Development and Validation of HPTLC Method For Simultaneous Estimation of Bioactives From Ayurvedic Formulation: Brahma Rasayan

Abstract

In Ayurveda, "Rasayana" medicine refers to treatments that rejuvenate the body, prevent ageing, and increase lifespan. Brahma Rasayan is a Medhya Rasayana having more than 35 constituents, principally from the plants Emblica officinalis (gooseberry) and Terminalia chebula, (Indian gall nut). Gallic acid, Quercetin, and Piperine were picked for the analytical method development. This technique has been developed in order to enable the quantitative estimation of the total phenolic, flavonoid, and alkaloid content in ayurvedic formulations. The method was developed using High Performance Thin Layer Chromatography (HPTLC). After trials, the HPTLC method was optimized using precoated silica as a stationary phase and Toluene: Ethyl acetate: Formic acid (4:5:2 v/v/v) as a mobile phase. The detection of spots is carried out at 254 nm wavelength. The Rf values observed were 0.54, 0.68, and 0.76 for Gallic acid, Quercetin, and Piperine respectively. The developed method was validated according to the ICH guidelines. Linearity range was found to be 200-1200 ng/band with a regression co-efficient(R2) of 0.9966, 0.9941, and 0.9952 for Gallic acid, Quercetin, and Piperine respectively. Accuracy, Precision, Specificity, and Robustness of the method were tested, and the results were in agreement with the specifications. Based on the analysis of extract, the total phenolics, flavonoids, and alkaloids was found to be 0.34%, 1.37%, and 0.22% w/w, respectively. Additionally, the specific content of gallic acid, quercetin, and piperine in the extract was determined through peak area, and found to be 0.09%, 0.14%, and 0.18% w/w, respectively. After successful development of the method, it was established that these bioactive constituents of the Brahma Rasayan can be successfully analysed by the HPTLC method.