Effect of Ethylacetate and Deionized Water Leaf Extracts of Diodia Scandens on Hematological and Reproductive Systems in Male Albino Rat

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Effect of Ethylacetate and Deionized Water Leaf Extracts of Diodia Scandens on Hematological and Reproductive Systems in Male Albino Rat

The staple needs of man such as food, shelter and fiber are gotten from plants and plant related products. Wood and timber obtain from plants still constitute major raw materials for construction while the cotton still remains the most preferred among clothing materials (Ashok and Pande, 2007).

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In the same vein, there has been a great deal of attention and breakthroughs in tapping plant kingdom for pharmaceutical application and the interest in plants as a means of potential therapeutic agents is increasing very fast (Sujatha et al., 2011). Current studies suggest that several thousands of plants are now known to haves medicinal applications in different cultures (Lira et al., 2014). Natural substances of plant source have been used throughout the universe for human health care (Enzo, 2003). Approximately 80% of the world population with 90% of this coming from African countries are involved in the use of medicinal plants in addressing their primary health care requirements, (Osonug et al., 2004) due to increasing poverty level and affordability of herbal medicines as well as the belief that it has minimum or zero side effects (Igoli et al., 2005).

Medicinal plants are plants which contain substances used for the prevention and or treatment of diseases or infection and other health disorders in human and other animals (Nwachukwu et al., 2010). Phytochemical screening methods have disclosed the chemicals responsible for these functions (Araz et al., 2003). Analysis of the structures of these phytochemicals has supplied the ground for their phytotherapeutic potencies (Stintzing et al., 2002). These substances are majorly secondary metabolites such as aromatic substances made up of phenols and their derivatives tannins, at least, 12000 of these secondary metabolites have been isolated, other phytochemicals contained in plants are alkaloids, polyphenols, anthocyanins, isoflavones, terpenoid, carotenoids, glycosides (Goldman, 2001). The use of medicinal plants varies from species to species as diseases differ from one form to another in different localities (Nwachukwu et al., 2010). Currently, research found one hundred and twenty-two compounds employed in main stream medicine which were derived from ethno medical plant origin, 80% of these compounds were employed in the same or related way as the traditional ethno medical use (Elvin-lewis, 2001). Some of the phytotherapeutic agents or bioactive molecules used as drugs include insulin, quinine, morphine, codeine, salicin and aspirin, opium, mint, garlic, castor oil e.t.c. (Vicker and Zollman, 1999).

Reproductive system is responsible for the continuation of species, master reproductive organs are the gonads which produce the gametes: a couple of testes produces sperm in male and a couple of ovaries produces ovum in female. (Sembulingan and Sembulingan, 2012). The biological procedure of reproduction is affected by multiple factors (Irvine, 2001). Reproductive power in the male includes the production of semen having normal spermatozoa (quality) in the right number (quantity), alongside the desire and ability to mate (Oyeyem et al., 2008).
Infertility is described as the unfitness to conceive after twelve months of sexual intercourse without using contraception (Bashir et al., 2009).

The male factor contributes to over 50% of the infertile couples (Irvine, 2001). Based on the report of the World Health Organization (WHO), about 15 to 10 percent of couples have been involved in some forms of infertility problems which 40% of these problems are traced to male factor (Kashani et al., 2012; Henkel et al.2005). Wane in male fertility has been a great concern from time since male infertility explains over 30% of fertility problems all over the world (WHO, 1991). Male infertility is a crucial matter and is a common problem occurring worldwide, though not given much attention in Nigeria (Okonofua et al., 2005). Infertility is a known clinical problem, touching people medically and psychologically (Raghuveer et al., 2010).

Male infertility may be attributed to different reasons such as genetic disorders, genital duct obstruction, and varicocele, decreased sperm production, decreased semen quality parameters (sperm count, motility, morphology and viability), erectile dysfunction and male impotence (Kashani et al. ,2012). Normality or abnormality of semen is considered a reference point of the function of the male reproductive system and semen quality is of crucial importance in foretelling male fertility (Wijeratna et al., 2005). Separated seminal plasma abnormalities are abnormalities in the physical composition, biochemical composition and presence of pathogens, though, the significance of these abnormalities in the onset of infertility is unknown (Comhaire, 2003). Since seminal plasma is the contiguous environment of spermatozoa, the constituents of the seminal plasma are believed to have effect on the quality of the sperm (Comhaire, 2003). Sperm quality can be affected by endocrine factors through two possible mechanisms: by affecting spermatogenesis or by directly affecting sperm functions through receptors thus, the sperm quality (Wijeratna et al., 2005). Malproduction and malfunction of hormones are also implicated in pathological changes in male reproductive function and male infertility (Wijeratna et al., 2005).

