The in vitro cytotoxic activity of ethno-pharmacological important plants of Darjeeling district of West Bengal against different human cancer cell lines

Fresh leaves of plants as listed in Table 1 were collected from Darjeeling district of West Bengal, India. The specimens were identified and the voucher specimens were assigned specific reference number (Table 1).The leaves were washed thoroughly, air-dried and crushed. The crushed samples were extracted with 95% ethanol (1 g in 10 ml ethanol) for 72 h at 37°C with occasional shaking. After 72 h, the extract was filtered through a Whatman no. 1 filter paper and the filtrate was evaporated to dryness using a rotary evaporator (RV 10 Digital, IKA, Germany). The dried samples were stored at −20°C until use.

The human breast adenocarcinoma cell line (MCF 7), human hepatocarcinoma cell line (HepG₂) and human cervix adenocarcinoma cell line (HeLa) was obtained from National Centre for Cell Science, Pune, India. All cell lines were cultured in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 mg/ml streptomycin, 0.14% sodium bicarbonate and 0.1 mM sodium pyruvate. The cell lines were maintained in CO₂ incubator (N-Biotech) at 37°C in a 5% CO₂ atmosphere with 95% humidity.

Stock solutions at a concentration of 10 mg/ml were prepared by reconstituting the dried alcoholic extracts in dimethyl sulfoxide (DMSO, Hi-Media). The cytotoxic effect of each of the plant leaf extract was evaluated by tetrazolium- dye, MTT, assay [17,18] with slight modifications. Briefly, the cancerous cells (MCF 7, HepG₂ and HeLa) were seeded in 96-well plates at a density of 5×10³ cells/well in 200 μl culture medium. Following 24 h incubation and attachment, the cells were treated with different concentrations of extracts and similar concentration of diluents (DMSO) for further 24 h. After treatment, media was replaced with MTT solution (10 μl of 5 mg/ml per well) prepared in PBS and incubated for 3 h at 37°C in a humidified incubator with 5% CO₂. The yellow MTT dye was reduced by succinate dehydrogenase in the mitochondria of viable cells to purple formazan crystals. To solubilize the formazan, 50 μl of isopropanol was added to each well. The plates were gently shaken for 1 min and absorbance was measured at 600 nm, with reference 490 nm, by microtiter plate reader (MIOS Junior, Merck). The percentage of cytotoxicity was calculated as (Y-X)/Y x 100, where Y is the mean optical density of control (DMSO treated cells) and X is the mean optical density of treated cells with plant extracts.

Cells were seeded in 35 mm polyvinyl coated cell culture plates and allowed to attach at 37°C for 24 h in CO₂ incubator. The following day, cells were treated with either 30 μg/ml plant leave extract or DMSO alone, serving as control, and incubated again at the same conditions. The morphological changes of cancerous cells under treated and control conditions were compared by monitoring with phase contrast inverted microscope (Olympus, CK40-SLP) at 200X magnification. The images were photographed at 8, 16, and 24 h of incubation.

After morphological assessment, the cell viability was simultaneously assessed by Trypan blue dye exclusion assay [19]. For this, the cells were trypsinized with 0.25% trypsin-EDTA solution, resuspended in phosphate buffer saline (PBS) and stained with 0.4% Trypan blue dye solution (v/v in PBS). Within two minutes, the cells were loaded in a Neubauer chamber and the number of viable and non-viable cells per 1 x 1 mm square was counted under phase contrast microscope. The dead cells, because of losing the semi permeability of membrane, retained the blue dye and hence are coloured whereas viable or live cells remained unstained. The cells/ml was calculated as average cell count x dilution factor x 10⁴ cells/ml. The % cell viability was determined as [(no. of viable cells/ total no. of viable + non-viable cells) x 100. The percentage of growth inhibition was represented as {cell viability (control) – cell viability (with extract)}.

Phytochemical screening by means of TLC was carried out for selected plants following the method of Wagner and Bladt [20]. For this, the dried extracts were reconstituted in ethanol to a concentration of 10 mg/ml. 20 μl of the extracts were spotted, in triplicate, on aluminium-backed TLC plates (Merck, silica gel 60 F254). The chemical constituents were separated using any one of the three different eluent systems. For polar/neutral eluting system, solvents used were ethyl acetate/methanol/water (40:5.4:5); for intermediate polarity/acidic elution, solvents ethyl acetate/formic acid/glacial acetic acid/water (10:1.1:1.1:0.5) were used; and for non-polar/basic elution, hexane/ethyl acetate (3:1) solvents were used. The TLC plates were dried under a stream of cold air until there was no solvent smell remaining to ensure complete removal of the eluting solvents. The plates were examined under UV light (365 nm) to detect coumarins that appear as blue, violet or yellow fluorescent spots. The specific groups present in the extracts were identified using specific developers. Vanillin-sulphuric acid spray reagent (0.1 g vanillin:28 ml methanol:1 ml sulphuric acid) were then sprayed on the dried plates and heated at 110°C for colour development for detecting the presence of monoterpene alcohol, bitter principle and saponin. Sprinkling the plates with 5% ethanolic solution of aluminum Chloride (AlCl₃) resulting in the appearance of yellow or greenish fluorescent spot under UV light at 365 nm indicated the presence of flavonoids. A brown or yellowish coloration with Lieberman-Burchard’s reagent reveals the presence of triterpenes and steroids. A 10% vanillin ethanol solution was used for detecting saponins, the presence of which results in blue, violet and sometimes yellow spots. Using a 2% ferric chloride solution, development of chestnut, violet, green and blue colour reveals the presence of polyphenols, whereas a bluish or greenish black colour would indicate a positive test for tannins.

Results were presented as Mean ± SD of triplicates from three independent experiments. To calculate the concentration required to produce 50 % reduction in cell viability (IC₅₀) regression analysis was performed using Microsoft Excel 2010. Data were analyzed and compared by one-way analysis of variances (ANOVA) by using the software SPSS 15.0 for windows (SPSS Inc. Chicago, IL, USA) and differences with p < 0.05 were considered significant.