently, 

psocids are perhaps the most important category of 

emerging pests in stored grains and related commodities 

(Turner, 1999). Infestations of stored product insects 

could be controlled by fumigation or insecticidal treatment 

of commodities and surfaces, which has led to problems 

such as disturbances of the environment, increasing 

costs of application, pest resurgence, pest resistance to 

pesticides and lethal effects on non-target organisms in 

addition to direct toxicity to users (Zettler and Arthur, 

2000).

Essential oils or their constituents may provide an 

alternative to currently used fumigants/pesticides to 

control stored-food insects (Isman, 2000). Investigations 

in several countries confirm that some plant essential oils 

not only repel insects, but possess contact and fumigant 

toxicity against stored product pests as well as exhibited 

feeding inhibition or harmful effects on the reproductive 

system of insects (Isman, 2006). The toxicity of a large 

number of essential oils and their constituents has been 

evaluated against a number of stored-product insects 

(Rajendran and Srianjini, 2008). 

Botanical pesticides have the advantage of providing 

novel modes of action against insects that can reduce the 

risk of cross-resistance as well as offering new leads for 

design of target-specific molecules (Isman, 2006). During 

the screening program for new agrochemicals from 

Chinese medicinal herbs, the essential oil of Lonicera 

japonica Thunb. (Family: Caprifoliaceae) flower buds was 

found to possess strong insecticidal toxicity against the 

two grain storage insects, S. zeamais and L. 

bostrychophila. 

L. japonica is a medicinal plant widely used in China. 

As a traditional Chinese medicine with a wide spectrum 

of biological and pharmacological properties, the dried 

flowers of L. japonica have been used in clinical practice 

for thousands of years for their antibacterial, antiviral and 

antioxidant activities and in the treatment of 

exopathogenic wind-heat, epidemic febrile diseases, 

sores, carbuncles and furuncles (Jiangsu New Medical 

College, 1977; Peng et al., 2000). The essential oil and 

chlorogenic acid are reported to be effective components 

of L. japonica (Li et al., 2006). The essential oil of L. 

japonica is an edible natural perfume that is often used in 

foods, cigarettes and cosmetics (Schlotzhauer et al., 

1996, Shang et al., 2011). Previous phytochemical 

studies on L. japonica resulted in the identification of 

several saponins, triterpenoid saponins, cerebrosides, 

caffeoylquinic acids and esters, flavoids, alkaloids and 

iridoid glucosides (Kawai et al., 1988a, b; Son et al., 

1992; 1994; Kakuda et al. 2000; Teng et al., 2000; Kumar 

et al., 2005; 2006; Lin et al., 2008; et al., 2008). 

The chemical composition of L. japonica essential oil 

was also studied previously (Ikeda et al., 1994; 

Schlotzhauer et al., 1996; Rahman and Kang, 2009; 

wang et al., 2009). The methanol extract of leaves and 

twigs of L. japonica possessed insecticidal and acaricidal 

activity against the cotton aphid (Aphis gossypii), the 

green peach aphid (Myzus persicae), the greenhouse 

whitefly (Trialeurodes vaporariorum), the two-spotted 

spider mite (Tetranychus urticae), and the citrus red mite 

(Panonychus citri) (Kim et al., 2005) while repellency of 

the n-hexane, ethyl acetate, n-butanol and water extracts 

of L. japonica were observed against the Asian tiger 

mosquito, Aedes albopitus (Yoon and Kyung, 2002). 

However, insecticidal activity of L. japonica essential oil 

against grain storage insects was not determined. This 

study analyses the chemical composition and toxicity of 

Zhou et al. 913 

essential oil of L. japonica flower buds against the two 

grain storage insects. 



Three kilogram of fresh flower buds of L. japonica were collected 

from Pingyi Country (35.35° N latitude and 117.37° E longitude), 

Shangdong Province, China in May 2011. The samples were airdried 

and identified by Dr. Liu, Q.R. (College of Life Sciences, 

Beijing Normal University, Beijing 100875, China) and a voucher 

specimen ((BNU-DuShushan-Jinyinghua-shangdong-2011-05) was 

deposited in the State Key Laboratory of Earth Surface Processes 

and Resource Ecology, Beijing Normal University. The samples 

were ground to a powder using a grinding mill (Retsch Mühle, 

Germany). Each 600 g portion of powder was mixed in 1,800 ml of 

distilled water and soaked for 3 h. The mixture was then boiled in a 

round-bottom flask, and steam distilled for 6 to 8 h. Volatile 

essential oil from distillation was collected in a flask. Separation of 

the essential oil from the aqueous layer was done in a separatory 

funnel, using the non-polar solvent, n-hexane. The solvent was 

evaporated using a vacuum rotary evaporator (BUCHI Rotavapor 

R-124, Switzerland). The sample was dried over anhydrous Na2SO4 

and kept in a refrigerator (4°C) for subsequent experimen ts. 

Estragole (98%) and linalool (98%) were purchased from Aladdin- 

Reagent (China) Co. (Shanghai 201206, China). 

Insects 

The maize weevils (S. zeamais) and the booklouse, L. 

bostrychophila were obtained from laboratory cultures in the dark in 

incubators at 29 to 30°C and 70 to 80% relative humid ity. The 

maize weevils were reared on whole wheat at 12 to 13% moisture 

content in glass jars (diameter 85 mm, height 130 mm) and the 

booklouse were reared on a 1: 1: 1 mixture, by mass, of milk 

powder, active yeast, and flour. Unsexed adult weevils and 

booklous used in all the experiments were about one week old. All 

containers housing insects and the petri dishes used in experiments 

were made escape proof with a coating of polytetrafluoroethylene 

(Fluon, Blades Biological, UK). 

Gas chromatography-mass spectrometry (GC-MS) 

The essential oil of L. japonica was subjected to GC-MS analysis 

on an Agilent system consisting of a model 6890N gas 

chromatograph, a model 5973 N mass selective detector (EIMS, 

electron energy, 70 eV), and an Agilent ChemStation data system. 

The GC column was an HP-5 ms fused silica capillary with a 5% 

phenyl-methylpolysiloxane stationary phase, film thickness of 0.25 

µm, a length of 30 m, and an internal diameter of 0.25 mm. The GC 

settings were as follows: the initial oven temperature was held at 

60°C for 1 min and ramped at 10°C min −1 to 180°C held for 1 min, 

and then ramped at 20°C min −1 to 280°C and held for 15 min. The 

injector temperature was maintained at 270°C. The sa mple (1 µl) 

was injected neat, with a split ratio of 1: 10. The carrier gas was 

helium at flow rate of 1.0 mL min −1 . Spectra were scanned from 20 

to 550 m/z at 2 scans s −1 . Most constituents were identified by gas 

chromatography by comparison of their retention indices with those 

of the literature (Ikeda et al., 1994; Schlotzhauer et al., 1996; 

Rahman and Kang, 2009) or with those of authentic compounds 

available in our laboratories. The retention indices were determined 

in relation to a homologous series of n-alkanes (C8–C24) under the 

same operating conditions.


Further identification was made by comparison of their mass 

spectra with those stored in NIST 08 and Wiley 275 libraries or with 

mass spectra from literature (Adams, 2007). Component relative 

percentages were calculated based on normalization method 

without using correction factors. 

Contact toxicity by topical application 

Range-finding studies were run to determine the appropriate testing 

concentrations of the essential oil of L. japonica flower buds. A 

serial dilution of the essential oil was prepared in n-hexane. 

Aliquots of 0.5 µl per insect were topically applied dorsally to the 

thorax of the weevils, using a Burkard Arnold microapplicator. 

Controls were determined using 0.5 µl n-hexane per insect. Ten 

insects were used for each concentration and control, and the 

experiment was replicated six times. Both the treated and control 

weevils were then transferred to glass vials (10 insects/vial) with 

culture media and kept in incubators at 29 to 30°C a nd 70 to 80% 

relative humidity. Mortality was observed after 24 h. Results from all 

replicates were subjected to probit analysis using the PriProbit 

Program V1.6.3 to determine LD50 values (Sakuma, 1998). 

Contact toxicity by filter paper impregnation 


concentrations of L. japonica flower buds essential oil and the 

constituent compounds. A 3.5 cm diameter filter paper was treated 

with 150 µl of the solution of the essential oil/compounds. The filter 

paper after treated with solid glue (Glue Stick, Jong Ie Nara Co., 

Ltd. Hong Kong) was placed in a 3.5 cm diameter petri dish and 10 

booklice were put on the filter paper. A cover was put and all the 

peteri dishes were kept in incubators at 27 to 29°C, 70 to 80% 

relative humidity. Acetone was used as a negative control and 

pyrethrum extract was used as a positive control. Five 

concentrations (in acetone) and five replicates of each 

concentration were used. Mortality of insects was observed after 24 

h and results from all replicates were subjected to probit analysis 

using the PriProbit Program V1.6.3 to determine LC50 values 

(Sakuma, 1998). Pyrethrum extract (25% pyrethrine I and 

pyrethrine II) was purchased from Fluka Chemie. 

Fumigant toxicity bioassay 


concentrations of the pure compounds and L. japonica flower buds 

essential oil. A filter paper strip (3.5 ´ 1.5 cm) treated with 10 µl of 

an appropriate concentration of the test essential oil/compounds. 

