Effects of Ethanolic Extract of Sesamum Indicum Seeds on the Histology and SpermConcentration in Ketoconazole-Induced Testicular Damage in Male Wistar Rats

Aim: The study was aimed at evaluating the ef﻿fects of ethanolic extract of Sesamum indicum seeds on the histology and sperm concentration in ketoconazole induced testicular damage in Wistar rats. Methods: Forty eight (48) adult male Wistar rats were divided into eight (8) Groups of six (6) rats each. Groups 1, 2, 3, 4, 5, 6, 7 and 8 were administered 1 ml of normal saline, 100 mg/kg body weight (bwt) of Ketoconazole only, 500 mg/kg bwt of Ethanolic Extract of Sesamum Indicum (EESI) seeds then100 mg/kg bwt of Ketoconazole,100 mg/kg bwt of Ketoconazole then 250 mg /kg body weight of EESI seeds,100 mg/kg bwt of Ketoconazole then 500 mg/kg bwt of EESI, 100 mg/kg body weight of Ketoconazole then 0.36 mg/kg body weight of Mesterolone, 0.36 mg/kg body weight of Mesterolone, 500 mg/kg body weight of EESI respectively. All administrations were carried out orally once per day. The testes were processed for histopathological and semen analyses. Result: Significant increase in the sperm concentration of rats administered EESI before the administration of ketoconazole and enhancement of the histo-architecture of testicular cells were noted when compared with rats in the control Group and those administered ketoconazole only (p ≤ 0.05). Conclusions: Ethanolic extract of sesamum indicum seeds enhances sperm cell concentration and ameliorates the toxic ef﻿fects of ketoconazole on the testes by improving the cyto-architecture of the treatment group.


Introduction
Toxins such as drugs, chemicals, endocrine disruptors, biogenetically engineered western diets and life style among other factors are known to affect normal body functions and most especially reproductive health [1]. One of such toxins (Drugs) which possess adverse health hazard despite its advantages is Ketoconazole, an antifungal drug used in the treatment of fungal infections such as blastomycosis, histoplasmosis, coccidioidomycosis and para coccidioidomycosis [2].
Ketoconazole is packaged pharmaceutically in the form of tablets, creams and injections and has been reported to inhibit the stimulation of testicular testosterone production by Human Chorionic Gonadotropine in a dose dependent manner [3]. Steroids such as dihydrotestosterone and estradiol are equally, selectively displaced from serum-binding globulin by ketoconazole [4]. The suppression of testicular testosterone synthesis and the displacement of oestrogen from sex hormone binding globulin may decrease the androgen/ and vegetables could be attributed to their phyto chemical constituents such as flavanoids, anthocyanins, Vitamins C and E, phenolic compounds, dietary fibers and carotinoids [9]. One of such vegetable plants is Sesame indicum whos taxonomic order is lamiales, Family is pedialiaceae, Genus is Sesamum and Specie is indicum. Sesame seeds exist in brown, black and yellow forms and are called ridi in Hausa, Ekuku in Yoruba, Isasa in Ibo and go-ri-go in Ebira languages that are ethnic groups in Nigeria.
Sesame seeds oil has been used as healing oil for thousands of years, it naturally possess antibacterial properties for common skin pathogens such as staphylococcus and streptococcus species. It also possesses anti-fungal, antiviral and anti-inflammatory effects [2]. The entire sesame plant is very valuable and serves as staple food in most ethnic groups in North-Central and South-Western part of Nigeria and this could be attributed to the high level of fecundity among adult males in these regions [10].

Materials and Method
Seed procurement and identification 470 g of Sesamum indicum seeds were obtained from Samaru market-Zaria, Kaduna State, Nigeria and authenticated in the herbarium of the Department of Biological Sciences, Ahmadu Bello University Zaria and assigned voucher no. 4. The seeds were macerated using 95% ethanol to obtain the extract with a yield weight of 46.42 g in solvent form.

Methods
Extraction of sesame seed: Four hundred and seventy (470) g of sesame seed was macerated in a maceration apparatus. The sesame seeds were poured into 2 ml maceration apparatus (Pyrex) and the seeds coats were manually removed. The macerated seeds were later transferred into a three and half (3.5) L beaker containing 95%v/v of ethanol to cause the removal of flavanoids and lignans. It was stirred and left for six hours to allow the extraction of polar substances. The supernatant was sieved off using a metallic sieve and the filtrate was collected into a separate beaker. The filtrate was collected into an evaporating dish with the use of a spatula and placed on a water bath, heated to a temperature of 60°C to completely remove the ethanol. The extract was kept for eighteen hours to allow for evaporation to take place. The yield weight of the extract was 46.42g in solvent form. (Figure 1)

