ISSN (Print) ISSN (Online) Asian J Beauty Cosmetol 2017; 15(4): R E S E A R C H

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1 ISSN (Print) ISSN (Online) Asian J Beauty Cosmetol 2017; 15(4): R E S E A R C H A R T I C L E Open Access Anti-oxidant Activities of Phytol on Keratinocytes Sun-Hee Jeong Department of Beauty Arts, Suwon Women s University, Suwon-si, Gyeonggi-do, Korea Corresponding author: Sun-Hee Jeong, Department of Beauty Arts, Suwon Women s University, 72 Onjeong-ro, Gwonseon-gu, Suwon-si, Gyeonggi-do 16632, Korea Tel.: Fax: sunheejeong@swc.ac.kr Received June 11, 2017 Revised September 21, 2017 Accepted September 28, 2017 Published December 30, 2017 Abstract Purpose: The aim purpose of this study is to evaluate anti-oxidant and cytoprotective effects of phytol as an ingredient of cosmetic against hydrogen peroxide ( ) in keratinocytes. Methods: Cell viability assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, 2,7 -dichlorofluorescin diacetate (DCFDA) assay, reactive oxygen species (ROS) scavenging activity assay, flow cytometry analysis, quantitative real-time polymerase chain reaction (qrt-pcr), and glutathione assay were performed to verify the cell effectiveness of phytol. Results: To determine the experimental concentration of and phytol in HaCaT keratinocytes, water-soluble tetrazolium salt (WST-1) assay was examined with treatments of various and phytol concentrations in HaCaT keratinocytes. As a results 750 µm and 10 µm phytol treatment was chosen for the experiment. The expression of copper-zinc superoxide dismutase (CuZn-SOD; SOD1), manganese superoxide dismutase (Mn-SOD; SOD2), catalase (CAT) mrna, very well-known as anti-oxidant genes, was increased by phytol in a dose-dependent manner. DPPH radical scavenging activity and intracellular reduced form of glutathione (GSH) levels were increased by phytol in a dose-dependent manner, which showed that anti-oxidant effects are recovered by phytol. Conclusion: It suggests possibility of phytol as a cosmetic ingredient which enhances a defense system and slows an ageing process for skin. Keywords: Phytol, Anti-oxidant, Cytoprotective, Cosmetics, Ageing Introduction 신체에서피부는다양한환경적인요인에대해개체를보호하는일차적인물리적장벽으로서의중요한역할을담당하고있다. 이러한피부는크게표피, 진피, 피하지방조직으로구성되며, 최외각층에있는표피는대부분각질형성세포 (keratinocytes) 로구성되어있다. Keratinocytes는표피의기저층에서각질층까지단계를거치면서분화되고, 이러한과정을통해피부장벽기능을수행한다. 하지만생체의최외각층에존재하고있어, ultraviolet (UV), 열, 환경독소등과같은외적요인에의해영향을받기쉽다. 이와같은산화적스트레스 (oxidative stress) 는피부에축적되어궁극적으로피부노화로이어지게된다. 피부노화의주된원인중하나인활성산소종 (ROS) 은대표적인예로 superoxide anion (- ), nitric oxide (NO), hydroxyl radical (OH - ), singlet oxygen ( 1 ), 등이있다 (Thannickal & Fanburg, 2000; Park & Shim, 2016). 