Tropical Journal of Natural Product Research

leucorrhoea, and constipation. 6-9 Primary screening of the secondary metabolites, mineral contents and amino acid profile of the aqueous extracts of P. amarus leaves showed the presence of alkaloids, flavonoids, saponins, steroids, tannins, calcium, potassium, iron, zinc, chromium, copper, glutamine and methionine. 10 In another study, Nurudeen and Yakubu 11 reported that the aqueous extract of Phyllanthus amarus leaves restored sexual adequacy in female rats induced into sexual dysfunction by the selective serotonin reuptake inhibitor (fluoxetine). It has also been reported that the aqueous extract possesses hypotensive, 12 antioxidant, 13 anti-viral, 14,15 analgesic, 16 anti-arthritic, 17 anti-plasmodial, 18 anti-nociceptive, 19 anti-hepatitis, 20 anti-amnesic 21 and anti-convulsant 22 activities.

amarus leaves on fluoxetine-induced anti-oestrogenic activity in female Wistar rats regarding the synthetic and secretory functioning indices of the ovaries and the uterus. Consequently, this study seeks to investigate and elucidate the effects of the aqueous extract of P. amarus leaves on fluoxetine-induced anti-oestrogenic activity in female rats. This study is furtherance to the efficacious study of the P. amarus leaves as a sex-enhancing plant in fluoxetine-induced sexual dysfunction in female rats previously reported by Nurudeen and Yakubu. 11

Plant material and authentication
Fresh Phyllanthus amarus leaves were collected within the premises of the Department of Biochemistry, University of Ilorin, Ilorin, Nigeria around July, 2016. The leaves were identified and authenticated by Mr. Bolu Ajayi of the University of Ilorin Herbarium, Ilorin, Nigeria. The plant was assigned a voucher number UIH 001/1109.

Experimental animals
Thirty healthy, in-bred, sexually active, female Wistar rats weighing 141.52 ± 6.26 g were obtained from the Animal Holding Unit of the Department of Biochemistry, University of Ilorin, Ilorin, Nigeria. The animals which were contained in their respective cages were placed in a well-ventilated Animal House and maintained at a temperature of 22 ± 3 o C, 12:12 h light and dark cycle, relative humidity of 45-50%. The animals were maintained on rat pellets (Premier Feeds, Ibadan, Nigeria) and tap water (pH 6.54 ± 0.20) ad libitum. The ethical review committee of the University of Ilorin, Ilorin, Nigeria approved this study in a letter referenced UERC/ASN/2015/210. All the animals were handled strictly according to prescribed guidelines. 23 Assay kits Fluoxetine and Tadalafil were products of Tillomed Laboratories Limited, Herdfordshire, UK and Tuyil Pharmaceuticals Industries Limited, Ilorin, Nigeria respectively. Assay kits for glycogen, cholesterol, acid phosphatase and alkaline phosphatase were products of Randox Laboratories, Co-Atrim, UK, while that of oestrogen was from Inteco Diagnostics UK Ltd., London, United Kingdom. All other reagents used were of analytical grade and were prepared in volumetric flask using all glass-distilled water.

Preparation of extracts
The method described by Nurudeen and Yakubu 11 was used in the preparation of leaf extracts of the test plant. Prior to use, leaf stalks of Phyllanthus amarus were removed. The leaves were thereafter rinsed in distilled water. The leaves were later oven-dried at 40 o C for 48 h and pulverized in a blender (Master Chef Blender, Model MC-BL 1980, China). A known weight (50 g) of the powder was extracted in 500 mL of distilled water for 48 h at room temperature with constant shaking. The lyophilized (Vir Tis Benchtop K, Vir Tis Co., Gardiner, NY) filtrate yielded 5.25 g (percentage yield of 10.5%). This was reconstituted in distilled water to give the required doses of 20, 40, and 80 mg/kg body weight before administration to the experimental animals as previously used by Nurudeen and Yakubu. 11 Experimental design Thirty female rats, after two weeks of acclimatization were randomly assigned into six groups (A-F) of five animals each. Rats in group A (control group) were orally administered 0.5 mL of distilled water, once daily with the aid of a metal oropharyngeal cannula. Those in groups B, C, D, E, and F apart from being treated with 15 mg/kg of fluoxetine suspension (prepared daily in distilled water), once daily (08:00 -08:45 h) for 14 days, also received 0.5 mL each of distilled water, 10 mg/kg body weight of Tadalafil, 20, 40 and 80 mg/kg body weight of the extract, respectively for 7 days.

