SCREENING OF SELECTED MEDICINAL PLANTS FOR ANTICANCER AND BIOLOGICAL ACTIVITIES BY KHUSHNOOD UR REHMAN DEPARTMENT OF BOTANY FACULTY OF CHEMICAL AND LIFE SCIENCES ABDUL WALI KHAN UNIVERSITY MARDAN SESSION: 2013-18 SCREENING OF SELECTED MEDICINAL PLANTS FOR ANTICANCER AND BIOLOGICAL ACTIVITIES BY KHUSHNOOD UR REHMAN A Thesis Submitted to the Faculty of Chemical and Life Sciences, Abdul Wali Khan University Mardan in Partial Fulfillment of the Requirement for the Degree of DOCTOR OF PHILOSOPHY IN BOTANY DEPARTMENT OF BOTANY FACULTY OF CHEMICAL AND LIFE SCIENCES ABDUL WALI KHAN UNIVERSITY MARDAN SESSION: 2013-18 میں نے جب لکھنا سیکھا تھا پہلے تیرا نام لِکھا تھا DEDICATION Success always Solicits for two things Exertion and fortune. If I am successful, then my Success is the exertion of my parents and teachers Those they made to fulfill my Wishes in every critical time, Moreover, fortune is due to their prayers. Hence, I dedicate my humble effort, the fruit of my thoughts and study to my teachers and parents. Specially to my supervisor Prof. Dr. Muhammad Hamayun Author’s Declaration I Khushnood Ur Rehman hereby state my PhD Thesis titled ―Screening of Selected Medicinal Plants for Anticancer and Biological Activities‖ is my own work and has not been submitted previously by me for taking any degree from this university ―Abdul Wali Khan University Mardan‖ or anywhere else in country/world. At any time, if my statement is found to be incorrect even after my Graduate the University has the right to withdraw my PhD degree. Signature__________ Khushnood Ur Rehman Date June 20, 2019 ACKNOWLEDGEMENT The first and most important acknowledgement is to almighty Allah Who enabled me to complete this tiresome job. In the beginning, it appeared to me a hard nut to crack but Allah, appeared in grace and led me to the assigned destination. The author is highly indebted to his supervisor Professor Dr. Muhammad Hamayun Chairman Department of Botany AWKUM who is not less than a hero to me, for his invaluable assistance and guidance. While feeling the sin qua non I consulted the following personalities and I am obliged for their proud and great education and due to this persuasion, my task become possible. The generously given moral support by my co supervisor Prof. Dr. Sumera Afzal khan, Director Center of Biotechnology, UOP In a one way or another is a source of inspiration for me. I have no words to thank her, but for her immense help I am very thankful. I owe a very special debt to Prof. Dr. Sadiq Azam, Prof Dr. Gul jan, Prof Dr. Muhib shah, Prof. Dr. Humaira, Dr. M. Irshad, Dr. Abid Ali khan, Dr. Imtiaz ahmad, Prof. Dr. Sher Wali and Prof. Dr. Barakat Ullah for their immense help to complete this task. Prof. Dr. Amjad Iqbal, Prof. Dr. Muhammad Ibrar and Prof. Dr. Zahid Ali Butt are those shining stars who enlightened my way to the target and troubled their selves much for my favor. I am greatly obliged for the AWKUM office and laboratory staff, for their immense help during my research. At the last but not the least, I acknowledge all my teachers who continuously inspired me for the research work. Khushnood Ur Rehman TABLE OF CONTENTS LIST OF TABLES VI LIST OF FIGURES VII LIST OF ABBREVIATIONS X CHAPTER 1 INTRODUCTION 