Asian Pacific Journal of Tropical Medicine (2010)696-701 696

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Hepatoprotective activity of brown alga Padina boergesenii against CCl4 induced oxidative damage in Wistar rats Rajamani Karthikeyan1, ST Somasundaram1, T Manivasagam2, T Balasubramanian1, P Anantharaman1*

1Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai-608 502, Tamil Nadu, India 2Department of Bio-chemistry and Bio-technology, Annamalai University, Chidambaram-608 002, Tamil Nadu, India

ARTICLE INFO ABSTRACT

Article history: Objective: Padina boergesenii P. boergeseii To investigate the protective effects of brown alga ( ) Received 13 August 2010 Methods: against carbon tetrachloride (CCl4) induced oxidative damage and liver fibrosis in rats. Received in revised form 27 August 2010 To assess the hepatic damage liver weight, the activities of TBARS level, glutathione, SOD, CAT Accepted 1 September 2010 Results: and GPx in circulation and liver. The group of rats induced with CCl4 alone (2 mL/kg Available online 20 September 2010 body weight), showed noticeable increase in the liver weight and TBARS level. Followed by, the level of antioxidantP enzymes glutathione peroxidase, Superoxide dismutaseP. boergesenii and catalase was Keywords: also significantly ( <0.01) diminished. Where as, the rats pretreated with (150 mg/ kg body weight) modulated the CCl4 induced liver fibrosis. The level of antioxidant enzymes and PadinaHepatotoxicity boergesenii P lipid peroxidation products was found to be significantly ( <0.01) attenuated to near normal level, Carbon tetrachloride when compared with rats induced with CCl4 alone. In order to assess the role of carotenoids in Liver fibrosis the/ relevant activity, total carotenoid content of the extract was analysed and found to be 0.59 FW) Carotenoids mg g fresh weight ( . Further, the histopathogicalP. boergesenii. studiesConclusions: provide a supportive evidence for this study to show the protective nature of The protective role P. boergesenii of brown alga extract has confirmed its potential activity through its antioxidant sparing actions against CCl4 induced free radical damage. However, the possible mechanism of hepatoprotection is rather speculative at thisP. stage boergesenii and investigations are underway to isolate and characterize the bioactive compounds from .

