Anticarcinogenic And Antioxidant Activities Of Methanolic Extract And Lophirones B And C Derived From Lophira Alata Stem Bark
Anticarcinogenic and Antioxidant Activities of Methanolic Extract and Lophirones B and C Derived from Lophira alata Stem Bark
BY
Ajiboye, Taofeek Olakunle
B.Sc., M.Sc. (Ilorin)
Matric No: 02/55EH191
A thesis submitted to the Department of Biochemistry, Faculty of Science, University of Ilorin, Ilorin, Nigeria,
In fulfillment of the requirements for the award of Doctor of Philosophy, Ph.D. in Biochemistry
September, 2012
CERTIFICATION
We certify that this work was carried out by Mr. Ajiboye, Taofeek Olakunle in the Department of Biochemistry, University of Ilorin, Ilorin, Nigeria under our supervision.
__________________________ _________________
Yakubu, M. T., Ph.D. Date
(Supervisor)
__________________________ _________________
Prof. A. T. Oladiji Date
(Co-Supervisor)
APPROVAL PAGE
This thesis has been read and approved as having met the requirements of the Department of Biochemistry and the Postgraduate School, University of Ilorin, Ilorin, Nigeria, for the award of the degree of Doctor of Philosophy (Ph.D.) in Biochemistry.
__________________________ _________________
Yakubu, M. T., Ph.D. Date
(Supervisor)
__________________________ _________________
Prof. A. T. Oladiji Date
(Co-Supervisor)
__________________________ _________________
Yakubu, M. T., Ph.D. Date
(Acting Head of Department)
__________________________ _________________
External Examiner Date
DEDICATION
This thesis is dedicated
to my parents, Mr. and Mrs. S.B Ajiboye
through whom I am made what I am today.
To my siblings,
Olasunkanmi, Omowunmi, Oluwabukola and Olamilekan
for their love, care and encouragement
To my fiancée,
Motajo, Kaliyat Yetunde
for her moral support and understanding
that saw me through the successful completion of this research.
ACKNOWLEDGEMENTS
All praises and honours are due to Allah, the Almighty who through His infinite mercies saw me through this programme in good health, wisdom and accident free.
The past few years that I have spent at the University of Ilorin have been challenging and rewarding. As my Ph.D. programme comes to an end, I would like to thank several people that have made this research possible and my time here in Ilorin truly special.
My unreserved and profound gratitude goes to my supervisor, Dr. M. T. Yakubu for creating time out of your busy schedules as the Sub-Dean (Student Affairs) to supervise this work thoroughly. Your valuable suggestions and constructive criticism eased the successful completion of this research. Sir, I am really grateful for mentoring me.
Also to my co-supervisor, Prof. A. T Oladiji, who despite her tight and busy schedule took time to go through my work at different stages of the research. Her suggestions and encouragement immensely contributed to the success of this work.
I wish to acknowledge the staff of the Department of Biochemistry, starting from the Ag. Head of Department, Dr. M. T. Yakubu, the lecturers and the technologist, who have impacted on me academically and morally. I appreciate you all and pray Almighty Allah reward you abundantly as what you have impacted on me is been appreciated by the society. Thank you all.
I want to specially appreciate my Vice-Chancellor, Professor O. B Oloyede and all the staff of the Department of Chemical Sciences, Fountain University, Osogbo for their various encouragement and assistance throughout the course of this programme. I thank you all.
I cannot forget to appreciate the Special Consultant, Professor of Medicinal Plant Research and Traditional Medicine Department of National Institute for Pharmaceutical Research and Development, Abuja, Professor J.I Okogun who equipped me with the necessary resources and skills needed in isolation of natural product. He is a great man I will ever be grateful to God I came across. Also in the same Department is Dr. Iliya, a Natural Product Scientist who consolidated the foundation laid by Professor J.I Okogun in me. The skill of preparatory thin layer chromatography and running of column chromatography were acquired from him. I am grateful sir.
The assistance rendered by Mr. J. Nvau towards obtaining the 1H and 13C NMR data for the isolated compounds at National Institute of Health, Bethesda, United States is appreciated.
I wish to appreciate the special assistance of Mr. R. A. Lawal, who engineered the evaluation of my extract for the cytotoxicity at the Molecular Biology Laboratory of the Department of Biology, Faculty of Arts and Science, University of Gaziantep, Gaziantep, Turkey.
I am also indebted to my friends: Kehinde Olorunimbe, Rolake Popoola, Tosin, Okemute Okpalafe, Wunmi, Folake, Maryham, Martin Alabi, Rapheal Adeoye among others. I really appreciate your friendship support all through the time of my study. Thanks a lot, I appreciate you all.
I wish to recognise all the students of Biochemistry and Nutrition Unit, Department of Chemical Sciences, Fountain University, Osogbo for their encourgament and prayers during this programme.
Special appreciation goes to my fiancée, the love of my life for her moral support during this programme. ‘Sugar”, you are the best.
My utmost appreciation and profound gratitude goes to my family most especially my Mum. Their encouragement, parayers and support were never found wanting during this research. I appreciate you all and will always do.
Ajiboye, T. O.
September, 2012
TABLE OF CONTENTS
Title page i
Certifcation page ii
Approval page iii
Dedication iv
Acknowledgements v
Table of contents viii
List of figures xix
List of tables xxiv
List of plates xxvii
Abstract xxxii
Chapter One
Introduction 1
Chemical carcinogenesis 4
Classification of chemical carcinogens 4
Metabolic activation of chemical carcinogens 6
Oxidative stress associated carcinogenesis 8
Metabolism of reactive oxygen species 8
Oxidative effects of reactive oxygen species on cellular and
biological molecules 12
Antioxidant defense mechanism in oxidative stress associated carcinogenesis 17
Reactive oxygen species detoxifying enzymes 17
Non-enzymic antioxidant 22
Role of redox transcription factor, nuclear erythroid related factor-2
in oxidative stress associated carcinogenesis 27
Mechanism of induction of antioxidant responsive elements
regulated genes by nuclear erythroid related factor-2 31
Nuclear erythroid related factor-2 regulated genes 31
Aflatoxin B1 hepatocarcinogenesis 34
Chemistry and occurrence of aflatoxin B1 34
Metabolism of aflatoxin B1 37
Reactive oxygen species and oxidative stress associated
aflatoxin B1 hepatocarcinogenesis 39
Role of flavonoids in oxidative stress associated carcinogenesis 40
Flavonoids 40
Chalcones 42
Medicinal plants 44
Lophira alata 44
Statement of research problem and justification of the study 47
Aims 48
Objectives 48
Chapter Two
Materials and methods 50
Materials 50
Plant material and authentication 50
Ehrlich ascite carcinoma cells 50
Experimental animals 50
Chemical and solvents 51
Assay kits 51
Antibodies 51
Methods 52
Isolation of lophirones B and C from Lophira alata stem bark 52
2.2.1.1 Preparation of the extract 52
2.2.1.2 Thin layer chromatography 52
2.2.1.2 Dry column chromatography 52
Cytotoxic study of methanolic extract and lophirones B and C from Lophira alata stem bark against Ehrlich ascites carcinoma cells 55
In vitro antioxidant study of the methanolic extract of Lophira alata
stem bark, lophirones B and C 57
2, 2 diphenyl-1-picrylhydrazyl scavenging assay 57
Superoxide anion radical scavenging assay 59
Hydrogen peroxide scavenging assay 60
Hydroxyl radical scavenging assay 61
Reducing power assay 62
Nuclear erythroid related factor-2 study 63
Dose dependent study 63
Time dependent study 64
Cytoprotective enzymes induction study 64
Methanolic stem bark extract of Lophira alata 64
Lophirones B and C 64
Aflatoxin B1 induced hepatocarcinogenesis study 65
Preparation of serum, tissue homogenates and microsomes 65
Determination of protein concentration 67
Western blot analysis 68
Enzyme assay 72
Antioxidant enzymes 72
Superoxide dismutase 72
Catalase 74
Glutathione peroxidase 75
Glutathione reductase 77
Determination of electrophilic species detoxifying enzymes 79
Glutathione S- transferase 79
Epoxide hydrolase 80
Quinone oxidoreductase-1 82
Uridyl glucuronosyl transferase 83
Hepatic marker enzymes 85
Alkaline phosphatase 85
Alanine aminotransferase 87
Aspartate aminotransferase 89
Antioxidant proteins 90
Reduced glutathione 90
Oxidised glutathione 91
Thiol proteins 92
Lipid peroxidation 93
Malondialdehyde 93
Lipid hydroperoxides 94
Conjugated dienes 96
Protein oxidation 98
Protein carbonyl 98
DNA fragmentation 99
Hepatic and kidney function indices 101
Total protein concentration 101
Albumin 102
Globulin 103
Urea 103
Creatinine 104
Lipid profile 106
Total cholesterol 106
Triglycerides 108
High density lipoprotein 110
Low density lipoprotein 111
Haematological parameters 112
Statistical analysis 112
Chapter Three
Results 113
Isolation of lophirones B and C 113
Cytotoxic effects of methanolic stem bark extract of Lophira
alata, lophirones B and C on Ehrlich ascites carcinoma 119
Cytotoxic effects of methanolic stem bark extract of Lophira
alata on Ehrlich ascites carcinoma 119
Cytotoxic effects of lophirones B and C on Ehrlich ascites
carcinoma 119
In vitro electrophilic, free radical and reactive oxygen species
scavenging effects of methanolic extract and chalcone dimers
(lophirones B and C) derived from Lophira alata stem bark 137
Scavenging effect of methanolic extract and chalcone dimers
(lophirones B and C) derived from Lophira alata stem bark on
2, 2 diphenyl-1-picrylhydrazyl radicals 137
Scavenging effect of methanolic extract and chalcone dimers
(lophirones B and C) derived from Lophira alata stem bark on
superoxide anion radical 137
Scavenging effect of methanolic extract and chalcone dimers
(lophirones B and C) derived from Lophira alata stem bark on
hydrogen peroxide 140
Scavenging effect of methanolic extract and chalcone dimers
(lophirones B and C) derived from Lophira alata stem bark on
hydroxyl radical 140
Reducing effect of methanolic extract and chalcone dimers
(lophirones B and C) derived from Lophira alata stem bark on 143
In vivo electrophilic and reactive oxygen species detoxifying potentials
of Lophira alata 143
Antioxidant enzymes 143
Phase II detoxifying enzymes 143
Effects of lophirones B and C on redox transcription factor,
nuclear erythroid related factor-2 and its inhibitor, Kelch ECH
associating protein-1 149
Effects of lophirones B and C on redox transcription factor, nuclear
erythroid related factor-2 149
Effects of lophirones B and C on redox transcription factor, nuclear
erythroid related factor-2 inhibitor, Kelch ECH associating protein-1 149
In vivo electrophilic and reactive oxygen species detoxification
effects of lophirones B and C 157
In vivo electrophilic detoxification effects of lophirones B and C 157
In vivo reactive oxygen species detoxification effects of lophirones B
and C 157
Effects of lophirones B and C on aflatoxin B1 carcinogenesis induced
redox status perturbation 169
Reactive oxygen species detoxifying enzymes 169
Non-enzyme reactive oxygen species detoxifying proteins 169
Lipid peroxidation 172
Protein oxidation 172
DNA fragmentation 172
Effect of lophirones B and C on electrophilic species detoxifying
enzymes and proteins of aflatoxin B1 treated rats 176
Aflatoxin aldehyde reductase 176
Glutathione S-transferase 176
NADH: Quinone oxidoreductase-1, epoxide hydrolase and uridyl
glucuronosyltransferase 182
Effects of lophirones B and C on hepatocellular damaging activity
of aflatoxin B1 182
Hepatic marker enzymes 182
Effects of lophirones B and C on some selected liver and kidney
function indices of aflatoxin B1-treated rats 189
Effects of lophirones B and C on lipid profile of aflatoxin B1-treated
rats 189
Effects of lophirones B and C on selected hematological parameters
of aflatoxin B1-treated rats 193
Chapter Four
Discussion 195
Isolation of lophirones B and C 195
Cytotoxic activity of methanolic extract and chalcone dimers
(lophirones B and C) derived from Lophira alata stem bark 196
In vitro free radicals and reactive oxygen species scavenging
activities of methanolic extract and chalcone dimers (lophirones B
and C) derived from Lophira alata stem bark 196
Effect of lophirones B and C on redox transcription factor, nuclear erythroid related factor-2 and its regulator, Kelch ECH associating protein-1 198
Nuclear erythroid related factor-2 198
Kelch ECH associating protein-1 199
Effects of methanolic extract of L. alata and lophirones B and C on electrophilic and reactive oxygen species detoxifying enzymes 200
Reactive oxygen species detoxifying enzymes 200
Electrophilic species detoxifying enzymes 201
Effects of lophirones B and C on redox and electrophilic status in
AFB1 hepatocarcinogenesis 204
Reactive oxygen species detoxifying enzymes 204
Non-enzymic antioxidant proteins 206
Electrophilic species detoxifying enzymes 207
Lipid peroxidation 209
Protein oxidation 210
DNA fragmentation 211
Effects of lophirones B and C on aflatoxin B1-induced hepatocellular damage 212
Hepatic damage maker enzymes 212
Liver and kidney function indices 213
Effects of lophirones B and C on aflatoxin B1-induced lipid profile perturbation 215
Effects of lophirones B and C on aflatoxin B1-induced hematological disturbances 216
Summary
Conclusion
Recommendations
Suggestions
References
Appendix A
Appendix B
Appendix C
LIST OF FIGURES
Figure Title Page
1 Multistage carcinogenesis 2
2 Metabolic activation pathways of chemical carcinogens 7
3 Role of glutathione in oxidation of protein sulphydryl groups 26
4 Activation of antioxidant responsive element by Nrf-2 inducers 29
5 Structures of naturally occurring aflatoxin 36
6 Metabolic pathway of aflatoxin B1 in the liver 38
7 Reactive oxygen species production during AFB1 –
hepatocarcinogenesis 41
8 Structure of chalcone 43
9 Schematic representation of the isolation procedure of
lophirones B and C 56
10 Hydrolysis of cholesterol ester by cholesterol esterase 107
11 Structure of lophirone B 117
12 Structure of lophirone C 118
13 Effect of methanolic extract and lophirones B and C derived
from Lophira alata stem bark on DPPH radical 138
14 Effect of methanolic extract and lophirones B and C derived
from Lophira alata stem bark on superoxide anion radical 139
15 Effect of methanolic extract and lophirones B and C derived
from Lophira alata stem bark on hydrogen peroxide 141
16 Scavenging effect of methanolic extract and lophirones B and
C derived from Lophira alata stem bark on hydroxyl radical 142
17 Reducing power of methanolic extract and lophirones B and C
derived from Lophira alata stem bark on K3Fe(CN)6 144
18 Nrf-2 expression following daily administration of lophirones
B and C for 48 hours 154
19 Time dependent Nrf-2 expression following daily administration
of lophirones B and C for 48 hours 155
20 Keap-1 expression following daily administration of lophirones
B and C for 48 hours 156
21 GST- expression following daily administration of 20 mg/kg
body weight of lophirones B and C for 28 days 158
22 GST- expression following daily administration of 20 mg/kg
body weight of lophirones B and C for 28 days 159
23 GST- expression following daily administration of 20 mg/kg
body weight of lophirones B and C for 28 days 160
24 Specific activity of GST in liver of rats treated with 20 mg/kg
body weight lophirones B and C once daily for 28 days 161
25 NQO-1 expression following daily administration of 20 mg/kg
body weight of lophirones B and C for 28 days 162
26 Specific activity of NQO-1 in liver of rats treated with 20 mg/kg
body weight lophirones B and C once daily for 28 days 163
27 Epoxide hydrolase expression following daily administration of
20 mg/kg body weight of lophirones B and C for 28 days 164
28 Specific activity of epoxide hydrolase activity in liver of rats
treated with 20 mg/kg body weight lophirones B and C once
daily for 28 days 165
29 UGT 1A1 expression following daily administration of 20 mg/kg
body weight of lophirones B and C for 28 days 166
30 Specific activity of UGT in liver of rats treated with 20 mg/kg
body weight lophirones B and C once daily for 28 days 167
31 Aflatoxin aldehyde reductase expression following daily treatment
of rat with 20 mg/kg body weight of lophirones B and C for 6
weeks and 20 µg/day of aflatoxin B1 for 3 weeks 177
32 Glutathione S-transferase- expression following daily treatment
of rat with 20 mg/kg body weight of lophirones B and C for 6
weeks and 20 µg/day of aflatoxin B1 for 3 weeks 178
33 Glutathione S-transferase- expression following daily treatment
of rat with 20 mg/kg body weight of lophirones B and C for 6
weeks and 20 µg/day of aflatoxin B1 for 3 weeks 179
34 Glutathione S-transferase- expression following daily treatment
of rat with 20 mg/kg body weight of lophirones B and C for 6
weeks and 20 µg/day of aflatoxin B1 for 3 weeks 180
35 Specific activity of GST in liver of rats treated with 20 mg/kg
body weight of lophirones B and C for 6 weeks and 20 µg/day
of aflatoxin B1 for 3 weeks 181
36 NADH: Quinone oxidoreductase-1 expression following
daily treatment of rat with 20 mg/kg body weight of lophirones
B and C for 6 weeks and 20 µg/day of aflatoxin B1 for 3 weeks 183
37 Specific activity of NADH: Quinone oxidoreductase-1 in liver of
rats treated with 20 mg/kg body weight of lophirones B and C for
6 weeks and 20 µg/day of aflatoxin B1 for 3 weeks 184
38 Epoxide hydrolase expression following daily treatment of rat
with 20 mg/kg body weight of lophirones B and C for 6 weeks
and 20 µg/day of aflatoxin B1 for 3 weeks 185
39 Specific activity of epoxide hydrolase in liver of rats treated with
20 mg/kg body weight of lophirones B and C for six weeks and
20 µg/day of aflatoxin B1 for 3 weeks 186
40 Uridyl glucuronosyl transferase 1A1 expression following
daily treatment of rat with 20 mg/kg body weight of lophirones B
and C for 6 weeks and 20 µg/day of aflatoxin B1 for 3 weeks 187
41 Specific activity of uridyl glucuronosyl transferase in liver of
rats treated with 20 mg/kg body weight of lophirones B and C
for 6 weeks and 20 µg/day of aflatoxin B1 for 3 weeks 188
42 Proposed mechanism of action of lophirones B and C as
anticancer agent 218
43 Calibration curve for protein concentration 254
44 Calibration curve for superoxide dismutase 255
45 Calibration curve for reduced glutathione concentration 256
46 Calibration curve for oxidised glutathione concentration 257
LIST OF TABLES
Tables Title Pages
Environmental and pharmaceutical carcinogens that can
induce oxidative stress 10
Retention factor values for the spots on thin layer chromatogram
of isolated compounds from Lophira alata stem bark 115
3 1H and 13C NMR data of lophirones B and C 116
4 Cytotoxic activity of methanolic stem bark extract of Lophira
alata on Ehrlich ascites carcinoma cells 120
5 Cytotoxic activity of methanolic stem bark extract of lophirones
B and C on Ehrlich ascites carcinoma cells 126
6 Specific activity of superoxide dismutase in the liver of rats
administered methanolic stem bark extract of Lophira alata 145
7 Specific activity of catalase in the liver of rats administered
methanolic stem bark extract of Lophira alata 146
8 Specific activity of glutathione peroxidase in the liver of rats
adminsitered methanolic stem bark extract of Lophira alata 147
9 Specific activity of glutathione reductase in the liver of rats
administered methanolic stem bark extract of Lophira alata 148
10 Specific activity of NADPH:Quinone oxidoreductase in the liver
of rats administered methanolic stem bark extract of Lophira alata 150
11 Specific activity of glutathione S transferase in the liver of
rats administered methanolic stem bark extract of Lophira alata 151
12 Specific activity of uridyl diphosphoglucuronysl transferase in
the liver of rats administered of methanolic stem bark extract
of Lophira alata 152
13 Specific activity of epoxide hydrolase activity in the liver of rats
administered of methanolic stem bark extract of Lophira alata 153
14 Specific activities of antioxidant enzymes following administration
of lophirone B and C for 28 days 168
15 Effects of lophirones B and C on the hepatic antioxidant enzymes
of aflatoxin B1-treated rats 170
16 Effects of lophirones B and C on the hepatic non-enzymic
antioxidant proteins of aflatoxin B1-treated rats 171
17 Effects of lophirones B and C on the hepatic lipid peroxidized
products of aflatoxin B1-treated rats 173
18 Effects of lophirones B and C on the hepatic protein carbonyl
levels of aflatoxin B1-treated rats 174
19 Effects of lophirones B and C on the fragmented DNA in the liver
of aflatoxin B1-treated rat 175
20 Effects of lophirones B and C on the hepatic marker enzymes of
aflatoxin B1-treated rats 190
21 Effects of lophirones B and C on some liver and kidney function
indices of aflatoxin B1-treated rats 191
22 Effects of lophirones B and C on the lipid profile of aflatoxin
B1-treated rats 192
23 Effects of lophirones B and C on the haematological parameters
of aflatoxin B1-treated rats 194
24 Protocol for the determination of protein calibration curve 258
25 Dilution factors for the liver homogenates, microsomes and
serum 259
LIST OF PLATES
Tables Title Pages
1 Lophira alata plant 46
2 Dry column chromatography 54
3 TLC Chromatogram of the fractionated Lophira alata stem bark 114
4 Cytotoxic effect of methanolic stem bark extract of Lophira alata
(0.1 g) on Ehrlich ascites carcinoma cells for 3 hours 121
5 Cytotoxic effect of methanolic stem bark extract of Lophira alata
(1 g) on Ehrlich ascites carcinoma cells for 3 hours 121
6 Cytotoxic effect of methanolic stem bark extract of Lophira alata
(10 g) on Ehrlich ascite carcinoma cells for 3 hours 122
7 Cytotoxic effect of methanolic stem bark extract of Lophira alata
(100 g) on Ehrlich ascite carcinoma cells for 3 hours 122
8 Cytotoxic effect of methanolic stem bark extract of Lophira alata
(1000 g) on Ehrlich ascite carcinoma cells for 3 hours 123
9 Cytotoxic effect of methanolic stem bark extract of Lophira alata
(0.1 g) on Ehrlich ascite carcinoma cells for 24 hours 123
10 Cytotoxic effect of methanolic stem bark extract of Lophira alata
(1 g) on Ehrlich ascite carcinoma cells for 24 hours 124
11 Cytotoxic effect of methanolic stem bark extract of Lophira alata
(10 g) on Ehrlich ascite carcinoma cells for 24 hours 124
12 Cytotoxic effect of methanolic stem bark extract of Lophira alata
(100 g) on Ehrlich ascite carcinoma cells for 24 hours 125
13 Cytotoxic effect of methanolic stem bark extract of Lophira alata
(1000 g) on Ehrlich ascite carcinoma cells for 24 hours 125
14 Cytotoxic effect of lophirone B (0.1 g) on Ehrlich ascite
carcinoma cells for 3 hours 127
15 Cytotoxic effect of lophirone B (1 g) against Ehrlich ascite
carcinoma cells for 3 hours 127
16 Cytotoxic effect of lophirone B (10 g) on Ehrlich ascite
carcinoma cells for 3 hours 128
17 Cytotoxic effect of lophirone B (100 g) on Ehrlich ascite
carcinoma cells for 3 hours 128
18 Cytotoxic effect of lophirone B (1000 g) on Ehrlich ascite
carcinoma cells for 3 hours 129
19 Cytotoxic effect of lophirone C (0.1 g) on Ehrlich ascite
carcinoma cells for 3 hours 129
20 Cytotoxic effect of lophirone C (1 g) on Ehrlich ascite
carcinoma cells for 3 hours 130
21 Cytotoxic effect of lophirone C (10 g) on Ehrlich ascite
carcinoma cells for 3 hours 130
22 Cytotoxic effect of lophirone C (100 g) on Ehrlich ascite
carcinoma cells for 3 hours 131
23 Cytotoxic effect of lophirone C (1000 g) on Ehrlich ascite
carcinoma cells for 3 hours 131
24 Cytotoxic effect of lophirone B (0.1 g) on Ehrlich ascite
carcinoma cells for 24 hours 132
25 Cytotoxic effect of lophirone B (1 g) on Ehrlich ascite
carcinoma cells for 24 hours 132
26 Cytotoxic effect of lophirone B (10 g) on Ehrlich ascite
carcinoma cells for 24 hours 133
27 Cytotoxic effect of lophirone B (100 g) on Ehrlich ascite
carcinoma cells for 24 hours 133
28 Cytotoxic effect of lophirone B (1000 g) on Ehrlich ascite
carcinoma cells for 24 hours 134
29 Cytotoxic effect of lophirone C (0.1 g) on Ehrlich ascite
carcinoma cells for 24 hours 134
30 Cytotoxic effect of lophirone C (1 g) on Ehrlich ascite
carcinoma cells for 24 hours 135
31 Cytotoxic effect of lophirone C (10 g) on Ehrlich ascite
carcinoma cells for 24 hours 135
32 Cytotoxic effect of lophirone C (100 g) on Ehrlich ascite
carcinoma cells for 24 hours 136
33 Cytotoxic effect of lophirone C (1000 g) on Ehrlich ascite
carcinoma cells for 24 hours 136
34 Nrf-2 expression following daily administration of lophirones
B and C for 48 hours 154
35 Time dependent Nrf-2 expression following administration of
20 mg/kg body weight of lophirones B and C for 48 hours 155
36 Keap-1 expression following administration of 20 mg/kg body
weight of lophirones B and C for 48 hours 156
37 GST- expression following daily administration of 20 mg/kg
body weight of lophirones B and C for 28 days 158
38 GST- expression following daily administration of 20 mg/kg
body weight of lophirones B and C for 28 days 159
39 GST- expression following daily administration of 20 mg/kg
body weight of lophirones B and C for 28 days 160
40 NQO1 expression following daily administration of 20 mg/kg
body weight of lophirones B and C for 28 days 162
41 Epoxide hydrolase expression following daily administration of 20
mg/kg body weight of lophirones B and C for 28 days 164
42 UGT 1A1 expression following daily administration of 20 mg/kg
body weight of lophirones B and C for 28 days 166
43 Aflatoxin aldehyde reductase expression in AFB1, lophirones
B and C treated rats 177
44 Glutathione S-tranferase- expression in AFB1, lophirones
B and C treated rats 178
45 Glutathione S-transferase- expression in AFB1, lophirones
B and C treated rats 179
46 Glutathione S-transferase- expression in AFB1, lophirones
B and C treated rats 180
47 NADH: Quinone oxidoreductase-1 expression in AFB1,
lophirones B and C treated rats 183
48 Epoxide hydrolase expression in AFB1, lophirones B and C
treated rats 185
49 Uridyl glucuronosyl transferase 1A1 expression in AFB1,
lophirones B and C treated rats 187
ABSTRACT
This study evaluates the anticarcinogenic and antioxidant activities of methanolic extract as well as lophirones B and C derived from Lophira alata stem bark. The chalcones dimers (lophirones B and C) were isolated from Lophira alata by the combination of solvent-solvent extraction and dry column chromatographic techniques. The isolated compounds were further subjected to 1H and 13C Nuclear Magnetic Resonance (NMR) analysis. Cytotoxicity of the methanolic extract and the chalcone dimers at the doses of 0.1 – 1000 g/mL were evaluated against Ehrlich ascites carcinoma cells. In vitro antioxidant activity was assessed using models such as 2, 2-diphenyl-1-picrylhydrazyl (DPPH), superoxide anion radical, hydrogen peroxide, hydroxyl radicals and iron (II) ion (Fe2+) reducing power. The in vivo antioxidant activity of methanolic extract and the chalcone dimers was investigated in rats by assaying the levels of antioxidant enymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GSH-Red) in a 28 days study. Also, the effects of methanolic extract and lophirones B and C at the dose of 5, 10 and 20 mg/kg body weight on redox transcription factors and cytoprtotective enzymes [nicotinamide adenine dinucleotide reduced: quinone oxidoreductase-1 (NQO-1), epoxide hydrolase (EpH), glutathione-S-transferase (GST), and uridyl glucuronosyl transferase (UGT)] were evaluated in male rats. In addition, the effects of lophirones B and C on the redox perturbation of aflatoxin B1 (AFB1) -induced hepatocarcinogenesis was investigated for six weeks in rats by assaying the levels of the reactive oxygen species detoxifying enzymes, electrophilic detoxifying enzymes, oxidative stress biomarkers [malondialdehyde (MDA), lipid hydroperoxide, conjugated dienes, protein carbonyl, fragmented deoxyribonucleic acid (DNA)], lipid profile and haematological parameters and hepatic and renal indices. The data generated were subjected to analysis of variance followed by Tukey-Kramer test to account for significant (P<0.05) differences between the variables. Isolation of lophirones B and C was achieved with dry column chromatography, while the NMR produced data similar to previously isolated lophirones B and C. The methanolic extract and lophirones B and C at the doses investigated significantly (P<0.05) reduced the viability of Erhlich ascites carcinoma cells. The extract and the chalcone dimers dose-dependently scavenged DPPH radical, superoxide anioin radical, hydrogen peroxide, hydroxyl radicals, as well as reduced ferric ion in the potassium hexacyanoferrate III reducing system. While lophirones B and C significantly (P<0.05) reduced the expression of keap-1, it significantly (P<0.05) enhanced the expression of nuclear erythroid related factor- 2 (Nrf-2). Also, the methanolic extract and lophirones B and C significantly (P<0.05) increased the expression and specific activity of the cytoprotective enzymes (GST, NQO-1, EpH and UGT). There was a significant (P<0.05) reduction in the level of antioxidant system in aflatoxin B1-induced hepatocarcinogenesis. Furthermore, lophirones B and C significantly (P<0.05) attenuated the aflatoxin B1-mediated decrease in the specific activity of the reactive oxygen species detoxifying enzymes. The oxidative stress biomarkers, malondialdehyde, lipid hydroperoxides, conjugated dienes, protein carbonyl, fragmented DNA were significantly (P<0.05) increased in aflatoxin B1 hepatocarcinogenesis in rats. Although lophirones B and C did not significantly (P<0.05) alter the oxidative biomarkers, the chalcone dimers significantly attenuated the AFB1-mediated increase in the biomarkers of oxidative stress. AFB1 significantly (P<0.05) decreased the hepatic marker enzymes (alkaline phosphatase and alanine and aspartate aminotransferase) in the liver with corresponding increase in the serum. Also, hepatic and renal indices (total protein, albumin, globulin, urea and creatinine) were significantly (P<0.05) increased by AFB1 hepatocarcinogenesis. The alterations were significantly attenuated by lophirones B and C. Also, AFB1 hepatocarcinogenesis caused a significant (P<0.05) alteration in the lipid profile and haematological parameters of rats. These alterations were significantly (P<0.05) reversed by lophirones B and C. Results obtained show that the methanolic extract, lophirones B and C derived from Lophira alata stem bark posses anticancer properties, as it is evident from the cytotoxicity against Ehrlich carcinoma cells and restoration of normal redox homeostasis in AFB1–induced hepatocarcinogenesis. The in vitro antioxidants activity, in vivo antioxidant activity, induction of Nrf-2 as well as the inhibition of keap-1 proffer the possible mechanism of action of lophirones B and C. This study thus justifies the acclaimed use of Lophira alata in the management of tumour in folk medicine of some African countries including Nigeria.
CHAPTER ONE
INTRODUCTION
Fertility is the state of being fertile; capable of producing offspring. It is the ratio of live births in an area to the population of that area; expressed per 1000 population per year.
Reproduction, a fundamental feature of all known life is a process by which organisms replicate themselves. It is a biological process by which new individual organisms – "offspring" – are produced from their "parents". There are two forms of reproduction: sexual and asexual. While asexual reproduction is a process by which organisms create genetically similar or identical copies of themselves without the contribution of genetic material from another organism, sexual reproduction occurs when a new individual is formed by the union of two sex cells, or gametes, a term that includes sperm and eggs (or ova).
The reproductive rate of human race throughout the world is a direct reflection on the population growth which is complemented with the advancement of modern medical science that has ensued declined infant mortality rate but increased birth rate with improved life average life expectancy. Progressive increase in birth rate as well as gradual decline in death rate has caused a huge population burst in the world (Amit et al., 2011). In Nigeria, the relatively high birth rate in which has been accompanied by steady declines in death rates has resulted in high rates of population growth (Ebigbola and Ogunjuyigbe, 1998).
This growing and increasingly affluent world population is increasing demand for and pressure on vital natural resources and services whereas population growth has been one of the biggest problems facing developing and developed countries lately with its inevitable consequences on all aspects of development, especially employment, education, housing, health care, sanitation and environment (Ghulam et al., 2009).
It is apparent that the increase in the world population has become a serious and threatening issue in this century owing to the exponential growth in the world population as 75 million more people are added annually to the world population that is over seven billion as at 2010. It was estimated that the world population will double by 2050 (United Nations Population Division, 2002; UNDESAPD, 2010).
With this exponential increase of world population, it becomes pertinent that fertility control is an issue of global and national public health concern. Even though methods to proffer solutions to the high human number has surface, current methods of contraception still result in an unacceptable rate of unintended pregnancies, significant numbers of failure in the usage and are seriously associated with one side effect or the other. In recent years, there have been concerns about the use of plant products in affecting fertility of humans with the prospect that these potential damages of long term use of hormones of fertility control to be curtailed by use of nonsteroidal plant compounds.
It therefore becomes absolutely important to explore botanicals with antifertility activity for their fertility regulation potentials as well as identify and characterize the active principle(s) to be a useful guide towards the formulation of cheaper, affordable, easily available antifertility agents and contraceptives (spermicidal, anti-ovulatory, anti-implantation, or abortifacient) that are nontoxic even on prolonged use. Accordingly, the continuous search for drug of plant origin possessing the aforementioned attributes necessitated the choice of Spondias mombin, a plant claimed in traditional medicine to possess antifertility actions.
1.1 Problem Statement
Birth and death are the two ends of life; even though man cannot control death but to some extent can control birth. The advance in medicine and science has decreased infant mortality where by birthrate is on the increase. This progressive increase in birth rate as well as gradual decline in death rate cause a huge population burst in the world (by 2050 the world’s population is projected to increase from 7 billion in 2011 to 9.6 billion; in the same period the population living in urban areas will grow from 3.6 billion to 6.3 billion) (UN Population Division, 2013). In Nigeria, there is a report for an estimated doubling period of less than 25 years at the current rate of population growth of about 2.87 percent and an average total fertility rate of 6.0 lifetime births per women (Oyedokun, 2007). With this progressive increase in birth rate and population (which has been a major cause of concern for population experts and policy makers); consequences on all aspects of development, especially employment, education, housing, health care, sanitation and environment (Ghulam et al., 2009) etc. are inevitable; there is therefore a critical need of regulation of births so as to forms the main basis of the various population control and family welfare programmes.
1.2 Aims and Objectives
The overall objective of the present study will be to isolate and characterize the male and female fertility regulating agent(s) from ethanolic extracts of Spondias mombin leaves using guinea pigs.
1.2.1 Specific Objectives
The specific objectives of this study include:
To chemically analyze the leaves of the plant and extract for: amino acid components, mineral constituents and secondary metabolites (Qualitative and quantitative);
To investigate the ethanolic extract of Spondias mombin leaves for its acclaimed antifertility potential in male and female guinea pigs using anti-ovulation, contraception, anti-implantation, estrogenicity/antiestrogenicity, abortifacient, androgenic and anti-spermicidal models;
To isolate and characterize the active agent(s) of anti-ovulation, contraception, anti-implantation, estrogenicity/antiestrogenicity, abortifacient, androgenic and anti-spermicidal activities.
1.3 Significance of the Study
In the attempt to control population outburst and accordingly preventing conception, scientists have made attempts both on male and female counterparts. Efforts on finding spermicidal agents for male contraception are being made but on the female side; since conception consist of different stages (ovulation, fertilization of the ovum, implantation of the fertilized ovum and ultimate maturation of the foetus to term which makes them more vulnerable to drug action), there has been attempts to barricade fertilization directed towards these stages by various agents claimed to be anti-ovulatory, anti-implantation, or abortifacient. Till date, steroidal pills and injections, intra uterine devices (IUDs), barrier methods, sterilization devices and oral agents (most popular and acceptable due to their simplicity of application) are available means of contraception; but all these means has been named with one/more side effects like thromboembolic manifestation, hypertension, liver disease, uterine and breast cancer as well as gastrointestinal problems, severe and painful uterine contractions, systemic illness, permanent sterility or even death (Yakubu et al., 2010). Moreover, studies have reported significant numbers of failure in the usage of these modern methods/steroidal pills (Trusell et al., 1998; Kost et al., 2008). These potential damages of long term use of hormones may be curtailed by use of nonsteroidal plant compounds. Therefore, the screening of plants with antifertility activity and the subsequent identification and characterization of the active principle(s) to be a useful guide towards the formulation of cheaper, affordable, easily available antifertility agents and contraceptives (spermicidal, anti-ovulatory, anti-implantation, or abortifacient) that are nontoxic even on prolonged use. Accordingly, the continuous search for drug of plant origin possessing the aforementioned attributes necessitated the choice of Spondias mombin, a plant claimed in traditional medicine to possess antifertility actions.
CHAPTER TWO
LITERATURE REVIEW
2.1 Male Reproductive System
The structures of the human male reproductive system, typical of mammals, are illustrated in Figure 1. The reproductive role of the male is to produce and deliver sperm to impregnate the female. To carry out these functions, a male has internal and external sexual organs. These structures include the testes, several tubules that carry sperm out of the testes, various glands, and the penis. In most mammalian species, including human, the male's external reproductive organs are the scrotum and penis. The internal reproductive organs consist of gonads that produce gametes (sperm cells) and hormones, accessory glands that secrete products essential to sperm movement, and ducts that carry out the sperm and glandular secretions (Campbell and Reece, 2005). Inside the testis is a network of fine-diameter tubes called seminiferous tubules. Sertoli cells, nourish, support, and protect developing germ cells, which undergo cell division by meiosis to form spermatozoa (immature sperm). Prostate secretions are rich in zinc, citric acid, antibiotic like molecules, and enzymes important for sperm function. During sexual excitation, the bulbourethral glands produce a droplet of alkaline fluid that neutralizes residual urine in the urethra, protecting the sperm from its acidity (Robinson, 2001). Table 1 summarizes the function of the male reproductive system.
Fig. 1: Organization of the human male reproductive system.The penis and scrotum are the external genitalia, the testes are the gonads, and the other organs are sex accessory organs, aiding the production and ejaculation of semen
Source:
2.1.1 Hormonal Regulation in Males: Hormonal Control of Testes
The anterior pituitary gland secretes two gonadotropic hormones: FSH and LH. Although these hormones are named for their actions in the female, they are also involved in regulating male reproductive function (McPhaul and Young, 2001). In males, FSH stimulates the Sertoli cells to facilitate sperm development, and LH stimulates the Leydig cells to secrete testosterone. The principle of negative feedback inhibition applies to the control of FSH and LH secretion (Figure 2). The hypothalamic hormone, gonadotropin releasing hormone (GnRH), stimulates the anterior pituitary to secrete both FSH and LH (McPhaul and Young, 2001). FSH causes the Sertoli cells to release a peptide hormone called inhibin that specifically inhibits FSH secretion. Similarly, LH stimulates testosterone secretion, and testosterone feeds back to inhibit the release of LH, both directly at the anterior pituitary gland and indirectly by reducing GnRH release. The importance of negative feedback inhibition can be demonstrated by removing the testes; in the absence of testosterone and inhibin, the secretion of FSH and LH from the anterior pituitary is greatly increased (McPhaul and Young, 2001).
Fig. 2: Hormonal interactions between the testes and anterior pituitary. LH stimulates the Leydig cells to secrete testosterone, and FSH stimulates the Sertoli cells of the seminiferous tubules to secrete inhibin. Testosterone and inhibin, in turn, exert negative feedback inhibition on the secretion of LH and FSH, respectively
Source: McPhaul and Young (2001)
2.1.2 Male Reproductive Hormones
Hormones are chemical substances synthesized in small amounts by an endocrine tissue and carried in the blood to another tissue where it acts as a messenger to regulate the function of the target tissue or organ (Nelson and Cox, 2005). Cells respond to hormone when they express a specific receptor for that hormone. The hormone binds to the receptor protein, resulting in the activation of a signal transduction mechanism that ultimately leads to cell type-specific responses. Their interference with the synthesis, secretion, transport, binding, action, or elimination of natural hormones in the body can change the homeostasis, reproduction, development, and/or behaviour, just as endogenously produced hormones do (Crisp et al., 1998).
2.1.2.1 Testosterone
Testosterone is a steroid hormone from the androgen group. It is found in mammals, reptiles (Cox and John-Alder, 2005), birds (Reed et al., 2006), and other vertebrates. In mammals, testosterone is primarily secreted in the testicles of males and the ovaries of females, although small amounts are also secreted by the adrenal glands. It is the principal male sex hormone and an anabolic steroid.
2.1.2.1.1 Distribution of testosterone
On average, in adult human males, the plasma concentration of testosterone is about 7–8 times as great as the concentration in adult human females' plasma, (Torjesen and Sandnes, 2004) but as the metabolic consumption of testosterone in males is greater; the daily production is about 20 times greater in men (Dabbs and Dabbs, 2000).About 97-99% of the circulating testosterone in plasma is bound to globulin (GSBG) of sex steroid binding globulin (SSBG), and 33 % to albumin, while only a small fraction of the hormone is circulating in the free (biologically active) form (Murray et al.,2000).
