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International Conference Quality and Safety Issues Related to Botanicals,August 15-18th 2005, University, MS

Sponsored by CFSAN/FDA and the International Society for Horticultural Sciences (ISHS)

The National Center for Natural Products Research (NCNPR) within the School of Pharmacy at The University of Mississippi is pleased to announce a conference on Quality and Safety Issues Related to Botanicals. The conference is supported by a cooperative agreement between the NCNPR and the Center for Food Safety and Applied Nutrition (CFSAN) of the U.S. Food and Drug Administration. This conference is held in conjunction with the International Society for Horticultural Sciences (ISHS), and will take place at the NCNPR in University, MS.

The purpose of this conference is to review, discuss, and explore methods for determining the identity, purity, quality, and strength of medicinal and aromatic plants, commonly known as botanicals. Topic areas will include such issues as authentication, cultivation, collection, and post-harvest practices for producing quality plant material, and chemical, toxicological methods for quality/safety assessment. Contributed presentations, both oral and poster, are invited. Each session will open with a plenary speaker outlining the current approaches, limitations, and research needs of the topic area.

The proceedings of the conference will be published in Acta Horticulturae, the official publication of the ISHS. Speakers will be leading researchers from industry, academia, nonprofit institutions, and government. Each speaker will address current approaches, limitations, and research needs.



Advisory CommitteeSam Page, Ph.D. World Health OrganizationLarry Walker, Ph.D. Director, NCNPR, The University of MississippiSusan Walker, M.D. Director, Division of Dietary Supplement Programs

ONPLDS, CFSAN, FDA

Organizing CommitteeJoseph M. Betz, Ph.D. Office of Dietary Supplements of NIHLyle E. Craker, Ph.D. Professor, Department of Plant and Soil Sciences

University of MassachusettsJeanine Davis, Ph.D. North Carolina State University Steven Dentali, Ph.D. Vice President, Scientific and Technical Affairs,

American Herbal Products AssociationStephen O. Duke, Ph.D. Research Leader, USDA, ARS, NPURU

NCNPR, The University of MississippiIkhlas Khan, Ph.D. Director of FDA Program, Assistant Director NCNPR,

The University of MississippiSteven Musser, Ph.D. Chief, Instrumentation and Biophysics Branch, CFSAN, FDATroy Smillie, Ph.D. Research Scientist, NCNPR, The University of MississippiLuis G. Valerio Jr., Ph.D., Toxicologist, Division of Biotechnology and GRAS Notice

Review, OFAS, CFSAN, FDAJason Woo, M.D., MPH, FACOG Division of Dietary Supplement Program, ONPLDS,

CFSAN, FDA

Scientific Program CommitteeK. Hüsnü C. Baser, Ph.D. Professor, Head of the Department of Pharmacognosy,

Anadolu University, Eskisehir, Turkey Mark Blumenthal Executive Director, American Botanical CouncilCharles Burandt, Ph.D. Research Assistant Professor, NCNPR,

The University of MississippiJosé A. da Silva Cabral Lab. Fitoquímica – Coordenador SUFRAMA John Cardellina, Ph.D. National Institutes of HealthShaw T. Chen, M.D., Ph.D. Associate Director, ODE-V, CDER, FDAEdward Croom Jr., Ph.D. Adjunct Associate Professor, Pharmacognosy,

The University of MississippiKent E. Cushman, Ph.D. Assistant Professor, SWFREC, University of Florida Mahmoud A. ElSohly, Ph.D. Research Professor RIPS, Professor of Pharmaceutics.

The University of MississippiTrish Flaster Executive Director, Botanical LiaisonsEdward J. Fletcher Strategic Sourcing, Inc., COO/Botanicals Division



Scientific Program Committee (continued): Chlodwig Franz, Ph.D. Professor and Director, Institute for Applied Botany,

University of Veterinary Medicine, Vienna, AustriaStefan Gafner, Ph.D. Team Leader of Analytical Chemistry, Tom’s of MaineDe-An Guo, Ph.D. Modern TCM Research Center, Peking Univ., ChinaPeter Griffee Senior Officer (Industrial Crops) Plant Production and

Protection Division. FAO of the United NationsMahabir P. Gupta, Ph.D. Director, Centro de Investigaciones Farmacognósticas de la

Flora Panameña (CIFLORPAN)Jana Hildreth AOAC - InternationalLoren Israelsen, J.D. President, LDI Group, Inc., Executive Director, Utah

Natural Products AllianceHemant Lata, Ph.D. Associate Research Scientist, NCNPR,

The University of MississippiShiyou Li, Ph.D. Research Scientist, Arthur Temple College of ForestryRadha K. Maheshwari, Ph.D. Department of Pathology, Uniformed Services University

of the Health Sciences.Susan Manly, Ph.D. Associate Director, NCNPR, The University of MississippiAkos Mathe, Ph.D. Professor of Botany, University of West Hungary, Faculty

of Agriculture and Food ScienceJim Miller, Ph.D. Missouri Botanical GardenRita M. Moraes, Ph.D. Research Assistant Professor, NCNPR

The University of MississippiDavid S. Pasco, Ph.D. Assistant Director, NCNPR. The University of MississippiLinda S. Pellicore, Ph.D. Division of Dietary Supplement Program, ONPLDS,

CFSAN, FDAG.N. Qazi, Ph.D. Regional Research Laboratory, Jammu, CSIR, India Jeanne Rader, Ph.D., Director, Division of Research and Applied Technology,

ONPLDS, CFSAN, FDAIiya Raskin, Ph.D. Professor II, Plant Science, Cook College,

Rutgers University James Rushing, Ph.D. Professor of Horticulture, Postharvest Specialist,

Clemson University James E. Simon, Ph.D. Director of The New Use Agriculture and Natural Plant

Products Program, Rutgers UniversityBarbara Steinhoff, Ph.D. German Medicines Manufacturers‘ Association (BAH)Roy Upton Executive Director, American Herbal PharmacopoeiaAruna Weerasooriya, Ph.D. Research Scientist, NCNPR, The University of Mississippi



Day 1 (August 15th) 8:00 - 8:45 Registration and Continental Breakfast – Oxford Conference Center 8:45 - 9:00 Introductory Remarks: Ikhlas Khan, Ph.D. and Lyle Craker, Ph.D.

Welcome: Alice M. Clark, Ph.D., Vice-Chancellor for Research and Sponsored Programs, The University of Mississippi.

9:00 - 9:30 Opening Address: Barbara O. Schneeman – CFSAN. Claims Used on Food and Dietary Supplements

Session 1: Cultivation, Collection, Post Harvest and Processing Practices to Produce Quality Botanical Material

Session Chair: Lyle E. Craker, Ph.D., Professor, Department of Plant and Soil SciencesUniversity of Massachusetts

9:30 - 10:00 Peter Griffee Senior Officer (Industrial Crops) - FAO of the United Nations. An Overview of FAO’s Work on Medicinal and Aromatic Plants (MAPs) with Particular Reference to Ecocrop and EcoPort.

10:00 - 10:30 Ed Fletcher, Chief Operating Officer, Botanicals Division of SSI. GAP and GMP Initiatives that Assure the Safety and Quality of Botanicals.

10:30 - 10:50 Break 10:50 - 11:20 K. Hüsnü C. Baser, Ph.D., Professor, Head of the Department of

Pharmacognosy, Faculty of Pharmacy, Anadolu University. Aromatic Plants as a source for Botanicals.

11:20 - 11:50 José A. da Silva Cabral Ph.D. Lab. Fitoquímica, Coordenador, CBA/SUFRAMA. The Role of CBA - The Amazon Biotechnology Center in the Safety of Amazonian Botanicals.

11:50 - 12:20 G.N. Qazi, Ph.D. Director, Regional Research Laboratory, Jammu Tawi, India. Safety and Efficacy Validation of Indian Botanical Drugs.

12:20 - 2:00 Lunch – Oxford Conference Center Dining HallSession 2: Authentication, Identification and Purity of BotanicalsSession Chair: Edward Croom Jr., Ph.D. Adjunct Associate Professor,

Pharmacognosy, The University of Mississippi 2:00 - 2:30 Roy Upton, President and Chief Executive Officer, American Herbal

Pharmacopoeia. Authentication, quality, and safety assessment of botanicals and botanical products used in clinical research.

2:30 - 3:00 James Miller, Ph.D., Director, Missouri Botanical Garden. The Special Issues of Test Materials in Botanical Dietary Supplement Research.

3:00 - 3:30 Ann Hirsch, Ph.D. Professor, Department of Moleclular, Cell and Developmental Biology, UCLA. Authenticating Botanicals through Molecular and Anatomical Methods.

3:30 - 4:00 Vaishali Joshi, Ph.D. University of Mississippi. Microscopic Techniques for the Identification and Authentication of Botanicals.

4:00 - 6:00 Tour of the National Center for Natural Products Research. (please sign up) 6:00 - 8:30 Reception/Dinner – Memory House (Held in Yerby Center if raining)



Day 2 (August 16th)Session 3: Quality Assessment of BotanicalsSession Chair: Mahmoud A. ElSohly, Ph.D., Research Professor RIPS, Professor of

Pharmaceutics. The University of Mississippi 8:30 - 9:00 Michael Andreas Karl Popp, Ph.D., CEO of Bionorica. Quality, safety and

efficacy assurance: from plant culture to the final product. 9:00 - 9:30 Stefan Gafner, Ph.D., Team Leader of Analytical Chemistry, Tom’s of

Maine. Chromatographic fingerprinting as a means of quality control: Distinction between Actaea racemosa and four different Actaea species.

9:30 - 10:00 Ikhlas Khan, Ph.D., Assistant Director, National Center for Natural Products Research. Challenges in Assessing the Quality and Safety of Botanicals: A Global Co-operation is needed.

10:00 - 10:20 Break 10:20 - 10:50 De-An Guo, Ph.D., Modern Traditional Chinese Medicine Research, Peking

University, China. Quality control and in vivo process of traditional Chinese medicine.

10:50 - 11:20 Aviva Romm, Herbalist perspective on safety of botanicals in humans. Blue cohosh: An evaluation of use prevalence and safety in pregnancy.

11:20 - 11:50 Mark Blumenthal, Executive Director, American Botanical Council. Determining Safety Information for Herb Product Labels: The American Botanical Council's Safety Assessment Program.

11:50 - 12:20 Steven Dentali, Ph.D., Vice President, Scientific and Technical Affairs, American Herbal Products Association. Building the Global Consortium for the Science of Botanicals.

12:20 - 2:00 Lunch – Oxford Conference Center Dining Hall

Session 4: Poster Session – 2:00 - 4:00 Oxford Conference Center Lobby 4:00 - 6:00 Tours of the Medicinal Plant Garden (please sign up) 6:00 - 8:30 Dinner – Oxford University Club



Day 3 (August 17th)Session 5: Safety Assessment of BotanicalsSession Chair: Steven Musser, Ph.D., Chief, Instrumentation and Biophysics Branch,

CFSAN, FDA. 8:30 - 9:00 Klaus Reif, Ph.D. Phytolab. Contamination in Herbal Drugs in View of

European Regulations and Future Developments. 9:00 - 9:30 Mary Trucksess, Ph.D. FDA/CFSAN. Determination of Mycotoxins in

Botanical Roots. 9:30 - 10:00 Jon Wong, Ph.D. FDA/CFSAN. Pesticides in Botanical Dietary

Supplements. 10:00 - 10:30 Bill Gurley, Ph.D. UAMS. In Vivo Assessment of Potential Herb/Drug

Interactions. 10:30 - 10:50 Break 10:50 - 11:20 Christopher Borgert, Ph.D. President and Principal Scientist, Applied

Pharmacology & Toxicology, Inc. Causal Assessment of Herbal-Drug Interactions: Scientific Data vs. Diagnostic Scales.

11:20 - 11:50 Radha K. Maheshwari, Ph.D., Department of Pathology, USUHS. Enhancement of Wound Healing and Angiogenesis by Novel Botanicals.

11:50 - 12:20 David Pasco, Ph.D., Assistant Director, National Center for Natural Products Research. Using Bioassays to Improve Botanical Consistency and Safety: From The Standardization Of Immune Enhancing Botanicals To The Identification Of Toxic Principles.

12:20 - 1:30 Lunch – Oxford Conference Center Dining Hall

Session 6: Safety Assessment of Botanicals Session Chair: Jeanne Rader, Ph.D., Director, Division of Research and Applied

Technology, ONPLDS, CFSAN, FDA. 1:30 - 2:00 David Kroll, Ph.D. Research Triangle Institute. Simple and Inexpensive

Methods to Prevent Botanical Misfortune. 2:00 - 2:30 Paul But, Ph.D., Chinese University of Hong Kong. Trouble-shooting in

the cases of adverse reactions to botanical products. 2:30 - 3:00 Armando Cáceres, M.D., Farmaya, S.A. Multidisciplinary Development of

Phytotherapeutic Products From Native Central American Plants. 3:00 - 4:00 GCSB Discussion group. OCC Dining hall 5:00 - 9:00 Reception and Dinner – Ground Zero Blues Club, Clarksdale, MS. (please

sign up)



Day 4 (August 18th)Session 7: Regulatory Aspects for Safety Assessment of Botanical ProductsSession Chair: Sam Page, Ph.D., World Health Organization (Retired). 8:30 - 9:00 Susan Walker, M.D., Director, Division of Dietary Supplement Programs,

Office of Nutritional Products, Labeling, and Dietary Supplements, CFSAN, FDA. Regulation and Safety Assessment of Botanical Dietary Supplement Products.

9:00 - 9:30 Shaw T. Chen, M.D., Ph.D., Associate Director, ODE-V, CDER, FDA. Safety Evaluation of Botanicals as New Drugs: A Report from the CDER Botanical Team.

9:30 - 10:00 Loren Israelsen President, J.D., LDI, Group Inc. Quality and Safety Issues Related to Botanicals.

10:00 - 10:30 Edmund Lee, Ph.D., Executive Director, Hong Kong Jockey Club Institute of Chinese Medicine Limited. Development of Chinese medicine and related products: safety and quality perspectives.

10:30 - 10:50 Break 10:50 - 11:20 Robin Marles, Ph.D. Manager, Natural Health Products Directorate 11:20 - 11:50 Sam Page, Ph.D., World Health Organization (Retired). Safety

Assessment of Botanicals in Canada. 11:50 - 12:20 Closing Remarks - Comments 12:20 - 2:00 Lunch – Oxford Conference Center Dining Hall (box lunch)



Bio-sketch forDr. Barbara O. Schneeman

Dr. Barbara Schneeman is currently Director of the Office of Nutritional Products, Labeling, and Dietary Supplements in the Center for food Safety and Applied Nutrition at FDA. Prior to FDA she was faculty member at the University of California, Davis from 1976 to 2004. She holds a professorial appointment in the Departments of Nutrition, Food Science and Technology, and Internal Medicine in the School of Medicine and served as the Associate Vice Provost for University Outreach. For 5 years she served as chair of the Department of Nutrition and for 6 years she was the Dean of the College of Agricultural and Environmental Sciences. Dr. Schneeman was a visiting scientist at the Cardiovascular Research Institute, University of California, San Francisco and completed an 18-month term, on leave from UC Davis, as Assistant Administrator for Nutrition in the Agricultural Research Service of the U.S. Department of Agriculture.

Dr. Schneeman received her Bachelor of Science degree from the University of California, Davis in food science and technology, her Ph.D. in nutrition from the University of California, Berkeley and postdoctoral training in gastro-intestinal physiology at Children’s Hospital in Oakland.

Dr. Schneeman’s professional activities include membership on both the 1990 and 1995 Dietary Guidelines Committee as well as serving as a consultant for FAO on food-based dietary guidelines. She has been a member of the Food and Nutrition Board of the Institute of Medicine-National Academies of Science and chaired committees for IOM on nutritional issues for women in the military and for the development of a safety framework for dietary supplement ingredients. She has served on the California State Board of Food and Agriculture, the USDA Research, Extension and Education Advisory Board and on committees for FAO, WHO, the American Society for Nutritional Sciences, and the Institute of Food Technologists. She has served as Associate Editor for the Journal of Nutrition and on the editorial board for Proceedings of the Society of Experimental Biology and Medicine, Food and Nutrition Series of Academic Press, Nutrition Reviews, Journal of Nutrition, and California Agriculture.

Professional honors for Dr. Schneeman include Fellow of the American Association for the Advancement of Science, Carl Fellers Award from the Institute of Food Technology, the FDA Commissioner’s Special Citation and the Harvey W. Wiley Medal, the Samuel Cate Prescott award for research, the Future Leader Award, and several honorary lectureships. She currently has over 120 publications and is recognized for her research contributions in the areas of gastrointestinal function, dietary fiber, lipid metabolism and food-based dietary guidelines.



Claims Used on Food and Dietary Supplements

Barbara O. Schneeman, Ph.D., DirectorOffice of Nutritional Products, Labeling, and Dietary Supplements

Center for Food Safety and Applied Nutrition, Food and Drug AdministrationWashington DC USA

In 1990, the Nutrition Labeling and Education Act (NLEA) was signed into law (Public law 101-535). This amendment to the Federal Food, Drug and Cosmetic Act (FDCA) led to significant changes in food labeling and resulted in major revisions to nutrient labeling. Through rule-making, the Food and Drug Administration (referred to as FDA or the agency) promulgated regulations that specified the requirements for nutrition labeling as well as the criteria for nutrient content and health claims. In 1994 the FDCA was further amended by the Dietary Supplement Health and Education Act (DSHEA), which defined dietary supplements and provided for the use of claims on these products. There are currently 3 types of claims allowed on products that are intended for use as foods or dietary supplements; these include health claims, structure-function claims, and nutrient content claims. Nutrient content claims characterize the level of nutrient in a food; structure/function claims describe the role of a nutrient or dietary substance that is intended to affect normal structure or function of the body; and health claims describe the relationship between a food, food component, or dietary supplement ingredient and reducing the risk of a disease or health-related condition. Health claims that meet the standards of significant scientific agreement are authorized through regulations. In response to several court decisions, FDA has developed an interim process to allow qualified health claims for foods and dietary supplements through enforcement discretion. Because these claims are based on emerging science, they contain language to qualify the quality and strength of scientific evidence to support the claim. The criteria used to review and evaluate qualified health claims have been published as interim guidance. In November, 2004, FDA published draft guidance for substantiation of structure-function claims on dietary supplements and we are currently reviewing the comments submitted.



Bio-sketch forMr. Peter Griffe

Mr. Griffee represented FAO on the WHO editorial committee in Geneva to do the final revision of ‘Good Agricultural and Field Collection Practices for Medicinal Plants’ (published in 2004) and the "Model Monograph on Good Agricultural and Collection Practices for Artemisia annua".

He is a member of the Bureau of The International Council for Medicinal and Aromatic Plants (ICMAP) and of the Scientific Committee of The World Congresses of Medicinal and Aromatic Plants (WOCMAP). He was involved in the planning of WOCMAP II (Mendoza, Argentina) and WOCMAP III (Chiang Mai, Thailand); presented papers in both and helped lead the preparation of the resolutions for both. FAO provides funds for the ICMAP Newsletter.

The IFAD/IDRC/Ford project ‘Organic Production of Medicinal, Aromatic and Dye Plants for Livelihood Support in Southern Asia’ was prepared with his help and he will lead operations in India, Sri Lanka, Nepal and Bhutan beginning in 2005 He is also a key player in the FAO Technical Cooperation Project on medicinal plants in India.

Mr. Griffee represented FAO on the committee for the development of ‘MEDUSA’ (The Mediterranean Network on the Conservation and Use of Medicinal Plants) for 3 years until established.

Networking is one of his principal strengths. In this respect he is leading the development of ‘Ecocrop’- the Crop Environmental Requirements Database (http://ecocrop.fao.org) - also on CD-ROM – which includes searches for several hundred MAPs related to their environmental needs and medicinal uses.

Ecocrop is an associated database of ‘EcoPort’ (http://www.EcoPort.org) which has data on many MAPs. EcoPort is managed by contributors globally (including editors from Brazil) who contribute, own and share their information. In 2004 900 pages per day were updated and peer-reviewed. He represents FAO in the EcoPort plant entity.

The abstracts of the Rabat (Morocco) International Medicinal Plants Seminar were prepared by him in EcoPort. He gave a keynote address at the Budapest MAP Conference and made presentations at many MAP conferences.

CV Highlights (May 2005): 38 years of post-graduate multidisciplinary experience in production of annual and perennial crops generally including major and underutilized species. Email; [email protected] , work tel.; +39-06-570-56763 (FAO). Home address; Via del Forno 2, 00060 Formello, RM, Rome, Italy.

mailto:[email protected]



An Overview of FAO’s Work on Medicinal and Aromatic Plants (MAPs) with Particular Reference to Ecocrop and EcoPort.

Peter J. GriffeeIndustrial Crops, Crop and Grassland service

Plant Production and Protection DivisionAgriculture Department

FAO, Viale delle Terme di Caracalla00100 Rome, Italy.

An overview of FAO’s work on medicinal and aromatic Plants (MAPs) with particular reference to Ecocrop and EcoPort.

The PowerPoint presentation gives the scope of the multidisciplinary involvement with MAPs and associated projects and institutions and relates to the safety aspects where appropriate. Emphasis is placed on ‘Ecocrop’- the Crop Environmental Requirements Database (http://ecocrop.fao.org) which includes a search function for several hundred MAPs related to their environmental needs and medicinal uses and on the associated knowledge management system ‘EcoPort’ (http://www.EcoPort.org) which has data on over 550 MAPs including precautions. EcoPort is managed by contributors globally who contribute, own and share their information. In 2004 900 pages per day were updated and peer-reviewed.

EcoPort’s pledge is to: Establish and sustain a "Knowledge Commons" where individuals and communities can work and learn together to develop sustainable ways to manage the Earth's natural resources; Facilitate access to information through a public service that will enable participants to own and update the knowledge created by their collective effort through application of EcoPort's procedures; Promote the availability and use of EcoPort's information and procedures as a Global Public Good to provide education to communities and individuals engaged in natural resources management and conservation; and to Make provision to ensure data quality through peer review and to preserve and display individual ownership of shared information.



Biosketch forMr. Edward J. Fletcher

Mr. Ed Fletcher, COO/Botanicals Division of Strategic Sourcing, Inc., has over 20-plus years of direct experience in botanical cultivation (most recently with Wilcox Natural Products) and oversees a large network of experienced growers. His combined experience makes him uniquely qualified in his field.

Mr Fletcher oversees SSI’s significant grower network of over 200 years of combined experience with over 150 different types of botanicals and its Certified Organic Growing (COG) capabilities. All crops Produced by SSI follow Good Agricultural Practices (G.A.P.)



GAP and GMP Initiatives that Assure the Safety and Quality of Botanicals

Mr. Edward J FletcherCOO/Botanicals Division of Strategic Sourcing, Inc.,

This presentation will be an overview of GAP and GMP initiatives in the United States botanical industry that are used to assure the quality and safety of botanical products.



Bio-sketch forDr. K. Hüsnü C. Baser

K. Husnu Can Baser Pharmacist, Professor of Pharmacognosy

PhD in Pharmacognosy Research Laboratories, Department of Pharmacy, Chelsea College, University of London.

Founding Director of the Medicinal and Aromatic Plant and Drug Research Centre (TBAM) in Anadolu University, Eskisehir, Turkey (1980-2002).

Dean, Faculty of Pharmacy of Anadolu University (1993-2001). Secretary General (1997-2003) and Vice-President (2003-) of the International Council for Medicinal and Aromatic Plants (ICMAP).

Member, Turkish Pharmacopoeia Commission (since 1985). Expert, Group 13B (Phytotherapeuticals) of the European Pharmacopoeia (1999)

Consultant, United Nations Industrial Development Organization (UNIDO), served in several African and Asian countries.

Consultant, International Federation of Essential Oils and Aroma Trades (IFEAT) since 1995.

Author of over 400 papers, recipient of 5 awards.

General research interests: Botanical, ethnobotanical, chemical, analytical, technological and biological activity aspects of medicinal and aromatic plants.

Specific research interests: Essential oils, alkaloids



Aromatic Plants as a Source of Botanicals

K. Husnu Can BaserAnadolu University, Faculty of Pharmacy, Department of Pharmacognosy, 26470 Eskisehir,

Turkeywww.khcbaser.com [email protected]

Aromatic plants have been used in botanicals, food additives, dietary supplements and as a source of essential oils and aromatic extracts.

As most aromatic plants are wildcrafted, standardization to assure their quality and safety is important for their safe and effective utilization in health products. Recent years have witnessed the issuance of more and more pharmacopoeial monographs and standards to overcome possible constraints.

The paper will focus on these aspects as well as discuss ways to assure their appropriate utilization especially from the quality and safety standpoint.



Bio-sketch forDr. José Augusto da Silva Cabral

José Augusto da Silva Cabral, born on May 26, 1946, in Manaus, Amazonas, Brasil to Abílio Cabral and Maria do Carmo da Silva Cabral. In Dec 1975 received a BS degree in Pharmacy from the Universidade Federal do Amazonas. In Jun 1881 received a MS degree in Organic Chemistry from the Universidade Federal do Ceará. In May 1989 received a PhD Degree under the direction of Prof. Dr. James D. McChesney from the University of Mississippi. I am retaired from INPA– National Research Institute of Amazonia. Actually I am the head of the Phytochemical and Vegetal Tissue Culture Laboratories of CBA – Amazon Biotechnology Center – SUFRAMA in Manaus-Amazonas-Brasil.



The Role of CBA - The Amazon Biotechnology Center in the Safety of Amazonian Botanicals

José Augusto da Silva CabralCBA - Centro de Biotecnologia da Amazônia /SUFRAMA - Superintendência da Zona Franca

de Manaus - Amazonas - Brasil.

