Protective effect of fucoxanthin isolated fromIshige okamurae against high-glucose inducedoxidative stress in human umbilical veinendothelial cells and zebrafish model

Min-Cheol Kang a,☆, Seung-Hong Lee b,☆, Won-Woo Lee a, Nalae Kang a,Eun-A Kim a, Seo Young Kim a, Dae Ho Lee c, Daekyung Kim d,You-Jin Jeon a,*a Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Koreab Division of Food Bioscience and Korea Nokyong Research Center, Konkuk University, Chungju 380-701,Republic of Koreac Department of Internal Medicine, Wonkwang University School of Medicine & Hospital, Jeonbuk 570-711,Republic of Koread Marine Bio Research Team, Korea Basic Science Institute (KBSI), Jeju 690-140, Republic of Korea

A R T I C L E I N F O

Article history:

Received 5 March 2014

Received in revised form 10

September 2014

Accepted 10 September 2014

Available online

A B S T R A C T

High glucose induced oxidative stress is implicated in intracellular toxicity in tissue and

blood vessel such as oxidative stress, lipid peroxidation, and cell death. In this study, we

attempted to investigate protective effects of fucoxanthin isolated from Ishige okamurae against

high glucose induced oxidative stress in human umbilical vein endothelial cells (HUVEC)

and zebrafish model. The ROS generation, lipid peroxidation and cell death were signifi-

cantly reduced in both the fucoxanthin treated HUVEC and zebrafish in vivo model, compared

to those of both negative controls. This study indicates that fucoxanthin could protect cells

and organ injury against oxidative stress induced by high glucose in vitro HUVEC and in vivo

zebrafish model.

© 2014 Elsevier Ltd. All rights reserved.

Keywords:

High glucose

Fucoxanthin

Oxidative stress

Zebrafish model

1. Introduction

The chronic diseases have attracted much attention in recentyears, especially for diabetes, obesity, hypertension and hy-perlipidemia (Vizoris & Tarasuk, 2003). Among them, diabetes

is one of the most serious metabolic diseases that have beenknown to be caused by metabolic disorders (Niazi & Kalra, 2012).Diabetes is characterized by an increase in the blood glucoselevel by insulin resistance (Soumya & Srilatha, 2011). Re-cently, many researchers have reported that the high glucoselevel in blood induces a wide range of disorders such as heart

* Corresponding author. Department of Marine Life Science, Jeju National University, Jeju 690-756, Republic of South Korea. Tel.: +82-64-754-3475; fax: +82-64-756-3493.

E-mail address: Youjinj@jejunu.ac.kr (Y.-J. Jeon).☆ These authors equally contributed to this work.

http://dx.doi.org/10.1016/j.jff.2014.09.0071756-4646/© 2014 Elsevier Ltd. All rights reserved.

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disease, kidney damage and stroke (Kawahito, Kitahata, &Oshita, 2009). Moreover, high glucose level in blood can causeoxidative damage to vessel, brain and tissue, leading to disease(Ceolotto et al., 2007). Previously reported studies have shownthat antioxidants could protect oxidative damage induced byhigh glucose in vitro and in vivo (Tsubouchi et al., 2005).

Moreover, several studies have reported that antioxidativeeffects improve dysfunction of the metabolisms and oxida-tive damage by high glucose level in blood (Sheu et al., 2008).Recently, many researchers have reported that radix hedysari,Cordyceps militaris and olea europaea L. have the protective effectsagainst oxidative damage caused by the treatment of highglucose in vitro and in vivo (Chu, Chien, & Duh, 2011; Kaeidi et al.,2011; Liu et al., 2012a). The main components of brown algaeinclude polyphenols, pigments, polysaccharides, minerals andamino acids (Kang et al., 2012a, 2012b). In previous studies, pig-ments of brown algae have been reported to exhibit variousbiological activities such as antioxidant, anti-inflammation andanti-tumor effect (Budhiyanti, Raharjo, Marseno, & Lelana, 2012).The extract of Ishige okamurae has shown various biological ac-tivities in vitro and in vivo such as antioxidant, anti-diabetesand neuro-protective effect (Heo et al., 2012; Min, Kim, Jeon,& Han, 2011). Among these pigment, fucoxanthin are found inbrown algae and chrysophyta, have been reported to improveoxidative stress and metabolic syndrome such as anti-diabetesand anti-obesity (Kang et al., 2011; Kumar, Hosokawa, &Miyashita, 2013; Liu, Liang, & Hu, 2011; Maeda, Hosokawa,Sashima, Funayama, & Miyashita, 2005; Maeda, Hosokawa,Sashima, & Miyashita, 2007; Nishikawa, Hosokawa, & Miyashita,2012; Woo et al., 2010). Therefore, in the present study, we in-vestigated the protective effects of fucoxanthin against highglucose level induced oxidative damage in human umbilicalvein endothelial cells (HUVEC) and zebrafish model.

