Pc

MKa

b

c

d

e

f

a

A

R

R

1

A

A

K

C

E

D

L

H

P

1

T(rcbbi

T

1h

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 517–523

Available online at www.sciencedirect.com

jo u r n al hom ep age: www.elsev ier .com/ locate /e tap

rotective effect of a marine polyphenol, dieckol againstarbon tetrachloride-induced acute liver damage in mouse

in-Cheol Kanga, Sung-Myung Kanga, Ginnae Ahnb, Kil-Nam Kimc, Nalae Kanga,alpa W. Samarakoona, Myung-Cheol Ohd, Jung-Suck Leee, You-Jin Jeona,f,∗

Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of KoreaComparative Animal Medicine, Division of Animal Life Science, Institute of Agriculture, Tokyo Univ. of Agri. & Technol., JapanJeju Center, Korea Basic Science Institute (KBSI), Jeju, Republic of KoreaDepartment of Food Science & Food Service Industry, Jeju International University, Jeju 690-714, Republic of KoreaIndustry-Academy Cooperation Foundation, Jeju National University, Jeju 690-756, Republic of KoreaAqua Green Technology Co. Ltd., 209 Jeju Bio-Industry Center, 102 Jejudaehakno, Jeju 690-121, Republic of Korea

r t i c l e i n f o

rticle history:

eceived 17 September 2012

eceived in revised form

4 February 2013

ccepted 15 February 2013

vailable online 24 February 2013

eywords:

arbon tetrachloride (CCl4)

a b s t r a c t

In this study, the hepatoprotective effect of dieckol on carbon tetrachloride (CCl4) induced

hepatic damages in ICR mice liver was investigated. Mice were randomly divided into 4

groups such as saline treated (negative control), CCl4 treated (positive control), CCl4 + dieckol

(5 mg/kg mouse) and CCl4 + dieckol (25 mg/kg mouse), respectively. The body weights and

survival rates of mice, followed by dieckol treatments were significantly increased compared

to the positive control. The level of GOT, GPT and MDA in the serum of the dieckol treated

groups were reduced dose dependently than the control, significantly. The antioxidant

enzymes including CAT, and GSH-px levels were increased significantly compared to the

positive control. However, no significant differences were observed on hepatic histophatho-

cklonia cava

ieckol

iver

epato-protetive effect

hlorotannins

logical analysis in dieckol treated groups dose dependently. Down-regulation of Bax and

up-regulation of Bcl-xl protein expressions were observed in liver tissues of the dieckol

administered groups. These results suggested that, dieckol can be developed as a therapeutic

agent for liver disease by oxidative stress.

© 2013 Elsevier B.V. All rights reserved.

apoptosis and is involved in harmful effects such as cirrho-

. Introduction

he liver is an organ which plays an important role in the bodyWang et al., 2008), which is functioning extensively as theegulation of blood sugar levels, protein synthesis and detoxifi-ation (Bhardwaj et al., 2011). The liver diseases can be caused

y toxic substances such as abuse of alcohol, drug and car-on tetrachloride (CCl4). The CCl4 has been widely used in the

nduction of acute liver damage in experiment of mice model

∗ Corresponding author at: Department of Marine Life Science, Jeju Natel.:+82 64 754 3475; fax: +82 64 756 3493.

E-mail address: youjinj@jejunu.ac.kr (Y.-J. Jeon).382-6689/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.etap.2013.02.013

(Olorunnisola et al., 2011; Toyin et al., 2008). According to theprevious studies CCl4 assumed to be a typical poison caus-ing oxidative stress (Dolai et al., 2012; Basu, 2003). Acute liverdiseases are associated with the causing of CCl4 and character-ized by increasing apoptosis and oxidative stress in the liver.Importantly, the oxidative stress caused by CCl4 which induces

ional University, Jeju 690-756, Republic of Korea.

sis and fibrosis in the liver (Palmieri and Sblendorio, 2007).It is known that many human diseases are associated withfree radicals and natural antioxidants could be used as free

d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 517–523

518 e n v i r o n m e n t a l t o x i c o l o g y a n

radical scavengers (Hamid et al., 2010). Recently both in vitroand in vivo studies had been examined for the new antiox-idants and the effect of hepato-protective substances fromnatural resources. Previous reports have showed that sub-stances like, resveratrol have both the hepato-protectiveand antioxidant effect (Chumbhale and Upasani, 2012;Olorunnisola et al., 2011; Rivera et al., 2008).

