Screening and validated semi - quantification high-performance thin layer chromatography method development for lupeol, lupeol acetate, β-sitosterol, ρ-coumaric acid and proto-catechuic acid in the root extracts of Hemidesmus indicus (L.) R.Br. &Decalepis hamiltonii Wight & Arn.

Abstract

H. indicus (L.) R. Br. roots are widely used in traditional medicine systems in India. D. hamiltonii Wight & Arn. It looks like H. indicus and is substituted in the traditional herbal market. Five marker compounds, namely lupeol, lupeol acetate (LA), β-sitosterol (BS), ρ-coumaric acid (PC) and protocatechuic acid (PCA) were quantified, and the method was validated. Separation of lupeol (Rf max 0.48), LA (Rf max 0.75) and BS (Rf max 0.36) was achieved using hexane: ethyl acetate (8:2, v/v) and scanned at λ520 nm after derivatization with vanillin–sulphuric acid reagent (VSR). For PC (Rf 0.59) and PCA (Rf 0.47), the separation was performed using toluene: ethyl acetate: formic acid (7:5:0.5, v/v/v) and scanned at λ254 nm. The validation parameters include linearity, accuracy, precision, the limit of detection and quantification (LOD and LOQ), repeatability, specificity and recovery as per ICH guidelines. Lupeol, LA and BS were found to be present in both plants; PC was present in H. indicus, and PCA was present in D. hamiltonii. The calibration plots were linear in the range (μg/band) of 5–15 for lupeol; 20–45 for LA; 1–5 for PC; 5–20 for BS, and PCA. The LOD were 0.0181, 0.023, 0.080, 0.0021 and 0.0019 (μg/band) for lupeol, LA, BS, PC and PCA, respectively, concerning area, correlation coefficients (r2) were 0.9980, 0.9988, 0.9958, 0.9998 and 0.9938 for lupeol, LA, BS, PC and PCA respectively. The validated high-performance thin layer chromatography (HPTLC) method provided an excellent linear relationship for all the quantified analytes; hence it may be used for quantitative estimation of the above markers to assess the quality of H. indicus and D. hamiltonii or herbal formulations containing them.