Cocculus hirsutus is a perennial climbing shrub having a broad spectrum activity against several ailments. Flavonoids present in a crude ethanolic extract of Cocculus hirsutus leaves were analysed. Rutin, liquirin and quercetin were used as calibration standards. Objective of this research was to develop and validate HPLC method for simultaneous determination of rutin, liquiritin and quercetin in Cocculus hirsutus leaves. The analysis was performed by reverse phase chromatography using Sil C-18 column (250 x 4.6 mm I. D., 10 μm particle size) as a stationary phase and methanol, water and 1 % acetic acid (40:60:2 v/v/v) as a mobile phase with an isocratic flow rate of 1 ml/min for 20 minutes and detection at a wavelength of 360 nm. Chromatographic conditions were optimized and results showed good peak resolution among rutin, liquitin and quercetin with retention time of 2.85, 3.79 and 15.44 min. respectively. Calibration curves were acquired with r2 > 0.997 and relative standard deviation values for intra- and inter-day precision were less than 2.0%. The recovery rate of each component in Cocculus hirsutus leaves in the range of 97.71 - 99.49 %. The contents of rutin, liquirin and quercetin in Cocculus hirsutus leaves were 48.11±0.03, 0.038±0.05 and 17.828±0.04%. Thus the proposed method was statically validated as per ICH guidelines. The proposed HPLC method can be implemented for simultaneous estimation of rutin, liquirin and quercetin in herbal medicinal plants and their products.
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