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Byline: Abdullah H. Al-Assaf


The present study is aimed to evaluating the protective effect of corosolic acid (CRA) on mitochondrial enzymes and DNA damage in carbon tetrachloride (CCl4)-induced hepatotoxic rats. The activities of hepatic mitochondrial enzymes such as isocitrate dehydrogenase (ICH) a-ketoglutarate dehydrogenase (KDH) succinate dehydrogenase (SH) malate dehydrogenase (MDH) NADPH dehydrogenase and cytochrome C oxidase were significantly decreased in CCl4- hepatotoxic rats and administration of corosolic acid brought back these parameters towards normal. The lipid peroxidative index in hepatic mitochondrial fractions was increased in CCl4-treated groups which were decreased significantly on treatment with CRA. The antioxidants which were depleted in CCl4-treated groups were improved significantly by CRA. An increase in the severity of DNA damage was observed in liver mitochondrial fractions of CCl4 induced rats. Pretreatment with CRA shows a significant decrease in tail length and tail moment of comet.

Thus the results demonstrate that CRA has potent antioxidant properties improves mitochondrial enzymes and protective effect against DNA damage in CCl4 induced liver damage in rats.

Key words: Hepatotoxicity carbon tetrachloride (CCl4) corosolic acid DNA damage.


Liver diseases represent a serious health problem throughout the world induced by chemicals alcohol virus and various toxic intermediates. Oxidative stress plays an important role in the pathogenesis of CCl4-induced liver injury (Muhtaseb et al. 2008). The CCl4 induced hepatotoxicity is a well proven experimental model for inducing acute liver injury is bio-transformed by hepatic microsomal cytochrome P450 to trichloromethyl-free radical (CCl3 or CCl3OO) (Weber et al. 2003). These metabolites react with antioxidant enzymes such as glutathione (GSH) catalase and superoxide dismutase (SOD) lead to lipid peroxidation and liver damage. CCl4 causes mitochondrial stress and its subsequent dysfunction can activates signaling cascades involving the activation of caspases resulting in apoptosis or necrosis (Jo et al. 2001); Kowaltowski and Vercesi 1999).

DNA damage is one of the most sensitive biological markers for evaluating the oxidative stress direct breakage of DNA strands occurs when ROS interacts with DNA. Superoxide radicals can directly or indirectly damage DNA whereas hydrogen peroxide mediates DNA damage by the production of hydroxyl radical via events such as the Fenton reaction (Imlay 1998). Modern medicine offers few effective treatments apart from natural medicines. Polyphenolic compounds which are antioxidants of natural origin have generated considerable interest as potential therapeutic agents for a wide variety of chronic diseases. Corosolic acid (CRA) a triterpenoid which is derived from Actinidiavalvata Dunn and also has been identified in many Chinese medicinal herbs such as the Lagerstroemia speciosaL (Fukushima et al. 2006) and banaba leaves (Yamaguchi et al. 2006). It has been reported that CRA exhibited hepatoprotective (Abdullah 2013) and anti-diabetic activity in animal models and clinical trials includes the improvement of glucose metabolism by reducing insulin resistance in a mice model and lowers the post-challenge plasma glucose levels in human (Fukushima et al. 2006; Miura et al. 2006). It was also reported that CRA displayed cytotoxic activity against human cancer cell lines (Ahn et al. 1998; Yoshida et al. 2005). However no study has been investigated the mitochondrial protective nature of CRA on CCl4 hepatotoxicity.

Hence the present study aims to investigate the effect of CRA on mitochondrial enzymes antioxidants status and DNA damage in liver tissue from CCl4 induced hepatotoxicity in rats.


Chemicals: CCl4 was purchased from Sigma-Aldrich Co. St. Louis Missouri USA. CRA was purchased from Mansite Pharmaceutical Co. Ltd (Chendu China). All other chemicals used were of analytical grade obtained from E. Merck or HIMEDIA Mumbai India.

