Cumhuriyet Üniversitesi Fen Fakültesi Cumhuriyet University Faculty of Science Fen Bilimleri Dergisi (CFD), Cilt:36, No: 3 Özel Sayı (2015) Science Journal (CSJ), Vol. 36, No: 3 Special Issue (2015) ISSN: 1300-1949 ISSN: 1300-1949   Cytotoxic Activities of Salvia Sahendica Extract on HeLa Cells Fatemeh SINA1, Farkhondeh NEMATI1,* 1 Department of Biology, Qaemshahr Branch, Islamic Azad University, Qaemshahr, IR Iran Received: 01.02.2015; Accepted: 05.05.2015 ______________________________________________________________________________________________ Abstract. The study was aimed to evaluation of the anticancer activity of the Salvia sahendica on the HeLacell line. The cytotoxicity of S. sahendicaon HeLacell was evaluated by the MTT assay.Results showed thatS. sahendicaethanolic extract has significant cytotoxiceffect on HeLacell line in concentration range between 0.156 mg/ml to 1.25 mg/ml. Maximum cytotoxiceffects were found in HeLa cells after incubation with the S. sahendica extract at 0.625 mg/ml (68.02%). Also results showed the concentrations producing 50% growth inhibition (IC50) of the S. sahendica extracts was 3.45 mg/ml. The S. sahendica extract was found dose inhibits the proliferation of cancerous HeLa cells possibly couses through rich secondary methabolits. Based on the results of this experiment extract of S. sahendica has cytotoxiceffect on HeLa cell line, and could be usefull in variety fields as the anticancer complex. Keywords: Salvia sahendica, ethanolic extract, cytotoxic, MTT assay 1. INTRODUCTION Cancer is major health problem in both developed and developing countries. Cancer aftercardiovascular disease is the second leading cause of death. It is the abnormal growth ofcells in our bodies and is a complex genetic disease that is caused primarily by environmental factors, that can lead to death. Because of high death rate associated with cancer and because of serious side effects of chemotherapy and radiation therapy, many cancer patients seek alternative complementary methods of treatment. Plants have been used for treatingdiseases since time immemorial. Species of the genus Salvia (Lamiaceae) are well known throughout the world for theirmedicinal properties which have made it an attractive choice for many researchers. Although some Salvia species have been scientifically studied in many parts of the world and are reported to have various biological activities including antibacterial (Ulubelen etal., 2001), anti-oxidant (Couladis et al., 2003), anti-inflammatory (Perry et al., 2003, anticancer (Liu et al., 2000) and anticholinesterase (Perry et al., 2003), but there is little scientific basis to validate the claims by traditional medicine practitioners about the effectiveness of local indigenous Salvia species.A literature search indicated that the genus Salvia has been a popular topic in phytochemical and ethnobotanical research. The solvent extracts, essential oils and compounds isolated from Salvia species revealed that they displayed a broad range of pharmacological properties, both in vitro and in vivo.Salvia sahendica, also known as Maryam Goli sahandy, is one of the native plants used to Persian medicinal herbs.From 900 Salvia species which are distributed in the world, about 17 species are endemic of Iran (Mozafarian, 1996). S.sahendica extract (endemic of Iran) is traditionally used for antibacterial, anti-fungi proposes and treatment of dyspepsia in many part of Iran (Lotfipour et al., 2007). In addition, the essential oils and various extract of S. sahendica were found to have antioxidant potential (Salehi et al., 2007). _____________ *Corresponding author. Email address: farkhondehnemati@gmail.com. Special Issue: The Second National Conference on Applied Research in Science and Technology http://dergi.cumhuriyet.edu.tr/cumuscij ©2015 Faculty of Science, Cumhuriyet University Cytotoxic Activities of Salvia Sahendica Extract on HeLa Cells The objective of this study was to examine the in vitro cytotoxic activities of ethanol standardized extract, onHeLa cell lines, using a MTT cytotoxicity assay. 