Euphorbiaceae is a large family of flowering plants including 300 genera and over 5000 species . Euphorbia is the largest genus in this plant family with about 2000 known species . In the flora of Iran, the genus is represented by 70 species, of which 17 species are endemic .
Species of Euphorbia have been used in the treatment of cancer, asthma, leukemia, and some warts [4-6]. The plant is also used as a
laxative and diuretic in different parts of the world . Some of the Euphorbia species have cytotoxic, antiviral, antibacterial, and antifungal activities [8-10]. Latex of some species of Euphorbia (Farfion in Persian) has traditionally been used in the treatment of skin diseases, gonorrhea, migraines, intestinal parasites and warts . In several studies different extracts and isolated compounds from Euphorbia showed cytotoxic activity against cell lines such as KB, K562, CNE2, ANA-1, B16, CHO, Hep-2, and Hela [12-15].
In recent decades a number of species have been tested for their antitumor effects, partly on the basis of references to traditional usage. These bio-evaluations resulted in finding new compounds that are biologically active against tumor cells. To our best knowledge there is no published data regarding the cytotoxicity of E. macroclada Boiss. Therefore following our investigations on the characteristics of Iranian native Euphorbia, it was decided to evaluate cytotoxic activity of different extracts and latex of this plant against MDA-MB-468 as a breast cancer cell line using the in vitro colorimetric MTT assay.
2. Materials and methods
2.1. Plant material
E.macroclada Boiss. was collected at full flowering stage from Isfahan province in May 2005 at altitude of 2400m. The plant was identified by Prof. Rahiminejad, Department of Biology, University of Isfahan, Isfahan, Iran. A voucher specimen (no. 1117) is deposited in the Herbarium of Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran.
All chemicals and reagents used in extractions were of analytical grade and obtained from Merck (Darmstadt, Germany). Chemicals used in tissue culture assays were purchased from Gibco (Scotland) via local vendors. Taxol was obtained from Bristol-Myers Squibb Co. (USA).
2.3. Preparation of the extracts
Aerial parts of the plant were air-dried in shade at room temperature. The dried parts were powdered mechanically and 170 g was extracted by 500 ml of dichloromethane using maceration method. The residue was subjected to more extraction using 500 ml of ethylacetate and methanol, respectively. The obtained extracts were evaporated to dryness under reduced pressure. Crude white milky latex of E. macroclada was obtained through cutting and squeezing the stem of the fresh plants and dried in vacuum oven at 40 °C. Dried latex and extracts were stored at 4 °C until used. To evaluate the biological activity, 1 mg of the dried crude extracts of dichloromethane, ethylacetate, methanol, and dried latex were dissolved in 100, 80, 50, and 100 μl of DMSO, respectively. RPMI 1640 was added to the total volume of 1 ml and stored at 4 °C as stock solutions. To evaluate the cytotoxicity of each extract 5 different concentrations (10, 20, 30, 50 and 100 μg/ml) were prepared by diluting the stock solution in RPMI 1640, so that the highest concentration was 1 mg/ml except for latex which the highest concentration was 2 mg/ml. For each individual experiment final solutions were prepared freshly from stock solution.
Figure 1. Dose response curves for MDA-MB-468 cell line following 72 h continuous exposure to dichloromethane (a), ethylacetate (b), methanol (c) extracts and latex (d) of Euphorbia macroclada. Cells were pre-incubated for 24 h prior to extract addition.Negative control: Grey line; cells (1×106 cell/ml) were incubated with extract solvent and assumed as 100 % survival. Results are the mean of five determinations (Mean±SD) and are expressed as % cell survival.
2.4. Cell line
MDA-MB-468 (human breast adenocarcinoma) was purchased from Pasture Institute of Iran, Tehran. It was grown in RPMI 1640 supplemented with 10% (v/v) heat inactivated fetal calf serum, penicillin-streptomycin (100 IU/ml and 100 μg/ml, respectively), sodium pyruvate (1 mM), NaHCO3 (1 g) and L-glutamine (2 mM) and sterilized using 0.22 μ microfilters and stored at 4 °C before use.