CBA – The Amazon Biotechnology Center was inaugurated in 2002 and began in 2003 the installation of equipment and infrastructure, which will be completed this coming December. So far the principal sectors that are installed are Phytochemistry, Plant Tissue Culture, Biochemistry and Molecular Biology, Microbiology, Physical-chemical Analysis, Spectroscopy, Animal Experimentation, Behavioral Studies and Nuclear Magnetic Resonance laboratories, besides a Decontamination, Washing and Sterilization Unit. Other facilities set up are a Pilot Plant, an Extract Production Nucleus, an Animal House, a Business Enterprise Incubator and a Biotechnology Information Nucleus. Services the CBA will offer include: Preclinical Pharmacological and Toxicological Assays; Physical, Chemical and Biochemical Analyses; Access to Amazonian Biodiversity; Genomics, Proteomics and Metabolomics Applied to Biotechnological Innovation; Breeding and Provision of Germ Free Laboratory Animals (Rats and Mice); Development of Products and Bio Industrial Processes; Production, Standardization and Certification of Extracts, Intermediate and Finished Products; Microbiological and Contaminant Control; Creation of Technological Spin-off Companies; and the Adaptation and Development of Bio Industrial Processes.

Among the first botanicals to be studied are “Carapanaúba” (Aspidosperma carapanauba and other spp.), Saracura Mirá” (Ampelozizyphus amazonicus), “Moruré” (Brosimum sp.), “Uxi-amarelo”(Endopleura uchi), “Unha de Gato” (Uncaria guianensis, U. guianensis), “Casca Preciosa”(Aniba canelilla) , “Pau d’arco” (Tabebuia serratifolia), “Marapuama”, (Ptychopetalum olacoides) e “Sucuúba”(Himatanthus sucuuba), plants that are utilized by locals for the treatment of various diseases and conditions.



Bio-sketch forDr. Ghulam. N. Qazi

Dr. G. N. Qazi is a Biotechnologist of international repute, with more than 37 years research experience in the area of Biochemistry & Microbial Biotechnology and Bioprospecting of Natural Products.

Presently he is Director RRL (CSIR), Jammu, a multidisciplinary Research Institute engaged in R & D on – Bioprospecting of natural molecules; Biotechnology – fermentation and enzyme technology, microbial biodiversity, molecular biology; Natural Products Chemistry; Cultivation & Utilization of drugs and essential oil bearing plants, standardization of Botanicals.

He is leading a large group of Scientists, Technologists, Post Doctoral Associate, Research Fellows and Technicians engaged in multidisciplinary research. He has more than 100 publications in the journals of international repute and has filed 65 Patents both in India and abroad.

He has developed a strong International collaboration in the area of Biochemical engineering, Enzymology, Genetic engineering and quality control & standardization of botanicals with a number of reputed Institutes in Europe, (Germany, Britain and Italy) and USA.

Dr. Qazi is a recognized Ph.D. guide for 8 Indian universities and has supervised large number of Ph. D. thesis. He is a widely traveled scientist and is member of number of national and international academic / research bodies. As a fermentation technologist, Dr Qazi & his team has developed a number of important processes some of which are already commercialized.

He is the recipient of highly prestigious CSIR Technology Award and VASVIK Industrial Research Award for his contributions in the area of Fermentation technology.



Safety and Efficacy Validation of Indian Botanical Drugs

Ghulam N. Qazi,

Regional Research Laboratory (CSIR), Jammu-Tawi 180 001, India

Globally, there has been an unparalleled growth in the plant-derived medicinally useful formulations, drugs and health care products, with annual growth rates between 10-20% in most of the countries, thus providing window of opportunity to India. The country bears a competitive edge in the international botanical market as Indian Traditional drugs, have their roots in time tested systems of medicine namely, Ayurveda, Unani and Siddha.

However, the major hindrance in the introduction of herbal medicines into modern medical practices is the lack of scientific and clinical data, and better understanding of efficacy and safety of the herbal products. To ensure the quality and safety of such products and practices standardization is of vital importance. Thus with the growing emphasis on quality control, safety has become a key factor in assessing the value of botanicals.

Efficacy testing of the traditional and new herbal products in appropriate experimental screening models (in vitro / in vivo) is paramount to establish the activity and bio-active component with the appropriate extract of the plant. There is therefore, a need to establish the pharmacological activities of herbals for identifying and comparing the various preparations for their potency.

Since the botanical medicines have long histories of traditional use, the proof of efficacy through contemporary scientific process can follow a “ Reverse Pharmacology” path and reduce time and cost of development.

Quality control of botanicals has to be dealt from cultivation of the plant to the finished product. The WHO guidelines on good agricultural and collection practices (GACP) for medicinal plants demands the quality of herbal medicines with ecologically sound cultivation practices.

In India, the National Medicinal Plant Board coordinates and implements policies relating to medicinal plants both at central and state level to facilitate inter ministry, inter state and institutional collaboration and to avoid duplication of efforts.

Implementing GMP for herbal drugs, as per the recent amendment of Drugs and Cosmetic Act of Government of India, is a great challenge for indigenous botanical industry. The presentation will illustrate the Indian perspective on herbal medicine and cultivation efforts with some light on Government initiatives in this direction.



Bio-sketch forMr. Roy Upton

Roy Upton is the President, Executive Director, and Editor of the American Herbal Pharmacopoeia, a non-profit organization dedicated to the development of quality control standards for botanical products. He is trained in both Western and Traditional Chinese herbalism and has been working professionally as an herbalist for more than 22 years. He is the past president and current vice-president of the American Herbalists Guild (AHG). The AHP monographs have been regarded as many as being the most comprehensive and critically reviewed works on herbal medicine worldwide. Roy is also involved with the development of quality control standards for the herbal products industry as general manager of the California-based herbal company Planetary Formulas and as a member of the Standards Committee of the American Herbal Products Association. Roy has also authored several books including St. John's Wort and Echinacea in the Good Herb Series of Keat's Publishing and the Botanical Safety Handbook published by CRC Press.



Authentication, quality, and safety assessment of botanicals and botanical products used in clinical research

Roy Upton, Registered HerbalistExecutive Director, American Herbal Pharmacopoeia

A challenge facing clinical researchers is how to ensure the authenticity, quality, and safety of the botanicals or botanical products used in clinical trials. Herbal products are not required to be manufactured according to independent standards. Because of this there is great variation in the composition of herbal products, even among those using the same botanical(s). In order to assure the highest degree of safety, efficacy, and reproducibility of a study, it is critical to fully characterize the herbal product being used. This often requires the combination of physical and chemical testing, as well as a review of specific batch records to ensure the product was made according to appropriate Good Manufacturing Practices (GMPs). This presentation will address the primary issues regarding botanical product characterization introducing researchers to both physical and chemical assessment methodologies, an overview of independent standards that can be used as a guiding post, and practical sources of information for sourcing appropriate labs who can perform such testing.



Bio-sketch forDr. James S. Miller

Dr. James S. Miller is the William L. Brown Curator at the Missouri Botanical Garden and Head of the William L. Brown Center for Plant Genetic Resources. Activities of the Center include efforts in ethnobotany, the study and conservation of medicinal plants, natural products discovery, management of databases on plant use and other areas of economic botany.

The WLB Center’s efforts in natural products discovery include programs to discover new agricultural, nutritional, or pharmaceutical products from plants through cooperative ventures with governmental, university, or corporate partners. A project with the National Cancer Institute searches for new anti-cancer drugs in Madagascar. The NIH-funded International Cooperative Biodiversity group looks for new medicines and agricultural products from plants in Suriname and Madagascar in partnership with six other institutions. Programs with the University of Mississippi and Sequoia Sciences look for new applications of plants to human health in a variety of countries. Dr. Miller has conducted field research and managed international programs throughout Africa, Asia, and Latin America. He is currently involved in a medicinal plant conservation program in the Republic of Georgia, an effort aimed at survey, ex-situ conservation, and reintroduction of critically endangered species of medicinal plants in the Caucasus region.

Dr. Miller has written and lectured extensively on the methodology, ethics, and legal issues relating to agricultural and pharmaceutical uses of plants, systematics of Boraginaceae, and the floras of Madagascar and Latin America. His interest in the floristics of Madagascar continues and he is completing a botanical inventory in collaboration with P.-J. Rakotomalaza and J. Raharilala of the Reserve Naturelle de Marojejy, a 50,000-hectare protected area in northeastern Madagascar, a project that has been supported by the National Geographic Society and the World Wide Fund for Nature. He also studies systematics of tropical Boraginaceae and continues to describe new species from both the old and new world tropics. His current research interests include generic delimitation and work with post-doc Marc Gottschling on the systematics of the genus Bourreria.

Dr. Miller earned his Ph.D. in biology in 1985 from St. Louis University and his M.S. in 1979 and B.S. in 1975 from the University of Maryland. He is a member of the Botanical Society of America, the American Society of Plant Taxonomists, the International Association for Plant Taxonomy, the Society for Economic Botany, the Association for the Taxonomic Study of the Flora of Africa, and the honor societies Pi Alpha Xi and Sigma Xi. He is an adjunct faculty member in biology at the University of Missouri, St. Louis.



The Special Issues of Test Materials in Botanical Dietary Supplement Research

James S. Miller and Wendy Applequist, William L. BrownCenter for Plant Genetic Resources, Missouri Botanical Garden, St. Louis, Missouri 63166-

0299

Understanding the efficacy and safety of botanical dietary supplements has been difficult because of poor description of the substances being tested. Botanical dietary supplements are complex mixtures of chemical compounds. Presence or absence and concentration of multiple constituents may vary depending on source, environmental conditions where the plants are grown, and the methods by which materials are processed. This presentation will review criteria for ensuring that research on dietary supplements can 1) be repeated and confirmed and 2) that results can be compared with other studies, in both cases with confidence that similar substances are being tested. The three critical issues to ensure consistency in test materials are: 1) Source, 2) Characterization, and 3) Communication. It is necessary to confirm the identity of the constituent species used to manufacture dietary supplements and document the precise population from which they were collected or region in which they were cultivated. Chemically consistent test materials require validated methods for harvest, processing, and formulation. Furthermore, these must be coupled with methods to detect chemical differences from batch to batch to minimize variation and produce reliable test substances for rigorous research. Finally, if the source, methods for preparation, and information on chemical composition are not communicated in publications, readers of research articles may not be able to compare results with those of other studies.



NAME: Ann M. Hirsch POSITION TITLE: Professor

EDUCATION/TRAINING

INSTITUTION AND LOCATION DEGREE YEAR(s) FIELD OF STUDY

Marquette University, Milwaukee, WI B.S. 1969 Biology

University of California, Berkeley, CA Ph.D. 1974 Botany

Harvard University, Cambridge, MA Postdoctoral 1976-1978 Plant Biology/MicrobiologyPositions and Honors. List in chronological order previous positions, concluding with your present position. List any honors. Include present membership on any Federal Government public advisory committee.Employment and Positions Held1974-1976. Assistant Professor, Dept. of Botany, University of Minnesota, St. Paul, MN.1976-1978. Postdoctoral Fellow, Maria Moors Cabot Foundation, Harvard University, Cambridge, MA.1978-1982. Assistant Professor, Department of Biology, Wellesley College, Wellesley, MA.1983-1988. Associate Professor, Department of Biology, Wellesley College, Wellesley, MA.1988-1992. Associate Professor, Dept. of Biology, University of California, Los Angeles, CA.1996-1999. Visiting Associate in Biology, California Institute of Technology, Pasadena, CA.1992-present. Professor, Dept. of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA.Honors Maria Moors Cabot Fellow, Harvard University, 1976-1977.Brachman-Hoffman Fellowship, Wellesley College, 1981-1982.O.E.C.D. (Organization for Economic Cooperation and Development) Fellowship, summers 1987, 1988.NSF Faculty Award for Women, 1990-1995.Research Prize, Instituto Politecnico Nacional de México en el Programma Institucional de Medio Ambiente y Desarrollo Sustentable, 1996-1997.

Selected peer-reviewed publications (in chronological order). Do not include publications submitted or in preparation.(Publications selected from a total of >105 publications) van Rhijn, P., Goldberg, R.B. and Hirsch, A.M. 1998. Lotus nodulation specificity is changed by the presence of a soybean lectin gene. Plant Cell 10:1233-1249.

Brill, L.M., Evans, C.J. and Hirsch, A.M. 2001. Expression of MsLEC1- and MsLEC2-antisense genes in alfalfa plant lines causes severe developmental and reproductive abnormalities. Plant J. 25:453-461. Woo, H.-H., Kuleck, G., Hirsch, A.M., and Hawes, M.C. 2002. Flavonoids: signal molecules in plant development. Adv. Exp. Med. Biol. 505: 51-60. Leroy, A., Potter, E., Woo, H.-H., Heber, D., and Hirsch, A.M. 2002. PCR-based identification of degraded DNA and monitoring of flavonoids isolated from alfalfa and red clover commercial preparations used as botanicals in dietary supplements. J. Agr. Food Chem. 50:5063 –5069. Valdés, M., Pérez, N.-O., Estrada-de los Santos, P., Caballero-Mellado, J., Peña-Cabriales J.J, Normand, P., and Hirsch, A.M. 2005. Non-Frankia actinomycetes isoalted from surface-sertilized roots of Casuarina equisetifolia fix nitrogen. Appl. Environ. Microbiol. 71: 460-466. Lum, M., Potter, E., Dang, T., Heber, D., Hardy, M., and Hirsch, A.M. 2005. Identification of botanicals and potential contaminants through RFLP and sequencing. Planta Med. Published on line 29 July, 2005.



Authenticating Botanicals through Molecular and Anatomical Methods

Michelle R. Lum1, Barry A. Prigge2 and Ann M. Hirsch1,3. 1

Dept. of Molecular Cell and Developmental Biology, 2UCLA Herbarium, and 3Molecular Biology Institute, University of California-Los Angeles, CA 90095-1606

In the U.S., it is estimated that over 80 million people use some type of herbal medicine or botanical supplement. The number of botanical species that are exploited for their potential health benefits is enormous. Although an increasing amount of information is being obtained on the medicinal properties of the phytochemicals contained in botanicals, the efficacy of the botanical supplements used to deliver these compounds relies on the quality of the starting and processed products. Analysis of different commercial products has found them to be highly variable in pharmacologically relevant substances. An even greater concern facing both the industry and the consumer is the authenticity of the plant material in botanical supplements. Other species may be included in the preparation as a result of misidentification or the substitution, either intentionally or unintentionally, of related species. Several incidents have arisen where the presence of undesirable contaminants resulted in illness. Traditionally, identification of the plants ground into powders for packaging into capsules or for use as infusions or teas has been done by direct observation of the anatomical and morphological features of the preparation. This can be subjective and requires considerable expertise in distinguishing the desired plant material from any adulterants. To verify the identity of plant material in botanical supplements, we have utilized a PCR-based approach that incorporates the repair of degraded DNA and molecular biological methods to separate contaminating plant species from the botanical. A particular advantage of the methods we have developed is that they do not rely on having prior knowledge of the species expected to be present in the botanical supplement. We will discuss the development and application of these molecular methods to facilitate monitoring the authenticity of botanicals as well as to identify contaminants in botanical supplements.



Bio-sketch forDr. Vaishali C. Joshi

Dr. Vaishali C. Joshi is a Plant Systematics. Her field of specialization includes herbal medicine, phytogeography and biodiversity conservation.

She received her M.Sc (1996) and Ph.D. degrees (2001) from the Department of Botany, Goa University, India. For her doctoral program she worked on the taxonomy and phytogeography of endemic and medicinal plants of the Western Ghats.

She worked as a Research Associate in the Conservation Research group, FRLHT, Bangalore, India. Were she was involved in developing an “Eco-systematic” and “Distribution” databases for medicinal plants, which are highly traded/endemic to the Western Ghats, India. She has also developed ecodistribution Maps using GIS for medicinal plants. She has worked on Biodiversity Characterization at Landscape level using Remote Sensing and GIS, project sponsored by DBT-DOS, Space application Center, Space application Center as a part of biodiversity characterization of the Western Ghats, India. She was Junior Research fellow and then a Senior Research fellow at the Department of Botany Goa University, were she worked on Plant Biodiversity of Goa, India. She also worked as visiting lecturer, and conducted classes for Botany graduates.

She joined the National Center for Natural Products Research, Research Institute of Pharmaceutical Science, School of Pharmacy, Thad Cochran Research Center, University of Mississippi, MS, in the year 2002 as Postdoctoral Research Associate, project funded by USA FDA. She is involved in macroscopic and microscopic authentication of Botanicals, used in herbal preparation and in developing database/repository for “Authenticated botanicals” used in herbal preparation”. She has published quite a few articles in review journals.

http://www.umich.edu/



Microscopic Techniques for the Identification and Authentication of Botanicals

Vaishali C Joshi, Ph.DNational Center for Natural Products Research

Research Institute of Pharmaceutical Science, School of Pharmacy, Thad Cochran Research Center, University of Mississippi

MS 38677, USA

Authentication of botanical is the starting point for any herbal preparation. Microscopy is an effective tool for authentication of botanicals, by comparing them with an authenticated reference standard. Botanicals may be in the form of cuts, shifts or in powder form. Microscopy detects substitution/contamination of botanicals, closely related species as well as cheap substitutions. It also assists in detecting microbial contamination. The conventional light microscopy, along with advance and sophisticated Fluorescence, Phase contrast and Electron microscopy are very effective and reliable tools for authentication of botanicals.



Bio-sketch forProf. Dr. rer. nat. Michael A. Popp

Prof. Dr. rer. nat. Michael Andreas Karl PoppBorn on July 28, 1959 in Nuremberg, Germany.Nationality: German.

Study of chemistry; winter semester 1980/81 Study of food chemistry: summer semester 1981/winter semester 1981/82 Study of pharmacy: until winter semester 1985/86 State examination in pharmacy 1987 (all studies at Friedrich-Alexander-Universität,

Erlangen, Germany)

November 1987 - June 1991: Dissertation for doctorate at the school of natural sciences of Leopold-Franzens-Universität, Innsbruck, Austria: Topic ”Neue hochdruckflüssigkeitschromatographische und kapillarelektrophoretische Methoden zur Trennung von Kationen, Flavonoiden, Proteinen und Kohlehydraten” [New methods in high pressure liquid chromatography and capillary electrophoresis for the separation of cations, flavonoids, proteins and carbohydrates].

May 1987 to present: CEO of the company formerly known as Pharmamed, later Plantamed Arzneimittel GmbH in Neumarkt, Germany

January 1989 to present: CEO of Bionorica Arzneimittel GmbH in Neumarkt, Germany March 1994 to present: Member of the Board of the German Pharmaceutical Industry

Association (BPI) June 1998 to present: Deputy Chairman of the German Pharmaceutical Industry Association

(BPI) June 1999 to present: Honorary professor lecturing in analytical phytochemistry at Leopold-

Franzens-Universität, Innsbruck, Austria 1999 to present: Chairman of the Committee for Research into Natural Medicine (CRNM) November 2001 to present: Vice-president of Europharm SMC Winter semester 2001/2002: Honorary professor lecturing in pharmaceutical biology at

Leopold-Franzens-Universität, Innsbruck, Austria.



Quality, safety and efficacy assurance: from plant culture to the final product

Prof. Dr. rer. nat. Michael A. Popp, CEO of Bionorica AG, Neumarkt, Germany

“Bionorica – The phytoneering company“ is one of the leading manufacturers of plant-based pharmaceuticals in Germany. A third-generation family company in the Bavarian town of Neumarkt, it is market leader in phytomedicines both in terms of prescriptions as well as in terms of the number of packs sold. The 70-year-old company specializing in respiratory diseases and gynecology engages in enhancing existing products and developing new preparations.

Phytoneering research:Bionorica AG has its own research and development department, and our investments in this field distinctly exceed the norm for the sector. We also work together closely with some 70 external scientists at (international) universities and institutes who work exclusively for Bionorica: Growth and cultivation of medicinal plants: For the manufacture of our highly effective plant-based

medicines we use only the best starting materials. We cultivate medicinal plants which were previously collected from wild stocks and develop new seed with the aim of cultivating homogeneous plants whose complex patterns of constituents conform to the latest pharmacological findings and clinical requirements.

Quality assurance and control: For the exact determination of the drug quality, seed, growing conditions, further processing, extract production and for the extensive quality control of the finished medicinal product we use highly modern analysis methods and techniques. This activity embraces not only individual constituents but the plants and our special extracts as a whole with all their different constituents and relevant constituent groups.

Engineering : Together with engineers and mechanical engineering companies we elaborate new production techniques for the manufacture of liquid extracts, concentrates and dry extracts in order to preserve the large content of native constituents of the plant in the finished medicinal product.

Environmentally friendly production : We have applied for patents throughout Europe, in the USA and other countries for the energy-saving concentration methods we have developed for liquid extracts and for the manufacture of dry extracts.

Innovative pharmacological models: We test our special extracts and preparations in pharmacological and toxicological studies and in this way obtain new findings about medicinal plants long familiar to mankind. In our research work we use not only new pharmacological models but also findings from the field of molecular biology.

We research new plants for future use in phytotherapy and meanwhile have patents in this area in Europe, the USA and Japan. The pharmacological foundations – obtained mainly from human cell lines – are subsequently substantiated in clinical studies. In this way we create the conditions for the registration of our medicinal products with health authorities the world over.Our internationalization strategy

Owing to our consistent implementation of the philosophy of phytoneering, developing only high-quality, researched and effective pharmaceuticals, we have numerous documentations for marketing authorization the world over.

Rational phytotherapy:Not only can our highly effective plant-based pharmaceuticals bear comparison in the various indications to synthetic chemical pharmaceuticals, they are often superior to them in their treatment value. What is more, phytomedicines have far fewer side effects. Their excellent efficacy and the safety of our special extracts and pharmaceuticals are based on the large number and combination of highly potent active substances. We are, of course, aware that acute treatment requires a combination with synthetic chemical pharmaceuticals or even highly-effective natural substances.



Bio-sketch ofDr. Stefan Gafner

Stefan Gafner received his Bachelor of Pharmacy degree from the University of Bern, Switzerland, and earned his PhD at the Institut of Pharmacognosy et Phytochemistry, University of Lausanne, Switzerland under the direction of Kurt Hostettmann. He continued his education as a post-doctoral fellow in John Pezzuto’s group at the University of Illinois at Chicago, where he was involved in the screening of natural products and identification of the mode of action of actives in the chemoprevention of cancer.

His is currently director of the analytical group with Tom's of Maine, where he is responsible for the development and validation of quality control methods for herbal products and OTC drugs. His research interests are the identity, quality and stability of herbal preparations as well as the mode of action of the botanicals. To date, he has published more than 20 papers in numerous scientific journals.



Chromatographic fingerprinting as a means of quality control: Distinction between Actaea racemosa and four different Actaea species

Stefan Gafner1, Sidney Sudberg2, Robert Gauthier3 and Chantal Bergeron1

1Tom’s of Maine, 302 Lafayette Center, Kennebunk, ME 04043, USA 2Alkemists Pharmaceuticals, 1260 Logan Ave., # B-3, Costa Mesa, CA 92626, USA 3Herbier Louis-Marie,

Pavillon Marchand, Université Laval, Québec, Qc, Canada G1K 7P4

Over the past decade, manufacturers have increasingly focused on the issue of quality, and standardization in particular, as the key feature differentiating otherwise generic herbal products. Marketing campaigns have emphasized the use of modern scientific techniques to ensure that products contain specified amounts of marker compounds, asserting that this process of standardization guarantees identity, safety, efficacy and stability. For the vast majority of botanicals, however, it is impossible to determine potency measuring such marker compounds, since the active principles have neither been conclusively identified nor can they be accurately measured.

In order to overcome this issue, some manufacturers have attempted to measure an herbal product as a whole. The evaluation of a product in its entirety, so-called “fingerprinting” can be determined by appropriate methods, which may include HPLC-UV(DAD), HPLC-ELSD, HPLC-MS, GC-MS, HPTLC-densitometry, FT-NIR, high-field NMR or a combination of these techniques. The usefulness of fingerprinting is especially evident in the quality control of black cohosh (Actaea racemosa L.), which has been reportedly adulterated with other Actaea species, mostly imported from China. The majority of the triterpene glycosides of A. racemosa occur in several other Actaea species, therefore, an identification procedure based on a few marker compounds may not be enough to rule out every adulterant. We have compared the HPLC-UV, HPLC-MS, HPLC-ELSD and HPTLC fingerprints of five Actaea species (A. racemosa, A. rubra, A. pachypoda, A. podocarpa and A. heracleifolia). While the distinction between black cohosh and the other Actaea species was difficult using HPLC-UV or HPTLC, a marked difference was observed in the HPLC-MS and HPLC-ELSD fingerprints. Interestingly, the chromatography traces of A. rubra and A. pachypoda were so similar that none of the techniques allowed distinguishing the two species.



Bio-sketch forDr. Ikhlas Khan

Dr. Ikhlas Khan is the Director of the FDA Program, a Research Professor, as well as Professor in the Department of Pharmacognosy at the National Center for Natural Products Research at the University of Mississippi. He received a B.S. in Chemistry in 1980 and a M.S. in Organic Chemistry in 1982 from the Aligarh Muslim University in Aligarh, India. Dr. Khan then received a Ph.D. in Pharmacy from the Institute fuer Pharmaceutische Biology in Munich, West Germany in 1987 and postdoctoral studies at Swiss Federal Institute of Technology (ETH) Zurich. After completing his education, Dr. Khan began his career with the University of Mississippi in 1992 as a research scientist. He became a research assistant professor in 1995, and was promoted to research associate professor and director of the FDA program in 2001, assistant director of the center in Oct. 2002 and to professor in July 2005.

Dr. Khan’s primary research interests include analytical fingerprinting for standardization of herbal products, and bio-analytical approaches to improvement of product quality and safety.

