Effect of Hibiscus sabdariffa calyx extract on reproductive hormones in normal rats

Medicinal plants contain physiologically active principles that over the years had been exploited in traditional medicine for the treatment of various ailments. The present study was undertaken to investigate the effects of ethanolic extract of Hibiscus sabdariffa calyx on rat reproductive hormones. The effects on the basal levels of estradiol, testosterone, prolactin and follicle stimulating hormone were conducted in experimental animals. H. sabdariffa calyx extract at a dose of 250 mg/kg produced minor effects on rat reproductive hormones, namely testosterone and estradiol while no change was observed on both prolactin and follicle stimulating hormone levels. Moreover, no histological changes were detected on both testes and ovaries of the experimental animals after 28 days of administration. It can be concluded that H. sabdariffa calyx extract at a dose of 250 mg/kg caused mild effects on rat reproductive hormones.


INTRODUCTION
Endocrine disrupting compounds (EDCs) are natural or synthetic compounds that have the ability within the body to alter endocrine functions often through mimicking or blocking endogenous hormones (James et al., 2013).These actions on the endocrine system have resulted in developmental deficits in various invertebrate and aquatic species (Crain et al., 2007;Elango et al., 2006) and mammals (Christopher et al., 2012).Exposures in adulthood have consequences but fetal and early life exposures appear to have more severe effects that persist through life (Rubin and Soto, 2009).Among these classes of chemicals are phytoestrogens that show effects suggestive of estrogenicity, such as binding to the estrogen receptors, induction of specific estrogen-responsive gene products, stimulation of estrogen receptor(s) and positive breast cancer cell growth (James et al., 2013).Through these interactions by acting as agonists or antagonists, EDCs are able to alter the activity of response elements of genes, block natural hormones from binding to their receptors, or in some cases increase the perceived amount of endogenous hormone in the body by acting as a hormone mimic to its receptor (Ze-hua et al., 2010).
Hibiscus sabdariffa Linn (Roselle) is an annual shrub commonly used to make jellies, jams and beverages.The brilliant red colour of its calyx makes it a valuable food product, a part from its multitude of traditional medicinal uses.Infusions of the calyces are considered as diuretic, cholerectic, febrifugal and hypotensive, decreasing the viscosity of the blood and stimulating intestinal peristalsis (Salleh et al., 2002).Roselle calyx extract is a good source of antioxidants from its anthocyanins and associated with antitumor and inhibitory effects on the growth of several cancer cells (Ajiboye et al., 2011).
Extracts of H. sabdariffa calyces have been reported to be rich in phytoestrogens (Adigun et al., 2006;Orisakwe et al., 2004;Brian et al., 2009;Omotuyi et al., 2011) and some reports indicated that H. sabdariffa calyces have estrogenic effects, although exact estrogen-like ingredient is not determined (Ali et al., 1989).This study was undertaken to determine to which extent H. sabdariffa calyces extract alters the basal levels of selected reproductive hormones: estradiol, testosterone, prolactin and follicle stimulating hormone as well as the histological features of both testes and ovaries of rats.

Plants
The dried calyces of H. sabdariffa were purchased from the local market in Wad-Medani, Sudan.The plant material was identified by the Department of Pharmacognosy, Faculty of Pharmacy, University of Gezira, Sudan.

Extraction of plant material
One hundred grams of coarsely powdered calyces of H. sabdariffa were extracted by maceration using ethanol (70%) in a conical flask for 72 h, kept away from light throughout the extraction period, then filtered and evaporated by a rotary evaporator at 60°C.The resulting solution was freeze dried and placed into a refrigerator until use.

Experimental animals
The effect of ethanolic extract of H. sabdariffa calyx on rat reproductive hormones was conducted based on the method described by Omotuyi et al. (2011).A total of 20 rats (10 each for males and females) were housed in a clean animal house subjected to an intensive nutritional program.Rats were acclimatized for a period of 14 days under standard environmental conditions.The ethical committees, University of Gezira and Ministry of Health, Gezira State, ethically approved the experimental protocol.

Experimental design
Albino wistar rats were divided into four groups each of five.Water control groups for both males and females and the other two groups (either males or females) received 250 mg/kg of plant extract via gastric tube daily for 28 days.

Collection of blood samples
Blood samples were collected from conjunctival veins using capillary tubes at 7-day intervals for 28 days.

Hormonal assay
The hormones were estimated using the standard protocols of enzyme-linked immunosorbent assay (ELISA) kits (Roche, Switzerland) for determination of estradiol, testosterone, prolactin and follicle stimulating hormone (FSH) levels.

