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.

Hyperglycemia induced reactive species trigger structural changes in human serum albumin of type 1 diabetic subjects.

Chronic oxidative stress fuels pathogenesis of a large set of diseases. Oxidative stress is the cause and consequence of numerous diseases including type 1 diabetes mellitus (T1DM), in which there is selective destruction of insulin producing pancreatic ß-cells. Studies have documented that hyperglycemia produces profound stress. In vivo production of numerous reactive oxygen, nitrogen, chlorine species and lipid/sugar oxidation products in T1DM patients may be the result of persistent hyperglycemia. Post-translational modifications by reactive species may create new antigenic epitopes and play a role in the development of autoimmune response. In this paper our main focus was to establish the effect of existing hyperglycemia induced oxido-nitrosative stress in T1DM patients on the integrity of human serum albumin. Raised nitric oxide, carbonyl, RBC hemolysis, lowered ferric reducing antioxidant power (FRAP), thiol and deformed RBC in T1DM are all highly suggestive of persistent oxido-nitrosative stress. Hyperglycemia induced generation of advanced glycation end products (AGEs) was established by LCMS. Chronic oxido-nitrosative stress can modify HSA in T1DM patients, producing immunologically active albumin. Therefore, it is speculated that the aberrant HSA may play a role in the initiation/progression of T1DM.

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.

A single molecular marker to distinguish between species of Dioscorea.

Yams are species of the genus Dioscorea (family Dioscoreaceae), which consists of approximately 630 species. The majority of the world production of yams occurs in Africa with 58.8 million t annually, but they are also produced in the Americas and Asia. The saponins in yams have been reported to possess various properties to improve health. The tuber and aerial parts of various species often share morphological similarities, which can cause problems in the proper identification of sample material. For example, the rootstocks and aerial parts of Dioscorea villosa L. share similarities with Dioscorea polystachia Turcz. Dioscorea bulbifera L. may be mistaken for Dioscorea alata L. owing to similar morphologies. Various molecular analyses have been published to help with the identification of species and varieties within the genus Dioscorea. The multi-loci or single-locus analysis has resulted in varying success, some with only a limited discrimination rate. In the present study, a single nuclear genomic region, biparentally inherited, was analyzed for its usefulness as a molecular marker for species identification and discrimination between D. bulbifera, D. villosa, D. nipponica, D. alata, D. caucasica, and D. deltoidea samples. The results of this study show that the LFY genomic region can be useful as a molecular marker to distinguish between samples.

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.

Investigating sesquiterpene biosynthesis in Ginkgo biloba: molecular cloning and functional characterization of (E,E)-farnesol and α-bisabolene synthases.

Ginkgo biloba is one of the oldest living tree species and has been extensively investigated as a source of bioactive natural compounds, including bioactive flavonoids, diterpene lactones, terpenoids and polysaccharides which accumulate in foliar tissues. Despite this chemical diversity, relatively few enzymes associated with any biosynthetic pathway from ginkgo have been characterized to date. In the present work, predicted transcripts potentially encoding enzymes associated with the biosynthesis of diterpenoid and terpenoid compounds, including putative terpene synthases, were first identified by mining publicly-available G. biloba RNA-seq data sets. Recombinant enzyme studies with two of the TPS-like sequences led to the identification of GbTPS1 and GbTPS2, encoding farnesol and bisabolene synthases, respectively. Additionally, the phylogenetic analysis revealed the two terpene synthase genes as primitive genes that might have evolved from an ancestral diterpene synthase.

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.

The loci recommended as universal barcodes for plants on the basis of floristic studies may not work with congeneric species as exemplified by DNA barcoding of Dendrobium species.

BACKGROUND: Based on the testing of several loci, predominantly against floristic backgrounds, individual or different combinations of loci have been suggested as possible universal DNA barcodes for plants. The present investigation was undertaken to check the applicability of the recommended locus/loci for congeneric species with Dendrobium species as an illustrative example. RESULTS: Six loci, matK, rbcL, rpoB, rpoC1, trnH-psbA spacer from the chloroplast genome and ITS, from the nuclear genome, were compared for their amplification, sequencing and species discrimination success rates among multiple accessions of 36 Dendrobium species. The trnH-psbA spacer could not be considered for analysis as good quality sequences were not obtained with its forward primer. Among the tested loci, ITS, recommended by some as a possible barcode for plants, provided 100% species identification. Another locus, matK, also recommended as a universal barcode for plants, resolved 80.56% species. ITS remained the best even when sequences of investigated loci of additional Dendrobium species available on the NCBI GenBank (93, 33, 20, 18 and 17 of ITS, matK, rbcL, rpoB and rpoC1, respectively) were also considered for calculating the percent species resolution capabilities. The species discrimination of various combinations of the loci was also compared based on the 36 investigated species and additional 16 for which sequences of all the five loci were available on GenBank. Two-locus combination of matK+rbcL recommended by the Plant Working Group of Consortium for Barcoding of Life (CBOL) could discriminate 86.11% of 36 species. The species discriminating ability of this barcode was reduced to 80.77% when additional sequences available on NCBI were included in the analysis. Among the recommended combinations, the barcode based on three loci - matK, rpoB and rpoC1- resolved maximum number of species. CONCLUSIONS: Any recommended barcode based on the loci tested so far, is not likely to provide 100% species identification across the plant kingdom and thus is not likely to act as a universal barcode. It appears that barcodes, if based on single or limited locus(i), would be taxa specific as is exemplified by the success of ITS among Dendrobium species, though it may not be suitable for other plants because of the problems that are discussed.

DNA barcoding of endangered Indian Paphiopedilum species.

The indiscriminate collections of Paphiopedilum species from the wild for their exotic ornamental flowers have rendered these plants endangered. Although the trade of these endangered species from the wild is strictly forbidden, it continues unabated in one or other forms that elude the current identification methods. DNA barcoding that offers identification of a species even if only a small fragment of the organism at any stage of development is available could be of great utility in scrutinizing the illegal trade of both endangered plant and animal species. Therefore, this study was undertaken to develop DNA barcodes of Indian species of Paphiopedilum along with their three natural hybrids using loci from both the chloroplast and nuclear genomes. The five loci tested for their potential as effective barcodes were RNA polymerase-ß subunit (rpoB), RNA polymerase-ß' subunit (rpoC1), Rubisco large subunit (rbcL) and maturase K (matK) from the chloroplast genome and nuclear ribosomal internal transcribed spacer (nrITS) from the nuclear genome. The intra- and inter-specific divergence values and species discrimination rates were calculated by Kimura 2 parameter (K2P) method using mega 4.0. The matK with 0.9% average inter-specific divergence value yielded 100% species resolution, thus could distinguish all the eight species of Paphiopedilum unequivocally. The species identification capability of these sequences was further confirmed as each of the matK sequences was found to be unique for the species when a blast analysis of these sequences was carried out on NCBI. nrITS, although had 4.4% average inter-specific divergence value, afforded only 50% species resolution. DNA barcodes of the three hybrids also reflected their parentage.