이러한 ROS는미토콘드리아의 전자전달계에서산소가수소이온과전자를받아물로치환되어 adenosine triphosphate (ATP) 를형성하게되는데이때대략 1 2% 의홀전자가산소에전해져생성된다 (Halliwell & Gutteridge, 1989). 이와같이 ROS는세포내정상적인활성작용과정에의하여생성되며, 세포신호와증식, DNA 복제, 사이토카인에대한반응정도를포함한다양한생물학적과정에연관되어있다 (Gao et al., 2001; Rhee, 2006). 따라서 ROS의항상성을유지하는것은세포성장과생존에매우중요하다. 하지만약물, 자외선, 공해물질, 감염등과같이외부인자들에의해서 ROS 생성이촉진되기도한다. ROS 노출량과지속시간이증가되고 ROS 방어기작이돌연변이를일으키게되면항산화제양이감소하여산화-환원항상성이무너져산화적스트레스가유발된다. 여기서산화적스트레스란 ROS 발생과체내항산화효소의활동과같은항산화반응이균형을이루지못해활성산소가축적되는상태를말한다. 이러한산화적스트레스는세포내지질과산화, 단백질변성, Copyright c Korea Institute of Dermatological Sciences. 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 Anti-oxidant Activities of Phytol Figure 1. Anti-oxidant pathways of ROS. ROS comprise both free radical and non-free radical oxygen containing molecules such as and -. Cellular redox homeostasis is maintained by an endogenous anti-oxidant defense system, which includes endogenous anti-oxidant enzymes such as SOD, CAT, GPx, and GSH. ROS, reactive oxygen species;, hydrogen peroxide; -, superoxide; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GSH, reduced form of glutathione; GSSG, oxidized form of glutathione; GR, glutathione reductase. Figure 2. Analysis of cytotoxic effects of in HaCaT keratinocytes. The cytotoxicity of on HaCaT cells was tested by performing WST-1 assay. HaCaT cells ( cells/well) were seeded on 96- well plates and then treated with each of indicated concentration of for 6 h. The data are expressed as M±SD from three independent experiments. * p<0.05., hydrogen peroxide; WST-1, water-soluble tetrazolium salt; M±SD, mean±standard deviation. DNA 손상, 세포괴사등을일으켜결국노화를일으키게된다 (Ozben, 2007; Rhee, 2006). 산화적스트레스로부터생체를보호하기위해세포에는내재적방어기전인 superoxide dismutase (SOD), CAT, glutathione peroxidase (GPx) 등의항산화효소와 ascorbic acid, glutathione 등의비효소적항산화제가있다 (Slater, 1984). 항산화효소와비효소적항산화제는상호의존적으로활동하며세포내다양한항산화제시스템 (endogenous antioxidant system) 을작동시키고있다. 건강한상태의세포내에서는이러한다양한메커니즘을통해해로운산화경로를억제함으로써세포상해로부터스스로를보호하고, 항상성을유지한다. 인체내대표적인 1차적초기항산화효소인 SOD는활성산소를 와 로전환시키고, 생성된 는 CAT와 GPx 에의해소거된다. GPx는 reduced form of glutathione (GSH) 을 oxidized form of glutathione (GSSG) 로산화시키면서 를제거한다 (Figure 1). 여기서 GSH는생체내의주요항산화성분으로자신의 thiol을이용하여활성산소를제거하며세포내해독작용을한다 (Dickinson & Forman, 2002; Lee et al., 2017). 산화적스트레스로부터세포를보호하는데강한항암작용및면역증강효과가있다고알려진 phytol은식물조직에서녹색의색소성분인클로로필의구성성분으로존재하며식물조직파괴시가수분해되어생성된다 (Gross, 1991). Phytol이강한면역효과가있다는선행연구결과를바탕으로인간각질 형성세포 (HaCaT keratinocyte cell line) 에서항산화효능을확인함으로써, phytol의화장품소재로서의가능성을평가하였다. Methods 1. 