ABSTRACT
This study evaluates the effect of aqueous stem bark extract of Anogeissus leiocarpus on the ovaries and ovarian function through investigating its effect on the histomorphologic changes in the ovaries using Hematoxylin and eosin for light microscopy, and the associated changes in the serum levels of estradiol and progestin hormones using the enzyme-linked-immunosorbent assay (ELISA). Twenty-four young adult Wistar rats were used for the study. The experimental animals were synchronized using 0.5ml injection of prostaglandin PGF2α to ensure uniformity in the estrous cycle before extract administration. Four experimental groups were assigned and received an extract concentration of 600mg/kg, 400mg/kg, and 200mg/kg for groups I, II and III, while the control group (IV) received distilled water for the period of six (6) weeks respectively. The extract has no effect on the microscopic slides of the ovaries prepared at the end of the experiment. There was an increase in the concentration of progesterone in groups II, and group III following the extract administration. The progesterone level in the orally treated group was 19% higher, and statistically significant (p< 0.05), and an increase in the level of serum estradiol in groups II, and III was also observed after extract administration (p< 0.05).These results suggest that Anogeissus leiocarpus has a potential effect on the ovarian function indirectly via its inhibitory effect on PDEs; an effect which is established to promotes developmental competence of mice oocytes retrieved from small antral follicles.
TABLE OF CONTENTS
Title page i
Approval page ii
Declaration iii
Certification iv
Dedication v
Acknowledgement vi
Abstract viii
Table of content ix
List of figure xiv
List of table xiv
List of plates xv
CHAPTER ONE
1.0 INTRODUCTION 1
1.1 Background of the study 1
1.2 Statement of research problem 3
1.3 Justification of the study 3
1.4 Significance of the study 3
1.5 Research Hypothesis 3
1.6 Aim and Objectives 3
CHAPTER TWO
2.0 LITERATURE REVIEW 5
2.1.0 Medicinal plants 5
2.2.0 The plant Anogeissus leiocarpus 6
2.2.1 Scientific classification of Anogeissus leiocarpus 7
2.2.2 Properties of Anogeissus leiocarpus 9
2.2.3 Phytochemical analysis of Anogeissus leiocarpus 10
2.2.4 Plant extract and active principles 10
2.2.5 Phytochemical constituents of plants 10
2.2.5.1 Alkaloids 11
2.2.5.2 Cardiac glycosides 11
2.2.5.3 Tannins 12
2.2.5.4 Saponins 12
2.2.5.5 Flavonoids 12
2.2.5.6 Phytoestrogen 13
2.2.6 Toxicological studies 15
2.2.7 Medicinal properties and uses of Anogeissus leiocarpus 15
2.2.7.1 Antihypertensive activity 16
2.2.7.2 Antibacterial activity 16
2.2.7.3 Antihelminthic activity 17
2.2.7.4 Antiplasmodial activity 17
2.2.7.5 Hypoglycemic activity 17
2.2.7.6 Antifungal activity 18
2.3.0 The Ovaries 18
2.3.1 Development of the ovaries 18
2.3.2 Relationship of the ovary 19
2.3.3 Mesovarium 20
2.3.4 Histological structure of the ovary 20
2.3.5 Blood supply of the ovary 21
2.3.6 Lymphatic drainage to the Ovaries 22
2.3.7 Nerve supply to the Ovaries 22
2.4.0 The Oocyte 23
2.4.1 Origin and development of the Oocyte 23
2.4.2 Oocyte growth and differentiation 24
2.4.3 Oocyte granulosa cell connection 26
2.4.4 The secondary follicle 27
2.4.5 Theca development 28
2.4.6 Oocyte meiotic competence 29
2.4.7 The Antral follicle 29
2.4.8 Histological architecture of the Graafian follicle 30
2.4.9 Atresia 31
2.5.0 Ovulation 33
2.6.0 Reproductive System of the Female Rat 34
2.6.1 Estrous cycle 34
2.6.2 Phases of the Estrous Cycle 35
2.6.3 Proestrus Phase 35
2.6.4 Metestrus Phase (diestrus I) 35
2.6.5 Diestrus 36
2.6.6 Characteristics of the estrus cycle in the rats 36
2.7.0 Hormones of the Gonads 37
2.7.1 Effect of progestins on the uterus 37
2.7.2 Effect of estrogen on the uterus 38
CHAPTER THREE
3.0 MATERIALS AND METHOD 39
3.1 Experimental animals 39
3.2 Collection and Identification of Plant material 39
3.2.1 Preparation of plant extract 39
3.2.2 Choice of solvent for extract administration 40
3.2.3 Determination of concentration and volume of extract 40
3.2.3 Route of administration of extract 40
3.3 Chemical reagents 41
3.4 Experimental design 41
