Integrated ITS2 DNA Barcoding and LC-QTOF/MS Metabolomics for Authentication of Ngueak Pla Mo (Acanthus spp.) and Differentiation of Pharmacopoeial Species from A. montanus L.

Ngueak Pla Mo is a Thai medicinal crude drug derived from the pharmacopoeial Acanthus species A. ebracteatus Vahl and A. ilicifolius L.; however, morphological similarity and the occurrence of non-pharmacopoeial substitutes such as A. montanus (Nees) T. Anderson may complicate authentication and quality control. This study aimed to develop an integrated molecular-chemical approach for differentiating pharmacopoeial Ngueak Pla Mo species from A. montanus. Twenty authenticated Acanthus samples were analyzed using ITS2 DNA barcoding, including nucleotide variation, pairwise genetic distance, barcode gap, haplotype network, phylogenetic analysis, and predicted ITS2 secondary structure. Representative samples were further examined by untargeted LC-QTOF/MS metabolite profiling combined with hierarchical clustering, PCA, PLS-DA, VIP analyses, fold-change evaluation, and specificity scoring. ITS2 analyses clearly separated A. montanus from A. ebracteatus and A. ilicifolius, supported by diagnostic sequence variation, positive barcode gaps, distinct haplotypes, phylogenetic clustering, and secondary-structure differences, whereas A. ebracteatus and A. ilicifolius could not be reliably differentiated from each other at the species level using ITS2. LC-QTOF/MS profiling revealed species-consistent metabolite patterns and prioritized gallic acid, catechol, 3,4-dihydroxybenzoic acid, trigonelline, and ursolic acid as preliminary candidate markers for A. montanus discrimination. These results indicate that combining ITS2 barcoding with metabolite profiling provides complementary evidence for Ngueak Pla Mo authentication and supports future quality-control development.