OUTLINE OF RESEARCH WORK

OUTLINE OF RESEARCH WORk

9. TITLE OF THESIS:  EVALUATION OF HEPATOPROTECTIVE ACTIVITY OF AQUEOUS AND METHANOLIC EXTRACTS OF Careya arborea STEM BARK IN CARBON TETRACLORIDE INTOXICATED RATS.

10. INTRODUCTION:

            India has an ancient heritage of traditional medicine. The traditional uses of therapeutically important herbs including Careya arborea are documented in Materia Medica , Ayurveda ,Siddha and Unani systeme of medicines. The World Health Organization has defined Traditional medicine as comprising therapeutic practices that have been in existence for hundreds of years (Kamboj 2000). The traditional preparations comprise medicinal plants, minerals and organic matter.

             Many herbs and herbal preparation are reported to possess the antioxidant and hepatoprotective properties. Herbal drugs have been used in the treatment of liver diseases for a long time. Herbal drugs are frequently considered to be less toxic and free from side effect than the synthetic ones.

             Medicinal plants, which form the backbone of traditional medicine, have in the last few decades been the subject for very intense pharmacological studies; this has been brought about by the acknowledgement of the value of medicinal plants as potential sources of new compounds of therapeutic value and as sources of lead compounds in the drug development. In developing countries, it is estimated that about 80% of the population rely on traditional medicine for their primary health care.

             The liver is the most important organ in the body. It plays a pivotal role in regulating various physiological processes. It is also involved in several vital functions, such as metabolism, secretion and storage. It has great capacity to detoxicate toxic substances and synthesize useful principles (Shanani, 1999; Subramoniam and Pushpangadan, 1999). It helps in the maintenance, performance and regulating homeostasis of the body. It is involved with almost all the biochemical pathways to growth, fight against disease, nutrient supply, energy provision and reproduction. In addition, it aids metabolism of carbohydrate, protein and fat, detoxification, secretion of bile and storage of vitamins (Ahsan, et al., 2009). Any abnormality and or insult with this organ therefore adversely affect the production and heath of animals.

          

            Liver diseases remain one of the major threats to animal health and are worldwide problem.  Liver diseases have become one of the major causes of morbidity and mortality in man and animals all over globe and hepatotoxicity due to drugs appears to be the most common contributing factor. Most of the hepatotoxic chemicals damage liver cells mainly by inducing lipid peroxidation and the other oxidative damages (Wendel, et al., 1987; Dianzani, et al., 1991).

         Exposure of animals to the environmental pollutants, insecticides, pesticides, mycotoxins, toxic plants and synthetic drugs such as anticancer, corticosteroids, antibiotics result in the hepatic dysfunction and damage (Gupta and Solunkhe, 1985. Michel, 1984.). Despite, enormous development in synthetic drugs, there is a hardly any drug that found to be absolutely safe to cure diseases of hepatic origin. CCL₄  is also known to induce fatty liver and liver cells necrosis in variety of mammalian species  (Smuckler, 1976).   

         Careya arborea Roxb. Commonly known as “Kumbi” in Hindi, “Kumbha” in Marathi belongs to the family Lecythidaceae medium sized deciduous tree, bark dark grey exfoliating in thin strip. Widely available in India, Ceylon, Malay and Peninsula. The plant has been extensively investigated and chemical constituents from the barks, leaves and seeds of the plant have previously been reported to include triterpe-noids (Das, et al., 1982), flavonoid (Gupta, et al., 1975), cumarin (Basak, et al.,1976), saponins and tannins (Kulakkattolickal, 1987).

          Stem bark of Careya arborea is traditionally used in the treatment of tumours, bronchitis, epileptic fits, astringents, antidote to snake-venom and skin disease (Kirtikar and Basu, 1975). It is also used as remedy for diarrhoea, dysentery with bloody stools and ear pain. Antipyretic, leech repellant, fish poison and antivenin activities were also reported in literature. Pharmacological activities and mode of action of this plant is yet to be established.

