감태함유자외선차단필름의보호효과 715 (Park et al., 214).. (Ecklonia cava) 1 m 1, 2. 2 phloratannins,,,,,, (Wijesekara et al., 21), (Ko et al., 21).. 4. HaCaT cell,,,

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1 한수지 5(6), , 217 Original Article Korean J Fish Aquat Sci 5(6),714-72,217 감태 (Ecklonia cava) 를이용한자외선차단필름의 UV-B 조사에의한광손상으로부터보호효과 이효근 원유선 1 고은별 2 김윤아 2 김정은 2 김윤정 2 한채원 2 최민우 3 김재일 3 * 전유진 * 제주대학교해양생명과학과, 1 제주대학교수산생명의학과, 2 신성여자고등학교, 3 부경대학교식품영양학과 Protective Effects of Ecklonia cava Film on UV-B-induced Photodamages Hyo Geun Lee, Yu Sun Won 1, Eun Byeol Koh 2, Kim Yoon Ah 2, Jeong Eun Kim 2, Yoon Jeong Kim 2, Chae Won Han 2, Min-Woo Choi 3, Jae-Il Kim 3 * and You-Jin Jeon* Department of Marine Life Science, Jeju National University, Jeju 63243, Korea 1 Department of Aquatic Life Medicine, Jeju National University, Jeju 63243, Korea 2 Sinseong Girls' High School, Jeju 6338, Korea 3 Department of Food Science and Nutrition, Pukyong National University, Busan 48513, Korea Exposure to ultraviolet (UV) radiation is associated with the development of adverse effects in skin. Among the three types of UV rays, UV-B causes the most damaging effects, inducing sunburn and penetrating the outer skin, resulting in DNA mutations and skin cancer. The objective of this study was to formulate a UV-protective film by incorporating Ecklonia cava extracts. Cells covered with the film were exposed to UV-B (5, 8, and 1 mj/cm 2 ). To determine the protective effects of the film, we evaluated cell viability, intracellular ROS generation, and apoptosis. We found that all E. cava extracts absorbed UV light and exhibited protective effects against UV-B-induced photodamage. Among the protective films examined in this study, that incorporating an E. cava 7% ethanol extract (7EX) formed the most effective protection against UV-B in HaCaT cells. These findings suggest that the application of film containing E. cava extract could prevent UV-B-induced photodamage, and offer protection against the detrimental effects of UV radiation, thus maintaining physiological condition. Key words: Ecklonia cava, UV Protective effects, Photodamages, Algae, Ocean 서론 (ultraviolet, UV) (32-4 nm) UV-A, (28-32 nm) UV-B, (2-28 nm) UV-C (Verschooten et al., 26), UV-A. UV-B UV-B,,, (F'guyer et al., 23; Bowden, 24; Afaq et al., 25; Halliday, 25; Moriwaki and Takahashi, 28). UV-C. UV-A UV-B. reactive oxygen species (ROS) matrix metalloproteinase-1 (MMP-1) (Kim et al., 216).,. HaCaT cells (Lee et al., 214), (Kim and Park, 215), (Kim et al., 216), human dermal fibroblast (HDF) cells Korean J Fish Aquat Sci 5(6) , December 217 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 2 November 217; Revised 22 November 217; Accepted 26 November 217 *Corresponding author: Tel: Fax: address: jikim@pknu.ac.kr; youjin@jejunu.ac.kr Copyright 217 The Korean Society of Fisheries and Aquatic Science 714 pissn: , eissn:

2 감태함유자외선차단필름의보호효과 715 (Park et al., 214).. (Ecklonia cava) 1 m 1, 2. 2 phloratannins,,,,,, (Wijesekara et al., 21), (Ko et al., 21).. 4. HaCaT cell,,, apoptosis. 재료및방법 실험재료및기기,. potassium sodium tartrate tetrahydrate, Folin & Ciocalteu s phenol reagent, dimethyl sulfoxide (DMSO), 2',7'-dichlorofluorescin diacetate (DCF-DA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin (BSA), glucose, galic acid Sigma-Aldrich (St, Louis, MO, USA). Sodium hydrogen carbonate, sodium carbonate anhydrous, copper (II) sulfate pentahydrate, ethylenediaminetetraacetic acid (EDTA) Yakuri (Japan), Dulbecco s modified eagle s medium (DMEM) fetal bovine serum (FBS) Gibco/BRL (Canada). ultraviolet irradiator (CL-1 series UV Crosslinker, OPTIZEN, UK), microplate reader (Synergy HT Multi-Detection microplate reader, Bio-Tek, USA) UV/Vis spectrophotometer (OPTIGEN POP, OPTIZEN, UK). apoptosis Immunofluorescence analyzer (BD FACS caliber, BD bioscience, UK). 추출물제조.. distilled water (DW) (), 5% ethanol (EtOH) (5EX), 7% EtOH (7EX), 1% EtOH (1EX). : 5:1 5%. 5 g 1 ml shaking incubator (37, 24 h) (12 rpm, 4, 1 min) pore size 25 µm 4. 감태에탄올추출물의수율및함유성분함량분석, 1. AOAC (199), phenol-sulfuric acid method (Kuda et al., 22) Lowry protein assay (Harrington, 199) Folin-Ciocalteu method (Chandler and Dodds, 1983). 세포배양및계대배양 HaCaT cells (, keratinocyte). HaCaT cells 1% FBS 1% penicillin (1 unit/ml) DMEM CO 2, incubator (5%, 37 ) (21) 자외선차단감태필름제조 gelatin (López-Caballero et al., 25)..1% (w/w),.1% (w/w), 1% (w/w) 1 mg, 1 mg, 1 mg gelatin 9 mg ml % EtOH (1EX) 1% (v/v) EtOH. UV-Vis Spectrometer Analysis.125 mg/ml, (Roleda et al., 26) UV/Vis Spectrophotometer (Optizen POP, Optizen, UK). UV-A, UV-B, UV-C 19 nm-

