Spermatogenic and Haematological effects of Aqueous and Ethanolic Extracts of Hymenocardia acida Stem Bark on Aluminium-Induced Toxicity in Male Wistar Rats

This study was designed to determine the spermatogenic and haematological effects of aqueous and ethanolic stem bark extracts of Hymenocardia acida on Aluminium chloride-induced toxicity in male wistar rats. Thirty male albino Wistar rats were used and toxicity was induced using 100 mg/kg body weight (bw) AlCl3. The rats were distributed into six groups of five rats each. The drug and extract administration lasted for seven days, thereafter the animals sacrificed. The results indicated that oral dose of 100 mg/kg bw extracts of H. acida did not show any significant (p>0.05) change in testosterone levels when compared with both normal and negative control. Ethanolic extract showed non-significant (p>0.05) increase on Luteinizing hormone (LH) when compared with both normal and negative control group. Administration of AlCl3 caused a significant (p<0.05) decrease in Follicle Stimulating Hormone (FSH), while the plants extracts caused a non-significant increase when compared with the negative control. Thiobarbituric Acid Reactive Substance (TBARS) increased significantly (p<0.05) in negative control, but decreased significantly in groups treated with aqueous and ethanolic extract of H. acida when compared with the negative control. Induction of AlCl3toxicity caused a non-significant (p>0.05) alterations of most of the haematological parameters evaluated, but administration of the plant extracts was able to mildly ameliorate these effects (especially on RBC and Hb). The result of this study shows AlCl3 toxicity causes alterations of LH, FSH and TBARS, but aqueous and ethanolic stem bark extracts of H. acida may possibly ameliorate the alteration. Also, AlCl3toxicity mildly influences the synthesis of certain haematological indices, but the extracts may modulate some of this interference.


Introduction
In Nigeria, the folkloric uses of plant preparations for reproduction related purposes are well documented [1]. Herbal contraceptives are used because of affordability, ease of availability from local sources and less side effects [2]. Large number of plants used as abortifacient, contraceptive or sterility agents prevent implantation [3], suppress spermatogenesis [4], or are spermicidal [5].
Medicinal plants are used in Folkoric medicine in treatment of tumor ailment. Information obtained from traditional medicinal practioners in the South-Western Nigeria revealed that drinking decoctions of Paquetina nigrescens leaf, Moringa oleifera, Bradystegia eurocoma and Struchium sparganophora could significantly arrest the progression of tumor proliferation in patient [6].

Insights in Biomedicine ISSN 2572-5610
Hymenocarda acida (euphorbiaceae) is a small brows tree or shrub with palatable foliage, widely distributed within the savannah region of Nigeria. The antimicrobial activities of H. acida leaves and roots have been reported [7]. There are literatures which have reported it to be anti-sickling [8], anti-ulcer [9], antidiarrhoea [10], and to possess anti-inflammatory activities [11]. Another study has investigated the antioxidant activity of the aqueous and methanolic extracts of H. acida. [12]. Phytochemical studies of H. acida showed the presence of saponins, tannins [13] flavonoids, flavonols, phenols, proanthocyanidins, steroids and triterpenoids. This study is designed to evaluate the spermatogenic and haematological effects of aqueous and ethanolic stem bark extracts of this Hymenocardia acida on Aluminium chlorideinduced toxicity in male wistar rats. The result obtained will help to know the possible effects of the plant extracts in human.

Experimental animals
Thirty healthy male albino wistar rats of weight 100-150 g were obtained from Animal house, Federal University Wukari, Taraba State. The animals were kept in clean cages (plastic bottom and wire mesh top) under room temperature, with 12 hours light and 12 hours dark cycle and were allowed to acclimatize for one week before the experiment. The animals were allowed free access to standard rat chow and water ad libitum.

Plant specimen
The stem bark of H. acida were collected within the premises of Federal University Wukari Taraba State. They were identified and authenticated in the Department of Biological Sciences, Federal University Wukari. The stem bark was rinsed, air dried at room temperature and pulverized using pestle and mortar. Water was used for the aqueous extract and stored in refrigerator, while exactly 270 g of the sample was weighed and dissolved in 1350 ml of ethanol (for ethanolic extraction) for 48 hrs after which it was filtered: first using a clean white sieving mesh and further with Whatman No. 1 filter paper. The ethanolic filtrate was concentrated using a thermostat water cabinet at 40°C for 7 days. The concentrated extracts were then transferred to air-tight containers, corked and preserved in the refrigerator at 4°C until required for the experiment.