          In view of the above context and the meager literature available on Careya arborea prompted us to select the same for the study. Thus the present study is aimed at evaluation of hepatoprotective activity of stem bark extract of Careya arborea inCCL₄ intoxicated rats with the objectives mentioned below.

11. OBJECTIVES:

The objective of proposed research work:

1.    To prepare aqueous and methanolic extract of bark of Careya arborea and determine its extractibity percentage.

2.    Phytochemical analysis of extracts of stem bark of Careya arborea for presence of active principles.

3.    Estimation of haematological parameters: Hemoglobin, PCV, TEC, TLC.

4.    Estimation Serum biochemical parameteres: Serum SGOT, Serum SGPT, Serum alkaline phosphatase and Bilirubin. 

5.    To study gross and histopathological investigation of liver.

12. REVIEW OF LITERATURE:

            Sambhat kumar et al., (2005) evaluated the hepatoprotective and antioxidant effect of methanolic extract of Careya arborea Robx (MECA) stem bark in wistar albino rats. The hepatotoxicity was induced by carbon tetrachloride (30% CCL₄, 1 ML/Kg b .w. in liquid paraffin 3 doses (i.p.) at 72 h interval. The MECA at the doses of 50,100 and 200 mg/kg and silymarin 25 mg/kg were administered to the CCL₄ treated rats .analytical parameters like GOT, GPT, ALP, bilirubin, uric acid, and total protein were measured in the rats induced hepatotoxicity by CCL₄ .the effect of the extract on Lipid Peroxidation  (LPO), enzymatic antioxidents (Superoxide Dismutase(SOD)) and Catalase , and nonenzymatic antioxidents (Glutathione (GOT,GPT), vitamine C and vitamine E) were estimated . The MECA and silymarine produced significant (p < 0.05) hepatoprotective effect by decreasing the activity of serum enzymes bilirubin , uric acid and lipid peroxidatiion and significantly (p <  0.05) increased the level of SOD, CAT, GSH, vitamin C, vitamin E and protein in a dose dependant manner. From these results, it was suggested that MECA possess potent hepatoprotective and antioxidant agents.

            Senthilkumar et al., (2008) studied Antioxidant and hepatoprotective activity of the methanol extract of Careya arborea bark in Ehrlich ascites carcinoma (EAC) bearing mice. Tumor control animals inoculated with EAC showed a significant alteration in the levels of antioxidant and hepatoprotective parameters. The extract treatment at 50, 100 and 200 mg/kg body weight doses given orally caused a significant reversal of these biochemical changes towards the normal in serum, liver and kidney when compared to tumor control animals indicating the potent antioxidant and hepatoprotective nature of the standardized extract.

           Sambath Kumar (2008) studied The Antioxidant Defense System Induced by Methanol Extract of Careya arborea in N-Nitroso- diethylamine (NDEA) Induced Hepatocarcinogenesis. NDEA was used to induce cancer in animal causing oxidative stress and cellular injury due to the enhanced formation of free radicals and is associated with rise in biochemical parameters like serum glutamyl pyruvate transaminase (SGPT), serum glutamyl oxalacetic acid (SGOT), serum alkaline phosphatase (SALP), bilirubin, as well as decrease in the levels of total protein and uric acid. The methanol extract of stem barks of Careyaarborea Roxb (MECA) was administered at the doses of 100 and 200 mg/kg body weight/rat in alternative days for 8 weeks. At the end of the drug schedule, rats were sacrificed and the effect of MECA on visible Macroscopic (Morphometry) liver lesion (neoplastic nodules) and the level of SGPT, SGOT, SALP, bilirubin, total protein, uric acid and liver antioxidant defense parameters like lipid peroxidation (LPO), nonenzymatic antioxidant such as glutathione content (GSH), vitamin C, vitamin E and enzymatic antioxidant like superoxide dismutase (SOD) and catalase (CAT) were estimated. The MECA was found to be protective against NDEA-induced carcinoma by decreasing the activity of serum enzymes, bilirubin and increase the protein and uric acid levels. MECA significantly decreased the levels of LPO, while it significantly increased the levels of GSH, vitamin C, vitamin E, SOD and CAT. The results indicate that MECA exhibited significant chemopreventive effects by suppressing nodules development and enhancing the antioxidants in NDEA carcinogenesis by reducing the formation of free radicals.