2. MATERIALS AND METHODS 2.1. Preparation of plant extract Salvia sahendica plants were collected from the west part of Iran (tabriz) in April 2013. Mr. Eslami from the Department of Biology of Qaemshahr, Iran identified the plant. The whole parts of the plant were shade dried and grinded into powder. Extraction of ethanolic extract was carried out by macerating 40 g of powdered dry plant in 120 ml of 96% ethanol for 48 h at room temperature. Then, the macerated plant material was filtered through Whatman filter paper (NO.4) and dried under reduced pressure at 37 ºC with rotator evaporator and then evaporated to dryness. Briefly, the concentrated plant extracts were dissolved in dimethyl sulphoxide (DMSO) (SIGMA, USA) to obtain appropriate solutions of the extracts.The sub-stock solutions of 0.156, 0.312, 0.625, 1.25, 2.5, 5, 7.5 and 10 mg/ml was prepared by diluting the stock solution into serum-free culture medium, RPMI 1640 (the percentage of DMSO in the experiment should not exceed 1). The stock and sub-stock solutions were both stored at 4 ºC. 2.2. Cell cultures Human cervical cancer cell line (HeLa) were purchased from Pasteur Institute of Iran (NCBI C115). The cells was grown and maintained in a humidified incubator at 37 ºC and in 5% CO2 atmosphere. RPMI-1640 medium (SIGMA) was supplemented with 0.01 mg/ml heat inactivated Fetal Calf Serum (FCS), 100 units/ml penicillin, and 100 µg/ml streptomycin (ALL FROM INVITROGEN GIBCO) was used for cell cultures. Upon reaching confluency, the cells were passaged. After being harvested from sterile T75 culture flasks, the cells were counted using a hemocytometer and cell viability was determined by trypan blue exclusion. Three thousand cells from log phase cultures were seeded in 100 µl of RPMI medium supplemented with 10% fetal bovine serum per well of 96-well flat-bottom culture plates (Phelan, 1998). Cells were incubated with the S.sahendica extract for a defined time (72 hours). Proliferative response and cell death of the S.sahendicaextract-treated cells were determined using MTT assay and cell death ELISA Cell Viability Assay, respectively. 2.3. MTT Cell viability assay Growth of tumoral cells quantitated by the ability of living cells to reduce the yellow dye 3(4,5-dimethyl- 2-thiazolyl)-2,5-diphenyl-2H-terazolium bromide (MTT) to a blue formazan product. At the end of 72 hours incubation, the medium in each well was replaced by MTT solution (20 cell/well, 5 mg/ml in phosphate-buffered saline), the plates were incubated for 4 hours under 5% CO2 and 95% air at 37ºC. MTT reagent was removed and the formazan crystals produced by viable cells were dissolved in 100 DMSO and gently shaken. The absorbance was then determined by ELISA reader at 570 nm. The percentage growth inhibition was calculated using following formula, % cell inhibition = 100- [(At-Ab)/(Ac-Ab)] × 100 Where, At = absorbance value of test compound, Ab = Absorbance value of blank and Ac = Absorbance value of control. The effects of extracts were expressed by IC50 values (the drug concentration reducing the absorbance of treated cells by 50% with respect to untreated cells). 2333     SINA, NEMATI 3. STATISTICAL ANALYSIS The data are expressed as mean ± standard deviation (SD) for at least three independent determinations in triplicate for each experimental point. The data were analyzed using IBM SPSS Statistics 20 software. For all the measurements, Tow-way ANOVA followed by Duncan’s New Multiple Range Test (P≤0.05) was used to assess the statistically significance of difference between control and treatments. 4. RESULTS To investigate the cytotoxic potential of extracts were prepared according to thetraditional use in Iran from Iranian plants used in traditional medicine for the treatment of various diseases such as cancer, inflammation or infectious diseases. We collected the plant of S. sahendicain order to screen them for possible cytotoxic activity against human cervical cancer cell lines (HeLa) at different concentrations to determine the IC50 (50% growth inhibition) by MTT assay. 4.1. Effects of ethanolic extract of S.sahendicaon proliferation of HeLa cells Results of different concentrations ofS. sahendica including 0.156, 0.312, 0.625, 1.25, 2.5, 5, 7.5 and 10 mg/ml are tabulated in Table 1. MTT assay of S. sahendica shows significant effect on HeLa cell in concentration range between 0.156 mg/ml to 1.25 mg/ml compared with control. The highest cytotoxicity of this extract against HeLa cell was found in 0.625 and 0.156 mg/ml concentration with 68.02 and 66.16 percent of cell growth inhibition. It was found that the percentage of growth inhibition to be increasing with increasing concentration of test compounds, and IC50 value of this assay was 3.45 mg/ml, thus S. sahendica extract at 0.156 to 10 mg/ml exhibited dose-dependent inhibitory effects on the proliferation of HeLa cells (Figure 1). Table 1. Cytotoxicity activity of S. sahendicaextracts against HeLa cell line at different concentrations by MTT assay Concentrations of sahendica (mg/ml )S. 0.156 0.312 0.625 1.25 2.5 5 7.5 10 Control Monocyte DMSO Absorbance Inhibition (%) 0.308 ± 0.248* 0.360 ± 0.288* 0.300 ± 0.214* 0.355 ± 0.225* 0.541 ± 0.249 0.635 ± 0.250 0.786 ± 0.151 0.787 ± 0.100 0.800 ± 0.098 0.503 ± 0.140 0.746 ± 0.071 66.16 57.65 68.02 61.23 33.66 21.14 0.500 0.100 38.72 5.97 2334     IC50 (mg/ml) 3.45 % Cell Growth Inhibition Cytotoxic Activities of Salvia Sahendica Extract on HeLa Cells Concentration (mg/ml) Figure 1. Effect of different concentrations of S.sahendicaHeLa cell line in 72 h. Values represent the mean of three experiments. Overall, this study evaluate that ethanolic extract of S. sahendica has potential cytotoxic activity on Hela cell, indicating the presence of cytotoxic compounds in these extracts. This study provides only basic data, further studies are necessary for isolation and identification of biologically active substances from these extracts. 