2.1.2.1.2 Mechanism of action of testosterone
The effects of testosterone in humans and other vertebrates occur by way of two main mechanisms: by activation of the androgen receptor (directly or as DHT), and by conversion to estradiol and activation of certain estrogen receptors (Mooradian et al., 1987). Free testosterone (T) is transported into the cytoplasm of target tissue cells, where it can bind to the androgen receptor, or can be reduced to 5α-dihydrotestosterone (DHT) by the cytoplasmic enzyme 5-alpha reductase. DHT binds to the same androgen receptor even more strongly than testosterone, so that its androgenic potency is about 2.5 times that of T (Breiner et al., 1996). The T-receptor or DHT-receptor complex undergoes a structural change that allows it to move into the cell nucleus and bind directly to specific nucleotide sequences of the chromosomal DNA. The areas of binding are called hormone response elements (HREs), and influence transcriptional activity of certain genes, producing the androgen effects. Androgen receptors occur in many different vertebrate body system tissues, and both males and females respond similarly to similar levels.
2.1.2.1.3 Biochemical roles of testosterone
Testosterone is necessary for normal sperm development. It activates genes in Sertoli cells, which promote differentiation of spermatogonia, regulates acute (Hypothalamic–pituitary–adrenal axis) HPA response under dominance challenge (Mehta et al., 2008), cognitive and physical energy, the population of thromboxane A2 receptors on megakaryocytes and platelets and hence platelet aggregation in humans (Ajayi and Halushka, 2005), and maintains muscle trophism. High androgen levels are associated with menstrual cycle irregularities in both clinical populations and healthy women (Van Anders and Watson, 2006).
2.1.2.2 Follicle stimulating hormone (FSH)
Follicle-stimulating hormone (FSH) is a hormone found in humans and other animals. It is synthesized and secreted by gonadotrophs of the anterior pituitary gland. Like other glycoproteins, FSH consists of alpha and beta subunits (Radu et al., 2010).
2.1.2.2.1 Distribution of follicle stimulating hormone
The gene for the alpha subunit is located on chromosome 6p21 and expressed in different cell types. The gene for the beta subunit is located on chromosome 11p13, and is expressed in gonadotropes of the pituitary cells, controlled by Gonadotropin-releasing hormone (GnRH), inhibited by inhibin, and activated by activin (Radu et al., 2010).
2.1.2.2.2 Mechanism of action of follicle stimulating hormone
Follicle-stimulating hormone (FSH) plays a key role in male and female reproduction. In females, it regulates ovarian follicular growth and development through FSH receptors on granulosa cell membrane surface. This signal is transmitted through several intracellular mechanisms like the activation of cAMP/protein kinase A (PKA)- dependent, mitogen-activated protein kinase (MAPK)- dependent and other signalling pathways. Transcription factor CREB (cAMP response element binding protein) is one of their substrates. CREB regulates expression of some genes related to proliferation and steroidogenesis of granulosa cells (GC). In males, FSH induces Sertoli cells to secrete inhibin and stimulates the formation of sertoli-sertoli tight junctions (zonula occludens). FSH enhances the production of androgen-binding protein by the Sertoli cells of the testes by binding to FSH receptors on their basolateral membranes, and is critical for the initiation of spermatogenesis (Boulpaep and Boron, 2005).
2.1.2.2.3 Biochemical roles of follicle stimulating hormone
FSH regulates the development, growth, pubertal maturation, and reproductive processes of the human body. In both males and females, FSH stimulates the maturation of germ cells. Administration of FSH to humans and animals induces ‘superovulation’, or development of more than the usual number of mature follicles, hence increases the number of mature gamates. FSH is also critical for sperm production. It supports the function of Sertoli cells, which in turn support many aspects of sperm cell maturation (Radu et al., 2010).
2.1.2.3 Luteinizing hormone (LH)
Luteinizing hormone (LH), also known as lutropin and sometimes lutrophin is a hormone produced by gonadotroph cells in the anterior pituitary gland. It is a transmembrane receptor found in ovary, testis and extragonodal organs like the uterus. The activation of LH is necessary for the hormonal functioning during reproduction (Rousseau-Merck et al., 1991). In females, an acute rise of LH (LH surge) triggers ovulation and development of the corpus luteum. In males, LH has been called interstitial cell-stimulating hormone (ICSH), because it stimulates Leydig cell for the production of testosterone (Louvet et al., 1975). It acts synergistically with FSH.
2.1.2.3.1 Distribution of luteinizing hormone
There are two kinds of LH and hCG-like immunopositive cells. The LH and hCG-like immunopositive cells existed in early gonads (ovaries and testis) in protochordata. Positive substances are distributed in the cytoplasm, nucleoplasm and nucleolar membrane of oogonia as well as early spermatogenic cells in testis (Kaplan and Pesce, 1996).
2.1.2.3.2 Mechanism of action of luteinizing hormone
The mechanism of action of Luteinizing hormone is involved in the interaction with its receptor in the Leydig cells. This interaction initiates a signal through the cyclic adenosine monophosphate pathway via guanosine triphosphate (GTP) binding proteins. Signal transduction occurs through the proteinkinase A (PKA) pahway. Intracellular calcium concentration induced by the action of luteinizing hormone involves activation of phospholipases in the lipoxygenase pathway (Cooke, 1990). This process results in stimulation of the side-chain cleavage enzyme with the subsequent release of testosterone (Payne et al., 1996).
2.1.2.2.3 Biochemical roles of luteinising hormone
Luteinising hormone (LH) is a type of glycoprotein that is produced in the anterior pituitary via gonadotroph cells and regulates the function of the gonads. In males, LH stimulates the production and secretion of testosterone from the testes via Leydig cells. In both sexes, LH stimulates secretion of sex steroids from the gonads.
2.1.3 Male Organs Studied
2.1.3.1 Penis
The penis is the male external excretory and sex organ. The penis contains the external opening of the urethra, which is used for urination and to deliver semen into the vagina of a female sexual partner. Erectile tissue inside the penis allows the penis to increase in size and become rigid during sexual stimulation. A penis' erection helps to deliver semen deeper into the female reproductive tract during sexual intercourse.
The penis is located in the pubic region superior to the scrotum and inferior to the umbilicus along the body’s midline.
2.1.3.1.1 Anatomy of the Penis
The penis is an external genital organ. The distal end of the penis is called the glans penis and is covered with a fold of skin called the prepuce or foreskin. Within the penis are masses of erectile tissue. Each consists of a framework of smooth muscle and connective tissue that contains blood sinuses, which are large, irregular vascular channels.
It is made up of 3 major regions: the root, body, and glans.
1. The root of the penis connects the penis to the bones of the pelvis via several tough ligaments.
2. Roughly cylindrical in shape, the body of the penis is the largest region. Large masses of erectile tissue in the body allow this region to harden and expand greatly during sexual stimulation.
3. The glans is the enlarged tip of the penis that contains the urethral orifice where semen and urine exit the body. Erectile tissue in the glans causes this region to harden and expand in width during sexual stimulation.
The penis is an organ made of several distinct tissue layers. The outside of the penis is covered with skin that is continuous with the skin of the surrounding pubic region. Many sensory receptors in the penis' skin allow it to receive sensory stimulation during sexual intercourse. Deep to the skin of the penis is a layer of subcutaneous tissue containing blood vessels and protein fibers that loosely anchor the skin to the underlying tissue. Under the subcutaneous tissue is a tough and elastic layer of fibrous connective tissue known as the tunica albuginea. The tunica albuginea plays an important role by providing strength and support to the penis when it becomes erect. Inside the tunica albuginea are three masses of erectile tissue: the two corpora cavernosa and corpus spongiosum. The corpora cavernosa (singular: corpus cavernosum) fill the left and right dorsal regions of the penile body, while the corpus spongiosum surrounds the urethra on the ventral side of the body and in the glans. These regions of erectile tissue fill with blood to harden and enlarge the penis during times of sexual excitement.
2.1.3.1.2 Functions of the Penis
The penis functions as both a reproductive organ and an excretory organ. As a reproductive organ, the penis becomes erect during sexual intercourse in order to deliver semen more effectively into the vagina. Semen travels through the urethra to the tip of the penis where it is ejaculated out of the body.
As an excretory organ, the penis delivers urine out of the body through the urethra.
Fig. 12:
Source: Monga (1999)
2.1.3.2 Testes
The testes (singular, testis) are located in the scrotum (a sac of skin between the upper thighs). In the male fetus, the testes develop near the kidneys, then descend into the scrotum just before birth. Each testis is about 1 1/2 inches long by 1 inch wide. Testosterone is produced in the testes which stimulates the production of sperm as well as give secondary sex characteristics beginning at puberty.
2.1.3.2.1 Structure and Anatomy of the Testes
Located in the hollow sac of the scrotum, each testis is about 1.5 to 2 inches long along its long axis and around 1 inch in diameter
The testes are connected to the vital organs of the ventral body cavity via the spermatic cords. Nerves, blood vessels, and lymphatic vessels travel through the spermatic cords to support the testes. The vas deferens also passes through the spermatic cord carrying sperm out of the testes toward the prostate and urethra. The cremaster muscle wraps around the exterior of the spermatic cord to lift the testes closer to the body or permit them to descend.
The testes are wrapped by the tunica vaginalis, an extension of the peritoneum of the abdomen, and the tunica albuginea, a tough, protective sheath of dense irregular connective tissue. Each testis is divided by invaginations of the tunica albuginea that divide it into several hundred small segments called lobules. Each lobule contains several tightly coiled tubes called seminiferous tubules.
The walls of the seminiferous tubules contain the germ cells, Sertoli cells, and Leydig cells that give the testes their function. Millions of germ cell in the walls of the seminiferous tubules multiply and differentiate to produce spermatocytes from the onset of puberty until death. The spermatocytes develop into spermatids and eventually spermatozoa, or sperm cells. The immature sperm cells are supported and protected by Sertoli cells as they travel the length of the seminiferous tubules and slowly mature. Leydig cells at the ends of the seminiferous tubules produce the male hormone testosterone that produces the secondary sex characteristics associated with males.
Each sperm produced by the testes takes about seventy-two days to mature and its maturity is overseen by a complex interaction of hormones. The scrotum has a built-in thermostat that keeps the testes and sperm at the correct temperature. It may be surprising that the testes should lie in such a vulnerable place outside the body, but it is too hot for them inside. Spermatogenesis requires a temperature that is three to five degrees Fahrenheit below body temperature. If it becomes too cool on the outside, the cremaster muscle will contract to bring the testes closer the body for warmth.
Fig. 12: Vertical section of the testis, to show the arrangement of the ducts
Source:
2.1.3.2.2 Functions of the Testes
The testes are responsible for the production of sperm cells and the male sex hormone testosterone. The testes produce as many as 12 trillion sperm in a male's lifetime, about 400 million of which are released in a single ejaculation.
Testes perform dual functions generally, as the site of spermatogenesis and hormone production. The sperm is the exocrine product while the hormone is the endocrine product (Moore and Dalley, 1999). The testis produces large number of spermatozoa in sexually mature males as well as secretion of androgens for the maintenance of male reproductive system, libido, and potency (Marieb, 2003). Other functions of the testes include stimulation of metabolism particularly those involved in protein synthesis and muscle growth, maintenance of secondary sexual characteristics such as fully developed male genitals, deepening of the voice, increased bone density and calcium retention, increased basal metabolic levels and increased number of red blood cells (Standring, 2005).
2.1.3.3 Epididymis
The epididymis (plural, epididymides) is a tightly coiled mass of thin tubes that carries sperm from the testes to the ductus deferens in the male reproductive system. Sperm matures as it passes through the epididymis so that it is ready to fertilize ova by the time it enters the ductus deferens.
2.1.3.3.1 Structure and Anatomy of the Epididymis
The epididymis is a crescent-shaped coil of thin tubules located inside the scrotum and posterior to the testis. The entire mass of the epididymis is actually a single, 20-foot-long (six-meter) tubule that has been coiled upon itself so tightly that the entire mass of the epididymis is only around 1.5 inches (4 cm) long The seminiferous tubules join together to become the epididymis. The epididymis is a tube that is about 20 feet long that is coiled on the posterior surface of each testis. Within the epididymis the sperm complete their maturation and their flagella become functional. This is also a site to store sperm until the next ejaculation. Smooth muscle in the wall of the epididymis propels the sperm into the ductus deferens. Vasa efferentia from the rete testis open into the epididymis which is a highly coiled tubule. The epididymis has three parts:
head or caput epididymis- it is the proximal part of the epididymis. It carries the sperms from the testis.
body or corpus epididymis- it the highly convoluted middle part of the epididymis.
tail or cauda epididymis- it is the last part that takes part in carrying the sperms to the vas differens.
Epididymis keeps sperms for sometimes, gives nourishment to it. The cauda epididymis continues to form less convoluted vas differens.
Fig. 12:
Source:
Adult human testicle with epididymis: A. Head of epididymis, B. Body of epididymis, C. Tail of epididymis, and D. Vas deferens
2.1.3.3.2 Functions of the Epididymis
The role of epididymis in sperm functions such as sperm survival, maturation, motility, capacitation and fertilizing ability cannot be overemphasized. The molecular basis of these functions is beginning to be elucidated in recent time with the discovery of some epididymis-specific proteins and their functions. The epididymis performs three basic functions which include sperm conduct, sperm reservoir and sperm maturation (Jones, 1999). The caput receives spermatozoa via the efferent ducts of the mediastinium of the testis and stores sperm until it is ready to undergo maturation and the concentration of the sperm is diluted here. Sperm matures in the corpus and the process takes approximately one week. The cauda is involved in absorbing fluid to make the sperm more concentrated and it is from here the sperm is transported to the ejaculatory duct.
2.1.3.4 Seminal vesicles
The seminal vesicles, more commonly referred to as the seminal gland, holds the liquid that mixes with sperm to form semen.
Semen combines fluid elements from the epididymis, seminal vesicles, prostate gland, and vas deferens. Each body part plays a key role in semen production. The fluids help the sperm swim towards the egg and keep the sperm nourished during the transit process.
2.1.3.4.1 Structure and Anatomy of the Seminal vesicles
This gland is located behind the bladder, above the prostate gland, and in front of the rectum. It is about two inches long, on average. This gland releases a fluid rich in sugars (especially fructose), which feeds the sperm. The fluid also has clotting properties that make the semen sticky. This ensures that the semen clings inside the vagina long enough for the sperm to travel to the egg.
The seminal vesicles are bilateral, lobulated glands. They are soft and approximately 5-7 cm long. The vesicles are blind pouches and are rounded on their most superior aspects and taper to their inferior aspects, where they constrict to ultimately form short ducts. They descend inferomedially while lying on the fundic portion of the posterior surface of the urinary bladder. The seminal vesicles are immediately inferior and lateral to the ampullary portions of the ductus deferentes.[2] This bilateral arrangement most closely resembles the letter "V." The ureters pass in between the superior, rounded aspects of the seminal vesicles and the superior portions of the ampullae of the ductus deferentes. The short ducts of the seminal vesicles join the lateral aspects of the ductus deferentes at an acute angle, creating the ejaculatory ducts at the base of the prostate gland.
The seminal vesicles lie below the inferior-most aspect of the peritoneum in the pelvic cavity. They are covered by endopelvic fascia, which is an abundant extraperitoneal connective tissue.[2, 1, 3]
Fig. 12: Structure of the male reproductive organs typically showing the seminal vesicles
Source:
2.1.3.4.2 Functions of the Seminal vesicles
The pair of seminal vesicles is posterior to the urinary bladder. They secrete fructose to provide an energy source for sperm and alkalinity to enhance sperm mobility. The duct of each seminal vesicle joins the ductus deferens on that side to form the ejaculatory duct. The vesicle produces a substance that causes the semen to become sticky/jelly-like after ejaculation. The thick secretions from the seminal vesicles contain proteins, enzymes, fructose, mucus, vitamin C, flavins, phosphorylcholine and prostaglandins. The high fructose concentrations provide nutrient energy for the spermatozoa.
2.1.3.5 Prostate gland
The prostate is a walnut-sized gland that grows throughout a man’s life and may eventually interfere with or prevent urination by blocking the urethra. The prostate gland is a muscular gland that surrounds the first inch of the urethra as it emerges from the bladder. The smooth muscle of the prostate gland contracts during ejaculation to contribute to the expulsion of semen from the urethra. The prostate makes a significant contribution to the production and ejaculation of semen during sexual intercourse.
2.1.3.5.1 Structure and Anatomy of the Prostate gland
The prostate is a small muscular gland located inferior to the urinary bladder in the pelvic body cavity. It is shaped like a rounded cone or a funnel with its base pointed superiorly toward the urinary bladder. The prostate surrounds the urethra as it exits the bladder and merges with the ductus deferens at the ejaculatory duct.
Several distinct lobes make up the structure of the prostate:
On the anterior end of the prostate are the two lateral lobes, which are rounded and shaped like orange slices when viewed in a transverse section. The lateral lobes are the largest lobes and meet at the midline of the prostate.
Posterior and medial to the lateral lobes is the much smaller anterior lobe, a triangle of fibromuscular tissue just anterior to the urethra. The fibromuscular tissue of the anterior lobe contracts to expel semen during ejaculation.
The median lobe is found just posterior to the urethra along the midline of the prostate. The median lobe contains the ejaculatory ducts of the prostate.
The posterior lobe forms a thin layer of tissue posterior to the median lobe and the lateral lobes.
The prostate contains two main types of tissue: exocrine glandular tissue and fibromuscular tissue. Exocrine glandular tissue in the prostate is epithelial tissue specialized for the secretion of the components of semen. Most of the prostate is made of exocrine glandular tissue, as the prostate’s primary function is the production of semen.
Fibromuscular tissue is a mixture of smooth muscle tissue and dense irregular connective tissue containing many collagen fibers. The collagen fibers of the tissue provide strength to the tissue while the smooth muscle permits the tissue to contract to expel fluids. Fibromuscular tissue forms the outermost layer of the prostate and the tissue surrounding the urethra.
Fig. 12: Structure of the male reproductive organs typically showing the Prostate
Source:
2.1.3.5.2 Functions of the Prostate gland
The function of the prostate is to secrete a slightly alkaline fluid, milky or white in appearance. The alkalinity of semen neutralises the acidity of the vaginal tract, prolonging the lifespan of sperm. The prostate also contains some smooth muscles that expel semen during ejaculation. It also produces a protein called protein-specific antigen (PSA) that turns the semen into liquid.
2.1.3.6 Adrenal
The supraneal, or adrenal, glands are a pair of glands that secrete hormones directly into the bloodstream.
2.1.3.6.1 Structure and Anatomy of the Adrenal
Each gland can be divided into two distinct organs. The outer region, the adrenal cortex, secretes hormones which have important effects on the way in which energy is stored and food is used, on chemicals in the blood, and on characteristics such as hairiness and body shape. The smaller, inner region – the adrenal medulla – is part of the sympathetic nervous system and is the body's first line of defense and response to physical and emotional stresses. The adrenal glands are shaped like the French Emperor Napoleon's hat and, just as Napoleon's three-cornered hat sat on his head, so each gland is perched on each of the kidneys. These glands are about one to two inches in length; they weigh only a fraction of an ounce each yet are among the most productive of all of the body's glands, secreting more than three dozen hormones. The adrenal cortex takes instruction from the pituitary glands and have important effects on physical characteristics, development and growth. The adrenal gland has two parts. The cortex, or outer, yellow layer, takes its instructions from the pituitary hormone ACTH. The hormones secreted here are called steroids and have three main types: those which control the balance of sodium and potassium in the body; those which raise the level of sugar in the blood; and sex hormones. The inner, reddish brown layer of the adrenal gland (the adrenal medulla) makes two types of hormones; this part of the adrenal gland takes its instruction from the nervous system, producing chemicals which react to fear and anger and are sometimes called fight or flight hormones.
Fig. 12: Structure of the male reproductive organs typically showing the Adrenal
Source:
2.1.3.6.2 Functions of the Adrenal
The main purpose of your adrenals is to enable your body to deal with stress from every possible source, ranging from injury and disease to work and relationship problems. They largely determine the energy of your body's responses to every change in your internal and external environment. Whether they signal attack, retreat or surrender, every cell responds accordingly, and you feel the results. It is through the actions of the adrenal hormones that your body is able to mobilize its resources to escape or fight off danger (stress) and survive. In a more primitive society that would mean being able to run away quickly, fight or pursue an enemy or game, endure long periods of physical challenge and deprivation, and store up physical reserves when they are available.
The adrenals produce hormones that help balance your blood sugar and manage your daily ebbs and flows of energy. They keep your body’s reactions to stress in balance. Adrenal hormones have protective anti-inflammatory and anti-oxidant activity and closely affect the utilization of carbohydrates and fats, the conversion of fats and proteins into energy, the distribution of stored fat (especially around the waste and the sides of the face), normal blood sugar regulation and proper cardiovascular and gastrointestinal function.
Cortisol is a steroid hormone that is produced by the adrenals. It is also known as the stress hormone and is involved in a wide range of metabolic processes like:
* Mobilizing and increasing amino acids, the building blocks of protein, in the blood and liver.
* Stimulating the liver to convert amino acids to glucose, the primary fuel for energy production.
* Stimulating increased glycogen in the liver (the stored form of glucose).
* Mobilizing and increasing fatty acids in the blood (from fat cells) to be used as fuel for energy production.
* Counteracting inflammation and allergies.
* Preventing the loss of sodium in urine and thus helps maintain blood volume and blood pressure.
* Maintaining resistance to stress (e.g., infections, physical trauma, temperature extremes, emotional trauma, etc.).
* Maintaining mood and emotional stability
Vas Deferens
2.1.3.7 Liver
The liver is a vital organ present in vertebrates and some other animals. It is the largest organ of the body, constituting 2-5 % of the adult body weight (Guyton and Hall, 2000). The liver is a reddish brown organ with four lobes of unequal size and shape. A human liver normally weighs 1.44–1.66 kg. It is a soft, pinkish-brown traingular organ situated in the right upper quadrant of the abdominal cavity, resting just below the diaphragm. It lies to the right of the stomach and overlies the gall bladder (Cotran et al., 2005). The positioning of the liver allows it to carry out diverse functions in the body. It is a principal organ of metabolism and has a part to play in many processes in the body (Guyton and Hall, 2000).
2.1.3.7.1 Structure and Anatomy of the Liver
The liver is a large, reddish-brown organ with two lobes. It is located just below the diaphragm to the right side, partly overlapping the stomach. Leading from the liver to the small intestine is the bile duct to which the gall bladder is attached. It is a soft organ with a rich and extensive blood supply (Ramalingam, 1997).
The liver consists of four sections, or lobes (Figure 5). There are two main lobes-the right lobe, which is by far the larger, and the left lobe. Two small lobes lie behind the right lobe. Each lobe is made up of multisided units called lobules. Most liver have between 50,000 and 100,000 lobules. Each lobule consists of a central vein surrounded by tiny liver cells grouped in sheets or bundles; these cells perform the work of the liver. Cavities known as sinusoids separate the groups of cells within a lobule. The sinusoids give the liver a spongy texture and enable it to hold large amounts of blood (Arthur and John, 1995). A hepatocyte is a cell of the main tissue of the liver. Hepatocytes make up 70-80 % of the liver's cytoplasmic mass (Hamel et al., 2006). The liver receives a dual blood supply from the hepatic portal vein and arteries. Supplying approximately 75 % of the liver's blood supply, the hepatic portal vein carries venous blood drained from the spleen, gastrointestinal tract, and its associated organs. The hepatic arteries supply arterial blood to the liver, accounting for the remainder of its blood flow (Maton et al., 1993). Bile can either drain directly into the duodenum via the common bile duct or be temporarily stored in the gallbladder via the cystic duct. The common bile duct and the pancreatic duct enter the second part of the duodenum together at the ampulla of Vater (Bramstedt, 2006).
Fig. 5: Structure of the liver
Source: Patel (2009)
2.1.3.7.2 Functions of the Liver
As the metabolic crossroads of the body, the liver helps in filtering circulating blood, removal and destruction of toxic substances, conversion of protein metabolism products into urea for excretion by the kidneys, manufacture of plasma protein, inactivation of polypeptide hormones, regulation of blood-clotting mechanisms and mobilization and metabolism of chemical and cellular arsenal for self-protection. The ability of the liver to regenerate this important organ survives the wear and tear of a lifetime (Diehl, 1993; Ganong 1997; Guyton and Hall, 2000).
2.1.3.8 Kidney
The kidneys are organs that serve several essential regulatory roles in most animals, including vertebrates and some invertebrates (Ramalingam, 1997; Glodney et al., 2009).
2.1.3.8.1 Structure and Anatomy of the Kidney
The kidney is a brownish, red bean shaped organ, which are paired in the mammalian system (Figure 6). Each kidney is covered by strong connective tissue membrane called the capsule which can be peeled off being loosely connected to the kidney (Kamath, 1972). When the kidney is cut longitudinally, two major
Fig. 6: Structure of the kidney
Source: Patel (2009)
regions that can be visualized are the outer cortex and the inner region called medulla as shown in Figure 6. The kidney contains one to two million nephrons closely packed in the connective tissue and amply supplied with blood. The kidneys have three basic mechanisms of filtration, reabsorption, and secretion separating the various components of the blood. These three processes occur in the nephron. The nephron contains a cluster of blood vessels known as the glomerulus, surrounded by the hollow Bowman's capsule. The glomerulus and Bowman's capsule together are known as the renal corpuscle. Bowman's capsule leads into a membrane-enclosed, U-shaped tubule that empties into a collecting duct. The collecting ducts from the various nephrons merge together, and ultimately empty into the bladder.
Functions of the Kidney
The mammalian kidney is the major organ of excretion in vertebrates. The major functions of the kidneys are:
1. Excreting unwanted substances by purifying the blood through the process of ultra-filtration and re-absorption.
2. Regulating a constant internal environment (homeostasis).
3. Synthesizing hormones from ductless and endocrine glands and transports it through the blood to their target site where they exert their effect.
4. Carrying out gluconeogenesis by synthesizing glucose from amino acids and other precursors during fasting (Murray et al., 2000).
2.1.3.9 Heart
The heart is a muscular organ about the size of a closed fist that functions as the body’s circulatory pump (Bowman et al., 1971). It takes in deoxygenated blood through the veins and delivers it to the lungs for oxygenation before pumping it into the various arteries (which provide oxygen and nutrients to body tissues by transporting the blood throughout the body).
2.1.3.9.1 Structure and Anatomy of the Heart
The heart is located in the thoracic cavity medial to the lungs and posterior to the sternum. On its superior end, the base of the heart is attached to the aorta, pulmonary arteries and veins, and the vena cava. The inferior tip of the heart, known as the apex, rests just superior to the diaphragm. The base of the heart is located along the body’s midline with the apex pointing toward the left side. Because the heart points to the left, about 2/3 of the heart’s mass is found on the left side of the body and the other 1/3 is on the right (Barcoft, 1963).
The heart sits within a fluid-filled cavity called the pericardial cavity. The walls and lining of the pericardial cavity are a special membrane known as the pericardium. Pericardium is a type of serous membrane that produces serous fluid to lubricate the heart and prevent friction between the ever beating heart and its surrounding organs. Besides lubrication, the pericardium serves to hold the heart in position and maintain a hollow space for the heart to expand into when it is full (Bowman et al., 1971). The pericardium has 2 layers—a visceral layer that covers the outside of the heart and a parietal layer that forms a sac around the outside of the pericardial cavity.
The heart wall is made of 3 layers: epicardium, myocardium and endocardium. The heart contains 4 chambers: the right atrium, left atrium, right ventricle, and left ventricle. The atria are smaller than the ventricles and have thinner, less muscular walls than the ventricles (Schwartz et al., 2008). The atria act as receiving chambers for blood, so they are connected to the veins that carry blood to the heart. The ventricles are the larger, stronger pumping chambers that send blood out of the heart. The ventricles are connected to the arteries that carry blood away from the heart (Schwartz et al., 2008).
The chambers on the right side of the heart are smaller and have less myocardium in their heart wall when compared to the left side of the heart (Guyton and Hall, 2000). This difference in size between the sides of the heart is related to their functions and the size of the 2 circulatory loops. The right side of the heart maintains pulmonary circulation to the nearby lungs while the left side of the heart pumps blood all the way to the extremities of the body in the systemic circulatory loop (Guyton and Hall, 2000).
Fig. 5: Schematic Diagram of the Heart Structure
Source: Patel (2009)
Fig. 8: Anatomy of the Human Heart
Source: Ties van Brussels (2010)
2.1.3.9.2 Functions of the Heart
The major function of the heart is to pump blood to all parts of the body, a process that keeps organisms active. Blood enters the right atrium from the body and the left atrium from the lungs. It also provides additional homeostatic function by producing a hormone, arterial natriuretic peptide (Robert, 1976). The heart functions by pumping blood both to the lungs and to the systems of the body.
2.1.4 Male Biological Evaluation Indices
Biological screening entails monitoring sexual activities of the animals. This may be achieved by evaluating the male rat sexual behaviour and male fertility.
2.1.4.1 Male Rats Sexual Behaviors
Many of the behavioural functions are elicited from the hypothalamus and other limbic structures and are mediated through the reticular nuclei in the brain stem and their associated nuclei (Guyton and Hall, 2000). Male rat sexual behaviour parameters include: Mount Frequency, Intromission Frequency, Mount Latency,
Intromission Latency, Ejaculatory Latency, Post Ejaculatory Interval (PEI), Mean Interintromission Interval (MIII) and Hit Rate (HR).
2.1.4.1.1 Mount frequency
Mounting is defined as the climbing of one animal by another usually from the
posterior end with the intention of introducing one organ into another. Mount may also be operationally defined as the male assuming the copulatory position but failing to achieve intromission. Mount Frequency (MF) is therefore defined as the number of mounts without intromission from the time of introduction of the female until ejaculation (Gauthaman et al., 2002).
2.1.4.1.2 Intromission frequency
Intromission is the introduction of one organ or parts into another, for example the penis into the vagina. Intromission Frequency (IF) is therefore defined as the number of intromissions from the time of introduction of the female until ejaculation.
2.1.4.1.3 Mount latency
Mount Latency (ML) is defined as the time interval between the introduction of the female and the first mount by the male (Gauthaman et al., 2002).
2.1.4.1.4 Intromission latency
Intromission Latency (IL) is the time interval from the time of introduction of the female to the first intromission by the male. This is usually characterized by pelvic thrusting, and springing dismounts (Gauthaman et al., 2002).
2.1.4.1.5 Ejaculatory latency
Ejaculation is the act of ejecting semen brought about by a reflex action that occurs as the result of sexual stimulation. Ejaculatory Latency (EL) is defined as the time interval between the first intromission and ejaculation. This is usually characterized by longer, deeper pelvic thrusting and slow dismount followed by a period of inactivity or reduced activity (Gauthaman et al., 2002).
2.1.4.1.6 Post Ejaculatory Interval (PEI)
This is the time interval between ejaculation and erection of the male copulatory organ for the next phase of sexual cycle.
2.1.4.1.7 Mean Interintromission Interval (MIII)
This is the mean intervals in seconds separating the intro-missions of the series.
2.1.4.1.8 Hit rate (HR)
This is the number of intromissions divided by the number of intromissions plus the number of mounts.
2.1.4.2 Male fertility
The evaluation of male fertility can be done using parameters that focus on conception in the females. These include index of libido, quantal frequency, fertility index, implantation index, pre- and post- implantation losses.
2.1.4.2.1 Index of libido
Index of Libido is defined as the ratio of number mated to number paired expressed in percentage (Ratnasooriya and Dharmasiri, 2000).
This can be expressed mathematically as:
% Index of Libido = Number mated x 100
Number paired
2.1.4.2.2 Quantal pregnancy
Quantal Pregnancy is defined as the ratio of number pregnant to number mated expressed in percentage unit (Ratnasooriya and Dharmasiri, 2000).
% Quantal Pregnancy = Number pregnant x 100
Number mated
2.1.4.2.3 Fertility index
Fertility index can be described as the ratio of number pregnant to number paired expressed in percentage (Ratnasooriya and Dharmasiri, 2000).
% Fertility Index = Number pregnant x 100
Number paired
2.1.4.2.4 Implantation index
Implantation Index is the ratio of total number of implantations to number mated expressed in percentage (Ratnasooriya and Dharmasiri, 2000).
% Implantation Index = Total number of implantation x 100
Number mated
2.1.4.2.5 Pre-implantation loss
Pre-implantation loss is the ratio of the difference between the number of corpora lutea and number of implantations to number of corpora lutea expressed in
Percentage (Ratnasooriya and Dharmasiri, 2000).
% Pre-implantation loss = Number of corpora lutea – number of implantation x 100
Number of corpora Lutea
2.1.4.2.6 Post-implantation loss
Post-implantation loss is the ratio of the difference between the total number of implants and number of viable implants to total number of implants expressed in percentage (Ratnasooriya and Dharmasiri, 2000).
% Post-implantation loss = Total no. of implants – no. of viable implants x 100
Total number of implants
2.1.4.3 Weights of reproductive organs
2.1.4.3.1 Organ body weight ratio
The organ-body weight ratio is the weight of the organ to the overall body weight. The weight of various organs in the body varies considerably and depends on their location and function. The higher the number of functions carried out by an organ, the larger the weight of the organ (George et al., 1994). The organ-body weight ratio is used in predicting clinical conditions like inflammation or constriction of organ. It is a 'marker' of cell constriction and inflammation (Moore and Dalley, 1999). Organ-body weight ratios are normally investigated to determine whether the size of the organ has changed in relation to the weight of the whole animal. Organ body weight is also used to indicate atrophy and hypertrophy (Adebayo et al., 2003). It is used to indicate hypertrophy, atrophy or cellular constriction (Amresh et al., 2009).
2.1.5 Male Biological Fluids
2.1.5.1 Blood
Blood is the extracellular fluid within the cardiovascular system. It is also the red viscid fluid that circulates through the heart and blood vessels supplying nutritive materials to all the parts of the body, carrying away waste products. The blood consists of plasma in which cellular constituents are suspended. These are mostly erythrocytes (red blood cells), leukocytes (white blood cells) and platelets (Bray et al., 1999). The average adult human has 5-6 litres of blood, half of which is occupied by the three types of blood cells: erythrocytes filled with hemoglobin, leukocytes which include lymphocytes, granulocytes (neutrophils and monocytes) and platelets (Nelson and Cox, 2000).
2.1.5.1.1 Hematological Parameters
Hematological parameters are related to the blood and blood forming organs (Stenesh, 1975). Assessment of hematological parameters can be used to determine the extent of deleterious effect on blood constituents of an animal (Muhammad et al., 2004; Ashafa et al., 2009). It can also be used to functions of chemical compounds/plant extract (Yakubu et al., 2007). Hematological Parameters include the following:
2.1.5.1.1.1 Packed Cell Volume
Packed cell volume (PCV) is the volume of red blood cells in 1litre of whole blood. PCV or hematocrit measures the percentage of blood volume taken up by red blood cells. Since 3-4% of the plasma is entrapped among cells, the true packed cell volume is only about 96% of the measured value (McKnight et al., 1999).
2.1.5.1.1.1.1 Clinical significance
The PCV is a measurement of the fractional volume of red blood cells. This is a key indicator of hydration, anemia or severe blood loss, as well as the blood’s ability to transport oxygen. A decreased PCV can be due to either over hydration, which increases the plasma volume, or a decrease in the number of red blood cells caused by anemias or blood loss. An increased PCV can be due to loss of fluids, such as in dehydration, diuretic therapy, and burns, or an increase in red blood cells, such as in cardiovascular and renal disorders, polycythemia vera, and impaired ventilation (McKnight et al., 1999).
2.1.5.1.1.2 Red Blood Cell
The human Red Blood Cell (RBC) is non-nucleated, biconcave disc, with a mean diameter of about 8μm, about 2.5μm thick (Waugh and Grant, 2001). It is produced in the bone marrow and has a life span of about 120 days during which it travels about 280Km through the circulation. The erythrocyte membrane is composed of 40% protein, 44% lipids and 7% carbohydrates. The red blood cell membrane contains antigenic components, which allow the human blood to be classified into various groups namely A, AB, B and O. The major function of the red blood cells is to transport hemoglobin, which in turn carries oxygen from the lungs to the tissues (Waugh and Grant, 2001). Very low values of RBC, hemoglobin and hematocrit can indicate anemia (Waugh and Grant, 2001).
2.1.5.1.1.2.1 Clinical significance
The RBC count is most useful as raw data for calculation of the erythrocyte indices MCV and MCH (Waugh and Grant, 2001). Decreased RBC is usually seen in anemia of any cause with the possible exception of thalassemia minor, where a mild or borderline anemia is seen with a high or borderline-high RBC. Increased RBC is seen in erythrocytotic states, whether absolute (polycythemia vera, erythrocytosis of chronic hypoxia) or relative (dehydration, stress polycthemia), and in thalassemia minor (Tietz, 1983, Waugh and Grant, 2001).
2.1.5.1.1.3 Haemoglobin Content
Hemoglobin is the red respiratory pigment of erythrocytes. It is a conjugated protein of molecular weight of approximately 67,000 composed of four polypeptide chains of globin (Bray et al., 1999). Haem, which constitute about 4% of the hemoglobin molecule, consists of protoporphyrin ring with a central atom of ferrous, which combines reversibly with a molecule of oxygen. Four different types of globin which can be differentiated on the basis of their amino acid constituents include α, β, γ and δ. The tetrameric structure of hemoglobin allows for conformational changes in the molecule, which accounts for important characteristics of oxygen transport (Bray et al., 1999).
2.1.5.1.1.3.1 Clinical significance
Haemoglobin the main component of the red blood cells, functions in the transportation of oxygen and CO2. Haemoglobin consists of one molecule of globin and four molecules of haeme (each containing one molecule of iron in the ferrous state). Globin consists of two pairs of polypeptide chains. In the haemoglobin molecule, each polypeptide chain is associated with one haeme group; each haeme group can combine with one molecule of oxygen or CO2 (Lotspech-Steininger et al, 2009). Haemoglobin carries oxygen from places of high oxygen pressure (lung) to places of low oxygen pressure (tissues), where it readily releases the oxygen. Haemoglobin also returns CO2 from the tissues to the lungs (Turgeon, 2009).
Haemoglobin in circulating blood is a mixture of haemoglobin, oxyhaemoglobin, carboxyhaemoglobin and minor amounts of other forms of this pigment. Measurements of haemoglobin from venous or capillary blood aids in the detection of a variety of conditions that alter the normal haemoglobin concentration of the blood, e.g. anemia or polycythemia (Turgeon, 2009; Lotspech-Steininger et al., 2009).
2.1.5.1.1.4 Mean corpuscular volume
Mean Corpuscular Volume (MCV) measures the average volume (size) of individual red blood cells (Dacie and Lewis, 1995). The measure is obtained by multiplying the proportion of blood that is cellular (haematocrit), and dividing that product by the number of erythrocytes in that volume. The mean corpuscular volume is a part of a standard complete blood count.
2.1.5.1.1.5 Mean corpuscular haemoglobin
Mean Corpuscular Haemoglobin (MCH) is the average amount of haemoglobin in each cell measured in pictogram (Guyton and Hall, 2006). It is the average mass of haemoglobin per red blood cell in a sample of blood. The MCH is calculated by dividing total hemoglobin by the total number of red blood cells (Ganong, 2001).
2.1.5.1.1.6 Mean corpuscular haemoglobin concentration
Mean Corpuscular Haemoglobin Concentration (MCHC) measures the concentration of haemoglobin in the average cell. MCHC is the amount of haemoglobin in 100 cm3 of the red cell (Dacie and Lewis, 1995; Ganong, 2001). It is calculated by dividing the haemoglobin by the haematocrit. Reference ranges for blood tests are 32-36 g/dL, or between 19.9-22.3 mmmol/L (Wallach, 2007).
2.1.5.1.1.7 White Blood Cells
White blood cells (WBC) or leukocytes are the mobile units of the body’s protective system (Robert, 1976). There are six types of WBC normally found in the blood. These are polymorphonuclear neutrophils, eosinophils, basophils, monocytes, lymphocytes and occasionally plasma cells (Robert, 1976).
2.1.5.1.1.7.1 Clinical significance
The main function of white blood cell is to combat and prevent infection. A high WBC usually means the body is fighting an infection. A very low WBC can be caused by problems in the bone marrow. This condition, called cytopenia or leucopenia means the body is less able to fight of infections (Robert, 1976).
2.1.5.1.1.7.1 Neutrophils
Neutrophils are normally found in the blood stream during the acute phase of inflammation, due to bacterial infection, environmental exposure (Jacobs, 2010). Neutrophils are one of the first-responders of inflammatory cells to migrate towards the site of inflammation. They migrate through the blood vessels, then through interstitial tissue, following chemical signals such as Interleukin-8 (IL-8) and C-5 in a process called chemotaxis. They are the predominant cells in pus, accounting for its whitish/yellowish appearance. Neutrophils are recruited to the site of injury within minutes following trauma and are the hallmark of acute inflammation (Cohen and Richard, 2002). Low neutrophil counts are termed neutropenia. This can be congenital (genetic disorder) or it can develop later, as is the case of aplastic anaemia or some kinds of leukemia. It can also be a side-effect of medication, most prominently chemotherapy. Neutropenia makes an individual highly susceptible to infections (Bagby, 2007). Neutropenia can be the result of colonization by intracellular neutrophilic parasites (Bagby, 2007). Increase in number of neutrophils is termed neutrophilia which is an indication of infection or tissue damage, particularly when accompanied by circulating neutrophils precursor cells (Monica, 2004).
