This article was downloaded by: [Marongiu, Bruno] On: 19 November 2010 Access details: Access Details: [subscription number 929825550] Publisher Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 3741 Mortimer Street, London W1T 3JH, UK Natural Product Research Publication details, including instructions for authors and subscription information: http://www.informaworld.com/smpp/title~content=t713398545 Essential oil composition of leaves of Stachys yemenensis obtained by supercritical CO2 Nasser A. Awadh Alia; Bruno Marongiub; Alessandra Pirasb; Silvia Porceddab; Danilo Falconierib; Paola Molicottic; Stefania Zanettic a Department of Pharmacognosy, Sana'a University, Sana'a, Yemen b Dipartimento di Scienze Chimiche, Università degli Studi di Cagliari, Cittadella Universitaria di Monserrato, 09042 Cagliari, Italy c Dipartimento di Scienze Biomediche, Sezione di Microbiologia Sperimentale e Clinica, Università di Sassari, 07100 Sassari, Italy Online publication date: 19 November 2010 To cite this Article Awadh Ali, Nasser A. , Marongiu, Bruno , Piras, Alessandra , Porcedda, Silvia , Falconieri, Danilo , Molicotti, Paola and Zanetti, Stefania(2010) 'Essential oil composition of leaves of Stachys yemenensis obtained by supercritical CO2', Natural Product Research, 24: 19, 1823 — 1829 To link to this Article: DOI: 10.1080/14786411003754272 URL: http://dx.doi.org/10.1080/14786411003754272 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material. Natural Product Research Vol. 24, No. 19, 20 November 2010, 1823–1829 Essential oil composition of leaves of Stachys yemenensis obtained by supercritical CO2 Nasser A. Awadh Alia, Bruno Marongiub*, Alessandra Pirasb, Silvia Porceddab, Danilo Falconierib, Paola Molicottic and Stefania Zanettic Downloaded By: [Marongiu, Bruno] At: 06:48 19 November 2010 a Department of Pharmacognosy, Sana’a University, Sana’a, Yemen; bDipartimento di Scienze Chimiche, Università degli Studi di Cagliari, Cittadella Universitaria di Monserrato, SS 554, Km 4,500, 09042 Cagliari, Italy; cDipartimento di Scienze Biomediche, Sezione di Microbiologia Sperimentale e Clinica, Università di Sassari, Viale San Pietro 47b, 07100 Sassari, Italy (Received 20 November 2009; final version received 16 February 2010) This article reports the composition of the essential oil from the leaves of Stachys yemenensis. The essential oil was extracted by supercritical CO2 (90 bar; 40 C) and its chemical composition was determined by gas chromatography and gas chromatography–mass spectrometry. The major components of the sample were -phellandrene (13.9%), -phellandrene (11.7%), elemol (12.0%), spathulenol (6.7%), -eudesmol (5.0%), eudesmol (4.75%) and squalene (4.8%). On the exhausted matrix, deprived of the volatiles, we carried out a high-pressure (250 bar) treatment for the extraction of squalene (49.7%). The antimicrobial activity of the essential oils has been assayed by using the broth dilution method on two American Type Culture Collection (ATCC) strains, Escherichia coli ATCC 35218 and Staphylococcus aureus ATCC 43300, and two clinical strains, Candida albicans and Candida glabrata. Keywords: Stachys yemenensis; supercritical carbon dioxide; essential oil; antibacterial activity; antifungal activity 1. Introduction The genus Stachys is one of the largest genera of the Lamiaceae family and is distributed in the Mediterranean regions and south-west Asia. About 300 Stachys species are recorded. Two species of this genus are found in Yemen, Stachys yemenensis endemic (Wood, 1997) and Stachys aegyptiaca (Trease & Evans, 1989). Stachys species have been reported in folk medicine to treat genital tumours, sclerosis of the spleen, inflammatory tumours and cancerous ulcers. Antimicrobial activity of Stachys oils has been reported (Skaltsa, Lazari, Chinou, & Loukis, 1999). To date, many Stachys species have been investigated for their essential oil. In some of the more recent works, Javidnia, Rezai, Miri and Jafari (2006) analysed the essential oil obtained from the aerial parts of Stachys obtusicrena, finding spathulenol (11.5%) and 10-epi- -eudesmol (6.8%) as the main components. *Corresponding