ORIGINAL PAPER
Cytoprotective effect of fucoxanthin isolated from brown algae Sargassum siliquastrum against H 2O 2-induced cell damage
Soo-Jin Heo ÆSeok-Chun Ko ÆSung-Myung Kang ÆHahk-Soo Kang ÆJong-Pyung Kim ÆSoo-Hyun Kim ÆKi-Wan Lee ÆMan-Gi Cho ÆYou-Jin Jeon
Received:26March 2008/Revised:9June 2008/Accepted:23June 2008/Published online:22July 2008ÓSpringer-Verlag 2008
Abstract In this study,the cytoprotective effect of fuco-xanthin,which was isolated from Sargassum siliquastrum ,against oxidative stress induced DNA damage was investi-gated.Fucoxanthin,a kind of carotenoid,was pretreated to the medium and the protective effect was evaluated via 20,70-dichlorodihydrofluorescein diacetate,3-(4,5-dimeth-ylthiazol-2-yl)2,5-diphenyltetrazolium bromide,and comet assays.Intracellular reactive oxygen species were over produced by addition of hydrogen peroxide (H 2O 2),but the production was significantly reduced by the treatment with fucoxanthin.The fucoxanthin strongly enhanced cell via-bility against H 2O 2induced oxidative damage and the inhibitory effect of cell damage was a dose-dependent manner.Furthermore,a protective effect against
oxidative stress-induced cell apoptosis was also demonstrated via nuclear staining with Hoechst dye.These results clearly
indicate that fucoxanthin isolated from S.siliquastrum possesses prominent antioxidant activity against H 2O 2-mediated cell damage and which might be a potential ther-apeutic agent for treating or preventing several diseases implicated with oxidative stress.
Keywords Fucoxanthin ÁSargassum siliquastrum ÁCell damage ÁOxidative stress ÁReactive oxygen species
Introduction
Reactive oxygen species (ROS),which include free radi-cals such as superoxide anion radical (O 2-),hydroxyl radicals (ÁOH)and non free-radical species such as H 2O 2and singlet oxygen (1O 2),formed during normal metabolic processes,which can easily initiate the peroxidation of membrane lipids,leading to the accumulation of lipid peroxides.Free radical scavengers and antioxidants can reduce lipid peroxidation and the generation of ROS.The importance of antioxidants in human health has become increasingly clear due to spectacular advances in under-standing the mechanisms of their reaction with oxidants.Furthermore,interest in employing antioxidants from nat-ural sources to increase
the shelf-life of foods is considerably enhanced by consumer preference for natural ingredients and concerns about the toxic effects of syn-thetic antioxidants [1–3].
Algae,as photosynthetic organisms,are exposed to a combination of light and high oxygen concentrations what induces the formation of free radicals and other oxidative reagents.The absence of structural damage in the algae leads to consider that these organisms are able to generate the necessary compounds to protect themselves against oxidation [4].In this respect,algae can be considered as an
S.-J.Heo ÁS.-C.Ko ÁS.-M.Kang ÁK.-W.Lee ÁY.-J.Jeon Faculty of Applied Marine Science,Cheju National University,Jeju 690-756,South Korea
e-mail:skysjheaven@paran
H.-S.Kang ÁJ.-P.Kim
KRIBB,52Eoeun-dong,Yuseong,Daejeon 305-806,South Korea
S.-H.Kim
Department of Food Bioengineering,Cheju National University,Jeju 690-756,South Korea
M.-G.Cho
Food and Biotechnology,Dongseo University,Pusan 617-716,South Korea
Y.-J.Jeon (&)
Marine and Environmental Research Institute,
Cheju National University,Jeju 695-814,South Korea e-mail:youjinj@cheju.ac.kr
Eur Food Res Technol (2008)228:145–151DOI 10.1007/s00217-008-0918-7
important source of antioxidant compounds that could be suitable for protecting our bodies against the ROS by our metabolism or induced by external factors(as pollution,stress,UV radiation,etc.).
Fucoxanthin is found in edible brown algae and,along with b-carotene,is one of the most abundant carotenoids found in nature[5].There have recently been several reports that fucoxanthin showed biological activities such as radical scavenging,antiproliferative,antiobesity,anti-tumor,and antiangiogenic activities[6–10].Nonetheless, the protective effects of cell damages related to antioxida
nt activities of fucoxanthin have not yet been reported. Accordingly,the present study isolated the marine natural carotenoid fucoxanthin from Sargassum siliquastrum and evaluated its cytoprotective effects against H2O2-induced cell damage.
Materials and methods
Materials
The marine alga S.siliquastrum was collected along the coast of Jeju Island,Korea,between October2005and March2006.The samples were washed three times with tap water to remove the salt,epiphytes,and sand attached to the surface,then carefully rinsed with fresh water,and maintained in a medical refrigerator at-20°C.Therefore, the frozen samples were lyophilized and homogenized with a grinder prior to extraction.
