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1 Journal of Nutrition and Health (J Nutr Health) 2016; 49(6): 429 ~ pissn / eissn Research Article 두릅순에탄올추출물의인간유래피부각질형성세포와피부섬유아세포에서의자외선에의한광노화억제효과 * 양지원 곽충실 서울대학교의과대학노화고령사회연구소 Inhibitory effect of Aralia elata ethanol extract against skin damage in UVB-exposed human keratinocytes and human dermal fibroblasts* Yang, Jiwon Kwak, Chungshil Institute on Aging, Seoul National University College of Medicine, Seoul 03080, Korea ABSTRACT Purpose: Solar ultraviolet (UV) radiation causes inflammation and matrix metalloproteinase (MMP) overexpression and extracellular matrix depletion, leading to skin photoaging such as wrinkle formation, dryness, and sagging. Activation of MMP is influenced by various molecules such as reactive oxygen species (ROS), proinflammatory cytokines, and transient receptor potential vanilloid type (TRPV)-1, which are increased in UV-irradiated skin cells. Aralia elata (AE) ethanolic extract was reported to inhibit ROS generation caused by UVB-irradiation in keratinocytes. In this study, we investigated the photoprotective effect of AE ethanolic extract on UVB-irradiated human keratinocytes (HaCaT) and human dermal fibroblasts (HDF). Methods: AE was freeze-dried, extracted in 70% ethanol, and concentrated. Skin cells were treated with AE extract for 24 h and then exposed to UVB (55 mj/cm 2 ). After 48 h of incubation, proinflammatory cytokines, MMP-1, type-1 procollagen, and TRPV-1 levels were measured by ELISA or Western blotting. Results: Treatment with AE extract (100 µg/ ml) significantly inhibited UVB-induced IL-6, IL-8, and PGE 2 production in HaCaT by 25.6%, 5.3%, and 70.2%, respectively, and also inhibited elevation of MMP-1 and TRPV-1 caused by UVB irradiation by 20.0% and 41.9%, respectively (p < 0.05). In HDF, AE extract treatment significantly inhibited both elevation of MMP-1 and reduction of type-1 procollagen caused by UVB irradiation (p < 0.05). In addition, type-1 procollagen was elevated by AE extract treatment in normal HDFs (p < 0.05). Conclusion: AE 70% ethanol extract has photoprotective ability via reduction of proinflammatory mediators, TRPV-1 and MMP-1 production, and elevation of collagen synthesis. Our findings suggest that AE extract might be a good natural material to protect against UVB-induced premature skin aging. KEY WORDS: Aralia elata, MMP-1, type-i procollagen, inflammatory cytokines, ultraviolet B 서론 노인인구의증가와고령화사회로의진입으로인체의노화에대응하기위한방법들이모색되고있다. 노화현상은신체의성장과발달이중지되면서나타나는피할수없는과정으로서체성분및신체각기관의기능에변화가생기는것을의미한다. 1 피부는이러한인체의노화를잘보여주는기관중하나이다. 노화된피부는주름형성과처짐, 거칠어짐, 건조함등의특징을나타내며, 이러한피부의노화는나이가들어감에따라나타나지만외부요인에의해서노화가촉진되기도하는데그중자외선이가장큰요인이다. 