Hepatoprotective Effects of 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone on CCl 4 -Induced Acute Liver Injury in Mice

Hepatoprotective Effects of 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone on CCl4-Induced Acute Liver Injury in MiceWan-Guo Yu,† Jie Qian,‡ and Yan-Hua Lu*,†

†State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, PR China‡School of Life Sciences and Technology, TongJi University, Shanghai, PR China

ABSTRACT: In this paper, the hepatoprotective effects of 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC) on CCl4-induced acute liver injury in Kunming mice were investigated. DMC was administered intraperitoneally (ip) (5, 10, or 20 mg/kgof body weight) for 7 days prior to the administration of CCl4 (0.1%, ip). Pretreatment with DMC significantly decreasedactivities of serum hepatic enzymes, namely alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, alkalinephosphatase, γ-glutamyl transferase, and total bilirubin, and decreased the elevation of lipid peroxidation, malondialdehyde,reactive oxygen species, and protein carbonyl content. Pretreatment with DMC markedly increased activities of enzymaticantioxidants such as superoxide dismutase, catalase, glucose-6-phosphate dehydrogenase, glutathione peroxidase, glutathioneS-transferase, and glutathione reductase and increased levels of nonenzymatic antioxidant markers such as reduced glutathione,total sulfhydryl groups, vitamin C, and vitamin E in liver. These results combined with liver histopathology demonstrate thatDMC has potential hepatoprotective effects, which may be related to the attenuation of oxidative stress, accelerating theantioxidant cascade and inhibition of lipid peroxidation.

KEYWORDS: 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone, hepatoprotective effects, hepatotoxicity, lipid peroxidation, mice,oxidative stress

■ INTRODUCTIONLiver disease is one of the most serious health problems all overthe world, because the liver is an important organ for thebiotransformation and detoxification of endogenous andexogenous harmful substances.1 Steroids, vaccines, and antiviraldrugs, which have been used to treat liver diseases, havepotentially adverse effects, especially when administered longterm.1,2 Therefore, it is necessary to develop more natural, safe,and effective drugs for the treatment of liver disease.The most common in vivo model used in the investigation of

new hepatoprotective agents has been a well-characterized rodentmodel of liver injury induced by carbon tetrachloride (CCl4), achemical hepatotoxin that causes free radical-mediated hepatocel-lular damage.3 CCl4 is metabolized by cytochrome P450 2E1(CYP2E1) in becoming a trichloromethyl radical (•CCl3) and aproxy trichloromethyl radical (•OOCCl3), which are thought toinitiate free radical-mediated lipid peroxidation, leading to theaccumulation of lipid-derived oxidation products that cause liverinjury.4−7 A number of studies have shown that various herbalsand natural remedies could protect liver against CCl4-inducedoxidative stress by altering the levels of increased lipidperoxidation, including malondialdehyde (MDA), reactive oxygenspecies (ROS), and protein carbonyl content (PCC), enhancingthe decreased activities of antioxidant enzymes such as superoxidedismutase (SOD), catalase (CAT), glucose-6-phosphate dehydro-genase (G6PD), glutathione peroxidase (GPx), glutathioneS-transferase (GST), and glutathione reductase (GR), anddecreasing the levels of hepatic reduced glutathione (GSH),total sulfhydryl groups (TSH), vitamin C, and vitamin E.8−11

Recently, herbal products are widely used in the treatment ofhepatic disorders in the world, because they exhibit one or acombination of antioxidant, antifibrotic, immunomodulatory,

and antiviral activities.12,13 Natural antioxidants, includingpolyphenols, flavonoids, and terpenes, could prevent thedeleterious effects of toxic agents by scavenging reactiveoxygen species (ROS) and free radicals, attenuating oxidativeand/or nitrosative stress, and inhibiting the inflammatoryresponse.14,15 Cleistocalyx operculatus (Roxb.) Merr. et Perry(Myrtaceae) is a well-known pharmaceutical and food plantwhose buds are commonly used as an ingredient for tonicdrinks in Southern China. Our previous phytochemicalattention to this species has led to the characterization ofsterol, flavanone, chalcone, and triterpene acid from its buds.16

The structure of 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchal-cone (DMC) has been reported in our previous study,17 themain compound from the buds of C. operculatus has beenreported to have neuroprotective, antidiabetic, spasmolytic,antitumoral, and anti-inflammatory properties.17−21 In thisstudy, we have investigated the protective effects of DMC onCCl4-induced acute liver injury in vivo.