1. Introduction pigments-chlorophylls, carotenes and other compounds while carotenoids are powerful antioxidants. Fucoxanthin is a major carotenoid of [3], which reported to have Brown algae are rich sources of various bioactive beneficial effects on cancer as chemopreventive agent[4]. compounds, including polyphenols, carotenoids and It is commonly recognized that antioxidants can able to polysaccharides with different physiological effects (toxic neutralize harmful free radicals in body cells before they or curative) on human health. The components of various cause lipid and protein oxidation. Excessive production of brown algae (Phaeophyceae) have been widely ROS and the related oxidative stress have been proposed investigated and it has rich inorganic composition. More to play a several roles in the pathogenesis of chronic- than 1 140 secondary metabolites have been reported degenerative conditions, such as neurodegenerative from Phaeophyceae. The brown algae are a rich source of diseases, cancer, arteriosclerosis, malaria, rheumatoid various natural antioxidants such as polyphenols, which arthritis & diabetes[5]. Hence, the development of play an important role in preventing lipid peroxidation[1]. antioxidants from natural origins has drawn more attention In addition to potential role of polyphenols Lewin[2] have amongPadina the researchers against free radicals. The brown stated that, the lipids of algae comprise photosynthetic alga isPadina widely distributed along the Indian coast, and species of arePadina important boergesenii source ofP. mannitol boergesenii and iodine. The brown alga ( ) *Corresponding author: Dr. P. Anantharaman, Associate Professor, CAS-In Marine Biology, Faculty of Marine Sciences, Annamalai University, Portonovo-608 502 Tamil is a fan-shaped plant body, segmented, ruffled, and light Nadu India. brown in colour. The presenceP. boergeseniiof phenolic compounds andin Tel: +914144 243223 its anticancer activity from has proven on Fax: +914144 243555 vivo [6] E-mail: [email protected] model . Since, there was no report of carotenoids and Rajamani Karthikeyan et al./Asian Pacific Journal of Tropical Medicine (2010)696-701 697 P. boergesenii. 2.5. Animal and housing its antioxidant activity of this particular alga Therefore, the aim of theP. present boergesenii study was to investigate the ameliorative effect of against CCl4 induced The male albino rats of the Wistar strain (4-6 weeks old), liver fibrosis in Wistar rats. Hence, we expect to detect the weighing 125-150 g, were obtained from Central Animal higher levels of lipid peroxidation and its products in blood House, Faculty of Medicine, Rajah Muthiah Medical and liver organ. This investigation not only provides the College and Hospital, Annamalai University, India. information about the therapy or prevention of such liver Animal experiments were approved by the Institutional disease, but also could promote an understanding of its Ethical Committee Regulations. The Central animal 160/1999/ CPCSEA mechanisms, especially to control the role of free radicals house registration number is ad libitum. The rats or oxidative stress in the pathogenesis of acute toxic liver were fed with pellet diet and water . During injury. experimentation they were housed in the respective polypropylene cages and allowed free access to food and water. The animals were maintained in a controlled 2. Materials and methods environment under standard conditions of temperature and humidity with alternating 12 h light/dark cycles. 2.1. Chemicals 2.6. Experimental induction of hepatotoxicity Reduced glutathione (GSH), oxidized glutathione (GSSG), 4 glutathione peroxidase, thiobarbituric acid (TBA), CCl and All rats were allowed to acclimatize for 1 week prior P.to diethyl ether were purchased from Sigma Aldrich. All other boergeseniiexperimentation. The treatment regimen for brown inalga vitro chemicals and solvents of analytical grade were obtained extract was based on the preliminary [6] CCl4 from S.D. Fine chemicals, Chennai, India. studies carried out inet our al laboratory . dose was selected according to Janbaz [10]. A total of 24 rats were used the 2.2. Collection of seaweeds animals were randomized and had free access to standard laboratory feed. The rats are grouped into experimental and Padina boergesenii n In the present study the seaweed control rats ( =6 in each group). Group I served as saline- belonging to the class Phaeophyceae, family , treated negative control and was administered with saline (0.85% NaCl) collected from Vaan island,º Gulf of Mannarº Biosphere in the alone. Group II subcutaneously injected with intertidal region (Lat 11 29`N; Long 79 46`E) during April CCl4 (2 mL/kg body weight) every day for 1 month. Groups III and May 2007. The algal species were hand picked and were pretreated with diethyl ether extract with dose of 150 washed thoroughly with seawater to remove all the impurities mg/kg body weight orally by a tube, which is inserted into like sand particles and epiphytes. Morphologically distinct the mouth and down the oesophagus of the rat, where the thallus of algae was placed separately in new polythene substance is subsequently injected into the stomach by the bags and were kept in an icebox containing slush ice and syringe connected to the gavage tube for one month before CCl4 transported to the laboratory. Finally, the seaweeds were one hour of administrationP. boergesenii of . Group IV. Rats were rinsed carefully in freshwater and shade dried then the orally administered with diethyl ether extract 2 (150 mg/kg body weight) alone for one month. At 24 h after samples were pulverized into powder (size P. mm) boergesenii using a grinder before extraction. The brown alga theth last treatment of extracts or pellet feed animals on the were identified by their taxonomical features[7]. 30 day from group II and III received an i.p. injection of CCl4 (2 mL/kg body weight). 2.3. Preparation of seaweed extract 2.7. Collection of blood In general, the principle of soxhlet extraction is an [8] 5 At the end of the experiment, all the animals were extractionPadina methodboergesenii by using chemical solvents . The g of grinded powder was extracted with sacrificed to collect blood samples by cardiac puncture. 100 mL of diethyl ether (Non polar solvent) using a soxhlet Blood plasma is prepared by spinning a tube of fresh blood extractor for 8 hours at 35 曟. The extraction was repeated containing an anti-coagulant in a centrifuge until the blood many times to obtain a sizable quantity of extract. The cells fall to the bottom of the tube. The obtained plasma extracts were then concentrated under reduced pressure was kept at -70 曟 before determination of the biochemical 4 parameters for GSH, GPx, SOD and CAT. and the resultant residues were stored in avivo dark at 曟 until further use. For the investigation of study diethyl 2.8. Preparation of hemolysate ether residue were dissolved in 0.3% Carboxyl Methyl Cellulose as a vehicle. After the collection of plasma, the buffy coat was removed 2.4. Estimation of carotenoids and the packed cells were washed with cold physiological saline. A known value of the RBC was lysed with cold P. boergesenii The presence of carotenoids in extract was hypotonic phosphate buffer, pH 7.4. The hemolysate were estimated according to the protocol prescribed by Parsons separated by centrifugation at 2 000 g for 10 minutes at 20 曟 to and Strickland[9]. determine the activity of antioxidant enzymes SOD, CAT, GPx. Rajamani Karthikeyan et al./Asian Pacific Journal of Tropical Medicine (2010)696-701 698 2.9. Evaluation of liver damage in plasma as units/mL and in liver as units/mg protein. 2.12.2. Estimation of catalase Livers of all rats were excised immediately after collection of the blood. Some of the liver tissue samples were washed The catalase activities were determined by the method of twice with a cold saline solution and were placed into the conversion of dichromate in acetic acid to perchromic acid glass labeled bottles. The tissue samples from the group and then to chromic acetate in the presence of H2O2 when of rats were used for determination of differences in liver subjected to heat[16]. Thus formed chromic acetate was weight and to assess biochemical parameters as an index measured at 620 nm and the activities were expressed as of lipid peroxidation. The other liver tissue specimens were 毺M of H2O2 consumed units/mL and in liver as units/mg used for histopathological examination. protein. 2.10. Preparation of tissue samples 2.12.3. Estimation of glutathione peroxidase Glutathione peroxidase was estimated by the reaction The part of the liver tissue sample was rinsed in the sample with hydrogen peroxide in the presence of GSH GSH physiological saline, and homogenized. The homogenate for a specified time period. Then theet remainingal were (25% w/v) was in ice-cold 0.25 M sucrose, then the measured by the method of Rotruck [17]. The activities homogenate was deproteinized with an equal volume of ice- were expressed as 毺g of GSH consumed units/mL and in cold (10% w/v) trichloroacetic acid. The ensuing supernatant liver as units/mg protein. fraction was analyzed for enzymatic and non-enzymatic 2.12.4. Histopathological examination biochemical assays. 2.11. Biochemical investigations After animal experimentation the liver were dissected immediately and fixed in 10% neutral formalin solution for 24 2.11.1. Vitamin E in liver at least for h and was embedded in paraffin wax (melting 56-58 4 point 曟). Tissue sections were’ made with 毺m The level of vitamin3+ 2+ E were determined by the method of thickness and were stained with Mayer s hematoxylin and (H&E) reduction in Fe to Fe by the2+ tocopherolsα α with the formation eosin for histopathological examination under light [11,12] [18] of a red complex of the Fe with , ` -dipyridyl ’ . microscope . The transmissions were measured by calorimeter and it s 2.13. Statistical analysis expressed in terms of 毺g/mg protein. 2.11.2. Estimation of thiobarbituric acid reactive substances The results were analyzed among the experimentalP P group (* <0.05, ** <0.01 (TBARS) of animals and statistical significance ) between controls and treated groups were evaluated using TBARS (ANOVA) The level of in liver were estimated in liver by multivariate’ analysis of variance followed by measuring malondialdehyde and TBARS reactivity with Duncan s multiple range test. thiobarbituric acid (TBA) to generate a pink coloured 535 [13] chromophore, which was read at nm . The’ 3. Results transmissions were measured by calorimeter and it s 100 expressed in terms of mM/ g wet tissue. 3.1. Effects of P. boergesenii on liver weight 2.11.3. Estimation of reduced glutathione P. boergesenii The total carotenoid content-1 of extract was 0.63 FW CCl4 2 Reduced glutathione was estimated in plasma and liver found to be mg毺g . Induction Rattus of novergicus ( mL/kg, by the method of Ellman[14]. The yellow colour developed S.C.) results in acute liver injury in . The 412 5.85依0.39) g CCl4 was read at nm. A series of standards were treated in liver weight was P.increased boergessenii to ( in induced a similar manner along with a blank containing 3.5 mL of group, where as extract treated group (150 buffer. The amount of glutathione was expressed as mg/dL mg/kg, orally) showed a decrease in the liver weight to (3.57依 plasma and mg/100 g tissue. 0.33) g. 2.12. Quantitative analysis of enzyme activities 3.2. Effects of P. boergesenii extract on vitamin-E

