Evaluation of the Protective Effect of Moringa oleifera Leaf Extract against Aluminium Induced Liver Damage in Male Albino Wistar Rats

Aluminium is a hepatotoxic element that is extensively used in the production of household cookware, storage utensils, water purification and in the preparation of some drugs. Conversely, the leaf extract of Moringa oleifera is hepatoprotective amongst other medicinal and nutritional benefits.The present study evaluated the protective effect of ethanol leaf extract of Moringa oleifera on Aluminium induced hepatotoxicity in male albino wistar rats. Eighteen (18) male albino wistar rats weighing between140 and 180 g were divided into 3 groups of 6 animals per group. Group 1 served as control and was given normal rat chow and distilled water; Group 2 was administered 100 mg of Aluminium chloride per kg body weight whereas animals in Group 3 received 300 mg/kg body weight of ethanol leaf extract of Moringa oleifera and 100 mg per kg body weight of Aluminium chloride by oral gavage. The rats were sacrificed after 28 days of treatment. Blood and liver samples were obtained and used for the analyses of some marker enzymes (ALP, AST, ALT); haematological indices; lipid profile and histopathological assessment using standard techniques. The results show that Aluminium chloride increased the activities of ALP, ALT and AST significantly (P<0.05). TC, TG and LDL also increased significantly whereas HDL showed a significant decrease (P<0.05). The RBC count, Hb, PCV, lymphocytes and platelets decreased while WBC count and neutrophils increased significantly (P<0.05). Aluminium chloride caused alterations in the normal histology of the hepatocytes consistent with observed changes in enzyme activities. Administration of ethanol leaf extract of Moringa oleifera moderated the deleterious effects of Aluminium chloride.


Introduction
Moringa oleifera belongs to the Moringaceae family of perennial angiosperm plants. It is a fast growing tree that can attain a height of about 10 -12 m with a diameter of about 45 cm (Bosch, 2004;Parrotta, 2005). Although native to the Sub-Himalayan tracts of India, Pakistan, Bangladesh and Afghanistan, it is now cultivated throughout the tropical and subtropical regions of the world because of its numerous benefits (Odee, 1998;Anwar and Bhanger, 2003;Hsu et al., 2006). In Nigeria, Moringa oleifera is planted in all parts of the country and is identified by a variety of local names incuding, 'Zogale' (Hausa); 'ewe igbale' (Yoruba) and 'ikwa oyibo' (Ibo) (Thilza et al., 2010).The leaves, fruit, flowers and immature pods of Moringa oleifera are highly nutritious and have also been utilized in ethnomedicine for the treatment of various human ailments (Anwar et al., 2007;Kumar et al., 2010). Specifically, the leaves are reported to be rich in proteins, mineral elements,  (Yokel, 2000) and tooth paste (Abbasali et al, 2005). Aluminium is also used extensively in the manufacture of various household cookware and storage utensils (Sorenson et al., 1974). Therefore, its abundance and widespread use underscores the potential for human exposure and susceptibility to harm (Zhang and Zhou, 2005). Most individuals ingest 1 -10 mg of Aluminium per day (Greger, 1992) mainly through the oral route and by inhalation (Hanchez-Leroy, 2013). In adults, the tolerable weekly intake of Aluminium is 7 mg/kg bw (WHO/ FAO, 1989). However, this tolerable limit can be exceeded as a consequence of continuous exposure (Gauthier et al., 2000). Aluminium could be toxic if ingested in amounts greater than 40 mg/day (Dolara, 2014). The liver is one of the target organs for Aluminium toxicity (Klein et al., 1984;Galle et al., 1987;Spencer et al., 1995 In view of the reported hepatoprotective properties of Moringa oleifera leaves, the present study was carried out to evaluate the protective role of ethanol leaf extract of Moringa oleifera against Aluminium chloride induced damage to the liver of male albino wistar rats.

