Cancers belong to a group of disorders with difficulty in treatment and sometimes are incurable. Nowadays, considerable scientific and commercial interests have been increased for discovering new anticancer agents with natural sources [1-4]. Plants have been sources of the well known anticancer drugs such as camptothecin, podophyllotoxin and paclitaxel . The potential of natural products as anticancer agents was recognized for the first time in the 1950s by the U.S. National Cancer Institute (NCI), and after that many investigations have been performed to the discovery of new natural anticancer agents . Different methods are used for screening of anticancer agents. One of the techniques is MTT [3-(4,5-dimethylthiazol-2yl)-2,4-diphenyltetrazolium bromide] assay which is a simple and reliable method for preliminary evaluation of anti-cancer agents .
Ferula genus (Apiaceae) is one of the most important genders in Iran. These plants were used in traditional medicine as anticonvulsion, anti-swelling, anti-spasm and expectorant . The genus has been represented in Iran by 31 species of which 16 are endemic to Iran . Many investigations have proved different biological activity of these plants . It has been established that some Ferula species have cytotoxic activity [11, 12]. F. persica is one of the endemic plants to Iran , and some investigations have been carried out to isolate, purify and elucidate the structure of its compounds [13- 15]. But, there is no report on cytotoxic effects of the plant on MDBK, A549, HT29, HepG2 and MCF7 cell lines. F. hezarlalehzarica has been identified for the first time in 2008 from Iran  and no phytochemical and biological study has been done on the plant so far. In this investigation, cytotoxic activity of F. persica var. persica and F. hezarlalehzarica aerial parts on some cell lines has been determined by using MTT method.
2. Material and methods
Methyl Thiazol Tetrazolium (MTT) was obtained from Sigma (Germany). Methanol,dimethyl sulfoxide (DMSO) and all other organic solvents (analytical grade) were purchased from Merck (Germany).
2.2. Plant extraction and fractionation
Total plant extracts were obtained by extraction of dried and milled aerial parts of the plants with 80% methanol (1:10) using maceration method for 4 days. After every 24h, the mixture was filtered and new solvent was added to the plant powder. The combined extracts were concentrated to dryness under vacuum pressure.
Different solvents containing hexane, chloroform, ethyl acetate, 100% and 50% methanol were used for fractionation. Each solvent was used for extraction during four days as same as total extract method. After 4 days, the plant powder was dried and new solvent was added. Combined extracts of each solvent were mixed and dried.
Figure 1. Viability (%) of tested cell lines against different concentrations of F. persica var. persica hexane fraction.
Figure 2. Viability (%) of tested cell lines against different concentrations of F. persica var. persica chloroform fraction.
2.3. Preparing the extracts for MTT assay
All samples were dissolved in DMSO to make the stock solution (100 μg/ml). But 50% methanolic extract of F. hezarlalehzarica was dissolved in water. Serial dilutions were prepared accordingly from the above stock solution to get the final concentrations (50, 25, 12.5, 6.25 and 3.125 μg/ml) with DMSO or water.
2.4. Cell cultures
HepG2 (human hepatocellular liver carcinoma), MCF7 (human breast adenocarcinoma), HT29 (human colon adenocarcinoma), A549 (human lung adenocarcinoma) and MDBK (bovine kidney cells) cell lines were obtained from Pasteur Institute of Iran. Each cell line was cultured in suitable medium for desired growth, completed with FBS (5 or 10%) in ahumidified incubator at 37 °C in an atmosphere of 5% CO2. Then the growth curve of each cell line was plotted
2.5. MTT assay
Cytotoxic activity was assessed by MTT assay according to the method which proposed by Alley et al.  using some modification, with different concentrations of the plants extracts. The cells were seeded in 96-well plates. Three wells for each concentration were seeded and triplicate plates were used for each cell line. Then, the cells were incubated in CO2 incubator. After 24 h, the medium was replaced by fresh medium containing different concentrations of the plants extracts and incubated for further 72 h. The initial concentration of samples was 100 g/ml in DMSO, which was serially diluted in the medium with two fold dilutions to give six concentrations. The medium was changed by fresh medium containing MTT with a final concentration of 0.5 mg/ml. The cells were incubated for another 4 h in a humidified atmosphere at 37 °C and after that the medium containing MTT was removed and remaining MTT-formazan crystals were dissolved in DMSO. The absorbance was measured at 570 nm using a microplate reader and viability of the cells was investigated relative to the negative control which was exposed to the solvent without extract. Tamoxifen was included as positive control.
