ANTIOXIDANT AND HEPATOPROTECTIVE ACTIVITY OF PIMENTA DIOICA LEAVES EXTRACT

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1 Journal of Cell and Tissue Research Vol. 8(3) (2008) ISSN: (Available online at Original Article ANTIOXIDANT AND HEPATOPROTECTIVE ACTIVITY OF PIMENTA DIOICA LEAVES EXTRACT? NAYAK, Y. 1, ABHILASH, D. 2, VIJAYNARAYANA, K. 3 AND FERNANDES, J. 4 1 Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal , 2 Department of Pharmacology, Shree Devi College of Pharmacy, Mangalore , 3 Department of Pharmacology and 4 Department of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical Sciences, Mangalore, India E. mail: yogendranayak@gmail.com Received: July 7, 2008; Accepted: August 30, 2008 Abstract: The berries of Pimenta dioica (L) Merril (Family Myrtaceae) are commonly known as allspice in culinary. The natives of Kerala and Mangalore use Allspice leaves as medicine for pain, arthritis, fever and stress. In all these pathological conditions oxidative stress is one of the causes, hence we tried extract the leaves for in vitro antioxidant activity. 20% yield of alcoholic extract obtained from the dry leaves showed total phenolic content (TPC) equivalent to 20 ± 1.63 mg/g of the dry leaves powder. The extract showed significant antioxidant activity by reducing power, DPPH radical scavenging and Lipid peroxidation assays. The antioxidant activity was proportional to the total phenolic content in the leaves. The high content of phenolic antioxidants in the extract urged us to screen for hepatoprotective activity in carbon tetrachloride ( ) intoxicated Wistar rats. The oral doses (250, 500, 750 mg/kg/day) showed statistically significant protection of liver when investigated by histopathological and biochemical parameters. Key words: Antioxidant, Hepatoprotection, Pimenta dioica leaves INTRODUCTION Antioxidants are substances which retard or prevent deterioration, damage or destruction caused by oxidation/free radicals [1]. Human body has got endogenous antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase and glutathione reductases that prevent free radical damage [2]. When ever there is high free radical level in the tissue and simultaneously dearth of endogenous antioxidant levels, body will be vulnerable to many diseases. This condition is generally called oxidative stress. In such circumstances exogenous application of antioxidants is required to prevent unhealthy conditions leading to serious disease [3]. Antioxidants from natural or synthetic origin act by preventing and scavenging free radical formation, and simultaneously facilitating the repair of damage caused by free radicals [4]. Plant sources like turmeric, ginger, garlic, onion, clove, grape seeds, apple, ginkgobiloba and fruit peels like lemon, orange have got strong antioxidants [5]. Tea has got the highest antioxidant strong inhibitor of lipid peroxidation [6]. Antioxidants are beneficial in preventing several disease such as cardiovascular, diabetes, cancer, rheumatoid arthritis, inflammatory bowel disease, pancreatitis, haematological and neurodegenerative diseases [7-8]. Vitamins (A,C,E), glutathione, lycopene, α-lipoic acid, L-Cystine, L-methionine and micronutrients such as zinc and selenium are beneficial in oxidative stress induced diseases for prevention and improvement [9]. Berries of Pimenta dioica (L.) Merril are commonly known as allspice in culinary. It takes its name from the aroma of dried berries, which smell like the combination of spices, especially cinnamon, cloves, ginger and nutmeg. Allspice owes its characteristic odour due to the presence of essential oil in the 1571

