Effects of Ethanolic Seed Extract of Dacryodes Edulis on the of Paraquat Induced on Testicular Toxicity in Male Adult Wistar Rats

INTRODUCTION Dacryodes edulis (African pear tree) is a tropical oleiferous fruit tree that possesses enormous potential in Africa (Kengué, 1990). It is commonly known as Ube by the Igbos, Mzembe by the Tivs of Nigeria(Burkill, 1985). Various parts of the plantare used in traditional medicine to treat several diseases in different areas (Okafor, 1983; Duru et al, 2012). The fruits are edible, and the bark, leaves, stem, and roots are employed for a variety of purposes (Neuwinger 2000; Jirovetz et al., 2003,and Waruhiu et al., 2004). The bark resin is used in Nigeria to treat parasitic skin disease and jiggers (Hutchinson, 1963). Seeds of Dacryodes edulis are chewed by the Tiv people of Nigeria as a remedy for stomach problems like diarrhoea, dysentery etc (Ajibesin, 2008), the wood serves for firewood and carpentry (Ndoye et al., 1997), while the entire tree is used in agroforestry systems for soil conservation, fertility, shade and apiculture (Ndangang, 1989). Dacryodes edulis fruit or safou is popular in the diets of many Africans. It can be eaten raw, roasted or boiled in hot water, and is eaten alone or used in garnishing cooked or roasted maize. It could also be used as spread to eat bread (Duru et al., 2012). Dacryodes edulis has a potential to improve nutrition andfood security (Ayuku et al., 2000). Paraquat (1, 1′-dimethyl-4, 4′-bipyridilium dichloride PQ), is one of the most widely used herbicides and holds a large share of the global herbicide market till today, it is a nonABSTRACT ARTICLE DETAILS


INTRODUCTION
Dacryodes edulis (African pear tree) is a tropical oleiferous fruit tree that possesses enormous potential in Africa (Kengué, 1990). It is commonly known as Ube by the Igbos, Mzembe by the Tivs of Nigeria (Burkill, 1985). Various parts of the plantare used in traditional medicine to treat several diseases in different areas (Okafor, 1983;Duru et al, 2012). The fruits are edible, and the bark, leaves, stem, and roots are employed for a variety of purposes (Neuwinger 2000;Jirovetz et al., 2003,and Waruhiu et al., 2004. The bark resin is used in Nigeria to treat parasitic skin disease and jiggers (Hutchinson, 1963). Seeds of Dacryodes edulis are chewed by the Tiv people of Nigeria as a remedy for stomach problems like diarrhoea, dysentery etc (Ajibesin, 2008), the wood serves for firewood and carpentry (Ndoye et al., 1997), while the entire tree is used in agroforestry systems for soil conservation, fertility, shade and apiculture (Ndangang, 1989). Dacryodes edulis fruit or safou is popular in the diets of many Africans. It can be eaten raw, roasted or boiled in hot water, and is eaten alone or used in garnishing cooked or roasted maize. It could also be used as spread to eat bread (Duru et al., 2012). Dacryodes edulis has a potential to improve nutrition andfood security (Ayuku et al., 2000). Paraquat (1, 1′-dimethyl-4, 4′-bipyridilium dichloride -PQ), is one of the most widely used herbicides and holds a large share of the global herbicide market till today, it is a non-selective quaternary nitrogen herbicide, commonly used as a desiccant and defoliant in a variety of crops all around the world (Dasta, 1978;Bismuth et al., 1982, Bismuth et al., 1990Raghu et al., 2013).Paraquat is also known as methyl viologen because of its dark blue-green colour (Dinis-Oliveira et al.,2008).It has been considered as a toxic compound over the past 60 years, which is why it is classified as a moderately hazardous herbicide and placed in class II poison for acute toxicity (WHO, 2009). Paraquat was found to be highly toxic towards animals and humans with fatalities being reported by Kelly et al., 1978 andFlorkowski et al., 1992. The main risks are due to deliberate dose dependent ingestion resulting in multiple organ failure and death (Florkowski et al., 1992). Other routes of toxic exposure are inhalation, ocular and skin contacts (Bataller et al., 2000;Baharuddin et al., 2011). Toxicity resulting from skin exposure is more common in concentrated forms and causes irritation while prolonged contact leads to severe systemic toxicity or even death (Bataller et al., 2000;Marrs and Adjei, 2003). Paraquat mainly affects the lungs, where it accumulates at up to 6-10 times the plasma concentration, sequestered in pulmonary type I, type II and Clara cells (Krieger and Krieger, 2001;Cope et al., 2004;Shuler et al, 2004;Dinis-Oliveira et al., 2008,). Oxygen-free radicals are formed resulting in acute alveolitis 1-3 days' post-exposure. Tachypnoea, dyspnoea and cyanosis begin from 2 to 7 days' post-exposure. If the affected animal or human survives, diffuse alveolar septal fibrosis and compensatory type II pneumocyte hyperplasia develop followed by pulmonary fibrosis (chronic phase). Refractory hypoxaemia and eventual death occur from 5 days to several weeks later (Gfeller and Messonnier, 1998;Cope et al., 2004;Dinis-Oliveira et al., 2008;Gawarammana and Buckley, 2011).