■ MATERIALS AND METHODSMaterials. 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone

(DMC) was isolated from C. operculatus in our laboratory as describedby Ye et al.16 Silymarin (≥98.0% high-performance liquid chromatog-raphy grade) was purchased from Sigma-Aldrich Co. (St. Louis, MO).Carbon tetrachloride (CCl4), olive oil, and dimethyl sulfoxide(DMSO) were of analytical grade and were obtained commercially.Diagnostic kits used for the determination of alanine aminotransferase(ALT) activity, aspartate aminotransferase (AST) activity, lactate

Received: July 30, 2011Revised: November 18, 2011Accepted: November 18, 2011Published: November 19, 2011

Article

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© 2011 American Chemical Society 12821 dx.doi.org/10.1021/jf2042032 | J. Agric.Food Chem. 2011, 59, 12821−12829

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dehydrogenase (LDH) activity, alkaline phosphatase (ALP) activity,γ-glutamyl transferase (GGT) activity, superoxide dismutase (SOD)activity, catalase (CAT) activity, glucose-6-phosphate dehydrogenase(G6PD) activity, glutathione peroxidase (GPx) activity, glutathioneS-transferase (GST) activity, glutathione reductase (GR) activity, totalbilirubin (T-Bili) content, reduced glutathione (GSH) content, totalsulfhydryl group (TSH) content, malondialdehyde (MDA) content,protein carbonyl content (PCC), reactive oxygen species (ROS)percentage, and vitamin C and vitamin E levels were obtained from theNanjing Jiancheng Institute of Biotechnology (Nanjing, China). Allother chemicals were analytic reagents (AR).Animals and Experimental Design. Male Kunming mice (KM

mice, body weight of 28 ± 2 g) used in this study were purchased fromShanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China) (catalogno. 0046813). Experiments with animals were performed on the basisof animal ethics guidelines of the Institutional Animal EthicsCommittee. All animals were acclimatized to the laboratory environ-ment for 1 week before experiments were performed. Mice wereallowed free access to standard food and drinking water and weremaintained at a constant room temperature of 22 ± 2 °C and 50−60%relative humidity conditions with an automatic 12 h light−12 h darkcycle. All experimental animals were randomly divided into sevengroups with 10 mice in each group. DMC [5, 10, and 20 mg/kg ofbody weight (bw)] and silymarin (20 mg/kg of bw) dissolved inDMSO and diluted with saline [final 5% (v/v) DMSO solution].Animals from the first group (control) and the second group (a CCl4hepatotoxicity pathological model) were given the same volumeinjection of vehicle only for seven consecutive days. Animals from thethird group (DMC hepatotoxicity model) were injected intra-peritoneally (ip) with DMC (20 mg/kg of bw) only once daily forseven consecutive days and used for evaluation of the potentialhepatotoxicity of DMC. Animals from the fourth group (positivecontrol) were injected intraperitoneally with reference drug silymarin(20 mg/kg of bw) once daily for seven consecutive days prior to CCl4intoxication. Animals from the other three groups were pretreated withDMC at three different daily doses (5, 10, and 20 mg/kg of bw,respectively) over the seven days. On the seventh day, all mice exceptthose in the control group and DMC hepatotoxicity model groupwere simultaneously intoxicated ip with the single dose of CCl4(10 mL/kg of bw, 0.1% in olive oil) 1 h after the last administration,at different sites of the abdomen to avoid cross-reaction of chemicals,while animals from the control group and DMC hepatotoxicity modelgroup were injected intraperitoneally with olive oil (10 mL/kg of bw)alone.Collection and Preparation for Analysis of Samples. All the

animals were sacrificed by decapitation 24 h after the administration ofCCl4. Blood samples were separated for serum aliquots bycentrifugation at 4 °C and 4000g for 15 min. Livers were excised

immediately, washed with an ice-cold physiologic saline solution,blotted dry, and weighed. They both were stored at −20 °C until theywere further analyzed. Liver tissues were ground in liquid nitrogen andthen homogenized in buffer containing 50 mM Tris-HCl (pH 7.4),150 mM NaCl, 50 mM saccharose, and 2 mM ethylenediaminetetra-acetic acid on ice. After centrifugation at 4 °C and 16000g for 30 min,supernatants were collected. For the determination of toxicity, relativevalues of body and liver weights were calculated as shown in thefollowing equations:22

where BWB is the body weight before administration of CCl4,BWA is the body weight after administration of CCl4, LW is theliver weight, and BW is the body weight.