General experimental procedures
Optical rotations were measured on a JASCO P-1020 polarimeter.The UV and FT-IR spectra were recorded on a Pharmacia Biotech Ultrospec3000UV/Visible spec-trometer and a SHIMAZU8400s FT-IR spectrometer, respectively.NMR spectra were recorded on a Varian INOVA400MHz NMR spectrom
eter.CD3OD was used as a solvent for the NMR experiments,and the solvent signals were used as an internal reference.ESI and HREI mass spectra acquired using a Finnigan Navigator30086and JMS-700MSTATION high resolution mass spectrometer system,respectively.The HPLC was carried out on a Waters HPLC system equipped with a Waters996photo-diode array detector and Millenium32software using C18 column(J’sphere ODS-H80,150mm920mm,4l m, YMC Co.).
Extraction and isolation
The powdered S.siliquastrum(400g)was extracted with 80%aqueous MeOH,and was evaporated under vacuo.Then,the MeOH extract was partitioned with CHCl3.The chloroform extract(9g)was fractionated by silica column chromatography using stepwise elution with CHCl3–MeOH mixture(100:1?1:1)to afford separated active fractions.A combined active fraction was further subjected to a Sephadex LH-20column saturated with100%MeOH, and thenfinally purified by reversed-phase HPLC to give fucoxanthin(74.9mg).1H and13C NMR data were assigned in Table1and the structure was illustrated in Fig.1.
Cell culture
Cells of a monkey kidneyfibroblast line(Vero)were maintained at37°C in an incubator with humidified at
mosphere of5%CO2.Cells were cultured in Dulbecco’s modified Eagle’s medium containing10%heat-inactivated fetal calf serum,streptomycin(100l g/mL),and penicillin (100U/mL).
Intracellular ROS measurement
For detection of intracellular ROS,Vero cells were seeded in96-well plates at a concentration of19105cells/mL. After16h,the cells were treated with various concentra-tions of fucoxanthin,and incubated at37°C under a humidified atmosphere.After30min,H2O2was added at a concentration of1mM,and then cells were incubated for additional30min at37°C.Finally,20,70-dichlorodihy-drofluorescein diacetate(DCFH-DA;5l g/mL)was introduced to the cells,and20,70-dichlorofluorescein(DCF)fluorescence was detected at an excitation wavelength of 485nm and an emission wavelength of535nm,using a Perkin-Elmer LS-5B spectrofluorometer.The percentage of intracellular ROS scavenging activity was calculated in accordance with the following equation:
Intracellular ROS scavenging activity%
ðÞ
¼1ÀC1=C0
ðÞ
½ Â100
where C1is thefluorescence intensity of cells treated with H2O2and fucoxanthin,and C0is thefluorescence intensity of cells treated with H2O2and distilled water instead of fucoxanthin.
Assessment of cell viability
Cell viability was then estimated via an3-(4,5-dimethyl-thiazol-2-yl)2,5-diphenyltetrazolium bromide(MTT) assay,which is a test of metabolic competence predicated upon the assessment of mitochondrial performance.It is colorimetric assay,which is dependent on the conversion of yellow tetrazolium bromide to its purple formazan derivative by mitochondrial succinate dehydrogenase in
viable cells[11].The cells were seeded in96-well plate at a concentration of19105cells/mL.After16h,the cells were treated with fucoxanthin at difference concentrations. Then,10l L of H2O2(1mM)was added to the cell culture medium,and incubated for24h at37°C.MTT stock solution(50l L;2mg/mL)was then applied to the wells, to a total reaction volume of200l L.After4h of incu-bation,the plates were centrifuged for5min at8009g,and the supernatants were aspirated.The formazan crystals in each well
were dissolved in150l L of dimethylsulfoxide (DMSO),and the absorbance was measured via ELISA at a wavelength of540nm.Relative cell viability was evalu-ated in accordance with the quantity of MTT converted to the insoluble formazan salt.The optical density of the formazan generated in the control cells was considered to represent100%viability.The data are expressed as mean percentages of the viable cells versus the respective control.
