Identification and Functional Characterization of Oxidosqualene Cyclases from Medicinal Plant Hoodia gordonii.

Oxidosqualene cyclases (OSCs) are the key enzymes accountable for the cyclization of 2,3-oxidosqualene to varied triterpenoids and phytosterols. Hoodia gordonii (from the family Apocynaceae), a native of the Kalahari deserts of South Africa, Namibia, and Botswana, is being sold as a prevalent herbal supplement for weight loss. The appetite suppressant properties are attributed to P57AS3, an oxypregnane steroidal glycoside. At the molecular level, the enzymes involved in the biosynthesis of triterpenes and phytosterols from H. gordonii have not been previously reported. In the current study, predicted transcripts potentially encoding oxidosqualene cyclases were recognized first by searching publicly available H. gordonii RNA-seq datasets. Two OSC-like sequences were selected for functional analysis. A monofunctional OSC, designated HgOSC1 which encodes lupeol synthase, and HgOSC2, a multifunctional cycloartenol synthase forming cycloartenol and other products, were observed through recombinant enzyme studies. These studies revealed that distinct OSCs exist for triterpene formation in H. gordonii and provided opportunities for the metabolic engineering of specific precursors in producing phytosterols in this plant species.

Deoxyribonucleic Acid Barcoding for the Identification of Botanicals.

The Natural Herbal Products industry uses botanicals or herbs as raw materials for production of herbal products or dietary supplements. Recently, the demand for natural herbal products has increased tremendously and this has led to adulteration and to counterfeit herbal products. The present chapter deals with currently used molecular methods from "simple" single genomic regions to high-throughput whole genome or transcriptome sequencing methods used in the identification of botanicals.

Is Baikiain in Tara Flour a Causative Agent for the Adverse Events Associated with the Recalled Frozen French Lentil & Leek Crumbles Food Product? - A Working Hypothesis.

The French Lentil & Leek Crumbles frozen food product was recently recalled due to reports of gastrointestinal issues. So far, 393 adverse illness complaints and 133 hospitalizations have been reported from consumption of this food, and the tara (Tara spinosa) protein flour ingredient is hypothesized to be responsible. A multipronged approach resulted in identification of (S)-(-)-baikiain in tara as a compound of interest due to its abundance, possible metabolic fate, and close resemblance to irreversible inhibitors of L-pipecolate oxidase. Oral administration of baikiain in ND4 mice showed a statistically significant increase in blood ALT levels and a reduction in liver GSH.

The Low Copy Nuclear Gene Region, Granule Bound Starch Synthase (GBSS1), as a Novel Mini-DNA Barcode for the Identification of Different Sage (Salvia) Species.

Morphological similarity within species makes the identification and authentication of Salvia species challenging, especially in dietary supplements that contain processed root or leaf powder of different sage species. In the present study, the species discriminatory power of 2 potential DNA barcode regions from the nuclear genome was evaluated in 7 medicinally important Salvia species from the family Lamiaceae. The nuclear internal transcribed spacer 2 and the exon 9 - 14 region of low copy nuclear gene WAXY coding for granule-bound starch synthase 1 were tested for their species discrimination ability using distance, phylogenetic, and BLAST-based methods. A novel 2-step PCR method with 2 different annealing temperatures was developed to achieve maximum amplification from genomic DNA. The granule-bound starch synthase 1 region showed higher amplification and sequencing success rates, higher interspecific distances, and a perfect barcode gap for the tested species compared to the nuclear internal transcribed spacer 2. Hence, these novel mini-barcodes generated from low copy nuclear gene regions (granule-bound starch synthase) that were proven to be effective barcodes for identifying 7 Salvia species have potential for identification and authentication of other Salvia species.

A Comprehensive Workflow for the Analysis of Bio-Macromolecular Supplements: Case Study of 20 Whey Protein Products.

The presence of bio-macromolecules as major ingredients is a primary factor in marketing many biologically derived macromolecular supplements. Workflows for analyzing these supplements for quality assurance, adulteration, and other supply-chain difficulties must include a qualitative assessment of small-molecule and macromolecular components; however, no such integrated protocol has been reported for these bio-macromolecular supplements. Twenty whey protein supplements were analyzed using an integrated workflow to identify protein content, protein adulteration, inorganic elemental content, and macromolecular and small-molecule profiles. Orthogonal analytical methods were employed, including NMR profiling, LC-DAD-QToF analysis of small-molecule components, ICP-MS analysis of inorganic elements, determination of total protein content by a Bradford assay, SDS-PAGE protein profiling, and bottom-up shotgun proteomic analysis using LC-MS-MS. All 20 supplements showed a reduced protein content compared to the claimed content but no evidence of adulteration with protein from an unclaimed source. Many supplements included unlabeled small-molecule additives (but nontoxic) and significant deviations in metal content, highlighting the importance of both macromolecular and small-molecule analysis in the comprehensive profiling of macromolecular supplements. An orthogonal, integrated workflow allowed the detection of crucial product characteristics that would have remained unidentified using traditional workflows involving either analysis of small-molecule nutritional supplements or protein analysis.