세포배양본연구에서는인간각질형성세포주인 HaCaT keratinocyte cell line을 American Type Culture Collection (ATCC; USA) 에서구매하여사용하였다. HaCaT 세포주의배양은 Dulbecco s modified Eagles medium (DMEM; Hyclone TM, GE Healthcare Life Sciences, UK) 에 10% fetal bovine serum (FBS; Hyclone TM ), 1% penicillin/streptomycin (penicillin 100 IU/mL, streptomycin 100 μg/ml; Invitrogen TM, Thermo Fisher Scientific, USA) 가첨가된배지를사용하였고, 37, 5% C 조건에서배양하였다. 2. 세포생존율측정 HaCaT keratinocytes에서 phytol과 의세포독성을확인하기위하여 WST-1 assay를이용하였다. WST-1 assay는세포내미토콘드리아의탈수소효소에의해 tetrazolium salt 가 formazan으로환원되는방식을이용하여세포생존율을측정하는시험방법이다. Phytol (Sigma-Aldrich, USA) 은순수정제 (>97%) 된 458

3 powder 형태로구매하였고, dimethyl sulfoxide (DMSO; Sigma-Aldrich) 에용해하여실험에사용하였다. Phytol과 각각의세포독성을확인하기위하여 96-well plate에 HaCaT keratinocytes ( cells/well) 을 100 µl씩접종하여 24 h 배양하였다. Phytol은 HaCaT keratinocytes에 1, 2, 5, 10, 20 µm의농도로처리하여 24 h 동안배양하였고, 는 100, 250, 500, 750, 1000 µm의농도로 6 h 동안배양하였다. EZ-Cytox Cell Viability Assay Kit Reagent (ItsBio, Korea) 를 10 µl씩첨가하고 1 h 배양후 microplate reader (Bio-Rad Laboratories, USA) 를사용하여 490 nm 에서흡광도를측정하였다. 세포생존율은 3회반복하여세포생존율의평균값과표준편차를도출하였다. 3. 세포보호효과측정 에대한 phytol의세포보호효과를확인하기위하여 96-well plate에 HaCaT keratinocytes ( cells/ well) 를 100 µl씩접종하여 24 h 배양하였다. Phytol을 1, 5, 10, 20 µm의농도로 24 h 동안배양한다음, phosphate buffered saline (PBS; Sigma-Aldrich) washing 후 750 µm 를 6 h 동안처리한후 WST-1 assay를수행하여흡광도를측정하였다. 세포생존율은 3회반복하여세포생존율의평균값과표준편차를도출하였다. 4. DPPH 라디칼소거활성측정항산화효과를측정하기위해화학적으로안정화된 DPPH free radical이항산화활성이있는물질을만나면색상이변화하는원리를이용하여실험을진행하였다. 96-well plate 에최종농도 1, 5, 10 µm의 phytol 희석액를 100 µl씩분주하고여기에 50 µl DPPH ( 최종농도 0.2 mm) 을넣은후암실에서 30 min 동안방치하였다. Microplate reader를이용하여흡광도 514 nm에서측정하였으며, 대조군으로는항산화물질로잘알려진 L-ascorbic acid를사용하여비교실험하였다. 실험측정은 3회반복하여흡광도의평균값과표준편차를도출하였다. 5. 세포내 ROS 정량분석세포내 ROS 변화를측정하기위해 DCFDA assay를이용하였다. HaCaT를 cells/well로 60 mm 배양접시에접종하여 24 h 배양한다음, 5, 10 µm 농도의 phytol을 24 h 전처리하고, PBS washing 후 750 µm 를 6 h 후처리하여추가배양하였다. 세포내 ROS을측정하기위한 dye인 DCFDA (Sigma-Aldrich) 를 10 µm 첨가하여 30 min 배양후세포를수확하여 PBS를첨가하여세포를풀어준후 BD FACSCalibur TM Flow Cytometer (BD Biosciences, USA) 를이용하여 ROS의변화량을측정하였다. Phytol의 ROS 제거효능을검증하기위하여 ROS scavenger 역할을하는 L-ascorbic acid 역시동일한방식으로측정하여비교 분석하였다. 6. 항산화관련유전자의발현분석산화적스트레스상황에서 phytol이항산화효소인 SOD 와 CAT의유전자발현에미치는영향을확인하기위하여 HaCaT를 cells/well로 60 mm 배양접시에접종하여 24 h 배양하였다. Phytol을 1, 5, 10 µm 농도로 24 h 전처리하고, PBS washing 후 750 µm 를 6 h 후처리하여추가배양하였다. qrt-pcr 분석을위해세포배양후수확한세포를 TRIzol reagent (Invitrogen TM ) 에용해한다음, 0.2 ml chloroform (Biopure, Austria) 를첨가하여상온에방치하였다. mrna는 diethylpyrocarbonate (DEPC; Biopure) water로녹여실험에사용하였으며, 추출된 RNA 는 Nanodrop (MaestroGen, Taiwan) 을이용하여 260/280 nm의 ratio 1.8 이상순도의 RNA만을실험에사용하였다. SOD1, SOD2 및 CAT mrna에대한 qrt-pcr 분석을실시하였으며, PCR의유효성은 melting curve로검증하였고, 각유전자의발현은 actin beta (ACTB) 로표준화하여비교 분석하였다 (Table 1). 