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 Pleuropterus multiflorus (Thunb.) Turcz. ex Nakai (PM), is a fundamental traditional Chinese medicine utilized for treating sleep disorders. Despite its medicinal importance, species confusion exists due to the taxonomic synonymy of Polygonum multiflorum var. hypoleucum (PMH) and Pleuropterus angulatus (PA) with PM. Furthermore, conventional quality standards often restrict PMC collection to specific stem diameters. This study evaluatedthe chemical and genetic diversity of PMC and its related taxa and assessed the authenticity of commercial PMC products. 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- O - β -D-glucoside (THSG), emodin, and physcion. The ITS2 region was sequenced to evaluate genetic diversity and identify single nucleotide polymorphisms (SNPs). We examined the impact of stem diameter (2.0 to 25.1 mm) on constituent levels in PM and surveyed eleven commercial PMC samples from Taiwanese markets. 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.