Comparative effects of curcumin and an analog of curcumin in carbon tetrachloride induced hepatotoxicity in rats

C Basic & Clinical Pharmacology & Toxicology 2005, 97, 15–21.Printed in Denmark . All rights reserved

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Comparative Effects of Curcumin and an Analogue ofCurcumin in Carbon Tetrachloride-Induced Hepatotoxicity

in RatsNarasimhanaidu Kamalakkannan1, Rajagopalan Rukkumani1, Penumathsa Suresh Varma1, Periyasamy Viswanathan2,

Kallikat Narayanan Rajasekharan3 and Venugopal Padmanabhan Menon1

1Department of Biochemistry, Faculty of Science, 2Department of Pathology, Faculty of Medicine, Rajah MuthiahMedical College, Annamalai University, Annamalainagar-608002, Tamil Nadu, and 3Department of Chemistry,

Faculty of Science, Kerala University, Kariavattom, Kerala, India

(Received October 28, 2004; Accepted January 4, 2005)

Abstract: We have evaluated the comparative effect of curcumin (diferuloyl methane) and its analogue [bis-1,7-(2-hydroxy-phenyl)-hepta-1,6-diene-3,5-dione] (BDMC-A) on carbon tetrachloride-induced hepatotoxicity in rats. Administration ofcarbon tetrachloride (3 ml/kg/week) for three months significantly (P�0.05) increased the levels of marker enzymes suchas aspartate transaminase (AST), alkaline phosphatase (ALP) and g-glutamyl transferase (GGT). The levels of plasmathiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides were also significantly (P�0.05) increased. Wehave observed a significant (P�0.05) decrease in the levels of plasma reduced glutathione (GSH), vitamin C and vitaminE. There was a significant (P�0.05) increase in the levels of TBARS and hydroperoxides in liver and kidney and asignificant (P�0.05) decrease in the activities of enzymic antioxidants- superoxide dismutase (SOD), catalase and GSHperoxidase along with GSH in CCl4-treated rats. Oral administration of curcumin and BDMC-A to CCl4-induced ratsfor a period of three months significantly (P�0.05) decreased the levels of marker enzymes, plasma TBARS and hydroper-oxides and increased the levels of plasma and tissue antioxidants. Histopathological studies of liver also showed protectiveeffect of curcumin and BDMC-A. We have observed thickening of blood vessels and microvesicular fatty changes aroundthe portal triad in CCl4-treated rat liver. Treatment with curcumin showed only mild sinusoidal dilatation while withBDMC-A there was only mild portal inflammation. The effect exerted by BDMC-A was found to be more promisingthan curcumin.

Liver diseases constitute a major problem of worldwide pro-portions. Carbon tetrachloride is a well known hepatotoxinthat is widely used to induce toxic liver injury in a rangeof laboratory animals. Damage by CCl4 is regarded as theanalogue of liver damage caused by a variety of hepatotox-ins in man (Muriel 1998). CCl4 hepatotoxicity depends onthe reductive dehalogenation of CCl4 catalyzed by cyto-chrome P450 in the liver cell endoplasmic reticulum leadingto the generation of an unstable complex of trichloromethyl(CCl¡3) radical (Packer et al. 1978). Covalent binding of thetrichloromethyl radical to cell protein is considered the ini-tial step in a chain of events that eventually leads to lipidperoxidation of the cell membrane and endoplasmic retic-ulum.

