RESEARCH ARTICLE 과산화수소 (H 2 O 2 ) 에의해유도된인간각질형성세포주손상에대한키네틴 (Kinetin) 의항산화효과 김수연 1, 김민정 2, 안규중 2, 안인숙 3, 안성관 3 * 1 두원공과대학교뷰티아트과, 2 건국대학교의학전문대학원피부과학교실, 3 건국대학교한국피부임상과학연구소 Antioxidant Effects of Kinetin on HaCaT Human Keratinocytes Damaged by Hydrogen Peroxide (H 2 O 2 ) Soo-Yeon Kim 1, Min Jung Kim 2, Kyu Joong Ahn 2, In-Sook An 3, Sungkwan An 3 * 1 Department of Beauty Art, Doowon Technical University College 2 Department of Dermatology, Konkuk University School of Medicine 3 Korea Institute for Skin and Clinical Sciences, Konkuk University Abstract This study aimed to verify functions of kinetin as a raw material for cosmetics products in skin through the cell efficacy test. Kinetin, plant cytokinins, regulates plant growth. It has been reported that kinetin has antioxidant effects, synthesis promotion of DNA, and senescence delay on dermal fibroblast in cellular function. But there is no study reported in effects of kinetin on keratinocytes. Hydrogen peroxide (H 2O 2) is a species of reactive oxygen, affects to fate of cells in growth, senescence, and extinction. H 2 O 2 is unstable property because of free radicals. As unstable property, H 2 O 2 reacts strong with other molecules and damage to cells and DNA. To verify the efficacy of kinetin on HaCaT human keratinocyte cells, the cell viability essay, and reactive oxygen species (ROS) assay were performed. As kinetin with concentrations of 200 μm and above were toxic in HaCaT cells, this study used kinetin with concentrations of less than 200 μm. Cell viability assay showed that kinetin efficiently rescued HaCaT cells from H 2 O 2 -induced oxidative stress damages in a concentration-dependent manner. Furthermore, flow cytometry analysis revealed that kinetin decreased the intracellular ROS levels in H 2 O 2 -treated HaCaT cells. These data indicated that kinetin can be used as a raw effective natual material for for functional cosmetic products which can reduce facial wrinkles, came from ROS, and improve the condition and function of skin. Keywords: Kinetin, Antioxidant, Keratinocyte, Reactive oxygen species, Hydrogen peroxide 급속한경제성장과사회 문화적환경변화로여성들의사회 진출이활발해지고각분야로다양하게확산됨에따라아름다 움에대한관심과욕구가더욱강해지고있다. 