5. DISCUSSION On a whole, our goal was to determine whether the extracts of these plants exerted an inhibitory effect on cancer cell proliferation and caused cell death. The results of our studies suggest that ethanolic extract of S. sahendica possess the strongest cytotoxic effects on human cancer cell. In the present study, we observed that the extract of S. sahendica caused marked cell growth inhibition in the human cervical cancer HeLa cell line in a dose –dependent, but this effect is caused in the lower concentration.Studies have shown differential sensitivities to several natural compounds between tumor and normal cells in vitro, and the results obtained from the present study show that the ethanolic extract from S. sahendica is cytotoxic to HeLa cell lines. In this preliminary study, we have focused our interest on crude plant extracts, the cytotoxic activity couldbe due to the presence in the ethanolic extract of active products that could probably have highly anti-growth effects. On the other hand, some studiesrevealed the presence of terpenoid, flavonoids and alkaloids in the extract of S. sahendica, which could be responsible for this activity (Esmaeili et al., 2009; Salehi et al., 2007; Shaerzadeh et al., 2011a). Flavonoids have been found to possess antimutagenic and antimalignant effects (Mosmann, 1983; Brown, 1980). Moreover it has protective effect against cancer by their effect on signal transduction in cell proliferation and angiogenesis. Several studies have been reported on the phytochemical and other biological properties of S. sahendica(Salehi et al., 2007; Shaerzadeh et al., 2011b).For example Esmaeili et al. (2009) reported that S. sahendica has potent hepato-protective effect against alcohol induced tissues damage in experimentalanimals. This study also suggests that possible mechanism of this activity may be due to the presenceof flavonoids and phenolics compound(s) in the methanolic extract of S. sahendicawhich may be responsible to hepato-protective activity. They showed methanolic extract of S. sahendica had a potent increaseing effect on GSH level and vitamin C and E contents on liver and kidney tissues compared to alcohol treated rats. Also, Shaerzadeh et al. (2011b) investigated the protective effect of S.sahendica against H2O2-induced cell death in rat pheochromocytoma (PC12) cells. they results indicate that S. 2335     SINA, NEMATI sahendica protects PC12 cells treated with H2O2 via suppression of upstream factors of apoptosis pathway. Therefore, this plant could be as a source for new lead structures in drug design to combat cancer. It also justifies the folklore medicinal uses and claims about the therapeutic values of this plant as curative agent against cancer and we therefore, suggest further, the purification and characterization of the phytochemicals and isolatation the active constituent. In addition, it can be subjected to pharmacological screening of this plant along with investigations that are needed to provide some additional insight into the in vivo cytotoxic activity of the plants with a view to obtaining useful chemotherapeutic agent. Thus, further studies are required to elucidate the precise molecular mechanisms and targets for cell growth inhibition which will allow the rationale design for more effective molecules for the eventual use as cancer chemopreventive and/or therapeutic agents. The current study has demonstrated that ethanolic extract of a commonly used in Persian medicinal herb, S.sahendica in its natural form, could significantly suppress the proliferation of cervical cell line (HeLa) in vitro by means of the MTT assay.Based on the results of this experiment extract of S. sahendica has inhibitory effect on HeLa cell line, and could be usefull in variety fields as the anticancer complex. REFERENCES [1] Brown, J.P. 1980. A review of the genetic effect of naturally occurring flavonoids anthraquinones and related compounds. Mutat. Res., 75:243-247. [2] Couladis, M., Tzakou, O., Verykokidou, E., Harvala, C. 2003. Screening of some Greek aromatic plants for