Preparation of serum and tissue supernatants
The serum and tissue supernatants were prepared according to the procedure described by Yakubu et al. 24 Rats were anesthetized in diethyl ether fumes. When they became unconscious, the jugular veins were cut, and 5 mL of the blood was collected into clean, dry centrifuge tubes. The samples were left for 15 min at room temperature for the blood to clot. Clear serum was then collected using Pasteur pipette after centrifuging at 503 × g for 10 min using Uniscope Laboratory Centrifuge (Model SM800B, Surgifriend Medicals, Essex, UK). Furthermore, the ovaries and uterus were blotted with blotting paper, cut very thinly with sterile scalpel blade and then homogenized in ice-cold 0.25 M sucrose solution (1:5 w/v) to maintain the integrity of the organs. The homogenates were centrifuged at 894 × g for 15 min, while the resulting supernatant was frozen at −20°C until use for the biochemical assays. The biochemical assays were carried out within 24 h of preparation.

Determination of oestrogenic parameters
The ovarian and uterine activities of acid phosphatase, alkaline phosphatase and the concentration of oestrogen were determined using standard methods. [25][26][27] Other biochemical parameters of the ovaries and uterus were determined for ovarian and uterine protein, 28 ovarian and uterine glycogen 29 and ovarian and uterine total cholesterol. 30 Statistical analysis Data were expressed as the mean ± SEM of five replicates. Means were analyzed using one-way analysis of variance followed by Duncan Multiple Range Test. Statistical Package for Social Sciences, version 20.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analyses. Differences were considered statistically significant at p < 0.05.