7. GSH 측정세포내항산화지표인 GSH 발현변화를측정하기위해발광기법에기초하여측정하는방법 (luminescence-based assay), GSH-Glo Glutathione Assay (Promega, USA) Table 1. Lists of primers used in this study Gene Forward primer (5 3 ) Reverse primer (5 3 ) SOD1 GGGAGATGGCCCAACTACTG CCAGTTGACATGCAACCGTT SOD2 GCCCTGGAACCTCACATCAA GGTACTTCTCCTCGGTGACGTT CAT ATGGTCCATGCTCTCAAACC CAGGTCATCCAATAGGAAGG ACTB GGATTCCTATGTGGGCGACGA CGCTCGGTGAGGATCTTCATG SOD1, CuZn-SOD, copper-zinc superoxide dismutase; SOD2, Mn-SOD, manganese superoxide dismutase; CAT, catalase; ACTB, actin beta

4 Anti-oxidant Activities of Phytol Figure 3. Cytoprotective effects of phytol against in HaCaT keratinocytes. Protective effects of phytol on cell viability in -treated HaCaT keratinocytes were tested by performing WST-1 assay. After seeding HaCaT cells ( cells/well) on 96-well plates with indicated concentrations of phytol, the cells were incubated for 24 h. The cells were washed with PBS, then they were exposed to 750 μm for 6 h. The data are expressed as M±SD from three independent experiments ( * p<0.05)., hydrogen peroxide; WST-1, water-soluble tetrazolium salt; PBS, phosphate buffered saline; M±SD, mean±standard deviation. Figure 4. Anti-oxidant effects of phytol via DPPH radical scavenging activity. DPPH free radical scavenging activities were estimated to evaluate anti-oxidant activity of phytol. HaCaT cells on 96-well plates with indicated concentrations of phytol were incubated in the dark with DPPH for 30 min. Each sample was measured at 514 nm absorbance. The L-ascorbic acid was used as a positive control. The data are expressed as M±SD from three independent experiments ( * p<0.05). DPPH, 2,2-diphenyl-1-picrylhydrazyl;, hydrogen peroxide; M±SD, mean±standard deviation. 를사용하였다. HaCaT 를 cells/well 로 96-well plate 에접종하여 24 h 배양하였다. Phytol 을 1, 5, 10 µm 농도로 24 h 전처리하고, PBS washing 후 750 µm 를 6 h 후처리하여추가배양하였다. 배양접시에시료를처리한세포에 GSH-Glo reagent 100 μl/well를첨가하여 30 min 동안상온에서반응시켰다. 그후에 15 min 동 µm 농도로 6 h 동안처리한후 WST-1 assay로측정하였다. 무처리대조군의세포생존율 (100%) 을기준으로하고, 100, 250, 500, 750, 1000 µm 처리시 98%, 92%, 85%, 76%, 59% 의세포생존율을확인하였다 (Figure 2). 따라서본연구에서는세포생존율이적어도 70% 이상인 750 µm을실험에사용하기로결정하였다. 안 luciferin detection reagent 100 μl/well 를이용하여반 응시키고, Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, USA) 를이용하여 404/526 nm에서발광정도를측정하였다. 2. 에대한 phytol의세포보호효과 에대한 phytol의세포보호효과를확인하기위하여 HaCaT keratinocytes에 phytol을농도별로배양한다 음 을처리한후, WST-1 assay 를실시하였다. Phytol 8. 통계처리본연구의모든실험은동일한조건하에독립적으로 3 회이상실시하였고, 각실험은 paired t-test를이용하여 p-value를구하고모든실험결과 p-value 값이 0.05 이하인경우에통계적으로유의하다고분석하였다. 과 무처리군세포생존율 (100%) 을기준으로 750 µm 단독처리시세포생존율이 71% 로감소함을확인하였다. Phytol을 1, 5, 10 µm 농도로처리한후 처리된 HaCaT keratinocytes에서는세포생존율이 76%, 86%, 94% 로세포생존율이농도의존적으로증가하였으나, 20 µm phytol 처리된 HaCaT keratinocytes 는세포생존율이 87% Results and Discussion 1. 인간각질형성세포에서 에의한세포독성 HaCaT keratinocytes에 를사용하여산화적스트 로 phytol 10 µm에비해감소하였다 (Figure 3). 따라서향후실험은 1, 5, 10 µm 농도를사용하기로결정하였으며, 이상의결과를통해 로인해유발된산화적스트레스에의한 HaCaT keratinocytes 손상으로부터 phytol의세포보호효과를확인하였다. 레스를유도하고세포생존회복률의변화를확인하기위해, HaCaT keratinocytes 에 를 100, 250, 500, 750, Phytol 의 DPPH 라디칼소거활성 460

