Concurrent analysis of the biologically active markers β-amyrin and β-sitosterol by applying a validated high-performance thin-layer chromatography method In the aerial parts of tinospora cordifolia and calotropis gigantia

In this study, we have developed a validated high-performance thin-layer chromatography (HPTLC) method for the concurrent estimation of the biomarkers β-amyrin and β-sitosterol in di-chloromethane and ethanol extracts of the aerial parts of Tinospo-ra cordifolia (TCDC and TCET) and Calotropis gigantia (CGDC and CGET). Chromatographic estimations were carried out on HPTLC (glass-backed silica gel 60 F₂₅₄) plates with solvents hexane and ethyl acetate in the ratio of 7.5:2.5, v/v (as the mobile phase). Post development, the plate was derivatized with p-anisaldehyde reagent to furnish compact spots of β-amyrin and β-sitosterol and scanned at λ_max = 530 nm. Well-resolved, compact as well as intense peaks of β-sitosterol (R = 0.26 ± 0.001) and β-amyrin (R = 0.39 ± 0.001) were found. The linear regression equation and the correlation coefficient square (r²) forβ-amyrin (Y = 6.118x + 460.76 and 0.9959) and β-sitosterol (Y = 7.109x + 1069.1 and 0.9967) in the concentration range of 100-1400 ng spot^-1 indicated good linear relationship. The low values of the percent relative standard deviation (% RSD) for intra-day and inter-day precisions for β-amyrin (1.003-1.148 and 0.993-1.105) and β-sitosterol (0.578-0.969 and 0.513-0.813) suggested that the method is precise. The % recovery and % RSD values were found to be 98.42-99.29% and 1.103-2.103, respectively, for β-amyrin and 98.33-99.39% and 1.375-2.346, respectively, for β-sitosterol, which confirms the good accuracy of the proposed method. The quantity of β-amyrin in TCDC, CGDC, TCET, and CGET was found to be 70.14, 10.76, 4.85, and 0.87 μg mg^-1, respectively, of the dried weight of the extracts, while the β-sitosterol content was found to be 19.4, 18.5, 1.18, and 0.27 μg mg^-1, respectively. The highest quantity of β-amyrin and β-sitosterol in the dichloromethane fractions of T. cordifolia and C. gigantia validated the wide range of their proved activities including antioxidant, anticancer, and hepatoprotective features. The above developed method can be further employed in the analysis of these markers in marketed preparations.