2.12.1. Estimation of superoxide dismutase CCl4 Since administrationP caused hepatic injury with (SOD) ( <0.01) A superoxide dismutase activity was determinedet significantP. boergesenii decrease in vitamin-E, on treatmentP ( <0.01) alin plasma and liver based on the method of Kakkar with extract there was a significant [15]. SOD removes the super-oxide radicals and inhibits increase in the level of vitamin-E (Figure 1). the formation of formazan blue. The intensity of colour is 3.3. Effects of P. boergesenii extract on TBARS & reduced inversely proportional to the activity of the enzyme. One unit GSH content of the enzyme activity is defined as the enzyme reaction, which gave 50% inhibition of NBT reduction in one minute. Thus obtained values could be expressed as specific activity The induction of CCl4 (2 mL/kg body weight, S.C.) was Rajamani Karthikeyan et al./Asian Pacific Journal of Tropical Medicine (2010)696-701 699 associated with alteration of lipid peroxidation products peroxidase (GPX) in circulation as well as in liver tissue. illustrating a significant increase in TBARS content to that After intoxication of CCl4, the level of SOD, CAT and GPX (Table 3) (Table 4) of control animals. On treatmentP with extract, the level was decreased bothP. boergesenii in circulation and liver . TBARS ( <0.01) 150 of was significantlyP. boergesenii lowered in the liver Treatment with extract ( mg/kg, orally) was CCl4 (Figure 2). Treatment with extract was able to able to overcome the induced hepatocellularP damage, ameliorate CCl4 induced hepatocellular damage as evidenced which was evidenced by significant ( <0.01) increase in through the activity of glutathione. The level of GSH in the activity of SOD. Since CCl4 induction caused significant hepatic injury and inflammation, the toxicity of CCl4 and circulationP as well as in tissues was significantly decreased P. boergesenii ( <0.01) after the rats induced with CCl4, where as the rat protective effects of extract was revealed by 150 enzymatic assay of catalase. It showed a significant increase treated with extract ( Pmg/kg, orally) showed considerable P GSH ( <0.01) in catalase activity ( <0.01) both in circulation and liver as increase in P. level boergesenii as shown in Table 1. This tendency of the extract reflects its potential shown in Table 2 & 3. Simultaneously, the enzymatic assay antioxidant nature. of glutathioneP peroxidase activity was also significantly increased ( <0.01). 3.5. Histopathological changes 3.5 Vit-E in Liver 3.0 The tissue sections stained with H&E were examined for 2.5 the extent of liver damage, inflammation, and liver cell 2.0 necrosis. Extensive liver damage areas were observed in CCl4 1.5