2 적당량의자외선 (UV) 노출은인간이피부에서비타민 D를합성하는데꼭필요하기때문에유용하지만장기간의과도한 UV 노출은피부, 눈, 면역계등에해로운영향을미친다. 더구나, 최근에는태양으로부터오는 UV를흡수하 Received: November 2, 2016 / Revised: November 22, 2016 / Accepted: November 29, 2016 *This study was supported by a grant of the Ministry of Science, ICT and Future Planning through the National Research Foundation, Republic of Korea (NRF-2014R1A2A2A ), and Seoul National University (2015). To whom correspondence should be addressed. tel: , kwakcs@snu.ac.kr 2016 The Korean Nutrition Society This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 430 / 두릅순에탄올추출물의피부광노화억제효과 는역할을하는오존층의급격한감소로인하여지구표면에도달하는 UV의양이증가하고있다. UV의강도는지역과시간, 기후등에따라달라지는데일반적으로여름이겨울보다높고, 고도가높은곳이낮은곳보다높으며하루중에도시간에따라변한다. 3 UV는 UVA (320~400 nm) 와 UVB (280~320 nm), UVC (100~280 nm) 로나뉘는데지구표면에도달하는햇빛자외선의대부분은 UVA 이며 UVB는대기권에서대부분흡수되고 10% 정도만도달한다. UVA 와 UVB 모두피부손상을초래하지만 UVA 는피부세포의 DNA에직접적으로손상을주지는못하는반면 UVB는직접적으로세포의 DNA에작용함으로써화상, 광노화, 피부암등을유발할수있어 UVB가더해롭다고볼수있다. 4 UVB에의한피부손상은직접적으로피부세포내의 DNA의손상을초래하는경로와간접적으로활성산소종 (reactive oxygen species, ROS) 생성으로인한세포의지질막손상과세포염증반응, 피부구성단백질의손상등에의한피부광노화가있다. 5 UVB에의한피부세포의산화스트레스증가는 matrix metalloprotease (MMP) 발현을가속시켜피부 collagen의감소로인한피부노화로이어지는것으로알려져있다. 6,7 표피세포의 80% 를차지하는피부각질형성세포 (HaCaT) 는주로피부외측에위치하여피부각질을형성하고장벽의기능을담당하는것으로알려졌으나, UV에의한면역학적반응을조절하는사이토카인분비와염증성인자를유도하는생리조절물질을생성하고분비하는역할도하는것으로알려져있다. 8 HaCaT 세포에서 UV에의한염증반응이증가할경우 transient receptor potential vanilloid type (TRPV)-1도함께증가하는것으로알려져있는데, TRPV-1은칼슘이온채널로 UV 조사시활성화되어세포내칼슘유입을증가시켜염증반응이증가되도록하고이로인한 MMP-1의증가를가져오는것으로알려져있다. 8,9 실제로항산화물질들을섭취함으로써 UVB에의한광노화로부터피부를보호할수있다는보고들이있는데, 그작용기전은 ROS를직접적으로제거하거나스트레스신호를조절하거나 UV에의한염증반응을줄이는것으로보고되었다. 예를들어카로티노이드, 비타민 E, 비타민 C, 폴리페놀과같은많은항산화물질들이 UV로인하여발생한 ROS를줄임으로써피부방어에기여하여피부손상을막아주는것으로알려져있다. 10 자연적피부노화는나이가들어가면서시간경과에의해일어나는불가피한현상인반면, UV에의한피부광노화는어느정도예방이가능하기때문에피부광노화의예방및개선을위한기능성화장품과다양한식이소재의개발에대한관심과연구가증가하고있다. UV 조사와병행 된녹차식이공급이무모생쥐에서자외선조사로증가된피부 ph를정상화시켰고, 11 단삼추출물은 UVB에의한섬유아세포의광노화를보호하는효과를보였다. 5 또한, Hwang 등은무모생쥐에서 UVB에의한피부광노화를억제하는 gallic acid의효과 12 를보고하는등 UV에의한피부노화개선과관련한식품소재들이탐색되고있지만아직은그연구가부족한실정이다. 두릅나무 (Aralia elata Seem) 는두릅나무과에속하는식물로아시아전역의산에분포하는식물의하나로 13 예로부터민간과한방에서당뇨병, 신장병, 급만성간염, 위장질환개선과강장제로이용되어져왔다. 두릅나무의약효성분은사포닌을비롯하여올리에놀산, 트리테르핀, 시스토스테롤, 콜린, 헤데라제닌, 알칼로이드, 팔미틴산, 리놀렌산, 메틸아이코사논산, 3,4-디히드록시벤존산및헥사코졸등이알려져있다. 4 우리나라에서는두릅나무의어린잎과줄기를식용으로이용하고있는데특유의향과약간쓴맛이있다. 14 또한두릅순에는아스코르빈산, 레티놀, 베타카로틴과같은비타민과폴리페놀이풍부하여높은항산화효능을가지고있다. 15 두릅의사포닌성분은칼슘조절을통한심장수축을조절하거나, 16 당뇨병성망막병증에서신경손상을예방하는효과가있었으며, 17 유방암세포의사멸효과 18 도있었다는연구보고가있다. 최근, 본연구팀은두릅순에탄올추출물이비교적높은폴리페놀과플라보노이드함량을갖고있었으며, UVB를조사한 HaCaT에처리하였을때 ROS 생성을억제시켰고 nuclear factor erythroid 2-related factor 2 (Nrf-2) 항산화시스템을활성화시킴으로써피부의주요항산화효소인 superoxide dismutase-1 (SOD-1) 의합성을증가시켰음을보고한바있다. 4 이에본연구에서는두릅순 70% 에탄올추출물을전처리한 HaCaT과피부섬유아세포 (HDF) 에 UVB를조사한후염증성매개인자및피부세포의주요구성단백질인 collagen에영향을주는생화학적지표들의변화를측정함으로써두릅순추출물이피부광노화에대한방어효과를나타내는지알아보고자하였다. 연구방법 시료의준비본연구에사용한두릅순추출물은이전의연구 4 와같은방법으로준비하였다. 전라북도순창군에서 4월에채취한두릅순을씻어물기를제거한후 3일동안동결건조 (Samwon, Korea) 하였다. 동결건조한두릅을분쇄기로갈아분말화하였고, 분말시료 50 g에 10배부피의 70% 에탄올 (Duksan, Korea) 을넣고 24 시간씩 2 회교반하여

