DNA barcoding coupled with high resolution melting (BAR-HRM) analysis for differentiation white kwao khruea (pueraria candollei), black kwao khruea (mucuna macrocarpa), and red kwao khruea (butea superba)

Kwao Khruea is the herbal product that known as a rejuvenation herb. In Thai traditional medicine, three species of Kwao Khruea including white Kwao Khruea (Pueraria candollei Wall. ex Benth.), black Kwao Khruea (Mucuna macrocarpa Wall.) and red Kwao Khruea (Butea superba Roxb. ex Willd.) have been used. Each Kwao Khruea accumulates different chemical constituents. White Kwao Khruea contains estrogenic biological activities while black and red Kwao Khruea accumulate testosterone-like activities. Black Kwao Khruea was reported to be potent than red Kwao Khruea. Because of similar common names with the difference in chemical compositions of Kwao Khruea herbs, it is necessary to differentiate those herbs for efficiency and safety purposes. Therefore, this study aims to differentiate the herbal species using genetic information by barcoding of the four core DNA regions including matK gene, rbcL gene, psbA-trnH transgenic spacer and internal transcribed spacer (ITS). The result showed that all four core barcode regions can differentiate those three Kwao Khruea. In addition, DNA barcoding coupled with high resolution melting (Bar-HRM) analysis of the PCR amplicons by real-time PCR was performed to investigate a particular DNA sequence in the matK gene and ITS. The melting temperature (Tm) of PCR products amplified by matK gene of red, white and black Kwao Khruea were 73.6ºC, 74.7ºC and 74.8ºC, respectively while the Tm of PCR amplicons from the ITS were 88.1ºC, 85.6ºC and 84.5ºC, correspondingly. In conclusion, DNA barcode coupled with HRM analysis has been successful to identify Kwao Khruea species in dietary supplements and traditional medicines containing Kwao Khruea as the main mixture. Our developed method is potentially used for the authentication of Kwao Khruea products prior to registration.