Phytochemical Composition, Antiviral Activity and Toxicity of Dichrocephala Integrifolia Kuntze

101 Pages·2017·1.99 MB·English

PHYTOCHEMICAL COMPOSITION, ANTIVIRAL ACTIVITY AND TOXICITY OF
DICHROCEPHALA INTEGRIFOLIA KUNTZE. CRUDE EXTRACTS

By

Dr. Abdi Hussein Hadun, B Pharm.

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF REQUIREMENTS FOR
MASTERS OF SCIENCE DEGREE IN PHARMACOLOGY AND TOXICOLOGY,
UNIVERSITY OF NAIROBI

2017
DECLARATION
This thesis is my original work and has not been presented in any other institution for
examination or any other purposes.

Signature....................................................... Date…………………………………….

Abdi Hussein Hadun

This thesis has been submitted for examination with our approval as Supervisors

Signature………………………………….. Date…………………………...

Prof. James Mbaria, (BVM ,Msc ,Ph.D)

Department of Public Health Pharmacology and Toxicology, University of Nairobi

Signature………………………………….. Date…………………………...

Dr. Gabriel Oluga Aboge (BVM, Msc , Ph.D.)
Department of Public Health Pharmacology and Toxicology, University of Nairobi

ii
DEDICATION
To my lovely wife Dr. Siraja Ibrahim for her immeasurable love and support, she gave
throughout the period. She has been my inspiration and motivation by continually encouraging
me to work on the project.
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ACKNOWLEDGEMENTS
For most, all praise and thanks be to Allah the Almighty for the wisdom, health, and
perservearnce bestowed on me in my entire life. I would also like to express my sincere gratitude
to my supervisors Prof. James Mbaria and Dr. Gabriel Oluga Aboge for their overwhelming
support, great advice, guidance, mentorship and painstaking proofreading of my drafts. Without
their guidance, this research would not have been possible.

My special and sincere thanks go to the Director of the Center for Traditional Medicine and Drug
Research, KEMRI, for the attachment at the Center and also Mr. Chrispus Ngule for taking me
through the virological techniques during my attachment at the center.

My uttermost appreciation and thanks to Mr. Nderitu and Mr. Maloba for their guidance and
invaluable assistance every time it was required during the experiments. I also thank animal
house attendants in the Department of Public Health, Pharmacology and Toxicology, Mr. Asava
and Mr. Mwakio for availing the mice and always being available to assist at times outside the
normal working hours.
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TABLE OF CONTENTS
Page
DECLARATION........................................................................................................................... ii
DEDICATION.............................................................................................................................. iii
ACKNOWLEDGEMENTS ........................................................................................................ iv
TABLE OF CONTENTS ............................................................................................................. v
LIST OF TABLES ....................................................................................................................... ix
TABLE OF FIGURES ................................................................................................................. xi
LIST OF APPENDICES ............................................................................................................ xii
LIST OF ABBREVIATIONS AND ACRONYMS ................................................................. xiii
ABSTRACT ................................................................................................................................. xv
CHAPTER ONE: INTRODUCTION ......................................................................................... 1
1.1 Background information ........................................................................................................... 1
1.2 Statement of the Problem .......................................................................................................... 3
1.3 Study justification ..................................................................................................................... 4
1.4 Study objectives ........................................................................................................................ 5
1.4.1 General objective ............................................................................................................. 5
1.4.2 Specific objectives ........................................................................................................... 5

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CHAPTER TWO: LITERATURE REVIEW ............................................................................ 6
2.1 Ethnobotany and Ethnopharmacology ...................................................................................... 6
2.2 Traditional Medicine and Medicinal plants .............................................................................. 7
2.2.1 Natural products and drug development ........................................................................ 7
2.2.2 Active principles in medicinal plants ............................................................................. 9
2.3 Chemotherapy of Viral diseases ............................................................................................. 11
2.3.1 Aetiology and Classification ........................................................................................ 11
2.3.2 Herpes Viruses and their pathology ............................................................................. 14
2.3.3 Structure and Life Cycle of Herpes Simplex Virus ...................................................... 15
2.3.4 Epidemiology of HSV infections ................................................................................. 17
2.4 Antiviral chemotherapy .......................................................................................................... 19
2.5. Plants as sources of antiviral medicines................................................................................. 20
2.6 Asteraceae family.................................................................................................................... 23
2.6.1 Taxonomic Classification of D. integrifolia ............................................................... 23
2.6.2 Phytochemical Compounds isolated from D. integrifolia .......................................... 26
2.6.3 Ethnomedicinal information of D. integrifolia ........................................................... 26
2.6.4 Biological activity of D. integrifolia ........................................................................... 27