g/mg of protein challenged group. Hepatocytes surrounding the central vein 毺 1.0 and perivenular regions exhibited coagulative type of necrosis 0.5 against various oxidative stress conditions (Figure 3). CCl4 (2 mL/ 0 kg body weight, S.C.) administration produced intense necrotic Normal CCl4 CCl4+Extract Extract zones, edema, acute inflammatory infiltration, and hepatocyte Groups degeneration characterized by cytoplasmic swelling and Figure 1. nuclear damage. These changes in the hepatic architecture Activity of Vit-E in liver. Values are expressed in Mean 依 n P were P.not boergesenii observed in any of the control rats or animals treated S.E.M. ( =6); * significantly ( <0.01) different from control group. with extract (150 mg/kg, orally) alone. Treatment Table 1 of this extract prevented CCl4-induced morphological changes in some zones like edematous hepatocytes and showed less Changes in the level of reduced glutathione in plasma and tissues. cytoplasmic degeneration along with less inflammatory Plasma Liver infiltration. There was no necrosis observed in the extract No Groups (mM/100 mL) (mM/100 g tissue) treated group of animals. 1 Normal 18.66依1.43 114.37依8.71 2 CCl4 10.05依0.23* 75.66依0.23* 3 CCl4+Extract 16.45依1.26* 99.07依4.10* 4 Extract 22.06依1.68 118.60依4.13 n P Values are expressed in Mean 依 SEM ( =6); *significantly ( <0.01) different from control group.

