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Journal of Ethnopharmacology 138 (2011) 683– 690

Contents lists available at SciVerse ScienceDirect

Journal of Ethnopharmacology

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epatoprotective effect and its possible mechanism of Coptidis rhizoma aqueousxtract on carbon tetrachloride-induced chronic liver hepatotoxicity in rats

ibin Fenga,∗, Ning Wanga, Xingshen Yea, Huangyun Lib, Yigang Fengb, Fan Cheunga, Tadashi Nagamatsuc

School of Chinese Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong, PR ChinaGuanhua School of Stomatology, Hospital of Stomatology, Sun Yat-Sen University, Guangzhou, PR ChinaDepartment of Pharmacobiology and Therapeutics, Faculty of Pharmacy, Meijo University 150 Yagotoyama, Tenpakuku, Nagoya 468-8503, Japan

r t i c l e i n f o

rticle history:eceived 17 April 2011eceived in revised form 3 September 2011ccepted 18 September 2011vailable online 22 September 2011

eywords:optidis rhizomahronic liver damageepatoprotective effectnti-oxidant agentrk1/2 inhibition

a b s t r a c t

Ethnopharmacological relevance: Coptidis rhizoma is traditionally used for heat-clearing and toxic-scavenging and it belongs to liver meridian in Chinese medicine practice. Clinically, Coptidis rhizomacan be used for hepatic and biliary disorders, yet details in the therapies of liver diseases and underlyingmechanism(s) remain unclear. Our previous study demonstrated that Coptidis rhizoma aqueous extract(CRAE) against CCl4-induced acute liver damage was related to antioxidant property. In the presentstudy, the protection of CRAE on chronic liver damage induced by carbon tetrachloride (CCl4) in rats andits related mechanism were explored.Materials and methods: The CCl4-induced chronic liver damage model was established, and CRAE’s protec-tive effect was examined. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT)activity, serum and liver superoxide dismutase (SOD) activity were then measured. The histologicalchanges were observed under microscopy and then computed in numerical score. The normal or damagedcells were isolated and related signaling pathway was evaluated.Result: Serum AST and ALT activities were significantly decreased in rats treated with different doses of

CRAE, indicating its protective effect against CCl4-induced chronic liver damage. Observation on serumSOD activity revealed that CRAE might act as an anti-oxidant agent against CCl4-induced chronic oxidestress. Histological study supported these observations. Erk1/2 inhibition may take part into CRAE’s effecton preventing hepatocyte from apoptosis when exposed to oxidative stress.Conclusion: CRAE showed protective effect against CCl4-induced chronic liver damage in rats and itspotential as an agent in the treatment of chronic liver diseases by protecting hepatocyte from injury.

. Introduction

Hepatocellular carcinoma (HCC), amounting for 80–90% of liverancer, has nowadays become one of the most common and preva-ent human malignancies in the world (Gramantieri et al., 2007;udhu et al., 2008). One of the risky factors that can induceCC is extended and chronic liver damage, the process causes

emolishment of the normal liver blood system, leading to shortupply of blood circulation in liver cells and consequently inducingypoxia, the condition that normal cells are exposed to tremendous

Abbreviations: HCC, hepatocellular carcinoma; CRAE, Coptidis rhizoma aqueousxtract; CCl4, carbon tetrachloride; AST, aspartate aminotransferase; ALT, alanineminotransferase; SOD, superoxide dismutase; BW, body weight; SD rats, Sprague-awley rats; H&E staining, hematoxylin and eosin staining.∗ Corresponding author. Tel.: +852 2589 0482; fax: +852 2872 5476.

E-mail addresses: yfeng@hku.hk, jamesfeng852@hotmail.com (Y. Feng).

378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2011.09.032

© 2011 Elsevier Ireland Ltd. All rights reserved.

oxidative stress (Wu et al., 2007). One of the effective treatmentsin suppressing HCC is to control progress of chronic liver damage(Feng et al., 2009b).

Recent years, herbal medicines as a resource for liver diseaseshave been attracted by the world wide scientists (Schuppan et al.,1999; Feng et al., 2009b; Seeff et al., 2001), among which Cop-tidis rhizoma (CR, Huanglian in Chinese) is one of the potentialherbs (Feng et al., 2009b; Ye et al., 2009). CR was a Chinese medic-inal herb that is widely used for clearing heat and scavengingtoxics during thousand years of clinical utilization. CR comprisesvarious kinds of chemicals including berberine, palmatine and jatr-orrhizine (Deng et al., 2008), among which berberine is the majoringredient representing a variety of bioactivities. Extensive stud-ies in recent years have displayed that CR has various kinds of

bioactivities including antibacterial, antiviral, antiinflammatory,antineoplastic, antihypertensive, antioxidative, antihyperglycemicand cholesterol-lowering effects (Fukutake et al., 1998; Li et al.,2000; Chang et al., 2001; Sanae et al., 2001; Yokozawa et al., 2003,

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84 Y. Feng et al. / Journal of Ethno

