Estimation of Rhein from Cassia fistula Linn. using validated HPTLC method

Abstract

Background: Rhein is the major anthraquinone found in Cassia fistula Linn, which is traditionally used as an important component of plant based medicine. Aims and Objective: To develop and validate a High Performance Thin Layer Chromatographic method for the estimation of Rhein from C. fistula Linn. Materials and Methods: Chromatographic separation was achieved on silica gel 60 F254 plates with n‑hexane: Ethyl acetate: Formic acid (5:5:0.2, v/v/v) as a mobile phase. Detection of Rhein was carried out by derivatizing the plate with 10% ethanolic KOH reagent followed by heating at 110°C for 10 min. CAMAG TLC scanner 4 equipped with winCATS software was used for densitometric scanning at 550 nm. The accuracy of the method was checked by conducting various validation parameters according to ICH (International Conference on Harmonization) guidelines. Results: The method was found applicable to evaluate the content of Rhein in different plant parts of C. fistula and the impact of different geographical regions on the Rhein content in fruit pulp of C. fistula was also studied. Estimation of Rhein from a herbal formulation containing C. fistula fruit pulp was also carried out using the validated HPTLC method. The developed method was found sensitive and accurate for the estimation of Rhein from various plant matrices. Conclusion: The developed method was found useful for the quantitation of bioactive marker Rhein and can be used as a routine quality control tool for the assessment of botanicals. Key words: Cassia fistula, fruit pulp, geographical variation, HPTLC, Rhein