Table 1. Relative Body Weights and Liver Weights in CCl4-Induced Liver Injury in Micea

treatmentrelative body weight

(%)relative liver weight

(%)

control 99.92 ± 1.61 3.88 ± 0.11DMC (20 mg/kg of bw) 99.87 ± 1.73 4.09 ± 0.14CCl4 98.17 ± 3.91 6.21 ± 0.35b

CCl4 and DMC (5 mg/kg ofbw)

99.13 ± 1.56 5.29 ± 0.18c


99.12 ± 2.14 5.15 ± 0.14c


99.26 ± 2.94 4.98 ± 0.18c

CCl4 and silymarin (20 mg/kg ofbw)

98.71 ± 2.69 5.23 ± 0.29d

aEach value represents the mean ± the standard error for 10 mice.bP < 0.001 compared with the control group. cP < 0.001 com-pared with the CCl4-induced group. dP < 0.01 compared with theCCl4-induced group.

Figure 1. Effects of DMC on the hepatic (A) ALT and (B) ASTactivity in experimental groups. Mice were treated ip with DMC (5,10, and 20 mg/kg of bw) or silymarin (20 mg/kg of bw) once daily forseven consecutive days prior to ip treatment with CCl4 [10 mL/kg ofbw, 0.1% (v/v) in olive oil], except for the control group and DMC-only group, which received vehicle. Each value represents the mean ±SD for 10 mice. ###P < 0.001 compared with the control group.***P < 0.001, **P < 0.01, and *P < 0.05 compared with the CCl4-induced group.

Journal of Agricultural and Food Chemistry Article

dx.doi.org/10.1021/jf2042032 | J. Agric.Food Chem. 2011, 59, 12821−1282912822

Biochemical Parameters of Liver Function. Levels of ALT,AST, LDH, ALP, GGT, and T-Bili in serum were measured bydiagnostic kits (Nanjing Jiancheng Institute of Biotechnology).Determination of Enzymatic Antioxidant Activities. Activ-

ities of SOD, CAT, G6PD, GPx, GST, and GR in liver homogenatewere evaluated by assay kits according to the manufacturer’sinstructions (Nanjing Jiancheng Institute of Biotechnology).Determination of Nonenzymatic Antioxidant Activities. The

contents of GSH, TSH, vitamin C, and vitamin E in liver homogenatewere evaluated by assay kits according to the manufacturer’sinstructions (Nanjing Jiancheng Institute of Biotechnology).Estimation of Lipid Peroxidation. The levels of MDA, ROS,

and PCC in liver tissue samples were evaluated by assay kits accordingto the manufacturer’s instructions (Nanjing Jiancheng Institute ofBiotechnology).Histopathological Studies. Liver samples for histopathological

analysis were placed in 10% buffered formalin saline, processed byroutine histology procedures, and embedded in paraffin. Tissue

sections (6−7 μm thick) were stained with hematoxylin and eosin dye(H&E) and observed under a light microscope (Nikon, Tokyo, Japan),and images were recorded.Statistical Analysis. All experiments were conducted in triplicate,

and data were presented as means ± standard deviations (SD) for10 mice in each group. The results were evaluated by one-way analysisof variance (ANOVA), and Duncan’s test was used to determinesignificant differences of multiple comparisons. Calculations wereperformed using SPSS version 17.0 (SPSS, Chicago, IL). A P value of<0.05 was taken to be statistically significant.

■ RESULTS

DMC Inhibits CCl4-Induced Hepatotoxicity in Mice. Treat-ment with CCl4 significantly increased (P < 0.001) liver weightbut did not affect body weight. However, administration ofDMC at three different doses (5, 10, and 20 mg/kg of bw)

Figure 2. Effects of DMC on the hepatic (A) LDH and (B) ALPactivity in experimental groups. Mice were treated ip with DMC (5,10, and 20 mg/kg of bw) or silymarin (20 mg/kg of bw) once daily forseven consecutive days prior to ip treatment with CCl4 [10 mL/kg ofbw, 0.1% (v/v) in olive oil], except for the control group and DMC-only group, which received vehicle. Each value represents the mean ±SD for 10 mice. ###P < 0.001 compared with the control group.***P < 0.001, **P < 0.01, and *P < 0.05 compared with the CCl4-induced group.