            Natesan et al., (2007) studied the methanol extract of Careya arborea Roxb bark for the anticancer potentials against Dalton’s lymphoma ascites (DLA)-induced ascitic and solid tumors. The methanol extract of its bark given orally to mice at the dose of 250 or 500 mg/kg body weight for 10 days caused significant reduction in percent increase in body weight, packed cell volume, and viable tumor cell count when compared to the mice of the DLA control group. Restoration of hematological and biochemical parameters towards normal was also observed. Histological observations of liver and kidney also indicated repair of tissue damage caused by tumor inoculation. The extract at the dose of 5 or 25 mg/kg body weight given i.p. daily for 14 days significantly reduced the solid tumor volume induced by DLA cells.

              Ahmad et al., (2002) reported hepatoprotective potential of jigrin a polypharmaceutical herbal formulation containing aqueous extracts of 14 medicinal plants including Careya arborea Roxb was one of the constituent. The hepatoprotective potential of jigrine post-treatment at the dose of 0.5 ml/kg per day p.o. for 21 days was evaluated against thiocetamide induced liver damage in rats. Biochemical parameters like AST, ALT in serum and TBARS and glutathione in tissues were estimated to assess liver function. Data on the biochemical parameters revealed hepatoprotective potential of jigrine post-treatment against thioacetamide induced hepatotoxicity in rats.

               Ghosh et al., (2011) studied the antioxidant and hypolipidemic activity of Kumbhajatu a polyherbal preparation containing Careya arborea in hypocholesterolemic rats. Hypercholestremia was induced in normal rats by including 2 % w/w cholesterol, 1% w/w sodium cholate and 2.5% w/w coconut oil in the normal diet. Powdered form of Kumbhajatu was administered as a feed supplement at 250 and 500 mg/kg dose levels to the hypercholesterolemic rats. Plasma lipid profile, SOD, catalase activity, reduced glutathione and extend of lipid peroxidation in the form of malondialdehyde were estimated using standard methods. Feed supplementation with 250 and 500 mg/kg of Kumbhajatu observed a significant decline in plasma lipid profile. Catalase, SOD, glutathione and HDL-c significantly increased in both treatment groups. On the other hand, the concentration of malondialdehyde, cholesterol, triglycerides, LDL-C and VLDL were decreased significantly in treatment groups. Study reported that addition of Kumbhajatu powder (250 and 500 mg/kg) level as a feed supplement reduces the plasma lipid levels and also decreases lipid peroxidation significantly.

            Kumar et al., (2008) evaluated the antimicrobial and antioxidant activities of methanol extract of Careya arborea (MECA) stem barks (Myrtaceae) in various in-vitro systems. Antimicrobial activities of MECA were carried out using disc diffusion methods with Gram positive and Gram negative bacteria and some fungal species. MECA showed broad spectrum antimicrobial activity against all tested microorganisms. Antioxidant and free radical scavenging activities of MECA was assessed by using 1, 1-diphenyl-2-picryl-hydrazyl radical (DPPH), superoxide anion radical, nitric oxide radical and hydroxyl radical scavenging assays. The antioxidant activity of MECA increased in a concentration dependent manner. The result indicates that the MECA can be a potential source of natural antimicrobial and antioxidant agents.

