A simple validated high-performance thin-layer chromatographic-densitometric analysis method for simultaneous quantitation of betulin, lupeol, stigmasterol, and β-sitosterol in Asteracantha longifolia (L.) Nees

Background: Asteracantha longifolia (L.) Nees is an essential and versatile ingredient in traditional medicine and herbal formulations, owing to their in curing ability against numerous diseases and disorders. There are forms of utilization like hot water extract, Methanol and ethanol extracts, as well as in some eastern parts of India, the plant is used daily as a leafy vegetable for human consumption during the medical treatment2,3. Also, it is considered a nutritious fodder for cattle. With many essential vitamins and minerals, available literature also reports vital phytoconstituents like Betulin, Lupeol, Stigmasterol, and β-sitosterol as therapeutic activity enhancers for Asteracantha longifolia (L.) Nees. Standardizing these vital phytoconstituents in the raw material will control the quality of the herbal formulations, extracts, and nutritional value. Aim: To develop and validate a HPTLC method-densitometric analysis method for simultaneous quantitation of four phytoconstituents of Asteracantha longifolia (L.) Nees. Objective: The method should be simple, capable of separating Betulin, Lupeol, Stigmasterol, and β-sitosterol to simultaneously estimate the contents at 366nm and 540nm. Further, the method is validated following the guidelines laid by ICH, Q2 (R1). Materials and Methods: Precoated F254 silica gel TLC plates of Merck were utilized as stationary phase to carry out the separation in combination with n-Hexane: Ethyl acetate (8:2) as mobile phase. After development phytoconstituents were derivatized with Anisaldehyde Sulphuric Acid Reagent and TLC plates are dried at 100 °C for 3 min before visualization at 366nm and 540nm. Results: In summary, the method separates all the phytoconstituents adequately and is specific, the limit of detection–limit of quantification for betulin is 0.8 ng and 2.4 ng, lupeol is 0.3 ng and 1.02 ng, and total sterols is 0.2 ng and 0.7 ng the coefficient of variance for precision (Intraday and Interday) is Not more than (NMT) 5%, Linearity and Range is 0.5ng to 6ng, and Recovery ranges from 88.5% to 96.8%. Conclusion: It is evidential from the obtained results of the study that method is selective, sensitive, and reproducible for simultaneous analysis of Betulin, Lupeol, Stigmasterol, and β-sitosterol in Asteracantha longifolia (L.) Nees.