5 다. Phytol이 에의해세포내발생하는 ROS의억제효능을확인한결과, HaCaT keratinocytes에 를 750 µm 처리시 를처리하지않았을때보다 ROS가 143% 로증가하였으나 phytol를 5, 10 µm 처리시 119%, 94% 로 ROS가감소되었다. 양성대조군인 L-ascorbic acid를 10 µm 처리시에는 ROS가 107% 측정되어, phytol이 L-ascorbic acid보다높은항산화효과를나타내는것을확인하였다 (Figure 5). Figure 5. Effects of phytol on the induced intracellular ROS levels using the DCFDA in HaCaT keratinocytes. ROS scavenging effects of phytol in -treated HaCaT keratinocytes were tested by performing DCFDA assay. HaCaT cells ( cells/well) were seeded and then treated with the indicated concentrations of phytol and L-ascorbic acid for 24 h. Then, the cells were washed with PBS and exposed to 750 μm for 6 h. They were placed with 10 μm DCFDA for 30 min. Later, ROS scavenging of phytol and L-ascorbic acid were measured by flow cytometer. The L-ascorbic acid was used as a positive control. The data are expressed as M±SD from three independent experiments ( * p<0.05). ROS, reactive oxygen species; DCFDA, 2,7 -dichlorofluorescin diacetate;, hydrogen peroxide; PBS, phosphate buffered saline; M±SD, mean±standard deviation. Phytol 의항산화효능을확인하기위하여가장보편적인 항산화측정방법인 DPPH radical 소거활성을이용하였다. DPPH는화학적으로안정화된 free radical을가지며, 514 nm에서특이적인광흡수를나타내는진한보라색화합물로, 항산화활성이있는물질과만나면전자를내어주고 free radical이소멸되어정량적으로탈색된다. 본연구에서 에의한 HaCaT keratinocytes 생장저해및사멸을억제하는 phytol의항산화효과를확인한결과, phytol을 1, 5, 10 µm 처리시 DPPH 라디칼소거활성이 6%, 17%, 36% 로농도의존적으로증가하였다. 양성대조군인 L-ascorbic acid를 1, 5, 10 µm 처리시에는 DPPH 라디칼소거활성이 7%, 12%, 29% 로증가하여 phytol이양성대조군에비해항산화효과가높음을확인하였다 (Figure 4). 4. Phytol의세포내 ROS 억제활성 Phytol이 HaCaT keratinocytes 세포내 ROS에미치는영향을알아보기위해 DCFDA assay를이용하였다. ROS 측정도구인 DCFDA assay는세포막투과후활성산소와반응하여형광을나타내며산화적스트레스에의한세포손상을측정하기위한방법으로보편적으로사용되고있다. ROS 소거효능으로잘알려진 L-ascorbic acid과 phytol을 HaCaT keratinocytes에처리하여 ROS 소거효과를비교확인하였 5. Phytol의항산화유전자발현촉진효과인체에서대표적인 1차항산화효소인 SOD는산화적스트레스를일으키는 ROS 중 - 을 와산소로전환시키는효소이다 (Allen & Tresini, 2000; Shull et al., 1991). 포유류에서 SOD family는 SOD 활성중심부위에존재하는산화 환원금속이온의종류에따라 SOD1, SOD2, extracellular superoxide dismutase (EC-SOD; SOD3) 로분류된다. SOD1은세포질에서, SOD2는미토콘드리아에서, SOD3는세포바깥쪽에존재하며, 세가지 SOD의기능은비슷하지만염색체위치나단백질구조는서로다르다 (Frederiks & Bosch, 1997; Zelko et al., 2002). CAT는항산화효소로산소호흡으로생명을유지하는생물들의생체내에서필연적으로발생하는 ROS 중하나인 를물과산소로분해한다. CAT는고등동물이나식물에서부터미생물에이르기까지광범위하게발견된다 (Brioukhanov & Netrusov, 2004). 