Experimental animals: Male albino Wistar rats (180 to 200 g) were housed in clean cages at a temperature of 20 to 24C with 12-hr light/12-hr dark cycle and 52% relative humidity in the animal house (College of Medicine King Saud University). Ethical approval was obtained from the ethics committee of the College of Medicine Research Center at King Saud University Riyadh Saudi Arabia (11/3215/IRB).

CCl4-induced hepatotoxicity: Hepatotoxicity was induced by a single oral dose of CCl4 (1:1 in liquid paraffin) at 1.25 ml/kg BW at an interval of 6 hr after the administration of the last dose of CRA on the 7th day

Experimental design: The animals were divided into four groups of six animals each as given below. CRA was suspended in 0.1% DMSO and fed to rats via an oral route at 20 mg/kg BW for 7 days. Then a single oral dose of CCl4 (1:1 in liquid paraffin) at 1.25 ml/kg BW (Saba et al. 2010) was given at an interval of 6 hr after the administration of last dose of CRA. Groups IV was administered CRA at 20 mg/kg BW and also administered CCl4 at an interval of 6 hr after the administration of last dose of CRA on the 7th day.

Group I: Control rats received 0.1% DMSO only Group II: Control rats received CRA (20 mg/kg BW) in 0.1%DMSO Group III: CCl4 (1:1 in liquid paraffin) at 1.25 ml /kg BW Group IV: CCl4+CRA (20 mg/kg BW) in 0.1%DMSO On 8th day morning the animals were killed by cervical dislocation. The blood was collected in clean dry test tubes and the serum was separated by centrifugation at 175 g for 10 min. The liver and kidney were immediately removed and washed in ice-cold saline. The tissues were sliced and homogenized in 0.1 M Tris-HCl buffer (pH 7.0). The homogenates were centrifuged at 48 g for 10 min at 0C in a cold centrifuge. The supernatants were separated and used for the determination of various parameters.

Biochemical estimations

Isolation of liver mitochondria: The liver mitochondria were isolated from cell debris nuclei microsomes soluble components and contaminant RBC using differential centrifugation by the method of Johnson and Lardy (1981). A 20% (w/v) homogenate was prepared in 0.25 M sucrose containing 0.05 M TrisHCl buffer and 5.0 mM EDTA. To remove cell debris tissue fragments and cell nuclei (nuclei pellet) the homogenate was centrifuged at 600g for 10 min. The supernatant fraction was centrifuged in a refrigerated centrifuge at10000g for 5 min at 4C to bring down the mitochondrial pellet and used for the estimation of various parameters.

Tricarboxylic acid cycle enzymes analysis: The enzyme activity of isocitrate dehydrogenase was assayed by the method of Bell and Baron (1960) a calibration curve was established with a-ketoglutarate as standard. The of a -ketoglutarate formed/h/mg protein. a-ketoglutarate dehydrogenase was assayed by the method of Reed and Mukherjee (1969) a standard containing potassium ferrocyanide was assayed simultaneously. The activity of a-ketoglutarate dehydrogenase was expressed as nmol of ferrocyanide formed/h/mg protein. Succinate dehydrogenase was assayed according to the method of Slater and Bonner (1952) The succinate dehydrogenase activity was expressed as nmol of succinate oxidised/min/mg protein. Malate dehydrogenase was assayed by the method of Mehler et al. (1948). The activity of the enzyme was expressed as nmol of NADH oxidised/min/mg protein.

Cytochrome c oxidase was assayed by the method of Pearl et al. (1963). The enzyme activity was expressed as change in absorbance/min/mg protein. NADH dehydrogenase was assayed according to the method of Minakami et al. (1962). The activity of NADH dehydrogenase was expressed as nmol of NADH oxidised/min/mg protein

Lipid peroxidative marker and enzymic antioxidant assay: Thiobarbituric acid reactive substance (TBARS) was estimated by the method described by Niehaus and Samuelson (1968). Value was expressed nmol/mg protein. Superoxide dismutase (SOD) was estimated by the method of Kakkar et al. (1984). The enzyme concentration required to inhibit the chromogen produced by 50% in one min under standard conditions was taken as one unit. The specific activity of the enzyme was expressed as units/min/mg protein chronological. Glutathione peroxidase (GPx) was estimated by the method of Rotruck et al. (1973). The tubes were centrifuged and the supernatants were assayed for GSH by the method of Ellman (1959).The specific activity of GPx was expressed as micromoles of GSH consumed/ min/ mg protein.

