¹ Department of Anatomy, University of Medical Sciences, Ondo State. Nigeria

² Department of Anatomy, Edo State University Uzairue, Edo State. Nigeria

Corresponding Author: C. A. Idaguko, Department of Anatomy, Faculty of Basic Medical Sciences, Edo State University Uzairue. Edo State Nigeria. Tel: +2348054919134; E-mail: annachi67@yahoo.com. https://orcid.org/0000-0002-0799-527X

Running title: Effect of X. aethiopica on AICI3 - induced toxicity in female rats

	ABSTRACT
	INTRODUCTION
	MATERIALS AND METHODS
	RESULTS
	DISCUSSION
	CONCLUSION
	FUNDING
	COMFLICT OF INTERESTS
	AUTHOR CONTRIBUTION
	REFERENCES

	ABSTRACT

Introduction: Reproductive disorders in females as a result of exposure to heavy metals are considered an important public health and social problem. Aim: The study was to determine the level of influence of Xylopia aethiopica extract on the female reproductive hormone, and histological changes in the uterus following administration of Aluminum chloride (AICI₃). Materials and methods: Twenty female Wistar rats were randomly divided into 4 groups of five rats. Group 1 served as normal control, groups 2, 3, 4 were administered 150mg/kg Aluminum chloride (AICI₃). Group 2 served as positive control, while groups 3 and 4 were also administered 50 mg/kg and 100 mg/kg extract of Xylopia aethiopica respectively. Administration of extract was done through the oral route for 21 days, after which the animals were sacrificed. Hormonal assay such as Luteinizing hormone, Progesterone, Follicle Stimulating Hormone, Estradiol and histology of the uterus were evaluated. Results: This revealed that (AICI3) decreased the levels of FSH, LH, Estradiol and Progesterone. These results were further supported by histological observation that showed the thickenings and degeneration in the endometrial epithelial in the uterus caused by Aluminum chloride. Xylopia aethiopica treated groups showed significant (p<0.05) increase in FSH, LH and Progesterone levels, while the Estradiol level was increase but it was not significant. Histologically, Xylopia aethiopica reduced thickenings and degeneration in the endometrial epithelium of the uterus caused by Aluminum chloride. Conclusion: The finding demonstrated that treatment using Xylopia aethiopica may minimizes the toxic effect of Aluminum chloride on the female reproductive hormones and the uterus.

	INTRODUCTION

   Aluminum is a toxic metal and is present everywhere in the environment. Most of the population is exposed to it through the use of many manufactured foods e.g. food additives, medicines such as (antacids and buffered aspirin), tooth paste, cosmetic products such as antiperspirants, first aid antibiotics, antiseptics, anti-inflammatory and anti-inflammatory agents (1) Furthermore minor exposure can occur through ingestion of aluminum in drinking water and inhalation of ambient air (2). Aluminum accumulates in living organisms, disrupts balance, thereby causing toxic effects (3). Aluminum chloride (AlCl3) is sometimes called aluminum trichloride or aluminum(III) chloride; prolonged exposure to these chemicals can cause skin, eye and respiratory irritation (4). However, knowledge of the effects of aluminum on the female reproductive system is limited.

   The reproductive system is one of the most significant characteristics for the continuity of human life; and is faced with danger as a result of continued exposure from environmental agents. One of the challenges of reproductive system is infertility and it has resulted in large cases of marital problems (5). This high burden of infertility has led couples, who yearn but are unable to realize and sustain desired pregnancy to seek for assistance in tradomedicine especially in low resource countries.

   The key hormones of the reproductive system are the Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH), which are released by the anterior pituitary as a result of stimulation by Gonadotropin-Releasing Hormone (GnRH). FSH and LH regulates the ovary gonadal function by enhancing sex steroid production and gametogenesis. In women, follicle-stimulating hormone stimulates the growth of ovarian follicles in the ovary before the release of an egg at ovulation, and promotes oestra-diol production (6). Common major underlining factor of female infertility are hormonal imbalance (7, 8). In the bid to manage infertility, various medicinal plants were often locally consumed and there have been reports of effective activities of such plants (9, 10).