004; Choi et al., 2007; Kim et al., 2008). Our clinical study showedhat CR had a promising potential as a drug for treatment of liveriseases, such as liver fibrosis and cancer (Feng et al., 2008). More-ver, it has been demonstrated in our previous studies that CRAEnd its active compound, berberine exhibited potent protectiveffect on CCl4-induced acute liver damage in rats (Ye et al., 2009;eng et al., 2010). However, no observation and investigation haseen proceeded to study CRAE’s effect on chronic liver damage and

ts underlying mechanism.In this study, CCl4-induced chronic liver injury in rats was intro-

uced as an animal model to investigate the protective effect ofRAE. HPLC was used in this study to analyze the chemical com-osition of CRAE. Serum AST and ALT was measured to observehe liver function in different treatment groups, and serum andissue SOD activities were determined. Histological study was con-ucted to observe the morphological changes among differentroups. To elucidate the underlying mechanism of CRAE’s protec-ion against chronic liver damage induced by CCl4, rat hepatocyteas isolated and the apoptosis was evaluated by DNA fragmenta-

ion and caspase-3 activity. The related signaling transduction waslso examined in this study.

. Materials and methods

.1. Chemicals

Berberine hydrochloride and palmatine hydrochloride wereurchased from Sigma (Sigma–Aldrich, USA). Jatrorrhizineydrochloride was purchased from National Institute for theontrol of Pharmaceutical and Biological Products (Beijing, China).agnolflorine chloride was purchased from Tauto Biotech (Shang-

ai, China). Coptisine was purchased from Chromadex (USA).arbon tetrachloride (CCl4) and liquid paraffin were purchasedrom Sigma (Sigma–Aldrich, USA).

.2. Herbs and sample preparation

CR was collected in the GAP authorized field for Huanglian cul-ivation in Shizu county of Chongqing city of People Republic ofhina. Plants were cleaned under distilled water, dried and cut

nto small pieces. The roots of Coptidis were first boiled in distilledater at 100 ◦C for 1 h. The solution was percolated through filteraper (Whatman, pleated filter grade 597 1/2, 4–7 �m) and thenterilized by filtration through a 0.2 �m pore filter (Minisart®-plus,artorius). The collected extraction was evaporated to dryness byacuum at temperature. The dry extract powder obtained is storedn −20 ◦C freezer and used in following experiments.

.3. Phytochemical analysis

High performance liquid chromatography (HPLC) was intro-uced to analyze the active components in CRAE. The phytochemi-al analysis was performed under the following conditions: Reserve18 column (Symmetry®, 250 mm × 4.0 mm, 5 �m) was used asolid phase; elution was performed using acetonitrile–25 mMotassium dihydrogen phosphate (23:77) as mobile phase. Detect-

ng wavelength and flow rate was 254 nm and 1.0 ml/minespectively. The analysis was performed under room temperature.

.4. Animals

Male SD rats weighing about 200 g were used. All animals were

ed a standard diet ad libitum and housed at the temperature of0–25 ◦C under a 12 h light/dark cycle throughout the experiment.he rats were randomly assigned into different groups: normalroup, control group, CRAE low dose (400 mg/kg body weight, BW)

acology 138 (2011) 683– 690

group, CRAE medium dose (600 mg/kg BW) group, CRAE high dose(800 mg/kg BW) group and berbeine (120 mg/kg BW) group. All ani-mals received humane care and study protocols complied with theguidelines of the animal center of the University of Hong Kong.

2.5. Animal treatment

Control group was given an intraperitoneal injection of CCl4at 1 ml/kg per rat (diluted 1:1 in liquid paraffin. The following isthe same) twice a week for 8 weeks. The dose and duration wereadopted from the methods of previous studies (Luo et al., 2004;Chung et al., 2005). CRAE treatment groups were given an intraperi-toneal injection of CCl4 at 1 ml/kg per rat twice a week for 8 weeks,and were given CRAE once per day at the dose of 400, 600 and800 mg/kg BW during the same period. Berberine treatment groupreceived daily oral administration of BW 120 mg/kg berberine. Nor-mal group was given an intraperitoneal injection of liquid paraffinat 1 ml/kg per rat twice a week for 8 weeks, and was given double-distilled water orally once per day in the same period. At the endof the period, the rats were anesthetized with ether, blood sampleswere collected by cardiac puncture and serum was obtained by cen-trifugation (3000 rpm, 12 min). Liver tissues were washed quicklyin situ with ice-cold isotonic saline.

2.6. Biochemical assay and histological study

Serum was collected as mentioned above. ALT and AST activitieswere then determined under the manufacturer’s instruction (Biovi-sion US). ALT and AST activities were reported in terms of units perliter (U/L). Serum was collected and the SOD activity was measuredusing the manufacturer’s instruction (Biovision US).

