Simultaneous quantification of five bioactive markers for standardization of ayurvedic polyherbal formulation Jwarahara Kwatha Choornam using HPTLC

Jwarahara Kwatha Choornam (JKC) is a polyherbal coded Ayurvedic formulation developed by the Central Council for Research in Ayurvedic Sciences (CCRAS), New Delhi, India. Traditionally used for managing chronic fever, cold, and malaria, JKC has gained recognition for its therapeutic benefits, such as enhancing digestion, stimulating appetite, detoxifying blood, modulating the immune response, and offering protection against common bacterial infections. The medicinal plant used in JKC is widely utilized by Ayurvedic practitioners and the general population in the Kerala region, where it holds a longstanding place in traditional health practices. Notably, during the COVID-19 pandemic, both practitioners and users have reported the formulation's supportive role in treatment, further highlighting its therapeutic relevance. To ensure the quality, safety, and efficacy of this important Ayurvedic preparation, CCRAS has undertaken standardization efforts, including the development of a novel High-Performance Thin-Layer Chromatography (HPTLC) method for the simultaneous estimation of five key bioactive marker compounds. The study establishes a robust High-Performance Thin-Layer Chromatography (HPTLC) method for the simultaneous estimation of five key bioactive markers-Andrographolide (AG), Piperine (PP), Picroside-I (P-I), Picroside-II (P-II), and α-Cyperone (AC) present in the plants Andrographis paniculata, Cyperus rotundus, Piper longum, Piper nigrum, Zingiber officinale, Hedyotis corymbosa, and Picrorhiza kurroa. Used in the Jwarahara Kwatha Choornam (JKC) formulation. Effective separation of these compounds was achieved using a carefully optimized mobile phase comprising Toluene, Ethyl Acetate, Methanol, and Formic Acid in a 4:4:1:1 (v/v/v/v) ratio. The developed HPTLC method, resolved the five targeted bioactive markers-Andrographolide (AG), Piperine (PP), Picroside-I (P-I), Picroside-II (P-II), and α-Cyperone (AC)-with distinct R_f values of 0.563 ± 0.005, 0.706 ± 0.015, 0.280 ± 0.0173, 0.180 ± 0.0115, and 0.803 ± 0.005, respectively, using a mobile phase of Toluene: Ethyl Acetate: Methanol: Formic Acid (4:4:1:1, v/v/v/v). The method was rigorously validated, demonstrating excellent linearity (r² = 0.97-0.99), precision, accuracy (RSD < 2%), robustness, and ruggedness under optimized analytical conditions. Quantitative analysis of JKC revealed the presence of AG (3.638 ± 0.0234 mg/g), PP (3.360 ± 0.0792 mg/g), P-I (0.1426 ± 0.0031 mg/g), P-II (0.6025 ± 0.0198 mg/g), and AC (0.2102 ± 0.0023 mg/g). This study demonstrates that the developed HPTLC method is a rapid, precise, and reliable analytical tool for simultaneously quantifying five key bioactive markers in individual plant materials and polyherbal formulations. Owing to its robustness and reproducibility, this method offers a practical and efficient approach for routine quality control and standardization of JKC formulations.