Hepatoprotective Effect of Cinnamon Extracts against Carbon Tetrachloride Induced Oxidative Stress and Liver Injury in Rats

Cinnamon is used to flavor most foods in Arabian countries. The aim of this study was to evaluate the medicinal importance, reflecting an important trend in research. The hepatoprotective activity of aqueous and ethanolic extracts of cinnamon was investigated against carbon tetrachloride (CCl 4) induced lipid peroxidation and hepatic injury in rats. The elevated serum AST and ALT enzymatic activities induced by CCl 4 were significantly restored to near normal by oral administration of 200 mg/kg of either extracts once daily for 7 days, as compared to untreated rats. There was a significant elevation in the level of liver malondialdhyde (MDA), while the activities of antioxidant enzymes superoxide dismutase and catalase (SOD and CAT) were significantly decreased in CCl 4 intoxicated rats. The results obtained indicated that ethanolic extract has more potent hepatoprotective action than water extract against CCl 4 by lowering the MDA level and elevating antioxidants enzymes activities (SOD and CAT). The possible mechanism of this activity may be free radical-scavenging polyphenol compounds. The hepatoprotective properties were documented by the histopathological data obtained. Consequently, this extract can be used as a therapeutic regime in treatment of some hepatic disorders without any side effects. Further study will be done for separation and identification of active components and for testing antitumor activity.


INTRODUCTION
Free radicals in the form of reactive oxygen and nitrogen species are an integral part of normal physiology.Over-production of these reactive species can occur, due to oxidative stress brought about by the imbalance of the bodily antioxidant defense system and free radical formation.These reactive species can react with biomolecules, causing cellular injury and even death.They can lead to the development of chronic diseases such as cancers and those that involve the cardioand cerebrovascular systems (Gurdip et al., 2007).The consumption of fruits and vegetables (Peschel et al., 2006) containing antioxidants has been found to offer protection against these diseases.Antioxidants are often added to foods to prevent the radical chain reactions of oxidation, and they act by inhibiting the initiation and propagation step leading to the termination of the reaction and delay the oxidation process (Shahidi et al., 1992).
Cinnamon is a popular flavoring ingredient, widely used in food products.It has exhibited beneficial properties to health, such as antimicrobial activity, for controlling glucose intolerance and diabetes, inhibiting the proliferation of various cancer cell lines, and for treating the common cold (Anderson & Broadhurst, 2004;Murcia et al., 2004).Cinnamon extracts can reduce lipid peroxidation in the β-carotene-linoleic acid system, (Mancini-Filho & Van-Koiij, 1998).
Cinnamon extracts exhibit a protective capacity against irradiation induced lipid peroxidation in liposomes, and quench hydroxyl radicals and hydrogen peroxide (Murcia et al., 2004).Extracts on lard and vegetable oils demonstrated that they could stabilize lard against oxidation and showed antioxidative properties when tested on vegetable oils during storage or frying conditions (Vekiari, & Thomopoulos, 1993).
In spite of the tremendous advances in modern medicine no effective drugs are available, which stimulate liver functions and offers protection to the liver from damage or help to regenerate hepatic cells (Chattopadhyay, 2003).In the absence of reliable liver-protective drugs in modern medicine, a large number of medicinal preparations are recommended for the treatment of liver disorders (Chatterjee, 2000) and quite often it is claimed they offer significant relief.The goal of the present study was to examine the antioxidant and hepatoprotective activity of water and ethanolic extracts of cinnamon against oxidative stress induced by CCl 4 in rats.

Animals
Forty male albino Wister rats weighing (60-80gm) were used in this study .The animals were kept at 27±2 C, for 1 week before and during the experiments.Animals were provided with standard diet and water ad libitum.

Tested cinnamon
Cinnamon powder was obtained from the local market at Jeddah, Saudi Arabia.
All reagents and solvents used in this study were purchased from Aldrich Company unless otherwise stated.

Preparation of cinnamon extract
The dried powder was defatted with petroleum ether (100 gm in 200 ml ether).The defatted material was extracted with 95% ethanol and then vacuum dried.One part of powder was extracted in boiling water and then filtered and vacuum dried.

Hepatotoxicity and treated groups.
Animals were divided into four groups (n = 10).Group I (control).Group II (CCl4) rats were injected with a single dose of CCl4 in corn oil (1mL/kg body weight, s.c.) (Avijeet et al.,2008).Groups III and IV were administered 100 mg/kg of aqueous or ethanolic extracts orally by gastric tube, respectively, in the form of aqueous suspension once daily for 7 days.The animals were then simultaneously administered a single dose of CCl4 (1mL/ kg body weight, s.c.).The animals were sacrificed 24 h after the last treatment.Blood was collected, allowed to clot and serum was separated at 2500 rpm for 15 min.The livers was dissected out and divided into two parts.The first was kept at -20 o C for analysis, while the second part was used for histopathological studies.

Estimation of MDA, SOD, CAT in liver tissue
Liver samples were dissected out and washed immediately with ice cold saline to remove as much blood as possible.Liver homogenates (5% w/v) were prepared in cold 50mM potassium phosphate buffer (pH 7.4) using glass homogenizer in ice.The cell debris was removed by centrifugation at 5000 rpm for 15 at 4 0 C using refrigerated centrifuge.The supernatant was used for the estimation of malondialdehyde (MDA) (Yagi and Rastogi, 1979), superoxide dismutase (SOD) (Kakkar et al., 1972) and catalase (Smna, 1972) levels.

