HEPATOPROTECTIVE ACIVITY OF THE ETHANOLIC EXTRACT OF DOG FRUIT RIND ( Pithecellobium lobatum Benth . )

The present study was conducted to investigate the hepatoprotective activity of ethanolic extract of dog fruit rind (Pithecellobium lobatum benth.) against carbon tetrachloride (CCl4) induced hepatotoxicity in rats. Male Wistar albino rats were randomly divided into four groups and administered orally with 50 mg/200 g body weight of dog fruit rind extract (K1), 100 mg/200 g body weight (K2) of dog fruit rind extract, 5 mg/200 g body weight of silymarin (K3/positive control), and 0.4 mL/200 g body weight of distilled water (K4/negative control), for seven days The levels of alanine transaminase (ALT) of each K1; K2; K3; and K4 were 143.40±83.75 U/L, 94.80±93.77 U/L, 130.20±58.54 U/L and 147.25±107.97 U/L, respectively, while the aspartate transaminase (AST) levels were 304.20±128.67 U/L; 213.20±88.93 U/L; 333.00±128.31 U/L; and 239.25 ± 94.90 U/L, respectively (P>0.05). Group K2 showed better histological pattern than other groups with 60% of mild and 40% of moderate liver damage. Our findings revealed the hepatoprotective activity of the ethanolic extract of dog fruit rind. ____________________________________________________________________________________________________________________


INTRODUCTION
Liver is the most important vital organ in the human body.It plays major role in metabolism of nutrition i.e. carbohydrate, protein, and lipid.In addition, drugs and xenobiotic are also metabolized and excreted via liver, making it vulnerable to damage.Some studies reported that acetaminophen, carbon tetrachloride (CCl 4 ), and alcohol produced liver damage (Dash et al., 2007;Arun and Balasubramanian, 2011;Panjaitan et al., 2013).Liver damage is indicated by changes of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase, bilirubin, total protein levels, a necrotic, and inflammation microanatomy pattern of the liver (Dash et al., 2007;Arun and Balasubramanian, 2011;Domitrović et al., 2011;Sengupta et al., 2011;Eidi et al., 2012;Panjaitan et al., 2013).

Experimental Animals
The study was carried out using male Wistar albino rats (200-250 g) aged 2-2.5 months old.The animals were acclimated for seven days before the experiment.We observe the rat health by weighing their body weight and feeding them ad libitum.

Extraction and Partition
Five kg of dog fruits were collected from traditional market in Pontianak and the seed was separated from the rind.A total of 2.43 kg of the rind was obtained and dried under the sunlight until the weight was 630 g, and it was then crushed into pieces.The pieces of dog fruit rind were macerated for 48 h in ethanol 96% at room temperature based on Harborne (1998).Maceration was performed twice by adding new ethanol.The extract filtrate was concentrated until it weighs 72.75 g.

Hepatoprotective Activity Assessment
The rats were randomly divided into four groups of five animals per each group.Treatment was carried out according to the following groups for seven days: Group K1-K2 (the extract groups) received 50 mg/200 g body weight (K1) and 100 mg/200 g body weight (K2) of ethanolic extract of dog fruit rind; Group K3 (positive control) received silymarin at the dose of 5 mg/200 g body weight; and Group K4 (negative control) received 0.4 mL/200 g body weight of distilled water.On 8 th day, as much as 0.02 mL/200 g of CCl 4 was administered to all group.Twenty-four hours after CCl 4 administration (the 9 th day), blood and liver organ were obtained from all groups.Parameters analysed were ALT and AST levels, as well as liver histopathology.

Biochemical Liver Function Analysis
The blood samples were collected from the heart.Serum was separated by centrifugation at 4000 rpm for 5 min and collected into eppendorf tube for further biochemical parameters analysis using a certain kit (Analyticon ® Biotechnologies AG, Germany).

