INTRODUCTION:

The liver is largest glandular organ in the body and weight 1.5kg making up about 2-3% of the total body weight. It is responsible for detoxifying the poisonous substances in the body by transforming and removing toxins and wastes. The liver serves a variety of functions. The most crucial is its role in the body’s metabolism. There is no organ is more important to healthy metabolism than the liver in many ways (Robbins et al., 2003).

Liver disease is a collective term for a whole group of problems that afflict the tissues, structures and cells of the human liver. The liver performs a multitude of important functions, so there’s plenty of opportunity for something to go wrong. One of the most common causes of liver disease is inflammation, which often results from abuse of alcohol, poor diet or even malnutrition (Arias et al., 1989).

Drug induced liver injury is the major health problem that challenges not only care health professional but also the pharmaceutical industry and drug regulatory agencies. According to the United States Acute Liver Failure Study Group, drug induced liver injury accounts for more than 50% of acute liver failure, including hepatotoxicity caused by over dose of acetaminophen (39%) and idiosyncratic liver injury triggered by other drugs (Micheale and Cynthiya, 2006) .

Medicinal herbs are significant source of pharmaceutical drugs. Latest trends have shown increasing demand of phyto drugs and some medicinal herbs have proven hepato protective potential. Medicinal herbs and extracts prepared from them are widely used in the treatment of liver diseases like hepatitis, cirrhosis, and loss of appetite (Nadkarni and Nadkarni, 1954).

Terminalia arjuna is a deciduous and ever green tree, standing 20-30m above ground level. It belongs to Combretaceae family (Chopra and Ghosh, 1929). It is found in Uttar Pradesh, South Bihar, Madhya Pradesh, Delhi and Deccan region near ponds and livers. Ancient Indian physicians used the powdered tree bark of Terminalia arjuna for alleviating “Hritshool” (angina) and other cardiovascular conditions. Its stem bark possesses glycosides, large quantities of flavonoids, tannins and minerals. Flavonoids have been detected to exert antioxidant, anti inflammatory and lipid lowering effects while glycosides are cardiotonic, thus making Terminalia arjuna unique amongst currently used medicinal plants.

MATERIALS AND METHODS:

Collection of plant materials:

The plant material Terminalia arjuna was collected from Trichy district which was carefully identified with the help of regional floras.

Extraction of plant material:

Aqueous extract was prepared according to the methodology of Indian pharmacopoeia. The shady dried plant materials were subjected to pulverization to get coarse powder. The coarse powder material was subjected to sox let extraction separately and successively with distilled water. These extract was concentrated to dryness in flash evaporator under reduce pressure and controlled temperature (40 – 50^oC). The aqueous extract put in air tight containers stored in refrigeration.

Phytochemical analysis:

Phytochemical analysis for major phytoconstituents of the plant extract was undertaken using standard methods .The plant was screened for the presence of biologically active compounds like sugars, amino acids, proteins, phenols, terpenoids, etc.

Collection of animal species:

The healthy young adult female rats (120-200gm sex) were kept separately in individual polypropylene cages with stainless steel hopper. The females was nulliparous and non pregnant, at the commencement of the study, the weight of the animals were minimal and not exceed ± 20% of the mean weight.

Experimental hepatotoxicity:

Isoniazid (100 mg/kg body weight) solution was prepared separately in sterile distilled water. Rats were treated with Isoniazid, administered for 10 days by IP route (Jing et al., 2004).

In order to the study the effect of aqueous extract of bark of Terminalia arjuna in rat 200mg/kg.bwt (Gupta et al., 2005). Rats were divided into four groups.

Experimental design:

Albino rats of either sex between 120-200g were randomly assigned into 4 groups of 6 animals each. Group-I (Negative control) received 1ml/kg normal saline, Group-II (Isoniazid 100mg/kg), Group-III (200mg/kg aqeous extract only) and Group-IV (treated 200mg/kg aqeous extract after induction with Isoniazid) were treated with respective treatments for 20 days. The blood samples were drawn from all the animals by puncturing retro-orbital plexus on 20^th day of the treatment. The blood samples were centrifuged immediately to get clear serum and subjected for estimation of various biochemical parameters namely SGPT (Reitman and Frankel, 1957), SGOT (King and King, 1954), ALP, ACP (Committee on enzymes of the Scandinavian society), and Serum protein (Lowry et al., 1951), Serum Bilirubin (Malloy and Evelyn, 1937), SOD (Kakkar and Viswanathan, 1998) GSH (Griffith1980).