Antioxidants and radical scavengers have been employedto study the mechanism of CCl4 toxicity as well as to protectliver cells from CCl4-induced damage by breaking the chainreaction of lipid peroxidation (Weber 2003). Curcumin (fig.1) is a component of turmeric, a yellow spice extracted fromthe rhizome of the herb Curcuma longa (Zingiberaceae).This spice is used as a food preservative and herbal medi-

Author for correspondence: Venugopal P. Menon, Department ofBiochemistry & Center for Micronutrient Research, AnnamalaiUniversity, Annamalainagar – 608 002, Tamil Nadu, India (faxπ91 4144 238343, e-mail biocmr/sify.com;cmrana/sify.com).

cine in India (Kelloff et al. 2000). Curcumin and its ana-logues have a variety of physiological and pharmacologicalactivities such as antiinflammatory, anticarcinogenic andantioxidant properties (Osawa et al. 1995; Sreejayan etal.1997). Preliminary studies have shown a hepatoprotectiveeffect of curcumin inCCl4-induced toxicity in experimentalanimals (Zhao & O’Brien 1996; Park et al. 2000; Liu et al.2003; Soudamini et al. 1992). Reports from our laboratoryhave shown the hepatoprotective effect of curcumin in alco-hol (Rukkumani et al. 2004) and also in alcohol and heatedpolyunsaturated fatty acid-induced hepatotoxicity in rats(Rukkumani et al. 2003).

Bisdemethoxy curcumin (BDMC) is a natural curcuminanalogue and a potent antioxidant (Sreejayan & Rao 1997).A synthetic analogue of bisdemethoxy curcumin (BDMC-A) (fig. 2) is a phenolic compound which possesses anticar-cinogenic (Devasena et al. 2002 & 2003), antioxidant

Fig. 1. Curcumin.

NARASIMHANAIDU KAMALAKKANNAN ET AL.16

Fig. 2. Bis-1,7-(2-hydroxyphenyl)-hepta-1,6-diene-3,5-dione.

(Rukkumani et al. 2004) and antidiabetic (Anusuya et al.2003) effects. In spite of the variety of pharmacological ac-tivities of curcumin and BDMC-A reported earlier, thepresent study was undertaken to evaluate the comparativeeffect of curcumin and BDMC-A on the antioxidant statusin a CCl4 rat model, with specific emphasis on BDMC-A.

Materials and Methods

Experimental animals. Male albino Wistar rats of body weight 150–180 g were obtained from the Central Animal House, Rajah Muthi-ah Medical College and Hospital, Annamalai University, India andwere maintained there. The rats were housed in polypropylene cageslined with husk. They were fed on a standard pellet diet (Agro Cor-poration Private Ltd., Bangalore, India) and water was freely avail-able.

Chemicals. Curcumin was obtained from Sigma Chemical Com-pany, St. Louis, MO, USA. BDMC-A was synthesised as describedby Dinesh Babu & Rajasekharan (1994). CCl4 was purchased fromMerck Ltd., Mumbai, India. All other chemicals and biochemicalsused in our study are of high analytical grade.

Experimental design. A total of 36 rats were used in our study. Therats were divided into 6 groups of 6 rats each.

Group I: Normal control rats; Group II: Normal rats orally ad-ministered with curcumin (80 mg/kg body weight) (Rukkumani etal. 2004); Group III: Normal rats orally administered with BDMC-A (80 mg/kg body weight) (Anusuya et al. 2003); Group IV: Ratssubcutaneously injected with CCl4 (3 ml/kg body weight/week) (Ak-ila et al. 1998); Group V: Rats orally administered with curcumin(80 mg/kg body weight) along with subcutaneous injection of CCl4(3 ml/kg body weight/week); and Group VI: Rats orally adminis-tered with BDMC-A (80 mg/kg body weight) along with subcutane-ous injection of CCl4 (3 ml/kg body weight/week).

The experiment was carried out for a period of three months.The experimental protocols were approved by the Ethical Commit-tee of Annamalai University. After the last treatment, the animalswere fasted overnight and sacrificed by cervical dislocation. Bloodwas collected in heparinised tubes. Plasma was separated and usedfor various biochemical estimations. Liver and kidney were col-

Table 1.

Effect of curcumin and BDMC-A on the activities of ALP, GGT and AST in normal and CCl4-treated rats. Each value is mean∫S.D. for6 rats in each group. Values not sharing a common superscript (a,b,c,d) differ significantly at P�0.05.