author. Email: maronb@unica.it ISSN 1478–6419 print/ISSN 1029–2349 online ß 2010 Taylor & Francis DOI: 10.1080/14786411003754272 http://www.informaworld.com Downloaded By: [Marongiu, Bruno] At: 06:48 19 November 2010 1824 N.A. Awadh Ali et al. The major components of the essential oils of the dried flowering aerial parts of Stachys byzantina, Stachys inflata, Stachys lavandulifolia and Stachys laxa, collected in the north of Iran, were piperitenone (9.9%) for S. byzantina, hexadecanoic acid (9.1%) and germacrene D (8.9%) for S. inflata, 4-hydroxy-4-methyl-2-pentanone (9.3%) and -pinene (7.9%) for S. lavandulifolia and germacrene D (17.1%) and 4hydroxy-4-methyl-2-pentanone (12.3%) for S. laxa (Morteza-Semnani, Akbarzadeh, & Changizi, 2006). The essential oils from the aerial parts of Stachys schtschegleevii and Stachys balansae were both rich in sesquiterpenes (54.2% and 37.2%), with germacrene D (25.8% and 16.4%) as the major component (Rezazadeh, Hamedani, Dowlatabadi, Yazdani, & Shafiee, 2006). Skaltsa, Demetzos, Lazari and Sokovic (2003) analysed the different Stachys species endemic to Greece and made a chemotaxonomic investigation of the volatile constituents of this genus; sesquiterpene hydrocarbons were shown to be the main group of constituents of all taxa. Grujic-Jovanovic, Skaltsa, Marin and Sokovic (2004) analysed different Stachys species from Serbia, finding that sesquiterpene hydrocarbons were the major components of all samples except that of Stachys plumosa, which was rich in monoterpene hydrocarbons. A species from Turkey, Stachys aleurites, was studied by Flamini et al. (2005) and was found to be rich in sesquiterpene hydrocarbons, while germacrene D was the main component of Stachys sylvatica, an Italian species (Tirillini, Pellegrino, & Maleci Bini, 2004). In spite of the large size of Stachys, the composition of its volatile compounds is known in only a small number of species. In this work, we report for the first time the chemical composition of the essential oil from the endemic Yemeni Stachys extracted by supercritical carbon dioxide and hydrodistillation (HD). 2. Results and discussion The essential oil composition of S. yemenensis is reported here, for the first time. Table 1 shows the constituents of the essential oils extracted by supercritical CO2 and by HD. Both oils were light yellow, with a yield of 0.9% (w/w) for supercritical fluid extraction (SFE) and 0.8% (w/w) for HD. The essential oil consisted mainly of oxygenated sesquiterpenes (41% SFE and 32% HD) and hydrocarbon monoterpenes (30% SFE and 45% HD). Nevertheless, some differences in the amounts of the major constituents were found; all the samples showed the presence of -phellandrene and -phellandrene as the dominant constituents (from 20.7% to 13.9% and from 16.8% to 11.7%). Other minor constituents of the oils were elemol (12.0% SFE and 7.5% HD), spathulenol (6.7% SFE and 4.7% HD), -eudesmol (5.0% SFE and 5.1% HD), -eudesmol (4.7% SFE and 6.4% HD) and squalene (4.8% SFE). Then, on the exhausted matrix, deprived of the essential oil, a further supercritical CO2 extraction at 250 bar was performed. S. yemenensis gave a yield of 4.4% with respect to charged material. Squalene represented 49.7% of the total extract that also contained a significant amount of volatiles not fully extracted at 90 bar. As expected, the essential oil composition of S. yemenensis is rather different from those obtained from other Stachys species. The compounds which characterise Natural Product Research 1825 Table 1. Main components found in S. yemenensis extracts. Downloaded By: [Marongiu, Bruno] At: 06:48 19 November 2010 IR 938 977 980 992 1006 1027 1032 1090 1100 1302 1339 1392 1409 1419 1434 1439 1451 1454 1461 1474 1477 1481 1485 1491 1496 1499 1503 1514 1524 1550 1557 1566 1590 1618 1631 1638 1642 1650 1653 1668 1689 1736 1808 2299 2500 2700 2850 Y% Compound HD SFE 90 bar SFE 250 bar -Pinenea Sabinenea -Pinenea Myrcene -Phellandrenea o-Cymene -Phellandrene Terpinolenea Linaloola Carvacrola -Elemene -Elemene -Gurjunenea -Caryophyllenea -Elemene Aromadendrenea cis-Muurola-3,5-diene -Humulenea