1. INTRODUCTION:

Free radicals, from both endogenous and exogenous sources are implicated in the etiologic of several degenerative diseases such as coronary artery diseases, stroke, rheumatoid arthritis, diabetes and cancer.¹ High consumption of fruits and vegetables is associated with low risk for these diseases, which is attributed to the antioxidant vitamins and other phytochemical.^2-4 There is a great deal of interest in edible plants that contain antioxidants and health promoting phytochemicals, in view of their health implications.

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 diseases.⁵ 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. It is cardinal to treat liver disorders since it directly affects the biochemistry of the cell through participating events like oxidative stress, redox change, etc.⁶ which induces lipid peroxidation.

Peroxidation of membrane phospholipids not only alters the lipid mileu and structural as well as functional integrity of cell membranes, but also affects the activities of various membrane-bound enzymes including total ATPase, Mg2+ATPase, Ca2+ATPase and Na+K+ATPase.^7-8 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.⁹ Therefore; there is a need to develop satisfactory hepatoprotective drugs. Ardisia solanacea (Myrsinaceae) is a large, evergreen shurb growing up to 1.5 – 4 m tall. Leaves are 10-20 cm long oblanceolate to obovate, entire, tip narrow with short stalks. Its bark is used in Arthritis, Rheumatism where as its leaf is used in Fabrifuge generally healing, stomach troubles.

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 and 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), alcohol (CDH, Mumbai) anesthetic ether (CDH, Mumbai) and CCl₄ (Ranbaxy, Delhi) Thiobarbituric acid, Trichloro acetic acid, H₂O₂, Epinephrine, EDTA, Tric Hcl buffer, GSH, NADP and other phytochemical reagents were obtained from Institute.

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).

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.¹⁰

2.5 Acute toxicity study (AOT): -

Acute oral toxicity study was performed according to the procedure OECD guideline no. 425.¹¹ 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.^12-15

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. After the treatment animals were sacrificed by cervical decapitation under excessive anaesthesia on the 8^th day. Immediately after sacrifice, the liver was dissected out, washed in the ice-cold saline, and the homogenate was prepared in 0.1M Tris–HCl buffer (pH 7.4). The homogenate was centrifuged and the supernatant liquid was used for the assay of marker enzymes, namely Glutathione peroxidise by Paglia, D.E., Valentine, W.N., (1967), superoxide dismutase by Saggu et al., (1989)., Misra and Fridovich, (1972), catalase by Chance and Maehly., (1955) and MDA by Esterbauer and Cheeseman (1990).

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 and 100 mg/kg body weight of AEAS for oral dose was select as therapeutic dose for pharmacological activity screening.

The effects of AEAS on rat liver Glutathione peroxidase (GP_X), Malondialdehyde (MDA) and enzyme antioxidants namely SOD and CAT levels are shown in Table 1. and Fig.1-4. A significant decrease in the level of glutathione and enzyme antioxidants (SOD and CAT) and increase in MDA were noted after single administration of CCl₄. Upon administration of AEAS, the activities of glutathione and enzymic antioxidants were significantly (P<0.05) reversed to near normal (Fig. 6.23 to 6.25). The effects of AEAS were comparable with that of standard reference drug silymarine.

4. DISCUSSION:

In biological systems, lipid peroxidation (oxidative degradation of polyunsaturated fatty acid in the cell membranes) generates a number of degradation products, such as malondialdehyde (MDA), and is found to be an important cause of cell membrane destruction and cell damage .¹⁶ CCl₄ is commonly used to induce hepatoxicity in animal models. Metabolic processes convert CCl₄ in to the trichloromethyl radical (CCl₃^-) which interacts with O₂ to yield the highly reactive trichloromethylperoxy radical (CCl₃O₂^-). Both radicals are capable of binding to protein and lipid or abstracting a hydrogen atom from unsaturated lipid, which induced lipid peroxidation and leads to changes in the endoplasmic reticulam, reduction in protein synthesis and elevation of serum transaminase enzyme levels.^17-18

Table No. – 3.1 Chemically intoxicated and its associated antioxidant activity of alcoholic extract of Ardisia solanacea leaves.

Group	SOD	CAT	MDA	GP_X
Normal control Group-1	0.09 ± .00	128.3± 1.13	6.16 ± 0.59	139.42 ± 0.44
Toxic control Group-2	0.04± 0.00	94.89± .60	19.70 ± 0.55	95.378 ± 0.52
Standard Drug Group-3	0.08± 0.00	122.5± .44	9.82 ± 0.32	130.55 ± 0.29
Test-1 (Group-4)	0.06± 0.00	107 ± 0.48	15.29 ± 0.41	109.76 ± 0.29
Test-2 (Group-5)	0.07± 0.00	113.5 ±.38	12.31 ± 0.49	126.26 ± 0.89

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.

Fig -1 Superoxide dismutase (SOD) activity of Ardisia solanacea leave extract on CCl₄-intoxicated rats.

Fig -2 Catalase (CAT) activity of Ardisia solanacea leaves extract on CCl₄ intoxicated rats.

Fig -3 Malondialdehyde (MDA) activity of Ardisia solanacea leaves extract on CCL₄- intoxicated rats.

Fig -4 Glutathione peroxidise (GPX) activity of Ardisia solanacea leaves extract on CCl₄-intoxicated rats.