Various treatment which include surgery, chemical and herbal drugs and laboratory methods are accessed to increase fertility (Sinclair, 2000). Investigations and researches have shown that herbal or botanical medicine has a positive effect on sperm parameters (Sinclair, 2000). Traditional medicines were employed for fertility control in ancient time (Ogbuewu, et al., 2011). For this reason numerous plant species have been screened for their fertility efficacy (Yama et al., 2011). Ladan et al., (2013) reported that a study carried out in a fertility clinic in Iran recorded that about 43.3% of the attendants preferred to use herbal medicinal remedies. These medicinal remedies may act by affecting sperm motility and viability, modification of uterine lining functions, implantation of fertilized egg or a rejection effect within the uterus (Yakubu and Afolayan, 2009). Plants like Fodogia agrestis, Piper geineese Afromonum medlegueta and Lepidium meyenii and the root of L. meyenii has been shown to have fertility enhancing properties (Yakubu and Afolagyan, 2009).

Diodia scandens belong to the family of Rubiaceae and is a perennial herb having straggling branches, slender angular simple leaves and clustered white flowers, stem is measuring up to 3m high (Akobundu and Agyakwa, 1987). Diodia scandens is wide spread in Tropical Africa and also in Mascarene Island, Tropical Asia and Tropical America. Its native name includes umunyovo (Burundi), Onaedi (Igbo) and Ewe idatcha (Yoruba) (Chifundera, 2001). In folk medicine which gave rise to traditional systems of medicine, various parts of the plants, including sap, stem, leaf and roots have been employed in different medicinal purposes (Ogu et al., 2011).

The most reported use of the plant in West Africa is its use as an antidote (Venomous stings, bites and pain killers) (Burkill, 1985). Etukudo (2003), reported the traditional use of the leaf extract or sap for curing eczema, stop bleeding, treat ear problems and also act as an antiabortifacient used to prevent bleeding during pregnancy. Diodia scadens leaf extract is also used to treat infertility in male and female and also act as antioedema and analgesic, oxytoxic agent, laxative and antifungal agent for treating skin diseases by traditional medicine healers in South-eastern part of Nigeria. Diodia scandence may contain the following phytochemicals; tannins, saponins, cardiac glycosides, flavonoids and steroid (Ogu et al., 2011).

Normal levels of vital hemato-biochemical constituents are of utmost importance for maintaining functional integrity of the reproductive system. Any change in hematological and biochemical parameters may adversely affect the reproductive efficiency. The assessment of hematological parameters could be used to reveal the deleterious effect of plant extracts on the physiological and pathological status of man and animal (Magalhaes et al., 2008). The effect of various parts of Diodia scandens on hematology and reproductive systems has not been fully established.

Aim and Objectives
Aim
This study was designed to investigate the effect of ethylacetate and deionized water leaf extracts of Diodia scandens on hematological and reproductive systems in male albino rat.
Specific Objectives

1. To conduct proximate analyses on ethylacetate and deionized water leaf extracts of Diodia scandens.

2. To conduct phytochemical analyses on ethylacetate and deionized water leaf extracts of Diodia scandens.

3. To conduct vitamin (A, C, E, B2, B6, Niacin, Thiamin and K) analyses on ethylacetate and deionized water leaf extracts of Diodia scandens.

4. To conduct mineral (Mg, K, Ca, Na, P, Zn, Cu, Fe, Mn, Co and Se) analyses on ethylacetate and deionized water and leaf extracts of Diodia scandens.

5. To conduct LD50 (acute toxicity) on Diodia scandens leaf- extracts.

6. To determine the effect of the leaf extracts on hematological system using the following hematological indices; hemoglobin concentration, WBCT, PCV and platelet count in albino rats.

7. To determine the effect of the leaf extracts on the reproductive system using sialic acid, cholesterol and total protein, GGT , ACP, testosterone, , luteinizing hormone and follicle stimulating hormone as biomarkers in albino rats.

8. To determine the effect of the extracts on antioxidant indices: MDA, SOD, CAT and GR in albino rats.

9. Statistical comparison of extracts.

Materials and Methods

Materials: The reagents and chemicals used were of a good analytical standard.
Collection of sample (plant material): Fresh leaves of Diodia scandens were collected from the out sketch of Amuro in Afikpo-North local government area of Ebonyi state, the plant was identified in the department of Applied Biology Ebonyi State University, Abakaliki.