Seaweeds contain biologically active substances includ-ing minerals, polyphenols, polysaccharides and amino acids(Lordan et al., 2011; Yang et al., 2011; Kang et al., 2012). Espe-cially, brown algae are known to have various kind of bioactivecompounds including pigments, steroids, phycocolloids andphlorotannins (Shilpi and Nissreen, 2011). In particular, thephlorotannins isolated from brown algae have been exhib-ited a variety of biological activities such as antioxidant,anti-cancer, anti-inflammation and protective effect againstoxidative stress (Li et al., 2011). Among them, dieckol (DK)isolated from brown seaweed Ecklonia cava, exhibits variousbiological activities such as antioxidant (Ahn et al., 2007), ACEinhibitory activity (Wijesinghe et al., 2011), anti-inflammation(Jung et al., 2009) and protective effect against oxidative stress(Lee et al., 2010). Especially, dieckol isolated from E. cavashowed excellent properties of antioxidant activity. Therefore,the objective of the present study is to evaluate the in vivohepato-protective effect of dieckol isolated from brown sea-weed E. cava against CCl4-induced liver damage.

2. Materials and methods

2.1. Materials

Male ICR mice (6 weeks of age; purchased from Joong Ang LabAnimal Co., Seoul, Korea) were used. The thiobarbituric acid-reactive substances (TBARS), superoxide dismutase (SOD),catalase (CAT), reduced glutathione peroxidase (GSH-px) wasdetermined in the liver using a commercial available kit fromSigma Chemical Co. (St. Louis, MO, USA).

All chemicals and reagents used were of analyticaland obtained from commercial sources. Antibodies againstphosphor-Bax, phosphor-Bcl-xl and �-actin were purchasedfrom Cell signaling Technology (Bedford, MA, USA).

2.2. Isolation of dieckol from Ecklonia cava

The powdered E. cava was extracted with 80% aqueous EtOH,and was evaporated under vacuum. The EtOH extract wasthen partitioned with EtOAc. The EtOAc extract was frac-tionated via silica column chromatography with the stepwiseevolution of CHCl3–MeOH mixture (100:1–1:1) to generate andseparate the active fractions. The combined active fractionwas then further subjected to a Sephadex LH-20 column(GE Healthcare, USA) saturated with 80% MeOH, and finallypurified via reverse-phase HPLC (ThermoFisher Scientific,USA) using a Waters HPLC system equipped with a Waters

996 photodiode array detector and C18 column (J’sphereODS-H80, 150 × 20 mm, 4 �m, YMC Co.) by stepwise elutionwith methanol–water gradient (UV range: 230 nm, flow rate:0.8 ml/min). The purified compound, dieckol was confirmed by

Fig. 1 – Chemical structure of dieckol isolated from E. cava.

comparing their LC/MS, 1H NMR data to the literature report(Li et al., 2009)

Dieckol: LC/MS data (M+, m/z: 742.0 Calcd. For C36H22O18).1H NMR (400 MHz, DMSO-d6) � 9.71(1H, s, OH-9), 9.61 (1H, s,OH-9′′), 9.51 (1H, s, OH-4′′), 9.46 (1H, s, OH-4), 9.36 (2H, s, OH-3′′, 5′′), 9.28 (1H, s, OH-2′′), 9.23 (1H, s, OH-2), 9.22 (1H, s, OH-7′′),9.15 (2H, s, OH-3′, 5′) 6.17 (1H, s, H-3′′), 6.14 (1H, s, H-3), 6.02 (1H,d, J = 2.7 Hz, H-8), 5.98 (1H, d, J = 2.7 Hz, H-8′′), 5.95 (1H, s, H-2′′′,6′′′), 5.82 (1H, d, J = 2.7 Hz, H-6), 5.81 (1H, d, J = 2.7 Hz, H-6′′), 5.80(1H, t, J = 2.0 Hz, H-4′), 5.78 (2H, d, J = 2.0 Hz, H-2′, 6′).

The purity of dieckol (Fig. 1) was >95%, based on the peakarea of all components absorbed at each specific wavelengthin HPLC analysis. Dieckol was dissolved in DMSO and was usedfor experiments adjusting the final concentration of DMSO inthe culture medium to <0.01%.

2.3. Animals

Male ICR mice, weighing 25–30 g, were acclimated to tem-perature (22 ◦C) and humidity (55%) controlled rooms witha 12-h light/dark cycle for 1 week prior to use. Male ICRmice of Six-week-old were randomly divided into 4 groups.A negative control is described as saline oral administratedmice (saline, 200 �l), for 6 days daily during the experimen-tal period. A positive control group is described as saline oraladministrated mice (saline, 200 �l) for 6 days, followed by CCl4oral administrated (0.5 mg/kg, mouse) on 6th day. In addi-tion, dieckol (5 mg/kg, mouse) oral administration for 6 daysfollowed by CCl4 oral administration (0.5 mg/kg, mouse) on6th day and dieckol (25 mg/kg, mouse) oral administrationfollowed by CCl4 oral administration (0.5 mg/kg, mouse) on6th day mouse groups were described as experimented twogroups, respectively. Each group consisted of 5 mice. Duringthe experimental period, the survival rates were investigateddaily. All the animals during 6 days pre-treated by dieckoland CCl4 oral administrated after 24 h (7th day) were killed.The body weights and survival rates were investigated daily.After 7 days, the mice were anesthetized and blood sam-