3.5 Synchronization of experimental animals 43
3.6. Method of taking Vaginal smear 45
3.7 Hormonal assay 47
3.8 Histological studies 47
3.8.1 Tissue processing procedure 48
3.9 Histometrical Studies 48
3.10 Statistical analysis 49
CHAPTER FOUR
4.0 RESULTS 50
4.1 Physical observation 50
4.2 Synchronization of Experimental Rats 50
4.3 Effect of Anogeissus leiocarpus extract on body weight of the animals 53
4.4 Effect of different doses of extract on the weight of the ovaries, uterus, and Fallopiantubes 54
4.5 Histological changes on the Ovary 61
4.6 Hormonal assay for Estradiol and Progestin 61
CHAPTER FIVE
5.0 DISCUSSION 72
CHAPTER SIX
6.0 SUMMARY, CONCLUSSION AND RECOMMENDATION 76
6.1 Summary 76
6.2 Conclusion 76
6.3 Recommendations 77
REFERENCE 78
LIST OF FIGURES
Figure1. The molecular weight of Dinoprost 46
LIST OF TABLES
Table 4.1 Effect of oral administration of Anogeissus leiocarpus extract On body weight of animals 52
Table 4.2 Weight of the Uterus, Tubes, and Ovaries across the experimental groups 53
Table 4.3 Number of follicular cells across each experimental group 54
Table 4.4 Mean serum level of Progesterone of mice pre and post administration of Anogeissus leiocarpus extract by 42 days 55
Table 4.5 Mean serum level of Estrogen of mice pre and post- administration of Anogeissus leiocarpus extract by 42 days 56
LIST OF PLATES
Plate i: Anogeissus leiocarpus (DC) Guill and Perr (Steentoft, 1988)
Plate ii: Photomicrograph showing the cross section of the medulla of the ovary from the group treated with 200mg/kg body weight, showing some developing follicles and rete ovaries. (H&E x 100) 58
Plate iii: Photomicrograph showing a cross section of the ovary of the group receiving 200mg/kg body weight showing normal architecture of the ovary with primary oocyte (O); theca folliculi (TF); vesicular spaces (VS); zona granulosa (ZG). (H&E x 100) 59
Plate iv: Photomicrograph showing a cross section of the ovary of the group receiving 200mg/kg body weight showing normal architecture of the ovary with primary oocyte (O); outer surface epithelium (OSE); tunica albuginea (TA). (Pas x 250) 60
Plate v: Photomicrograph showing the cross section of the ovaries of the group treated with 200mg/kg body weight showing normal architecture of the ovary with oocytes at different stages of development (O); and rete ovaries. (Pas x 250) 61
Plate vi: Photomicrograph showing the cross section of the ovaries of the group treated with 400mg/kg body weight showing normal architecture of the ovary with secondary oocytes and developing follicles at different stages of development. (H&E x 250) 62
Plate vii: Photomicrograph showing the cross section of the ovary in the group treated with 400mg/kg showing normal architecture developing follicles at different stages of development. (H&E x 40) 63
Plate viii: Photomicrograph showing the cross section of the ovary of the group treated with 400mg/kg body weight, showing normal architecture developing follicles at different stages of development, and a degenerating corpus luteum (PAS x 40)
64
Plate ix: Photomicrograph showing the cross section of a mature follicle, with large follicular antrum (FA), corona radiate (CR) and a centrally situated ovary from the group receiving 600mg/kg (H&E x 100) 65
Plate x: Photomicrograph showing the cross section of a secondary follicle, and a centrally situated ovary from the group treated with 600mg/kg (PAS x 250) 66
Plate xi: Photomicrograph showing the cross section of a follicle from the control group, a matured ovary (O), follicular antrum (FA), and corona radiate (CR) (H&E x 250) 67
Plate xii: Photomicrograph showing the cross section of the ovary with a primary follicle from the control group (PAS x 250) 68
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of the Study
Anogeissus leiocarpusis referred to by many vernacular names in different languages across Africa. It is referred to as Orin-odanin Yoruba, Kojoli in Fulbe, and Markein Hausa, is a common plant distributed across the African savanna region, mostly across the West and Central Africa (Takahashi, 1978). It is mostly employed for agricultural purposes for woods, charcoal, and production of agricultural implements.