Animal Material: Adult male albino rats were procured from Department of Veterinary Medicine University of Nigeria Nsukka. The rats were acclimatized for one week in the animal house of the Department of Biochemistry, Ebonyi State University, Abakaliki.

Extraction: The leaves were washed with distilled water, air- dried and pulverized for extraction. Extraction method according to Sofowora and Olutimsyin, (2001) was used. Deionized water and ethyl acetate served as solvents for extraction.

Organic extraction: Exactly 400g of the pulverized sample was soaked in 1500ml of ethyl acetate and allow stand for 48 hours with constant shaking using magnetic shaker. The mixture was filtered using a piece of muslin cloth and final concentration done using rotary evaporator.

Aqueous extraction: The same method as above was followed using deionized water as solvent.
Chemical Analysis of Extracts:

Proximate composition: The procedures for the determination of the proximate composition of the samples were those of the Association of Official Analytical Chemists (AOAC, 1990).
Phytochemical Screening: Quantitative measurement of phytochemicals: saponins, alkaloids, tannins, cardiac glycosides, terpenoids, Anthraquinones and flavonoids was carried out according to standard procedures of Trease and Evans (1989), Harbone (1973) and Sofowora (1993).

Determination of Mineral Composition: Exactly 2g of the plant sample was subjected to dry ashing at 5500C in a muffle furnace and the ash was dissolved in 2M Hcl. The solution was diluted to 100ml with deionized water. The resulting solution was used for determination of Na, Ca, Mg, Mn, Fe, Se, Co, K, P, Zn and Cu using atomic absorption spectrophotometer (Shahidi et al., 1999 and Nahapetian and Bassiri, 1975)
Measurement of Vitamin Contents: The level of vitamins A, C, E, K, B2, B6 Niacin and Thiamin in the extracts was measured according to methods described by AOAC (1990).
Measurement of LD50 (Acute Toxicity Dose) of the leaf extracts

A modified method of Lorke (1983) was used to measure the LD50. The study was done in two phases with 75 adult male albino rats. Phase one was made up of five groups of five rats each, A, B, C, D and E. Groups A, B, C and D were treated orally with graded doses of 1000,1500, 2000 and 2500 mg/Kg body weight of ethyl acetate leaf extract respectively while group E received olive oil and serve as control. Second phase of the study comprised of three groups of five rats each, F, H and H. These was treated with the doses; 3000, 3500 and 4000 mg/kg body weight of the extract respectively. Animal was observed for the first 24 hours and daily for three days. Circulatory signs, respiratory rate, changes in eyes and skin and nasal tract were monitored. This procedure was repeated until 100 percent death was recorded. The LD50 was calculated as the square root of the product of the lowest lethal dose and the highest non lethal dose that is the geometric mean of consecutive doses for which 0 to 100% survival rates was recorded.

Experimental Design:

Effect of Ethylacetate and Deionized water Leaf-Extracts of Diodia scandens on Some Hematological Parameters in Albino Rat.
A total seventy male albino rats placed into fourteen groups of five rats each were used in the study. Groups A, B, C and D were treated with graded doses of 200, 400, 600 and 800mg/kg body weight of ethylacetate leaf extract while E, F, G and H received same doses of deionized water leaf extract. Negative controls I and J received normal saline and olive oil respectively. Standard controls K and L received 10 and 14.3mg/kg body weight vitamin C and menevit respectively. Positive controls M and N received normal saline and olive oil respectively. Treatment lasted for 14 days and all animals were allowed free access to feed and water. One hour after the last administration on day 14, groups A, B, C, D, E, F, G, H, K, L, M and N were given 2.5ml/Kg of carbon tetrachloride (in olive oil 1:1) intraperitonealy. The animals were fasted for 24 hours and sacrificed. Blood sample was collected from each rat by cardiac puncture into a clean EDTA and plain – containers for hematological analysis and reproductive hormone assay respectively. Hemoglobin concentration and Packed Cell Volume were measured using the method described by Baker et al., (2001). Total White Blood Cell Count and Platelets were done using the method described by Dacie and Lewis(2001). The testes were prepared into homogenate for biochemical parameters.

