INTRODUCTION:

Liver is one of the largest organ in the human body and perform several vital activities and its role in metabolism makes it susceptible to various types of hepatic diseases.¹Liver damage is always associated with cellular necrosis, increase in lipid peroxidation and reduction in the tissue GSH levels. In addition serum levels of many biochemical markers like AST, ALT, ALP and bilirubin levels are elevated². After numerous developments in medical science, liver diseases are still a worldwide health problem^3. Unfortunately conventional or synthetic drugs used in the treatment of liver diseases are inadequate and sometimes can have serious side effects^4.Therefore it is necessary to search for alternative drugs in order to replace currently used drugs. A single drug may not be effective for all types of severe liver diseases. So an effective formulation has to be developed using indigenous medicinal plants^5.

Liver injuries induced by D-galactosamine are the best characterized system of xenobiotics induced hepatotoxicity and commonly used models for the screening of anti–hepatotoxic and/or hepatoprotective activities of drugs^7.

In this study, an attempt has been made to develop and evaluate the hepatoprotective activity of a polyherbal preparation consisting of methanolic extracts of dried rhizomes of Curcuma longa (C ), dried leaves of Murraya koenigii (M), Nyctanthes arbortristis (N) and Occimum sanctum (O), against d-galactosmine induced hepatotoxicity in albino rats. Curcuma longa which consists of curcumin is known to possess antioxidant, hepatoprotective, antimicrobial, anti-inflammatory effects.^{8-9. .}Murraya koenigii consists of carbazole alkaloid and is used for its antibacterial, analgesic, hepatoprotective, antidiabetic, hypolipidemic activities^10-11.Nyctanthes arbotristis which contains flavanol glycosides is used for its antipyretic, hepatoprotective antimicrobial, immunomodulating properties^12-13.Occimum sanctum consists of mainly eugenol and is also known as Queen of Herbs. Its extracts are used in ayurvedic remedies for common cold, headache, stomach disorders, heart disease, and hepatic disorders etc^14-15

MATERIALS AND METHODS:

Collection and preparation of plant extract

The plant material was procured from a market in Old Delhi and authenticated by Dr. H.B. Singh at NISCAIR, New Delhi. The quality of plant material was established as per monographs in Indian Herbal Pharmacopoeia, Indian Pharmacopoeia and Ayurvedic Pharmacopoeia of India. Plant material was dried and coarsely powdered. Weighed quantity of the drugs was first defatted with petroleum ether and then extracted separately in soxhlet with methanol. The extracts were concentrated to dryness in a rotary vacuum evaporator and dried in lyophilizer under reduced pressure.

Phytochemical screening and standardization of extracts

After concentration and drying of each extract, identification of phytoconstituents was carried out using thin layer chromatography with different detecting reagents¹⁶.The extracts were further standardized using TLC.

Experimental animals

Swiss albino rats and mice of wistar strain with body weight 110-150 gm and 20-25 gm, respectively of either sex were used. They were kept in departmental animal house at 26+ 2 degree Celsius and relative humidity 44-56% with light and dark cycle of 12 hour in polypropylene cages. Animals were provided with standard rodent pallet diet and water ad libitum. All the procedures were reviewed and approved by IAEC.

Acute toxicity studies

Acute toxicity studies were performed according to organization for economic cooperation and development (OECD) guidelines¹⁷.

Mice weighing between 20-25 gm were used. The animals were fasted overnight with free access to water only. All the extracts were administered orally in dose range of 50 to5000 mg/kg to different groups of mice and observed for over 48 hours for mortality and physical/behavioral changes

Since no mortality was observed at any of these dose level, 1/10 th of maximum dose could be used.

Hepatoprotective activity

Optimization of doses

Preliminary testing was done in mice by performing pentobarbitone sleeping time(PST) ¹⁸.Hepatotoxicity was induced by paracetamol(2 gm/kg) This functional parameter was used to determine metabolic activity of liver

All the four extracts were mixed in different ratios .These extracts were administered to mice for seven days Hepatotoxicity was induced by paracetamol. Pentobarbitone (45 mg/kg) was administered and pentobarbitone sleeping time was determined

On the basis of PST, extracts (CMNO) in the ratio of 1:1:2:1 were chosen. Hepatotoxicity was induced with d-galactosamine (400mg/kg i.p.) in rats

Experimental Design

All the plant extracts were individually weighed and mixed properly. The drugs were administered by oral route, suspended in 1% CMC. Animals were divided into six groups, each group containing 6 animals.