   Xylopia aethiopica (X. aethiopica) commonly known as “African guinea pepper” or “Ethiopian pepper, is an angiosperm of the Annonaceae family, and grows predominantly in humid forest zones of West Africa (11). Its common names include; African pepper, Guinea pepper, Spice tree, Negro pepper, West African pepper and Senegal pepper (12). In Nigeria it is found all over the lowland rain forest and most fringe forest in the savannah zones; it is called “Ata” (Efic), “Chimba” (Hausa), “Uda” (Igbo), “Eeru” (Yoruba), “Urhirien” (Urhrobo) (13). According to ethnomedicinal survey, the plant is used in managing a myriad of ailments and the fruits are widely consumed within many localities in Nigeria. These includes antiplasmodial (14), analgesic (11), anti-inflammatory (15), antidiabetic (16), antimicrobial (17), antioxidant (14), membrane stability (18) among others. Aside the aforementioned benefits of the fruits of X. aethiopica, the fruits are also used as spices and the aqueous decoctions are used especially after child birth, probably due to its haemostatic potential to arrest bleeding (19).

   Some phytochemical compounds have been characterized and isolated from different parts of X. aethiopica and have been alleged to be responsible for its diverse therapeutic and pharmacological properties. These includes alkaloids, phenolics compounds, flavonoids, tannins, glycosides (terpene, sterols and deoxy-sugar), saponins, anthraquinone, steroid, carbohydrates, mucilage, balsams, phlobatannins, volatile aromatic oils, (18, 20), rutin and fixed oils (21, 20). Xylopia aethiopica have also been reported to possess minerals (manganese and zinc), vitamins (A, B, C, D and E) and proteins (22, 20). Also, acidic compounds like kaurine, kolavane and trachylobanediterpenes have been isolated from the stem, bark and fruit of X. aethiopica (23).

   The aim of this research is to determine the level of influence of X. aethiopica extract on the female reproductive hormones, and to determine if any histological changes occur in the histology of the uterus of the female rat following administration.

	MATERIALS AND METHODS

   Chemical Reagents

   Aluminum Chloride was procured from Pascal Enterprise opposite Akure South Local Government, Akure. The ELIZA kits for hormone profile were bought from Alums Diagnostic Center, Suleja, Niger State.

   Plant collection and preparation

   The dried fruit of Xylopia aethiopica was purchased from Ondo Central Market, Ondo, Ondo State, Nigeria. It was identified and authenticated by Mr Ojo Samuel of the Department of Biological Science, Plant Biology and Biotechnology, University of Medical Sciences, Ondo with Voucher number UNIMED P.B.T.H No 008. The dried fruits were carefully de-seeded and the seeds were grinded into a coarse powdered form using grinding machine.

   Plant extract and preparation

   A total of 200 g of the coarse powdered seed was weighed and extracted in 70% of ethanol via maceration for 72 hours in an air tight container. The mixture was filtered with Whatman No 3-filter paper. Using a water bath, the ethanol filtrate was concentrated at low temperature of 45 ºC under reduced pressure, which yielded 6g of the jelly-like extract using the formula:

   Percentage yield = mass of extra(g) ÷ mass of powdered-sample(g) × 100

   Experimental Animals

   The experiment was performed with twenty (20) adult female Wistar rats. The rats weighing (120 - 200 g) were obtained from the Animal Housing Facility of Department of Anatomy, University of Medical Sciences, Ondo State, Nigeria. The rats were kept under standard laboratory condition and fed normal rat feed pellet and water. The animals were kept in standard cage. They were acclimatized for two weeks before starting the experiment.

   Ethical approval

   All the experimental procedures and protocols used in this study were in accordance with the National Institution of Health Guideline Principles of Laboratory Animals in Biomedical Research (24). Ethical approval was obtained from University of Medical Sciences Ethical Review Committee Ondo State.

   Determination of Dosage of Aluminum chloride to administer

   The Aluminum chloride solution was prepared using 1g of Aluminum chloride salt dissolved in 100mL of distilled water, while 50 and 100 mg/kg bw of X. aethiopica extract were administered orally using an oral cannula. The body weight of the rats was measured every week of the experimental work.

   Experimental Design and Animal grouping

   Twenty female rats were randomly divided into four (4) groups.

   Group 1- control received distilled water orally.

   Group 2- received 150 mg/kg bw. of Aluminum chloride.

   Group 3- received 150 mg/kg bw. of Aluminum chloride + 50mg/kg bw. of X. aethiopica extract

   Group 4- received 150 mg/kg bw. of Aluminum chloride + 100mg/kg bw. of X. aethiopica extract

   Animal sacrifice and sample collection

   The extracts was administered for 21 days after which, the animals were sacrificed. Blood samples were taken by cardiac puncture from all groups for hormonal assay (Follicle Stimulating Hormone, Luteinizing Hormone, Progesterone and Estradiol). The uterus was harvested after abdominal incision, and then fixed in 10% buffered formalin. Tissue sections were prepared using routine histological tissue preparation. Photomicrographs were taken using the 5 mega pixel Amscope digital scope, mounted on an Olympus microscope.

