1. INTRODUCTION:

Liver is the key organ of metabolism and excretion is constantly endowed with the task of detoxification of xenobiotics, environmental pollutants and chemotherapeutic agents. Thus, disorders associated with this organ are numerous and varied¹. Liver disease has become a global concern worldwide. Liver is often abused by environmental toxins, poor eating habits, alcohol and over-the-counter drug use, that damage and weaken the liver leading to important public health problems like hepatitis, cirrhosis and alcoholic liver diseases². The conventional drugs used in the treatment of liver diseases viz., corticoasteroids, antiviral and immunosuppressant agents are sometimes inadequate and may lead to serious adverse effects. In India, numerous medicinal plants and their formulations are used for liver disorders in traditional systems of medicine. Some of these plants are evaluated for their hepatoprotective actions against hepatotoxins.

However, the readily available hepatoprotective herbal drugs are not sufficiently active to effectively combat severe liver disorders. In view of lack of synthetic agents for the treatment of hepatic disorder, there is a growing focus to evaluate traditional herbal medicines for hepatoprotective activity.³ Therefore; there is a need to develop satisfactory hepatoprotective drugs.

2. MATERIALS AND METHODS:

2.1 Plant Materials: - The leaves of Ardisia solanacea (Myrsinaceae) were collected from Thakur Chedilal Barristor Agriculture College and Research Centre, Bilaspur, India, in the month of September 2011, and air dried at room temperature after wash with tape water. The Plant identification was done by Dr. H. B. Singh Chief Scientist Head of the Raw Materials Herbarium & Museum, NISCAIR, New Delhi (Ref.-NISCAIR/RMHD/Consult/ 2011-12/1812/112).

2.2 Drugs and Chemicals: - Analytical grades Chemical were used in this study. Silymarin (Micro labs, Bangalore) was purchased from local market. Chemical like ethanol (CDH, Mumbai), anesthetic ether (CDH, Mumbai), CCl₄ (Ranbaxy, Delhi) and other phytochemical reagents were obtained from Institute. For estimation of biochemical parameter; biochemical kits like SGOT, SGPT, ALP, albumin, total protein, direct bilirubin and total bilirubin were obtained from Span Diagnostics ltd. Surat, India were procured from Matushri Trading Company, Bilaspur.

2.3 Animals: - Each experiment had separate set of animals and care was taken to ensure that animals used for one response were not employed elsewhere. Animals were habituated to laboratory conditions for 48 hours prior to experimental protocol to minimize if any of non-specific stress. The approval of the Institutional Animal Ethical Committee (IAEC) of SLT Institute of Pharmaceutical Sciences, Bilaspur (Chhattisgarh) was taken prior to the experiments (Reference No. IAEC/Pharmacy/2012/36). All the protocols and the experiments were conducted in strict compliance according to ethical principles and guidelines provided by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) (Approval No. 994/a/GO/06/CPCSEA).

2.4 Preparation of plant Extracts: - About 300 g of the leaves powder of Ardisia solanacea was extracted with 1.2 L of alcohol using Soxhlet apparatus for 72 hrs at 40-50°C. The extract was concentrated to ¼ of its original volume by distillation as it was adapted to recover the solvent, which could be used again for extraction.⁴

2.5 Acute toxicity study (AOT): - Acute toxicity study was performed according to the procedure OECD guideline no. 425.⁵ AOT was performed on Swiss albino mice and the animal were kept fasting for overnight providing water ad libitum, after which the alcoholic extract of Ardisia solanacea (AEAS) was administered orally 2000 mg/kg and observed the mortality of animals.

2.6 Preliminary Phytochemical Analysis: - The extracts obtained were subjected to various chemical tests to detect the chemical constituents present in extracts of Ardisia solanacea.^6-9

2.7 Assessment of liver function: - The alcoholic extract of Ardisia solanacea was evaluated for their hepatoprotective activity by using CCl₄ induced acute hepatotoxicity model. Either sex of Wistar rats, weighing (180-220) was divided into 5 groups consisting of 6 animals in each group. Group 1 received distilled water (6 ml/kg, p.o.) for 7 days. Group 2 were treated with vehicle (0.5% of Tween 40, 1 ml/kg, p.o.) for 7 days. Group 3 received silymarin (50 mg/kg, p.o.) for 7 days. Group 4, 5 pretreated with alcoholic extract of Ardisia solanacea 100 mg and 200 mg/kg body weight respectively for 7 days. Food was withdrawn 16 hrs before administration to enhance the acute liver toxicity. Group 2, 3, 4 and 5 were treated with CCl₄ was administered (2 ml/kg, s.c.) diluted in olive oil (1:1) was administered on 7^th day after 1 hrs of extracts treatment and sacrificed 24 hours after administration of CCl₄.⁵ Animals were anesthetized using anesthetic ether and blood sample were collected by cardiac puncture method and serum was used for estimation of AST, ALT, ALP, albumin, total protein, total and direct bilirubin. Immediately after the collection of blood, their livers were separated. The liver was washed by normal saline were preserved in 10% formalin for histopathological studies.