Figure 3. Effects of DMC on the hepatic (A) GGT activity and (B)total bilirubin content in experimental groups. Mice were treated ipwith DMC (5, 10, and 20 mg/kg of bw) or silymarin (20 mg/kg ofbw) once daily for seven consecutive days prior to ip treatment withCCl4 [10 mL/kg of bw, 0.1% (v/v) in olive oil], except for the controlgroup and DMC-only group, which received vehicle. Each valuerepresents the mean ± SD for 10 mice. ###P < 0.001 compared withthe control group. ***P < 0.001 and **P < 0.01 compared with theCCl4-induced group.



significantly reversed (P < 0.001) the CCl4-induced increase inliver weight to the levels of the control group. Administration ofDMC (20 mg/kg of bw) to normal mice did not have any effecton body weight or liver weight (Table 1). All these changesinduced by CCl4 intoxication were significantly (P < 0.001)reduced upon administration of DMC in a dose-related manner.Effect of DMC on Hepatic Markers. The results of

hepatic marker enzymes and T-Bili in experimental mice areshown in Figures 1−3. Treatment with CCl4 caused abnormalliver function in mice. The activities of serum hepatic enzymesALT (Figure 1A), AST (Figure 1B), LDH (Figure 2A), ALP(Figure 2B), GGT (Figure 3A), and T-Bili (Figure 3B) weresignificantly (P < 0.001) increased, compared with the controlgroup. However, pretreatment of DMC at three different doses(5, 10, and 20 mg/kg of bw) significantly (P < 0.05) decreasedthe levels of serum hepatic marker enzymes and T-Bili,

compared with the pathological model group. Pretreatmentwith DMC (10 mg/kg of bw) was found to be comparable tothat with the reference drug silymarin (20 mg/kg of bw). Therestoration of hepatic marker enzymes and T-Bili was maximalat the highest dose (20 mg/kg of bw) of DMC when comparedwith the other two dose groups. Administration of DMC(20 mg/kg of bw) to normal mice did not have any effect onhepatic markers.Effect of DMC on Enzymatic Antioxidants. The

activities of hepatic enzymatic antioxidants in the liver tissueare shown in Figures 4−6. After treatment with CCl4, theactivities of enzymatic antioxidants SOD (Figure 4A), CAT(Figure 4B), G6PD (Figure 5A), GPx (Figure 5B), GST(Figure 6A), and GR (Figure 6B) were significantly (P < 0.001)decreased, compared with the control group. However,administration of DMC at three different doses with CCl4

Figure 4. Effects of DMC on the hepatic (A) SOD and (B) CATactivity in experimental groups. Mice were treated ip with DMC (5,10, and 20 mg/kg of bw) or silymarin (20 mg/kg of bw) once daily forseven consecutive days prior to ip treatment with CCl4 [10 mL/kg ofbw, 0.1% (v/v) in olive oil], except for the control group and DMC-onlygroup, which received vehicle. Each value represents the mean ± SD for10 mice. ###P < 0.001 compared with the control group. ***P < 0.001and **P < 0.01 compared with the CCl4-induced group.

Figure 5. Effects of DMC on the hepatic (A) G6PD and (B) GPxactivity in experimental groups. Mice were treated ip with DMC (5,10, and 20 mg/kg of bw) or silymarin (20 mg/kg of bw) once daily forseven consecutive days prior to ip treatment with CCl4 [10 mL/kg ofbw, 0.1% (v/v) in olive oil], except for the control group and DMC-onlygroup, which received vehicle. Each value represents the mean ± SD for10 mice. ###P < 0.001 compared with the control group. ***P < 0.001and **P < 0.01 compared with the CCl4-induced group.



