Development of betulin as phytochemical reference standard for the analysis of Hygrophila schulli plant by a validated high-performance thin-layer chromatography method

Hygrophila schulli (HS) plant has been used in traditional systems of medicine to cure several ailments. According to the World Health Organization, development of phytochemical reference standard (PRS) is essential to standardize the medicinal plants. An attempt was, therefore, made to develop betulin as a PRS for the analysis of HS plant by a validated high-performance thin-layer chromatography (HPTLC) method. The laboratory-isolated and -identified betulin from the HS roots was used to develop and validate the proposed method with respect to linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, and robustness. The mobile phase containing toluene-methanol (8:1, V/V) and an HPTLC aluminum plate, pre-coated with silica gel 60 F-254, were used. The post-chromatographic derivatization using Liebermann-Burchard reagent was performed to enable the quantification of betulin in the test samples at 366 nm in fluorescent mode. Data for the calibration plots of betulin showed good linearity with y = 4.475x + 546.73 (r(2) = 0.9953) regression equation in the concentration range of 200-1000 ng band(-1) (R-F = 0.494 +/- 0.016). The slope and intercept (mean +/- SD) values were 4.475 +/- 0.0045 and 546.73 +/- 45.58, respectively. The LOD and LOQ for betulin were found to be 33.62 and 101.88 ng band(-1), respectively. The stability study suggested that betulin is stable in the chloroform solution during 24 h at room temperature. The proposed method showed good precision and robustness. The recovery values of betulin from the chloroform extracts of HS stems and roots were 97.95 +/- 0.46% and 99.21 +/- 0.69%, respectively, with % w/w contents 0.56 +/- 0.01 and 1.14 +/- 0.01 on dry weight basis of the extracts. The proposed method can be employed to standardize HS plant using betulin as a PRS for its quality control.