2 1.0 Orientation of the study 2 1.1 Medicinal Plants 3 1.2 Medicinal Plants as a Safe Source of Medicines 5 1.3 Uses of Medicinal Plants 6 1.4 Medicinal Plants of Pakistan 8 1.5 Medicinal Plants and Modern Medicine 9 1.6 Cancer and Medicinal Plants 13 1.7 Phytochemistry of Medicinal Plants 17 1.8 Phytochemical Constituents of Phytomedicine 19 CHAPTER 2 MATERIALS AND METHODS 33 2.1 Collections and Drying of Selected Plants Materials 33 2.2 Extraction and Fractionation 33 2.3 Fractionation of Dried Filtrate on the Basis of Solubility 34 2.4 Antifungal Activity 34 2.4.1 Media preparation for fungal growth 34 2.4.2 Antibacterial Activity 35 2.4.3 Microorganisms Used 35 2.4.4 Media for Bacterial Culture 35 2.4.5 Preparation of Inoculum 35 2.4.6 Preparation of Agar Plates 35 2.4.7 Extract Preparation for Activity 35 2.4.8 Incubation and Measurement of Zone of Inhibition 36 2.5 Phytotoxicity Activity 36 2.5.1 Bioassay 36 2.6 Anticancer Activity 36 2.6.1 Principle of MTT Assay 36 2.6.2 Media Preparation 37 2.6.3 Defrosting Cells 37 2.6.4 Trypsinisation of Cells 37 2.6.5 Freezing Cells 38 2.6.6 Cell Counting 38 2.6.7 Preparing MTT Solution: 38 2.6.8 Dilution of Plant Extracts 38 2.6.9 MTT Assay Methodology 38 2.6.10 Statistical analysis 39 2.7. Antioxidant Assays 39 2.7.1 DPPH free radicals scavenging activity 39 2.8 Heavy Metals Analysis 39 2.8.1 Acid Digestion of Samples 39 2.9 Proximate Analysis 40 2.9.1 Determination of Moisture 40 2.9.2 Determination of Ash 40 2.9.3 Determination of Curd Fat 40 2.9.4 Determination of Protein 41 i. Digestion 41 ii. Distillation 41 iii. Titration 41 2.9.5 Determination of Carbohydrate 42 2.10 Phytochemical Analysis of Plant Material 42 2.10.1 Test for Alkaloids (Mayer’s reagent) 42 2.10.2 Test for Flavonoids 42 2.10.3 Test for Saponins 43 2.10.4 Test for Glycosides 43 2.10.5 Test for Phenols 43 2.11 Isolation of Compounds 43 2.11.1 Methodology of Column Chromatography 43 2.11.2 Thin layer chromatography 44 2.11.3 Spotting and developing the TLC plate 44 2.12 Identification and Spectral Analysis of Compounds 45 2.12.1 Nuclear Magnetic Resonance Spectroscopy (NMR) Analysis 45 2.13 Anticancer Activity of Pure Isolated Compounds 45 2.13.1 Chemicals and reagents 45 2.13.2 Cell culture 45 2.13.3 MTT assay 46 2.13.4 Measurement of ROS generation 46 2.13.5 Annexin-V-FITC apoptosis detection assay 46 2.13.6 Caspase-3 activity assay 46 2.13.7 Western immunoblotting 46 CHAPTER 3 RESULTS 48 3.1 Antibacterial Activity 48 3.3 Phytotoxicity of Selected Medicinal Plants 80 3.4 Heavy Metal Analysis 87 3.5 Antioxidant Activity 91 3.6 Proximate analysis 95 3.7 Phytochemical Analysis of Selected Medicinal Plants 101 3.8 Anticancer Activity 102 3.9 Results of Isolated Compound 108 3.10 Anticancer Activity of Isolated Compound 113 3.10.1 Adlumidine Reduce the Cell Viability of HCT 116 And PC3 Cells 113 CHAPTER 4 DISCUSSION 118 4.1 Antifungal Activity 118 4.2 Anti-Bacterial Activities 120 4.3. Heavy Metals in Medicinal Plants 124 4.5 Proximate Analysis of Selected Medicinal Plants 127 4.6 Phytotoxicity of Medicinal Plants 129 4.7 Antioxidant Activity 131 4.8 Phytochemical Analysis of Selected Medicinal Plants 133 4.9 Anticancer Activity 135 4.10 Isolation of Compound 139 CONCLUSION 141 FUTURE PROSPECTS 142 REFERENCES 143 LIST OF TABLES Table 3. 