Profiling of adulterant in tongkat ali herbal product using dna barcoding in combination with high resolution melting analysis

Abstract

Eurycoma longfiolia or known as Tongkat Ali is popular in Malaysia for its aphrodisiac and therapeutic properties. However, the increasing demand of this herbal remedy make it prone to adulteration due to the limited availability of Tongkat Ali plant resources. Hence, to fulfil the market demands, unscrupulous manufacturers may intentionally add Tongkat Ali herbal product with cheaper plant species as substitute to increase their profit. Misidentification of plant species during collection or false mixing with other plant species during processing stage also contribute to unintentional adulteration of herbal products. Therefore, this study was conducted to assess the authenticity of E. longifolia herbal products by using DNA Barcode combined with a new sensitive method of High Resolution Melting Analysis (Bar-HRM). In order to obtain high quality genomic DNA, extraction was done using modified Cetyl Trimethyl Ammonium Bromide (CTAB) method and Nucleospin Plant II kit. The rbcL and ITS2 was chosen as the plant DNA Barcode region in PCR amplification for E. longifolia root and four selected herbal products samples (P1, P2, P3, P4). For sensitivity evaluation of HRM analysis to detect traces of targeted DNA in admixture sample, E. longifolia DNA was mixed with Camellia sinensis DNA in increasing percentages of 0%, 1%, 5%, 10%, 30%, 50%, 75% and 100%. DNA melting profiles recorded from the different percentages of admixture was then used as a standard to detect traces of E. longifolia DNA in three admixture herbal tea products containing E. longifolia and C. sinensis on the packaging label (P5, P6, P7). Results showed that genomic DNA obtained using Nucleospin Plant II kit method recorded better DNA quality as compared to modified CTAB method. From HRM analysis result, P1 and P4 herbal products were authentic while P2 and P3 herbal products were not genuine based on the DNA melting profiles of rbcL. On the other hand, ITS2 DNA melting profile successfully detected three herbal products (P1, P2 and P4) that were genuine containing E. longifolia. However, E. longifolia was undetectable in P3 herbal product. The results of bioinformatics analysis including BLASTn, Multiple Sequence Alignment and Phylogenetic tree also supported the HRM analysis data for both genes. The result suggested that ITS2 primer used in this study was more specific and suitable to detect E. longifolia in herbal products up to species level, while rbcL managed to identify only up to genus level. From the HRM sensitivity results, rbcL can only detect E. longifolia DNA at 5% level while ITS2 managed to detect at lower than 1%. This proved that ITS2 had more DNA sequence variations and discrimination power than rbcL. In conclusion, Bar-HRM analysis is a reliable, fast and sensitive method to detect the true targeted plant species in herbal products and can be used to monitor food product authenticity issue in the future.