Dr. Khan has authored or co-authored over 190 original research articles, publications, or reviews. He has been an invited speaker at numerous events. Dr. Khan serves as reviewer for several journals. Co-editor of planta medica and foreign editor for J. Traditional Chinese medicine.

Dr. Khan is a fellow with the American Institute of Chemists. He is a member of expert panel of United States of Pharmacopoeia (USP) Committee. He is a member of Sigma Xi, the Scientific Research Society, and Rho Chi, the Pharmaceutical Honor Society. Dr. Khan is also an active member of the American Chemical Society, the Society of Medicinal Plant Research, the American Association for the Advancement of Sciences, and the New York Academy of Sciences. He is a member of the American Society of Pharmacognosy , where he served as Chair of the Constitution of By-laws Committee in 1998. He also served as a WHO consultant in Vietnam in 2002.



Challenges in Assessing the Quality and Safety of Botanicals:A Global Co-operation is needed

Ikhlas A. Khan

National Center for Natural Products Research and Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, MS 38677, U.S.A.

Nature has provides human with products for good health since the beginning of time. 80% of the people in developing countries rely on traditional medicines for primary health care. The traditional medicines are slowly being integrated into modern medicine in the form of dietary and nutritional supplements and the scientific community is challenged to address the issue of safety, quality and efficacy.

A study cannot be considered scientifically valid if the material tested was not authenticated and characterized such that the material can be reproduced. In the case of botanicals, there may be misidentification of the collected plant, adulteration with other species, or contamination with extraneous ingredients. The scientific integration of herbal medicine into modern medical practices must take into account the interrelated issues of quality, safety and efficacy. Quality is the paramount issue since it can affect the efficacy and/or safety of the herbal products being used.



Bio-sketch forDr. De-an Guo

Modern Traditional Chinese Medicine Research, Peking University, China



Quality control and in vivo process of traditional Chinese medicine

De-an Guo, Min Ye, Lie Li, Jinlan Zhang and Rongxia LiuSchool of Pharmaceutical Sciences, Peking University, Beijing 100083, P.R. China

Safety, efficacy and quality are the mainly considered issues for any newly developed drugs. Traditional Chinese medicine (TCM) has long been practiced in the Chinese clinics and played an important role in the health system of Chinese people. Unlike the chemical drugs, TCM is composed of a number of herbal drugs and is a multi-component preparation. Therefore, quality control for TCM is especially essential to assure the efficacy of the herbal combinations. The quality control techniques including fingerprint for several Chinese herbal medicines and their related preparations were discussed and expected to set up a model for the generalized control for the TCM quality. Several commonly used traditional Chinese medicines including the roots of Salvia miltiorrhiza (Danshen), the roots of Panax notoginseng (Sanqi), the skin secreation of Bufo Bufo gargarzans (Chansu), the roots of Paeonia lactiflora (Shaoyao), seeds of Cuscuta chinensis (Tusizi) and the heart wood of Dalbergia odorifera (Jiangxiang) have been investigated by means of phytochemical analysis and their quality control standards were established. The HPLC fingerprints of the above mentioned crude drugs and their representative preparations were formulated and the major peaks were designated. From the roots of Salvia miltiorrhiza, 15 salvianolic acids and 10 tanshinones ere unambiguously identified by HPLC-MS-MS analysis and comparing with the authentic compounds. The qualitative and quantitative analyses of bufadienolides in the Chinese drug ChanSu were carried out by HPLC/DAD/APCI-MS/MS technique. The APCI-MS fragmentation behaviors of bufadienolides were studied for the first time. A total of 35 bufadienolides were identified in the crude drug including four new constituents. And the eight major bufadienolides were simultaneously quantified by a well-established HPLC-UV method. The phenolic compounds in Tusizi were thoroughly analyzed. From the methanol extracts of two derived species (Cuscuta chinensis and Cuscuta australis), a total of 50 compounds, including 23 flavonoids, 20 lignans and 7 quinic acid derivatives were identified or tentatively characterized based on UV spectra and MS fragmentation behaviors. Contradictory to the previous reports, the phenolic patterns of these two Cuscuta species were found to be very different. The phenolic constituents of C. chinensis and C. australis were comprehensively compared for the first time, and the great differences strongly encouraged further comparison of the bioactivities of these two species. Similarly, the saponin compounds in the Panax ginseng, the flavanoids in Dalbergia odorifera, the active principles of Paeonia lactiflora were also phytochemically analyzed and their major active principles were quantified. In order to obtain more information about the active constituents of TCM in biological system, the in vivo process of several commonly used TCMs including Danshen, Notoginseng etc. was also investigated by HPLC-UV and HPLC-MS method.



Bio-sketch forMs. Aviva Romm

Aviva Romm has been a midwife and herbalist since 1985. She is a recognized expert in the areas of obstetric, gynecologic, and pediatric herbal medicine. Amongst her numerous publications are the forthcoming Textbook of Botanical Medicine for Women (Churchill 2006) and Pediatric Botanical Medicine (Thieme 2006). Aviva is the President of the American Herbalists Guild and a medical student (M1) at the Yale School of Medicine (class of 2009).



Abstract: Blue cohosh: An evaluation of use prevalence and safety in pregnancyAviva Romm

Caulopyhyllum thalictroides (blue cohosh), a native of the eastern and central woodlands of the United States, has been used traditionally and historically as an anticonvulsant, antirheumatic, febrifuge, emetic, sedative, and most notably, a gynecologic aid.1, 2 It has been used for labor induction, amenorrhea, dysmenorrhea, menorrhagia, and to induce abortion.1 Blue cohosh was official in the United States Pharmacopoeia from 1882-1905 for labor induction, and in the National Formulary from 1916-1950.3 The practice of labor induction with blue cohosh remains a popular choice both amongst self-prescribers and obstetric professionals in the US and abroad, with one large survey indicating widespread use amongst nurse- midwives in North Carolina.4 Maternal ingestion has been associated with a range of fetal and neonatal side-effects and adverse outcomes, including fetal tachychardia, increased meconium, profound neonatal congestive heart failure, and perinatal stroke.4-7 Alkaloid and glycoside components in blue cohosh suggest possible mechanisms for these effects, as well as teratogenicity and mutagenicity.8, 9 This presentation reports on the goals, process, and preliminary findings of a study currently being conducted by Aviva Romm and Tieraona Low Dog in conjunction with Larry Walker and Ikhlas Khan of the National Center for Natural Products Research., The study seeks to determine use volume of blue cohosh by midwives through a national midwife survey, prevalance and contents of blue cohosh literature on the internet, volume of bulk herb and product sales in the US through tonnage and manufacturer sales information, and finally, direct toxicological and chemical studies with products obtained through internet purchases. The goals of the primary researchers, Romm and Low Dog, both health care providers and formerly practicing midwives, are to determine whether and to what extent there is a public health threat to neonates from maternal of blue cohosh prenatally, and ultimately to provide a basis for establishing use guidelines and warnings.

1. Moerman D. Native American Ethnobotany. 3 ed. Portland, OR: Timber Press; 2000.2. Felter H, Lloyd J. King's American Dispensatory. Cincinnati: reprinted by Eclectic Medical

Publications, 1983; 1898.3. Low Dog T. Women's Health in Complementary and Integrative Medicine: A Clinical Guide. St

Louis, MO: Elsevier; 2004.4. McFarlin B, Gibson M, O'Rear J, Harman P. A national survey of herbal preparation use by nurse-

midwives for labor stimulation: Review of the literature and recommendations for practice. J Nurse Midwifery. 1999;44(3):205-216.

5. Finkel R, Zarlengo K. Blue cohosh and perinatal stroke [correspondence]. NEJM. 2004;351(3):302-303.

6. Jones T, Lawson B. Profound neonatal congestive failure caused by maternal consumption of blue cohosh herbal medication. J Pediatr. 1998;132:550-552.

7. Wright I. Neonatal effects of maternal consumption of blue cohosh. J Pediatr. 1999;134(3):384-385.8. Rao R, Hoffman R. Nicotinic toxicity from tincture of blue cohosh (Caulophyllum thalictroides)

used as an abortifacient. Vet Hum Toxicol. 2002;44(4):221-222.9. Kennelly E, Flynn T, Mazzola E, Roach J, McCloud T, Danford D, et al. Detecting potential

teratogenic alkaloids from blue cohosh rhizomes using an in vitro rat embryo culture. J Nat Prod. 1999;62(10):1385-1389.



Bio Sketch forMr. Mark Blumenthal

Mark Blumenthal is the Founder and Executive Director of the American Botanical Council (ABC); an independent, nonprofit organization dedicated to disseminating accurate, reliable, and responsible information on herbs and medicinal plants. He is the Editor/Publisher of HerbalGram, an international, peer-reviewed quarterly journal, the contents of which reflect the educational goals of ABC. For six years he was an Adjunct Associate Professor of Medicinal Chemistry at the University of Texas at Austin, College of Pharmacy, teaching a course on herbal products in today’s pharmacy. Mark has served as Co-Founder and former Vice-President of the Herb Research Foundation (HRF) and President of the Herb Trade Association, the former organization that represented the interest of the herb industry in the 1970’s. He was also a founding board member of the American Herbal Products Association (AHPA). Recently, his name appeared with some of the most prestigious names in the natural health movement when he was awarded Natural Health Magazine’s Hall of Fame Award for “…opening America’s eyes to the healing powers of herbs”.

He is the senior editor of the English translation of The Complete German Commission E Monographs-Therapeutic Guide to Herbal Medicines, a rational system for evaluating the safety and efficacy of herbal medicines. This publication was ranked second of the medical books published in 1998. Mark is the senior editor of The ABC Clinical Guide to Herbs, a new reference work and continuing education module.



Determining Safety Information for Herb Product Labels: The American Botanical Council's Safety Assessment Program

Mark BlumenthalFounder & Executive Director, American Botanical Council

Editor, HerbalGram & HerbClip

One of the largest concerns related to herbal dietary supplements during recent years has been over the safety of these products. In November 2004 the Food and Drug Administration announced new guidelines for the regulation of dietary supplements, including the announcement that it would begin to review dietary supplement product labels to determine if they provide adequate warnings for known risks. The Institute of Medicine released a proposed mechanism for the FDA’s evaluation of the safety of dietary supplements but the proposed procedure would presumably require considerable cost for each supplement evaluated. Funding for such a program is not currently available to the Agency. Consequently, much of the safety evaluation of herbs and other supplements must originate in the private sector. Since 2002 the American Botanical Council, an independent nonprofit research and education organization, has been conducting the Safety Evaluation Program in which ABC has produced authoritative, peer-reviewed safety evaluations of several dozen of the most popular herbs in the dietary supplement market. These Safety Assessment Reports are intended for use by herb manufacturers and marketers as guides for potential warnings and precautions on product labels, as well as information for product information, websites, customer service centers, etc. This presentation will describe ABC’s Safety Assessment Program.



Bio-sketch forDr. Steven Dentali

Steven Dentali, Ph.D., Vice President, Scientific and Technical Affairs at the American Herbal Products Association works to further acceptance of the concept that rational and responsible use of herbs is a legitimate health care option.

Formerly, he served as Senior Director of Botanical Sciences for Rexall Sundown and Quality Assurance Director at Trout Lake Farm, the largest cultivator and supplier of certified organically grown medicinal plants in North America. In addition, as President of his own natural products consulting company for over a decade, he has facilitated the exchange of information on botanicals among organizations, regulators, scientists, practitioners, and the media.

Dr. Dentali earned his doctorate in Pharmaceutical Sciences with a specialization in Natural Products Chemistry from the University of Arizona, Tucson. An American Foundation for Pharmaceutical Education Fellow, he has written a book chapter on the uses of black cohosh in menopause and chaste tree in PMS, a book on ginkgo and memory, and safety reviews of kava and ephedra.

Recognized as a foremost expert in the natural products industry, Dr. Dentali is an Advisory Board Member for the American Botanical Council and a reviewer for the National Center for Complementary and Alternative Medicine at NIH, the Institute of Medicine at the National Academy of Sciences, AOAC INTERNATIONAL, and the American Herbal Pharmacopoeia, among other organizations. He has been appointed as a member of the United States Pharmacopeial 2000–2005 Convention and elected to the USP Expert Committee on Dietary Supplements, General Chapters.



Building the Global Consortium for the Science of Botanicals

Steven Dentali, Ph.D.Vice President, Scientific and Technical Affairs

American Herbal Products Association8484 Georgia Ave. Suite 370

Silver Spring, MD 20910, USA

The first organizational meeting of the GCSB on September 10, 2004 at the University of Mississippi explored the problem of duplicated effort and lack of communication between various groups involved in modern medicinal plant research and commercialization. The need for cooperative effort that includes resource document sharing was recognized and an open membership consortium involving academia, government, and industry was proposed. The development of an international ‘clearing house’ of botanical science information, particularly as related to herbal product issues, was envisioned and several ‘areas of interest’ were identified.

Continued progress in establishing standardization practices has been evident in many national and international arenas. Advances have occurred in the establishment of good agricultural and collection practices, the validation of methods of analysis, the availability of analytical reference materials, the creation of pharmacopoeial monographs, the continuation of biological and clinical testing, and the development of regulatory strategies for botanically derived dietary supplement and medicinal products. Facilitating the exchange of ideas and resources is of interest in countries with mature drug plant industries, others that may be attempting to modernize ancient traditional products, and those building their medicinal plant industries and their country’s regulatory framework.

This talk will discuss the identification and collection of resource documents and placing them in an internet accessible location. The formation of ad hoc botanical science interest groups to provide resources to individuals and organizations that need those resources will be opened for audience discussion and participation.



Bio-sketch forDr. Klaus Reif

Familienname: Reif

Vorname: Klaus

Studium:

1983 - 1989 Chemistry, diploma at the Friedrich-Alexander-University Erlangen

1989 University degree as chemist

1989 - 1991 PhD at Friedrich-Alexander-University Erlangen, Institute for physical

chemistry (group Prof. Dickert) and in the research center of Siemens AG

Erlangen (development of chemosensors for solvent vapours)

Sep. 1991 PhD of natural science(Dr. rer. nat.)

since Oct. 1991 employed by PhytoLab GmbH, Vestenbergsgreuth

Field of activity: Head of Analytical Development

Head of HPLC-department

Head of LC/MS-department

Chair of the AOAC Working Group on Herbal Products

Responsible for: method development and method validation for the registration procedure

of phytopharmaceuticals, residue analysis, analysis of food and cosmetics,

product release and stability tests with HPLC of herbal raw materials and

finished herbal products



Contamination in Herbal Drugs in View of European Regulations and Future Developments

Klaus Reif PhytoLab GmbH & Co. KG, Germany

The term Herbal Drug refers to whole, comminuted or powdered plants or parts of plants usually in dried form which are mainly used for medicinal purposes. The EMEA has published a Note for Guidance CPMP/QWP/2820/00: Test Procedures and Acceptance Criteria for Herbal Drugs ... providing general principles on setting specifications for herbal drugs. One of the sources of methods and criteria mentioned there is the Ph.Eur. But the Note for Guidance itself describes acceptance criteria applicable to all herbal drugs. Contaminants resulting from environmental factors, improper handling and microbial spoilage occurring during cultivation, harvesting, drying and storage of herbal drugs require our special attention.

Furthermore Herbal Drugs can be adulterated by other species, which may be toxic. For this purpose qualitative and quantitative methods for the Quality Control of the raw herbal materials are needed.

1. AdulterationExamples for the QC of different raw herbal materials are presented. Star anise can be

adulterated by toxic Illicium species like I. anisatum or I. floridianum. TLC and HPLC-MS/MS-methods have been developed. Within the EC there have been alerts about the occurrence of carcinogenic colours (Sudan I – IV etc.) in spices like red pepper, curcuma etc. An HPLC-MS/MS-method has been developed for the sensitive detection of a variety of artificial colouring agents.

2. Inorganic impurities, toxic metalsPotential soil contamination and air pollution must be diligently considered and taken into

account when selecting the site of cultivation, as they influence the quality of the herbal drugs with regard to the content of heavy metals and residues of chemicals. PhytoLab developed an multielement ICP-MS-technique for the detection of traces of different heavy metals and elements.

3. Microbial limitsSludge and harvesting technology influence the microbiological growth and thus the

microbiological quality of herbal drugs. But the microbiological load also depends on the drying process and the temperature applied.

4. MycotoxinsHumid conditions in herbal drugs promote the growth of moulds potentially forming

mycotoxins. Especially mycotoxins of Aspergillus, Penicillium and Fusarium species are detrimental to human health because of their serious chronic toxic effects. Methods and results of the screening of herbal raw materials on a variety of mycotoxins will be presented.

5. Pesticides, Fumigation AgentsApproved plant protection products should be applied at the minimum effective level, in

accordance with the regulations of the grower and the end-user countries. If the use of chemical plant protection products is not limited to the necessary minimum, while cultural measures are applied, their excessive use results in not acceptable quantities of residues. By using multi residue methods a detection of almost 300 substances can easily be achieved.



Bio-Sketch forDr. Mary W. Trucksess

Dr. Mary W. Trucksess is a research chemist at the Food and Drug Administration, Center for Food Safety and Applied Nutrition. She is well known internationally in the field of mycotoxins. She has developed methods for analysis of mycotoxins of diverse chemical structures in grain, grain products, cheese, animal tissue, and juice. She has been the AOAC International General Referee for Mycotoxins for more than 10 years and has published more than one hundred papers on mycotoxins, food allergens and transgenic plants.



Determination of Mycotoxins in Botanical Roots

Mary W. Trucksess, Carol M. Weaver, Carolyn J. Oles, Jeanne I. Rader,

US Food and Drug Administration, Center for Food Safety and Applied Nutrition, College Park, MD

Mycotoxins are toxic secondary metabolites produced by certain molds and are common contaminants of many important food crops, such as grains, nuts, and spices. Some mycotoxins are found in fruits, vegetables, and botanical roots. These contaminants have a broad range of toxic effects, including carcinogenicity, immunotoxicity, neurotoxicity, and reproductive and developmental toxicity. The public health concerns related to both acute and chronic effects of mycotoxins in animals have prompted more than 100 countries to establish regulatory limits for some of the well-known mycotoxins such as the aflatoxins (AFL). Our research focused on method development for three of these toxins: AFL, ochratoxin A (OTA) and fumonisins (FB) in botanical roots, specifically on ginseng and other selected roots. Methods using an immunoaffinity column (IAC) cleanup, liquid chromatographic separation, and fluorescence detection were modified and evaluated. Two types of IAC were evaluated: IAC for a single analyte and IAC for both AFL and OTA. Three derivatization techniques to enhance the fluorescence of the AFL were compared: pre-column trifluoroacetic acid, post-column bromination and post-column UV irradiation. Pre-column derivatization was used for FB and no derivatization was needed for OTA. Results for AFL using the single analyte IAC and the three derivatization techniques were all comparable for ginseng and for other roots such as ginger, licorice, and kava-kava. Recoveries of added AFL for ginseng at levels from 2 ng/g to 16 ng/g were about 80%. Recoveries of added toxins for ginger, licorice and kava-kava were about 60%. Recoveries of added FB and OTA for ginseng at various levels and preliminary results of using commercially available ELISA kits for the three mycotoxins will also be presented.



Bio-sketch forDr. Jon W. Wong

Jon W. Wong is a Chemist at the Center for Food Safety and Nutrition, U.S. Food and Drug Administration (FDA) in College Park, MD. He received B.S. degrees in Chemistry and Chemical Engineering from the University of California, Davis (UC Davis), a M.S. in Chemical Engineering from Purdue University, and a Ph.D. in Agricultural and Environmental Chemistry from UC Davis. He was a National Institutes of Health postdoctoral researcher at the University of California School of Medicine, UC Davis, and a Chemist with the Bureau of Alcohol, Tobacco, and Firearms prior to his current FDA position. His research has included lipid peroxidation processes in foods and biological systems, environmental effects (ultraviolet radiation, chemical oxidants, heat) in skin and lung cells, bioactive compounds in wines, and pesticides and industrial contaminants in foods and beverages.

Dr. Wong is a co-editor of the American Journal for Enology and Viticulture and is a member of various professional organizations, such as the AOAC International, the American Chemical Society, the American Association for the Advancement of Science, and the American Society of Enology and Viticulture.



Pesticides in Botanical Dietary Supplements

Jon W. Wong, Mark S. Wirtz, Michael K. Hennessy, Alexander J. Krynitsky, and Stephen G. Capar

U.S. Food and Drug AdministrationCenter for Food Safety and Nutrition

5100 Paint Branch ParkwayHFS-336

College Park, Maryland 20740 USA

Current U.S. Food and Drug Administration (FDA) procedures to analyze pesticides in botanical dietary supplements are described. The procedures are from FDA protocols for pesticide analysis in foods, which involve organic solvent extraction (usually with acetone:water or acetonitrile:water) of the sample, followed by cleanup steps to remove interfering coextractives, and subsequent analysis primarily by gas chromatography-mass spectrometry. From a 2004 FDA assignment, pesticides were found in 43 out of 81 samples of botanical dietary supplements (the majority of these being ginseng products) suspected of containing pesticides. Over 30 different types of organochlorine, organophosphorus, and organonitrogen pesticides were present in these samples, with pentachloroaniline, pentachlorobenzene, and quintozene being the most abundant. A majority of these products contained more than one pesticide; one such sample was found to contain as many as 12 organochlorine pesticides. Concentrations of pesticides ranged from trace levels (< 0.01 mg/kg for pentachlorobenzene and dacthal) to 5.57 mg/kg (quintozene). The presence and concentration of pesticides found in dietary supplements indicate the need for increased monitoring. However, current and new procedures need to be validated for pesticides in botanical dietary supplements and expanded to include other pesticide classes.



NAME: Bill J. Gurley POSITION TITLE: Professor of Pharmaceutical Sciences

INSTITUTION AND LOCATION DEGREE YEAR(s) FIELD OF STUDYTennessee Technological University, Cookeville, TN BS 1980 ChemistryUniversity of Tennessee at Memphis, TN BS 1983 PharmacyUniversity of Tennessee at Memphis, TN PhD 1989 Pharmaceutics

PROFESSIONAL EXPERIENCE:1989 to 1994 Assistant Professor of Pharmaceutics, University of Arkansas for Medical Sciences (UAMS) College of Pharmacy.1995 to present Associate Professor of Pharmaceutics, UAMS College of Pharmacy1996 to1999 Chairman (interim) Department of Pharmaceutics, UAMS College of Pharmacy1992 to present Director of the Clinical Pharmacokinetics Research Laboratory, UAMS College of Pharmacy2003 to present Professor of Pharmaceutical Sciences, UAMS, College of PharmacyAWARDS AND OTHER PROFESSIONAL ACTIVITIES:1995 Lloyd R. Thompson Memorial Award for "Most Outstanding Professor, UAMS Dept. of Pharmaceutics"1997 Lloyd R. Thompson Memorial Award for "Most Outstanding Professor, UAMS Dept. of Pharmaceutics"2000 Outstanding Faculty Award, "Most Outstanding Professor, UAMS Dept. of Pharmaceutical Sciences"2004 Outstanding Faculty Award, "Most Outstanding Professor, UAMS Dept. of Pharmaceutical Sciences"1986 to present member of the American Association of Pharmaceutical Scientists1990 to present member of the American Association for the Advancement of Science1991 to present member of the American Association of Colleges of Pharmacy2001 to present member of the American Society of Clinical Pharmacology & Therapeutics

SELECTED PUBLICATIONS (25 OF 96):1. Barone GW, Gurley BJ, Anderson KE, Ketel BL, Abul-Ezz SR. The tolerability of newer immunosuppresive medications in a patient with

acute intermittent porphyria. J. Clin Pharmacol 2001;41:113-115.2. Barone GW, Gurley BJ, Ketel BL, and Abul-Ezz SR. Herbal dietary supplements: a source for drug interactions in transplant recipients.

Transplantation. 2001;71:239-241.3. Kommuru TR, Gurley B, Khan MA, and Reddy IK. Self-emulsifying drug delivery systems (SEDDS) of coenzyme Q10: formulation

development and bioavailability assessment. Int. J. Pharm. Sci. 2001;212:233-246.4. Penzak SR, Jann MW, Cold JA, Hon YY, Desai HD, and Gurley BJ. Seville (sour) orange juice: synephrine content and cardiovascular effects

in normotensive adults. J. Clin. Pharmacol. 2001;41:1-5.5. Zaghloul A, Gurley B, Kahn M, Bhagavan H, Chopra R, and Reddy I. Bioavailability assessment of oral coenzyme Q10 formulations in dogs.

Drug Dev. Ind. Pharm. 2002; 28:1195-1200. 6. Gurley BJ, Gardner SF, Hubbard M, Gentry W, Williams D, Ang C, and Cui Y. Cytochrome P-450 phenotypic ratios for predicting herb-drug

interactions in humans. Clin Pharmacol Ther. 2002; 72:276-287.7. Cui YY, Gurley BJ, Ang CYW, Leakey J. Determination of hyperforin in human plasma using solid-phase extraction and high-performance

liquid chromatography with ultraviolet detection. Journal of Chromatography B 2002;780: 129-135.8. Gardner SF, Franks AM, Gurley BJ, Haller CA, Singh BK, Mehta JL. Effect of a multi-component, ephedra-containing dietary supplement

(Metabolife 356) on holter monitoring and hemostatic parameters in healthy volunteers. Am J. Cardiol. 2003; 91:1510-1513.21. Barone GW, Gurley BJ, Abul-Ezz S, Gokden N. Sirolimus-induced thrombotic microangiopathy in a renal transplant recipient. American

Journal of Kidney Diseases. 2003; 42: 1-5.22. Gubbins PO, McConnell SA, Gurley BJ, Fincher TK, Franks AM, Williams DK, Penzak, Saccente M. Influence of grapefruit juice on the

systemic availability of itraconazole oral solution in healthy adult volunteers. Pharmacotherapy 2004; 24: 460-467.23. Gurley BJ, Gardner SF, Hubbard MA, Williams K, Gentry WB, Carrier J, Edwards D, Khan I, ad Shah A. In vivo assessment of botanical

supplementation on human cytochrome P450 phenotypes: Citrus aurantium, Echinacea purpurea, milk thistle, and saw palmetto. Clin Pharmacol Ther 2004;76:428-440.