Chemicals and Reagents
Aluminium chloride was obtained from Guangdong Guanghua Sci-Tech Company Limited, China. Reagent kits used for the determination of biochemical parameters were products of Randox Laboratories Ltd., England. All the other chemicals used in this study were of analytical grade (AR).

Collection of Leaf Samples and Preparation of Extract
Fresh leaves of Moringa oleifera were obtained from a local plantation in Uyo, Akwa Ibom State, Nigeria. They were identified and authenticated by the curator in the Pharmacognosy Herbarium, Faculty of Pharmacy, University of Uyo. The leaves were washed clear of dust particles and air dried at room temperature for two weeks. The dried leaves were ground into a powder using a kitchen blender. The powdered sample was extracted in 80% ethanol. The extract was filtered through Whatman No. 1 filter paper. The resulting extract was concentrated and evaporated to dryness using a Rotary Evaporator at 40°C. The extract was further reconstituted in distilled water and preserved in a refrigerator maintained at 4°C.

Experimental Animals
Eighteen (18) male albino Wistar rats were procured from the animal house facility of Faculty of Basic Medical Sciences, University of Uyo, Uyo, Nigeria.
They were housed in well ventilated wire-wooden cages and maintained under standard conditions (temperature, 28 ± 3°C; relative humidity, 67 ± 3%; 12 hours light/dark cycle). The animals were allowed unrestricted access to drinking water and rat chow (Livestock Feeds Plc, Lagos, Nigeria). The animals were cared for in accordance with the United States National Institute of Health Guidelines for the Care and Use of Laboratory Animals in Biomedical Research (NRC, 1985). Institutional approval for the study was obtained from the Postgraduate School, University of Uyo, Uyo.
The rats were allowed to acclimatize for a period of fourteen days after which they were randomly divided into three groups of six animals per group. Group 1 served as control and received 1.0 ml of distilled water; Group 2 received 100 mg of AlCl 3 per kg body weight of rat; Group 3 was treated with 300 mg of the ethanol leaf extract of Moringa oleifera and 100 mg of AlCl 3 per kg body weight of rat. Administration of extract and AlCl 3 was carried out by oral garage, once daily, between the hours of 8 and 10 am for a period of 28 days.

Collection of Blood Samples
At the end of the experimental period, the animals were allowed to fast for 12 hours and thereafter sacrificed under chloroform anaesthesia. Blood samples were collected by cardiac puncture using sterile needles and syringes. One portion of blood was transferred into EDTA sample bottles and used for the determination of haematological parameters. The second portion of blood was collected in sterile plain bottles and allowed to clot. Serum was separated from the clot by centrifugation at 3000g for 5 mins using a bench top centrifuge (MSE minor, England). The serum samples were stored frozen (−20°C) until required for analysis.

Determination of Haematological Parameters
The automated haematologic analyzer (Sysmex KX-21) was used to analyze the haematological parameters at the Department of Haematology, University of Uyo Teaching Hospital, Uyo, Nigeria.

Determination of Biochemical Parameters
AST, ALT and ALP were analysed using reagent kits from Randox Laboratories, England. Estimation of total serum cholesterol was carried out using the cholesterol oxidase phenol aminosphenazone (CHOD-PAP) method whereas high density lipoprotein cholesterol was analysed by the polyethylene glycol cholesterol oxidase phenol aminophenazone (PEG-CHOD-PAP) method. Triacylglycerols (TGs) were determined using the glycerol phosphate oxidase, phenol aminophenozone (GPO-PAP) end point assay. LDL-cholesterol was obtained by calculation (Friedwald et al., 1972).
Reagent kits for lipid analyses were obtained from Randox Laboratories, England.

Histopathological Examination of Liver Samples
A portion of the liver was removed, fixed in buffered formalin and embedded in paraffin wax. Tissue sections (5 m) were prepared and stained with Hematoxylin and Eosin (H&E) for microscopic examination (Bancroft et al., 1996).