TBARS-LIVER 8 7 6 5 4

mg/100 g tissue mg/100 3 2 1 0 Normal CCl4 CCl4+Extract Extract

Groups Figure 2. Level of TBARS in liver. Values are expressed in Mean 依 n P SEM ( =6); * significantly ( <0.01) different from control group. 3.4. Changes in the activities of SOD, CAT and GPX Figure 3. Histopathology of liver. A: Shows normal central vein and portal triad, B: CCl4 toxicity in rats The activities of hepatic enzymes were altered by (2 mL/kg body weight, S.C.,)P. boergesenii show macro and micro vesicular changes, induction of CCl4 (2 mL/kg body weight, S.C.) in rats, C: Rats pretreated with (150 mg/kg bodyweight, orally) which was quantified by biochemical markers like super- one hour before CCl4 induction. D: Rats treated with extract alone (150 oxide dismutase (SOD), catalase (CAT) and glutathione mg/kg bodyweight, orally) show normal histology with central vein. Rajamani Karthikeyan et al./Asian Pacific Journal of Tropical Medicine (2010)696-701 700 Table 2 P. boergesenii Effect of extract on antioxidant enzymes in circulation. No Groups Superoxide dismutase(units/mL) Catalase (毺M of H2O2) Glutathione peroxidase(units/mL) 1 Normal 2.17依0.17 5.86依0.56 8.48依0.36 2 CCl4 1.48依0.11* 3.59依0.16* 5.88依0.62* 3 CCl4+Extract 1.86依0.28* 4.90依0.34* 7.96依0.40 4 Extract 2.14依0.17 5.79依0.42 8.38依0.46 n P Values are expressed in Mean 依 SEM ( =6); significantly different from control (* <0.01).

Table 3 P. boergesenii Effect of extract on antioxidant enzymes in liver. No Groups Superoxide dismutase(units/mg protein) Catalase(units/mg protein) Glutathione peroxidase(units/mg protein) 1 Normal 17.95依0.52 50.24依4.64 11.58依0.86

2 CCl4 6.58依0.81** 40.99依2.39* 5.34依0.35**

3 CCl4+Extract 9.87依1.36** 45.24依1.19* 8.38依0.46** 4 Extract 16.67依0.69 52.13依5.33 11.69依0.86 n P P Values are expressed in Mean & SEM ( =6); significantly different from control (* <0.05, ** <0.01).