2.1.5.1.1.7.2 Eosinophils
Eosinophil granulocytes, usually called eosinophils or eosinophiles (or, less commonly, acidophils), are one of the immune system components responsible for combating multicellular parasites and certain infections in vertebrates. Along with mast cells, they also control mechanisms associated with allergy and asthma. They are granulocytes that develop during haematopoiesis in the bone marrow before migrating into the blood (Shi, 2004).
The increases in eosinophil cells (eosinophilia) are indication of helminth infections, and also in allergic disorders such as asthma, hay fever, eczema (Monica, 2004).
2.1.5.1.1.7.3 Basophils
Basophil granulocytes, sometimes referred to as basophils, are the least common of the granulocytes, representing about 0.01% to 0.3% of circulating white blood cells. Basophils contain large cytoplasmic granules which obscure the cell nucleus under the microscope. However, when unstained, the nucleus is visible and usually has 2 lobes. The mast cell has many similar characteristics. For example, both cell types store histamine, a chemical that is secreted by the cells when stimulated in certain ways (histamine causes some of the symptoms of an allergic reaction). Like all circulating granulocytes, basophils can be recruited out of the blood into a tissue when needed (Schroeder, 2009). Normal range of basophils in human is 0.5% to 1% (Bagby, 2007).
Increase in circulating basophils (basophilia) is found in myeloproliferative disorders, some leukaemia (especially chronic myeloid leukaemia), and occasionally in allergic disorders (Monica, 2004).
2.1.5.1.1.7.4 Lymphocyte
Lymphocyte, is a type of leukocyte (white blood cell) that is of fundamental importance in the immune system because lymphocytes determine the specificity of the immune response to infectious microorganisms and other foreign substances (Britannica, 2011). In humans, lymphocytes make up 25 – 33 % of the total number of leukocytes (Britannica, 2011). They are found in the circulation and are also concentrated in central lymphoid organs and tissues, such as the spleen, tonsils, and lymph nodes, where the initial immune response is likely to occur, the two primary types of lymphocytes are B lymphocytes and T lymphocytes (Britannica, 2011). The three major types of lymphocyte are T cells, B cells and natural killer (NK) cells (Paul et al., 2001). A lymphocyte count is usually part of a peripheral complete blood cell count and is expressed as percentage of lymphocytes to total white blood cells counted. An increase in the number of lymphocytes is known as lymphocytosis whereas a decrease is lymphocytopenia. An increase in lymphocyte concentration is usually a sign of a viral infection. A low lymphocyte concentration is associated with increased rates of infection after surgery or trauma (Kumar et al., 2004).
2.1.5.1.1.7.5 Monocyte
Monocyte is a type of white blood cell that plays multiple roles in immune function. These replenishing resident macrophages and dendritic cells under normal states, and responding to inflammation signals (Swirski et al., 2009). A monocyte count is part of a complete blood count and is expressed either as a ratio of monocytes to the total number of white blood cells counted, or by absolute numbers. Both may be useful in determining or refuting a possible diagnosis.
A high count of CD14+ and CD16+ monocytes is found in severe infection (sepsis) (Fingerle et al., 1993) and a very low count of these cells is found after therapy with immuno-suppressive glucocorticoids (Fingerle-Rowson et al., 1998).
2.1.5.1.1.8 Platelets
Platelets are fragments of another type of cell similar to the white blood cell that are found in the bone marrow. They are minute, round, oval disc, 1-4 μm in diameters (Pasternak, 1979). The unactivated platelets are biconvex discoid structures, shaped like a lens, 2-3 μm in diameter. They have no nucleus: they are fragments of the cytoplasm which are released by megakaryotes of the bone marrow, and then enter circulation. The function of platelets is mainly to activate the blood clotting mechanism (Yip et al., 2005) and prevent loss of blood from the blood vessels through coagulation. Platelets are found only in mammals, an adaptation that may have evolved to offset the risk of death from haemorrhage at childbirth-a risk unique to mammals (Yip et al., 2005). Low platelet concentration referred to as thrombocytopaenia, is due to either decreased production or increased destruction. Elevated platelet concentration is thrombocytosis and is either congenial, reactive (to cytokines), or due to unregulated production (Machlus, 2014).
2.1.5.2 Serum
Serum is the clear liquid that can be separated from clotted blood. Serum differs from plasma, the liquid portion of normal unclotted blood containing the platelets, red and white blood cells. It is the clot that makes the difference between serum and plasma. When the blood is left undisturbed without the addition of anticoagulants (heparin, EDTA, etc.), it clots. Within a few minutes after a clot, it begins to contract and usually expresses most of the fluid from the clot within 20 – 60 minutes. The fluid expressed is called serum because all its fibrinogen and most other clotting factors have been removed (Guyton and Hall, 2000). Though, serum retains albumin and globulin that are plasma proteins (Murray et al., 2000). Blood could be centrifuged, that is after clotting or may be refrigerated for easy collection of serum (Wilson and Walker, 1995).
2.1.5.2.2 Functions of the Serum
The blood, apart from transporting various materials like oxygen and carbon dioxide, also dissipates heat generated by oxidative reactions in cells. It also distributes hormones, thus helping to maintain homeostasis and co-ordinate the activities of various organs of the body (Bray et al., 1999). It also contains specialized substances, which play important roles in immunity and blood clotting.
2.1.5.3 Seminal fluid
Seminal fluid also known as semen is an organic fluid that may contain spermatozoa. It is secreted by the gonads (sexual glands) and other sexual organs of male or hermaphroditic animals and can fertilise female ova (Mann, 1954). In humans, seminal fluid contains several components besides spermatozoa. The seminal vesicles produce a yellowish viscous fluid that makes up 70 % of the human semen and contains proteolytic enzymes as well as fructose which promote the survival of spermatozoa and provide a medium through which they can move or "swim". Semen is produced and originates from the seminal vesicle, which is located in the pelvis. The prostatic secretion, influenced by dihydrotestosterone, is a whitish (sometimes clear), thin fluid containing proteolytic enzymes, citric acid, acid phosphatase and lipids. The bulbourethral glands secrete a clear secretion into the lumen of the urethra to lubricate it (Guyton, 1991).
2.1.6 Semen analysis/Spermicidal studies
A semen analysis evaluates certain characteristics of a male's semen and the sperm so as to know the quantity and quality of semen and sperm. Semen analysis comprises a set of descriptive measurements of spermatozoa and seminal fluid parameters that help to estimate semen quality (Campana et al., 1995). Conventional semen analysis includes measurement of particular aspects of spermatozoa such as concentration, motility and morphology and of seminal plasma. Quantification and identification of non-spermatozoidal cells and detection of antisperm antibodies are also part of basic semen analysis (Comhaire and Vermeulen, 1995). Semen is the thick, white fluid released during ejaculation that contains sperm. Normal semen is a mixture of spermatozoa suspended in secretions from the testis and epididymis which are mixed at the time of ejaculation with secretions from the prostate, seminal vesicles, and bulbourethral glands. The final composition is a viscous fluid that comprises the ejaculate (WHO, 1992). A semen analysis measures three major factors of sperm health which include number of sperm (sperm count), shape of sperm (sperm morphology) and mobility of sperm (sperm motility). The analysis is done to help evaluate male fertility, whether for those seeking pregnancy or verifying the success of vasectomy (Kippley et al., 1996).
2.1.6.1 Semen pH
The pH is determined by acidic secretions of the prostate and alkaline secretions of the seminal vesicles. It should normally be in the range of 7.2-8.0 (WHO, 1992). If the pH exceeds 8.0, infection should be suspected with decreased secretion of acidic products by the prostate, such as citric acid. Abnormal pH may also be recorded in cases of incomplete ejaculation. Acidic ejaculate (lower pH value) may indicate one or both of the seminal vesicles are blocked. Extremely acidic pH (<6.5) is found in cases of agenesis (or occlusion) of the seminal vesicles (Comhaire and Vermeulen, 1995).
2.1.6.2 Semen Volume
The major component of the ejaculate volume is made up of secretions from the accessory glands. The bulk of the volume is secreted by the seminal vesicles and between 0.5 and 1 ml originates from the prostate (Comhaire and Vermeulen, 1995). A low ejaculate volume can reflect abnormalities in accessory sex gland fluid synthesis or secretion (Siegel, 1993). It can also be indicative of a physical obstruction somewhere in the reproductive tract (Siegel, 1993), or may occur in cases of incomplete or (partially) retrograde ejaculation (Comhaire and Vermeulen, 1995). Large volumes are sometimes found in association with varicocele or after relatively long periods of sexual abstinence (Comhaire and Vermeulen, 1995).
2.1.6.3 Sperm Count
Sperm count, or sperm concentration, measures the concentration of sperm in a man's ejaculate, distinguished from total sperm count, which is the sperm count multiplied with volume. Over 20-40 million sperm per milliliter ejaculate is considered normal (WHO, 1992). A lower sperm count is considered oligozoospermia. The total number of spermatozoa per ejaculate reflects spermatogenesis and is related to the duration of sexual abstinence (Campana et al., 1995). Perhaps the most widely utilised semen parameter is sperm count. Men with <20×106 spermatozoa per ml are typically deemed sub-fertile, and men with counts <5×106 spermatozoa/ml are often considered infertile (Seibel and Zilberstain, 1995). However, it must be emphasised that patients with sperm counts <20×106 are not infertile but it simply takes them a substantially longer period of time to achieve pregnancies (Seibel and Zilberstain, 1995).
2.1.6.4 Sperm Morphology
Sperm morphology gives information for the function of the reproductive tract and is a predictor of man’s fertility potential (Ayala et al., 1996). Physical sperm aberrations may occur during the production of sperm or during storage in the epididymis. Sperm cells represent a unique population in which up to 50% of the cells can have morphological defects in normal fertile individuals (Campana et al., 1995). The normal head should be oval in shape whose length should be 4.0-5.5 µm, and the width 2.5-3.5 µm. There should be a well-defined acrosomal region comprising 40-70% of the head area. There must be no neck, midpiece or tail defects and no cytoplasmic droplet more than one-third the size of a normal sperm head (WHO, 1992; Campana et al., 1995). The increased number of immature spermatozoa may be due to epididymal dysfunction or a consequence of frequent ejaculations. The increased number of spermatozoa with tapering heads is found in association with varicocele.
2.1.6.5 Sperm Motility
Sperm motility describes the ability of sperm to move properly through the female reproductive tract (internal fertilisation) or through water (external fertilisation) to reach the egg (Yanagimachi, 1994). The extent of progressive sperm motility is related to pregnancy rates (Yanagimachi, 1994). The World Health Organization has a value of 50 % and this must be measured within 60 minutes of collection. WHO also has a parameter of vitality, with a lower reference limit of 60 % live spermatozoa (Cooper et al., 2010). A man can have sperm counts greater than 20 million sperm cells per milliliter with few motile sperm cells. However, if the sperm count is very high, then a low motility (for example, less than 60 %) might be irrelevant, because the fraction might still be more than 8 million per millilitre. A man with sperm count that is less than 20 million sperm cells per millilitre may have good motility, if more than 60% of the sperm cells show good forward movement (Cooper et al., 2010). A more specified measure is motility grade, where the motility of sperm is divided into four different grades:
Grade a: Sperm with progressive motility. These are the strongest and swim fast in a straight line. Sometimes it is also denoted motility IV.
Grade b: (non-linear motility): These also move forward but tend to travel in a curved or crooked motion. Sometimes also denoted motility III.
Grade c: These have non-progressive motility because they do not move forward despite the fact that they move their tails. Sometimes also denoted motility II.
Grade d: These are immotile and fail to move at all. Sometimes also denoted motility I.
2.1.6.6 Sperm Viability (life/dead ratio)
Viable sperm is sperm that is capable of fertilising an egg. When combined with a viable egg, it results in offspring. Sperm viability is measured when the motility test is abnormal. Reduced percentage of motility with a high percentage of viable sperm may reflect structural or metabolic abnormalities of sperm that are derived from abnormalities in testicular function or antimotility factors in the seminal plasma (Siegel, 1993). This technique also provides a check on the accuracy of the motility evaluation, since the percentage of dead cells should not exceed the percentage of immotile spermatozoa (WHO, 1992).
Sperm density/rough cell estimation
A general sperm count as part of a fertility evaluation should include the total density or count (20 million per ml or above), and the motile density (8 million per ml or higher). The motile density is perhaps the most important part of the semen analysis, as it reports the total number of sperm thought capable of progressing from the site of sperm deposition to the site of fertilization.
2.1.6.7 Sperm density/rough cell estimation
A general sperm count as part of a fertility evaluation should include the total density or count (20 million per ml or above), and the motile density (8 million per ml or higher). The motile density is perhaps the most important part of the semen analysis, as it reports the total number of sperm thought capable of progressing from the site of sperm deposition to the site of fertilization.
2.1.6.8 Sperm colour
Semen is normally translucent or whitish-gray opalescent in colour. Blood found in semen (hematospermia) can colour the semen pink to bright red to brownish red. The presence of blood in semen is abnormal.
2.1.6.9 Sperm turbidity
Semen is normally produced as a coagulum. The specimen will usually liquefy within 30 minutes. The failure to liquefy within one hour is abnormal (Ohl and Menge, 1996).
2.1.7 Male Function Indices
2.1.7.1 Prostatic Function Indices
The function indices of the prostate gland are secretory substances produced by the prostate. These include: acid phosphatase (ACP), calcium ions, citrate ions and phosphate ions. All these and many more makes up the fluid secreted by the prostate.
2.1.7.1.1 Calcium
Calcium is the first messenger for the prompt, plasma membrane-mediated action of thyroid hormone that increase cellular sugar uptake. Calcium is involved in almost every biological function. This amazing mineral provides the electrical energy for the heart to beat and for all muscle movement. It is the calcium ion that is responsible for feeding every cell including the sperm cells. It does this by latching on to seven nutrient molecules and one water molecule and pulls them through the nutrient channel. It then detaches its load and returns to repeat the process (Segal, 1990).
Calcium is one of the most important elements in the body. About 99% can be found in the bones and teeth where it gives structural strength in the form of calcium salts. Ionized calcium (Ca2+) is very active physiologically. It is important for nerve
cells, enzyme activity and muscle contraction, where the utilization of high-energy bonds of ATP involves its hydrolysis by a Mg2+-activated myofibrilar enzyme, myosin ATPase (Bell et al., 1972). Calcium also acts as an intracellular second messenger for hormones. In addition, the ionized calcium ion (Ca2+) is required for blood coagulation (Factor IV).
Parathyroid hormone and vitamin D regulate the concentration of Ca2+ in extracellular fluid by increasing the rate at which calcium is absorbed from the
intestine while vitamin D increases the efficiency of the process. Calcitonin also
causes a decrease in the reabsorption of calcium from bones by decreasing the activity of osteocast and also preventing their formation (Kamath, 1972; Rose, 1994; Guyton and Hall, 2000).
2.1.7.1.2 Citrate
One of the major functions of normal prostate gland is synthesis, accumulation and secretion of large amounts of citrate (Costello and Franklin, 2000). Intracellular citrate is important for generation of energy as well as synthesis of fatty acids, isoprenoids and cholesterol (Inoue et al., 2002).
Citrate is a key tricarboxylic acid (TCA) cycle intermediate formed by the addition of oxaloacetate to the acetyl group of acetyl-CoA. Citrate is transported out of the mitochondria via the citrate-malate shuttle and converted back to acetyl-CoA for fatty acid synthesis. Citrate is an allosteric modulator of both fatty acid synthesis via its actions on acetyl-CoA carboxylase and of glycolysis via its actions on phosphofructokinase. Citrate metabolism and disposition can vary widely due to sex, age, and a variety of other factors including disease states. Cellular citrate levels are decreased in prostrate cancer cells and citrate levels may be a marker of prostrate cancer growth rate.
Citric acid (HOOC-CH2-C(-OH)(-COOH)-CH2-COOH) is a key intermediate in the TCA cycle which occurs in mitochondria. It is formed by the addition of oxaloacetate to the acetyl group of acetylCoA derived from the glycolytic pathway. Citrate can be transported out of mitochondria and converted back to acetyl CoA for fatty acid synthesis. Citrate is an allosteric modulator of both fatty acid synthesis (acetyl-CoA carboxylase) and glycolysis (phosphofructokinase). Citrate is widely used industrially in foods, beverages and pharmaceuticals. Citrate metabolism and disposition can vary widely due to sex, age and a variety of other factors.
2.1.7.1.3 Phosphate
Phosphate is one of the most important of the inorganic ions in biological systems. It functions in a variety of roles (Webb and Hunter, 1992). One of the most important roles is as a molecular switch, turning enzyme activity on and off through the mediation of the various protein kinases and phosphatases in biological systems. Phosphate is also of great importance in mineralization processes and is a primary stimulus of algal blooms frequently found in bodies of fresh water, due to run-off from areas of high fertilizer use (Webb, 1992)
2.1.7.1.4 Acid phosphatase
Prostatic acid phosphatase is one of the two antigenic markers of prostatic carcinoma, the other being prostate specific antigen. It belongs to the kallikrein family of serine proteases and is suggested to function as a transferase and as a hydrolase that split phosphatidyl choline in semen (Wolff et al., 1998).
2.1.7.2 Seminal Vesicles Function Indices
The function indices of the seminal vesicles are secretory substances produced by this organ. These include fructose and glutamate dehydrogenase. These and many more can serve as tools for determing the secretary role of the seminal vesicles.
2.1.7.2.1 Seminal vesicle – body weight ratio
This is the weight of the seminal vessicle divided by the weight of the animal. The seminal vessicles secrete fructose to provide an energy source for sperm and alkalinity to enhance sperm mobility. The high fructose concentrations provide nutrient energy for the spermatozoa. Therfore, perturbation in the weight of the seminal vesicle and/or the seminal vessicle–body weight ratios may affect the secretary content of the organ.
2.1.7.2.2 Fructose
Fructose is a monosaccharide found in many foods and is one of the three most important blood sugars along with glucose and galactose (Southgate, 1976). Fructose is the sweetest naturally occurring sugar, estimated to be twice as sweet as sucrose (AOAC, 1995). Fructose is phosphorylated by adenosine triphosphate (ATP) in the reaction catalyzed by hexokinase (Bergmeyer and Bernt, 1974). Fructose 6-phosphate is converted to glucose 6-phosphate by phosphoglucose isomerase (PGI). Glucose-6-phosphate (G6P) is then oxidized to 6-phosphogluconate in the presence of nicotinamide adenine dinucleotide (NAD) in the reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PDH). During this oxidation, an equimolar amount of NAD is reduced to NADH (Beuter et al., 1985).
2.1.7.2.3 Seminal Fluid Glutamate Dehydrogenase
Glutamate dehydrogenase (GDH) is an enzyme that converts glutamate to α-Ketoglutarate, and vice versa. It represents a key link between catabolic and metabolic pathways and is therefore ubiquitous in eukaryotes. Glutamate Dehydrogenase (GDH) is a mitochondrial enzyme that catalyzes the reversible oxidative deamination of glutamate to α -ketoglutarate and serves as a key link between anabolic and catabolic pathways. In mammals, GDH is subject to allosteric regulation and has high activity in liver, kidney, brain, and pancreas. GDH activity in serum can be used to differentiate between liver diseases due to liver inflammation, which do not show elevated serum GDH activity, and diseases that result in hepatocyte necrosis, which results in elevated serum GDH. 2.1.7.4.2
2.1.7.3 Testicular Function Indices
The testicular function indices are parameters that are used to measure the functional activities of the testes. Alteration, in the testicular function indices has become a diagnostic tool for the evaluation of various disease states and for testing organ functions (Burtis and Ashwood, 2001). The various testicular secretory constituents like total protein, cholesterol, sialic acid, glycogen and testosterone can be used to determine the functional capacity of the testes (Gupta et al., 2004).
2.1.7.3.1 Total protein
Proteins are the most abundant biological macromolecules occurring in all cells and all parts of the cells. Testicular proteins, which are androgen dependent, are one of the constituents that ensure the maturation of spermatozoa (Kasturi et al., 1995).
2.1.7.3.2 Sialic acid
Sialic acid is widely distributed throughout human tissues like the testes, kidney, brain and adrenal gland and acts as a ‘lubricant ‘to facilitate the movement of sperm and to reduce friction among spermatozoa (Riar et al., 1997). Increased sialic acid contents of the testis may enhance the structural integrity of acrosomal membrane, which ultimately may enhance the motility and fertilising capacity of spermatozoa (Chinoy and Bhattachary, 1997b).
2.1.7.3.3 Cholesterol
Cholesterol is a 27-carbon molecule with 46 hydrogen atoms and a lone oxygen atom. It has a fundamental framework of steroid which is a tetracyclic unit. It is a molecule of four rings of carbon and hydrogen attached to an eight-carbon tail on one end Cholesterol is the precursor in the synthesis of steroid hormone and its requirement for normal testicular activity has been well established (Bedwall et al., 1994; Watcho et al., 2004). Their synthesis requires relatively small amounts of cholesterol. A significant increase in testicular cholesterol period could imply stimulation of steroidogenesis, thereby leading to increased androgen concentration (Bedwall et al., 1994).
2.1.7.3.4 Glycogen
Glycogen is the main source of energy in the animal reproductive system and the largest fraction is contained in the seminiferous tubule (Re, 1984). However, its distribution is not uniform and varies according to the degree of maturity of seminiferous epithelium (Re, 1984). It plays an important role in the maturation of germ cells. Intra-tubular glycogen is abundant in the prepubertal stage, diminishes strikingly with the beginning of puberty, and reappears during the period of sexual maturation. The glycogen level in the seminiferous tubule shows a cyclic behaviour during spermatogenesis at puberty acting as a source of energy in the synthesis of DNA (Re, 1984). The activity of active phosphorylase, low in prepubertal life (Re, 1984), rises during the pubertal phase and is very high during spermatogenesis. In prepubertal testes, in the absence of DNA synthesis, glycogen is not used because of the lack of activation of phosphorylase (Re, 1984).
2.1.7.3.5 Testosterone
Testosterone (T) is ubiquitous among male vertebrates and plays a central role in the expression of numerous sexually selected traits and it is strongly associated with reproductive aggression and sexual behaviour (Wingfield et al., 1990). Hence, individual variation in testosterone production appears likely to be a key factor underpinning differential reproductive success in male vertebrates. It is also responsible for the establishment of secondary sexual characteristics and the promotion of spermatogenesis (Orth, 1993). In vertebrates, about 95% of the body’s testosterone is produced by Leydig cells that are located within the interstitial compartments of the testes, intimately associated with the seminiferous tubules (Thibault et al., 1993) while the remaining 5% body testosterone concentration is produced by the adrenal gland. Therefore, testicular testosterone is the level of testosterone that the testis synthesises at a given period. It is an important index to evaluate the synthetic capacity of the testis. Not all testosterone in the body is available to tissues for action. About 40 % is bound tightly to a protein called “sex hormone binding globulin” and is not available for action. About 58 % is bound to albumin and is available to most tissue. The remaining 2 % circulates freely in the blood (Wingfield et al., 1990). The levels of male testosterone change throughout the day with the highest in the morning (Brian et al., 2012).
2.1.7.3.6 Testes-body weight ratio
This is the weight of the testes divided by the weight of the animal. Testes are androgen-dependent organs, relying on testosterone for growth and function (Klinefelter and Hess, 1998). Therefore, increases in the testes–body weight ratios often reflect a bioavailability and production of androgens (Sujatha et al., 2001). It may also be a consequence of hypertrophy, spermatogenesis, steroid synthesis in the Leydig cells, and/or reduced tubule size.
2.1.7.3.7 Ascorbic acid
Ascorbic acid (Vitamin C) is an electron donor, water- soluble antioxidant vitamin in humans (Sebastian et al., 2003). Under stress conditions, it acts as a primary antioxidant in plasma and within cells that quenches ROS and serves as a cofactor for enzymes involved in the synthesis of collagen, neurotransmitters and carnitine (Sebastian et al., 2003). This helps in the strengthening of tissues. It is an abundant component of plants that is capable of neutralising ROS in the aqueous phase before lipid peroxidation is initiated. As a scavenger of reactive oxygen and nitrogen oxide species, ascorbic acid has been shown to be effective against the superoxide radical ion, hydrogen peroxide, hydroxyl radical, and singlet oxygen (Padayalty et al., 2003). Under physiological conditions, Vitamin C predominantly exists in its reduced form, Ascorbic Acid or Ascorbate. It also exists in trace quantities in the oxidised form, DHA (Dehydroascorbic Acid) (Padayalty et al., 2003).
Ascorbic acid serves as an antioxidant and may be beneficial for reducing the risk of developing chronic diseases such as cancer, cardiovascular disease, and cataracts (Padayalty et al., 2003). Ascorbic acid is also frequently used in the food industry as an antioxidant to prevent undesirable changes in colour, taste, and odour (Vislisel et al., 2007).
2.1.7.3.8 Fructose
2.1.7.2.2
2.1.7.3.9 Other Parameters for Testicular Function Evaluation
2.1.7.3.9.1 Bile acid
Bile is a complex mixture of lipids, protein, carbohydrates, mineral salts, vitamins, and various trace elements, with bile acids making up about 67 % of the total composition (Reshetnyak, 2013). Bile acids are produced from excess cholesterol, secreted from the liver, absorbed into the small intestines, and returned to the liver with portal blood. While bile acid synthesis is critical for the removal of cholesterol from the body, bile acids are also needed for proper uptake of dietary lipids, fat soluble vitamins, and other nutrients into the small intestines (Ambros-Rudoph et al., 2007). Under physiological conditions, newly synthesized bile acids are conjugated to glycine or taurine to form bile salts, and not much free bile acid is actually found in bile (Angelin et al., 1978).
Determining circulatory levels of bile acids can be used to identify or diagnose certain liver diseases (Setchell et al., 1997). In addition, elevated serum bile levels have been observed in intrahepatic cholestasis of pregnancy cases (Mashige et al., 1981).
2.1.7.3.9.2 17-ketosteroids
17-ketosteroids are substances that form when the body breaks down male steroid sex hormones called androgens and other hormones released by the adrenal glands in males and females, and by the testes in males (Williamson and Snyder, 2011).
2.1.7.3.9.2.1 Clinical Application
Normal values of 17-ketosteroids in males and females are 8 to 20 milligrams (mg) per 24 hours and 6 to 12 mg per 24 hours repectively (Zumoff et al., 1980). Normal value ranges may vary slightly among different laboratories. Increased levels of 17-ketosteroids may be due to adrenal gland problems such as tumor, Cushing syndrome; imbalance of sex hormones in females (polycystic ovary syndrome); ovarian cancer; testicular cancer (McPherson and Pincus, 2011). Decreased levels of 17-ketosteroids may be due to adrenal glands not making enough of their hormones (Addison's disease), kidney damage, pituitary gland not making enough of its hormones (hypopituitarism), and removal of the testicles (castration) (McPherson and Pincus, 2011).
2.1.7.4 Testicular enzymes
Enzymes are studied by measuring their activity to provide useful information on tissue/cellular damage (Coodley, 1970). It has been established that the site of injury and the extent of damage to the cell could be correlated and determined by assaying the level of activities of the “marker’ enzymes in such tissue (Ngaha, 1981).
2.1.7.4.1 Lactate dehydrogenase (EC 1.1.1.27)
Lactate dehydrogenase has the systemic name: L-lactate: NAD Oxidoreductase. Meyerhof first described its action in 1919. Lactate dehydrogenase is an anaerobic glycolytic enzyme which catalysis the reversible reduction of pyruvate to lactate utilizing nicotinamide adenine dinucleotide (reduced) as the redox co-enzyme (Figure 11). It was also reported that NADP might act as hydrogen donor though at a much lower rate (Rodwell, 1985). It is a tetramer of molecular weight of 140,000.
In the reverse direction, this reaction represents the last step in the process of anaerobic glycolysis and provides a means of regeneration of NAD+ required for the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase. The enzyme can also reduce α-keto and α, δ-di-keto acids. However, α-ketoglutarate, δ-keto and δ-keto acids are reduced at negligible rates (Lehninger, 1987). The optimum pH varies with the source of enzyme and depends on the temperature, substrate and concentration of buffer.
Fig. 12: Oxidoreduction Reaction Catalyzed by Lactate Dehydrogenase
Source: Rodwell (1985)
2.1.7.4.1.1 Distribution
Lactate dehydrogenase is present in all tissues of vertebrates particularly in cardiac and skeletal muscle, kidney, liver, brain, heart, eryhtrocytes and it has long been shown to be located in the extractable fraction in these tissues (Johnson, 1960).
Human red blood cells are rich in lactate dehydrogenase, having approximately 150 times the activity of plasma (Johnson, 1960). It has also been shown that the activity of the enzyme is highest in the proximal convoluted tubules and least in the thin parts of the loop of Henle with the glomeruli showing well -marked activity in the epithelial cells only (Davidson and Conning, 1968).
2.1.7.4.1.2 Activators and inhibitors
Lactate dehydrogenase activity is inhibited by reagent with reactivity against groups such as mercuric ions and p-chloromercuricbenzoate (Anderson et al., 1974). Borate and oxalate inhibit by competing with lactate for its binding site on the enzyme. Both pyruvate and lactate in excess inhibit lactate dehydrogenase activity, although, the effect of pyruvate is greater. Inhibition by either substrate is greater for the H-type than the M-type, and substrate inhibition decreases with increase in pH. It is activated by compounds such as dimethylsulfoxide, ethanol, methanol which stabilizes the enzyme (Burgner and Ray, 1984).
2.1.7.4.1.3 Isoenzymes
The heterogeneity of lactate dehydrogenase was first demonstrated by Wieland and Delederer (1957) who obtained two protein components from the beef heart. Later, Wiemer (1975) succeeded in separating five distinct lactate dehydrogenase forms in human serum based on their electrophoretic motilities and are designated as LDH-1, LDH-2, LDH-3, LDH-4 and LDH-5. The fastest electrophoretically moving isoenzymes LDH-1 and LDH-2 are predominant in cells of cardiac muscle, erythrocytes and kidney while the slowest moving isoenzymes, LDH-4 and LDH-5 are predominant in the liver and in skeletal muscle. A different, sixth LDH isoenzyme, LDH-X (also called LDC) is present in post-pubertal human testes. A seventh LDH isoenzyme called LDH-6 has been identified in the sera of severally ill patients, its identity remains uncertain (Carl and Edward, 1994). The different lactate dehydrogenase isoenzymes differ significantly in the maximum velocities (Vmax), in the Michelis constant (Km) for their substrates and the degree of their allosteric inhibition by pyruvate (Lehninger, 1987).
All the lactate dehydrogenase isoenzymes contain four polypeptide chains; each of
molecular weight 33,500, but the five isoenzymes contains varying ratios of two kinds of polypeptide, which differ in composition and sequence. The A-chains (also designated M for muscle) and the B-chain (also designated as H for heart) are coded by two different genes. In the skeletal muscle, the lactate dehydrogenase isoenzyme that predominates contains four A-chains and in heart the predominating isoenzymes contains four B-chains (Lehninger, 1987).
2.1.7.4.1.4 Clinical application
Lactate dehydrogenase levels in various tissues are very high compared with those of the serum. Thus, tissue levels are about 500 times greater than those normally found in the serum. However, leakage of the enzyme from damaged tissue can result in increased level in the serum.
It has been discovered that human sera contains several lactate dehydrogenase
isoenzymes and their relative proportion change significantly in certain pathologic conditions (Rodwell, 1985).
Erythrocytes are known to be rich in lactate dehydrogenase and therefore, the
slightest haemolysis can cause a significant rise in the serum level of this enzyme. Increase in lactate dehydrogenase activity in serum and other biological fluids have also been observed in pathological conditions of the heart, liver and the determination of the enzyme activities in these conditions has been of immense diagnostic value to the investigators (Wroblewski and La Due, 1955).
Urinary lactate dehydrogenase activity in humans and animals has been extensively investigated in normal and diseased conditions and its activity has been found to increase in infective hepatitis, infectious mononucleosis and toxic jaundice (Wroblewski et al., 1956).
However, toxic renal damage may be accompanied by an impairment of tubular re-absorptive capacity. This leads to the additional explanation for the elevated enzyme activity in the urine as reported by Wright and Plummer (1974) who found high levels of urinary lactate dehydrogenase activity in rats infected with urinyl nitrate. However, serum lactate dehydrogenase is normal in patient with acute febrile
and chronic infectious infarction, localized neoplastic disease and chronic disease processes (Rodwell, 1985).
2.1.7.4.2 Glutamate dehydrogenase (EC 1.4.1.3)
Glutamate dehydrogenase is a zinc containing enzyme that catalyses the reversible oxidative deamination of L-glutamate to α-ketoglutarate and ammonia (Figure 12).
The enzyme is of major importance because it occupies a pivotal position between carbon and nitrogen metabolism. There are three basic types: those that are specific for NAD+ (EC 1.4.1.2), those specific for NADP+ (EC 1.4.1.4) and those that can use both (EC 1.4.1.3) (dual coenzyme specific).
Most glutamate dehydrogenase is a homopolymer consisting of two to six subunits of molecular weight 40,000-60,000D. The most common number of subunit is six. In general, glutamate dehydrogenase catalyses the deamination of glutamate optimally at alkaline pH (8-10), while the pH optimum for reductive amination is 0.5-2 pH units lower. The glutamate dehydrogenases from vertebrate are dual coenzyme specific. Their in vivo reaction direction may be dependent on the biochemical status of the cell or on the tissue where they are found. Intracellular compartmentation of nicotinamide co-factors and of the glutamate dehydrogenase enzyme themselves, is a feature which further control their reaction direction and the destination of their end products (Smith et al., 1975)
Fig 12: Reaction catalysed by glutamate dehydrogenase
Source: Smith et al. (1975)
The properties of these enzymes are significantly different (with respect to their stability and susceptibility to guanosine triphosphate inhibitor), which suggest that they play different roles in controlling levels of ammonia and glutamate, the latter being important as a neurotransmitter (Shoemaker and Haley, 1993).
α-ketoglutarate L-glutamate Glutamate dehydrogenase from bovine liver is undoubtedly the most extensively studied. It has the molecular weight of 332,000D, has six subunits (Smith et al. 1975), and undergo polymerization process which may appear to be an in vivo control mechanism since the concentration of glutamate dehydrogenase in some tissues is high enough (>2mg/ml) to cause polymerization. In other tissues such as skeletal muscle, it is so low that polymerization is not feasible (Hudson and Daniel, 1993).
The enzyme has four binding sites per subunit and these include Site I (the active site), Site II (the adenine nucleotide binding site), Site III (the guanine binding site) and Site IV (the reduced coenzyme) (Shoemaker and Haley, 1993). Glutamate dehydrogenase from other vertebrate is similar in some respect to the bovine enzyme. The molecular weight of rat and chicken glutamate dehydrogenase are similar to bovine and they have also been shown to be able to aggregate (Smith et al., 1975).
2.1.7.4.2.1 Distribution
Glutamate dehydrogenase is widely distributed throughout the eukaryotic, eubacteria and archaebacteria kingdoms with few organisms being known to lack them. In mammals, it is found mainly in the liver, muscle and the kidney while a small amount occur in other tissues like the brain, skeletal muscle and leukocyte. In vertebrate cells, glutamate dehydrogenase appears to be localized primarily in the mitochondria matrix (Smith et al., 1975), but Colon et al. (1986) have isolated both a soluble (mitochondria matrix) and a particulate (membrane bound) glutamate dehydrogenase from rat brain.
2.1.7.4.2.2 Activators and inhibitors
They have complex regulatory effect mediated by purine nucleoside phosphate and a variety of other metabolic intermediate including hormones. Glutamate dehydrogenase of bovine liver is also inhibited by guanosine and inosine nucleotide while adenosine diphosphate and adenosine monophosphate activates it. ATP has very little stimulatory effect (Hudson and Daniel, 1993).
2.1.7.4.2.3 Isoenzymes
Glutamate dehydrogenase enzyme found in human tissue has been shown to contain isoenzymes. The isoenzyme has been separated by agar gel electrophoresis into as many as six isoenzymes (Vanderhelm, 1962).
2.1.7.4.2.4 Clinical application
Owing to low absolute levels of activity of glutamate dehydrogenase in serum, low precision of assay and particularly the precision of the transaminase: glutamate dehydrogenase ratio used in diagnosis, there are difficulties in making use of the enzyme (Tietz, 1995).
However, despite low levels of glutamate dehydrogenase in the serum normally, elevations of its level have been noticed in uncomplicated viral hepatitis, chronic hepatitis and cirrhosis. Large-scale increase in serum glutamate dehydrogenase occurs in halothane toxicity and in response to some other hepatotoxic agents. Compared with other hepatocellular enzymes, increase in the activity of glutamate dehydrogenase in serum is disproportionately large and may reach ten to twenty times the normal value.
2.1.7.4.3 Alkaline Phosphatase (E.C. 3.1.3.1)
Alkaline phosphatase (ALP) refers to a group of phosphomonoesterases that hydrolyze phosphate esters at a pH of 10 (Walker et al., 1990). It is an enzyme that catalyzes the hydrolysis of phosphate monoesters (e.g. glycerophosphate and para-nitrophenyl phosphate) with the release of inorganic phosphate and alcohol (Figure 8).
OPO3H3 OH
ALP
+ H2O + PO42-
pH 10.1 Mg2+
NO2 NO2
P-nitrophenyl phosphate P-nitrophenol
Fig. 8: Hydrolysis of P-nitrophenyl phosphate by alkaline phosphatase
Source: Wright et al. (1972)
2.1.7.4.3.1 Distribution
It is widely found in mammals, fish, amphibians, reptiles, birds and bacteria (Yola and Sakagishi, 1986). In humans, alkaline phosphatase is present in all tissues throughout the entire body, but is particularly concentrated in the liver, bile duct, kidney, bone, reticuloendothelial tissue and placenta (Walker et al., 1990).
2.1.7.4.3.2 Activators and Inhibitors
Alkaline phosphatase is activated by some divalent ions such as magnesium ion (Mg2+), manganese ions (Mn2+), and cobalt ion (Co2+). In addition, glycine and alanine at low concentrations increases serum alkaline phosphatase activity (Stigbrand, 1984).
Zinc ion (Zn2+) is a constituent metal ion but is inhibited by calcium ion, phosphate ion, borate, cyanide ion, adrenalin, adrenochrome, beryllium, copper ion, mercuric ion, sulphide and oxidizing agents (Anderson, 1968).
2.1.7.4.3.3 Isoenzymes
Humans and most other mammals contain the following alkaline phosphatase isozymes: ALPI – intestinal, ALPL – tissue non-specific (liver/bone/kidney). ALPP–placental (Regan isozyme). Heating for approximately 2 hours at 650C inactivates most isoenzymes except the Placental isoforms (PALP and SEAP) (Garen and Levinthal, 1960).
2.1.7.4.3.4 Clinical significance
Bile ducts are blocked when there is a high ALP level (Lanzer and Leb, 1983). Elevated ALP indicates that there could be active bone formation occurring as ALP is a by-product of osteoblast activity (such as the case in Paget's disease of bone). Lowered levels of ALP are less common than elevated levels. Placental alkaline phosphatase is elevated in seminomas (Lange et al., 1982; Berk and Korenblat, 2007).
Reduced levels of alkaline phosphatase can be seen in conditions or diseases such as hypophosphatasia, an autosomal recessive disease. Postmenopausal women receiving estrogen therapy because of osteoporosis, men with recent heart surgery, malnutrition, magnesium deficiency, hypothyroidism or severe anemia, children with achondroplasia and cretinism, children after a severe episode of enteritis, pernicious anemia, aplastic anemia, chronic myelogenous leukemia (Schiele et al., 1998).
2.1.7.4.4 Acid phosphatase (EC 3.1.3.2)
Acid phosphatase (orthophosphoric monoester phosphohydrolase) includes all phosphatase with optimal activity below pH value of 7.0 and the majority of instance
at pH values between 3.8 and 6.0 (Tietz, 1995). However, the optimum pH varies with the substrates; the more acidic the ester, the greater is the hydrolysis and further from neutrality is the optimum pH. Thus, the name acid phosphatase refers to a group of similar or related enzymes rather than that derived from prostate, which has a pH optimum in the range of pH 5-6. The enzyme can hydrolyse a variety of phosphate esters as shown in Figure 14 and indeed every substrate utilized in measuring alkaline phosphatase activity has also been used to determine acid phosphatase (Tietz, 1995).
Acid phosphatase is present in lysosmes and thus used as a ‘marker’ enzyme for the lysosomal membrane (de Duve et al., 1962; Collins and Lewis, 1971). Extralysosomal acid phosphatases are also present in many cells (Tietz, 1995). Davidson and Conning (1968) showed that acid phosphatase has intense activity in the convoluted tubules of rat kidney and moderate activity in all part of glomerulus. It is mainly excreted in urine (Ctiktin and Trujilo, 1970).
Davis (1934) has shown that the acid phosphatase of red cell differs from that of spleen in that the former hydrolyses α-glycerolphosphate much more readily than the β compound, while the spleen acid phosphatase hydrolyses β-glycerolphosphate more quickly.
OPO3H3 OH
ACP
+ H2O + PO42-
pH 4.5 Mg2+
NO2 NO2
Paranitrophenyl phosphate paranitrophenol
Fig 14: Hydrolysis of paranitrophenyl phosphate by acid phosphatase
Source: Wright et al (1972b)
2.1.7.4.4.1 Distribution
Acid phosphatase has been found to be present in many tissues which include kidney (Shibko and Tappel, 1965), erythrocyte (Abdulfadl and King, 1949), prostatic fluid (Wilkinson, 1963), liver, bone, milk and saliva. The greatest concentrations of
acid phosphatase activity occur in liver, spleen, milk, erythrocyte, platelets, bone marrow and prostate gland (Tietz, 1995). The presence of acid phosphatase has also been reported in higher plants (Axelrod, 1974), and yeast cells.