2.5. Cytotoxic assay
The cytotoxic effects of extracts were determined against MDA-MB-468 cell line by a rapid colorimetric assay using 3- (4, 5-dimethylthiazol -2-yl)-2, 5- diphenyl tetra-zolium bromide (MTT) and compared with the untreated control . This assay is based on the metabolic reduction of soluble MTT by mitochondrial enzyme activity of viable cells into an insoluble colored formazan product, which can be measured spectrophotometrical-ly after dissolving in DMSO . For experiments, cells were plated in 96 -well micro plates (8000 cells / well in 200 μ l of medium) and incubated for 24 h (37 °C, 5% CO2, air humidified), then 20 μl of prepared concentration of each extract was added and incubated for further 72 h in the same condition.
Taxol was used as a positive control. The second column of each microplate was assumed as negative control (containing cell suspension with a concentration of 1×106 cell/ml incubated with extracts solvent). To evaluate cell survival, 150 μl of old media was replaced by fresh RPMI and 20 μl of MTT solution (5 mg/ml in phosphate buffer solution) was added to each well and incubated for 3 h. Then supernatant was removed and 150 μl of DMSO was added and pipetted to dissolve formazan crystals formed. Absorbance was then determined at 540 nm using an ELISA plate reader (startfix-2100, Awareness, USA). Each extract and latex concentration was assayed in 6 wells and each experiment was repeated at least 4 times(n ≥4).
Cell survival percentage was calculated using MTT assay, via following Equation.
% cell survival = Abs in drug treated wells-Abs in blank
Abs in control well-Abs in blank
Percentage of cell survival in the negative control was assumed as 100.
2.6. Statistical analysis
SigmastatTM was used to perform statistical test. Analysis of variance followed by post Hoc test was used to distinguish the differences among groups. Significance was assumed at 5% level.
Extracts which caused at least 50% growth inhibition were accounted as cytotoxic. The dichlormethane and ethylacetate extracts had shown cytotoxic effects on MDA-MB-468 cell line at concentrations of 30 and 50 μg/ml respectively (Figure 1); whereas methanol extract and latex were not cytotoxic even at highest applied concentrations (100 and 200 μg/ml, respectively). In these studies dichloromethane extract showed most cytotoxic effect against tested cell line (IC50 = 30 μg/ml).
Cytotoxic effects of some Euphorbia species have been already evaluated on different cell lines. Betancur-Galvis and co-workers  showed that dichloromethane extract (using soxhlet) of leaves of E. cotinifolia with IC50 of 35 and 18 μg/ml had most cytotoxic effect on HEP -2 and CHO cell lines, respectively, which is consistent with the results of this study. Our results showed that methanol extract of E. macroclada was not cytotoxic. This was consistent with Javidnia and co-worker's results  that showed the methanol extract of E. hebecarpa. was not cytotoxic against KB cell line, although this extract inhibited K562 and U937 growth by 66.3 and 56.1%, respectively, that can be related to various sensitivity of different cell lines.
Jassbi showed that polycyclic diterpenoids such as ingenol-type diterpenoids and triterpenoids isolated from some Iranian Euphorbia species are biologically active constituents that contribute to cytotoxic activity of Euphorbia genus . Ravikanth et al.  revealed that from 8 indol diterpenes isolated from the latex of E. nivulia, only 3 of them were cytotoxic against 3 tested cell lines, using MTT assay. Darwish et al.  in their studies used E. macroclada Bioss. latex against two different strains of Staphylococcus aurous (standard and resistant) and showed it was effective against standard strain but the growth of S. aurous was not affected significantly. Furthermore, Mucsi et al.  demonstrated that cytotoxicity of diterpenes was in a dose dependent manner.
Terpenoids as lipophilic compounds are efficiently extracted by nonpolar solvents like ethyl acetate, hexane and dichloromethane . This is consistent with our results showing cytotoxicity of ethyl acetate and dichloromethane extracts of E. macroclada with lower IC50.
In summary, cytotoxic compounds in this plant mostly belong to nonpolar constituents such as terpenoids and can be extracted by nonpolar solvents. Further investigations are underway for the isolation and identification of the active constituents of the extracts.
This study was financially supported by the research council of Isfahan University of Medical Sciences (Project No. 384110), Isfahan, Iran.