24. Gurley BJ, Gardner SF, Hubbard MA, Gentry WB, Williams DK, Khan I, Shah A. In vivo effects of goldenseal, kava kava, black cohosh, and valerian on human cytochrome P450 1A2, 2D6, 2E1, and 3A4 phenotypes. Clin Pharmacol Ther 2005;77:415-426.

25. Gurley BJ, Gardner SF, Hubbard MA, Williams DK, Gentry WB, Cui Y, Ang CYW. Clinical assessment of botanical supplementation on cytochrome P450 phenotypes in the elderly: St. John’s wort, garlic oil, Panax ginseng, Ginkgo biloba. Drugs & Aging 2005;22:525-539

RESEARCH PROJECTS ONGOING OR COMPLETED DURING THE LAST 5 YEARS: "Aging and CYP-mediated Herb-drug Interactions."Principal Investigator: Bill Gurley, Ph.D. "Clinical Assessment of Herb-Drug Interactions."Principal Investigator: Bill Gurley, Ph.D. (active, 22% effort) Agency: National Institute of General Medical SciencesType: RO1 (GM71322-01A2) Period: October 1, 2003 – September 30, 2006The hypothesis to be tested in this proposal is that patients are at an increased risk for herb-drug interactions secondary to herb-mediated changes in hepatic cytochrome P-450 (CYP) activity. The goal of the project is to assess the effect in humans of the top-ten selling botanical supplements in the U.S. on the activity of CYP1A2, CYP2D6, and CYP3A4 as well as the drug efflux pump, P-glycoprotein. There is no overlap with the current proposal.



In Vivo Assessment of Potential Herb/Drug Interactions

Bill Gurley, Ph.D.University of Arkansas for Medical Sciences, College of Pharmacy

Objectives: Phytochemical-mediated modulation of cytochrome P-450 (CYP) and/or P-glycoprotein (P-gp) activity may underlie many herb-drug interactions. Single time-point, phenotypic metabolic ratios were used to determine if single-ingredient botanical dietary supplements could affect the activity of various CYP isoforms in vivo. Changes in the disposition of digoxin, a P-gp substrate, were used to assess supplement effects on P-gp-mediated drug transport in vivo. Supplements evaluated included St. John’s wort, Panax ginseng, Ginkgo biloba, garlic oil, Citrus aurantium, Echinacea purpurea, milk thistle, saw palmetto, kava kava, goldenseal, and black cohosh,. Clinical relevance was also assessed using rifampin and clarithromycin, two known modulators of CYP and P-gp.

Methods: In a series of studies, healthy volunteers (young and elderly) were randomly assigned to receive each botanical supplement for 14-28 days. In some instances, subjects also received rifampin and clarithromycin, as positive controls for CYP3A4/P-gp induction or inhibition, respectively. A 30-day washout period was interposed between each supplementation/control phase. Probe drug cocktails of midazolam, caffeine, chlorzoxazone, and debrisoquine were administered before and at the end of each supplementation/control period. Pre- and post-supplementation phenotypic trait measurements were determined for CYP3A4, CYP1A2, CYP2E1, and CYP2D6 using 1-hydroxymidazolam/midazolam serum ratios (1-hour), paraxanthine/caffeine serum ratios (6-hour), 6-hydroxychlorzoxazone/chlorzoxazone serum ratios (2-hour), and debrisoquine urinary recovery ratios (8-hour collection), respectively. Using an identical supplementation/control scheme but different groups of volunteers, digoxin pharmacokinetics were used to evaluate supplement-mediated effects on P-gp activity. Phytochemical content was determined for each supplement.

Results: Comparisons of pre- and post-supplementation phenotypic ratios indicated that St. John’s wort significantly increased CYP3A4 and CYP2E21 activity in both young and elderly subjects. Garlic oil inhibited CYP2E1 activity in these age groups. Goldenseal significantly inhibited CYP3A4 and CYP2D6 activity while kava inhibited CYP2E1. None of the other supplements affected the activity of these specific CYP isoforms to a degree that would be considered clinically relevant. Milk thistle exhibited a mild inductive effect on P-gp.

Conclusions: If taken concomitantly, botanical supplements containing St. John’s wort, are likely to reduce the efficacy of medications whose metabolism is mediated by CYP3A4 and CYP2E1. Goldenseal may increase the toxicity of drugs metabolized by CYP3A4 and/or CYP2D6. However, most of the botanicals tested did not appear to pose a significant risk for causing CYP- or P-gp-mediated herb-drug interactions.



Bio-sketch forDr. Christopher J. Borgert

Christopher J. Borgert, Ph.D. is President of applied pharmacology and toxicology, inc. (apt), a consulting firm that specializes in applied research in the areas of causation analysis, safety assessment and study design. He also holds a courtesy faculty appointment in the Department of Physiological Sciences, University of Florida College of Veterinary Medicine. He received a bachelor of arts from Kenyon College, Gambier, Ohio, a doctorate in Pharmacology and Therapeutics from the University of Florida College of Medicine, and completed a postdoctoral fellowship in toxicology at the University of Florida Center for Environmental and Human Toxicology. He has served on the U.S.EPA Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) as the general representative for Small Business stakeholders and on numerous national and international expert and peer-review panels, including the current Society of Toxicology Expert Panel on Chemical Mixtures. His recent publications address methods for evaluating chemical mixtures in human milk, cumulative risk assessments for human exposure to drugs and chemicals, the pharmacology and toxicology of dietary supplements and interactions with drugs, and mechanistic dose-response evaluation for chemicals in human tissues, as well as conceptual and basic research papers that address the use of interaction data in mixture risk assessment.



Causal Assessment of Herbal-Drug Interactions: Scientific Data vs. Diagnostic ScalesChristopher J. Borgert, PhD

Kathleen Cullen Findley, PharmD, MSTSusana Ulloa, MD

Susan A. Borgert, RPh, MS

The clinical management of pharmacological interactions, including interactions between herbal supplements and drugs, is possible when objective, reliable information is available regarding the type, the expected magnitude, and the potential clinical impact of the interaction. Providing this information, however, requires not only the presence of high-quality interaction data, but also objective, transparent procedures for evaluating those data and their clinical relevance. The clinical literature is replete with diagnostic probability scales and scoring systems for evaluating adverse drug effects, and recently, similar scales have been proposed for evaluating drug-supplement interactions. Such scales are often proposed as tools for evaluating “the likely cause” of particular adverse outcomes, but causal propositions based on such scales warrant scrutiny because the assessment of cause requires a degree of rigor commensurate with the purpose of the evaluation, which can vary widely among public health, clinical medicine and pharmacology, experimental biology, epidemiology, statistics, and law. Diagnostic probability scales are most consistent with mosaic theories of causality, which rely on professional judgment about the degree to which various factors are satisfied. Causal conclusions based on such scales are inherently fluid and malleable, and thus, cannot be considered to be scientific conclusions. In contrast, we have proposed evaluative criteria that are more consistent with concepts of causal determinism and that are designed to be objectively testable. We argue that these criteria are more firmly rooted in the foundational tenets of scientific analysis, including demonstration of the identity of the tested outcome, the error surrounding all measurements, the elimination of spurious variables that could affect the parameters under study, and independent replication of results.



Bio-sketch forDr. Radha Maheshwari

Radha Maheshwari, Ph.D is Professor of Pathology at the Uniformed Services University of the Health Sciences (USUHS, Bethesda, Maryalnd . He did his Masters from Birla Institute, Pilani, Ph.D from the Central Drug Research Institute (CDRI), Lucknow, India and Post doctoral fellowship at NIH with Dr. Robert Friedman and worked on the mechanism of Interferon Inhibition of enveloped viruses including VSV and HIV. He has published more than 100 papers and edited books and written chapters and invited articles. His work has been published in prestigious Journals such as Science, Nature, PNAS, Virology, Journal of Immunology, Cell Growth and Differentation etc. Dr. Maheshwari has created useful, innovative, comprehensive and productive partnerships with several Institutions in India such as CDRI, Industrial Toxicology Research Center, BITS and Indian Armed Forces Medical Services and several others. Over the last 23 years, through these efforts, the Indian and American scientists were able to address some of the important problems affecting human health, such as studies on Inhibitory potential of Gamma Interferon against malaria, role of natural products and botanicals in Wound healing, Cancer chemoprevention and angiogenesis, Prevention of Ischemia/hemorrhage and hypoxia-induced injury. Encephalitic viruses are a threat to military personnel worldwide. His laboratory has extensively studied the pathogenesis of encephalitic and neuroinvasive viruses such as Semliki Forest (SFV) and Encephalomyocarditis (EMCV) viruses. During neuroinvasion, encephalitic viruses cross or bypass the blood brain barrier (BBB) and gain entry to the brain. His laboratory has shown that exposure with certain chemicals and environmental agents enhance SFV, EMCV and Venezuelan Equine Encephalitis virus (VEE) pathogenesis leading to enhanced neurodegeneration in animals. These finding are significant as VEE has been weaponized and can be used a biowarfare virus.



Enhancement of Wound Healing and Angiogenesis by Novel Botanicals

Radha K Maheshwari1, Anuj Sharma1,2, Rajesh Thangapazham1,2 and Anoop K Singh1.1 Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda,

Maryland, USA2 Birla Institute of Technology and Sciences, Pilani, India

Wound healing is a complicated biological process that involves the interactions of multiple cell types, various growth factors, their mediators and the extracellular matrix proteins. Earlier, we reported that curcumin, an active ingredient derived from the root of the Curcuma longa plant (Sidhu, et al 1998, Wound Repair and Regeneration, 6:67; Sidhu, et al. 1999, Wound Repair and Regeneration, 7: 362, Mani et. al. 2002, Bio Factors, 16: 29); and arnebin-1, derived from the Arnebia nobilis plant (Sidhu, et al. 1999, J. Invest. Dermatology, 113: 101, and Mani et.al. 2004, Skin Pharmacology and Physiology, 17: 49), resulted in a faster restoration of tissue integrity in both full skin punch biopsy and skin incision models of normal as well as in several models of impaired healing including streptozotocin induced diabetes in rats, hydrocortisone sodium succinate in rats, and transgenic diabetic female C57/Ks J db+/db+ mice.

Recently, we have studied the effect of picroliv, obtained from the roots of Picrorhiza

kurrooa, in normal and impaired wound healing models in rats and the differential gene expression pattern in normal and transgenic diabetic mice. Animals were treated with picroliv following wounding with a 8mm punch. Wound tissues were studied at 4, 7 and 11 days post wounding and compared to vehicle treated controls using histology and histochemistry. Data showed that picroliv treatment resulted in earlier re-epithelialization, increased neovascularization, higher collagen content and increased migration of a number of cells into the wound bed. Zymogram analysis of granulation tissue homogenates indicated differential regulation of matrix metalloproteinases (MMPs) in picroliv treated group. The pathway specific gene array analysis shows the differential regulation of growth factors, transcription factors and other related genes such as fibroblast growth factors and their receptor in normal and diabetic mice. A detailed understanding and importance of these differentially expressed gene(s) during wound healing and use of non-toxic, phytochemicals which enhances multiple growth factors thus could be developed as therapeutics for the enhancement of wound healing and other biological activities. Therefore, it will be important and highly desirable to develop sensitive in-vitro and in vivo assays to test the various biological activities of botanicals and relate it to their quality and safety issues.



Bio-sketch forDr. David Pasco

Dr. David Pasco received his bachelor’s degree in biology from Thiel College and PhD degree in physiology in 1980 from Roswell Park Cancer Institute division of the University of Buffalo. He then completed 2 years of postdoctoral training at the Cancer Research Laboratory at University of California at Berkeley.

He joined the faculty of the National Center for Natural Products Research at the University of Mississippi upon its completion in 1995. He has served as Assistant Director of the Center overseeing Biological Evaluation for the past two years. His laboratory is focused on the isolation and identification of immunostimulatory agents found within botanicals and this research has been funded by several grants from the National Center for Complementary and Alternative Medicine of the NIH.



Using Bioassays to Improve Botanical Consistency and Safety: From The Standardization Of Immune Enhancing Botanicals To The Identification Of Toxic Priciples

David S. Pasco, National Center for Natural Products Research, Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, MS 38677

One factor contributing to the difficulty of assuring that immune enhancing botanicals and botanical preparations exhibit consistent therapeutic efficacy is that large molecular weight agents often contribute to their activity. In contrast to the relatively straightforward process of determining the content of small molecular weight actives within a botanical or botanical preparation, predicting biological activity based, for example, on the “amount of an active polysaccharide” is laced with difficulties especially with extremely large polysaccharides. Over the past 5-6 years we have identified several potent large molecular weight immune enhancing compounds within botanicals and have developed standardized preparations containing these agents. Several of these technologies have been licensed to commercial partners and product development (including sourcing, extraction optimization and stability testing) as well as standardization schemes assuring batch-to-batch consistency have been based on biological activity. Examples will be given of how we use this approach for Spirulina polysaccharide extracts in addition to botanical melanin, a newly identified class of immune stimulants. Examples will also be given to show how bioassay information can be used to identify potentially toxic principles or activities within botanicals with respect to Aristolochia species.



Bio-sketch forDr. David J. Kroll

David J. Kroll, Ph.D. is Senior Research Pharmacologist for the Natural Products Laboratory at Research Triangle Institute (RTI) in Research Triangle Park, NC, the nation’s second-largest independent, not-for-profit research and development organization. Dr. Kroll also holds adjunct associate professor appointments in Medicine at the Duke University Medical Center and Duke Comprehensive Cancer Center and in Pharmacotherapy and Medical Journalism at The University of North Carolina at Chapel Hill.

Dr. Kroll earned a B.S. in Toxicology, magna cum laude, from the Philadelphia College of Pharmacy & Science in 1985 where he was also a research intern in the Department of Drug Metabolism at the company then known as SmithKline & French Laboratories. Dr. Kroll then took his Ph.D. in Pharmacology & Therapeutics at the University of Florida College of Medicine in 1989, training under Dr. Thomas C. Rowe on the molecular pharmacology of DNA topoisomerases and the anticancer natural products that target this enzyme class. Dr. Kroll conducted postdoctoral research in medical oncology and molecular endocrinology at the University of Colorado School of Medicine and, in 1992, joined the faculty of the University of Colorado School of Pharmacy. At Colorado, Dr. Kroll’s research on DNA topoisomerase II and interactions with herbal dietary supplements was funded by the National Cancer Institute and the American Cancer Society. Dr. Kroll also pursued his pharmacy and medical teaching assignments as a scholarly effort, receiving 11 teaching awards between 1992 and 2001, including tenured-faculty peer selection as a President’s Teaching Scholar of the University of Colorado, due in large part to public and health professions education on thesafe and effective use of botanical and non-botanical dietary supplements. Following a sabbatical at Duke University Medical Center and the Duke Center for Integrative Medicine, Dr. Kroll assumed his current position at RTI in 2002, to work together with the co-discoverers of the natural product antitumor drugs, taxol and camptothecin, and assist in the continuity of research contributions by the RTI Natural Products Laboratory.

Dr. Kroll has served as a research or training grant reviewer for NCI and NCCAM and is currently a member of the NCI Cancer Complementary and Alternative Medicine PDQ editorial advisory panel. Dr. Kroll also serves on the drug and supplement safety advisor to the ABC News Medical Unit. In addition, Dr. Kroll is an editorial board member for the AACR journal, Molecular Cancer Therapeutics, and the international review journal, FACT: Focus on Alternative and Complementary Therapies



Simple and Inexpensive Methods to Prevent Botanical Misfortune

David J. Kroll,Natural Products Laboratory, Research Triangle Institute (RTI), Research Triangle Park, NC

27709 [email protected]

Botanical dietary supplements are often targeted by the mainstream media and scientific/medical communities as being unscientific and unsafe relative to prescription pharmaceuticals and over-the counter drugs. However, the relative risk of botanical medicines is largely overestimated and causal relationships between botanical remedies and significant adverse reactions pale in comparison to the frequency and magnitude of those observed with normal use of FDA-approved pharmaceuticals. Historically, a greater premium has been placed on demonstrating the safety of health-related products as opposed to confirming therapeutic efficacy; in fact, the required standards for prescription drug efficacy were implemented in the U.S. more than 50 years after standards for purity and safety were firmly in place. An argument will be made that botanical manufacturers can support their place in the national health agenda and generate significant goodwill by voluntarily subjecting supplements to relatively simple and inexpensive preclinical studies, and even limited phase I clinical trials, employing the basic tenets of pharmacology and toxicology (i.e., dose-response relationships) to support the relative safety of specific products. Moreover, many of the unfortunate adverse events of drug-herb interactions over the last 10 years could have been prevented by relatively simple hepatic metabolism and drug transport studies that have become commonplace in even the smallest of academic laboratories, due in large part to the increased availability and lower cost of previously scarce and cumbersome biological reagents. Demonstrating the safety of semi-pharmaceutical formulations of herbal products now requires more than simple reliance on the historic folk record of safe use; real health risks and negative publicity can be successfully mitigated with a modest, prospective investment in basic preclinical toxicology testing.



Bio-sketch forDr. Paul But

Prof Paul But is a professor in the Department of Biology, The Chinese University of Hong Kong (CUHK). He obtained his B.Sc. and MBA from CUHK, and his M.A. and Ph.D. from the University of California, Berkeley. He served as the Director of the Chinese Medicinal Material Research Centre, CUHK, from 1992-99. He is now the Coordinator of the Laboratory for Quality Control of Chinese Medicines; Co-Convener of the Section for Standardization and Safety of Chinese Medicines, Institute of Chinese Medicine; and Webmaster of the website on ‘Hong Kong Flora and Vegetation (http://www.hkflora.com)’. His interest focuses on the authentication, quality control, safety and efficacy of traditional Chinese medicines. He published 13 books, 200 papers, and seven U.S. and China patents.



Trouble-shooting in the cases of adverse reactions to botanical products

Paul Pui-Hay ButDepartment of Biology and Institute of Chinese Medicine,

Chinese University of Hong Kong, Hong Kong([email protected])

The use of traditional Chinese medicines and botanical products is widespread in Hong Kong. Occasionally, adverse reactions to such products do take place. The causes of such adverse reactions can be grouped under nine categories: (a) wrong herb, (b) poor quality of herb due to inadequate curing, (c) excessive dosage, (d) prolonged usage, (e) wrong prescription, (f) interaction with Western medicines, (g) intentional contamination with Western medicines, (h) individual errors, and (i) idiosyncratic responses. As most of these cases were due to human errors, proper regulation and training may help minimize them. Research is much needed to gain better understanding of the chronic toxicity of botanicals and their interaction with Western medicines. The general public should also be educated concerning the proper use of and potential adverse reactions to botanicals.



Bio-sketch forDr. Armando Cáceres

Armando Cáceres is Chemical Biologist from the University of San Carlos (USAC), Guatemala, with specialization training in Immunology (University of Wisconsin and Lausanne) and Pharmacognosy (USAC and Kitasato University). Since 1972 is Professor of Immunology at the School of Biological Chemistry, and is presently Director of Natural Products Research Laboratory (LIPRONAT), and Professor of Phytotherapy at the Pharmacy Graduate School, both at USAC. He is also Director of Research at Farmaya Phytopharmaceutical Laboratory in Guatemala. During 1996-2003 he was international coordinator of the Ibero-american Network on Phytopharmaceutical Products (RIPROFITO) in the framework of the Ibero-american Program on Science and Technology for Development (CYTED). He has been awarded with the Panamerican Pharmacy Award, the Science and Technology Congressional Medal and the University Medal. Is author of over 100 papers and 5 books on multidisciplinary research of medicinal plants (detection, validation, production, regulation and safe utilization).



Multidisciplinary Development of Phytotherapeutic Products From Native Central American Plants

Armando CáceresFaculty of Chemical Sciences and Pharmacy, University of San Carlos

(Coordinator of a Regional Project supported by OAS/AICD, Washington)

Based on ethnopharmacological research, 10 plants native to Central America were chosen for technology development for the sustainable utilization of regional biodiversity in the production of phytopharmaceutical products. The plants chosen were: Arrabaidea chica (anti-inflammatory and colorant), Eclipta alba (hepatoprotective), Lippia dulcis (expectorant), Lippia graveolens (antimicrobial), Passiflora edulis (sedative), Petiveria alliacea (anti-inflammatory), Phlebodium decumanum (immunomodulator), Phlebodium pseudoaureum (solar protector), Quassia amara (digestive and insecticide) and Smilax domingensis (antifungal and antipsoriatic). Since 2001-2004, the interaction of an academic institution and a private enterprise from each of the five participating countries (Costa Rica, Guatemala, Honduras, Nicaragua, Panama), permitted the multidisciplinary approach for an integral evaluation of the chosen plants. Activities being conducted for achieving such aim are: Regional market study of the selected plants, specific agrotechnology generation (conservation, propagation, cultivation, post harvest), phytopharmaceutical technology (extract characterization, markers selection, product development and formulation), six training and transfer seminars (three in Agrotechnology and three in Phytopharmacy), and the production of two technical manuals, one for cultivation and post harvest technologies for medicinal plants, and another for characterization and analysis of herbal drugs and phytotherapeutic products, in both cases including specific monographs of each of the species. By the middle of 2006 it is expected that two products will be developed by the enterprise involved in each country, based on the data, biological materials, knowledge, and experience generated during the project. International cooperation is requested for further development and standardization in order to initiate pilot production for clinical validation.



Bio-sketch forDr. Susan Walker

Dr. Walker is director of the Division of Dietary Supplement Programs, Food and Drug Administration (FDA), and Lead Scientist for Dietary Supplements at FDA’s Center for Food Safety and Applied Nutrition (CFSAN), U.S. Food and Drug Administration. The Division has primary responsibility for developing and implementing policy, regulations, guidance documents, position papers, advisory opinions, preclinical and clinical evaluation, and compliance activities intended to ensure the safety and proper labeling of dietary supplements and works closely with other units in FDA to accomplish these tasks. The division reviews safety information submitted in 75-day pre-market notifications for new dietary ingredients to ensure that such products are reasonably expected to be safe, reviews 30 day notices of structure-function claims, and conducts post-marketing surveillance of dietary supplement safety. Divisional teams include Regulations and Review, Compliance and Enforcement, and Clinical Review.

Dr Walker’s prior positions include Medical Team Leader in the Center for Drug Evaluation and Research (CDER/FDA), with responsibilities for the evaluation, review, and approval of investigational new drug applications (IND’s) and new drug applications (NDA’s). She has extensive experience in the review of adverse event reports and post-market surveillance issues. While at CDER she served on several committees, including the Women’s Health Sub-Committee, the Topical Anti-Microbial working group, the Thalidomide Working Group, and the Photo sciences Working Group. Dr Walker served as Deputy Director II in the Division of General, Reconstructive, and Neurological Devices in the Center for Devices and Radiologic Health (CDRH/FDA), and was responsible for review management within the General Surgery and Plastic/Reconstructive devices branches.

Prior to joining FDA, Dr Walker served as Chief of the Dermatology Section at the Veterans Administration Northern California in Martinez, CA and as Assistant Professor at the University of California at Davis. She served on the Veteran’s Administration Residency Review Committee, the Pharmacy and Therapeutics Committee, the Managed Care Educational and Training Committee, the Clinical Executive Board, the Institutional Review Board (IRB), Scientific Review Sub-committee, and as chair of the Animal Studies Sub-Committee of the IRB.

Dr Walker received her bachelor’s degree from the University of Michigan in Ann Arbor, and her M.D. from the Uniformed Services University of the Health Sciences in Bethesda, Maryland. She is board certified in Dermatology, and maintains active clinical staff privileges at National Naval Medical Center, Bethesda, MD and is a faculty member at the Uniformed Services University School of Medicine.



Regulation and Safety Assessment of Botanical Dietary Supplement Products

Susan Walker, M.D., DirectorDivision of Dietary Supplement Programs

ONPLDS, CFSAN. FDALead Scientist for Dietary Supplement Safety

Dietary Supplement Products are regulated under the Federal Food Drug and Cosmetic Act, which was amended in 1994 by the Dietary Supplement Health and Education Act in order to provide a definition of dietary supplements and to provide specific considerations for the regulation of dietary supplement products.

The Dietary Supplement Health and Education Act of 1994 (DSHEA) requires a pre-market safety notification for any New Dietary Ingredients (NDIs) introduced into commerce after October 15, 1994. A dietary supplement that contains an NDI is deemed to be adulterated unless one of two conditions is met; either: (1) The dietary supplement must contain only dietary ingredients that have been present in the food supply as an article used for food in a form in which the food has not been chemically altered or (2) there must be a history of use or other evidence of safety establishing that the dietary ingredient when used under the conditions recommended or suggested in the labeling of the dietary supplement will reasonably be expected to be safe, and the manufacturer or distributor of the dietary supplement containing the NDI must provide FDA with information, including any citation to published articles, which is the basis on which the manufacturer or distributor has concluded that a dietary supplement containing

DSHEA established the pre-market notification process for NDIs that have not been present in the food supply as articles used for food without chemical alteration, placing upon the manufacturer or distributor the pre-market responsibility for providing to FDA the basis upon which the firm has concluded that the NDI is reasonably expected to be safe. This information must be submitted to FDA 75 days prior to introduction of the ingredient into the marketplace. There are frequently novel and complex issues of scientific and statutory interpretation and implementation that must be managed during the review of these notifications. In May of 2005 FDA issued a public notice seeking comments from industry, consumers, and other interested members of the public concerning the type, quantity, and quality of information that a notifier should provide in notifications addressing reasonable assurance of safety under the new dietary ingredient provision of the act.



