Chemicals and reagents
Gallic acid, BHA (butylated hydroxyanisole), ascorbic acid, DPPH (1,1-diphenyl-2-picrylhydrazyl), potassium ferricyanide, Folin-Ciocalteu’s phenol reagent, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], RPMI 1640 medium and McCoy’s 5A medium were obtained from Sigma-Aldrich Company. Trichloroacetic acid, ferric chloride, ethanol, hexane and ethyl acetate were purchased from Merck Company. Foetal bovine serum, penicillin, streptomycin and fungizone were from PAA Lab (Austria). SOD (superoxide dismutase) kit was purchased from Sigma-Aldrich Company.
Plant sample collection and identification
The fresh leaves of L. indica were collected from Seremban, Negeri Sembilan, Malaysia in February 2011. The plants were identified by Dr Yong Kien Thai of Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia and a voucher specimen (herbarium no: KLU47724) was deposited at the herbarium of the Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.
Preparation of extracts
The extracts were prepared as previously described [15]. Briefly, the leaves of L. indica (2.70kg) were washed, dried (38°C) and ground to fine powder (1.60kg, 59.26%). The dried, ground leaves (300.30g) were extracted with ethanol (3x 1.5L) at room temperature yielding a dark green crude ethanol extract (27.80g, 9.26%). The ethanol extract (24.80g) was further extracted with hexane to give a hexane-soluble extract (5.80g, 23.39%) and a hexane insoluble residue. The hexane-insoluble residue was further partitioned between ethyl acetate–water (1:1, 100ml: 100ml) to give an ethyl acetate-soluble extract (3.60g, 14.52%). The water layer was freeze-dried to give a brown coloured fractionated water extract (3.60g, 14.52%). All the extracts (ethanol, hexane, ethyl acetate and water) were kept in the dark at 4°C for not more than one week prior to evaluation of total phenolic content, antioxidant effect and cytotoxicity.
Determination of total phenolic content
The concentrations of phenolic compounds in the extracts of L. indica leaves were measured according to the Folin-Ciocalteu method as previously described [16]. Briefly, extract solution (0.02ml) at different concentrations (concentrations ranging from 0 to 20mg/ml) was mixed with 1.58ml of distilled water. Folin-Ciocalteu’s phenol reagent (0.1ml) was then added to each test tube. After 3min, 0.3ml of saturated sodium carbonate solution was added to the mixture. The reaction mixtures were incubated in dark at 40°C for 30min. The absorbance was measured at 765nm with a spectrophotometer. All extracts were assayed in triplicate. Gallic acid solutions with concentrations ranging from 25 to 1000mg/l were used for calibration. A dose response linear regression was generated by using the gallic acid standard absorbance and the levels in the samples were expressed as gallic acid equivalents (mg of GAEs/g of extract).
Scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals
The scavenging activity of the extracts of L. indica on DPPH radicals was measured according to the method as previously described [16]. Briefly, extract solution with different concentrations (concentrations ranging from 0 to 5mg/ml) was mixed with 0.8% of DPPH solution. The reaction mixtures were incubated at room temperature and allowed to react for 30minutes in the dark. All measurements were done in dim light. The absorbance was measured at 520nm with a spectrophotometer. All assays were conducted in triplicate. The scavenging activity (%) on DPPH radical was calculated according to the following equation:
Scavenging activity (%)=[(Acontrol-Asample)/Acontrol] x 100%; where Acontrol is the absorbance of the control and Asample is the absorbance of the tested extract.
The scavenging ability of the extracts was expressed as EC50 value, which is the effective concentration at which 50% of DPPH radicals were scavenged. The EC50 value was obtained from the graph of scavenging activity (%) versus concentration of samples. Ascorbic acid was used as positive reference standard.
Reducing power assay
The reducing power of the prepared extracts was determined according to method as previously described [16]. Briefly, extract solution at different concentrations (concentrations ranging from 0 to 0.8mg/ml) was added with 2.5ml of 0.2M phosphate buffer (pH 6.6) and 2.5ml of 1% (w/v) solution of potassium ferricyanide. The mixture was incubated in a water bath at 50°C for 20min. Following this, 2.5ml of 10% (w/v) trichloroacetic acid solution was added and the mixture was then centrifuged at 1000rpm for 10min. A 2.5ml aliquot of the upper layer was combined with 2.5ml of distilled water and 0.5ml of a 0.1% (w/v) solution of ferric chloride. The absorbance was measured at 700nm with a spectrophotometer. All assays were conducted in triplicate. Ascorbic acid was used as positive reference standard.
Detection of superoxide dismutase (SOD) activity
SOD activity was measured using water-soluble tetrazolium salt (WST) according to the method described by [17]. This method utilizes Dojindo’s WST-1, which can produce a water soluble formazan dye upon reduction with superoxide anion. After addition of all the working solution and extract solution with different concentrations (concentrations ranging from 0 to 20mg/ml) in each well as described in the SOD kit manual, the ninety-six-well microplate was agitated and incubated at 37°C for 20min. Absorbance was taken using microplate reader (Oasys UVM340) at 450nm. Percentage inhibition of each sample was calculated by using following equation: {[(B1 – B3) - (S – B2)]/(B1 – B3)} x 100 where B1, B2, B3 and S were the absorbance at 450nm for Blank 1, Blank 2, Blank 3 and sample, respectively. BHA was used as positive reference standard.
Cell lines and culture medium
The colon cancer cell lines HT-29, HCT-15 and HCT-116 were purchased from American Type Culture Collection (ATCC, USA). The HCT-15 cells were maintained in RPMI 1640 medium; HCT 116 and HT-29 cells in McCoy’s 5A medium, supplemented with 10% foetal bovine serum, 2% penicillin or streptomycin and 1% of fungizone. The cells were cultured in a 5% CO2 incubator (Shel Lab water-jacketed) kept at 37°C in a humidified atmosphere.
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay
The cytotoxic activities of samples were evaluated using MTT assay according to the method described by Mosmann [18]. Cytotoxicity of each extract was expressed as IC50 value, which is the concentration of extract that reduced the viability of the cells by 50% compared to the control, which were treated with 0.5% DMSO. Three replicate plates were performed for each sample. Cis-platin was used as positive reference standard.
Statistical analysis
The antioxidant data in the present study were subjected to one-way analysis of variance (ANOVA) and the significance of the difference between the means was determined by the Duncan’s multiple range tests at 95% least significant difference (p<0.05). The Pearson correlation analysis was performed to determine the correlation between total phenolic content and antioxidant activity of the extracts. Statistical significance was set at p<0.05. The IC50 values for cytotoxic activity were obtained by non-linear regression using GraphPad Prism statistical software.