Groups ALP (IU/l) GGT (IU/l) AST (IU/l)

Normal 73.10∫4.13a 0.57∫0.04a 73.13∫5.47a

Curcumin 74.66∫6.12a 0.55∫0.03a 74.89∫7.02a

BDMC-A 74.36∫5.80a 0.57∫0.05a 73.61∫6.71a

CCl4 193.13∫13.28b 1.65∫0.11b 139.02∫9.42b

CCl4πcurcumin 117.30∫6.54c 0.91∫0.04c 94.62∫4.18c

CCl4πBDMC-A 98.85∫5.68c 0.72∫0.04d 83.91∫4.76d

Abbreviations: ALP-alkaline phosphatase; GGT-g-glutamyl transferase; AST-aspartate transaminase.

lected in ice-cold containers, washed with saline, homogenised withappropriate buffer and used for various estimations.

Biochemical estimations. In plasma, the activites of marker enzymessuch as AST (Reitman & Frankel 1957), ALP (King & Armstrong1988), GGT (Fiala et al. 1972) were measured and the levels ofTBARS (Fraga et al. 1988), hydroperoxides (Jiang et al. 1992), GSH(Ellman 1959), vitamin C (Omaye et al. 1979) and vitamin E (Bakeret al. 1951) were also measured.In tissues (liver and kidney), theconcentration of GSH (Ellman 1959), superoxide dismutase (Kak-kar et al. 1984), catalase (Sinha 1972) and glutathione peroxidase(Rotruck et al. 1973) were estimated.

Histopathological studies. For histopathological studies, livers fromanimals of different groups were perfused with 10% neutral form-alin solution. Paraffin sections were made and stained using haema-toxylin-eosin stain. After staining, the sections were observed underlight microscope and photographs were taken.

Statistical analysis. Statistical analysis was performed using oneway analysis of variance (ANOVA) followed by Duncan’s MultipleRange Test (DMRT). The values are mean∫S.D. for 6 rats in eachgroup. P value �0.05 was considered as significant.

Results

The effect of oral administration of curcumin and BDMC-A on plasma AST, GGT and ALP activities in normal andCCl4-induced rats is presented in table 1. A significant in-crease in the activities of these marker enzymes were ob-served in CCl4-treated rats. On treatment with both curcu-min and BDMC-A, the activities of these enzymes werefound to be significantly decreased.

Table 2 shows the changes in the levels of plasmaTBARS, hydroperoxides, glutathione, vitamin C and vit-amin E. Treatment with CCl4 significantly increased thelevels of TBARS and hydroperoxides and decreased thelevels of non-enzymatic antioxidants in plasma. But oraladministration of curcumin and BDMC-A decreased thelevels of plasma TBARS and hydroperoxides and improvedthe levels of nonenzymic antioxidants.

Table 3 shows the levels of TBARS and hydroperoxidesin the tissues (liver and kidney) of normal and CCl4-treatedrats. An increase in the levels of TBARS and hydroperox-ides were noted in the CCl4-treated rats. These increasedlevels were brought down by treatment with curcumin andBDMC-A.

The activities of superoxide dismutase and catalase in the

17CURCUMIN AND ITS ANALOGUE ON HEPATOTOXICITY

Table 2.

Effect of curcumin and BDMC-A on plasma TBARS, HP, GSH, vitamin C and vitamin E in normal and CCl4-treated rats. Each value ismean∫S.D. for 6 rats in each group. Values not sharing a common superscript (a,b,c,d) differ significantly at P�0.05.

