2.1 Preparation of extracts
Fresh fruits of Terminalia chebula were collected from Kannur district of Kerala, India, and were taxonomically identified by Dr. Sujanapal P, Scientist, Kerala Forest Research Institute (KFRI), Thrissur, Kerala, India. They were washed with running water followed by double-distilled water. It was air-dried in the dark and was powdered coarsely. Shade-dried fruit powder of the plant was weighed and extracted with 80% methanol using the Soxhlet method, for 24 h. The methanol extract was decanted, and the extract was filtered through Whatman filter paper No 1. The solvent extract was concentrated, and the dried extract was stored at 4 °C for further analysis.
2.2 Extraction and isolation of phenolics from Terminalia chebula
Eighty percent of methanolic extracts were washed with petroleum ether to remove fatty matter. The filtrate was partitioned with diethyl ether and ethyl acetate. Diethyl ether and ethyl acetate fractions were concentrated by vacuum oven and further subjected to column chromatographic purification. The column was eluted with hexane to ethyl acetate and ethyl acetate to methanol in varying ratios [20]. Fractions of 20 ml with each solvent system were collected and were analyzed by TLC. The fractions showing similar Rf values were pooled together and concentrated in vacuum to isolate the active principles. All the pooled fractions were tested for the presence of phenolics.
2.3 Tests for phenolics
2.3.1 Ferric chloride test
The sample was treated with few drops of ferric chloride solution; the formation of bluish-black color indicated the presence of phenolic compounds.
2.4 Structural characterization of the compound
Many fractions showing positive results for phenolics were collected and were also subjected to in vitro antioxidant activity studies. The active fraction which showed the highest antioxidant activity was selected for further structural elucidation. The chemical structure of the isolated compound was determined by proton nuclear magnetic resonance spectroscopy (1H NMR) and mass spectroscopy. NMR spectra were taken on a Bruker Ascend 500 MHz spectrometer for 1H NMR and 125 MHz for 13C NMR. The spectra were recorded using CD3COCD3 (deuterated acetone) as solvent and TMS (tetramethyl silane) as the internal standard. Mass spectrum of the purified compound was recorded on a Thermo Fisher Scientific high-resolution mass spectrometer (HRMS).
2.5 Cytotoxicity of phenolic compound isolated from diethyl ether extract of Terminalia chebula towards HeLa cell line
Cytotoxicity of isolated phenolic compound from diethyl ether extract of Terminalia chebula fruit towards HeLa cell lines was evaluated by MTT assay. Initially, cervical cancer cells line HeLa was obtained from NCCS, Pune, India, and maintained in DMEM (Sigma Aldrich, USA) supplemented with L-glutamine, 10% FBS, sodium bicarbonate, penicillin (100 U/ml), streptomycin (100 μg/ml), and amphotericin B (2.5 μg/ml). Cells were cultured in 25 cm2 tissue culture flasks and kept at 37 °C in a humidified 5% CO2 incubator (NBS Eppendorf, Germany). Sub-culturing of the cells was performed by trypsinization, and the cells were maintained in DMEM. The experiment was repeated twice in triplicates.
2 × 103 cells were seeded in a 96-well plate and incubated for 24 h. After 24 h treatment, the cells were treated with different concentrations of extracts. Untreated cells served as negative control, and the cells were incubated for 24 h at 37 °C and 5% CO2. At the end of the opted time of incubation, 10 μl MTT (5 mg/ml) was added to each well and the plates were incubated for 4 h. Supernatants were removed, and the resultant formazan crystals formed were dissolved in 100 μl DMSO. The extent of MTT reduction was measured at 590 nm with reference wavelength at 620 nm using a microwell plate reader [2, 21]. All absorbance values were corrected against blank wells which contained growth media alone. For each test concentration, the mean absorbance of the triplicated wells was noted. Mean absorbance of the cells grown in the absence of test compound was taken as 100% cell survival.
Percentage cell viability was calculated by using the following formula:
$$ \%\mathrm{Cell}\ \mathrm{viability}=\frac{\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{sample}\times \kern0.36em 100}{\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{control}} $$
2.6 In vitro antigenotoxic activity of phenolic compound isolated from Terminalia chebula fruit by comet assay
Initially, the cell lines (cervical cancer cell HeLa) were obtained from NCCS, Pune, India, and maintained in DMEM (Sigma Aldrich, USA) supplemented with L-glutamine, 10% FBS, sodium bicarbonate, penicillin (100 U/ml), streptomycin (100 μg/ml), and amphotericin B (2.5 μg/ml). Cells were cultured in 25 cm2 tissue culture flasks and kept at 37 °C in a humidified 5% CO2 incubator (NBS Eppendorf, Germany). Sub-culturing of the cells was performed by trypsinization, and the cells were maintained in DMEM. The experiment was repeated twice in triplicates.
The cells were treated with the isolated compound for 1 h at 37 °C. The concentration of compounds was chosen from the range of non-cytotoxic concentration as assessed by MTT assay. DNA damage was induced by 50 μM H2O2 for 15 min and incubated for 24 h. The adherent cells were then trypsinized, centrifuged, and resuspended in ice-cold PBS and were mixed well with 50 μl of 0.5% low melting point agarose (LMPA) at pH 7.4 at 40 °C. Microscopic slides were cleaned using alcohol and cleared in flame. Then, the slides were frosted and coated with 1% normal melting point agarose (NMA) and stored at 4 °C. 1.6 ml sample was immediately pipetted onto a frosted glass slide pre-coated with a layer of 1% normal melting point agarose prepared in PBS and covered with a glass coverslip. The agarose was immediately placed in a refrigerator for 5 min to allow complete agarose solidification. The coverslip was removed, and slides were immersed in a lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, NaOH to pH 10 and 1% Triton X-100) at 4 °C for 1 h. DNA was allowed to unwind for 20 min in freshly prepared alkaline electrophoresis buffer (1 mM Na2EDTA, 0.3 N NaOH, pH 13). The slides were then placed in a horizontal electrophoresis tank, and electrophoresis was performed at 12 V/cm for 20 min at an ambient temperature of 4 °C. The slides were then washed three times with 1× Tris for 5 min with neutralizing buffer (0.4 M Tris-HCl buffer, pH 7.4) before staining with 20 μl ethidium bromide (20 μg/ml) [10, 13].
