Phase 4 (2009 Deadline)
Standardization and Quality Assurance of Medicinal Plants
Allan Showalter, Ohio University
Zabta Khan Shinwari, Quaid-i-Azam University
Pakistani Funding (HEC): $104,502
US Funding (Department of State): $281,000
Project Dates: November 15, 2010 - November 14, 2013 (Extended through October 31, 2014)
Project Overview
Pakistan has more than 6,000 species of higher plants, of which at least 12 percent are used medicinally. The country is among the top ten in exporting raw herbs, with more than 400 local herbal product companies involved in the industry. Due to over-collection, however, several plant species have become extinct in the Hindukush-Himalayan regions. The major reason for this loss of plant biodiversity is that most people involved in gathering plant materials (mainly women and children) harvest natural resources thoughtlessly to subsidize their meager incomes as best they can. Due to inadequate collection methods, more than half of all plant material gathered is lost before even reaching the processing stage. Moreover, due to lack of training, the harvesters frequently collect plants that may look similar but are actually different species. Serious mistakes have been reported when the wrong plant material has been used. This project will help herbal product processors identify medicinal plant species through the use of molecular markers (i.e., DNA barcoding and DNA fingerprinting) that can be used to uniquely “fingerprint” and “barcode” plant species. Databases of such genetic barcode and fingerprint data will allow for simple and conclusive identification of medicinal plant species. Application of the methods to be used in this project will also determine the heavy metal content and nutritional value of these medicinal plants to ascertain whether the material meets international standards. In the process of testing and identifying the most appropriate barcode genes and fingerprinting sequences for plants for this purpose, the US partners will help their Pakistani counterparts to learn and apply these new techniques, thus strengthening human resources and research infrastructure at Quaid-i-Azam University (QAU). Improving the quality of herbal products will also help to increase their value and should ultimately increase the earnings of the poor communities involved in collection and cultivation as those involved are trained in proper techniques for collection, cultivation, and processing. In time, enhanced exports of standardized, semiprocessed, and later fully processed herbal drugs could become an increasing source for earnings for Pakistan in the international marketplace.
Quarterly Update
During the last quarter, the US research staff received voucher specimens from Pakistan. Kirk Emch and Dr. Melanie Schori carried out DNA extractions and a PCR analysis of these samples. They have also downloaded almost all of the GenBank sequence data available for comparisons in alignments for each of the genera under investigation. These sister sequences will allow analyses of intra- and interspecific variation, and provide information on which gene region(s) is most informative for each genus.
The project staff on the US side has sequenced market material of Fagonia and voucher material of fig family specimens for a student research project, with the results sent back to Pakistan. The Fagonia data indicate that any of the three gene regions (rbcL, matK, psbA-trnH) can be used to distinguish species that occur in Pakistan. They also confirm that multiple species are sold in the marketplace under the same common name. Sequence data has also been sent to Dr. Helen Griffiths and Dr. Kirsten Wolff, two researchers in the UK who have investigated anti-cancer properties of Fagonia from Pakistan, to assist them in determining which species they are studying.
On the Pakistani side, two PhD students, Nadia Batool Rizvi and Ikram Ullah, have collected different species samples and are conducting tests, including DNA extraction. Ms. Rizvi made a trip to Khyber Pakhtunkhwa, where she collected Lamiaceae and Rosaceae samples. She is working on DNA extraction of these species. Mr. Ullah brought in more samples of Fagonia from Peshawar and has conducted protein kinase inhibition, antifungal, and cytotoxicity assays. He is preparing an article on the results. Furthermore, five M. Phil students continue to assist in the project by gathering samples in the field and performing tests. Some of these tests include analyzing anti-cancerous properties.
In addition to field and lab work, the research team has submitted a review article on the ethnomedicinal importance of the Apiaceae family to the Pakistan Journal of Botany. Moreover, a joint US-Pakistan manuscript has been started to highlight the need for vouchers and DNA barcoding for Fagonia research. Dr. Schori presented the results of the barcoding project at the Botanical Society of America conference in Boise, ID.