   Estimation of hormones

   Estimation of serum Luteinizing hormone (LH), serum Progesterone Hormone, serum Follicle Stimulating Hormone (FSH), serum Estradiol was done according to the instruction on the ELIZA kits for hormone profile by the manufacturer.

   Statistical Analysis

   The data was presented as mean values ± SEM. ANOVA was carried out on the Statistical Package for the Social Sciences (SPSS package version 17) and the occurrence of significant differences between the results was checked. Differences were considered significant at p<0.05.

	RESULTS

   Data on FSH level showed a significant increase (P<0.05) in groups 3 and 4 (7.60 ± 0.42 and 8.44 ± 0.25) in animals administered with 50 and 100, mg/kg of X. aethiopica when compared to the group 2 (4.05 ± 0.49). However group 1 (12.68±0.33) was significant increase (P<0.05) when compared to the treated groups (3 and 4). Data on LH level showed an increase in groups 3 and 4 (3.39 ± 0.18 and 4.22 ± 0.31) in animals administered with 50 and 100, mg/kg of X. aethiopica when compared to the group 2 (2.16 ± 0.07). However group 1 (4.22 ± 0.31) was not significant increase (P<0.05) when compared to the treated groups (3 and 4). Data on Progesterone level showed an increase in group 4 (5.19 ± 0.95) in animals administered with 100mg/kg of X. aethiopica when compared to the group 2 (1.37 ± 0.06). However group 1 (17.19 ± 1.68) was significant increase (P<0.05) when compared to the treated groups (3 and 4). Data on Estradiol level showed a non-significant increase (P<0.05) in groups 3 and 4 (2.37 ± 0.07, and 2.42 ± 0.05) in animals administered with 50 and 100, mg/kg of X. aethiopica when compared to the group 1 (3.82 ± 0.10). However, there was decrease in group 2 (1.00 ± 0.15) when compared to group 1 (3.82 ± 0.10) as shown in Table 1.

   Effect on the uterus histology

   The uterine histology of the tissue sections from the control rats showed Endometrium (E), Perimetrium (P), and Myometrium(M), a normal regular columnar epithelial cells lining the uterine lumen with elongated nuclei at the base of the cells, highly folded epithelial lining, numerous and tortuous endometrial glands, edematous stroma (Group 1a, 1b). The uterine histology of the aluminum chloride group revealed several thickenings and increase in the epithelial degeneration and vacuolation in the lining epithelium of the endometrium, invaginations of the uterine lumen with dilated endometrial glands (Group 2a, 2b). Xylopia aethiopica treated groups showed reduced thickenings and reduction in the degeneration of the endometrial epithelium (Groups 3a, 3b and 4a, 4b) as shown in Figure 1.

View larger version : [in a new window] Figure 1. Representative uterine micrograph of Wistar rat of control group 1 (a, b), group 2 (a, b) group 3 (a, b) and group 4 (a, b) showing Perimetrium (P); Myometrium (M); Endometrium (E), Surface Epithelium (SE); Stroma (S) Lumen (L), Uterine gland(UG), Endometrial gland (GL), Necrosis(N) a= (H&E×40) and b = (H&E ×100)

	DISCUSSION

   In an industrialized world, there are different types of metals in use, and humans are exposed to them at work as a result of environment, food and water contamination (25). Aluminum is one of the most abundant metal and the third common chemical element in the earth’s crust and it poses as a relevant risk to population health when expose to it (26). The use of medicinal plants have been used in treating disease and health conditions including reproductive disorders. In developing countries like those in the sub-Sahara Africa, reproductive disorders pose a major problem and it is considered as the second most prevalent health care problem in Africa (27). The application of some plants constituents in pharmaceutical industries has gone a long way in the elevation of the status of the traditional herbal medicine in Africa and particularly in Nigeria (28).