Groups TBARS (nM/ml) HP (values ¿10ª5 mM/dl) GSH (mg/dl) Vitamin C (mg/dl) Vitamin E (mg/dl)

Normal 0.14∫0.01a 7.13∫0.51a 23.25∫1.61a 1.89∫0.05a 1.33∫0.04a

Curcumin 0.14∫0.01a 7.02∫0.60a 23.44∫1.40a 1.91∫0.05a 1.35∫0.06a

BDMC-A 0.13∫0.01a 7.00∫0.44a 24.64∫1.88a 1.98∫0.08a 1.38∫0.04a

CCl4 0.52∫0.04b 28.64∫2.13b 10.71∫0.92b 0.81∫0.06b 0.67∫0.05b

CCl4πcurcumin 0.34∫0.02c 20.20∫1.70c 16.43∫1.16c 1.42∫0.09c 0.93∫0.07c

CCl4πBDMC-A 0.20∫0.02d 13.22∫1.07d 20.02∫1.49d 1.69∫0.07c 1.18∫0.06c

tissues of normal and CCl4-treated rats is shown in table 4.CCl4 treatment significantly decreased the activities of theseenzymes. Upon administration of curcumin and BDMC-A,the activities of these enzymes were found to be significantlyincreased.

A significant decrease in the activity of glutathione per-oxidase and decreased concentration of glutathione was ob-served in the tissues of CCl4-treated rats in the present study(table 5). Treatment with curcumin and BDMC-A exerteda significant effect on these parameters.

Oral administration of BDMC-A to CCl4-treated ratsshowed a better effect than curcumin. Administration ofboth curcumin and BDMC-A to normal rats did not showany significant effect in any of the parameters studied.

Studies carried out on liver histopathology supports ourfindings that both curcumin and BDMC-A exerted protec-tive effects. Examination of the liver slices from normal ratsshowed normal parenchymal architecture (fig. 3). The liverof rats treated with curcumin or BDMC-A alone did notshow any noticeable alterations (fig. 4 and 5). In rats treatedwith CCl4 alone, the liver sections showed thickening ofblood vessels (fig. 6) and microvesicular fatty changesaround the portal triad (fig. 7). In rats treated with CCl4πcurcumin, only mild sinusoidal dilatation was observed (fig.8) and in the CCl4πBDMC-A group only mild portal in-flammation was seen (fig. 9).

Discussion

Lipid peroxidation damages cellular functions by compro-mising membrane function and by covalent binding of reac-tive intermediates formed during the CCl4 toxication

Table 3.

Effect of curcumin and BDMC-A on TBARS and hydroperoxides in tissues of normal and CCl4-treated rats. Each value is mean∫S.D. for6 rats in each group. Values not sharing a common superscript (a,b,c,d) differ significantly at P�0.05.

TBARS (mM/100 gtissue) Hydroperoxides (mM/100 g tissue)

Groups Liver Kidney Liver Kidney

Normal 1.06∫0.05a 1.13∫0.09a 83.81∫7.17a 69.08∫6.37a

Curcumin 1.12∫0.05a 1.08∫0.08a 79.89∫7.85a 61.40∫6.22a

BDMC-A 1.01∫0.04a 1.15∫0.08a 82.05∫6.33a 73.60∫5.21a

CCl4 3.10∫0.22b 5.27∫0.43b 249.07∫20.66b 209.51∫16.73b

CCl4πcurcumin 1.67∫0.08c 2.06∫0.20c 150.80∫13.40c 121.32∫11.80c

CCl4πBDMC-A 1.58∫0.08d 1.70∫0.14d 127.67∫10.62d 95.33∫7.64d

(Weber et al. 2003). The peroxidative products induce hypo-function of the membrane and cytosolic enzymes appear inthe blood (Recknagel et al. 1989). The increase in the activ-ities of these enzymes in plasma suggest enhanced hepato-cellular damage by CCl4. The activities of these enzymeswere found to be decreased after treatment with curcuminand BDMC-A.

Curcumin restores the antioxidant status by its ability toscavenge or neutralize free radicals, inhibits peroxidation ofmembrane lipids and maintains cell membrane integrityand their function (Rukkumani et al. 2004). The presenceof hydroxyl group in the ortho position of the aromatic ringin BDMC-A may be responsible for the inhibitory effect(Anto et al. 1996). The o-hydroxyl group, because of its res-onance property, easily donates eª to free radicals and ef-fectively neutralizes them. Curcumin and BDMC-A stabil-ize cell membrane integrity and prevent the increase of thesemarker enzymes.