3 Journal of Nutrition and Health (J Nutr Health) 2016; 49(6): 429 ~ 436 / 431 추출한다음추출액을모아 whatman paper no.2 (GE Healthcare, UK) 로여과하였다. 여과액을 rotary vacuum evaporator (EYELA, Tokyo, Japan) 로 35 o C에서감압농축시키고, 24시간동안동결건조시켜분말상태의에탄올추출시료를얻었다. 세포및세포배양실험에사용된세포는 HaCaT과 HDF로서울대학교의과대학피부과에서제공받았다. 세포배양은 Dulbecco's modified Eagle Medium (DMEM, Welgene, Korea) 에 10% (V/V) fetal bovine serum (FBS, Hyclone, USA) 와 1% penicillin-streptomycin solution을첨가한후 37 o C, 5% CO 2 의배양기 (HERA Cell incubator, Thermo Scientific, USA) 에서배양하였고 2~3일간격으로계대배양하여유지하였다. 19 추출시료의세포독성평가동결건조분말형태의두릅순 70% 에탄올추출물을 dimethyl sulfoxide (DMSO, Sigma-Aldrich, MO, USA) 에먼저녹인후세포배양배지에희석하여농도별로처리하였다. 시료의세포독성평가는 Hwang 등 12 의연구를참조하여 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) 를이용한방법으로측정하였다. 96 well plate에 cell/ml의농도로 HaCaT 세포또는 HDF 세포를분주하여세포배양기 (37 o C, 5% CO 2 ) 에서안정화시킨후농도별로두릅순에탄올추출물을처리하여 48시간배양하였다. 그후 MTT시약을처리하고생존세포의효소작용에의해환원되도록 3시간더배양한후배양액을제거하고각 well에 DMSO를첨가하여생성된 formazan 결정을녹인다음 microplate reader (Power wave XS, BioTek, US) 를이용하여 560 nm에서흡광도를측정하였다. 추출시료의처리및 UVB 조사 UVB 조사를위해자체제작한 UV 조사장치에 UV lamp (TL20W/12RS UV-B, Philips, Netherlands) 와 UVC 제거필터인 TA 401/407 Kodacel filter (Kodak, USA) 를장치한후 UVX Radiometer (Ultra-Violet Products Ltd., US) 로 UVB의강도를측정하였다. UVB 조사강도는사전연구결과를바탕으로세포생장률에영향을주지않는안전한수준인 55 mj/cm 2 로설정하였다. 세포를 cells/ well의밀도로 6 well plate에분주하여안정화시킨후각 well에 AE 추출물을무혈청배양액에녹여농도별로처리하여 24시간배양시킨후배지를제거하고세포를살짝덮을정도로 PBS를첨가한다음 UVB를조사하였다. 효소면역측정법 (ELISA) 에의한 IL-6, IL-8, PGE 2 의측정 HaCaT 세포에두릅순추출물과 UVB를처리후 48시간배양한다음배양액을수거하여 4 o C, 13,000 rpm에서 10 분간원심분리후상층액만을모아서 enzyme linked immunosorbent assay (ELISA) kit (RnD system, Minneapolis, MN, USA) 을이용하여 prostaglandin (PG) E 2, interleukin (IL)-6, IL-8의농도를측정하였다. 단백질발현측정 (Western blotting analysis) MMPs는세포에서합성된후배지로분비되기때문에배양한세포의배지를수거하여 sodium dodecyl sulfate (SDS) sample buffer를넣어준비하였고, TRPV-1과 type-i procollagen의단백질량을측정하기위해서는세포의배지를제거한후차가운 PBS로세척한후 cell lifter (Costar, Corning, USA) 로세포를긁어모았다. RIPA buffer (intron, Korea) 를넣고세포를용해시켜 4 o C, 13,000 rpm에서 15분간원심분리한다음상층액을모은후 BCA 방법으로 kit (Thermo SCIENTIFIC, IL, USA) 을이용하여단백질농도를측정한다음 sample buffer를첨가하여동일농도의단백질을함유하는시료를준비하였다. Mini-PROTEIN system (BIO-Rad, USA) 를이용하여 8% SDS polyacrylamide gel의각 well에단백질양이동일하도록시료를넣고전기영동후 Amersham Hybond P 0.45 PVDF membranes (GE Healthcare Life Sci., Germany) 에 blotting 하여, 5% non-fat milk에서 1시간동안 blocking 하였다. Type I procllagen과 MMP-1 1차항체및 anti-trpv1 (NOVUS Biological, USA), anti-β-actin (Sigma-Aldrich) 을 4 o C에서 18시간반응시킨다음 2차항체 (Santa Cruz Biotech, TX, USA) 를실온에서 2시간동안반응시킨후 ECL solution (Pierce, USA) 을뿌려 LAS-4000 Luminescent Image Analyzer (Fujifilm, Japan) 을이용하여단백질 band를확인하였다. 