Icates. Hydroxyl radicalAbrahim et al. BMC Complementary and Alternative Medicine 2012, 12:220 http
Icates. Hydroxyl radicalAbrahim et al. BMC Complementary and Alternative Medicine 2012, 12:220 http://www.biomedcentral.com/1472-6882/12/Page 4 ofscavenging activity was calculated using the following equation: h of inhibition?Absorbance lank ? bsorbance ample ??=Absorbance lank ?X 100 Results were expressed as IC50, i.e. concentration of the plant extract required to inhibit 50 of the hydroxyl radicals.Analyses of phenolic compounds using high performance liquid chromatography (HPLC)Cell viability following treatment with the P. betle extracts was measured using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT is reduced by mitochondrial succinate dehydrogenase in viable cells, forming purple formazan crystals that can be measured at 595 nm. Briefly, 5 mg/ml MTT reagent in PBS were added to each treatment well and were incubated for 4 h. The media containing the MTT reagent were subsequently removed and replaced with 100 l of acidic isopropanol. The resulting absorbance was measured at 595 nm and cell viability was calculated based on the formula: Percentage of inhibition ??? total cells iable cells?total cellsX 100 All analyses were performed in triplicate.Analyses of antioxidant enzyme activitiesThe dried powder of P. betle leaf was subjected to acid hydrolysis to release free polyphenols from their glycosides, following the procedure described in Razali, MatJunit, Abdul-Muthalib, Subramaniam, Abdul-Aziz (2012) [22]. Briefly, In a glass vial, 20 mg of the dried P. betle powder in 1.2 M HCl and 50 methanol was heated at 90 for 2 h. The mixture was left to cool and was Cynaroside cost centrifuged at 3000 g for 10 min. The supernatant was collected and kept at -20 for the HPLC analysis. The samples were analysed using a Shimadzu HPLC system. Reverse phase separation was performed at 30 using a Waters C18 column (3.9 X 150 mm) (Waters, USA). The mobile phase consisted of trifluoroacetic acid (TFA) in water PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/29072704 at pH 2.6 (solvent A) and acetonitrile (solvent B). The flow rate was kept at 1 ml/min and the gradient programme consisted of: 7 to 40 B for 20 min, 40 to 100 B for 6 min and 100 to 7 B for 9 min. The eluted peaks were monitored at 260 nm. Data acquisition and processing was performed using a Lab Solution chromatography manager. 200 l of sample was injected into the HPLC. All samples were analyzed in triplicate.Cell cultureMCF-7 cells (1 x 106) were seeded in T-25 flasks containing RPMI 1640 medium supplemented with 10 FBS and allowed to attach for 24 h before treatment. Cells were treated with the ethyl acetate extract of P. betle at a final concentration of 64 g/ml (IC50 concentration determined from the MTT assay), at varying time points (0, 24 and 48 h). Following incubation, the cells were washed with PBS and detached using a scraper. Cells were then lysed in 1 ml of cold PBS using a sonicator, centrifuged for 10 min at 10000 rpm at 4 and the resulting supernatant was used for the antioxidant enzyme assays.Catalase (CAT) assayMCF-7 human breast cancer cells were utilised for the anti-proliferation study. The cells were maintained in RPMI 1640 culture medium supplemented with 10 (v/v) foetal bovine serum (FBS) (Flowlab, Australia), 10 g/ml bovine serum albumin and antibiotics and kept at 37 in T-25 tissue culture flasks (TPP, Switzerland). Cell were grown to confluence in a humidified atmosphere containing 5 CO2.Cell viability using MTT assayThis assay was performed using the Catalase Assay Kit.
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