Authenticity Testing and Detection of Eurycoma longifolia in Commercial Herbal Products Using Bar-High Resolution Melting Analysis

Abstract

The present study demonstrated High Resolution Melting (HRM) analysis combined with DNA barcode (Bar-HRM) as a fast and highly sensitive technique for detecting adulterants in Eurycoma longifolia commercial herbal products. Targeting the DNA barcoding of the chloroplastic region-ribulose biphosphate carboxylase large chain (rbcL) and the nuclear ribosomal region- internal transcribed spacer 2 (ITS2), PCR amplification and HRM analysis using saturated Eva green dye as the source of fluorescence signals, was accomplished by employing a real-time cycler. The results were further validated by sequencing to identify unknown sequence from Genbank database and to generate phylogenetic tree using neighbour joint (NJ) analysis. Both of the DNA markers exhibited a distinguishable melting temperature and shape of the normalised curve between the reference and the adulterants. In the case of species identification, ITS2 was more successful in differentiating between species. Additionally, detection of admixture sample containing small traces of targeted E. longifolia DNA (w/v) can be detected as low as 5% for rbcL and less than 1% for ITS2, proving the sensitivity and versatility of the HRM analysis. In conclusion, the Bar-HRM analysis is a fast and reliable technique that can effectively detect adulterants in herbal products. Therefore, this will be beneficial for regulatory agencies in order to regulate food safety issues.

Differentiation of Mitragyna Speciosa, a Narcotic Plant, From Allied Mitragyna Species Using DNA Barcoding-high-resolution Melting (Bar-HRM) Analysis

“…The advantage of HRM is its capacity to screen variations in speci c regions that would not be identi ed by Sanger sequencing; moreover, using HRM analysis of melting temperatures can allow any different nucleotides in a sequence to be detected 31 . Recent studies have shown that Bar-HRM analysis can be effective in providing consistent shapes and pro les of melting curves that correspond to very closely related medicinal plant species and allow the authentication of herbal products 19,[23][24][25] . Nucleotide composition and DNA length are affected by the dissociation step of double-stranded amplicons in HRM analysis.…”

“…However, the shortcomings of the PCR-RFLP approach are that it is time consuming and very limited in its ability to identify species when the samples are from incomplete specimens or damaged. More recently, several studies have shown that very closely related medicinal plant species can also be distinguished accurately by using DNA barcoding combined with highresolution melting (Bar-HRM) analysis 19,[23][24][25] .…”

Cannabis Seeds Authentication by Chloroplast and Nuclear DNA Analysis Coupled with High-Resolution Melting Method for Quality Control Purposes

“…ITS2F 5¢-ATGCGATACTTGGTGTG AAT-3¢ ITS3R 5¢-GACGCTTCTCCAGACTACAAT-3¢ rbcLa-F: 5¢-ATGTCACCACAAACAGAGACTAAAGC-3¢, rbcLa-R: 5¢-GTAAAATCAAGTCCACCRCG-3¢. 14,30 For rbcL, the amplification conditions were as follows: an initial denaturation at 95°C followed of 30 cycles of 55°C for 1 min, and 72°C for 1 min. 32 The fluorescent data were acquired at the end of each extension step during PCR cycles.…”

“…25,26 In addition, the chloroplast ribulose bisphosphate carboxylase large chain gene (rbcL) is another barcode region used in most land plants due to the ease of amplification, the availability of databases 21,[27][28][29] and its potential application for authenticity testing. 30 Several other cannabis chloroplast markers have been used for forensic purposes (e.g., to establish the geographical origin of seizures). 18 Therefore, we proposed a method based on the ITS2 nuclear DNA region and rbcL, a chloroplast marker, combined with HRM-PCR, for detection of adulterants in cannabis seeds commercial strains.…”

A review of the phytochemical compounds and pharmacological activities of Eurycoma longifolia

“…Due to high demand of this plant there are various formula of E. longifolia are available in health food market, in crude drug powder form or capsules which contain crude drug powder or E. longifolia extract (Effendy et al, 2012). It can be found as health supplements in capsules, tablets and beverages (Fadzil et al, 2018). Nowadays, root extract of E. longifolia used as pre-mixed in tea, coffee and carbonated drink which sales in commercial market (Low et al, 2013).…”