Comparative evaluation of different DNA extraction methods from E. Longifolia herbal medicinal product

The aphrodisiac property of Eurycoma longifolia has led to an increase in the demand for its Herbal Medicinal Products (HMPs). However, the efficiency of such HMPs depends on the usage of their genuine raw materials. The conventional methods cannot identify species in processed form. The authentication of HMPs can be achieved effectively using DNA barcoding as the method species-specific. However, the use of this method solely relied on the extraction of high-quality DNA from the HMPs. Therefore, it is necessary to establish a satisfactory method for extracting high-quality DNA from the HMPs. Here, four DNA extraction methods were compared to evaluate the best protocol in yield, purity, polymerase chain reaction (PCR) amplification, sequencing, and species identification. The spectrophotometer analysis showed that the Nucleospin Plant II extraction kit has the best purity as this can be severely affected by the presence of various contaminants in the HMPs. Our findings reveal that DNA purity was more important as a predictor for PCR amplification than yield. Therefore, the present study results demonstrate that the Nucleospin Plant II extraction kit is the best because it produces the purest, amplifiable, and sequenceable DNA for identification and authentication of E. Longifolia HMPs

Review: DNA Barcoding and Chromatography Fingerprints for the Authentication of Botanicals in Herbal Medicinal Products

In the last two decades, there has been a tremendous increase in the global use of herbal medicinal products (HMPs) due to their claimed health benefits. This has led to increase in their demand and consequently, also, resulted in massive adulteration. This is due to the fact that most of the traditional methods cannot identify closely related species in a process product form. Therefore the urgent need for simple and rapid identification methods resulted in the discovery of a novel technique. DNA barcoding is a process that uses short DNA sequence from the standard genome for species identification. This technique is reliable and is not affected by external factors such as climates, age, or plant part. The difficulties in isolation of DNA of high quality in addition to other factors are among the challenges encountered using the DNA barcoding in the authentication of HMP. These limitations indicated that using DNA barcoding alone may ineffectively authenticate the HMP. Therefore, the combination of DNA barcoding with chromatographic fingerprint, a popular and generally accepted technique for the assessment and quality control of HMP, will offer an efficient solution to effectively evaluate the authenticity and quality consistency of HMP. Detailed and quality information about the main composition of the HMPs will help to ascertain their efficacy and safety as these are very important for quality control

Exploring DNA quantity and quality from raw materials to botanical extracts

Objectives: The aim of this study was to explore the variability in DNA quality and quantity along a gradient of industrial processing of botanical ingredients from raw materials to extracts. Methods: A data matrix was assembled for 1242 botanical ingredient samples along a gradient of industrial processing commonly used in the Natural Health Product (NHP) industry. Multivariate statistics was used to explore dependant variables for quality and quantity. The success of attaining a positive DNA test result along a gradient of industrial processing was compared among four biotechnologies: DNA barcoding, NGS, Sanger sequencing and qPCR. Results: There was considerable variance in DNA quality and quantity among the samples, which could be interpreted along a gradient from raw materials with greater quantities (50–120 ng/μL) of DNA and longer DNA (400-500bp) sequences to extracts, which were characterized by lower quantities (0.1–10.0 ng/μL) and short fragments (50-150bp). Conclusions: Targeted molecular diagnostic tests for species identity can be used in the NHP industry for raw and processed samples. Non-targeted tests or the use of NGS for any identity test needs considerable research and development and must be validated before it can be used in commercial operations as these methods are subject to considerable risk of false negative and positive results. Proper use of these tools can be used to ensure ingredient authenticity, and to avert adulteration, and contamination with plants that are a health concern. Lastly these tools can be used to prevent the exploitation of rare herbal species and the harvesting of native biodiversity for commercial purposes

DNA Metabarcoding Authentication of Ayurvedic Herbal Products on the European Market Raises Concerns of Quality and Fidelity

Ayurveda is one of the oldest systems of medicine in the world, but the growing commercial interest in Ayurveda based products has increased the incentive for adulteration and substitution within this herbal market. Fraudulent practices such as the use of undeclared fillers and use of other species of inferior quality is driven both by the increased as well as insufficient supply capacity of especially wild plant species. Developing novel strategies to exhaustively assess and monitor both the quality of raw materials and final marketed herbal products is a challenge in herbal pharmacovigilance. Seventy-nine Ayurvedic herbal products sold as tablets, capsules, powders, and extracts were randomly purchased via e-commerce and pharmacies across Europe, and DNA metabarcoding was used to assess the ability of this method to authenticate these products. Our analysis reveals that only two out of 12 single ingredient products contained only one species as labeled, eight out of 27 multiple ingredient products contained none of the species listed on the label, and the remaining 19 products contained 1 to 5 of the species listed on the label along with many other species not specified on the label. The fidelity for single ingredient products was 67%, the overall ingredient fidelity for multi ingredient products was 21%, and for all products 24%. The low level of fidelity raises concerns about the reliability of the products, and detection of threatened species raises further concerns about illegal plant trade. The study highlights the necessity for quality control of the marketed herbal products and shows that DNA metabarcoding is an effective analytical approach to authenticate complex multi ingredient herbal products. However, effort needs to be done to standardize the protocols for DNA metabarcoding before this approach can be implemented as routine analytical approaches for plant identification, and approved for use in regulated procedures

