Evaluation of liver protective activity of Moringa oleifera bark extract in paracetamol induced hepatotoxicity in rats

Background Moringa oleifera has been used in folk medicine to alleviate several diseases. In the present study, ethanolic extract of Moringa oleifera bark has been investigated to study its potential on paracetamol induced hepatotoxicity on model rats. Methods Rats (150–200 gm) were divided into 5 groups containing 6 animals each. Acute hepatotoxicity was induced by paracetamol (600 mg/kg body weight) administered once daily for one week whereas the extract of investigated plant was given orally throughout the whole experiment at 250 and 500mg/kg body weight. Silymarin (100mg/kg body weight) was given orally as standard hepatoprotective drug. The level of hepatic injury recovery was determined by the estimation of liver enzymes like SGPT, SGOT, ALP, Bilirubin, Total protein and Albumin. Results Treatment with MO extract as well as standard hepatoprotective agent silymarin ameliorated the increased plasma levels of these hepatic enzymes and indicated the hepatoprotective potential of the extract. Conclusion The biochemical parameters provide evidence that the ethanolic extract of of Moringa oleifera bark has shown hepatoprotective activity.


Introduction
Liver is the major organ which plays a key roles in processing critical biochemical and physiological phenomena including metabolism and Detoxification of endogen and exogenous compounds, such as drugs and xenobiotics, homeostasis, growth, energy and nutrient supply [1,2 ] Hepatic injury could be occurred by hepatotoxic agents including drugs , alcohol and viral infections. [ 1,3 ]Liver diseases like jaundice, cirrhosis and fatty liver have been public health concern across the world. Prevalence of chronic liver disease worldwide is 18  Deoxy-niazimicine [ 10,11,12] A great number of pharmacological activity has also been reported .Aqueous and alcoholic extracts of leaves and roots of Moringa oleifera has been reported to have a strong in-vitro anti-oxidant and radical scavenging activity [12] while Methanolic and ethanolic extract of leaves against pentylenetetrazole and maximal electroshock induced convulsions, demontrated significant anti-convulsant activity [13,14] Potent antidiabetic activity from different extracts of leaves , seed and pod, has also been reported [ ,15,16, 17] an investigation against seed power also revealed its Anti-asthmatic activity [ 19,18] Study on Ethanolic extracts of Moringa oleifera exhibited potent anti-tumor [ 19] and Anthelmintic activity [21 , 22] Another study of alcoholic extracts of leaves as well as seeds has showed effectiveness on isoniazid, rifampicin, and pyrizinamide induced liver damage. [23] However , our present study was concentrated on the bark extract of Moringa oleifera on paracetamol induced liver damage on Sprague Dawley rats .

Plant collection and Extraction
The plant Moringa oleifera (MO) bark was collected from Savar area and was identified and

Toxicity studies
Toxicity studies of the extracts were carried out in Swiss Albino mice of either sex weighing between 20 and 25 g. The extract was found to be safe till 5000 mg/kg p.o. Therefore, doses were selected as 250 mg/kg and 500 mg/kg b.w. [24].

Experimental design for the assessment of liver functions
Animal study was performed at Pharmacology Laboratory, Department of Pharmacy, Jahangirnagar University, Savar, Dhaka-1342. The rats were housed in polypropylene cages at room temperature (27±2ºC). The rats were divided into five groups of 6 animals (n = 6) each [25].
Group I: received water (10 mL/kg p.o.) once daily for 7 days, and served as normal control Group II: received water (10 mL/kg p.o.) once daily for 7 days and served as paracetamol control.
Group III: received standard drug silymarin (100mg/ kg p.o.). once daily for 7 days, serving as STD.
Group IV and V: received Moringa oleifera bark extract (250 and 500 mg/kg respectively) once daily for 7 days. In all groups except group-I paracetamol 600mg/kg bw p.o was administered once daily with respective treatment according to Tabassum N and Agrawal SS (2004) with slight modification with error and trial [26].
Rats were anesthetized using ketamine (500mg/kg, i.p.). After sacrifice, blood samples from each group of rats were collected and the serum was separated by centrifugation. Serum samples were subjected to liver function tests of enzymes such as glutamate pyruvate transaminase (GPT/ALT), glutamate-oxaloacetate tranaminase (GOT/AST) [27], alkaline phosphatase (ALP) [28], total bilirubin [29] and total protein by standard enzymatic colorimetric method.

Statistical Analysis
Statistical analysis for animal experiments was carried out using One way ANOVA following Bonferroni's post hoc test using SPSS 16.0 for windows. Data were presented as Mean ± SEM.
The results obtained were compared with the vehicle treated paracetamol control group. p values <0.05, <0.01 and <0.001 were considered to be statistically significant, highly significant and very highly significant respectively.

Result and Discussion
Acetaminophen (AAP) is a frequently used analgesic that causes the formation of NAPQI and hepatic damage by GSH depletion. At high doses, more NAPQI will bind covalently to cellular macromolecules [30,31,32,33]. Therefore, macromolecules like enzymes will leak from the damaged tissues into the bloodstream, [34] and a study of these enzyme activities in plasma has been found to be of great importance in the assessment of liver damage [35].
The increased levels of AST and ALT indicate cellular damage and loss of functional integrity of the hepatocytes [36]. The increase in ALP in liver disease reflects the pathological alteration in biliary flow [37].
The present study demonstrated that the MO extract decrease the SGPT very highly significantly (p<0.001), SGOT level highly significantly (p<0.01) at 500 mg/kg dose and also the ALP level significantly at 250 mg/kg (p<0.01) and 500 mg/kg dose (p<0.001) (table 2 and figure 2).
Reduction of the enhanced level of serum SGPT, SGOT, ALP and total bilirubin by MO extract seemed to offer protection and maintain the functional integrity of hepatic cells.
An abnormal increase in the levels of bilirubin in plasma indicates hepatobiliary disease and severe disturbance of hepatocellular function [38]. Prior oral administration of Moringa oleifera extract exhibited significant protection against AAP-induced hepatotoxicity. It decreased the levels of bilirubin although it was insignificant at both the doses which is an indication of protection against hepatic damage caused by AAP ( figure 2.4).
Plasma proteins are mainly produced by the liver, the principle exception being immunoglobulins. Severe liver damage decreases the production of various proteins resulting in reduced serum levels of total protein, albumin, and/ or globulin [39,40]. Decreased protein production may render other abnormal test values. e.g. depletion of coagulation factors (all are globulins) may result in prolonged prothrombin or activated partial thromboplastin times [41].
The results indicate that protein level was slightly increased at 250 mg/kg and 500mg/kg dose which was insignificant. The serum albumin level was also slightly and insignificantly increased at both the doses although the globulin level was decreased (table 3 and figure 3).
Through this study also the consumption of MO for 7 days was found to reduce the body weight as well as the liver weight of rats (table 1 and figure 1).

Conclusion
The above study showed that, the treatment with Moringa oleifera bark extract was able to protect the changes induced by AAP. On the basis of the above results it can be concluded that Moringa oleifera has significant hepatoprotective value against paracetamol induced liver injury.
Further studies are recommended to determine the possible mechanism(s) involved.

ACKNOWLEDGEMENTS
The author wishes to thank Pharmacology Laboratory, Jahangirnagar University, Savar, Dhaka for contribution to this research.