2.1.7.4.4.2 Activators and inhibitors
Abdulfadl and King (1949) have shown that magnesium, calcium, cobalt, manganese, chromium, nickel and zinc all have variable inhibitory effect on acid phosphatase, particularly at optimum pH. However, metal complexing compound do not inhibit acid phosphatase, but may slightly activate them (Abdulfadl and King, 1949). Similarly, mammalian erythrocyte acid phosphatase has been reported to be activated by magnesium (Davis, 1934).
The major inhibitor of the enzyme is tartarate (Zimmerman and Seefi, 1970).
Copper ions in acetate buffer strongly inhibit acid phosphatase of red blood cells, but
with a slight effect on the prostatic enzyme (Abdulfadl and King, 1949). It is also inhibited by acetyl salicylic acid (Anderson, 1968).
2.1.7.4.4.3 Isoenzymes
In normal serum, as many as five isoenzymes have been identified (Grundig et al., 1965). Use of differential inhibition of acid phosphatase isoenzyme has also been employed in the identification of diseased organs (Abdulfadl and King, 1949).
2.1.7.4.4.4 Clinical application
Variations in serum acid phosphatase activity have been widely used in the diagnosis of many diseased states. For example, increase in serum acid phosphatase level has been used in the diagnosis of prostatic cancer (Zimmerman and Seefi, 1970). The highest concentration is present in the prostate and detection of prostatic carcinoma by monitoring elevated activity of acid phosphatase has been a very useful tool in the diagnosis of the disease (Horder and Wilkinson, 1979).
Elevation of acid phosphatase may arise as a result of increased erythrocyte destruction (Ryman, 1978). The elevated urinary acid phosphatase in patients suffering from chronic renal failure could be used as an important index of kidney disease (Kobayashi et al., 1971) and can also be used in the investigation of rape and similar offences (Tietz, 1995).
2.1.7.4.5 Gamma glutamyl transferase (EC 2.3.2.2)
Gamma glutamyl transferase (GGT) or (γ-GT) is a membrane localized enzyme that plays a major role in glutathione metabolism and resorption of amino acids from the glomerular filterate and from the intestinal lumen (Kaplan and Pesce, 1996). It is an enzyme derived from the endoplasmic reticulum of the cells of the hepatobiliary Tract (Mayne, 1998). This enzyme was originally termed a transpept idase, but the more appropriate term is transferase (Burtis and Ashwood, 2001).
This enzyme acts only on peptides or peptide-like compounds containing a terminal glutamate residue joined to the remainder of the compound through the terminal carboxyl (Tietz, 1995). They catalyze the transfer of amino acids from one peptide to another amino acid or peptide (Burtis and Ashwood, 2001), as shown in Figure 15.
Fig 15: Reaction catalysed by Gamma glutamyl transferase
Source: Burtis and Ashwood (2001)
Beyond the fact that glycylglycine is five times more effective as an acceptor than is either glycine or the tripeptide (gly-gly-gly), little is known about the optional properties of the acceptor co-substrate (Burtis and Ashwood, 2001).
2.1.7.4.5.1 Distribution
The enzyme is found in a number of tissues. It occurs mainly in the cells of the liver, kidney, pancreas and prostrate. It is also present in the plasma membrane of renal tubular cells and in the endoplasmic reticulum of the hepatocytes (Murray et al., 2000). Also, GGT was detected intensively in epithelial cells of the epididymis and seminal vesicles (Kohdaira et al., 1986).
2.1.7.4.5.2 Activators and inhibitors
The activity of the enzyme increases with sexual maturation in epididymis and
seminal vesicles, but not in prostate (Kohdaira et al., 1986). Several inhibitors of GGT in vitro [L-serine plus borate, 6-diazo-5-oxo-norleucine, and L- and D-γ-glutamyl- (O-carboxy) phenylhydrazine] are also active in vivo. The hydrizides
(both L and D isomers) are the m ost potent inhibitor of gamma glutamyl transferase
in vivo and in vitro and this is always accompanied by extensive glutathionuria (Komlosh et al., 2002).
2.1.7.4.5.3 Isoenzymes
Five distinct zones of the enzyme have been demonstrated on acrylamide gel electrophoresis, in normal adult serum, serum from normal pregnant women and from the cord blood (Huseby, 1982). The enzyme is in two molecular forms in the serum (the predominant amphiphilic form and the hydrophilic form) (Huseby, 1982).
2.1.7.4.5.4 Clinical applications
Even though, tissue has the highest level of GGT, the enzyme present in serum
appears to originate primarily from hepatobiliary system, and GGT activity is elevated in any and all forms of liver disease. Measurement of plasma GGT activity is probably the most sensitive test for liver disease (Tietz, 1995). Since the plasma GGT activity is not raised in bone disease, its measurement allows the determination of the
likely origin of a raised plasma alkaline phosphatase activity (Kaplan and Pesce,1996). Although, plasma GGT may be increased after myocardial infarction and other diseases, this is thought to be attributable to secondary liver involvement (Mayne, 1998). It is more sensitive than alkaline phosphatase, leucine aminopeptidase, and the transaminases in detecting obstructive jaundice, cholangitis and cholecystitis (Mayne, 1998).
Gamma glutamyl transferase in urine probably originates from the kidney and genitourinary tract. Elevated enzyme activity is found in the urine of patient with acute urorenal infections and in diseases involving renal tissue destruction. However, in chronic renal diseases and in older individuals, urine enzyme levels may be depressed (Burtis and Ashwood, 2001).
High levels of GGT are also present in the prostate, and this may account for the fact that the activity of GGT in sera of males is approximately 50% higher than in sera from females (Kaplan and Pesce, 1996).
2.1.7.4.6 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase (EC 1 .1 .1 .34)
3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is a transmembrane glycoprotein, located on the endoplasmic reticulum (Koning et al., 1996). This enzyme catalyzes the four-electron reduction of HMG-CoA to coenzyme A (CoA) and mevalonate, which is the rate-limiting step in sterol biosynthesis (Holdgate et al., 2003). The activity of HMGR is controlled through synthesis, degradation, and phosphorylation in order to maintain the concentration of mevalonate derived products. In addition to the physiological regulation of HMGR, the human enzyme has been targeted successfully by drugs in the clinical treatment of high serum cholesterol levels (Istvan, et al., 2000; Istvan and Deisenhofer, 2000). Controlling serum cholesterol levels has an important therapeutic role as hypercholesterolemia often leads to the development of atherosclerosis and consequently to cardiovascular pathologies, which might result in myocardial infarction and stroke. Recent evidence suggests that a disturbance of cholesterol homeostasis contributes to the development of a chronic inflammatory state (Kleemann and Kooistra, 2005)
2.1.7.4.6.1 Distribution
3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase is the rate-controlling enzyme of the mevalonate pathway, the metabolic pathway that produces cholesterol from acetyl-CoA. In an NADPH-dependent reaction, HMG-CoA reductase reduces HMG-CoA to generate mevalonate and CoA. The enzyme is target of a group of cholesterol-lowering drugs known as statins. Inhibition of HMG-CoA reductase induces expression of LDL receptors in the liver, which lowers plasma concentration of cholesterol.
2.1.7.4.6.2 Inhibitors
Drugs that inhibit HMG-CoA reductase, known collectively as HMG-CoA reductase inhibitors (or "statins"), are used to lower serum cholesterol as a means of reducing the risk forcardiovascular disease (Farmer, 1998). These drugs include rosuvastatin (CRESTOR), lovastatin (Mevacor), atorvastatin (Lipitor), pravastatin (Pravachol), fluvastatin (Lescol), pitavastatin (Livalo),and simvastatin(Zocor). HMG-CoA reductase is active when blood glucose is high. The basic functions of insulin and glucagon are to maintain glucose homeostasis. Thus, in controlling blood sugar levels, they indirectly affect the activity of HMG-CoA reductase, but a decrease in activity of the enzyme is caused by an AMP-activated protein kinase, which responds to an increase in AMP concentration, and also to leptin.
2.1.7.4.6.3 Clinical significance
Since the reaction catalysed by HMG-CoA reductase is the rate-limiting step in cholesterol synthesis, this enzyme represents the sole major drug target for contemporary cholesterol-lowering drugs in humans. The medical significance of HMG-CoA reductase has continued to expand beyond its direct role in cholesterol synthesis following the discovery that statins can offer cardiovascular health benefits independent of cholesterol reduction (Arnaud et al., 2005). Statins have been shown to have anti-inflammatory properties (Sorrentino and Landmesser, 2005) most likely as a result of their ability to limit production of key downstream isoprenoids that are required for portions of the inflammatory response. It can be noted that blocking of isoprenoid synthesis by statins has shown promise in treating a mouse model of multiple sclerosis, an inflammatory autoimmune disease (Stüve et al., 2003).
HMG-CoA reductase is an important developmental enzyme. Inhibition of its activity and the concomitant lack of isoprenoids that yields can lead to germ cell migration defects (Thorpe et al., 2004) as well as intracerebral hemorrhage (Eisa-Beygi et al., 2013).
2.1.7.4.7 Sorbitol dehydrogenase (EC 1.1.1.14)
This enzyme, L-iditol dehydrogenase or sorbitol dehydrogenase catalyzes the reversible oxidation-reduction reaction between sorbitol and fructose. Sorbitol dehydrogenase has been identified in several human and animal tissues (Dooley et al., 1979). It is located primarily in the cytoplasm and mitochondria of the liver, kidney and seminal vesicles. SDH activity in serum is usually low but increases during acute episodes of liver damage (Dooley et al., 1979). Measurement of SDH is a specific indicator of liver cell damage andparenchymal hepatic diseases (Secchi et al., 1962). SDH activity rises rapidly in liver damage and decreases very shortly after peaking.
Sorbitiol dehydrogenase [SDH] catalyzes the reversible oxidation reduction of sorbitol, fructose and NADH. It is primarily found in the cytoplasm and mitochondria of liver, kidney and seminal vesicles. It is a specific marker of acute hepatocellular injury (Ozer et al., 2008) that functions across preclinical species like rodents, rhesus monkey and beagle dogs. Normal levels in the plasma are in the range of 1-3 U/L.
2.1.7.4.7.1 Clinical significance
In tissues where sorbitol dehydrogenase is low or absent, such as in the retina, lens, kidney, and nerve cells, sorbitol can accumulate under conditions of hyperglycemia. In uncontrolled diabetes, large amounts of glucose enter these tissues and is then converted to sorbitol by aldose reductase. Sorbitol then accumulates, causing water to be drawn into the cell due to the increased osmotic pressure, impairing tissue function. Retinopathy, cataract formation, nephropathy, and peripheral neuropathy seen in diabetes are partly due to this phenomenon Harvey and Ferrier, 2011).
2.1.7.4.8 Malic enzyme (E.C. 1.1.1.40)
Malic enzyme (Malate dehydrogenase decarboxylating) catalyzes the oxidative carboxylation of L-malate to pyruvate using NAD as a co-substrate (Spina and Bright, 1966).
(L)-malate + NADP+ pyruvate + CO2 + NADPH
Thus, the two substrates of this enzyme are (L)-malate and NAD+, whereas its three products are pyruvate, CO2, and NADH. Malate isoxidized to pyruvate and CO2, and NAD+ is reduced to NADH.
This enzyme belongs to the family of oxidoreductases, to be specific, those acting on the CH-OH group of donor with NAD+ or NADP+ as acceptor. The systematic name of this enzyme class is (L)-malate: NAD+ oxidoreductase (decarboxylating). This enzyme participates in pyruvate metabolism and carbon fixation. NAD-malic enzyme is one of three decarboxylation enzymes used in the inorganic carbon concentrating mechanisms of C4 and CAM plants. The others are NADP-malic enzyme and PEP carboxykinase (Christopher and Holtum, 1996; Kanai and Edwards, 1999).
2.1.7.4.8.1 Distribution
The absolute activity in the cytoplasm is greatest in liver followed by heart, skeletal muscle and brain (Bergmeyer et al., 1974). MDH is also a periportal enzyme that is released into the serum indicating tissue damage.
2.1.7.4.8.2 Clinical Significance
Normal value of MDH in healthy human plasma is 23.5-47.7 U/L. MDH activity was utilized as a biochemical index of acetaminophen induced liver injury that coincided with histological evidence of necrosis in rats (Zieve et al., 1985). Elevations in MDH activity was found to correlate with morphological changes aftr dosing with thioacetamide, dimethylonitrosamine and diethanolamine (Korsrud et al., 1972). The measurements of MDH activity were reported to be more useful in estimating the severity of liver injury than similar AST measurements (Kawai and Hosaki, 1990). Ozer et al. (2008) suggested that MDH can be utilized as a novel enzymatic serum liver biomarker.
2.1.7.4.9 17-β-hydroxy steroid dehydrogenase (EC 1.1.1.51)
17β-Hydroxysteroid dehydrogenases (EC 1.1.1.51, beta-hydroxy steroid dehydrogenase, 17-ketoreductase, 17beta-hydroxy steroid dehydrogenase, 3beta-hydroxysteroid dehydrogenase, 3beta-hydroxy steroid dehydrogenase, 17β-HSD, 17-ketosteroid oxidoreductases, HSD17B, 17-ketosteroid reductases, 17-KSR), are a group of alcohol oxidoreductases which catalyse thedehydrogenation of 17 hydroxysteroids in steroidogenesis (Dahm and Breuer, 1964). This includes interconversionof DHEA and androstenediol,androstenedione and testosterone,and es rone and estra iol, respectively (Labrie et al., 1997; Charles et al., 2011).
2.1.7.4.10 3-β-hydroxysteroid dehydrogenase (EC 1.1.1.145)
3-β-HSD (or 3-β-hydroxysteroid dehydrogenase/Δ-5-4 isomerase) (EC 1.1.1.145) is an enzyme that catalyzes the synthesis of progesterone from pregnenolone, 17 hydroxyprogesterone from 17-hydroxypregnenolone, and androstenedione from dehydroepiandrosterone in the adrenal gland. It is the only enzyme in the adrenal pathway of corticosteroid synthesis that is not a member of the Cytochrome P450 family (Cravioto et al., 1986). In humans, there are two 3-β-HSD isozymes encoded by the HSD3B1 and HSD3B2 genes.
2.1.7.4.10.1 Isozymes
Humans express two 3-β-HSD isozymes, HSD3B1 (type I) and HSD3B2 (type II) (Simard et al., 2005). The type I isoenzyme is expressed in placenta and peripheral tissues, whereas the type II 3β-HSD isoenzyme is expressed in the adrenal gland, ovary, and testis. Testosterone production in Leydig cell utilizes cholesterol as a substrate. Conversion of the cholesterol substrate to testosterone occurs in a series of reactions catalyzed by four enzymes: cytochrome P450 cholesterol side-chain cleavage enzyme, 3β hydroxysteroid dehydrogenase (3β-HSD) isoform, cytochrome P450 17α-hydroxylase/17-20 lyase, and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3). Hydroxysteroid dehydrogenases (3β-HSD and 17β-HSD3) are localized to the smooth endoplasmic reticulum in Leydig cells. There are two 3β-HSD isoforms in humans, types 1 and 2. Type 1 is expressed in the placenta (Le Bail et al., 2000) and type 2 is expressed in the testis (Simard et al., 2005).
2.1.7.4.10.2 Clinical significance
A deficiency in the type II form through mutations in HSD3B2 is responsible for a rare form of congenital adrenal hyperplasia (Rhéaume et al., 1992). No human condition has yet been linked to a deficiency in the type I enzyme. Its importance in placental progesterone production expression suggests that such a mutation would be embryonically lethal.
The fetal adrenal cortex lacks expression of the enzyme early on, thus mineralocorticoids (i.e. aldosterone) and glucocorticoids (i.e. cortisol) cannot be synthesized. This is significant because cortisol induces type II pneumocytes of the lungs to synthesize and secrete pulmonary surfactant; without pulmonary surfactant to reduce the alveolar surface tension, premature neonates may die of neonatal respiratory distress syndrome. If delivery is unavoidable (i.e. because of placental abruption, or pre-eclampsia/HELLP syndrome), then glucocorticoids (i.e. cortisol) can be administered to induce type II pneumocytes to synthesize and secrete pulmonary surfactant, improving the chances of newborn survival (by preventing neonatal respiratory distress syndrome).
2.1.7.4.11 Glucose-6-phosphate dehydrogenase (EC 1.1.1.49)
Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the conversion of glucose-6-phosphate to 6-phosphoglucono-δ-lactone, the first and rate-limiting step of the pentose phosphate pathway (PPP). The PPP pathway is critical for maintaining the cofactor nicotinamide adenine dinucleotide phosphate (NADPH) and for the production of pentose sugars. The NADPH produced is critical for redox regulation via the regeneration of GSH and for providing reducing equivalents for fatty acid biosynthesis. Deficiencies in G6PDH predisposes individuals to non-immune hemolytic anemia.
2.1.7.4.11.1 Distribution
G6PD is widely distributed in many species from bacteria to humans. In higher plants, several isoforms of G6PDH have been reported, which are localized in the cytosol, the plastidic stroma, and peroxisomes (Corpas et al., 1998). Among humans, G6PD is common in certain insular groups, such as Parsis (G6PD Enzyme Deficiency, 2014).
2.1.7.4.11.2 Clinical Significance
G6PD is remarkable for its genetic diversity. Many variants of G6PD, mostly produced from missense mutations, have been described with wide ranging levels of enzyme activity and associated clinical symptoms. Two transcript variants encoding different isoforms have been found for this gene.
Glucose-6-phosphate dehydrogenase (G6PD), the principal source of NADPH, serves as an antioxidant enzyme to modulate the redox milieu and nitric oxide synthase activity. Deficient G6PD activity is associated with increased endothelial cell oxidant stress and diminished bioavailable nitric oxide (NO). G6PD, the first and rate-limiting enzyme of the pentose phosphate pathway, catalyzes the synthesis of riboses for nucleic acid production and is the principal intracellular source of NADPH.
2.1.7.5 Antioxidant Assay System
2.1.7.5.1 Total antioxidant capacity (TAC)
Reactive oxygen species (ROS) are produced as a consequence of normal aerobic metabolism. Unstable free radical species attack cellular components causing damage to lipids, proteins, and DNA which can initiate a chain of events resulting in the onset of a variety of diseseases (Halliwell, 1996). Living organisms have developed complex antioxidant systems to counteract ROS and to reduce their damage. These antioxidant system includes enzymes such as superoxide dismutase, catalase, and glutathione peroxidase; macromolecules such as albumin, ceruloplasmin, and ferritin; and array of small molecules, including ascorbic acid, α-tocopherol, β-caratene, reduced glutathione, uric, and bilirubin (Benzie and Strain, 1996). The serum of endogenous and food-derived antioxidants represents the total antioxidant activity of the system. The cooperation among different antioxidant provides greater protection against attack by reactive oxygen or nitrogen species, than any single compound alone (Miller et al., 1993). Thus, the overall antioxidant capacity may provide more relevant biological information compared to that obtained by the measurement of individual components, as it considers the cumulative effect of all antioxidants present in plasma and body fluids (Rice-Evans and Miller, 1994).
2.1.7.5.2 Malondialdehyde (MDA)
MDA is one of the products of lipid peroxidation, which is mutagenic in bacterial and mammalian cells and carcinogenic in rats. MDA can react with DNA bases G, A and C to form adducts M1G, M1A and M1C respectively (Marnett, 1999). M1G adducts were found to range in tissue at levels ranging from below the limit of detection to as high as 1.2 adducts per 10 raise to power 6 nucleosides (which corresponds approximately 6000 adducts per cell). M1G has also been detected in human breast tissue by 32P-post-labelling as well as in rodent’s tissues (Wang et al., 1996). Site- specific experiments confirmed that M1G is mutagenic in E. coli, inducing transversion to T and transitions to A (Fink, Reddy & Marnett, 1997).
2.1.7.6 Histology
Histology is the study of the microscopic structure of cells and tissues and the ways in which individual components are structurally and functionally related (Anthony, 2013). Histopathology is the study of tissue’s architecture in a diseased state. It involves the use of microscopes in monitoring manifestations of diseases. Several processes involved include: tissue sampling and identification, fixation, dehydration, clearing, infiltration and embedding.
2.2 Female reproductive system
The structures of the reproductive system in a human female are shown in Figure 2. The primary function of the female reproductive system is to produce gametes, the
specialized cells that contribute half of the total genetic material of a new individual. The female reproductive system has several additional functions: to be the location for
fertilization, to protect and nourish the new individual during the gestation period, and to nourish the newborn postpartum, through lactation and nursing (Weck, 2002). The female's external reproductive structures include: the clitoris and two sets of labia
which surround the clitoris and vaginal opening. The internal organs are a pair of gonads (ovaries) and a system of ducts and chambers that carry gamates and house the embryo and fetus (Campbell and Reece, 2005). The functions of the female reproductive organs are summarized in Table 2.
Fig. 3: Organization of the human female reproductive system.The ovaries are the gonads, the fallopian tubes receive the ovulated ova, and the uterus is the womb, the site of development of an embryo if the egg cell becomes fertilized
Source:
2.2.1 Hormonal Regulation in Females: Hormonal Control of Ovaries
The overall process of conception is controlled by hormones of the pituitary and hypothalamus (Sophie, 2008). Hypothalamus control the release of the hormone gonadotropin releasing hormone which stimulates the pituitary gland from releasing luteinizing hormone (LH) and follicle stimulating hormones (FSH) which enters the blood stream and are carried to the ovaries (Anne and John, 2000). LH and FSH act synergistically to regulate reproduction. FSH stimulates the growth of the follicles, one of which eventually developed into an egg (Dickerson et al., 2008). As follicles grow, they release estrogen into the blood stream, a hormone that significantly causes changes in the physiology of women; responsible for enlargement of breast and give them a more rounded shape than men (Roberts et al., 2004). A high level of estrogen halts production of FSH by the pituitary gland via negative feedback inhibition loop and stimulates the release of luteinizing hormone (Anne and John, 2000). Luteinizing hormone (LH) causes the ‘ripening’ of the follicle and the release of the egg. The follicles under the influenced of LH, form a yellow area in the area called ‘corpus luteum’ which secretes large amount of progesterone in addition to the estrogen. Progesterone like estrogen plays several roles in women’s body but its principal function is to prepare the uterus for implantation of fertilized egg. This is achieved in two ways; first is the thickening of the uterus lining and secondly is to increase the number of glands in the lining (Anne and John, 2000). These glands increase the fluid content of the uterus and provide nourishment for the embryo. In addition to estrogen, another hormone, inhibin, down-regulates FSH synthesis (Chen et al., 2006). Prolactin plays an important role in the production of breast milk during lactation (Stallings et al., 1996). It is generally supposed that there is a standard normal menstrual cycle which last for 28 days, from the first day of one period to the first day of the next. However, unless taken hormones or supplements, the menstrual cycle varies in length from one month to the other depending on the emotional and physiological state of the woman (Anne and John, 2000). The varying length of the cycle will thus causes differences in the day at which the egg is released from the ovary (ovulation) which is always at interval of 14 days before menstruation. A simplified outline of the interrelationship of these substances in the physiological control of the menstrual cycle is given in Figure 2. This is how long it takes for the corpus luteum to disappear and for the lining to begin to break down and this is why the ‘rhythm’ method of family planning is liable to failure since the date of ovulation cannot be known in advance due to susceptibility of the cycle to variations.
2.2.2 Female Reproductive Hormones
2.2.2.1 Progesterone
Progesterone (4-pregnene-3, 20-dione) is a C21 steroid hormone containing a keto-group (at C-3) and a double bond between C-4 and C-5. Like other steroids, it is synthesized from cholesterol via a series of enzyme-mediated steps (Charles et al., 1973). Progesterone is a female sex hormone of primary importance in ovulation, fertility and menopause. It is particularly important in preparing the endometrium for the implantation of the blastocyte and in maintaining pregnancy (Ross et al., 1983). The rate of progesterone secretion may be affected by the degree of progestational activity of the uterus and the level of circulating LH (Pedersen et al., 2003). Analyses suggest that progesterone acts as an anti-glucocorticoid in rat adipose tissue in vivo, attenuating the glucocorticoid effect on adipose tissue metabolism (Pepe and Rothchild, 1974). Furthermore it could be demonstrated that progesterone alone may be a valuable agent for management of postmenopausal osteoporosis (Barengolts et al., 1990). In female rodents, the determination of progesterone is a useful marker in evaluating and monitoring the state of the reproductive functions and pregnancy as well.
Progesterone is synthesized from both tissue and circulating cholesterol. Cholesterol is transformed to pregnenolone which is then converted via a combined dehydrogenase and isomerase to progesterone (Chattoraj, 1976). The principle production sites are the adrenals and ovaries and the placenta during pregnancy. The majority of this steroid is metabolized in the liver to pregnanediol and conjugated as a glucuronide prior to excretion by the kidneys (Tietz, 1995).
2.2.2.1.1 Biochemical Role of Progesterone
Progesterone exhibits a wide variety of end organ effects. The primary role of progesterone is exhibited by the reproductive organs (Tietz, 1995). In males, progesterone is a necessary intermediate for the production of corticosteroids and androgens. In females, progesterone remains relatively constant throughout the follicular phase of the menstrual cycle (Ross et al., 1981). The concentration then increases rapidly following ovulation and remains elevated for 4-6 days and decreases to the initial level 24 hours before the onset of menstruation. In pregnancy, placental progesterone production rises steadily to levels of 10 to 20 times those of the luteal phase peak.
Progesterone measurements are thus performed to determine ovulation as well as to characterize luteal phase defects. Monitoring of progesterone therapy and early stage pregnancy evaluations comprise the remaining uses of progesterone assays (Radwanska et al., 1978).
2.2.2.2 Estradiol
Estradiol (17β-estradiol) is the major estrogen secreted by the premenopausal ovary. Estrogens direct the development of the female genotype in embryogenesis and at puberty. In addition, estradiol is an important luteolytic agent in humans (Erickson, 1995). Estradiol is synthesized from testosterone primarily in the ovarian granulosa cells and plancenta, but small amounts can be produced in the adrenal gland (Erickson, 1995; Miller and Tyrrell, 1995). The conversion of testosterone to estradiol is accomplished by the aromatase system, which consists of the three enzyme activities localized to the endoplasmic reticulum of these tissues (Vance, 1988; Erickson, 1995). Plasma estradiol levels increase gradually between days 1 – 7 of the menstrual cycle followed by a sharp increase to a peak value of about 300 pg/ml on day 12 just prior to ovulation (Munro et al., 1991; Erickson, 1995).
Estradiol (E2) is the most potent natural estrogen, produced mainly by the ovary, placenta, and in smaller amounts by the adrenal cortex, and the male testes (Tsang et al., 1980) Estradiol is secreted into the blood stream where 98% is bound to sex hormone binding globulin (SHBG). Estrogenic activity is affected via estradiol-receptor complexes, which trigger the appropriate response at the follicles, uterus, breast, vagina, urethra, pituitary, hypothalamus, and to a lesser extent the liver and skin (Gore-Langton and Armstrong, 1988).
In non-pregnant women with normal menstrual cycles, estradiol secretion follows a cyclic, biphasic pattern with the highest concentration found immediately prior to ovulation (Baird, 1976). During pregnancy, maternal serum estradiol levels increase considerably, to well above the pre-ovulatory peak levels and high levels are sustained throughout pregnancy (Abraham et al., 1972). Serum estradiol measurements are a valuable index in evaluating a variety of menstrual dysfunctions such as precocious or delayed puberty in girls, and primary and secondary amenorrhea and menopause. Estradiol levels have been reported to be increased in patients with feminizing syndromes, gynaecomastia, and testicular tumors (Simpson and MacDonald, 1981). In cases of in fertility, serum estradiol measurements are useful for monitoring induction of ovulation following treatment (Tietz, 1995).
2.2.2.3 Estrogen
Estrogen or oestrogen is the primary female sex hormone and is responsible for development and regulation of the female reproductive system and secondary sex characteristics. Estrogen may also refer to any substance, natural or synthetic that mimics the effects of the natural hormone (Raloff, 1997). The steroid 17β-estradiol is the most potent and prevalent endogenous estrogen, but several metabolites of estradiol also have estrogenic hormonal activity. Synthetic estrogens are used as part of some oral contraceptives, inestrogen replacement therapy for postmenopausal women, and in hormone replacement therapy for trans women (Nelson and Bulun, 2001).
Distribution
Like all steroid hormones, estrogens readily diffuse across the cell membrane. Once inside the cell, they bind to and activate estrogen receptors (ERs) which in turn modulate the expression of many genes (Whitehead and Nussey, 2001). Additionally, estrogens bind to and activate rapid-signalingmembrane estrogen receptors (mERs) (Micevych and Kelly, 2012; Soltysik and Czekaj, 2013), such as GPER (GPR30) (Prossnitz et al., 2007). Estrogens are synthesized in all vertebrates (Ryan, 1982) as well as some insects (Mechoulam et al., 2005). Their presence in both vertebrates and insects suggests that estrogenic sex hormones have an ancient evolutionary history. Estrogen, otherwise known as oestrogen, is a primary female sex hormone that is vital in the development and functioning of females. The name is derived from estrus, the period of fertility for female mammals, and gen, meaning to generate. However, males too contain estrogen in lower quantities. Production of estrogen occurs primarily in the ovaries, more specifically the theca internal cells. Estrogen secretion is stimulated by another hormone, the luteinizing hormone (LH). Estrogen is also produced, in smaller quantities, in the liver, adrenals glands, fat cells and the breasts.
Biochemical Roles of Estrogen
In males, estrogen helps maintain a healthy libido and aids in the maturation of the sperm. In females, estrogen serves to develop secondary sexual characteristics such as breasts, endometrium, and regulation of menstural cycle. It does this by accelerating burning of body fat, reducing muscle bulk, and decelerating height increase during puberty (Raloff, 1997).
Other functions of estrogen include reducing bone resorption, increasing bone formation, increasing platelet adhesiveness, increasing good cholesterol and triglycerides, improving lung functions, and causing salt and water retention (Hess et al., 1997).
2.2.2.4 Prolactin
Human prolactin (lactogenic hormone) is secreted from the anterior pituitary gland in both men and woman. Human prolactin is a single chain polypeptide hormone with a molecular weight of approximately 23,000 daltons. The release and synthesis of prolactin is under neuroendocrinal control, primarily through Prolactin Releasing Factor and Prolactin Inhibiting Factor (Uotila et al., 1981).
Women normally have slightly higher basal prolactiin levels than men; apparently, there is an estrogen-related rise at puberty and a corresponding decrease at menopause. The primary functions of prolactin are to initiate breast development and to maintain lactation. Prolactin also suppresses gonadal function (Shome and Parlow, 1977).
During pregnancy, prolactin levels increase progressively to between 10 to 20 times normal values, declining to non-pregnant levels by 3-4 weeks post-partum. Breast-feeding mothers maintain high levels of prolactin, and it may take several months for serum concentrations to return to nonpregnant levels (Cowden et al., 1979).
The determination of prolactin concentration is helpful in diagnosing hypothalamic-pituitary disorders. Microadenomas (small pituitary tumors) may cause hyperprolactinemia, which is sometimes associated with male impotence. High prolactin levels are commonly associated with galactorrhea and amenorrhea (Frantz, 1978).
Prolactin concentrations have been shown to be increased by estrogen, thyrotropin-releasing hormone (TRH), and several drugs affecting dopaminergic mechanism. Prolactin levels are elevated in renal disease and hypothyroidism, and in some situations of stress, excercise, and hypoglycemia (Trantz et al., 1978). Additionally, the release of prolactin is episodic and demonstrates diurnal variation. Mildly elevated prolactin concentrations should be evaluated taking these considerations into account. Prolactin concentrations may also be increased by drugs such as chloropromazine and reserpine, and may be lowered by bromocyptine and L-dopa (Jacobs et al., 1978).
2.2.2.5 Oxytocin
Oxytotocin is a nine amono acid hypothalamic peptide hormone that is stored in the posterior pituitary gland (Gimpl and Fahrenholz, 2001). In females, oxytocin is relaeased from the pituitary in to the bloodstream in larger quantities during parturition and lactation, facilitating uterine contraction and milk letdown reflex (Kuwabara et al., 1987; Gimpl and Fahrenholz, 2001). In both males and females, oxytotocin is involved in social and sexual behavior and may play a role in nueropsyciatric disorders like autrism and postpartum depression (Carmichael et al., 1994; Kosfeld et al., 2005; Hollander et al., 2007; Felman, 2012). Oxytocin and related hormone arginine vasopressin are involved in the maintaining water and sodiem homeostatsis.
The actions of oxytocin are mediated by stimulation of a tissue G protein-coupled receptor (OXTR) expressed in myoepithelial cells, mammary gland, both myometrium and endometrium of the uterus, and also in the cental nervous system (Gimpl and Fahrenholz, 2001). In some mammals, oxytocin receptors are also found in the kidnry and heart (Gimpl and Fahrenholz, 2001).
Reported levels of oxytocin in plasma vary depending on the method of measurement (EIA, RIA, and LC-MS) (Zhang et al., 2011; Szeto et al., 2011). Most common reported levels for EIA are nholz1 – 250pg/ml.
Maxey et al., 1992
2.2.2.6 Follicle stimulating hormone (FSH)
2.1.2.2
2.2.2.7 Luteinizing hormone (LH)
2.1.2.3
2.2.3 Females Organs Studied
2.2.3.1 Ovaries
The ovaries are two small organs located on either side of the uterus in a woman’s body. They make hormones, including estrogen, which trigger menstruation. Every month, the ovaries release a tiny egg. The egg makes its way down the fallopian tube to potentially be fertilized. This cycle of egg release is called ovulation. The ovaries are paired, oval organs attached to the posterior surface of the broad ligament of the uterus by the mesovarium (a fold of peritoneum, continuous with the outer surface of the ovaries) (Daftary and Chakravarti, 2011).
2.2.3.1.1 Structure and Anatomy of the Ovaries
Each ovary is a small glandular organ about the shape and size of an almond. The ovaries are located on opposite sides of the uterus in the pelvic cavity and are attached to the uterus by the ovarian ligament (Daftary and Chakravarti, 2011). The open ends of the fallopian tubes rest just beyond the lateral surface of the ovaries to transport ova, or egg cells, to the uterus.
The ovaries are connected to the uterus by the fallopian tubes, or oviducts, which carry the eggs into the uterine cavity. Each ovary contains numerous Graafian follicles, egg-containing tubes that grow and develop between puberty, sexual maturation, and menopause, when the monthly menstrual cycle stops. When a woman is fertile, each month a Graafian follicle travels to the surface of the ovary, bursts, and releases an egg and its fluid contents into a fallopian tube.
The Graafian follicles are fixed in a network of supporting tissue (stroma) and blood vessels. They are covered by a clear, smooth, plasma-like membrane that develops from the peritoneum—lining of the abdominal cavity. Also within the ovaries are small numbers of corpus lutea—the remains of Graafian follicles that have released an egg and are in the process of being reabsorbed by ovarian tissue. Each month the corpus luteum (the scar tissue of a Graafian follicle) is responsible for the production of progesterone. Progesterone is the pregnancy hormone that readies the lining of the uterus for the arrival of a fertilized egg.
The tissues of the ovaries are arranged into several distinct layers:
The outmost layer of simple epithelium, known as the germinal epithelium, forms a soft, smooth covering for the ovary.
The tunica albuginea is a thick band of tough fibrous connective tissue just below the germinal epithelium. It supports and protects the delicate underlying tissues.
Deep to the tunica albuginea is the ovarian cortex, which contains follicles and their supporting connective tissues. The follicles contain oocytes that mature into ova throughout a woman’s reproductive years.
The innermost layer, the ovarian medulla, contains most of the vascular tissue that supports the other layers of the ovary (Rzepka-Górska et al., 2006).
The ovary has 3 components;
Surface: The surface layer of the ovary is formed by simple cuboidal epithelium, known as germinal epithelium.
Cortex: The cortex (outer part) of the ovary is largely comprised of a connective tissue stroma. It supports thousands of follicles. Each primordial follicle contains an oocyte surrounded by a single layer of follicular cells.
Medulla: The medulla (inner part) is composed of supporting stroma and contains a rich neurovascular network which enters the hilum of ovary from the mesovarium (Harris et al., 2011).
Fig. 1: Cross section of an ovary typically showing the three major components of the ovary.
Source:
2.2.3.1.2 Functions of the Ovaries
The ovaries play two central roles in the female reproductive system by acting as both glands and gonads (Rzepka-Górska et al., 2006). Acting as glands, the ovaries produce several female sex hormones including estrogens and progesterone. Estrogen controls the development of the mammary glands and uterus during puberty and stimulates the development of the uterine lining during the menstrual cycle. Progesterone acts on the uterus during pregnancy to allow the embryo to implant and develop in the womb (Ross and Pawlina, 2011).
At birth the ovaries contain anywhere from several hundred thousand to several million circular bundles of cells known as follicles. Each follicle surrounds and supports a single oocyte that has the ability to mature into an ovum, the female gamete (Ross and Pawlina, 2011). Despite this large number of potential ova, only around 4,000 oocytes survive to puberty and only 400 oocytes mature into ova in a woman’s lifetime (Melmed et al., 2011). During each menstrual cycle around 10-20 follicles and their oocytes begin to develop under the influence of the pituitary hormone follicle-stimulating hormone (FSH). Of these follicles, only one cell completes its development and becomes a mature ovum.
Around the middle of the menstrual cycle the mature ovum is released to the surface of the ovary. Fingerlike projections of the fallopian tubes, known as fimbriae, sweep the ovum from the surface of the ovary and into the fallopian tube to be transported to the uterus (Hansen et al., 2008).
2.2.3.2 Uterus
Uterus, also called womb, an inverted pear-shaped muscular organ of the female reproductive system, located between the bladder and rectum, in the pelvic area (Strauss and Lessey, 2004). This complex organ is quite small in size, actually comparable to the size of a pear. However, this pear-sized organ is responsible for what can arguably be the most important aspect of human life: continuity of the human species (Speroff et al., 1999).
2.2.3.2.1 Structure and Anatomy of the Uterus
The anatomy of the uterus consists of the following 3 tissue layers:
The inner layer, called the endometrium, is the most active layer and responds to cyclic ovarian hormone changes; the endometrium is highly specialized and is essential to menstrual and reproductive function
The middle layer, or myometrium, makes up most of the uterine volume and is the muscular layer, composed primarily of smooth muscle cells
The outer layer of the uterus, the serosa or perimetrium, is a thin layer of tissue made of epithelial cells that envelop the uterus (Behera et al., 2005)
Fig. 2: The female reproductive organs typically showing the position of the uterus
Source: Speroff et al. (1999)
2.2.3.2.2 Functions of the Uterus
The uterus is a dynamic female reproductive organ that is responsible for several reproductive functions, including menses, implantation, gestation, labor, and delivery (Speroff et al., 1999). It is responsive to the hormonal milieu within the body, which allows adaptation to the different stages of a woman’s reproductive life. The uterus adjusts to reflect changes in ovarian steroid production during the menstrual cycle and displays rapid growth and specialized contractile activity during pregnancy and childbirth. It can also remain in a relatively quiescent state during the prepubertal and postmenopausal years (Strauss and Lessey, 2004).
2.2.3.3 Fallopian Tubes
The fallopian tubes, also known as oviducts or uterine tubes, are the female structures that transport the ova from the ovary to the uterus each month. In the presence of sperm and fertilization, the uterine tubes transport the fertilized egg to the uterus for implantation (Katz et al., 2007).
2.2.3.3.1 Structure and Anatomy of the Fallopian Tubes
The fallopian tubes are uterine appendages located bilaterally at the superior portion of the uterine cavity (Feng et al., 2014). These tubes exit the uterus through an area referred to as the cornua, forming a connection between the endometrial and peritoneal cavities. Each uterine tube is approximately 10 cm in length and 1 cm in diameter and is situated within the mesosalpinx. The mesosalpinx is a fold in the broad ligament. The distal portion of the uterine tube ends in an orientation encircling the ovary. The primary function of the uterine tubes is to transport sperm toward the egg, which is released by the ovary, and to then allow passage of the fertilized egg back to the uterus for implantation (Ezzati et al., 2014).
A uterine tube contains 3 parts. The first segment, closest to the uterus, is called the isthmus. The second segment is the ampulla, which becomes more dilated in diameter and is the most common site for fertilization. The final segment, located farthest from the uterus, is the infundibulum. The infundibulum gives rise to the fimbriae, fingerlike projections that are responsible for picking up the egg released by the ovary (Ezzati et al., 2014).
The arterial supply to the uterine tubes is from branches of the uterine and ovarian arteries; these small vessels are located within the mesosalpinx. The nerve supply to the uterine tubes is via both sympathetic and parasympathetic fibers. Sensory fibers run from thoracic segments 11-12 (T11-T12) and lumbar segment 1 (L1). Lymphatic drainage of the uterine tubes is through the iliac and lateral aortic nodes (Gray, 1999; Chung, 2000). Both ultrasonography and hysterosalpingography can be useful in diagnosing uterine anomalies (Szkodziak et al., 2014).
Histologically, the uterine tubes are composed of 3 layers—the mucosa, muscularis, and serosa. The 3 different cell types within the mucosa of the uterine tubes include the columnar ciliated epithelial cells (25%), secretory cells (60%), and narrow peg cells (< 10%)(Junqueira et al., 1998). The mucosa has many folds, called plicae, which are most evident in the ampulla. The next layer is the muscularis, which is a layer of smooth muscle that surrounds the mucosa. The serosa is the outermost layer; it is primarily visceral peritoneum (Junqueira et al., 1998).