2 J. Cell Tissue Research pericarp of the seeds. The plant Allspice and its characterstics are well mentioned in Wealth of India [10]. The dried leaves contain 0.7 to 2.9 % of oil which is called pimento oil. Like berry oil it contains eugenol as its main constituent but has an inferior odour and flavour. Phytochemistry and pharmacology of berries is well reported in literature [11-16]. is a potent toxicant that induced hepatotoxicity and Allspice leaves have medicinal properties to cure pain, arthritis, fever and oxidative stress. Since Allspice leaves contain stress relieving charecterstics, we have used the extract of the leaves in the protection of liver damaged by intoxication in rats. MATERIALS AND METHODS Chemicals: Chemicals and reagents used for the experiments were of AR grade, purchased from Sigma, HiMedia, NICE and Loba chemicals. Animals: In-bred Wistar rats maintained under controlled conditions of temperature (23 ± 2 C) and humidity (50 ± 5%) and a 12-hour light dark cycle, weighing between 150 to 200 g were used for the experiment. The animals were housed in sanitized polypropylene cages containing sterile paddy husk as bedding. They had free access to standard rat food and water ad libitum. The studies were conducted with the prior approval of Institutional Animal Ethics Committee in accordance with guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. Plant material: Pimenta dioica (L.) Merril (Fam: Myrtaceae) was identified by Dr. Pradeep, Dept of Botany, NSS College, Vazhoor, Kerala. Fresh leaves collected were cleaned and washed with water. Weighed initially and dried under shade at room temperature for constant weight. It was found to loose 70% weight and the leaves became brittle in nature. Dried leaves were then powdered using mixer grinder and stored at 4 C in refrigerator before it was used for the alcoholic extraction. Alcoholic extraction: Powdered leaves were extracted with 70% ethanol in soxhlet apparatus. Each batch was extracted for 40 cycles, and then concentrated in a flash evaporator under reduced pressure and temperature. The semisolid product was stored at 4 C. Further, phytoconstituents were tested by standard methods [17]. Total phenolic content: The herbal extracts are standardized on the basis of their phytoconstituents such as proteins, carbohydrates and total phenolic content (TPC). The alcoholic extract prepared did not contain carbohydrate and protein. Hence, for each lot of extracts TPC was estimated by Folin-Ciocalteu colorimetric method using Gallic acid as a standard and absorbance was measured at 765 nm [18]. Reducing power assay: The assay is based on the principle of oxidation-reduction reaction of potassium ferricyanide and ferric chloride. The reducing power of the potassium ferricyanide increases in the presence of the antioxidants [19]. Aliquots of the extract were mixed with 500 μl of 1% potassium ferricyanide, incubated at 50 C for 20 min. 50 μl of 10% TCA was added and centrifuged at 6000 rpm for 10 minutes. 0.5 ml of 0.1 % ferric chloride was added to supernatant, absorbance was measured at 700 nm (V-630 Jasco Spectrophotometer). Increase in the absorbance indicates the increased reducing power. Ascorbic acid was used as standard and the experiment was repeated in triplicates. DPPH radical scavenging activity: Diphenyl Picryl Hydrazyl (DPPH ) radical scavenging activity was determined as reported earlier [20]. Different aliquots of extract in methanol (10 to 200 µg/ml) were mixed with 1 mm of methanolic DPPH solution to a final volume of 2.0 ml, incubated for 20 minutes at room temperature and absorbance measured at 517 nm (V-630 Jasco Spectrophotometer). Blank was carried out in the same manner without the drug and butylated hydroxyltoluene (BHT) was taken as the standard. The experiment was performed in triplicate and the % DPPH radical scavenging was calculated by the formula: % DPPH radical scavenging = [(Blank absorbance Test absorbance)/ Blank absorbance] x 100. IC50 was calculated using the Microsoft Excel. Lipid peroxidation assay using rat liver homogenate: Wistar rats of 3-4 month old weighing g were killed by cervical dislocation and liver was perfused with cold saline, isolated and 10% w/v homogenate was prepared in ice-cold 0.15 M potassium chloride using homogenator (INCO, India). Homogenate was then centrifuged (Remi, India) at 6000 RPM for 20 min. The supernatant was used for in vitro lipid peroxidation assay [21]. To 0.5 ml of liver homogenate different aliquots of allspice leaves alcoholic extract were added and peroxidation was initiated by 100 μl of ferric chloride, incubated at