항산화효소 SOD에의해생성된 는 GPx가 GSH 를 GSSG로산화시키면서소거된다. GSSG는 glutathione reductase (GR) 에의해다시 GSH로환원되어인체내에서 glutathione은 GSH와 GSSG의두가지형태로균형을이루면서존재하게된다. 일반적으로세포내에서 98% 이상이 GSH로존재하고 2% 가산화적스트레스에의해서산화형으로변환되어 GSSG로존재한다 (Aslund et al., 1997). 세포내에서독성또는산화적손상이일어난경우에는 GSSG 가서서히증가하여, GSH/GSSG의균형이파괴되어방어기전으로서의역할이소실된다. 이로써세포의 GSH과 GSSG의비율은종종세포독성의지표로사용된다 (Hayes & McLellan, 1999). GSH의생체내에서의생리학적기능은매우다양하지만크게두가지의주요기능으로나누어볼수있다. 첫번째, GSH는생체내의주요항산화성분으로자신의 thiol를이용, 과산화수소와반응하여활성산소를환원하여생체내에서보다쉽게활성산소를제거한다 (Siliprandi et al., 1978). 두번째주요생리학적기능은세포내 cysteine의제공자로서다양한독극물, 마약, 전달물질등이 cysteine 잔기의 thiol 과결함됨으로서이결합체의수용성을증가시켜세포밖으로 461

6 Anti-oxidant Activities of Phytol Figure 7. Effects of phytol on induced the expression level of intracellular GSH in HaCaT keratinocytes. HaCaT cells ( cells/well) on 96-well plates were seeded, then treated with the indicated concentrations of phytol for 24 h. The cells were washed with PBS, and then they were exposed to 750 μm for 6 h. GSH-Glo TM reagent was applied to HaCaT cells in thiol-free buffer to stain them. The approximate fluorescence excitation and emission wavelength for GSH-GloTM reagent is 404/526 nm, and flow cytometer was used. The data are expressed as M±SD from three independent experiments ( * p<0.05). GSH, reduced glutathione;, hydrogen peroxide; PBS, phosphate buffered saline; M±SD, mean±standard deviation. Figure 6. Effects of phytol on induced anti-oxidant enzyme expression in HaCaT keratinocytes. HaCaT cells ( cells/well) were seeded and then treated with the indicated concentrations of phytol for 24 h. Then, the cells were washed with PBS and exposed to 750 μm for 6 h. The expression of SOD1, SOD2 (A), and CAT (B) mrna was determined using the qrt-pcr. The data are expressed as M±SD from three independent experiments ( * p<0.05). SOD1, CuZn-SOD, copper-zinc superoxide dismutase; SOD2, Mn-SOD, manganese superoxide dismutase; CAT, catalase;, hydrogen peroxide; PBS, phosphate buffered saline; qrt-pcr, quantitative real-time polymerase chain reaction; M±SD, mean±standard deviation. 빠르게배출시키는역할을한다. 따라서 GSH는세포내의해독작용을한다 (Dickinson & Forman, 2002). 본실험에서는세포내에존재하는항산화유전자인 SOD1, SOD2, CAT mrna 및 GSH의발현변화를관찰하여 phytol 의 HaCaT keratinocytes에서항산화효과를알아보았다. HaCaT keratinocytes에서 phytol이 SOD1, SOD2 mrna 의유전자발현변화에미치는영향을확인한결과, 를 750 µm 처리시 SOD1, SOD2 mrna의발현량이대조군에비해각각 0.76배, 0.63배정도로감소하였으나 phytol을 1, 5, 10 µm 처리시 SOD1는 0.84배, 0.91배, 1.02배로 SOD2 는 0.72배, 0.75배, 0.89배로모두농도의존적으로증가하여 ROS가소거되고있음을확인하였다 (Figure 6A). HaCaT keratinocytes에서노화억제기전에있어대표적항산화효소인 CAT mrna의유전자발현변화를확인한결과, 를 750 µm 처리시대조군에비해 0.72배로감소하였으나 phytol을 1, 5, 10 µm 처리시 CAT mrna의유전자발현량이 0.86배, 0.89배, 0.92배로농도의존적으로회복되 HaCaT keratinocytes에서 phytol이노화억제기전에있어대표적항산화지표인 GSH 발현변화를확인한결과, 를 750 µm 처리시대조군에비해 0.68배로감소하였으나 phytol을 1, 5, 10 µm 처리시 GSH 발현량이 0.81배, 0.84 배, 1.20배로농도의존적으로회복되어 phytol의항산화효과를확인하였다 (Figure 7). Conclusion 본논문은 HaCaT keratinocytes에서의 phytol의항산화효능을연구하였다. Phytol을 HaCaT keratinocytes에 24 h 전처리후산화적스트레스를유발하는 를 6 h 후처리하였다. 