significantly (P < 0.05) increased the activities of enzymaticantioxidants in liver. Pretreatment with DMC (10 mg/kg ofbw) yielded activities almost comparable to those of thepositive group treated with silymarin (20 mg/kg of bw). Thelargest dose (20 mg/kg of bw) of DMC had a beneficial effecton enzymatic antioxidants because the activities of theseenzymes were found to be significantly (P < 0.001) and dose-dependently increased, compared with the pathological modelgroup. Administration of DMC (20 mg/kg of bw) to normalmice did not have any effect on enzymatic antioxidants.Effect of DMC on Nonenzymatic Antioxidants. The

changes in the levels of hepatic nonenzymatic antioxidants inthe liver tissue are shown in Figures 7 and 8. After treatmentwith CCl4, the levels of nonenzymatic antioxidants GSH(Figure 7A), TSH (Figure 7B), vitamin C (Figure 8A), and

vitamin E (Figure 8B) were significantly (P < 0.001) decreased,compared with the control group. However, administration ofDMC at three different doses with CCl4 significantly (P < 0.05)increased the levels of nonenzymatic antioxidants in liver.Pretreatment with DMC (10 mg/kg of bw) yielded activitiesalmost comparable to those of the positive group treated withsilymarin (20 mg/kg of bw). The largest dose (20 mg/kg of bw) ofDMC had a beneficial effect on nonenzymatic antioxidants becausetheir levels were found to be significantly (P < 0.001) and dose-dependently increased, compared with the pathological modelgroup. Administration of DMC (20 mg/kg of bw) to normal micedid not have any effect on nonenzymatic antioxidants.Effect of DMC on Lipid Peroxidation. Figure 9 illustrates

the levels of lipid peroxidation product in livers of experi-mental mice. After treatment with CCl4, the contents of MDA

Figure 6. Effects of DMC on the hepatic (A) GST and (B) GR activityin experimental groups. Mice were treated ip with DMC (5, 10, and20 mg/kg of bw) or silymarin (20 mg/kg of bw) once daily for sevenconsecutive days prior to ip treatment with CCl4 [10 mL/kg of bw,0.1% (v/v) in olive oil], except for the control group and DMC-onlygroup, which received vehicle. Each value represents the mean ± SD for10 mice. ###P < 0.001 compared with the control group. ***P < 0.001,**P < 0.01, and *P < 0.05 compared with the CCl4-induced group.

Figure 7. Effects of DMC on the hepatic (A) GSH and (B) TSHcontent in experimental groups. Mice were treated ip with DMC (5,10, and 20 mg/kg of bw) or silymarin (20 mg/kg of bw) once daily forseven consecutive days prior to ip treatment with CCl4 [10 mL/kg ofbw, 0.1% (v/v) in olive oil], except for the control group and DMC-onlygroup, which received vehicle. Each value represents the mean ± SD for10 mice. ###P < 0.001 compared with the control group. ***P < 0.001compared with the CCl4-induced group.



(Figure 9A), ROS (Figure 9B), and PCC (Figure 9C) weresignificantly (P < 0.001) increased, compared with the controlgroup. However, administration of DMC at three differentdoses with CCl4 significantly (P < 0.05) lowered the levelsof lipid peroxidation product in liver. Pretreatment with DMC(20 mg/kg of bw) was found to be comparable to that withthe reference drug silymarin (20 mg/kg of bw). The largestdose of DMC (20 mg/kg of bw) was found to be compar-able to those of two other groups. Administration of DMC(20 mg/kg of bw) to normal mice did not have any effect onlipid peroxidation.Histopathological Results. According to the light micro-

scope evaluation of the experimental animal liver tissue, aregular morphology of the liver parenchyma with well-designated hepatic cells was evident (Figure 10). The

histological examination showed no pathological abnormalitiesin the liver of the control group and DMC hepatotoxicitymodel group (Figure 10A,B). However, treatment with CCl4

Figure 8. Effects of DMC on the hepatic (A) vitamin C and (B)vitamin E content in experimental groups. Mice were treated ip withDMC (5, 10, and 20 mg/kg of bw) or silymarin (20 mg/kg of bw)once daily for seven consecutive days prior to ip treatment with CCl4[10 mL/kg of bw, 0.1% (v/v) in olive oil], except for the control groupand DMC-only group, which received vehicle. Each value representsthe mean ± SD for 10 mice. ###P < 0.001 compared with the controlgroup. ***P < 0.001, **P < 0.01, and *P < 0.05 compared with theCCl4-induced group.