Progress Reports
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2011 Show summary || Hide summary
The researchers on this project have been busy during the spring and summer of 2011, following up on their initial planning discussions at the Pakistan-U.S. Science and Technology Conference in Dubai in late March. Dr. Shinwari and his graduate students have shipped their first batch of medicinal plant samples to Ohio University, and Dr. Showalter’s postdoctoral research associate, Dr. Melanie Schori-Bragg, has begun DNA barcoding work on the materials. The QAU PhD student who is supervising the sampling team has collected 30 species of the tribe Anthemideae of the family Asteraceae, a group with many medicinal plants that may be confused with similar non-medicinal species. He is working with one of the M.Phil students on random amplified polymorphic DNA (RAPD) and barcode analysis of the samples. Another M.Phil student has successfully completed her experimental work on antibacterial activity and heavy metal content determination of 10 medicinal plants traditionally used to treat throat and chest infections and is currently writing her thesis on these findings. With guidance from the PhD student, two other M.Phil students are conducting their experimental work after completing an initial ethno-botanical survey and collecting plant samples. One is working on the antimicrobial potential of medicinal plants used to treat skin infections, and the other is studying the antimicrobial potential of medicinal plants traditionally used to treat gynecological diseases and infections.
In December 2011, Dr. Showalter participated in the 4th International Barcode of Life Conference in Adelaide, Australia, after which he joined his postdoctoral associate Dr. Melanie Schori in Islamabad for a visit to their counterpart Dr. Shinwari at QAU. The highlight of the visit was the International Workshop on Medicinal Plants: Conservation & Sustainable Use, held at QAU December 8-10. Nearly 200 attendees from universities and research institutes across Pakistan took part, and 29 papers presented at the workshop were published in a special supplemental issue of the Pakistan Journal of Biology. During the trip, Drs. Schori and Showalter visited Dr. Shinwari's laboratories and the university's herbarium and met with Dr. Shinwari's students to learn more about their research and provide suggestions. Dr. Schori also conducted a half-day seminar for 60 participants on how to write a scientific paper and provided training to two QAU students on proper procedures for collecting leaf material to ensure that future samples will yield high-quality DNA for barcoding. On the final day of the workshop, most of the participants visited Qarshi Industries, one of the leading companies in Pakistan's herbal products industry.
2012 Show summary || Hide summary
Collection of plant material in Pakistan was temporarily suspended during the winter dry season, but additional materials will be shipped in the coming months after the plants begin blooming again. A memorandum of understanding is currently being drafted in preparation for Qarshi Industries, a leading company in the Pakistani herbal products industry, to begin submitting samples from their collections for barcoding. So far, 17 barcodes have already been produced by Dr. Schori for medicinal plant samples previously received from Pakistan. In July 2012, Dr. Shinwari will visit the United States to join his colleagues in the annual conference of the Botanical Society of America in Columbus, OH, after which he will visit Ohio University for collaboration with Drs. Showalter and Schori. One of Dr. Shinwari’s PhD students, Nadia Rizvi, is also being invited to spend one month at Ohio University this summer receiving hands-on training on barcoding and related procedures. Meanwhile, back at QAU, two M.Phil. students involved in the project (both female) received their degrees, and three other female M.Phil. students are currently carrying out project-related research toward their degree requirements. A PhD scholar on the project, Mr. Muhammad Nadeem, is involved in specimen collections and will travel to the United Kingdom this year for barcoding training.
An interesting side benefit of this project on the U.S. side has been the involvement of a very young scientist, eighth grader Winter Wilson, who worked with Dr. Schori in November 2011 to extract and sequence DNA barcodes from a locally purchased sample of dill (Anethum graveolens), which were compared with samples from the same species sent from Pakistan. Winter presented her results at her middle school science fair, and based on her strong showing there she has been invited to participate in the Ohio state science fair in May 2012.
During spring 2012, 39 plant samples for DNA barcoding were received from Pakistan, and AlexaRae Kitko, an undergraduate Honors Tutorial College apprentice in Dr. Showalter’s lab, has prepared extractions for all the samples and begun amplifying and sequencing selected genes. Meanwhile, Dr. Schori gave a colloquium on the DNA barcoding project to Ohio University’s Department of Environmental and Plant Biology on April 27, 2012. She has been working with a scientist at Kapa Biosystems, Dr. Maryke Appel, on an optimization protocol for a PCR kit made by Kapa. This recently developed kit appears to work well for many of the medicinal plants from Pakistan. Dr. Schori and Dr. Appel will be submitting a manuscript with the protocol to BioTechniques, an open-access research journal.