2.2.3.3.2 Functions of the Fallopian Tubes
When an oocyte is developing in an ovary, it is encapsulated in a spherical collection of cells known as an ovarian follicle. Just prior to ovulation the primary oocyte completes meiosis I to form the first polar body and a secondary oocyte which is arrested in metaphase of meiosis II. This secondary oocyte is then ovulated. The follicle and the ovary's wall rupture, allowing the secondary oocyte to escape. The secondary oocyte is caught by the fimbriated end and travels to the ampulla of the uterine tube where typically the sperm are met and fertilization occurs; meiosis II is promptly completed. The fertilized ovum, now a zygote, travels towards the uterus aided by activity of tubal cilia and activity of the tubal muscle. After about five days the new embryo enters the uterine cavity and on about the sixth day implants on the wall of the uterus (Hirst et al., 2009).
The release of an oocyte does not alternate between the two ovaries and seems to be random. After removal of an ovary, the remaining one produces an egg every month (Daftary and Chakravarti, 2011).
Occasionally the embryo implants into the Fallopian tube instead of the uterus, creating an ectopic pregnancy, commonly known as a "tubal pregnancy" (Kodaman et al., 2004).
2.2.3.4 Grafian follicles
The Graafian follicles are fixed in a network of supporting tissue (stroma) and blood vessels. They are covered by a clear, smooth, plasma-like membrane that develops from the peritoneum—lining of the abdominal cavity.
Ovarian follicle is a roughly spheroid cellular aggregation set found in the ovaries. It also secretes hormones that influence stages of the menstrual cycle. Women begin puberty with about 400,000 follicles (David, 2010), each with the potential to release an egg cell (ovum) atovulation for fertilization (David, 2010). These eggs are developed only once every menstrual cycle
Stucture and Anatomy of the Grafian follicles
Ovarian follicles are the basic units of female reproductive biology. Each of them contains a single oocyte (immature ovum or egg cell). These structures are periodically initiated to grow and develop, culminating in ovulation of usually a single competent oocyte in humans (Luijkx, 2015). They also consists of granulosa cells and theca of follicle.
Clinical Significance
Ovarian function may be measured by gynecologic ultrasonography of follicular volume. Presently, ovarian follicle volumes can be measured rapidly and automatically from three-dimensionally reconstructed ultrasound images (Salama et al., 2010). Rupture of the follicle can result in abdominal pain (mittelschmerz) and is to be considered in the differential diagnosis in women of childbearing age.
Cryopreservation of ovarian tissue is of interest to women who want to preserve their reproductive function beyond the natural limit, or whose reproductive potential is threatened by cancer therapy (Isachenko et al., 2009), for example in hematologic malignancies or breast cancer ( Oktay and Oktem, 2009).
For in vitro culture of follicles, there are various techniques to optimize the growth of follicles, including the use of defined media, growth factors and three-dimensional extracellular matrix support (Smitz et al., 2010). Molecular methods and immunoassay can evaluate stage of maturation and guide adequate differentiation (Smitz et al., 2010). Animal studies have generally showed correct imprinted DNA methylation establishment in oocytes resulting from follicle culture (Anckaert et al., 2012).
2.2.3.5 Corpora lutea
2.2.3.6 Atretic follicles
2.2.4 Female Biological Evaluation Indices
Biological screening entails monitoring sexual activities of the female animals. This may be achieved by evaluating relevant biological indices and computation or recording of relevant parameters.
2.2.4.1 Organ/body weight ratio
Organ/body weight ratio is defined as the ratio of weight of a particular organ to weight of the animals expressed in percentage (Yakubu and Bukoye, 2009).
Organ/body weight = Weight of organ x 100
Weight of the animals
2.2.4.2 Vaginal opening
Vaginal opening is defined as the ratio of number of rats with open vagina to number of treated rats expressed in percentage (Yakubu and Bukoye, 2009).
Vaginal opening = Number of rats with open vagina x 100
Number of treated rats
2.2.4.3 Post-natal viability index
Post-natal viability index is defined as the ratio of the number of pups alive on day 35 to the number of alive pups expressed in percentage (Yakubu and Bukoye, 2009).
Post-natal viability index = Number of pups alive on day 35 x 100
Number of alive pups
2.2.4.4 Weaning viability index
Weaning viability index is defined as the ratio of the number of pups alive at day 65 to the number of pups alive at day 35 expressed in percentage (Yakubu and Bukoye, 2009).
Weaning viability index = Number of pups alive at day 65 x 100
Number of pups alive at day 35
2.2.4.5 Birth live index
Birth live index is defined as the ratio of the number of live offspring to number of offspring delivered expressed in percentage (Yakubu and Bukoye, 2009).
Birth live index = Number of live offspring x 100
Number of offspring delivered
2.2.4.6 Delivery index
Delivery index is defined as the ratio of number of females delivering to number of pregnant females expressed in percentage (Yakubu and Bukoye, 2009).
Delivery index = Number of females delivering x 100
Number of pregnant females
2.2.4.7 Gestation index
Gestation index is defined as the ratio of number of females with alive pups to number of pregnant females expressed in percentage (Yakubu and Bukoye, 2009).
Gestation index = Number of females with alive pups x 100
Number of pregnant females
2.2.4.8 Implantation index
Implantation Index is the ratio of total number of implantations to number mated expressed in percentage (Ratnasooriya and Dharmasiri, 2000).
% Implantation Index = Total number of implantation x 100
Number mated
2.2.4.9 Pre-implantation loss
Pre-implantation loss is the ratio of the difference between the number of corpora lutea and number of implantations to number of corpora lutea expressed in percentage (Ratnasooriya and Dharmasiri, 2000).
% Pre-implantation loss = Number of corpora lutea – number of implantation x 100
Number of corpora Lutea
2.2.4.10 Post-implantation loss
Post-implantation loss is the ratio of the difference between the total number of implants and number of viable implants to total number of implants expressed in percentage (Ratnasooriya and Dharmasiri, 2000).
% Post-implantation loss = Total no. of implants – no. of viable implants x 100
Total number of implants
2.2.4.11 Mating index
Mating index defined as the ratio of the number of sperm positive females to number of mated females expressed in percentage (Yakubu and Bukoye, 2009).
Mating index = Number of sperm positive females x 100
Number of mated females
2.2.4.12 Pregnancy index
Pregnancy index defined as the ratio of the number of pregnant females to the number of sperm positive females expressed in percentage (Yakubu and Bukoye, 2009).
Pregnancy index = Number of pregnant females x 100
Number of sperm positive females
2.2.4.13 Fertility index
Fertility index is the ratio of the number of pregnant females to the number of females with successful copulation expressed in percentage (Yakubu and Bukoye, 2009).
Fertility index = Number of pregnant females x 100
Number of females with successful copulation
2.2.4.14 Resorption index
Resorption index is the ratio of the total number of resorption sites to the total number of implantation sites expressed in percentage (Yakubu and Bukoye, 2009).
Resorption index = Total number of resorption sites x 100
Number of implantation sites
2.2.4.15 Survival ratio (%)
Survival ratio (%) is the ratio of the number of live foetuses to the number of live plus the dead foetuses (Yakubu and Bukoye, 2009).
Survival ratio (%) = Number of live foetuses x 100
Number of live + the dead foetuses
2.2.4.16 Percentage of animals that aborted
Percentage of animals that aborted is the ratio of the number of animals that aborted to the number of rats assessed expressed in percentage (Yakubu and Bukoye, 2009).
Percentage of animals that aborted = Number of animals that aborted x 100
Number of rats assessed
2.2.4.17 Number of resorption sites
Number of resorption sites is the difference between the number of implantation sites in the control animals and number of implantations in the test animals (Yakubu and Bukoye, 2009).
Number of resorption sites = Number of implantation sites in the control animals – Number of implantations in the test animals.
2.2.4.18 Other Parameters
The weight of uterus and luminal fluid (g), vagina cornification (%), length of epithelium, number of uterine glands, number of litters, pituitary weight (g), number of implants, and corpora lutea, implantation site, the weight of uterus and ovaries (g), length of right uterine horn (cm), number of implants, aborted implants and corpora lutea, implantation site, uterine diameter, thickness of the endometrium, epithelial cell height, number of live fetuses, number of dead fetuses, average weight of live fetuses, number of animals that aborted, number of animals with vaginal bleeding, number of implantation sites; number of corpora lutea, reduction endometrial height, uterine glands, % no of animals with opened vagina and cornificated vagina, weight of fetuses and placenta; uterine contractility. The weights of the animals both before pairing and prior to sacrifice, as well as feed and water intake will also be recorded.
2.2.5 Female Biological Fluid
2.2.5.1 Blood
2.1.5.1
2.2.5.2 Serum
2.1.5.2
2.2.6 Female Functional Indices
2.2.6.1 Ovarian Function Indices
2.2.6.1.1 Cholesterol
2.1.7.3.3
2.2.6.1.2 Ascorbic Acid
2.1.7.3.7
2.2.6.1.3 Protein Content
Serum total protein is a measure of the total amount of protein in serum. The serum protein make up of an individual is of important diagnostic significance because of the involvement of the protein in enzymes, hormones and antibodies as well as osmotic pressure balance, maintaining acid-base balance and as a reserve source of nutrition for the body tissues and muscles (Eastham, 1985). Protein measurement reflects nutritional state, kidney disease, and liver disease.
2.2.6.1.3 Biochemical Relevanve of Protein Content
Proteins of different types exist and have diverse functions including:
(i) Maintenance of oncotic pressure in the blood plasma by albumin.
(ii) Immune defence by immunoglobulins (antibodies)
(iii) Transport of bound substance e.g. fatty acids by albumin.
(iv) Inflammatory response by acute phase proteins
(v) Blood clotting and fibrinolysis by fibrinogen (Eastham, 1985).
Increased levels of total protein in the serum results in dehydration; conditions of relatively greater increase in serum albumin include chronic infection, autoimmune disease, hepatic cirrhosis and paraproteinaemia (Eastham, 1985). Decreased levels of serum total protein results in over hydration i.e. excess intravenous fluid infusion compared with renal output and other body fluid losses (Eastham, 1985). Decreased levels of serum total proteins may be due to poor nutrition, liver disease, malabsorption or severe burns.
2.2.6.2 Ovarian Steroidal Enzymes
2.2.6.2.1 3-β-hydroxysteroid dehydrogenase
2.1.7.4.10
2.2.6.2.2 Glucose-6-phosphate dehydrogenase
2.1.7.4.11
2.2.7 Biochemical Assayed Parameters
2.2.7.1 Nitric oxide
The free radical nitric oxide (NO) is produced by a number of different cell types for a variety of biological functions. Nitric oxide is a product of the oxidation of L-arginine to L-citrulline in a two-step process catalyzed by the enzyme nitric oxide synthase (NOS).
2.2.7.1.1 Isoforms
Two major isoforms of nitric oxide synthase have been identified. The constitutive isoform found in neurons and endothelial cells, produces very low amounts of nitric oxide in a calcium and calmodulin dependent fashion. NO activates soluble guanlyate cyclase in target cells, resulting in increased levels of cGMP, which in turn facilitates neuronal transmission and vascular relaxation, and inhibits platelet aggregation (Misko et al., 1993). The inducible isoform, found in macrophages, fibroblasts, and hepatocytes, produces NO in relatively large amounts in response to inflammatory or mitogenic stimuli and acts in a host defensive role through its oxidative toxicity (Nathan, 1992). Regardless of the source or role, the free radical NO has a very short half life (T½= 4 seconds), reacting with several different molecules normally present to form either nitrate (NO3-) or nitrite (NO2-). Because the ratio of nitrite to nitrate is variable and unpredictable, the best index of NO production is the total of both nitrate and nitrite.
2.2.7.1.2 Biochemical Role of Nitric Oxide
Nitric oxide is an endogenous mediator which plays an important role promoting mucosal defence. It performs functions similar to that of prostaglandins and there is a co-operative modulation of prostaglandins and nitric oxide synthesis (Wallace, 2001). It has been reported that nitric oxide increase prostaglandin E2 synthesis in vivo through cGMP- independent mechanism and thus regulate the release and/or the synthesis of PGE2 in the stomach after ulceration (Wallace, 2001). Nitric oxide (NO) influences various biochemical and physiological reactions that are key to preventing or repairing injury to the gastrointestinal tract, as stimulating mucus secretion from the mucus membrane of the stomach and intestine, regulating the blood flow in the wall of the gastrointestinal tract and the mucus membrane and controlling inflammatory cell activation in the inflammatory process (Wu and Morris, 1998).
Nitric oxide (NO) also plays an important role in neurotransmission, vascular regulation, immune response and apoptosis. NO is rapidly oxidized to nitrite and nitrate which are used to quantitate NO production.
Nitric oxide is synthesized by the enzyme nitric oxide synthase in a variety of different cell types at levels that vary according to the intended function. Because of its role in normal and pathophysiological processes, the determination of nitric oxide is of tremendous importance. Most importantly, in all instances nitric oxide is converted in vivo to either nitrite or nitrate.
Nitric oxide (NO) plays essential roles in mammalian life [1,2]. Synthesis of this seemingly most simple molecule involves one of the most complicated enzymes in nature, the nitric oxide synthase (NOS), which contains several cofactors and is highly regulated [3]. Multiple physiological and pathophysiological functions of nitric oxide are achieved by using diverse classes of NOS (neuronal NOS, endothelial NOS, inducible NOS and mitochondria NOS). Unregulated production of nitric oxide can
cause nitrosative stress, leading to damages of proteins/DNA and to cell injury and death [4,5].
2.2.7.2 Glucose
Glucose is the main form by which sugar is used by the cells of the body (Waugh and Grant, 2001). Carbohydrates are the main dietary sources of glucose. Examples of carbohydrate rich foods include rice, potatoes, bread, etc. (Waugh and Grant, 2001). After a carbohydrate–rich meal, glucose molecules are absorbed into the blood stream and carried into the cells where they are used to generate energy.
Serum glucose levels stay within narrow limits of 4 to 8mmol/L throughout the day but are higher after meals and usually lowest in the morning (Waugh and Grant, 2001).
2.2.7.2.1 Biochemical Relevance of Glucose
Glucose, a form of sugar is an important fuel for generating energy for the cells of the body. A greater-than-normal value of serum glucose may be an indication of diabetes which occurs when any two of the following conditions are met:
1. A fasting blood glucose level of 7.0 mmol/L or higher (normal fasting blood glucose level is less than 6.1 mmol/L)
2. A-two hour oral glucose tolerance test value of 11.1 mmol/L or higher (a normal 2 hour post prandial is less than 7/8 mmol/L).
3. A random blood glucose test value of 11.1 mmol/L or higher (normal result for random blood glucose test is usually less than 7.0mml/L (Waugh and Grant, 2001).
2.2.7.3 Total Cholesterol
Cholesterol is a lipid sterol that is produced in and transported throughout the bloodstream in eukaryotes (Admundson et al., 1999) Cholesterol is a critical compound used in the structure of cell membranes, hormones, and cell signaling. It is an essential component of animal cell structure in order to maintain permeability and fluidity (Fossati et al., 1982). Cholesterol is a precursor for steroid hormones including the adrenal gland hormones cortisol and aldosterone, sex hormones progesterone, estrogens, and testosterone, and bile acids and vitamin D (Ledwozyw et al., 1986). Cholesterol is transported throughout the body within lipoproteins, which have cell-specific signals that direct the lipids they transport to certain tissues (Lee et al., 2008). For this reason, lipoproteins exist in different forms within the blood based on their density. These include chylomicrons, very-low density lipoproteins (VLDLs), low-density lipoproteins (LDLs), intermediate-density lipoproteins (IDLs), and high-density lipoproteins (HDLs) (Prévéraud et al., 2014). The higher the lipid content within a lipoprotein, the lower its density. Cholesterol exists within a lipoprotein as a free alcohol and as a fatty cholesteryl ester, which is the predominant form of cholesterol transport and storage (Raveendran et al., 2014).
Determining circulatory levels of lipoproteins is critical to the diagnosis of lipid transport disorders (Marino et al., 2014). High levels of cholesterol and cholesteryl esters (hypercholesterolemia) have been associated with cardiovascular disease such as atherosclerosis and heart disease, although lower levels (hypocholesterolemia) may be associated with cancer, depression, or respiratory diseases (Mathews et al., 2014).
2.2.7.3.1 Clinical significance
Total cholesterol has been found to correlate with cardiovascular mortality in the 30-50 year age group (Emma-Leah, 2009). Cardiovascular mortality increases 9 % for each 10 mg/dL increase in total cholesterol over the baseline value of 180 mg/dL. Approximately 80 % of the adult male population has values greater than this, so the use of median 95 % of the population to establish normal range has no utility for this test. Excess mortality has been shown not to correlate with cholesterol levels in the >50 years age group, probably because of the depressive effects on cholesterol levels expressed by various chronic diseases to which older individuals are prone. Increases may be seen with a variety of metabolic disturbances including diabetes mellitus, hypothyroidism, Cushing’s disease, pancreatitis and some types of kidney disease; decreases may be seen with liver insufficiency and intestinal disease (Emma-Leah, 2009).
2.2.7.4 High Density Lipoprotein-Cholesterol
High-density lipoprotein cholesterol (HDL-C) is one of the five major groups of lipoproteins which, in order of sizes, largest to smallest, are chylomicrons, VLDL, IDL, LDL and HDL, which enable lipids like cholesterol and triglycerides to be transported within the water-based bloodstream (Superko et al., 2002). In healthy individuals, about thirty percent of blood cholesterol is carried by HDL (Barter et al., 2007). Blood tests typically report HDL-C, the amount of cholesterol contained in HDL particles. It is often contrasted with low density or LDL cholesterol or LDL-C. HDL particles are able to remove cholesterol from atheroma within arteries and transport it back to the liver for excretion or re-utilization, which is the main reason why the cholesterol carried within HDL particles, termed HDL-C, is sometimes called "good cholesterol" (Segrest et al., 2001; Toth, 2005).
2.2.7.4.1 Clinical significance
HDL-cholesterol is "good" cholesterol, in that risk of cardiovascular disease decreases with increase of HDL. An HDL-cholesterol level of <35 mg/dL is considered a coronary heart disease risk factor independent of the level of total cholesterol. One way to assess risk is to use the total cholesterol/HDL-cholesterol ratio, with lower values indicating lower risk (Barter et al., 2007).
2.2.7.5 Triacylglycerol
A triacylglycerol, TAG, (triglyceride or triacylglyceride) is an ester derived from glycerol and three fatty acids. It is the main constituent of vegetable oil and animal fats (Nelson and Cox, 2000).
Triacylglycerol, as major components of very-low-density lipoprotein (VLDL) and chylomicrons, play an important role in metabolism as energy sources and transporters of dietary fat. They contain more than twice as much energy (9kcal/g or 38 kJ/g) as carbohydrates and proteins (Hemat, 2003). In the intestine, triacylglycerols are split into monoacylglycerol and free fatty acids in a process called lipolysis, with the secretion of lipases and bile, which are subsequently moved to absorptive enterocytes, cells lining the intestines (Hemat, 2003). The triacylglycerols are rebuilt in the enterocytes from their fragments and packaged together with cholesterol and proteins to form chylomicrons. These are excreted from the cells, collected by the lymph system and transported to the large vessels near the heart before being mixed into the blood. Various tissues can capture the chylomicrons, releasing the triacylglycerols to be used as a source of energy. Fat and liver cells can synthesize and store triacylglycerols (Hemat, 2003). When the body requires fatty acids as an energy source, glucagon signals the breakdown of the triglycerides by hormone-sensitive lipase to release free fatty acids. As the brain cannot utilize fatty acids as energy source (unless converted to a ketone), the glycerol component of triacylglycerols can be converted into glucose, via glycolysis by conversion into dihydroxyacetone phosphate and consequently into glyceraldehyde 3-phosphate, for brain fuel when it is broken down. Fat cells may also be broken down for that reason, if the brain's needs ever outweigh the body's (Parks, 2002). Triacylglycerol cannot pass through cell membranes freely. Special enzymes on the walls of blood vessels called lipoprotein lipases must break down triacylglycerols into free fatty acids and glycerol. Fatty acids can then be taken up by cells via the fatty acid transporter (FAT) (Balch and Phyllis, 2006).
2.2.7.5.1 Clinical significance
Markedly increased triglycerides (>500 mg/dL) usually indicate a non-fasting patient (i.e., one having consumed any calories within 12-14 hour period prior to specimen collection). If patient is fasting, hypertriglyceridemia is seen in hyperlipoproteinemia types I, IIb, III, IV, and V. (Hemat, 2003) Exact classification theoretically require lipoprotein electrophoresis, but this is not usually necessary to assess a patient's risk to atherosclerosis. Cholestyramine, corticosteroids, estrogens, ethanol, miconazole (intravenous), oral contraceptives, spironolactone, stress, and high carbohydrate intake are known to increase triglycerides. Decreased serum triglycerides are seen in abetalipoproteinemia, chronic obstructive pulmonary disease, hyperthyroidism, malnutrition, and malabsorption states (Balch and Phyllis, 2006).
2.2.7.6 Low density Lipoprotein-Cholesterol
Low-density lipoprotein cholesterol (LDL-C) is one of the five major groups of lipoproteins, which in order of size, largest to smallest, are chylomicrons, VLDL, IDL, LDL and HDL, that enable lipids like cholesterol and triglycerides to betransported within the water-based bloodstream (Superko et al., 2002). Blood tests typically report LDL-C, the amount of cholesterol contained in LDL.
2.2.7.6.1 Clinical significance
In clinical context, mathematically calculated estimates of LDL-C are commonly used to determine how much low density lipoproteins are driving progressions of atherosclerosis (John et al., 2008). Since current theory holds that higher levels of LDL particles promote health problems and cardiovascular disease, they are often called the bad cholesterol particles, (as opposed to HDL particles, which are frequently referred to as good cholesterol or healthy cholesterol particles) (Segrest et al., 2001).
2.2.7.7 Atherogenic Index
The atherogenic index of serum is defined as the base 10 logarithm of the ratio of serum triacylglycerol to serum high-density lipoprotein cholesterol. It has been employed as a predictor of cardiovascular risk (Vnitr-Lek, 2006).
2.2.8 Enzymes Studied
2.2.8.1 Na+-K+ ATPase (EC 3.6.1.3)
The Na+-K+ ATPase belongs to a class of transporters, all of which are reversibly phosphorylated as part of the transport cycle. Virtually every animal cell maintains a lower concentration of Na+ and a higher concentration of K+ intracellularly than is found in its surrounding medium. This is achieved by the action of the Na+-K+ ATPase, which couples breakdown of ATP to the simultaneous movement of both Na+ and K+ against their concentration gradients (Lehninger, 1993). For each molecule of ATP converted to ADP and Pi, this transporter moves two K+ inward and three Na+ outward, across the plasma membrane. The Na+-K+ ATPase is an integral membrane protein and was first isolated in 1957 by Jen Skou. It consists of two types of subunits: A110-KD non glycosylated α-subunit that contains the enzyme’s catalytic and ion-binding sites, and a 55-KD glycoprotein subunit of unknown fraction (Voet and Voet, 1990).
The mechanism by which ATP hydrolysis is coupled to transport remains to be established, but a working model supposes that the ATPase cycles between two conformations-conformation II, a phosphorylated form with high affinity for K+ and low affinity for Na+, and conformation I, a dephosphorylated form with high affinity for Na+ and low affinity for K+ (Lehninger, 1993). The conversion of ATP to ADP and Pi occurs in two steps catalysed by Na+-K+ATPase:
(1) Formation of phosphoenzyme.
ATP + Enz I ADP + P-EnzII
(2) Hydrolysis of Phosphoenzyme
P-Enz II + H2O Enz + Pi
Since three Na+ ions move outward for every two K+ ions that move inward, this process is electrogenic (it creates a net separation of charge across the membrane, making the inside of the cell negative relative to the outside). In the binding of free ATPase to ATP, phosphorylation is controlled by Na+, while dephosphorylation is controlled by K+. Fig 14 shows a kinetic scheme for the active transport of Na+ and K+ by Na+-K+ATPase.
In testing for the effects of ATP, vanadate and ouabain on the uptake of Na+
Overall ion Movement
3Na + (in) + 2k + (out) + ATP 3Na + (out) + 2K+ (in) + ADP + Pi
Fig 14: Postulated mechanism of transport by Na+-K+ATPase across the membrane
Source: Voet and Voet (1990)
(Boumendil- Podevin and Podevin, 1983), addition of ATP in the presence of Mg2+ to the outside of K+ loaded membrane vesicle stimulated Na+ uptake. ATP-dependent uptake of Na+ was progressively abolished by external vanadate, while ouabain had no effect. When ouabain was however trapped internally during formation of vesicles, this agent inhibited completely the ATP-dependent Na+ uptake. This showed that ATP dependent uptake of Na+ by inside-out vesicles required the presence of intravesicular K+ i.e at the plasma or external membrane surface, while K+ at the cytoplasmic surface (extravesicular) inhibited Na+ uptake.
Studies carried out on the biosynthesis of the Na+-K+ATPase in the Mardin Darby Canine kidney cells, suggested that post-synthetic processing is required before the newly synthesized Na+-K+ ATPase can display its full array or catalytic functions (Anderson et al., 1988). This processing seems to be completed prior to the arrival of the newly synthesized sodium pump at the cell surface. The extrusion of Na+ enables animal cells to control their water content osmotically and without functional (Na+-K+) pumps, animal cells which lack cell walls, would swell and burst. Moreover, the electrochemical potential gradient generated by the (Na+-K+) pump is responsible for the electrical excitability of nerve cells and provides the free energy for the active transport of glucose and amino acids into some cells (Voet and Voet,1990).
Its diagnostic importance in pathological conditions such as hypertension, abnormal erythrocytes and nutritional deficiency states have been reported (Olorunsogo et al., 1985). For example, the activity of Na+-K+ ATPase is increased in hypertensive patients (Olorunsogo et al., 1985).
2.2.8.2 Ca+ Mg+ ATPase
The concentration of Ca2+ in extracellular spaces (approximately 1500 µM) is four order of magnitude higher than in the cytosol (approximately 0.1µM).This large concentration gradient is maintained by the active transport of Ca2+ across the plasma membrane, the endoplasmic reticulum (the sarcoplasmic reticulum in muscle), and the mitochondrial inner membrane. Plasma membrane and endoplasmic reticulum each contains a Ca2+- ATPase that actively pumps Ca2+ out of cytosol at the expense of ATP hydrolysis (Fig. 15) (Voet and Voet, 1990).
Fig. 15: Postulated mechanism for Ca2+-Mg2+ATPase
Source: Voet and Voet (1990)
The protein (with molecular weight of 115KD) can be solubilized in monomeric complexes in the membranous state (Anderson et al., 1988). Reconstitution of the Ca2+-ATPase has provided information for the dispersion of the Ca2+ pumps. Findings by Anderson et al. (1988) indicate that the protein vesicles within the membrane do not participate in Ca2+ transport. This is consistent with the idea that the intravesicular volume per Ca2+ transporting unit is an important determinant of the Ca2+-uptake capacity. For the Ca2+-ATPase activity in the heart, there is first an influx of Na+, causing a rapid action potential. There is then a slow influx of Ca2+ causing a plateau phase of action potential, followed by depolarization and a rise in Ca2+concentrations, which triggers Ca2+ release from the endoplasmic reticulum. In addition there is a 3Na+:1Ca2+ antiport system, which moves three positive charges out of the cell for each Ca2+; the resulting negative inside charge of the resting cell therefore favours calcium efflux (Anderson et al., 1988). Lipid intermediates such as palmitoylcarnitine increased the uptake of Ca2+ by increasing the activity of the Ca2+-Mg2+ ATPase by 90 %. This is due to the fact that there is an alteration of the vesicle by the diacylcarnitine (Anderson et al., 1988). The acyl carnitine did not have any effect on the Na+-K+-ATPase of native sarcolemma but inhibited the enzyme markedly if the enzyme was measured in sodium dodecyl sulphate (SDS)-treated vesicles. SDS detergent allowed the lipid to enter the lipid bilayer to affect the enzyme. Calmodulin is an endogenous protein activator that stimulates the Ca2+-Mg2+ATPase in erythrocytes. This activation of the enzyme by calmodulin is strongly dependent on Ca2+.
In vitro studies by Anderson et al. (1988) showed that Ca2+ besides its effects via calmodulin (Olorunsogo et al., 1985) activates the ATPase and this has been used to purify the Ca2+-Mg2+ATPase of human erythrocytes and of heart sarcolemma by means of the calmodulin-affinity chromatography (Rasmussen,1989). It has also been reported that ivermectin reduces the Ca2+ uptake into the sarcoplasmic reticulum of rat and rabbit skeletal muscle (Ahern et al., 1999). The enzyme activity has been found to be reduced in sickle cell disease resulting in an elevated level of intracellular Ca2+ which results in some of the pathological conditions experienced in red blood cells (Eaton et al., 1978). The enzyme activity in erythrocytes of protein malnourished subjects has also been reported to be lowered (Olorunsogo, 1989).
2.2.8.3 Cyclooxygenase-2 (COX-2)
COX (cyclooxygenase, also known as Prostaglandin G/H synthase) is a membrane-bound enzyme responsible for the oxidation of arachidonic acid to Prostaglandin G2 (PGG2) and the subsequent reduction of PGG2 to PGH2 (Smith et al., 1993; Vane and Botting, 1995) These reactions are the first steps in the formation of a variety of prostanoids. COX has been shown to be expressed in at least two different isoforms: a constitutively expressed form, COX1, and an inducible form, COX2. COX1 is thought to regulate a number of "housekeeping" functions such as vascular hemostasis, renal blood flow and maintenance of glomerular function (Mene et al., 1989). Inflammation mediators such as growth factors, cytokines and endotoxin induce COX2 expression in a number of cellular systems (Herschman, 1993; Meade, 1993).
2.2.8.4 Protein Kinase C-α
The phosphorylation of serine and threonine residues in proteins by Protein Kinase C (PKC) family members is critical to the normal regulation of many biological mechanisms, including the modulation of membrane structure and cytoskeletal reorganization, receptor desensitization, transcriptional control, cell growth and differentiation, and mediation of immune response. PKCs also play a role in memory, learning, and long-term potentiation. The PKCs influence cellular events via their activation by second messenger pathways that involve the production of diacylglycerol (Nishizuka, 1886; 1992). The in vivo regulation of PKC family members involves a combination of the subcellular location of the enzyme(s) and their substrate(s). Identification of specific functions of the different isoforms is dependent on the development of isoform-specific inhibitors (Wilkinson and Hallam, 1994; Conrad et al., 1994)
2.2.8.4.1 Isoforms
PKCα, PKCβI, PKCβII, PKCγ, PKCδ, PKCε, PKCζ, PKCηand PKCθ (alpha, betaI, betaII, gamma, delta, epsilon, zeta, eta and theta, respectively).
2.2.8.5 Alkaline phosphatase
2.1.7.4.3
2.3 Medicinal Plant
Medicinal plants are herbs or botanicals that have been recognized for therapeutic uses. They range from plants which are used in the production of mainstream pharmaceutical products to those used in herbal medicine preparations. Medicinal plants can be found growing in numerous settings all over the world (Smith, 2003).
There are three ways in which plants have been found useful in medicine. First, they may be used directly as teas or in other extracted forms for their natural chemical constituents. Secondly, they may be used as agents in the synthesis of drugs while lastly, the organic molecules found in plants may be used as models for synthetic drugs (Smith, 2003 and Bennett, 2007). Historically, the medicinal value of plants was tested by trial and error using the guiding principle of ‘Doctrine of Signature’. Doctrine of Signature states that herbs that resemble various parts of the body can be used to treat ailments of that part of the body. Examples include the plants liverwort; snakeroot, an antidote for snake venom; lungwort; bloodroot; toothwort; and wormwood, to expel intestinal parasites (Bennett, 2007). Modern approaches to determining the medicinal properties of plants involve collaborative efforts that can include ethnobotanists, anthropologists, pharmaceutical chemists, and physicians. Many modern medicines had their origin in medicinal plants (Smith, 2003).
2.3.1 Spondias mombin
Spondias mombin is a tree, in Nigeria known by various names – Ibo: Ichikara, Hausa: Tsardarmasar, Yoruba: Akika etikan, Iyeye (Chukwuka and Thomas, 2008). This plant is readily common in South West of Nigeria (Yoruba). It belongs to the family Anacardiaceae.
Spondias mombin leaves (Plate 1) alternate, once pinnate with an odd terminal leaflet; stipules absent; rachis 30−70 cm long; leaflets 5−10 pairs, elliptic, 5−11 x 2−5 cm; apex long acuminate, asymmetric, truncate or cuneate; margins entire, glabrous or thinly puberulous (Orwa et al., 2009).
The various parts of Spondias mombin has been acclaimed in folkloric practice for the management of several ailments especially its leaves in regulation of fertility (Orwa et al., 2009). Scientifically validated pharmacological activities of Spondias mombin extracts is provided in Table 1.
Plate 1: Spondias mombin Leaves
Table 1: Scientifically validated pharmacological activities of Spondias mombin extracts
2.4 Phytochemicals
Phytochemicals are natural bioactive compounds which are present in plants. These natural compounds work with nutrients and dietary fibres to protect animals and man against diseases. Since time immemorial, these plant products which are derived from plant parts such as stem, barks, leaves, fruits and seeds have been part of phytomedicine, thus indicating that any part of a plant may contain important active compounds (Inavova et al., 2005). Extraction and characterisation of several active phytochemicals from green plants have led to the production of some high activity profile drugs (Sonibare et al., 2009).
2.4.1 Secondary metabolites
Secondary metabolites are organic compounds that are not directly involved in the normal growth, development, or reproduction of an organism (Fraenkel, 1959; Bidlack, 2000). Unlike primary metabolites, absence of secondary metabolites does not result in immediate death, but rather in long-term impairment of the organism's survivability, fecundity, or aesthetics, or perhaps in no significant change at all (Bidlack, 2000). Secondary metabolites are often restricted to plant species and can be classified on the basis of their chemical structure, composition, solubility in various solvents, or the pathway by which they are synthesised (Bidlack, 2000). A simple classification according to their biosynthetic pathways includes three main groups: the terpenes (mevalonic acid), phenolics (from simple sugars), and alkaloids (Harborne, 1999). Secondary metabolites often play an important role in plant defense against herbivory (Stamp, 2003) and other interspecies defenses (Samuni-Blank et al., 2012). Secondary metabolites are used by human beings as medicines, flavouring agents and recreational drugs (Nafiseh and Mohammad, 2013).
2.4.1.1 Saponins
Saponins are a class of chemical compounds, one of many secondary metabolites found in natural sources. More specifically, they are amphipathicglycosides grouped, in terms of phenomenology, by the soap-like foaming they produce when shaken in aqueous solutions, and, in terms of structure, by their composition of one or more hydrophilic glycoside moieties combined with a lipophilictriterpene derivative (Hostettmann and Marston, 1995; Francis et al., 2002). Saponins have historically been understood to be plant-derived, but they have also been isolated from marine organisms (Riguera, 1997). Saponins are indeed found in many plants (Hostettmann and Marston, 1995) and derive their name from the soapwort plant (Genus Saponaria, Family Caryophyllaceae), the root of which was used historically as a soap (Liener, 1980).
Saponins have been reported to possess a wide range of biological activities. Saponins are used medically as an expectorant, emetic, contraceptive, and for treatment of excessive salivation, epilepsy, chlorosis, and migraines. Saponins inhibit some kinds of cancer cell tumor growth in animals, particularly lung and blood cancers, without killing normal cells (Dharmananda, 2003). It is used to treat albuminuria and diabetes (Gbolade, 2009).
2.4.1.2 Alkaloids
Alkaloids are a group of naturally occurring chemical compounds that contain mostly basicnitrogen atoms. They consist of some related compounds with neutral (McNaught and Wilkinson, 1997) and even weakly acidic properties (Manske, 1965). Also, some synthetic compounds of similar structure are attributed to alkaloids (Robert, 1998). In addition to carbon, hydrogen and nitrogen, alkaloids may also contain oxygen, sulphur and more rarely other elements such as chlorine, bromine, and phosphorus (Arnold, 1989).
Generally, alkaloids are amalgams that do not have any scent and boast of a distinctive outcome on the animals' body mechanism or function. They are exuded in particular cells or tubes and can be of great use in safeguarding against plant predators as they have a bitter flavor. However, their characteristic bitter taste and accompanying toxicity generally help to repel insects and herbivores (Hamilton-Miller, 1995). Owing to these properties of bitter taste and toxicity, alkaloids have significant therapeutic value and form the ingredients of many important medicines (Rhoades and David, 1979).
Alkaloids are known for their potent pharmacological activities, and thus have clinical uses like analgesic, antimalarial, treatment of hypertension, mental disorder, tumours (Swaminathan and Jain, 1973), local anaesthesia in ophthalmology, central nervous stimulant, Alkaloids such as aconitine, anisodamine, charantine, leurosine show anti-diabetic effects (Li et al., 2004). The potential glucose-lowering effect of alkaloids was noted when it was used for diarrhea in diabetic patients. In vitro and in vivo studies have then showed its effects on hyperglycemia and dyslipidemia (Yifei et al., 2008).
2.4.1.3 Flavonoids
Flavonoids are plant-based compounds with powerful antioxidant properties. They are widely distributed in plants fulfilling many functions including producing yellow or red/blue pigmentation in flowers and protection from attack by microbes and insects. The widespread distribution of flavonoids, their variety and their relatively low toxicity compared to other active plant compounds meaning that many animals, including humans, ingest significant quantities in their diet (Lotito and Frei, 2006).
They are synthesized in phenylpropanoid metabolic pathway in which the amino acid phenylalanine is used to produce 4-coumaroyl-CoA. This can be combined with malonyl-CoA to yield the true backbone of flavonoids, a group of compounds called chalcones, which contain two phenyl rings. Conjugate ring-closure of chalcones results in the familiar form of flavonoids, the three-ringed structure of flavones (Lotito and Frei, 2006).
Flavonoids have been referred to as "nature's biological response modifiers" because of strong experimental evidence of their inherent ability to modify the body's reaction to allergens, viruses, and carcinogens. They show anti-allergic, anti-inflammatory, anti- microbial and anti-cancer activity. Flavonoids are most commonly known for their antioxidant activity. Flavonoids are poorly absorbed by the human body (less than 5%), and most of what is absorbed is quickly metabolized and excreted from the body (Lotito and Frei, 2006).
Flavonoids have been shown to have antibacterial, anti-inflammatory, antiallergic, antimutagenic, antiviral, antineoplastic, anti-thrombotic, and vasodilatory activity (Galeotti et al., 2008). The potent antioxidant activity of flavonoids—their ability to scavenge hydroxyl radicals, superoxide anions, and lipid peroxy radicals—may be the most important function of flavonoids, and underlies many of the above actions in the body. Oxidative damage is implicated in most disease processes, and epidemiological, clinical, and laboratory research on flavonoids and other antioxidants suggest their use in the prevention and treatment of a number of these. It is also used to treat albuminuria and diabetes (Galeotti et al., 2008).
2.4.1.4 Anthraquinones
Anthraquinones are group of secondary plant metabolites with over 170 natural compounds that make up the largest group of natural quinines, formed via the acetate-malonate pathway. They are usually found in plants as glycoside (sugar containing molecules) (Hans-Samuel et al., 2002; Samp, 2008). The term anthraquinone, however, almost invariably refers to one specific isomer, 9, 10-anthraquinone wherein the keto groups are located on the central ring (Samp, 2008).
Anthraquinones are organic compounds found in some plants. Anthraquinones have a laxative effect on the body, but are generally not recommended for regular use due to concerns about the risk of habit-forming dependence and adverse side effects, including a higher risk for colorectal cancer (Muller-Lissner, 1993). Anthraquinone laxatives irritate the bowel wall, provoking increased muscle contractions and peristaltic movements. Examples include senna, cascara, sagrada, rhubarb, yellow dock, and aloe.
Anthraquinones may also have antiviral, antibacterial, and cytotoxic properties (Mills et al., 2006). They found wide application as immunosuppressive, immune-stimulant, antiulcer, antioxidant (Yen et al., 2000), antitumor, cardiac stimulant (Sun et al., 2000) and anti-microbial activity (Wang and Chung, 1997).
2.4.1.5 Tannins
Tannins are astringent, bitter plant polyphenols that either bind and precipitate or shrink proteins (Harold, 2004). The term 'tannin' refers to the use of tanning animal hides into leather; however, the term is widely applied to any large polyphenolic compound containing sufficient hydroxyl and other suitable groups (such as carboxyls) to form strong complexes with proteins and other macromolecules. Tannins have molecular weights ranging from 500 to over 3,000 (Van-Burden et al., 1996). Tannins are mainly located in the vacuoles or surface wax of the plants. These sites are where tannins do not interfere with plant metabolism, and it is only after cell breakdown and death that the tannins are active in metabolic effects. Tannins are found in leaf tissues, bud tissues, seed tissues, root tissues and stem tissues. They are also found in the heartwood of conifers and may play a role in inhibiting microbial activity, thus resulting in the natural durability of the wood (Van-Burden et al., 1996).
