VALIDATION OF MARKETED MEDICINAL PLANTS OF DISTRICT LAHORE, BASED ON CLASSICAL AND MOLECULAR MARKERS _____________________________________________________________________ SEHRISH RAMZAN ________________________________________________________________________ DEPARTMENT OF BOTANY LAHORE COLLEGE FOR WOMEN UNIVERSITY, LAHORE 2018 VALIDATION OF MARKETED MEDICINAL PLANTS OF DISTRICT LAHORE, BASED ON CLASSICAL AND MOLECULAR MARKERS _____________________________________________________________________ A THESIS SUBMITTED TO LAHORE COLLEGE FOR WOMEN UNIVERSITY IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BOTANY By SEHRISH RAMZAN ________________________________________________________________________ DEPARTMENT OF BOTANY LAHORE COLLEGE FOR WOMEN UNIVERSITY, LAHORE 2018 MY WORK IS DEDICATED TO MY PARENTS, MY HUNSBAND AND KIDS FOR THEIR PRAYERS, LOVE AND ENDLESS SUPPORT AND ENCOURGEMENT ACKNOWLEDGMENTS First of all I bow my head before Almighty Allah, the most Beneficent and the most Merciful, who is the entire and only source of every knowledge and wisdom endowed to mankind and who blessed me with the ability to do this work. It is the blessing of Almighty Allah and His Prophet Hazrat Muhammad (Sallallaho Alaihe Wasallam) which enabled me to achieve this goal. My special gratitude goes to our ever active Vice Chancellor Prof Dr. Farkhanda Manzoor for her facilitations and patronage during the whole course of this research. I am profoundly thankful to Prof. Dr. Farah Khan, Chairperson of Botany Department, for providing the light under which I dare to think of conducting the research and struggle to complete it. I am humbly thank you for affording an opportunity to aspire for the valuable academic degree through this study. I would like to take this opportunity to convey my cordial gratitude and appreciation to my supervisor Dr. Shabnum Shaheen, and co-supervisor Prof. Dr. Farah Khan, for their patience, motivation, enthusiasm, and immense knowledge. I am really indebted to them for their accommodative attitude, thought provoking guidance, immense intellectual input, patience and sympathetic behavior. I am highly obliged to Dr. Khadim Hussain, Assistant Professor in GCU, Faisalabad who provided valuable guidance throughout my research. I am greatly thankful for his extend and endless help, which I took as much as I could without fear of any reluctance. I am thankful to Nazish, Ammara and Kamran of Dept. of Bioinformatics in GCU, Faisalabad for their great cooperation. I offer my great regards to Dr. Mushtaq Ahmed, Associate Professor and Dr. Muhammad Zafar, Herbarium Curator in Department of Plant Sciences, Quaid-i-Azam University, for their sincere cooperation and for providing me access to the reference specimens of under study plants. My sincere thanks also go to my best friends Nidaa, Nadia Khalid, Mehreen Jalal and Hina Afzal for their support, guidance and prayers throughout my studies and research work. Furthermore my heartfelt thanks to my lab fellows Mehwish Jaffer, Sidra Younas and Tahira Illyas for their help and moral sustenance. I am thankful to Sarfaraz, Nadeem, Asif, Tariq, Hassan and Ms. Shaheen, attendants of Dept. of Botany for their cooperation. Last but not least, I am really thankful to my loving, caring and inspiring parents Mr Muhammad Ramzan and Ishrat Ramzan. Thank you both of you for always paving the way to my destinations and for strengthening the resoluteness of my dreams by sacrificing yours. I acknowledge and offer my heartiest gratitude to all my family members especially my sister Faiza for their moral support and cooperation. I also owe a special thanks to my Husband Mr. Waseem Abbas for his encouragement, patience, tolerance and motivation which enabled me to achieve this goal with excellence. His kind involvement gave me the knowledge that will continue to benefit me all my life. I thank him for the kindness towards me. SEHRISH RAMZAN CONTENTS Title Page No. List of Table i List of Figures v List of Plates xiii List of Abbreviations xiv Abstract xv Chapter 1 : Introduction 1.1: Introduction to medicinal plants 1 1.2: Importance of medicinal plants in healthcare 1 system 1.3: Adulteration in medicinal plants – A Burning 3 Issue 1.4: Reasons of adulteration 3 1.4.1: Adulteration caused due to the similar 3 morphology 1.4.2: Adulteration caused due to confusion in 4 Vernacular names 1.4.3: Lack of elementary knowledge about the 5 authentic plant source 1.5: Significance of taxonomic approaches in 5 resolving adulteration issue of medicinal plants 1.5.1: Morphological Analysis 5 1.5.2: Microscopic Analysis 6 1.5.3: Organoleptic Analysis 6 1.5.4: Molecular approaches in Medicinal 7 Plants Identification 1.6: Justification of the Current Research 10 1.7: Aims and objective 