Simultaneous estimation of lupeol, stigmasterol and betulin in Desmodium oojeinensis bark and roots by a validated instrumental thin-layer chromatography method

Abstract

Desmodium oojeinensis (Roxb.) H. Ohashi (DO) plant has been traditionally known to possess astringent, anti-inflammatory, hepatoprotective, wound healing, antimicrobial, rejuvenating, etc., activities. Since standardization of medicinal plants is essential to assess their quality by determining the active chemical constituents, the present work was aimed to estimate lupeol, stigmasterol, and betulin in DO bark and roots by a validated thin-layer chromatography (TLC) method. The proposed instrumental TLC method was validated in terms of linearity and range, limit of detection (LOD) and limit of quantification (LOQ), accuracy, precision, robustness and specificity. The mobile phase containing hexane‒ethyl acetate (8.5:1.5, V/V) and TLC aluminum plate, precoated with silica gel 60 F₂₅₄ as stationary phase were used. Anisaldehyde‒H₂SO₄ reagent was used for post-chromatographic derivatization. Data for the calibration plots of lupeol, stigmasterol and betulin showed linearity with y = 20.097x + 5124.9 (r² = 0.9906), y = 12.466x + 1518.5 (r² = 0.99472), and y = 10.902x + 545.3 (r² = 0.9944) regression equations, respectively, in 100–500 ng band⁻¹ concentrations. Lupeol, stigmasterol, and betulin were eluted at R_F [± standard deviation (SD)] 0.42 ± 0.01, 0.27 ± 0.01, and 0.19 ± 0.01, respectively. The LOD and LOQ for lupeol, stigmasterol, and betulin were found to be 12.75 and 38.64 ng band⁻¹, 18.02 and 54.59 ng band⁻¹, and 13.35 and 40.46 ng band⁻¹, respectively. Stability study revealed that lupeol, stigmasterol, and betulin were stable in chloroform at 25 ± 2 °C for 24 h with %RSD value of 0.69, 1.07, and 1.64, respectively. The proposed work showed good precision, robustness and specificity of the method. Average percentage recoveries of lupeol, stigmasterol, and betulin from the chloroform extracts of DO bark and roots were 97.43 ± 0.47, 97.69 ± 0.59; 97.02 ± 1.04, 97.89 ± 0.65, 98.18 ± 0.73, and 97.68 ± 1.09, respectively. The contents (% w/w; n = 3) of lupeol, stigmasterol, and betulin in the chloroform extracts of bark and roots of DO were found to be 2.15 ± 0.03 and 0.45 ± 0.06, 0.43 ± 0.03 and 0.40 ± 0.05, and 2.65 ± 0.04 and 2.13 ± 0.05, respectively, on dry weight basis of the extracts.