Tannins may be employed medicinally as antidiarrheal, hemostatic, and antihemorrhoidal agents (Van-Burden et al., 1996). The anti-inflammatory effect of tannins helps control all indications of gastritis, esophagitis, enteritis, and irritating bowel disorders (Van-burden et al., 1996). Tannins not only heal burns and stop bleeding, but also stop infection. The ability of tannins to form a protective layer over the exposed tissue keeps the wound from being infected even more. The tannins help draw out all irritants from the skin because tannin is an astringent that tightens pores and pulls out liquids (Van-Burden et al., 1996). Tannins such as tannic acid stimulate the transport of glucose and inhibit adipocyte differentiation (Xueqing et al., 2005). Tannins have been recognized as functionally active molecules, possessing antioxidant, anticancer, antimutagenic properties, hypoglycemic agents, as well as exerting protective effects against cardiovascular and other diseases (Chung et al., 1997; Shahidi, 1997; Bravo, 1998). Additionally, considerable studies recommended the use of tannins in the diet because of their known beneficial properties, their metabolism, their interactions with nutrients, and their safety intake (Diplock et al., 1998 and Yumiko et al., 2001). Tannins have also been described as anti-hyperglycemic agents in diabetic rats (Pinent et al., 2004).
2.4.1.6 Terpenoids
The terpenoids, sometimes called isoprenoids, are a large and diverse class of naturally occurring organic chemicals similar to terpenes, derived from five-carbon isoprene units assembled and modified in thousands of ways. Most are multicyclic structures that differ from one another not only in functional groups but also in their basic carbon skeletons. Some authors will use the term terpene to include all terpenoids. Terpenes are hydrocarbonsresulting from the combination of several isoprene units. Terpenoids can be thought of as modified terpenes, wherein methyl groups have been moved or removed, or oxygen atoms added (Michael, 2009). Just like terpenes, the terpenoids can be classified according to the number of isoprene units used: hemiterpenoids-1 isoprene unit (5 carbons), monoterpenoids-2 isoprene units (10C), sesquiterpenoids-3 isoprene units (15C), diterpenoids-4 isoprene units (20C) (e.g. ginkgolides), sesterterpenoids-5 isoprene units (25C), triterpenoids-6 isoprene units (30C), tetraterpenoids-8 isoprene units (40C) (e.g. carotenoids), polyterpenoid with a larger number of isoprene units (Kolawole et al., 2006). Plant terpenoids are used extensively for their aromatic qualities. Terpenes and terpenoids are the primary constituents of the essential oils of many types of plants and flowers. Essential oils are used widely as natural flavor additives for food, as fragrances in perfumery, and in traditional and alternative medicines such as aromatherapy. Synthetic variations and derivatives of natural terpenes and terpenoids also greatly expand the variety of aromas used in perfumery and flavors used in food additives. Vitamin A is an example of a terpene (Michael, 2009).
Terpenoids are synthesized through two potential pathways, the mevalonate and, morerecently identified, deoxy-d-xylulose pathways (Rohmer, 1999). Terpenes can be used for the prevention and/or treatment of diabetes type II, obesity and neuropathy (Rohmer, 1999).
2.4.1.7 Phlobatannins
Phlobatannins (phlobaphenes) can be defined as the reddish coloured material extracted from plant that are alcohol soluble and water insoluble. It is the reddish coloured, water insoluble products that result from treatment of tannin extracts with mineral acids (tanner's red) (Richard et al., 1992). Natural phlobaphenes are the common bark, pericarp, cob glume and seed coat (testa) pigments. They have not been found in flowers, unless the brown and black pigments in the involucrum of certain compositae are found to be of the phlobaphen type (Karl, 1955). In bark, phlobaphenes accumulate in the phellem layer of cork cambium, part of the suberin mixture (Rompp and Georg, 2006).
They are common in redwoods barks like Sequoia sempervirensin oak barks where the chief constituent, quercitannic acid, a molecule also present in quercitron. It is an unstable substance, having a tendency to give off water to form anhydrides (phlobaphenes), one of which is called oak-red (C28H22O11) (Buchanan, 1944; Hager's, 1979). Phlobaphens can be formed under action of acids or heating of condensed tannins or of the fraction of tannins called phlobatannins. Water containing soda can be used for the conversion of hop tannins into phlobaphens (Etti, 1978). When heated with hydrochloric acid, tannins in cocoa solids yield glucose and a phlobaphene (Warden, 1985).
2.4.1.8 Steroids
A steroid is a type of organic compound that contains a characteristic arrangement of four cycloalkane rings that are joined to each other (Moss, 1989). The steroids vary by the functional groups attached to these rings and by the oxidation state of the ring. Sterols are special forms of steroids, with a hydroxyl group at position-3 and a skeleton derived from cholestane (Kuzuyama and Seto, 2003). Hundreds of distinct steroids are found in plants, animals, and fungi. All steroids are made in cells either from the sterols lanosterol (animals and fungi) or cycloartenol (plants). Both lanosterol and cycloartenol are derived from the cyclization of the triterpene squalene. Steroids play a role as essential hormones in plants as well as in animals. Plants produce numerous steroids and sterols, some of which are recognized as hormones in animals (Geuns, 1978; Jones and Roddick, 1988). Examples of steroids include cholesterol, the sex hormones-estradiol and testosterone, and the anti-inflammatory drug dexamethasone (Rossier, 2006).
2.4.1.9 Cardiac Glycosides
Cardiac glycosides are secondary metabolites found in several plants and in some animals, such as the milkweed butterflies. Cardiac glycosides are drugs used in the treatment of congestive heart failure and cardiac arrhythmia. Glycosides work by inhibiting the Na+/K+ pump. This causes an increase in the level of sodium ions in the myocytes, which then leads to a rise in the level of calcium ions. This inhibition increases the amount of Ca2+ ions available for contraction of the heart muscle, improves cardiac output and reduces distention of the heart. Glycosides do this by stabilizing the E2-P transition state of the Na+/K+ pump (Kaplan, 2005).
The therapeutic use of cardiac glycosides primarily involves the treatment of congestive heart failure (Lapostelle and Borron, 2007).
2.4.1.10 Phenolics
Phenolics are a huge and diverse group of aromatic compounds containing benzene ring usually with hydroxyl groups. Many plant phenolics have three carbon side chains called “phenyl-propanoids”. Hydroxylbenzoic acid is one of the simple plant phenolics, whereas coumarin, a phenyl-propanoid is found in some grasses (Walker, 1975).
Most phenolics are glycosylated, thereby rendering them water soluble, and non-toxic (Michael, 2008). Phenolics are substances present in most plants, but their functional significance to plant is only known in few cases and may be toxic (Walker, 1975). Plant phenolics have been considered classic defense compounds for protection from herbivores (Close and McArthur, 2002). Plant phenolics may act as anticarcinogens or antimutagens by blocking or trapping ultimate carcinogen electrophiles in a nucleophilic chemical reaction, to form innocuous products. A continuous input of phenolics could serve as an additional buffer against DNA damage, supplementing the endogenous systems qualitatively and quantitatively. Certain plant phenolics can be effective inhibitor of chemical mutagens and (or) carcinogens. Tetrapyrroles and porphyrins, both plant and animal, can also act as blocking agents (Newmark, 1987).
Phenolics have promising antioxidant and type II diabetes related enzyme inhibition properties (Sabu et al., 2002; Hiroshi et al., 2004). Polyphenols decrease the blood glucose levels (Sabu et al., 2002 and Hiroshi et al., 2004). Shimizu et al. (2000) reported that the reduction in glycemia (blood glucose levels) caused by phenolic compounds has been attributed to such actions as a reduction in the absorption of nutrients (i.e. tea catechins inhibit intestinal glucose absorption). Kao et al. (2000) reported the reduction in food intake (i.e. green tea epigallocatechin gallate significantly reduces food intake), induction of cell regeneration (Kim et al., 2003) and a direct action on adipose cells that enhances insulin activity (Anderson and Polansky, 2002). Phenolic compounds also modify enzymatic and transcriptional activities. However, the ability of nuclear receptors to modulate a wide variety of genes reveals them to be targets in the treatment of such disorders as diabetes or dyslipemia. Nuclear receptors are implicated in the control of lipid homeostasis. They establish a coordinated net of metabolic sensors which integrates lipid metabolism, inflammation, drug metabolism, bile acid synthesis and glucose homeostasis among other processes (Eloranta and Kullak-Ublick, 2005; Beaven et al., 2006).
Amino acids
Amino acids are biologically important organic compounds composed of amine (-NH2) and carboxylic acid (-COOH) functional groups, along with a side-chain specific to each amino acid.
In the form of proteins, amino acids comprise the second largest component (water is the largest) of human muscles, cells and other tissues (Hertweck, 2011). There are 23 proteinogenic ("protein-building") amino acids (Hertweck, 2011), which combine into peptide chains ("polypeptides") to form the building-blocks of a vast array of proteins. Twenty of these are encoded directly by triplet codons in the genetic code and are known as "standard" amino acids. Nine proteinogenic amino acids are called "essential" for humans because they cannot be synthesised from other compounds by the human body but can be obtained from dietary sources (Hertweck, 2011). Amino acids play critical non-protein roles in processes such as neurotransmitter transport and biosynthesis within the body. In the human brain, glutamic acid and gamma-amino-butyric acid (GABA) are the main excitatory and inhibitory neurotransmitters respectively (Petroff, 2002). The amino acid, arginine is the precursor for nitric oxide which aids penile erection, while glycine is a precursor for the synthesis porphyrins used in red blood cells (Petroff, 2002).
Mineral elements
Minerals are inorganic substances, present in all body tissues and fluids and their presence is necessary for the maintenance of certain physicochemical processes which are essential to life (Ozcan, 2003). Minerals may be broadly classified as macro (major) or micro (trace) elements. The third category is the ultra trace elements. The macro-minerals include calcium, phosphorus, sodium, chlorine, while the micro-elements include iron, copper, cobalt, potassium, magnesium, iodine, zinc, manganese, chromium, selenium, sulphur and molybdenum (Eruvbetine, 2003). The macro-minerals are required in amounts greater than 100 mg/dl while the micro-minerals are required in amounts less than 100 mg/dl (Murray et al., 2000). The ultra trace elements include boron, silicon, arsenic and nickel. Cadmium, lead, tin, lithium and vanadium are non-essential in animals (Albion Research Note, 1996). The trace elements are essential components of the enzyme systems; deficiencies therefore affect metabolism and tissue structure and may cause certain diseases (Eruvbetine, 2003). Minerals have important roles to play in many activities in the body. They are involved in a large number of digestive, structural, catalytic (in enzyme and hormone systems), regulatory, physiological and other biosynthetic processes within the body (Eruvbetine, 2003). They therefore fulfill important functions for the maintenance of animal growth and reproduction as well as health status (Close, 1998).
2.3 Regulation of the reproductive system
In females, the secretion of hormones and the reproductive events they regulate are cyclic. Whereas males produce sperm continuously, females release only one egg or a few eggs at a specific time during each cycle (Campbell and Reece, 2005).
The secretory and gametogenic functions of the gonads are both dependent on the secretion of the anterior pituitary gonadotropins, FSH, and luteinizing hormone (LH) (Figure 1). The sex hormones and inhibin B feedback to inhibit gonadotropin secretion. In males, gonadotropin secretion is noncyclic; but in postpubertal females an orderly, sequential secretion of gonadotropins is necessary for the occurrence of menstruation, pregnancy, and lactation (Barrett et al., 2010). Sperm production and androgen synthesis are controlled by a complex feedback loop involving the testes, hypothalamus, and pituitary gland. The pituitary controls the function of the testis by producing follicle-stimulating hormone (FSH) and luteinizing hormone (LH).
FSH stimulates spermatogenesis, in part by affecting Sertoli cells, while LH stimulates androgen production by interstitial cells. Pituitary production of these hormones depends on secretion of gonadotropin-releasing hormone (GnRH) by the hypothalamus which can be stimulated by the cerebral cortex. Elevated levels of GnRH initiate puberty. The production of LH is controlled by the actions of testosterone on the hypothalamus and pituitary. The testis can control brain function. If testosterone concentration is elevated, this hormone inhibits production of GnRH by the hypothalamus; subsequently, LH and FSH production decreases (Palladino, 2002). Hormones also coordinate functions in several different organs at the same time. Considerable coordination among the organs of the female reproductive tract is required. Reproduction will not be successful unless ovulation at the ovary occurs near the time when the uterus is prepared to receive the pre-embryo and, soon thereafter, begin forming the placenta.
Without a functional placenta the pregnancy will not continue very long after implantation of the blastocyst. Surrounding the tubules are clusters of interstitial cells, which synthesize testosterone secretion into the bloodstream. Testosterone is present in infant boys, although synthesis increases dramatically at puberty around the age thirteen. This increase stimulates the onset of spermatogenesis and development of accessory sex glands. All male reproductive organs require testosterone for functions such as protein synthesis, fluid secretion, cell growth, and cell division. Androgens also play an important role in the male sexual response and stimulate secondary sex characteristics such as skeletal development, facial hair growth, deepening of the voice, increased metabolism, and enlargement of the testes, scrotum, and penis.
CHAPTER THREE
2.0 MATERIALS AND METHODS
2.1 Materials
2.1.1 Plant Material and authentication
The plant was obtained from Isaba Ekiti in Ikole LG of Ekiti state, Nigeria, and was authenticated at the Herbarium of Department of Plant Biology, University of Ilorin, Ilorin, Nigeria, where a voucher specimen ddd was deposited.
2.1.2 Drugs, chemicals and assay kits
Aspartate transaminase (AST), Alanine transaminase (ALT), Alkaline phosphatase (ALP), Acid phosphatase (ACP), 3-β-hydroxy steroid dehydrogenase, Glucose-6-phosphate dehydrogenase, Total protein, nitric oxide, Ca+ ATPase, Na+K+ ATPase, Cyclooxygenase 2 (COX-2), Protein Kinase C (PKC)-α, Total-cholesterol, triglycerides, HDL- cholesterol, and glucose assay kits were obtained from Randox Laboratory, Co-Atrim, United Kingdom. Progesterone, estradiol, follicle stimulating hormone (FSH), lutenizing hormone (LH), estrogen, prolactin, and oxytocin assay kits were products of Diagnostics Laboratories, Freiburg, Germany. Mifepristone tablets will be a product of Redson group, Gujarat, India.
2.1.3 Other Chemicals and Reagents
All other chemicals and reagents used which were of analytical grade were products of Sigma Aldrich Ltd., Buchs, Canada.
2.2 Methodology
The chart in Figure 3 gives the summary of the procedural approach in the course of the studies.
Figure 1: Scope of work of the proposed research study
2.2.1 Preparation of chemicals and reagents
The chemicals and reagents were prepared in volumetric flask using glass wares with distilled water. The reagents were stored in neat, airtight reagent bottles except for the Biuret reagent which was stored in plastic containers.
2.2.2 Preparation of extract and mifepristone
The leaves of Spondias mombin were collected, washed under running tap and thereafter oven-dried at 40 ℃ for 72 hours to a constant weight using Uniscope SM9053 Laboratory Oven (Surgifriend Medicals, England). The dried material was pulverized with an electric blender (Crown Star Blender CS- 242B, Trident (H.K.) Ltd, China). Briefly, 2 kg of the powder was extracted in 1 L of 98% ethanol for 48 hours using the cold extraction method (Adome et al., 2003). The contents were later filtered and ethanol evaporated under reduced pressure in a rotary vacuum evaporator. This filtrate was then dried at room temperature and dried mass was stored at 4 °C. The crude yield was later reconstituted in physiological saline to give the required doses of 300, 500 and 800 mg/kg body weight that was used in this study. Information from ethnobotanical survey was put together to arrive at the most frequently mentioned dose of 500 mg/kg body weight while the doses of 300 and 800 mg/kg body weight were computed from the calculated dose of 500 mg/kg body weight.
Mifepristone shall also be reconstituted in physiological saline to give the equivalent of the recommended doses of 2 and 160 mg/kg body weight that was used in this study for the contraceptive and abortifacient role respectively.
2.2.3 Determination of the nutritional/chemical constituents of Spondias mombin leaves
2.2.3.1 Proximate analysis
The proximate constituents of the leaves of Spondias mombin were determined using the method of Pearson (1976) for moisture and ash content. The methods described by Association of Official Analytical Chemists (A.O.A.C) (1980) was used for crude protein, fibre and lipid while carbohydrate was determined using the method described by Oyeleke (1984).
2.2.3.1.1 Determination of Moisture Content
A clean Petri dish was dried in an oven at 1050 C for 30 minutes. It was allowed to cool in a desiccator and weighed (W1). The test sample was measured into the empty, clean Petri dish and weighed (W2). The sample was dried in an oven at 1050 C for 30 minutes and thereafter transferred into a desiccator to cool and weighed again (W3). The weight loss in each case represented the amount of the moisture in the sample. The percentage moisture was calculated using the expression:
Calculation:
Percentage moisture = Weight loss due to drying x 100
Weight of original sample
= (W2 – W3) g x 100
(W2 – W1)g
W1 = Weight of crucible
W2 = Weight of crucible + sample
W3 = Weight of crucible + dried sample
2.2.3.1.2 Determination of Ash Content
A dried porcelain crucible that has been heated in a muffle furnace for 1 minute was transferred to a desiccator to cool and thereafter weighed (W1). A known weight of the sample (2 g) was placed into the crucible and weighed (W2). The crucible with sample was gently heated on the Bunsen flame until smoke ceased, and then transferred into a Muffle furnace where it was burnt at 6000 C into white ashes. The crucible and its contents were then removed and placed in a desiccator to cool after which it was weighed to constant weight (W3).
Calculation:
Ash (%) = Weight of ash (W2 – W3) x 100
Weight of sample (W2 – W1)
W1 = Weight of crucible
W2 = Weight of crucible + sample
W3 = Weight of crucible + ash
2.2.3.1.3 Determination of Crude Protein Content
The determination was carried out in four stages:
Digestion
The sample (2 g) was carefully placed in into a digestion flask, 0.5 g of copper sulphate pentahydrate, 5 g of anhydrous Na2SO4, a speck of selenium and 25 ml of concentrated sulphuric acid was added to the flask. The mixture was slowly heated for 2 hours using a Bunsen burner, until sample frothed. When frothing subsided, the solution changed from black to brilliant green colouration, indicating a complete digestion. The digest was diluted to 250 ml with distilled water in a volumetric flask after cooling as represented by the equation:
R-NH2 + H2SO4 Selenium (NH4)2 SO4 + CO2 + H2O + other sample matrix
(Organic N) Catalyst by-products
Distillation
Kjedhal (Markam) distillation apparatus was set up for the distillation process. An aliquot (5ml) of the digest was pipetted and delivered into the distillation apparatus. A known volume (10 ml) of 60 % NaOH was added and distilled with 5 ml of 2 % boric acid (purple) in the receiving flask to trap the liberated ammonia. The distillate was collected in a 50- ml flask as represented by the equation:
(NH4)2SO4 + 2 NaOH Na2SO4 + 2NH3 + 2H2O
Absorption
The liberated ammonia was trapped by boric acid to form ammonium borate (green).
3NH3 + H3BO3 (NH4)3 BO3
Titration
The distillate obtained was titrated against 0.01M hydrochloric acid (HCl) and the titre value recorded. Boric acid was once more produced as well as ammonium chloride giving the original colour of boric acid. The reaction is represented by the equation:
(NH4)3BO3 + 3 HCl 3NH4Cl + H3BO3
Calculation:
1000 ml of 1 M HCl = 14 g Nitrogen, 1 ml of 0.01 M HCl = 0.00014g N2. 5 ml of digest contains (0.00014 x 5) g of N2. If the titre value is Vml, then 250 ml of the digest will contain:
(1.4 x 10-4 x 250 x V x 1)g x N2 ;
5 w
Weight (g) of sample will contain
(1.4 x 10-4 x 250 V x 1)g x N2 ;
5 w
6.25 = Crude protein conversion factor for food materials
% protein = 1.4 x 10-4 x 250 V x 100 x 6.25
5 x W
2.2.3.1.4 Determination of lipid Content
A known weight of the dried sample (2 g) was placed into a filter paper that has been previously weighed (W1) and the weight (Dried sample + filter paper) was noted as W2. It was then placed in a Soxhlet extractor and fixed with a reflux condenser and a round bottom flask. The flask was half-filled with petroleum ether. This was heated at 50 to 600 C and allowed to reflux for 6 hours. The filter paper was removed from the extractor and dried in the oven at 400 C after which it was transferred into a desiccator to cool after which the final weight (W3) was noted.
Calculation:
Lipid (%) (w/w) = W3 – W2 x 100
W2 – W1
W1 = Weight of filter paper = W1
W2 = Weight of filter paper + test sample (Before fat extraction)
W3 = Weight of filter paper + test sample (After fat extraction)
2.2.3.1.5 Determination of Fibre Content
The defatted sample was used for the determination of fibre content. The sample (2 g) was boiled under reflux for 30 minutes with 200 ml (1.25 %) of H2SO4. It was further filtered and washed with boiling water until the washing was no longer acidic. The residue was boiled in a round bottom flask with 200 ml of (1.25 %) NaOH for another 30 minutes filtered and washed with boiling water until the washing was no longer alkaline. The residue was scrapped into a previously weighed crucible (W1) and dried at 1000 C. It was left in a desiccator to cool and weighed (W2). It was thereafter incinerated in a Muffle furnace at about 600o C for 3 hours, put in a desiccator to cool and then weighed (W3).
Calculation:
Weight of fibre = (W2 – W3)g
% Fibre = W2 – W3 x 100
Weight of Original sample
2.2.3.1.6 Estimation of Carbohydrate content
The procedure described by Oyeleke (1984) was employed. When the total protein and lipid content is subtracted from organic matter, the remainder gives the approximate carbohydrates and nucleic acid.
Calculation:
Carbohydrate = Organic matter (%) – [Protein (%) +Lipid (%) + Nucleic Acid]
This is referred to as the method of Estimation by Difference
Carbohydrate = 100 – (% moisture + % ash + % protein + % lipids + % fibre).
2.2.3.2 Chemical analysis of the leaves of Spondias mombin
2.2.3.2.1 Determination of the amino acid composition
The amino acid profile of Spondias mombin leaves was determined using the method described by Spackman et al. (1958). The sample was dried to a constant weight, defatted (so as to remove non-polar component of the sample), hydrolysed with 7 ml of 6 N HCl, evaporated using a rotatory evaporator and ten microlitre of the hydrolysate was loaded into the Technicon Sequential Multi-sample Acid Analyzer (TSM). The TSM analyzer was designed to separate and analyze free acidic, neutral and basic amino acids of the hydrolysate. The period of an analysis lasted for 76 minutes. The chromatogram obtained showed amino acids peaks corresponding to the magnitudes of their concentrations. The net height of each peak produced by the chart-recorder of TSM (each representing an amino acid) was measured. The half-height of the peak on the chart and width of the peak on the half-height were measured and recorded. Approximate area of each peak was then obtained by multiplying the height with the width at half-height. Finally, the amount of each amino acid present in the sample was calculated in g/100g protein.
2.2.3.2.2 Determination of mineral content
Perchloric acid digestion (wet oxidation) was used for the determination of the mineral content of Spondias mombin leaves. The powdered sample (2 g) of the fruit pulp was placed in a 250 cm3 Erlenmeyer flask which was previously rinsed with acid and distilled water. Perchloric acid (4 cm3), 25 cm3 of concentrated HNO3 and 2 cm3 of concentrated H2SO4 were added under a fume hood. The contents were mixed and heated gently on a hot plate until dense white fumes appeared. The intensity of the heat was increased for 30 minutes and then allowed to cool. A known volume (50 cm3) of distilled water was added and then boiled for 30 seconds. The resulting solution was allowed to cool, filtered into a 100 cm3 Pyrex volumetric flask and made-up to the marked level with distilled water before analysis. The minerals were then analyzed using Atomic Absorption Spectrophotometer (AAS) (Solar 969 Unicam) (AOAC, 1990).
2.2.3.2.3 Determination of the secondary metabolites in Spondias mombin leaves
Phytochemical screening carried out on Spondias mombin leaves included qualitative and quantitative analyses.
2.2.3.2.3.1 Qualitative analysis
A known quantity (1.0 g) of the powdered Spondias mombin leaves was extracted in 100.0 cm3 of distilled water (1% w/v) and used for phytochemical screening. A portion of the extract was subjected to standard chemical tests as described for alkaloid (Harborne, 2008); steroids, anthraquinones, cardenolides and dienolides, phlobatannins (Trease and Evans, 1996); saponins (Wall et al., 1954), phenolics and flavonoids (Awe and Sodipo, 2001), cardiac glycoside (Sofowora, 2006); tannins and terpenes (Odebiyi and Sofowora, 1990).
This was carried out using standard methods described for alkaloids (Harborne, 1973); steroids (Trease and Evans, 1989); phenolics and flavonoids (Awe and Sodipo, 2001); saponins (Wall et al., 1954), glycosides and anthraquinones (Sofowora, 1993); tannins (Odebiyi and Sofowora, 1978). Quantitative analysis of the detected phytochemicals will be carried out using methods described for flavonoids (El-Olemy et al., 1994), alkaloid (Obadoni and Ochuko, 2001), saponins (Brunner, 1984), phlobatannins (Edeoga et al., 2005), anthraquinones, and tannins (Van-Burden and Robinson, 1981).
2.2.3.2.3.1.1 Alkaloids
Exactly 1.0 cm3 of the extract was stirred with 5.0 cm3 of 1% v/v aqueous HCl on a steam bath and filtered while hot. Distilled water was added to the residue and 1.0 cm3 of the filtrate was treated with two drops of Mayer’s reagent (Potassium mercuric iodide- solution), Wagner’s reagent (solution of iodine in Potassium iodide) and Dragendorff’s reagent (solution of Potassium bismuth iodide). The formation of a cream colour with Mayer’s reagent and reddish-brown precipitate with Wagner’s and Dragendorff’s reagents give a positive test for alkaloids.
2.2.3.2.3.1.2 Anthraquinones
Exactly 3.0 cm3 of the extract was shaken with 10.0 cm3 of benzene, filtered and 5.0 cm3 of 10 % v/v NH4OH was added to the filtrate. The presence of a pink colour in the ammonical (lower) phase indicated the presence of anthraquinones.
2.2.3.2.3.1.3 Cardenolides and dienolides
A portion (5.0 cm3) of the extract was added to 2.0 cm3 of glacial acetic acid containing one drop of 5 % w/v FeCl3 solution. This was then followed by the addition of 1.0 cm3 of concentrated H2SO4. A brown ring at the interface indicated the presence of a deoxy sugar characteristic of cardenolides.
2.2.3.2.3.1.4 Cardiac glycosides
A known volume (1.0 cm3) of the extract was added to 2.0 cm3 of chloroform. Thereafter, 2.0 cm3 H2SO4 was carefully added. A reddish brown colour at the interface indicated the presence of aglycone portion of cardiac glycosides.
2.2.3.2.3.1.5 Chalcones
A known volume (2.0 cm3) of NH4OH was added to 5.0 cm3 of extract. Formation of a reddish colour confirmed presence of chalcones.
2.2.3.2.3.1.6 Flavonoids
Exactly 3.0 cm3 of the filtrate was mixed with 4.0 cm3 of 1 % potassium hydroxide in a test tube. A dark yellow colour indicated the presence of flavonoids.
2.2.3.2.3.1.7 Phenolics
Two drops of 5 % w/v of FeCl3 was added to 1.0 cm3 of the plant extract. Presence of a greenish precipitate indicated the presence of phenolics.
2.2.3.2.3.1.8 Phlobatannins
Boiling 3.0 cm3 of the extract with 1 % aqueous hydrochloric acid which resulted in the formation of red precipitate indicated the presence of phlobatannins.
2.2.3.2.3.1.9 Saponins (Frothing Test)
A known volume (5.0 cm3) of the extract was boiled in 20 cm3 of distilled water in a water bath and filtered. The filterate (10.0 cm3) was mixed with 5.0 cm3 of distilled water and shaken vigorously for a stable persistent froth which confirms the presence of saponins.
2.2.3.2.3.1.10 Steroids
Five drops of concentrated H2SO4 was added to 1.0 cm3 of the extract. Red colouration indicated the presence of steroids.
2.2.3.2.3.1.11 Tannins
Exactly 1.0 cm3 of freshly prepared 10% w/v ethanolic KOH was added to 1.0 cm3 of the extract. A white precipitate indicated the presence of tannins.
2.2.3.2.3.1.12 Terpenes
A known volume (1.0 cm3) of the extract was added to 5 drops of acetic acid anhydride followed by a drop of concentrated H2SO4. The mixture was steamed for 1 hour and neutralised with NaOH followed by the addition of chloroform. Presence of bluish-green colour indicated the presence of terpenes.
2.2.3.2.3.2 Quantitative analysis of secondary metabolites
Qualitative analysis of the detected phytochemicals was carried out using methods described for flavonoids (Boham and Kocipai, 1974), anthraquinones (El-Olemy et al., 1994), alkaloid (Adeniyi et al., 2009), saponins (Obadoni and Ochuko, 2001), phlobatannins (Edeoga et al., 2005), phenolics (Harborne,1973), terpernoids (Sofowora, 1993), steroids (Wall et al., 1954) and tannins (Van-Burden and Robinson, 1981).
2.2.3.2.3.2.1 Determination of alkaloids content
Quantitative determination of the alkaloid was carried out by the procedure described by Adeniyi et al (2009). Briefly, 20 g of the powdered sample of Spondias mombim leaves was weighed into 200 ml of 10% (v/v) acetic acid solution in ethanol. The mixture was shaken and left undisturbed for 4 hours before being filtered using a Whatman No.1 filter paper. The filtrate was then concentrated to one quarter of its original volume on the water bath. Concentrated NH4OH was added drop-wise in order to precipitate the alkaloid. A pre-weighed filter paper was used to filter off the precipitate and was thereafter washed with 1% NH4OH solution. The filter paper containing the precipitate was oven-dried at 60o C for 30 minutes to constant weight and cooled in a dessicator. The percentage weight of the alkaloid was determined using the expression:
Concentration of alkaloid (%) = W2 – W1 x 100
W0
Where: W2 = weight of crude alkaloid on filter paper
W1 = weight of the filter paper
W0 = weight of powdered sample used for extraction
2.2.3.2.3.2.2 Determination of tannins content
This was carried out following the procedure described by Van-Burden and Robinson (1981). Briefly, 500 mg of the powdered sample (Spondias mombin leaves) was weighed into a 50 cm3 plastic bottle. Exactly 50 ml of distilled water was added and shaken for 1hour in a mechanical shaker. This was filtered into a 50cm3 volumetric flask. Then, 5 cm3 of the filtrate was pipetted out into a test tube and mixed with 2 cm3 of a mixture of 0.l M FeCl3, 0.1N HCl and 0.008M potassium ferrocyanide. The absorbance was read at 120 nm within 10 minutes. Tannin concentration was then extrapolated from the calibration curve of standard tannin solution (gallic acid) (Figure 15) (Appendix).
2.2.3.2.3.2.3 Determination of anthraquinone content
The procedure described by El-Olemy et al (1994) was used. The powdered sample of Spondias mombim leaves (0.5 g) was weighed into 100 cm3 volumetric flask and 0.2 % zinc dust was added followed by the addition of 50.0 cm3 of hot 5 % w/v NaOH. The mixture was boiled at 80o C for five minutes and rapidly filtered. The filtrate was again heated with another 50.0 cm3 of 5 % w/v NaOH for a red colour to develop. The absorbance of the sample as well as that of the standard was read at 640 nm. The average gradient obtained from calibration curve for anthraquinones (Fig.16) was used to calculate the percentage of anthraquinones using the expression:
% Anthraquinone = Absorbance of sample x Average of gradient x Dilution factor
Weight of sample x 10,000
2.2.3.2.3.2.4 Determination of sapanins content
The procedure described by Obadoni and Ochuko (2001) was adopted for the determination of saponins. A known value (100 cm3) of 20 % aqueous ethanol was added to 20 g of the sample. The sample was boiled at 550 C for 4 hours with continuous stirring. The mixture was filtered and the residue re-extracted with another 200 ml of 20 % ethanol. The combined extracts were reduced to 40 cm3 over water bath at about 900 C. The concentrate was transferred into a 250 cm3 separating funnel after which 20 cm3 of diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. The extraction process was repeated two times after which 60 cm3 of n-butanol was added. The combined n-butanol extracts were washed twice with 10 cm3 of 5 % aqueous sodium chloride. The remaining solution was boiled at 55o C for 30 minutes in a water bath. After evaporation, the sample was dried in the oven to a constant weight; the saponin content was calculated using the expression:
Concentration of saponin (%) = Experimental yield x 100
Theoretical yield
2.2.3.2.3.2.5 Determination of flavonoids content
The procedure described by Boham and Kocipai (1974) was used. Briefly, 0.5 g of the powdered sample of Spondias mombin leaves was weighed into a 100 ml beaker and 80 ml of 95 % ethanol was added and stirred with a glass rod to prevent lumping. The suspension was filtered using Whatman No.1 filter paper into a 100 ml volumetric flask and made up to the marked level with ethanol. The extract (1 ml) was pipetted into a 50 cm3 volumetric flask, and 4 drops of concentrated HCl was added via a dropping pipette after which 0.5 g of magnesium turnings was added to develop a magenta red colouration. The calibration curve (Figure 17) was prepared from standard flavonoid solutions (quercetin) treated with Neu reagent of range 0-5ppm from 100 –ppm stock flavonoid solution and treated in a similar way like the sample. The absorbance of magenta red colouration of the sample and standard solution were read on a Spectronic 21D digital spectrophotometer at a wavelength of 520 nm against the reagent blank. The percentage flavonoid was calculated using the expression:
% Flavonoid = Absorbance of Sample x Gradient factor x Dilution factor
Weight of Sample x 10,000
2.2.3.2.3.2.6 Determination of phenolics content
The procedure described by Harborne (1973) was used. The finely grounded Spondias mombim leaves (0.2 g) was placed in a 50 cm3 beaker and 20 cm3 of acetone added and homogenized for 1 hour to prevent lumping. The mixture was filtered through a Whatman No.1 filter paper into a 100 cm3 volumetric flask. 5 ml of Acetone was then used to rinse, made up to the marked level with distilled water and mixed thoroughly. The mixture (1 ml) was pipetted into 50 cm3 volumetric flask and 20 cm3 of distilled water, 3 ml of phosphomolybdic acid and 5 ml of 23 % Na2CO3 were added and mixed thoroughly. This reaction mixture was left undisturbed for 10 minutes to develop bluish-green colour. Standard phenol of concentration range 0 – 10 mg/ml were prepared from a 100 mg/ml stock phenol solution. (Figure 18). The absorbance of sample and standard phenol was read on a Spectronic 21D digital spectrophotometer at a wavelength of 510 nm. The percentage phenol was calculated using the expression:
% Phenolics = Absorbance of Sample x Gradient factor x Dilution factor
Weight of Sample x 10,000
2.2.3.2.3.2.7 Determination of terpenoids content
The procedure described by Sofowora (1993) was used. The grounded sample of Spondias mombim leaves (0.5 g) was placed in a 50 cm3 conical flask and 20 cm3 of 2.1 (v/v) chloroform: methanol mixture was added, shaken thoroughly and left undisturbed for 15 minutes at room temperature. The suspension was later centrifuged at 1398 g x 15 minutes. The supernatant was discarded, and the precipitate re-washed with 20 ml chloroform: methanol and then re-centrifuged again. The resultant precipitate was dissolved in 40 ml of 10 % sodium deodocyl sulphate solution. The solution of 0.01 M ferric chloride (1 ml) was then added at 30 seconds interval, shaken properly, and left undisturbed for 30minutes. Standard terpenoids (alpha terpineol) of concentration range 0 – 5 mg/ml were prepared from 100 mg/ml stock terpenoids solution and was used to draw the calibration curve (Figure 19). The absorbance of sample and standard terpenoids were read on a Spectronic 21D digital spectrophotometer at a wavelength of 510nm against reagent blank. The percentage terpenoids was calculated using the expression:
% Terpenoids = Absorbance of Sample x Gradient factor x Dilution factor
Weight of Sample x 10,000
2.2.3.2.3.2.8 Determination of steroids content
The method described by Wall et al (1954) was used to quantify the steroid content of the sample. The sample of Spondias mombim leaves (0.5 g) was placed in a 100 cm3 beaker, 20 cm3 of chloroform- methanol (2:1) mixture was added to dissolve the extract upon shaking for 30 minutes on a shaker. The mixture was filtered through a Whatman No 1 filter paper into a dry clean 100ml conical flask. The resultant residue was repeatedly washed with chloroform-methanol (2:1) mixture until free of steroids. A known volume (1 ml) of the filtrate was pipetted into a 30ml test tube and 5.0 ml of alcoholic KOH was added and shaken to obtain a homogenous mixture, which was then heated in a water bath at 37 – 40o C for 90 minutes, cooled to room temperature after which 10 ml petroleum ether was added, followed by 5.0 cm3 of distilled water. This was evaporated to dryness on a water bath, 6.0 cm3 of Liebermann-Burchard reagent was added to the residue and the absorbance of the resulting solution was read at 620 nm on a 21D Spectrophotometer. Standard steroid concentration range (0 – 4 mg/ml) of stock solution were prepared and treated as described above and values obtained for the standard were used to construct calibration curve (Figure 20) used to calculate the steroid content in the sample.
% Steroid = Absorbance of Sample x Gradient factor x Dilution factor
Weight of Sample x 10,000
2.2.4 Preliminary studies of crude extract
2.2.4.1 Female reproductive studies
2.2.4.1.1 Anti-ovulatory studies
2.2.4.1.1.1 Experimental animals
Sexually matured female guinea pigs of proven fertility weighing 700 – 900 g were used for the study. The animals were obtained from the Animal Breeding Unit of the Department of Biochemistry, University of Ilorin, Ilorin, Nigeria. The animals were acclimatized: housed in clean, aluminum cages placed in well-ventilated room conditions as recommended by Sutherland and Festing (1987) (temperature: 18 – 22 ℃; 8 – 20 air changes/hour; 12 – 16 hour light/dark cycle; relative humidity: 45 – 70 %) and they were allowed free access to rat pellets (Premier Feed Mills Company Limited, Ibadan) and tap water. The cages were cleaned daily and the study was conducted following the guidelines on the care and use of laboratory animals of the Ethical Committee of the European Convention and other scientific purposes-ETS-123 as well as that of the National Academies, Washington DC, USA (The National Academies, 1996). Ethical clearance was also obtained from the University of Ilorin Ethical Committee.
2.2.4.1.1.2 Animal grouping, treatment and confirmation of estrus cycle
The female animals were randomized in to 5 groups (A – E) each group containing 5 animals:
Group A: Animals treated with 5 ml physiological saline
Group B: Animals treated with 5 ml of 300 mg/kg body weight of extract
Group C: Animals treated with 5 ml of 500 mg/kg body weight of extract
Group D: Animals treated with 5 ml of 800 mg/kg body weight of extract
Group E: Animals treated with 5 ml of mifepristone solution containing 2 mg/kg body weight.
Confirmation of estrous was done by vaginal smear method of Shresta et al. (2010) − introducing 2 − 3 drops of physiological saline in the vagina of the guinea pigs. Then final drop obtained in the dropper was taken on the slide and under the high power of the microscope.
The animals were held with ventral side up. Drops of 0.9 % w/v normal saline were inserted carefully in to the vagina with a dropper, without damaging the vagina to avoid false positive smears. The drop of normal saline was aspirated and introduced twice, before withdrawing from vagina the withdrawn fluid was transferred on to a microscopic glass slide. A cover slip was then placed carefully on the smear avoiding the entry of air bubbles. The slide was later observed under microscope.
Stages of estrus cycle of each female animal were determined by taking vaginal smears daily between 9 – 10 a.m. for 55 days. The 55 days smear observation was to cover three regular estrus cycles and those animals that showed regular estrus cycles were chosen for the study (Allen, 1922). The length of oestrus cycles and duration of each phase of the cycle were recorded as described by Makonnen et al. (1997).
2.2.4.1.1.3 Preparation of serum and tissue homogenates
On the 56th day, the body weights of the animals were recorded. The serum, uterine and ovarian homogenates were prepared according to the procedures described by Yakubu and Bukoye (2010). Briefly, under ether anaesthesia, the guinea pigs were made to bleed through their cut jugular veins which were slightly displaced (to prevent contamination of the blood by interstitial fluid) into clean, dry centrifuge tubes. The blood was left for 10 minutes at room temperature to clot. The tubes were then centrifuged at 33.5× g for 15 minutes using Hermle Bench Top Centrifuge (Model Hermle, Z300, Hamburg, Germany). The sera were then aspirated with Pasteur pipettes into sample bottles and were used within 12 hour of preparation for the hormonal assay. The guinea pigs were thereafter quickly laparotomised in the cold; the uteri excised and were carefully removed with their luminal fluid and their weights noted; and later transferred into ice-cold 0.25 M sucrose solution. Ovaries and uteri were dissected out, freed from extra deposition and weighed on a sensitive balance. Fimbriated part of the oviduct was dissected out from the rats, suspended in normal saline placed on microscopic slide with cover slip to count number of ova in the oviduct. Ovary and uterus were processed for biochemical analysis (Makonnen et al., 1997).
2.2.4.1.1.4 Computation of some biochemical parameters
The weight of ovaries (g) ovarian/body weight ratio (%) = (weight of ovary/weight of the animals ×100) were computed.
2.2.4.1.1.5 Determination of some biochemical parameters
Ovarian cholesterol, ascorbic acid and protein content were determined by adopting the procedure outlined by Fredrickson et al. (1967), Koshiishi and Imanari (1997), Plummer, (1978) respectively.
2.2.4.1.1.5.1 Determination of ovarian cholesterol
The concentration of total cholesterol in the ovaries of the animal was carried out using the CHOD-PAP reaction described by Friedrickson et al. (1967).