Experimental procedure
The weight of the animals was recorded weekly throughout the experimental period and the mean weight of each group was calculated. LD 50 the ethanolic extract of sesamum indicum seeds in wistar rats was done and was found to be above 5000 mg/kg bwt [11].
Group 1 was administered 1 ml of normal saline throughout the experimental period to keep the rats in normal physiologic state while Group 2 was administered 100 mg/kg bwt of ketoconazole in accordance with the method of Amin [2]; Group 3 was the Prophylactic group and was administered 500 mg/kg body weight of ethanolic extract of Sesamum indicum seeds and 100 mg/kg bwt of ketoconazole while Groups 4 and 5 which were the therapeutic low and high dose. These groups were administered 100 mg/kg bwt of Tablet ketoconazole each, and (250/500) mg/kg body weight of ethanolic extract of Sesamum indicum seeds, respectively. Group 6 was administered 100 mg/kg bwt of ketoconazole and 0.36 mg/kg bwt of Mesterolone (testosterone) which was used as a standard drug. Group 7 was administered with 0.36 mg/kg bwt of mesterolone to study effect of mesterolone in the testes of Wistar rats and for comparision. Group 8 was administered with 500 mg/kg body weight of ethanolic extract of sesame seeds ( Table 1).
The equivalent dose of the drugs and the sesame seed extract; in ml (milliliters) were derived using the formulae: The equivalent dose in ml of ethanolic extract of Sesamum indicum seeds was calculated as follows: 500 mg/kg bwt=500 mg=1000 g, the average weights of the rats at the time of commencement of the experiment was 180 g; therefore, the administrable dose (×) for a rat that weighed 180 g was calculated as:

500
/ 0.180 128.3 / mg kg kg Volume mg ml × = 0.70 ml was obtained as the volume for a dose of 500 mg/kg bwt which was the high dose group. Similar method was used to calculate the volume of the low dose group (250 mg/kg bwt) and the value 0.35 ml was obtained. Tablet mesterolone was prepared as 25 mg and it was the recommended dose for an adult person (Average weight=70 kg).
From above, the administrable dose per 1 kg was calculated as shown below. 25 mg (mesterolone) =70; kg=70000 g; therefore, the dose (x) for every 1 kg=1000 g is calculated as follows: Earlier, it was stated that the packaged form of Mesterolone is 25 mg per tablet.
The tablet was dissolved in 10 ml of distilled water to obtain a concentration of 2.5 mg/ml. from above, 2.5 mg is contained in 1 ml, x ml will contain 0.065 mg. By calculation, X =0.065/2.5=0.026 ml, therefore, 0.026 ml which contains 0.065 mg of Mesterolone tablet was measured using a micro pipette and was administered to the rats.

Animal sacrifice
The animals were humanely sacrificed through perfusion technique; the left testis was fixed in Bouin's fluid for routine histological analysis and stained with haematoxylin and eosin (H&E). Periodic Acid Schiff (PAS) and Masson Trichrome (MT) stains. The epididymis of the left testis was removed and immersed in 5 ml of formal saline for the determination of the sperm parameters. Semen analysis was done in accordance with [12] with aid of improved Neubar haemocytometer counting chamber.

Sperm concentration
The results from table 2 showed that the amount of sperm concentration in Groups 3, 4, 5, 6, 7 and 8 significantly increases when compared with group 2 (P ≤ 0.05). Similarly, the amount of sperm concentration in Group 1 significantly increases when compared with that in group 2 (P ≤ 0.05). i.e. the amount of sperm concentration in Group 2 significantly decreases when compared with that in Groups 1, 3,4,5,6,7 & 8 (P ≤ 0.05).

Discussion
From the present study, it was observed that ketoconazole induced testicular damage such as reduction of sperm concentration and severe histopathological lesions. The reduction of sperm concentration was characterized by decrease in the number and concentration of sperm cells in the group administered ketoconazole alone which resulted in decreased sperm concentration. This observation was in line with the findings of Amin [2], who observed that ketoconazole induced testicular damages in male Wistar rats by reducing the sperm indices and serum testosterone level.
The administration of ethanolic extract of Sesamum indicum appeared to have minimized the damages caused by ketoconazole in testes which may be due to its chelating and anti-oxidant properties.
Follicle Stimulating Hormone (FSH) has been shown to have a synergistic effect with testosterone hormone and stimulating synthesis of the androgen receptor at the receptor level [13]. It may then be assumed that the extract of sesame seeds may have caused the FSH to instigate increase in testosterone level which led to the observed increase in the sperm concentration as indicated in the present study.
It has been reported that endocrine disruptors are wide spectrum ion blockers [14]. This also suggests that ketoconazole disrupted the ion channel leading to the disturbance in ionic equilibrium across membrane and ultimately to a disruption or distortion in the cellular morphology. From the present study, it was observed that disruption of the tubular morphology was also accompanied by general destruction of testicular interstitium and spermatogenesis.

Conclusion
The ethanolic extract of sesamum indicum seeds has enhanced sperm cell concentration and ameliorated the toxic effect of ketoconazole on the testes by improving the cyto-architecture of the histology of the treatment groups and the ameliorative effect was most prominent on the prophylactic administration of the extract to the experimental animals.