Integrated chemical and genetic authentication of Polygoni Multiflori Caulis: Refuting diameter restrictions and exposing market adulteration.

Background: Polygoni Multiflori Caulis (PMC), the dried stem of Methods: Authenticated samples of PM, PMH, and PA were analyzed using high-performance liquid chromatography (HPLC) to quantify three key markers, including 2,3,5,4'-tetrahydroxystilbene-2- Results: Chemical analysis revealed that PMC samples across the entire diameter range (2.0 to 25.1 mm) consistently fulfilled the regulatory standards of the Taiwan Herbal Pharmacopoeia (THP), Chinese Pharmacopoeia (CP), and Hong Kong Chinese Materia Medica Standards (HKCMMS), effectively challenging the previously recommended 4 to 7 mm limit. In contrast, authenticated PMH samples lacked both the characteristic marker THSG and the enlarged tuberous roots typical of PM, and PA stem samples exhibited variable chemical phenotypes, with only a small subset meeting official standards. ITS2 sequencing successfully resolved the three taxa by identifying 13 SNPs and 16 distinct SNP genotypes. Notably, the market survey revealed that 82% of commercial samples were substituted with PA, of which 72% contained undetectable levels of the required analytes. Conclusions: The findings demonstrate that PMH and PA represent distinct chemotypic lineages and should not be considered interchangeable botanical sources for PMC. The integration of HPLC quantification and ITS2 SNP barcoding provides a robust, multifaceted framework for quality control. Furthermore, our findings suggest that stems across the 2.0-25.1 mm diameter range can meet current pharmacopoeial requirements, providing preliminary evidence that the traditional 4-7 mm restriction may be unnecessarily narrow.