            Kumar et al., (2008) studied the methanol extract of barks of Careya arborea (Myrtaceae) to investigate central nervous system (CNS) activity in Swiss albino mice and Wistar albino rats. General behavior, exploratory behavior, muscle relaxant activity and phenobarbitone sodium–induced sleeping time were studied. The results revealed that the methanol extract of barks of Careya arborea at 100 and 200 mg/kg caused a significant reduction in the spontaneous activity (general behavioral profile), remarkable decrease in exploratory behavioral pattern (Y–maze and head dip test), a reduction in muscle relaxant activity (rotarod and traction tests), and also significantly potentiated phenobarbitone sodium–induced sleeping time, the result showed significant CNS depressant activity of careya arborea in tested animal models.

            Garg (2006) documented oxidative liver damage and formation of reactive free radicals such as trichloromethyl phenoxy reactive peroxides from membrane lipids (polyenoic) and proteins and irreversible inhibition of cytochrome  oxydase P₄₅₀  by carbon tetrachloride intoxication. They mentioned loss of appetite, gastrointestinal pain, diarrhoea, blood stained feces, biliary obstruction, jaundice, drowsiness, depression, stumper, incoordination, convulsions, hydropic degeneration, centrilobular necrosis in the liver tissue were of pathognomic importance.

            Sambathkumar et al., (2005) studied the anti-inflammatory and analgesic effect of methanolic extract Careya arborea (MECA) stem bark in experimental animal models. The effect of MECA on the acute and chronic phases of inflammation were studied in carrageenan, dextran and mediators (histamine and serotonin) induced paw oedema and cotton pallet induced granuloma respectively. Analgesic effect of MECA was evaluated in acetic acid induced writhing and hotplate test. The antioedema effect was compared with indomethacin 10mg/kg orally. In acute phase of inflammation a maximum inhibition of 50.56, 48.86, 47.12 and 48.23 % (p < 0.05) was noted at the dose rate of 200 mg/kg b.w. after 3 hours of treatment with MECA in carrageen, dextran, histamine and serotonine induced paw oedema respectively. Administration of MECA (200mg/kg b.w.) and indomethacine (10 mg/kg b.w.)  Significantly (p < 0.05) decreased the formation of granuloma tissue induced by cotton pellet method at a rate of 53.91%  and 57.60% respectively. The MECA revealed significant (p<0.01) analgesic activity in both models.

                Rahman et al., (2002) reported the methanol extract of the Careya arborea Roxb.bark significantly reduced castor oil induced diarrhoea in mice. This effect supports the local traditional use of the plant against diarrhoea.

13. PROGRAMME OF RESEARCH WORK:

        Present study is planned to evaluate hepatoprotective effect of aqueous and  methanolic extract of Careya arborea stem  bark in carbon tetracloride intoxicated wistar rats.

·         Collection and identification of plant material.

·         Preparation of aqueous and methanolic extract of stem bark of Careya arborea and to determine its extractibity percentage.

·         Qualitative phytochemical analysis of aqueous and methanolic extract of stem bark of Careya arborea for presence of active principles.

·         Aqueous and methanolic extract of Careya arborea stem bark will be evaluated for hepatoprotective property in rats.

·         Experimental animals:  Fourty two wistar albino rats will be procured from registered animal house for experimental work. Animals will be acclimatized for a period of 15 days to reduce the stress before the start of experiment period.

·         Drugs and chemicals: Carbon tetracloride_, Silymarine , and diagnostic kits will be purchased from authorized chemical supplier.

·         Experimental design:  Rats will be divided into seven groups T_1, T₂, T₃, T₄, T_5,T₆ and T₇ respectively and each group comprising of six rats. GroupT₁ serve as a vehicle control, Group T₂ as positive control and Group T₃ serve as standard group will receive only CCL₄ and silymarine. The experimental design is as follows.

