Determination of Oleanolic Acid, Ursolic Acid, Lupeol, and Stigmasterol by High-Performance Thin-Layer Chromatographic Method in Oldenlandia corymbosa Linn.

Oldenlandia corymbosa Linn. (Rubiaceae) is an important herb traditionally used as a febrifuge and liver tonic. In this study, a high-performance thin-layer chromatography (HPTLC) method has been established for the quantification of four bioactive markers, oleanolic acid (OA), ursolic acid (UA), lupeol (LU), and stigmasterol (ST), in the whole plant of O. corymbosa. Separation was achieved on silica gel 60 F-254 HPTLC plates using hexane-ethyl acetate-methanol (8.2:1.8:0.5, v/v) for oleanolic acid and ursolic acid; and toluene-methanol (9.4:0.6, v/v) for lupeol and stigma-sterol as the mobile phases. The quantitation of the four markers was carried out using the densitometric scanning at 540 nm after derivatization using sulfuric acid reagent. The linear regression analysis data for the calibration plots showed a good linear relationship (r(2) = 0.9831-0.9979) in the concentration range of 1200-4200 ng for oleanolic acid, 400-1400 ng for ursolic acid, 100-500 ng for lupeol, and 500-2500 ng per spot for stigmasterol with respect to area. The method was validated for linearity, inter-day precision, intra-day precision, repeatability, accuracy, specificity, limit of detection, and limit of quantification. The average recoveries for oleanolic acid, ursolic acid, lupeol, and stigmasterol were 98.77 to 99.12%, indicating the good reproducibility. Stigmasterol 1.19 +/- 0.04% w/w was present at high concentration, and oleanolic acid 0.012 +/- 0.006% w/w was present at low concentration in the whole plant powder. The proposed HPTLC method was found to be simple, precise, sensitive, accurate, reproducible, and robust.