500 5th St NW - KWS 502
Washington, DC 20001
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Phase 4 (2009 Deadline)
Bioactive Genes and Peptides/Proteins from Medicinal Plants
Leslie M. Hicks, Donald Danforth Plant Science Center
Amer Jamil, University of Agriculture, Faisalabad
Pakistani Funding (HEC): $117,565
US Funding (Department of State): $317,500
Project Dates: November 15, 2010 - November 14, 2013 (Completed)
The LTQ Orbitrap Velos, the world's fastest and most sensitive hybrid ion trap-Orbitrap mass spectrometer, is utilized to routinely deliver ultra-high resolution and accurate mass data for peptide and protein identification and characterization (photo courtesy of Dr. Hicks).
Although plants have been used as a source of medicine since ancient times, only a very small percentage of plants have been studied to date. This project focuses on investigating unexplored medicinal plants of Pakistan to isolate bioactive peptides and proteins with antimicrobial, anticancer, and antiviral activities. Collaboration with US colleagues at the Donald Danforth Plant Science Center will enhance the capacity of Pakistani researchers to carry out cutting-edge research aimed at discovering promising natural compounds. The long-term objective of the project is to deliver some bioactive compounds for commercialization after further characterization. Because the private companies that have been created in Pakistan’s growing herbal medicine industry generally have poorly developed research and development capacities and are thus unable to isolate and test new compounds themselves, the results of this project could also help in attracting corporate interest in commercializing new products.
The project continues to focus on the separation (fractionation) of black cumin (N. sativa) extracts into simpler components and reproduced bioactivity seen with N. sativa extracts in the fractionation. The Hicks lab moved to UNC Chapel Hill in September 2013, with a new graduate student overlapping with the previous postdoctoral associate at the Danforth Center. The following species are growing in UNC’s Greenhouse Facility (http://greenhouse.bio.unc.edu/): black cumin, dill, basil, castor, fennel, Indian mustard. A new collaboration was established with UNC’s Center for Integrative Chemical Biology and Drug Discovery (CICBDD) (https://pharmacy.unc.edu/research/centers/center-for-integrative-chemical-biology-and-drug-discovery) in replicating assays using a high throughput format to quickly screen other plant extracts using the black cumin as positive control to assess broad antimicrobial activity. Mass spectrometric acquisition and data analysis parameters are being optimized to efficiently detect and characterize active components for further sequencing. A female graduate student from the Univ. of Agriculture, Faisalabad, will join the Hicks lab Oct-Jan 2013 as a visiting scientist and will work on various aspects of this project and receive training in analytical characterization.
Plant species now growing in our 210 sq. ft of space in the UNC Greenhouse.
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2011 Show summary || Hide summary
In the spring and summer of 2011 both the US and Pakistani PIs worked to recruit postdoctoral research associates. Until an appropriate candidate is hired on the US side, Dr. Hicks is working with a lab technician already employed in her lab to begin analyzing the first shipment of plant materials, which were received from Pakistan in mid-July. Five plant species that have control, 2-4 hour, and 6-hour post-inoculation samples were sent, as well as two plant species that have control and 4 or 5 day post-inoculation samples. Proteins will be extracted and preliminary comparison assessments will be done to try to identify any changes between the control and treated samples for any peptides and/or proteins that are increased relative to the control. Dr. Hicks has also contacted several colleagues in the United States and Canada to obtain existing sequence information for the Pakistani plants and related family members to aid in peptide and protein identification efforts. Other colleagues based at the Danforth Center have agreed to help in setting up and conducting antifungal screening and in providing some relevant seeds of species of interest to begin growing so that plant material will be readily available at any time for testing and methods development even if relevant Pakistani plant materials are delayed for any reason.
2012 Show summary || Hide summary
On the Pakistani side, Dr. Jamil reports that seeds of 16 plant species were collected from the Botanical garden at the University of Agriculture Faisalabad for the study during first year of the project. A memorandum of understanding was signed between UAF and Qarshi Industries Ltd. (a leading Pakistani firm in the herbal products industry), and seeds were obtained for 8 additional plant species from the Qarshi herbarium. The seeds were cultivated in growth chamber and subjected to antifungal assays. Most of the plants exhibited good antifungal activities, and seven plants with the highest activity levels were selected for the first round of investigations. Protein extractions were made from the selected plants and the samples were sent for further analysis to Dr. Hicks at the Danforth Center. Thanks to funds provided under this grant (supported by HEC on the Pakistani side), Dr. Jamil has acquired an Experion automated electrophoresis system, a vertical gel electrophoresis system, a dry-heat bath, and a nanophotometer, along with essential chemicals and labware. Intermittent power supply problems has made the research work all the more challenging. Low salary levels have also made it difficult to recruit researchers with the desired qualifications.