Bio-sketch forDr. Shaw T. Chen

Dr. Chen was originally from Taiwan; he received his Ph.D. in Chemistry from Johns Hopkins, M.D. from University of Miami, pediatric residency training from University of Minnesota and a clinical/research fellowship from National Institutes of Health. He is currently Associate Director, Office of Drug Evaluation I (ODE-I), Center for Drug Evaluation and Research, FDA, and the Leader of the Center’s Botanical Review Team, coordinating the review of all botanical drug applications and serving as the Agency’s leading expert in botanical related policies. Prior to his current assignment, he was a Medical Team Leader at FDA’s Division of Cardiorenal Drug Products, supervising a group of medical reviewers and multidiscipline scientists. Dr. Chen has near 18 years of extensive experiences in regulations of new drug development, clinical trial design & analysis, risk/benefit assessment in drug reviews, post-marketing safety surveillance and general regulatory affairs.



Safety Evaluation of Botanicals as New Drugs: A Report from the CDER Botanical Team

Shaw T. Chen, M.D., Ph.D.

This presentation will summarize the experience of safety evaluation for botanical products to be used as drugs, from the perspective of CDER’s Botanical Review Team. The current regulatory environment will be reviewed first, with a report on the status of botanical drug applications in FDA’s CDER. Regulatory experience and related issues in the safety reviews of botanical applications will then follow. More emphasis will be placed on the safety assessment of botanical INDs; basic principles in safety review of botanical NDAs will also be discussed.



Bio-sketch forMr. Loren Israelsen

Loren Israelsen is president of LDI Group, Inc., a consulting firm that specializes in dietary supplements, functional foods and phytomedicine issues. He began his career as general counsel of Nature’s Way Products, Inc., and served in various positions of increasing responsibility, including president. While acting as vice president of strategic development, he engineered agreements with Efamol Ltd., Madaus AG, and Dr. Willmar Schwabe, GmbH, to bring phytomedicines to the United States.

He has been heavily involved in the ongoing political and regulatory issues facing the supplement industry. He has served in various capacities, including past general counsel to the American Herbal Products Association, counsel to the Bee Products Association, co-counsel to the European American Phytomedicine Coalition, advisory board member to the Office of Technology Assessment Working Group on Dietary Supplement Regulation, and advisor to the Natural Products Quality Assurance Alliance, a dietary supplement industry group devoted to establishing standards and trade practices for dietary supplements. Mr. Israelsen has also served as industry liaison to the Food and Drug Administration’s Expert Advisory Committee to Ephedra and as advisor to the National Institutes of Health’s Office of Dietary Supplements.

Since 1992, he has served as executive director of the Utah Natural Products Alliance, which was instrumental in the development and passage of the Dietary Supplement Health and Education Act of 1994. He recently served as the U.S. issue manager for dietary supplements to the TransAtlantic Business Dialogue. He advises, lectures and writes extensively on the emerging issues facing the dietary supplement and functional foods industry. Mr. Israelsen received his B.S. in Political Science, Magna Cum Laude, from Utah State University. He attended the Oxford University Queens College Jurisprudence Program in Oxford, England and received his J.D. from J. Rueben Clark Law School, Brigham Young University.



Quality and Safety Issues Related to Botanicals

Loren IsraelsenExecutive Director, Utah Natural Products Alliance

Assessing the safety of botanicals encompasses a wide range of disciplines. From an industry perspective, none is more immediate than appropriate good manufacturing practices (GMP). Great attention is being paid to six key issues within the GMP compliance arena. They are (1) Vendor certification and specification of raw materials; (2) Competent and informative certificates of analysis; (3) Official methods of analysis for botanicals; (4) Management of contract analytical laboratories; (5) Sterilization technologies for botanicals, and (6) FDA inspection and enforcement of GMPs.

This presentation will review the current state of industry efforts and perspectives on global efforts to further manage botanical product quality and safety.



Bio-sketch forDr. Edmund T Y Lee

Dr Lee received his B.Sc and Ph.D in Microbiology at King's College, University of London. His Ph.D was financed by an Overseas Research Award and supervised by late Professors S. John Pirt and Michael Bazin in 1990.

Before his return to Hong Kong, Dr Lee was Group Leader at British Biotech Pharmaceuticals (now Vernalis after the merge of British Biotech and Vernalis Group) and Group Manager at Medeva Pharma (now acquired by Celltech). He specialised in fermentation, cell culture-based bioprocess and clinical trial manufacture. Dr Lee was involved in numerous recombinant DNA projects, spanning vaccines, chemokines and antithrombotics and, in various stages of development from CTX, IND through Phase III to pre-market regulatory inspection. Having participated also in a series of infrastructural projects, Dr Lee is well versed in technology transfer, cGMP, pilot facility operations and quality related and regulatory disciplines. He was awarded a full company sponsorship from British Biotech Pharmaceuticals and obtained his Master of Business Administration (MBA) from Warwick Business School.

In late-1999, Dr Lee joined the new Innovation and Technology Commission (formerly the Industry Department) of the Hong Kong Special Administrative Region Government as Senior Biotechnology Officer, advising the Commissioner for Innovation and Technology on strategic matters related to the development of biotechnology and Chinese medicine. He also served as Secretary of the Biotechnology and Environment Projects Vetting Committees. Dr Lee has championed two new funding solicitation themes on "Applied Genomics" and "Biosensor Technologies", setting directions for the biotechnology and biomedical development in Hong Kong.

Since October 2001, Dr Lee heads the Hong Kong Jockey Club Institute of Chinese Medicine Limited as Executive Director and serves as Convener of the Strategic Advisory Panel. He is also member of the Chinese Medicine Working Party of the Mainland-Hong Kong Technology Collaboration Committee set up between the Mainland and HKSAR Governments. Formerly, he was member of the Government's Admission of Talents Scheme and Biotechnology Projects Vetting Committee. Dr Lee currently holds the European Professional Biologist and Chartered Biologist status, and is a Fellow of the Institute of Biology and the Chartered Management Institute in the UK, and the Hong Kong Biotechnology Industry Association. Dr Lee is also a member of the Institute of Directors.



Development of Chinese medicine and related products: safety and quality perspectives

Edmund LeeExecutive Director

Hong Kong Jockey Club Institute of Chinese Medicine

The HKJCICM is set up as the local focal point to spearhead and coordinate the development of Chinese medicine (CM). It pursues a public mission that embraces (i) enhancing quality, science and evidence base of CM; (ii) assisting companies developing higher quality CM-based medicines and products for international markets; and (iii) facilitating exchange and commercialization of R&D outputs.

With the aim to accelerate research for applications, facilitate applicability of R&D outputs, the HKJCICM has established a few technology and R&D platforms for researching the quality of CMs and for developing CM-based medicines and products. In my presentation, I’ll take you through related safety and quality issues, drawing experience from latest regulatory developments and funded research cases.

The HKJCICM will expand its networks of advisors, business and research partners in major international markets.



Bio-sketch forDr. Robin Marles

Dr. Robin Marles is the Manager of the Research and Science Division, and Acting Manager for the Product Assessment and Clinical Trials Divisions, at the Natural Health Products Directorate, Health Products and Food Branch, Health Canada. Since joining the federal government in 2004, he has been assigned responsibility for the safety and efficacy assessment of natural health products (e.g. herbs, vitamins, minerals, and probiotics) that are the subject of applications for pre-market Product Licences, Clinical Trial authorizations, or post-market health hazard evaluations. He holds adjunct professor positions at the Universities of Manitoba and Saskatchewan, and was a professor of Botany at Brandon University in Manitoba from 1992-2004. His academic training includes a B.Sc. in Biology (University of Victoria, 1977), an M.Sc. in Biology (University of Saskatchewan, 1984), a Ph.D. in Pharmacognosy (University of Illinois at Chicago College of Pharmacy and World Health Organisation Collaborating Centre for Traditional Medicine, 1988), and two Postdoctoral Fellowships (University of Ottawa Biology Department, 1988-90, and Health Canada Bureau of Drug Research, 1990-92). His scientific research experience includes ethnobotany in Canada and Latin America, herbal medicine traditional uses, chemistry, pharmacology, standardization and quality control, and development of medicinal and nutraceutical plants as alternate crops or non-timber forest products for diversification and sustainable development of the farm and forest industries.



Safety Assessment of Botanicals in Canada

Robin J. Marles, Ph.D., Manager, Research & Science Division, Acting Manager, Product Assessment and Clinical Trials Divisions, Natural Health Products Directorate, Health Canada,

2936 Baseline Road (A/L 3302C), Ottawa, ON, CANADA K1A 0K9

Botanical health products require a pre-market Product License under the Canadian Natural Health Products (NHP) Regulations. Product safety, efficacy, and quality evidence is assessed according to standards of evidence modeled after those of the U.S. Agency for Healthcare Research and Quality. Unique is our mandate to ensure that Canadians have ready access to NHPs that are safe, effective, and of high quality, while respecting freedom of choice and philosophical and cultural diversity. This is realized by assessing benefits versus risks not only from the pharmacological perspective but also with respect to health claims and risk information from other paradigms such as Traditional Chinese Medicine, Ayurveda, or homeopathy. The mandatory Site License for an importer, manufacturer, packager or labeler is granted on the basis of compliance to Good Manufacturing Practices developed specifically for NHPs that take into account their distinctive differences compared to conventional pharmaceuticals, and the capacity of the NHP industry in Canada. Clinical Trial Authorizations involve assessment of the study products and the protocol according to Good Clinical Practices and the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. Allowances are made for the possible substitution of data from previous (e.g. traditional) use in humans for preclinical data that may be limited or unavailable for some botanicals. Research Ethics Boards approving the conduct of NHP trials must have expertise in Complementary and Alternative Medicine. For products already on the market prior to the NHP Regulations (2004), a Compliance Policy based on levels of risk sets priorities to bring unlicensed products into compliance by 2008. The Adverse Reaction Reporting system has been improved for including data on NHPs. When an NHP safety issue is identified, a Health Hazard Evaluation indicates the appropriate level of action for inspectors and border services.



Bio-sketch forDr. Sam Page

Sam Page retired from the World Health Organization in Geneva, Switzerland, in July 2005. At WHO he was a scientist in International Programme on Chemical Safety, where his primary function was Coordinator for the Project to Update the Principles and Methods for the Risk Assessment of Chemicals in Food. He was interim WHO Secretary for the Joint Expert Committee on Food Additives (JECFA) and the Joint Meeting on Pesticide Residues (JMPR). At WHO he also participated in the traditional medicines activities of the Division of Essential Drugs and Medicines Policies and in the Global Alert and Response Operations. Following the completion of his Ph.D. under the direction of the late Professor S. William Pelletier at the University of Georgia, he served his active military service in the Division of Medicinal Chemistry at Walter Reed Army Institute of Research. He joined the U.S. Food and Drug Administration in 1974 as a research scientist. Prior to his secondment to WHO in June 2001, he was a member of the U.S. Senior Biomedical Research Service and held a joint position of Lead Scientist for Chemistry in the Center for Food Safety and Applied Nutrition, and Scientific Director of the Joint Institute for Food Safety and Applied Nutrition at the University of Maryland. Among his professional affiliations, he was formerly a member of the Council of Experts of the U.S. Pharmacopoeia and of the Board of Directors of AOAC International. He has authored or co-authored more than one hundred research publications, primarily on the chemistry and toxicology of food components and contaminants. He is currently a private consultant in international food regulatory toxicology and continues to work with the traditional medicines and risk assessment programmes of WHO. He and his wife Jane plan to divide their time between Sixt-Fer-à-Cheval, France, and New Castle, New Hampshire, USA.



International Regulatory Perspectives on Botanicals

Samuel W. Page

Englène, Sixt-Fer-à-Cheval, 74740 FRANCE

The widespread and resurgent use of botanicals as traditional medicine and dietary supplements has created public health challenges in terms of policy, safety, efficacy and quality, access, and rational use. Accordingly, the World Health Organization (WHO) launched a comprehensive traditional medicine strategy in 2002. This strategy was endorsed by the Fifty-sixth World Health Assembly in May 2003. More than 70 countries have national regulations on herbal medicines. However, plant materials with a history of use as traditional medicines are defined differently in different countries. Diverse approaches, from prescription drugs to foods, have been adopted for the regulation of these products. The international food-trade related considerations are the responsibility of the Codex Alimentarius Commission, particularly the Codex Committee for Nutrition and Foods for Special Dietary Uses. In all regulatory approaches, safety should be the overriding criterion. Toward this objective, WHO has developed technical guidelines for ensuring the safety, efficacy, and quality of medicinal plants. The incorporation of safety monitoring into regulatory frameworks is a key need for traditional medicines. In order to meet the need for authoritative information for traditional medicines, WHO collaboratively has developed monographs on selected medicinal plants and encourages the development of national pharmacopoeia and monographs to meet the more specific needs of Member States. The nineteen WHO collaborating centres on traditional medicine are playing an important role in WHO's traditional medicine strategy, particularly in providing scientific information. In addition, with the Food and Agricultural Organization of the United Nations (FAO), WHO is developing general guidelines for risk assessment, which are applicable to botanicals, particularly in those cases where they are used as dietary supplements. The WHO Global Strategy on Diet, Physical Activity and Health, adopted in 2004, also has implications for the use of botanicals. From its global perspective, after safety considerations, perhaps the most important role for WHO is to strongly encourage the development of the evidence base for the use of botanicals to ensure that they are neither blindly accepted nor rejected outright.

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American Mayapple as a Potential Domestic and Renewable Source of Podophyllotoxin: Progress to Date

Kent E. Cushman1, Ikhlas Khan2, Rita Moraes2, Hemant Lata2, Ebru Bedir3, Pat Gerard4, and Muhammad Maqbool

1 Southwest Florida Res. & Educ. Ctr., Univ. of Florida, Immokalee, FL 34142; 2 Nat. Ctr. Natural Prod. Res., Univ. of Mississippi, University, MS 38677

3 Dept. Pharmacology, Faculty of Medicine, Ege Univ., Izmir, Turkey, 35100 and 4 Exptl. Statistics Unit, Mississippi State Univ., Mississippi State, MS 39762

Leaves of American mayapple (Podophyllum peltatum) contain podophyllotoxin, which is used as a precursor in the manufacture of several drugs. Canel et al. (2001) reported podophyllotoxin content in leaves of P. peltatum almost equal to that of roots and rhizomes of Indian mayapple, P. emodi, the current commercial source of the compound. Surveys of wild populations of P. peltatum revealed genotypes with podophyllotoxin contents ranging from zero to 56 mg/g (Moraes et al., 2000). Genotypes with high podophyllotoxin contents were isolated and propagated via tissue culture (Moraes-Cerdeira et al., 1998). Research reported here was funded by NRI grant 02-01525 to explore the use of American mayapple as a potential domestic and renewable source of podophyllotoxin. For greenhouse and field plantings, two-node rhizome segments were considered excellent, one-node segments good, and terminal-node segments fair for use as propagules for planting, and rhizomes transplanted during fall or spring performed better than those transplanted during summer. (Cushman and Maqbool, 2005). Further work showed that dormancy of rhizome segments harvested from the wild could be broken by chilling for 60 days or more at 4º C (Maqbool et al., 2004). A rhizome planting depth of 5 cm in combination with 7.5 or 15 cm of straw or pine bark mulch was considered excellent in establishing field plantings (Cushman et al., 2005). Plants grown in full sun performed well and had higher podophyllotoxin content than plants grown under increasing levels of shade (Cushman et al., 2005). Leaf removal from plants every year during early spring was too severe and greatly reduced regrowth of shoots (Cushman et al., in prep.). Plants tolerated leaf removal every year when removed during late spring. In conclusion, these studies indicate that domestication of American mayapple is possible and the species is relatively easy to manipulate using ordinary horticultural techniques.

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Comparative Analysis of Artemisinin and related compounds by Evaporative Light Scattering Detection (HPLC-ELSD) and Flame Ionizatin Detection

(GC-FID)

CONGYUE PENG, JORGE F.S. FERREIRA*. ANDREW J. WOOD

*Usda-Ars/Afsrc, 1224 Airport Rd., Beaver, Wv 25813, [email protected]

Currently, Artemisia annua cultivation and artemisinin analysis has boomed around the world due to the world demand and the plead by WHO. Simplified and affordable chromatographic methods are desperately needed meet the needs of developing nations afflicted by malaria. Artemisinin has been analyzed by thin-layer chromatography (TLC), HPLC, GC, radioimmunoassay, and enzyme immunoassay. The lack of chromophores indicates that artemisinin needs derivatization before HPLC-UV detection. This work compared the analyses of artemisinin, dihydroartemisinin, arteannuin B, and artemisinic acid from crude plant samples by GC with flame ionization detection (GC-FID) and HPLC with evaporative light scattering detector (HPLC-ELSD). Artemisia annua plants from a Brazilian cultivar (CPQBA, Campinas, Brazil) were field-grown in Illinois, dried under 40 ºC, and refluxed with petroleum ether for one hour. GC-FID and HPLC-ELSD were chosen due to their low cost compared to GC or HPLC coupled with mass spectrometry, and to their ease of operation compared to HPLC with electrochemical detection. Both GC-FID and HPLC-ELSD provided sensitive (ng level) and reproducible results when compared for the analysis of artemisinin, the main compound found in plants field-grown in Southern Illinois. The methods had a correlation coefficient of r2= 0.87, significant at p=0.001. Both methods could be used for the analysis of plants, commercial artemisinin and dihydroartemisinin capsules with minimal sample preparation and no derivatization.

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Do we need proper Identification & Quality Control of Herbs?

Madan M.R. Guntupalli*, Madan M. Pandey and Pushpangadan Palpu

Pharmacognosy and Ethnopharmacology DivissionNational Botanical Research Institute, Rana Pratap Marg, Lucknow

Uttar Pradesh – India, 226 001

Quality Control is a term that refers to processes involved in maintaining the quality of a manufactured product. Currently, there is no organization that certifies an herb as labeled correctly. Without proper quality control, there is no assurance that the herb contained in the bottle is the same as what is stated on the outside label. The widespread disregard for quality control in the health food industry has tarnished the reputation of many important medicinal herbs. For example, it has been estimated that more than 50% of the Echinacea sold in the United States from 1908 through 1991 was actually Parthenium integrifolium. This highlights the importance proper plant identification of using the Latin name as well as based upon organoleptic, microscopic, and chemical analyses.

The solution to the quality control problem that exists for manufacturers and suppliers of herbal products is to adhere to quality control standards and good manufacturing practices. Consumers, health food stores, pharmacists, and physicians who use or sell herbal products should ask for information from the suppliers of herbal products on their quality control processes. Currently, only a few manufacturers adhere to complete quality control and good manufacturing procedures. Companies supplying standardized extracts currently offer the greatest degree of quality control, hence these products typically offer the highest quality. Currently, in many countries, numerous standardized extracts fulfill requirements for marketing as drugs. These extracts have typically gone through many thorough quality control steps. In contrast, we should not put our heads in the sand and hope that concerns about herbs quality & safety will go away, because they will not. We must do something about it. Fortunately, bridges are now being formed between academia, government, the herbal industry, and the pharmaceutical industry. These bridges must result in practical concepts that actually help to improve the identification, quality & safety of today's herbal market.

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Genetic assessment of Podohyllum peltatum L. resources and evaluation of synthetic seed technology as an option for conservation.

Hemant Lata1, Bianca Bertoni2, Bladimiro Silva1, Ana M.S. Pereira2 and Rita M. Moraes1

1 National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS 38677, USA and 2 Departamento de Biotechnologia,

Universidade de Ribeirao Preto, Ribeirao Preto, Sao Paulo, 14,100, Brazil.

Podophyllum peltatum L. is an important anticancer medicinal plant, due to presence of lignans. The objective of the present study was to investigate inter and intra- population genetic variation present in 11 accessions using AFLP (amplified fragment length polymorphism) technique. The information determined by AFLP analysis on P. peltatum genetic diversity was employed to estimate similarity among these accessions based on the Jaccard Index. The dendrogram generated by UPGMA (unweighted pair group method of arithmetic averages) divided eleven accessions into two main clusters. Chemical analysis revealed that these clusters might gather two biosynthetically distinct groups of P. peltatum lignans. One having a 4-hydroxy-3, 5-dimethoxy substituted aryl group, which yield primarily -peltatin and trace amounts of podophyllotoxin. The other cluster gathers genotypes that produce compounds having 3, 4, 5-trimethoxy substituted aryl group such as podophyllotoxin. This data confirms our previous results, that suggested that P. peltatum chemical diversity is a genetic trait. Subsequently, conservation protocols to store elite genotypes at low temperature using synthetic seed technology and different osmotic agents were investigated. Excised buds from in vitro cultures were encapsulated in calcium alginate beads and cultured on different substrates then stored at 5° C, 10° C and 25° C for 8 months. Survival and vigor in the re-growth were the parameters used to evaluate the germplasm storage conditions. Cultures could be stored effectively for 8 months without subcultures. However, a mutation was detected by the AFLP analysis suggesting that further research is needed for conservation of elite genotypes.

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A Comprehensive Echinacea Germplasm Collection Located at the North Central Regional Plant Introduction Station, Ames, Iowa.

Joe-Ann H. McCoy, Mark P. Widrlechner and Jeff D. Carstens

USDA/ARS North Central Regional Plant Introduction Station, Iowa State University, Ames, IA 50011

Echinacea is a well-established, high value crop, both as an ornamental and dietary supplement. A comprehensive collection of Echinacea germplasm is currently held at the NCRPIS and is available via seed distribution for research purposes (www.ars-grin.gov/npgs). Representing all nine species collected throughout their respective North American geographic ranges, the Echinacea collection includes 179 accessions. Extensive morphological characterization data associated with the collection have been collected and are available to researchers to aid in selection criteria. The collection has been used extensively for various research projects ranging from HPLC analysis of metabolites of interest for the phytopharmaceutical industry to ornamental breeding studies for the horticulture trade. The mission of the USDA North Central Regional Plant Introduction Station includes: 1) the conservation of genetically-diverse crop germplasm through collection and acquisition; 2) conducting a variety of germplasm-related research; and 3) encouraging the use of the germplasm collections and associated information for research, crop improvement and product development

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Phytochemical variation in naturally-occurring chemotypes of black cohosh

Hussein Al-Amier1, Khaled A. Nasr2, Lorna Lueck1, Zoë E. Gardner1

and Lyle E. Craker1

1Medicinal Plant Program, Department of Plant and Soil Sciences and 2Department of Chemistry University of Massachusetts, Amherst, MA 01003

Black cohosh (Actaea racemosa L. [syn. Cimicifuga racemosa Nutt.]), a medicinal plant used primarily to relieve menopausal symptoms, is one of the best-selling medicinal plants in the United States. Approximately 96 percent of the black cohosh in trade is harvested from the wild, suggesting that the quality (level of bioactive constituents) of the plant tissue on the market may vary with source site. To assess the phytochemical variation occurring in wild populations of black cohosh, rhizome cuttings of 29 individual plants representing 11 populations of black cohosh were grown in a common garden setting. Methanolic extracts of the roots and rhizomes of these individual plants were analyzed by HPLC-PDA to determine the content of triterpenes (cimiracemoside A, cimicifugoside H-1, 26-deoxyactein, actein, cimicifugoside), phenolic acids (caffeic acid, ferulic acid, isoferulic acid), flavonoids (kaempferol, formononetin) and sugars (methyl-beta-L-arabinopyranoside). The greatest variations in constituent levels were seen in isoferulic acid (0.04-2.23mg/g D.W.), actein (0.9-13.43mg/g D.W.), formononetin (0-0.851mg/g D.W.). The data indicates that significant phytochemical variation exists among wild populations of black cohosh.

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The Special Issues of Test Materials in Botanical Dietary Supplement Research

James S. Miller, Wendy Applequist and William L. Brown

Center for Plant Genetic Resources, Missouri Botanical Garden, St. Louis, Missouri 63166-0299

Understanding the efficacy and safety of botanical dietary supplements has been difficult because of poor description of the substances being tested. Botanical dietary supplements are complex mixtures of chemical compounds. Presence or absence and concentration of multiple constituents may vary depending on source, environmental conditions where the plants are grown, and the methods by which materials are processed. This presentation will review criteria for ensuring that research on dietary supplements can 1) be repeated and confirmed and 2) that results can be compared with other studies, in both cases with confidence that similar substances are being tested. The three critical issues to ensure consistency in test materials are: 1) Source, 2) Characterization, and 3) Communication. It is necessary to confirm the identity of the constituent species used to manufacture dietary supplements and document the precise population from which they were collected or region in which they were cultivated. Chemically consistent test materials require validated methods for harvest, processing, and formulation. Furthermore, these must be coupled with methods to detect chemical differences from batch to batch to minimize variation and produce reliable test substances for rigorous research. Finally, if the source, methods for preparation, and information on chemical composition are not communicated in publications, readers of research articles may not be able to compare results with those of other studies.

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A Data Management System for Medicinal Herbs

Qiang Zheng

Center for Pharmaceutical Information and Engineering Research, Peking Universityand National Engineering Research Center for Beijing Biochip Technology

To help the State Food and Drug Administration of China to implement the new regulation of Good Agriculture Practice (GAP) for medicinal herbs, we have developed a Data Management System to help the herb producers to collect, storage, analyze and report all types of original and processed data generated through the entire process of herbal production. The system is also aimed to help regulatory agencies, researchers and buyers to inspect and review all the QA/QC procedures and data associated with herbal production. The system has a built-in auditing function to faithfully record all data input and modifications, where the auditing record can never be altered by anyone. The system also provides a bilingual capability to allow, for example, a Chinese herbal producer to enter the data in Chinese, and a US FDA reviewer to view the data in English, in real time.

