Curcuma longa normalized cimetidine‐induced pituitary‐testicular dysfunction: Relevance in nutraceutical therapy

Abstract Background The increasing incidence of chemically induced infertility is both a social threat and a threat to the continuation of life itself. Treatment or management therapy is often expensive. This study investigated the effects of acetone extract of a local plant (Curcuma longa) in a Wistar rat model of cimetidine‐induced pituitary‐testicular dysfunction. Methods Thirty‐five male Wistar rats were divided into 7 groups of 5 rats. After a phytochemical screening of an acetone extract of C. Longa, cimetidine and the extract at three doses, 200, 400 and 600 mg/kg, were orally co‐administered to the rats for 28 consecutive days. Comparisons were made (at P < 0.05) against a control (2 mL/kg distilled water), a standard treatment group (cimetidine + 50 mg/kg vitamin C), a toxic group (60 mg/kg cimetidine) and a group receiving extract alone. Results Cimetidine administration was associated with deleterious alterations to sperm motility, sperm count and sperm viability, as well as derangements in the plasma levels of FSH, LH and testosterone (P < 0.05). Both brain and testicular GSH and TBARS levels were significantly altered following cimetidine administration, and distortions were seen in the pituitary and testicular histoarchitecture. These changes were significantly normalized by co‐administration of graded doses of the extract, with an associated improvement of both pituitary and testicular histology. Conclusion Acetone extract of C. Longa normalized cimetidine‐induced pituitary‐testicular dysfunction in Wistar rats. This presents the extract as a potential nutraceutical choice against chemically induced reproductive toxicity.

body by eliciting hormonal changes; hence they are referred to as the hypothalamic-pituitary-gonadal axis. 6 This axis plays a crucial role in the control of development, reproduction, and aging. 6,7 Some drug regimens are known to cause fluctuations in this axis with consequent deleterious derangement of reproductive functions. [8][9][10] Cimetidine is a globally prescribed potent drug that is commonly used for the treatment of gastric and duodenal ulcers. [11][12][13] It is also readily available without prescription, 13 thereby increasing its chances of being abused. Cimetidine is an H 2 -receptor antagonist that blocks histamine action on H 2 -receptors in the parietal cells of the stomach, consequently inhibiting acid production. [13][14][15] Its administration has been found to be associated with reproductive toxicity and it is thus described as a reproductive toxicant. 8,9,13 Some of the deleterious reproductive effects of cimetidine include distortion of testicular histoarchitecture with marked degeneration of the seminiferous tubules and maturation arrest of spermatogenic cells, 8,9,16 decreases in sperm motility and count, 17,18 as well as impotence, with a resulting decrease in sexual drive and desire. 19 Although the precise mechanism of cimetidine-induced reproductive toxicity is uncertain, in males, cimetidine is known to target the hypothalamic-pituitarytesticular axis with associated structural changes in the histology of the testes. 16 We therefore hypothesized that the normalization of reproductive function in subjects with cimetidine-induced reproductive toxicity may be possible with intervention(s) that provide beneficial effects on both gastrointestinal and reproductive functions.
Even today, traditional medicine is a mainstay of primary health care in underdeveloped and developing countries. [20][21][22][23] Natural and herbal products from some plants are still very relevant in folk medicine for pharmaceutical formulations either as pure compounds or as extracts. 24 Plant-derived medicines are relatively cheap and readily available compared with their synthetic alternatives. They are also an important source of drug discovery and can inspire novel drug development. 21,23 Curcuma longa (Linn.), commonly called turmeric, is a perennial plant belonging to the Zingiberaceae family and is widely cultivated throughout the tropical parts of the world including India, China, Pakistan, Kenya, Ghana and Nigeria, [25][26][27] making it readily accessible and cheap. It has a characteristic yellow colour that is conferred by its curcumin component. 24 Some of the documented health benefits of this plant include anti-inflammatory, 28 antioxidant, 29 anti-carcinogenic, 30 anti-HIV, 31 anti-diabetic, 32 lipid-lowering, 33 anti-obesity, 34 hepato-protective, 33 anti-malarial 35 and immunomodulating 36 effects. The United States Food and Drug Administration (US FDA) classifies turmeric as a nutraceutic that is generally recognized as safe (GRAS). 37,38 According to a glossary produced by the American Diabetics Association, nutraceutics are substances that are considered as food or a part of food that offer medicinal health benefits, which include the prevention and treatment of diseases. 39,40 Despite the favorable ethnopharmacological properties of C. Longa, our literature survey revealed a dearth of information on the effects of its acetone extract on cimetidine-induced pituitarytesticular dysfunction. This study aimed to bridge this gap in our knowledge by assessing the nutraceutical effect of an extract of C. Longa in a Wistar rat model.

