Simultaneous densitometric determination of β-sitosterol and lupeol through validated HPTLC method in different plant parts of Uraria picta (Jacq.) Desv. ex DC. - A dashmool species

Abstract

β-sitosterol (BS) and lupeol (LU) exhibit a number of biological activities and are the important bioactive marker compounds in pharmaceutical science. In the present study, a simple, precise, accurate and validated high performance thin layer chromatographic (HPTLC) method was developed for simultaneous quantification of these two compounds in leaves, stem and roots of Uraria picta, a critically endangered medicinal plant and one of the important constituents of ten plants ayurvedic formulation called Dashmoola. Standards and test samples were applied on TLC aluminum plate precoated with 0.2 mm layer of silica gel 60F254. The plate was run in a twin glass chamber comprising toluene: methanol: chloroform (8:1:1, v/v/v) as a mobile phase. The plates were immersed in anisaldehyde-sulfuric reagent and then heated at 105 °C for 5 min in CAMAG TLC plate heater for appearance of bands. Densitometric scanning was performed at λmax = 525 nm using tungsten light source in CAMAG TLC Scanner4 armed with WinCATS software. RF values of BS and LU standards and those of test samples were found to be 0.53 ± 0.01 and 0.63 ± 0.01 respectively. The method was further validated by following the International Conference of Harmonization (ICH) guidelines. For BS and LU, the linear regression data for the calibration plots revealed a satisfactory linear association with r2 = 0.995 and 0.998, respectively. Linear range for both BS and LU was 200-600 ng/band. Accuracy of the method was evaluated by performing recovery study at three different levels by standard addition methods with an average recovery of 99.86% and 101.07%. The results revealed that the leaf samples of U. picta contained highest concentration of BS (0.150 ± 0.02%) while its root samples confined the highest concentration of LU (0.149 ± 0.01%). The developed method can be applied for routine and quality control analysis in different herbal formulations containing U. picta species.