Figure 3. Viability (%) of tested cell lines against different concentrations of F. hezarlalehzarica hexane fraction.
3. Results and discussion
The results of cytotoxic assay of F. persica var. persica and F. hezarlalehzarica aerial parts showed that among different samples, only hexane and chloroform fractions of the plants exhibited cytotoxic effects up to concentration of 100 μg/ml (Figures 1-4), in the manner that both fractions of F. persica var. persica were more cytotoxic than F.ezarlalehzarica fractions against MCF7, HepG2, MDBK, A549 and HT29 cell lines, except that hexane fraction of F. persica var. persica, which showed no cytotoxicity against MDBK cells up to concentration of 100 μg/ml (Figures 5 and 6). The highest cytotoxicity was observed in MDBK cell line by chloroform fraction of F. persica var. persica (IC50, 22.3 μg/ml). In order to consider a compound as cytotoxic agent in treatment of cancers, it is best to have no cytotoxicity on normal cell lines such as MDBK. Regarding cytotoxicity of the chloroform fraction of F.persica var. persica and hexane fraction of F.hezarlalehzarica on MDBK cell line, it seems that they are not the first choices for further evaluations in cancer researches. Of course, it is possible to purify a compound without cytotoxicity on MDBK but with cytotoxic effect on other cell lines. The results also demonstrated that F. hezarlalehzarica chloroform extract is toxic only against A549cell line (IC50, 105.3 μg/ml which obtained from extrapolation), therefore, it is a suitable choice for more investigations in lung cancer.
Figure 4. Viability (%) of tested cell lines against different concentrations of F. hezarlalehzarica chloroform fraction.
Lack of cytotoxicity of the total extracts of the plants and cytotoxicity of the non-polar fractions (hexane and chloroform fractions) shows that in order to evaluate of the plant for further experiments, fractionation is necessary.
Several investigations demonstrated the cytotoxic activity of Ferula species. Bagheri et al.  determined the cytotoxicity of some Ferula species on Artemia salina as a model for evaluating general cytotoxicity. They proved that methanolic extract of F. diversivittata, F. persica, F. ovina, F. badrakema, F. latisecta and oleo gum resin ofF. assa-foetida were cytotoxic with LC50 values in the range of 6-321 μg/ml. Anotherstudy on F. assa-foetida showed that some compounds were toxic against HepG2, Hep3B and MCF7 cancer cell lines . Ferula species are rich in coumarin compounds. Many biological activity of Ferula genus such as antibacterial, antiviral, cytotoxicity and anti-inflammatory effects have been attributed to sesquiterpene coumarins .
Iranshahi et al. have studied cancer chemoprotective activity of some terpenoid coumarins purified from Ferula species through inhibition of Epstein - Barr virus early antigen (EBV-EA) activation in Raji cells. Auraptene (7-gerany loxycoumarin) and umbelliprenin (7-farnesy loxycoumarin) were found as antitumor-promoting agents. They proposed that prenyl moiety in terpenoid coumarins plays an important role in anti-tumor promoting activity as previously reported for flavonoids, coumarins, phenylpropanoids and xanthones . An investigation on farnesiferol A (fromF. persica) and galbanic acid (from F. szowitsiana) with sesquiterpene coumarin structure showed that they could inhibit Pglycoprotein-mediated rhodamine efflux in a doxorubicin resistant breast cancer cell line(MCF7/Adr). These two compounds were considered for further studies on the reversal of multi-drug resistance phenotype in chemotherapy of cancer patients . Regarding the reported cytotoxicity of terpenoid coumarins and similarity of their structures in different Ferula species, these compounds may be considered as cytotoxic agents in F. persica var. persica and F. ezarlalezarica as well, but further studies are necessary to establish this idea.
Figure 5. The IC50s of hexane and chloroform fractions of Ferula persica var. persica
Figure 6. The IC50s of hexane and chloroform fractions of Ferula hezarlalehzarica
The authors wish to thank Traditional Medicine and Materia Medica Research Center, Shahid Beheshti University of Medical Sciences for supporting the project (No. 121) and Mr. H. Moazzeni and Mrs. A. Pirani for collection and identification of the plants. This manuscript has been obtained from Pharm. D. student thesis.