Protective Influence of Ficus asperifolia Miq Leaf Extract on Carbon tetrachloride ( CCL 4 )-Induced Testicular Toxicity in rat ’ s Testes

Article history: Received on: 16/10/2015 Revised on: 13/12/2015 Accepted on: 09/02/2016 Available online: 28/06/2016 The aim of this study was to investigate the ability of the polyphenolic rich Ficus asperifolia (Miq.) leaf extract in protecting rat testes against carbon tetrachloride-induced testicular damage in male Wistar rats. Thirty rats (weighing 140 180 g) were divided into five groups. In each treatment groups, aqueous extract of F. asperifolia (100, 200 and 400 mg/kg bw) administered by oral gavage for 21 days before exposure to carbon tetrachloride (CCl4) 3 mL kg -1 i.p. were used to test protective influence of the plant extract. Protective influence were observed on antioxidant marker enzymes such as reduced glutathione (GSH) levels, catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), and malonaldehyde (MDA) and histological examination. Animal exposure to the CCl4 resulted in significant elevation in the MDA with concomitant depletion (p < 0.05) in the level of GPx, CAT and SOD activities compared with control. Daily oral administration of F. asperifolia showed beneficial and ameliorative effects in all biochemical parameter evaluated. Histopathological alteration in testes was observed in CCl4 untreated rats and was ameliorated inCCl4 rats treated with F. asperifolia. Result shows that the aqueous leaf extract of F. Asperifolia has ameliorative effect against carbontetrachloride-induced testicular toxicity.


INTRODUCTION
Infertility is a major clinical problem, affecting majority of the people medically and psychosocially (Rowe, 2006).In recent years, oxidative stress has been implicated in the progression of male infertility.Evidence has shown that these damaging events are caused by free radicals (Halliwell and Gutteridge, 1993).Oxidative stress results from either a decrease of natural cell antioxidant capacity or an increased amount of reactive oxygen species (ROS) in organisms.Carbon tetrachloride (CCl 4 ), is a clear, colourless, volatile, heavy and non-flammable industrial liquid, widely used to induce free radical toxicity in various tissues of experimental animals such as liver, kidneys, heart, lung, testis, brain and blood (Khan et al., 2010).CCl 4 is converted through hepatic microsomal cytochrome P 450 into tri-chloromethyl-free radical (•CCl 3 or •CCl 3 OO) (Preethi et al., 2009) which in turn, initiate lipid peroxidation process (Adewole et al., 2007;Adewole et al., 2012).The most widely accepted mechanism of CCl 4 is the formation of free radicals which is a rate limiting process in tissue peroxidative damage (Sahreen et al., 2011;Khan et al., 2011).
Maintaining the balance between reactive oxygen species and natural antioxidants could serve as a major mechanism in preventing damage by oxidative stress induced by toxic agents.Antioxidant defense systems that protect the body from free radical damage include the antioxidant nutrients and enzymes (Sreelatha et al., 2009).Antioxidant and radical scavengers have been used to study the mechanism of CCl 4 toxicity as well as to protect tissue cells from CCl 4 induced damage by breaking the chain of lipid peroxidation (Weber et al., 2003).
Ficus asperifolia Miq belonging to the Moraceae family is a small or average size tree, terrestrial or epiphyte which can reach 20 m in height.It is found in Nigeria, Senegal, Uganda, Tanzania, Natal (South Africa), Madagascar and Cameroon.Ficus asperifolia is abundant in the savannah regions, especially along river banks and marshy areas at an altitude of up to 1100 m.The leaves are enormous and displayed spirally, the limb is largely oval or has a form of ellipse and the roots are most often fibrous (Adjanohoun et al., 1996).In many traditional medicines of Africa, the leaf extract of Ficus asperifolia is used as an antihelmintic and a purgative (Soforowa, 1996).Ficus asperifolia has been shown to possess many pharmacological and physiological activities such as antioxidants (Ojo and Akintayo, 2014).In view of the fact that CCl 4 has been shown to induce testicular toxicity, the effect being through oxidative stress and the antioxidant properties of phytochemical constituents of Ficus asperifolia influence oxidative stress in tissue of rats, we therefore aimed at investigating the protective efficacy of Ficus asperifolia on CCl 4 induced testicular derangement in rat.

Plant material
Fresh sample leaves of Ficus asperifolia was procured from local suppliers in Ado-Ekiti (Ekiti State) and authenticated at the Department of Plant Sciences, Ekiti State University, Nigeria.A voucher specimen (U.A.H.E 23) has been deposited in the herbarium of the Plant Science Department, Ekiti State University.The fresh leaves were air dried and finely powdered with an electric grinder.

Extraction
The plant leaves was air dried in the laboratory at ambient temperature (30±2°C) for 10 days, pulverized using a laboratory mechanical grinder (Christy and Norris limited, machine type 8) and the fine powders obtained stored until further use.50 g of the powdered sample was extracted with distilled water of 500 ml for 48 hours.The mixture was decanted and filtered using sterile whatman paper No 1.The filtrate measured up to 425 ml and evaporated to dryness using a freeze dryer to obtain 12% yield.

