ABSTRACT:
The presence of diverse phytoconstituents in herbal medicines enhances their therapeutic efficacy, especially when synergistic herbs are combined in polyherbal formulations. However, the complexity of herbal matrices, the coexistence of multiple active compounds, and their occurrence in trace amounts pose challenges in standardization and quality assurance. Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) is a powerful analytical tool for addressing these issues and ensuring batch-to-batch consistency. This study aims to develop and validate a novel, selective, and sensitive RP-HPLC method for the simultaneous quantification of three key phytomarkers—E-Guggulsterone, Glycyrrhizic acid, and Gallic acid—in a polyherbal formulation composed of thirteen ethnomedicinal herbs with reported cardioprotectiveactivity.Gallic acid, E-guggulosterone, and glycyrrhizic acid were selected as marker compounds for both their pharmacological relevance and functional roles in silver nanoparticle synthesis. Gallic acid acts as a potent reducing and stabilizing agent, E-guggulosterone contributes to nanoparticle formation while enhancing bioactivity, and glycyrrhizic acid provides capping and therapeutic potential. Their inclusion ensures analytical standardization, reproducibility, and regulatory compliance. The developed RP-HPLC method employed a reverse-phase C18 column (250mm × 4.6 mm, 5µm), with a mobile phase of acetonitrile and 0.05% orthophosphoric acid in water (50:50), delivered at a flow rate of 1 mL/min. Detection was carried out at 224nm. The method was applied to the evaluation of Collasyntablets, a marketed polyherbal formulation by Dr. Palep’s Medical Research Foundation Pvt. Ltd., India. The quantified contents were E-Guggulsterone (0.63%), Glycyrrhizic acid (1.05%), and Gallic acid (11.47%). Method validation was conducted in strict accordance with ICH guidelines, and all analytical parameters—including linearity (50–150mcg/mL), precision, accuracy, robustness, and specificity—complied with the recommended criteria. This validated method is a significant advancement in phytochemical standardization, enabling simultaneous, accurate assessment of multiple bioactive components. It supports quality control, enhances therapeutic consistency, and provides a foundation for broader pharmaceutical adoption of standardized herbal formulations.