3 716 이효근ㆍ원유선ㆍ고은별ㆍ김윤아ㆍ김정은ㆍ김윤정ㆍ한채원ㆍ최민우ㆍ김재일ㆍ전유진 7 nm Survey scan mode (SUR mode). 감태필름을적용한 HaCaT cells 에자외선조사후생존율변화 HaCaT cells HaCaT cells..1% (w/w),.1% (w/w), 1% (w/w). HaCaT cells (1 1 5 ) 24 well plate seeding CO 2 incubator (5%, 37 ) 24 4 UV-B 5, 8, 1 mj/cm 2 ). UV-B 1% penicillin (1 unit/ ml) DMEM CO 2 incubator (5%, 37 ) 24 2 mg MTT 1 µl. CO 2 incubator (5%, 37 ) 3 DMSO microplate reader (Synergy HT Multi-Detection microplate reader, Bio Tek, USA) 54 nm. gelatin. 감태필름을적용한 HaCaT cells 에자외선조사후활성산소발생량변화 HaCaT cells (5, 8, 1 mj/ cm 2 ), (intracellular) ROS. Intracellular ROS DCF-DA 2',7'-dichlorofluorescin (DCFH) ROS (Rosenkranz et al., 1992). HaCaT cells (1 1 5 ) 24 well plate seeding CO 2 incubator (5%, 37 ) 24 4 UV-B. UV-B 1% penicillin (1 unit/ml) DMEM CO 2 incubator (5%, 37 ) 3 5 µg/ml DCF-DA 25 µl CO 2 incubator (5%, 37 ) 3 microplate reader (Synergy HT Multi-Detection microplate reader, Bio Tek, USA) 485/53 nm (excitation/emission). (A) (B) (C) Cell viability (% of control) Cell viability (% of control) Cell viability (% of control) UV-B irradiation (5 mj/cm 2 ) * UV-B irradiation (8 mj/cm 2 ) UV-B irradiation (1 mj/cm 2 ).1%.1% 1% E. cava film 5EX 7EX 1EX Fig. 1. UV absorption spectrum of Ecklonia cava extracts [distilled water (DW) (), 5% ethanol (EtOH) (5EX), 7% EtOH (7EX), 1% EtOH (1EX)]. Each extracts (.125 mg/ml) were dissolved in DW. Specturm was measured using UV-Vis spectrophotometer. Table 1. The yields and proximate chemical components of Ecklonia cava extracts [distilled water (DW) (), 5% ethanol (EtOH) (5EX), 7% EtOH (7EX), 1% EtOH (1EX)]. Each value is the average of triplicate and each bar indicates mean±standard derivation (SD) 5EX 7EX 1EX Yield (%) 21.7 ± ± ± ±.56 Polysaccharides (%) 2.15 ± ± ± ±.35 Polyphenols (%) 9.21 ± ± ± ±.28 Protein (%) 7.21 ± ± ± ±.4 (A) ity ( 1 3 ) 6 4.1% E. cava film