Experimental animals
Thirty Male Wistar albino rats (140-180 g) were maintained in the Laboratory Animal Unit of the College of Sciences, AfeBabalola University.They were housed in metallic cages and fed with standard chow diet and water ad libitum.The animals were exposed to alternate cycle of 12 hours of darkness and light.Male rats were used because of their constant metabolism compared to the variation in the female physiology.Animals were allowed to adapt to the laboratory environment for one week before experimentation.The care and handling of the animals were in accordance with the internationally accepted standard guidelines and were approved by AfeBabalola institutional review board with ethical approval number (ABUAD/ACA/324).

Doses
The dose selection for the aqueous extract of F. asperifolia was based on the acute toxicity study, which did not show any adverse effect following oral administration of doses up to 4000 mg/kg.Accordingly, with a slight modification by (Ojo et al., 2014) experimental oral doses of 100, 200 and 400 mg/kg that equal to one-fortieth, one-twentieth and one-tenth of the maximum possible dose of the extract that did not cause mortalities in rats were selected.

Experimental induction of Testes damage
CCl 4 was dissolved in groundnut oil in the ratio 1:1 v/v.Testes damage was induced in rats following subcutaneous (SC) injection of CCl 4 in the lower abdomen at a dose of 3 mL/kg (Theophile et al., 2006).

Biochemical parameters
Thirty male Wistar albino rats were randomly divided into five groups of six animals, each.Rats of the 1 st group (normal control) and 2 nd group (CCl 4 -intoxicated control) groups received 3 mL/kgCCl 4 .The 3 th , 4 th and 5 th groups were treated with the aqueous extract of F. asperifolia in doses of 100, 200 and 400 mg/kg, respectively.All medications were administered orally through an oral gavage for 7 consecutive days.Two hours after the last dose, normal control rats were given a single dose of groundnut oil (3 mL/kg, SC), while animals of the 2 nd to 5 th groups received a single dose of CCl 4 (3 mL/kg, SC).After 24 h of CCl 4 injections, blood sample from each rat (2 mL) was withdrawn by puncturing their retro-orbital plexus of veins and collected in previously labeled centrifuging tubes and allowed to clot for 30 min at room temperature.Serum was separated by centrifugation at 10,000 rpm for 5 min.Testes were dissected out and divided into two parts.One part was kept in liquid nitrogen for determination of antioxidant status and the other part was immediately fixed in buffered formalin 10% and was used for histopathological examination.

Assessment of CCl 4 mediated oxidative stress
Protein contents of the samples were tested by the method of Lowry et al. (1951) using bovine serum albumin as the standard.The lipid peroxidation level was tested by the reaction between 2-thiobarbituric acid (TBA) and malondialdehyde (MDA), a product of lipid peroxides as described by Buege and Aust (1978).The tissue SOD was measured by the nitro blue tetrazolium (NBT) decrease method of McCord and Fridovich (1969) CAT was tested spectrophotometrically by measuring the rate of decomposition of hydrogen peroxide at 240 nm as described by (Aebi, 1974).Reduced GSH level was measured by the method of Beutler et al. (1963) This method is on developing a stable (yellow) color when 5',5'-dithiobis-(2-nitrobenzoic acid) (Ellman's reagent) mix to sulfhydryl compounds.The chromophoric product resulting from Ellman's reagent with the reduced GSH (2-nitro-5-thiobenzoic acid) holds a molar absorption at 412 nm, which is part to the reduced GSH in the test sample.The GPx was tested by the method of Rotruck et al. (1973) When this substance is mixed with reduced GSH, its absorption shifts to a longer wavelength (340 nm), and increase at this wavelength provides a direct measurement of the enzymatic reaction.

Histopathology of tissues
The testes from the control and experimental groups were fixed with 10% formalin and embedded in paraffin wax and cut into longitudinal section of 5 μm thickness.The sections were stained with hemotoxylin and eosin dye for histopathological observation.

Statistical analysis
All the data are expressed as mean ± SEM.The significance of difference in means between the control and treated animals was determined by one-way analysis of variance (ANOVA) followed by the Duncan multiple range test for analysis of biochemical data using SPSS (20.0).Values were considered statistically significant at P < 0.05.

Acute Oral Toxicity Studies
All rats treated with different doses (1000-4000 mg/kg) of F. asperifolia extract survived during the 48 h of observation.The animals did not show visible signs of acute toxicity (Table 1).