이에따라외모 *Corresponding author: Sungkwan An, Korea Institute for Skin and Clinical Sciences, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea Tel.: +82 2 3436 3777, Fax: +82 2 3436 3778 E-mail: ansfgrc@konkuk.ac.kr Received January 5, 2015; Revised February 6, 2015; Accepted February 13, 2015; Published February 28, 2015 를비롯한신체적아름다움을표현하는기법의하나로미용이발전변화되어왔다 (Kim, 2012). 오늘날의미용은헤어, 피부미용, 메이크업 (make-up) 의개념에서한단계더발전된네일에이르기까지다양해졌으며 (Woo, 2005), 아름답게가꾸고자하는미적본능에따라자신의외모와자기표현방식에서도다양한개성을선호하게되었기때문에미용산업의발달이점점가속화되고세분화되고있다 (Kim, 2012). 미용분야가운데서도특히피부에대한관심이커지면서깨끗한피부를선호하는경향이매우높아졌으며부드럽고매끄러운피부는아름다움의상징으로인식되고있다 (Kim, 2003). 피부는단순히신체를덮고있는것같지만실제로는외부환경으로부터신체를보호해주며땀배출등유용한기능도겸하 www.kosac.or.kr 59
고있다 (Sim, 2007). 피부조직은바깥에서부터표피, 진피, 피하지방층으로구성되어있고표피는각질형성세포가대부분을차지하고있으며분화정도에따라기지층, 유극층, 과립층, 각질층으로나뉘고, 그외멜라닌세포, 랑게르한스세포, 메켈세포로형성되어있다 (Jang, 2014). 피부표피층에있는각질층은단백질이풍부한각질세포와각질세포사이를채우고있는지질로구성되어있으며피부장벽을이루는기본구조를가지고있다. 각질층의주성분은케라틴단백질과지질그리고천연보습인자가차지하고있으며 (Song, 2011), 각질층지질의주성분에는표피의층판소체 (lamellar body) 에서유래한세라마이드, 에스테르, 왁스에스테르, 스쿠알렌, 콜레스테롤, 중성지질, 자유지방산등이있다 (Kim, 2014). 상부과립층에있는층판소체는각질층전체무게의 6 10% 를차지하며이곳에서는지질이분비되어각질세포사이로퍼져나가게된다. 각질세포사이지질의층판이형성되면이층판은체액의손실을막고경피흡수를조절하는장벽기능을갖게되며각질형성세포들간의순조로운결합과이탈에관여하는중추적인역할을한다 (Blanken et al., 1989; Elias et al., 1992). 각질형성세포의증식과분화를조절하는인자로는칼슘, 레티놀산, 비타민D 등이있으며 (Kwon et al., 2007), 칼슘은세포의생장과분화그리고유전자발현을조절하는중요한물질이다. 표피내칼슘기울기는피부장벽의기능유지여부와직접적인관계가있으며피부장벽이손상될경우상부표피에서칼슘이온이빠져나가층판소체의분비와생성을활성화시켜피부장벽을회복시킨다 (Penn et al., 1987). 건강한피부에대한기준은각질층의수분이항상 30% 정도로유지되는상태를말하며 (Kim et al., 2013), 적정한수분유지는각질세포와각질세포사이지질의수분손실을막는데필수적인요건이다. H 2 O 2 는활성산소종의하나로세포의생장과소멸그리고노화등그운명을결정짓는중요한요소이다. 활성산소란자유라디칼 (free radicals) 을포함한생체내의불안정한산소를지칭하며이러한활성산소는불안정하기때문에강하게활성화하는특성을가지고있다 (Frippiat et al., 2001). 불안정한특성에따라주위의물질과매우강하게반응하기때문에 (Beckman et al., 1988), UV와같은외부자극요인의지속적피부노출은 H 2 O 2 와같은활성산소의지나친생성으로산화적스트레스에의해세포손상을일으키고더나아가유전자와단백질구조를변화시켜피부노화를가속화한다 (Kim, 2012; Son et al., 2013; Jung et al., 2013). 피부가건조하다는것은피부보습에중요한각질층의수분함량이떨어진상태이며이럴경우건조증상과피부장벽기능이상이나타나는데특히수분함량이 10% 이하로내려가면 (Lee, 2009), 피부색이칙칙해지고윤기와탄력이떨어져주름 Figure 1. The chemical structure of kinetin. 이생기면서점차노화가시작된다 (Kang et al., 1993). 노화된피부에서장벽기능이손상되면당연히회복되는속도가현저히늦어지는데이때보습제를도포하면피부장벽기능회복에많은도움이된다 (Choi et al., 2008). 따라서대부분의보습제는피부표면의수분공급은물론각질세포사이지질의생성과분비를원활하게함으로써, 천연보습인자의수분기능과피지막의밀폐기능그리고지질의장벽기능을보다효율적으로보완할수있다 (Park et al., 2003). 