ples were collected to determine biochemical parameters. Theliver (from three animals of each group) immediately fixedin 10% formalin, and then stained with hematoxylin and

p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 517–523 519

egsgttae

2

Esiai1asafid1nsb

ss1

w((ttm

us

2

Llcs(

2

TTStt(due

Su

rviv

al r

ate

s (%

)

0

20

40

60

80

100

0 1 2 3 4 5 6 7

Saline-treated mou se

CCl4-treated mouse

CCl4 + DK 5mg/kg-treated mou se

CCl4 + DK 25mg/kg-treated mou se

Saline-trea ted mou se

CCl4-treat ed mo use

CCl4-treat ed mo use + DK 5mg/kg-t reat ed mo use

CCl4-treat ed mo use + DK 25 mg/kg-trea ted mou se

Days

e n v i r o n m e n t a l t o x i c o l o g y a n d

osin (H&E). The remaining livers were frozen in liquid nitro-en and stored at −70 ◦C for biochemical assays. The bloodamples of mouse were collected to determine the serumlutamic oxaloacetic transaminase (GOT), glutamic pyruvicransaminase (GPT) concentration and total cholesterol con-ent respectively, and livers were excised from the animals tonalyze malondialdehyde (MDA) formation and antioxidantnzyme activity.

.4. Biochemical assays

arly acute hepatic damages were determined by detectingerum GOT and GPT activities. The activities of GOT and GPTn the plasma samples were determined using an enzymaticnalysis kit (Asan Pharmaceuticals, Hwasung, Korea), accord-ng to the Reitman-Frankel method (Reitman and Frankel,957). The livers were weighed, minced into small pieces,nd rinsed twice with ice-cold homogenization buffer [250 mMucrose, 50 mM Tris–HCl, pH 7.4, 1 mM ethylenediaminetetra-cetic acid (EDTA)] at 4 ◦C. The livers were homogenized inve volumes of ice-cold homogenization buffer with a motor-riven Teflon pestle. The homogenates were centrifuged at000 × g for 10 min. The pellets were discarded, and the super-atants were centrifuged at 12,000 × g for 30 min. The finalupernatant protein contents were determined using theicinchoninic acid protein assay reagents.

Lipid peroxidation productions were estimated by mea-uring the concentration of thiobarbituric acid-reactiveubstances (TBARS) in fluorescence at 530 nm (Fraga et al.,988; Olorunnisola et al., 2011).

Activities of superoxide dismutase (SOD) and catalase (CAT)ere measured according to the previously described method

Kakkar et al., 1984). The liver homogenates were diluted 1:20g ml−1) in 5% metaphosphoric acid. The homogenates werehen centrifuged at 2500 × g and 4 ◦C, for 10 min. Reduced glu-athione peroxidase (GSH-px) is expressed as micro-molar per

illigram of protein (�M mg protein−1) (Bakan et al., 2003).The total cholesterol content in the serum was determined

sing a commercial available kit (Asan Pharmaceuticals, Hwa-ung, Korea) according to the method of Allain et al. (1974).

.5. Hematoxylin and eosin staining

iver tissues were fixed in 10% (v/v) phosphate buffer forma-in and embedded in paraffin wax. Sections (3 �m thick) wereut, and each section was stained with hematoxylin and eosintain. All the sections were examined by light microscopyOlympus DP70, Olympus Optical Co., Japan).

.6. Protein extraction and Western blot analysis

he liver lysates were prepared by using lysis buffer (50 mmol/lris–HCl (pH 7.4), 150 mmol/l NaCl, 1% Triton X-100, 0.1%DS and 1 mmol/l EDTA). The protein concentrations ofhe cell lysates were determined by using a BCATM pro-ein assay kit (Thermo Scientific, Rockford, USA). The protein

30 �g/well) preparations were loaded into each lane of sodiumodecyl sulfate-polyacrylamide gels and electrophoresednder denaturing conditions. Subsequently, the proteins werelectro-transferred onto nitrocellulose transfer membranes

Fig. 2 – Survival rates of experimental mice.