2. Materials and methods

2.1. Extraction and isolation of fucoxanthin

The marine brown alga, Ishige okamurae, was collected alongthe coast of Jeju Island, Korea, between October 2011 and March2012. The sample was washed three times with tap water toremove salt, epiphytes, and sand attached to the surface. Theywere then carefully rinsed with fresh water, and maintainedin a medical refrigerator at −20 °C.Thereafter, the frozen sampleswere lyophilized and homogenized using a grinder prior to ex-traction. The algal powder was extracted three times with 80%aqueous methanol, and was evaporated under vacuum at 40 °C.The methanol extract was partitioned with chloroform, and

the chloroform extract was fractionated via silica column chro-matography with the stepwise elution of a chloroform–methanol mixture (100:1–1:1) to generate the separated activefractions. The combined active fraction was then applied to aSephadex LH-20 column (GE Healthcare, Uppsala, Sweden) satu-rated with 100% methanol, and finally purified via reversed-phase HPLC (Thermo Fisher Scientific, San Jose, CA, USA) usinga Waters HPLC system equipped with a Waters 996 photodi-ode array detector and a C18 column (150 × 20 mm, 4 µm, J’sphereODS-H80; YMC Co., Kyoto, Japan) via stepwise elution with amethanol–water gradient (UV range: 440 nm, flow rate: 0.8 ml/min). The purified compounds were definitively identified viacomparisons of their LC/MS, 1H, and 13C NMR data with thosein the relevant literature (Heo et al., 2008). The purity of fuco-xanthin (Fig. 1) was >95%, based on the peak area of allcomponents absorbed at each specific wavelength in HPLCanalysis. Fucoxanthin was dissolved in dimethyl sulphoxideand employed in experiments in which the final concentra-tion of dimethyl sulphoxide in culture medium was adjustedto <0.01%.

2.2. Cell culture

HUVEC cultures were maintained at 37 °C in an humidifiedatmosphere containing 5% CO2, in an endothelial cell growthmedium-2 EBM-2 supplemented with ascorbic acid, 2% fetalbovine serum (FBS), hydrocortisone, human fibroblast growthfactor (hFGF), long R insulin-like growth factor-1 (R3-IGF-1),gentamicin sulphate (CA-1000, ph of 7) and heparin as de-scribed by the manufacturer (Clonetics, Walkersville, MD,USA).

2.3. Assay of intracellular ROS levels in HUVEC cells

Intracellular ROS levels were measured by the dichloro-fluorescein assay (Wang & Joseph, 1999). 2′,7′-Dichloro-dihydrofluorescein diacetate (DCF-DA) can be deacetylated incells, where it can react quantitatively with intracellular radi-cals to be converted into its fluorescent product, DCF, whichis retained within the cells. Therefore, DCF-DA was used toevaluate the generation of ROS in oxidative stress. Cells (4 × 104

cells/well) were seeded to 24-well plates and preincubated withglucose (5.5 mM or 30 mM) for 48 h, and then incubated withoutor with the indicated concentrations of fucoxanthin for 20 h,after that the cells were washed with phosphate buffered saline(PBS) and incubated with 5 µM DCF-DA for 30 min at room tem-perature. Fluorescence was measured using a fluorescence platereader.

