Evaluation of Antiproliferative effect of Grewia hirsuta on HepG2 cell ...

Journal <strong>of</strong> Academia and Industrial Research (JAIR)Volume 2, Issue 1 June 2013 2Radical scavenging activity was expressed as theinhibition percentage <strong>of</strong> free radical by the sample andwas calculated using the formula:% DPPH activity = (Control OD–Sample OD/Control OD) × 100.Phosphomolybdenum assay: Antioxidant activity <strong>of</strong>samples was evaluated by green phosphomolybdenumcomplex formation according to Prieto et al. (1999).An aliquot <strong>of</strong> 100 μL <strong>of</strong> sample solution was combinedwith 1 mL <strong>of</strong> reagent solution (0.6 M sulphuric acid,28 mM sodium phosphate and 4 mM ammoniummolybdate) in a 4 mL vial. The vials were capped andincubated in a water bath at 95C for 90 min. Aftercooling, the absorbance <strong>of</strong> the mixture was measured at695 nm against a blank. Ascorbic acid (10 mg/mLDMSO) was used as standard. The results were reportedas % phosphomolybdenum reduction potential.Hydroxyl radical scavenging activity: The scavengingactivity <strong>of</strong> the methanol extracts on hydroxyl radical wasmeasured according to Klein et al. (1992). Variousconcentrations (250, 500, 750 and 1000 μg/mL) <strong>of</strong>extracts were added with 1.0 mL <strong>of</strong> iron‐EDTA solution,0.5 mL <strong>of</strong> EDTA solution and 1.0 mL <strong>of</strong> dimethylsulphoxide (DMSO). The reaction was initiated by adding0.5 mL <strong>of</strong> ascorbic acid and incubated at 80-90C for15 min in a water bath. After incubation the reaction wasterminated by the addition <strong>of</strong> 1.0 mL <strong>of</strong> ice‐cold TCA.Nash reagent (3 mL) was added and left at roomtemperature for 15 min. The reaction mixture withoutsample was used as control. The intensity <strong>of</strong> the colorformed was measured spectroscopically at 412 nmagainst reagent blank. The % hydroxyl radicalscavenging activity (HRSA) is calculated by the followingformula:% HRSA = [(Abs control‐Abs sample)/Abs control] × 100Where, Abs control is the absorbance <strong>of</strong> the control;Abs sample is the absorbance <strong>of</strong> the extract/standard.Metal chelating activity: The chelating <strong>effect</strong> <strong>of</strong> ferrousions by the extracts was estimated according to Diniset al. (1994). Hundred μL <strong>of</strong> the extract was added to0.05 mL <strong>of</strong> 2 mM FeCl 2 . The reaction was initiated by theaddition <strong>of</strong> 0.2 mL <strong>of</strong> ferrozine (5 mM) and the mixturewas shaken vigorously and left at room temperature for10 min. Absorbance <strong>of</strong> the solution was then measuredspectrophotometrically at 562 nm. The chelating activity<strong>of</strong> the extracts was evaluated using EDTA as standard.The results were expressed as % metal chelatingactivity. The ratio <strong>of</strong> inhibition <strong>of</strong> ferrozine Fe 2+ complexwas calculated as follows:% inhibition = (Control OD–Sample OD/Control OD) × 100.Qualitative and quantitative phytochemical screening:The extracts were subjected to preliminaryphytochemical screening to identify the presence <strong>of</strong>phytoconstituents such as alkaloids, flavonoids,saponins, tannins, phenols, glycosides and steroidsaccording to Harborne (1973).Estimation <strong>of</strong> total free phenolics: Total phenolicconstituents <strong>of</strong> plant extracts were estimated byFolin-Ciocalteau’s method using Folin-Ciocalteu reagent.The estimation was done spectrometrically at 760 nmand the results were expressed as gallic acid equivalents(GAE) (Sengul et al., 2009).Estimation <strong>of</strong> total flavonoids: Aluminium chloride methodwas employed to quantify the total flavonoid content inthe plant extracts. The results were expressed asquercetin equivalents (QE) (Chang et al., 2002).Estimation <strong>of</strong> total alkaloids: Total alkaloid content <strong>of</strong> theplant extracts was determined according to Sutharsinghet al. (2011). Five gram <strong>of</strong> the sample was filtered andconcentrated to one quarter <strong>of</strong> the original volume on awater bath after treatment with 200 mL <strong>of</strong> 10% aceticacid in ethanol. Concentrated NH 4 OH was addeddrop wise to the extract until the precipitation wascomplete. The whole solution was allowed to settle andthe precipitate was collected and washed with diluteNH 4 OH, filtered and weighed.Estimation <strong>of</strong> total saponins: Powdered sample (20 g)was treated with 100 mL <strong>of</strong> 20% aqueous ethanol,heated over a hot water bath for 4 h at about 55°C withcontinuous stirring. The mixture was filtered and theresidue re-extracted. The combined extracts werereduced to 40 mL over water bath at about 90°C and theconcentrate was transferred into a separating funnel and20 mL <strong>of</strong> diethyl ether was added and shaken vigorously.The aqueous layer was recovered while the ether layerwas discarded. The purification process was repeatedand 60 mL <strong>of</strong> n-butanol was added to the combinedextracts and washed twice with 10 mL <strong>of</strong> 5% aqueousNaCl. The remaining solution was heated in a waterbath, dried in an oven to a constant weight and thesaponin content was calculated as percentage(Sutharsingh et al., 2011).Thin layer chromatography: The plant extract was loadedon pre-coated silica plates which were then developedusing methanol, chlor<strong>of</strong>orm in the ratio <strong>of</strong> 1:9. The spotswere identified both in the UV light and in the iodinechamber. Then R f value was calculated as the ratio <strong>of</strong>distance travelled by the solute to the distance travelledby the solvent (El<strong>of</strong>f, 1998).Bioautography: The extract which showed DPPHinhibition <strong>of</strong> more than 90% was examined by thin layerchromatography (TLC) bioautography.©Youth Education and Research Trust (YERT) jairjp.com Ashana Ema et al., 2013