Abstract
Curcumin and its analogue (bis.demethoxy curcumin analogue [BDMC-A]) were studied for their possible lipid-lowering properties in carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. Carbon tetrachloride (3 ml kg−1 wk−1) administration to albino Wistar rats increased the levels of hepatic marker enzymes such as aspartate transaminase (AST), alkaline phosphatase (ALP), and γ.-glutamyl transferase (GGT) in the plasma. The levels of lipids cholesterol, triglycerides, and free fatty acids were also increased in plasma and tissues (liver, kidney, heart, and brain). Phospholipid levels increased in plasma, heart, and brain but decreased in liver and kidney. Curcumin (80 mg/kg) and BDMC-A (80 mg/kg) administration to CCl4-treated rats for a period of 3 months significantly decreased the lipid levels. The effect exerted by BDMC-A was more prominent than that of curcumin. Studies on the histopathology of the liver are also in line with the biochemical parameters studied. These observations show the lipid-lowering efficacy of curcumin and its analogue in CCl4-induced hepatotoxicity.
Introduction
Carbon tetrachloride (CCl4) is an important model agent to study the pathogenesis of liver injury (Boll et al., Citation2001). It is known to cause damage to the liver, lungs, adrenals, and central nervous system in humans and experimental animals (McGregor & Lang, Citation1996). CCl4 requires biotransformation by hepatic microsomal P450 to produce the hepatotoxic metabolite, trichloromethyl radical (CCl3·) which, in the presence of oxygen, is further converted to a peroxy radical (CCl3OO·). These radicals may interact with membrane lipids leading to peroxidation (Muriel, Citation1997). CCl4 causes an imbalance between the synthesis and degradation of lipids (Boll et al., Citation2001).
Curcuma longa. Linn. (Zingiberaceae) is a medicinal plant widely cultivated in tropical regions of Asia. Curcumin (diferuloyl methane) () is extracted from the rhizomes of Curcuma longa.. Curcumin possesses a wide variety of pharmacological properties (Quiles et al., Citation2002). The lipid-lowering effect of curcumin in CCl4-induced hepatotoxicity (Akila et al., Citation1998) and in alcohol and polyunsaturated fatty acid–induced hyperlipidemia (Rukkumani et al., Citation2002) were reported. bis.demethoxycurcumin (BDMC) is a natural curcuminoid that also possess lipid-lowering properties in high fat diet–induced lipid accumulation (Asai & Miyazawa, Citation2001). An analogue of bis.demethoxycurcumin (BDMC-A) () has been reported for its hypolipidemic properties (Rukkumani et al., Citation2004a) along with its antioxidant (Rukkumani et al., Citation2004b) and antidiabetic (Anusuya et al., Citation2003) properties.
Figure 1 Curcumin.
Figure 2 BDMC-A.
There are no available reports on the hypolipidemic properties of BDMC-A in CCl4-induced hepatotoxicity. Hence, we made an attempt to study the effect of BDMC-A and to compare it with curcumin on lipid profile in CCl4-induced hepatotoxicity in rats.
Materials and Methods
Drugs and chemicals
Curcumin was obtained from Sigma Chemical Company (St. Louis, MO, USA). BDMC-A was synthesised as described by Babu and Rajasekharan (Citation1994). CCl4 was purchased from Merck Ltd. (Mumbai, India). All other chemicals and biochemicals used in our study were of high analytical grade.
Experimental animals
Male albino Wistar rats of body weight 150–180 g were obtained from the Central Animal House, Rajah Muthiah Medical College and Hospital, Annamalai University. The rats were housed in polypropylene cages lined with husk. They were fed on a standard pellet diet (Agro Corporation Private Ltd., Bangalore, India), and water was freely available. The standard pellet diet comprised 21% protein, 5% lipids, 4% crude fiber, 8% ash, 1% calcium, 0.6% phosphorus, 3.4% glucose, 2% vitamin, and 55% nitrogen-free extract (carbohydrates). It provides metabolizable energy of 3600 kcal/kg..
Experimental design
A total of 36 rats were used in our study. The rats were divided into 6 groups of 6 rats each.
Group I Normal control rats injected with saline subcutaneously (3 ml/kg body weight per week) and orally administered with saline.
Group II Normal rats injected with saline subcutaneously (3 ml/kg body weight per week) and orally administered curcumin (80 mg/kg body weight) (Rukkumani et al., Citation2002).
Group III Normal rats injected with saline subcutaneously (3 ml/kg body weight per week) and orally administered BDMC-A (80 mg/kg body weight) (Anusuya et al., Citation2003).
