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Pakistan-US Science and Technology Cooperation Program                                                            
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 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 

Research on this project has produced multiple DNA sequences, called barcodes, which can be used to identify medicinal plants. The researchers have been comparing sequences from dried plant material sold in open-air markets in Pakistan with sequences produced from voucher specimens, which are identified by experts and kept as reference samples for further study. One of the project’s objectives has been to demonstrate that DNA barcodes can be used to identify to medicinal plants, and another aim has been to use DNA barcodes to help identify market material to make sure it is sold under the correct names and is not adulterated. So far, most of the market material from Pakistan has been successfully barcoded, and most of the material has been sold under the correct names. They have documented a few instances of adulteration or outright substitution. In several cases, market material contained a mixture of two plants, and in others, entirely different plants were sold under the same common name. Three of the substitutions were noted before barcodes were produced because the plant material did not look right. Other samples consisted of seeds or twigs, which were much harder to identify by sight and were only identified with the DNA barcodes. These results have helped to highlight which medicinal plants are most likely to be sold under the wrong name, which may lead to an effort to cultivate these plants within Pakistan so that a guaranteed source is available to the herbal products industry.

In addition to generating barcodes, the team has published a set of protocols for improved DNA amplification from medicinal plants. Medicinal plants often have compounds that can interfere with separating and copying pieces of DNA, both of which are essential for generating barcodes. The team has been working with Kapa Biosystems to develop guidelines for using one of their amplification kits with medicinal plant samples. The combination of the kit with the protocols led to much higher success rates than we had with traditional amplification kits. To analyze the DNA barcodes and select which ones are most useful for different groups of plants, the project team collaborates with researchers in Ohio University’s mathematics and bioinformatics programs.

On the US side, an undergraduate student who was trained last summer continued work on DNA extractions and amplifications. A different undergraduate was trained in preparing DNA extractions. Two PhD students and an undergraduate math major have contributed to implementing analysis methods for DNA barcodes. Dr. Schori gave a lecture to a bioinformatics class about plant systematics, DNA barcoding, and challenges in data analysis. The lecture provided background information for the students who have been working on implementing the analysis methods.

On the Pakistan side, a partnership was developed between Quaid-i-Azam University and Qarshi Industries (Pvt) Ltd which will supply voucher and market samples of medicinal plants for barcoding in the US lab. Two new students have begun PhD research on DNA barcoding. Two current PhD students have continued their work on Artemisia and species of Lamiaceae. Six other students are working on M. Phil. Projects, conducting ethnobotanical surveys, antimicrobial screenings, proximate analyses, and phytochemical investigations.

Progress Reports

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