Simultaneous Quantification of Gymnemic Acid as Gymnemagenin and Charantin as β-Sitosterol Using Validated HPTLC Densitometric Method

Gymnemic acid and charantin are well-established antidiabetic phytosterols found in Gymnema sylvestre and Momordica charantia, respectively. The fact that these plants are often used together in antidiabetic poly-herbal formulations lured us to develop an HPTLC densitometric method for the simultaneous quantification of their bioactive compounds. Indirect estimation of gymnemic acid as gymnemagenin and charantin as beta-sitosterol after hydrolysis has been proposed. Aluminum-backed silica gel 60 F-254 plates (20 3 10 cm) were used as stationary phase and toluene-ethyl acetate-methanol-formic acid (60 : 20 : 15 : 5, v/v) as mobile phase. Developed chromatogram was scanned at 550 nm after derivatization with modified vanillin-sulfuric acid reagent. Regression analysis of the calibration data showed an excellent linear relationship between peak area versus concentration of the analytes. Linearity was found to be in the range of 500-2,500 and 100-500 ng/band for gymnemagenin and beta-sitosterol, respectively. The suitability of the developed HPTLC method for simultaneous estimation of analytes was established by validating it as per the ICH guidelines. The limits of detection and quantification for gymnemagenin were found to be approximate to 60 and approximate to 190 ng/band, and those for beta-sitosterol approximate to 30 and approximate to 90 ng/band, respectively. The developed method was found to be linear (r(2) = 0.9987 and 0.9943), precise (relative standard deviation <1.5 and <2% for intra-and interday precision) and accurate (mean recovery ranged between 98.43-101.44 and 98.68-100.20%) for gymnemagenin and beta-sitosterol, respectively. The proposed method was also found specific and robust for quantification of both the analytes and was successfully applied to herbal drugs and in-house herbal formulation without any interference.