Preparation of aluminium chloride
Exactly 2g of Aluminium Chloride salt was weighed and dissolved in 5 ml of distilled water. The solution was prepared daily. The Aluminium Chloride solution was administered 100 mg/kg bw of the rats.

Experimental design
The rats were distributed into six groups of five (5) rats each: Group 1 served as normal control: received only normal feed and water daily. Group 2 served as negative control (received only Alumimium Chloride: 100 mg/kg bw daily). Group 3 was administered Aluminium chloride + aqueous extract of Hymenocardia acida stem bark: The rats were administered with 100 mg/kg bw of the extracts one hour after administration of 100 mg/kg bw of AlCl 3 . Group 4 was administered Aluminium chloride + ethanolic extract of Hymenocardia acida stem bark: The rats were administered with 100 mg/kg bw of the extracts one hour after administration of 100 mg/kg bw of AlCl 3 Group 5: Received normal feed + aqueous extract of Hymenocardia acida stem bark bark daily, while group 6 received normal feed + ethanolic extract of Hymenocardia acida stem bark daily.

Animal sacrifice and sample collection
After the experiment, the animals were sacrificed under chloroform anesthesia. Blood samples were collected by cardiac puncture and the liver was harvested, weighed and rinsed in ice cold normal saline. The blood samples were collected in to EDTA collection tubes (for haematological analysis) and plain tubes for serum. The liver was homogenized using a standard laboratory mortar and pestle. The homogenate was centrifuged and the supernatant collected for Thiobabituric Acid Reactive Substances (TBARS) analysis.

Determination of Thio-barbituric Acid Reactive Substances (TBARS)
Thiobabituric Acid Reactive Substance formation was measured by using 10% trichloroacetic acid (TCA) solution and 0.6% of thiobabituric acid (TBA) solution. Exactly 50 µL of tissue homogenate was added to a test tube containing 2 ml of TCA, followed by 2 ml of TBA and incubated at 80 o C for 30 mins in a water bath. It was cooled immediately under ice and centrifuged at 3500 rpm for 10 mins. The absorbance of the solution was read at 535nm against a blank solution containing all except the sample. The TBARS was calculated using a molar extinction coefficient of 1.56 × 105 M -1 cm -1 .

Hormonal assay
Luteinizing hormone (LH), Follicle stimulating hormone (FSH) and Testosterone were estimated using the method of Enzyme Linked Immunosorbent assay (ELISA)

Ethics
All animals received humane care according to the criteria outlined in the Institution's Guide for the Care and Use of Laboratory Animals.

Thiobarbituric Acid Reactive Substance (TBARS)
Results in Table 1 show the level of Thiobarbituric acid reactive substance in liver of the rats. The result show a significant (p<0.05) increase in animals of negative control group when compared with normal control. There was also a significant change in group administered normal rats feed + aqueous extract and ethanolic extract of H. acida when compared with normal control. There was a significant (p<0.05) reduction of TBARS level in animals Insights in Biomedicine ISSN 2572-5610 treated with AlCl 3 +aqueous and AlCl 3 +ethanolic extract of H. acida when compared with the negative control. Table 2 shows the result of hormonal assay in the male albino rats. The result shows that there was a significant (p<0.05) decrease in luteinizing hormone (LH) and follicle stimulating hormone (FSH) in the negative control animals (intoxicated with aluminium chloride only) when compared with normal control group. The LH non-significant (p>0.05) increased in normal rats administered the aqueous and ethanolic extract of H. acida. There was no alteration in testosterone level of all groups when compared with normal control and negative control. There was a non-significant (p>0.05) decrease in follicle stimulating hormone in animals treated AlCl 3 +aqueous and AlCl 3 +ethanolic extract, and also a non-significant (p>0.05) decrease in luteinizing hormone in animals treated AlCl 3 +ethanolic extract: when compared with the negative control. Table 3 shows the result of the haematological parameters evaluated. There was a non-significant (p>0.05) alterations of all the parameters in the negative control, when compared with the normal control. White blood cell (WBC) increased, while there was a decrease in haemoglobin (Hb), red blood cell (RBC) and platelet levels of rats induced with AlCl 3 . The administration of the plant extracts moderated most of these alteration, although some are not statistically significant.

