HEPATOPROTECTIVE EFFECT OF METHANOLIC LEAVE EXTRACTS OF V. ALBUM ON PARACETAMOL-INDUCED LABORATORY ANIMALS

Hepatoprotective assay was carried out on laboratory animals in which paracetamol (oral dose,2 g/kg /body weight) was used to induce hepatotoxicity. The animals were orogastrically fed with the Viscum album (growing on cocoa and cola host trees) methanolic leave extracts. The activities of serum enzyme biomarkers showed no significant difference (P< 0.05) for V. album growing on cocoa (1000-5000mg/kg) but there were corresponding significant increase in the serum enzymes (P< 0.05) for V. album growing on cola at 4000 and 5000 mg/kg doses. Similarly, the photomicrograms of the animals’ vital organs (liver, stomach, small intestine and kidneys) studied revealed that V. album extract growing on cocoa was more hepatoprotective efficacious than that growing on cola tree on all the biochemical parameters that were screened for when compared with sylimarin (a standard heptoprotective drug).

analysis. Some of the leaves were also ground fresh using electric blender and were stored (at 4 o C) in refrigerator in a well labeled air-tight containers for analysis. A 100 g of each sample was weighed (using electric weighing balances) into different beakers and 1000 ml of 60% methanol was added separately. This was homogenized in a warring blender until a homogenate results and then concentrated in vacuo using a rotary evaporator. It was reconstituted using 35 % dimethyl sulphoxide (DMSO) 1:50(w/v) for the analysis. The extracts were sterilized using 0.45 µm millipore membrane filter.

2.2
Hepatoprotective studies Animals divided into five groups of six rats (albino rats, Wister's strain) each were used for the study using the method of Setty et al. (2007). Groups 1 and 2 were maintained only on the basal diet and sterile water for 7 days. Groups 3, 4 and 5 were orogastrically dosed, each with 1000 mg/kg of silymarin, V.album extracts grown on cocoa and that grown on cola respectively, once a day for 7 days. On the fifth day, after the administration of the respective treatments, all the animals in groups 2, 3, 4, and 5 were administered 2 g/kg paracetamol (PCM) orally. On the seventh day, the blood samples were collected via cervical dislocation for the estimation of biochemical marker enzymes. The percentage of the protection is calculated as 100 X (values of PCM control -values of test sample) ÷ (values of PCM control -values of normal control).

Biochemical Assay of Blood Serum
The blood samples were allowed to coagulate for 30 min and the clear serum was separated by centrifuging at 2500 rpm for 10 min and was then used for the analysis of biochemical hepatic markers-total bilirubin (Jendrassik et al.,1938),aspartateaminotransferase (AST) (Reitman and Frnakel, 1957), alanine aminotransferase (ALT) (Reitman and Frnakel, 1957) and alkaline phosphatase (ALP) (Roy, 1970). Reflotron (Boehringer Marnherm Company, Germany) was used for the analysis of some major serum biochemical markers that can reveal the effect of the extracts on the internal organs of rats. Serum total protein and Albumin/Globulin ratio level of the serum were also determined. The general procedure involves pipetting standardized amount of sample that was then applied on the test zone of the appropriate test strip. The strips were inserted into the test chamber and the flap closed. The results were then displayed after some seconds on the frame computer monitor. Test was carried out at 25 o C.

2.4
Histopathological studies of selected organs Post-mortem examination was carried out on section of the organs; liver, kidney, stomach and small intestine of albino rats. The blood was rinsed in normal saline and preserved in 10 % formalin solution. Thereafter, sub-sections were taken from each selected organ, fixed in 10% formol saline and dehydrated in ascending grades of ethanol (60%, 70%, 80% and 90%). The specimens were cleared in xylene and embedded in paraffin. The tissues were then processed into 4-5 µm thick sections with the aid of a microtome and thereafter stained with hematoxylin and Eosin, viewed under the light microscope, and the photomicrograms were taken (Carleton, 2007).

