Performing Department
CARC
Non Technical Summary
PROJECT SUMMARYThis proposed project is focused on establishing a comprehensive reference system for identification, verification and authentication of plant species used in dietary supplements for male health, weight loss and cardiovascular health. The specific objectives of this project are: 1.To establish a reference living collection for morphological verification /authentication, 2. To investigate and establish a DNA based procedures for identification authentication, and verification of the selected species, 3. To compile, sort and verify existing analytical data and develop analytical methods, if necessary for active marker compounds in processed raw materials, 4. To centralize all above 1-3 data of selected plant species into a web based searchable database for better use and disseminate the information. Successful completion of this project could yield a comprehensive reference system that has a collection of accurately identified living collection of selected medicinal plant species, DNA-barcode or other DNA-based identification information along with standard reference material libraries, and a compiled set of analytical assays for signature bioactive compounds. The output information will be organized into a web-base searchable database for public access. This reference system will provide a powerful tool for manufacturers, trade associations, regulators and consumer advocacy groups to accurately identify plant materials to ensure the quality and safety of herbal products. The successful completion of the project could also build essential capacity in medicinal plant research so as to further strengthen and open new teaching and research avenues in plant sciences, natural products and human nutrition in the College.
Animal Health Component
0%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Goals / Objectives
The goals of this project are:1. To establish a reference garden with a focused living collection of medicinal plants for species identification in raw and processed ingredients that is used for dietary supplements for male health, weight loss and cardiovascular health. These reference plants along with their closely related species will be accurately identified and vouchered for morphological verification/authentication of plant species, as well as for providing standard reference materials.2. To research and establish DNA-based procedures for identification, verification and authentication of raw materials and/or process ingredients for selected species.3. To compile, sort/sieve and verify, and to develop, if necessary, analytical assay for plant-derived bioactive compounds in processed materials.4. To centralize the morphological/image, DNA-based-identification and analytical assay information on these selected plants in a searchable web-based database for best use of these data.
Project Methods
The following methodologies will be used:For Goal 1. The reference garden will host a collection of selectedmedicinal plant species that are used in popular dietary supplements for male health, weight loss and cardiovascular health, respectively. These species, their derived product formats and relevant information already in BOLD are summarized in Appendix1. Depending on the need for forming the Barcode Index Number (BIN) System that clusters sequences to produce operational taxonomic units that closely correspond to a species, at least 2 to 3 representative varieties of each species, and related species for controls will be collected and hosted in the garden. All germplasm will be acquired by PI through the Botanical Garden Conservation International and the International Plant Exchange Network, to ensure the identity and also further taxonomically authenticated by PI after the species are grown at PVAMU. A collection of complete reference voucher specimens will be made, and their morphological (macro and micro) characters will be characterized. A seed bank will be established from all the species in cultivation and to share with other partnership institutes in the International Plant Exchange Network (IPEN). One of the two greenhouses outlined in the CAHS 2014-2019 strategic plan will be dedicated to hosting of these selected reference plant species.For Goal 2: There are two lines of research works for this goal.i. To develop barcodes and establish the Barcode Index Number (BIN) sequence cluster for each selected species. The Barcode Index Number (BIN) System clusters sequences using well-established algorithms to produce operational taxonomic units that closely correspond to species. The guidelines of BOLD will be followed and procedures adapted for barcode and BIN development. The matK and rbcL locus will be used as a combined universal barcode for the selected plant species, with augmentation of other recommended loci (psbA-trnH, atpF-atpH, psbK-psbI or ITS) if necessary. Procedures for major steps of these works are discussed below.• Sampling and sample archiving: Fresh leaf samples of ~1-2g will collected induplicates, one sample for immediate DNA isolation and the other for storage forfuture use and reference. The sample for storage will be dried using silica-geldesiccation, and archived. Briefly, the leaf samples will be placed in a tea filter bag,and then tightly sealed in a plastic bag containing indicating silica gel beads (>10:1gel to leaf weight ratio). The silica gel will be replaced every 6 hour or so untilcomplete drying of the sample. After adding fresh silica gel, the plastic bag will be inturn sealed in a light-proof aluminum bag, and archived in a storage bin under cooland dry environment.