PHYTOCHEMICAL AND PHARMACOLOGICAL PROFILING OF Dysphania botrys L. BY MUHAMMAD NAEEM KHAN A dissertation submitted to The University of Agriculture Peshawar, in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY AND GENETIC ENGINEERING INSTITUTE OF BIOTECHNOLOGY & GENETIC ENGINEERING FACULTY OF CROP PRODUCTION SCIENCES THE UNIVERSITY OF AGRICULTURE PESHAWAR, PAKISTAN AUGUST, 2018 PHYTOCHEMICAL AND PHARMACOLOGICAL PROFILING OF Dysphania botrys L. BY MUHAMMAD NAEEM KHAN A dissertation submitted to The University of Agriculture Peshawar, in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY AND GENETIC ENGINEERING APPROVED BY: Chairman Supervisory Committee Dr. Asad Jan Associate Professor Co-Supervisor for Research Dr. Inamullah Khan Assistant Professor Pharmacy (UOP) Member Major Field Dr. Safdar Hussain Shah Member Minor Field Prof. Dr. Farhatullah Chairman and Convener Board of Studies Prof. Dr. Iqbal Munir Dean Faculty of Crop Production Sciences Prof. Dr. Zahir Shah Director Advanced Studies and Research Dr. Shahid Sattar INSTITUTE OF BIOTECHNOLOGY & GENETIC ENGINEERING FACULTY OF CROP PRODUCTION SCIENCES THE UNIVERSITY OF AGRICULTURE PESHAWAR, PAKISTAN AUGUST, 2018 TABLE OF CONTENTS S. No. Title Page No. List of Tables .......................................................................................... i List of Figures ....................................................................................... iii Abbreviations ......................................................................................... iv Acknowledgments .................................................................................. vii Abstract .................................................................................................. ix I INTRODUCTION .............................................................................. 1 II. REVIEW OF LITERATURE ............................................................ 13 III. MATERIALS AND METHODS ....................................................... 32 3.1 Plant collection and identification ....................................................... 32 3.2 Extraction of plant material .................................................................. 32 3.3 Fractionation procedure ........................................................................ 33 3.4 Phytochemical investigation ................................................................. 35 3.4.1 Quantitative analysis of phytochmeicals ............................................. 35 3.4.1.1 Stock solution ....................................................................................... 35 3.4.1.2 Test for crude alkaloids......................................................................... 35 3.4.1.3 Test for saponins ................................................................................... 35 3.4.1.4 Test for phenols .................................................................................... 35 3.4.1.5 Test for flavonoids ................................................................................ 35 3.4.1.6 Test for tannins ..................................................................................... 36 3.4.1.7 Test for sterols ...................................................................................... 36 3.4.2 Qualitative analysis of phytochemicals ............................................... 36 3.4.2.1 Determination of total phenol ............................................................... 36 3.4.2.2 Determination of total saponins ............................................................ 36 3.4.2.3 Determination of total flavonoids ......................................................... 37 3.4.2.4 Determination of total alkaloids ........................................................... 37 3.4.3 Proximate composition ........................................................................ 37 3.4.3.1 Moisture content ................................................................................... 37 3.4.3.2 Inorganic matter .................................................................................... 38 3.4.3.3 Crude lipid ............................................................................................ 38 3.4.3.4 Dietary fiber .......................................................................................... 38 3.4.3.5 Crude protein ........................................................................................ 39 3.4.3.6 Nitrogen˗free extract ............................................................................. 39 3.4.4 Minerals analysis .................................................................................. 39 3.5 In-vitro studies ...................................................................................... 41 3.5.1 Antimicrobial activity ........................................................................... 41 3.5.2 Strains and culture media ...................................................................... 41 3.5.3 Antibacterial activity............................................................................. 42 3.5.4 Antifungal activity ................................................................................ 42 3.5.5 Phytotoxic activity ................................................................................ 42 3.5.6 Antioxidant assay .................................................................................. 43 3.5.6.1 1, 1-diphenyl-2-picrylhidrazyl (DPPH)) radical scavenging activity .................................................................................................. ..43 3.5.6.2 ABTS (2, 2˗azinobis˗3-ethylbenzothiozoline-6-sulfonic acid) radical scavenging assay ....................................................................... 43 3.5.7 Lipoxygenase-inhibitory assay (LOX) ................................................. 44 3.6 In-vivo studies ....................................................................................... 44 3.6.1 Acute toxicity study .............................................................................. 44 3.6.2 Anti-inflammatory effect ...................................................................... 45 3.6.2.1 Carrageenan induced paw edema model ............................................. 45 3.6.2.2 Xylene˗induced ear edema.................................................................... 45 3.6.3 Analgesic effect .................................................................................... 46 3.6.3.1 Formalin test ........................................................................................ 46 3.6.3.2 Hot plate test ......................................................................................... 46 3.6.4 Antipyretic effect ................................................................................. 47 3.6.5 Anti-diarrheal effect .............................................................................. 47 3.6.6 Anti-diabetic effect ............................................................................... 47 3.6.7 Hepatoprotective effect ......................................................................... 48 3.6.7.1 Carbon tetra chloride (CCl4) induced hepatotoxicity Model ................ 48 3.6.7.2 Biochemical investigations ................................................................... 48 3.6.8 Sedative/hypnotic effect ...................................................................... 49 3.6.9 Anticonvulsant effect ........................................................................... 49 3.6.10 Antidepressant effect ........................................................................... 49 3.7 Statistical analysis ................................................................................. 50 IV RESULTS ............................................................................................ 51 4.1 Phytochemical investigation ................................................................. 51 4.1.1 Qualitative analysis of phytochemicals ................................................ 51 4.1.2 Quantitative analysis ............................................................................. 51 4.1.2.1 Total phenols......................................................................................... 51 4.1.2.2 Total alkaloids....................................................................................... 52 4.1.2.3 Total saponins ....................................................................................... 53 4.1.2.4 Total flavonoids ................................................................................... 53 4.1.3 Proximate composition ......................................................................... 54 4.1.4 Mineral analysis .................................................................................... 55 4.2 In-vitro activities ................................................................................... 55 4.2.1 Antibacterial activity............................................................................. 55 4.2.2 Antifungal activity ................................................................................ 56 4.2.3 Phytotoxic activity ................................................................................ 57 4.2.4 Antioxidant activity .............................................................................. 58 4.2.4.1 DPPH radical