The increased levels of plasma and tissue TBARS andhydroperoxides in rats treated with CCl4 were decreased byadministration of curcumin and BDMC-A. Curcumin in-hibits lipid peroxidation by its antioxidant action (Reddy &Lokesh 1994) or by its ability to induce drug-detoxifyingenzymes (Yokota et al. 1998). Curcumin has both polyphen-olic structure and b-diketone functional group and is astronger antioxidant inhibitor of lipid peroxidation thanother flavonoids which have a single phenolic hydroxygroup. The antilipoperoxidative effect of BDMC-A can berelated to its phenolic nature. BDMC-A, as a phenolic com-pound acts as a powerful counter measure against lipid per-oxidation by scavenging free radicals and quenching thelipid peroxidative side chain. The significant reduction in


Table 4.

Effect of curcumin and BDMC-A on the activities of superoxide dismutase and catalase in tissues of normal and CCl4-treated rats. Eachvalue is mean∫S.D. for 6 rats in each group. Values not sharing a common superscript (a,b,c,d) differ significantly at P�0.05. Superoxidedismutase units: Enzyme concentration required to inhibit the optical density at 560 nm of chromogen production by 50% in 1 min.

SOD (units/mg protein) Catalase (mmol of H2O2 consumed/min./mg protein)


Normal 15.77∫1.20a 16.24∫1.47a 61.31∫5.22a 64.64∫5.20a

Curcumin 15.02∫1.33a 16.03∫1.30a 59.24∫4.35a 65.00∫5.54a

BDMC-A 15.26∫1.04a 16.20∫1.55a 58.87∫5.09a 64.57∫4.68a

CCl4 6.18∫0.70b 8.11∫0.84b 40.36∫3.34b 38.06∫3.17b


CCl4πBDMC-A 13.73∫1.25c 14.51∫1.51d 56.08∫5.28c 57.62∫3.30d

the levels of TBARS and hydroperoxide confirms that cur-cumin and BDMC-A could effectively protect against freeradicals induced by CCl4.

Carbon tetrachloride-induced hepatotoxicity is associ-ated with reduced antioxidant levels (Basu 2003). Glutathi-one acts as an antioxidant both intracellularly and extracel-lularly in conjuction with various enzymatic processes thatreduce hydrogen peroxide and hydroperoxides (Kadiska etal. 2000). Vitamin E is a major lipophilic antioxidant andvitamin C is also a potent scavenger of reactive oxygen spe-cies in plasma. CCl4 dosing with its generation of the trich-loromethyl radical and the resultant lipid peroxidationmight have resulted in a decrease of antioxidants in theplasma. Curcumin and BDMC-A administration enhancedthe antioxidant system by scavenging free radicals therebypreventing the depletion of plasma non-enzymatic antioxi-dants.

The role of free radicals in CCl4-induced toxicity hasbeen shown to be a major pathway of non-enzymaticallyinduced lipid peroxidation which subsequently affects vari-ous enzyme activities of the body (Basu et al. 2003). Theobserved significant decrease in the activities of SOD, cata-lase, GSH peroxidation and the concentration of glutathi-one in the present study is related to the excessive produc-tion of free radicals generated by the breakdown of CCl4.Superoxide dismutase and catalase are easily inactivated bylipid peroxides or reactive oxygen species which result indecreased activities of these enzymes in CCl4-toxicity. Thedecreased activities of these enzymes can also be related tothe exhaustion as a result of increased oxidative stresscaused by CCl4.

Table 5.

Effect of curcumin and BDMC-A on GSH and glutathione peroxidase in tissues of normal and CCl4-treated rats. Each value is mean∫S.D.for 6 rats in each group. Values not sharing a common superscript (a,b,c,d) differ significantly at P�0.05.

