Simultaneous Quantitative Estimation of Scopoletin And Β-Sitosterol in Girardinia Diversifolia Plant Extracts by HPTLC and HPLC Methods

Girardinia diversifolia is a traditionally important medicinal plant recognized for its therapeutic benefits, primarily attributed to bioactive phytoconstituents such as scopoletin and β-sitosterol. Despite their pharmacological significance, reliable and validated analytical methods for the simultaneous estimation of these compounds are limited. Hence, the present study was designed to develop and validate simple, rapid, precise, and accurate HPTLC and HPLC methods for the concurrent quantification of scopoletin and β-sitosterol in Girardinia diversifolia extracts. The whole plant material was collected, authenticated, shade-dried, and powdered, followed by successive extraction using petroleum ether, ethyl acetate, and ethanol. Preliminary phytochemical screening confirmed the presence of key secondary metabolites including flavonoids, tannins, and triterpenoids. Initial TLC profiling indicated that the ethyl acetate extract produced well-resolved spots when developed in a mobile phase consisting of toluene:ethyl acetate:acetic acid:formic acid (7:2:0.5:0.5 v/v/v/v). HPTLC analysis was performed using a CAMAG system with densitometric scanning at 366 nm and after derivatization using anisaldehyde–sulphuric acid reagent at 525 nm. For HPLC analysis, a Shimpack RP-C18 column (50 mm × 4.6 mm, 5 µm) was employed with an isocratic mobile phase of acetonitrile:water (85:15 v/v). The HPTLC method showed distinct and well-resolved peaks with Rf values of 0.30±0.03 for scopoletin and 0.54±0.03 for β-sitosterol, while HPLC analysis revealed retention times of 1.04 and 1.40 minutes, respectively. Both methods demonstrated satisfactory linearity, precision, and accuracy. Overall, the validated HPTLC and HPLC methods proved to be reliable, efficient, and suitable for routine quality control and standardization of Girardinia diversifolia extracts.