(Schleicher and Schuell, Keene, NH, USA). After blockingwith 5% bovine serum albumin (BSA) for 1 h, the blots wereincubated with phosphorylation of Bax and Bcl-xl (1:1000dilution, all from Cell Signaling Technology, Beverly, MA,USA) protein antibody for 60 min followed by incubation withhorseradish peroxidase-conjugated anti-mouse or anti-rabbitIgG (1:2000, Santa Cruz Biotechnology, Inc.) as secondary anti-body for 45 min. Visualization was achieved by using ECLreagents (Amersham Life Science, Buckinghamshire, UK). Sig-nal intensities were determined by densitometric analysisusing Odyssey IR imaging system software (Scion Image soft-ware, version beta 4.0.3; Frederick, MD, USA).

2.7. Statistical analysis

Data are analyzed using the statistical package for the socialscience (SPSS) package for Windows (Version 8). Values wereexpressed as means ± standard error (SE). A p-value of lessthan 0.05 was considered significant.

3. Results

3.1. Changes in the survival rates of experimentalmice

Fig. 2 shows the survival rates of all mice were adminis-trated saline (negative control), CCl4 (positive controle), andtwo groups with CCl4 + dieckol (5 and 25 mg/kg, mouse) dur-ing 7 days. At after the day 7, the survival rate was higher andshowed 100% survivability in the saline-treated group than inCCl4-treated group. As shown in Fig. 2, when the mice weretreated with CCl4 as the positive group, the survival rate was60%. In case of administrated CCl4 + dieckol (5 and 25 mg/kg,mouse) treated groups, the survival rates were 60% and 80%,respectively. This results indicated that a high concentrationof dieckol (25 mg/kg) improved the survival rate by 20% com-pared the positive control and might have a protective effectagainst CCl4 caused toxicity.

3.2. Effect of dieckol on GOT and GPT levels in

CCl4-treated mice

The effect of dieckol on GOT and GPT levels in serum wereshown in Table 1. The level of GOT and GPT in serum was the

520 e n v i r o n m e n t a l t o x i c o l o g y a n d p h

Table 1 – Effect of the dieckol on serum glutamicoxaloacetic transaminase (GOT), glutamic pyruvictransaminase (GPT) levels in CCl4 treated mice.Experiments were performed in triplicate and the dataare expressed as mean ± SE (*P = 0.01).

Groups GOT (Karmen/ml) GPT (Karmen/ml)

Saline-treatedmouse

194.8 ± 8.97 217.49 ± 16.08

CCl4-treated mouse 255.33 ± 11.11* 285.75 ± 10.87*CCl4 + DK

5 mg/kg-treatedmouse

237.06 ± 18.60* 296.57 ± 10.20*

CCl4 + DK25 mg/kg-treatedmouse

203.41 ± 20.60 236.86 ± 13.74

end product and common bio-marker of liver damage. The lev-els of GOT and GPT in the serum of saline administrated micewere low as reported as 194.8 and 217.5 Karmen/ml, respec-tively. However, after administrated the CCl4 in mice groupwere reported (GOT and GPT) as 255.33 and 285.75 Karmen/ml,respectively. Pretreatment of dieckol (25 mg/kg mouse) wasattenuated the levels of GOT and GPT significantly comparedto the positive control group. However, a low concentration ofdieckol (5 mg/kg mice) administration was not showed a sig-nificantly difference in the decreasing of GPT level, althoughGOT level was decreased. Therefore, a high concentration ofdieckol (25 mg/kg mice) can protect the liver by lowering thelevels of GOT and GPT in serum induced toxicity by CCl4.

3.3. Antioxidant enzyme activities and MDA levels ofliver

The effects of dieckol on antioxidant enzymes including SOD,CAT, and GSH-px in the liver of CCl4-exposed mice is presentedin Table 2. The antioxidant enzymes including SOD, CAT, andGSH-px levels in the CCl4-treated group were decreased sig-nificantly than that of the saline-treated group. With thetreatment of different concentrations of dieckol followedby CCl4-treated groups were increased the activities of CATand GSH-px, significantly compared to the only-CCl4-treatedgroup. However, pretreatments of dieckol did not affect to theactivity of SOD which was decreased by CCl4 treatment. More-over, as indicated in Table 2, CCl4 treatment was increasedthe level of MDA which is an indicator of lipid peroxidationin oxidative damaged of liver tissues, compared to the saline-treated group. Interestingly, the pretreatments with different

concentrations of dieckol (5 and 25 mg/kg, mouse) followedby CCl4 treatment, shown to be decreased the levels of MDAsignificantly compared to positive control.

Table 2 – Activities of catalase, SOD, GSH-px and MDA levels indata are expressed as mean ± SE (*P = 0.01).