Discussion
Aluminium chloride has been shown to affect spermatogenesis in experimental animals and humans which can lead to low sperm count, abnormal sperm morphology and poor semen quality [14]. Hymenocardia acida on the other hand has an antioxidant activity [12] and pro-oxidant activity [15]. The result of this study show that aluminum chloride did not cause significant (p<0.05) difference in testosterone levels of the rats. This may be as a result of antioxidant capacity of Hymenocardia acida extract on Aluminum chloride-induced toxicity or may be that the plant extracts do not possess the ability to interfere in the processes involved in the production of testosterone.
The decrease in Luteinizing hormone of the negative control group when compared with normal control is in accordance with the previous studies [16] which reported that aluminium chloride contents in humans and animal testes, spermatozoa, blood and urine were associated with impaired sperm quality, viability and alternation in histology of testes, alternation in spermatogenesis, enhancement of free radicals and alternation in antioxidant enzymes. The decrease in LH that occurred in the group induced with aluminium chloride (negative control) may be due to oxidative damage to histo-archicture of hypothalamus caused by AlCl 3 toxicity. There was an increase in the level of luteinizing hormone in animals treated with aqueous and ethanolic extracts of H. acida when compared with the negative control. The shows that the plant extracts could encourage the process involved in the synthesis of luteinizing hormone.
There was significant (p<0.05) decrease in follicle stimulating hormone (FSH) in animals induced with AlCl 3 -toxicity (negative control) when compared with normal control. This may be due to interaction of AlCl 3 with the anterior pituitary gland thereby resulting in oxidative damage to architecture of the anterior pituitary gland responsible for the production of follicle stimulating hormone (FSH) (glycoprotein hormone) and Luteinizing hormone. The increase in the groups treated with the aqueous and ethanolic extracts of the plant show that its chemical content may promote the effect of pituitary gland and the production of luteinizing hormone.
The result also show that AlCl 3 -toxicity caused significant (p<0.05) increase in the TBARS levels in liver of albino wistar rats (negative control). The increase of TBARS concentration in the liver can be a consequence of critical accumulation of AlCl 3 , thereby causing hepatic lipid peroxidation. This result is in agreement with previous studies [17,18] that reported significant increase in TBARS levels of AlCl 3 treated rats. However, treatment with the plant extracts show that the plant possesses the ability to ameliorate the negative effects caused by AlCl 3 -toxicity. This suggests that H. acida possess antioxidant properties.
Red blood cells (RBC) and haemoglobin (Hb) are important in transporting respiratory gases. Administration of AlCl 3 mildly altered some of the haematological parameters analyzed. This shows that AlCl 3 -toxicity could cause reduction of Hb and red blood cell count, and therefore could possibly induce anaemia. The effect of the stem bark extracts of H. acida was able to moderate some of the haematological parameters altered by AlCl 3 . Platelets play important role in the process of haemostasis and its reduction might adversely affect thrombopoietin. The modulation of the platelet count following oral administration of H. acida stem bark extract indicates that the extract may not cause any coagulation problem, but has the potential to enhance clotting and prevent haemorrhages.

Conclusion
This result of this study provides significant evidence for the oxidative damage caused by AlCl 3 in Wistar rats using testosterone, follicle stimulating hormone (FSH), luteinizing hormone, TBARS and some haematological parameters (WBC, RBC, PLT, PCV and Hb) as biomarkers. It is therefore, concluded that AlCl 3 toxicity causes alterations of LH, FSH and TBARS, but aqueous and ethanolic stem bark extracts of H. acida may possibly ameliorate the alteration. Also, AlCl 3 -toxicity mildly influences the synthesis of certain 0.11 ± 0.02 b, c N. + H.A Eth 0.13 ± 0.02 c Note: Each value represents the mean of 5 rats ± SD Mean in the same column, having different letters of the alphabet are statistically significant (p<0.05). Table 1 Effect of aqueous and ethanolic extract of H. acida on TBARS levels in liver of Aluminum chloride treated male rats.