DNA barcoding and chemical analysis for evaluation of authenticity of selected herbal medicinal products in Malaysia

Abstract

The increasingly demand of Ficus deltoidea (Mas Cotek) and Eurycoma longifolia (Tongkat Ali) medicinal plants due to antidiabetic and aphrosidiac therapeutic properties respectively have resulted in an increase in demand for their herbal medicinal products (HMPs) in Malaysia. The safety and efficacy of such HMPs which are in the form of tea, capsules or tablets relies on the authenticity of the plant raw material used as most of them are exclusively sourced from wild population. Consequently, commercial HMPs claiming to be authentic may be adulterated or contaminated with other plants species. Current identification methods such as organoleptic, microscopic and macroscopic examinations cannot identify plant species in a processed herbal product form due to lacking of indicative morphological features and plants taxonomy. Evaluation of DNA analysis such as the direct DNA sequence analysis (BLASTn and Neighbour-Joining) of the chloroplastic (rbcL and psbA-trnH) and nuclear (ITS2) regions for rapid detection of F. deltoidea and E. longifolia was evaluated. A standard reference materials (SRM) from all barcode regions were successfully cloned into p-Easy-T3. The sequence analysis of these 3 barcode regions showed that same species or closely related species shared high percentage of DNA identity. The phylogenetic analysis showed that rbcL had high discriminatory power in F. deltoidea var. kunstleri and ITS2 for E. longifolia. Identification of F. deltoidea HMPs using DNA barcoding showed that 67% were authentic, 11% were substituted while the identity of 22% could not be conclusively determined. For E.longifolia HMPs, 36% of the tested HMPs were authentic, 27% were substituted and the identity of 37% could not be conclusively determined. Further analysis using High Performance Liquid Chromatography (HPLC) revealed that 100% of the tested F. deltoidea HMPs were authentic as all samples contained the expected vitexin marker. This include 14% of tested HMPs identified as substitutions using DNA barcoding. Eurycomanone was detected in 43% of E. longifolia HMPs but at lower concentration than root extract In contrast, 14% of the tested HMPs which were authentic using DNA barcoding were found not to contain the expected marker. The ITS2 proved to be the ideal marker for the authentication of both plants HMPs. Using DNA barcoding, the overall study showed that there was fraudulence activities occurred in the HMPs tested. Even though HPLC of herbal compounds require different protocol and different standards for each biomarker, DNA sequence methods were the same for all HMPs. Hence, DNA barcoding should be utilised in authenticating other HMPs available in the Malaysia market. ABSTRACT