Liver tissues were collected from animals in different groups andwere fixed in 10% buffered formaldehyde solution for at least 24 h.The paraffin sections were then prepared (Automatic Tissue Proces-sor, Lipshaw) and cut into 5 �m thick sections by a Leica RM 2016rotary microtome (Leica Instruments Ltd., Shanghai, China). Thesections were stained with hematoxylin and eosin staining (H&Estaining) and then mounted with Canada balsam (Sigma, USA).The degree of liver damage was examined under the microscope(Leica Microsystems Digital Imaging, Germany). The images weretaken using Leica DFC 280 CCD camera at original magnificationof 10 × 10. Chronic liver injury was then evaluated by grading theliver sections numerically to assess their histological features. Vac-uolation, nuclei, hepatocyte necrosis, inflammatory cell infiltrationand central vein and portal triad were used as criteria, and a com-bined score of histological features was given for each liver section.The parameters were graded from score 0 to 6, with 0 indicating noabnormality, 1–2 indicating mild injury, 3–4 indicating moderateinjury and 5–6 with severe liver injury (Wang et al., 2008).

2.7. Cells

The rat hepatocyte was isolated by in situ perfusion method aspreviously described (Seglen, 1973). Isolated cells were culturedin Williams’ medium E (WME, Sigma–Aldrich, USA) supple-mented with 10% FBC (Invitrogen, USA), 0.9 M dexamethasone(Sigma–Aldrich, USA) and 10 M insulin (Sigma–Aldrich, USA).Medium was replaced with serum- and hormone-free WME (Ikedaet al., 2007).

2.8. DNA fragmentation assay

The cell apoptosis was determined by DNA fragmentation assay.Briefly, isolated hepatocyte was lysed in 1% NP-40 buffer contain-ing 20 mM EDTA, 50 mM Tris–HCl, pH 7.5 for 1 min with thorough

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Y. Feng et al. / Journal of Ethno

ortex. The lysate was centrifuged at 3000 × g for 5 min and super-atant was collected. 10 �L of Proteinase K (25 mg/ml, Sigma, USA)as added to the supernatant followed by 2 h incubation at 37 ◦C.

5 �L of 10 M ammonium acetate and 500 �L ice-cold ethanol weredded and thoroughly mixed. Then samples were stored at −80 ◦Cor 1 h to precipitate the DNA. Centrifugation at 14,000 rpm for0 min was conducted to collect the DNA from supernatant andhe white pellet was obtained by washing with 200 �L 80% ice-old ethanol and air-drying for 10 min at room temperature. Theesidues were reconstituted in 30 �L of TE buffer and the DNAragment was separated by 2% agarose gel and visualized UV tran-illuminator (Bio-Rad, USA).

.9. Caspase-3 activity assay

The caspase-3 activity was determined by the Caspase-3 Activityssay Kit (Merck, Germany) according to manufacturer’s instruc-

ion. The caspase-3 activity was normalized by total protein levelf each sample by BSA assay (Bio-Rad, USA).

.10. Immunoblotting

The isolated hepatocyte was lysed with RIPA buffer supple-ented with proteinase inhibitor (1% PMSF, 0.5% apotinin and 0.5%

eupitine) and phosphatase inhibitor (1 mM Na3VO4 and 1 mM NaF)n ice for 30 min and then centrifuged at 14,000 rpm at 4 ◦C for5 min. The supernatant was transferred to a new tube and pro-ein concentration was determined using BSA as standard. Equalmounts of protein were resolved by SDS–PAGE and transferrednto a polyvinylidene fluoride membrane (PVDF, Biorad). Thenhe membrane was blocked with 5% BSA in buffer containingris (10 mmol/L, pH 7.4), NaCl (150 mmol/L) and Tween 20 (1%)vernight at 4 ◦C. The membrane was then incubated with primaryntibodies at 4 ◦C overnight followed by incubation with appro-riate secondary antibody (Abcam, UK) at room temperature for

h. The immunoreactivites were detected using ECL advanced kitGE Healthcare, UK) and visualized using a chemiluminenescencemaging system (Bio-Rad, USA).

.11. Statistical analysis

The data obtained were analyzed by one-way of varianceANOVA) and Student–Newman–Kelus post hoc tests for the sig-ificant interrelation between the groups. Data were expressed asean ± SD of the mean and values of p < 0.05 were considered to

e statistically significant.

. Results

.1. Phytochemical analysis of CRAE by HPLC

According to the China Pharmacopeia (Edition 2005), the crudeerb of CR was authenticated by morphological characteristics ofR (Coptis chinensis Franch) and was compatible with the voucherpecimens (no. CR20060616) deposited in the Herbarium of Schoolf Chinese Medicine, The University of Hong Kong. The plant andrude herb were presented in Fig. 1A. CRAE stored in −20 ◦C freezeras the same batch as that of our previous study (Ye et al., 2009).

he chemical fingerprint of the CRAE was conducted by HPLC anal-sis again to confirm whether CRAE is still normal as before. Thisime, we used five external standards for HPLC analysis. HPLC chro-

atograms of Standard compounds and CRAE were displayed in

ig. 1B. Five peaks were identified in CRAE with retention timend UV spectra comparison. Using standard curve, the yields ofve compounds were calculated and the result showed that CRAEontains 2.33% Magnolflorine, 4.81% Jateorrhizine, 8.21% Coptisine,

acology 138 (2011) 683– 690 685

9.76% Palmatine and 24.49% Berberine. The data in Fig. 1C show agood linearity in standard calibration curves of the five standardcompounds over different five dosages in this study. Based on theabove results, the CRAE was compatible with that of our previousstudy (Ye et al., 2009) and can be used in this study.