4. Discussion P. boergesenii the one hand, the pretreatment of rats with extract prevented CCl4 toxicity on lipid peroxidation, GSH Hence, the data of this present study show that the brown and antioxidant enzyme levels in circulation as well as liver P. boergesenii P. boergesenii alga showed hepatoprotective activity tissue. These results suggest that, the act as an against CCl4 induced liver fibrosis in Wistar rats. It is well effective antioxidant or a free radical scavenger was able to recognized that, the CCl4 is a lipid-soluble and is thus stabilize membrane structures, thus, to preserve the cellular well distributed through out the body. The biochemical integrity and to restrain the severity of CCl4 induced cellular P P mechanisms involved in the development of CCl4 injury at significant level ( <0.05, <0.01). On the other hand, P. boergesenii hepatotoxicity have long been investigated. It is believed the brown alga showed its protective role that, it is due to lipid peroxidation caused by the carbon against cellular damage as evidenced by histopathological trichloromethyl radical, CCl3 . CCl4 is biotransformed by sections. Based on these results, it is reasonable to suggest P. boergesenii cytochrome P450 to the trichloromethyl-free radical that that the presence of carotenoids from extract 2+ induces membrane lipid peroxidation and disturbs Ca might exert their mechanism different from its antioxidant homeostasis to produce hepatocellular injury. Therefore, action or free radical scavenging activity. the administration of CCl4 causes necrosis, fibrosis in liver. In conclusion, as far as the present data is concerned, this The CCl4 induced hepatotoxicity lead to rapid increase in study is the first investigation to show that the presence P. boergessenii the serum enzyme levels, which have been attributed to of carotenoids from brown alga possesses damage in the structural integrity of cell membrane in liver a potent antioxidant activity against CCl4 induced acute and leakage of transaminases from the cytoplasm into the liver damage in rats. This was revealed by changes in blood circulation[18,19]. The toxicity of CCl4 has been well level of TBARS and GSH concentration in liver, in addition attributed in assessing changes in body weight of the rats. to the elevation of SOD, CAT and GPx activity. Whereas, P. boergesenii In addition to liver weight, we assessed the changes in able to ameliorate acute liver damage body weight of the rats during the experimental periods[20]. to a high degree, as evidenced by the improvement in P. boergesenii Free radical catalyzed lipid peroxidation is thought to be histopathological changes. The brown alga one of the major processes involved in hepatotoxicity or extract proved its activity either through stabilization of xenobiotic hepatotoxicity. Free radicals are the essential cellular membrane or through antiperoxidase activity. The components of energy and healthy immune function; they outcome of the present study reveals that, there is a strong must be controlled to prevent cell and tissue damage. These correlation between antioxidant activity and carotenoid free radicals are concerned in around sixty negative health content. It is believed that, carotenoid has the ability to conditions, which include heart disease, cancer, macular act as reducing agents, hydrogen donors, and free radical degeneration, cellular degradation (ageing), and numerous quenchers which can also act as metal chelators. Further inflammatory diseases. investigations are in progress to analyze and characterize the P. boergesenii The brown alga has proven its potential bioactive compounds responsible for this antioxidant nature P. boergessenii nature in two ways against such free radical damages. On from . Rajamani Karthikeyan et al./Asian Pacific Journal of Tropical Medicine (2010)696-701 701 [7] Allender BM, Kraft GT. The marine algae of Lord Howe Island Conflict of interest statement (New South Wales): the and cutleriales (Phaeophyta). Brunonia 6 1983; : 73-130. We declare that we have no conflict of interest. [8] Lim SN, Cheung PCK, Ooi VEC, Ang PO. Evaluation of antioxidative activity of extracts from brown seaweed, Sargassum Acknowledgements J Agric Food Chem 50 siliquastrum. 2002; : 3862-6. [9] Parsons TR, Strickland JD. Discussion of spectrophotometric determination of marine plant pigments with revised equations We are grateful to Prof. Venugopal P. Menon, Director J Mar Res for ascertaining chlorophylls and carotenoids. 1963; of Research, Annamalai University for purveying this 21 opportunity to do this research work. The authors wish to : 155-63. thank Department of Biotechnology (DBT), India for the [10]Janbaz KH, Saeedb SA, Gilani AH. Studies on the protective effects of caffeic acid and quercetin on chemical-induced financial support. Finally, we would like to thank Dr. P. Phytomedicine 11 Subramanian for his inspiring support to carry out this work. hepatotoxicity in rodents. 2004; : 424-30. [11]Emmerie A, Engel C. Calorimetric determination of alpha References Rev Trav Chim tocopherol (Vitamin E). 1938a; 57: 1351. 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