| Plants, drugs, chemicals, and biochemical kits
Fresh rhizomes of C. Longa were purchased from a commercial supplier at Sabo market in Ile-Ife and certified by a Taxonomist at the   Department of Botany, Obafemi Awolowo University (OAU), Ile-Ife, where a voucher specimen (IFE-17700) was deposited.
Cimetidine tablets were procured from Shandong Shenglu Pharmaceuticals, China. Vitamin C (analytical standard ascorbic acid) was from Nevada, USA. Acetone was of analytical grade. Standard laboratory hormone assay (biochemical) kits for testosterone, luteinizing hormone and follicle-stimulating hormone for experimental rodents were supplied by Accu-Bind Elisa (Monobind Inc).

| Plant extraction process
The extraction process for obtaining an acetone extract of C. Longa rhizome (AECUL) followed the standard procedure described by Imafidon et al 41

| Phytochemical screening of the extract
Phytochemical screening of the extract was carried out according to standard laboratory protocols. Alkaloids, flavonoids and tannins was detected by the method of Halilu et al, 43 saponin was identified using the froth test as described by Benmedhdi et al, 44 and phenolics were identified as described by Edeoga et al 45 (Table 1).

| Ethics statement
All experimental protocols were in strict compliance with the guidelines for animal research, as detailed in NIH Guidelines for the Care and use of Laboratory Animals 46 and were approved by the local institutional research committee.

| Preparation of stock solutions of the extract
According to existing literature, the oral lethal dose of C. Longa is greater than 5000 mg/kg. 47 Experimental dosage is usually taken to be less than or equal to 10% of the oral LD 50 . 41,48,49 Therefore, the therapeutic doses of AECUL adopted for this study were 200, 400 and 600 mg/kg. In order to avoid deleterious biological effects due to fluid overload, stock solutions of the extract were prepared such that each 100 g rat received 0.02 mL (2 mL/kg).
The stock solutions were prepared by dissolving 2, 4 and 6 g of AECUL in 20 mL of distilled water, to provide the respective 200, 400 and 600 mg/kg doses in 0.02 mL.

| Animal management
Thirty-five male Wistar rats, weighing 150-180 g, were used for this study. These were purchased from the Animal Holdings Unit of the College of Health Sciences, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria, where the study was carried out. The rats were kept in standard plastic cages under a natural light and dark cycle and were allowed access to standard rodent pellets and water ad libitum.

| Experimental design
The rats were divided into seven groups of five rats each and treated as follows. All groups received treatment for 28 consecutive days before they were euthanized. Group 1 received 2 mL/kg of distilled water; group 2 received oral cimetidine at 60 mg/kg; group 3 (standard treatment group) received co-administration of cimetidine (60 mg/kg) and vitamin C (50 mg/kg); groups 4, 5 and 6 received oral co-administration of cimetidine (60 mg/kg) and graded doses of AECUL at 200, 400 and 600 mg/kg, respectively; and group 7 received a single medium oral dose (400 mg/kg) of the extract (Table 2).
After euthanasia, blood samples were collected by cardiac puncture into separate EDTA bottles and centrifuged at 4000 rpm using a cold centrifuge (Centrium Scientific, Model 8881) at −4°C. The plasma obtained was decanted into separate plain bottles. Thereafter, the caudal epididymis of each rat was excised and minced in 2 mL of normal saline and the resulting suspension was used for sperm characterization. The brain and testis of each rat were excised and weighed. The right testis and 1 g of the excised brain where transferred to a cooler for homogenate preparation, while the left testis and pituitary were fixed in 10% formal saline solution for histological examination using hematoxylin and eosin (H&E) staining.

| Determination of percentage weight change, relative brain weight, and relative testicular weight
Assessment of weekly weight change was carried out using a Hanson digital weighing scale (Hanson, China), while organ weights were determined using a Camry sensitive weighing balance (Camry, China).
Thereafter, percentage weight change, relative brain weight and relative testicular weight were calculated using the formulae below 50 :

| Sperm characterization
From the caudal epididymis of each rat, sperm fluid was squeezed onto a microscope slide. Sperm motility was assessed by counting the number of motile spermatozoa per unit area and was expressed as motility per unit area. Sperm counts were made with the aid of a hemocytometer and expressed as millions/ml of suspension. Sperm viability was determined by preparing a uniform smear of spermatozoa on the slides using eosin-nigrosin stain according to the method of Bloom, 51 as described by Raji et al. 52

| Hormone assays
The concentrations of reproductive hormones (follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone) were determined using standard laboratory kits involving the enzyme-linked immunosorbent assay (ELISA) technique, according to the manufacturer's instructions.

| Assessment of oxidative stress and lipid peroxidation indicators
A 10% homogenate of the tissue in phosphate buffer (100 mmol/L; 7.4 pH) was prepared using an electric homogenizer (S1601001).

| Histological examination
The pituitary and testis of the rats were dehydrated in graded alcohol and embedded in paraffin wax. Sections >4 µm thick were stained with hematoxylin-eosin and photomicrographs were taken with a Leica DM750 Camera Micro at ×40 and ×100 magnifications for pituitary and testis, respectively.

| Statistical analysis
The results were expressed as means ± standard error of mean using one-way analysis of variance (ANOVA) followed by a Neuman-Kuels post hoc test. Values at P < 0.05 were considered statistically significant. Data were analyzed using GraphPad Prism 5.03 statistical package (GraphPad Software Inc).