The impregnated filter paper was then placed in the bottom cover of 

a 250 ml volume of glass bottle. Ten unsexed adults of the 

booklouse in a small glass bottle (8 ml) were put into the glass 

bottle and exposed for 24 h. Five concentrations of the 

oil/compounds were used in the experiments and each 

concentration with five replicates. Acetone was used as a negative 

control and dichlorvos was used as a positive control. The LC50 

values were calculated by using Probit analysis (Sakuma, 1998). 

Dichlorvos (99.9%) was purchased from Aladdin-reagent Co. 

(Shanghai, China). 

The fumigant toxicity of L. japonica flower buds essential 

oil/constituent compounds against the maize weevils was 

determined by used the method of Liu and Ho (1999) with some 

modifications. A Whatman filter paper (diameter 2.0 cm) was placed 

on the underside of the screw cap of a glass vial (diameter 2.5 cm, 

height 5.5 cm, volume 24 ml). Ten microliters of the essential oil/ 

compounds was added to the filter paper. The solvent was allowed 

to evaporate for 15 s before the cap was placed tightly on the glass 

vial (with 10 unsexed insects) to form a sealed chamber. They were 

incubated at 27 to 29°C and 70 to 80% relative humid ity for 24 h. 

Mortality of insects was observed and results from all replicates 

were subjected to probit analysis using the PriProbit Program 

V1.6.3 to determine LC50 values (Sakuma, 1998). 


The yellow essential oil yield of L. japonica flower buds 

was 0.07% (V/W) and the density of the concentrated 

essential oil was determined to be 0.96 g/ml. A total of 25 

components of the essential oil were identified, 

accounting for 94.35% of the total oil. The principal 

compounds in L. japonica flower buds essential oil were 

estragole (80.17%) and linalool (6.05%) followed by 

germacrene D (3.17%) (Table 1). Monoterpenoids 

represented 10 of the 25 compounds, corresponding to 

82.62% of the whole oil while also 10 of the 25 

constituents were sesquiterpenoids (5.88% of the crude 

essential oil). The result is quite different from the 

previous report. For example, the major compounds 

detected in the oil of L. japonica floral parts harvested 

from Korean were trans-nerolidol (16.31%), 

caryophyllene oxide (11.15%), linalool (8.61%), ρcymene 

(7.43%), hexadecanoic acid (6.39%), eugenol 

(6.13%), and geraniol (5.01%) (Rahman and Kang, 

2009). 

Twenty-seven compounds were identified among the 

three developmental stages of L. japonica flowers. 

Germacrene D was a major component at all stages; 

linalool and α-farnesene appeared in high concentrations 

in fresh and 24 h flowers but were greatly reduced in 

overnight flowers (Schlotzhauer et al., 1996). Moreover, 

Wang et al. (2009) demonstrated that the primary 

components of the volatile oil were linalool (0.15 to 

15.35%), linalool oxide (0.09 to 6.53%), geraniol (0.24 to 

8.17%) and α-terpineol (0 to 10.57%) and obvious 

variations in components of the volatile oil were observed 

at six different developmental stages. The foregoing 

findings suggest that there are great variations in 

chemical composition of the essential oil of different 

population and even of different development stage of the 

same population. Further studies on plant cultivation and 

essential oil standardization are needed. 

The essential oil of L. japonica flower buds exhibited 

contact toxicity against S. zeamais adults with a LD50 

value of 21.54 mg/adult (Table 2). When compared with 

the positive control pyrethrum extract, the essential oil 

demonstrated 5 times less toxic against S. zeamais. L. 

japonica essential oil also possessed contact toxicity 

(LD50 = 64.04 mg/cm 2 ) against the booklouse and the two 

constituent compounds, estragole and linalool acting 

against the booklouse with LD50 values of 49.95 and 

172.54 mg/cm 2 , respectively (Table 3). When compared 

with the positive control, pyrethrum extract, the essential 

oil and its constituent compounds, estragole and linalool, 

showed 3, 2.5 and 9 times less active against the 

booklouse (Table 3). However, compared with the other

Table 1. Chemical constituents of essential oil derived from Lonicera japonica. 

Compounds RI* Peak area (%) 

1-Octen-3-ol 978 0.04 

β-Pinene 981 0.03 

(d)-Limonene 1029 0.17 

1, 8-Cineole 1032 0.31 

Benzyl alcohol 1034 0.08 

Acetophenone 1065 0.34 

Linalool 1094 6.05 

Phenylethyl alcohol 1116 0.25 

α-Terpineol 1189 0.12 

Estragole 1197 80.17 

Linalool acetate 1248 0.28 

(Z)-β-Damascenone 1347 0.16 

α-Cubebene 1350 0.12 

Eugenol 1361 0.23 

β-Geranyl acetate 1379 0.12 

β-Caryophyllene 1420 0.25 

β-Gurjenene 1435 0.36 

Germacrene D 1480 3.17 

α-Selinene 1492 0.31 

α-Farnesene 1508 1.01 

β-Cadinene 1519 0.12 

Elemol 1551 0.22 

Caryophyllene oxide 1583 0.15 

α-Cadinol 1654 0.11 

Junipher camphor 1695 0.18 

Total 94.35 

Monoterpenoids 87.62 

Sesquiterpenoids 5.88 

Others 0.85 

*RI, retention index as determined on a HP-5MS column using the homologous series of n-hydrocarbons. 


Table 2. Contact toxicity of Lonicera japonica essential oil and its main constituent compounds against Sitophilus zeamais adults. 

Treatment LD50 (μg/adult) 95% FL Slope ± SE Chi square (χ 2 ) 

L. japonica 21.54 19.56-23.42 4.11 ± 0.42 20.72 

Estragole* 17.63 15.56-19.97 3.09 ± 0.32 12.04 

Linalool* 13.90 13.05-14.83 5.86 ± 0.55 9.80 

Pyrethrum extract* 4.29 3.86-4.72 - - 

* from Wang et al. (2011). 

essential oils in the literature, the essential oil of L. 

japonica possessed stronger contact toxicity against S. 

zeamais adults, for example, essential oils of Artemisia 

lavandulaefolia, Artemisia sieversiana, Artemisia 

capillaries, Artemisia mongolica, and Artemisia vestita 

(LD50 = 55.2, 113.0 106.0, 87.9, and 50.6 µg/adult, 

respectively) (Liu et al. 2010a, 2010b; Chu et al., 2010a), 

essential oil of Schizonpeta multifida (30.2 µg/adult) (Liu 

et al. 2011), essential oil of Illicium simonsii fruits (LD50 = 

112.7 µg/adult) (Chu et al., 2010b). 

The essential oil of L. japonica and its constituent 

compounds, estragole and linalool exhibited fumigant 

toxicity against the maize weevils with LC50 values of 

13.36, 14.10 and 10.46 mg/L, respectively (Table 4). The 

commercial grain fumigant, methyl bromide (MeBr) was 

reported to have fumigant activity against S. zeamais adults 

with a LC50 value of 0.67 mg/L (Liu and Ho, 1999), thus the 

essential oil and its constituent compounds were 15 to 21 

times less toxic to S. zeamais adults compared with 

MeBr. Moreover, the essential oil of L. japonica (LC50 = 

0.20 mg/L) and its constituent compounds, estragole 

(LC50 = 0.16 mg/L) and linalool (LC50 = 0.41 mg/L) 

possessed fumigant toxicity against the booklouse. 

Compared with the positive control, dichlorvos (LC50 =


Table 3. Contact toxicity of Lonicera japonica essential oil and its main constituent compounds against Liposcelis bostrychophila. 

Treatment LD50 (μg/cm 2 ) 95% FL Slope ± SE Chi square (χ 2 ) 

L. japonica 64.04 60.44-69.85 14.98 ± 1.42 27.42 

Estragole 49.95 48.04-52.14 19.65 ± 2.01 30.36 

Linalool 172.54 157.71-186.94 27.86 ± 2.53 9.43 

Pyrethrum extract 18.99 17.56-20.06 7.64 ± 1.05 22.96 

Table 4. Fumigant toxicity of essential oil of Lonicera japonica and its main constituent compounds against Sitophilus zeamais (SZ) and 

Liposcelis bostrychophila (LB) adults. 

Insects Treatment LC50 (mg/L air) 95% FL Slope ± SE Chi square (χ 2 ) 

SZ 

LB 

L. japonica 13.36 11.63-14.96 2.44 ± 0.30 21.23 

Estragole* 14.10 12.45-15.97 3.09 ± 0.32 12.04 

Linalool* 10.46 9.58-11.55 4.20 ± 0.47 10.36 

MeBr** 0.67 - - - 

L. japonica 0.20 0.18-0.22 4.97 ± 0.46 33.59 

Estragole 0.16 0.13-0.19 5.01 ± 0.45 67.39 

Linalool 0.41 0.37-0.43 8.39 ± 0.93 22.54 

Dichlorvos 1.35×10 -3 1.25×10 -3 -1.40×10 -3 6.87 ± 0.77 19.78 

* From Wang et al. (2011). ** From Liu and Ho (1999). 