Group IV Rats subcutaneously injected with CCl4 (3 ml/kg body weight per week) (Akila et al., Citation1998) and orally administered saline.
Group V Rats orally administered curcumin (80 mg/kg body weight) along with subcutaneous injection of CCl4 (3 ml/kg body weight per week).
Group VI Rats orally administered BDMC-A (80 mg/kg body weight) along with subcutaneous injection of CCl4 (3 ml/kg body weight per week).
The experiment was carried out for a period of 3 months. All the experimental protocols were approved by the Ethical Committee of Annamalai University. After the last treatment, the animals were fasted overnight and sacrificed by cervical dislocation. Blood was collected in heparinized tubes. Plasma was separated and used for various biochemical estimations. Liver, kidney, heart, and brain were collected in ice-cold containers, washed with saline, homogenized with appropriate buffer, and used for various estimations.
Biochemical estimations
In plasma, the levels of marker enzymes such as AST (aspartate transaminase), ALP (alkaline phosphatase), and GGT (γ.-glutamyl transferase) were estimated by the methods of Reitman and Frankel (Citation1957), King and Armstrong (Citation1988), and Fiala et al. (Citation1972), respectively.
Lipids were extracted using a chloroform-methanol mixture (2:1 v/v) by the method of Folch et al. (Citation1957). Total cholesterol (Zlatkis et al., Citation1953), triglycerides (Foster & Dunn, Citation1973), free fatty acids (Falholt et al., Citation1973), and phospholipids (Zilversmit et al., Citation1950) were estimated in plasma and tissues.
For histopathological studies, livers from animals of different groups were perfused with 10% neutral formalin solution. Paraffin sections were made and stained using hematoxylin-eosin (H&E) stain. After staining, the sections were observed under a light microscope and photographs were taken.
Statistical analysis
Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT). The values are mean ±SD for 6 rats in each group. p values < 0.05 were considered as significant.
Results
The effect of oral administration of curcumin and BDMC-A on plasma AST, GGT, and ALP levels in normal and CCl4-induced rats is presented in . A significant increase in the levels of these marker enzymes was observed in CCl4-treated rats. On treatment with both curcumin and BDMC-A, the levels of these enzymes were found to be significantly decreased.
Table 1.. Effect of curcumin and BDMC-A on ALP, GGT, and AST in normal and CCl4-treated rats.
presents the levels of cholesterol, triglycerides, free fatty acids, and phospholipids in normal and CCl4-treated rats in plasma. The levels of these lipids significantly increased in CCl4-treated rats. Administration of curcumin and BDMC-A to these rats significantly decreased the lipid levels.
Table 2.. Effect of curcumin and BDMC-A on plasma lipids in normal and CCl4-treated rats.
The levels of cholesterol, triglycerides, free fatty acids, and phospholipids in the tissues of normal and CCl4-treated rats are presented in Tables . In CCl4-administered rats, the levels of these lipids significantly increased while phospholipids decreased in liver and kidney. Treatment with curcumin and BDMC-A exerted a significant effect on the lipid levels.
Table 3.. Effect of curcumin and BDMC-A on cholesterol level in tissues of normal and CCl4-treated rats.
Table 4.. Effect of curcumin and BDMC-A on triglycerides level in tissues of normal and CCl4-treated rats.
Table 5.. Effect of curcumin and BDMC-A on free fatty acids level in tissues of normal and CCl4-treated rats.
Table 6.. Effect of curcumin and BDMC-A on phospholipids level in tissues of normal and CCl4-treated rats.
In all the parameters studied, administration of curcumin and BDMC-A to normal rats did not show any significant effect. Results obtained with BDMC-A were found to be more effective than those of curcumin.
Discussion
Lipids are more easily attacked by the activated metabolites of CCl4 resulting in damage to intracellular membranes and the plasma membrane (Cheeseman et al., Citation1985). As a result of membrane damage, the levels of marker enzymes such as AST, ALP, and GGT increase in plasma. This increase is mitigated by treatment with curcumin and BDMC-A. Curcumin, by scavenging or neutralizing free radicals, inhibits peroxidation of membrane lipids and maintains cell membrane integrity and their function (Rukkumani et al., Citation2003). The presence of hydroxyl group in the ortho. position of the aromatic ring in BDMC-A may be responsible for the inhibitory effect (Anto et al., Citation1996). The o.-hydroxyl group, because of its resonance property, easily donates e− to free radicals and effectively neutralizes them. Curcumin and BDMC-A stabilize cell membrane integrity and prevent the increase of these marker enzymes.
