EFFECTS OF HYDROETHANOLIC EXTRACT OF THE SEEDS OF Sesamumindicum ON SOME REPRODUCTIVE PARAMETERS OF FEMALE ALBINO RATS


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EFFECTS OF HYDROETHANOLIC EXTRACT OF THE SEEDS OF Sesamumindicum ON SOME REPRODUCTIVE PARAMETERS OF FEMALE ALBINO RATS  

ABSTRACT

Phytoestrogens are natural estrogenic agents present in plants. Phytoestrogens are any plant compounds structurally and/or functionally similar to ovarian and placental estrogens and their active metabolites. Sesamumindicum is one of the phytoestrogen containing seeds used by man for food and medicinal purposes. Researches have shown that phytoestrogens present in plants might have both fertility-enhancing and anti-fertility effects. Present studies evaluated the mean lethal dose of the hydroethanolic seed extract of sesame and the effects of the extract on some reproductive parameters of female albino rats. Cycling female rats weighing 120-140g were used for the studies. For the ovulation study, twenty (20) female rats were divided into four (4) groups of five (5) rats groups II, III and IV received 100, 300 and 500 mg/kg of seeds extract of S. indicumorally respectively. The animals were dosed at 4 hourly interval for 24 hours. Animals were sacrificed the following morning. For the hormonal level study, the extract was administered orally to three (3) experimental groups of five (5) female rats at doses of 100, 300 and 500 mg/kg once daily while control group received distilled water at dose of 1ml/kg for 30 days. The animals were subsequently sacrificed in the morning of the day after the last administration. For studies on pregnancy outcome, the extract was administered orally four (4) groups of five (5) pregnant rats each. Administration was done once daily from day 1-7 of pregnancy as follows: group I: distilled water (1 ml/kg), group II: 100 mg/kg, group III: 300 mg/kg and group IV 500 mg/kg respectively, with seeds extract of S. indicum.

The number of ovarian follicles was significantly higher (p < 0.05) in group IV than control rats (31.40 + 3.20 vs 22.80 + 4.22), while groups II and III had lower number of follicles than control rats (14.80 + 2.40, 19.80 + 3.40 vs 22.80 + 4.22).

The values of gonadosomatic index were similar in group III and control rats (0.83 + 0.12% vs 0.83 + 0.11%). However, the gonadosomatic indices in groups II and IV were significantly lower (p < 0.05) than control rats (0.66 + 0.03, 0.75 + 0.14 vs 0.83 + 0.12, respectively).

The mean (+ SEM) serum estrogen concentration was significantly lower (p<0.05) in group III than in groups II, IV and control rats (349.60 + 10.20 pg/ml vs 422.40 + 28.19 pg/ml,

414.17 + 17.49pg/ml, 431. 80 + 9.47 pg/ml respectively). The mean serum progesterone followed a similar pattern, with relatively lower values in group III than groups II, IV and control rats (14.70 + 1.39 ng/ml vs 15.10 + 0.74 ng/ml, 19.12 + 1.91 ng/ml and 17.22 + 4.22, respectively).

The number of fetuses delivered by pregnant rats was not significantly different (p < 0.05) between treated and control rats (group I: 6.0 + 0.71, group II: 7.00 + 0.90, group III: 6.20 +

1.07 and group IV: 7.40 + 1.78, respectively; p<0.05).

It is therefore concluded that hydroethanolic seeds extract of S. indicum possess both anti- and pro-estrogenic properties in a dose-dependent fashion in adult female albino rats.

Table of Contents

Content Page No.