3 C for 1 h. The reaction was stopped by adding 2 ml of ice cold 0.25 N hydrochloric acid containing 15% trichloroacetic acid, 0.38% thiobarbituric acid and 0.5% BHT. Reaction mixture was then heated and maintained at 80 C for 1 h, cooled, centrifuged and absorbance of supernatants was measured at 532 nm (V-630 Jasco Spectrophotometer). An identical experiment was performed in absence of extract as blank. Each experiment was carried out in triplicate. Gallic acid was taken as standard antioxidant for comparison. % antilipid peroxidation = [1- (absorbance of sample/absorbance of blank)] x 100. IC 50 was calculated using Microsoft Excel. Protection of induced hepatotoxicity in Wistar rats: Animals of both sexes were randomly divided into five groups (n = 6). Group I and II received 0.5% CMC, group III to V received alcoholic extract at the dose of 250, 500 and 750 mg/kg/day respectively for 15 days. On the 15 th day, one hour after the treatment with the extract, animals in group II to VI were given (1.2 ml/kg i.p) [22]. After 24 h animals were sacrificed by cervical dislocation, blood collected from the carotid bleeding, serum was separated and stored at 4 C till enzyme estimation. The liver was perfused with cold saline, dried, stored at 4 C until used for the enzyme estimation. For the histopathological studies a small part of liver was fixed in 10% buffered formalin solution. The fixed tissues are processed for histopathology. Slides were prepared and stained with haematoxylin and eosin. Preparation of the liver homogenates: 5% liver homogenates was prepared using ice-cold 0.15 M potassium chloride buffer and centrifuged at 6000 rpm for 10 minutes. The supernatant obtained was collected and used for the estimation of thiobarbituric acid reactive substances (TBARS), catalase (CAT), and total proteins (TP) [23-24]. Similarly, 5% liver homogenate in 0.25% sucrose phosphate buffer (ph 7.4) was prepared, centrifuged at 6000 rpm for 10 minutes and the supernatant was used for estimation of superoxide dismutase (SOD) [24]. Protein was estimation by Lowry s method [25]. Serum glutamate oxaloacetate transaminase (SGPT), glutamate oxaloacetate pyruvate transaminase (SGOT), and alkaline phosphatase (ALP) were estimated spectrophotometrically by the standard procedures [26-27]. Statistical analysis: Results were analysed by ANOVA and Dunnet s t test (the level of significance p < 0.05) using the software Graphpad prism version 2. RESULTS Phytochemical findings: 20% yield of alcoholic extract was obtained from the dried leaves of allspice. The extract showed the presence of flavonoids, alkaloids, tannins, triterpenoids and phytosterols. The phenolic content in the alcoholic extract was found to be ± 1.80 mg/ g of extract equivalent to 20 ± 1.63 mg/g of dry leaves. In vitro antioxidant activity: The antioxidant activity is reported to be concomitant with the reducing power of the medicinal plant extracts. The reducing power of alcoholic extract of allspice leaves was compared with the ascorbic acid, using the potassium ferricyanide reduction method. The 6 mg of alcoholic extract showed the reducing power of which was equivalent to of 2 mg of ascorbic acid (Table 1). The Allspice leaf extract showed graded increase in the DPPH radical scavenging activity. It showed the IC 50 of 70 μg/ml which was at par with the synthetic antioxidant BHT (IC 50 = 11 μg/ml) (Table 2). Table 1: Reducing power assay Concentrations Reducing power (mg) Alcoholic extract of allspice leaves Ascorbic acid Table 2: DPPH radical scavenging assay Tested material Alcoholic extract of allspice leaves BHT Table 3: Lipid peroxidation assay Concentrations % DPPH Radical (μg/ml) scavenging IC 50 (μg/ml) % Anti-lipid peroxidation activity Concentrations (μg/ml) Alcoholic extract Gallic acid of Allspice leaves IC 50 (μg/ml) More than 120μg/ml IC 25 is 82.1 μg/ml