Radical formation and lipid peroxidation are the predominant cellular mechanisms involved in the development of fatty liver caused by CCl4 (Tribble et al., Citation1987). Extensive accumulation of lipids is regarded as a pathological condition, and when the accumulation becomes chronic, fibrotic changes occur in the cells that progress to cirrhosis and impaired liver function (Murray et al., Citation1993). An increase in the levels of cholesterol, triglycerides, and free fatty acids were noted in plasma and tissues. CCl4 increases the synthesis of fatty acids and triglycerides from acetate. This could be due to the transport of acetate into the liver cell, resulting in increased substrate (acetate) availability. In CCl4 toxicity, the synthesis of cholesterol is also increased (Boll et al., Citation2001).
On the other hand, CCl4 lowers β-oxidation of fatty acids and hydrolysis of triglycerides. This increases the availability of fatty acids to esterification (Lieber, Citation2000). Reports have also shown that during CCl4 toxicity, fat from the peripheral adipose tissue is translocated to the liver and kidney leading to its accumulation (Devarshi et al., Citation1986). Moreover, the synthesis of apolipoproteins is inhibited by CCl4 (Honma & Suda, Citation1997) subsequently resulting in the decreased synthesis of lipoproteins. A decrease in the secretion of bile acids is also reported (Boll et al., Citation2001).
Phospholipids are the vital components of biomembranes. They are more susceptible to CCl4-induced lipid peroxidation than other lipid classes (Morrow et al., Citation1992). A decrease in the levels of phospholipids in liver and kidney is probably due to an increase in phospholipase activity (Lamb et al., Citation1988). During normal lipoprotein metabolism, phospholipids are extensively converted into triglycerides (Wiggins & Gibbons, Citation1996). CCl4-induced inhibition of lipoprotein-associated triglyceride export may also result in increased release of phospholipids from these tissues. An increase in phospholipid levels in brain and heart could be due to increased phospholipid content of their membranes.
Oral administration of curcumin to CCl4-treated rats significantly decreased the lipid levels in plasma and tissues. It has been reported that curcumin possesses hypocholesterolemic action (Soni & Kuttan, Citation1992) and this could be due to a decrease in absorption of cholesterol (Rao et al., Citation1970) or an increase in HDL cholesterol (Soudamini et al., Citation1992). This indicates that curcumin mobilizes excess cholesterol from extrahepatic tissues to liver where it is catabolized. Curcumin also increases the activity of 7α.-hydroxylase, which converts cholesterol to bile acids, facilitating the biliary cholesterol excretion (Babu & Srinivasan, Citation1997). The decrease in the levels of triglycerides and phospholipids might be due to decreased free fatty acid (FFA) synthesis by curcumin, which may suppress the enzymes involved in FFA synthesis (Rukkumani et al., Citation2002).
Treatment with BDMC-A resulted in a significant decrease in the lipid profile in CCl4-administered rats. The structure of BDMC-A resembles that of curcumin, and this may have an impact on lipid metabolism. BDMC-A is a potent antioxidant, and the lipid-lowering activity might be due to its antioxidant property. Phenolics have been recognized as a powerful countermeasure against lipid peroxidation (Schroeter et al., Citation2000). Phenolic compounds are capable of scavenging free radicals and quenching the lipid peroxidative side chain. Pan et al. (Citation1999) have proposed that hydroxy and hydroperoxy radicals initiate H+ abstraction from a free phenolic substrate to form phenoxy radical that can rearrange to quinone methide radical intermediate, which is excreted via bile. BDMC-A inhibits lipid peroxidation, as well membrane damage and thus prevent the release of free fatty acids. A decrease in the levels of FFA might result in the decreased levels of other lipids.
Histopathological observations of the liver of CCl4-administered rats showed thickening of blood vessels (). CCl4-administered rats when treated with curcumin showed mild sinusoidal dilatation (), and treatment with BDMC-A showed a near normal histology of the liver (). These observations show the protective effect of both curcumin and BDMC-A in CCl4-induced hepatotoxicity in rats. No histological alterations were observed in liver of normal rats and curcumin or BDMC-A treated rats ()
Figure 6 Thickening of blood vessels caused by CCl4 administration (H&E, × 10).
Figure 7 CCl4 + curcumin administration showing mild sinusoidal dilatation (H&E, × 10).
Figure 8 Mild portal inflammation seen in CCl4 + BDMC-A treated rats (H&E, × 10).
Figure 3 Liver section of normal rat showing normal parenchymal architecture (H&E, × 10).
Figure 4 No histological alterations were observed in rats treated with curcumin or BDMC-A (H&E, × 10).