Title Page i

Declaration… ii

Certification… iii

Dedication… iv

Acknowledgement… v

Abstract… vi

Table of Contents… viii

List of Tables… xi

List of Figures… xii

List of Appendices… xiii

List of Abbreviations xiv

Chapter One

Introduction…1

Statement of Research Problems…6

Justification for the Stud…7

Aim and Objectives…8

Hypothesis…8

Chapter Two

Literature Review…9

Introduction…9

2..1.1 Processes taking place in the ovary 10

Estrogen11

Progesterone14

2.1.2 The physiological changes of the ovaries (The Ovarian Cycle) 17

Fertile Window21

Infertility22

Prevalence22

Causes and factors…22

Treatment…29

Herbal Treatment for Infertility30

Chapter Three

Materials and Methods32

Materials…32

Plant Collection…32

Experimental Animals…32

Laboratory Materials…32

Chemicals and Solutions33

Methodology33

Preparation of Extract…33

Acute Toxicity Study33

Experimental Design…34

Ovulation Studies…34

3.3.2. Effect of Extract on Serum Concentration of Estrogen and

Progesterone 35

3.3.3 Effect of Extract on Fetal Number 36

Statistical Analysis37

Chapter Four

4.0 Results… 38

Chapter Five

Discussions…44

The Effects of Sesamumindicum on Follicular

Numbers Ovulation… 44

Effect of Sesamumindicumon

Gonadosomatic Index 46

Effect of Sesamumindicumon Serum Concentration of

Estrogen and Progesterone 46

Effect of Sesamumindicumon Fetal Number…48

Chapter six

Conclusion and Recommendations…49

Conclusion…49

Recommendations…49

Contributions to Knowledge49

References… 50

CHAPTER ONE

INTRODUCTION

Hormones are intimately involved in our sex lives, as well as all other aspects of ourlives, from intrauterine life to death. Of these, the most important are the socalledsex hormones. Mammalian sexual behavior is controlled by these gonadal steroids actingat the level of the central nervous system. They are divided into the male hormones (orandrogens) and the female hormones. While, both sexes produce both types, those corresponding to the appropriate sex predominate (in most cases) (Wierman, 2007). Women produce a number of hormones specific to them, particularly during their childbearing years. The two best-known female hormones are estrogen and progesterone, sometimes also referred to as sex hormones. These are produced predominantly in the ovaries, although the adrenal glands and the placenta of pregnant women also secrete them. Some research has indicated that estrogen can be manufactured in the brain as well (Haybach, 2010).

The main female hormone is estrogen. Estrogens display one common biologic activity, the ability to stimulate growth and maintain the female sex characteristics (Goldzieher and Castracane, 2008). Estrogen stimulates the development of the female sex organs both within the fetus and at puberty, creating her femaleness, her rounded breasts andcurvy hips. It also enhances pheromone secretion and 'invitational' behavior and her yearning for sexual contact. Estrogen levels peak just prior to ovulation. They drop off precipitously during menopause, with consequent vaginal dryness and atrophy and increased risk of osteoporosis and heart disease (Wierman, 2007).The actions of estrogen are mediated by the estrogen receptor (ER), a

dimericnuclear protein that binds to DNA and controls gene expression (Cookeet al.1998;Psarra and Sekeris, 2008.).

Progesterone is an essential regulator of normal human female reproductive function in the uterus, ovary, mammary gland and brain, and also plays an important role in non-reproductive tissues such as the cardiovascular system, bone and the central nervous system, highlighting the widespread role of this hormone in normal physiology. The effects of progesterone are mediated through the nuclear progesterone receptor (PR), which interacts with transcriptional co-regulators, moves into nuclear aggregates and regulates gene expression. Although, progesterone plays a pivotal role in normal physiology, exposure to its analogues in exogenous hormone formulations is associated with deleterious effects, most notably an increase in breast cancer risk (Scarpinet al., 2009).

Sesame, SesamumindicumL., is an annual herb native tothe tropics, which was formerly classified as SesamumorientaleL.It belongs to the Pedaliaceae family (Crewset al., 2010; Mahabadiet al. 2013). All parts of the sesame plant such as the seed, oil and leaves are useful and are locally consumed as a staple food by subsistence farmers in the Northern, South-west and Middle - belt regions of Nigeria and celebrated also in folkloric medicine in Asia and Africa (Shittu and Shittu, 2012). The local names of the plants depend on the source areas of cultivations in the world, such as ekuku–gogoro (Yoruba- Sesamumradiatum), yanmoti(Yoruba-S. indicum), ridi (Hausa) and beni (Tiv/Idoma and English) or gingelly (English) (Shittuet al, 2009).