Histological Studies
The rats were sacrificed by cervical dislocation and their liver was carefully removed followed the routine process.The slides were stained with hematoxylin and eosin for pathological analysis (Kiernan, 1990).The score was examined under light microscope (Table 1).

Data Analysis
The ALT and AST data were analyzed using analysis of variance then followed by Duncan test.Liver histophatology was analysed descriptively.

RESULTS AND DISCUSSION
The levels of ALT and AST of rats in K1, K2, K3, and K4 is presented in Table 2 As illustrated in Figure1, histological assessment of the liver sections revealed that injection of CCl 4 induced pathological changes with different level of damage.About 60% of K4 group showed moderate damage (score 2), whereas the rest 40% showed severe damage (score 3).The rats treated with the extract at dose of 50 mg/200 g produce similar protection as silymarin group, in which mild and moderate damage developed in 40% rats, while 20% of rats showed severe damage.Administration of 100 mg/200 g of ethanolic extract of dog fruit rind attenuate liver damaged, and was indicated by mild damage in 60% rats and moderate damage in the other 40% rats.The score was significantly lower than the group given standard drug, silymarin, which generate mild damage only in 20% of rats, whereas 60% had moderate liver damage, and the other 20% had severe liver damage.
Histopathological examination of liver sections confirmed our biochemical finding.This research describes the potential protective effect of ethanolic extract of dog fruit rind against carbon tetrachloride (CCl 4 ) induced hepatotoxicity.Domitrović et al. (2011) stated that CCl 4 induced liver damaged involved  ).Free radicals bind to hepatocyte membrane and cell organelles leading to lipid peroxidation and unbalanced calcium that further cause cell death.Moreover, Stockham and Scott (2002) revealed one of the techniques to detect, determine the cause, and assessing the severity of liver disease is by analyzing liver biochemical enzyme, including AST and ALT.In addition, liver abnormality can be detected by histopathological examination.
Alanine transaminase is a cytosolic enzyme involved in gluconeogenesis.The increase in ALT levels in the blood indicates hepatocyte damage.Aspartate transaminase is also involved in gluconeogenesis and elevated levels of AST in the blood indicates advanced hepatocyte damage accompanied with necrosis which release the mitochondrial enzyme (Shanmugasundaram and Venkataraman, 2006;Panjaitan et al., 2013).In relation with ALT and AST levels in blood, Panjaitan et al. (2007) revealed that administration of 0.1 mL/kg CCL 4 induce multifocal cells degeneration and necrosis in the liver and hepatocyte alteration is directly proportional to the dose.Moreover, the administration of 0.1 mL/kg CCl 4 produce extensive and severe hepatocyte damage that lead to very low availability of ALT and AST levels in hepatocyte.
Hepatoprotector is an agent that produces protective and regenerative effects on toxic induced liver damage.It has been reported that there is a relationship between hepatoprotective and antioxidant effect.Moreover, the activity of medicinal substances related to their chemical compounds (Panjaitan et al., 2013;Singh et al., 2014).The chemical compounds in dog fruit rind encompass alkaloid, flavonoid, tannin, quinone (Syafnir et al., 2014) and polyphenol (Syafnir et al., 2014;Yanti et al., 2015).Particularly, flavonoid (Syafnir et al., 2014) and polyphenol (Syafnir et al., 2014;Yanti et al., 2015) have antioxidant activity against free radicals.Therefore, the hepatoprotective activity of ethanolic extract of dog fruit rind is associated to the antioxidant activity of the compound they contain.

CONCLUSION
Ethanolic extract of dog fruit rind at dose of 100 mg/200 g have hepatoprotective activity against CCl 4 induced liver damage in rat.
necrosis and steatosis since the process of CCl 4 biotransformation in the liver by P450 cytochrome reductase, with NADPH as cofactor, formed free radicals i.e. trichloromethyl (

Table 1 .
Liver damage scoring system

Table 2 .
Mean ALT and AST levels in blood serum of male albino rats administered with ethanolic extract of dog fruit rin .