DNA barcodes and chemical profiles of Aristolochia plants for examination of krai-krua herbs

The genus Aristolochia belongs to the Aristolochiaceae family. The major chemical constituents in the whole part of Aristolochia plant are aristolochic acids I (AAI) and AAII, which are classified as human carcinogens. In Thailand, 2013, The National Drug Committee have issued an order that demands the removal of crude drugs derived from Aristolochia plants from all registered formulas. Dried roots of A. pothieri Pierre ex Lecomte, A. pierrei Lecomte and A. tagala Cham., have been reported as sources of medicinal crude drugs called “Krai-Krue”. However, Krai-Krue can also be derived from dried roots of Raphistemma pulchellum (Roxb) Wall, Jasminum spp and Gymnopetalum integrifolium Kurz. Authentication of Krai-Krue by morphological examination is quite difficult and can cause confusion. For the protection of consumer’s safety, reliable and effective tools for identification of raw herbal materials have been continuously developed. In this study, genetic assessment of 11 Aristolochia plants by DNA barcoding technique was conducted based on four DNA regions including rbcL, matK, ITS and trnH-psbA. The nucleotide variations of the four regions are useful to differentiate the eleven Aristolochia species. Multiplex PCR based on nucleotide sequences of ITS2 region combining with HPTLC using AAI as standard substance were used for the identification of Krai-Krue herbs. This method can be used as a preliminary AAI-screening test for safety control by the herbal industries as well as the regulatory authorities. The results from these studies indicated that the combination of genetic and chemical assessment would be useful for the identification and discrimination of Aristolochia plants and Krai-Krue herbs.