3.2. Effect of CRAE on ALT and AST activities in serum of rats withchronic liver damage induced by CCl4

As shown in Fig. 2A and B, serum ALT and AST activities in ratsinjected with 0.15 ml CCl4 twice a week for 8 weeks displayed a sig-nificant increase in comparison to the normal rats (p < 0.01). Withoral administration of CRAE in the same period of CCl4 treatment, adose-dependent decrease of serum ALT and AST activities and theactivities restored to normal level in rats treated with 600 mg/kgand 800 mg/kg was observed (p > 0.05), when compared with thenormal and CCl4 control groups (Fig. 2A and B). As the major activecompound in CRAE, berberine 120 mg/kg showed similar effect onserum ALT and AST activity as 600 mg/kg CRAE treatment (Fig. 2Aand B).

3.3. Effect of CRAE on SOD activities in serum of rats with chronicliver damage induced by CCl4

CCl4 treatment induced a response to oxidative stress, and dropof serum SOD activity was observed in rats in CCl4 control group(Fig. 2C). The activity elevated to normal level when CRAE was givenorally in the same period of CCl4 treatment and a dose-dependenteffectiveness of CRAE on the restoration of serum SOD activitywas observed (Fig. 2C). Berberine exhibited similar effect as CRAE(Fig. 2C).

3.4. Effect of CRAE on histopathological changes of chronic liverdamage in rats

Liver damage in rats was evaluated by histological methodof H&E staining, and liver sections from rats in treatment groupshowed the effect of CRAE on chronic liver injury induced by CCl4.The histological analysis of the liver from the normal rats indi-cated normal architecture (Fig. 3A). CCl4-induced demolishmenton hepatocellular architecture was evidenced by disruption of tis-sue architecture, extension of fibers, large fibrous septa formation,pseudolobe separation and collagen accumulation in liver sectionsfrom rats in CCl4 control group (Fig. 3B). These alterations wereremarkably improved in the liver sections of the rats co-treatedwith CRAE and CCl4 for 8 weeks (Fig. 3C–E), while the liver damagewas also greatly attenuated when treated with berberine (Fig. 3F).Numerical scores were computed in Table 1 to evaluate the histo-logical changes after CRAE and berberine treatment.

3.5. CRAE attenuates CCl4-induced hepatocyte apoptosis in rats

Chronic and sustained treatment of CCl4 may induce hepato-cyte apoptosis in rats. In this study, we detected the cell apoptosisby DNA fragmentation assay in isolated hepatocyte from rats withor without CCl4 administration. Significant DNA fragment could beobserved in hepatocytes isolated from CCl4 exposed rats (Fig. 4A),indicating that hepatocyte under apoptosis in rat liver underwentchronic hepatic injury. CRAE treatment could dose-dependentlyattenuate the CCl4-induced hepatocyte apoptosis, and high doseof CRAE could completely prevent the hepatocyte from cell deathand therefore protected liver from CCl4-induced chronic injury.

Consistently, the caspase-3 activity was significantly increasedin hepatocyte isolated from CCl4-treated rats, and CRAE couldreduce the CCl4-induced caspase-3 activation in hepatocyte indose-dependent manner (Fig. 4B). This result indicates that CRAE

686 Y. Feng et al. / Journal of Ethnopharmacology 138 (2011) 683– 690

Fig. 1. Coptidis rhizoma identification and phytochemical analysis of CRAE. (A) Photos of Coptidis rhizoma: left: the original plant of CR; right: the dried raw rhizoma of coptis.(B) HPLC chromatogram of standards and CRAE. (C) Standard curves of active compounds.

Y. Feng et al. / Journal of Ethnopharmacology 138 (2011) 683– 690 687

Fig. 2. Biomedical assay of serum ALT, AST and SOD activities in rats with or without treatment. (A) Serum ALT activities in rats from different groups. (B) Serum ASTa ent grw

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ctivities in rats from different groups. (C) Serum SOD activities in rats from differhen compare with CCl4 model group.

nd berberine may prevent chronic liver injury in rats by attenuat-ng hepatocyte apoptosis.

.6. Erk1/2 inhibition involves in CRAE’s protective effect onhronic liver injury

Potent activation of Erk1/2 signaling was observed in CCl4-reated hepatocyte when compared with the normal and CCl4ontrol groups. Interestingly CRAE and berberine could inhibit therk1/2 pathway. Though low dose of CRAE shows no remarkeduppression of Erk1/2 signaling, activation of Erk1/2 pathway byCl4 treatment was suppressed by medium dose (600 mg/kg) andigh dose (800 mg/kg) of CRAE, and berberine 120 mg/kg (Fig. 5).hese results indicate that inhibition of Erk1/2 signal transductionay be correlated to protective effect of CRAE’s and berberine on

Cl4-induced chronic liver injury in rats.