Sr. no	Experiment Group	No   of animals	Treatment	Dose
1	T1	6	Vehicle control	Distilled water     (2 ml/kg, p.o.)
2	T2	6	Receive CCL4  alone	30 % CCL4 in liquid paraffin( 1 ml/kg, s.c.) at every 72 h.
3	T3	6	CCL 4 + SILYMARINE	25 mg /kg b.w. p.o. once a day.
4	T4	6	CCL 4  +  Plant extract         (Aqueous)	100 mg /kg b.w. p.o. once a day.
5	T5	6	CCL 4  +  Plant extract         (Aqueous)	200 mg /kg b.w. p.o. once a day.
6	T6	6	CCL 4    +  Plant extract        (Methanolic)	100 mg /kg b.w. p.o. once a day.
7	T7	6	CCL 4    +  Plant extract        (Methanolic)	200 mg /kg b.w. p.o. once a day.

14. METHODOLOGY:

·         Protocol:

            The experiment will be conducted for period of 14 days. Group T₁  serves as negative control. Group T₂ serves as a positive control and will receive only CCL₄. (30 %)1 ml /kg b.w. Three doses repeated after 72 hrs. Group T₃ serves as standard receiving  CCL₄+ Sylimarine 25 mg /kg  p.o. once a day). Group T₄ and T_{5 ,} will receive  CCL₄ +  Aqueous Extract @ 100 and 200 mg/kg b.w. p.o.respectively. T₆ and  T₇ will receive  CCL₄ +  Methanolic Extract @ 100 and 200 mg/kg b.w. p.o.respectively.

·         The aqueous and methanolic extract will be prepared by as per standered laboratory procedure.

·         Haematological parameters:

Estimation of haemoglobin level by Sahli’s Haemocytometer, Benjamin 2001.

Packed cell volume (PCV), TEC and TLC count will be done.

·         Biochemical parameters:

Serum sample of all rats will be collected for the biochemical investigation. Following biochemical parameters will be studied using standard diagnostic kits.

1. SGOT

2. SGPT

3. Serum alkaline Phosphatase.

4. Bilirubin.

·         Histopathology:

Routine paraffin embedded sections with H and E staining Luna (1968).

15. REFERENCES:

Kamboj, V.P.(2000): Herbal medicine. Current Science. 78:35-7.

Ahmad A.; Pillai, K. k.; Najmi, A. K.; Ahmad, S. J.; Pal, S. N. and Balani, D. K.(2002): Evaluation of hepatoprotective potential of jigrine post-treatment against thioacetamide induced hepatic damage.Journal of Ethnopharmacology. 79:35-41.

Ahsan, M.R.; Islam, K.M.; Bulbul I.J. (2009): Hepatoprotective activity of Methanol Extract of some medicinal plants against carbon tetrachloride-induced hepatotoxicity in rats. Eur. J. Sci. Res. 37(2): 302-310.

Basak, A.; Banerjee, B. L.; Basu, K. (1976): Chemical examination of the leaves of Careya arborea. J Indian Chem Soc . 53:639-40.

Das, M.C.and Mahato, S.B. (1982): Triterpenoid sapogenols from the leaves of Careya arborea structure of careyagenolide. Phytochemistry.21:2069-73.

Dianzani, M.U.; Muzia, G.; Biocca,M.E.; Canuto, R.A. (1991): Lipid peroxidation in fatty liver induced by caffeine in rats. Int. J. Tissue React. 13: 79-85.

Garg, S. K. (2006): “ Carbon tetrachloride “ , In veterinary Toxicology, Reprint Edn. PP.12, 16, 28, 30, 144, 192-194. CBS Publishers and Distributors, New Delhi-110002 (India).

Ghosh, R.; Kadam, P. P. and Kadam, V. J. (2010): Antioxident and hypolipidemic activity of Kummbhajatu in hypercholesterolemic rats. 1(3):159-162.

Gupt, R.K.; Chakraborty, N.K.and Dutta, T.R. (1975): Crystalline constituents from Careya arborea Roxb. Indian J Pharm . 37 (6):161-2.