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Influence of irradiance on gas exchanges and enzymes of antioxidative response system of Pothomorphe umheHata L. Miquel

Jose Abramo Marchese1,2,4, Raquel Souza Mattana2, Lin Chau Ming2, Fernando Broetto2,3, Patricia Vendramini3 and Rira Maria Moraes4

1Laboratory of Biochemistry and Plant Physiology, Agronomy College, CEFET-PR, Pato Branco, PR CEP 85503-390, Brazil; 2Agronomic Sciences College, Sao Paulo State

University, Botucatu, SP CEP 18603-970, 3Brazil Biosciences Institute, Sao Paulo State University, Botucatu, SP CEP 18603-970, Brazil and 4National Center for Natural Products Research, University of Mississippi, University, MS 38677-1848, USA

Pothomorphe umbeltata (L.) Miquel, is a Brazilian medicinal species that belongs to the Piperaceae. Although P. umbellata produces bioactive compounds of great importance for the pharmaceutical industry, little is known about the species growth requirements. Thus, this work attempts to evaluate growth and development of P. umbellate under different levels of irradiance and their influence on gas exchange and the activity of antioxidant enzymes. Plants were cultivated under different shade levels 30%, 50%, and 70%, and full sun. Photosynthetically active radiation (PAR) measured under these shade conditions varied from 835.7 mol m-2 s-1 at 30 % shade 580.6 mol m-2 s-1

at 50 % shade and at 70 % shade 284.8 mol m'2 s"1 to 1254.3 mol m'2 s"1 under full sun. CO2 assimilation, stomatal conductance and chlorophyll content reached maximum capacity when plants were cultivated under 30 % shade. Plants grown under full sun showed chlorosis, necrosis symptoms, and decreased chlorophyll content, indicating a photodynamic degradation of chlorophyll associated with the generation of reactive oxygen species (ROS). Consequently stress related antioxidant enzymes, Mn-SOD, Fe-SOD, and CuZn-SOD were at their highest activities in plants under full sun, whereas catalase had its lowest activity. The combined results lead us to conclude that P. umbellata is an obligate shade plant (sciophyte). P. umbellata agronomic studies stilt under investigation to determine which forest species could effectively provide shade in a consortium that could benefit both species.

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Carbon isotope composition as a tool for quality control of botanicals.

Jose Abramo Marchese1,2,* , Lin Chau Ming2, Carios Ducatti3; Fernando Broetto2,Evandro Tadeu da Silva3 and Marcelo Leonardo2

1CEFET-PR, Agronomy College, Laboratory of Biochemistry and Plant Physiology, Pato Branco 85503-390, Brazil; 2São Paulo State University, Agronomic Sciences College, Botucatu 18603-970, Brazil and 3São Paulo State University, Isotopes

Stables Center, Botucatu 18618-000. Brazil.

Modern ecophysiological research makes abundant use of the fact that the isotope composition of plant biomass differs from that of the atmosphere. It is particularly interesting that the isotope composition of plants with different photosynthetic pathways (i.e., C3, C4 and CAM) can be differentiated. Therefore, isotopic I composition can be used to control the quality of phytomedicines and botanicals, allowing the identification of frauds or contamination through the analysis of stable isotope composition of medicinal plants. Also, information on the photosynthetic mechanism of plants is important when one wants to introduce and cultivate exotic and wild herbs, spices, and medicinal plants. In general, plants with photosynthetic mechanism of the type C4 adapt better in hot climates, white the C3 will be better in milder climates. Isotopic screening is a simple test for determining the photosynthetic pathway used by plants. The scope of this work was to classify the photosynthetic mechanism of some herbs, spices, and medicinal plants through studies of the carbon isotope composition (13C). 13C of 54 species was investigated. For studies of 13C (13C% = [R (sample)/R (standard) - 1] x 10-3), dry leaves and powdered in cryogenic mill were analyzed in a mass spectrometer coupled with an elemental analyzer for determining the ratio R (R = 13CO2/12CO2). As results, 23 botanical families and 43 species possessed a type C3 photosynthetic mechanism; 6 species found among the botanical families Apocynaceae and Poaceae were C4, and 5 species found among the botanical families Agavaceae, Euphorbiaceae, and Liliaceae possessed a CAM type photosynthesis,

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Effect Of Altitude On Photosynthetic And Biochemical Characteristics of Podophyllum Hexandrum And Aconitum Balfourii, Two Alpine Endangered Medicinal Herbs From

Central Himalayas

Suman Chandra12 and Suresh C. Joshi2

1High Altitude Plant Physiology Research Centre, HNB Garhwal University, India; 2 National Center for Natural Product Research, Research Institute of Pharmaceutical Sciences, School of

Pharmacy, University of Mississippi, MS-38677; 2G.B.Pant Institute of Himalayan Environment Development, Garhwal Unit, Uttaranchal, India

In the recent past, ecological disturbances and over exploitation of natural resources have resulted in considerable decline in the Himalayan biodiversity. As a result, frequency of medicinal plants is decreasing sharply in nature and many of them have already been declared threatened, rare or endangered. Aconitum balfourii (distribution 2200 – 4000 m) and Podophyllum hexandrum (distribution 2000 – 4000 m) are two among the list of top 37 species requiring priority attention for conservation. Therefore, to explore the conservation and cultivation possibilities of these two species at comparatively lower altitude, a study on morphological, physiological and biochemical characteristics was conducted at two different altitudes. Plants of same age were collected from their natural habitat and grown in Alpine Medicinal Crop Garden, Tungnath at 3600 m and High Altitude Plant Physiology Research Centre at 550 m altitudes. After well acclimatization and growth, plants were examined for morphological, biochemical and photosynthetic studies during there active growth period. Our results show higher leaf length, width and area at lower altitude whereas, leaf thickness was found high at higher altitude in both the species. Leaf pigments (Chl a, Chl b, total Chl), Carotenoides concentration and PEP carboxylase activities were found higher whereas, Chl a/b ratio, soluble Protein and total Rubisco was found lower at 550 m altitude as compared to high altitude. An increase in the concentration of phosphorus, potassium and carbon and, a decrease in leaf nitrogen were observed in plants grown at lower altitude as compared to alpine plants. The temperature optima for photosynthesis in A. balfourii was observed around 20 oC at both the altitudes whereas, it was observed at 20 oC in alpine plants of P. hexandrum and around 25oC in the plants grown at lower altitude. Both the species have shown higher mesophyll efficiency at 550 m elevation, which reflects their better adaptability potential at lower altitude. The study reveals that these species can easily be cultivated at relatively lower altitudes. However, proper agronomical methodology will need to be developed for better yields.

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PhytomicsQC: A Quality Control Platform for Botanical Drugs.

Rajendra Marathe1, Winfried van Eyndhoven1, Jing Guan1, Hailong Wang1, Shwu-Huey Liu1, Robert Tilton1 and Yung-chi Cheng2

1 Phytoceutica Inc., 5 Science Park, Suit 13 New Haven CT 06311 and 2 Yale School of Medicine, Yale University, New Haven, CT 06520

PhytomicsQC, an analytical platform system, has been developed for assessing botanical extract consistency that integrates a phytochemical constituent fingerprint and a biological response fingerprint with a quantitative fingerprint assessment metric. Combining high resolution LC/MS and genomic bioresponse fingerprinting as general, well-accepted high resolution methodologies with novel pattern comparison and datamining informatics, provides an information-rich approach to addressing the critical issue of botanical quality control that remains as a central problem for regulatory approval of prescription botanical drugs.

In this poster, we will illustrate the utility of this QC approach with our botanical drug candidate, PHY906, an extract of four individual herbal ingredients, that is under an IND and is in clinical studies as an adjuvant with conventional cancer chemotherapies. Our experience indicates:

1. PhytomicsQC is broadly applicable, robust and affordable.2. Chemical and genomic bioresponse fingerprints can serve as unique, sensitive and

quantifiable metrics for botanical quality control.3. The integration of chemical and bioresponse fingerprints provides an information rich

approach to provide insight into active compound identification and potential mechanism of action

4. Botanical drugs can be manufactured to a high standard of consistencyWhile no single approach can completely define the subtle, complexities of botanical

drug action, a combination of high resolution methods focused on patterns of individual phytochemical compounds and the resulting biological responses, offers a powerful, general and objective approach to define comprehensive and quantifiable metrics for assessing the quality control of botanical drugs.

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Microscopic authentication of “Cuachalalate” preparation

Vaishali C. Joshi1, Andres Navarrete1, 3 and Ikhlas A. Khan1, 2

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, University of Mississippi, MS 38677; 2Department of

Pharmacognosy, School of Pharmacy, The University of Mississippi, MS 38677, USA and 3Facultad de Química, Departamento de Farmacia. Universidad Nacional Autónoma de México. Ciudad Universitaria Coyoacan 04510, México D.F México.

Bark of Amphipterygium adstringens (Schltdl.) Schiede ex Standl. (Julianaceae/ Anacardiaceae), locally names as “Cuachalalate”, is an important anti-ulcer remedy in Mexican traditional medicine. The crude drug and commercial extracts are exported from Mexico to different countries. The Herbal Mexican Pharmacopoeia (Farmacopea Herbolaria de los Estados Unidos Mexicanos) cites a monograph for Cuachalalate describing TLC method for qualitative identification. Extensive studies have been published on the chemistry of this plant however no microscopy description was available. Macroscopic and microscopic analysis of the stem bark was performed as it would assist in authentication.

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Morphological and chemical variation between the unripe and ripe fruits of Poncirus trifoliatus Raf. (Rutaceae) used in the Traditional Chinese Medicine

Vaishali C. Joshi1, Bharathi Avula1, Aruna Weerasooriya1 and Ikhlas A. Khan1, 2

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences and 2Department of Pharmacognosy, School of Pharmacy. The University of

Mississippi, MS 38677, USA.

The fruits of Poncirus trifoliatus Raf. are used in the Traditional Chinese Medicine (TCM). The immature, unripe fruits are known as Chih Shih and the fully ripe, mature fruits as Chih Ko and have different therapeutic values. However there are no studies indicating the morphology and chemical variation between Chih Shih and Chih Ko forms of Poncirus trifoliatus Raf. fruits. We studied the morphological and chemical variation at different developmental stages in Poncirus trifoliatus Raf. fruits as it would assist in quality control of the raw material.

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Authentication of Valeriana procera Kunth and comparative account of five Valeriana species

Vaishali C. Joshi1, Andres Navarrete1,3 and Ikhlas A. Khan1,2

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, University of Mississippi, MS 38677; 2Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, MS 38677, USA and 3Facultad de Química,

Departamento de Farmacia. Universidad Nacional Autónoma de México. Ciudad Universitaria Coyoacan 04510, México D.F México.

Valeriana procera Kunth (Mexican Valerian) is one of the commercially important species, being used as substitute in commerce for Valeriana officinalis L an important sedative in herbal medicine. A detail macroscopic and microscopic account has been provided for Valeriana procera Kunth. A comparison between the wild and cultivated samples of Valeriana procera Kunth was also carried out. Macroscopic and microscopic comparative analysis has been performed to differentiate Valeriana procera Kunth from Valeriana officinalis L. and other commercially important Valeriana species such as Valeriana jatamansi Jones, Valeriana edulis Nutt. ex Torr. & A. Grey and Valeriana sitchenisis Bong.

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Detection of Melanin in Echinacea purpurea (L.) Moench roots

Vaishali C. Joshi1, Nirmal Pugh1, Hemant Lata1, Rita M. Moraes1, I. Khan1,2

and D Pasco1,2

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences and 2Department of Pharmacognosy, School of Pharmacy.

The University of Mississippi, MS 38677, USA.

The genus Echinacea consists of perennials, native to the Prairie region of North America. Echinacea purpurea (L.) Moench (roots/ herb), E. angustifolia DC. (root) and E. pallida (Nutt.) Nutt. (root) are used as herbal medicine. Echinacea is one of the best selling botanical supplements in American herbal market and is used extensive to treat common cold, bronchitis, influenza, bacterial, viral, respiratory and urinary tract infections. Recent studies suggest melanin to be a major immune stimulatory compound in Echinacea which went undetected due to its solvent-specific requirement for extraction/solubility. So we analyzed Echinacea purpurea (L.) Moench roots to detect the presence of phytomelanin using light and fluorescent microscopy. Phytomelanin is brown to black in colour and fluoresces when examined under UV light.

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Development of a streamlined, DNA-based diagnostic platform for black cohosh authentication

Fabricio Medina-Bolivar12, Maureen C. Dolan12 and Selester Bennett1

1 Nature Diagnostics Inc. Blacksburg, VA 24060 and 2 Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR 72401

The US natural products market has experienced tremendous growth over the past five years and has become a multi-billion dollar business. At the current growth rates, demand pressures may outstrip supply for authentic product, thus increasing the opportunity for adulteration. The lack of strict oversight to standardize the formulations of natural products has made the industry vulnerable to product adulteration and contamination and augmented the need for product monitoring with an effective, reliable and reproducible diagnostic platform. We have initially targeted black cohosh (Cimicifuga racemosa) authentication due to its global popularity as an alternative treatment for menopausal symptoms and its susceptibility to adulteration and mislabeling. While analytical chromatographic techniques (i.e. LC-MS, HPLC-UV) are the most widely used test system in the industry for black cohosh identification, there is a need for a more streamlined, reliable diagnostic platform. DNA-based testing methods, which leverage unique DNA sequences to differentiate a given species from a related organism, have provided a powerful diagnostic platform used in several fields including forensics and medicine. The unique challenges to DNA-based testing of natural products are the co-purification of phenolics and other PCR inhibitors that compromise template quality for downstream PCR testing. We are developing a diagnostic platform with an effective yet facile sample collection/extraction procedure and a single tube, PCR-based assay for black cohosh authentication. This platform has broad application in delivering a quality assurance diagnostic system that can be used for a wide array of commercially valuable natural products.

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The Use of Polymerase Chain Reaction (Pcr) For The Identification Of Ephedra In Dietary Supplements.

Natascha Techen1, Ikhlas A. Khan1,2 and Brian E, Scheffler3

1

National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences and 2Department of Pharmacognosy, School of Pharmacy, The University of

Mississippi, University, MS 38677, USA and 3USDA-ARS-NPURU, MSA Genomics Laboratory, 141 Experiment Station Rd. Stoneville,

MS 38776, USA

Plant material such as ground roots or leaves can be difficult to identify even for experienced taxonomist. Unique molecular markers such as intergenic spacers, the noncoding region between two loci, can help with the identification of taxa at a molecular genetic level. For several plants and animals, such molecular markers were identified by using the polymerase chain reaction (PCR) method.

Ephedra has been used for centuries in traditional Chinese medicine. Since the 20th century it became very popular in the US as dietary supplement because of its positive influence upon weight loss. But recently studies showed evidence of possible hazards caused by the misuse or abuse of the herb. FDA regulation went into effect on April 12, 2004 prohibiting the sale of dietary supplements containing Ephedra (Ephedrine alkaloids).

We have analyzed the chloroplast psbA-trnH spacer region in 21 species of the genus Ephedra. This region is highly conserved (98%) in all of the Ephedra species analyzed and has less homology to Ephedra’s closest relatives Gnetum gnosum (81%) and Welwitschia (85%). Based on the results presented here, we conclude that the psbA-trnH spacer sequence could be used as a molecular marker for the identification and detection of Ephedra DNA in dietary supplements.

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The Use of Polymerase Chain Reaction (Pcr) for the Identification of Illicium ssp.

Natascha Techen1 and Ikhlas A. Khan12

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences and 2Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS 38677, USA

Chinese star anis (Illicium verum Hook f.) is being used principally as a spice and in herbal teas, for the treatment of infant colic. It has been reported that after the consumption of Star anise tea persons showed symptoms of general malaise, nausea and vomiting. Morphological and organoleptic investigations of the herbal teas indicated that this possibly contained Japanise star anise (Illicium anisatum L., Shikimi fruit). Japanise star anise contains the neurotoxin anisatin which can cause hyperactivity on the central nervous system and tonic-clonic seizures.

Current methods to verify the authenticity of plants are based mostly on taxonomic characterization, which often requires considerable expertise in analyzing macro- and microscopically features, or on phytochemical parameters. We have developed a molecular method that can help to identify and verify Illicium verum and Illicium anisatum. We have isolated genomic DNA from dried plant samples and analyzed a genomic region that could be used as a molecular marker.

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Identification of Quercetin, Amentoflavone, Sesamin and GA-1 in Ginkgo Products by Liquid Chromatography

Bharathi Avula1, Sampath K. Upparapalli1, Peter Goldman3 and Ikhlas A. Khan1,2

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, 2Department of Pharmacognosy, School of Pharmacy, The University of

Mississippi, MS 38677, USA. 3Division of Research and Education in Complementary & Integrated Medical Therapies, Osher Institute, Harvard Medical School, Boston, MA, USA

Quercetin, the biflavone amentoflavone, sesamin, (Z,Z)-4,4’-(1,4-pentadiene-1,5-diyl)diphenol (GA-1) and 3-Nonadec-8-enyl-benzene-1,2-diol (GA-3) are significant in-vitro inhibitors of human cytochrome P 450 (CYP) in human liver microsomes. An HPLC method has been developed which permits the analysis of quercetin, amentoflavone, sesamin and GA-1 in various Ginkgo Products. The best results were obtained with a Phenomenex Gemini C18 column using gradient mobile phase of water (0.1 % acetic acid) and acetonitrile (0.1 % acetic acid). Elution was run at a flow rate of 1.0 mL per minute and a temperature of 30 C. The detection wavelength was 280 nm. Several batches of Ginkgo products were analyzed for the presence of quercetin, amentoflavone, sesamin and GA-1. Quercetin and amentoflavone were identified in all the products analyzed.

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Quantitative Determination of Triterpenes from Amphiptherygium adstringens by HPLC and HPTLC

Bharathi Avula1, Andrés Navarrete1,3, Xiuhong Ji1, Paul Hersh4 and Ikhlas A. Khan1,2

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences and 2Department of Pharmacognosy, School of Pharmacy. The University of

Mississippi, MS 38677, USA; 3Facultad de Química, Departamento de Farmacia. Universidad Nacional Autónoma de México. Ciudad Universitaria. Coyoacan 04510, México D.F., México and 4Instituto Nacional de Antropologia e Historia. Delegacion Morelos. Matamoros 14 Colonia Acapatzingo 62440, Cuernavaca Morelos, México.

Amphiptherygium adstringens (Anacardiaceae/Julianiacaee), local name ‘Cuachalalate’, is used in folk medicine for the treatment of cholelithiasis, fevers, fresh wounds, hypercholesterolaemia, gastritis, gastric ulcers and cancer of the gastrointestinal tract. The development of two analytical methods (HPLC-PDA and TLC-Densitometry) for the determination of masticadienonic acid and 3-hydroxymasticadienonic acid in Cuachalalate preparations is described in this paper. By two methods a satisfying separation of the compounds could be achieved. Each method might be preferable depending on the requirements. The HPLC separation was performed on a Synergi MAX-RP 80A reversed phase column (Phenomenex, 150 x 4.6 mm I.D.; 4 µm particle size) and operated at 40 ºC. The triterpenes present in the plant material and commercial extracts were separated with an acetonitrile-water-reagent alcohol isocratic system at a flow rate of 1.0 mL per minute. The limit of detection was 0.1-0.2 µg/mL. The relative standard deviation (RSD) values for the determination of triterpenes in plant extracts were less than 1.00 %. This is the first report of analytical method developed for the quantitative analysis of triterpenes from Amphiptherygium adstringens by HPLC with PDA and HPTLC detection. The stem bark showed the higher amount of triterpenes and low amounts in root and stem root. Detection was performed at 215 nm.

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Simultaneous Quantification of Benzethonium Chloride, Methyl Paraben and Triclosan in Grapefruit Seed Extracts by HPLC.

Bharathi Avula1 and Ikhlas A. Khan1,2

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences and 2Department of Pharmacognosy, School of Pharmacy, The University of

Mississippi, MS 38677, USA

It has been reported that the commercial Grapefruit Seed Extracts (GSE) are promoted as a natural product for having antibacterial and antiviral properties for both internal and external use. Recent reports showed the presence of preservatives in commercial grapefruit seed extracts. An HPLC method has been developed which further confirms and permits the analysis of methyl paraben, benzethonium chloride and triclosan in various GSE samples. The best results were obtained with a Phenomenex Gemini C18 column using gradient mobile phase of water (0.1 % acetic acid) and acetonitrile (0.1 % acetic acid). Elution was run at a flow rate of 1.0 mL per minute. The detection wavelength was 254 nm for methyl paraben, 275 nm for benzethonium chloride and triclosan. Preservatives such as methyl paraben, benzethonium chloride and triclosan have been identified and quantitated in various commercial grapefruit seed extract samples. The main constituent identified was benzethonium chloride, a synthetic antimicrobial agent in the concentration range from 0.29-21.39 %. Positive ion electrospray MS of the commercial GSE sample showed a molecular ion at m/z 412 [M+] that matched with that of a standard of benzethonium chloride. Triclosan detected in two samples was found in the concentration range from 0.009-1.13 % and methyl paraben was not detected in all the samples analyzed.

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Enantiomeric Separation and Determination of Adrenergic Amines in Citrus aurantium, Various Citrus Species, Related Genera and Dietary Supplements by

High Performance Capillary Electrophoresis

Bharathi Avula1, Sampath K.Upparapalli1 and Ikhlas A. Khan1,2


Mississippi, MS 38677, USA

Citrus aurantium L. (Rutaceae) fruit extracts have recently been used for weight loss. Among the adrenergic amines the most important active constituent is the sympathomimetic compound synephrine and commercially available extracts are standardized for their content of this active principle. A capillary electrophoresis method was developed for the quantitative and qualitative determination of d-synephrine, l-synephrine, d-octopamine, l-octopamine, tyramine, n-methyl tyramine and hordenine. The electrophoretic separation was performed using a 75 cm x 50 µm ID (66.5 cm effective length) fused silica capillary. The samples were injected by pressure for 5 seconds at 50 mbar and the running voltage was 30 kV at the injector end of the capillary. The method developed was successively applied to the determination of the adrenergic amines in dietary supplements, in various Citrus species including Citrus aurantium, Citrus jams and Citrus juices. Synephrine was the main component and present in the levels from 0.02-0.17 % in various Citrus species and 0.42-69.28 mg in dietary supplements claiming to contain Citrus aurantium. Parameters affecting the resolution between (+) and (-)-enantiomers, such as pH, cyclodextrin concentration, temperature, organic modifier, buffer concentration and capillary dimensions were reported.

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Simultaneous Quantification of Adrenergic Amines and Flavonoids in Various Citrus Species and Dietary Supplements by Liquid Chromatography

Bharathi Avula1, Sampath K.Upparapalli1, Andres Navarrete1 and Ikhlas A. Khan1,2



An analytical method was developed for the simultaneous quantitative analysis of six amines and twenty flavonoids in fruits and extracts of thirty Citrus species including C. aurantium, near-Citrus relatives and dietary supplements by HPLC with PDA detection. The separation was achieved with a Phenomenex Synergi Hydro reversed phase column using gradient mobile phase of sodium acetate buffer (pH 5.5) and acetonitrile. Elution was run at a flow rate of 1.0 mL per minute and ultraviolet detection at 254, 280 and 330 nm. Among the amines analyzed, synephrine was the main component and present in the levels from 0.11-2.0 mg/g dry weight in twenty-one Citrus species and 0.07-18.62 % in dietary supplements claiming to contain C. aurantium. The flavanones and flavones were analyzed in the same Citrus samples and were species specific. The levels of flavones were very low compared with the levels of flavanones. The method facilitated the simultaneous quantitation of six amines and twenty flavonoids in various Citrus species, the distinction between the different Citrus species, and the analysis of dietary supplements containing C. aurantium.

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Simultaneous Analysis of Synephrine, other Adrenergic Amines and Flavonoids in Citrus Peel Jams and Fruit Juices by Liquid Chromatography

Bharathi Avula1, Andres Navarrete1, Sampath K.Upparapalli1 and Ikhlas A. Khan1,2

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences and 2Department of Pharmacognosy, School of Pharmacy,

The University of Mississippi, MS 38677, USA.

Citrus species are rich sources for various bioactive compounds like flavonoids, adrenergic amines, limonoids and coumarins. Six adrenergic amines and twenty flavonoids in the jams and juices of Citrus species were analyzed by HPLC with a C18 reversed phase column using gradient mobile phase of sodium acetate buffer (pH 5.5) and acetonitrile at a flow rate of 1.0 mL per minute with detection at 254, 280 and 330 nm. The content of synephrine present in the levels from 3.65-60.66 mg/mL in forty-eight Citrus juices and 0.018-1.02 mg/serving in thirty-two Citrus jams. It has been observed that the content of these amines and flavonoids vary to a large extent depending on the type of the Citrus species used. The method has been successfully applied to the determination of adrenergic amines and flavonoids in several samples of Citrus fruit juices and jams.

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Quantification of Parthenolide in Tanacetum species by LC-UV/LC-MS and Microscopic Comparison of Mexican/US Feverfew Samples

Bharathi Avula1, Andres Navarrete1,3, Vaishali C. Joshi1 and Ikhlas A. Khan1,2

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences; 2Department of Pharmacognosy, School of Pharmacy, The University of

Mississippi, MS 38677, USA and 3Facultad de Química, Departamento de Farmacia. Universidad Nacional Autónoma de México. Ciudad Universitaria Coyoacan 04510,

México D.F., México.

Feverfew has been recommended and used for the prevention of migraine. An LC-UV and LC-MS methods have been developed which permits the analysis of parthenolide in different Mexican/US feverfew samples and commercial products. The best results were obtained with a Phenomenex Luna C18 (2) column using gradient mobile phase of water and acetonitrile:methanol (9:1). Elution was run at a flow rate of 1.0 mL per minute and ultraviolet detection at 210 nm. The results obtained using LC-UV were comparable to those obtained using LC-MS. Parthenolide was detected in all the samples analyzed. The samples collected in Oaxaca (0.28 %) and Puebla (0.25 %) showed the highest content of parthenolide. All parthenium samples were also examined under light and fluorescent microscopy.