• DNA Extraction: Silica based column methods (e.g. ZR Plant/Seed DNA Kits, fromZymo Research) will be used for DNA extraction from fresh or dried plant materials.Necessary modifications for tissue disruption and lysate clearing will be made, asleaves from some of these medicinal plants are rich in polyphenols, terpenes andpolysaccharides.• Primers, multiplex PCR, sequencing and data processing: Currently available best"universal" primers for matK and rbcL (also for other loci if necessary) recommendedby CBOL will be based for developing primers best for each taxonomic group. PCRor Multiplex-PCR (rbcL+ matK barcode) conditions will be optimized for eachtaxonomic group from those recommended by CBOL and BOLD. Gel-purifiedamplicon will be directly sequenced using commercial sequencing service. All dataprocessing for BOLD submission will use the "Workbench" of the BOLD system,following its guidelines.ii. To develop procedures for barcode-based or other DNA-based species identification inraw and processed materials. For barcode-based species identification and establishing aStandard reference material (SRM) herbal barcode library, the methodology and procedureoutlined by Newmaster, S. G., et.al. will be adapted and modified. The focus and the goalare to successfully identify target species, adulterants/contaminants, substitutions and fillers in processed products such as tablets, capsules and powders in the market. An effective extraction of residual DNAs from these products and PCR amplification procedures will be developed. Because of the highly-fragmented nature of the DNA in the processed ingredients, mini-barcodes will be developed, if necessary. For species-identification in highly processed ingredients in liquid product, we will attempt more selective and specific GQ-HCR-based or SNP-Multiplex-PCR-based identification procedures.For Goal 3: As all the selected medicinal plant species are being used as sole material or ingredients in popular dietary supplements, analytical methods for signature or chemical markers, or even for metabolite profiles in almost all these plants have been developed around the world. Some of the analytical tests may have already been the standards for manufacturers. Our task is to conduct literature search, screening various methods for effective but simple ones, compiling and centralizing all the selected analytical information in a database along with the morphological and DNA-based identification information. If necessary, some of the selected analytical methods will be screened and tested on the reference materials and processed products at the core lab in the college.For Goal 4: The IT specialists, Co-PI, Ashwani Srivastava and Ibuomi Abibo, willlead the effort in building, managing and hosting the database and website. The contents will be provided by the three teams on the project. The web page for each species will focus on the following information.• Distinct morphological characteristics along with images from reference plants, and unique identification-assisting organoleptic properties, in addition to taxonomicinformation.• DNA-barcode and BINs, which will be linked to BOLD and MMDBD, and DNA basedspecies identification in raw and processed products.• Analytical methods /procedures for identification of signature compounds.In future, information on popular dietary supplement product information may be added and linked to each plant species, which will be beneficial to extension work, especially in areas of agriculture & natural resources, and family and consumer sciences.Project Management Plan and EvaluationManagement TasksLeading PersonManagement SummaryEvaluationBudgetWeerasooriya, A.Understanding award terms and conditionsMonitoring expenses, billing and payments monthly.Re-budgeting if necessary in accordance with progress.Compliance.Proper resource allocationTimely purchasing.Planning & ImplementationAll participantsReview the proposed operation plan, identify potential issues or necessary modification.Re-affirm roles, and expectations of each research part.Make a detailed execution plan.Establish a meeting schedule for sharing progress, solving problems, and coordinating research operations.A detailed operation plan, and quality of execution, as reflected in execution. Quality ControlLeading researcher for each operation.Cross-reference check on plants with collections & voucher specimen at other botanical garden.Sequence verification of DNA barcoding data during BOLD submission.Data verification before being presented on the website.Peer review and evaluation by the proposed expert.Progress MonitoringWeerasooriya, A.Track progress according to the timetable.Identify any bottleneck issues, and coordinate a team effort in finding a solution.On time execution of the project.ReportingWeerasooriya, A. & Leading researcher for each operation.Gather progress information on each planned operation, and preparing for annual and final CRIS reports.Preparing presentation & manuscripts, web contentsTimely completion of CRIS reports.No. of presentations and publications.