Statistical Analysis:

The value are reported are means + SD (n=6). Experimental results were statistically analyzed using the variance (ANOVA) by student t’ test.

RESULTS AND DISCUSSION:

Preliminary phytochemical Screening:

The plant extract of Terminalia arjuna were screened for the presence of biologically active compounds like steroids, tannins, phenolics compound, quinone, terpinoids, sugar, alkaloids and flavonoids (Table 1).

Table I : Preliminary phytochemical investigation in the aqueous extract of bark of Terminalia arjuna

Phytochemical compound	Result of qualitative test
Sugar	+
Terpinoids	+
Alkaloids	+
Phenolic compounds	+
Tannins	+
Flavanoids	+
Volatile oil	-
Quinones	+
Steroids	+
Coumarins	-

+ indicates presence; - indicates absence

Serum Protein:

A significant increase (p<0.01) in serum protein level was noted in group III when compared to Group II. Also there was a significant increase (p<0.05) in serum protein level in group IV when compared to Group II. Thus the restoration of the level of serum and tissue protein after the administration of Terminalia arjuna confirmed the hepatoprotective nature of it (Table II).

Previous studies on the hepatoprotective effect of Terminalia chebula extract against antipyretic drug induced hepatotoxicity proved that the extract exhibited hepatoprotective nature by restoring the total serum protein level (Gujarti et al., 2006).

Serum Bilirubin:

A significant increase (p<0.05) in serum bilirubin levels in Group II was observed when compared to Group I. A significant decrease (p<0.05) in serum bilirubin level was noted in group IV when compared to Group II. But there was no significant change when Group I was compared with Group IV (Table II).

Bilirubin concentrations have been used to evaluate chemically induced hepatic injury (Giuseppe cooper et al., 2004). Treatment with Terminalia arjuna extract reduced the elevated level of bilirubin. This is probably due to the presence of highest content of phenolic derivatives that exert bile flow and liver protection. Thus Terminalia arjuna has cholertic activity.

Table II : Estimation of Serum Proteins and Bilirubin

S. No	Groups	Proteins (mg/g)	Bilirubin (µm/mg)
1.	Group I	0.91±0.4	1.59±0.91
2.	Group II	0.61±0.3	4.23±1.24
3.	Group III	0.79±0.9	1.88±0.45
4.	Group IV	0.88±0.1	2.02±1.31

Values are means ± SD from 6 rats in each observation.

*<0.05; **<0.01; as compared with Group II. +<0.05,

as compared with normal group.

Serum ALP:

Serum level of ALP were increased significantly in group II rat (p<0.05) when compared to Group I. A significant decrease in ALP levels were observed in Group III (p<0.01) and Group IV (p<0.05). (Table III). Elevated levels of serum and tissue alkaline phosphates are indicative of cellular leakage and loss of functional integrity of cell membrane in liver cells (Sethu mathavan et al., 2007). Damage to liver cells cause leakage of cellular enzymes into serum.

In the present study the ALP levels returned to normal by administration of Terminalia arjuna with healing of parenchyma and regeneration of hepatocytes. Previous study on Solanum nigrum reduced the elevated level of ALP in CCl₄included hepatotoxicity (Ruiz et al., 2008).

Serum ACP:

A significant increase (p<0.05) in the levels of serum acid phosphatase level was observed. A significant decrease (p<0.01) in serum acid phosphatase level was seen in Group III when compared to Group II. Similarly, a significant decrease (p<0.05) in ACP levels was noted in Group IV when compared to Group II. But no significant changes were observed between Group I and Group IV (Table III).

The abnormal higher level of marker enzymes such as ACP and bilirubin in the serum of CCl4 treated rat indicate damage to hepatic cells (Venukumar and Latha, 2002).In the present study, treatment with Terminalia arjuna extract reduced the elevated levels of ACP . Glycosmis arborea extract overcome the toxic effects of hepatotoxic agents by the lowering the levels of ACP (Gomes et al., 2003).

Table III : Estimation of Serum ALP and ACP

S. No	Groups	Serum ALP (KA Units)	Serum ACP(KA Units)
1.	Group I	13.96±0.78	5.68±0.29
2.	Group II	22.92±0.16	11.82±0.31
3.	Group III	15.28±0.98	5.93±0.11
4.	Group IV	19.96±0.21	9.86±1.05

Values are mean ± SD from 6 rats in each observation. *<0.05; **<0.01; as compared with Group II.

