Simultaneous Determination of Stilbenes, Flavones, Coumestans, Anthraquinones, and Chalcones in Ethanolic Extract of Pet-Sang-Kard Mixed Herbal Remedy Using HPLC-PDA Analysis

Abstract

The Pet-Sang-Kard mixed herbal remedy (PSKMHR) is a traditional Thai medicinal formulation used as an herbal supplement for the treatment of hemorrhoids. This remedy consists of four specific herbal ingredients in the following proportions: 50 parts Cissus quadrangularis L. stems, 15 parts Eclipta prostrata L. aerial parts, 10 parts Rheum sp. rhizome, and 10 parts Boesenbergia rotunda (L.) Mansf. rhizome. This study presents the development, validation, and application of a high-performance liquid chromatography with photodiode array detection (HPLC-PDA) method designed for the simultaneous quantification of 13 key bioactive compounds, including rhaponticin, rhapontigenin, quercitrin, wedelolactone, aloe-emodin, rhein, emodin, chrysophanol, physcion, alpinetin, pinocembrin, pinostrobin, and panduratin A, present in the 70% ethanolic extract of PSKMHR. Method validation was conducted in accordance with Association of Official Analytical Collaboration (AOAC) international guidelines, evaluating parameters such as the specificity, linearity, accuracy, precision, and limit of detection. The results demonstrated exceptional linearity (R > 0.9999), high precision (% RSD < 2), and recovery rates within acceptable limits (98–102%) for all analytes. This developed method was successfully applied to quantify the 13 target compounds in the crude extracts of PSKMHR formulated from 10 market raw material samples, providing a robust analytical framework for quality control of this herbal remedy.