Screening for Toxic and Haematological Effect of Ethanol Extract of Azadirachta Indica in Wistar Rats Using Some Serum Enzymes as Markers

تاريغتلا نع فشكلا مدلاو ةيمسلا تابنل لوناثيلإا صلختسمل ةي Azadirachta Indica تاميزنأ ضعب مادختساب ناذرجلا يف تارشؤمك لصملا ةصلاخلا ت تابن قاروأ صحف م Azadirachta indica لصم ميزنإ ةطشنأ نع فشكلل متو يذرج نيرشع مادختسا مت .نارئفلا يف مدلا اهميسقت عيماجم ىلا 4 5 كل نارئف ل ةعومجملا تناك .اهنم 1 لا ةعومجم ةرطيس رطقملا ءاملا تطعأو ةيبلسلا تناك امنيب . مجملا تاعو 2 و 3 و 4 ا تاعومجم يه تعرج ثيح ةلماعمل 200 و 400 و 800 صلختسمل مسجلا نزو نم مغك / مغلم A. indica مت .يلاوتلا ىلع اهعيرجت ةدمل 14 مت ، امًوي اهلتق بقث قيرط نع مدلا عمجو بلقلا ةيويحيميكلا تلايلحتلا ءارجلا مت . سايق صلختسم ريثأت A. indica ىلع جئاتنلا عيمج ةنراقم تمت .لصملا تاميزنإ ةطشنأو ةيومدلا تاملعملا ةلماعملا تاعومجمل ةرطيسلا عم ةيونعملا ىوتسم تحت 95 % نم صلختسملا ةعرج تداز ثيح ةيومدلا تاملعملا يف يجيردت ضافخنا كانه ناك 200 ، 400 ىلإ ، 800 .مسجلا نزو نم مغك / مغلم ، ةيومدلا تاملعملا ترهظأ PCV ، RBC ضافخنا نيبولجوميهلا ، ل يلكلا دعلا رهظا امنيب . ةيونعم نكت مل نكل ميقلاب تايركل مدلا ضيبلا للاخ نم اًيجيردت اعًافترا رهظأ ، صلختسملا ريثات نكلو ايونعم نكي مل . جئاتن تنيب امك رك ضيبلا تاي يقيرفتلا ىلإ ةدايز ةيوافمللا ايلاخلا تاضمحلا ةلقو تلادعلا ةلق ، و نكت مل يتلا ةيونعم ، ةيئايميكويبلا تاملعملا ترهظأ . نا صلختسم A.indica لا ةيعيبط تايوتسم لإ تاميزن ( لصملا AST و ALT و (ALP فيفط ضافخنا دوجو نم مغرلا ىلع نا . نيتوربلا يلكلا عملا نمض ناك .يعيبطلا لد امك ميقلا ريشت MCV و MCH و MCHC صلختسم ربتعي .ةيرايعملا مدلا رقف ةلاح ىلإ ا A. indica ةعرجب ىلكلا ودبكلا تاميزنإو مدلا ايلاخل اًنمآ نم لقأ 800 .مسجلا نزو نم مجك / مجم


Introduction
Many plants extracts are known to cause anaemia by destruction of RBCs, or may cause reduction in production of RBC in the bone marrow (1) and also cause liver and kidney damage.A. indica (Family, Meliaceae) known by common name Neem tree is native to Asian countries. It has long been used in India as remedy for sickness (2). The leaves, bark, stem, root have medicinal properties (3). A. indica is reported to be anthelmintic, antibacterial, antifungal, antihyperglycermic antiinflametory, antiviral, antipyretic, insecticidal, hypercholesteremic and hypoglycermic agent (4,5). Chemical compounds in plants mediate their effect on the human/ animal body through processes identical with compounds in conventional drugs, thus herbal medicine do not differ greatly from conventional drug with reference to their mechanism of action (6). Azadirachta indica is called 'Ogwu akom' by Ibo tribe in Nigeria meaning literally malaria drug. The Hausa tribe in Nigeria call it Dogo yaro. Malaria is a major disease that kills children in Nigeria and other tropical countries. Environmental temperature provides conducive ground for the arthropod vector (mosquitoes) to thrive (7). Malaria is responsible for about 750000 mortalities especially in children. (8). AST and ALT elevation in conditions of hepatocye damage in inflammatory condition of the liver, hypoxic states, hepatotoxicity by toxicants, trauma and some plant extracts, (9,10). Liver ALP elevation also in hepatocyte and biliary epithelial damage. They could also be ALP elevation in osteoblast, intestinal epithelial and corticosteroid stimulation when used for treatment (11,12). Hyperproteinaemia is associated with dehydration occasioned by vomiting, diarrhoea, impaired renal concentration ability, excessive sweating or decreased water intake (13). Elevated urea production is associated with intestinal heamorrhage, increased dietary urea or increased protein catabolism, (14). Elevated creatinine occurs in pathological processes that cause adecrease in glomerular filration rate which could be pre-renal, renal or post renal, (15). Hyperbilirubinaemia occur in diseases associated with heamolysis of blood as seen in babesiosis, anaplasmosis, trypanosomiasis, snake bite and some plant toxicants, (16). The aim of this work is to check the effect of A. indica on haematological changes and biochemical indices for liver and kidney status after using the extract for treatment.