Histopathological studies
The liver tissue was dissected out and fixed in 10% formalin, dehydrated in gradual ethanol (50-99%), cleared in xylene, and embedded in paraffin.Sections were prepared and then stained with hematoxylin and eosin dye.

2.8.Statistical analysis
Statistical analysis was performed on a PC using SPSS, V.13, (special package for social sciences).Data are presented as arithmetic mean ± S.D., The difference among means has been analyzed by oneway ANOVA followed by student t test.A value of P < 0.05 was considered as statistically significant.

RESULTS
The results of hepatoprotective effect of water or ethanol extracts on CCl4intoxicated rats are shown in Table 1.In the CCl4 intoxicated group serum AST and ALT were significantly increased as compared to control group (p<0.001).The elevated activities of serum AST and ALT were significantly reduced in the animal groups treated with either water or ethanolic extracts.Treatment with ethanolic extract showed significantly more activity (P < 0.001) than with water extract.Thus, the ethanol extract treated group was superior to the water extract.Results obtained revealed an increase in the level of liver MDA in CCl4-intoxicated rats compared to control group.Treatment with extracts significantly prevented this raise in levels.The activities of SOD and CAT were significantly reduced in the CCl4intoxicated group, while they were significantly elevated in the groups pretreated with either extracts.Ethanolic extract has been shown to be more protective than water extract.
Fig 1 .Histological examination of liver sections of control animals (Group I) showed normal hepatic cells with wellpreserved cytoplasm prominent nucleus.The livers of CCl4-intoxicated rats showed massive fatty changes, necrosis, and broad infiltration of the lymphocytes.The histological architecture of liver sections of the rats treated with either aqueous or ethanolic extracts showed more or less normal patterns, with a mild degree of fatty change, necrosis and lymphocyte infiltration, almost comparable to those of the control group 4. DISCUSSION Cinnamomum verum belongs to the family Lauraceae and possesses significant ant allergic, antiulcerogenic, antipyretic and anesthetic activities (Kurokawa & Shiraki, 1998).The bark yields an essential oil containing cinnamaldehyde and eugenol.Several biological activities, such as peripheral vasodilator, antitumor, antifungal, cytotoxic and antimutagenic, have been attributed to cinnamaldehyde (Shaughnessy& DeMarini, 2001).
Serum AST and ALT activities were used as a marker of liver damage.CC1 4 produces an experimental damage (James and Pickering, 1976).The toxic metabolite .CC1 3 radical is produced by cytochrome p 450 which further reacts with oxygen to produce trichloromethyl peroxy radicals.These radicals bind covalently with the macromolecule and cause peroxidative degradation of lipid membrane of the liver.The reduction of AST and ALT activities by the extracts is an indication of repair of hepatic tissue damage induced by CC1 4 .This is in agreement with Thabrew et al. (1987), who found that serum transaminases returned to normal activities with the healing of hepatic parenchyma and regeneration of hepatocytes, The ethanolic extract induced suppression of increased ALT and AST activities.Thus, administration of ethanolic or aqueous extracts of cinnamon revealed hepatoprotective activity against the toxic effect of CCl 4 , which is also supported by histological studies.
Oxidative stress induced due to the generation of free radicals and/or decreased antioxidant level in the target cells and tissues has been suggested to play an important role in carcinogenesis (Huang et al., 1999).During cell membrane damage, various enzymes leak down to the circulatory fluid and their assessment in serum serves as markers in clinical studies.SOD is the first antioxidant enzyme to deal with oxyradicals by accelerating the dismutation of superoxide to hydrogen peroxide, while CAT is a peroxisomal hem protein that catalyses the removal of  (Bhathal et al., 1983).A major defense mechanism involves the antioxidant enzymes, including SOD, catalase and glutathione peroxidase (GPx), which convert active oxygen molecules into nontoxic compounds.Lipid peroxidation is accelerated when free radicals are formed as the results of losing a hydrogen atom from the double bond in the structure of unsaturated fatty acids.The free radical scavenging activity of water or ethanolic extracts of cinnamon was evaluated.Lan et al., (2007) stated that 50% of acetone extract of cinnamon contained high levels of phenolic groups.Scavenging of free radicals is one of the major antioxidation mechanisms to inhibit the chain reaction of lipid peroxidation.
Reduced lipid peroxidation was revealed by a significant decrease in MDA level in groups pretreated with water or ethanol extracts, simultaneously with a significant elevation in SOD and CAT activities.
The present study revealed that SOD and CAT activities decreased in CCl 4 injected animal, which may be due to altered antioxidant status.This is in accordance with results that indicated a decreased CAT in CCl 4 injected animals may be due to the utilization of antioxidant enzymes in the removal of released H 2 O 2 released (Cerutti et al.,1994).SOD and CAT activities increased significantly in the treated group versus the untreated animals.

Fig. 1 :
Fig. 1: Light micrographs of mice liver treated with CC14, without and with pretreatment with either water or ethanol extract of cinnamon compared to normal control.Representative sections from (a) normal control liver (b) rats intoxicated with CC1 4 , showing extensive hepatocellular necrosis; and (c) pretreated treated with water E and (d) pretreated with ethanol extract showing absence of hepatocellular necrosis, magnification X 250.