4 J. Cell Tissue Research Fig. 1 Fig. 2 Fig. 3 Fig. 4 All figures are histopathology of liver sections stained with haematoxylin & eosin. For detail explanation see text. X 100. Fig. 5 Fig. 1: Liver section of control animal demonstrating normal structure. Fig. 2: Liver in intoxicated animal. Fig. 3: Liver of the animal treated with allspice leaves extract (250 mg/ kg) for 15 days + on 15 th day. Fig. 4: Liver of the animal treated with allspice leaves extract (500 mg/ kg) for 15 days + on 15th day. Fig. 5: Liver of the animal treated with allspice leaves extract (750 mg/ kg) for 15 days + on 15th day. Treatment Total Proteins SGPT SGOT ALP TBARS n=6 mg/dl IU/L IU/L IU/L U/mg of protein Group-I 6.5 ± ± ± ± ± Group-II 5.8 ± a ± 3.39 a ± 3.59 a ± 2.88 a ± 0.03 a Group-III 6.3 ± a ± 1.10 a ± 2.35 a ± 2.37 a ± a Group-IV 6.8 ± a, b ± 1.09 a, b ± 3.98 a, b ± 2.72 a, b ± a, b Group-V 6.4 ± b ± 1.98 b ± 3.88 b ± 3.02 b ± b Treatment Total Protein SOD CAT TBARS n=6 mg/ml U/mg of protein U/mg of protein U/mg of protein Group-I ± ± ± ± 0.29 Group- II ± ± 1.97 a ± 2.39 a ± 0.55 a Group-III ± ± 3.01 a, b ± 2.37 b ± 0.46 a Group-IV ± 3.17 a ± 1.96 a, b ± 2.83 b ± 0.37 a, b Group-V ± 2.90 a ± 2.43 b ± 3.11 b ± 0.52 b 1574 Table 4: Enzyme estimation in Serum: All values are Mean ± SEM; SGOT: Serum glutamate oxaloacetate transaminase; SGPT: serum glutamate Pyruvate transaminase; TBARS: Thiobarbituric Acid Reactive Species; ALP: Alkaline Phosphatase. For abbreviations see Table 5. Table 5: Enzyme estimation in liver homogenate: All values are Mean ± SEM, SOD: Superoxide dismutase; CAT: Catalase; TBARS: Thiobarbituric acid reactive substances; a = p<0.05 Vs normal control (group I); b = p<0.05 Vs intoxicated control (Group II); Treatments: Group- I: 0.5% CMC; Group-II: 0.5% CMC + on 15 th day; Group III to V: Extract at doses 250, 500, 750 mg/kg + (1.2 ml / kg i.p) on 15 th day.

5 120 μg/ml of alcoholic extract showed 42.8 % antilipid peroxidation activity. Concentration above 120 μg/ ml showed unlikely results, hence IC 25 value was calculated (82.1 μg/ml). All these results were at par with the antilipid peroxidation activity of gallic acid a standard antioxidant (IC 50 = 57.3 μg/ml) (Table 3). Hepatoprotection in intoxicated Wistar rats: Liver sections showed large area of damaged architecture characterized by the congestions, cloudy swelling, vacuolar degeneration, nuclear pyknosis, karyolysis and necrotised regions were found in the intoxicated animal (Fig. 2). The hepatic damage in the animals treated with allspice leaves extract (250 mg, 500 mg/kg or 500 mg/kg) for 15 days + on 15th day showed less damage in liver structure (Figs. 3-5). Further, preservation of structural and architectural frame was dose dependent. This was further substantiated by the enzyme estimation in liver homogenate and serum (Tables 4, 5). In the intoxicated rats significance increase in the serum TBARS (0.152 ± 0.03 U/mg protein) was found as compared to the normal control ( ± U/mg protein ). Where as in the animals, treated with three different doses of allspice leaves extract, there was no significant difference as compared to the normal control. Similar results were found in the liver homogenate (Table 5). The serum enzymes SGPT, SGOT and ALP levels were significantly elevated in the intoxicated animals (Table 4). In the treatment group V (750 mg/kg) there was no significant difference as compared with the normal