+<0.05, as compared with normal group

SGOT:

A significant increase in SGOT activity (p<0.05) was noted in Group II when compared to Group I. A significant decrease (P<0.05) in serum SGOT level was observed in Group III when compared to Group II. Also a significant decrease (P<0.05) in serum SGOT level in Group IV was noted when compared to Group II (Table IV).

The Isoniazid has got hepatotoxic potentical (Yew et al., 2006). Isoniazid metabolite hydrazine plays an important role in inducing hepatotoxicity (Al-Howiriny et al., 2004). Isoniazid hepatotoxicity results in hepatocellular damage, thus variety of enzymes normally located in the cytosol are released into blood stream. The elevated level of SGOT due to Isoniazid hepatotoxicity was normalized by treatment with Terminalia arjuna. This is probably because of free radical scavenging activity of flavonoids and polyphenols present in Terminalia arjuna.

SGPT:

An increased level of SGPT (p<0.05) was found in Group II rat when compared with Group I. A significant decrease (p<0.01) in serum SGPT level was noted in Group III when compared to Group II. Also there was a significant decrease (p<0.05) in serum SGPT level in Group IV when compared to Group II. But there was no significant change between Group I and Group IV (Table IV).

The normalization of plasma alanine amino transferase (SGPT) has been proved to be a strategy for preventing the development of hepatocellular carcinoma in hepatitis C virus (HCV) infection. In the present study treatment with Terminalia arjuna extract normalized the elevated SGPT levels. Osthole, a simple coumarin caused strong reduction of plasma SGPT and exhibited hepatoprotective nature (Okamoto et al., 2005).

Table IV: Estimation of Serum SGOT and SGPT

S. No	Groups	Serum SGOT (IU/L)	Serum SGPT (IU/L)
1.	Group I	16.28±2.09	6.96±0.75
2.	Group II	32.54±1.28	12.33±0.58
3.	Group III	17.29±1.33	6.99±0.55
4.	Group IV	19.36±2.92	7.38±1.29

Values are mean ± SD from 6 rats in each observation. *<0.05; **<0.01; as compared with Group II. +<0.05, as compared with normal group.

SOD:

There was a significant decrease (p<0.05) in SOD levels in Group II when compared to Group I. A significant increase (p<0.01) in serum SOD level was noted in Group III when compared to Group II. Also there was a significant decrease (p<0.05) in serum level was noted in Group IV when compared to group II. But there was no significant change between Group I and Group IV (Table V).

SOD is very effective antioxidant enzyme and responsible for catalytic disputation of highly reactive and potentially toxic superoxide radicals to H2O2.The H2O2 is further metabolized either by catalase or peroxidase. The non enzymatic antioxidants such as GSH, Vit.E and Vit.C act as scavengers (Foyer et al., 1993).

GSH:

There was a significant decrease (p<0.05) in level of Reduced Glutathione in Group II when compared to Group I. A significant increase (p<0.01) in level of Reduced Glutathione was noted in Group III when compared to Group II. Also there was a significant increase (p<0.05) in level of reduced glutathione in Group IV. When compared to Group II. But there was no significant change between Group I and Group IV.

GSH in the cytosolic pool consists of 85% hepatocellular GSH and 15% mitochondrial GSH. Hepatic GSH depletion or even extra hepatic GSH depletion can provide useful information on the protective role of GSH against toxic foreign compounds. Thus GSH, be regarded as an endogenous protective agent against drugs (Chattopadhyay, 1992).

Table V : Estimation of Serum SOD and GSH

S. No	Groups	Serum SOD (mg/dl)	Serum GSH (mg/dl)
1.	Group I	5.33±0.97	4.38±0.22
2.	Group II	4.14±0.28	2.23±0.24
3.	Group III	5.30±0.21	4.78±0.45
4.	Group IV	5.16±2.01	5.15±1.31

Values are mean ± SD from 6 rats in each observation. *<0.05; **<0.01; as compared with Group II. +<0.05, as compared with normal group.

CONCLUSION:

The present findings demonstrated the hepatoprotective and antioxidant effect of Terminalia arjuna aqueous bark extract against Isoniazid induced hepatotoxicity. According to traditional indigenous medicinal systems of India this plant has got several medicinal effects without producing any severe side effects. This plant could be very well used as hepato protectant. However this is only a tentative research and more work is to be done to identify the extract phytochemical responsible for this curative effect.

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Received on 30.12.2011 Accepted on 10.01.2012

Asian J. Pharm. Tech. 2(1): Jan.-Mar. 2012; Page 15-18

P.G. Department of Biochemistry, S.T.E.T Women’s College, Mannargudi – 614 601.

*Corresponding Author E-mail: elangani576@ gmail.com