Groups SOD (%) CAT (�mol/mgprotein/min)

Saline-treated mouse 52.71 ± 2.13 27.47 ± 0.52

CCl4-treated mouse 46.22 ± 0.50* 25.37 ± 0.488*

CCl4 + DK 5 mg/kg mouse 47.33 ± 0.10* 28.94 ± 0.77*

CCl4 + DK 25 mg/kg mouse 47.61 ± 0.47 27.42 ± 0.20

a r m a c o l o g y 3 5 ( 2 0 1 3 ) 517–523

3.4. Histopathological studies

H&E staining is most widely used stain in medical diagnosis.The morphological changes in the extracted liver tissues wereshown in Fig. 3. As shown in Fig. 3, CCl4 treatment causedthe marked liver damages, compared to the saline-treatedmice. However, when the mice were given by the dieckol atthe two concentrations, these CCl4-induced hepatic patholog-ical changes were reduced and recovered completely when thetreatment of dieckol at 25 mg/kg, mouse.

3.5. Western blot analysis of Bax and Bcl-xL proteinlevels in CCl4-treated mice

Western blot analysis was performed to understand themechanism of action of dieckol against CCl4-induced hepatictoxicity, and determined the apoptosis signaling pathwaysthrough the protein expressions such as Bax and Bcl-xL (Fig. 4).According the results, the pretreatments of dieckol was down-regulated the expression levels of Bax at the concentrationsof 5 and 25 mg/kg mouse compared to the CCl4-treated mice,dose-dependently. In addition, the expression levels of Bcl-xLwere markedly up-regulated at all the concentrations, com-pared to the CCl4-treated cells. Especially, a high concentrationof dieckol (25 mg/kg mouse) profoundly decreased the Baxexpression, and increased the Bcl-xL as the anti-apoptotic pro-tein expression in comparison with the CCl4-treated mice.These data indicated that dieckol can be inhibited the CCl4treatment caused apoptosis by modulating apoptosis-relatedmolecules.

4. Discussion

Acute liver disease caused by CCl4 treatment is a potentinducer of oxygen free radicals which plays an importantrole in the modulation of apoptosis and necrosis in livercells. Previous studies have reported that acute CCl4-inducedliver injury has been metabolized by the antioxidant enzymedefense system in the liver (Domitrovic et al., 2009; Dolai et al.,2012). Thus, hepato-protecitve effects are considered to per-form a crucial role in the reduction of oxidative stress in liver.The liver of mice treated with CCl4 exhibited the distinct char-acteristics of acute liver disease such as increased level of GOT,GPT, lipid peroxidation, apoptosis and decreasing of antioxi-dant enzymes in the liver.

Previous studies have reported that the isolation ofphlorotannins from E. cava could be utilized as promisingagents for antioxidant and cytoprotective capacities (Kanget al., 2005; Ahn et al., 2007). Among them, dieckol isolated

liver. Experiments were performed in triplicate and the

GSH-px (�mol/mg protein) Malondialdehyde(mmol/g tissue)

0.94 ± 0.06 0.74 ± 0.520.78 ± 0.06* 3.95 ± 0.21*0.91 ± 0.03 1.29 ± 0.780.92 ± 0.01 1.11 ± 1.04

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 517–523 521

Fig. 3 – Hepatic histopathologic changes of central vein and portal area of liver in CCl4 treated mice. The liver sections weres

faGpeeiudctslC

dliCai

tained with hematoxylin and eosin (H&E stain, 200×).

rom E. cava has interested and indicated the protective effectgainst oxidative stress (Ko et al., 2011). Therefore, the level ofOT, GPT, antioxidant enzymes, lipid peroxidation and apo-tosis regulatory pathways, as well as the hepato-protectiveffect on histological changes of mice livers and serums werevaluated with the treatment of dieckol for the CCl4 toxicity-nduced ICR mice. Hepatic GOT and GPT are the most widelysed biomarkers for screening of hepato-protective effect onrugs and natural products. Many reports have shown CCl4ould increase the levels of GOT and GPT, which can provokehe acute liver disease (Sun et al., 2007; Basu, 2003). This isuggested as the phagocytic activity as the function of reticu-oendothelial system (RES) suppression more strongly due toCl4 toxicity (Okazaki et al., 1985).