Figure 5 No histological alterations were observed in rats treated with curcumin or BDMC-A (H&E, × 10).
Curcumin and BDMC-A effectively reduced the lipid levels altered by CCl4 metabolism. The effect exerted by BDMC-A was more pronounced than curcumin. This could be due to the presence of hydroxyl group at the ortho. position. Because of its positional isomerism, the ortho. hydroxyl group easily donates the e−to the free radicals and effectively neutralizes them. Hence, BDMC-A is more effective than curcumin in treating hyperlipidemia.
Acknowledgments
We thank UGC for sanctioning the project. The first author is a junior research fellow in the project.
Related Research Data
References
- Akila GV, Rajakrishnan V, Viswanathan P, Rajasekharan KN, Menon VP (1998): Effects of curcumin on lipid profile and lipid peroxidation status in experimental hepatic fibrosis. Hepatol Res 11: 147–157. [CSA]
- Anto RJ, George J, Babu DKV, Rajasekharan KN, Kuttan R (1996): Antimutagenic and anticarcinogenic activity of natural and synthetic curcuminoids. Mutat Res 17: 127–131. [CSA]
- Anusuya S, Viswanathan P, Rajashekaran KN, Menon VP (2003): Protection of pancreatic β.-cell by the potential antioxidant bis.-O.-hydroxycinnamoyl methane, analogue of natural curcuminoid in experimental diabetes. J Pharm Pharmaceut Sci 6: 327–333. [CSA]
- Asai A, Miyazawa T (2001): Dietary curcuminoids prevent high-fat diet-induced lipid accumulation in rat liver and epididymal adipose tissue. J Nutr 131: 2932–2935. [PUBMED], [INFOTRIEVE], [CSA]
- Babu KV, Rajasekaran KN (1994): Simplified conditions for the synthesis of curcumin 1 and other curcuminoids. Org Prep Procedure Int 26: 674–677. [CSA]
- Babu PS, Srinivasan K (1997): Hypolipidemic action of curcumin, the active principle of turmeric (Curcuma longa.) in streptozotocin induced diabetic rats. Mol Cell Biochem 166: 169–175. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Boll M, Weber LWD, Becker LE, Stampfl A (2001): Pathogenesis of carbon tetrachloride in hepatocyte injury. Bioactivation of CCl4 by cytochrome P450 and effects on lipid homeostasis. Z Naturforsch 56c: 111–121. [CSA]
- Cheeseman KH, Albano EF, Tomasi A, Slater TF (1985): Biochemical studies on the metabolic activation of halogenated alkanes. Environ Health Perspect 64: 85–101. [PUBMED], [INFOTRIEVE], [CSA]
- Devarshi P, Kanase A, Kanase R, Mane S, Patil S, Varuthe AT (1986): Effect of mandur bhasma on lipolytic activities of liver, kidney and adipose tissue of albino rat during CCl4-induced hepatic injury. J Biosci 10: 227–234. [CSA]
- Falholt K, Falholt W, Lund B (1973): An easy colorimetric method for routine determination of free fatty acids in plasma. Clin Chim Acta 46: 105–111. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Fiala S, Fiala AE, Dixon B (1972): Gamma glutamyl transpeptidase in transplantable chemically induced rat hepatomas and spontaneous mouse hepatomas. J Natl Cancer Inst 481: 1393–1409. [CSA]
- Folch J, Lees M, Solane SGH (1957): A simple method for isolation and purification of total lipids from animal tissues. J Biol Chem 26: 497–509. [CSA]
- Foster CS, Dunn O (1973): Stable reagents for determination of serum triglycerides by a colorimetric Hantzsch condensation method. Clin Chim Acta 19: 338–340. [CSA]
- Honma T, Suda M (1997): Changes in plasma lipo-proteins as toxicity markers for carbon tetrachloride, chloroform and dichloromethane. Ind Health 35: 519–531. [PUBMED], [INFOTRIEVE], [CSA]
- King EJ, Armstrong AR (1988): Calcium, phosphorus and phosphatases. In: Practical Clinical Biochemistry. New Delhi, CBS Publishers, pp. 431–467.
- Lamb RG, Snyder JW, Coleman JB (1988): New trends in the prevention of hepatocellular death. Modifiers of calcium movement and of membrane phospholipid metabolism. In: Testa B, Perrissaud D, eds., Liver Drugs: From Experimental Pharmacology to Therapeutic Application. Boca Raton, CRC Press, pp. 53–66.