On the U.S. side, Dr. Hicks hired a postdoctoral associate, Brian Gau, who began work in her lab on January 16, 2012. With partial support provided by this grant, her lab has purchased an AB Sciex TripleTOF 5600 mass spectrometer, which will allow samples for this project to be analyzed by Dr. Hicks and Dr. Gau instead of being sent to an outside facility for processing. The researchers are continuing to work on sample preparation for the 2D gels from the plant extract materials already in hand from Pakistan, and they have received a second round of plant materials that will be extracted with optimized methods. In the coming months they will work on the 2D gel analysis of the inoculated material from Pakistan, and they are beginning to work on liquid chromatography-mass spectroscopy strategies for profiling and identifying potential bioactive target peptides/proteins. They have also started growing some bulk plant material at the tissue culture facility of the Danforth Center to have on hand for optimization of methods.
Dr. Hicks reports that she and her postdoc received training on their new Triple TOF 5600 mass spectrometer during the first week of April 2012. They have already optimized LC separation and MS data acquisition methods for protein identification and have carried out two-dimensional DIGE experiments on protein extracted from three inoculated plant species sent by Dr. Jamil. They have obtained MS data on the spots that were statistically significantly different from the control and are working on identification based on database searching and further data mining strategies due to the fact the genomes for their plants of interest are not sequenced. In the coming months they will continue to work on the 2D gel analysis of the inoculated material from Pakistan and will use a 2D DIGE approach for that work. They will also begin to work on enrichment strategies for profiling and identification of potential bioactive target peptides/proteins. Dr. Hicks and her team have done their own growth and mock treatment to have on hand for optimization of methods, and they have material from Pakistan available in freezer storage to use once the method is optimized. Meanwhile, in Pakistan Dr. Jamil and his team have prepared extracts from two plant species, Ricinus cummunis (castor bean) and Datura stramonium (jimson weed), that were exposed to fungal stress, and they are currently working on similar extracts with another species, Psoralea corylifolia (babchi, a plant used in traditional medicine). They have initiated some peptide purification work, although their efforts continue to be hindered by frequent power failures. A backup power supply has recently been acquired, so this should help support the reliable operation of key pieces of equipment in the lab at UAF. Later in August, Dr. Hicks, Dr. Jamil, and their colleagues on the project are preparing for their first videoconference meeting, during which they will present their work and discuss their results and next steps.
2013 Show summary || Hide summary
In spring 2013, the UAF team investigated the induced antibacterial and antifungal properties of Nigella sativa (black cumin). While the fungus Fusarium solani is pathogenic to black cumin and many other plants (compare seedlings in Fig 1A to infected seedlings in 1B), exposure to the fungus promotes the increased production of some antimicrobial compounds (Fig 1C-H). (See Figure 1).
Figure 1. The induction of bioactive biomolecules in Nigella sativa by Fusarium solani inoculation. A) N. sativa seedlings growing on wetted filter paper; B) N. sativa seedlings post 72 h inoculation with F. solani. Disc-diffusion bioassays were conducted on the extracts of seedlings collected at different time points post inoculation. Antimicrobial and buffer controls reside at the center and top discs for each plate, respectively. By virtue of the zones of growth inhibition, bactericidal activity is seen for C) Escherichia coli, D) Pasteurella multocida E) Staphylococcus aureus, andF) Bacillus subtilis. Fungicidal activity is seen forG) F. solani andH) Trichoderma harzianum.
The US team has been following the lead from the Pakistani team and optimized growth condition for Nigella sativa, developed a mass spectrometry-friendly extraction protocol for antimicrobial compounds of N. sativa, onsite antimicrobial assays, and confirmed that the antimicrobial activity of N. sativa extracts for compounds are smaller than most proteins. (See Figure 2).
Figure 2. The peptidome extraction and bioassay of N. sativa. The aerial tissue of 100 three-weak-old seedlings was cold-pulverized, and the endogenous soluble peptides and biomolecules extracted in a special 10% acetic acid solution. Centrifugation, filtration, dialysis, and concentration steps gave a crude peptidomic extract that exhibited E. coli bactericidal activity in a microtiter plate assay.