MATERIALS AND METHOD
This study was conducted in the Department of Anatomy, Faculty of Basic Medical Sciences, College of Health Sciences, Nnamdi Azikiwe University, Nnewi Campus, Anambra State. The animals were acclimatized for two weeks and the actual experimental protocol lasted for 4 weeks.
Fifty-Four (54) Male Albino Wistar Rats weighing between 130-180g (22 for LD50 determination and 32 for the experiment proper), were purchased from Animal House, University of Nigeria, Nsukka, and housed in Nnamdi Azikiwe University Animal Farm, Nnewi, Nnewi North Local Government Area, Anambra state. Paraquat in the form of Paraquat dichloride was be purchased from Agro-allied division of new market Owerri, Imo State 2kg of Dacryodes edulis seed was purchased from Nkwo market at Nnewi, Nnewi North L.G.A of Anambra State. Identification of this seed was carried out in the Department of Pharmacognosy of the Faculty of Pharmacy, Nanmdi Azikwe University (NAU). Twenty-four hours after the last administration, the animals were anesthetizied with diethyl ether in a close jar, blood samples were collected through ocular puncture using heparinized capillary tube and put into plain serum bottle, and then serum were separated by centrifugation and was stored in a refrigerator of temperature -18 o c for biochemical analysis. Thereafter, the animals were anesthetized using diethyl ether. Each animal was placed on the dissecting board, pinned to the board and dissecting set (sharp scalpel on scalpel holder for making incision; scissors for cutting and dissecting forceps for harvesting) were used to harvest the testes which was immediately weighed before transferring into 10% formal saline for proper fixing for histological sectioning. Small slices of testes tissue were taken and passed through several stages of tissue processing before embedding in paraffin. Five-micron thick sections were stained with hematoxylin and eosin (H & E) as described by Carleton, (1976); Bancroft and Gamble, 2002 for demonstrating histoarchitecture of the liver, kidney and testes tissues. Data were analysed using SPSS version 25. Data were subjected to inferential statistics, and values were presented as Mean ± Standard error of Mean (SEM) using tables. hormonal test, semen quality and relative organ weight was analysed using one way Anova followed by post hoc LSD multiple comparism. Body weight was analysed using t-test. Data was considered significant at p <0.05.