A. leiocarpus has many applications locally in Nigeria. It is used medicinally for the treatment of ascaris, gonorrhea, pain, blood clotting, and coughing (Mann et al., 2003). The Yoruba’s of South-Western Nigeria illustrated that the plant is also used as an antimicrobial agent to treat bacterial infection (Dweek, 1996).
A. leiocarpus is also locally used for problems associated with reproduction and to enhance fertility in many parts of Northern Nigeria. The effect of A. leiocarpus on reproduction and fertility has not been investigated, and was the motivating factor for this study.
Phytochemical studies conducted on A. leiocarpus revealed that the stem bark extracts has various chemical constitutions such as alkaloids, glycosides, steroid, anthraquinone, phenol, tannins, saponins, calcium, and fluorides (Adekunle et al., 2006).
The World Health Organization (WHO) in 2008 estimated that 80% of the world population; particularly in Asian and African countries depend on traditional medicine for primary health care. The use of herbal medicine in developed countries has expanded sharply in the latter half of the twentieth century, and the WHO produce monographs on selected herbs and their sources (WHO, 1999).
The problem of infertility has been one of the major concerns affecting most couples all over the world. In Africa, impaired fertility is a well-known public health issue (Larsen, 1994). Despite advancement in medicine and the accessibility of reproductive health clinic, the problem of infertility continues to affect many couples thereby serving as a common source of marital problem and disharmony, a problem more aggravated in the African society.
The International Family Planning perspectives in 2005 declared that a substantial proportion of women suffer from infertility in Nigeria, making about 33% of women aged 20-44yrs.
Several studies indicate that infertility is the most frequent reason for gynecological consultation in Nigeria (Okonofua, 2003).More than 50% of gynecological case loads are as a result of infertility (Otuba,1990), and over 80% of laparoscopic investigations are as a result of infertility (Okoye, 2002).
1.2 Statement of research problem
Anogeissus leiocarpus extract has been used locally for a long time to treat different kind of ailment generally and infertility problem. Clinical studies regarding its use are few; therefore a clear understanding through which it exert its effect has not been established.
1.3 Justification of the study
Various studies conducted (invitro and in vivo) using crude and refined extract of A. leiocarpus have shown it to have some antimicrobial activities. The clinical significance of A. leiocarpus and its use for problems associated with reproduction and fertility is yet to be determined.
The present study is aimed at determining the effect of consuming A. leiocarpusextracts on the microscopic structure of ovary, and the associated changes in the serum levels of female reproductive hormones in mature female adult wistar rats.
1.4 Significance of the study
The result of this study could confirm the effectiveness of A. leiocarpus in the treatment of some problems associated with reproduction and infertility through its effect on the ovary.
1.5 Research Hypothesis
Aqueous extract of Anogeissus leiocarpus improves the function of the ovaries and the output of ovarian hormones
1.6 Aim and objectives
The aim the study was: -
To study the effect of aqueous stem bark extract of Anogeissus leiocarpus onthe ovaries and ovarian function.
The objectives of the study were:-
1. To investigate the effect of aqueous stem bark extract of A. leiocarpus on the histomorphologic changes in the ovaries of the adult Wistar rats exposed, using Hematoxylin and eosin for light microscopy.
2. To determine the associated changes on the serum levels of estradiol and progestin in the ovaries of the mature Wistar rats exposed, using the enzyme-linked-immunosorbent assay (ELISA).
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