해당 band의 intensity는 Multi Gauge Ver. 3.0 (Fujiflim, Japan) image densitometer을이용하여정량하였다. 통계분석모든실험결과는 IBM SPSS Statistics 22 (IBM, NY, USA) 통계프로그램을이용하여분석하였고, 모든실험은 3회이상반복하여그결과를평균과표준편차 (mean ± SD) 로나타내었다. 각군간의평균값의비교는 one-way ANOVA 로분석후 p < 0.05 수준에서 Duncan s multiple range test 또는 t-test로대조군에비하여각실험군이통계적으로유의한가를평가하였다.

4 432 / 두릅순에탄올추출물의피부광노화억제효과 결 두릅순시료의세포독성평가 HaCaT 과 HDF 에두릅순 70% 에탄올추출물을각각 48 시간처리하였을때 100 μg/ml까지는세포생장에영향을주지않는것으로나타났고 (Fig. 1), 200 μg/ml 이상에서는세포생장이떨어지는것으로나타나이들세포에대한두릅순 70% 에탄올추출물의안전범위는 100 μg/ml 이하로추정되었다. 한편, UVB를조사하기전추출시료를농도별로각세포에 24시간동안전처리하고 55 mj/cm 2 의 UVB를조사한후 48시간동안배양한뒤측정한세포생장률도 100 μg/ml 이하에서독성이없는것으로나타났다 (Fig. 2). 주요염증성사이토카인의변화 HaCaT 세포에서 UVB 조사로인하여염증성매개인자인 IL-6, IL-8, PGE 2 의생성이크게증가하였다 (p < 0.05). 그러나, 두릅순추출물을 10, 50, 100 μg/ml의농도로전처리한경우 50 μg/ml과 100 μg/ml 처리농도에서 UVB 에의해증가한 IL-6, IL-8, PGE 2 생성을모두유의하게감소시켰으며, IL-6와 PGE 2 는시료농도가증가할수록더감 과 소하였다 (p < 0.05) (Fig. 3). 두릅순추출물을 100 μg/ml 로처리시 IL-6는 UVB 대조군에비하여 25.6% 감소하였고, IL-8은 5.3%, PGE 2 는 70.2% 감소함으로써 (p < 0.05) 특히 PGE 2 의농도는정상대조군과비슷한수준으로크게감소됨을관찰하였다 (Fig. 3). TRPV-1, MMP-1, type-i procollagen의단백질량에미치는영향 HaCaT에서는 UVB 조사로인해세포내칼슘의유입을증가시키는 TRPV-1 단백질이 67.2% 증가하였으며피부 collagen 을분해하는효소인 MMP-1 의단백질발현량도 62.3% 증가하였다 (p < 0.05). 그러나, 두릅순추출물을전처리한경우 UVB 대조군에서의 TRPV-1과 MMP-1 단백질발현량과비교하여각각 41.9% 와 20.0% 감소하였다 (Fig. 4). HDF에서도 UVB 조사로인해 MMP-1 단백질의발현은 56.5% 증가하였는데, 두릅순에탄올추출물을 50 μg/ml과 100 μg/ml 농도로처리시정상대조군과비슷한수준으로유의하게감소하였다 (p < 0.05). 또한, type-i procollagen 단백질은 UVB 조사시단백질발현량이 23.0% 감소하였 Fig. 1. Cell viability of HaCaT and HDF treated with Aralia elata (AE) extract. Cell viability of HaCaT and HDF were measured at 48 h treatment of AE extract. Results were shown as mean ± SD (n = 3). *Significantly different at p < 0.05 compared to the control Fig. 2. Viability of HaCaT and HDF cells treated with Aralia elata (AE) extract and UVB. Cells were treated with AE extract for 24 h, exposed to UVB (55 mj/cm 2 ) and cell viability were measured at 48 h incubation. Results were shown as mean ± SD (n = 3). *Significantly different at p < 0.05 compared to UVB(-) control. NS: not significant Fig. 3. Effect of Aralia elata (AE) extract on proinflammatory mediator secretion in UVB irradiated HaCaT cells. Cells were treated with AE extract for 24 h, exposed to UVB (55 mj/cm 2 ) and IL-6, IL-8 and PGE 2 concentrations at 48 h incubation were measured in the culture medium by ELISA. Results were shown as mean ± SD (n = 3). Means sharing the same alphabet on the bar are not significantly different at p < 0.05 by ANOVA and Duncan s multiple range test.