In our study, the mice treated with CCl4 induced liveramages were attenuated by the pre-treatment of dieckol, as

owering the GOT and GPT levels significantly. This results

ndicated that dieckol could prevent liver damages induced byCl4 administration. Acute liver damage caused by CCl4 plays

vital role in oxidative stress processes and have been exam-ned the oxidative stress parameters including SOD, GSH-Px,

CAT and MDA, recently (Olorunnisola et al., 2011). In addition,previous very recent in vivo studies have reported that theliver damage caused by CCl4 and decreased the expressionsof antioxidant enzymes (SOD, GSH-Px and CAT) (Chumbhaleand Upasani, 2012; Khan et al., 2012). These results suggestedthat dieckol can increase the antioxidant enzymes whichdecreased the effect of toxicity by CCl4 administration. More-over, previous studies have shown that lipid peroxidation isclosely related to oxidative stress (Yoshida et al., 2005; Selmanet al., 2002; Zarghami and Khosrowbeygi, 2004). Thus, Ellah(2011) have reported that oxidative stress causes to damageliver through lipid peroxidation. In this study, the level of MDAwas increased due to the lipid peroxidation in liver of mice,induced toxicity by CCl4 in the positive control group. However,the level of MDA in the dieckol treated mice was signifi-cantly reduced dose dependently. These results indicate thatthe pre-treatment of dieckol positively improved the levels of

antioxidant enzyme and markedly reduced the lipid peroxida-tion in liver. A histopathological change in liver was visualizedusing of hematoxylin and eosin staining. The hematoxylinand eosin staining is widely using an application to view the

522 e n v i r o n m e n t a l t o x i c o l o g y a n d p h

Fig. 4 – Dieckol treatment results in modulation of anti- andpro-apoptotic proteins in liver of mice analyzed by westernblot. The liver in mouse lysates were analyzed via westernblotting and signal intensities were determined bydensitometric analysis using Odyssey IR imaging systemsoftware (Scion Image software, version beta 4.0.3;Frederick, MD).

r

carcinoma. World J. Gastroenterol. 8 (6), 1059–1062.

diagnosis of abnormal tissues such as those found in hepatictissue (Fig. 3). In this study, the hepatic damages due to theCCl4 administration-induced acute toxicity were apparentlyappeared and attenuation effect of the damages was observedin the dieckol treated mice groups. Many previous studieshave demonstrated that regulatory effect of protein expres-sions such as Bcl-xL and Bax are being inducing the apoptosisin liver tissue (Tien et al., 2011; Guicciardi and Gores, 2005;Guo et al., 2002). Mitochondria are known to be a susceptibletarget due to various toxins and oxidative stress conditions.The mitochondrial apoptotic pathway is regulated by variousproteins such as Bcl-2 family proteins, which consist of bothanti-apoptotic and pro-apoptotic proteins (Lee et al., 2006). Ithas been shown that Bax can translocate from the cytosol tomitochondria and exhibit conformational change under theapoptotic process (Ding and Nam, 2003). Our results also indi-cating that the dieckol used to inhibit the apoptosis causedby CCl4 treatment as modulating along the apoptosis-relatedmolecules.

In conclusion, dieckol isolated form E. cava can act as aneffective hapetoprotective agent via apoptosis pathway by up-regulating the expression of Bcl-xL and down-regulation of

a r m a c o l o g y 3 5 ( 2 0 1 3 ) 517–523

Bax in the liver. Thus, dieckol could be used as an effectiveagent for preventing CCl4 related liver diseases.

Conflict of interest statement

The authors declare that there are no conflict of interest.

Acknowledgements

This research was supported by a grant from Marine Biopro-cess Research Center of the Marine Biotechnology Programfunded by the Ministry of Land, Transport and Maritime,Republic of Korea.

e f e r e n c e s

Ahn, G.N., Kim, K.N., Cha, S.H., Song, C.B., Lee, J., Heo, M.S., Yeo,I.K., Lee, N.H., Jee, Y.H., Kim, J.S., Heu, M.S., Jeon, Y.J., 2007.Antioxidant activities of phlorotannins purified from Eckloniacava on free radical scavenging using ESR and H2O2-mediatedDNA damage. Eur. Food Res. Technol. 226, 71–79.

Allain, C.C., Poon, L.S., Chan, C.S.G., Richmond, W., Fu, P.C., 1974.Enzymatic determination of total serum cholesterol. Clin.Chem. 20 (4), 470–475.

Bakan, N., Taysi, S., Yilmaz, O., Bakan, E., Kuskay, S., Uzun, N.,Gundogdu, M., 2003. Glutathione peroxidase glutathionereductase, Cu–Zn superoxide dismutase activities,glutathione, nitric oxide, and malondialdehydeconcentrations in serum of patients with chronic lymphocyticleukemia. Clin. Chim. Acta 338, 143–149.

Basu, S., 2003. Carbon tetrachloride-induced lipid peroxidation:eicosanoid formation and their regulation by antioxidantnutrients. Toxicology 189, 113–127.

Bhardwaj, A., Khatri, P., Soni, M.L., Ali, D.J., 2011. Potent herbalhepatoprotective drugs – a review. J. Adv. Sci. Res. 2 (2), 15–20.

Chumbhale, D.S., Upasani, C.D., 2012. Hepatoprotective andantioxidant activity of Thespesia lampas (Cav.) Dalz & Gibs.Phytomedicine 2 (1), 114–122.