그후 phytol의세포보호효과를검증하기위해서세포생존율, DPPH assay, ROS scavenging activity assay, qrt-pcr, flow cytometry analysis, glutathione assay를실시하였다. Phytol의항산화효능을알아보기위해 DPPH, ROS 정량 462

7 분석, SOD1, SOD2, CAT mrna qrt-pcr, GSH 측정실험을실시하였다. 그결과 DPPH 라디칼소거정도가양성대조군인 L-ascorbic acid와유사한정도로나타났으며, 세포내에서발생하는 ROS는 phytol에의해농도의존적으로소거되었음을확인하였다. 항산화유전자로알려진 SOD1, SOD2, CAT mrna는 phytol에의해농도의존적으로발현이증가하였고, GSH 또한 phytol 농도의존적으로증가하여 phytol의항산화효능을확인하였다. 본연구를통해 phytol 의항산화활성을검증하였고, 이러한연구결과를통해잠재적인노화예방화장품소재로서 phytol의활용가능성을알아보았다. This work is part of the Sun-Hee Jeong s Ph.D. thesis at the Konkuk University, Seoul, Korea. Acknowledgements 본논문이완성되기까지많은조언을주신안성관지도교수님께감사드립니다. References Allen RG, Tresini M. Oxidative stress and gene regulation. Free Radical Biology and Medicine, 28: , Aslund F, Berndt KD, Holmgren A. Redox potentials of glutaredoxins and other thiol-disulfide oxidoreductases of the thioredoxin superfamily determined by direct protein-protein redox equilibria. The Journal of Biological Chemistry, 272: , Brioukhanov AL, Netrusov AI. Catalase and superoxide dismutase: distribution, properties, and physiological role in cells of strict anaerobes. Biochemistry (Moscow), 69: , Dickinson DA, Forman HJ. Glutathione in defense and signaling: lessons from a small thiol. Annals of the New York Academy of Sciences, 973: , Frederiks WM, Bosch KS. Localization of superoxide dismutase activity in rat tissues. Free Radical Biology and Medicine, 22: , Gao Z, Huang K, Xu H. Protective effects of flavonoids in the roots of Scutellaria baicalensis Georgi against hydrogen peroxide-induced oxidative stress in HS-SY5Y cells. Pharmacological Research, 43: , Gross J. Pigments in vegetables: chlorophylls and carotenoids. Springer Publishing, New York, pp29-30, Halliwell B, Gutteridge JM. Free radicals in biology and medicine. Clarendon Press, Oxford, pp86-89, Hayes JD, McLellan LI. Glutathione and glutathionedependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radical Research, 31: , Lee NK, Ku JE, Han HS. Cytoprotective and antiinflammatory effects of 6-shogaol on human dermal fibroblasts. Asian Journal of Beauty and Cosmetology, 15: , Ozben T. Oxidative stress and apoptosis: impact on cancer therapy. Journal of Pharmaceutical Sciences, 96: , Park S, Shim JH. Anti-aging effect of Psoraleae Fructus extract in UVA-irradiated HaCaT Cells. Asian Journal