GSH (mM/100 g tissue) GP peroxidase (mg of GSH consumed/min./mgprotein)


Normal 160.30∫12.11a 132.85∫11.07a 10.65∫0.92a 8.93∫0.77a

Curcumin 156.87∫10.20a 126.15∫10.45a 10.80∫0.74a 9.02∫0.82a

BDMC-A 156.18∫15.68a 134.45∫7.11a 11.10∫0.85a 8.87∫0.64a

CCl4 80.98∫9.23b 68.49∫6.03b 3.84∫0.40b 4.11∫0.50b


CCl4πBDMC-A 134.70∫12.67d 108.55∫7.54d 9.10∫0.81d 6.80∫0.55c

Glutathione peroxidase and glutathione content in theliver play a primary role in the protection against trichloro-methyl radical-induced liver damage. It has been suggestedthat lipid peroxides generated after CCl4 exposure is elimin-ated by glutathione peroxidase in the presence of glutathi-one resulting in the decreased propagation of lipid peroxi-dation. Kojima et al. (1998) have also reported that underoxidative stress, glutathione is largely consumed by the glu-tathione-related enzymes thereby resulting in the intoxi-cation of toxic metabolites. Administration of curcuminand BDMC-A to rats exposed to CCl4 resulted in increasedactivities of tissue enzymic antioxidants and glutathioneconcentration.

Histopathological observations of the liver of CCl4-ad-ministered rats when treated with curcumin and BDMC-Ashowed mild sinusoidal dilatation and mild portal inflam-mation. The damage caused by CCl4 was minimised ontreatment with curcumin and BDMC-A. These obser-vations show protective effects of both curcumin andBDMC-A in CCl4-induced hepatotoxicity in rats.

Curcumin by its ability to scavenge free radicals, inter-acting with oxidative cascade, quenching oxygen, inhibitingoxidative enzymes and chelating metal ions inhibits lipidperoxidation (Rukkumani et al. 2004) and restored the anti-oxidant status. The protective effect exerted by BDMC-Awas more prominent than by curcumin. Normally phenoliccompounds act by scavenging free radicals and quenchingthe lipid peroxidative side chain. It has been proposed thathydroxy and hydroperoxy radicals initiate Hπ abstractionfrom a free phenolic substrate to form phenoxy radical thatcan rearrange to quinonemethide radical intermediate (Pan


Fig. 3. Liver section of normal rat showing normal parenchymalarchitecture (haematoxylin and eosin, magnification ¿10).

Fig. 4. No histological alterations were observed in rats treated withcurcumin or BDMC-A (haematoxylin and eosin, magnification¿10).

Fig. 5. No histological alterations were observed in rats treated withcurcumin or BDMC-A (haematoxylin and eosin, magnification¿10).

Fig. 6. Thickening of blood vessels caused by CCl4 administration(haematoxylin and eosin, magnification ¿10).

Fig. 7. Microvesicular fatty changes around portal triad in ratstreated with CCl4 (haematoxylin and eosin, magnification ¿10).

Fig. 8. CCl4πcurcumin administration showing mild sinusoidal di-latation (haematoxylin and eosin, magnification ¿10).


Fig. 9. Mild portal inflammation seen in CCl4πBDMC-A treatedrats (haematoxylin and eosin, magnification ¿10).

et al. 1999) which is excreted via bile. Thus BDMC-A beinga phenolic compound, might have inhibited lipid peroxi-dation.

The increased efficacy of BDMC-A may be attributed tothe presence of a hydroxyl group at the ortho position. Theo-hydroxyl group, because of its resonance property, easilydonates to free radicals and effectively neutralizes them.Also, the presence of a hydroxyl group in the ortho positionincreases its antioxidant potential through intermolecularhydrogen bonding involving the ªSH group of non-proteinthiols and enzymes resulting in the restoration of the anti-oxidant system.

AcknowledgementsWe thank UGC for sanctioning the project. Mr. N. Ka-

malakkannan is a Junior Research Fellow in the project.

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Comparative effects of curcumin and an analog of curcumin in carbon tetrachloride induced hepatotoxicity in rats