Allo-aromadendrene trans-Cadina-1(6),4-diene -Muurolene Germacrene D -Selinene trans-Muurola-4(14),5-diene Bicyclogermacrene -Muurolene Premnaspirodiene -Cadinene -Cadinene Elemol Germacrene B Spathulenol Viridilflorol 10-epi- -Eudesmol -Eudesmol Hinesol -Muurolol -Eudesmol -Eudesmol Bulnesol Shyobunol Oplopanone Cryptomeridiol Tricosanea Pentacosanea Heptacosanea Squalenea 4.6 0.2 0.1 2.8 20.7 8.5 16.8 0.1 0.1 0.2 tr 0.4 0.3 0.4 tr 0.2 0.1 0.3 0.1 0.2 0.2 0.2 0.4 0.2 3.4 0.6 0.2 0.6 3.4 7.5 0.6 4.7 0.4 0.3 3.2 0.5 1.4 5.1 6.4 0.8 1.5 – – tr – – – 0.8 2.4 0.2 tr 1.8 13.9 5.3 11.7 tr tr tr 0.2 0.6 0.5 0.5 tr 0.2 – 0.3 0.2 – tr 0.2 0.4 tr 4.3 0.3 0.3 1.3 1.2 12.0 0.7 6.7 0.4 – 1.7 0.7 0.4 5.0 4.7 3.1 6.0 0.3 tr 0.5 0.6 tr 4.9 0.9 0.3 tr – tr 1.7 0.6 1.5 – – tr tr tr tr tr – tr – tr tr – tr tr tr – 1.1 tr tr 0.5 tr 5.9 tr 3.3 tr – 1.6 0.4 tr 5.1 5.2 3.8 6.2 0.5 0.8 0.6 1.6 0.7 49.7 4.4 Notes: IR, GC retention indices relative to C9–C25 n-alkanes on the HP-5 column; tr, traces50.1%; and Y%, percentage of yield. aPeaks identified by comparison with respective pure standards. 1826 N.A. Awadh Ali et al. Table 2. Biological activity of S. yemenensis extracts. HD Strains Downloaded By: [Marongiu, Bruno] At: 06:48 19 November 2010 S. aureus ATCC E. coli ATCC C. albicans C. glabrata SFE 90 bar MIC% (v/v) MCC% (v/v) MIC% (v/v) MCC% (v/v) 42.5 0.15 42.5 42.5 42.5 42.5 42.5 42.5 0.6 0.3 42.5 2.5 2.5 42.5 42.5 42.5 the essential oil of S. yemenensis are absent, or present in very small quantities, in other Stachys essential oils, and vice versa. The minimum inhibitory concentrations (MICs) of the S. yemenensis oil, both from HD and SFE, obtained by the microdiluition method are shown in Table 2. In particular, MIC values shown against the E. coli ATCC strain were 0.15 % (v/v) and 0.3 % (v/v), respectively; S. aureus ATCC showed different sensitivity to HD and SFE forms: the MIC values were 0.6% (v/v) in SFE and 42.5% (v/v) in HD, respectively. The MIC values revealed against the Candida strains were 42.5% (v/v), with the exception of C. glabrata, for which the MIC was 2.5% (v/v) in the SFE oil. Minimum cidal concentration (MCC) values for both fungal and bacterial strains were 42.5% (v/v) in SFE and HD oils. Since S. yemenensis essential oil showed a good antimicrobial activity against E. coli and S. aureus, it would be interesting to test its activity against other bacteria, both Gram positive and negative, and particularly clinical strains. 3. Experimental 3.1. Materials The leaves of S. yemenensis were collected from Ashmor district, Hajah province, Yemen, in April 2008. The plant was identified by Mr Hassan M. Ibrahim of the Department of Botany, Faculty of Sciences, Sana’a University. A voucher specimen (S. yemenensis, YMP – La 13) of the plant material has been deposited at the Department of Pharmacognosy, Sana’a University, Yemen. Vegetal material was air dried in a hot-air drier at 40 C with forced ventilation for two days. Before utilisation, the plant matter was ground with a Malavasi mill (Bologna, Italy), taking care to avoid overheating. 3.2. Supercritical fluid extraction Supercritical CO2 (purity 99%, Air Liquide Italia, Cagliari, Italy) extractions were performed, according to the method of Marongiu, Piras, Porcedda and Scorciapino (2005), in a laboratory apparatus equipped with a 320 cm3 extraction vessel and two separator vessels of 300 and 200 cm3, respectively, connected in series. Experiments to obtain the essential oil were carried out at 90 bar and 40 C in the extraction section. In the first separator, the temperature was set at 10 C and the Natural Product Research 1827 pressure at the same value as the extraction section. The second separator was set at 15 bar and 10 C. On the exhausted matrix, a further extraction at a higher pressure of 250 bar and 40 C with a single separator was performed to obtain an extract rich in squalene. Extractions were carried out in a semi-batch mode: batch charging of vegetable matter and continuous flow solvent. About 250 g of material was charged in each run. 3.3. Hydrodistillation Downloaded By: [Marongiu, Bruno] At: 06:48 19 November 2010 HDs