anti-oxidant activity. Phytotherapy Research 17, 194−195. [3] Esmaeili, M.A., Sonboli, A., Kanani, M.R. 2009. Salvia sahendicaprevents tissue damages induced by alcohol in oxidative stressconditions: Effect on liver and kidney oxidative parameters. J MedPlants 34: 276-283. (Persian). [4] Liu, J., Shen, H.M., Ong, C.N. 2000. Salvia miltiorrhiza inhibits cell growth and induces apoptosis in human hepatoma (HepG-2) cells. Cancer Letter 153, 85–493. [5] Lotfipour F, Samiee M, Nazemiyeh H 2007. Evaluation of the antibacterial activity of Salvia sahendica and Phlomis caucasica. Pharmaceutical sciences, Journal of Faculty of Pharmacy, Tabriz University of Medical Sciences 1: 29-34. [6] Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods;65(1-2):55-63. [7] Mozafarian, V. 1996. A dictionary of Iranian plant names (Latin English Persian) Farhang Moaser Publication, Tehran, pp. 477-479. [8] Perry, N.S., Bollen, C., Perry, E.K., Ballard, C. 2003. Salvia for dementia therapy: review of pharmacological activity and pilot tolerability clinical trial. Pharmacology,Biochemistry and Behavior 75, 651–659. [9] Phelan MC. 1998. Basic Techniques for Mammalian to Cell Tissue Culture. Current protocols in cell biology; 7: 1-10. [10] Salehi P, Sonboli A, Ebrahimi SN, Yousefzadi M 2007. Antibacterial and antioxidant activities of the essential oils and various extracts of Salvia sahendica in different henological stages. Chem. Nat. Comp. 43: 328-330. [11] Shaerzadeh, F., Ahmadiai, A., Esmaeili, M.A., Ansari, N., Asadi, S., Khoramian Tusi, S., Sonboli, A., Ghahremanzamane, M., Khodagholi, F. 2011b. Antioxidant and antiglycating activities of Salvia sahendica and its protective effect against oxidative stress in neuron-like PC12 cells. J. Nat. Med. 65:455-465. [12] Shaerzadeh, F., Alamdary, S.Z., Esmaeili, M.A., Sarvestani, N.N.,Khodagholi, F. 2011a. Neuroprotectiveeffect of salvia sahendica ismediated by restoration of mitochondrial function and inhibition ofendoplasmic reticulum stress. Neurochem Res. 36: 2216-2226. 2336     Cytotoxic Activities of Salvia Sahendica Extract on HeLa Cells [13] Ulubelen, A., Oksuz, Kolak U., Johansson, C., Celik, C., Voelter, W. 2000. Antibacterial diterpenes from roots of Salvia viridis. Planta Medica 66, 458–462. [14] Brown, J.P. 1980. A review of the genetic effect of naturally occurring flavonoids anthraquinones and related compounds. Mutat. Res., 75:243-247. [15] Couladis, M., Tzakou, O., Verykokidou, E., Harvala, C. 2003. Screening of some Greek aromatic plants for anti-oxidant activity. Phytotherapy Research 17, 194−195. [16] Esmaeili, M.A., Sonboli, A., Kanani, M.R. 2009. Salvia sahendicaprevents tissue damages induced by alcohol in oxidative stressconditions: Effect on liver and kidney oxidative parameters. J MedPlants 34: 276-283. (Persian). [17] Liu, J., Shen, H.M., Ong, C.N. 2000. Salvia miltiorrhiza inhibits cell growth and induces apoptosis in human hepatoma (HepG-2) cells. Cancer Letter 153, 85–493. [18] Lotfipour F, Samiee M, Nazemiyeh H 2007. Evaluation of the antibacterial activity of Salvia sahendica and Phlomis caucasica. Pharmaceutical sciences, Journal of Faculty of Pharmacy, Tabriz University of Medical Sciences 1: 29-34. [19] Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods;65(1-2):55-63. [20] Mozafarian, V. 1996. A dictionary of Iranian plant names (Latin English Persian) Farhang Moaser Publication, Tehran, pp. 477-479. [21] Perry, N.S., Bollen, C., Perry, E.K., Ballard, C. 2003. Salvia for dementia therapy: review of pharmacological activity and pilot tolerability clinical trial. Pharmacology,Biochemistry and Behavior 75, 651–659. [22] Phelan MC. 1998. Basic Techniques for Mammalian to Cell Tissue Culture. Current protocols in cell biology; 7: 1-10. [23] Salehi P, Sonboli A, Ebrahimi SN, Yousefzadi M 2007. Antibacterial and antioxidant activities of the essential oils and various extracts of Salvia sahendica in different henological stages. Chem. Nat. Comp. 43: 328-330. [24] Shaerzadeh, F., Ahmadiai, A., Esmaeili, M.A., Ansari, N., Asadi, S., Khoramian Tusi, S., Sonboli, A., Ghahremanzamane, M., Khodagholi, F. 2011b. Antioxidant and antiglycating activities of Salvia sahendica and its protective effect against oxidative stress in neuron-like PC12 cells. J. Nat. Med. 65:455-465. [25] Shaerzadeh, F., Alamdary, S.Z., Esmaeili, M.A., Sarvestani, N.N.,Khodagholi, F. 2011a. Neuroprotectiveeffect of salvia sahendica ismediated by restoration of mitochondrial function and inhibition ofendoplasmic reticulum stress. Neurochem Res. 36: 2216-2226. [26] Ulubelen, A., Oksuz, Kolak U., Johansson, C., Celik, C., Voelter, W. 2000. Antibacterial diterpenes from roots of Salvia viridis. Planta Medica 66, 458–462. 2337