Non-enzymic antioxidant assay: GSH was estimated by the method of Ellman (1959). The amount of GSH was expressed as g/mg protein. Vitamin C was estimated by the method of Roe and Kuether (1943). The results were expressed as g/mg protein. Vitamin E was estimated by the method of Baker et al.(1940). The value was expressed as g/mg protein.

Single-cell gel electrophoresis (comet assay) Lymphocyte preparation: Lymphocytes were isolated using FicollHistopaque (Sigma) using the method of Boyum (1968). Blood was diluted 1:1 with PBS and layered on to histopaque with the ratio of blood and PBS to histopaque maintained at 4:3. The blood was centrifuged at 175g for 35 min. The lymphocyte layer was removed and washed twice in PBS at 140.3 g for 10 min each and irradiated to an average value of 1.52 A- -3 at room temperature for 30 min and were then subjected to biochemical assays.

Isolation of hepatocytes: Hepatocytes were isolated from the liver of rats by collagenase perfusion method described by Santhosh (1996). The viability of the cells was tested by trypan blue exclusion and only cell preparations with more than 90% viability were used for experiments and retained their viability in culture. Hepatocytes in minimum essential medium (MEM) were seeded on 35 mm plastic dishes passively coated with collagen I (50 g/ ml) and after 4 h unattached cells were removed and the attached cells were incubated with MEM medium. Typically each culture consisted of an initial density of 1A-106 cells in 2 ml culture medium.

Comet assay: DNA damage was estimated by alkaline single-cell gel electrophoresis (comet assay) according to the method of Singh et al. (1988). In this method the cells were first lysed to form nucleoids. During electrophoresis DNA fragments (from damaged DNA) stream towards anode while the undamaged DNA is trapped within the nucleus. When they are stained with propidium iodide damaged DNA gives the appearance of a comet tail and undamaged DNA gives as spherical appearance. To prevent additional DNA damage all steps were conducted under dimmed light or in the dark.

Twenty five images were randomly selected from each sample and were examined at 200 magnification in a fluorescence microscope connected to a personal computer-based image analysis system Komet v. 5.0 (Kinetics Imaging Ltd. Liverpool UK). The relative amount of DNA appearing in the tail of the comet (percent tail DNA) tail length and tail moment (% tail DNA x tail length) were linearly related to DNA break frequency.

Statistical analysis: Statistical evaluation was done using one-way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT) using SPSS 10.0 for Windows. The significance level was set at pless than 0.05.


Carbon tetrachloride has been extensively used for decades to explore the response of liver toxicity since injury caused by this hepatotoxin replicate those seen in most cases of human liver diseases which makes it a reliable model to study both signal transduction and cell cycle events in vivo (Zoo 2000; Brennan 1998). Table 1and 2 represents the restorative effect of CRA on CCl4 induced mitochondrial tricarboxylic acid (TCA) cycle enzymes and respiratory chain enzymes alterations in control and experimental rats. A significant decrease was observed in the activities of ICH KDH SDH and MDH after CCl4 administration (pless than 0.05). CRA treatment reversed the altered values of ICH KDH SDH and MDH with a significant improvement towards normal (pless than 0.05). ICH is also involved in the supply of NADPH needed for GSH production which acts against cytosolic and mitochondrial oxidative damage (Bailey 2002).