| Effects of acetone extract of Curcuma longa (AECUL) on percentage weight change, relative brain weight and relative testicular weight of Wistar rats with cimetidine-induced pituitary-testicular dysfunction
Cimetidine administration (group 2) was not associated with significant changes in the percentage weight change (%), relative brain weight (%) or relative testicular weight (%) of the rats (P > 0.05). The same was true for the administration of AECUL alone (group 7) compared with the control (P > 0.05) ( Table 3).

| Effects of AECUL on sperm motility, sperm count and sperm viability of Wistar rats with cimetidine-induced pituitary-testicular dysfunction
Sperm motility was significantly lower (%) in group 2 compared with the standard treatment group 3 and the AECUL-treated groups 4, 5 and 6 (P < 0.05). Sperm motility was also significantly reduced in group 3 compared with the control, and was insignificantly lower compared with the AECUL-treated groups 4, 5 and 6. However, there was no significant difference in sperm motility in group 7 compared with the group 1 control (P > 0.05) ( Table 4).
Sperm count (millions/ml) and sperm viability (%) followed the same trend in our study. These parameters were significantly lower in group 2 compared with all other groups (P < 0.05). The standard treatment group 3 had an insignificantly lower sperm count and sperm viability compared with the AECUL-treated groups 5 and 6.
However, these parameters were not significantly different in group 7 compared with group 1 (Table 4).

| Effects of AECUL on plasma levels of FSH, LH and testosterone of Wistar rats with cimetidineinduced pituitary-testicular dysfunction
The plasma level of FSH (mIU/mL) was significantly lower in group were compared with group 1 (0.37 ± 0.01) (P > 0.05) ( Figure 1C).

| Effects of AECUL on oxidative stress and lipid peroxidation indicators in the brain and testis of Wistar rats with cimetidineinduced pituitary-testicular dysfunction
The brain GSH level (µg/mg protein) was significantly lower in group 2 (1.98 ± 0.10) compared with group 1 (2.52 ± 0.12) (P < 0.05).
However, no significant differences were recorded when groups 3,  Figure 2D).

| Histological effects of AECUL on the pituitary and testis of Wistar rats with cimetidineinduced pituitary-testicular dysfunction
Cimetidine administration (group 2) was associated with histoarchitectural distortion of the pituitary interstitium, with micrographic TA B L E 3 Effects of acetone extract of Curcuma longa on percentage weight change, relative brain weight and relative testicular weight of Wistar rats with cimetidine-induced pituitary-testicular dysfunction F I G U R E 1 Effects of acetone extract of Curcuma longa on plasma levels of FSH, LH and testosterone in Wistar rats with cimetidine-induced pituitary-testicular injury. FSH, follicle stimulating hormone; LH, luteinizing hormone; CIM, cimetidine; Vit C, vitamin C; AECUL, acetone extract of C. Longa. Each bar represents mean ± standard error of mean at P < 0.05. * Significant difference compared with group 1 (Control); µ Significant difference compared with group 2 (CIM); β Significant difference compared with group 3 (CIM + Vit. C); δ Significant difference compared with group 4 (CIM + 200 mg/kg AECUL) evidence of sparsely distributed pituitary cells compared with the control and the AECUL-treated groups. The micrographic evidence from group 7 (treated with AECUL alone) showed features of apparently intact pituitary histoarchitecture compared with the control (Figure 3).
The micrographic evidence also showed features of cimetidineassociated distortion of testicular histoarchitecture, characterized by ballooned/abnormally shaped seminiferous tubules and mild vacuolation of the testicular interstitium compared with micrographs from the control and AECUL-treated groups (Figure 4).

| D ISCUSS I ON
Our study demonstrated that cimetidine-induced reproductive toxicity is not accompanied by disturbances in body weight or relative brain and testicular weights of Wistar rats. This supports the findings of Qamar et al, 55 who reported that cimetidine had no effect on testicular and body weights of Wistar rats. However, in our study, cimetidine induced deleterious alterations in the reproductive function of Wistar rats without causing significant changes in their relative testicular weight.
Cimetidine is involved in the control of multiple hormone secretory patterns by blocking the receptors for these hormones, thereby altering hormone profiles. 56,57 Furthermore, it has been reported that cimetidine penetrates the blood-brain barrier 58 to inhibit the synthesis of reproductive hormones. 59 The micrographic evidence in

ACK N OWLED G EM ENTS
The authors wish to express their profound gratitude to members of staff of Department of Physiological Sciences, Obafemi Awolowo University (OAU, Ile-Ife, Osun State, Nigeria), as well as to Professor Obuotor's Laboratory, Department of Biochemistry and Molecular Biology, OAU, for their kind support and technical assistance.

CO N FLI C T O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
AAO initiated the research idea and supervised the study. ONJ and ICE conceptualized the study. NJO was responsible for fund acquisition.
ICE and ONJ performed the data analyses. All authors participated in the acquisition and interpretation of data. All authors were involved in drafting, revising and proof-reading the article for intellectual content.