Effects of Ethanolic Seed Extract of Dacryodes Edulis on the of Paraquat Induced on Testicular Toxicity in Male Adult Wistar Rats
Result from table 4.1 below showed that there was a significant (p<0.05) increase in the body weight in group A, as the final weight was 53.33% greater than the initial weight. Group B showed a decrease in weight that was not significant (p>0.05) when the initial weight was compared to the final weight to the tone of 23.43%. Group C showed a decrease in weight that was not significant (p>0.05) when the initial weight was compared to the final weight to the tone of 4.29%. Group D showed an increase in weight that was not significant (p>0.05)as the final weight was 16.42% greater than the initial weight. Group E showed a significant increase (p<0.05) in the weight when the initial weight was compared to the final weight to the tone of 29.85%. Group F showed a significant increase (p<0.05) in the weight when the initial weight was compared to the final weight to the tone of 37.25%. Group G showed a significant increase (p<0.05) in the weight when the initial weight was compared to the final weight to the tone of 22.95%. Group H showed a significant increase (p<0.05) in the weight when the initial weight was compared to the final weight to the tone of 20%. 1.45 Data was analyzed using ANOVA, followed by Post Hoc LSD multiple comparism, and data was considered significant at (p<0.05).
Result from table 4.2 Results of the relative testicular weight showed an increase in organ weight that was not significant (p>0.05) in groups C, D, E, F, G and H, while a significant increase in organ weight (p<0.05)in group A when compared to group B. Result from table 4.6 below showed a significant increase (p<0.05) in luteinizing hormone level in groups A, C, D, E, F, G, and H when compared to group B. Result of Follicular Stimulating hormone showed a significant (p<0.05) increase in group D, G, and H, while an increase that was not significant (p>0.05) in groups A, C, and E when compared to group B. Testosterone result showed a significant (p<0.05) increase in groups D, E, F, G, and H, and increase that was not significant (p>0.05) in groups A and Cwhen compared to group B. 37 Data was analyzed using ANOVA, followed by Post Hoc LSD multiple comparism, and data was considered significant at (p<0.05).

Table 4.7 shows the effect of ethanoic seed extract Dacryodes edulis on paraquat-induced toxicity on Active motility, Sluggish motility, and Non-motile Semen Groups Active Motility (%) P-Value Sluggish Motility (%) P-Value Non-Motile (%) P-Value
Result from table 4.7 showed that active motility revealed a significant increase (p<0.05) in-groups A, C, D, E, F, G, and H when compared to group B. Sluggish motility, result revealed a decrease that was not significant (p>0.05) in groups A and C, while a significant increase (p<0.05) in groups D, E, F, G, and H when compared to group B. Non-motile sperms showed a significant decrease (p<0.05) in groups A, C, D, E, F, G, and H when compared to group . 17.09 Data was analyzed using ANOVA, followed by Post Hoc LSD multiple comparism, and data was considered significant at (p<0.05).

Table 4.8 shows the effect of ethanoic seed extract Dacryodes edulis on paraquat-induced toxicity on Normal and Abnormal Sperm cells Groups Normal Sperm Cells (%) P-Value Abnormal Sperm cells (%) P-Value
Result from table 4.8 showed a significant increase (p<0.05) in Normal sperm cells in group A, C, D, E, F,G, and H when compared to group B. Abnormal sperm cell result showed a significant decrease (p<0.05) in group A, C, D, E, F,G, and H when compared to group B. The precise mechanism of action is due to the presence of flavonoids and polyphenolic compounds present in Dacryodes Edulis attenuating oxidative damages caused by PQ intoxication. Although, in-group E, PQ intoxication showed a reverse effects of semen motility changes. However, paraquat control group when compared to normal control showed a significant decrease (p<0.05) in normal sperm cell and significant increase (p<0.05) in abnormal sperm. This is attributed to generation of ROS production by PQ intoxication. This study agrees with Chen et al., (2017) who reported a significant decrease in sperm viability following paraquat administration. Eduardo et al., (2018) findings agrees with report of this present study on viability of normal sperm cell. Findings from table 6 showed a significant increase (p<0.05) total sperm count in groups D, E, F, & H when compared to paraquat control (group B). This is attributed to polyphenols and flavonoids present inDacryodes edulis. However, when paraquat control was compared to normal control, there was a significant decrease (p<0.05) in total sperm count. This is present of ROS generation by PQ intoxication. This study agrees with Chen et al., (2017) who reported a significant decrease in sperm count following paraquat administration. Eduardo et al., (2018) findings agrees with report of this present study on sperm count, which showed a significant decrease following paraquat administration