1.35 µg/L), the essential oil and its constituent 

compounds show 100 to 300 times less fumigant toxicity 

against the booklouse. However, by considering the 

currently used fumigants are synthetic insecticides, the 

foregoing findings suggest that fumigant activity of the 

essential oil of L. japonica and its constituent compounds 

are quite promising and they show potential to be 

developed as possible natural fumigants/insecticides for 

control of stored product insects. For the practical 

application of the essential oil and its constituent 

compounds as novel insecticides/fumigants, further 

studies on the safety of the essential oil/compounds to 

humans and on development of formulations are 

necessary to improve the efficacy and stability and to 

reduce cost. 

Conclusion 

The composition of the essential oil derived from L. 

japonica flower buds was determined by GC-FID and 

GC-MS. The essential oil and its constituent compounds 

exhibited strong contact and fumigant toxicity against the 

two grain storage insects. 

These findings suggest that the essential oil of L. 

japonica flower buds and its constituent compounds 

possess potential for development as novel natural 

insecticides/fumigants for stored products. Further 

studies are required to evaluate the safety of the 

essential oil/compounds to humans. 


This work was funded by the Hi-Tech Research and 

Development of China 2011AA10A202, 2006AA10A209 

and National New-drug Innovation Project 2009ZX09501- 

014. The authors thank Dr. QR Liu from College of Life 

Sciences, Beijing Normal University, Beijing 100875, for 

the identification of the investigated plant. 

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aJournal of Medicinal Plants Research Vol. 6(5), pp. 918-925, 9 February, 2012 


DOI: 10.5897/JMPR11.1684 



Research on herbal combinations of traditional Chinese 

medicine for chronic gastritis based on network biology 

Peng Lu 1# , Qingqiong Deng 2# , Chenghe Shi 3# , Yibao Gao 1 , Jianqiang Yi 1 , Mingquan Zhou 2 , 

Yiping Yang 1 and Yuhao Zhao 4 * 

1 Institute of Automation, Chinese Academy of Sciences, Beijing 100190, P. R. China. 

2 College of Information Science and Technology, Beijing Normal University, No. 19 Xin-Jie-Kou-Wai Street, 

Beijing 100875, China. 

3 Department of Traditional Chinese Medicine, Peking University Third Hospital, Beijing 100191, P. R. China. 

4 School of Traditional Chinese, Medicine, Capital Medical University, 100069, Beijing, P. R. China. 


Herbal combinations are important for traditional Chinese medicine physicians to treat diseases. Based 

on Professor GAO Zhongying’s medical records, combination laws are explored in order to study and 

inherit Professor GAO’s academic thoughts and improve the level of clinical treatment from multiple 

perspectives in treating chronic gastritis. Based on an entropy clustering method of complex systems, 

Professor GAO’s formula data is mined to draw the combinations which have good effects on the 

treatment of chronic gastritis. Meanwhile, the software Pajek developed for complex network was 

applied in the process. It not only provides a group of fast and efficient algorithms for to analyze 

complex networks, but also presents a visual interface to facilitate the understanding on the structural 

characteristics of complex networks from a visual point of view. Through analyzing the commonness of 

Professor GAO’s formulas, we found the compatibility structure that reflected formula thinking and core 

clinical features supported the arrangement of Professor GAO’s experiences. Through the procedure 

mentioned earlier, we analyzed and screened 730 formulas in the database and found 30 herbs most 

frequently used in the treatment of chronic gastritis. By applying the measure of modified mutual 

information, we got 94 commonly-used herbs with correlation coefficients above 0.05 and through the 

entropy clustering method of complex systems, we found 11 core combinations. The entropy clustering 

method of complex systems was used to build the association among 80 herbs commonly used to treat 

chronic gastritis, and then 122 associations were obtained. We draw out the complex network graph of 

herbs commonly used for chronic gastritis. These results are completely in line with clinical practices, 

and they are essentially the commonly used herbs employed by Professor GAO Zhongying for chronic 

gastritis. 

Key words: Herbal combination, chronic gastritis, entropy clustering of complex systems, complex network. 

INTRODUCTION 

Chronic gastritis is caused by different chronic 

inflammations of gastric mucosa or atrophic lesions. 

Besides, chronic gastritis is one of the most common is 

usually divided into chronic superficial gastritis (CSG) 

*Corresponding author. E-mail: yuhao.zhao2011@gmail.com. 

# These authors contributed equally to this work. 

and chronic atrophic gastritis (CAG). CSG is caused by a 

digestive diseases; its incidence rate is the highest 

among the various types of stomach diseases, to account 

about 80 to 90% of gastroscopy patients. Chronic gastritis 

variety of chronic inflammation of gastric mucosa 

superficial, more abdominal pain, fullness after eating, 

loss of appetite and belching and other bits main 

symptoms. Unfortunately, there is no effective therapy for 

CSG in modern medicine. 

According to the taxonomy in Traditional Chinese

Table 1. Distributions of age and gender. 

Type Frequency Male Female Age 

CSG 416 169 247 45.25±7.8 

CAG 314 130 184 48.50±8.6 

Medicine, CSG belongs to stomach swelling of the liver, 

stomach category in Traditional Chinese Medicine. On 

the other hand, CAG is a gastric glands atrophy, mucosal 

thinning, or with intestinal metaplasia, dysplasia of the 

pathological features of Digestive Disease Branch of 

Chinese Medical Association (2006). In western 

medicine, there exist many treatments including the 

general, Helicobacter pylori (Hp) eradication, mucosal 

protective agent, acid, acid-suppressing agents, 

prokinetic agent therapies and so on for chronic gastritis. 

The treatment method for Hp-related gastritis has a firstline 

therapy, second-line treatment, re-called remedial 

treatment, and sequential therapy. 

In Traditional Chinese Medicine, treatment of chronic 

gastritis achieves a good effect. However herbal 

combination laws play an essential role in the process of 

therapy. Therefore, the current research on chronic 

gastritis is focused to select the appropriate methodology 

to discover the herbal combination laws. Complex 

network is one of the methods of data mining which 

discovers the potential value of the relationship between 

decision-making, patterns and trends from a large 

amount of implicit, previously unknown data (Chen et al., 

2007). In many cases, data mining is essential to 

accidentally discovery unexpected and valuable 

knowledge. 

In recent years, complex network is in the stage of 

development, and models of complex network have been 

applied in many fields of natural and social sciences. 

Complex network can be used to describe the social 

relations among people, the prey species relationships, 

the topological structures of computer network, semantic 

relations of words, cooperative relationships of scientists, 

interactive relations of protein effect, reference 

relationships of scientific research articles, link relations 

of webs, and so on. 

In conclusion, complex network is used widely from 

World Wide Web and social network to the food chain 

and neural system of organisms. Now complex network 

of qualitative and quantitative study of science has 

become a major trend. Although, the types of complex 

network are vastly different, we can use a common model 

called graph theory to characterize their shared feature. 

Compared with the complexity of general graph, complex 

network has a large number of nodes and edges. 

Therefore, effective and efficient software is required to 

analyze and simulate complex networks. Pajek can be 

used for realizing the functions of complex network 

(Batagelj and Mrvar, 2003). And Pajek, which means 

Lu et al. 919 

spider in Slavic, not only provides a group of fast and 

efficient algorithms for analysis of complex networks, but 

also provides a visual interface that can be more intuitive 

from a visual point of view to understand the structural 

characteristics of complex networks. Pajek has a quick 

calculation, simple visualization and abstract features 

(Andrew, 2008; Shi et al., 2010). Professor GAO 

Zhongying has 58 years of experiences in clinical 

medicine, teaching and research work and especially has 

contributed to Chinese medicines which treat chronic 

gastritis. 

In order to study and inherit Professor GAO’s academic 

thoughts, and improve the level of clinical treatment, his 

clinical medication laws are summarized and investigated 

in treating chronic gastritis from multiple perspectives. By 

means of entropy clustering method of complex systems 

and complex network, Professor Gao’s formula data is 

mined, and afterwards we explored the laws of herbal 

combination in formulas which have good effects in the 

treatment of chronic gastritis. 


Clinical data 

All the medical records of chronic gastritis were collected from 

Professor GAO Zhongying’s recipes in the Dashanlan Clinic and the 

Dongshi Tenth Road Clinic of Beijing Tong Ren Tang, and the 

Famous Physician Clinic of Gulou Traditional Chinese Medicine 

(TCM) Hospital from March 2008 to July 2009. Totally, 730 records 

were included and all the cases meet the diagnostic criteria for 

chronic gastritis stipulated in the Guiding Principles of Clinical 

Studies of New Chinese Drugs in 2002. All the patients were 

diagnosed as chronic gastritis by measures of endoscopy or biopsy 

in first class hospitals. In totally, there are 416 chronic superficial 

gastritis cases and 314 chronic atrophic gastritis cases. The 

distributions of age and gender are shown in Table 1. 

The construction of prescription database 

It is necessary to standardize prescription information in order to 

meet the requirements of data mining. First, the medical named 

entities of symptoms, signs and checked terms are unified to create 

the related term specification. Secondly, the terms of diagnosis, 

syndromes, and therapies are standardized by means of unifying, 

summarizing and splitting into relatively independent evidences of 

the meaning of elements in accordance with unified textbooks and 

finally the related database is created. Thirdly, herbal names are 

unified by the way of summarizing their categories, functions, 

natures, flavors and channel tropisms according to the current 21st 

century curriculum materials. Based on Access database, the 

structural medical record template is built for Professor GAO


Zhongying clinical diagnosis. The patient information was written 

into the database in accordance with national standard format. 