. Discussion

CR is a fork remedy with cold property and bitter taste in Chi-ese medicine. It is used for heat-clearing and toxic-scavengingccording to the Chinese medicine theories. In Chinese ancientharmacopeia <Ben Cao Gang Mu>, Coptidis rhizoma and its for-ulae are recorded as therapeutic agents against eye diseases,hich are also considered to be closely related with liver abnor-ity in Chinese medicine theories (Feng et al., 2009a). Recent

tudies showed that Coptidis rhizoma extract orally could protecthe kidney from damage induced by oxidative stress-medicated

poptosis and therefore ameliorated renal function impairmentCho et al., 2004). We previously reported that CRAE and its activeompound, berberine exhibited potent protective effect on CCl4-nduced acute liver damage in rats (Ye et al., 2009; Feng et al., 2010).

oups. #p < 0.05, ##p < 0.01, when compared with normal group; *p < 0.05, **p < 0.01,

However, no experimental evidence was reported to demonstratethe hepatoprotective activity and the mechanism of Coptidis rhi-zoma on chronic liver diseases. In this study, CCl4-induced chronicliver damage rats were orally administrated with different dosesof CRAE, and the serum ALT and AST enzyme activities of liverdamage rats significantly decreased after CRAE treatment, indi-cating its potential as a drug candidate for treating chronic liverdiseases.

CCl4-induced hepatotoxicity model was widely reported instudies on therapies against various hepatic diseases in thatCCl4-induced liver damage shares similar mechanism with viralhepatitis (Rubinstein, 1962), drug/chemicals-induced hepatopa-thy and oxidative stress (Recknagel et al., 1989; Kadiiska et al.,2000). The oxidative stress-induced overproduction of free radicalscause lipid peroxidation of hepatocellular membrane, leading to aseries of cascades of cellular events involving the massive release ofinflammatory mediators and cytokines (Pessayre, 1995; Dizdarogluet al., 2002; Higuchi and Gores, 2003). The continuous adminis-tration of CCl4 therefore results in extended oxidative stress andeventually chronic liver damage. Superoxide dismutase (SOD) isone of the most important antioxidant enzymes, which catalyzethe dismutation of the superoxide anion into hydrogen peroxideand molecular oxygen (McCord and Fridovich, 1969; Ukeda et al.,1997). In this study, dose-dependent increases of serum SOD activ-ities were observed in rats with chronic liver damage induced byCCl4 when rats were treated CRAE and berberine during the sameperiod, indicating the reconstruction of anti-peroxidative systemand the scavenging of free radicals produced by CCl4. It is therefore

deduced that the hepatoprotective effect of CRAE on CCl4-inducedchronic liver damage results from its anti-oxidant action. Con-sistent results are observed from histopathological studies. Thedisruption of tissue architecture, extension of fibers, large fibrous

688 Y. Feng et al. / Journal of Ethnopharmacology 138 (2011) 683– 690

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ig. 3. Histological observation on liver sections from CCl4-induced chronic liver da00 mg/kg CRAE treatment group. (D) 600 mg/kg CRAE treatment group. (E) 800 mg

epta formation, pseudolobe separation and collagen accumula-ion produced by CCl4 treatment in rats ameliorated after CRAEdministration, indicating the protection of CRAE could signifi-antly prohibit hepatocyte from peroxidative damage caused byCl4.

Oxidative stress may induce hepatocyte apoptosis both in vitrond in vivo (Singh et al., 2009; Cui et al., 2010). CCl4 is a classi-al hepatotoxicant which could induce reactive oxygen formationnd reduce anti-oxidant enzyme activity. Therefore CCl4 is ableo induce oxidative stress which is an important factor for liverhronic injury (Lin et al., 2008). Several signaling transductionsay be involved in this process, where the extracellular receptor

inase (Erk1/2) has been demonstrated to play a role (Qian et al.,010). In this study, we found that Erk1/2 signaling was activatedy CCl4 exposure in rats, consistent with the remarked apop-otic phenotype in isolated hepatocyte. This indicates that Erk1/2

ig. 4. CRAE prevents damaged hepatocyte from oxidative stress-induced apoptosis. (A)ith normal group; *p < 0.05 when compare with CCl4 model group.

rats with or without CRAE treatment. (A) Normal group. (B) CCl4 model group. (C)RAE treatment group.

activation may serve as pro-apoptotic mediator in CCl4-inducedchronic liver injury in rats. Treatment of CRAE dose-dependent sup-presses the CCl4-induced activation of Erk1/2 signaling, reducingphosphorylation of Erk1/2 expression and attenuating CCl4-induced liver apoptosis, indicating the Erk1/2 inhibition may serveas the major mechanism in CRAE’s protective action. Several stud-ies have reported that inhibition of Erk1/2 signaling pathway playsan important role in the protective effect of Chinese medicines andtheir active compounds on physical- or chemical-induced hepaticinjury, including Ophiopogonin D (Qian et al., 2010), Piper betelleaves (Young et al., 2007), Baicalein (Peng et al., 2008) and mag-nesium lithospermate B (Hur et al., 2008). Our study reveals that

CRAE may be one of the therapeutic agents on chronic liver injuryand Erk1/2 may be the potential target. Our study also indicatesthat, with normalized dose treatment of berberine, the chronic liverdamage induced by CCl4 in rats was significantly eliminated and

DNA fragmentation assay. (B) Caspase-3 activity assay. #p < 0.05 when compared

Y. Feng et al. / Journal of Ethnopharmacology 138 (2011) 683– 690 689

Tab

le

1Pr

otec

tive

and

pre

ven

tive

effe

cts

of

CR

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ach

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de-

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n

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8).