Testicular Oxidative stress
As shown in table 2, the activities of testicular enzyme glutathioneperoxidase (GPx) in CCl 4 treated groups decreased significantly (p <0.05) when compared to the control treated groups.Following pre-administration with F. asperifolia, the activity of GPx increased significantly (p <0.05) in the CCl 4 + F. asperifolia treated groups compared to the CCl 4 treated groups but as shown in table 2, the changes in the activities ofGPx in the CCl 4 + F. asperifoliatreated groups was not significant when compared to the control treated groups.The testicular content of Malondialdehyde (MDA) in theCCl 4 treated groups was significantly elevated (p <0.05) when compared to the control treated groups.
There was a significant (p <0.05) decrease in the MDA level of the CCl 4 + F. asperifoliatreated groups when compared to CCl 4 treated groups.However, the changes were not significant when compared to the control treated groups (Table 2).The oxidative stress caused by CCl 4 in the testes was assessed by measuring the activity of antioxidant defense enzymes (SOD, GSH and CAT), GSH (Table 3).Results presented in Table 3 showed that subcutaneous injection of CCl 4 -induced significant reduction in the activities of SOD and CAT enzymes with a decreased level of GSH content as compared to the normal control group.On the other hand, it increased the MDA level in testes (Table 2).Pre-administration of aqueous extract F. asperifolia (100, 200 and 400 mg/kg) reduced the severity of CCl 4 toxicity, as evident from the non-significant differences observed in the oxidative stress indicators and antioxidant enzyme levels in these groups.Histopathological examination revealed severe necrosis, loss of villi and haemorrhagic lesions in CCl 4 treated group (Figure -1).

DISCUSSION
Reproductive toxicity from heavy and non-flammable industrial liquid in males is one of the areas of concern in toxicology today and our result in this present study showed that exposure to carbon tetrachloride (CCl 4 ) (3 mL kg -1 CCl 4 orally for 21 days) in rats caused stern testicular toxicity resulting in the obstruction of spermatogenesis and steroidogenesis, evidenced by the increase in the activities of GPx, GSH, CAT and testicular content of MDA of the CCl 4 treated group.Carbon tetrachloride toxicity is very well reported in literature and one of the main mechanisms behind this toxicity has been ascribed to oxidative stress (Sahreen et al., 2011;Khan et al., 2011).
Evaluation of lipid peroxidative activities of antioxidative enzymes such as SOD, CAT, GSH and GPx in tissues has always been used as biomarkers for tissue damage (Stohs and Bagchi, 1995).Hence, increase in the activities of CAT, SOD, GSH and GPx and testicular level of MDA from this study proved that CCl 4 caused oxidative and testicular injury.Pretreatment with aqueous extracts of F. asperifolia leaf extract significantly (p < 0.05) ameliorated the activities of tissue parameters and CAT, SOD and GSH with significant (p < 0.05) increase levels when compared to CCl 4 treated group (Table 3).Furthermore, the levels of LPO were significantly (p<0.05)increased in CCl 4 treated group when compared with normal group (Table 2).Treatment with F. asperifolia significantly (p<0.05)decreased the levels as compared to CCl 4 treated group suggesting that F. asperifolia which has been shown to contain flavonoid and other effective antioxidants (Ojo and Akintayo, 2014) resulted in a notable amelioration of the unbalanced sperm parameters of the testis as seen in the CCl 4 + F. asperifolia treated group.
Ojo and Akintayo (2014) reported that F. asperifolia contain fundamental antioxidant and phenolic compounds that helps in protecting the test is against spermatogenic and oxidative changes brought about by toxic materials and certain antineoplastic agents.
Knowing that toxic actions of CCl 4 are oxidative in nature, which has been shown by (Sahreen et al., 2011); it is indicative that F. Asperifolia was able to attenuate the toxicity ofCCl 4 due to its antioxidative potential.
These have shown the antioxidative properties of F. asperifolia and its ability to elevate a variety of antioxidant enzymes and testicular biomarkers.This could provide a rationalization for the findings in this study why the experimental group treated withCCl 4 + F. asperifolia showed a decrease in activities of GSH, CAT, GPx and testicular MDA.In this reverence, our results showed an increase in the antioxidant enzyme system levels; which implies that F. asperifolia treatment can favor reproductive potentials.
Histological examination of the testes revealed that CCl 4 treatment causes the seminiferous tubules to be closely packed with wider lumen and sparsely populated Leydig cells; however treatment with F. asperifolia showed noticeable improvement in the histological changes induced by CCl 4 .These histological changes seen in the testes of animals exposed to CCl 4 are in agreement with Khan and Ahmed (Khan and Ahmed, 2009) who reported alterations in the seminiferous tubules and reduction in spermatogenic cells in rats following F. asperifolia administration.

Fig. 1 :
Fig. 1: Histology of Testes tissues.(a) Testes section of normal control rat showing cross section of seminiferous tubules (ST), wit h stratified epithelium consisting of spermatogenic cells (SG), moderated lumen (L) and Leydig cells (LC), (b) Testes section of CCl4-treated rats showing closely packed seminiferous tubules, with a wider lumen and the Leydig cells are sparsely separated, degenerated intestinal tissues (c) Testes section of rats treated with CCl4 and 100 mg/kg of F. asperifolia showing seminiferous tubules with closing lumen, (d) Testes section of rats treated with CCl4 and 200 mg/kg of F. asperifolia showing seminiferous tubules with closing lumen and spermatogenic cells, (e) Testes section of rats treated with CCl4 and 400 mg/kg of F. asperifolia showing seminiferous tubules with closing lumen, increase in spermatogenic cells and less sparsely Leydig cells.

Table 1 :
Acute toxicity studies (LD50) of the aqueous extract of Ficus asperifolia.

Table 3 :
Changes in the levels of testicular antioxidant enzymes in CCl4induced rats treated with aqueous extract of Ficus asperifolia.