Detailed records of 730 patients were enrolled into Professor GAO 

Zhongying medical records database in order to discover the 

knowledge based on entropy clustering method of complex systems 

and complex network. 

Method of data mining 

Entropy clustering of complex systems is proposed to mining valued 

knowledge from formulas. Formula laws can be drawn from the 

three aspects. The first aspect is referred to the single herb, and it 

was mainly achieved through the frequency method; the second 

aspect is referred to the analysis on couplet herbs, mainly through 

mutual information and association rules, etc., to discuss the 

commonly used couplet herbs for spleen and stomach diseases; 

and the last aspect is referred to the combination laws of many 

herbs, which were discussed through the entropy clustering of 

complex systems. These three aspects mutually supplemented and 

corresponded with each other, revealing Professor GAO 

Zhongying’s formula laws together (Shi et al., 2010). The formula 

database is mined from the multi-levels and multi-dimensions 

perspectives so that single herb, couplet herbs and many complex 

herbs are picked up. Compared with the traditional mutual 

information, the modified mutual information can differentiate 

positive correlation and negative correlation, and express more 

positive correlation between couplet herbs. 

The core thought is to relatively punish couplet herbs occurred in 

formulas. Thus, the information, that the original positive couplet 

herbs are not lost and the negative couplet herbs are deleted, 

ensures the efficacy of mining. The principle of modified mutual 

information is as follows: 

Suppose n X 

herbs are expressed with 1,X 2, �Xi�Xnin which i X is expressed by 

1 2 j m 

Xi � x i, x i , �x i, �, 

xi 

� � 

. And the probability density 

functions of herbal i X are, respectively 

1 2 j m 

p(x i ),p(x i ), �p(x i ), �,p(x 

i ) 

, the joint probability 

distribution density functions of i X X j 

and are, 

respectively 

1 1 2 2 k k m m 

p(x i,x j),p(x i ,x j ), �p(x i ,x j ), �,p(x 

i ,x j ) 

(respectively). Shannon entropy of 

shown as formula (1): 

m 

i X is 

� � 

k k 

� � � � 

H( X ) � �p 

x log p x 

i i i 

k�1 

The joint entropy is expressed as formula (2): 

m m 

� � 

k l k l 

� � � � 

H(X ,X ) � ��p 

x , x log p x , x 

i j i j i j 

l�1k�1 The mutual information between and is defined as formula (3): 

(1) 

(2) 

� � � � � � 

I(X ,X ) � H X � H X � H X ,X 

i j i j i j 

This formula can be equal to formula (4) 

� � � � � � 

� i � � i j � 

I(X ,X ) � H X � H X � H X ,X 

i j i j i j 

� H X �HX 

| X 

Formula (4) depicts the correlations between two herbs, but it could 

not distinguish positive correlation and negative correlation. 

Therefore, modified mutual information has been proposed to 

resolve this problem. Positive-correlative herbs or negativecorrelative 

herbs can be differentiated by using the positive 

occurrence rate (Chen et al., 2007). The positive occurrence rate is 

referred to the probability when two variables are 0 at the same 

time. The positive occurrence rate between positive-correlative 

herbs is very large, while the occurrence rate between negativecorrelative 

herbs should be 0 theoretically, that is, it is impossible 

that there are two negative-correlative herbs in a formula at the 

same time. So, the definition of mutual information and correlation 

coefficient are anew expressed as: 

' 

��( Xi, X j) 

�� 

� H ( X i ) �H( X j ) �H( 

X i, X j ) 

� 

Po( i, j) 

� � 

� H( X j ) 

� H ( X i ) �H( X j ) �2 

H ( X i, X j ) 

Po( i, j) 

� � 

� 

� H( X j ) 

(5) 

Based on entropy clustering of complex systems, correlation 

coefficients of herbs are computed by the way of the modified 

mutual information. And the modified mutual information can define 

positive-correlative herbs that belong to the convergence of the 

proposed clustering principle to summarize it three-three relevant 

from two-two positive correlation. The clustering is performed with a 

fast convergence while the number of classes is not set. 

Construction of Chinese herbal complex network 

Chinese herbal complex network is constructed by Pajek. This 

software not only provides a group of fast and efficient algorithms 

for analysis of complex networks, but also presents a visual 

interface to facilitate the understanding on the structural 

characteristics of complex networks from a visual point of view. 

Through the analysis on the commonness of Professor GAO 

Zhongying’s formulas, we found the compatibility structure that 

reflected formula thoughts and core clinical features, thus 

supporting the arrangement of Professor GAO Zhongying’s 

experiences. Complex system entropy clustering method is used to 

build 80 associations between herbs of Chinese medicine treatment 

of chronic gastritis and 122 associations are achieved. These data 

are transformed to the adjacency matrix and this matrix is converted 

into Pajek format required. Node degrees and edge weights are 

computed separately by using Pajek 2.0. Node degree is a most 

simple but most important property in the complex network node 

properties. The degree of a node is defined as the number of nodes 

connected to it. Therefore, from an intuitive point of view, the 

degree of a node indicates that the node is more important. Using 

Pajek 2.0 Software Layout-Energy-Kamada-Kawai-Separate- 

Compinents command, the different types of nodes map can be 

drawn out, combined with manual operation of the node to mediate. 

RESULTS 

Through analyzing the screened 730 prescriptions from 

(4) 

(3)

Table 2. The frequency of herbs. 

The herb frequency (sorted index, herb) 

Index Herb, frequency 

1 OS swpiae (OS SEPIAE), 355 

2 Trichosanthes, 347 

3 Pseudostellaria heterophylla, 324 

4 Thunberg fritillary, 300 

5 Largehead Atractylodes Rh, 296 

6 Fermented Pinellia, 244 

7 Rhizoma Zedoariae, 201 

8 Gallus gallus domesticus Brisson, 196 

9 Amomum villosum, 144 

10 Semen Raphani, 142 

11 Root of Herbaceous Peony, 126 

12 Rhizoma Coptidis, 125 

13 Medicinal Evodia Fruit, 116 

14 RHIZOMA ZINGBERIS PREPARATA, 94 

15 Endothelium Corneum Gigeriae Galli, 80 

16 Radix codonopsis,76 

17 Semen Arecae Prepareta, 74 

18 Angelica, 71 

19 Fructus Aurantii Immaturus, 67 

20 Ophiopogon japonicus, 61 

21 Immature Trifoliate-orange Fruit, 60 

22 Cortex Magnoliae Officinalis, 59 

23 Bulbus fritillariae cirrhosae, 57 

24 Elecampane, 52 

25 Salvia Miltiorrhiza, 51 

26 Angelica dahurica, 47 

27 Baikal Skullcap Root, 47 

28 Dioscorea opposita, 44 

29 Radix Curcumae, 44 

30 Rehmanniae(raw), 38 

database, 30 most frequent herbs are use for chronic 

gastritis after 730 prescriptions were analyzed to screen 

the database. 30 herbs are shown in Table 2. Based on 

the modified mutual information, 94 common herbal pairs 

are obtained and their correlation coefficients are above 

0.05. Results are shown in Table 3. 11 core herbal 

combinations are achieved by using complex system 

entropy clustering methods, and these results are shown 

in Table 4. Based on the results of entropy clustering, 

herbal complex network is constructed by Pajek. It is 

shown is Figure 1. 

DISCUSSION 

Results of entropy clustering of complex systems 

Formula in TCM was found to be effective in treating 

disease (Jianxin et al., 2011; Jianxin et al., 2011). The 

Lu et al. 921 

mining of its herbal combination rules play a key role in 

understanding its action mechanism. Traditional statistics 

and data mining methods are hardly used to make a 

distinctions between positive correlation and negative 

correlation because Chinese medicine prescriptions often 

have the characteristics of high dispersion degree and 

nonlinear and so on (Selecting Biomarkers for Primary 

Hyperlipidemia and Unstable Angina in the Context of 

Neuro-endocring-immune Network by Feature Selection 

Methods, 2010). The complex system entropy clustering 

method is proposed to use to be appropriate for data 

characteristics of TCM clinical four diagnostic methods 

and Chinese medicine prescriptions. It is a kind of 

unsupervised data mining methods and accords with the 

non-linear relation among the graded variable such as 

four diagnostic information. And the variables are 

clustered by self-organization, and the variable number in 

each cluster is automatically determined. Through the 

methods mentioned above, we analyzed and screened


Table 3. Couple-herb and its correlation coefficient. 