Identification of Species in the Aromatic Spice Family Apiaceae Using DNA Mini-barcodes.

The species of the aromatic plant family Apiaceae are mainly used as spices and foods, but the family also includes medicinal and some poisonous plant species. Due to the similar chemical compounds or aroma and morphology, the poisonous species are often mistaken for the edible aromatic species. It is therefore imperative to correctly identify the species present at the initial raw stage samples to ensure product safety and efficacy. At the molecular level, plant species can be identified using DNA loci either from nuclear or plastid genome with easily available universal oligonucleotides, a technique called DNA barcoding. However, this is possible when single-species plant material is present but may not work on a mixture of plants species. Another disadvantage is that using universal oligonucleotides is of limited help, especially if the adulterating material is present in low quantities. On the other hand, if using the species-specific oligonucleotides, only single specific adulterating plant material could be detected and, consequently, the unexpected adulterants may go undetected. Therefore, in the current work, four degenerated oligonucleotides from ITS1 and ITS2 regions of the nuclear genome were designed that can bind to a variety of Apiaceae genera only and not to other genera belonging to different plant families. These family-specific oligonucleotides were able to amplify a diagnostic PCR product from 16 Apiaceae species that, upon sequencing, revealed the identity of the plant it was derived from. The size of these products is around 140 bp for ITS1 and approximately 80 bp for the ITS2 region. The size range of the amplified products falls in the category of a desired mini-barcode size to be used for damaged/fragmented DNA and next generation sequencing.

Evaluating five different loci (rbcL, rpoB, rpoC1, matK, and ITS) for DNA barcoding of Indian orchids.

Orchidaceae, one of the largest families of angiosperms, is represented in India by 1600 species distributed in diverse habitats. Orchids are in high demand owing to their beautiful flowers and therapeutic properties. Overexploitation and habitat destruction have made many orchid species endangered. In the absence of effective identification methods, illicit trade of orchids continues unabated. Considering DNA barcoding as a potential identification tool, species discrimination capability of five loci, ITS, matK, rbcL, rpoB, and rpoC1, was tested in 393 accessions of 94 Indian orchid species belonging to 47 genera, including one listed in Appendix I of CITES and 26 medicinal species. ITS provided the highest species discrimination rate of 94.9%. While, among the chloroplast loci, matK provided the highest species discrimination rate of 85.7%. None of the tested loci individually discriminated 100% of the species. Therefore, multi-locus combinations of up to five loci were tested for their species resolution capability. Among two-locus combinations, the maximum species resolution (86.7%) was provided by ITS+matK. ITS and matK sequences of the medicinal orchids were species specific, thus providing unique molecular identification tags for their identification and detection. These observations emphasize the need for the inclusion of ITS in the core barcode for plants, whenever required and available.

DNA Barcoding for the Identification of Botanicals in Herbal Medicine and Dietary Supplements: Strengths and Limitations.

In the past decades, the use of traditional medicine has increased globally, leading to a booming herbal medicine and dietary supplement industry. The increased popularity of herbal products has led to a rise in demand for botanical raw materials. Accurate identification of medicinal herbs is a legal requirement in most countries and prerequisite for delivering a quality product that meets consumer expectations. Traditional identification methods include botanical taxonomy, macroscopic and microscopic examination, and chemical methods. Advances in the identification of biological species using DNA-based techniques have led to the development of a DNA marker-based platform for authentication of plant materials. DNA barcoding, in particular, has been proposed as a means to identify herbal ingredients and to detect adulteration. However, general barcoding techniques using universal primers have been shown to provide mixed results with regard to data accuracy. Further technological advances such as mini-barcodes, digital polymerase chain reaction, and next generation sequencing provide additional tools for the authentication of herbs, and may be successful in identifying processed ingredients used in finished herbal products. This review gives an overview on the strengths and limitations of DNA barcoding techniques for botanical ingredient identification. Based on the available information, we do not recommend the use of universal primers for DNA barcoding of processed plant material as a sole means of species identification, but suggest an approach combining DNA-based methods using genus- or species-specific primers, chemical analysis, and microscopic and macroscopic methods for the successful authentication of botanical ingredients used in the herbal dietary supplement industry.

DNA barcoding of medicinal plant material for identification.

Because of the increasing demand for herbal remedies and for authentication of the source material, it is vital to provide a single database containing information about authentic plant materials and their potential adulterants. The database should provide DNA barcodes for data retrieval and similarity search. In order to obtain such barcodes, several molecular methods have been applied to develop markers that aid with the authentication and identification of medicinal plant materials. In this review, we discuss the genomic regions and molecular methods selected to provide barcodes, available databases and the potential future of barcoding using next generation sequencing.