Determination of DNA damage by comet assay
Comet assay was performed to determine the oxidative DNA damage[12].The cell suspension was mixed with 75l L of0.5%low melting agarose(LMA),and added to the slides precoated with1.0%normal melting agarose. After solidification of the agarose,slides were covered with another75l L of0.5%LMA and then immersed in lysis solution(2.5M NaCl,100mM EDTA,10mM Tris,and
Table1NMR spectroscopic data for fucoxanthin in CDCl3
Position13C1H(mult.J=Hz)Position13C1H(mult.J=Hz) 135.81035.2
247.1  1.36(1H,dd,J=8.7,14.2)
1.49(1H,dd,J=14.2)2045.4  1.41(1H,dd,J=10.4,14.9)
2.00
(1H,dd,J=2.9,14.9)
364.3  3.80(1H,m)3068.0  5.37(1H,tt,J=8.8,12.0)
441.6  1.77(1H,dd,J=8.7,14.2)
2.29(1H,dd,J=2.9,17.8)4045.2  1.53(1H,dd,J=10.4,14.9)2.29
(1H,dd,J=2.9,17.8)
566.25072.7
667.160117.5
740.8  2.59,3.64(2H,d,J=20.4)70202.4
8170.580103.4  6.04(1H,s)
reactive oxygen species (ros)9134.590132.5
10139.17.14(1H,d,J=12.8)100128.5  6.12(1H,d,J=11.6) 11123.4  6.58(1H,m)110125.7  6.71(1H,t,J=12.0) 12145.0  6.66(1H,t,J=12.8)120137.1  6.34(1H,d,J=11.6) 13135.4130138.1
14136.6  6.40(1H,d,J=11.6)140132.2  6.26(1H,d,J=11.6) 15129.4  6.67(1H,m)150132.5  6.71(1H,t,J=12.0,14.2) 1625.0  1.37(3H,s)16029.2  1.37(3H,s)
1728.10.95(3H,s)17032.1  1.06(3H,s)
1821.2  1.34(3H,s)18031.3  1.34(3H,s)
1911.8  1.80(3H,s)19014.0  1.80(3H,s)
2012.8  1.98(3H,s)20012.9  1.98(3H,s)
30OAc,CH321.4  2.03(3H,s)
30OAc,C=O197.9
Data were obtained using400MHz for1H and100MHz for13
C
1%sodium lauryl sarcosine;1%Triton X-100and10% DMSO)for1h at4°C.The slides were next placed into an electrophoresis tank containing300mM NaOH and 10mM Na2EDTA(pH13.0)for40min for DNA unwinding.For electrophoresis of the DNA,an electric current of25V/300mA was applied for20min at4°C. The slides were washed three times with a neutralizing buffer(0.4M Tris,pH7.5)for5min at4°C,and then treated with ethanol for another5min before staining with 50l L of ethidium bromide(20l g/mL).Measurements were made by image analysis(Kinetic Imaging,Komet5.0, UK)andfluorescence microscope(LEICA DMLB,Ger-many),determining the percentage offluorescence in the tail(tail intensity;50cells from each of two replicate slides).
Nuclear staining with Hoechst33342
The nuclear morphology of the cells was evaluated using the cell-permeable DNA dye,Hoechst33342.
Cells with homogeneously stained nuclei were considered viable, whereas the presence of chromatin condensation and/or fragmentation was indicative of apoptosis[13,14].The cells were placed in24-well plates at a concentration of 19105cells/mL.Sixteen hours after plating,the cells were treated with various concentration of fucoxanthin,and further incubated for1h prior to expose to H2O2(1mM). After24h,1.5l L of Hoechst33342(stock10mg/mL),a DNA-specificfluorescent dye,were added to each well, followed by10min of incubation at37°C.The stained cells were then observed under afluorescence microscope equipped with a CoolSNAP-Pro color digital camera,in order to examine the degree of nuclear condensation.Statistical analysis
The data are expressed as the mean±standard error.A statistical comparison was performed via the SPSS package for Windows(Version10).p-Values of\0.05were con-sidered to be significant.
Results and discussion
Cells are protected from ROS-induced damage by a variety of endogenous ROS-scavenging enzymes,chemical com-pounds,and natural products.Recently,interest has been increased in the therapeutic potential of natural products to be used as natural antioxidants in the reduction of such free
radical-induced tissue injuries,thereby suggesting that many natural products may prove to possess therapeuti-cally useful antioxidant compounds [15–17].
H 2O 2has been extensively used as an inducer of oxi-dative stress in in vitro models.It readily crosses the cellular membranes giving rise to the highly reactive hydroxyl radical,which has the ability to react with mac-romolecules,including DNA,proteins,and lipids,and ultimately damage to whole cell [18,19].S
everal studies have shown that oxidative stress is a major cause of cellular injuries in a variety of human diseases including cancer,neurodegenerative,and cardiovascular disorders [20–22].Recently,many researchers have made considerable efforts to search natural antioxidants.Many studies have revealed that seaweeds have potential to be used as a candidate for natural antioxidant.Potent antioxidative compounds have already been isolated from seaweeds and identified as phylopheophytin in Eisenia bicyclis (arame),fucoxanthine in Hijikia fusiformis (hijiki),phlorotannin in Ecklonia stolonifera ,bromophenols in Polysiphonia urceolata ,and sulfated polysaccharides [6,23–27].Among these antiox-idants,fucoxanthine,a kind of carotenoid,showed radical scavenging activity.In this study,we investigated the antioxidant effects of natural carotenoid fucoxanthin,after the administration of H 2O 2in cell lines.
20,70-Dichlorodihydrofluorescein diacetate was used as a probe for ROS measurement.DCFH-DA crosses cell membranes and is hydrolyzed enzymatically by intracel-lular esterase to nonfluorescent DCFH.In the presence of ROS,DCFH is oxidized to highly fluorescent DCF.It is well known that H 2O 2is the principal ROS responsible for the oxidation of DCFH to DCF [28].In the present
study,
Fig.5Photomicrographs of DNA damage and migration observed under fucoxanthin isolated from S.siliquastrum where the tail moments were decreased.a control,b cells treated with 50l M H 2O 2,c cells treated with 5l M compound +50l M H 2O 2,d cells treated with 50l M compound +50l M H 2O 2,e cells treated with 200l M compound +50l M H 2O 2

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