Hence the decline in ICH activity may result in the disturbance of the balance between oxidants and antioxidants and subsequently lead to a pro-oxidant state. Decrease activities of Kreb's cycle enzymes in CCl4 treated rats might be due to distress by increased levels of free radicals produced by CCl4 and also deficiency in one or more electron transport chain resulted in decreased activities of these enzymes. However the intake of CRA increases the activities of TCA cycle enzymes probably by improving the mitochondrial antioxidant defense system and overcome the complications associated with the decrease TCA cycle function.

The mitochondrial respiratory chain is the major site of ROS production within the cell. Superoxide is thought to be produced continually as a byproduct of normal respiration through the one electron reduction of molecular oxygen. Superoxide itself can react with nitric oxide to form the damaging oxidant peroxynitrite which is more reactive than superoxide anion (Beckman et al. 1990) CCl4 is activated by cytochrome P450 (CYP) 2E1 2B1/B2 and possibly 3A to form trichloromethyl radicals (CCl3) and trichloromethyl peroxy radicals (CCl3OO) that cause lipid peroxidation and subsequent tissue damage (Weber et al. 2003). The levels of TBARS increase significantly and the activities of the antioxidant enzymes decrease significantly (pless than 0.05) in CCl4 treated group when compared to control. Treatment with CRA

there was a significant decrease in the levels of TBARS and increase in the activities of the enzymatic antioxidants (pless than 0.05) when compared to CCl4 treated group. The results are shown in table-3. Table 4 shows the levels of non-enzymatic antioxidants (vitamin C vitamin E and GSH) in the mitochondrial fraction of liver in control and CCl4 rats. The levels of non-enzymatic antioxidants were significantly decreased (pless than 0.05) in CCl4 rats. Oral administration of CRA improves these parameters towards normal. The increase of lipid mitochondrial peroxidative markers and diminish antioxidant defense as observed in CCL4 treated rats. Treatment with CRA reduces the oxidative damage in mitochondria which is evidence from the reduce levels of TBARS and enhance antioxidant status. These decrease mitochondrial antioxidants could be due to a feed-back inhibition or oxidative inactivation of enzyme protein caused by excess ROS generation (Ohoto 2004). It has been suggested that reduction in

mitochondrial GSH results in lessening of other antioxidants such as ascorbic acid a-tocopherol and aggravate the cells to further damage. This condition may exacerbate the overproduction of mitochondrial ROS (Gao et al. 2004). peroxidation augments antioxidant status and thereby it improves mitochondrial function. This is probably due to antioxidant sparing action of CRA. Previous reports have also supported that CRA possesses antioxidative and free radical scavenging effects (Abdullah 2013).

The single cell gel electrophoresis or comet assay is a state-of-the-art technique for quantitating DNA damage and repair in vivo and in vitro. Figures 1 and 2 represent the DNA damage in the lymphocytes and hepatocytes of experimental animals. The extent of DNA damage measured as percentage of DNA present in the comet tail (% tail DNA) tail length and tail moment is shown in Tables 5 and 6. CCl4 rats show about two fold increases in DNA damage compared to that of control rats. Administration of CRA diminishes the DNA damage. CRA treatment to control animals did not show any effect on DNA damage. Determination of DNA damage in this study includes all types of damage detected by comet assay. Treatment with CRA significantly decreases the DNA damage when compared to CCl4 rats. The genoprotective effect of CRA may be due to the scavenging of free radicals before they damage to DNA as CRA is a potent antioxidant.

However further studies are needed at the molecular level to find out the exact mechanism of genoprotective effect of CRA.

In conclusion the results reveal that CRA exhibits beneficial effect on mitochondrial enzymes and DNA damage against CCl4-induced rats which is mainly due to free radical scavenging and enhances antioxidant properties. These protective effects of CRA characterize a possible development of novel therapeutic agents for liver injury.