Results and Discussion
Administration of fluoxetine to sexually active female rats significantly (P < 0.05) reduced the levels of oestrogen by 31.14%, when compared with the distilled water-treated animals ( Figure 1). The reduced level of oestrogen in the fluoxetine-treated animals were significantly (p < 0.05) increased following the administration of the aqueous extract of P. amarus, but the significant increase was more pronounced in the fluoxetine-induced sexual dysfunction animals administered 80 mg/kg body weight of the extract, and compared favourably (p > 0.05) both with those administered the reference drug (tadalafil) and those of the distilled water-treated animals. The ovaries are the sites of production and periodical release of egg cells for possible fertilization. The ovaries also secrete hormones, principally oestrogen (which is responsible for the appearance of secondary sex characteristics for females at puberty and for the maturation and maintenance of the reproductive organs in their mature functional state) and progesterone (which prepares the uterus for an eventual implantation of the embryo). 1 Whereas, the reproductive function of the uterus is not only to accept a fertilized ovum and nourish the developing foetus prior to birth, but also essential in sexual response by directing blood flow to the pelvis and to the external genitalia, which is crucial for the maintenance of the female reproductive system. 31,32 The secretory constituents of the ovaries and uterus include total protein, glycogen, cholesterol, acid phosphatase and alkaline phosphatase. These constituents can be used to assess the normal functioning of the ovaries and uterus as well as the oestrogenic and anti-oestrogenic properties of a chemical compound in a living system. 33 The administration of fluoxetine to sexually active female rats significantly reduced (p < 0.05) the concentrations of the ovarian secretory constituents: total protein, glycogen, cholesterol, alkaline phosphatase and acid phosphatase of the animals, when compared with the distilled water-treated animals ( Table 1). The reduced levels of these ovarian secretory constituents in the fluoxetine-treated animals were significantly increased (p < 0.05) following the administration of the aqueous extract of P. amarus at all the doses evaluated, but only the fluoxetine-induced sexual dysfunction animals  Table 1). Administration of fluoxetine to sexually active female rats significantly (p < 0.05) reduced the levels of the uterine secretory constituents: total protein, glycogen, cholesterol, alkaline phosphatase and acid phosphatase of the experimental animals when compared with the distilled water-treated animals ( Table 2). In contrast, all the doses of the extract (20, 40 and 80 mg/kg body weight) evaluated produced a significant (p < 0.05) increase in the concentrations of the uterine total protein, glycogen, cholesterol, alkaline phosphatase and acid phosphatase of the sexual dysfunction female rats, when compared with the distilled water-treated fluoxetine-induced sexual dysfunction female rats (Table 2). Although, the significant increase produced by 20 and 40 mg/kg body weight of the extract did not compare favourably (p > 0.05) with those of the distilled water-treated animals, the experimental animals that were administered 80 mg/kg body weight of the extract showed a significant increase that does not only compared favourably (p > 0.05) with those administered with the reference drug (Tadalafil), but also with those administered with distilled water only. However, the extract at all the doses evaluated (20, 40 and 80 mg/kg body weight) produced a significant (p < 0.05) increase in the concentrations of uterine alkaline phosphatase, but did not compare favourably (p < 0.05) with those of the distilled water-treated animals ( Table 2). Ovarian proteins are required for ovarian folliculogenesis, corpus luteum functions, oocyte maturation and fertility as well as for managing some reproductive disorders. 34 In the present study, the significant reversal in the concentration of ovarian protein in fluoxetine-treated female rats following oral administration of the aqueous extract of P. amarus might contribute to the enhanced maturation of the oocytes and improved control of ovarian functions. 34 Uterine proteins are required to enable the uterus provides an environment in which remarkable rates of growth are achieved and in the maintenance of pregnancy. 35 Thus, the significant reduction in the concentration of uterine protein in fluoxetine-treated rats could limit the growth of the conceptus. The restoration of the concentration of uterine protein following the administration of the aqueous extract of P. amarus leaves could play a part in directing the growth and enhancing the development of the foetus. 36 Figure 1: Oestrogen concentrations of sexual dysfunction female rats following oral administration of aqueous extract of P. amarus leaves.
Data are mean of five replicates ± SEM. Bars with superscripts different from the control across the group for each parameter are significantly different (p < 0.05).
Glycogen, an energy source for general metabolism and constant supply of glucose, has been reported to be directly proportional to the steroid hormones. 37 Therefore the decrease in glycogen levels in the ovaries and uterus of the fluoxetine-treated rats may suggest depleted carbohydrate reserve. 38 The restoration of the concentration of uterine glycogen following the administration of the aqueous extract of P. amarus leaves may be an indication that hormonal secretions were increased since oestrogen levels were enhanced in the present study. Such increased uterine glycogen content may seem to enhance the availability of substrates of energy required for uterine and preembryonic growth through implantation and early pregnancy. 38 Furthermore, the significant reversal of the ovarian glycogen contents by the aqueous extract of P. amarus leaves suggests that the extract enhanced the utilization of available fuel by increasing hormonal secretion and improving ovulation reproductive behaviour. 39 Cholesterol is the precursor in the synthesis of steroid hormone and its requirement for normal ovarian and uterine activity has been well established. [40][41][42] Thus, the significant reduction in the level of ovarian and uterine cholesterol in the fluoxetine-treated animals could be an indication of hindered oestrogenic activity in the present study. However, the significant reversal in the levels of ovarian and uterine cholesterol in all the animals treated with various doses of P. amarus suggests that the extract enhanced the biosynthesis of cholesterol required for the steroidogenesis, which could also be responsible for the elevated levels of oestrogen in the ovaries of the animals in the present study. Ovarian and uterine alkaline phosphatases are involved in mobilizing carbohydrates and lipid metabolites to be utilized either within the cells of the accessory sex structure or by the oocytes. 43,44 The decreased ovarian and uterine alkaline phosphatase in the fluoxetine-treated rats could compromise the transport of necessary materials needed for folliculogenesis. Administration of P. amarus significantly attenuated the fluoxetine-mediated decrease, indicating the capability of the extract to enhance mobilization of necessary materials needed for steroidogenesis. The restorations of the ovarian and uterine ALP levels also suggest that the extract could enhance the development of fertilized eggs and provide a niche for maturation. 43 Acid phosphatase (ACP) is a lysosomal enzyme which contributes to the ovarian and uterine metabolic functions such as oocyte maturation, resumption of mitotic divisions, germinal vesicle breakdown, ovulation and preparation of the uterine lining for implantation. 45 The attenuation of fluoxetine-mediated decrease in acid phosphatase following the administration of the aqueous extract of P. amarus shows the capability of the plant extract to enhance the process of steroidogenesis. 45

Conclusion
In conclusion, this research has revealed that fluoxetine; a SSRIs can be used to induce anti-oestrogenicity in female rats. Besides, the potentiating effect of Phyllanthus amarus leaves on endogenous oestrogen's activity and induction of gonadotrophin synthesis or secretion which restored the alterations in the functioning parameters of the ovaries and the uterus has been established. This may further lend support to the popular use of Phyllanthus amarus leaves in handling sexual inadequacies in folkloric medicine in Nigeria.

Conflict of interest
The authors declare no conflicting interest

Authors' Declaration
The authors hereby declare that the work presented in this article is original and that any liability for claims relating to the content of this article will be borne by them.

Concentrations pg/mL
Distilled water Fluoxetine + distilled water Fluoxetine + 10 mg/kg body weight of Tadalafil Fluoxetine + 20 mg/kg body weight of extract Fluoxetine + 40 mg/kg body weight of extract Fluoxetine + 80 mg/kg body weight of extract