Journal of Nutrition and Health (J Nutr Health) 2016; 49(6): 429 ~ 436 / 433 Fig. 4. Effect of Aralia elata (AE) extract on TRPV-1 and MMP-1 protein levels in UVB-irradiated HaCaT cells.

Protein level of MMP-1 (medium), TRPV-1 and β-actin (cell) determined by western blotting (A) and quantified (B). Results were shown as mean ± SD (n = 4).

5 Journal of Nutrition and Health (J Nutr Health) 2016; 49(6): 429 ~ 436 / 433 Fig. 4. Effect of Aralia elata (AE) extract on TRPV-1 and MMP-1 protein levels in UVB-irradiated HaCaT cells. Cells were treated with AE extract for 24 h, exposed to UVB (55 mj/cm 2 ) and the culture medium and the cells were collected at 48 h incubation. Protein level of MMP-1 (medium), TRPV-1 and β-actin (cell) determined by western blotting (A) and quantified (B). Results were shown as mean ± SD (n = 4). Means sharing the same alphabet on the bar are not significantly different at p < 0.05 by ANOVA and Duncan s multiple range test. Fig. 5. Effect of Aralia elata (AE) extract on MMP-1 and type I- procollagen protein levels in normal HDFs or UVB-irradiated HDFs. Cells were treated with AE extract for 24 h, exposed to UVB (55 mj/cm 2 ) and the culture medium and the cells were collected at 48 h incubation. The protein level of MMP-1 (medium), type I procollagen and β-actin (cell) determined by western blotting (A) and quantified (B). Results were shown as mean ± SD (n = 3~8). Means sharing the same alphabet on the bar are not significantly different at p < 0.05 by ANOVA and Duncan s multiple range test. NS: not significant 으나 (p < 0.05) 두릅순추출물을전처리한경우감소하지않고정상대조군수준을유지하였다 (Fig. 5). UVB를조사하지않은정상 HDF에두릅순추출물을 50 μg/ml과 100 μg/ml 농도로처리하였을때에도 type-i procollagen 단백질발현량이유의하게증가하였다 (p < 0.05). 따라서, 두릅순추출물은 UVB에노출된피부세포에서 UVB에의한 collagen 손상을억제하는동시에정상피부세포에서도 collagen의생성을촉진하는효과가있었다. 고찰 피부는장시간에걸쳐 UV에노출되면홍반이나열감등의임상적증상을느낄수있으나세포수준에서는 UV에의한피부세포의즉각적인반응을확인할수없다. 따라서, 본연구에서는 HaCaT과 HDF에서두릅순 70% 에탄올추출물의전처리가 UVB 조사에의한피부광노화관련인자에어떤영향을미치는지분석하였다. 본실험에앞서적정한 UVB 강도를결정하기위하여 0~100 mj/cm 2 범위로세포에 UVB를조사하고 48시간배양한후에세포생장률을측정한결과 75 mj/cm 2 까지는세포의생장률에영향 을주지않았기때문에보다안전한수준인 55 mj/cm 2 로모든실험을시행하였다. 또한, 두릅순추출물의처리농도도세포독성이나타나지않는 100 μg/ml 이하에서수행하였다. 피부가장시간에걸쳐 UV에노출되어일어나는피부광노화의경우조직학적변화로는각질형성세포가불규칙적으로변하고, 섬유모세포와염증세포는증가하고혈관벽은얇아지며진피부분의 type I과 III collagen의감소가가속화된다. 20 피부 collagen의파괴는주로상피의각질세포와피부섬유아세포에서분비하는 MMPs에의해일어나는데 MMPs 수준은자외선, 산화적스트레스, 사이토카인들을비롯하여다양한자극에의해서증가한다. MMPs 는피부의 extracellular matrix를분해시키는효소로염증반응, 암전이, 피부노화등에서그역할이중요한것으로알려져있다. 9 UV 조사에의해생성된 ROS는 MAPK (mitogen-activated protein kinase) 경로를활성화시키고이는다시전사인자인 AP-1 (activator protein-1) 을활성화시킨다. 21 AP-1은핵안으로들어가 collagenase (MMP-1) 와 stromelysin (MMP-3), 그리고 gelatinase (MMP-9) 의발현을유도한다. MMP-1이콜라겐분해를시작되게되면,