11 Chapter 2: Review of Literature 12 2.1. Adulteration; a burning issue 12 2.2. Use of classical taxonomic markers in 13 medicinal plant identification 2.3. Use of classical taxonomic markers in 17 medicinal plant identification Chapter 3: Materials and Methods 24 3.1: Survey of herbal markets and data collection 24 3.2: Collection of herbal and fresh plant samples 24 3.3: Taxonomic investigation 32 3.3.1: Morphological investigation 32 3.3.2: Leaf epidermal anatomical investigation 32 3.3.3: Palynological investigation 33 3.3.4: Light microscopic photography 33 3.3.5: Scanning electron microscopic 33 photographs 3.3.6: Organoleptic analysis 33 3.3.7: Solubility analysis 33 3.4: DNA barcoding 36 3.4.1: Preparation of stock solutions of DNA 36 extraction 3.4.2: Preparation of DNA extraction solutions 37 from the stock solutions 3.4.3: DNA extraction from plant material 39 3.4.4: DNA analysis techniques 39 3.4.5: Amplification of DNA 40 3.4.6: Purification of DNA 42 3.4.7: Application of biostatiscal tools 42 Chapter 4: Results Taxonomy results 44 4.1a: Cinnamomum verum Persl. vs Canella 44 winterana 4.2a: Cinnamomum Tamala (Buch-Ham.) T. Nees & 58 Eberm. Vs Cinnamomum Obtusifolium (Roxb.) Nees 4.3a: Gymnema sylvestre (Retz.) ex Sm. vs Gymnema 71 lactiferum (L.) R.Br. ex Schult 4.4a: Sphaeranthus indicus Linn. vs Sphaeranthus 84 africanus L. 4.5a: Artemisia maritima Linn. vs Artemisia 98 absinthium L 4.6a: Butea monosperma (Lam.) Taub. vs Averrhoa 111 carambolaL. 4.7a: 4.7a: Achillea millefolium L. vs Adhatoda 124 vasica Nees 4.8a: Morus nigraL. vs Morus albaL. 137 DNA Barcoding results 150 4.1b: Cinnamomum verum 156 4.2b: Cinnamomum tamala 149 4.3b: Gymnema sylvestre 161 4.4b: Sphaeranthus indicus 166 4.5b: Artemisia maritima 171 4.6b: Butea monosperma 176 4.7b: Achillea millefolium 182 4.8b: Morus nigra 187 Chapter 5: Discussion 192 5.1: Authentication of selected medicinal plants on 192 the basis of classical and molecular markers 5.1.1: Authentication of Cinnamomum verum 192 vs Canella winterana 5.1.2: Authentication of Cinnamomum 194 tamala vs Cinnamomum obtusifolium 5.1.3: Authentication of Gymnema sylvestre vs 196 Gymnema lactiferum 5.1.4: Authentication of Sphaeranthus indicus 197 vs Sphaeranthus africanus 5.1.5: Authentication of Artemisia maritima vs 199 Artemisia absinthium 5.1.6: Authentication of Butea monosperma vs 201 Averrhoa carambola 5.1.7: Authentication of Achillea millefolium vs 203 Adhatoda vasica 5.1.8: Authentication of Morus nigra vs Morus 204 alba 5.2: Authentication of selected medicinal plants on 206 the basis of advanced markers (DNA barcoding) 5.2.1: Cinnamomum verum (Darchini) 206 5.2.2: Cinnamomum tamala (Tezpath) 207 5.2.3: Gymnema sylvestre (Gurmar 207 booti) 5.2.4: Sphaeranthus indicus (Gul mundi) 208 5.2.5: Artemisia maritime (Afsathine) 208 5.2.6: Butea monosperma (Kamarkus) 209 5.2.7: Achillea millefolium (Branjasaf) 209 5.2.8: Morus nigra (Toth siyah) 210 5.3: Conclusion 211 5.4: Future recommendation 212 References 213 Annexures x vii Plagiarism Report xviii List of Publications and reprints xix i List of Tables Table No. Title Page No. 1.1: Most commonly used DNA markers in plants 8 identification 1.2 Disadvantages of most commonly used DNA markers in 9 plants identification 3.1 Category and ages of informants 24 3.2 Collection sites for selected medicinal plants 30 3.3 Stock solution preparation 36 3.4 2X CTAB buffer (Resuspension buffer) 37 3.5 Preparation of washing buffer 38 3.6 Preparation of TE buffer 38 3.7 Short oligonucleotide primers 40 3.8 PCR reagents for each reaction 42 4.1a Qualitative morphological comparison of Cinnamomum 50 verum and Canella winterana 4.1b Quantitative morphological comparison between 51 Cinnamomum verum and Canella winterana 4.1c Qualitative leaf epidermal anatomical comparison 52 between Cinnamomum verum and Canella winterana 4.1d Quantative leaf epidermal anatomyical comparison 53 between Cinnamomum verum and Canella winterana 4.1e Qualitative and quantitative palynological comparison 54 between Cinnamomum verum and Canella winterana 4.1f Solubility analysis of different samples of Cinnamomum 55 verum in various solvents 4.2a Qualitative morphological comparison between 63 Cinnamomum tamala and Cinnamon obtusifolium 4.2b Quantitative morphological comparison between 64 Cinnamomum tamala and Cinnamon obtusifolium 4.2c Qualitative leaf epidermal anatomical comparison 65 ii between Cinnamomum tamala and Cinnamon obtusifolium 4.2d Quantitative leaf epidermal anatomical comparison 66 between Cinnamomum tamala and Cinnamon obtusifolium 4.e Quantitative and qualitative palynological comparison 67 between Cinnamomum tamala and Cinnamon obtusifolium 4.2f Solubility analysis of different samples of Cinnamomum 68 tamalain various