Index Couple-herb, mutual information 

1 Rhizoma Coptidis, Medicinal Evodia Fruit, 0.64031 

2 OS swpiae (OS SEPIAE), Thunberg fritillary, 0.19216 

3 Endothelium Corneum Gigeriae Galli, Radix Trichosanthis, 0.17318 

4 Trichosanthes,OS swpiae (OS SEPIAE), 0.14874 

5 Tortoise Shell, Drgonsbones, 0.14749 

6 Tortoise Shell, Oyster, 0.14749 

7 Lignum Dalbergiae Odoriferae, Kaempferia galanga, 0.13394 

8 Semen Arecae Prepareta, elecampane, 0.13394 

9 RHIZOMA ZINGBERIS PREPARATA, Amomum villosum, 0.12569 

10 Largehead Atractylodes Rh, Rehmanniae (raw), 0.11946 

11 Largehead Atractylodes Rh, Baikal Skullcap Root, 0.11214 

12 Milkvetch root, Pseudostellaria heterophylla, 0.11102 

13 Rehmanniae (raw), Rehmanniae ( prepared ), 0.10788 

14 Rehmanniae (raw), Baikal Skullcap Root, 0.1005 

15 Angelica, Endothelium Corneum Gigeriae Galli, 0.096696 

16 Angelica, angelica dahurica, 0.096696 

Table 4. The core herbal combinations. 

Index Herbal combinations 

1 Pseudostellaria heterophylla, Largehead Atractylodes Rh, OS swpiae (OS SEPIAE), 

Thunberg fritillary and Trichosanthes 

2 Pseudostellaria heterophylla, Largehead Atractylodes Rh, Endothelium Corneum Gigeriae 

Galli, Trichosanthes and Thunberg fritillary 

3 OS swpiae (OS SEPIAE), Thunberg fritillary, Rhizoma Coptidis and Medicinal Evodia Fruit 

4 Angelica dahurica, Lignum Dalbergiae Odoriferae, elecampane and Kaempferia galanga 

5 Endothelium Corneum Gigeriae Galli, OS swpiae (OS SEPIAE), Rhizoma Coptidis and 

Medicinal Evodia Fruit 

6 Trichosanthes, OS swpiae (OS SEPIAE),Rhizoma Coptidis and Medicinal Evodia Fruit 

7 Thunberg fritillary, OS swpiae (OS SEPIAE) and Trichosanthes 

8 Largehead Atractylodes Rh, Rhizoma Zedoariae and Pseudostellaria heterophylla 

9 Largehead Atractylodes Rh, Baikal Skullcap Root and Pseudostellaria heterophylla 

10 Fermented Pinellia, OS swpiae (OS SEPIAE) and Trichosanthes 

11 Endothelium Corneum Gigeriae Galli, OS swpiae (OS SEPIAE) and Trichosanthes 

730 formulas in the database, and found most frequent 

30 herbs used in the treatment of chronic gastritis. By 

applying the method of modified mutual information, we 

got 94 commonly-used herbs with correlation coefficients 

of above 0.05; and through entropy clustering method of 

complex systems, we found 11 core combinations. 

Entropy clustering method of complex systems was 

used to build the associations among 80 herbs commonly 

used to treat chronic gastritis, and 122 associations were 

obtained. We draw out the classified graph of complex 

network of herbs commonly used for chronic gastritis. 

According to statistics, cuttlebone had the highest 

frequency of OS swpiae (OS SEPIAE) 355, Radix 

Trichosanthis 347, Heterophylla falsestarwort 324, 

Thunberg fritillary 300, Largehead Atractylodes Rh 296, 

Fermented Pinellia 244, Rhizoma Zedoariae 201, Gallus 

gallus domesticus Brisson 196, Amomum villosum 144, 

Semen Raphani 142, Root of Herbaceous Peony 126,

Figure 1. Herbal complex network node categories for chronicgastritis. 

Lu et al. 923


Rhizoma Coptidis 125, Medicinal Evodia Fruit 116, 

Rhizoma Zingberis Preparata 94 and Endothelium 

Corneum Gigeriae Galli80. The results completely 

conform to clinical practice, and basically they are the 

common herbs for treatment of chronic gastritis, with 

basically uniform with core combination data reflected in 

Table 3. And they are essentially Professor GAO 

Zhongying’s commonly used herbs for chronic gastritis. 

Results of complex network 

Complex network is an effective method to associate 

diverse kinds of information. The core prescription 

combinations are selected to reflect the experiences of 

famous physicians by means of analyzing the graph 

structures of entropy clustering result. 

The major core herbal combination in complex 

network 

The major herbal combination has Heterophylla false 

starwort (Radix codonopsis), Largehead Atractylodes Rh, 

Radix Trichosanthis, Thunberg fritillary (Bulbus fritillariae 

cirrhosae), OS swpiae (OS SEPIAE). Largehead 

Atractylodes Rh is mainly used to maintenance spleen; 

Radix Trichosanthis, Thunberg fritillary (Bulbus fritillariae 

cirrhosae), OS swpiae (OS SEPIAE) are used to repair 

erosions of stomach. The compatibility of core herbal 

combination reflects the key pathogenesis of spleen 

deficiency and stomach dryness for chronic gastritis. 

Compatibility mechanism of the herbal combination 

shows the core pathogenesis of spleen deficiency and 

stomach dry for chronic gastritis. This combination can be 

used either for chronic superficial gastritis or chronic 

atrophic gastritis. 

The secondary core herbal combinations in complex 

network 

The secondary core herbal combinations surround the 

major core herbal combination in complex network. 

These combinations are categorized in four groups. The 

first group is Zuojin pill composed of Medicinal Evodia 

Fruit and Rhizoma Coptidis. And its function is to treat 

heartburn, acid reflux embolism. Endothelium Corneum 

Gigeriae Galli, Fermented Pinellia and Gallus gallus 

domesticus Brisson can be added in order to promote 

gastric motility to help to digest. This five-herb 

combination can be used to cure spleen deficiency and 

stomach dry of chronic superficial gastritis or 

gastroesophageal reflux disease. The second group is 

Sini powder composed of Bupleurum, Radix Curcumae, 

Rot of Peony, Root of Herbaceous Peony and Fructus 

Aurantii Immaturus. It has the efficacy of liver and 

gallbladder. 

In clinical treatment, it is used for incompatibility of liver 

and stomach, incompatibility of gall bladder and stomach 

such as cholecystitis. The third group is Zhang Xichun’s 

Yuye soup which is composed of Adenophora stricta, 

Ophiopogon japonicus, Rhizoma polygonati odorati, 

Dioscorea opposita, Herba Dendrobii and Milkvetch root. 

Its core functionality is to strengthen spleen, invigorate 

Qi, stomach and promote fluid. This combination can be 

used to cure spleen deficiency and stomach dry of 

chronic superficial gastritis. With intestinal metaplasia or 

dysplasia, Salvia Miltiorrhiza and Rhizoma Zedoariae 

must be added to promote blood circulation and remove 

stasis. The fourth group includes Rhizoma Zingberis 

Preparata and Amomum villosum. It is used to cure 

hypofunction of Yang and deficiency and coldness of 

spleen. With diarrhea, Fructus Psoraleae, Gorgon fruit 

and Lotus meat must be added. 

Basic experience prescription of chronic gastritis 

Combining the above result of complex network with 

clinical practice, experience formula is summed up as 

follows: 

1. The basic formula for chronic superficial gastritis 

Heterophylla falsestarwort (Radix codonopsis), 

Largehead Atractylodes Rh, OS swpiae (OS SEPIAE), 

Thunberg fritillary (Bulbus fritillariae cirrhosae), Radix 

Trichosanthis, Gallus gallus domesticus Brisson, 

Endothelium Corneum Gigeriae Galli and Fermented 

Pinellia. 

2. The basic formula for chronic atrophic gastritis, 

Heterophylla falsestarwort (Radix codonopsis), 

Largehead Atractylodes Rh, OS swpiae (OS SEPIAE), 

Thunberg fritillary (Bulbus fritillariae cirrhosae), Radix 

Trichosanthis, Adenophora stricta, Ophiopogon 

japonicas, Rhizoma polygonati odorati, Dioscorea 

opposite, Herba Dendrobii and Milkvetch root. 

3. Basic formula addition and subtraction herbal laws; 

The prescription adds Medicinal Evodia Fruit and 

Rhizoma Coptidis accompanying with gastroesophageal 

reflux. If this reflux becomes severe, Concha Arcae, Inula 

flower and Hematite are added into this formula. If 

erosion or ulceration of stomach appears, Bletilla and 

Angelica dahurica are added and Bupleurum, Radix 

Curcumae, Fructus Aurantii Immaturus (Immature 

Trifoliate-orange Fruit) and Root of Herbaceous Peony 

are added when bile reflux. Rhizoma Zingberis Preparata 

and Amomum villosum are added if stomach cold 

becomes severe and pale tongue appears. Fructus 

Psoraleae, Gorgon fruit and Lotus meat are added if 

diarrhea appears. 

Conclusion 

Herbal combination laws are essential to treat the

diseases for traditional Chinese medicine. In this paper, 

we explore Professor GAO Zhongying’ experiences that 

they are reflected in his formulas of the treatment for 

chronic gastritis. And then entropy clustering and 

complex network are adopted in the process of 

discovering knowledge of herbs. The results show that 

herbal combinations are completely in line with clinical 

practices, and they are essentially the commonly used 

herbs employed by Professor GAO Zhongying for chronic 

gastritis. 


This work was supported by the National Science 

Foundation of China (30902020, 81173463 and 

61003133). 