Effect of Ethylacetate and Deionized water Leaf-Extracts of Diodia scandens on Some Fertility Parameters in Albino Rat Testis.
Protein concentration was measured using the method of Lowry et al., (1951) as modified by Peterson (1977). Tissue cholesterol level of testes was determined by the method described by Zlatkis and Zak (1968). Sialic acid level in the testes was determined by method described by Warren, (1959), while GGT and ACP assay were carried out according to methods by Szasz, (1969) and Wright et al., (1972) respectively.

Effect of Ethylacetate and Deionized water Leaf-Extracts of Diodia scandens on Some Fertility Hormones in Albino Rat Testis.
Serum levels of reproductive hormones were measured. Assay of testosterone was done based on guideline on the hormone assay kits according to the principle highlighted by Titz (1995) while assay for luteinizing and follicle stimulating hormones were done following the principle described by Uotila et al., (1987), Sinha-Hikim et al,(1995) and Wang et al.,(1993).
Preparation of Serum: Blood sample collected from the animals was centrifuged at 3000 revolution per minutes for 15 minutes using a bench centrifuge. The clear supernatant (serum) was obtained by decantation.

Effect of Ethylacetate and Deionized water Leaf-Extracts of Diodia scandens on Antioxidant Indices in Albino Rat Testis.
The gonad protective effect of the leaf extracts was studied using a total of sixty-five male albino rats placed in13 groups of 5 rats each. Groups I, II, III, and IV was given graded doses of 200, 400, 600 and 800mg/kg body weight of ethylacetate leaf extract while V, VI, VII and VIII received similar doses of deionized water leaf extract. Negative controls IX and X received normal saline and olive oil respectively, Positive controls XI and XII received normal saline and olive oil respectively while standard control XIII was given 10 mg/Kg body weight of Vitamin C. All the animals were allowed free access to feed and water for 14 days. One hour after the last administration on day 14, groups I, II, III, IV, V, VI, VII, VIII, XI, XII and XIII were given 2.5ml/kg of carbon tetrachloride (in olive oil 1:1) intraperitoneally. The animals were fasted for 24 hours and sacrificed.

Preparation of Testicular Homogenate: Testes were removed from the rats and immersed in ice-cold 0.25mol/L sucrose solution to maintain the integrity of the organs.

The testes was blotted with tissue paper cut and homogenize in ice-cold 0.25mol/L sucrose solution, the homogenate was centrifuged at 800rpm for 10 minutes at 40C and the resulting supernatant used for biochemical analysis (Nurudeen and Ajiboye, 2012). Measurement of MDA in testicular homogenate was done following the method by Ohkawa et al., (1979), the assay of SOD was based on the method described by Nishikimi et al., (1972). Catalyase (CAT) activity of the homogenate was determined by the method described by Albi (1984). Estimation of glutathione reductase (GR) was done according to the method described by Ellman, (1959)
Effect of Ethylacetate and Deionized water Leaf-Extracts of Diodia scandens on Sperm Count, Motility and Morphology in Albino Rat Testis.

Sperm Count: The concentration of sperm was done using a counting chamber (haemocytometer) as described by Ochei and Kalhartkar, (2007). The left cauda epididymidis was incised and semen that oozed was quickly sucked into a red blood pipette to the 0.5 mark, and then diluted with diluting fluid (Sodium bicarbonate: 5g, Formalin: 1ml and distilled water: 100ml) up to the 10L mark. A drop of the semen mixture was placed on the counting chamber and viewed under the magnification of ×40. The total numbers of sperm cells were counted and expressed as 106/mL.

Sperm Motility: Motility of sperm cells was determined by method described by Ochei and Kalhartkar, (2007). One drop of liquefied mixed specimen was placed on a clean slide and covered with a clean cover slip and then examined microscopically. Usual and unusual movements were noted and expressed as % motility.

Sperm Morphology: Morphology % was determined by method described by Ochei and Kalhartkar, (2007). Smear of specimen was made, stained with papanicolaou stain and finally examined under oil immersion objective. Result was expressed as % morphology.

 

Histological Examination
For histological examinations, the testes were quickly removed and fixed in 10% neutral buffered formalin solution. Following fixation, specimens were dehydrated in graded ethanol, embedded in wax, sectioned to 5 microns thickness. The sections were stained with Hematoxylin and Eosin (Banchroft et al., 1996) and examined using light microscope at X200.

Statistical Analysis
Data generated were expressed as mean + SD. Statistical significance of difference was determined using the programme SPSS 17(SPSS, USA). P<0.05 was considered statistically significant.

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