Histopathological examination
Twenty-eight days after oral administration of the extract, all experimental animals were anaesthetized using chloroform vapour and dissected.The ovaries and testes were collected and immediately fixed in Bouins fluid for 6 h and transferred to 70% alcohol for histological processing according to Drunny and Wallington (1990).Briefly, following fixation of the right side testes and ovaries from both control and test animals, tissue sections were processed by dehydration in 95% and absolute alcohol, cleared in xylene and embedded in pure clean moltenparaffin wax from which blocks of tissues were made for sectioning.Ribbon slices of about 5.0 μm in thickness were made with the aid of a microtome (delete machine) and the sections picked with slides, which were dried in oven.The slices were stained with haemotoxylin and eosin, and then mounted using DPX onto a light microscope (delete magnification 40× for testes and 10× for ovary) for histopathological and morphological changes.

Data analysis
All the obtained data were expressed as means ± standard deviation and analyzed using analysis of variance (ANOVA).Comparisons with the control groups were made using One-way ANOVA.Differences were considered significant if P-value < 0.05.

Effect of ethanolic extract of H. sabdariffa calyx on estradiol levels in female rats
The study revealed that the ethanolic extract of H. sabdariffa calyx in a dose of 250 mg/kg exhibited a mild increase (p-value < 0.05) in estradiol level in female rats in time-dependent manner (Table 1).On day 28, estradiol reached more than twice the value observed on day 0 compared to those of water control group.

Effect of ethanolic extract of H. sabdariffa calyx on testosterone levels in male rats
Following intragastric administration of 250 mg/kg of ethanolic H. sabdariffa calyx extract for 28 days, serum levels of testosterone were significantly reduced (P-value  < 0.05) in male rats throughout the experimental period compared to those of water control group (Table 2).

Effect of ethanolic extract of H. sabdariffa calyx on prolactin and FSH levels in female rats
The ethanolic extract of H. sabdariffa calyx in a dose of 250 mg/kg/ml did not cause changes in the serum levels of prolactin or FSH throughout the experimental period in female rats administered the plant extract for 28 days.

Histological effects of H. sabdariffa calyx extract on rat testes and ovaries
The extract did not cause histological changes on both testes and ovaries of the experimental animals when the plant extract was administered for 28 days.
The reduction of serum level of testosterone in male rats produced by ethanolic extract of H. sabdariffa calyx may be explained by the estrogenic activity of the plant, an evidence raised by Orisakwe et al. (2004).Furthermore, other studies had reported a statically significant decrease in testosterone levels in laboratory animals treated with phytoestrogens (Sharpe et al., 2002;Cline et al., 2004).The precise role that oestrogens play in male reproductive development is unclear, but generally, oestrogens tend to have 'demasculinising' or antiandrogenic effects.In foetal and neonatal life, this probably results from suppression of testosterone production (Haavisto et al., 2001), or loss of androgen receptors (McKinnell et al., 2001).Oestrogens are synthesised from androgens via the action of a single enzyme (aromatase), and there is a close relationship between the actions of these two hormones.Moreover, testosterone may be converted to estrogen by aromatase (Benassayag et al., 2002).
Although dietary phytoestrogens have been implicated in adverse effect upon fertility in various animals, there are few published reports of such effects in human populations consuming large amounts of these substances (Benassayag et al., 2002).In male rats, reduction of testosterone level might impair spermatogenesis and cause male infertility (Orisakwe et al., 2004).It should be noted that in the study of herbal extracts, one could not attribute the observed biological effects to a particular constituents because many other compounds are present in the plant extracts (Saied-Karblay et al., 2010).Factors such as species, age, gender, diet, dose, route of administration and metabolism strongly influence the ultimate biological response to phytoestrogen exposure (Shweta, 2009).

Conclusion
H. sabdariffa calyx extract caused mild effects on rat reproductive hormones and due to the pleiotropic effects of phytoestrogens in vivo, a broad panel of in vitro assays covering not only estrogenic action but also other regulating processes has to be used to assess the potential of plant-derived compounds to beneficially or adversely affect human health.

Table 1 .
Serum estradiol levels (pg/ml) in female rats (n=5) after oral administration of ethanolic extract of Hibiscus sabdariffa calyx and water (SD ± SEM).

Table 2 .
Serum testosterone levels (ng/ml) in male rats (n=5) after oral administration of ethanolic extract of Hibiscus sabdariffa calyx and water (SD ± SEM).