Principle:
The cholesterol is determined after enzymatic hydrolysis and oxidation. The indicator quinoneimine is formed from hydrogen peroxide and 4-aminoantipyrine in the presence of phenol and peroxidase according to the following reactions:
Cholesterol ester + H2O Cholesterol esterase Cholesterol + Fatty acids
Cholesterol + O2 Cholesterol esterase 4- cholesten-3-one + H2O2
2H2O2 + 4-aminoantipyrine + Phenol Peroxidase Quinoneimine (Red) + 4H2O
Procedure:
A known volume (2.0 cm3) of the working reagent (mixture of 4-aminoantipyrine, phenol, cholesterol esterase and peroxidase) was added to test tubes containing 0.02 cm3 of serum (appropriately diluted). The blank and standard were constituted by replacing the serum with 0.02 cm3 of distilled water and standard working reagent respectively. The reaction constituents were thoroughly mixed and incubated at 370 C for 5 minutes. The absorbance was spectrophotometrically read at 500 nm against the blank.
Calculation:
Concentration of cholesterol
(mg/dl) = Asample × 200
Astandard
2.2.4.1.1.5.2 Determination of ovarian ascorbic acid concentration
The ovarian ascorbic acid concentration was determined by the Folin–Ciocalteu reagent method described by Jagota and Dani (1982).
Principle:
This method is based on the reaction of ascorbic acid with Folin’s reagent to give a blue colour which has its maximum absorption at 760 nm.
Procedure:
The homogenate (0.5 cm3) was added to 0.8 cm3 of 10% trichloroacetic acid and shaken vigorously. The mixture was kept on ice for 5 minutes and then centrifuged at 3000 rpm for 5 minutes. This extract (0.2 cm3) was then diluted to 2.0 cm3 with double-distilled water. Commercially prepared 2.0 M Folin–Ciocalteu was diluted 10-fold with distilled water and 0.2 cm3 of this diluted reagent was added to the mixture and vigorously shaken. After 10 minutes at 250C, the absorbance was read at 760 nm against distilled water as a blank and the vitamin C content was estimated through the calibration curve of ascorbic acid (Figure 67).
2.2.4.1.1.5.3 Determination of ovarian protein content
The protein concentration in the ovaries of animals was assayed, using Biuret reagent as described by Gornall et al. (1949).
Principle:
Cupric ions form a purple coloured complex with compounds containing repeated amide group (-CONH-) in alkaline medium. The purple colour is due to the coordination between the cupric ions and the unshared electron pair of peptide nitrogen and the oxygen of water.
Procedure:
A known volume (4.0 ml) of Biuret reagent was added to 1.0 ml of the sample (appropriately diluted). This was mixed thoroughly by shaking and left undisturbed for 30 minutes at room temperature for colour development. The blank was constituted by replacing the sample with 1.0 ml of distilled water. The absorbance was read against blank at 540 nm.
Protein concentration of the sample was calculated from a calibration curve (Figure 68) obtained using Bovine Serum Albumin (BSA). For the calibration curve, different concentrations of BSA (1-10 mg/ml) were separately pipetted into ten test tubes. Each test tube was made up to 1 ml with appropriate volume of distilled water after which 4.0 ml of Biuret was added. The mixture was left undisturbed at room temperature for 30 minutes, after which absorbance was read at 540 nm against the blank. Thereafter, a calibration curve of absorbance was plotted against BSA concentration (Figure 62).
Calculation:
The concentration of the protein in the sample was extrapolated from the calibration curve and then multiplied by the appropriate dilution factor, using the expression:
Protein concentration (mg/ml) = Cs × F
Where:
Cs= corresponding protein concentration from the calibration
F= dilution factor
2.2.4.1.1.6 Assay of some ovarian steroidal enzymes
The assay methods Hu et al. (2009) and Noltmann et al. (1961) will be used to estimate the activities of 3-β-hydroxysteroid dehydrogenase and glucose-6-phosphate dehydrogense respectively.
2.2.4.1.1.6.1 3-β-hydroxysteroid dehydrogenase
2.2.4.1.1.6.2 Glucose-6-phosphate dehydrogenase
2.2.4.4.1.7 Determination of some reproductive hormones:
The procedure outlined in the manufacturers’ instruction manual as described for progesterone (Radwanska et al., 1978), estradiol (Smith et al., 1980), follicle stimulating hormone (FSH) (Kapen et al., 1973), lutenizing hormone (LH) (Uotila et al., 1981), Estrogen (Abraham et al., 1972), Prolactin (Frantz, et al., 1972), oxytocin (Porstmann and Kiessig, 1992) will be adopted. Protein concentration of the serum will be determined using the biuret method (Plummer, 1978).
2.2.4.1.1.7.1 Determination of Progesterone
The procedure described by Tietz (1995) will be adopted. A desired number of coated wells in the tube holder will be secured. 10 μl of standards, specimens and controls will be dispensed into appropriate wells. 10 μl of progesterone-HRP conjugate reagent and 50 μl rabbit anti-progesterone will be dispensed into each well. This will be thoroughly mixed for 30 seconds after which it will be incubated at 37° C for 90 minutes. The microwells will be rinsed and flicked 5 times with distilled water. 100 μl of 3, 3’, 5, 5’ Tetramethylbenzidine (TMB) reagent will be dispensed into each well, gently mixed for 5 seconds and incubated at room temperature (18 – 25°C) for 20 minutes. The reaction will be stopped by adding 100 μl of stop solution to each well. The resultant yellow colour formed from the blue colour will be read at 405nm with a microtiter well reader within 15 minutes.
Calculation of progesterone concentration
The serum progesterone concentration of test samples will be extrapolated from a calibration curve by correlating the absorbance of the sample with the corresponding absorbance on the calibration curve.
2.2.4.1.1.7.2 Luteinising hormone
The serum LH was quantitatively determined using the direct human serum luteinising enzyme immunoassay (EIA) kit as described by Tietz (1995).
Principle:
The microwell LH EIA is based on the sandwich principle following the general antibody-antigen reaction of the enzyme linked immunosorbent assay as described by Tietz (1995).
Procedure:
A desired number of coated mirowells were placed in well holders. Duplicates of sample, standard and control were chosen. 50 µl each of standards, samples and controls were dispensed into appropriate wells (excluding LH1) within 5 minutes. 100 µl of LH-HPR conjugate reagent was dispensed into each wells (excluding LH1), swirled gently for 30 seconds and covered with paraffin, then incubated at 370 C for 1 hour after which the incubation mixture was decanted and the plates blotted with absorbent paper. All the microwells were rinsed and flicked 5 times with 350 µl of 3, 3’, 5, 5’-tetramethylbenzidine (TMB) reagent (a surfactant) and dried. A known volume (50 µl) each of 3, 3’, 5, 5’-tetramethylbenzidine (TMB) reagent (a surfactant) and hydrogen peroxide were dispensed into the wells, gently mixed for 20 seconds and incubated at room temperature for 15 minutes. The reaction was stopped by adding 50 µl of 1N HCl to each well and then gently mixed for 20 seconds. The resultant yellow colour solution formed from blue colour was read at 405 nm with a microtitre within 15 minutes.
Calculation:
The mean absorbance value for each set of reference standard, control and sample was obtained. The corresponding serum LH concentration of the sample was extrapolated from the calibration curve for luteinising hormone using their absorbance values (Figure 60).
2.2.4.1.1.7.3 Follicle-stimulating hormone
The serum FSH was quantitatively determined using the direct human serum follicle-stimulating hormone enzyme immunoassay (EIA) kit as described by Tietz (1995).
Principle:
The microwell FSH EIA is based on the sandwich principle following the general antibody-antigen reaction of the enzyme linked immunosorbent assay as described by Tietz (1995).
Procedure:
The microwell test components and sample specimens were brought to room temperature. The number of coated microwells needed was determined and a data sheet with the appropriate information was marked. An extra well (FSH1) for substrate blank was included.
A desired number of coated mirowells were placed in well holders. A known volume (50 µl) each of standards, samples and controls were dispensed into appropriate wells (excluding FSH1) within 5 minutes. 100 µl of FSH-HRP conjugate reagent was dispensed into each well (excluding FSH1), swirled gently for 30 seconds and covered with paraffin, then incubated at 370C for 1 hour after which the incubation mixture was decanted and the plates blotted with absorbent paper. All the microwells were rinsed and flicked 5 times with 350 µl of 3, 3’, 5, 5’-tetramethylbenzidine (TMB) reagent (a surfactant) and dried. 50 µl each of 3, 3’, 5, 5’-tetramethylbenzidine (TMB) reagent (a surfactant) and hydrogen peroxide were dispensed into all well, gently mixed for 20 seconds and incubated at room temperature for 15 minutes. The reaction was stopped by adding 50 µl of 1N HCl to each well and then gently mixed for 20 seconds. The absorbance of each well was read at 405 nm with a microtitre within 15 minutes.
Calculation:
The mean absorbance value for each set of reference standard, control and sample was obtained. The corresponding serum FSH concentration of the sample was extrapolated from the calibration curve for follicle-stimulating hormone using their absorbance values (Figure 61).
2.2.4.1.1.7. 4Estradiol
2.2.4.1.1.7.5 Estrogen
2.2.4.1.1.7.6 Prolactin
2.2.4.1.1.7.7 Oxytocin
2.2.4.1.1.7.8 Protein concentration of the serum
2.2.4.1.1.8 Histology of some reproductive organs
For the histopathological changes in the ovary, fallopian tubes and uterus, as well as the number of developing follicles, grafian follicles, corpora lutea and atretic follicles; these organs were excised as previously described. The procedure described by Krause (2001) and as well as Drury and Wallington (1973) was used. Briefly, fixed organs in 10%v/v buffered formaldehyde were dehydrated through ascending grades of ethanol (70, 90 and 95% v/v). They were cleaned in xylene, impregnated and embedded in paraffin wax (melting point 56o C); sections were cut at 5 µm on a rotatory microtone. The sections were then floated out on clean microscope slides, which had previously been albumenized to prevent detachment from slides during staining procedure. They were later air-dried for 2 hours at 37o C. After staining, the slides were passed through ascending concentration of alcohol (20 – 100%) for dehydration and then cleaned with xylene. A permanent mounting medium (basalm) was put on the tissue section. A thin glass-covered slip was placed on the covering–mounting medium and underlying tissue sections were allowed to dry. This was later observed using the Leitz, DIALUX research microscope at x200 and photomicrographs weree taken in bright field at x200.
2.2.4.1.2 Contraceptive studies
2.2.4.1.2.1 Experimental animals
Sexually matured guinea pigs of both sexes (male, 900 − 1200 g; female, 700 – 900 g) for this study were obtained and used under the same care as earlier described.
2.2.4.1.2.2 Animal grouping and treatment
Briefly, female guinea pigs were paired overnight with the male guinea pigs in ratio 1∶2 in the aluminum cages that made free access to food and water for the animals. The day on which a vaginal plug and spermatozoa (detected with the aid of light microscope) appeared in the vaginal smear was assumed day 0 of copulation. The female animals were then completely randomized into 6 groups (groups A – F) of five animals each:
Group A: Non-mated animals treated with 5 ml of physiological saline
Group B: Mated animals treated with 5 ml of physiological saline
Group C: Mated animals treated with 5 ml of 300 mg/kg body weight of extract
Group D: Mated animals treated with 5 ml of 500 mg/kg body weight of extract
Group E: Mated animals treated with 5 ml of 800 mg/kg body weight of extract
Group F: Mated animals treated with 5 ml of mifepristone solution containing 2 mg/kg body weight
The animals were treated for 35 consecutive days (to cover two oetrous cycle) starting from the first day of detection of spermatozoa on the vagina. Physiological saline and the extracts were administered orally between 0800 and 0900 hours on daily basis using oropharyngeal cannula to respective groups of the animals for the experimental periods
2.2.4.1.2.3 Preparation of serum and tissue homogenates
On the 36th day, the body weights of the animals were recorded. The serum, uterine and ovarian homogenates will be prepared according to the procedures described by Yakubu and Bukoye (2009) as previously described in section 11. Briefly, under ether anaesthesia, the guinea pigs will be made to bleed through their cut jugular veins which were slightly displaced (to prevent contamination of the blood by interstitial fluid) into clean, dry centrifuge tubes. The blood will be was left for 10 minutes at room temperature to clot. The tubes will then be centrifuged at 33.5× g for 15 minutes using Hermle Bench Top Centrifuge (Model Hermle, Z300, Hamburg, Germany). The sera will later be aspirated with Pasteur pipettes into sample bottles and used within 12 hour of preparation for the hormonal assay. The guinea pigs will thereafter be quickly laparotomised in the cold; the uteri will be excised the uteri were carefully removed with their luminal fluid and their weights noted; and later transferred into ice-cold 0.25 M sucrose solution. The uteri and ovaries will be freed of surrounding tissues, blotted with clean tissue paper and then weighed. This will then be homogenized in ice-cold 0.25 M sucrose solution (1:5 w/v) (Akanji and Yakubu, 2000). The homogenates will further be centrifuged at 105.5× g for 15 minutes to obtain the supernatants which were kept frozen overnight at −20° C before being used for the various biochemical assays.
2.2.4.1.2.4 Computation of some biochemical parameters
Both uterine horns were examined for number of implants, and corpora lutea; implantation site, The weight of uterus and ovaries (g), uterine/body weight ratio (%) = (weight of uterus/weight of the animals) ×100, ovarian/body weight ratio (%) = (weight of ovary/weight of the animals) ×100), ovarian/uterine weight ratio, length of right uterine horn (cm), pituitary weight; implantation index=(total number of implantation sites/number of corpora lutea) ×100; pre-implantation loss=(number of corpora lutea– number of implantation sites/number of corpora lutea) ×100. Moreover, the following reproductive indices shall be calculated using the expression: Mating index defined as number of sperm positive females/number of mated females × 100; Pregnancy index defined as number of pregnant females/number of sperm positive females × 100; Fertility index = (number of pregnant females / number of females with successful copulation) x 100 (Zia-Ul-Haque et al., 1983).
2.2.4.1.2.5 Determination of some biochemical/enzyme concentrations
The ovarian and uterine concentration of nitric oxide will be determined by adopting the method of Wo et al. (2013). Protein content of the homogenate will also be determined using Biuret method described by Plummer (1978).
2.2.4.1.2.5.1 Determination of nitric oxide concentration
The method described by Wo et al. (2013) was used to assay for the concentration of nitric oxide in the ovary and uterus of animals.
Principle
The principle is based on reduction of nitrate by copper-coated cadmium, and the nitrite produced is determined by diazotization of fuchsin acid under acidic condition, coupling to resorcinol in a slightly alkaline medium. The change in absorbance at 436 nm was read spectrophotometrically.
Experimental procedures include:
i. Deproteinization of serum samples;
ii. Activation of cadmium granules and
iii. Reduction of Nitrate to Nitrite
i. Deproteinization of serum samples
A known volume (0.5 ml) of serum was added to 2 ml of 75 mmol/l ZnSO4 solution. The solution was mixed thoroughly and 2.5 ml of 55 mmol/l NaOH was added to the mixture. The solution was adjusted to a pH of 7.3 with 0.1N NaOH. After 10 minutes of incubation, the supernatant was collected by centrifugation at 25 x g for 10 minutes. This was used for the determination of nitrate and nitrite.
ii. Activation of cadmium granules
The acid from the cadmium granules (see appendix) was rinsed 3 times with distilled water. The cadmium granules were swirled for 2 minutes in 5 mmol/l CuSO4 in glycine-NaOH buffer, drained and re-rinsed 3 times with glycine-NaOH buffer. The activated, copper –coated granules were used within 10 minutes. Prolonged exposure of the granules to air diminishes their reductive ability.
iii. Nitrite assay
Glycine-NaOH buffer (1 ml) was added to each test tube of blanks and samples. A known volume (2 cm3) of deproteinized serum sample was added to each of the sample tubes, and the volume was adjusted to 4.0 cm3 in all the sample tubes using distilled water. Distilled water (3 cm3) was added to the blank tube. The reaction was initiated by the addition of 2.5 x g of freshly activated cadmium granules to each of the test tubes, stirring once. After 60 minutes, 2.0 cm3 of sample from each tube was transferred to an appropriately labelled tube for nitrite determination.
The concentration of nitrite was determined by the addition of 2.5cm3 of ethylenediaminetetracetic acid solution, 3.0 cm3 of 1.0 mol/l HCl and 0.3 cm3 of 1.0g/l fuchsin acid solution to each of the labelled tubes containing 2.0 cm3 of supernatant to yield a final volume of 7.8 cm3, mixed thoroughly followed by incubation for 2 minutes. Next, 0.2 cm3 of 0.05 mol/l resorcinol was added, followed by 3.0 cm3 of 1.0 mol/l NH4OH, mixed thoroughly. The absorbance of the samples was determined against the blank at 436 nm. The calibration curve (Figure 21) of the working standard solution was prepared using different concentration of NaNO2. The concentration series was made at 0, 22, 44, 66, 88,110, 132, 154, 176, 198 and 220 umol/l respectively, with each concentration repeated twice and the determination carried out as described previously. The concentration of serum nitrite was traced out from the calibration curve (Figure 21) and the result expressed in umol/l.
2.2.4.1.2.5.2 Determination of ovarian and uterine protein content
The protein concentration in the ovaries and uterus of the animals was assayed, using Biuret reagent as described by Gornall et al (1949). The protocol is the same with the one earlier described for serum total protein concentration (2.2.14.2.1.1) except that the samples in this regard are ovaries and uterus homogenate.
2.2.4.1.2.6 Determination of some reproductive hormones
The procedure outlined in the manufacturers’ instruction manual as described for progesterone (Radwanska et al., 1978), estradiol (Smith et al., 1980), follicle stimulating hormone (FSH) (Kapen et al., 1973), lutenizing hormone (LH) (Uotila et al., 1981), Estrogen (Abraham et al., 1972), Prolactin (Frantz, et al., 1972), oxytocin (Porstmann and Kiessig, 1992) will be adopted. Protein concentration of the serum will be determined using the biuret method (Plummer, 1978).
2.2.4.1.2.7 Histology of some reproductive organs
For the histopathological changes in the ovary, fallopian tubes and uterus, these organs were excised as previously described. The earlier described procedure described by Krause (2001) and as well as Drury and Wallington (1973) was adopted.
2.2.4.1.3 Anti-implantation studies
2.2.4.1.3.1 Experimental animals
Animals for this study were obtained and used under the same care as earlier described.
2.2.4.1.3.2 Animal grouping and treatment
Virgin female guinea pigs exhibiting normal oestrus cycle were selected for this study. The vaginal smear of each member of the group was daily examined for proestrus. Any member in which this was established were then removed from the others and caged overnight with a proven male for mating (in the ratio of 2 females to 1 male). Vaginal smear was examined for motile spermatozoa in the morning. The day on which the spermatozoa is found in the smear coupled with the use of pregnancy trip was considered the first day of pregnancy (Day 1). They were then randomly assigned into five groups of 5 animals each:
Group A: Non-pregnant animals treated with 5 ml of physiological saline
Group B: Pregnant animals treated with 5 ml of physiological saline
Group C: Pregnant animals treated with 5 ml of 300 mg/kg body weight of extract
Group D: Pregnant animals treated with 5 ml of 500 mg/kg body weight of extract
Group E: Pregnant animals treated with 5 ml of 800 mg/kg body weight of extract
Group F: Pregnant animals treated with 5 ml of 0.45 mg/kg body weight of Ethynyl oestradiol
The animals were treated for 35 days to cover two estrous cycle, during which the physiological saline and the extracts were administered orally between 0800 and 0900 hours on daily basis using oropharyngeal cannula to respective groups of the animals for the experimental periods.
2.2.4.1.3.3 Preparation of serum and tissue homogenates:
On the 36th day, the body weights of the animals will be recorded. The serum, uterine and ovarian homogenates were prepared according to the procedures described by Yakubu and Bukoye (2009). Briefly, under ether anaesthesia, the guinea pigs will be made to bleed through their cut jugular veins which were slightly displaced (to prevent contamination of the blood by interstitial fluid) into clean, dry centrifuge tubes. The blood will be was left for 10 minutes at room temperature to clot. The tubes will then be centrifuged at 33.5× g for 15 minutes using Hermle Bench Top Centrifuge (Model Hermle, Z300, Hamburg, Germany). The sera will later be aspirated with Pasteur pipettes into sample bottles and used within 12 hour of preparation for the hormonal assay. The guinea pigs will thereafter be quickly laparotomised in the cold; the uteri will be excised the uteri were carefully removed with their luminal fluid and their weights noted; and later transferred into ice-cold 0.25 M sucrose solution. The uteri and ovaries will be freed of surrounding tissues, blotted with clean tissue paper and then weighed. This will then be homogenized in ice-cold 0.25 M sucrose solution (1:5 w/v) (Akanji and Yakubu, 2000). The homogenates will further be centrifuged at 105.5× g for 15 minutes to obtain the supernatants which were kept frozen overnight at −20° C before being used for the various biochemical assays.
2.2.4.1.3.4 Computation of some biochemical parameters
Both uterine horns will be examined. The following parameters of implantation will be recorded or computed: number of implants, aborted implants and corpora lutea; implantation site, uterine diameter, thickness of the endometrium, epithelial cell height, resorption index resorption index = (total number of resorption sites/ total number of implantation sites) ×100; pre-implantation loss = (number of Corpora lutea – number of implantations/number of Corpora lutea) ×100; post-implantation loss = (number of implantations – number of life fetuses/number of implantations)×100; gestation index, defined as number of females with alive pups/ No of pregnant females × 100; delivery index defined as number of females delivering/number of pregnant females × 100; birth live index defined as number of live offspring/number of offspring delivered × 100; post-natal viability index defined as number of pups alive on day 35 / No of alive pups × 100; and weaning viability index defined as number of pups alive at day 65/ No of pups alive at day 35 × 100; the initial and final weights of the animals as well as the feed and water intake, pituitary weight.
2.2.4.1.3.5 Determination of some biochemical/enzyme concentrations
The ovarian and uterine concentration of nitric oxide and protein content will be determined by adopting the principle described by Schmidt et al. (1995) and Plummer (1978) respectively.
2.2.4.1.3.5.1 Nitric oxide
2.2.4.1.3.5.2 Protein content
2.2.4.1.3.6 Determination of some reproductive hormones
The procedure outlined in the manufacturers’ instruction manual as described for progesterone (Radwanska et al., 1978), estradiol (Smith et al., 1980), follicle stimulating hormone (FSH) (Kapen et al., 1973), lutenizing hormone (LH) (Uotila et al., 1981), Estrogen (Abraham et al., 1972), Prolactin (Frantz, et al., 1972), oxytocin (Porstmann and Kiessig, 1992) will be adopted. Protein concentration of the serum will be determined using the biuret method (Plummer, 1978).
2.2.4.1.4 Estrogenicity/Anti-estrogenic studies
2.2.4.1.4.1 Experimental animals
Animals for this study will be obtained and used under the same care as earlier described. For studies on estrogenic activity of the plant extract, 30 sexually immature female guinea pigs will be used. 25 of the animals will be prepared for ovariectomy, using intraperitoneal pentobarbital sodium (6 %) at the dose of 60 mg/kg to induce anaesthesia. The uterine horns will be exteriorized and the ovaries identified and excised via a laparotomy incision. The incision sites will be routinely closed.
2.2.4.1.4.2 Animal grouping and treatment
The in vivo estrogenic/anti-estrogenic response of the guinea pigs to the extract will be evaluated by adopting the procedure described by Kanno et al. (2001).
Fifteen days after ovariectomy, the animals will be completely randomized into five groups (A – E) and F for non-ovariectomized animals:
Group A: Ovariectomized animals treated with 5 ml of 0.1 mg/kg body weight of stilboestrol suspension in paraffin oil (positive control)
Group B: Ovariectomized animals treated with 5 ml of 0.1 mg/kg body weight of paraffin oil but without stilboestrol (negative control).
Group C: Ovariectomized animals treated with 5 ml of 300 mg/kg body weight of the extract
Group D: Ovariectomized animals treated with 5 ml of 500 mg/kg body weight of the extract
Group E: Ovariectomized animals treated with 5 ml of 800 mg/kg body weight of the extract
Group F: Non-Ovariectomized animals treated with 5 ml of physiological saline
The animals were treated for 17 days during which the physiological saline, stilboestrol, paraffin oil and the extracts were administered orally between 0800 and 0900 hours on daily basis using oropharyngeal cannula to respective groups of the animals for the experimental periods.
2.2.4.1.4.3 Preparation of serum and uterine homogenates
On the 33rd day, the body weights of the animals were recorded. The serum and uterine homogenates were prepared according to the procedures described by Yakubu and Bukoye (2009). Briefly, under ether anaesthesia, the guinea pigs will be made to bleed through their cut jugular veins which were slightly displaced (to prevent contamination of the blood by interstitial fluid) into clean, dry centrifuge tubes. The blood will be was left for 10 minutes at room temperature to clot. The tubes will then be centrifuged at 33.5× g for 15 minutes using Hermle Bench Top Centrifuge (Model Hermle, Z300, Hamburg, Germany). The sera will later be aspirated with Pasteur pipettes into sample bottles and used within 12 hour of preparation for the hormonal assay. The guinea pigs will thereafter be quickly dissected in the cold; the uteri will be excised the uteri were carefully removed with their luminal fluid and their weights noted; and later transferred into ice-cold 0.25 M sucrose solution. The uteri will be freed of surrounding tissues, blotted with clean tissue paper and then weighed. This will then be homogenized in ice-cold 0.25 M sucrose solution (1:5 w/v) (Akanji and Yakubu, 2000). The homogenates will further be centrifuged at 105.5× g for 15 minutes to obtain the supernatants which were kept frozen overnight at −20° C before being used for the various biochemical assays.
2.2.4.1.4.4 Computation of some biochemical parameters
The weight of uterus and luminal fluid (g), uterine/body weight ratio (%) = (weight of uterus/weight of the animals) ×100, length of right uterine horn (cm), vaginal opening (%) = (number of rats with open vagina/number of treated rats) ×100, vagina cornification (%), length of epithelium, number of uterine glands, number of litters, gestation index (defined as number of females with alive pups/ No of pregnant females × 100), delivery index (defined as number of females delivering/number of pregnant females × 100), birth live index (defined as number of live offspring/number of offspring delivered × 100), post-natal viability index (defined as number of pups alive on day 35 / No of alive pups × 100), weaning viability index (defined as number of pups alive at day 65/ No of pups alive at day 35 × 100), pituitary weight (g) will be computed.
2.2.4.1.4.5 Determination of some biochemical/enzyme concentrations
The uterine protein, glucose, total cholesterol, triacylglycerides, HDL-cholesterol, LDL-cholesterol, Ca+ ATPase, Na+-K+ ATPase as well as alkaline phosphatase activity will be determined using standard procedures described by Plummer (1978), Barham and Trinder (1972), Fredrickson et al. (1967), Tietz (1990), Lopes-Virella (1977), Friedewald et al. (1972), Pershadsingh and McDonald (1980), Esmann (1988), Wright et al. (1972) while the atherogenic index will be computed using the expression given by Ng et al. (1997) as LDL-C/HDLC
2.2.4.1.4.5.1 Determination of uterine protein concentration
The uterine protein concentration was assayed by using the biuret reagent as described as described by Gornall et al (1949). The protocol is the same with the one earlier described for ovarian total protein concentration (2.2.14.2.1.1).
2.2.4.1.4.5.2 Determination of uterine glucose concentration
Glucose was determined after enzymatic oxidation in the presence of glucose oxidase (Barham and Trinder, 1972).
Principle:
Glucose is oxidized enzymatically to gluconic acid and hydrogen peroxide in the presence of glucose oxidase. The resulting hydrogen peroxide reacts, under catalysis of peroxidase with phenol and 4-aminophenazone to form a red-violet quinoeimine dye as an indicator.
Glucose + O2 + H2O glucose oxidase gluconic acid + H2O2
2H2O2 + phenol + 4-aminophenazone peroxidase quinoeimine + H2O
Procedure:
2.00 ml of the working reagent was added to 20 µlof sample and 20 µl of standard reagent. The blank was constituted by replacing the sample with distilled water. The reaction constituents were thoroughly mixed and incubated at 37 oC for 10 minutes. The absorbance of the red-violet indictor from standard and samples were then measured against reagent blank at 500 nm within 60 minutes.
Calculation:
Concentration of glucose in the sample:
= ΔA sample x Concentration of standard (mmol/L)
_________________________________________________________
ΔA standard
Concentration of standard = 5.10 mmol/L
2.2.4.1.4.5.3 Determination of uterine total cholesterol concentration
The uterine total cholesterol concentration was determined by colorimetric method described in section 2.2.14.2.3.1. The only modification was that tissue homogenate was replaced by uterine sample.
2.2.4.1.4.5.4 Determination of uterine high density lipoprotein-cholesterol concentration
HDL cholesterol concentration was determined by CHOD-PAP method described by Friedwald et al. (1972).
Principle:
Low density lipoproteins (LDL and VLDL) and chylomicron fractions are precipitated quantitatively by the addition of phosphotungstic acid in the presence of magnesium ions. After centrifugation, the cholesterol concentration in the HDL (high density lipoprotein) fraction, which remains in the supernatant, is determined.
Procedure:
A known sample (0.2 cm3) of serum was added 0.5 cm3 of phosphotungstic acid/magnesium chloride solution, mixed and left undisturbed for 10 minutes at 250C after which it was centrifuged at 4000 rpm for 10 min in order to remove non-HDL lipoproteins. After that, samples were prepared by pipetting 0.05 cm3 of serum (supernatant) into test tubes. Standard and blank were prepared by pipetting 0.05 cm3 each of the standard and distilled water into appropriately labelled tubes. This was followed by the addition of 1 cm3 phosphotungstic acid (CHOL reagent) into each well. Samples were mixed, incubated at 370C for 5 minutes and absorbance read at 500 nm within 60 minutes.
Calculation:
Concentration of HDL-cholesterol (mg/dl) = Absorbance of sample x Conc. of standard
Absorbance of standard
2.2.4.1.4.5.5 Determination of uterine triacylglycerol concentration
The colorimetric reaction method described by Tietz (1995) was used to assay for the concentration of triglycerides in the uterus homogenate.
Principle:
The triglycerides are determined after enzymatic hydrolysis with lipases. The indicator is a quinoneimine formed from hydrogen peroxide, 4-aminophenazone and 4-chlorophenol under the catalytic influence of peroxidase as illustrated by these set of equations.
Triglycerides + H2O lipases glycerol + fatty acids
Glycerol + ATP glycerol kinase glycerol-3-phosphate + ADP
Glycerol-3-P + O2 G-3-P oxidase DHAP + H2O2
2 H2O2 + 4-aminophenazone + 4-chlorophenol peroxidase quinoneimine + HCl + 4H2O
Procedure:
Sample was prepared by pipetting 0.01 cm3 of serum and adding 1 cm3 of the enzyme reagent (Pipes buffer containing 4-chlorophenol, magnesium ion, 4-aminophenazone and various enzymes) in a test tube. Standard and blank were constituted by adding 1 cm3 of the enzyme reagent to 0.01 cm3 standard and distilled water respectively. Samples were mixed, incubated at 370 C for 5 minutes and absorbance read at 500 nm.
Calculation:
Concentration of triacylglycerol (mg/dl) = Absorbance of sample x Conc. of standard
Absorbance of standard
2.2.4.1.4.5.6 Determination of uterine low density lipoprotein-cholesterol concentration
The uterine LDL cholesterol was computed by adopting the expression of Friedwald et al. (1972) as follows:
LDL-cholesterol (mg/dl) = TC – TG/5 – HDLC
Where TC = Total cholesterol
TG = Triacylglycerol
HDLC = HDL-cholesterol
2.2.4.1.4.5.7 Atherogenic Index
The atherogenic index was computed using the expression given by Ng et al (1997) as LDL-C/HDLC.
2.2.4.1.4.5.8 Determination of uterine Ca+ ATPase activity
2.2.4.1.4.5.9 Determination of uterine Na+-K+ ATPase activity
2.2.4.1.4.5.10 Determination of uterine alkaline phosphatase activity
The method described by Wright et al. (1972a) was employed in this assay.
Principle:
The amount of phosphate ester split within a given period of time is a measure of the phosphatase enzyme. Para-nitrophenyl phosphate was hydrolysed to para-nitrophenol and phosphoric acid at a pH of 10.1. The para-nitrophenol conferred a yellowish colour on reaction mixture and its intensity was read spectrophotometrically at 400 nm.
Procedure:
A known volume, 2.2 cm3 of (0.1 M) carbonate buffer and 0.1 cm3 of (0.1 M) MgSO4.7H2O were added sequentially to the test tubes. Then 0.2 cm3 of the enzyme source (appropriately diluted) was added and incubated at 370C for 10 minutes. A known volume (0.5 cm3) of 10 mM p-nitrophenyl phosphate (substrate) was added and the assay mixture was incubated again for 30 minutes at 370 C. The reaction was terminated immediately by adding 2.0 cm3 of 1N sodium hydroxide. The blank was constituted by replacing the enzyme source with 0.2 cm3 of distilled water. The absorbance was read spectrophotometrically at 400 nm.
Enzyme activity was calculated using the following expression:
Enzyme activity (nM/min/ml) = ΔA/min × 1000 × TV × F
9.9 × SV × L
Where:
ΔA/min = Change in absorbance of reaction mixture per minute
TV = Total volume of the reaction mixture
F = Total dilution factor
SV = Volume of enzyme source
L = Light path length (1cm)
9.9 = Extinction co-efficient of 1 μm of p-nitrophenol in an alkaline solution of 1 ml and 1 cm path length
1000 = the factor introduced to enable the enzyme activity to be expressed in nM/min/mg protein.
The specific activity for alkaline phosphatase was calculated from the expression:
Specific enzyme activity (nM/min/mg protein) = Enzyme activity
Protein concentration
2.2.4.1.4.6 Determination of some reproductive hormones
The procedure outlined in the manufacturers’ instruction manual as described for progesterone (Radwanska et al., 1978), estradiol (Smith et al., 1980), follicle stimulating hormone (FSH) (Kapen et al., 1973), lutenizing hormone (LH) (Uotila et al., 1981), Estrogen (Abraham et al., +1972), Prolactin (Frantz, et al., 1972), oxytocin (Porstmann and Kiessig, 1992) will be adopted. Protein concentration of the serum will be determined using the biuret method (Plummer, 1978).
2.2.4.1.4.6.1 Luteinising hormone
The serum LH was quantitatively determined using the direct human serum luteinising enzyme immunoassay (EIA) kit as described by Tietz (1995) in section ssss
2.2.4.1.4.6.2 Follicle-stimulating hormone
The serum FSH was quantitatively determined using the direct human serum follicle-stimulating hormone enzyme immunoassay (EIA) kit as described by Tietz (1995) in section ssss
2.2.4.1.4.6.3 Progesterone
2.2.4.1.4.6.4 Estradiol
2.2.4.1.4.6.5 Estrogen
2.2.4.1.4.6.6 Prolactin
2.2.4.1.4.6.7 Oxytocin
2.2.4.1.4.6.8 Protein concentration of the serum
2.2.4.1.4.7 Histology of some reproductive organs
For the histopathological changes in the ovary, fallopian tubes and uterus; these organs will be excised as previously described. The earlier described procedure described by Krause (2001) and as well as Drury and Wallington (1973) will be used.
2.2.4.1.5 Abortifacient studies
2.2.4.1.5.1 Experimental animals
Animals for this study were obtained and used under the same care as earlier described
2.2.4.1.5.2 Animal mating, grouping and treatment analysis of abortifacient activity
The method described by Salhab et al. (1998) modified by Yakubu and Bukoye (2009) was adopted. Briefly, female guinea pigs will be paired overnight with the male guinea pigs (ratio 1:1) in the stainless steel cages that will provide free access to food and water. The day on which a vaginal plug or presence of spermatozoa appear in the vaginal smear which was observed under light microscope coupled with the use of pregnancy strip will be considered Day 0 of pregnancy. Six non-pregnant animals was used as positive control. The pregnant guinea pigs was thereafter randomized into five groups (B, C, D, E and F) consisting of six animals each as follows:
Group A: Non pregnant animals treated with 5 ml physiological saline
Group B: Pregnant animals treated with 5 ml physiological saline from the 35th to the 65th day of pregnancy
Group C: Pregnant animals treated with 5 ml of 300 mg/kg body weight of the ethanolic extract from the 35th to the 65th day of pregnancy
Group D: Pregnant animals treated with 5 ml of 500 mg/kg body weight of the ethanolic extract from the 35th to the 65th day of pregnancy
Group E: Pregnant animals treated with 5 ml of 800 mg/kg body weight of the ethanolic extract from the 35th to the 65th day of pregnancy
Group F: Pregnant animals treated with 5 ml of mifepristone solution containing160 mg/kg body weight from the 35th to the 65th day of pregnancy
The physiological saline, mifepristone and the extracts were administered orally between 0800 – 0900 hours on daily basis during the period of organogenesis in the guinea pigs using oropharyngeal cannula, to various groups of the animals for the experimental periods.
2.2.4.1.5.3 Preparation of serum and uterine homogenates
After 24 hours of their last dose (day 66 of pregnancy), all the animals in each of the groups were laparatomized ventrally under ether anaesthesia. The procedures described by Yakubu and Bukoye (2010) as earlier stated was replicated.
2.2.4.1.5.4 Computation of some biochemical parameters
The following parameters will be recorded/computed: number of live fetuses; number of dead fetuses; average weight of live foetuses; survival ratio (%) = (number of live fetuses/ number of live+dead fetuses) ×100; number of rats that aborted; percentage of rats that aborted= (number of rats that aborted/number of rats assessed) ×100; number of rats with vaginal bleeding; number of implantation sites; number of corpora lutea; implantation index=(total number of implantation sites/number of corpora lutea) ×100; pre-implantation loss=(number of corpora lutea– number of implantation sites/number of corpora lutea) ×100; post-implantation loss= (number of implantation sites–number of live fetuses/number of implantation sites) ×100; number of resorption sites=number of implantation sites in the control animals–number of implantations in the test animals; resorption index=(total number of resorption sites/total number of implantation sites) ×100; reduction endometrial height; uterine glands; % no of rats with opened vagina and cornificated vagina; weight of fetuses and placenta; uterine contractility. The weights of the animals both before pairing and prior to sacrifice, as well as feed and water intake will also be recorded.
2.2.4.1.5.5 Determination of some biochemical/enzyme concentrations:
The uterine protein, glucose, cholesterol, triacylglycerides, HDL-cholesterol, LDL-cholesterol, Ca+ ATPase, Na+-K+ ATPase as well as alkaline phosphatase activity will be determined using standard procedures described by Plummer (1978), Barham and Trinder (1972), Fredrickson et al. (1967), Tietz (1990), Lopes-Virella (1977), and Friedewald et al. (1972), Pershadsingh and McDonald (1980), Esmann (1988), Wright et al. (1972) while the atherogenic index will be computed using the expression given by Ng et al. (1997) as LDL-C/HDLC. Cyclooxygenase 2 (COX-2), Protein Kinase C (PKC)-α
2.2.4.1.5.5.1 Determination of uterine protein concentration
The uterine protein concentration was assayed by using the biuret reagent as described as described by Gornall et al (1949). The protocol is the same with the one earlier described in section (2.2.14.2.1.1).
2.2.4.1.5.5.2 Determination of uterine glucose concentration
Glucose was determined after enzymatic oxidation in the presence of glucose oxidase (Barham and Trinder, 1972). The protocol is the same with the one earlier described for uterine glucose concentration (2.2.14.2.1.1).
2.2.4.1.5.5.3 Determination of uterine total cholesterol concentration
The uterine total cholesterol concentration was determined by colorimetric method described in section 2.2.14.2.3.1. The only modification was that tissue homogenate was replaced by uterine sample.
2.2.4.1.5.5.4 Determination of uterine high density lipoprotein-cholesterol concentration
HDL cholesterol concentration was determined by CHOD-PAP method described by Friedwald et al. (1972). The protocol is the same with the one earlier described for high density lipoprotein-cholesterol concentration (2.2.14.2.1.1).
2.2.4.1.5.5.5 Determination of uterine triacylglycerol concentration
The colorimetric reaction method described by Tietz (1995) was used to assay for the concentration of triglycerides in the uterus homogenate as earlier described in sss.
2.2.4.1.5.5.6 Determination of uterine low density lipoprotein-cholesterol concentration
The uterine LDL cholesterol was computed by adopting the expression of Friedwald et al. (1972) as earlier described in section wwww
2.2.4.1.5.5.7 Atherogenic Index
The atherogenic index was computed using the expression given by Ng et al (1997) as LDL-C/HDLC.
2.2.4.1.5.5.8 Determination of uterine Ca+ ATPase activity
2.2.4.1.5.5.9 Determination of uterine Na+-K+ ATPase activity
2.2.4.1.5.5.10 Determination of uterine alkaline phosphatase activity
The method described by Wright et al. (1972a) was employed in this assay by following the same protocol earlier described in section www
2.2.4.1.5.6 Determination of some reproductive hormones
The procedure outlined in the manufacturers’ instruction manual as described for progesterone (Radwanska et al., 1978), estradiol (Smith et al., 1980), follicle stimulating hormone (FSH) (Kapen et al., 1973), lutenizing hormone (LH) (Uotila et al., 1981), Estrogen (Abraham et al., 1972), Prolactin (Frantz, et al., 1972), oxytocin (Porstmann and Kiessig, 1992) will be adopted. Protein concentration of the serum will be determined using the biuret method (Plummer, 1978).
