Ethnobotany, phytochemistry and DNA metabarcoding studies on Indian traditional medicine Seethapathy Gopalakrishnan Saroja Dissertation presented for the degree of Philoshiae Doctor (PhD) Section for Pharmaceutical Chemistry, Department of Pharmacy & Department of Research and Collections, Natural History Museum The Faculty of Mathematics and Natural Sciences University of Oslo 2019 © Seethapathy Gopalakrishnan Saroja, 2019 Series of dissertations submitted to the Faculty of Mathematics and Natural Sciences, University of Oslo No. 2140 ISSN 1501-7710 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission. Cover: Hanne Baadsgaard Utigard. Print production: Reprosentralen, University of Oslo. Table of contents List of papers……………………………………………………………………………....….1 Summary………………………………………………………………………………………2 Abbreviations………………………………………………………………………………....4 1) Introduction………………………………………………………………………………..6 1.1) Ethnobotany.……………………………………………………………………………...6 1.1.1) Ethnopharmacology and traditional medicine………………………………….8 1.1.2) Dioecy in angiosperms and traditional medicine……………………………….11 1.2) Herbal products and commercialization………………………………………...………..12 1.2.1) Authenticity issues of herbal products……………………………………….. .14 1.2.2) Regulatory status of herbal products…………………………………..……….16 1.2.3) Quality control methods………………………………………………………..17 1.2.3.1) Macroscopic and microscopic authentication………………………..18 1.2.3.2) Chromatographic and spectroscopic methods………………………..19 1.2.3.3) DNA based identification…………………………………………….21 2) Aim of the thesis…………………………………………………………………………..27 3) Materials and methods……………………………………………………………………28 3.1) International legislative framework……………………………………………...28 3.2) Ethnobotanical field studies……………………………………………………...29 3.2) Material collection…………………………………………………………….…29 3.3) Extraction, isolation, and characterization of chemical compounds………….…30 3.4) DNA barcoding methods…………………………………………………….…..31 4) Summary of results………………………………………………………………….……32 5) Discussion………………………………..………………………………………….…….40 6) Concluding remarks and perspectives……………………………………………….…50 Acknowledgements……………………………………………………………………….…52 References……………………………………………………………………………….…..54 Papers I-IV List of papers This thesis is based on the following four Papers, which are referred to in the text by their Roman numerals (I-IV). I. Gopalakrishnan Saroja Seethapathy, Kaliamoorthy Ravikumar, Berit Smestad Paulsen, Hugo J. de Boer and Helle Wangensteen. (2018). Ethnobotany of dioecious species: Traditional knowledge on dioecious plants in India. Journal of Ethnopharmacology, 221: 56-64. doi: 10.1016/j.jep.2018.04.011 II. Gopalakrishnan Saroja Seethapathy, Christian Winther Wold, Kaliamoorthy Ravikumar, Hugo J. de Boer and Helle Wangensteen. (2019). Ethnopharmacology, biological activities, and chemical compounds of Canarium strictum: an important resin-yielding medicinal tree in India. Manuscript. III. Gopalakrishnan Saroja Seethapathy, Margey Tadesse, Santhosh Kumar J. Urumarudappa, Srikanth V. Gunaga, Ramesh Vasudeva, Karl Egil Malterud, Ramanan Uma Shaanker, Hugo J. de Boer, Gudasalamani Ravikanth and Helle Wangensteen. (2018). Authentication of Garcinia fruits and food supplements using DNA barcoding and NMR spectroscopy. Scientific Reports, 8(1):10561. doi: 10.1038/s41598-018-28635-z IV. Gopalakrishnan Saroja Seethapathy, Ancuta-Cristina Raclariu, Jarl Andreas Anmarkrud, Helle Wangensteen and Hugo J. de Boer. (2019). DNA metabarcoding authentication of Ayurvedic herbal products on the European market raises concerns of quality and fidelity. Frontiers in Plant Science, 10(68). doi: 10.3389/fpls.2019.00068 1 Summary Medicinal plants form the basis of traditional medicine health systems and play an important role in meeting primary healthcare needs around the world. At the same time, traditional knowledge on medicinal plants is eroding and deteriorating due to ongoing cultural, ecological and socio- economical changes. This poses a serious threat to biodiversity-based cultural knowledge and can cause an attrition of leads for drug discovery. On the other hand, it is also important to validate traditional knowledge on plant use using scientific methods to promote or discourage their wider usage. Therefore, the first objective of the thesis is to document traditional knowledge on medicinal plants in Southern India, and validate traditional knowledge of selected medicinal plant using phytochemical methods and biological assays. Paper I aimed to document traditional knowledge on dioecious plants of India. Specifically it addressed the research questions: do folk healers have preference for plants of a specific gender? If so, what are those plants? In addition, do folk healers differentially utilize