Group I- Vehicle (1% CMC suspension)

Group II- Vehicle (1% CMC suspension)

Group III- CMNO, 50 mg/kg

Group IV- CMNO, 100 mg/kg

Group V- CMNO, 250mg/kg

Group VI- Sylimarin 100mg/kg

All the groups received their respective treatments for 10 days. On tenth day, after the last dose groups II-VI received galactosamine 400mg/kg by i.p. route. After 24 hours, blood was withdrawn from retro orbital plexus. Serum was separated out from whole blood by centrifugation at 3000 rpm for 15 minutes and was subjected to estimation of biochemical parameters like Aspartate aminitranferase (AST), Alanine aminotransferase (ALT), Alkalinephosphatase (ALP), total protein, total and direct bilirubin. Rats were then killed. Liver was removed and weighed. Part of the liver was fixed in 10% formalin solution for histopathological studies. Part was homogenized in cold phosphate buffer (PH-7.4) using homogenizer.MDH and GSH were estimated in liver homogenate.

Statistical analysis

All the values are expressed as mean + SEM. The data was analyzed by one way ANOVA followed by Dunnett’s t-test. p<0.05 was considered statistically significant.

RESULTS:

Intoxication with D-gal N significantly altered parameters when compared with normal rats (P<0.001).Treatment with methanolic extract of polyherbal preparation at 250 mg/kg body weight induced significant decrease in serum levels of AST, ALT, ALP, TB (P<0.001, P<0.01, P<0.05) and a significant elevation in serum level of total protein (P<0.001) when compared with D-galN treated rats (Table 1). Levels of GSH and MDH estimated in liver homogenate were also restored to normal. (p<0.001) (Table 2)

Histopahological examination of liver tissues of control group showed normal hepatic cells with a central vein.D-galactosamine treated group showed severe hepatic lesions which showed signs of necrosis. Administration of polyherbal preparation showed mild to moderate signs of necrosis (Fig. 1)

Treatment with silymarin (100 mg/kg) also exhibited similar results.

DISCUSSION:

Various xenobiotics are known to cause hepatotoxicity¹⁹.One among them is d-galactosamine. D-gal N induced liver toxicity is a useful model for the study of hepatic injury. It has been shown to produce liver damage resembling human viral hepatitis. D-galN induces a decrease in liver uracil nucleotides which causes a rapid inhibition of both RNA and protein synthesis. Rapid depletion of uridine diphosphate glucose appears to be the first biochemical lesion followed by defects in macromolecular glycoprotein synthesis which leads to progressive damage of cellular membranes and finally to liver cell necrosis. This cellular damage provokes inflammatory reactions resulting in a picture which resembles viral hepatitis²⁰.

Table 1: Effect of Polyherbal Preparation (PP) on biochemical parameters on D-galactosamine induced hepatotoxic rats

Experimental Groups	ALT U/L	AST U/L	ALP U/L	Total Bilirubin (mg/dl)	Direct Bilirubin mg/dl)	Total Protein (mg/dl)
Group I control	886.53+1.23	801.83+6.57	329.50+2.24	0.043+0.02	0.24+0.01	6.25+6.12
Group II D-Galac	1101.42+9.63+++	997.15+3.21+++	585.12+5.45+++	0.098+0.02++	0.78+0.07++	4.26+2.7+++
Group III PP 50mg/kg+D- Galac	940.12+5.11^*	952.23+4.15*	540.50+5.98^*	0.062+O.04**	0.71+0.05*	4.75+1.19*
Group IV PP 100 mg/kg + D-Galac	901.12+8.26^**	894.60+9.80**	506.01+1.58^*	0.051+0.09^**	0.49+0.03^**	5.75+6.53**
Group V PP 250 mg/kg + D-Galac	880.57+6.31^***	841.62+4.26***	413.47+4.66***	0.048+0.07***	0.33+0.09^***	6.10+8.76***
Group VI Silymarin100mg/kg+D-Galac	891.97+2.57^***	829.24+8.35^***	431.61+11.04^***	0.044+0.05^***	0.29+0.05***	6.29+9.43***

Values are mean +S.E.M n=6.Data was analyzed using one way ANOVA followed by Dunnets t –test