Sesame lignans, such as: sesamin, sesamolin, sesaminol, sesamolinol, pinorsinol, sesamol and gamma-tocopherol are isolated from S.indicumand

S.radiatumseeds. They have more tumorigenic, estrogenic or anti-estrogenic and antioxidant features compared with other plant species. The plant is rich in trace elements/minerals such as calcium, iron, magnesium, zinc, copper and phosphorus (Shittu and Shittu, 2012; Mahabadiet al., 2013). In terms of phytochemistry, these seeds have glycosides, alkaloids, flavonoids, tannins, polyphenols, steroids, and saponins (Sireeshaet al., 2013); non-protein amino acids, unsaturated fats and lipids with multiple double bonds, glazes, phospholipids and vitamins E, B1 and B2 (Mahabadiet al., 2013). Approximate analysis of sesame seed has made it clear that the seeds contain 50-60% oil, 8% protein, 5.8% water, 3.2% crude fiber, 18% carbohydrate and 5.7% ash

(Crewset al., 2010; Mahabadiet al., 2013).

Sesaminwas reported to be converted by intestinal microflora to enterolactone, a compoundwith estrogenic activity and also an enterometabolite of flaxseed lignans, which are known to be phytoestrogenic (Wen-Hueyet al.,2006). The phytoestrogens have attracted so much attention in the last decade in view of their reported health benefits and they include four broad classes of phytochemicals namely the lignans (grains e.g sesame), isoflavones (soybeans), stillbenes and coumestrol (Pradhanet al., 2012; Shittu and Shittu, 2012). These agents mimic endogenous estrogens depending on their concentrations, they either act agonistically or antagonistically by displacing theendogenous estrogens from their binding sites on the estrogen receptors ( ER1 and ER2), among its other mechanisms of action (Shittuet al., 2009; Shittu and Shittu, 2012).Lignans have beneficial effects on serum lipid levels and liver function and give sesame seed oil a marked antioxidant activity. The lignans are also responsible for the great stability of sesame seed oil to

oxidation (Crewset al., 2010). Based on the fact that they are consumed in large amounts in the diet, the effects noticed are usually antiestrogenic, thus, competing with the much more potent endogenous estradiol for the estrogen receptor binding sites and ultimately block its estrogenic activity (Shittu and Shittu, 2012).This led to the quest to undertake this research on the effects of Sesamumindicum on female fertility

Studies have investigated the multiple beneficial functions of sesame which includes antiaging, anticancer, antioxidation, antihypertensive, modulation of lipid metabolism and lipid peroxidation. Their effects on enhancement of liver function, and immunoregulatory and antithrombosis properties have also been investigated.These properties have made sesame seeds and their byproducts functional foods beneficial in the prevention and treatment of chronic diseases and metabolic disorders (Mirmiranet al., 2013). Sesamin, the major lignan found in sesame oil has been demonstrated to enhance hepatic detoxification, to protect against oxidative stress, and to prevent the development of hypertension (Fu-Chouet al., 2006).Ewe atura (one of the local names in southeast Nigeria) means leaves that provide comfort and health for the body because of being useful for treatment of constipation and digestive disorders. It has been reported that boiling a mixture of roots and leaves has antiviral and antifungal activity (Mahabadiet al., 2013). Shittuet al. (2007) evaluated the effects of fluid extract ofsesame leaves on epididymal spermatocyte reserves inadult rats and concluded that sesame leaves increase male fertility.Also researchers, who evaluated the effects of leavesextract of this plant on the fertility of hypoglycemic rats,found that this extract improves

parameters related to thetestis and increases reproductive potential in mice (Shittu et al., 2009).