male and female plant of a particular species for food, medicines or timber? The study found that informants recognize the phenomenon of dioecy in plants, and reported gender preferences for a number of species with respect to uses as timber, food and medicine. Paper II aimed to document the medicinal uses of Canarium strictum Roxb. (Burseraceae) by folk healers in India, and to investigate the chemical constituents and biological activities of the resin and stem bark. Our results revealed the presence of α-amyrin and β-amyrin as the major compounds in the resin. Whereas gallic acid, methyl gallate, scopoletin, 3,3'-di-O-methylellagic acid 4-O-α- arabinofuranoside, elephantorrhizol (3,3',4',5,6,7,8-heptahydroxyflavan) and procyanidins were isolated from the bark methanol extract. It is noteworthy that the finding of elephantorrhizol in C. strictum is of chemotaxonomic interest as it is the first report from the family Burseraceae. Furthermore, radical scavenging and 15-lipoxygenase inhibitory activities were tested in resin and bark extracts, but no toxicity towards Artemia salina nauplii was found. Similarly, dose dependent inhibition of NO production was observed in resin and dichloromethane bark extracts. Traditional medicine based herbal products have gained increasing popularity in developing countries as complementary therapies. However, herbal products are prone to contamination, adulteration and substitution, and this raises quality and safety concerns for the public. Therefore, the second part of thesis focused on molecular authentication of raw herbal drugs and marketed herbal products using NMR spectroscopic and DNA methods. The aim of Paper III was to assess 2 the adulteration of morphologically similar samples of Garcinia using DNA barcoding, and to quantify the content of (−)-hydroxycitric acid and (−)-hydroxycitric acid lactone in raw herbal drugs and Garcinia food supplements using NMR spectroscopy. Our DNA barcoding study revealed that there was no adulteration in raw herbal drugs of Garcinia. Whereas, analysis of ten Garcinia food supplements revealed a large variation in the content of (−)-hydroxycitric acid content per capsule or tablet. Paper IV aimed to test the composition and fidelity of Ayurvedic products marketed in Europe using DNA metabarcoding. Our analysis revealed that the fidelity for single ingredient products was 67%, and the overall ingredient fidelity for multi ingredient products was 21%, and for all products 24%. 3 Abbreviations ABS Access and Benefit Sharing ASE Accelerated Solvent Extraction AFLP Amplified Fragment Length Polymorphism API Ayurvedic Pharmacopoeia of India ARMS Amplification Refractory Mutation System CAM Complementary and Alternative Medicine CBD Convention on Biological Diversity CE Capillary Electrophoresis CTAB Cetyl trimethyl ammonium bromide DCM Dichloromethane DNA Deoxyribonucleic acid EFSA European Food Safety Authority EMA European Medicines Agency EtOAc Ethyl acetate EU European Union FDA Food and Drug Administration GC Gas Chromatography H2O Water HPLC High Performance Liquid Chromatography HPTLC High Performance Thin Layer Chromatography HTS High-Throughput Sequencing ITS Internal Transcribed Spacer MeOH Methanol MOTU Molecular Operational Taxonomic Unit MS Mass Spectrometry 4 NIR Near-Infrared Spectroscopy NMR Nuclear Magnetic Resonance PCR Polymerase Chain Reaction RAPD Random Amplified Polymorphic DNA rDNA Ribosomal DNA rbcL Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit RFLP Restriction Fragment Length Polymorphism SCAR Sequence Characterized Amplified Region SSR Simple Sequence Repeat TBGRI Tropical Botanical Garden and Research Institute of India TLC Thin Layer Chromatography TM Traditional Medicine WHO World Health Organization 5 1) Introduction 1.1) Ethnobotany The term Ethnobotany was suggested by John William Harshberger in 1896. Originally it was applied to the study of the utilitarian relationship between humans and the plant environment in primitive settings, and Harshberger (1896) noted that “ethnobotany aids in elucidating the cultural position of the tribes who used the plants for food, shelter or clothing”. However, ethnobotany has been given various definitions over time, and has now evolved into a much broader meaning that covers not only a utilitarian relationship, but also relationships that embrace the symbolic, ecological and cognitive, as well as the human–plant relationship in a modern setting (Soejarto et al., 2005; Leonti, 2011). Ethnobotany was defined by Catherine M Cotton in 1996 as “encompassing all studies which concern the mutual relationships between plants and traditional people” (Cotton, 1996). Albuquerque et al. (2017) defines ethnobotany as “the study of the direct