Gupta, P.K. and Salunkhe, D.K. (1985): Modern Toxicology. VOL.3.First Edn. Metropolitan Book Co. Pvt. Ltd., New Delhi,India.

Kirtikar, K.R and Basu, B.D.(1975): Indian Medicinal Plants. Vol. 2, 2nd edn, Bishen Singh Mahendra Pal Singh, Dehradun, India: pp. 894-5.

Kulakkattolickal, A.(1987): Piscicidal plants of Nepal, Preliminary toxicity screening using grass carp (Ctenopharyngodon Idella) Fingerlings. J Ethnopharmacol . 21 (1):1-9.

Kumar, R.S.; Sundram, R. S. ;Sivakumar, P.; Nethaji,  R.;Senthil, V.; Murthy, N. V. and Kanagasabi, R. (2008): CNS activity of methanol extract of Careya arborea in experimental animal model. Bangladesh J Pharmacol, 3:36-43.

Michael, J. T. (1984): Process in Pathology and Microbiology.2nd Edn. Blackwell Scientific Publication, Oxford, London, Edinburg, Melbourne.

Natesan, S.; Badami, S.; Dongre, S.H. and Godavarthi,  A. (2007): Antitumour activity and antioxidant status of the methanol extract of Careya arborea bark against dalton’s lymphoma ascites-induced ascetic  and solid tumour in mice . J Pharmacol Sci. 103:12-23.

Rahman, M.T.; Khan, O.F.;Saha, S.; Alimuzzaman, M.(2003):  Antidiarrhoeal activity of bark extract of Careya arboreya roxb.74: 116-118.

Sambath Kumar, R.; Sivakumar, T.; Sivakumar, P.; Nethaji, R.; Vijayabaskar. M.; Perumal, P.; Gupta, M. and Muzumdar, U. K.(2005):  Hepatoprotective and in vivo antioxidant effects of Careya arborea against carbon tetra chloride induced liver damage in rats. Int J Mol Med Adv Sci. 1(4): 418–424.

Sambath Kumar, Ramanathan (2008): The Antioxidant Defense System Induced by Methanol Extract of Careya arborea in N-Nitroso-diethylamine (NDEA) Induced Hepatocarcinogenesis. Journal of Complementary and Integrative Medicine: Vol. 5: Iss. 1, Article 10.

Sambathkumar, R.; Sivakumar, T.;Sundaram, R. S.;Sivakumar, P.;Nethaji, R.;Vijaybaskar, M.; Perumal, P.; Gupta, M. and Mazumdar, U.K. (2005): Anti-inflammatory and analgesic effects of careya arborea stem bark in experimental animal models.Nig J Pro and Med Vol.9:38-42.

Sambhat Kumar, R.; Sivakumar, T.; Sundaram, R. S.; Sivakumar, P.; Nethaji, R.; Gupta, M. and Mazumdar U. K. (2006): Antimicrobial and antioxidant activities of Careya arborea roxb.stem bark. Iranian J of Phar and Therap.5 (1): 35-41.

Shanani, S. (1999): Evaluation of hepatoprotective efficacy of APCL-A polyherbal formulation in vivo in rats. Indian Drugs. 36: 628-631.

Smuckler, E.A. (1976): Pan .Med. 18:292-309.

Subramoniam, A.; and Pushpangadan, P. (1999): Development of phytomedicine for liver diseases. Indian J. Pharmacol., 31: 166-175.

Wendel, A.; Feurensteins, S.; Konz, K.H. (1987): Acute paracetamol intoxication of starved mice leads to lipid peroxidation in vivo. Biochem. Pharmacol. 28: 2051-2053.

16. FACILITIES REQUIRED AND THEIR AVAILABILITY:

All required facilities are available in the Department Of Veterinary Pharmacology And Toxicology Nagpur.

17. PLACE OF WORK.

The research work will be conducted at department of Veterinary Pharmacology And Toxicology, Nagpur veterinary college, Nagpur Maharashtra.