Figure 1.1: Sesame Plant (Source: www.shutterstock.com)

Figure 1.2: Sesame seeds (Chemonics International Corporation, 2012)

Statement of Research Problem

Plant-derived chemicals that influence endocrine activities in both humans and animals have received a great deal of attention due to their possible beneficial as well as adverse effects (Yakubuet al., 2008). Since ancient times, mankind has used plants to cure diseases and relieve physical sufferings. The better cultural acceptability, better compatibility with the human body, lesser side effects and effectiveness of many, traditional medicine is now an accepted fact (Pradhan, et al., 2012). More than 35,000 plant species are being used in various human cultures around the world for medicinal purposes. Nearly 80% of the world population rely on traditional medicine for primary health care, most of which involve the use of plant extracts (Pradhanet al., 2012).

Fertility regulation with plants or plant preparations has been reported in the ancient literature of indigenous systems of medicine (Kauret al.,2011). The role of estrogen in female reproduction makes it to be a very important hormone whose level in the body has to be carefully regulated. Some traditional herbs consumed by females knowingly or unknowingly alter the levels of this hormone and other important aspects of the reproductive system. This might lead to beneficial effects by boosting the fertility level or produce anti-fertility effects.Sesamumindicumis one of the phytoestrogen containing edible seeds consumed by man as food and for medicinal purposes. Its high phytoestrogen content, about 373mg/100g (Peterson et al., 2010), makes it necessary to investigate its effects on the body, particularly the female reproductive system.

Justification

There are over 186 million couples in developing countries alone (excluding China) today who are affected by infertility (both primary and secondary). Rates of infertility vary considerably from country to country; in the worst- affected areas, over 25% of couples may be unable to have children (WHO, 2003). However, in developing countries of the world like Africa for example, with an infertility rate of more than 30% in some countries (WHO, 2011), there are more infertile cases with limited or no treatment options especially assisted reproductive technology available for the management of this medical condition (Shittu and Shittu, 2012). Hence, expressed worldwide is the search for an ideal folkloric phyto-chemical medicinal agent with a broad spectrum and proven potential of treating infertility/ subfertility conditions with minimal or no side effects as compared to their synthetic counterparts (Shittuet al, 2009).

The phytoestrogens appeared to be one of such natural estrogenic agents that have attracted so much attention in the last decade in view of their reported health benefits (Mahabadiet al., 2013). Sesame ingestion has been shown to improve blood lipids in humans and anti - oxidative ability in animals (Shittu and Shittu, 2012). The methanolic extract of the plant has antimicrobial effects on the male reproductive system (Shittu and Shittu, 2012). Sesame phytoestrogeniclignans tend to promote aromatization of testosterone to estradiol to favor spermatogenesis (Shittu and Shittu, 2012). In addition, sesame leaves extract consumption enhances the quality of the spermatozoa produced with improvement in the storage capacity of the epididymis for these spermatozoa in a dose related manner (Shittuet al., 2007). A research

conducted by Wen-Hueyet al.(2006) showed that sesame seeds tend to improve the conditions of post-menopausal women because of its estrogenic effects on the serum levels of dehydroepiandrosterone sulfate (DHEAS), sex hormone- binding globulin (SHBG) and follicle-stimulating hormone (FSH).

Aim andObjectives

This study was aimed at evaluating the effects of the hydroethanolic extract of seeds of Sesamunindicumon some reproductive parameters of matured female albino rats.

Specific Objectives were:

1. To determine the mean lethal dose (LD50) of the hydroethanolic extract of seeds ofSesamumindicum

2. To evaluate the effects of the hydroethanolic seed extract on ovulation and gonadosomatic index of female albino rats.

3. To determine the effects of the extract on serum levels of estrogen and progesterone in femalealbino rats.

4. To evaluate the effect of the extract on pregnancy outcome.

Research Hypothesis

H0: The hydroethanolic extract of Sesamumindicum seeds does not have pro- fertility effects on matured female albino rats.

HA:The hydroethanolic extract of Sesamumindicum seeds has pro-fertility effects inmatured female albino rats.

.

EFFECTS OF HYDROETHANOLIC EXTRACT OF THE SEEDS OF Sesamumindicum ON SOME REPRODUCTIVE PARAMETERS OF FEMALE ALBINO RATS


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