International Journal of Pharmacology, Phytochemistry and Ethnomedicine
Vol. 1

Discussion
Paracetamol is a known antipyretic, analgesic drug which produces hepatic necrosis at high doses and normally eliminated as sulfate and glucuronide conjugate. Results (Table 1) obtained from the hepatoprotective profile of the extract grown on cocoa, on biochemical parameters in albino rats show no significant difference (P < 0.05) when compared with the control at all concentration used (1000 -5000 mg/kg). However, there was a significant difference (P < 0.05) at concentrations 4000 and 5000 mg/ml of the extracts grown on cola. This is an indication that extracts from cola might create mild hepatic injury at a very high dose or if it is used 52 IJPPE Volume 1 indiscriminately (Yusuf, 2015). Eno et al. (2004) had also reported LD50 of 4170.5 mg/kg on albino rats injected with V. album leaves extract intraperitoneally. Though, they did not document the host plant.
In administration of toxic doses of paracetamol, the sulfation and glucuronidation routes become saturated and hence, higher percentages of paracetamol molecules are oxidized to highly reactive N-acetyl-p-benzoquinemine by cytochrome-450 enzymes.The Semiquinone radicals, obtained by one electron reduction of N-acetyl-p-benzoquineimine, can covalently binds to macromolecules of cellular membrane which increase the lipid peroxidation resulting in the tissue damage. Higher doses of PCM and N-acetyl-p-benzoquineimine can alkylate, oxidise intracellular glutathione (GSH), results in the depletion of liver GSH pool subsequently leads to increased lipid peroxidation thereby causes liver damage. In the assessment of liver damage by PCM, the determination of enzyme markers; Aspertateaminotransferase(AST), Alanineaminotransferase (ALT), Alkalinephosphatase (ALP) and total bilirubin were largely used. The results are shown in Tables 2 and 3. Liver necrosis or membrane damage releases the enzyme into circulation which can be measured in serum. A high level of AST indicates liver damage, as well as cardiac infarction and muscle injury. ALT catalyses the conversion of alanine to pyruvate and glutamate and is released in a similar manner. Therefore, ALT is more specific to the liver, and is thus a better parameter for detecting liver injury. Elevated levels of serum enzymes were indicative of cellular leakage and loss of functional integrity of cell membrane in liver (Oyetayo and Osho, 2004). Serum ALP and bilirubin level on other hand are related to the function of hepatic cell. Increase in serum level of ALP is due to increased synthesis, in presence of increasing biliary pressure. It is evident that several phytoconstituents have the ability to induce microsomal enzymes either by accelerating the excretion of the hepatotoxin or by inhibition of lipid peroxidation induced by it (Mehta et al., 1999). Phytoconstituents like flavonoids (Baek et al., 1996) and (Pandit et al., 2004), triterpenes (Xiong et al., 2003), saponins (Tran et al., 2001) and alkaloids (Vijyan et al., 2003) are known to possess hepatoprotective activities. These phytochemicals had been reported to be present in the extracts (Yusuf, 2013b).
The results of the comparative histopathology of the livers, stomachs, intestines and kidneys of the experimental animals are shown in the photomicrograms (Plates 1-4). It was observed that the V. album treated liver showed normal morphology without necrosis when compared with the control groups. In contrast, paracetamol treated liver section showed haemorrhagic hepatitis, leucocytic infiltration and contrasting features. Similarly, the stomach showed gastritis, section of the intestine (jejunum) showed ulceratic and infiltration of cells and section of kidney nephron showed interstitial dialation and conjested medulla. Results from this finding further give insight to the therapeutic effect of V. album leaves extracts. The plant extracts was able to improve the health of the experimental animals and found to be toxicologically safe (Yusuf et al., 2013).
V. album leaves extract demonstrated significant (P<0.05) liver protection against the hepatic injuries caused by the paracetamol hepatotoxins.