6 434 / 두릅순에탄올추출물의피부광노화억제효과 이어서 MMP-3 와 MMP-9 에의한연쇄반응이계속적으로일어난다고보고되었다. 22 UV에의한피부세포에서의 ROS 증가는피부광노화를촉진시키는요인으로잘알려져있기때문에자연식물의추출물을이용하여 UV에의한피부세포에서의 ROS 증가를낮추기위한다양한연구보고가있었다. 곽향잎 (Agastache rugosa leaf) 추출물은항산화효소인글루타치온과 SOD를증가시켜피부각질세포에서 UVB에의해증가된 ROS를낮추어피부를광노화로부터보호하는효과가있음이보고되었다. 23 다량의폴리페놀과안토시아닌을함유한빌베리 (Vaccinium myrtillus) 추출물은피부각질세포에서 UVB에의한세포독성과 DNA에손상을주는유전독성을감소시켰고피부세포에서의산화스트레스를낮추어세포막의지방산과산화를막아주는피부광노화효과가있음이보고되었다. 24 또한야생국화에탄올추출물은 HaCaT 세포에서 UVB 노출로인하여발생한 ROS를제거함으로써 p38 MAPK의인산화를저해하고피부 collagen을분해하는 MMP의증가를저해하는효과를보였다. 25 Ham 등은강향 (Dalbergia odorifera) 에탄올추출물의 HaCaT에서 UVB에의해의한 ROS 생성의감소가세포노화현상 (senescence) 과관련한유전자인 p53, p21 의발현의증가를막아세포노화를막음으로써피부광노화효과가있음을보고하였다. 26 이들식물추출물의항산화효능은비타민 C, E 등의영양성분과식물의녹황색색소성분인카로티노이드, 그리고안토시아닌색소등이포함된다양한폴리페놀등으로부터온것으로추정된다. 10 두릅순은 Lee 등의연구 15 와 Kwak과 Yang의이전연구 4 에서항산화효능을가진폴리페놀성분을다량함유하고있는것으로나타났다. 특히두릅 70% 에탄올추출물이 α,αdiphenyl-β-picrylhydrazyl (DPPH) 라디칼소거능과 2,2'- azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium sulfate (ABTS) 라디칼소거능, Fe +++ 에서 Fe ++ 로의환원력을측정하는 ferric reducing antioxidant power (FRAP) 등이우수하였고, HaCaT에서두릅순에탄올추출물이 UVB에의한 ROS 생성을감소시키는효과를확인된바있다. 따라서본연구에서두릅순추출물이 HaCaT과 HDF 세포에서 UVB에의한광노화관련인자의증가를억제시키는효과는그안에함유되어있는항산화효과를갖는폴리페놀의작용에의한것이크다고생각된다. UV 노출로인한피부손상기전의하나는염증반응이다. UV와같은외부자극은피부각질형성세포에서 IL-1β, IL-6, IL-8과 PGs와같은다양한사이토카인과염증반응매개물질을분비하여면역반응과염증반응을조절하는 데중요한역할을하는것으로알려져있다. 8 본연구에서도 HaCaT 세포에 UVB 조사로인해염증성사이토카인인 IL-6과 IL-8이증가하였는데이들염증성사이토카인은 PGE 2 와 nitric oxide (NO) 의생성을촉진함으로써통증, 부종, 기능장애, 홍반및발열등의염증반응을유발하고, 염증유발부위로면역세포의이동을활성화시는것으로알려져있다. 27 Lee 등은두릅추출물이대식세포에서 lipopolysaccharide (LPS) 에의해유도된 inducible nitric oxide synthesis (inos) 와 cyclooxygenase-2 (COX-2) 의합성을억제함으로써염증관련인자는 NO와 PGE 2 의생성을저해하고, 이로인해증가된 IL-1β와 IL-6 염증성사이토카인을거의정상수준으로떨어뜨렸다고하였다. 13 Suh 등은두릅의사포닌성분이염증반응에서활성화되는 nuclear factor kappa-light chain enhancer of activated B cells (NF-κB) 경로를저해하여대식세포에서의염증반응을억제하는효과가있었다고하였다. 28 본연구에서도두릅순추출물의전처리는 HaCaT에서 UVB 조사로인하여증가하는 IL-6와 IL-8을유의하게감소시켰으며, 특히 PGE 2 는거의정상수준과가깝게감소되었다. 또한, IL-6 와 IL-8은피부세포에서특히 MMPs의생성을촉진시켜피부를구성하는주요단백질인 collagen 감소를가속시키는인자로도잘알려져있기때문에 29 본연구에서두릅순추출물이 IL-6와 IL-8의합성을억제시킨것이 MMP-1의생성을감소시키는데기여한것으로생각된다. 한편, 피부세포가 UV를받으면칼슘이세포안으로유입되는데세포내칼슘수준이증가하면 NF-κB 경로가활성화되어염증반응및 MMP의증가가유도된다고보고되었다. 8 그런데, TRPV-1은 HaCaT 세포에있는주요칼슘유입채널로서접촉성피부염이나염증반응이증가할경우 TRPV-1의발현이증가하는것으로알려져있다. 8 본연구에서도 UVB를조사한세포에서 TRPV-1 단백질의증가가 IL-6, IL-8, PGE 2 및 MMP-1 증가와함께나타났기때문에 TRPV-1의증가가 UVB에의한염증반응및 collagen 감소와관계가있을것으로보인다. HaCaT 세포에서 UVB에의해민감하게증가하는것으로알려진 TRPV-1 9 이두릅순추출물처리에의하여감소함으로써 UVB에의한염증반응감소및피부 collagen의손상으로부터보호하는효과를유도할것으로사료된다. 덧붙여, 두릅순추출물은 UVB를조사한 HDF에서 type-i procollagen의감소를억제할뿐아니라 UVB를조사하지않은정상세포에서도 type-i procollagen 단백질발현을증가시켰는데이는두릅순추출물자체가피부세포에서 collagen 생성을촉진한다는것을의미한다. 본연구결과들에의하면두릅순에탄올추출물은 UVB에의해초래되는피부광노화로부터