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Complex system entropy cluster famous and old TCM physicians' 

experiences chronic gastritis (spleen deficiency and stomach 

dryness) drug selection law, J. Trad. Chin. Med., 30(4): 294.



DOI: 10.5897/JMPR11.1174 


Short Communication 

GC-MS analysis of volatile oils from Bupleurum 

chinense DC. f. vanheurckii (Muell.-Arg.) Shan et Y.Li 

LIU Ze-kun 1 and Chen-Haixia 2 * 

1 Marine College, Shandong University at Weihai, Weihai, Shandong 264209, China. 

2 Department of Pharmacy, Qilu Hospital of Shandong University, Jinan, Shandong 250012, China. 


The aim of this study was to analyze the volatile oil from Bupleurum chinense DC. f. vanheurckii (Muell.- 

Arg.) Shan et Y.Li. The volatile oil was extracted from B. chinense DC. f. vanheurckii (Muell.-Arg.) Shan 

et Y.Li by steam distillation, its components was analyzed and identified by gas chromatography-mass 

spectrometry (GC-MS) method. 27 compounds, which represent 93.3% of total oil, were identified. 

Predominant constituents over 5% were 4,7,10,13,16,19-Docosahexaenoic acid, methylester, (all-Z) - 

(19.1%); Germacrene D (14.8%); Retinyl acetate(7.3%); Cycloisolongifolene,8,9-dehydro-9-formyl-(6.9%); 

α-Cadinol(5.6%); and τ-Muurolo (5.3%). 

Key words: Bupleurum chinense DC. f. vanheurckii (Muell.-Arg.) Shan et Y.Li, volatile oil, gas chromatographymass 

spectrometry (GC-MS) analysis. 

INTRODUCTION 

Bupleurum chinense DC. f. vanheurckii (Muell.-Arg.) 

Shan et Y.Li is an herb of 20 to 30 cm height and belongs 

to the Apiaceae family. It is a variety of B. chinense DC. 

but rich in volatile oil. There are 40 species and 17 

varieties of Bupleurum in China and the majority of them 

are produced in Liaoning, Gansu, Hebei, Henan, 

Shandong, Hubei, Jiangsu, Sichuan, Anhui and other 

provinces (Sun and Zhu, 1994). B. chinense DC. f. 

vanheurckii (Muell.-Arg.) Shan et Y.Li differs from B. 

chinense DC. Its original species by its 4 to 5 ovatelanceolated 

green small bracts with white edge, slightly 

longer than umbel and the half of the fruit umbrella (Li 

and Shan 1974). In Chinese medicine, the plant is used 

as traditional drugs. The main active ingredients of 

Bupleurum are saponins and volatile oil. Up to now, there 

are no studies of its volatile oil. Therefore, the 

relationship between effective ingredients and the 

efficacy of B. chinense DC. f. vanheurckii (Muell.-Arg.) 

Shan et Y.Li should be discussed, so as to find out its 

clinical range and using standard and to apply it into 

clinical practice. Based on this, the volatile oils were 

extracted from the plant by steam distillation. Gas 

*Corresponding author. E-mail: 423603242@qq.com. 

chromatography-mass spectrometry (GC-MS) method 

was used to analyze and identify the components, in 

order to lay foundations for the further research and 

development of their medicinal value and for the 

expansion of clinical range. 


Research material 

Bupleurum chinense DC. f. vanheurckii (Muell.-Arg.) Shan et Y.Li 

were collected in Kunyu Mountain, Yantai City on August 25, 2010, 

and were identified by professor Zhang Qinde in Shandong College 

of Traditional Chinese Medicine. The plants were cut into decoction 

pieces of 1 cm and were dried under the sun. 

Main reagents 

Both anhydrous ether and anhydrous sodium sulfate were 

analytically pure sold in the market. 

Main instruments 

7890 to 5975 gas chromatography-mass spectrometry (GC-MS) is 

from Agilent Technologies and other instruments were conical flask, 

volatile oil extractor, electric heating jacket, injector, and 0.45 m

Ze-kun and Haixia 927 

Figure 1. GC-MS total ion chromatogram of volatile oil from Bupleurum chinense DC. f. vanheurckii (Muell.-Arg.) Shan et Y.Li. 

micro porous membrane. 

Procedure 

Extraction of volatile oil 

The method of steam distillation in appendix U of Chinese 

Pharmacopoeia was adopted for the extraction of volatile oil 

(Chinese Pharmacopoeia, 2000). 100 g decoction pieces of B. 

chinense DC. f. vanheurckii (Muell.-Arg.) Shan et Y.Li were grinded, 

sieved through 40 meshes, and then put into a steam distillation 

vessel. After heating reflux for 8 h and collecting the distilled fluid, 

NaCl was added until it reached saturation. Then ethyl ether was 

used for the extraction for three times. The extracts were dried by 

anhydrous sodium sulfate. And volatile oil was obtained by rotary 

evaporator after removing the ethyl ether. The volatile oil was 

sealed and stored in the refrigerator. Samples were sent to the 

Shandong Institute for Drug Control to carry out GC-MS analysis. 

Chromatographic conditions for GC-MS analysis 

GC conditions were as follows: HP-5MS 5% Phenyl Methyl 

Siloxane fused silica capillary column (30 m × 0.25 mm × 0.25 µm), 

injector temperature at 250°C, helium as the carrier gas, 72 kPa 

column pressure, 1 μl injection volume (n-hexane solution), and 1.2 

ml/min flow rate; the temperature programming started at 60°C for 2 

min, then increased to 180°C for 5 min at a rate of 15°C /min, and 

finally reached 250°C for 3 min at a rate of 5°C/min. The MS 

conditions were as follows: EI, ion source temperature at 230°C, 70 

eV electron energy, interface temperature at 270°C, solvent delay 

for 3 min, and mass scan range within 60 to 600 amu. NIST mass 

spectral database was used and compared with the standard 

spectrum, in order to identify the components peaks. According to 

the peak area normalization method, relative mass fraction of each 

component in volatile oil was calculated. 

RESULTS 

Total ion chromatogram of volatile oil components was 

obtained (Figure 1) based on the GC-MS analysis of 

volatile oil from B. chinense DC. f. vanheurckii (Muell.- 

Arg.) (Li and Shan, 1974). A total of 27 compounds, which 

represent 93.3% of total oil, were identified by computer 

retrieval of NIST05 mass spectral database and manual 

spectrogram analysis. Table 1 reported that the main 

components of volatile oils from the B. chinense DC. f. 

vanheurckii (Muell.-Arg.) (Li and Shan, 1974) were 

terpenoids, low-molecular aliphatic compounds, and a 

minority of alcohol compounds. According to the method 

of area normalization, predominant constituents over 5% 

were 4,7,10,13,16,19-Docosahexaenoicacid,methylester, 

(all-Z) - (19.1%); Germacrene D (14.8%); Retinyl acetate 

(7.3%); Cycloisolongifolene,8,9-dehydro-9-formyl - 

(6.9%); α - Cadinol(5.6%); and τ-Muurolo (5.3%). 

DISCUSSION 

A total of 27 compounds were identified, which were 

mainly terpenoids, low-molecular aliphatic compounds 

and alcohol compounds. It is reported that eight kinds of 

chemical compounds including τ-Muurolo, β- 

Caryophyllene, Aromadendrene, citronellyl acetate, 

Elemene, Guaiene and elemol are significantly related to 

the inhibition of Staphylococcus aureus and some other 

microbe, in which elemol is negatively correlated with the 

inhibition (Chen et al., 2010) and in this work,


Table 1. GC-MS analysis results of volatile oil from Bupleurum chinense DC. f. vanheurckii (Muell.-Arg.) Shan et Y.Li. 

S/N Compound Retention time Content (%) 

1 Elixene 9.439 0.598 

2 α-Elemene 9.964 0.607 

3 Bicylo[4.1.0]heptane,7-bicyclo[4.1.0]hept-7-ylidene- 10.166 1.088 

4 α-Cubebene 10.281 4.881 

5 β-Caryophyllene 10.314 1.262 

6 Cyclosativene 10.535 0.802 

7 τ-Cadinene 10.613 0.824 

8 τ-Muurolene 10.810 1.844 

9 Germacrene D 10.927 14.843 

10 α-Farnesene 10.983 0.910 

11 ç-Elemene 11.065 1.937 

12 ë-Cadinene 11.252 2.622 

13 Isoaromadendrene epoxide 11.937 0.757 

14 Ent-Spathulenol 12.072 3.451 

15 Caryophyllene oxide 12.580 2.681 

16 β-Farnesol 12.777 1.305 

17 τ-Muurolo 13.009 5.264 

18 α-Cadinol 13.217 5.609 

19 4-(2,2-Dimethyl-6-methylenecyclohexylidene)-3-methylbutan-2-one 

13.351 2.734 

20 Calarene epoxide 13.753 1.519 

21 Aromadendrene oxide-(1) 15.115 2.093 

22 Murolan-3,9(11)-diene-10-peroxy 15.576 3.618 

23 Spiro[tricyclo[4.4.0.0(5,9)]decane-10,2'-oxirane],1-methyl-4-isopropyl-7,8-dihydroxy- 

16.953 1.566 

24 Retinyl acetate 17.375 7.330 

25 4,7,10,13,16,19- Docosahexaenoic acid,methyl ester, (all-Z)- 17.508 19.1 

26 Cycloisolongifolene, 8,9-dehydro-9-formyl- 18.118 6.913 

27 Falcarinol 19.520 0.856 

Germacrene D, τ-Muurolo, Elemene and β- 

Caryophyllene are discovered in the volatile oil and they 

are totally more than 20%. Also, some of the other 

compounds have antibiotic effect. This fact maybe a good 

explanation of Chinese folk`s using B. chinense DC. f. 

vanheurckii (Muell.-Arg.) Shan et Y.Li as traditional 

medicine. However, the specific effect is not thoroughly 

cleared and further discussion is still needed on whether 

the volatile oil is the main active components on some 

efficacies such as dizziness and headache alleviation, 

blood activation, fever bring down, dampness and wind 

elimination. This research provided experimental basis for 

the further research on the development and utilization of 

physiological activity and medicinal resources of B. 

chinense DC. f. vanheurckii (Muell.-Arg.) Shan et Y.Li. 