Ding, W.X., Nam, O.C., 2003. Role of oxidative stress andmitochondrial changes in cyanobacterial-induced apoptosisand hepatotoxicity. FEMS Microbiol. Lett. 220, 1–7.

Dolai, N., Karmakar, I., Kumar, R.B.S., Kar, B., Bala, A., Haldar, P.K.,2012. Free radical scavenging activity of Castanopsis indica inmediating hepatoprotective activity of carbon tetrachlorideintoxicated rats. Asian Pac. J. Trop. Biomed. 12, S242–S251.

Domitrovic, R., Jakovac, H., Tomac, J., Sain, I., 2009. Liver fibrosisin mice induced by carbon tetrachloride and its reversion byluteolin. Toxicol. Appl. Pharmacol. 241, 311–321.

Ellah, M.R.A., 2011. The Role of Liver Biopsy in Detection ofHepatic Oxidative Stress. Vet. Med. Int., 1–7.

Fraga, C.G., Leibovitz, B.E., Tappel, A.L., 1988. Lipid peroxidationmeasured as thiobarbituric acid-reactive substances in tissueslices: characterization and comparison with homogenatesand microsomes. Free Radic. Biol. Med. 4 (3), 155–161.

Guicciardi, M.E., Gores, G.J., 2005. Apoptosis. A mechanism ofacute and chronic liver injury. Gut 54, 1024–1033.

Guo, X.Z., Shao, X.D., Liu, M.P., Xu, J.H., Ren, L.N., Zhao, J.J., Li, H.Y.,Wang, D., 2002. Effect of bax, bcl-2 and bcl-xl on regulatingapoptosis in tissues of normal liver and hepatocellular

Hamid, A.A., Aiyelaagbe, O.O., Usman, L.A., Ameen, O.M., Lawal,A., 2010. Antioxidants: its medicinal and pharmacologicalapplications. Afr. J. Pure Appl. Chem. 4 (8), 142–151.

p h a r

J

K

K

K

K

K

L

L

L

L

L

O

O

e n v i r o n m e n t a l t o x i c o l o g y a n d

ung, W.K., Heo, S.J., Jeon, Y.J., Lee, C.M., Park, Y.M., Byun, H.G.,Choi, Y.H., Park, S.G., Choi, I.W., 2009. Inhibitory effects andmolecular mechanism of dieckol isolated from marine brownalga on COX-2 and iNOS in microglial cells. J. Agric. FoodChem. 57 (10), 4439–4446.

akkar, P., Das, B., Viswanathan, P., 1984. A modified method forassay of superoxide dismutase. Ind. J. Biochem. Biophys. 21,131–132.

ang, K.A., Lee, K.H., Chae, S., Zhang, R., Jung, M.S., Lee, Y., Kim,H.S., Joo, H.G., Park, J.W., Ham, Y.M., Lee, N.H., Hyun, J.W., 2005.Eckol isolated from Ecklonia cava attenuates oxidative stressinduced cell damage in lung fibroblast cells. FEBS Lett. 579(28), 6295–6304.

ang, M.C., Kim, E.A., Kang, S.M., Wijesinghe, W.A.J.P., Yang, X.,Kang, N., Jeon, Y.J., 2012. Thermostability of a marinepolyphenolic antioxidant dieckol, derived from the brownseaweed Ecklonia cava. Algae 27 (3), 1–9.

han, M.R., Marium, A., Shabbir, M., Saeed, N., Bokhari, J., 2012.Antioxidant and hepatoprotective effects of Oxaliscorniculata against carbon tetrachloride (CCl4) inducedinjuries in rat. Afr. J. Pharm. Pharmacol. 6 (30), 2255–2267.

o, S.C., Cha, S.H., Heo, S.J., Lee, S.H., Kang, S.M., Jeon, Y.J., 2011.Protective effect of Ecklonia cava on UVB-induced oxidativestress: in vitro and in vivo zebrafish model. J. Appl. Phycol. 23,697–708.

ee, S.H., Han, J.S., Heo, S.J., Hwang, J.Y., Jeon, Y.J., 2010. Protectiveeffects of dieckol isolated from Ecklonia cava against highglucose-induced oxidative stress in human umbilical veinendothelial cells. Toxicol. Vitro 24 (2), 375–381.

ee, T.Y., Chang, H.H., Wang, G.J., Chiu, J.H., Yang, Y.Y., Lin, H.C.,2006. Water-soluble extract of Salbia miltiorrhiza amelioratescarbon tetrachloride-mediated hepatic apoptosis in rats. J.Pharm. Pharmacol. 58, 659–665.