7 Journal of Nutrition and Health (J Nutr Health) 2016; 49(6): 429 ~ 436 / 435 보호하는효과가있는것으로기대되며앞으로심도있는작용기전에대한후속연구와유효물질을확인하는연구가필요하다고본다. 요 약 본연구에서는피부의표피와진피에분포하는 HaCaT 세포와 HDF 세포를이용하여항산화효과가우수한두릅순추출물의처리가 UVB에의한피부광노화를억제할수있는지알아보기위하여피부염증반응과관련한사이토카인과피부의주요구성단백질인 collagen에영향을미칠수있는 MMP-1, type-i procollagen, TRPV-1 등의단백질발현에미치는영향을분석하였다. HaCaT에두릅순추출물을 24시간전처리한경우 UVB (55 mj/cm 2 ) 노출로인해증가한염증매개인자인 IL-6, IL-8, PGE 2 를유의하게감소시켰다. 또한, 피부 collagen의정상적인구조및양에영향을미치는단백질들의발현을측정한결과 HaCaT 에서는 UVB 조사로인해증가한 TRPV-1과 MMP-1 단백질의발현이두릅순에탄올추출물의전처리로모두감소하였고, HDF에서는 UVB를조사한대조군에비하여두릅순추출물처리가 MMP-1 단백질발현을감소시키는동시에 collagen의전구체인 type-i procollagen의발현을증가시키는효과를보였다. 이들결과들로부터항산화효과가우수한두릅순 70% 에탄올추출물은피부세포에서 UVB 에의한염증반응을억제시키는동시에피부 collagen의감소를억제시킴으로써피부광노화를예방할수있는천연소재로이용될수있다고본다. References 1. Lee JH, Jung JH, Kim HS. Modulation of immune parameters by aging process. Korean J Nutr 2010; 43(2): Jin XJ, Kim EJ, Oh IK, Kim YK, Park CH, Chung JH. Prevention of UV-induced skin damages by 11,14,17-eicosatrienoic acid in hairless mice in vivo. J Korean Med Sci 2010; 25(6): World Health Organization. Ultraviolet radiation and the INTER- SUN program: health effects of UV radiation [Internet]. Geneva: World Health Organization; [cited 2016 Nov 25]. Available from: 4. Kwak CS, Yang J. Suppressive effects of ethanol extract of Aralia elata on UVB-induced oxidative stress in human keratinocytes. J Nutr Health 2016; 49(3): Sun Z, Park SY, Hwang E, Zhang M, Jin F, Zhang B, Yi TH. Salvianolic acid B protects normal human dermal fibroblasts against ultraviolet B irradiation-induced photoaging through mitogenactivated protein kinase and activator protein-1 pathways. Photochem Photobiol 2015; 91(4): Oh MC, Kim KC, Ko C, Ahn YS, Hyun JW. Peptides-derived from scales of Branchiostegus japonicus inhibit ultraviolet B- induced oxidative damage and photo-aging in skin cells. J Life Sci 2015; 25(3): Jenkins G. Molecular mechanisms of skin ageing. Mech Ageing Dev 2002; 123(7): Huang J, Qiu L, Ding L, Wang S, Wang J, Zhu Q, Song F, Hu J. Ginsenoside Rb1 and paeoniflorin inhibit transient receptor potential vanilloid-1-activated IL-8 and PGE 2 production in a human keratinocyte cell line HaCaT. Int Immunopharmacol 2010; 10(10): Lee YM, Kang SM, Chung JH. The role of TRPV1 channel in aged human skin. J Dermatol Sci 2012; 65(2): Fernández-García E. Skin protection against