Figure 9. Effects of DMC on the hepatic (A) MDA content, (B) ROScontent of induced groups (%), and (C) PCC in experimental groups.Mice were treated ip with DMC (5, 10, and 20 mg/kg of bw) orsilymarin (20 mg/kg of bw) once daily for seven consecutive daysprior to ip treatment with CCl4 [10 mL/kg of bw, 0.1% (v/v) in oliveoil], except for the control group and DMC-only group, which receivedvehicle. Each value represents the mean ± SD for 10 mice. ###P < 0.001compared with the control group. ***P < 0.001, **P < 0.01, and*P < 0.05 compared with the CCl4-induced group.



produced histological changes in the liver tissue of intoxicatedmice. Histopathological analysis of the pathological modelgroup showed severe confluent, cytolysis, pyknosis, ballooningdegeneration, and infiltration of inflammatory cells into theportal tract in the necrotic lesion (Figure 10C). Pretreatmentwith DMC dose-dependently reversed the hepatic lesionsproduced by CCl4 (Figure 10D−F) as is evident from theabsence of cellular necrosis and inflammatory infiltrates in theliver section of mice pretreated with the largest dose tested,which were almost comparable to those of the pathologicalmodel group and positive group (Figure 10G). Histologicalexamination indicated that DMC can effectively protect liverfrom CCl4-induced injury.

■ DISCUSSION

Hepatotoxicity induced by CCl4 is the most commonly usedmodel system for the screening of hepatoprotective activity ofplant extracts and drugs.23 The purpose of this study is todetermine the hepatoprotective role of DMC against acute CCl4-induced changes in enzyme activities, oxidative damage, andimpairment of the antioxidant defense system in mice. DMCseems to possess considerably stronger activity against CCl4-induced liver damage than silymarin, which is widely recognizedas a potent hepatoprotective agent. All these morphologicalchanges observed in mice treated with CCl4 were attenuated bypretreatment with DMC.Several soluble enzymes of blood serum have been considered

as indicators of hepatic dysfunction and damage. The increase inthe activities of these enzymes in plasma is indicative of liverdamage, resulting in leakage of the cytosolic contents into thesystemic circulation, and thus causes alteration of liver function.24

In this study, the increased activities of serum ALT, AST, LDH,ALP, GGT, and T-Bili in serum obviously indicate that liver issusceptible to CCl4-induced toxicity. This increase could beattributed to the hepatic damage resulting in an increased rate ofrelease of functional enzymes from biomembranes or itsincreased level of synthesis.25 However, the increased levels ofthese markers were significantly decreased by pretreatment with

DMC, implying that DMC may stabilize the hepatic cellularmembrane and protect the hepatocytes against toxic effects ofCCl4, which may decrease the rate of leakage of the enzymes intothe bloodstream.26

Enzymatic antioxidants such as SOD, CAT, G6PD, GPx,GST, and GR that act as preventive antioxidants by playing amajor role in protecting the cells against oxidative stress are thefirst line of defense against oxidative injury.27 CCl4-inducedtoxicity might result in significantly decreased activities ofenzymatic antioxidants. Administration of DMC significantlyincreased the activities of enzymatic antioxidants, which may becaused by attenuation of oxidative stress. Indeed, DMC directlyexhibited a strong free radical scavenging effect that could havea beneficial action against pathological alterations caused by thegenerated free radical CCl3 induced by CCl4.The antioxidant defense is considered to be involved in

CCl4-induced toxic effects. Vitamin C is a dietary hydrophilicantioxidant that works in concert with vitamin E, which is achain-breaking antioxidant that prevents the free radical chainoxidation of lipids. A number of reports have shown thepositive effect of vitamin C as an antioxidant and scavenger offree radicals.28 GSH is the major nonenzymatic antioxidant andregulator of intracellular redox homeostasis, which has TSH inits peptide largely present in the biological system.29 Increasingthe rate of the antioxidant cascade of DMC is responsible forrestoring these levels by reducing the level of utilization ofnonenzymatic antioxidants that consequently leads to improve-ment of GSH, TSH, vitamin C, and vitamin E levels in plasma.Lipid peroxidation has been implicated in the pathogenesis of

hepatic injury by the free radical derivatives of CCl4 and isresponsible for cell membrane damage and the consequentrelease of marker enzymes of hepatotoxicity.29 Oxidative injuryinduced by CCl4 can be monitored in experimental animals bydetecting lipid peroxidative products such as thiobarbituric acidreactive substances, hydroperoxides, and conjugated dienes.30