***Values are statistically significant at p< 0.0001 compared to d- galactosamine treated group

** Values are statistically significant at p< 0.001 compared to d- galactosamine treated group

* Values are statistically significant at p< 0.05 compared to d- galactosamine treated group

+++p<0.0001 compared to control group

++ p<0.001 compared to control group

Table 2: Effect of Polyherbal Preparation(PP) on hepatic GSH and MDA levels in D-galactosamine induced hepatotoxic rats

Experimental groups	Hepatic MDA nmol/g	Hepatic GSH mg/g
Group I Control	29.58+3.57	11.47+ 3.57
Group II D-Galac	55.67+7.28⁺⁺⁺	4.1+ 5.54⁺⁺⁺
Group III PP 50 mg/kg+D- Galac	50.26+1.43^*	4.9 +6.75^*
Group IV PP 100 mg/kg + D-Galac	43.42+5.56^**	6.23+ 8.78^**
Group V PP 250 mg/kg + D-Galac	36.18 +6.53^***	9.56+1.76^***
Group VI Silymarin 100 mg/kg+D-Galac	34.25 + 7.41^***	9.45+ 2.43^***

Values are mean +S.E.M n=6.Data was analyzed using one way ANOVA followed by Dunnets t –test

***Values are statistically significant at p< 0.0001 compared to d- galactosamine treated group

** Values are statistically significant at p< 0.001 compared to d- galactosamine treated group

* Values are statistically significant at p< 0.05 compared to d- galactosamine treated group

+++p<0.0001 compared to control group

Fig 1: Effect of PP on acute liver injury induced by D-Galactosamine

A- Control: liver section with normal structure and architecture

B- D-GalN treated: disturbances in the lobular arrangement. Degenerative and early necrotic changes. Ballooning degeneration and steatotic changes.

C- PP 50 mg/kg :slightly reduced degenerative changes and steatosis

D- PP 100 mg/kg limited area of necrosis degenerative changes and steatosis

E- PP 250 mg/kg : typical lobular arrangement and intact architecture. Few hepatocytes showed steatotic accumulation and regenerative hepatocytes

Hepatocelular necrosis leads to increased serum levels of transaminases enzymes, AST and ALT which are released from liver into the blood. Alkaline phosphate (ALP) activity is related to functioning of hepatic cells. Increase in the serum level of ALP is due to increased synthesis in presence of increasing biliary pressure. In damaged liver bilirubin metabolism is disturbed due to increased formation of bilirubin, abnormal uptake of bilirubin, defective conjugation and failure of normal amounts of bile to reach duodenum, a condition called cholestasis²¹. Polyherbal preparation, 250mg/kg significantly reduced the levels of ALT, ASP, and ALP and Total biliurbin.

There is increasing evidence that D-gal also causes production of free hydroxyl radical leading to lipid peroxidation, which was seen as increased levels of MDA in liver homogenate of rat treated with D-gal²². GSH is sulphur containing nonenzymatic antioxidant molecule, which conjugates directly with free radicals. Reduction in concentration of GSH in the study may be related to excessive production of free radicals generated by D-gal. This antioxidant enzyme is exhausted by reactive oxygen species which results in decreased activity of this enzyme in D-gal induced liver toxicity.²³ Polyherbal preparation; 250mg/kg significantly restored the levels of MDA and GSH back to normal.

Histological profile of the liver of D-gal treated rats showed necrosis and fatty degeneration. The animals treated with polyherbal preparation (250mg/kg) showed significant improvement as is evident from the presence of normal hepatic cords, absence of necrosis and lesser degree of fatty infiltration. These observations are correlated with the biochemical results and gives clear evidence that there was not only improvement in liver functions with treatment of polyherbal preparation but also in hepatic architecture.

All the findings were comparable to sylimarin 100 mg/kg, which was taken as a standard drug.

CONCLUSION:

From the present studies it can be concluded that the polyherbal formulation designed and developed for the treatment of liver diseases has shown a significant hepatoprotective activity at the dose level of 250mg/kg p.o. Once daily.

ACKNOWLEDGEMENT:

The author is thankful to the management of R.K.G.I.T., Ghaziabad for providing facilities to carry out the studies.

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Received on 20.05.2013 Modified on 05.06.2013

Research J. Pharm. and Tech. 6(9): September 2013; Page 1079-1082