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High-Performance Liquid Chromatographic Method for the Determination of NPC1161, Primaquine and their Metabolites in Various Biological Systems

Bharathi Avula1, Lalit M. Tripathi1, Shabana I. Khan1, Babu L. Tekwani1, Dhammika Nanayakkara1, Waseem Gul1,3, Mahmoud ElSohly1,3 and Ikhlas A Khan1,2

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences; 2Department of Pharmacognosy, School of Pharmacy, The University of

Mississippi, MS 38677, USA; 3ElSohly Laboratories, Inc., 5 Industrial Park Drive, Oxford, Mississippi 38655, USA

NPC1161, an 8-aminoquinoline analog, has shown improved anti-malarial efficacy and favorable toxicity profile relative to other 8-aminoquinoline derivatives in rodents and primates models. High-performance liquid chromatographic method was developed for the determination of NPC1161, primaquine and their metabolites in the biological samples in order to facilitate metabolic and pharmacokinetic studies. The method includes extraction of the unchanged drugs and their metabolites from the biological samples. Separation was achieved by reversed-phase chromatography on a C18 column with water-acetonitrile (both containing 0.025 % TFA) as mobile phase. Recoveries of NPC1161 and its metabolites are greater than 60 % from various biological samples tested. No interference with the components of the biological matrix was observed. The detector response was linear with concentrations of NPC1161 and its metabolites (desalkyl NPC1161 and carboxy NPC1161) in the ranges from 0.5-100.0 µg/mL, 0.4-60.0 µg/mL and 0.3-70.0 µg/mL, respectively. A mass spectrometry coupled with electrospray ionization (ESI) interface method is described for the identification of NPC1161 and its metabolites in the biological samples. This method involved the use of the [M + H]+ ions of NPC1161, C3 analog (internal std. for the assay), desalkyl NPC1161 and carboxy NPC1161 at m/z 434, 406 349 and 449 in positive ion mode with extractive ion monitoring (EIM).

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Quantitative Determination and Separation of Adrenergic Amines and Flavonoids from Poncirus trifoliatus Raf. Fruits at Different Growth Stages by High Performance

Liquid ChromatographyBharathi Avula1, Vaishali C. Joshi1, Aruna Weerasooriya1 and Ikhlas A. Khan1, 2



The fruits of Poncirus trifoliatus Raf. (Rutaceae) have been used against allergic diseases for generations and still occupy an important place in traditional oriental medicine. The fruits have also been used to treat gastric and duodenal ulcers. The chemical analysis of the fruit extracts of Poncirus trifoliatus Raf. at different stages of maturation showed the variations in their concentration of flavonoids. Fourteen flavonoids (neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, neoponcirin, poncirin, naringenin, hesperetin, sinensetin, nobiletin, heptamethoxyflavone, 5-O-demethylnobiletin, tangeretin) and five amines (synephrine, octopamine, n-methyl tyramine, hordenine, tyramine) in the fruit extracts of Poncirus trifoliatus Raf. were analyzed by HPLC with a C18 reversed phase column. The four flavonoids viz naringin, poncirin, narirutin and neoponcirin showed maximum concentration during the first stage of the growth, gradually decreasing until the fruits reach maximum development. The paper also discusses about observations in the anatomical variations at different developmental stages in the fruit.

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Chemical Fingerprinting of Valeriana Species: Simultaneous Determination of Valerenic Acids, Flavonoids and Phenylpropanoids using HPLC with UV-detection

Bharathi Avula1, Andres Navarrete1,3, Young-Whan Choi1,4 & Ikhlas A. Khan1,2

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences; 2Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, MS 38677,

USA; 3Facultad de Química, Departamento de Farmacia. Universidad Nacional Autónoma de México. Ciudad Universitaria Coyoacan 04510, México D.F., México and 4Department of

Horticulture, Miryang National University, Miryang 627-702, Korea

The roots and rhizomes of various Valeriana species are currently employed as a sleeping aid or mild sedative. An HPLC method has been developed which permits the analysis of chlorogenic acid, lignans, flavonoids, valerenic acids and valpotrates in various Valerian samples. The best results were obtained with a Phenomenex Luna C18 (2) column using gradient mobile phase of water (0.05 % phosphoric acid) and acetonitrile:methanol (1:1) with 0.05 % phosphoric acid. Elution was run at a flow rate of 0.8 mL per minute and ultraviolet detection at 207, 225, 254, 280 and 325 nm. Different Valerian species and commercial products have shown remarkable quantitative variations. Chlorogenic acid (0.2-1.2 %), three lignans, linarin (0.002-0.24 %) and valepotriates were detected in all the Valeriana species analyzed. Highest amounts of valerenic acids were detected in V. officinalis L., trace amounts in V. sitchensis and absent in other species analyzed.

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Isobolographic analysis of the sedative interaction between six central nervous system depressant drugs and Valeriana edulis hydroalcoholic extract in mice

Martha Ugalde1, 2 , Victoria Reza1, Ma. Eva González-Trujano3, Bharathi Avula4, Ikhlas A. Khan4,5 and Andrés Navarrete1

1Facultad de Química, Departamento de Farmacia. Universidad Nacional Autónoma de México. Ciudad Universitaria Coyoacan 04510, México D.F., México; 2Facultad de Estudios Superiores Zaragoza. Universidad Nacional Autónoma de México. J.C. Bonilla 66 y Calzada

Ignacio Zaragoza, Colonia Ejercito de Oriente. Iztapalapa 09230, México D.F., México; 3Instituto Nacional de Psiquiatría “Ramón de la Fuente Muniz”, Av. México-Xochimilco

No. 101. Colonia Sn. Lorenzo Huipulco 14370, México D.F., México; 4National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences and 5Department of

Pharmacognosy, School of Pharmacy. The University of Mississippi, MS 38677, USA.

It has been declared frequently that valerian may potentiate the effect of other central nervous system (CNS) depressant drugs, however there has been a lack of experimental data. We have evaluated the profile of the interactions between the ethanol extract of Valeriana edulis spp procera and six CNS depressant drugs using an exploratory model to test the sedative effect in mice. All the compounds tested showed a dose-dependent sedative effect with the following ED50 values: valerian 181.62, diazepam 1.21, ethanol 1938, pentobarbital 11.86, buspirone 1.04, haloperidol 0.41 and diphenhydramine 17.06 mg/kg. An isobolographic analysis was used to evaluate the sedative interaction of the intraperitoneal co-administration of 1:1 fixed-ratio combination of equi-effective doses of valerian extract with each CNS depressant drug. The ED50 theoretical (Zadd) and experimental (Zexp) for each combination were: valerian + diazepam, Zadd = 1.41 mg/kg, Zexp = 81.64 mg/kg; valerian + ethanol, Zadd = 1060.22 mg/kg, Zexp = 687.89 mg/kg; valerian + pentobarbital, Zadd = 96.74 mg/kg, Zexp = 151.83 mg/kg; valerian + buspirone, Zadd = 91.33 mg/kg, Zexp = 112.73 mg/kg; valerian + haloperidol, Zadd = 91.01 mg/kg, Zexp = 91.52 mg/kg; valerian + diphenhydramine, Zadd = 99.34 mg/kg, Zexp = 123.52 mg/kg. Neither synergistic nor attenuate effects were found in any of the combinations evaluated. We concluded that the valerian extract did not potentiate the sedative effect of commonly prescribed CNS depressant drugs as was expected. The additive effect found through the isobolographic analysis suggested that the sedative effect of V. edulis resulted from the activation of common mechanisms of haloperidol, diazepam, buspirone, pentobarbital, diphenhydramine and ethanol.

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Validated HPTLC methods - a key element for identification of botanical raw material

Eike Reich, PhD1*, Anne Schibli1, Alison DeBatt2 and Kerry LeVanseler, PhD3

1CAMAG Laboratory, Muttenz, Switzerland. 2CSI, Wilmington, NC and 3NSF, Ann Arbor, MI

Modern High Performance Thin- Layer Chromatography (HPTLC) is an extremely powerful tool for identification of botanicals. HPTLC fingerprints are widely used to ensure quality of raw materials, to detect the presence of adulterants, and to monitor the stability of drugs and finished products. Consequently there is an increasing need of the botanical industry for suitable, reliable and state of the art methods, which are accepted by the authorities. For inclusion into the frame work of CGMP such methods have to be validated, but unfortunately there is no official guideline available for the validation of qualitative methods. This poster presents a pragmatic approach to validation of HPTLC fingerprints, including, aside of the selectivity as stipulated by the ICH guideline Q2A, also the parameters stability, precision (repeatability, intermediate precision, reproducibility) and robustness. Based on the example of Licorice the proposal is being discussed by NSF and AOAC.

Currently NIH supports validation of further qualitative methods. Methods for the identification of Ginseng, Eleuthero, Green Tea, Echinacea and Black Cohosh are completed. The validation of identification methods for Milk thistle, Feverfew, Saw palmetto, Ginger, and Kava is in progress. The methods can be obtained from the corresponding author and will be published soon in the Journal of AOAC.

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Influence of Particle size on Extraction Yield and Quantification of Parthenolide in Fevervew (Tanacetum parthenium)

Jorge M. Fonseca1*, James W. Rushing2, Ronald L. Thomas3, Melissa B. Riley4 and Nihal C. Rajapakse5

1The University of Arizona. Yuma Agricultural Center. Yuma, AZ 85364-9623; 2Clemson University. Coastal Research and Education Center. Charleston, SC 29414-5332;

3Clemson University. Department of Packaging Sciences. Clemson, SC 29634-0375; 4Clemson University. Department of Plant Pathology and Physiology. Clemson, SC 29634-0375;

and 5 Clemson University. Department of Horticulture. Clemson, SC 29634-0375.

The influence of particle size of feverfew powders on the parthenolide (PRT) extraction yield was investigated. Samples with 500 µm or smaller particle size yielded at least 5 times more PRT than samples with greater than 500 µm particles, independent of the method of extraction utilized (Soxhlet, bottle stirring, sonication). The negative correlation between extraction yield of PRT and particle size of powder followed an exponential pattern (R=0.96). The particle size of various commercial products was found to vary within large ranges. The stage of growth and development was found to influence the particle size obtained at grinding. Vegetative plants contained more PRT and yielded more coarse particles (>500 µm) than flowering and senescing plants. No major difference in particle size distribution of ground stems and leaves was observed. Our results revealed that uniformly small particle size is of paramount importance for reliable extraction of PRT from feverfew.

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HPLC Method for Analysis of Alkaloids in Blue Cohosh Samples.

Satchithanandam Subramaniam, Kevin D. White, Erich Grundel, Jeanne I. Rader.

ONPLDS, CFSAN, FDA, College Park, MD.

Blue Cohosh (Caulophyllum thalictroides) is a perennial plant whose extracts have been promoted for their estrogen-like benefits. Extracts of rhizomes of Blue Cohosh are sold as dietary supplements, but the safety and efficacy of this plant have not been systematically evaluated. Recent studies indicate that ingestion of Blue Cohosh can produce birth defects and neonatal heart failure. Anagyrine, an alkaloid present in Blue Cohosh, is a teratogen. Six commercial samples were analyzed for alkaloids and the profiles were compared against that of authenticated Blue Cohosh. The rhizomes of authenticated Blue Cohosh were obtained from Botanical Liaisons, Boulder CO. Samples of Blue Cohosh in powder, capsule and liquid form were obtained from commercial sources. The rhizomes were powdered and extracted in methanol. The contents of capsules were extracted in methanol. Liquid samples were diluted 1:10 with methanol. All samples were analyzed by HPLC using an XTerra Phenyl column with a gradient mobile phase consisting of water and acetonitrile, each with 20% mM ammonium bicarbonate and 0.1% ammonium hydroxide. HPLC chromatograms of all of the commercial samples were similar to that of authenticated Blue Cohosh. Four of the peaks obtained from HPLC analysis were identified as magnoflorine, N-methylcytisine, baptifoline and anagyrine by LC/MS. In all samples, area % accounted for by magnoflorine was 71-89 %. We are collecting fractions of magnoflorine from semi-preparative HPLC to prepare in-house standard.

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GC/MS Analysis of the 8-Aminoquinoline Antimalarial [NPC1161] and its Carboxy Metabolite in Plasma and Red Blood Cells of Primates

Mahmoud A. ElSohly1’2, Waseem Gul1,2, Shixia Feng1, N.P.D. Nanayakkara2, Alice M.

Clark2, Shabana I. Khan2, Frank B. Cogswell4 and Larry A. Walker2,3

1ElSohly Laboratories, Incorporated, 5 Industrial Park Drive, Oxford, Mississippi 38655, USA;

2National Center for Natural Products Research (NCNPR), School of Pharmacy, University of Mississippi, University, MS 38677, USA; 3Department of Pharmacology, School of Pharmacy, University of Mississippi, University, MS 38677, USA and 4Tulane Regional Primate Research

Center, Tulane University Health Sciences Center, Covington, LA 70433, USA

Among the 8-aminoquinoline antimalarial analogues, NPC 1161C (the racemic form) showed the best activity in prophylactic and treatment assays against both PCP and malaria with favorable toxicity profile. This study describes a GC/MS procedure developed for the determination of the concentration of its two enantiomers and their carboxy metabolites in plasma and red blood cells, following administration of the individual isomers to monkeys and dogs. Preliminary pharmacokinetic data were generated for the two enantiomers. The data suggest that the (-)- isomer is metabolized to its carboxy derivative at a much higher rate than the (+)- isomer.

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Phytochemical study of marker compounds from Actaea podocarpa

Zulfiqar Ali1, Rahul Pawar1, and Ikhlas A. Khan2

1National Center for Natural Products Research, The University of Mississippi, MS 38677, USA and 2Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, MS

38677, USA.

Actaea, a small genus in Ranunculaceae, consists of about twelve species distributed throughout East Asia, Europe, and North America.1 A. pachypoda is also called a white cohosh.2

Actaea racemosa (black cohosh) is one of the well known species in Actaea, which has been used for the treatment of a variety of ailments such as diarrhea, sore throat, and rheumatism by Native Americans. Now it has become a well-known alternative herbal medicine with health benefits in treating painful menstrual periods and menopausal disorders not only in the United States but also in European countries.3

A number of new as well as known cyclolanostane xylosides have been isolated and elucidated their structures during the course of our ongoing phytochemical investigation of A. pachypoda. A combination of normal and reverse phase (RP-18) silica was used for their isolation. The structures of all isolates were determined by using a combination of spectroscopic techniques including 1- and 2- Dimentional NMR.

1. Schopke et al., J. Nat. Prod. 2001, 64, 986.2. In, “World Economic Plants” Eds. Wiesema, J.H. and Leon B, CRC Press, Boca Raton, London, New York, Washington, D.C., 1999.3. Mimaki et al., Chem. Pharm. Bull. 2002, 50, 121.

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Phytochemical study of Actaea podocarpa

Zulfiqar Ali1, Rahul S. Pawar1, and Ikhlas A. Khan1,2

1National Center for Natural Products Research, and 2Department of Pharmacognosy, School of Pharmacy, University of Mississippi, MS 38677, USA

Actaea, one of the smallest genera in Ranunculaceae, consists of about twelve species distributed throughout East Asia, Europe, and North America.1 A. podocarpa is also called a yellow cohosh2 or summer cohosh.3 Actaea racemosa (black cohosh) is one of the well known species in Actaea. It has a long and diverse history of medicinal use dating back to native North American indigenous groups. It is presently used in the treatment of climacteric symptoms related to menopause, with a clinical history spanning over the last 40 years.4 Our ongoing phytochemical investigation of A. podocarpa resulted in the isolation and structure determination of a number of new cyclolanostane arabinosides, rarely found in nature. A combination of normal and reverse phase (RP-18) silica was used for their isolation. The structures of all isolates were determined by using a combination of spectroscopic techniques including 1- and 2- Dimentional NMR.

1. Schopke et al., J. Nat. Prod. 2001, 64, 986.2. http://www.nps.gov/plants/medicinal/pubs/2001appendixc.htm3. http://www.ibiblio.org/herbmed/php/get?id=161154. Fong et al., Phytochemistry 2002, 61, 409.

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Phytochemical Investigation of Valeriana Officinalis

Young-Whan Choi1,3, Jianping Zhao1,2 and Ikhlas A. Khan1,2*

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, 2Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, MS 38677, USA and 3Department of Horticulture, Miryang National

University, Miryang 627-702, Korea.

The dried underground parts (roots and rhizomes) of valerian (Valeriana officinalis L.) are used to prepare modern phytomedical products. Valerian is applied as antianxiety and digestive formulations, a tranquillizer for the treatment of nervousness and agitation and as a mild sedative for sleep promotion.

A methanolic extract of the roots was investigated for its phytochemical constituents. Two new compounds were isolated from the roots of Valeriana officinalis. Their structures were established on the basis of 1D and 2D NMR experiments including COSY, HMQC or HMBC as Berchemol-4--D-glucoside and 3-(hydroxymethyl)-2-(1-hydroxyprop-2-en-2-yl)-4-methylcyclopentanol-O--D-glucoside. Five lignans, namely pinoresinol-4-O--D-glucoside, 7,9'-monoepoxylignans massoniresinol-4'-O--D-glucoside, berchemol-4'-O--D-glucoside, 7,9':7',9-diepoxylignans pinoresinol-4,4'-di--O-D-glucoside and 8-hydroxypinoresinol-4'-O--D-glucoside, which had already been isolated from valerian in an earlier study, were isolated. In addition, adenosine, uridine, chlorogenic acid, chlorogenic acid, two different dicaffeoylquinic acid isomer, 4-[(1E)-3-hydroxy-1-propenyl]-2-methoxyphenyl-O--D-glucopyranoside, patiscabroside II, adoxosidic acid, trans-coniferin, vanilloloside and 1-(3',4'-dihydroxycinnamoyl)-cyclopenta-2,3-diol were isolated.

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Two New Constituents of Morinda citrifolia (Noni) Fruit Juice

Vladimir Samoylenko1, D. Chuck Dunbar1, James W. Rushing3, Ikhlas A. Khan12 and Ilias Muhammad1

1National Center for Natural Products Research and 2Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi,

University, MS 38677 and 3Department of Horticulture and Coastal Research and Education Center, Clemson University, Clemson, SC 29634-0319.

Morinda citrifolia L. (Rubiaceae), known as Noni, has a long history of traditional use in the Hawaiian and Tahitian islands. The fruit juice of this plant is reputed for anticancer and immunostimulant activities, while the various parts of this plant have been used to treat diabetes, hypertension, diarrhea, and topical infections. Today Noni products are widely available as a dietary supplement in health stores, supermarkets and the internet. Despite that great amount of research has been carried out on Noni, the active constituent(s) for its intended uses are still not unambiguously established. The increasing use of this product suggests an urgent need to isolate and identify marker compounds for quality control. In continuation of our work on the isolation of markers from dietary supplements, we now wish to report the isolation of two new markers, namely, 1-O-(3´-methylbut-3´-enyl)--D-glucopyranose (1) and 4-epi-borreriagenin (2), together with the known iridoid glycosides asperulosidic acid (3) and deacetylasperulosidic acid (4) from Noni fruit juice collected in Puerto Rico. The structure of compounds was based on 1H and 13C NMR, mainly 2D NMR COSY, HMQC, HMBC and NOESY experiments, and HRMS. Furthermore, samples from freshly squeezed Noni fruit juice from Japan revealed the presence of scopoletin, in addition to compounds 1-4.

(1) (2)

OHO

HOOH

O

OH

O

HOOH

HO O

H

H

HH

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Antiparasitic, Nematicidal and Antifouling Constituents from Juniperus procera Berries

Ilias Muhammad1, Vladimir Samoylenko1, Md. Abdul Gafur5, Samir A. Ross1,2, Jaber S. Mossa4, Babu L. Tekwani1, D. Chuck Dunbar1, Jan Bosselaers3, Mahmoud A. ElSohly,12 and Farouk S.

El-Feraly4

1National Center for Natural Products Research, and 2Departments of Pharmacognosy and Pharmaceutics, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University

of Mississippi, University, MS 38677, 3Plant- and Material Protection Division, Janssen Pharmaceutica, B-2340 Beerse, Belgium, 4Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia, 5Research Center for Ethnomedicines, Institute of

Natural Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan.

Juniperus procera Hochst. ex Endl. (Cupressaceae) is widely distributed throughout the Southern Arabian Peninsula and East Africa, and used locally for the traditional remedy of tuberculosis and jaundice. Juniper berry oil is reputed for a wide spectrum of pharmacological activities and its monographs are included in some national pharmacopoeias. A bioassay-guided fractionation of J. procera berries yielded antileishmanial, nematicidal and antifouling diterpenes, including a wide range of known abietane, pimarane and labdane diterpenes. The structure elucidation was based on 1H and 13C NMR data, mainly 2D NMR COSY, HMQC, HMBC and NOESY experiments, and HRMS. Among these, totarol (1), ferruginol (2) and 7-hydroxyabieta-8,13-diene-11,12-dione (3) exhibited significant in vitro antileishmanial activity against Leishmania donovani promastigotes (IC50 value 3.5-4.6 g/mL), and abieta-7,13-diene (4) inhibited Plasmodium falciparum D6 and W2 clones (IC50 value 1.9 g/mL). In addition, totarol demonstrated strong nematicidal and antifouling activity against Caenorhabditis elegans and Artemia salina, respectively.

(1) (2) (3) (4)

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Flavanones Flavonoids Isolated from Scutellaria baicalensis Georgi used as Makers for Quality and Safety Control

Jing Li1 and Ikhlas Khan12



Scutelllaria is a unique cosmopolitan genus of the subfamily Scutellarioideae belonging to Labiatae (Lamiaceae) family. About 360 species are found spread throughout the world and in different climate areas. There are above 100 species in China and over 90 species in North American. Many species in genus of Scutellaria are used in traditional medicinal system. For instance, S. baicalensis is well known in China by the name “Huang qin” in the traditional Chinese medicine. It was first listed in a Chinese herbal, Shen-Nong-Ben-Cao-Jing, which was written 2000 years ago, as a remedy for suppurative dermatitis, diarrhea and inflammatory diseases. S. baicalensis Georgi officially is now listed in the Chinese Pharmacopeia and Japanese Pharmacopeia JPXIII. It is one of the most widely used crude drugs for the treatment of bronchitis, hepatitis, diarrhea, and tumors. Some concentrated composite herbal preparations that contain S. baicalensis Georgi as a major ingredient in their prescriptions are widely used in oriental countries. Another example is Scutellaria lateriflora. It was introduced into American medicine in 1773 by Dr. Lawrence Van Derveer who used it to treat rabies; hence the common name mad-dog weed. Later it came to be recognized as a tonic, tranquilizer, and antispasmodic, and was therefore used as an ingredient in many “patent medicines” for “female weakness”. Other traditional uses include epilepsy, headache, insomnia, various other neurologic and psychiatric disorders, hypertension, fever, rheumatism, and stress.

In order to control the quality and safety of genus Scutellaria species, we have isolated fifteen flavonoids, including flavones, flavanones, isoflavones, and chalcone. There are baicalein, baicalin, wogonin, oroxylin A, viscidulin I, ganhuangenin, skullcapflavone II, gengkawanin, apgenin, quercetin, rutin, 7,8-dihydroxy-5-methoxyflavone, naringenin, daidzein, 2,6,2',4'-tetrahydroxy-6'-methoxychalcone,Using these compounds as markers we will analyze the contents of flavonoids in different species, different locations and different growing time.

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Microbial Metabolism of 3-Hydroxyflavone and 7-Hydroxyflavone

Wimal Herath1, Julie Rakel Mikell1, Daneel Ferreira1,2, Amber Lynn Hale1, and Ikhlas A Khan1,2

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, The University of Mississippi, University, MS 38677 and 2Department of Pharmacognosy,

School of Pharmacy, The University of Mississippi, University, MS 38677, USA.

As part of our program on microbial transformation studies of flavonoids, 3-hydroxyflavone and 7-hydroxyflavone were screened using 43 microorganisms. Fermentation of 3-hydroxyflavone(1) with Beauveria bassiana (ATCC 13144) yielded the glycoside, flavone 3-O--D-4-methoxyglucoside (3) and two minor metabolites. Beauveria bassiana (ATCC 7159) converted 7-hydroxyflavone (2) to the glycoside, flavone 7-O--D-4-methoxyglucoside(4) and 4’-hydroxyflavone 7-O--D-4-methoxyglucoside (5). Fermentation of 7-hydroxyflavone with Aspergillus alliaceus (ATCC 10060) yielded 4’,7-dihydroxyflavone(6) and 7-methoxyflavone(7) was produced from metabolism of 7-hydroxyflavone by Nocardia species (NRRL 5646). One of the minor metabolites of 3-hydroxyflavone was tentatively assigned a ’-chalcone structure (8) with the available data. Structure elucidation of the metabolites was based on spectroscopic data.

O

O

R3

R1

R2

R1 R2 R31 OH H H2 H OH H3 Glc H H4 H Glc H5 H Glc OH6 H OH OH7 H OCH3 H

OH

OGlc

OH

8

O

H

H3CO

HO

H

H

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New isoflavonoid from Iris songarica Shrenck

Mahmood Reza Moein 1,3, Seyd Albodlmajid Ayatollahi 1, Farzad Kobarfard 1, Muhammad I. Choudhary 2 and Ikhlas A. Khan 3,4.

1 School of Pharmacy, Shaheed Beheshti University of Medical Sciences, P.O. Box: 14155-6153, Tehran, Iran; 2 H.E.J. Research Institute of Chemistry, International Center for

Chemical Sciences, University of Karachi, Karachi, 75270, Pakistan; 3 National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences and 4Department of

Pharmacognosy, School of Pharmacy, University of Mississippi. University, MS 38677.