This study revealed increased levels of MDA, ROS, and PCC inthe liver of the CCl4-treated experimental animals. However, theincreased levels of these markers were significantly decreased by

Figure 10. Representative photomicrographs of livers (original magnification of 400×). The liver section was stained with H&E. Effect of DMC andsilymarin on CCl4-induced liver injury in mice: (A) control group and (B) animals treated with DMC (20 mg/kg of bw) only. (C) Animals treatedwith CCl4 only exhibited severe confluent, inflammatory cell infiltration and ballooning changes (arrow). (D) Animals pretreated with DMC (5 mg/kg of bw) and (E) DMC (10 mg/kg of bw) and then with CCl4 underwent a slight reduction in the number of necrotic lesions (arrow). (F) Animalspretreated with DMC (20 mg/kg of bw) and then with CCl4 showed mild necrosis and ballooning changes (arrow). (G) Animals pretreated withsilymarin (20 mg/kg of bw) and then with CCl4 did not have substantially ameliorated hepatic lesions (arrow).



pretreatment with DMC. This clearly demonstrates the ability ofDMC to directly interact with ROS that may initiate lipidperoxidation. It is possible that DMC effectively quenches thefree radicals because of the hydroxyl groups.In this study, the hepatic histoarchitecture of the CCl4-

treated mice resulted in severe confluence, cytolysis, pyknosis,ballooning degeneration, and inflammatory cell infiltration. Itmight be due to the formation of highly reactive radicalsbecause of the oxidative threat induced by CCl4. Theaccumulated hydroperoxides can cause cytotoxicity, which isassociated with peroxidation of membrane phospholipids bylipid hydroperoxides. The necrotic conditions coincide with ourbiochemical studies, which show increased levels of lipidperoxidation. Administration of DMC reduced the histologicalalterations induced by CCl4. It can be attributed to theantioxidant and anti-inflammatory ability of DMC, whichsignificantly reduced the oxidative threat leading to reduction ofpathological changes and restoration of normal physiologicalfunctions.In conclusion, this study demonstrates that DMC has a

protective effect against acute hepatotoxicity induced by theadministration of CCl4 in mice and that the hepatoprotectiveeffects of DMC may be related to the attenuation of oxidativestress, increasing the rate of the antioxidant cascade and inhibitionof lipid peroxidation in the liver. Further studies will be needed toinvestigate the possible hepatoprotective mechanisms of DMCand the metabolism in vivo. This study could serve as a usefulreference to allow the future exploitation of DMC as a potenthepatoprotective medication for the therapy and prevention ofoxidative stress-induced liver damage.

■ AUTHOR INFORMATIONCorresponding Author*State Key Laboratory of Bioreactor Engineering, East ChinaUniversity of Science and Technology, Box 283#, 130 MeilongRd., Shanghai 200237, PR China. E-mail: [email protected]. Phone: +86-21-64251185. Fax: +86-21-64251185.

■ ACKNOWLEDGMENTSThis work was supported by the National Natural ScienceFoundation of China (No. 30870253), and partially supportedby Shanghai Leading Academic Discipline Project (B505), theNational Special Fund for State Key Laboratory of BioreactorEngineering (2060204), and the 863 Project (2011AA090702).

■ ABBREVIATIONSDMC, 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone; KM,Kunming; DMSO, dimethyl sulfoxide; ALT, alanine amino-transferase; AST, aspartate aminotransferase; LDH, lactatedehydrogenase; ALP, alkaline phosphatase; GGT, γ-glutamyltransferase; T-Bili, total bilirubin; SOD, superoxide dismutase;CAT, catalase; G6PD, glucose-6-phosphate dehydrogenase;GPx, glutathione peroxidase; GST, glutathione S-transferase;GR, glutathione reductase; GSH, reduced glutathione; TSH,total sulfhydryl groups; MDA, malondialdehyde; ROS, reactiveoxygen species; PCC, protein carbonyl content

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Hepatoprotective Effects of 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone on CCl 4 -Induced Acute Liver Injury in Mice