i, Y., Qian, Z.J., Ryu, B.M., Lee, S.H., Kim, M.M., Kim, S.K., 2009.Chemical components and its antioxidant properties in vitro:an edible marine brown alga, Ecklonia cava. Bioorg. Med.Chem. 17, 1963–1973.

i, Y.X., Wijesekara, I., Li, Y., Kim, S.K., 2011. Phlorotannins asbioactive agents from brown algae. Process Biochem. Int. 46,2219–2224.

ordan, S., Paul Ross, R., Stanton, C., 2011. Marine bioactives asfunctional food ingredients: potential to reduce the incidenceof chronic diseases. Mar. Drugs 9, 1056–1100.

kazaki, M., Furuya, E., Kasahara, T., Sakamoto, K., 1985.Function of reticuloendothelial system on CCl4 induced liverinjury in mice. Jpn. J. Pharmacol. 39, 503–514.

lorunnisola, O.S., Akintola, A.O., Afolayan, A.J., 2011.

Hepatoprotective and antioxidant effect of Sphenocentrumjollyanum (Menispermaceae) stem bark extract againstCCl4-induced oxidative stress in rats. Afr. J. Pharm.Pharmacol. 5 (9), 1241–1246.

m a c o l o g y 3 5 ( 2 0 1 3 ) 517–523 523

Palmieri, B., Sblendorio, V., 2007. Oxidative stress tests: overviewon reliability and use Part 1. Riv. Eur. Sci. Med. Farmacol. 11,309–342.

Reitman, S., Frankel, S., 1957. A colorimetric method for thedetermination of serum glutamic oxaloacetate andglutamic pyruvic transaminases. Int. J. Sci. Technol. 28,56–62.

Rivera, H., Shibayama, M., Tsutsumi, V., Perez-Alvarez, V., Muriel,P., 2008. Resveratrol and trimethylated resveratrol protectfrom acute liver damage induced by CCl4 in the rat. J. Appl.Toxicol. 28, 147–155.

Selman, C., Mclaren, J.S., Collins, A.R., Duthie, G.G., Speakman,J.R., 2002. Antioxidant enzyme activities lipid peroxidation,and DNA oxidative damage: the effects of short-termvoluntary wheel running. Arch. Biochem. Biophys. 401,255–261.

Shilpi, G., Nissreen, A.G., 2011. Bioactive potential and possiblehealth effects of edible brown seaweed. Trends Food Sci.Technol. 22, 315–326.

Sun, W.Y., Wei, W., Wu, L., Gui, S.Y., Wang, H., 2007. Effects andmechanisms of extract from Paeonia lactiflora and Astragalusmembranaceus on liver fibrosis induced by carbon tetrachloridein rats. J. Ethnopharmacol. 112, 514–523.

Tien, Y.C., Liao, J.C., Chiu, C.S., Huang, T.H., Huang, C.Y., Chang,W.T., Peng, W.H., 2011. Esculetin ameliorates carbontetrachloride-mediated hepatic apoptosis in rats. Int. J. Mol.Sci. 12, 4053–4067.

Toyin, Y., Suru, F.S.M., Fafunso, M.A., Obioha, U.E., 2008.Hepatoprotective potentials of Phyllanthus amarus againstethanol-induced oxidative stress in rats. Food Chem. Toxicol.46, 2658–2664.

Wang, T., Sun, N.L., Zhang, W.D., Li, H.L., Lu, G.C., Yuan, B.J., Jiang,H., She, J.H., Zhang, C., 2008. Protective effects ofdehydrocavidine on carbon tetrachloride-induced acutehepatotoxicity in rats. J. Ethnopharmacol. 117, 300–308.

Wijesinghe, W.A.J.P., Ko, S.C., Jeon, Y.J., 2011. Effect ofphlorotannins isolated from Ecklonia cava on angiotensinI-converting enzyme (ACE) inhibitory activity. Nutr. Res. Pract.5 (2), 93–100.

Yang, X., Kang, M.C., Lee, K.W., Kang, S.M., Lee, W.W., Jeon, Y.J.,2011. Antioxidant activity and cell protective effect of loliolideisolated from Sargassum ringgoldianum subsp. Coreanum. Algae26 (2), 201–208.

Yoshida, Y., Itoh, N., Hayakawa, M., Piga, R., Cynshi, O., Jishage,K., Niki, E., 2005. Lipid peroxidation induced by carbontetrachloride and its inhibition by antioxidant as evaluated byan oxidative stress maker, Hode. Toxicol. Appl. Pharmacol.

208, 87–97.

Zarghami, N., Khosrowbeygi, A., 2004. Evaluation of lipidperioxidation as an indirect measure of oxidative stress inseminal plasma. Iran. J. Reprod. Med. 2, 34–39.