UV light by dietary antioxidants. Food Funct 2014; 5(9): Lee B, Kim J, Hwang J, Cho Y. Dietary effect of green tea extract on epidermal levels of skin ph related factors, lactate dehydrogenase protein expression and activity in UV-irradiated hairless mice. J Nutr Health 2016; 49(2): Hwang E, Park SY, Lee HJ, Lee TY, Sun ZW, Yi TH. Gallic acid regulates skin photoaging in UVB-exposed fibroblast and hairless mice. Phytother Res 2014; 28(12): Lee JH, Jeong CS. Suppressive effects on the biosynthesis of inflammatory mediators by Aralia elata extract fractions in macrophage cells. Environ Toxicol Pharmacol 2009; 28(3): Cha JY, Ahn HY, Eom KE, Park BK, Jun BS, Cho YS. Antioxidative activity of Aralia elata shoot and leaf extracts. J Life Sci 2009; 19(5): Lee YJ, Lee SW, Lee SC, Park EJ. Antioxidant activities and antigenotoxic effect of ethanol extracts of Acorus gramineus, bud of Aralica elata Seem, Capsella bursa-pastoris, and Taraxacum officinale. J Basic Sci 2014; 31: Wang M, Xu X, Xu H, Wen F, Zhang X, Sun H, Yao F, Sun G, Sun X. Effect of the total saponins of Aralia elata (Miq) Seem on cardiac contractile function and intracellular calcium cycling regulation. J Ethnopharmacol 2014; 155(1): Kim SJ, Yoo WS, Kim H, Kwon JE, Hong EK, Choi M, Han Y, Chung I, Seo S, Park J, Yoo JM, Choi WS. Aralia elata prevents neuronal death by downregulating tonicity response element binding protein in diabetic retinopathy. Ophthalmic Res 2015; 54(2): Ryu MJ, Chung HS. Effect of Aralia elata on apoptosis in MDA- MB-231 human breast cancer cells. Food Eng Prog 2015; 19(3): Hwang E, Park SY, Lee HJ, Sun ZW, Lee TY, Song HG, Shin HS, Yi TH. Vigna angularis water extracts protect against ultraviolet b- exposed skin aging in vitro and in vivo. J Med Food 2014; 17(12): Robert L, Labat-Robert J, Robert AM. Physiology of skin aging. Pathol Biol (Paris) 2009; 57(4): Kim M, Park YG, Lee HJ, Lim SJ, Nho CW. Youngiasides A and C isolated from Youngia denticulatum inhibit UVB-induced MMP expression and promote type I procollagen production via repression of MAPK/AP-1/NF-kB and activation of AMPK/Nrf2 in HaCat cells and human dermal fibroblasts. J Agric Food Chem 2015; 63(22): Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 2001; 17(1):

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Journal of Life Science 2011, Vol. 21. No μ μ

Journal of Life Science 2011 Vol. 21. No. 8. 1120~1126 ISSN : 1225-9918 DOI : http://dx.doi.org/10.5352/jls.2011.21.8.1120 μ μ μ α β Journal of Life Science 2011, Vol. 21. No. 8 1121 μ μ 1122 생명과학회지 2011,