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CHAPTER THREE: MATERIALS AND METHODS........................................................... 28
3.1 Study design ............................................................................................................................ 28
3.2 Study site ................................................................................................................................. 28
3.3 Medicinal plant collection....................................................................................................... 31
3.4 Preparation of plant parts ........................................................................................................ 31
3.5 Reagents, chemicals, and assay kits ........................................................................................ 31
3.6 Extraction of plant materials ................................................................................................... 32
3.6.1 Aqueous extraction ........................................................................................................ 32
3.6.2 Methanol extraction ....................................................................................................... 32
3.7 Evaluation of Phytochemical constituents of D. integrifolia .................................................. 33
3.7.1 Test for Tannins ............................................................................................................ 33
3.7.2 Test for Saponins .......................................................................................................... 33
3.7.3 Test for Alkaloids ......................................................................................................... 33
3.7.4 Test for Glycosides ....................................................................................................... 34
3.7.4.1 Cardiac glycosides- Keller-killiani test ............................................................ 34
3.7.4.2 Modified Borntrager’s test ............................................................................... 34
3.7.4.3 Keddie test ........................................................................................................ 34
3.7.5 Tests for flavonoids ...................................................................................................... 35
3.7.6 Test of Phenols ............................................................................................................. 35
3.7.7 Test for terpenoids ........................................................................................................ 35
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3.8 In vitro assay ........................................................................................................................... 35
3.8.1 Cell line and culture media ........................................................................................... 35
3.8.2 Cytotoxicity determination on Vero cell lines ............................................................. 36
3.8.3 HSV-1 isolate culture ................................................................................................... 36
3.8.4 Determination of Antiherpetic activity of Dichrocephala integrifolia in Vero cells ... 37
3.8.5 Treatment after virus infection (Post-infection Treatment) ......................................... 37
3.8.6 Treatment before virus infection (Pre-infection treatment) ......................................... 39
3.8.7 Investigation of Virucidal activities ............................................................................. 40
3.9 In-vivo assay............................................................................................................................ 40
3.9.1 Handling of the animals during experimentation and Personal Protective Equipment 40
3.9.2 Animal model ............................................................................................................... 41
3.9.3 Housing and feeding conditions ................................................................................... 41
3.9.4 Determination of in-vivo toxicity of the extract of D. integrifolia using Swiss albino
mice ............................................................................................................................. 41
3.10 Data analysis ......................................................................................................................... 42
3.11 Disposal of cells and experimental animals and Ethical considerations ............................... 43
CHAPTER FOUR: RESULTS .................................................................................................. 44
4.1 Phytochemical composition of different extracts of Dichrocephala integrifolia plant .......... 44
4.2 In vitro cytotoxicity of extract of D.integrifolia against Vero cell lines ................................ 45
viii
4.3 In vitro antiviral activity of extract of Dichrocephala integrifolia against Herpes simplex
virus......................................................................................................................................... 46
4.4 In vitro virucidal activity of extract of D. integrifolia against Herpes simplex virus ............. 47
4.5 In-vivo toxicity of the extract of Dichrocephala integrifolia using Swiss albino mice .......... 48
CHAPTER FIVE: DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS ......... 51
5.1 Discussion ............................................................................................................................... 51
5.2 Conclusions ............................................................................................................................. 53
5.3 Recommendations ................................................................................................................... 53
REFERENCES ............................................................................................................................ 55
APPENDICES ............................................................................................................................. 75

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LIST OF TABLES
Page
Table 1: Number of cases of some viral infections in the world in the year 2010 ...................... 13
Table 2: Cancer and associated viral infections .......................................................................... 13
Table 3: Taxonomic classification .............................................................................................. 24
Table 4: Working estimate of LD for use in Stage 2 of the sequential design derived
50
from mortality in a limit dose test at 2000 mg/kg-bwt ................................................. 43
Table 5: Phytochemical composition of methanolic extracts of Dichrocephala integrifolia
plant ............................................................................................................................ 44
Table 6: Phytochemical composition of aqueous extracts of D. integrifolia plant ..................... 45
Table 7: Cytotoxic effect of D. integrifolia extracts on Vero cells (normal). ............................. 46
Table 8: The IC values of D. integrifolia extracts from pre-treated and post-treated
50
Vero cells .................................................................................................................... 47
Table 9: The potential of various plant parts extracts and Acyclovir (positive control) to prevent
cell damage by HSV virus ............................................................................................ 48
Table 10: Changes in body weight of Swiss albino mice following administration of crude
extracts of D. integrifolia at 300 mg/kg .................................................................... 49
Table 11: Changes in body weight of swiss albino mice following administration of crude
extracts of D. integrifolia at 2000 mg/kg .................................................................. 50

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