Statistical Analysis
Results were expressed as mean ± standard deviation. The data obtained were analyzed by one-way ANOVA. Duncan's Multiple Range test (Duncan, 1955) was used to determine the significance of difference between means. Statistical significance was accepted at P< 0.05. Table 1 shows the effect of ethanol leaf extract of Moringa oleifera on Aluminium chloride induced increase in the activities of AST, ALT and ALP in male rats. This table indicates that treatment with Aluminium chloride (Group 2) precipitated a significant increase (p<0.05) in enzyme activities. Co-administration of ethanol leaf extract of Moringa oleifera with Aluminium chloride reversed enzyme activities towards normal values (Group 3).

RESULTS
The effect of ethanol leaf extract of Moringa oleifera on Aluminium chloride induced alterations in serum lipid profile of male rats is presented in Table 2. Aluminium chloride induced a generalized significant increases (p<0.05) in serum concentrations of total cholesterol (TC), triacylglycerol (TAG) and low density lipoprotein cholesterol (LDL-C), whereas there was a significant decrease (p<0.05) in HDL-C. Administration of aluminium chloride with ethanol leaf extract of Moringa oleifera ameliorated the dyslipidemia caused by Aluminium chloride (Group 3). Table 3 shows the effect of ethanol leaf extract of Moringa oleifera on Aluminium chloride induced changes in haematological indices of male rats. There was a significant decrease (p<0.05) in PCV, Hb, RBC, lymphocytes and platelets as a consequence of exposure to Aluminium chloride. The WBC and neutrophils increased significantly (p<0.05). The MCH and MCHC were not affected. Ethanol leaf extract of Moringa oleifera moderated most of the haematological effects of Aluminium chloride.

DISCUSSION
Aluminium is extensively used in daily life and it is potentially toxic to man. The present study evaluated the protective effects of ethanol leaf extract of Moringa oleifera against the toxicity of Aluminium chloride on the liver of male rats by the measurement of some biochemical parameters, haematological indices and histopathological examination. The neutrophils are the principal phagocytic and microbicidal responders in the immune system (Willey et al., 2013). Increase in neutrophil is usually considered as an index of tissue damage or the entry of foreign bodies into the blood stream (Sakthivel, 1988). A significant increase in neutrophil count as a consequence of exposure of African cat fish to Aluminium has also been reported by Selvi and Alagesan ( 2017).
The lymphocytes are the primary cells involved in specific immune reactions to foreign matter (Willey et al, 2013). In the present study, treatment with Aluminium chloride induced a significant decrease in lymphocyte count. Aluminium induced lymphopenia has been attributed to the capacity of this element to induce oxidative stress and exert genotoxic damage to the lymphocytes (Garcia -Medina et al, 2010).
The platelets are blood components involved in coagulation. A decrease in platelet count as observed in this study implies an inhibitory effect on thrombopoietin secretion (Malomo et al, 2002). A similar decrease in platelet count as a consequence of exposure to aluminium has been reported by Anacletus and Onyegeme-Okerenta, (2016).
Administration of ethanol leaf extract of Moringa oleifera was observed to restore to normal most of the immunological alterations induced by Aluminium chloride. The immunomodulatory effects of the leaf extract of Moringa oleifera is due to the wide range of macronutrients, micronutrient and phytochemicals found in this plant (Nfambi et al., 2015).
Histopathological evaluation plays an important role in the assessment of the adverse effects of xenobiotics (Reddy and Rawat, 2013

CONCLUSION
The present study has provided supportive evidence that the oral administration of Aluminium chloride in male rats at a dose of 100 mg/kg body weight daily for a period of 28 days induces hepatic dysfunction as evidenced in significant alterations in some biochemical, haematological and histological parameters. The use of ethanol leaf extract of Moringa oleifera in combination with Aluminium chloride was observed to attenuate some of the harmful effects of this element. Therefore, supplementation with Moringa oleifera leaves may prove useful as protective therapy against the hepatotoxic effects of Aluminium chloride.