REFERENCES 

Chen X, Liu X, Wu J (2010). Study on Chemical Composition and 

Antifungal Activity Volatile Oil of Alpinia oxyphylla Miq Fruits[J]. 

C.A.S.B., 26(22): 102-105. 

Chinese Pharmacopoeia (2000). pp. 232-233. 

Li Y, Shan R (1974). Study of Bupleurum Classification [J]. J.S.E., 12(3): 

293-294. 

Sun Y, Zhu Y (1994). Application of Radix Bupleuri at all times in China. 

J. Shandong Coll. Trad. Chin. Med., 18(6): 421-423.



DOI: 10.5897/JMPR11.1497 


Short Communication 

Isolation of cinnamic acid derivatives from the root of 

Rheum tanguticum Maxim.ex Balf. and its significance 

Liangliang Gao, Xudong Xu*, Haijiang Nan, Junshan Yang, Guangli Sun, Haifeng Wu 

and Mingliang Zhong 

Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, 

Beijing 100193, China. 


Eight compounds (1-8) including two naphthalene glycosides, three tannins and three cinnamic acid 

derivatives were isolated from Rheum tanguticum Maxim.ex Balf. for the first time, except compounds 

1, 6, 7 and 8. Their structures were determined mainly by NMR and MS techniques. The result is of 

significance for comparison and analysis between different rhubarbs by various chromatographic 

methods. 

Key words: Rhubarb, Rheum tanguticum, cinnamic acid. 

INTRUDUCTION 

Rhubarb, one of the oldest and best-known Chinese 

herbal medicines, has been used for thousands of years 

(Science Press, 1998). Its species are widely distributed 

in China (Chinese Pharmacopoeia Commission, 2005), 

Kirgheeze desert and Europe. More than 30 species of 

rhubarb grow in China. It contains biologically active 

compounds such as anthraquinone derivatives, anthrone 

derivatives, stilbenes and tannins etc (Nan et al., 2009) 

and has the effect of purging heat, loosening the bowels, 

removing heat from the blood, clearing away toxins, 

promoting blood circulation and removing blood stasis 

etc. All of foregoing is focused on previous studies of 

Rhubarb. However, few references are available on 

cinnamic acid derivatives from Rhubarb no matter 

constituent isolation, chromatographic analysis, or 

pharmacological studies. 

EXPERIMENTAL 

Collection and preparation of plant material 

The medicinal material was collected at Qinghai Province, China, in 

October 2007, and identified by Prof. Zhichuan Bai at the College of 

Horticulture and Landscape, Southwest University. 

*Corresponding author. E-mail: xdxu@implad.ac.cn. Tel: +86- 

10-57833296. Fax: +86-10-57833296. 

Extraction and Isolation 

The root of Rheum tanguticum (10 kg) were dried, powered, and 

exhaustively extracted with methanol (95%) three times. The 

methanol extract (2.35 kg) which was filtered and concentrated 

under reduced pressure was subjected to a column 

chromatography on silica gel (3 kg) using petroleum ether, CHCl3, 

EtOAc and methanol as the mobile phase and four fractions were 

obtained. The EtOAc fraction was repeatedly chromatographed 

over silica gel with stepped gradient of CHCl3-MeOH and 1000 ml 

fractions were collected. Combined fractions 19 to 36 were purified 

using Sephadex LH-20 eluting with CHCl3-CH3OH (1:1) and 

subfractions 51 to 58 and subfractions 136 to 138 were afforded. 

The two subfractions were further purified by semi-preparative 

HPLC using MeOH-H2O as the eluent to yield compounds 1 to 8 

(Figure 1). These compounds including three cinnamic acid 

derivatives, two naphthalene glycosides, three tannins and three 

cinnamic acid derivatives identified as trans- cinnamic acid (1), Pcoumaric 

acid gluside (2) (Xiang et al., 2007), 4-Hydroxybenzenepropanoic 

acid methyl ester (3), torachrysone -8-O-β-Dglucopyranoside 

(4) (Mashiko et al., 1977), 6-hydroxymusizin-8-O- 

β-D-glucopyranoside (5) (Mashiko et al., 1977), 4-(4′hydroxyphenyl)-2-butanone-4′-O-β-D-(2″-O-galloyl)glucopyranoside 

(6) (Gen-Ichiro and Itsuo 1983), 4-(4′hydroxyphenyl)-2-butanone-4′-O-β-D-(2″-O-galloyl-6″-O-cinnamoyl)glucopyranoside 

(7) (Yoshiki et al., 1984), 4-(4′-hydroxyphenyl)-2butanone 

4′-O-β-D-(6″-O-c innamoyl)-glucopyranoside (8) (Nonaka 

et al., 1981). Compounds 2 to 5 were isolated from R. tanguticum 

for the first time. Compound 1, NMR data: 1 HNMR (600MHz, 

MeOD) δ: 7.51 (2H, d, J = 7.2 Hz, H-2′,6′), 7.39 (1H, d, J = 15.6 Hz, 

H-3), 7.34 (2H, t, J = 7.2 Hz, H-4′), 7.30 (1H, t, J = 7.2 Hz, H-3′, 5′), 

6.51 (1H, d, H-2). 13 CNMR (150 MHz, MeOH) δ: 127.0 (C-1′), 130.0 

(C-2′,6′), 128.6 (C-3′,5 ′), 127.0 (C-4′ ), 137.6 (C-4), 140.8 (C-3), 

175.8 (C = O). Compound 2, NMR data: 1 HNMR (600 MHz, 

DMSO- d6)

1 


HO 

5 

3 

HO 

OH 

OH 

1 

OH 

HO 

O 

O 

OH 

O 

O 

O 

O 

OH 

OH 

OH 

O 

O 

OH 

HO 

O 

HO 

OH 

HO 

OH 

O 

OH 

O 

OH 

OH 

O 

O 

O 

O 

O 

O 

OH 

O O 

7 8 

Figure 1. Structures of compounds 1–8. 

δ: 10.05 (1H, s, 4-OH), 7.64 ( 1H, d, J = 16.2 Hz, H-7), 7.58 (2H, d, 

J = 8.4 Hz, H-2, 6), 6.80 ( 2H, d, J = 8.4 Hz, H-3, 5), 6.39 (1H, d, J = 

16.2 Hz, H-8), 5.46 (1H, d, J = 8.4 Hz, anomeric H), 4.60~3.10 

(6H, m, sugar H). 13 CNMR (150MHz, DMSO-d6 )δ: 124.9 (C-1), 

130.5 (C-2), 113.6 (C-3), 160.0 (C-4), 115.8 (C-5), 130.6 (C-6), 

145.9 (C-7), 115.8 (C-8), 165.3 (C-9), 94.2 (C-1'), 77.8 (C-3'), 76.4 

(C-5'), 72.5 (C-2'), 69.5 (C-4'), 60.6 (C-6'). Compound 3, NMR data: 

O 

O 

O 

OH 

OH 

2 

6 

O 

OH 

4 

Fig. 1 Structures of compounds 1–8 

OH 

OH 

HO 

O 

OH 

OH 

OH 

O 

O 

OH 

OH 

O 

OH 

OH 

O 

OH 

O 

1 HNMR (600MHz, DMSO-d6) δ: 7.61 (1H, d, J = 16.2 Hz, H-7), 7.44 

(2H, d, J = 8.4 Hz, H-2, 6), 6.79 (2H, d, J = 8.4 Hz, H-3, 5), 6.31 

(1H, d, J = 16.2 Hz, H-8), 3.76 (3H, s, OCH3), 4.60~3.10 (6H, m, 

sugar-H). 13 CNMR (150MHz, DMSO-d6) δ: 127.0 (C-1), 131.3 (C- 

2,6), 114.8 (C-3,5), 162.1 (C-4), 145.9 (C-7), 162.1 (C-8), 170.0 

(C=O). Compound 4, NMR data: 1 HNMR (600 MHz, MeOD) δ: 

