Qualitative and quantitative analysis of biologically active principles ...

adical generated by the Fe 3+ /ascorbate/H 2O 2 system (Fenton
reaction). The hydroxyl radical attacks deoxyribose eventually
resulting in the formation of thiobarbituric acid reacting substances
(Elizabeth and Rao, 1990). The reaction system contained
deoxyribose (2.8 mM), FeCl 3 (0.1 mM), KH 2PO 4 KOH buffer (20 mM;
pH 7.4) and from 10 mg to 1000 mg/ml of the test material in a final
volume of 1 ml. The reaction mixture was incubated for 37 o C for 1
hour. The scavenging activity of hydroxyl radicals was expressed
as:
% of inhibition = (OD of Control – OD of treated/OD of Control) x
100
Lipid peroxidation assay
Lipid peroxidation induced in rat liver homogenate by the method of
Bishayee and Balasubramonian (1971) and was estimated by
thiobarbituric acid reactive substances by the method of Ohkawa et
al (1979). Different concentrations of the extracts were incubated
with 0.1 ml of rat liver homogenate (25%) containing 30 mM KCl,
Tris­HCl buffer (0.04 M, pH 7.0), ascorbic acid (0.06 mM), ferrous
ion (0.16 mM) in a total volume 0.5 ml for 1 hour at 37 o C. After
incubation, 0.4 ml of the reaction mixture was treated with 0.2 ml
SDS (8.1%), 1.5 ml thiobarbituric acid (0.8%) and 1.5 ml acetic acid
(20%, pH 3.5). The total volume was made up to 4 ml by adding
distilled water and kept in water bath at 95 o C for 1 hour. After
cooling, 1 ml distilled water and 5 ml of butanol­pyridine mixture
(15:1, v/v) was added. After vigorous shaking, the tubes were
centrifuged and the upper layer containing the chromophore was
measured at 532 nm. The lipid peroxidation inhibition was
calculated as:
% of inhibition = (OD of Control – OD of treated/OD of Control) x
100
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