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vere

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mat

ory

cell

infi

ltra

tion

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yd

egen

erat

ion

Hae

mor

rhag

e

Hyd

rop

icd

egen

erat

ion

Fibr

osis

Mic

rove

sicu

lar

stea

tosi

sC

ombi

ned

scor

e

Nor

mal

0.2

±

0.4

0.7

±

0.1

0.9

±

0.2

0.3

±

0.4

1.0

±

0.8

0.5

±

0.3

1.0

± 0.

3 0.

5

±

0.3

CC

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2

±

0.5#

#5.

0

±

0.7#

#4.

3

±

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2

±

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#4.

5

±

0.8#

#4.

4

±

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9 ±

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2

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0.9#

#

Low

(400

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kg

BW

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8±

1.2#

,*3.

2±

0.5#

#,*

3.6

±

0.7#

#2.

8±

0.9#

,*3.

1±

0.7#

2.9

±

0.4#

#,*

2.7

± 0.

8#,

3.0

±

1.1#

#

Med

ium

(600

mg/

kg

BW

)1.

9

±

1.2#

,**

2.3

±

0.8#

,**

2.1

±

0.5#

,*1.

6

±

0.6**

1.2

±

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2.0

±

0.5**

1.9

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1.5

±

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Hig

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kg

BW

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0.9

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1.6

±

0.3**

1.2

±

0.6**

0.8

±

0.3**

0.5

±

0.2**

0.7

±

0.3**

1.2

±

0.9*

0.8

±

0.4**

Ber

beri

ne

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kg

BW

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4

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2.7

±

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,**

1.9

±

0.2#

,**

1.7

±

0.3#

,**

1.4

±

0.4**

2.5

±

0.8#

,*2.

3

±

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1.7

±

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com

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0.05

com

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com

par

ed

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Fig. 5. CRAE inhibits CCl4-induced Erk1/2 activation in dosed dependent manner.

Western blotting photos show expression of Erk1/2 in CCl4-treated hepatocyte afterCRAE treatment (upper panel is p-Erk1/2, middle panel is Erk1/2 and lower panel isbeta-actin).

action was similar with 600 mg/kg CRAE treatment. Combined withthe phytoanalysis result, the possible active component in CRAE forthe treatment of experimental chronic liver damage is berberine.

5. Conclusion

In conclusion, CRAE showed its potent effectiveness in thetreatment of chronic liver damage induced by CCl4 in rats sincesignificant decrease of serum AST and ALT activities were observedafter CRAE treatment. SOD activity restoration indicates that CRAEmight act as an anti-oxidative agent during the protection. The invitro study showed that CRAE could attenuate oxidative stress-induced hepatocyte apoptosis and Erk1/2 inhibition may play arole. Our study sheds light on the therapeutic potential of CRAEfor chronic liver diseases as a complementary therapy to protecthepatocyte from injury.

Acknowledgements

The study was financially supported by grants from the researchcouncil of the University of Hong Kong (project codes: 10400413,10400699), The Research Grants Council (RGC) of Hong Kong SAR ofChina (project code: 764708M), Hong Kong Government-MatchingGrant Scheme (4th phase, project code: 20740314). The authorsare grateful to the support of Professors Yung-Chi Cheng, Chi-MingChe, Yao Tong and Allan S.Y. Lau. The authors would like to expressthanks to Dr. Ka-Yu Siu, Ms. Cindy Lee, Mr. Keith Wong, and Mr.Freddy Tsang for their technical support.

References

Budhu, A., Jia, H.L., Forgues, M., Liu, C.G., Goldstein, D., Lam, A., Zanetti, K.A., Ye, Q.H.,Qin, L.X., Croce, C.M., Tang, Z.Y., Wang, X.W., 2008. Identification of metastasis-related MicroRNAs in hepatocellular carcinoma. Hepatology 47, 897–907.

Chang, H.M., But, Paul.P.H., 2001. Pharmacology and Applications of Chinese MateriaMedica. World Scientific Publishing, Hong Kong, pp. 1061–1077.

Cho, E.J., Yokzawa, T., Rhee, S.H., 2004. The role of Coptidis rhizoma extract in a renalischemia–reperfusion model. Phytomedicine 11, 576–584.

Choi, U.K., Kim, M.H., Lee, N.H., 2007. Optimization of antibacterial activity byGold-Thread (Coptidis rhizoma Franch) against Streptococcus mutans using evo-lutionary operation-factorial design technique. Journal of Microbiology andBiotechnology 17, 1880–1884.

Chung, H., Hong, D.P., Kim, H.J., Jang, K.S., Shin, D.M., Ahn, J.I., Lee, Y.S., Kong, G.,2005. Differential gene expression profiles in the steatosis/fibrosis model of ratliver by chronic administration of carbon tetrachloride. Toxicology and AppliedPharmacology 208, 242–254.