Genus Iris (Iiridaceae) is rich in isoflavone among monocotyledon. Isoflavones are known to have a wide range of biological activities including phytoesterogenic, anti-inflammatory, antioxidant and cancer chemopreventive properties. Iris songarica is distributed in Iran, Pakistan, Afghanistan, Uzbekistan, Kazakhstan, Soviet Union, Mongolia and North China. The under ground part materials of I.songarica were collected from Yazd Province, Iran. Ethanolic extract was subjected to column chromatography. A new minor isoflavone (1) was purified by recycling preparative HPLC. Also, a known peltygynoids (Irisoid A) (2) was purified by repeated column chromatography. The structures were identified through 1 & 2D NMR and MS spectroscopy.

(1)

(2)

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Five Marker Flavan-Phenylpropanoid Conjugates from Erythroxylum Catuaba

Nurhayat Tabanca3, Rahul Pawar1, Zulfiqar Ali1, David E. Wedge3

and Ikhlas A. Khan1,2

1National Center for Natural Products Research, Research Institute of Pharmaceutical

Sciences; 2Department of Pharmacognosy, School of Pharmacy, University of

Mississippi, University, MS 38677, USA and 3USDA-ARS-NPURU, The University

of Mississippi, University, MS 38677, USA

“Catuaba” is a popular herbal medicine used in Brazil. However, it comes from different botanical sources and its specific phytochemical contents vary widely. Its bark has been used as an analgesic, central nervous system stimulant, tonic and aphrodisiac. Our ongoing study on Erythroxylum catuaba stem bark showed two new and three known flavan-phenylpropanoid conjugates from EtOAc extract by using silica gel, sephadex LH-20, RP-18 and OPLC (Optimum Performance Laminar Chromatography). Structures of all compounds were determined by a combination of spectroscopic techniques including one and two-dimensional NMR.

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Minor triterpenoid constituents from Bacopa monniera.

Rahul S. Pawar1 and Ikhlas A. Khan1, 2

1National Center for Natural Products Research and 2Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, MS

38677, USA

Bacopa monniera is an Indian medicinal plant that is known for its adaptogenic and cognitive enhancing effects. The plant is reported to contain several compounds with jujobogenin and pseudojujubogenin skeleton that belong to dammarane family of triterpenoids. During our ongoing work on standardization of the plants and its marketed formulations, we have isolated three new and six known triterpenoids from a commercial extract of Bacopa monniera. The structures of the three novel minor compounds were established by the use of different spectroscopic methods.

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Phytochemical investigation of Hoodia gordonii, a South African succulent plant with anorectic activity

Yatin J. Shukla1, Brian Schaneberg3, Rahul S. Pawar1, Bharathi Avula 1

and Ikhlas A. Khan12

1 Department of Pharmacognosy, University of Mississippi, University, MS, 38677; 2National Center for Natural Product Research (NCNPR), Research Institute of

Pharmaceutical Sciences, University of Mississippi, University, MS, 38677 and 3Chromadex Inc., Santa Ana, CA 92705.

Hoodia gordonii, Fam. Apocyanaceae, is a succulent plant found in the summer rainfall regions of the Kalahari desert in South Africa, Namibia and Botswana. There are 21 reported species of the Hoodia genus, however H. gordonii is the only sought after species for trade due to its anorectic activity. It (H. gordonii) has been traditionally used by San tribe of S. Africa for its potential appetite suppressant and thirst quenching properties. Various uses of Hoodia have also been reported in African folklore such as for the treatment of abdominal cramps, hemorrhoids, tuberculosis, diabetes and as an aphrodisiac. In recent years, H. gordonii has gained popularity as a weight loss herb, and about 55 herbal preparations containing H. gordonii are available in the U.S. market. P57AS3, an Oxypregnane glycoside, is the only reported active constituent from this plant so far. It acts as an appetite suppressant by increasing the ATP content in hypothalamus neurons that regulate the food intake. P57AS3 is also claimed to prevent aspirin-induced stomach damage when combined with NSAIDS, and is reported to have anti-diabetic activity.

As a part of our ongoing research on H. gordonii, we have isolated P57AS3, and other related compounds. Structure elucidation for these compounds and development of an analytical method to standardize Hoodia extracts are discussed here.

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Phytochemical Investigation of Damiana – A Conventional Herbal Aphrodisiac

Jianping Zhao1 and Ikhlas A. Khan12

1Department of Pharmacognosy and 2National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, The University of Mississippi,

University, MS 38677, USA

Damiana (Turnera diffusa), a plant that grows in the Central American, as well as in parts of South America, was used by the ancient Mayan in treatment of giddiness and falling (loss of balance). Mexican Indians have used it as an aphrodisiac beverage with a long history. Although many other healing properties of this plant were reported, the aphrodisiac effect has been most commonly employed as its principle use. In 1874, the damiana product was marketed in the U.S. and was collected into the National Formulary from 1888 to 1947. The history of use of damiana is full of interesting stories, as well as controversy. Once it was said to be a marvelous plant, and later it was said to be a hoax. In recent years, plenty of botanical products containing damiana appear on the market. But until now, there have been no sufficient chemical and pharmacological studies to substantiate the alleged aphrodisiac effect. We report here some terpenoids and flavanoids isolated from this plant.

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Diterpenoid from Teucrium chamaedrys

Rangavalli B. Manyam1, Rahul S. Pawar1 and Ikhlas A. Khan 12

1National Center for Natural Products Research, Research Institute of PharmaceuticalSciences and 2 Department of Pharmacognosy, School of Pharmacy. University of

Mississippi, University, MS 38677. USA.

Teucrium chamaedrys (lamiaceae), commonly called as Wild germander, is a herbal plant that has been used since antiquity as folk medicine for its choleretic and antiseptic properties. Hydroalcoholic extracts of the plant are currently used in the preparation of flavoured wines, bitters and liqueurs. The plant was a subject of intense investigation as, Teucrin A, one of the major components of diterpenoid fraction was found to be hepatotoxic. As a result of our continuing study on the plant we have earlier isolated nine neo-clerodane diterpenoids, one was new and two known phenlypropenoids, and two new phenylethanoids compounds. In the present study we report the isolation and structure elucidation of another polar diterpenoid. The chemical structure was established by a combination of spectral techniques like ESI-M'S, IR, and NMR experiments.

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Synthesis of 1-(3,4-dihydroxyphenyl)-8Z-nonadecenean alkyl phenol from Ginkgo biloba

U. Sampath Kumar1, Jamal Mustafa1 and Ikhlas A. Khan1,2

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, and 2Department of Pharmacognosy, School of Pharmacy, The University of

Mississippi; University, MS-38677.

Ginkgo biloba is one of oldest plant in the plant kingdom and known for its resistance towards insects, bacteria and viruses. Extracts from the leaves of the Gingko tree are therapeutically used for the treatment of peripheral and cerebral vascular disorders as well as multi-infarct or Alzheimer-type dementia. A number of alkyl phenols have been isolated from this plant and have shown wide spectrum of biological properties like antitumor and inhibitors of glycerol-3-phosphate dehydrogenase. Some of the alkyl phenols are known to cleave DNA under oxidative conditions. The present work describes the total synthesis of 1-(3,4-dihydroxyphenyl)-8Z-nonadecene in eight steps starting from 3,4-dihydroxy benzaldehyde.

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Chemical investigation of Citrus aurantium

U. Sampath Kumar1 and Ikhlas A. Khan1,2

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, and 2Department of Pharmacognosy, School of Pharmacy, The University of

Mississippi; University, MS-38677.

As part of our continuing effort to establish the fingerprint of various plants phytochemical investigation of Citrus aurantium has been carried to isolate the marker compounds of the plant. The methanolic extract of the plant upon purification has yielded six flavonoids, flavonoid glycosides and two biogenic amines. The flavonoids were identified as nobiletin, tangeretin, sinensetin, 5-O-demethyl nobiletin, 3,5,6,7,8,3',4' Heptamethoxyflavone and 5,7,8,4'-Tetramethoxy flavone. The flavonoid glycosides were identified as hesperidin, neohesperidin, naringin, narirutin, 6-8-diglucosyl apigenin and 5,7,4' trihydroxy-8, 3'-dimethoxyflavone-3-O-{[3-hydroxy-3-methylglutaryl(1-6)]-ß-D-glucoside. Both the amines are identified as synephrine and methoxysinephrine. Details of the isolation using different chromatographic techniques are reported. The synthesis of N-methyl tyramine yet another marker compound is carried out.

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Variations in the constituents of two subspecies of Teucrium, Teucrium chamaedrys L. and T. canadense as determined by analytical reversed-phase HPLC.

P. Ramnathan Sundaresan, Erich Grundel, Kevin D. White, Jeanne I. Rader , Div Research & Appl. Tech., Office of Nutr.Products, Labeling & Dietary Supplements,

CFSAN/FDA, 5100 Paint Branch Parkway, College Park, Maryland, 20740

Neoclerodane diterpenoids are accepted as markers of the Teucrium species which are commonly known as germander. Preparations from germander have been used in traditional folk medicine as stimulants, tonics, diuretics, diaporetics and treatments for asthma and gout. Germander powder was marketed in France and Germany as an adjuvant to weight control. However, in 1992 these preparations were prohibited from sale in France and Germany because of severe hepatotoxicity resulting from their use. Germander has also been identified as an adulterant in several skull cap (Scutellaria lateriflora L.) herbal preparations. In the present report, we have demonstrated by analytical HPLC that teucrin A and teuflin (TC-5) are present in the aerial parts of T.chamaedrys L. in mg/g concentrations. However, teucrin A is absent in the subspecies T. canadense, while TC-5 and teucvidin (TC-6) are present at much higher concentrations. These results indicate that teucrin A alone cannot be used as a marker compound for the genus as a whole. Determination of teucrin A and TC-5, both of which are relatively abundant and at least one of which is present in both Teucrium species, is the preferred method for identifying the presence of germander in herbal preparations.

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Aristolochia Spp: Screening for cytotoxicity and apoptosis induction in vitro

Premalatha Balachandran1, Ikhlas A. Khan12 and David S. Pasco12.

National center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, and 2Department of Pharmacognosy, School of Pharmacy,

The University of Mississippi, MS 38677, USA

Aristolochia species have been administered by those trained in Traditional Chinese medicine for centuries. Consumption of Aristolochia spp. containing the renal toxin, aristolochic acid has been associated with permanent kidney damage, sometimes resulting in kidney failure and the development of certain types of cancers, most often occurring in the urinary tract. In 2001, USFDA issued an alert against the use of Aristolochia spp. for the possible presence of aristolochic acid. To evaluate the in vitro toxic nature of this plant species on proximal tubular cells from pig kidney called LLC-PK1, the unfractionated extracts from 12 different species of Aristolochia were analyzed for their cytotoxic effect using neutral red assay. Out of 55 extracts screened, 36 (65%) showed cytotoxicity (IC50

<300g/ml) in this kidney cell line. On further analysis of these extracts for their apoptosis inducing ability using caspase 3/7 assay, 13 out of 20 extracts (65%) showed statistically significant induction of caspase activity. These in vitro results suggest the potential risk of Aristolochia spp. on kidney cells, which may lead to severe nephrotoxic condition. Further studies on animal models will not only expand our knowledge about the in vivo toxic nature of Aristolochia Spp., but will also aid our ability to understand the kidney diseases caused by these botanicals.

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Botanicals of FDA concern: Screening for cytotoxicity and apoptosis induction in vitro

Premalatha Balachandran1, Ikhlas A. Khan12 and David S. Pasco12.

National center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy and 2Department of Pharmacognosy, School of Pharmacy, The University

of Mississippi, MS 38677, USA

Aristolochic acid found in certain botanicals is toxic to the kidneys and is a potent carcinogen. This chemical can cause serious kidney damage and the use of products that contain aristolochic acid has been associated with several occurrences of kidney failure and has also been linked to increased risk of kidney cancer in people who have consumed it. The FDA is warning consumers against the use of thirteen botanicals that are known or suspected to contain aristolochic acid because they may present a serious health hazard to consumers. 66 extracts from 8 botanicals of FDA concern belonging to different families were studied for the evaluation of cytotoxicity and apoptosis induction. The results of neutral red cytotoxicity assay have indicated that 5 out of 26 Clematis extracts (19.2%), 4 out of 11 Akebia extracts (36.36%), 5 out of 9 Asarum extracts (55.55%), 3 out of 4 Vladimiria extracts (75%), 5 out of 7 Stephania extracts (71.42%), 1 out of 2 Cocculus extracts (50%) and all the six Menispermum extracts (100%) exhibited cytotoxicity against a kidney cell line. Sinomelium was found to be non-toxic. Only one extract from each of Akebia, Menispermum and Clematis showed apoptosis induction when analyzed by Caspase 3/7 assay. These in vitro data suggest that these botanicals may have potential for nephrotoxicity although future studies such as testing in in vivo animal models are required.

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Structure-Antioxidant Activity Relationships of Lignans From Schisandra Chinensis

Young-Whan Choi1,3, Satoshi Takamatsu1, Shabana I. Khan1, Srinivas Pullela1, Daneel Ferreira1,2, Jianping Zhao1,2 and Ikhlas A. Khan1,2*

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, 2Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, MS 38677, USA and 3Department of Horticulture, Miryang National University, Miryang 627-702, Korea.

Phytochemical investigation of the fruits of Schindra chinensis led to the isolation of 13 lignans. One of them was indentified a new homolignan with a dibenzocyclooctadiene skeleton having an exocyclic double bond, schisandrene and its structure and absolute configuration have been established using extensive 1D and 2D NMR techniques including COSY, HMQC, HMBC, NOESY and MS and CD data. Antioxidant activity of lignans were evaluated using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) cellular-based assay. Shisandrene was identified as the predominant antioxidant constituent in the fruits of S. chinensis. The structure-activity relationships of the tested lignans were has demonstrated that the exocyclic methylene group on cyclooctadiene ring of the lignan structure were essential to antioxidant activity and the benzoyloxy group in the cyclooctadiene ring could enhance their antioxidant effect. The isolated compounds did not exhibit any anti-inflammatory, anticancer or cytotoxic activity when tested in a variety of in vitro cell based assays.

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Integrating Biotechnology, Phytochemistry, and Immunopharmacology to Explore Bioactivity of Scutellaria

Joshee, N1, Rimando, A. M2, Parajuli, P3 and Yadav, A. K1.

1Fort Valley State University, Fort Valley, GA 31030; 2USDA-ARS, Natural Products Utilization Research Unit, University, MS 38677 and 3Dept of Neurosurgery, Wayne State University and

Karmanos Cancer Institute, Detroit, MI 48201.

Scutellaria species have been used in the traditional medical systems of China and India and in Korea, Japan, some European nations, and North America. It is used as an anti-inflammatory, abortifacient, antispasmodic, emmenagogue, febrifuge, nervine, and sedative in alternative medicines. At Fort Valley State University (FVSU), we are working on the in vitro plant regeneration, hairyroot culture, and conservation of Scutellaria germplasm in our collection.

Extraction of plant samples (root, shoot, leaf) was performed to carry out phytochemical screening. Air dried Scutellaria samples (10g each tissue), received from FVSU were mixed with 10g purified sand and loaded in the extraction cartridge. The extracts were concentrated in vacuo. Concentrated/dried extract samples were weighed and an aliquot was sent for biological activity screening. Another aliquot was dissolved in 0.5%HCl-methanol, and analyzed by a Hewlett-Packard 1050 HPLC and monitored for baicalin, baicalein and wogonin at 270 nm. The endogenous flavonoids were quantitated from a calibration curve of the standards with 6-hydroxyflavone as an internalstandard. We have been studying the mechanism of induction of apoptosis/necrosis in brain tumor cells by crude Scutellaria extracts as well as by isolated active compounds. We are also studying their effect on the secretion of immuno-modulatory cytokines like IL-6, IL-10 or TGF-b by the tumor cells. Moreover, the presentation of tumor-associated antigens by dendritic cells and subsequent activation of cytotoxic T-cells in presence of Scutellaria extracts are also being studied in an effort to elucidating direct immunomodulatory functions. Significant observations obtained thus far by our team of collaborating researchers will be presented and discussed.

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Pharmacological Effects of Ephedrine Alkaloids on Human 1- and 2 –Adrenoceptors

Guoyi Ma1, Supriya A. Bavadekar1, Ikhlas A. Khan1,2 and Dennis R. Feller. 2

1National Center for Natural Products Research, Departments of Pharmacology and 2Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS.

Ephedra species of plants have both beneficial and adverse effects primarily associated with the presence of ephedrine alkaloids. Few studies have appeared that examine the direct actions of ephedrine alkaloids on human subtypes of adrenergic receptors (AR). Ephedrine alkaloids were evaluated for binding affinities and functional potencies on human 1A-, 1B-, 1D-, 2A-, 2B-, and 2C-AR subtypes expressed in HEK and CHO cells. Cell-based reporter gene assays were used to establish functional activity on 1A-, 2A- and 2C-ARs. These data showed that ephedrine alkaloids did not activate 1- and 2-ARs, and that they antagonized the agonist-mediated effects of phenylephrine and medetomidine on 1- and 2-ARs, respectively. As in the binding studies, 1R, 2S- and 1R, 2R–ephedrine showed greater antagonist activity than the 1S, 2R- or 1S, 2S-isomers.The rank order of affinity for the isomers was: 1R, 2R > 1R, 2S >1S, 2R > 1S, 2S. The rank order of potencies of alkaloids containing a 1R, 2S-configuration was norephedrine > ephedrine > N-methylephedrine. These studies have demonstrated that orientation of the beta-hydroxyl group on the ethylamino side chain and the state of N-methyl substitution are important for -AR binding and functional activity of the ephedrine alkaloids. In conclusion, the ephedrine isomers and analogs studied did not exhibit any direct agonist activity and were found to possess moderate antagonist potencies on cloned human -ARs. The blockade of pre-synaptic 2A- and 2C-ARs may have a pharmacological role in the direct actions of Ephedra alkaloids. Dependent upon the target tissue, the abundance and distribution of -ARs and the dose used, direct and/or indirect actions of Ephedra alkaloids on noradrenergic receptors may be responsible for observed in vivo responses. [Supported by NCCAM grant R21AT00510 and USDA-ARS Cooperative Agreement 58-6408-2-0009].

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Bioassay guided selection of Echinacea pallida var. tennesseensis: Characterization and in vitro storage of active clones

Rita M. Moraes1,, Hemant Lata1, Ana.M.S. Pereira2, Bianca. Bertoni2,Vaishali. C. Joshi1, Nirmal Pugh1, Bladimiro Silva1, Xiuhong Ji1, Ikhlas Khan1,3

and David Pasco1,3

1National Center for Natural Products Research, School of Pharmacy, The University of Mississippi University, MS 38677, USA; 2 Plant Biotechnology Center, Av. Costabile

Romano, 2201,University of Ribeirao Preto, Ribeirao Preto, SP, 14.096-380, Brazil and 3Department of Pharmacognosy, School of Pharmacy, The University of Mississippi,

University, MS 38677, USA

Tissue culture techniques for rapid clonal propagation and in vitro storage were carried out to establish and maintain the medicinal Echinacea germplasm repository. In vitro clones of Echinacea species were obtained from hypocotyls of germinated seeds. Genotypic diversity within varieties and species in the repository was detected by differential immune activation using a sensitive human monocyte assay and later detailed by macroscopic and microscopic examinations followed by molecular characterization. Echinacea pallida var. tenneseensis is the endangered variety within E. pallida species, reasoning for devoting special attention and optimizing storage conditions for its germplasm maintenance. Using AFLP markers the genetic variance within Echinacea pallida var. tenneseensis clonal collection was measured. The AFLP analysis confirmed that different melanin activities detected by the immuno assay was indeed of distinct genotypes. To further investigate maintenance of elite genotypes in storage, shoot cultures were transferred to media containing osmotic agents (sorbitol and mannitol or incubated under low temperature (5 ºC and 10 ºC). Data shows that incubation for 8 months at 5 ºC was highly promising with 100 % survival and minimum growth. After in vitro storage, the elite genotypes presented vigorous re-growth and no mutation was detected during germplasm multiplication and maintenance.

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Cytotoxicity Studies of Triterpenoids Isolated from Akebia trifoliata and Clematis ligusticifolia

Rahul S. Pawar1, Premlata Balachandran1, David Pasco1 and Ikhlas A. Khan1,2

1National Center for Natural Products Research and 2Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, MS

38677, USA

The extracts of two Chinese herbs, namely Clematis ligustifolia and Akebia trifoliata, were tested for their cytotoxic effects on the porcine renal cell line LLC-PK1. Using bioactivity directed fractionation the cytotoxic constituents hederagenin, quinatic acid, arjunolic acid, and 30-nor arjunolic acid along with six glycosides of hederagenin and one glycoside of oleanolic acid were isolated. In order to determine whether these compounds have the ability to induce apoptosis, caspase 3/7 assay was performed. Out of all the triterpenoids isolated, only collinsonidine, quinatic acid and 30-norarjunolic acid exhibited caspase activation indicating their apoptosis inducing nature. The apoptotic activity of these three compounds was further confirmed by microscopical observation of nuclear fragmentation pattern by staining with DAPI. Our findings suggest that the structural features of oleanane skeleton as well as the nature and type of saccharides linkages play an important role in the apoptosis inducing properties of these cytotoxic compounds.

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Bioavailability and Intestinal Transport of Triterpenoids from

Amphiptherygium adstringens

Shabana I. Khan1, Bharathi Avula1, Andrés Navarrete1 and Ikhlas A. Khan1,2

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences and 2Department of Pharmacognosy, School of

Pharmacy, University of Mississippi, MS 38677.

Amphiptherygium adstringens (Anacardiaceae/Julianaceae), commonly known as ‘Cuachalalate’ in Mexico, is used in folk medicine for the treatment of cholelithiasis, fevers, fresh wounds, hypercholesterolaemia, gastritis, gastric ulcers and cancer of the gastrointestinal tract. This study examined the bioavailability of the plant extract and the two major triterpenoids (masticadienonic acid and 3-hydroxymasticadienonic acid) isolated from Amphiptherygium adstringens. The intestinal epithelial transport was determined in Caco-2 model of intestinal absorption. Quantitation was performed by HPLC analysis on a Synergi MAX-RP 80A reversed phase column (Phenomenex, 150 x 4.6 mm I.D.; 4 µm particle size) operated at 40 ºC. The triterpenes were separated with an acetonitrile-water-reagent alcohol isocratic system at a flow rate of 1.0 mL per minute. Cumulative amounts in the receiver side were plotted against time. Apical to basolateral permeability coefficient and percent transport were determined and compared under identical conditions with atenolol. These transport parameters were also compared with reported values for mannitol, propranolol and glucose.

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Bioavailability and Intestinal Transport of Lignans from Schisandra chinensis

Shabana I. Khan1, Bharathi Avula1, Srinivas V. Pullela1, Young-Whan Choi1

and Ikhlas A. Khan1,2

1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences and 2Department of Pharmacognosy, School of

Pharmacy, University of Mississippi, MS 38677

Fruits and seeds of Schisandra chinensis (Schizandraceae) are used in traditional Chinese medicine possessing diverse biological activities. The pharmacological activities, including hepatoprotective, antioxidant, anticancer and anti-HIV have been attributed to the presence of lignans. This study examined the bioavailability of three major lignans (Schisandrin C, Gomisin A and Gomisin N) isolated from S. chinensis. The intestinal epithelial membrane transport across Caco-2 cell monolayers was determined. Quantitation of lignans was performed by an HPLC method using a Phenomenex Luna C18(2) (150 x 4.6 mm, 5 µm) reversed phase column with sodium dihydrogen phosphate-acetonitrile gradient at a flow rate of 1.0 mL per minute. Apical to basolateral permeability coefficient and percent transport were determined and compared under identical conditions with atenolol. Permeability coefficients were also compared with the reported values for mannitol, propranolol and glucose. Sodium fluorescein was used as the marker for paracellular leakage. These compounds, in the concentration range of 50-200 µM, demonstrated substantial linear transport across the monolayer in the apical to basolateral direction, with moderate to high efflux rates and permeability coefficients.

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Blood Pressure Lowering Action of Active Principle from Ocimum basilicum

Khalid Aftab, Ph.D.

Hamdard Institute of Pharmaceutical Sciences, Hamdard University, Islamabad Campus & H.E.J. Research Institute of Chemistry, University of Karachi, Karachi, Pakistan.

Ocimum basilicum belongs to the family labiatae and commonly has known as Basil (Tulsi). It is a widespread plant cultivated in the world. In Indo-China, the ashes of the roots are suggested as a remedy for skin disease. The plant is used as aromatic, anti-microbial, astringent in dysentery, while the leaves are antipyretic. The seeds are laxative, particularly in case of habitual constipation. The juice of the leaves and flowers are a treatment of cough. A decoction may be given after parturition as emmenagogue and febrifuge. The leaves are carminative, antispasmodic and sedative. Preparations of basil are used fir supportive therapy for feeling of fullness and flatulence, for the stimulation of appetite and digestion, and as diuretic.

In anaesthetized rats, methanolic extract, fractions, and pure compound Eugenol (0.3-3.0 mg/Kg) produced dose-dependent fall in blood pressure and heart rate. These effects were not blocked by atropine (1 mg/Kg) and Eugenol did not modify presser response of norepinephrine which rules out the possibility of cholinergic stimulation or -adrenergic blockade. In spontaneously beating atria, Eugenol caused decrease in force and rate of contractions. These effects remain unaltered in presence of atropine. In rabbit aorta, Eugenol caused relaxation of norepinephrine and potassium induced contractions in a concentration-dependent manner. These results suggest that the direct relaxant action of Eugenol on myocardium and on blood vessels may be responsible for its hypotensive and bradycardiac effects observed in the in vivo studies.

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Plants for a Better World

Gustavo [email protected]

Labfarve Inc., Bogota, Colombia.

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