Genomic DNA Isolation and DNA Barcoding of Constituent Plants in Athiyadhi Kashayam for Species Authentication

Siddha formulations form a major component of complementary and alternative medicine (CAM), but adulteration of traded medicinal plants threatens their safety and therapeutic reliability. Since morphological characters are often lost in processed raw drugs, DNA barcoding has emerged as a robust approach for authentication. In this study, we focused on Athiyadhi Kashayam, a classical Siddha polyherbal formulation, by authenticating five of its key ingredients—Ficus racemosa, Senna auriculata, Cassia fistula, Syzygium cumini, and Salacia oblonga. Genomic DNA was isolated from stem bark tissues using a modified CTAB protocol with β-mercaptoethanol. DNA quality and concentration were confirmed by agarose gel electrophoresis, Nanodrop spectrophotometry, and UV visualization. PCR amplification was performed with ITS and rbcL primers, both yielding successful amplification. The resulting sequences were analysed using BLAST against NCBI GenBank, confirming species identity. Validated sequences were submitted to GenBank, and species-specific barcodes were generated. The results highlight the effectiveness of ITS and rbcL markers in authenticating constituent plant species of Athiyadhi Kashayam. This study underscores the importance of DNA barcoding as a reliable tool to ensure authenticity of raw drugs, prevent adulteration, and enhance the credibility of traditional formulations. The genomic data generated also contribute to a growing reference database for medicinal plants, supporting regulatory compliance and quality assurance in herbal drug research and industry..