2.8 Statistical analysis: - The experimental results were expressed as the Mean ± SEM for six animals in each group. The biochemical parameters were analysed statistically using one-way ANOVA followed by Tukey Kramer’s post hoc test. P value of < 0.05 was considered as statistically significant.

3. RESULTS:

Preliminary phytochemical studies with extract revealed the phytoconstituents like cardiac glycoside, carbohydrates, phytosterols, saponins, phenolics and tannins. Different doses of alcoholic extract of Ardisia solanacea leaves (AEAS) was screened in albino mice for their acute oral toxicity. No mortality was recorded till 2000 mg/kg body weight. Hence the extract was found to be safe up to the dose levels of 2000 mg/kg. So 1/10^th and 1/20^th of these dose i.e. 200 & 100 mg/kg body weight of AEAS for oral dose was select as therapeutic dose for pharmacological activity screening. Effect of plant extract on pentobarbital sleeping time was studied in rats and the results are shown in Table: 3.1 Pentobarbital at a dose of 75 mg/kg. i.p. in normal control( group-1) caused sleep in rats for a period of 145 ± 4 min (mean ± SEM. n = 6). Whereas the sleeping time in the CCl₄ induced toxic control group (group-2) of animals was found to 229 ± 8 min. When sleeping time of toxic control group of animals was compared with test groups, the higher dose of AEAS (200mg/kg body wt) (Group-V) was highly significant189±3 min (P<0.001) which is very nearer to that of silymarine induced standard (Group-3) (177±5). The effects of AEAS on Serum glutamate oxaloacetate transaminase (SGOT), Serum glutamate pyruvate transaminase (SGPT), Alkaline phosphatase (ALP), Serum direct bilirubin(DBIL), Sreum total bilirubin(TBIL), Serum albumin(ALB) and Serum total protein(TLP) levels in CCl₄ induced liver damage in rats are summarized in Table - 3.2 and 3.3. Administration of CCl₄ (2 ml/kg, s.c.), after 24 hours of intoxication resulted a significant (P<0.05) elevation of hepatospecific serum enzymes markers like SGOT, SGPT and ALP and serum biochemicals markers like DBIL and TBIL in CCl₄treated groups, while seum biochemicals markers like albumin and total protein were found to be decreased in comparison with the normal control group. On administration of AEAS (Group IV & V) and Silymarin at the dose of 50mg/kg (Group III) the level of these enzymes and biochemicals were found retrieving towards normalcy. The hepatoprotective effect offered by AEAS (200 mg/kg p.o.) was found to be significantly greater than AEAS (100 mg/kg p.o.).

Table No. – 3.1 Effect of the Ardisia solanacea leaves extracts on pentobarbital Induced sleeping time in CCl₄ induced hepatotoxic rats.

Group	Treatment	Dose	Mean Sleeping time (min)
I	Solvent control	5ml/kg p.o	145±4
II	Vehicle + CCl₄	1 ml/kg p.o (2 ml/kg, s.c.)	229±8***
III	Silymarin + CCl₄	50mg/kg p.o (2 ml/kg, s.c.)	177±5***
IV	AEAS + CCl₄	100mg/kg p.o (2 ml/kg, s.c.)	199±4***
V	AEAS + CCl₄	200mg/kg p.o (2 ml/kg, s.c.)	189±3***

Values expressed as mean ± SEM, from six observations, ***p<0.001 when compared with normal control group. Using one-way ANOVA followed by Tukey Kramer’s post hoc test.

Table No. – 3.2 Effect of the Ardisia solanacea leaves extracts on serum enzyme in CCl₄ induced hepatic damage in rats.

Group	Treatment	SGOT (IU/L)	SGPT (IU/L)	ALP (IU/L)
I	Solvent control	96.38±4.02	48.28±3.63	107.45±8.98
II	Vehicle + CCl₄	436.16±7.45	196.48±2.88	283.10±3.12
III	Silymarin + CCl₄	218.40±2.77***	69.46±2.75***	186.23±9.85***
IV	AEAS + CCl₄	389.27±9.56***	116.14±4.89 ***	231.12±11.26
V	AEAS + CCl₄	283.10±3.52***	94.41±3.781 ***	205.16±8.46***

Values expressed as mean ± SEM, from six observations, **p<0.01, ***p<0.001 when compared with CCl₄ control group. Using one-way ANOVA followed by Tukey Kramer’s post hoc test.

Table No. -3.3 Effect of the Ardisia solanacea leaves extracts on serum biochemical parameters in CCl₄induced hepatic damage in rats.