Lipid peroxidation as measured by MDA formation increased in the liver tissue of rats treated with CCl₄.^19-20 MDA a secondary product of lipid peroxidation is a major reactive aldehyde, levels can lead to peroxidation of biological membrane. A major defense mechanism involves the antioxidant enzymes, GSH, CAT, and SOD.²¹ Decrease in SOD activity is a sensitive index of hepatocellular damage. SOD scanvenges the superoxide anion to form hydrogen peroxide, thus diminishing toxic effects caused the free radical. CAT is an enzymatic antioxidant widely distributed in all animals’ tissue, highest concentration are found in erythrocytes and liver cell. CAT decomposed H₂O₂ and protect the tissue from highly reactive hydroxyl radicals.²²

CCl₄- treated rats showed significant decreases in GP_X, CAT, and SOD level compare with control. In contrast GSH, CAT, and SOD levels were significantly increased in ASLE treated groups, suggesting enhanced antioxidant properties.²³

The qualitative analysis of Ardisia Solanacea leaves extract indicated the presence of flavonoids, polyphenols, are natural antioxidants but have also been reported to significantly increase SOD, Glutathion and catalase and decrease MDA level.

5. ACKNOWLEDGEMENTS:

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

6. REFERENCES:

1. Halliwell, B., Gutteridge, J. M. C., and Cross, C. E. (1992). Free radicals, antioxidants and human disease: Where are we now? Journal of Laboratory and Clinical Medicine, 119, 598–620.

2. Ames, B. N., Shigenaga, M. K., and Hagen, T. M. (1993). Oxidants, antioxidants and the degenerative diseases of aging. Proceedings of the National Academy of Sciences of the United States of America, 90, 7915–7922.

3. Prior, R. L. (2003). Fruits and vegetables in the prevention of cellular oxidative damage. American Journal of Clinical Nutrition, 78(suppl), 570s–578s.

4. Weisburger, J. H. (1999). Mechanisms of action of antioxidants as exemplified in vegetables, tomatoes and tea. Food and Chemical Toxicology, 37, 943–948.

5. Treadway, S., 1998. An ayurvedic approach to a healthy liver. Clinical Nutrition Insights 16, 1–4.

6. Kaplowitz, N., 2002. Biochemical and cellular mechanisms of toxic liver injury. Seminar in Liver Disposition 22, 137–144.

7. Rauchcova, H., Ledvinkova, J., Kalous, M., Drahota, Z., 1995. The effect of lipid peroxidation on the activity of various membrane-bound ATPases in rat liver kidney. International Journal of Biochemistry and Cell Biology 27, 251–255.

8. Hazarika, A., Sarkar, S.N., 2001. Effect of isoproturon pre-treatment on the biochemical toxicodynamics of anilofs in male rats. Toxicology 165, 87–95.

9. De S, Ravishankar B, Bhavsar GC. Plants with hepatoprotective activity-A review. Indian Drugs. 1993;30(8):355-63.

10. Kokate CK, Purohit AP, Gokhale SB. Text Book of Pharmacognosy, Nirali Prakashan, Pune. 1996; 4: 510-11.

11. OECD 2001-gudeline on acute oral toxicity (AOT) Environmental health and safety monograph series on testing and adjustment No.425.

12. Trease GE., Evans MC. Text book of Pharmacognosy London, BailliareTindall; 1983; 12:193,336.

13. Kokate CK, Purohit AP, Gokhale SB. Text book of pharmacognosy, Nirali Prakashan, Pune. 1996; 4: 510-11.

14. Khandelwal KR. Practical Pharmacognosy. Techniques and Experiments Pune, Nirali Prakashan, 2000; 2:149-155.

15. Oloyede OL. Chemical profile of Pulp of Carica papaya. Pakikistan J Nutrition. 2005; 4 (6) :379-381.

16. Yoshikawa, T., Naito, Y., and Kondo, M. (1997). Food and diseases. In M. Hiramatsu, T. Yoshikawa, and M. Inoue (Eds.), Free radicals and diseases (pp. 11–19). Plenum press: New York.

17. Mujumdar A.M. et al, Effect of Azedirachta indica leaf extract on CCl₄ induced hepatic damage in albino rats; Indian journal of pharmaceutical science; 1998 : 60 : 363-7.

18. Lee K.J., Jeong H.G. protective effect of platycod radix on CCl₄ induced hepatotoxicity; Food Chemistry Toxicology; 2002 : 40 :517-24.

19. Zimmermann R., et al. Inhibition of lipid peroxidation in isolated inner membrane of rat liver mitochondria by superoxide dismutase; Febs Lett; 1973 : 29 : 117-21.

20. Jain A. el al. Antioxidant and hepatoprotective activity of ethanolic and aqueous extracts of Momordica dioica Roxb. Leaves; Journal of Ethnopharmacology; 2008 :115 : 61-3.

21. Chance B., et al. The mechanism of catalase actions steady state analysis; Arch. Biochem. BIophys.; 1992 : 37 : 301-39.

22. Prakash J. et al. Chemopreventive activity of Withania somnifera in experimentally indused fibro sarcoma tumors in swiss albino rats. Phytother Res.; 2001 : 15 : 200-4.

Received on 28.04.2013 Modified on 06.05.2013

Research J. Pharm. and Tech. 6(5): May 2013; Page 585-588