Determination of DPPH free radical scavenging activity by reversed-phase HPLC: A sensitive screening method for polyherbal formulations

The colorimetric method of evaluation of antioxidant activity by scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical is with certain shortcomings like failure to indicate antioxidant activity of certain drugs and interference from color pigments of natural products. A specific HPLC method was developed for evaluating the DPPH free radical scavenging activity of commercial polyherbal formulations using a LiChrospher® 100 RP-18e column (250 mm × 4 mm, 5 μM). The mobile phase was a mixture of methanol and water (80:20, v/v) pumped at a flow rate of 1 mL/min. The DPPH peaks were monitored at 517 nm. The method was standardized using known antioxidants such as ascorbic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), probucol and α-tocopherol. The 50% radical scavenging activity (IC50) determined by the HPLC method correlated well with that of colorimetry. This HPLC method was applied for the estimation of free radical scavenging activity of Silymarin and a few commercial hepatoprotective polyherbal formulations. While the colorimetric method failed to estimate the free radical scavenging activity of polyherbal formulations, HPLC method was free from interferences and was specific. The HPLC method is sensitive and can be used as a quality control tool for the rapid determination of free radical scavenging activity of variety of products including plant extracts, foods, drugs and polyherbal formulations.