Group	Treatment	Albumin (mg/dl)	Total protein (mg/dl)	Direct bilirubin (mg/dl)	Total bilirubin (mg/dl)
I	Solvent control	4.67±0.29	14.78±0.54	0.28±0.01	0.39±0.01
II	Vehicle + CCl₄	2.47±0.0.27	7.089±0.15	0.81±0.06	1.09±0.05
III	Silymarin + CCl₄	3.94±0.18**	13.92±0.41***	0.391±0.026***	0.59±0.01***
IV	AEAS + CCl₄	2.49±0.35	10.21±0.75**	0.62±0.020***	0.85±0.02***
V	AEAS + CCl₄	3.68±0.19*	12.13±0.45 ***	0.58±0.072***	0.65±0.01***

Values expressed as mean ± SEM, from six observations, *p<0.05, **p<0.01 and ***p<0.001 when compared with CCl₄ control group. Using one-way ANOVA followed by Tukey Kramer’s post hoc test.

Fig. 1 Histopathology of solvent control group (Group-I)

Fig. 2 Histopathology of Toxic Control(Group-II)

Fig. 3 Histopathology of Standrad drug (Group-III)

Fig. 4 Histopathology of Test drug CETI (Group-IV)

Fig. 5 Histopathology of Test drug CETI (Group-V)

Histology of the liver sections of control animals (Group I) showed normal hepatic cells with well-preserved cytoplasm, prominent nucleus, nucleolus and visible central veins. The liver sections of CCl₄ intoxicated rats showed massive fatty changes, necrosis, ballooning degeneration and broad infiltration of the lymphocytes and the loss of cellular boundaries. The histological architecture of liver sections of the rats treated with alcoholic extract showed more or less normal lobular pattern with a mild degree of fatty change, necrosis and lymphocyte infiltration almost comparable to the control and silymarin treated groups (Fig. 1- 5).

4. DISCUSSION:

In recent years, many studies have been undertaken with traditional medicines, in an attempt to develop new drugs for hepatitis.¹⁰ In the present study alcoholic extract of Ardisia solanacea leaves were evaluated for the hepatoprotective activity using CCl₄ induced hepatotoxicity in rat model and find out the therapeutically better efficacious extract. CCl₄ is a well-known hepatotoxic agent and the preventive action of liver damage by CCl₄ has been widely used as an indicator of liver protective activity of drugs in general.¹¹ The changes associated with CCl₄-induced liver damage are similar to that of acute viral hepatitis .¹² CCl₄ is biotransformed by Cytochrome P-450 system to produce trichloromethyl free radicals. These free radicals may again react with oxygen to form trichloromethyl peroxyl radicals, which may attack lipids on the membrane of endoplasmic reticulum to elicit lipid peroxidation, finally resulting in cell necrosis and consequent cell death.¹³ As well known, phenolic antioxidants, such as flavonoids and tannins, are considered promising therapeutic agents for free radical pathologies due to their scavenging ability with ROS.¹⁴

SGOT is found in the liver, cardiac muscles, skeletal muscles, pancreas, lungs, kidney, brain, etc., whereas SGPT concentrationis highest in the liver and therefore, it appears to be a more sensitive test to hepatocellular damage than SGOT.¹⁵ Leakage of large quantities of enzymes into the blood stream is often associated with massive necrosis of the liver .¹⁶ CCl₄is known to cause marked elevation in serum enzymes (SGOT and SGPT). In the present study, a significant increase in the activities of SGOT and SGPT within 24 hr exposure to CCl₄ was observed, indicating considerable hepatocellular injury. Our results indicated that Ardisia solanacea (200 mg/kg) administration significantly alleviated the increased serum enzyme activity induced by CCl₄, indicating improvement of the functional status of the liver. The recovery towards normalization of serum enzymes and liver histological architecture caused by Ardisia solanacea was almost similar to that caused by silymarin, in the present study. Similar results have been reported.¹⁷ Silymarin is a known hepatoprotective compound. It is reported to have a protective effect on the plasma membrane of hepatocytes.¹⁸

In conclusion the possible hepatoprotective effect of Ardisia solanacea in CCl₄ induced liver injuries may be due to: (1) inhibiting Cytochrome P- 450 activity, (2) preventing the process of lipid peroxidation, (3) stabilizing the hepatocellular membrane and (4) enhancing protein and glycoprotein biosynthesis. However the exact hepatoprotective mechanism of Ardisia solanacea is still unknown. Further studies are warranted to isolate the active components.

5. ACKNOWLEDGEMENTS:

The authors wish to thank Prof. J.S. Dangi, Head of the Institute for facilities and Mr. Karteek Patra for technical assistance.

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Received on 01.02.2013 Accepted on 10.04.2013

Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 2, Pg 79-82