control, whereas at lower doses (250 and 500 mg/kg) there was significant difference but compared with the group II, the enzyme levels were much lower. Similarly, enzymes SOD and CAT in liver homogenate showed significant suppression in intoxicated (Group II) rats. The suppression was graded and at dose 750 mg/kg there was no significant difference compared to the normal control (Table 5). DISCUSSION The antioxidant property of plants is mostly due to phenolic compounds. These compounds also play an important role in auto-oxidation of lipids. The reducing power of medicinal herb extracts containing high levels of polyphenols is significantly correlated to the antioxidant activity [28]. These effects were compared with ascorbic acid which is a reducing agent as well as a reductone. Therefore, allspice leaf 1575 extract has electron donors and can react with free radicals to convert them to more stable products and terminates the radical chain reaction. The antioxidative activity of reductones is believed to break radical chains by donation of hydrogen atoms. The reductones also react with certain precursors of peroxide and prevent the peroxide formation. The allspice leaf extract showed graded increase in the DPPH radical scavenging activity. DPPH is nitrogen centered free radical. It reacts similar to the peroxyl radical. Antioxidants tested on DPPH were also found extremely effective in cell systems of oxidative stress [29]. The allspice leaves contain polyphenols as major constituent, which showed its DPPH radical scavenging activity by their ionisation potential. There may be proton donating and reducing components in the leaf extract other than the eugenol which adds up to the antioxidant activity. The cellular protection is mainly due to the intact cell membrane made up of poly unsaturated fatty acids. Malondialdehyde is the major reactive aldehyde resulting from the peroxidation of biological membrane polyunsaturated fatty acid (PUFA). Melandialdehyde (MDA), a secondary product of lipid peroxidation, can be used as an indicator of tissue damage by a series of chain reactions [30]. MDA is also a byproduct of prostaglandin biosynthesis in inflammation. It reacts with thiobarbituric acid and produces red coloured products measured as TBARS. Allspice leaves have substantial antilipid peroxidation activity in vitro but at par with the standard BHT. The toxic effect of on liver is due to its * conversion to the highly reactive free radical CCl 3 ( + e - * > CCl 3 + Cl - ) by p-450 enzyme system [31]. The oxidative decomposition of the lipid is initiated, and organic peroxides are formed after reacting with oxygen (lipid peroxidation). This reaction is autocatalytic so that new radicals are formed from the peroxide radicals themselves. Thus there is rapid breakdown of the structure and function of the endoplasmic reticulum and induced liver injury is both severe and extremely rapid in onset [32]. Within less than 30 minutes, hepatic protein synthesis declines and within 2 h there is a swelling of smooth endoplasmic reticulum and dissociation of ribosomes from the rough endoplasmic reticulum [33] Mitochondrial injury then occurs, and this is followed by progressive swelling of the cells due to increased permeability of the plasma membrane [34]. Plasma membrane damage is thought to be caused by

