Abstract

DEVELOPMENT AND VALIDATION OF HPTLC AND HPLC METHODS FOR THE STANDARDIZATION OF STIGMASTEROL FROM THE EXTRACT OF ACALYPHA INDICA AND ITS FORMULATION

Jayaprakasam R*, M. Francis Saleshier, T.K. Ravi

ABSTRACT

Two simple, sensitive and precise HPTLC and HPLC methods were developed and validated for the estimation of stigmasterol from Acalypha indica (Euphorbiaceae) root extracts and from its dosage form Bronchorest capsules. For the estimation of stigmasterol by HPTLC method, aluminium sheets precoated with silicagel 60F254 and a mobile phase system comprising of toluene: methanol, 9:1 % v/v were selected. After development, the plates were scanned and quantified at 525nm. Linearity was found in the concentration range of 100-900 ng/spot (r=0.9920). Limit of detection was found to be 70 ng/spot and limit of quantification was found to be 100 ng/spot. For HPLC method, the optimized mobile phase system used was methanol : water (99:1%v/v) with the flow rate of 1ml/min and the detection wavelength was 202 nm. The calibration curve was obtained in the range 20-60 μg/mL. The slope, intercept and correlation coefficient values were found to be 3264.29, - 4675.4 and 0.9927 respectively. Limit of detection and limit of quantification were found to be 5 μg/ml and 20 μg/ml respectively. Low Relative Standard Deviation and good percentage recovery values of both the methods showed that the developed methods were highly precise and accurate. The method of estimation of marker constituent present in the extract and formulation can be utilized in routine analysis and standardization of medicinal plant extracts.

Keywords: Acalypha indica, Euphorbiaceae, Stigmasterol, HPTLC, HPLC, Method validation