High-throughput sequencing of African chikanda cake highlights conservation challenges in orchids

Source at https://doi.org/10.1007/s10531-017-1343-7 .Chikanda is a traditional dish made with wild-harvested ground orchid tubers belonging to three orchidioid genera, Disa, Satyrium and Habenaria, all of which are CITES appendix II-listed. Identification of collected orchid tubers is very difficult and documentation of constituent species in prepared chikanda has hitherto been impossible. Here amplicon metabarcoding was used in samples of six prepared chikanda cakes to study genetic sequence diversity and species diversity in this product. Molecular operational taxonomic unit identification using similarity-matching reveals that species of all three genera were present in the chikanda samples studied. Disa was present in all of the samples, Satyrium in five out of six and Habenaria in one of the samples, as well as a number of other plants. The fact that each sample contained orchids and the presence of a wide variety of species from all genera in this traditional dish raise serious concerns about the sustainability of this trade and the future of wild orchid populations in the main harvest areas. This proof-of-concept study shows that Ion-Torrent PGM is a cost-effective scalable platform for metabarcoding using the relatively long nrITS1 and nrITS2 regions. Furthermore, nrITS metabarcoding can be successfully used for the detection of specific ingredients in a highly-processed food product at genus level, and this makes it a useful tool in the detection of possible conservation issues arising from commercialized trade or processed plant products

Identifying the Species of Seeds in Traditional Chinese Medicine Using DNA Barcoding

Seed is not only the main reproductive organ of most of herbal plants but also an important part of Traditional Chinese Medicine (TCM). Seed TCMs possess important medicinal properties and have been widely used as components of pharmaceutical products. In parallel with the increasing popularity and accessibility of seeds as medicinal products in recent years, numerous substitutes and adulterants have also appeared on the market. Due to the small volume and similar appearances of many seed TCMs, they are very difficult to accurately identify the constituent plant species through organoleptic methods. Usage of the wrong herb may be ineffective or may worsen the condition and even cause death. Correct identification of seed herbal medicines is therefore essential for their safe use. Here, we acquired 177 ITS2 sequences and 15 psbA-trnH sequences from 51 kinds of seed TCMs belonging to 64 species that have been described in the Chinese Pharmacopoeia. Tree-building analysis showed that the ITS2 sequences of 48 seed TCMs can be differentiated from each other, and they formed distinct, non-overlapping groups in the maximum-likelihood tree. Furthermore, all of the sequences acquired in this study have been submitted to the public DNA barcoding system for herbal medicine, and this integrated database was used to identify 400 seed TCM samples purchased from medicinal markets, drug stores, and the Internet, enabling the identification of 7.5% of the samples as containing non-declared species. This study provides a brief operating procedure for the identification of seed TCMs found in herbal medicine. In the future, researchers and traditional herbal medicine enterprises can use this system to test their herbal materials

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

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. ABSTRAC

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

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

Traditional Chinese medicine residues promote the growth and quality of Salvia miltiorrhiza Bunge by improving soil health under continuous monoculture

Continuous monoculture of crops has resulted in reduced yields and quality, as well as soil deterioration. Although traditional Chinese medicine residues (TCMRs) are known to promote plant growth and soil health, few studies have investigated their effectiveness in continuous monoculture soils. Here, we studied the impact of chemical fertilizers (CF) and four TCMRs with antibacterial activities on the growth of S. miltiorrhiza (a widely used medicinal plant in China), accumulation of active ingredients in plants, and soil health under continuous monoculture conditions. Compared with no fertilizer (CK) and CF, fermented Sophora flavescens radix residue (SFRf) and fermented and unfermented Moutan cortex residue (MCRf and MCRu, respectively) resulted in a reduction of the disease index of root rot, while CF did not. The CF and four TCMR treatments increased the accumulation of nitrogen (N) (42.8-124.6% and 17.0-101.7%), phosphorous (P) (19.8-74.7% and 8.3-27.4%), and potassium (K) (104.1-212.0% and 9.3-51.8%) in shoots and roots compared to CK. The differences in nutrient accumulation between the CF and TCMR treatments were statistically insignificant, excepted for the N accumulation in the roots. All fertilization treatments increased plant biomass compared to CK, with increases of 25.57-89.86% and 2.62-35.28% in shoots and roots, respectively. The SFRf treatment exhibited the most significant enhancement in both shoot and root biomass. CF significantly reduced the accumulation of seven active ingredients in roots by 23.90-78.95% compared to CK, whereas each TCMR increased accumulation of certain active ingredients. The TCMR treatments effectively improved the health of deteriorated soil by enhancing soil physicochemical properties, restoring the balance of the microbial community, recruiting beneficial bacteria, and reducing the relative abundance of the pathogen Fusarium. The SFRf treatment exhibited superior performance in improving soil health than other treatments. Overall, the TCMRs outperformed CF in restoring soil health and promoting the yield and quality of S. miltiorrhiza. These findings offer guidance for improving the health of continuous cropping soil and recycling TCMRs