were performed in a circulatory Clevenger-type apparatus according to the procedure described in the European Pharmacopoeia (Council of Europe, 1997) during 4 h. 3.4. Gas chromatography and gas chromatography–mass spectrometry analysis Analysis of the essential oil was carried out by gas chromatography (GC) and by gas chromatography–mass spectrometry (GC–MS). Analytical GC was carried out in a gas chromatograph (Agilent, Model 7890A, Palo Alto, CA) equipped with a flame ionisation detector (FID), an autosampler (Agilent, Model 7683B), an Agilent HP5 fused silica column (5% phenylmethylpolysiloxane), 30 m  0.25 mm i.d., film thickness 0.25 mm and a Agilent ChemStation software system. The oven temperature was set at 60 C, raising at 3 C min 1 to 250 C and then held for 20 min at 250 C; injector temperature: 250 C; carrier gas: helium at 1.0 mL min 1; splitting ratio 1 : 10; detector temperature: 300 C. GC–MS analyses were carried out in a gas chromatograph (Agilent, Model 6890N, Palo Alto, CA) equipped with a split–splitless injector, an autosampler Agilent model 7683 and an Agilent HP5 fused silica column; 5% phenylmethylpolysiloxane, 30 m  0.25 mm i.d., film thickness 0.25 mm. The GC conditions used were: programmed heating from 60 C to 250 C at 3 C min 1, followed by 20 min under isothermal conditions. The injector was maintained at 250 C. Helium was the carrier gas at 1.0 mL min 1; the sample (1 mL) was injected in the split mode (1 : 10). The GC was fitted with a quadrupole mass spectrometer (MS), Agilent model 5973 detector. MS conditions were as follows: ionisation energy 70 eV, electronic impact ion source temperature 200 C, quadrupole temperature 150 C, scan rate 3.2 scan s 1, mass range 30–480 units. The software adopted to handle mass spectra and chromatograms was ChemStation. NIST 02 (NIST, 2002) and LIBR (TP) (Adams, 2004) mass spectra libraries were used as references. Samples were run in chloroform with a dilution ratio of 1 : 100. Compounds were identified by matching their mass spectra and retention times with those reported in the literature. Moreover, whenever possible, identification was confirmed by the injection of pure compounds. The percentage of individual components was calculated based on GC peak areas. The response factors were estimates using standard compounds having the same molecular weight of the compound families that constitute the essential oil (hydrocarbon monoterpenes, oxygenated monoterpenes, hydrocarbon sesquiterpenes and oxygenated sesquiterpenes). Table 1 lists the oil composition in the percentage of chromatographic peak areas. 1828 N.A. Awadh Ali et al. Downloaded By: [Marongiu, Bruno] At: 06:48 19 November 2010 3.5. Antimicrobial activity The organisms tested in this study are as follows: Escherichia coli (ATCC 35218), Staphylococcus aureus (ATCC 43300), Candida albicans (clinical strain) and Candida glabrata (clinical strain). Bacteria were cultured in Luria-Bertani (LB) broth and fungi in Sabouraud dextrose agar plates, overnight. MIC values were determined as the lowest essential oil concentration that inhibits the visible growth of the isolates after 24–48 h incubation at 37 C. It was measured with the broth dilution method (microdilution using 96-well microplates) (Carson, Hammer, & Riley, 1995; Deriu et al., 2008). Nine different concentrations of each essential oil from 2.5% (v/v) to 0.001% (v/v) with 10% Tween 80 were used. The bacterial and fungal cultures were diluted with LB broth and Roswell Park Memorial Institute (RPMI) medium, respectively, to obtain 1.0  108 CFU mL 1 (0.5 MacFarland). MCC values were determined as the lowest essential oil concentration that kills both bacteria and fungi. It was measured with the broth dilution method starting from MIC as the lowest concentration to the maximum one (2.5% v/v). Positive and negative controls were also included in the test. References Adams, R.P. (2004). Identification of essential oil components by gas chromatography/mass spectroscopy. Carol Stream, IL: Allured Publishing. Carson, C.F, Hammer, K.A., & Riley, T.V. (1995). 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