2.2.4.1.5.6.1 Luteinising hormone
The serum LH was quantitatively determined using the direct human serum luteinising enzyme immunoassay (EIA) kit as described by Tietz (1995) in section ssss
2.2.4.1.5.6.2 Follicle-stimulating hormone
The serum FSH was quantitatively determined using the direct human serum follicle-stimulating hormone enzyme immunoassay (EIA) kit as described by Tietz (1995) in section ssss
2.2.4.1.5.6.3 Progesterone
2.2.4.1.5.6.4 Estradiol
2.2.4.1.5.6.5 Estrogen
2.2.4.1.5.6.6 Prolactin
2.2.4.1.5.6.7 Oxytocin
2.2.4.1.5.6.8 Protein concentration of the serum
2.2.4.1.5.7 Histology of some reproductive organs
For the histopathological changes in the ovary, fallopian tubes and uterus, these organs will be excised as previously described. The earlier described procedure described by Krause (2001) and as well as Drury and Wallington (1973) will be used.
2.2.4.2 MALE REPRODUCTIVE STUDIES
2.2.4.2.1 Experimental animals
Animals for this study were obtained and acclimatized under the same condition as earlier described. Sexually male and female guinea pigs of proven fertility weighing 900 – 1200 g and 700 – 900 g respectively were used for the study.
2.2.4.2.2 Animal grouping and extract administration
In the first category, male guinea pigs were randomly divided into four groups (A, B, C and D) of 8 animals each. Guinea pigs in groups B, C and D were treated with the plant extract once daily at 24 hours interval at the doses of 300, 500 and 800 mg/kg body weight respectively for 60 days. Group, A which served as the control, were treated with 5 ml of the vehicle (physiological saline) in a similar manner for the same number of days. 24 hours after their 60 daily doses, 5 rats from each group were sacrificed while the remaining 3 rats were not treated with any of their doses again but only maintained on their rat pellet and water ad libitum (these were used in the second category). They were later sacrificed for the recovery period spent for mating.
In the second category, fertility will be estimated in the 3 initial left over animals from the first category. These set of animals will be placed in an individual cage with virgin female guinea pigs (brought into behavioral estrus with sequential administration of suspension of oestradiol benzoate (10 μg/100 g body weight) orally and progesterone (0.5 mg/100 g body weight) through subcutaneous route, 48 and 4 hours respectively prior to pairing (Tajuddin-Ahmad et al., 2005) of the same strain (ratio 1:2). Mating tests will be conducted in a glass arena measuring 55 x 65 x 20 cm. The floor of the arena will be covered with absorbent paper. This paper will be changed after each session. Testing will be initiated approximately 3 hours into the dark phase of the 12 hours reversed day/night cycle. The arena will be illuminated by distant dim light. During the test, the male will be placed into the arena and the stimulus female will be introduced 5 minutes later. The test will be carried out until the male achieve one intromission after the first ejaculation, or terminated after 15 minutes if the male did not copulate. Oestrus phase in rats was confirmed by vaginal smear examination according to OECD-106 guideline (OECD, 2009). Success of matting will be confirmed by the presence of spermatozoa in the vaginal smear of the matted animals. They will left together for 33 days during which two estrous cycles had elapsed. On the 34th day after the mating, pregnant females will be sacrificed by as previously described.
All administrations were done daily at the same point time of between 0800 and 0900 hour. The experimental animals were allowed free access to rat pellets and tap water after the daily dose of the extract/ physiological saline.
2.2.4.2.3 Copulation/mating behavior
Standard measures of copulation/mating behaviour will be used as described by Dewsbury (1975) as well as Bialy and Beck (1993). These parameters of male sexual behaviour to be monitored on designated days include:
(i). Mount Frequency (MF): The number of mounts (climbing at copulatory positions) without intromission (no vaginal penetration) from the time of introduction of the female until ejaculation.
(ii). Intromission Frequency (IF): The number of intromissions (vaginal penetration) from the time of introduction of female until ejaculation.
(iii). Mount Latency (ML): The time interval between the introduction of the female and the first mount by the male.
(iv). Intromission Latency (IL): The time interval between the introductions of female to the first intromission by the male.
v). Ejaculation Latency (EL): The time interval between the first intromission and ejaculation.
vi). Post Ejaculatory Interval (PEI): the time interval between ejaculation and erection of the male copulatory organ for the next phase of sexual cycle.
vii). Mean Interintromission Interval (MIII): the mean intervals in seconds separating the intro-missions of the series.
viii). hit rate (HR): the number of intromissions divided by the number of intromissions plus the number of mounts.
2.2.4.2.4 Fertility test
After sacrificing the female animal in the second category, the number of implantation sites, the number of fetuses and the number of resorption sites if any, were recorded (Rugh, 1968). Their litter size, morbidity and mortality if any, were recorded. A fertility test was calculated using the following formula:
% Fertility Success = Pregnant Females × 100/Mated Females
2.2.4.2.5 Serum and Tissue preparation
The method described by Yakubu and Bukoye (2009) will be used for the preparation of serum supernatants. After 60 days of treatment, the animals will be anaesthetized using intraperitoneal pentobarbital sodium (6 %) at the dose of 60 mg/kg and sacrificed by simply incising the jugular vein; the blood was collected into EDTA and plain sample tubes for serum hematological analysis respectively. Blood samples will be left undisturbed at room temperature for 30 minutes to form clot after which the samples will be centrifuged at 1282 x g for 5 minutes. After centrifugation, the supernatant which is the serum will be collected using Pasteur pipette into clean sample bottles. The serum, thus obtained will be appropriately labelled and used within 12 hours of preparation for the biochemical assays and the hormonal assays. The testes, hearts, liver and kidneys, seminal vesicle, ventral prostate, vas deferens and adrenal will also be removed, freed from fat, blotted with tissue paper and weighed for the computation of organ-body weight ratio.
2.2.4.2.6 Weights of reproductive organs:
Body weights as well as organ weight and size
Initial and final body weights of the animals will be recorded. Testis and epididymis will be dissected out, blotted free of blood and fat as well as being weighed. Length and width of testes and epididymis will be measured. Moreover, the weights of the seminal vesicle, ventral prostate and vas deferens as well as weights of the liver, heart, kidney and adrenal will be taken after being cleared off fats.
The organ/body weight ratio will be computed as:
weight of organs × 100
weight of the animal
2.2.4.2.7 Prostatic functions
The method described by Yakubu et al. (2007b) for collection of the prostrate fluid from the prostate gland but with slight modification will be adopted. Using intraperitoneal pentobarbital sodium (6 %) at the dose of 60 mg/kg to induce anesthesia, the animals will be quickly dissected and the prostate glands will be excised from the animals. The prostate gland will be drained of its contents into sample bottles and then stored frozen at 0o C for 24 hours before being used for the various biochemical assay. The prostate gland will be later blotted with tissue paper and thereafter weighed.
The prostate body weight ratio will be determined according to the method described by Yakubu et al. (2007b). The concentrations of calcium, citrate, phosphate and pH of the prostatic fluid will be determined by the methods described by Biggs and Moorehead (1974), Petrarulo et al. (1995), Fiske and Subbarow (1925) as well as Comhaire and Vermeulen (1995) respectively. The assay methods described by Wright et al. (1972) will be used to determine the activity of ACP
2.2.4.2.7.1 Determination of calcium concentration in prostatic fluid
2.2.4.2.7.2 Determination of citrate concentration in prostatic fluid
2.2.4.2.7.3 Determination of phosphate concentration in prostatic fluid
2.2.4.2.7.4 Determination of pH of the prostatic fluid
2.2.4.2.7.5 Determination of Acid phosphatase activity in prostatic fluid
The method described by Wright et al (1972a) was used in assaying for the activity of acid phosphatase.
Principle:
The hydrolysis product, para-nitrophenol, has a characteristic yellow colour in alkaline solution. The initial phosphate incubation of the enzyme with acetate buffer served to inactivate microsomal phosphate (Shibko and Tappel, 1965).
Procedure:
A known volume (2.2 ml) of Sodium acetate buffer (Concentrated acetic acid; pH 4.5) was added to 0.2 ml of the samples (appropriately diluted) in the test tubes. The sample was mixed and left to equilibrate for 10 minutes. Then 0.5 ml of 10 mM p-nitrophenyl phosphate (substrate) was added. The mixtures were thoroughly mixed and incubated for 30 minutes at 370C. The reaction was terminated immediately by adding 2.0 ml of 1N sodium hydroxide. The blank was constituted by replacing the sample with 0.2 ml of distilled water. The absorbance was read spectrophotometrically at 400 nm.
Calculation: (a) Enzyme activity for acid phosphatase
Enzyme activity (nM/min/ml) = AB/min x 1000 x V x F
9.9 x L x SV
Where:
AB/min = Absorbance of the reaction mixture per minute
V = Total volume of reaction mixture
F = Total dilution factor
SV = Volume of enzyme source
L = Light path length (1cm)
9.9 = Extinction co-efficient of 1µm of P-nitrophenol in an alkaline solution of 1 ml and 1 cm light path
1000 = the factor introduced to enable enzyme activity be expressed in nM/min/ml
(b) Specific enzyme activity for acid phosphatase
The specific enzyme activity was calculated using the expression:
Specific activity (nM/min/mg protein) = Enzyme activity
Protein concentration
2.2.4.2.8 Seminal vesicles function
The method as described by Yakubu et al. (2007b) for the collection of the seminal fluid from the seminal vesicles but with slight modification will be replicated.
Percentage (%) seminal vesicle weight ratio will be determined according to the method described by Yakubu et al. (2007b). The method described by Anderson et al. (1979) will be used to estimate the seminal fluid fructose concentration. The assay methods described by Schmidt et al. (1965) will be used to determine the activity of GDH.
2.2.4.2.8.1 % seminal vesicle weight ratio
The organ-body weight ratio was determined by dividing the weight of the seminal vesicle by the body weight of the animal according to the expression:
% seminal vesicle weight ratio = Weight of organ x 100
Body weight of animal
2.2.4.2.8.2 Determination of seminal fluid fructose concentration
2.2.4.2.8.3 Determination of GDH activity in the seminal fluid
2.2.4.2.9 Semen analysis/Spermicidal studies:
The method of Amelar and Dublin (1973) will be used in collecting sperm cells. Briefly, epididymis samples will be removed from the freezer then allowed to thaw slowly in a refrigerator. Samples will be then carefully cut opened using clean sterile blade and placed in 1 ml of 0.1 M phosphate buffer of pH 7.4. It will then vigorously shaken for homogeneity and dispersal of sperm cells. The method of Biswas et al. (2002) will be used for the determination of sperm motility and sperm count while the method described by Magbagbeola et al. (2000) and Oyedeji et al. (2013) will be used to estimate the sperm density and viability respectively. The sperm morphology will be determined using the method described by Amelar and Dublin (1978) while the sperm viscosity will be evaluated using the method described by Barrat and John (1998). To test for semen pH, pH paper will be dipped into the sample, and the resulting colour will checked against known standards (Comhaire and Vermeulen, 1995).
2.2.4.2.9.1 Semen pH
The pH of the semen collected from the caudal epididymis was read using a pH cooperative paper (strip), ranged 5 – 9. This strip is capable of different colour change within this pH range which can be compared to a standard colour for a given pH value as supplied with the kit by the manufacturer.
2.2.4.2.9.2 Semen volume
The caudal epididymal duct on one side of the testis was exposed and incised. The connective tissue capsule around the caudal epididymis was teased out and the epididymal duct was uncoiled. The semen that oozed into the cavity block was quickly sucked into a graduated collecting tube and measured (Revathi et al., 2010).
2.2.4.2.9.3 Sperm count
This was done by removing the caudal epididymis from the right testes and blotted with filter paper. The caudal epididymis was immersed in 5 ml formol-saline in a graduated test-tube and the volume of fluid displaced was taken as the volume of the epididymis. The caudal epididymis and the 5 ml formol-saline were then poured into a mortar and homogenised into a suspension from which the sperm count was carried out using the improved Neubauer haemocytometer under the microscope at ×400 magnification (Oyedeji et al., 2013).
2.2.4.2.9.4 Sperm morphology
The fluid from the caudal epididymis was diluted with Tris buffer solution to 1.0 ml (Sonmez et al., 2005). Aliquot of this diluted mixture was further diluted (1:20) with 10 % neutral buffered formalin. The morphology of the spermatozoa was determined by adding two drops of warm Eosin/Nigrosin stain to the semen on a pre-warmed slide, a uniform smear was then made and air-dried; the stained slide was immediately examined under the microscope using x400 magnification (Laing, 1979). Five fields of the microscope were randomly selected and the types and number of abnormal spermatozoa were evaluated from the total number of spermatozoa in the five fields. The sperm cells were categorised based on the presence of one or more abnormal features, such as tail defects (short, irregular coiled or multiple tails); neck and middle piece defects (distended, irregular, bent middle piece, abnormally thin middle piece); and head defects (round head, small or large size, double or detached head). The number of abnormal spermatozoa was expressed as a percentage of the total number of spermatozoa (Saalu et al., 2010).
2.2.4.2.9.5 Sperm viability (life/dead ratio)
This was done by adding two drops of warm Eosin/Nigrosin stain to the semen on a pre-warmed slide, a uniform smear was then made and dried with air; the stained slide was immediately examined under the microscope using x400 magnification. The live sperm cells were unstained while the dead sperm cells absorbed the stain. The stained and unstained sperm were counted and the percentage was calculated (Laing, 1979).
2.2.4.2.9.6 Sperm motility
This was done immediately after the semen collection. Semen was squeezed from the caudal epididymis onto a pre-warmed microscope slide (270 C) and two drops of warm 2.9 % w/v sodium citrate was added. The slide was then covered with a warm cover slip and examined under the microscope using ×400 magnification. Ten fields of the microscope were randomly selected and the sperm motility of 10 sperms was assessed on each field. Therefore, the motility of 100 sperms was assessed randomly. Sperms were labelled as motile, sluggish, or immotile. The percentage of motile sperms was defined as the number of motile sperms divided by the total number of counted sperms (i.e. 100) (Mohammad-Reza et al., 2005).
2.2.4.2.10 Testicular parameters and testosterone
The method described by Yakubu et al. (2007) will be slightly modified for the preparation of the testicular and reproductive organ parameters. Using intraperitoneal pentobarbital sodium (6 %) at the dose of 60 mg/kg to induce anesthesia, the animals will be quickly dissected and the testes, epididymis, seminal ventral prostate and vas deferens will be excised from the animals. They will be cleaned of superficial fatty layer, weighed again and then transferred into 0.25 M sucrose solution. These organs will be later blotted with tissue paper, cut very thinly with sterile scapel blade and homogenized in ice cold 0.25 M sucrose solution (1:5, w/v). The homogenates will be further centrifuged at 1340 × g for 15 minutes to obtain the supernatant, which will later be aspirated with Pasteur pipette into sample bottle, stored overnight at 4◦ C before being used for the biochemical assays.
The concentrations of total cholesterol (in testes and liver) based on “CHOD-PAP” reactions; as well as glycogen, sialic acid and protein (in the testes, epididymis, seminal ventral prostate and vas deferens) will be estimated by the methods described by Fredrickson et al. (1967), Kemp et al. (1954),Warren (1959) and Gornall et al. (1949) respectively. The concentrations of fructose (in testes and seminal vesicles) will be estimated by the methods described by (Mann, 1964) Testosterone will be assayed in the serum of the animals following the procedure outlined in the manufacturers’ instruction manual as described by Tietz (1995). The concentrations of bile acid (in serum and feces) will be estimated by the methods described by Setchell et al. (1997).
2.2.4.2.10.1 Determination of sialic acid concentration
Sialic acid concentration was assayed in the testes, epididymis, seminal ventral prostate and vas deferens using the method described by Warren (1959).
Principle:
The principle is based on the periodate oxidation of the sialic acid and thiobarbituric acid, resulting in the formation of P-formylpyruvic acid. The reaction of periodate with strong acid solution and its eventual extraction in cyclohexanone results in intense deep-red colour and can be read spectrophotometrically at 549 nm.
Procedure:
Testicular supernatants were heated at 800C for 1 hour in 0.5N H2SO4. To 0.2 cm3 of the sample contained in a test tube, 0.1 cm3 of periodate solution was added. The test tube was shaken and left undisturbed at 250C for 20 minutes. Sodium arsenite solution (1.0 cm3) was added and the test tube shaken until yellow-brown colour disappears. Thiobarbituric acid solution (3.0 cm3) was added into the test tube, capped with glass beads, and then heated in a vigorously boiling water bath for 15 minutes. The test tube was then removed and placed in cold water for 5 minutes. Of this solution, 1.0 cm3 was transferred to another test tube containing 1.0 cm3 of cyclohexanone. The test tube was shaken twice and centrifuged at 4000 rpm for 5 minutes, using Uniscope Laboratory Centrifuge (Model SM800B, Surgifriend Medicals, England). The absorbance of the upper coloured cyclohexanone phase (supernatant) was read against the blank at 549 nm. The blank was constituted with distilled water instead of the supernatant.
Calculation:
Amount of sialic acid (µg/mg) = Tv x A549
Molecular extinction coefficient of sialic acid
Where:
Tv = Total volume of reaction mixture
A549= Absorbance at 549 nm
Molecular extinction coefficient of sialic acid= 57,000
2.2.4.2.10.2 Determination of glycogen concentration
The method described by Kemp et al. (1954) as modified by Yakubu et al. (2013) was adopted for the determination of glycogen content in the testes, epididymis, seminal ventral prostate and vas deferens.
Principle:
The principle is based on a colour development when a dilute solution of glucose is heated with concentrated sulphuric acid. The intensity of the pink colour measured at 520 nm is proportional to the concentration of glycogen.
Procedure:
A known volume (0.5 cm3) of appropriately diluted testicular homogenate was pipetted into a test tube. A known volume (5.0 cm3) of deprotenizing solution (i. e. trichloroacetic acid and AgNO3) was added. The test tube was then placed in a boiling water bath for 15 minutes and thereafter cooled in a running water, filled up to the marked level with deprotenizing solution to compensate for evaporation. The solution was then centrifuged at 100.5 x g for 5 minutes. A known volume (1.0 cm3) of the supernatant was added to 3.0 cm3 of diluted H2SO4 in a test tube and mixed vigorously. The mixture was heated in a boiling water bath for exactly 6.5 minutes and subsequently cooled in a running tap water. The intensity of the pink colour solution produced was read spectrophotometrically at 520 nm. The glycogen concentration was read from the calibration curve (Figure 63) in terms of glucose equivalent.
2.2.4.2.10.3 Determination of protein concentration
The protein concentration in in the testes, epididymis, seminal ventral prostate and vas deferens of the animals was assayed, using Biuret reagent as described by Gornall et al (1949). The protocol is the same with the one earlier described for serum total protein concentration (2.2.14.2.1.1) except that the samples in this regard are testes, epididymis, seminal ventral prostate and vas deferens homogenate instead of serum.
2.2.4.2.10.4 Determination of fructose concentration
2.2.4.2.10.5 Determination of total cholesterol concentration
The concentration of total cholesterol in the testes and liver of the animal was carried out using the CHOD-PAP reaction described by Friedrickson et al. (1967) as earlier described in section 222
2.2.4.2.10.6 Determination of ascorbic acid concentration
Ascorbic acid concentration in the testes and epididymis was determined by the Folin–Ciocalteu reagent method described by Jagota and Dani (1982). The protocol was the same as that described in section 345
2.2.4.2.10.7 Determination of fecal bile acid concentration
2.2.4.2.10.8 Determination of 17-ketosteroids concentration
2.2.4.2.10.9 Determination of testosterone concentration
The serum testosterone concentration was quantitatively determined using the direct human testosterone enzyme immunoassay (EIA) kit described by Tietz (1995).
Principle:
The testosterone Enzyme Immuno Assay (EIA) is based on the competitive binding between testosterone in the test sample and testosterone-Horseradish peroxide (HRP) conjugate for a constant amount of rabbit anti-testosterone.
Procedure:
A desired number of coated wells in the holders were secured. 10 µl each of standards, samples and controls were dispensed into appropriate wells. A known volume (50 µl) of testosterone-HPR conjugate reagent and 50 µl rabbit anti-testosterone were dispensed into all wells. This was then mixed for 30 seconds after which it was incubated at 370C for 1 hour. The microwells were rinsed and flicked 5 times with distilled water. 350 µl of 3, 3, 5, 5-tetramethylbenzidine (TMB) reagent (a surfactant) was dispensed into each well, gently mixed for 5 seconds and incubated at room temperature for 15 minutes. The reaction was stopped by adding 50 µl of 1N HCl to each well and then gently mixed for 20 seconds. The resultant yellow colour solution formed from blue colour was read at 405 nm with a microtitre within 15 minutes.
Calculation of testosterone concentration:
The serum testosterone concentration of the sample was extrapolated from the calibration curve for testosterone (Figure 59).
2.2.4.2.11 Testicular enzymes assay
The method described by Yakubu et al. (2007) will be slightly modified for the preparation of the testicular parameters as previously stated.
The assay methods described by Wright et al. (1972a), Wright et al. (1972b), Schmidt et al. (1965), Szasz (1969), Holdgate et al. (2003), Gerlach and Hiby (1974), Geer et al. (1980), Hu et al. (2009), Wroblewski and Due, (1995), Noltmann et al. (1961) will be used to estimate the activities of ALP, ACP, GDH, γ-GT, 3-hydroxy-3-methylglutaryl CoA (HMGCoA) reductase, sorbitol dehydrogenase, malic enzyme, 17-β-hydroxy sreroid dehydrogenase and 3-β-hydroxysteroid dehydrogenase, LDH, glucose-6-phosphate dehydrogense respectively.
2.2.4.2.11.1 Determination of testicular alkaline phosphatase activity
The method described by Wright et al. (1972a) was employed in this assay. The same protocol earlier described in section 2223 was replicated
2.2.4.2.11.2 Determination of testicular acid phosphatase activity
The method described by Wright et al (1972a) was used in assaying for the activity of acid phosphatase in the prostatic fluid. The protocol is the same as that described earlier in section 33
2.2.4.2.11.3 Determination of testicular lactate dehydrogenase activity
The assay method employed was that of kubowitz and Ott (1943) as modified by Wroblewski and LaDue (1955).
Principle:
Lactate dehydrogenase catalyses the reduction of pyruvate to lactate. In the process, NADH is oxidized to NAD+. The reaction can be monitored spectrophotometrically at 340 nm by measuring the rate of oxidation of NADH.
Pyruvate + NADH + H+ L-lactate + NAD+
Procedure:
A known volume (0.1 ml) of the appropriately diluted sample was pipetted into a test tube and 0.2 ml of the substrate reagent was added. The absorbance was read at 340 nm against air after 0.5s and for 3 minutes at equal interval of 1 minute.
Calculation: The activity of was obtained using the expression
(a) Enzyme activity for Lactate dehydrogenase
LDH activity (U/L) = 4127 x ∆A 340nm/min
(b) Specific enzyme activity for Lactate dehydrogenase
The specific enzyme activity was calculated using the expression:
Specific activity (nM/min/mg protein) = Enzyme activity
Protein concentration
2.2.4.2.11.4 Determination of testicular Gamma glutamyl transferase activity
The colorimetric method described by Szasz (1969) was used to assay for the activity of gamma glutamyl transferase.
Principle:
The substrate L-γ-glutamyl-3-carboxy-4-nitroanilide, in the presence of glycylglycine is converted by γ-GT in the sample to 5-amino-2-nitrobenzoate which can be read spectrophotometrically at 405 nm.
Procedure:
A known volume (1.0 cm3) of the reagent mixture (100 mmol/L,pH 8.25 Tris buffer/100 mmol/L glycylglycine and 2.9 mmol/L L-γ-glutamyl-3-carboxy-4-nitroanilide) was added to 0.1 cm3 of the enzyme source (appropriately diluted). The blank was constituted by replacing the enzyme source with distilled water. The solutions were mixed and the absorbance of the sample was spectrophotometrically read at 405 nm against the blank for 3 minutes at equal interval of 1 minute.
Calculation:
The enzyme activity was calculated using the following expression:
Enzyme activity (U/L) = 1158 × ΔA 405 nm/min
Specific enzyme activity (U/L protein) = Enzyme activity
Protein concentration
2.2.4.2.11.5 Determination of testicular glutamate dehydrogenase activity
2.2.4.2.11.6 Determination of testicular 3-hydroxy-3-methylglutaryl CoA (HMGCoA) reductase activity
2.2.4.2.11.7 Determination of testicular sorbitol dehydrogenase activity
2.2.4.2.11.8 Determination of testicular malic enzyme activity
2.2.4.2.11.9 Determination of testicular 17-β-hydroxy steroid dehydrogenase activity
2.2.4.2.11.10 Determination of testicular 3-β-hydroxysteroid dehydrogenase activity
2.2.4.2.11.11 Determination of testicular glucose-6-phosphate dehydrogenase activity
Principle:
The enzyme activity was determined by measurement of the rate of absorbance change at 340 nm due to the reduction of NADP+ (Nolltmann et al., 1961).
Glc-6-PD
Glc-6-P + NADP+ Gluconate-6-P + NADH+ + H+
Procedure:
Reagent R1, 1.00 ml, 0.03 ml of reagent R2 and 0.015 ml of tissue homogenate were added together in a test tube. The solution was allowed to mix and afterward it was incubated for 5 minute at 37oC, then 0.015 ml of reagent R3 was added and absorbance was read at 340 nm.
Calculation:
The enzyme activity was calculated using the following expression.
Enzyme Activity (nmol/min/ml)
= (A340 nm/min Sample – A340 nm/min blank) x 1000 xVx df
6.22 x v
6.22- Millimolar extinction coefficient of ᵦ- NADP at 340 nm
df: Dilution factor
V: Total volume (ml) of the reaction mixture
V: Volume (ml) of sample
1000: The factor introduced to enable enzyme activity is expressed in nmol/min/ml.
The specific enzyme activity (nmol/min/mg protein) = Enzyme Activity
Protein Concentration
2.2.4.2.12 Hormonal assay
The LH and FSH will be assayed in the serum of the animals following the procedure outlined in the manufacturers’ instruction manual as described for follicle stimulating hormone (FSH) (Kapen et al., 1973) and lutenizing hormone (LH) (Uotila et al., 1981).
2.2.4.2.12.1 Luteinising hormone
The serum LH was quantitatively determined using the direct human serum luteinising enzyme immunoassay (EIA) kit as described by Tietz (1995) in section ssss
2.2.4.2.12.2 Follicle-stimulating hormone
The serum FSH was quantitatively determined using the direct human serum follicle-stimulating hormone enzyme immunoassay (EIA) kit as described by Tietz (1995) in section ssss
2.2.4.2.13 Whole blood studies:
The red blood cells (RBC) and white blood cells (WBC) counts will be determined by the improved Neubauer haemocytometer method. The haemoglobin (Hb) concentration will be determined according to Jain (1986), using the cyanomethaemoglobin method. The packed cell volume (PCV) will be determined by the microhaematocrit method according to Dacie and Lewis (1991). Schilling method of differential lecukocyte count will be used to determine the distribution of the various white blood cells (Mitruka and Rawnsley, 1977). Mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) will be computed according to Jain (1986). Blood sugar and blood urea according to standard procedures of Barmer and Trinder (1972) as well as Bartels and Bohmer (1972) respectively.
Determination of haematological parameters
The Automated Haematological Analyser, Sysmex, KX- 21 (Japan) was used to determine the levels of haemoglobin (Hb), red blood cells (RBC), Packed cell volume (PCV), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), White blood cell (WBC) and Platelets.
Principle:
The Stromatolyser counts the red cells, lyses them to release the haemoglobin and estimates this concentration. The machine assumes that all nucleated cells are white cells and therefore counts all as white cells into their different forms i.e. lymphocytes and neutrophils, but will not differentiate between eosinophils, monocytes and basophils and as such, they are recorded as mixed. It lyses the white blood cells based on the size of the nucleus and counts the number of the white cells.
2.2.4.2.14 Other Biochemical studies
The serum will be analyzed to estimate acid phosphatase, alkaline phosphatase, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, total protein, cholesterol, triglycerides, phospholipids and bilirubin according to standard procedures as earlier described.
2.2.4.2.15 Histopathology changes and Light microscopy on the testes and three parts of epididymis (caput, corpus and cauda):
For the histology studies, the testes and three parts of epididymis (caput, corpus and cauda) will be excised as previously described. The earlier described procedure described by Krause (2001) and as well as Drury and Wallington (1973) will be used.
For the Light microscopic study, Five micrometer thick transverse sections of testis and epididymis will be studied under light microscope at 20, 40 and 100 magnifications. Slides of all the groups will be studied and photographed a digital camera. Mean ± SEM of the following morphological and histological data will be determined: Total (Right and left Testicular) weight, Total (Right and left Testicular) length, Total (Right and left Testicular) width, Total (Right and left, Epididymal) weight, Total (Right and left Epididymal) length.
The following histometerical parameters of testes will also be determined: Tunical thickness (µm), Leydig cell nuclear diameter (µm), Seminiferous Tubule Diameter (µm), Epithelial height (µm), Sertoli Cell Nuclear Diameter (µm).
Caput tubule diameter (m), Caput epithelial height (m), Corpus tubule diameter (m), Corpus epithelial height (m), Cauda tubule diameter (m), Cauda epithelial height (m) will be determined as histometerical parameters of epididymis.
2.2.4.2.16 Total antioxidant capacity (TAC) and Malondialdehyde (MDA) concentration
The Total antioxidant capacity (TAC) and Malondialdehyde (MDA) concentration will be assayed in the serum of the animals following the procedure outlined in the manufacturers’ instruction manual as described by Prieto et al. (1999) and Satoh (1978) respectively.
2.2.4.2.16.1 Malondialdehyde concentration
The concentration of malondialdehyde (MDA) was measured as an estimate of lipid peroxidation product, using the colorimetric method described by Satoh (1978).
Principle:
The assay is based on the reaction of MDA with thiobarbituric acid (TBA) under acidic condition; forming an MDA-TBA2, a coloured complex that gives maximum absorption at 532 nm (Satoh, 1978; Yagi, 1984) as depicted in Figure 18.
MDA TBA MDA-TBA2
Figure 18: Reaction of malondialdehyde with thiobarbituric acid
Source: Yagi (1984)
Procedure:
A known volume (2.0 cm3) of the sample (appropriately diluted testis homogenate) was pipetted into test tubes and 1.0 cm3 of 20 % w/v trichloroacetic acid was added, followed by 2.0 cm3 of 0.07 % w/v thiobarbituric acid. The mixture was heated in a tightly stoppered tube for 30 minutes in a boiling water bath at 800 C for colour development and cooled in running water. The resulting chromogen was extracted with 4.0 cm3 of n – butanol by vigorous shaking while the separation of organic phase was facilitated by centrifugation at 3000 rpm for 10 minutes. The absorbance of the organic phase (the upper layer) was determined at 532 nm. All MDA concentrations were expressed in nanomoles MDA per ml (nmoles/ml).
Calculation:
Malondialdehyde values (nanomoles per ml) was determined according to this expression:
MDA (nmol/ml) = A532 x M. Wt of MDA x Tv
t x E x 1000 x Sv
Where
A = Absorbance of test at 532 nm
M. Wt = Molecular weight of malondiadehyde = 72 gmol-1
Tv = Total volume of reaction mixture = 5 cm3
t = Time for colour development = 30 min
E = Molar extinction coefficient of MDA–TBA2 complex =1.56 x 105 M/cm
Sv = Volume of sample used = 2.0 cm3
1000 = Factor introduced for the conversion from litre to cm3
Thus, MDA (nmol/ml) = ______A532 x 360________
30 x 1.56 x 105 x 1000 x 2.0
= A532 X 360
9.36 X 109
= A532 X 3.846 X 10-8
2.2.5 Fractionation: Isolation and purification of ethanolic crude extract
2.2.5.1 Bioactivity Guided Fractionation
The method described by Mbaoji et al. (2014) was adopted. Briefly, solution of a known weight of ethanolic extract of S. mombin leaves was subjected to solvent guided fractionation by pouring into a glass column (60 x 4.5 cm) that has been packed with pretreated silica gel (60 – 120 mesh size). After complete absorption of the solution, the column was washed with enough distilled water to remove carbohydrates, and further washed with 65% ethanol to elute the flavonoid fraction. Thereafter, elusion was successively carried out with other solvents (dichloromethane, methanol, n-Hexane, chloroform) and their corresponding fractions collected separately. For example, the ethanol eluent (fraction) abundant in flavonoids will be collected and then concentrated at 40 0C with rotary evaporator (Heidolph, Schwabach, Germany) until the formation of sediment. The other fractions were treated in like manner.
2.2.5.2 Isolation bioactive agents of fraction
Thin Layer Chromatography (TLC) was used for the isolation. The procedure described by Singh and Sahu (2005) was adopted for the preparation of the TLC plates as well as determination of phytoconstituent present in the fraction. This is intended to identify the active compound contained in the fraction.
2.2.6 Determination of biological effects of fractions on female and male studies
All the female and male studies as previously described from sections 2223 -444 were repeated for all the resultant fractions.
2.2.7 Characterization of Bioactive Fraction
Using HPLC, components of the bioactive agent (fraction) obtained from TLC analysis was then identified. The identification of components in fraction was based on the comparison of retention indices (determined by retention time in stationary phase relative to how long it resides in the mobile phase), resolution and sensitivity (Knox et al., 1978; Kupiec, 2004).
Determination of malic enzyme concentration
The concentration of malic enzyme was determined by the continuous spectrophotometric rate determination method described by Geer (1982).
Principle:
This method is based on the reaction of ascorbic acid with Folin’s reagent to give a blue colour which has its maximum absorption at 760 nm.
Malic Enzyme
L-Malate + β-NADP Pyruvate + CO2 + β-NADPH
Abbreviations used:
β-NADP = β-Nicotinamide Adenine Dinucleotide Phosphate, Oxidized Form
β-NADPH = β-Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form
Procedure:
A known volume (2 ml) of the buffer (100 mM Triethanolamine HCl Buffer, pH 7.4 at 25°C) was mixed by inversion with 0.10, 0.05 and 0.75 ml of malic acid (100 mM L-Malic Acid Solution), NADP (20 mM b-Nicotinamide Adenine Dinucleotide Phosphate, Oxidized Form, Solution), MnCl2 (20 mM Manganese Chloride Solution) respectively and equilibrated to 25°C. The A340nm was monitored until when constant, using a suitably thermostatted spectrophotometer. Then 0.10 ml of the enzyme solution (malic enzyme solution) and deionized water was added to the test sample and blank respectively. The mixture was immediately mixed by inversion and monitored for increase in A340nm for approximately 5-10 minutes. The ΔA340nm/minute was obtained using the maximum linear rate for both the Test and Blank (Figure 11).
Calculations:
Units/ml enzyme = (ΔA340nm/min Test – ΔA340nm/min Blank)(3)(df)
(6.22)(0.1)
3 = Total volume (in milliliters) of assay
df = Dilution factor
6.22 = Millimolar extinction coefficient of b-NADPH at 340 nm
0.1 = Volume (in milliliter) of enzyme used
Units/mg solid = units/ml enzyme
mg solid/ml enzyme
Units/mg protein = units/ml enzyme
mg protein/ml enzyme
Geer, B.W., Krochko, D., Oliver, M.J., Walker, V.K. and Williamson, J.H. (1980) Comp. Biochem. Physiol. 65B, 25-34
Enzymatic Assay of SORBITOL DEHYDROGENASE (EC 1.1.1.14)
The concentration of sorbitol dehydrogenase was determined by the continuous spectrophotometric rate determination method described by Gerlach and Hiby (1974).
Principle:
Sorbitol Dehydrogenase
D-Fructose + ß-NADH D-Sorbitol + ß-NAD
Abbreviations used:
ß-NADH = ß-Nicotinamide Adenine Dinucleotide, Reduced Form
ß-NAD = ß-Nicotinamide Adenine Dinucleotide, Oxidized Form
Procedure:
A known volume (2.35 ml) of the buffer (100 mM Triethanolamine Buffer, pH 7.6 at 25°C) was mixed by inversion with 0.50 and 0.05 ml of (1.1 M D-Fructose Solution) and ß-NADH (12.8 mM ß-Nicotinamide Adenine Dinucleotide, Reduced Form, Solution) respectively and equilibrated to 25°C. The A340nm was monitored until when constant, using a suitably thermostatted spectrophotometer. Then 0.10 ml of the enzyme solution (Sorbitol Dehydrogenase Enzyme Solution) and 1.0% (w/v) Bovine Serum Albumin was added to the test sample and blank respectively. The mixture was immediately mixed by inversion and monitored for increase in A340nm for approximately 5 minutes. The ΔA340nm/minute was obtained using the maximum linear rate for both the Test and Blank (Figure 11).
Calculations:
Units/ml enzyme = (ΔA340nm/min Test – ΔA340nm/min Blank)(3)(df)
(6.22)(0.1)
3 = Total volume (in milliliters) of assay
df = Dilution factor
6.22 = Millimolar extinction coefficient of ß-NADH at 340nm
0.1 = Volume (in milliliter) of enzyme
Units/mg solid = units/ml enzyme
mg solid/ml enzyme
Units/mg protein = units/ml enzyme
mg protein/ml enzyme
Gerlach, U. and Hiby, W. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U. ed.) 2nd ed.,Volume II, 569-573, Academic Press Inc., New York, NY
Determination of total antioxidant capacity
The antioxidant activity of the extract was evaluated by the phosphomolybdenum method according to the procedure describe by Prieto et al. (1999). The assay is based on the reduction of Mo (VI)–Mo (V) by the extract and subsequent formation of a green phosphate/Mo (V) complex at acid pH. A 0.3 ml extract was combined with 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes containing the reaction solution were incubated at 950 C for 90 min. Then the absorbance of the solution was measured at 695 nm using a spectrophotometer (HACH 4000 DU UV – visible spectrophotometer) against blank after cooling to room temperature. Methanol (0.3 ml) in the place of extract is used as the blank. The antioxidant activity is expressed as the number of gram equivalents of ascorbic acid.
Antioxidant potential assay
The antioxidant power of the extracts has been assessed with the phosphomolybdenum reduction assay according to Prieto et al. (PRIETO P. et al (1999)[7]). The reagent solution contained ammonium molybdate (4 mM), sodium phosphate (28 mM) and sulfuric acid (0.6 M) mixed with the extracts diluted in 50% ethanol solution at the concentration of 5mg/ml. The samples were incubated for 90 min at 90 °C and the absorbance of the green phosphomolybdenum complex was measured at 695 nm. For reference, the appropriate solutions (0.2-2mM) of ascorbic acid have been used. The reducing capacity of the extracts has been expressed as the ascorbic acid equivalents
(milligrams per gram extract).
2.2.8 Dilution factor
The dilution factor for various parameters assayed in respective organ/tissue is as shown on Table 1.
Table 1: Dilution factors for the various assays
ALP-Alkaline phosphatase; ACP-Acid phosphatase; GGT- Gamma glutamyltransferase;
AST-Aspartate aminotransaminase;ALT-Alanine aminotransferase
2.2.9 Statistical analysis
All data will be expressed as the mean of three to six replicates + standard error of mean (S.E.M).
Statistical evaluation of data was performed by Graph pad prism version 5.02.
For comparison:
Values in frequencies or percentages, Chi-square goodness-of-fit statistic will be calculated to compare observed and expected frequencies.
Student’s t – test will be employed for comparison between two mean values.
For more than two mean values and variables, one and two ways analysis of variance (ANOVA) respectively, followed by Dunett’s posthoc test for multiple comparism will be used.
Values will be considered statistically significant at p < 0.05 (confidence level = 95 %) or p < 0.01 (confidence level = 99 %) depending on sample size and distribution.
The experiment was terminated 30 minutes after the day 7 pairing (Gauthaman et al., 2002).
2.2.8 Collection of biological fluids
Blood and semen were the biological fluids collected from the experimental animals for analysis.
2.2.8.1 Blood
Under ether anesthesia, the neck areas were quickly cleared of fur and skin to expose the jugular veins. The jugular veins were slightly displaced from the neck region (to prevent contamination of the blood with interstitial fluid) and then cut with a sharp sterile blade. Using the method described by Akanji and Ngaha (1989), the rats were held head downwards and allowed to bleed into a clean sample bottles containing EDTA.
2.2.8.2 Serum
The rats were made to bleed into clean, dry, EDTA-free sample bottles and left undisturbed at room temperature for 20 minutes. The bottles were centrifuged at 33.5 × g for 15 minutes using Uniscope Laboratory Centrifuge (model SM800B, Surgifriend Medicals, England). Using the method described by Yakubu et al. (2003), the sera were thereafter aspirated using Pasteur pipette into clean, dry, sample bottles and stored frozen until used for analysis.
2.2.8.3 Preparation of semen
After sacrifice, semen collection was done according to the method described by Raji et al. (2003). Briefly, the testis along with its epididymis was removed and fatty tissues trimmed off. The caudal epididymis was separated from the testis and lacerated to collect semen into a plain sample bottle containing normal saline for sperm characteristic analysis.
2.2.9 Preparation of tissue homogenate
After sacrifice, tissues of interest (liver, kidney, and testes) were removed from the dissected rats. The tissues were cleansed with blotting paper to remove blood stains and weighed, then collected in ice cold 0.25M sucrose solution. Known weight of the liver, pair of kidney, and testes were then subjected to homogenisation using Teflon homogeniser in ice-cold 0.25M sucrose solution (1:5w/v). The homogenates were centrifuged at 4000 rpm for 10 minutes and the supernatant was decanted and stored in the freezer (-50C).
2.2.10 Organ-body weight ratio
The organ-body weight ratio was determined by dividing the weight of the organ by the body weight of the animal according to the expression:
Organ-body weight ratio = Weight of organ x 100
Body weight of animal
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