- Lieber CS (2000): Alcoholic liver disease: New insights on pathogenesis lead to new treatment. J Hepatol 32: 113–128. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- McGregor D, Lang M (1996): Carbon tetrachloride: Genetic effects and other modes of action. Mutat Res 366: 181–195. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Morrow JD, Awad JA, Boss HJ, Blair IA, Roberts II LJ (1992): Non-cyclooxygenase-derived prostanoids (F2-isoprostanes) are formed in situ. on phospholipids. Proc Natl Acad Sci USA 89: 10721–10725. [PUBMED], [INFOTRIEVE], [CSA]
- Muriel P (1997): Peroxidation of lipids and liver damage. In: Baskin SI, Salem H, eds., Antioxidants, Oxidants and Free Radicals. Washington, DC, Taylor & Francis, p. 237.
- Murray RK, Granner DK, Mayes PA, Rodwell VW (1993): Harper's Biochemistry, 23rd ed. Appleton and Lange, Stamford, Connecticut, pp. 258–265.
- Pan GX, Spencer L, Leary GJ (1999): Reactivity of ferulic acid and its derivative towards hydrogen peroxide and peracetic acid. J Agric Food Chem 47: 3325–3331. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Quiles JL, Mesa DM, Ramirez-Tortosa CL, Aguilere CM, Battino M, Gil A, Ramirez-Tortosa MC (2002): Curcuma longa. extract supplementation reduces oxidative stress and attenuates aortic fatty streak development in rabbits. Arterioscler Thromb Vasc Biol 22: 1225–1231. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Rao DS, Chandrasekharan N, Sathyanarayanan MN, Srinivasan M (1970): Effect of curcumin in serum and liver cholesterol levels in rats. J Nutr 100: 1307–1315. [PUBMED], [INFOTRIEVE], [CSA]
- Reitman S, Frankel A (1957): A colorimetric method for the determination of serum glutamic oxaloacetic acid and glutamic pyruvic transaminases. Am J Clin Pathol 28: 56–63. [PUBMED], [INFOTRIEVE], [CSA]
- Rukkumani R, Balasubashini MS, Viswanathan P, Menon VP (2002): Comparative effects of curcumin and photo-irradiated curcumin on alcohol and polyunsaturated fatty acid-induced hyperlipidemia. Pharmacol Res 46: 257–264. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Rukkumani R, Balasubashini MS, Menon VP (2003): Protective effects of curcumin and photo-irradiated curcumin on circulatory lipids and lipid peroxidation products in alcohol and polyunsaturated fatty acid-induced toxicity. Phytother Res 17: 925–929. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Rukkumani R, Aruna K, Varma PS, Rajasekharan KN, Menon VP (2004a): Protective role of a novel curcuminoid in alcohol and PUFA induced hyperlipidemia. Toxicol Mech Meth (). [CSA]
- Rukkumani R, Aruna K, Varma PS, Rajasekharan KN, Menon VP (2004b): Comparative effects of curcumin and an analog of curcumin on alcohol and PUFA induced oxidative stress. J Med Food (). [CSA]
- Schroeter H, Williams RJ, Martin R, Iversen L, Evans R (2000): Phenolic antioxidants attenuate neuronal cell death following uptake of oxidised low density lipoprotein. Free Radic Biol Med 29: 1222–1233. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Soni KB, Kuttan R (1992): Effect of oral administration on serum peroxides and cholesterol levels in human volunteers. Indian J Physiol Pharmacol 36: 273–275. [PUBMED], [INFOTRIEVE], [CSA]
- Soudamini KK, Unnikrishnan MC, Soni KB, Kuttan R (1992): Inhibition of lipid peroxidation and cholesterol levels in mice by curcumin. Indian J Physiol Pharmacol 36: 239–243. [PUBMED], [INFOTRIEVE], [CSA]
- Tribble DL, Aw TY, Jones DP (1987): The pathophysiological significance of lipid peroxidation in oxidative cell injury. Hepatology 7: 377–387. [PUBMED], [INFOTRIEVE], [CSA]
- Wiggins D, Gibbons GF (1996): Origin of hepatic very-low-density lipoprotein triacylglycerol: The contribution of cellular phospholipid. Biochem J 320: 673–679. [PUBMED], [INFOTRIEVE], [CSA]
- Zilversmit DB, Davis AK (1950): Microdetermination of plasma phospholipids by trichloroacetic acid precipitation. J Lab Clin Med 35: 155–159. [PUBMED], [INFOTRIEVE], [CSA]
- Zlatkis A, Zak B, Boyl AJ (1953): A new method for the direct determination of serum cholesterol. J Lab Clin Med 45: 486–492. [CSA]