6 J. Cell Tissue Research relatively stable fatty aldehydes, which are produced by lipid peroxidation in the smooth endoplasmic reticulum but are able to act at distant sites [35]. As the result of the depletion in the cellular protein synthesis or destruction of the enzymes fatty degeneration takes place [36]. Degeneration of cells may leads to the release of liver enzymes (SGPT, SGOT and ALP), hence their levels are increased in the serum, as observed in the present study. Elevated level of TBARS observed in treated rats indicates excessive formation of free radicals and hepatic damage. On the contrary, allspice leaf extract treated groups showed significant low levels of SGPT, SGOT, ALP and TBARS in serum, signifing the protective effect against the liver toxicity. These treated group of animals, when compared with control, showed no statistical differences in the enzyme levels suggesting that the allspice leaves substantially protected liver damage. The antioxidant enzymes such as SOD, GPX and CAT are the best markers of the liver damage by free radicals and the conditions of oxidative stress. In the induced group the enzyme levels were significantly reduced revealing the oxidative stress. In leaf extract pre-treated groups the enzyme levels were nearer to the control group, demonstrating the protection of induced toxicity. The antioxidant and the hepatoprotective activity can be correlated to the high total phenolic content in the leaf. Thus these results warrant further phytopharmacological investigations on the allspice leaves. REFERENCES [1] McCord, J.M.: Am. J. Med., 108(8): (2000). [2] Valko, M., Leibfritz, D., Moncol. J., Cronin, M.T., Mazur, M. and Telser, J.: Int. J. Biochem. Cell Biol., 39(1): (2007). [3] Halliwell B.: Biochem. Soc. Trans., 35: (2007). [4] Bandyopadhyay, U., Dipak, D. and Ranajit, B.K.: Current Sci., 77 (5): (1999). [5] Pecker, L.: Antioxidant Food Supplement in Human Health. Acad. Press (1999). [6] Yen, G.C. and Chen, H.Y.: J. Agric. Food Chem., 43: (1995). [7] Irshed, M. and Chaudhuri, P.S.: Ind. J. Expt. Biol., 40(11): (2002). [8] Pecker, L. and Cadenas, E.: Hand Book of Synthetic Antioxidants. Marcel Decker (1997). [9] Rodrigo, R., Guichard, C. and Charles, R.: Fundam. Clin. Pharmacol., 21(2): (2007). [10] The Wealth of India - A Dictionary of Indian Raw Materials. CSIR, New Delhi, Vol-8: pp (1969). [11] Kikuzaki, H., Miyajima, Y. and Nakatani, N.: J. Nat. Prod., 71(5): (2008). [12] Miyajima, Y., Kikuzaki, H., Hisamoto, M. and Nikatani, N.: Biofactors., 21(1-4): (2004). [13] Suárez, A., Ulate, G. and Ciccio, J.F.: Rev. Biol. Trop., 48(1): (2000) [14] Kikuzaki, H., Sato, A., Mayahara, Y. and Nakatani, N.: J. Nat. Prod., 63(6): (2000). [15] Suárez, A., Ulate, G. and Ciccio, J.F.: J. Ethnopharmacol., 55(2): (1997). [16] Marzouk, M.S., Moharram, F.A., Mohamed, M.A., Gamal-Eldeen, A.M. and Aboutabl, E.A.: Z. Naturforsch., 62(7-8): (2007). [17] Gokhale, S.B., Kokate, C.K. and Purohit, A.P.: Pharmacognosy. 4 th ed., Nirali Prakashan, Pune (1996). [18] Aaby, K., Skrede, G. and Wrolstad, R.E.: J. Agric. Food Chem., 53(10): (2005). [19] Pin, D.D. and Yen, G.C.: Food Chem., 60(4): (1997). [20] Rajakumar, D.V. and Rao, M.N.: Free Radical Res., 22(4): (1995). [21] Ko, K.M. and Godin, D.V.: Mol. Cell Biochem., 95(2): (1990). [22] Rusu, M.A., Tamas, M., Puica, C., Roman, I. and Sabadas, M.: Phytother. Res., 19(9): (2005). [23] Ohkawa, H., Ohishi, N. and Yagi, K.: Anal. Biochem., 95: (1979). [24] Erlich, F.E.: Analytical Chem., 70: (1976). [25] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randal, R.J.: J. Biol. Chem., 193: (1951). [26] Reitman, S. and Frankel, S.: Am. J. Clin. Pathol., 28(1): (1957). [27] Kind, P.R.N. and King, E.J.: J. Clin. Pathol., 7(4): (1954). [28] Pak, E., Esrason, K.T. and Wu, V.H.: Prog. Transplant., 14(2): (2004). [29] Aruoma, O.I.: Mutat. Res., : 9-20 (2003). [30] Tuma, D.J.: Free Radical Biol. Med., 32(4): (2002). [31] Ruch, R.J., Klaunig, J.E., Schultz, N.E., Askari, A.B., Lacher, D.A., Pereira, M.A. and Goldblatt, P.J.: Environ. Health Perspect., 69: (1986). [32] Slatter, T.F.: Nature, 209 (1): (1996). [33] Archakov, A.I. and Karuzina, II.: Biochem. Pharmacol., 22(17): (1973). [34] Johnston, D.E. and Kroening, C.: Pharmacol. Toxicol., 83(6): (1998). [35] Le Page, R.N., Cheeseman, K.H., Osman, N. and Slater, T.F.: Cell Biochem. Funct., 6(2): (1988). [36] Weber, L.W., Boll, M. and Stampfl, A.: Crit. Rev. Toxicol., 33(2): (2003). 1576