1 Antibacterial potential of the crude extract of A. lebbeck 49 Table 3. 2 Antibacterial Potential of the Crude Extract of M. azedarach 51 Table 3. 3 Antibacterial Potential of the Crude Extract of Hedera helix 53 Table 3. 4 Antibacterial Potential of the Crude Extract of S. flagellaris 55 Table 3. 5 Antibacterial Potential of the Crude Extract of V. jatamansi 57 Table 3. 6 Antibacterial Potential of the Crude Extract of F. cretica 59 Table 3. 7 Antibacterial Potential of the Crude Extract of W. coagulans 61 Table 3. 8 Antibacterial Potential of the Crude Extract of M. oleifera 63 Table 3. 9 Antifungal Potential of the Crude Extract of A. lebbeck 65 Table 3. 10 Antifungal Potential of the Crude Extract of M. azedarach 67 Table 3. 11 Antifungal Potential of the Crude Extract of H. helix 69 Table 3. 12 Antifungal Potential of the Crude Extract of S. flagellaris 71 Table 3. 13 Antifungal Potential of the Crude Extract of V. jatamansi 73 Table 3. 14 Antifungal Potential of the Crude Extract of F. cretica 75 Table 3. 15 Antifungal Potential of the Crude Extract of W. coagulans 77 Table 3. 16 Antifungal potential of the crude extract of M. oleifera 79 Table 3. 17 Phytotoxicity of Selected Medicinal Plants 82 Table 3. 18 Heavy Metal Concentration of Selected Medicinal Plants 87 Table 3. 19 In vitro Antioxidant Activity of Different Extracts of Selected Medicinal Plants 92 Table 3. 20 Proximate Analysis of Different Parts of Selected Medicinal Plants all Values are Mean ± Sem of Three Values. 96 Table 3. 21 Phytochemical Analysis of Selected Medicinal Plants 102 Table 3. 22 Anticancer Activity of Different Fractions of Selected Medicinal Plants At 25 µm 103 Table 3. 23 Anticancer Activity of Different Fractions of Selected Medicinal Plants at 50 μM Concentrations 104 Table 3. 24 Anticancer Activity of Different Fractions of Selected Medicinal Plants at 100 μM Concentrations 106 LIST OF FIGURES Figure 1 Moringa oleifera Lam. 23 Figure 2 Albizia lebbeck (L.) Benth. 24 Figure 3 Withania coagulans (Stocks) Dunal 25 Figure 4 Melia azedarach L. 26 Figure 5 Hedera helix auct. non Linn 28 Figure 6 Fagonia cretica L. (Virgin's Mantle) 29 Figure 7 Valeriana jatamansi Jones 30 Figure 8 Saxifraga flagellaris Willdenow in C. M. von Sternberg 31 Figure 9 Antibacterial Activity of Albizia lebbeck 49 Figure 10 Antibacterial Activity of Melia azedarach 51 Figure 11 Antibacterial Activity of Hedera helix 53 Figure 12 Antibacterial Activity of Saxifraga flagellaris 55 Figure 13 Antibacterial Activity of Valeriana jatamansi 57 Figure 14 Antibacterial Activity of Fagonia cretica 59 Figure 15 Antibacterial Activity of Withania coagulans 61 Figure 16 Antibacterial Activity of Moringa oleifera 63 Figure 17 Antifungal Activity of Albizia lebbeck 65 Figure 18 Antifungal Activity of Melia azedarach 67 Figure 19 Antifungal Activity of Hedera helix 69 Figure 20 Antifungal Activity of Saxifraga flagellaris 71 Figure 21 Antifungal Activity of Valeriana jatamansi 73 Figure 22 Antifungal Activity of Fagonia cretica 75 Figure 23 Antifungal Activity of Withania coagulans 77 Figure 24 Antifungal Activity of Moringa oleifera 79 Figure 25 Phytotoxicity of 10/20/40 mg against Radicle of L.