Simultaneous Quantification of Bergenin, (+)-Catechin, Gallicin and Gallic acid; and Quantification of β-Sitosterol using HPTLC from Bergenia ciliata (Haw.) Sternb. Forma ligulata Yeo (Pasanbheda)

Abstract

In the present paper we report our work on development and validation of TLC densitometric method for simultaneous quantification of Bergenin, (+)-Catechin, Gallicin and Gallic acid, and quantification of ß-Sitosterol using HPTLC.To the best of our knowledge, this is the first report of simultaneous quantification of these four bioactive compounds viz. Bergenin, (+)-Catechin, Gallicin and Gallic acid using HPTLC from this plant. Simultaneous quantification of Bergenin, (+)-Catechin, Gallicin and Gallic acid was carried out from the methanolic and hydrolysed extract (2N HCl) using the Solvent System of Toluene: Ethyl acetate: Formic acid (6: 6: 1, v/v/v). ß-Sitosterol was quantified from petroleum ether extract using the Solvent System of Toluene : Methanol (9: 1, v/v). The methods were validated using ICH guidelines in terms of precision, repeatability and accuracy. The thin layer chromatography densitometric methods were found to be precise with RSD for intra-day in the range of 2.62–4.26, 0.35–2.5, 0.61–1.83, 1.14–1.57 and 0.15–0.52 and for interday in the range of 1.90–4.27, 0.85–3.05, 0.97–1.45, 0.58–1.27 and 0.26–0.61 for different concentrations of Bergenin, (+)-Catechin, Gallicin, Gallic acid and ß-Sitosterol respectively. The linearity ranges for Bergenin, (+)-Catechin, Gallicin and Gallic acid were found to be the same (160 –720 ng/spot) with correlation coefficients (r-values) of 0.999, 0.997, 0.998 and 0.996 respectively. The linearity range for ß-Sitosterol was 80–480 ng/spot with correlation coefficient (r-values) of 0.995. Instrumental precision was 3.39, 3.36, 3.05, 1.20 and 0.85 (% RSD) for Bergenin, (+)-Catechin, Gallicin, Gallic acid and ß-Sitosterol respectively. Accuracy of the method was checked by conducting recovery studies at three different levels for both the compounds and the average percentage recoveries obtained were 100.38, 101.45, 102.38, 99.9 and 99.92% respectively for Bergenin, (+)-Catechin, Gallicin, Gallic acid and -Sitosterol. The amount of Bergenin, (+)-Catechin and Gallic acid was 0.22, 0.063 and 0.25 % w/w respectively whereas Gallicin was not in detectable amount. Developed method permitted simultaneous quantification of Bergenin, (+)-Catechin, Gallicin and Gallic acid, and showed good resolution and separation from other constituents of extract and was found to be simple, precise, specific, sensitive and accurate. It can be adopted for routine quality control of herbal material and formulations containing Bergenia ciliata.