Plant Materials
Leaves of A. indica were collected from the University environment in Umudike, Nigeria and was identified by Prof. M. C. Dike at the Taxonomy section of College of Natural Resources and Environmental Management, Michael Okpara University of Agriculture, Umudike, Nigeria.

Preparation of Plant Extract
The identified leaves of Azadirachta indica was dried under shade for 10 days and grinded to a coarse powder using manual grinder (Corona-Landers C 1A SA). Extraction was done by Soxhlet method described by (17,18) and 35g of coarse powdered sample was introduced into the extraction chamber using ethanol as solvent. Throughout the extraction time of 48 hours, the temperature was kept at 70 0 C. The extract was concentrated in an oven at 30 0 C and the dried extract weighed and kept in a labelled sterile specimen bottle for the work. Trial toxicity test showed that at 2000 mg/kg the rats were still alive and safe for use experiment. Thus different doses of 200, 400 and 800 mg/kg body weight was prepared and administered to the rats. These doses were calculated from a stock solution dissolved in distilled water.

Haematology and Biochemical Investigation
For Haematological screening, PCV was examined by the micro-hematocrit method as described by (19) using capillary tubes while RBC and WBC were counted manually using an improved Neubauer counting chamber. The differential leukocyte was counted manually using a thin blood film stained with Leizhman stain. (Hb) concentrations was determined by cyanomathemoglobin method, (20). Using RBC, PCV and (Hb), the mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) were calculated using formulae; MCV = PCV x 10 fl RBC MCH = Hb x 10 pg RBC MCHC = Hb x 100 g/dl PCV (21,22,23,24).
Biochemical investigation was performed using ELISA reagent kits. The measure included, serum ALT, AST, ALP. (9,10). Total protein was determined by Biuret method as described by (15). Samples were analysed immediately to avoid artifactual changes (25).

Experimental Animals
Male albino rats (140 to 250 g) were purchased from University Farm. Approval was obtained from College of Vet Medicine of the University in line with the guidelines for the care and use of laboratory animals as provided by National Research Council (26). The rats were acclimatized and fed ad libitum.

Experimental Design
Twenty rats were used for the research and were grouped into four of five rats each. Group 1 was the normal control group and was administered distilled water. Groups 2, 3, and 4 were the treatment groups which received 200, 400 and 800 mg/kg body weight of the A. indica extract respectively. The rats were dosed for 14 days, thereafter was sacrificed and blood collected by cardiac puncture for analysis. The effect of A. indica extract was checked on haematological parameters and serum enzymes activities.

Statistical Analysis
Statistical analysis was done using Statistical Package for Social Sciences (SPSS) version 20. Values were expressed as mean ± Standard Error of Mean (SEM) and were further subjected to one -way analysis of variance (ANOVA) for comparison of doses with normal control. Duncan post-hoc test was used to separate the mean that showed significant difference. The statistical confidence was set at (p<0.05).       From the result of this study decreases in Hb, RBC and PCV was not significant at (p<0.05).

Results and discussion
There was mild decrease in TWBC value as the dose increased but was not significant at (p<0.05). The red cell indices MCV, MCH and MCHC was normal meaning that A. indica extract even at the highest dose did not alter the size and haemoglobin concentration.
The percentage proportion of lymphocytes, neutrophils, monocytes and the basophils were not significantly (p<0.05) varied in treated groups compared to normal control. Percentage proportion of eosinophil gradually decreased as the dose were increased and highest at 800 mg/kg body weight. There were no significant differences at (p<0.05) in values of different leukocytes counted though there was mild elevation Fig 3. Values of leukocytes fall within normal reference range.
Liver is vulnerable to attack because it is the major organ involved in biotransformation and detoxification the primary site of biotransformation and detoxification (28,29). kidneys the principal organ for the excretion of waist products and osmoregulation is prone to Vol. challenges by toxicants. When the cells of these organs are damage it results in elevation of clinical Biochemical parameters in serum, such as AST, ALT and ALP or Creatinine, Urea as markers of impaired renal function, (30). The increase in these enzymes is indicated in hepatic and nephrotic disorders. The result as presented in Fig 5, showed that the liver enzymes AST, ALT and ALP showed mild evaluation but was not significant (p<0.05). This observation suggests that A. indica extract was safe to liver or kidney which are responsible for metabolism, biotransformation, and elimination. Since A. indica did not cause any significant ((p<0.05)) increase in serum total protein (TP), AST, ALT and ALP, of treated Wistar rats compared with the control rats, it could be seen in this work that A. indica extract at dose <800 mg/kg body weight has no significant hepatotoxic or nephrotoxic effect on the liver and kidney respectively. This finding lay credence to the traditional claim of safety of the extract of A. indica to liver and kidney when used in the treatment of malaria at moderate doses < 800 mg/kg body weight. The mild reduction in value of Total protein by this plant extract had no significant (p<0.05), effect indicating its safety to the hepatosynthetic cells of the liver.

Conclusion
In this work, the extract of A. indica is safe to blood cells, liver and kidney at dose <800 mg/kg body weight.