Fig. 1 – Chemical structure of fucoxanthin from Ishige okamurae.

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2.4. Assay of lipid peroxidation in HUVEC cells

Lipid peroxidation was measured by production of thiobarbituricacid reactive substances (TBARS) (Fraga, Leibouitz, & Toppel,1988). Cells (4 × 104 cells/well) wells were seeded to 24-well platesand pre-incubated with glucose (5.5 mM or 30 mM) for 48 h,and then incubated without or with the indicated concentra-tions of fucoxanthin for 20 h. A volume of 200 µl of supernatantof each medium was mixed with 400 µl of TBARS solution thenboiled at 95 °C for 20 min. The absorbance was measured at532 nm and TBARS concentrations were extrapolated from the1,1,3,3-tetraethoxypropane serial dilution standard curve,TBARSvalues were then expressed as equivalent nanomoles ofmalondialdehyde (MDA).

2.5. Assay of cell viability in HUVEC cells

Cell viability was evaluated by the MTT reduction assay. Cells(4 × 104 cells/well) were seeded to 24-well plates and pre-incubated with glucose (5.5 mM or 30 mM) for 48 h, and thenincubated without or with the indicated concentrations of fu-coxanthin for 20 h.Thereafter, the cells were treated with 2 mg/ml of MTT solution and incubated for 3 h at 37 °C and 5% CO2.After the supernatant was removed and produced formazansalt by viable cells was dissolved in DMSO and measured theabsorbance at 540 nm with a microplate reader (PackardSpectrocountTM, Austria).

2.6. Origin and maintenance of parental zebrafish

Adult zebrafish were obtained from a commercial dealer (SeoulAquarium, Seoul, Korea) and 10 fish were kept in 3 l acrylic tankwith the following conditions; 28.5 °C, with a 14/10 h light/dark cycle. Zebrafish were fed three times a day, 6 days/week,with Tetramin flake food supplemented with live brine shrimps(Artemia salina). Embryos were obtained from natural spawn-ing that was induced in the morning by turning on the light.Collection of embryos was completed within 30 min.

2.7. Waterborne exposure of embryos to fucoxanthinand glucose

From approximately 3 h post-fertilization (3 hpf), embryos(group = 25 embryos) were transferred to individual wells of a24-well plate and maintained in embryo media containing 1 mlof vehicle (0.1% dimethyl sulphoxide) or fucoxanthin for 1 h,then treated with 150 mM glucose or co-treated glucose andfucoxanthin for up to 120 h post-fertilization (120 hpf).

2.8. Estimation of intracellular ROS generation, and lipidperoxidation inhibitory activity and cell death inzebrafish embryo

Embryos were treated with fucoxanthin after 3–4 hpf and 1 hlater, 150 mM glucose was added to the plate. Embryos wereincubated for 6 h, the embryo media were changed and theembryos developed up to 2 dpf. Intracellular ROS generation,lipid peroxidation inhibitory activity and cell death in zebrafishembryos were estimated according to the method describedby Kang et al., 2013a; Kang et al., 2014; Kim et al., 2014. After

incubation, the embryos were rinsed with fresh embryo mediaand anesthetized before observed under microscope. Fluores-cence intensity of individual embryo was quantified usinga spectrofluorometer (Perkin–Elmer LS-5B, Vienna, Austria)and the images of stained embryos were observed using afluorescent microscope, which was equipped with a CoolSNAP-Pro colour digital camera (Olympus, Tokyo, Japan).

2.9. Statistical analysis

The data are presented as the mean ± S.E.The statistical analy-ses were performed by using SAS software. The results weresubjected to an analysis of the variance using the Tukey testto analyze the difference. P < 0.01 was considered signifi-cantly different.