of Beauty and Cosmetology, 14: , Rhee SG., a necessary evil for cell signaling. Science, 312: , Shull S, Heintz NH, Periasamy M, Manohar M, Janssen YM, Marsh JP, Mossman BT. Differential regulation of antioxidant enzymes in response to oxidants. The Journal of Biological Chemistry, 266: , Siliprandi N, Siliprandi D, Bindoli A, Toninello A. Effect of oxidation of glutathione and membrane thiol groups on mitochondrial functions. In: functions of glutathione in liver and kidney. Sies H, Wendel A (ed.), Springer Science+Business Media, Berlin, pp , Slater TF. Free-radical mechanisms in tissue injury. Biochemical Journal, 222: 1-15, Thannickal VJ, Fanburg BL. Reactive oxygen species in cell signaling. American Journal of Physiology - Lung Cellular and Molecular Physiology, 279: , Zelko IN, Mariani TJ, Folz RJ. Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radical Biology and Medicine, 33: ,

8 Anti-oxidant Activities of Phytol 국문초록 정선희수원여자대학교미용예술과, 경기도수원시, 한국 목적 : 본연구는인간각질형성세포에서 phytol의항산화, 세포보호효능을검증하여화장품소재로서의가능성을알아보는데목적이있다. 방법 : Phytol의효능을검증하기위해세포생존율, 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, 2,7 -dichlorofluorescin diacetate (DCFDA) assay, reactive oxygen species (ROS) scavenging activity assay, flow cytometry analysis, quantitative real-time polymerase chain reaction (qrt-pcr), glutathione assay 를실시하였다. 결과 : 인간각질형성세포에서 와 phytol 의실험농도를결정하기위해, 다양한농도로 water-soluble tetrazolium salt (WST-1) assay 를실시하였다. 실험결과에따라 는 750 µm, phytol 은최대 10 µm을실험에사용하였다. 항산화유전자로잘알려진 SOD1, SOD2, CAT mrna 는 phytol 농도의존적으로발현이증가하였고, GSH 또한 phytol 농도의존적으로증가하여항산화기능이회복되는것을확인하였다. 결론 : 이상의실험들을통해 phytol 의항산화효과및인간각질형성세포보호효능을검증하였으며잠재적인노화억제화장품소재로서활용가능성을제시하였다. 핵심어 : Phytol, 항산화, 세포보호, 화장품, 노화 본논문이완성되기까지많은조언을주신안성관지도교수님께감사드립니다. 참고문헌 박선영, 심중현. 자외선조사에의해노화된 HaCaT 세포에서보골지추출물의항노화효능. 아시안뷰티화장품학술지, 14: , 이나경, 구정은, 한효선. 인간진피섬유아세포에서 6-Shogaol 의세포보호및항염증효과. 아시안뷰티화장품학술지, 15: ,

9 中文摘要人角质形成细胞中 phytol 的抗氧化效能鄭善熙水原女子大学美容艺术科, 京畿道水原市, 韩国目的 : 鉴定人角质形成细胞中 phytol 的抗氧化和细胞保护效能, 探索作为化妆品原料的可行性方法 : 为确定 phytol 的效能, 进行细胞生存率,2,2-diphenyl-1-picrylhydrazyl (DPPH) assay,2,7 -dichlorofluorescin diacetate (DCFDA) assay, reactive oxygen species (ROS) scavenging activity assay,flow cytometry analysis, quantitative real-time polymerase chain reaction (qrt-pcr),glutathione assay 等实验结果 : 在人角质形成细胞中, 为确定和 phytol 的实验浓度, 对不同浓度的 phytol 进行 water-soluble tetrazolium salt (WST-1) assay 实验结果显示, 的最佳实验浓度为 750 µm, phytol 的最大范围使用浓度为 10 µm 抗氧化遗传因子 SOD1 SOD2 CAT mrna 的表达程度根据浓度依赖性逐渐增加, GSH 也根据浓度依赖性逐渐恢复其抗氧化能力结论 : 通过以上的实验结果, 鉴定了 phytol 的抗氧化效能以及人角质形成细胞的保护效果, 因此作为潜在的抗衰老化妆品原料充分提出了活用可行性关键词 : Phytol, 抗氧化, 细胞保护, 化妆品, 衰老 465

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,