   In this study, AlCl3 exposure decreased the FSH, LH and Progestogen. Hence, the reproductive hormones of female rats is inhibited under aluminum chloride exposure. FSH and LH, are synthesized in the pituitary gland and are major components of the hypothalamic–pituitary–gonad axis; thereby regulating reproductive function in mammals (29). The results obtained for the hormonal analysis showed an increase in serum levels of FSH, LH and Progesterone in the groups treated with X. aethiopica when compared to the AlCl3 group. These findings may be as result of the X. aethiopica antioxidant ability to reduce the oxidative stress induced by aluminum chloride. Studies have revealed the detrimental role of oxidative stress in causing hormonal imbalance and dysfunctions (30). X. aethiopica has been found to contain some phytochemicals which exhibit a wide range of biological effects as a consequence of their antioxidant properties (31). Aside from antioxidant activity of flavonoids present in X. aethiopica, these molecules provides the following beneficial effects; antiviral, anticancer, anti-inflammatory and anti-allergic (31). The increase in the FSH at the dose of 100mg/kg of X. aethiopica could be as a result of the present of flavonoids and glycosides which have been reported to have a relationship with sex hormones (32. 33, 34). Flavonoids scavenge free radicals produced by ROS, thereby helping to prevent the negative effect of Aluminum that may have resulted in decrease of the hormones as seen in the Aluminum chloride untreated group. This increase in FSH level would most likely promote follicle production and help to keep the function of the ovary and uterus. It is possible that the extract might have exerted its effect on the anterior pituitary gland or the hypothalamus since the secretion of FSH is controlled by the concerted interaction between the gonadotropin releasing hormones of the hypothalamus on the anterior pituitary gland. However, some studies reported that the extract of X. aethiopica did not cause any significant change in FSH level even at high concentration of 500mg/kg in rats (35, 36).

   Luteinizing hormones stimulates the secretion of sex steroids from the gonads; hence, increase in LH secretion during the preovulatory periods in the female reproductive cycle results in the rupture of mature follicle in the ovary (37). It is also known that LH stimulates ovulation growth of corpus luteum and progesterone release (38). Progesterone is produced by the ovaries, placenta and adrenal glands; it regulates the monthly menstrual cycle, prepare the body for conception and pregnancy as well as stimulation of sexual desires (39). A study in literature reported an increase in FSH, Progesterone levels in male rats after 7 days of administration of the extract of X. aethiopica (40). A natural increase in blood estradiol concentrations during the menstrual cycle causes an egg to mature and be released; estradiol stimulates the growth of the uterine lining, causing it to thicken during the preovulatory phase of the cycle (41). Estradiol is responsible for the growth and development of reproductive organs and in synergy with FSH, estradiol stimulate granulosa cell proliferation during follicular development (42). In the present study, there was an increase in the estradiol in the X. aethiopica extracts treated groups when compared to the control group, but the increase was not significant. The estradiol slight increase indirectly may stimulates the hormone gonadotropin-releasing neurons in the hypothalamus and septum which, in turn, activates responsive cells in the anterior pituitary to release the luteinizing hormone and follicle-stimulating hormone into the circulation, as seen in this study. This probably may imply that X. aethiopica plays an important role in regulation of the hormonal levels.

   Tissue sections from the uterus of the female control group (Fig 1. 1a, 1b) show common uterine histology with regular columnar epithelial cells lining the uterine lumen. The histological alterations in Aluminum chloride group 2 (Fig 1. 2a, 2b), showed that apoptosis was demonstrated in the luminal epithelial cells inform of debris, infiltration of the endometrium with vascular congestion; this point out the existence of an inflammatory response in the endometrium. Also, the perimetrium layer showed apoptosis and squamation of luminar and glandular epithelium. Tissue sections from the treatment groups 3 and 4 shows moderate apoptosis and necrotic changes in the endometrium and perimetrium (Fig 1. 3a, 3b and 4a 4b). The work thus showed that Xylopia aethiopica may have an ameliorative effect on the toxicity caused by aluminum chloride on the uterus and exert a positive effect on the female fertility by enhancing the female reproductive hormones.

	CONCLUSION

   Aluminum chloride is capable of altering the histological structure of the uterus and the reproductive hormones. Treatment using Xylopia aethiopica could ameliorate the Aluminum chloride induced toxicity in the histological structure of the uterus and enhance the levels of female reproductive hormone.

	FUNDING

   The authors declare that no funds, grants, or other support were received during the preparation of this manuscript

	COMFLICT OF INTERESTS

   The authors declare that no conflicting interests exist.

	AUTHOR CONTRIBUTION

   The idea and design of the study was conceived by Leko J. Bankole. The experiment, data collection and analysis were performed by Aroge I. Precious and Idaguko C. Anna. The first draft of manuscript was written by Aroge I. Precious and all authors commented on the manuscript. All authors read and approved the final manuscript before submission.

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