4 감태함유자외선차단필름의보호효과 717 Absorbance (O.D.) Apoptosis apoptosis Immunofluorescence analyzer (BD FACS caliber, BD bioscience, UK) (cell cycle). 48 phosphate buffered saline (PBS) trypsin 7% EtOH CO 2 incubator (5%, 37 ) 3, 2 mm EDTA 37 3 propidium iodide (PI) Immunofluorescence analyzer. 통계학적분석 3 (mean standard deviation), Graph pad prism (virson 5.) (one-way ANOVA). 결과및고찰 감태추출물의수율및성분 Wavelnegth (nm) DW EX 5% EtOH EX 7% EtOH EX 1% EtOH EX Fig. 2. Effect of Ecklonia cava films [gelatin film (), distilled water (DW) extracts film (), 5% ethanol (EtOH) extracts film (5EX), 7% EtOH extracts film (7EX), 1% EtOH extracts film (1EX)] on UV-B radiation-induced cell viability in HaCaT cells. The cells were exposed to UV-B and their viability were measured by MTT assay. Each value is the average of triplicate and each bar indicates mean±standard derivation (SD). *P<.5, **P<.1 and ***P<.1 compared with group. (A) 5 mj/cm 2, (B) 8 mj/cm 2, (C) 1 mj/cm 2. Table 1,,,. 5% EtOH (A) (B) (C) Fluorescene intensity ( 1 3 ) Fluorescene intensity ( 1 3 ) Fluorescene intensity ( 1 3 ) % E. cava film.1% E. cava film 1% E. cava film UV-B irradiation (8 mj/cm 2 ) 5EX 7EX 1EX Fig. 3. Effect of Ecklonia cava films [gelatin film (), distilled water (DW) extracts film (), 5% ethanol (EtOH) extracts film (5EX), 7% EtOH extracts film (7EX), 1% EtOH extracts film (1EX)] on UV-B radiation-induced ROS (reactive oxygen speceis) generation in HaCaT cell. The cells were exposed to UV-B (8 mj/cm 2 ). ROS generation was measured by DCF-DA assay. Each value is the average of triplicate and each bar indicates the mean ± standard derivation (SD). *P<.5, **P<.1 and ***P<.1 compared with group. (A).1% film, (B).1% film, (C) 1% film. 7% EtOH 7% EtOH 1% EtOH. 5% EtOH. UV-Vis Spectrophotometer 를통한감태추출물의자외선파장흡수범위측정 Fig. 1. UV-A UV-B.

5 718 이효근ㆍ원유선ㆍ고은별ㆍ김윤아ㆍ김정은ㆍ김윤정ㆍ한채원ㆍ최민우ㆍ김재일ㆍ전유진 trol Sub G1=14.89% Sub G1=76.77% Sub G1=75.18% Sub G1=57.56% 5EX Sub G1=55.45% 7EX Sub G1=45.14% 1EX Sub G1=51.74% Sub G 1 content (%) EX 7EX 1EX 8 mj/cm 2 Film Fig. 4 Effect of Ecklonia cava films [gelatin film (), distilled water (DW) extracts film (), 5% ethanol (EtOH) extracts film (5EX), 7% EtOH extracts film (7EX), 1% EtOH extracts film (1EX)] on UV-B radiation-induced apoptosis in HaCaT cell. The cells were exposed to UV-B (5, 8 and 1 mj/cm 2 ). Apoptosis was measured by cell cycle analysis by propidium iodide (PI) staning method. Percentages of apoptotic cells are shown in each panel. The data represent the percentages of Sub-G1 cell populations in cell cycle distribution. trol;, film (); E.cava films [distilled water (DW) extracts film (), 5% ethanol (EtOH) extracts film (5EX), 7% EtOH extracts film (7EX) and 1% EtOH extracts film (1EX)]. DW () EtOH UV-B, EtOH 7% EtOH (7EX) 1% EtOH (1EX) UV-B. UV-B 조사후손상으로부터보호효과 HaCaT cells UV-B (Fig. 2, Fig. 3). 5 mj/cm 2 (Fig. 2A), 7-8% film 5%...1% 7% EtOH,.1% 1% 9% (Fig. 2A). 8 mj/cm 2 (Fig. 2B), film 4%..1% 7% EtOH (7EX).1% 85%, 1% 9% trol (Fig. 2B). 1 mj/cm 2 Fig. 2C.