Cui, Y., Gong, X., Duan, Y., Li, N., Hu, R., Liu, H., Hong, M., Zhou, M., Wang, L., Wang,H., Hong, F., 2010. Hepatocyte apoptosis and its molecular mechanisms in mice

caused by titanium dioxide nanoparticles. Journal of Hazardous Materials 183,874–880.

Dizdaroglu, M., Jaruga, P., Birincioglu, M., Rodrriguez, H., 2002. Free radical-induceddamage to DNA: mechanisms and measurement. Radical Biology & Medicine 32,1102–1115.

6 pharm

D

F

F

F

F

F

G

H

H

I

K

K

L

L

L

M

90 Y. Feng et al. / Journal of Ethno

eng, Y.T., Liao, Q.F., Li, S.H., Bi, K.S., Pan, B.Y., Xie, Z.Y., 2008. Simulta-neous determination of berberine, palmatine and jatrorrhizine by liquidchromatography–tandem mass spectrometry in rat plasma and its application ina pharmacokinetic study after oral administration of coptis–evodia herb couple.Journal of Chromatography B 863, 195–205.

eng, Y., Luo, W.Q., Zhu, S.Q., 2008. Explore new clinical application of Huanglian andcorresponding compound prescriptions from their traditional use. China Journalof Chinese Materia Medica 33, 1221–1225.

eng, Y., Siu, K.Y., Wang, N., Tsao, S.W., Nagamatsu, T., Tong, Y., 2009a. Bear bile:dilemma of traditional medicinal use and animal protection. Journal of Ethno-biology and Ethnomedicine 5, 2–10.

eng, Y., Cheung, K.F., Wang, N., Liu, P., Nagamatsu, T., Tong, Y., 2009b. Chinesemedicines as a resource for liver fibrosis treatment. Chinese Medicine 4, 1–11.

eng, Y., Siu, K.Y., Ye, X., Wang, N., Yuen, M.F., Leung, C.H., Tong, Y., Kobayashi, S.,2010. Hepatoprotective effects of berberine on carbon tetrachloride-inducedacute liver hepatotoxicity in rats. Chinese Medicine 5, 1–6.

ukutake, M., Yokota, S., Kawamura, H., Iizuka, A., Amagaya, S., Fukuda, K.,Komatsu, Y., 1998. Inhibitory effect of Coptidis rhizoma and Scutellariae radixon azoxymethane-induced aberrant crypt foci formation in rat colon. Biological& Pharmaceutical Bulletin 21, 814–817.

ramantieri, L., Ferracin, M., Fornari, F., Veronese, A., Sabbioni, S., Liu, C.G., Calin,G.A., Giovannini, C., Ferrazzi, E., Grazi, G.L., Croce, C.M., Bolondi, L., Negrini, M.,2007. Cyclin G1 is a target of mir-122a, a microrna frequently down-regulatedin human hepatocellular carcinoma. Cancer Research 67, 8–15.

iguchi, H., Gores, G.J., 2003. Mechanisms of liver injury: an overview. CurrentMolecular Medicine 3, 483–490.

ur, K.Y., Seo, H.J., Kang, E.S., Kim, S.H., Song, S., Kim, E.H., Lim, S., Choi, C., Heo, J.H.,Hwang, K.C., Ahn, C.W., Cha, B.S., Jung, M., Lee, H.C., 2008. Therapeutic effectof magnesium lithospermate B on neointimal formation after balloon-inducedvascular injury. European Journal of Pharmacology 586, 226–233.

keda, H., Kume, Y., Tejima, K., Tomiya, T., Nishikawa, T., Watanabe, N., Ohtomo, N.,Arai, M., Arai, C., Omata, M., Fujiwara, K., Yatomi, Y., 2007. Rho-kinase inhibitorprevents hepatocyte damage in acute liver injury induced by carbon tetra-chloride in rats. American Journal of Physiology – Gastrointestinal and LiverPhysiology 293, G911–G917.

adiiska, M.B., Gladen, B.C., Baird, D.D., Dikalova, A.E., Sohal, R.S., Hatch, G.E., Jones,D.P., Mason, R.P., Barrett, J.C., 2000. Biomarkers of oxidative stress study: areplasma antioxidants markers of CCl4 poisoning? Free Radical Biology & Medicine28, 838–845.

im, H.Y., Shin, H.S., Park, H., Kim, Y.C., Yun, Y.G., Park, S., Shin, H.J., Kim, K., 2008.In vitro inhibition of coronavirus replications by the traditionally used medic-inal herbal extracts, Cimicifuga rhizoma, Meliae cortex, Coptidis rhizoma, andPhellodendron cortex. Journal of Clinical Virology 41, 122–128.

i, X.K, Motwani, M., Tong, W., Bornmann, W., Schwartz, G.K., 2000. Huanglian, aChinese herbal extract, inhibits cell growth by suppressing the expression ofcyclin B1 and inhibiting CDC2 kinase activity in human cancer cells. MolecularPharmacology 58, 1287–1293.

in, H.M., Tseng, H.C., Wang, C.J., Lin, J.J., Lo, C.W., Chou, F.P., 2008. Hepatoprotectiveeffects of Solanum nigrum Linn extract against CCl(4)-induced oxidative damage

in rats. Chemico-Biological Interactions 171, 283–293.

uo, Y.J., Yu, J.P., Shi, Z.H., Wang, L., 2004. Ginkgo biloba extract reverses CCl4-inducedliver fibrosis in rats. World Journal of Gastroenterology 10, 1037–1042.

cCord, J.M., Fridovich, I., 1969. Superoxide dismutase. An enzymic function forerythrocuprein (hemocuprein). Journal of Biological Chemistry 244, 6049–6055.

acology 138 (2011) 683– 690

Pessayre, D., 1995. Role of reactive metabolites in drug-induced hepatitis. Journal ofHepatology 23, 16–24.

