DNA barcoding of Aristolochia plants and development of species-specific multiplex PCR to aid HPTLC in ascertainment of Aristolochia herbal materials

The anecdotal evidence is outstanding on the uses of Aristolochia plants as traditional medicines and dietary supplements in many regions of the world. However, herbal materials derived from Aristolochia species have been identified as potent human carcinogens since the first case of severe renal disease after ingesting these herbal preparations. Any products containing Aristolochia species have thus been banned on many continents, including Europe, America and Asia. Therefore, the development of a method to identify these herbs is critically needed for customer safety. The present study evaluated DNA barcoding of the rbcL, matK, ITS2 and trnH-psbA regions among eleven Aristolochia species collected in Thailand. Polymorphic sites were observed in all four DNA loci. Among those eleven Aristolochia species, three species (A. pierrei, A. tagala and A. pothieri) are used as herbal materials in Thai folk medicine, namely, in Thai Krai-Krue. Krai-Krue herbs are interchangeably used as an admixture in Thai traditional remedies without specific knowledge of their identities. A species-specific multiplex PCR based on nucleotide polymorphisms in the ITS2 region was developed as an identification tool to differentiate these three Aristolochia species and to supplement the HPTLC pattern in clarifying the origins of herbal materials. The combination of multiplex PCR and HPTLC profiling achieves accurate herbal identification with the goal of protecting consumers from the health risks associated with product substitution and contamination.