Table 1. Effect of corosolic acid on the activities of mitochondrial tricarboxylic acid cycle enzymes in CCl4- hepatotoxic rats



###(Ua/mg protein)###(Ub/mg protein)###(Uc/mg protein)###(Ud/mg protein)

Control###780.33 58.66a###176.81 13.39a###41.13 3.12a###364.41 27.14a

Control + corosolic acid (20 mg/kg body wt.)###775.11 53.55a###181.44 14.41a###39.17 2.54a###360.25 27.01a

CCl4-hepatotoxicity (1.25 mL/kg body wt.)###511.55 39.85b###105.18 7.40b###15.11 1.19b###214.15 19.22b

CCl4 + corosolic acid (20 mg/kg body wt.)###752.61 51.55 ac###157.14 12.01ac###33.10 2.19c###333.39 26.14c

Table 2. Effect of corosolic acid on the activities of mitochondrial respiratory chain enzymes in CCl4-hepatotoxic rats

###NADH dehydrogenase (Ua/mg###Cytochrome c-oxidase (Ub/mg



Control###42.44 3.05a###7.89 0.64a

Control + corosolic acid (20 mg/kg body wt.)###39.09 2.86a###7.84 0.57a

CCl4-hepatotoxicity (1.25 mL/kg body wt.)###11.16 1.02b###2.95 0.18b

CCl4 + corosolic acid (20 mg/kg body wt.)###30.11 2.11c###6.31 0.39c

Table 3. Effect of corosolic acid on the levels of TBARS and activities of enzymatic antioxidants in the mitochondrial fraction of the liver of CCl4-hepatotoxic rats

###TBARS (nmol/mg###SOD (Ua/mg###GPx (Ub/mg



Control###1.47 0.11a###2.34 0.13a###7.68 0.59a

Control + corosolic acid (20 mg/kg body wt.)###1.43 0.09a###2.35 0.12a###7.61 0.54a

CCl4-hepatotoxicity (1.25 mL/kg body wt.)###4.15 0.32b###1.03 0.06b###4.67 0.39b

CCl4 + corosolic acid (20 mg/kg body wt.)###1.95 0.14c###2.15 0.17ac###6.99 0.53c

Table 4: Effect of corosolic acid on the levels of non-enzymatic antioxidants in the mitochondrial fraction of the liver of CCl4-hepatotoxic rats

###Vitamin C (g/mg###Vitamin E (g/mg

###Groups###GSH (g/mg protein)


Control###13.36 0.98a###11.03 0.84a###6.02 0.41a

Control + corosolic acid (20 mg/kg body wt.)###13.27 0.94a###11.11 0.88a###5.97 0.37a

CCl4-hepatotoxicity (1.25 mL/kg body wt.)###5.89 0.49b###5.65 0.49b###2.16 0.12b

CCl4 + corosolic acid (20 mg/kg body wt.)###12.89 0.73ac###9.75 0.69c###5.83 0.36a

Table 5: Effect of corosolic acid on DNA damage in the blood lymphocytes of CCl4-hepatotoxic rats

Values are expressed as means S.D. for six rats in each group.

Values not sharing a common superscript differ significantly at pless than 0.05 (DMRT).

Table 6: Effect of corosolic acid on DNA damage in the liver of CCl4-hepatotoxic rats

###Groups###% Head DNA###% Tail DNA###Tail length###Tail moment

Control###98.56 4.22 a###1.44 0.13 a###3 0.29 a###0.11 0.02 a

Control + corosolic acid (20 mg/kg body wt.)###96.87 5.14 a###3.13 0.25 a###3 0.17 a###0.24 0.02 a

CCl4-hepatotoxicity (1.25 mL/kg body wt.)###76.68 5.41 b###24.32 1.38 b###24.86 1.77 b###2.58 0.20 b

CCl4 + corosolic acid (20 mg/kg body wt.)###91.73 5.57 c###8.27 0.34 c###6 0.40 c###0.68 0.05 c

Acknowledgment: The author would like to extend his sincere appreciation to the deanship of scientific research at King Saud University for its funding of this research through the research group project no: RGP-VPP-266.


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Publication:Journal of Animal and Plant Sciences
Date:Oct 31, 2014

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