Unfortunately, plans for Dr. Shinwari to visit the United States in July to deliver a joint presentation at the annual conference of the Botanical Society of America and spend some time working with Dr. Showalter and Dr. Schori at their lab had to be cancelled when his visa was delayed. Dr. Schori delivered the presentation herself at the conference on July 9, and Dr. Shinwari is hoping to reschedule the visit for this fall. One of his PhD students, Nadia Batool Zahra, is also awaiting issuance of a visa so that she can travel to Ohio in early September for one month of training in laboratory techniques in Dr. Showalter’s lab. Back at QAU, Dr. Shinwari’s PhD student Muhammad Nadeem is heading to the University of Edinburgh on a six-month scholarship from the Higher Education Commission of Pakistan. He will be DNA barcoding approximately 64 different species of the family Asteraceae. M.Phil scholar Ms. Khansa Jamil is working on DNA barcoding selected species (Acacia and Albizia). She has been working on rbcL sequences and regularly consults Dr. Schori for assistance. Another M.Phil scholar, Ms. Misbah Salima, is studying the antimicrobial activity of medicinal plants collected from Kohat District. Following completion of an ethnobotanical survey of medicinal plants, the collected specimens have been used to prepare methanolic extracts, which have been tested against six bacterial strains. Ms. Sumera Malik, also an M.Phil scholar, is screening medicinal plants used in the Ghazi Brotha Dam area. After an ethnobotanical survey of that area, plant extracts were prepared with three different solvents and tested for antimicrobial activity against nine bacterial strains and one fungal strain.
2013 Show summary || Hide summary
On the U.S. side, co-PI Dr. Schori and graduate student Ms. AlexaRae Kitko continue to sequence two gene regions from 51 samples obtained from the Royal Botanic Gardens, Kew’s DNA bank. The samples represent sister species to the medicinal plants under investigation, and the sequences are being used to compare the variation within the gene regions. Dr. Schori began collaboration with Dr. Lonnie Welch, a bioinformatics professor, Dr. Wei Lin, a mathematics professor, and Rami Al-Ouran, one of Dr. Welch’s PhD students to develop, test, and implement several algorithms for analyzing sequence variation. Dr. Schori wants to assess whether each gene region (rbcL, matK, or psbA-trnH) is suitably variable to distinguish the medicinal plant of interest from its sister species. No assessment method currently exists. Dr. Lin has written computer code for analyses, and Dr. Schori is currently comparing the analysis output to the original alignments of sequences to make sure the algorithms are analyzing the data appropriately. Dr. Schori has begun writing several manuscripts on the barcoding project. An overall paper on results will be submitted to PLOS ONE which is an open access international, peer-reviewed online publication. The data analysis will be submitted to Cladistics and a paper describing their work on species in six genera to be submitted to the American Journal of Botany. Dr. Showalter and Dr. Schori visited Islamabad from January 5-January 16 for a symposium on technology transfer, which was rescheduled after their arrival. However, they met with their industry counterpart, Dr. Altaf, from Qarshi Industries (well known natural product company) to report on progress and challenges with barcoding plant samples with additional 15 medicinal plants to be sent to the U.S. lab from Qarshi. They also joined Dr. Shinwari in presenting a lecture to students on writing scientific articles. In Pakistan, Dr. Shinwari’s PhD student, Ms. Nadia Rizvi (Zahra), is collecting more species of Lamiaceae for DNA barcoding to expand her studies to a wider range of medicinal species in the family. PI Dr. Zabta Shinwari collected seeds of Fagonia and sent them to Oregon State University in Corvallis for propagation. The Department of Biotechnology at QAU is also experimenting to determine the appropriate methods for germination and propagation. Fagonia has been shown to have an effect against breast cancer, although the species has (or have) not yet been fully identified. DNA barcoding should help determine which species are being used. Pakistan side. Dr. Shinwari presented on the “DNA barcoding of medicinal plants from Pakistan” at the First Pakistan-U.S. Science and Technology Cooperation Program Symposium on “Economic Growth through Technology Transfer” held on January 31- February 1, 2013. Muhammad Nadeem, a PhD student from the QAU lab has extended his stay at the University of Edinburgh, UK in order to complete DNA barcoding in a different gene region. He is the primary author along with Dr. Shinwari and Dr. Qaiser of an article published in the Pakistan Journal of Botany (http://www.pakbs.org/pjbot/PDFs?45%28S1%29/16.pdf) .
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