6 감태함유자외선차단필름의보호효과 719 film 2%..1%,.1% 7% EtOH 1% trol. 8 mj/cm 2 UV-B Fig. 3. film,.1% 1% trol (Fig. 3B, 3C).. UV-B 조사에대한세포자살 (apoptosis) 억제 HaCaT cells 48 Immunofluorescence analyzer (BD FACS caliber, BD bioscience, UK) Fig. 4. apoptotic cell accumulation Sub G1 phase, - apoptosis (Sitailo et al., 22). trol film Sub G1 75%. Sub G1 45-6% 7% EtOH (7EX) 45.14% Sub G1 (Fig. 4)., UV-B, HaCaT cells, intracellular ROS, apoptosis UV-B. UV,. UV.. 사사 (216 ). References Afaq F, Syed DN, Malik A, Hadi N, Sarfaraz S, Kweon MH, Khan N, Zaid MA and Mukhtar H. 27. Delphinidin, an anthocyanidin in pigmented fruits and vegetables, protects human hacat keratinocytes and mouse skin against UVBmediated oxidative stress and apoptosis. J Invest Dermatol 127, AOAC (Association of Official Analytical Chemists) Of ficial Methods of Analysis (15 th Edition). Helrich K, ed. Association of Official Analytical Chemists, Arlington, VA, U.S.A. Bowden GT. 24. Prevention of non-melanoma skin cancer by targeting ultraviolet-b-light signaling. Nat Rev Cancer 4, Chandler SF and Dodds JH The effect of phosphate, nitrogen and sucrose on the production of phenolics and solasodine in callus cultures of Solanum laciniatum. Plant Cell Reports 2, F guyer S, Afaq F and Mukhtar H. 23. Phochemoprevention of skin cancer by botanical agents. Photodermatol Photoimmunol Photomed 19, j x. Halliday GM. 25. Inflammation, gene mutation and photoimmunosuppression in response to UVR-induced oxidative damage contributes to photocarcinogenesis. Mutat Res 571, Harrington CR Lowry potein assay containing sodium dodecyl sulfate in microtiter plates for protein determinations on fractions from brain tissue. Anal Biochem 186, Kim TY and Park JP Antioxidant and protective effects of gagam-danguieumja on ultraviolet B-induced damage in human keratinocytes. J Physiol Pathol Korean Med 29, Kim TY, Jang SA, Chae YB, Bak JP. Antioxidant and protective effects of Leonurus sibiricus l. extract on ultraviolet b (uvb)- induced damage in human keratinocytes. Korean J Plant Res 1, Kim TY, Jang SA, Chae YB and Bak JP Antioxidant and protective effects of Leonurus sibiricus L. extract on ultraviolet B (UVB)-induced damage in human keratinocytes. Korean J Plant Res 29, kjpr Ko SC, Cha SH, Heo SJ, Lee SH, Kang SM and Jeon YJ. 21. Protective effect of Ecklonia cava on UVB-induced oxidative stress: in vitro and in vivo zebrafish model. J Appl Phycol 23, Kuda T, Taniguchi E, Nishizawa M and Araki Y. 22. Fate of water-soluble polysaccharides in dried Chorda filum a brown alga during water washing. J Food Compos Anal 15,

7 72 이효근ㆍ원유선ㆍ고은별ㆍ김윤아ㆍ김정은ㆍ김윤정ㆍ한채원ㆍ최민우ㆍ김재일ㆍ전유진 Lee SH, Ha SE, Lee JK and Park JK Effects of Rubus coreanus and Artemisia princeps extracts on the ultraviolet B-induced DNA damage responses in HaCaT cells. J Life Sci 2, Lo pez-caballero ME, Go mez-guille n MC, Pe rez-mateos M and Montero P. 25. A chitosan gelatin blend as a coating for fish patties. Food Hydrocolloid 19, dx.doi.org/1.116/j.foodhyd Moriwaki S and Takahashi Y. 28. Photoaging and DNA repair. J Dermatol Sci 5, jdermsci Park HC, Jung TK and Yoon KS Antioxidative activity of extract of Cornus walteri wanger leaves in human dermal fibroblast irradiated by UVB. KSBB J 29, dx.doi.org/1.7841/ksbbj Roleda MY, Clayton MN and Wiencke C. 26. Screening capacity of UV-absorbing compounds in spores of arctic laminariales. J Exp Mar Biol Ecol 338, org/1.116/j.jembe Rosenkranz AR, Schmaldienst S, Stuhlmeier KM, Chen W, Knapp W and Zlabinger GJ A microplate assay for the detection of oxidative products using 2',7'-dichlorofluorescin-diacetate. J Immunol Methods 156, dx.doi.org/1.116/ (92)98-h. Sitailo LA, Tibudan SS and Denning MF. 22. Activation of caspase-9 is required for UV-induced apoptosis of human keratinocytes. J Biol Chem 22, org/1.174/jbc.m2412. Verschooten L, Declercq L and Garmyn M. 26. Adaptive response of the skin to UVB damage: role of the p53 protein. Int J Cosmetic Sci 28, x. Wijesekara I, Yoon NY and Kim SK. 21. Phlorotannins from Ecklonia cava (Phaeophyceae): biological activities and potential health benefits. Biofactors 36, org/1.12/biof.114.

Lumbar spine

Lumbar spine CT 32 111 DOI : 10.3831/KPI.2010.13.2.111 Lumbar Spine CT 32 Received : 10. 05. 23 Revised : 10. 06. 04 Accepted : 10. 06. 11 Key Words: Disc herniation, CT scan, Clinical analysis The Clinical