Abstract

Hemidesmus indicus is misidentified in the raw-drug market. Because its dried roots resemble those of Decalepis hamilton in look and aroma. The Internal Transcribed Spacer 2 (ITS2) region was used to analyze authenticated samples. This made it possible to distinguish the two species clearly. The ITS2 sequences obtained in this study were submitted to GenBank with accession numbers PX495088 for H. indicus and PX495104 for D. hamiltonii. BLASTn analysis showed that H. indicus had more than 99% similarity with known sequences, while D. hamiltonii had about 98% similarity. This study shows that ITS2 is a practical and reliable tool for distinguishing H. indicus from its adulterant and can be effectively used for routine quality control in herbal supply chains. These findings show consistency in species identification. The two groups were easily identified by diagnostic nucleotide alterations, conserved motifs, and modifications to the secondary structure of ITS2 (including compensatory base changes). Neighbor-joining and maximum likelihood (ML) analyses consistently resolved two distinct clades corresponding to H. indicus and D. hamiltonii. The sequence generated in this study clustered within the H. indicus clade with strong bootstrap support, confirming its correct taxonomic identity. ITS2-based DNA barcodes generated using the Bio-Rad tool revealed distinct nucleotide patterns, enabling rapid visual discrimination between H. indicus and D. hamiltonii. While secondary structure analysis showed significant differences in folding and thermodynamic stability, enabling clear discrimination between H. indicus and D. hamiltonii. This study shows that ITS2 is a practical and reliable tool for distinguishing H. indicus from its adulterant and can be effectively used for routine quality control in herbal supply chains.

Key words: DNA barcoding; Hemidesmus indicus; Decalepis hamiltonii; ITS2 marker; Molecular authentication; Medicinal plant adulteration