3. Results

3.1. Inhibitory effect of fucoxanthin against high glucoseinduced ROS generation in HUVEC

ROS played an important role in HUVEC cells; we investi-gated ROS intracellular accumulation using DCFH-DA assay(Fig. 2).The ROS generation in high glucose-treated HUVEC cells

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Fig. 2 – Protective effect of fucoxanthin against highglucose induced intracellular ROS generation in HUVEC.Cells were exposed to high glucose (30 mM) and treatedwith fucoxanthin. Cells in wells of 96-well plates (4 × 104

cells/well) were preincubated with 5.5 of 30 mM glucose for48 h, and subsequently incubated for 24 h in the presenceor absence of 12.5, 25 or 50 µM fucoxanthin. The use of5.5 mM glucose was representative of normal glucoseconditions (negative control) and the 30 mM glucosetreatment represents high-glucose conditions (positivecontrol). Experiments were performed in triplicate and thedata are expressed as mean ± SE. *p < 0.01.

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was significantly increased to 169.2% than that of the non-treated cells. Pretreatment with different concentrations offucoxanthin significantly decreased the ROS generation levels.Especially, the treatments with 25 and 50 µM of fucoxanthinresulted in a significant decreasing of intracellular ROS to 145.5and 136.3%, respectively. This result indicates the protectiveeffect of fucoxanthin against high glucose caused oxidativestress in HUVEC.

3.2. Inhibitory effect of fucoxanthin against high glucoseinduced lipid peroxidation in HUVEC

For the inhibitory effect of fucoxanthin against lipid per-oxidation we investigated lipid peroxidation accumulation usingthiobarbituric acid reactive substances (TBARS) in the highglucose induced HUVEC and expressed as TBARS (nmol MDAeq/mg protein). As shown in Fig. 3, a significant increase ofTBARS level (0.826 nmol MDA eq/mg protein) was observed inthe absence of fucoxanthin. However, fucoxanthin inhibited thelipid peroxidation and especially the treatment with 50 µM offucoxanthin resulted in a significant decrease in the level ofTBARS to 0.728 nmol MDA eq/mg protein.

3.3. Protective effect of fucoxanthin against high glucoseinduced cell death in HUVEC

We investigated cell viability using MTT assay. As shownin Fig. 4, the effects of fucoxanthin on viability in high

glucose-treated HUVEC cells were evaluated. When the cellswere exposed to high glucose, in this study, the cell viabilitywas decreased to 74.72%. However, fucoxanthin treatment sig-nificantly increased the cell viability. Especially, the treatmentswith 25 and 50 µM of fucoxanthin resulted in a significantincrease in cell viability to 83.72 and 89.20%.

3.4. Effect of fucoxanthin on survival rate in high glucoseinduced zebrafish model

The survival rates of the experimental zebrafish were deter-mined (Fig. 5). In the non-treated group the survival rate was100% during the experiment period. The survival rate in thehigh glucose-treated group was significantly decreased to 78.57%than that of the non-treated group. Pretreatment with differ-ent concentrations of fucoxanthin significantly increased thesurvival rates. Especially, the treated-groups with 50 and 100 µMof fucoxanthin resulted in significant increases in survival ratesto 89.83 and 90%, respectively.

3.5. Inhibitory effect of fucoxanthin against high glucoseinduced ROS generation in zebrafish model

We investigated ROS accumulation in zebrafish using oxidation-sensitive fluorescent probe dye, DCFH-DA (Fig. 6). The ROS

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Fig. 3 – Effect of fucoxanthin on TBARS generation in thehigh glucose induced HUVECs. Cells were exposed to highglucose (30 mM) and treated with fucoxanthin. Cells inwells of 96-well plates (4 × 104 cells/well) werepreincubated with 5.5 of 30 mM glucose for 48 h, andsubsequently incubated for 24 h in the presence or absenceof 12.5, 25 or 50 µM fucoxanthin. The use of 5.5 mMglucose was representative of normal glucose conditions(negative control) and the 30 mM glucose treatmentrepresents high-glucose conditions (positive control).Experiments were performed in triplicate and the data areexpressed as mean ± SE. *p < 0.01.