2.28 (3H, s, Ar-CH3), 2.58 (3H, s, COCH3, 5.09 (1H, d, J = 8.4 Hz, 

O

anomeric H), 4.00~3.30 (6H, m, sugar H), 3.86 (3H, s, OCH3) , 

6.84,7.00 (each 1H, d, J = 2.4 Hz, aromatic H), 7.02 (1H, s, 

aromatic H). 13 CNMR (150 MHz, MeOD) δ: 20.4 (Ar-CH3), 32.7 

(COCH3), 56.2 (OCH3), 62.6 (C-6′), 71.5 (C-4′), 75.1 (C-3′), 78.3 (C- 

5′), 79.0 (C-6′), 102.7 (C-1′), 104.5 (C-5), 104.6 (C-7), 110.5 (C-9), 

120.5 (C-4), 124.2 (C-2), 135.6 (C-10), 139.4 (C-3), 153.9 (C-1), 

157.4 (C-8), 160.6 (C-6), 208.3 (CO). Compound 5, NMR data: 

1 HNMR (600 MHz, MeOD) δ: 2.27 (3H, s, Ar-CH3), 2.58 (3H, s, 

COCH3), 5.09 (1H, d, J = 8.4Hz, anomeric H), 4.00~3.30 (6H, m, 

sugar H), 6.69, 6.97 (each 1H, d, J = 1.8Hz, aromatic H), 6.91 (1H, 

s, aromatic H). 13 CNMR (150 MHz, MeOD) δ: 20.5(Ar-CH3), 32.7 

(COCH3), 62.6 (C-6′), 71.4 (C-4′), 75.1 (C-2′), 78.3 (C-5′), 79.0 (C- 

6′), 104.5 (C-1′), 104.7 (C-5), 105.6 (C-7), 109.8 (C-9), 120.0 (C-4), 

123.4 (C-2), 135.5 (C-10), 139.7 (C-3), 154.3 (C-1), 157.6 (C-8), 

158.4 (C-6), 208.4 (CO). Compound 6, NMR data: 1 HNMR 

(600MHz, MeOD) δ: 7.12 (2H, s, galloyl H), 7.03 (2H, d, J=8 Hz, H- 

2′, 6′), 6.87 (2H, d, J = 8 Hz, H-3′,5'), 5.13 (2H, m, anomeric H), 

4.55 (1H, dd, J = 2.5, 12.0 Hz, H-6″), 4.39 (1H, dd, J = 8.0, 12.0 Hz, 

H-6″), 3.5 to 4.0(5H, m, sugar-H), 2.70 (4H, m, H-3, 4), 2.07 (3H, 

s, H-1). 13 CNMR (150 MHz, MeOD) δ:211.4 (C-2), 167.8 (C-7″), 

157.5 (C-4′), 146.6 (C-3″′, 5″′), 140.1 (C-4″′), 137.0 (C-1′), 130.5 (C- 

2″, 6″), 121.6 (C-1″′), 118.2 (C-3′, 5′), 110.5 (C-2″′, 6″′), 101.5 (C-1″), 

78.5 (C-3″), 76.4 (C-5″), 75.5 (C-2″), 71.7 (C-4″), 62.6 (C-6″), 46.0 

(C-4), 30.1 (C-1, 3). Compound 7, NMR data: 1 HNMR (600 MHz, 

MeOD) δ: 2.03 (3H, s, COCH3), 2.64(4H, m, H-3,4), 3.5 to 4.0 (3H, 

m, sugar H), 4.43 (1H, dd, J = 6,12Hz, H-6″), 4.59 (1H, dd, J = 

2,12Hz, H-6″), 5.09 to 5.20 (2H, m, H-1″, 2″), 6.38,7.65 (each 1H, d, 

J = 16.2Hz, olefinic H), 6.84,6.95 (each 2H, d, J = 8.4 Hz, aromatic 

H), 6.84,7.49 (each 2H, d, J = 8.4 Hz, aromatic H), 7.09 (2H, s, 

galloyl-H). 13 CNMR (150 MHz,MeOH) δ: 30.5 (C-1), 30.9 (C-3), 

46.3 (C-2), 64.7 (C-6″), 72.4 (C-4″), 75.4 (C-5″), 75.8 (C-2″), 76.4 

(C-3″), 101.4 (C-1″), 110.5 (galloyl C-2,6), 115.3 (C-α), 117.1 (C-3"', 

C-5"'), 118.4 (C-3′, C-5′), 121.6 (galloyl C-1), 127.3 (C-1"') , 130.4 

(C-2′, C-6′), 131.5 (C-2"', C-6"'), 136.9 (C-1′), 139.1 (galloyl C-4), 

146.6 (galloyl C-3,5), 147.0 (C-β), 157.3 (C-4′), 161.6 (C-4"'), 167.8 

(COOH), 169.0 (COOH), 211.2 (C-2). Compound 8, NMR data: 

1 HNMR (600 MHz, MeOD) δ: 2.05 (3H, s, COCH3), 2.61 (4H, m, 

3,4-H), 3.3 to 3.8 (4H, m, sugar-H), 6.37,7.63 (each 1H, d, J = 

16.2Hz, olefinic-H), 6.84,7.49 (each 2H, d, J = 8.4Hz, aromatic-H), 

6.97,7.01 (each 2H, d, J = 8.4 Hz, aromatic-H). 13 CNMR (150 MHz, 

MeOH ) δ: 30.4 (C-1), 30.4 (C-3), 46.3 (C-2), 65.2 (C-6″), 72.6 (C- 

4″), 75.4 (C-5″), 75.9 (C-2″), 78.5 (C-3″), 102.9 (C-1″), 115.6 (C-α), 

117.5 (C-3"', C-5"'), 118.5 (C-3′, C-5′), 127.6 (C-1"'), 130.7 (C-2′, C- 

6′), 131.8 (C-2"', C-6"'), 136.8 (C-1′), 147.3 (C-β), 157.7 (C-4′), 

161.9 ( C-4"'), 169.4 (COOH), 211.4 (C-2). 


To date, about 200 compounds mainly in 6 different types 

of skeletons, including anthraquinones, anthrones, 

stilbenes, flavan, acylglucosides, and pyrones, have been 

isolated from the genus Rhuem. A number of researches 

have been done systematically, no matter their 

chromatographic analysis, or biological activities. But, 

their reports mainly focused on the most common 

compounds such as anthraquinone, anthrone, stilbenes 

and tannins etc from rhubarb. 

This paper describes cinnamic acid derivatives from the 

plant of rhubarb for the first time. So far, quality control 

and evaluation system of rhubarb on Pharmacopoeia of 

the People’s Republic of China have never referred to 

this class of compounds. The presence of them can be 

considered as a chromatographic marker for 

Gao et al. 931 

R. tanguticum compared with the other species of 

rhubarb, but more research is still needed to confirm it. 

The occurrence of cinnamic acid derivatives from R. 

tanguticum can be used as a very important 

phytochemical analysis of the components of the plant, 

and further chromatographic studies on rhubarb, too. 

Therefore, their quality control and evaluation system are 

preferably consummated. 


Authors would like to thank financial support from 

National Natural Science Foundation of China (No. 

81102770); Chinese Traditional Medicine Researches of 

Special Projects (No. 200707007); the technological large 

platform for comprehensive research and development of 

new drugs in the twelfth five-year ‘‘Significant New Drugs 

Created’’ Science and Technology Major Projects (No. 

2012ZX09301-002-001-026); the chemical composition of 

the digital library of traditional Chinese medicine for drug 

discovery in the twelfth five-year ‘‘Significant New Drugs 

Created’’ (No. 2011ZX09307-002 -01). 

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Chinese Pharmacopoeia Commission (2005). Pharmacopoeia of 

the People’s Republic of China[S], 1: 17. 

Gen-Ichiro N, Itsuo N (1983). Tannins and Related Compounds. X. 

Rhubarb(2): Isolation and Structures of a Glycerol Gallate, Gallic 

Acid Glucoside Gallates, Galloylglucoses and Isolindleyin. Chem. 

Pharm. Bull., 31(5): 1652-1658. 

Mashiko T, Masafumi M, Gen-ichro N, Itsuo N (1977). Studies on 

Rhubarb(Rhei Rhizoma). IV. Naphthalene Glycosides. Chem. 

Pharm. Bull., 25: 2708-2712. 

Nan HJ, XU XD, Chen SL (2009). Research Progress in Rheum 

Plants. Nat. Prod. Res. Dev., 21: 4. 

Nonaka GI, Nishioka I, Nagasawa T (1981). Tannins and Related 

Compouds I. Rhubarb(1). Chem. Pharm. Bull., 29(10): 2862- 

2870. 

Science Press (1998). Delectis Florae Reipublicase Pepularis 

Sinicase Agendae Acdemiae Sinicase Edita. Flora Reipublicase 

Popularis Sinicase Tomus, 25(1): 181-182. 

Xiang Y, Li YB, Zhang J (2007). Studies on chemical constituents of 

Sa lsola collina. China J. Mater. Med., 32(5): 409-413. 

Yoshiki K, Gen-ichiro N, ltsuo N (1984). Tannins and Related 

Compounds XX III. Rhubarb(4): Isolation and Struchtres of New 

Ciasses of Gallotannins. Chem. Pharm. Bull., 32(9): 3461.

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2013 

September 2013 

61st International Congress and Annual Meeting of the Society for Medicinal Plant 

and Natural Product Research, Muenster, Germany, 1 Sep 2013

Journal of 

Medicinal Plants Research 

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