Peng, C.Y., Pan, S.L., Huang, Y.W., Guh, J.H., Chang, Y.L., Teng, C.M., 2008. Baicaleinattenuates intimal hyperplasia after rat carotid balloon injury through arrestingcell-cycle progression and inhibiting ERK, Akt, and NF-kappaB activity in vas-cular smooth-muscle cells. Naunyn-Schmiedeberg’s Archives of Pharmacology378, 579–588.

Qian, J., Jiang, F., Wang, B., Yu, Y., Zhang, X., Yin, Z., Liu, C., 2010. OphiopogoninD prevents H2O2-induced injury in primary human umbilical vein endothelialcells. Journal of Ethnopharmacology 128, 438–445.

Rubinstein, D., 1962. Epinephrine release and liver glycogen levels after carbontetrachloride administration. American Journal of Physiology 203, 1033–1037.

Recknagel, R.O., Glende Jr., E.A., Dolak, J.A., Waller, R.L., 1989. Mechanisms of carbontetrachloride toxicity. Pharmacology & Therapeutics 43, 139–154.

Seglen, P.O., 1973. Preparation of rat liver cells, 3. Enzymatic requirements for tissuedispersion. Experimental Cell Research 82, 391–398.

Sanae, F., Komatsu, Y., Chisaki, K., Kido, T., Ishige, A., Hayashi, H., 2001. Effects ofSan’o-shashin-to and the constituent herbal medicines on theophylline-inducedincrease in arterial blood pressure of rats. Biological & Pharmaceutical Bulletin24, 1137–1141.

Seeff, L.B., Lindsay, K.L., Bacon, B.R., Kresina, T.F., Hoofnagle, J.H., 2001. Comple-mentary and alternative medicine in chronic liver disease. Hepatology 34,595–603.

Schuppan, D., Jia, J.D., Brinkhaus, B., Hahn, E.G., 1999. Herbal products for liverdiseases: a therapeutic challenge for the new millennium. Hepatology 30,1099–1104.

Singh, R., Wang, Y., Schattenberg, J.M., Xiang, Y., Czaja, M.J., 2009. Chronic oxida-tive stress sensitizes hepatocytes to death from 4-hydroxynonenal by JNK/c-Junoveractivation. American Journal of Physiology – Gastrointestinal and LiverPhysiology 297, G907–G917.

Ukeda, H., Maeda, S., Ishii, T., Sawamura, M., 1997. Spectrophotometric assay forsuperoxide dismutase based on tetrazolium salt 3′-1-(phenylamino)-carbonyl-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate reduc-tion by xanthine-xanthine oxidase. Analytical Biochemistry 251, 206–209.

Wang, T., Sun, N.L., Zhang, W.D., Li, H.L., Lua, G.C., Yuan, B.J., Jiang, H., She, J.H., Zhang,C., 2008. Protective effects of dehydrocavidine on carbon tetrachloride-inducedacute hepatotoxicity in rats. Journal of Ethnopharmacology 117, 300–308.

Wu, X.Z., Xie, G.R., Chen, D., 2007. Hypoxia and hepatocellular carcinoma: the ther-apeutic target for hepatocellular carcinoma. Journal of Gastroenterology andHepatology 22, 1178–1182.

Ye, X., Feng, Y., Tong, Y., Ng, K.M., Tsao, S., Lau, G.K., Sze, C., Zhang, Y., Tang, J., Shen, J.,Kobayashi, S., 2009. Hepatoprotective effects of Coptidis rhizoma aqueous extracton carbon tetrachloride-induced acute liver hepatotoxicity in rats. Journal ofEthnopharmacology 124, 130–136.

Yokozawa, T., Ishida, A., Cho, E.J., Nakagawa, T., 2003. The effects of Coptidis rhi-zoma extract on a hypercholesterolemic animal model. Phytomedicine 10,17–22.

Yokozawa, T., Ishida, A., Kashiwada, Y., Cho, E.J., Kim, H.Y., Ikeshiro, Y., 2004. Coptidisrhizoma: protective effects against peroxynitrite-induced oxidative damage and

elucidation of its active components. Journal of Pharmacy and Pharmacology 56,547–556.

Young, S.C., Wang, C.J., Lin, J.J., Peng, P.L., Hsu, J.L., Chou, F.P., 2007. Protection effectof piper betel leaf extract against carbon tetrachloride-induced liver fibrosis inrats. Archives of Toxicology 81, 45–55.