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Fig. 4 – Protective effect of fucoxantin against high glucoseinduced cytotoxicity in HUVEC. Cells were exposed to highglucose (30 mM) and treated with fucoxanthin. Cells inwells of 96-well plates (4 × 104 cells/well) werepreincubated with 5.5 of 30 mM glucose for 48 h, andsubsequently incubated for 24 h in the presence or absenceof 12.5, 25 or 50 µM fucoxanthin. The use of 5.5 mMglucose was representative of normal glucose conditions(negative control) and the 30 mM glucose treatmentrepresents high-glucose conditions (positive control).Experiments were performed in triplicate and the data areexpressed as mean ± SE. *p < 0.01.

307j o u rna l o f f un c t i ona l f o od s 1 1 ( 2 0 1 4 ) 3 0 4 – 3 1 2

intensity in the high glucose-treated zebrafish was 100% butpretreatment with different concentrations of fucoxanthin sig-nificantly decreased the ROS generation levels. The treatmentswith 25, 50 and 100 µM of fucoxanthin resulted in significantdecreases in ROS generation to 76.60, 65.11 and 45.14%,respectively.

3.6. Inhibitory effect of fucoxanthin against high glucoseinduced lipid peroxidation in zebrafish model

The lipid peroxidation is widely used as an indicator of oxi-dative stress in vitro and in vivo (Fig. 7). We investigated the lipidperoxidation using DPPP fluorescent dye assay. The DPPP in-tensity in the high glucose-treated zebrafish was increased.However, the fucoxanthin treatments with 25, 50 and 100 µMbrought the DPPP intensities in the tissue of zebrafish to lowerintensities of 89.59, 90.36 and 86.74%, respectively.

3.7. Protective effect of fucoxanthin against high glucoseinduced cell death in zebrafish model

We have investigated the protective effect of fucoxanthinagainst high glucose induced cell death in zebrafish model(Fig. 8). The protective effects of fucoxanthin in zebrafish areshown in Fig. 8. We investigated the cell death using acridineorange staining assay. The cell death in the high glucose-treated zebrafish was 100% but pretreatments with differentconcentrations of fucoxanthin significantly decreased the celldeaths. Especially, the treatments with 50 and 100 µM of fu-coxanthin resulted in significant decreases in cell death

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Fig. 5 – Survival rates after treatment with high glucose orco-treatment with fucoxanthin. The embryos were treatedwith 30 mM high glucose and co-treated with fucoxanthin.Experiments were performed in triplicate and the data areexpressed as mean ± SE. *p < 0.01.

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Fig. 6 – Inhibitory effect of fucoxanthin against high glucose induced ROS level in zebrafish embryos (A). Embryos weretreated with 30 mM glucose and co-treated with fucoxanthin. Photographs of high glucose induced ROS level in zebrafishembryos (B). ROS levels were measured by image analysis and fluorescence microscopy. Experiments were performed intriplicate and the data are expressed as mean ± SE. *p < 0.01.

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intensities to 69.88 and 54.41%, respectively. Our results indi-cate that fucoxanthin exhibited protective effects against highglucose caused oxidative damages in zebrafish model.

4. Discussion

Many reports have shown that increases in blood glucose andoxidative stress play an important role in complications of dia-betes mellitus (Phillips, Cataneo, Cheema, & Greenberg, 2004;Vanizor et al., 2001). Previous reports mentioned that diabe-tes is important to find ways to attenuate the oxidative stressinduced by hyperglycemia (Fiordaliso et al., 2004; Sundaram,Aggarwal, & Sandhir, 2013). Many researchers have reportedthat the protective effect of natural products against highglucose caused oxidative stress was affected by their antioxi-dant effects (Li et al., 2011; Qian, Huo, Wang, & Qian, 2011).Among the candidates, fucoxanthin has attracted extensive in-terest due to its excellent biological activities includinganticancer, anti-inflammation, antioxidant and anti-angiogenicactivity (Fung, Hamid, & Lu, 2013; Heo et al., 2010a; Kim et al.,2013; Sugawara, Matsubara, Akagi, Mori, & Hirata, 2006). Es-pecially, several evidences have demonstrated that fucoxanthinshows improvement of metabolic disease such as diabetes,obesity and hepatic lipid metabolism (Hosokawa et al., 2010;Hu et al., 2012; Maeda et al., 2005; Woo et al., 2010). However,protective effect of fucoxanthin against high glucose inducedoxidative stress has been examined on only mouse models

(Iwasaki et al., 2012). Therefore, the first aim of this study wasto investigate the protective effects of fucoxanthin against highglucose-induced oxidative damage in HUVEC and zebrafishmodels. The HUVEC has widely been used to investigate theprotective effect of natural product against high glucose causedoxidative damage (Chu et al., 2011; Lee, Han, Heo, Hwang, &Jeon, 2010). Hyperglycemia causes excessive ROS generation,which has been implicated in oxidative damage including oxi-dative stress, lipid peroxidation and cell death in HUVEC (Heoet al., 2010b; Sheu et al., 2008). Our results showed that expo-sure of HUVEC to high glucose (30 mM) resulted in a significantincrease of ROS generation. However, the treatment with fu-coxanthin significantly decreased the high glucose induced ROSlevels. These results imply that fucoxanthin obviously pro-tects cells against hydrogen peroxide induced oxidative stress(Heo et al., 2008).

Lipid peroxidation is oxidative of lipid and has been knownto be caused by free radicals (Oostenbrug, Mensink, Bar, &Hornstra, 1997). It is one of the most commonly used oxida-tive markers of natural products against oxidative stress in theexperimental study both in vitro and in vivo (Aalto, Raivio,Pietarinen, & Kinnula, 1996). Our results indicate that fucox-anthin can inhibit the lipid peroxidation which is induced byhigh glucose treatment. High glucose treatment has been re-ported to increase cell death by excessive formation of ROS inHUVEC (Liu et al., 2012b). Our results indicate that the highglucose increases free radical production, lipid peroxidation andcell death in the HUVEC. However, these results indicated thatfucoxanthin can protect the HUVEC against high glucose

Glucose (5.5 mM)- treated group

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Fig. 7 – Inhibitory effect of fucoxanthin against high glucose induced lipid peroxidation level in zebrafish embryos (A). Theembryos were treated with 30 mM high glucose and co-treated with fucoxanthin. After incubation, the lipid peroxidationwas detected by fluorescence spectrophotometer after DPPP staining. Photographs of high glucose induced lipidperoxidation level in zebrafish embryos (B). The lipid peroxidation levels were measured by image analysis andfluorescence microscopy. Experiments were performed in triplicate and the data are expressed as mean ± SE. *p < 0.01.

309j o u rna l o f f un c t i ona l f o od s 1 1 ( 2 0 1 4 ) 3 0 4 – 3 1 2

induced oxidative stress. Recently, zebrafish models havebeen widely used and investigated for in vivo studies includ-ing cancer, melanoma, cardiovascular disease, immune system,antioxidative stress (Berman, Skariah, Maro, Mingnot, &Mourrain, 2009; Kang et al., 2013b; Lam, Chua, Gonq, Lam, &Sin, 2004; Li, Huang, Huang, Du, & Hung, 2012; Yang et al., 2012).Zebrafish has numerous advantages for experimental studiessuch as low cost, large clutches, and physiological similarityto mammals (Dooley & Zon, 2000). Therefore, we investigatedthe protective effect of fucoxanthin against high glucose causedoxidative stress in zebrafish model.

From the results, this work demonstrated that high glu-cose caused significant increases in ROS generation, lipidperoxidation and cell death, however fucoxanthin has protec-tive effect against oxidative damage induced by the high glucosetreatment in the HUVEC and zebrafish model. Our results in-dicated that fucoxanthin may prove to be an effective mediatorto control oxidative stress for hyperglycemia.

Acknowledgments

This research was financially supported by the Ministry ofEducation (MOE) and National Research Foundation of Korea(NRF) through the Human Resource Training Project for Re-gional Innovation (NRF-2012H1B8A2025863)

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Glucose (5.5 mM)- treated group

Glucose (150 mM)-treated group

Glucose + Fucoxanthin- 25 µM treated group

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