건강하고매끄럽고윤기있는피부를유지하기위해서는지속적으로수분을충분히공급해주고적절한보습제를골라정기적으로사용하는것이대단히중요하며 (Park, 2007), 보습제를지속적으로사용할경우피부장벽의기능강화에상당한도움을준다 (Lee et al., 2011). Kinetin은식물생장조절기능을가지고있는사이토카이닌 (cytokinins) 중의하나이다 (Won, 2007; Figure 1). 1992년에진피섬유아세포의 DNA 합성을촉진하는 kinetin의기능이보고되었으며 (Olsen et al., 1997), 1994년에는진피섬유아세포의세포노화를지연시킨다는보고도나와 kinetin이항노화물질로학계에알려지면서 1997년과 2002년에는광노화피부에서진피를개선시키는효과까지확인된바있다 (Mccullough et al., 2002). 그러나 kinetin이표피에서는각질형성세포 (keratinocyte) 의분화를조절할수있다는가능성만제시되었을뿐 (Kligman et al., 1986), kinetin이표피에미치는영향이나효능에대한총체적인연구는제대로이루어지지못한상태이다. 이를위해본논문에서는 kinetin의세포효력시험을통해 kinetin의항산화효능을검증함으로써화장품원료로서의가능성을제시하고자한다. 1. 각질형성세포주배양본연구에서사용한각질형성세포주 (HaCaT keratinocytes) 는 ATCC (USA) 사에서구입하였으며세포의최적배양을위해 10% fetal bovine serum (FBS; Sigma-Aldrich, USA) 과 1% penicillin/streptomycin (Welgene, Korea) 이첨가된 DMEM 60 www.kosac.or.kr
키네틴 (Kinetin) 의항산화효과 Figure 2. Cytotoxicity of kinetin on HaCaT keratinocytes. Figure 3. Cytotoxicity of H 2 O 2 on HaCaT keratinocytes. 배지 (Welgene) 를사용하였고 37, 5% CO 2 조건에서세포배양기를통해서배양하였다. 2. 시료처리본연구에사용된 kinetin (Sigma-Aldrich) 은 dimethyl sulfoxide (DMSO) 에녹여사용하였다. Cell culture dish에 HaCaT 세포주 (1 10 6 ) 를 24 h 배양한후 kinetin을배지에첨가하여 3 h 동안전처리하였다. 전처리후배지를제거하고 phosphate-buffered saline (PBS) 로 2회세척한뒤, 배지제거후 2 h 동안처리한후 PBS로세척하였다. 그후 kinetin을새로운배지에첨가하여 24 h 배양해실험을실시하였다. 다. ROS의변화량측정은배양한세포를 PBS로세척하여 1% trypsin-edta 용액을처리하여세포수확한뒤다시 PBS로세척하여 Flow cytometry (Becton Dickinson, USA) 를이용하여변화량을측정하였다. 7. 통계처리본연구의모든실험은독립적으로 3회실시하였으며, 각실험에대하여 student t-test를이용하여 p-value를구하고, p-value 값이.05 이하인경우통계적으로유의하다고분석하였다. 3. 세포생존율 Cell viability를측정하는실험에는 WST assay 원리를이용한 EZ-Cytox cell viability assay kit (Itsbio, Korea) 을사용하였다. HaCaT 세포주 (3 10 3 ) 를 96-well plate에배양하고, 24 h이경과한후 well당 kinetin과 H 2 O 2 를각각 50, 100, 200, 400, 600 μm의농도로처리하여 24 h 동안배양하였다. 배양된세포에대해서는 kit reagent를 10 μl 첨가하여 1 h 배양한뒤 microplate reader (Bio-Rad, USA) 를이용하여 490 nm의파장에서흡광도를측정하는방법을사용하였으며 Cell viability 측정은 3회반복수행하여평균값을도출하였다. 4. ROS 생성도분석세포내활성산소의생성을측정하기위한방법으로는형광 probe DCF-DA를이용하였다. 비형광물질인 DCF-DA (Sigma-Aldrich) 는세포내 hydrogen peroxide (H 2 O 2 ) 와관련된물질이있을경우 ROS에의해산화되어녹색의형광을띠게되므로 ROS의측정은 DCF-DA를통해결과를얻을수있다. 2 10 5 개의 HaCaT cell을 60 mm culture dish에접종하여 24 h 동안배양하였고세포를수확하기전에 10 μm DCF-DA를처리하여 37 의조건에서 30 min 동안배양하였 1. Kinetin의세포독성평가 Kinetin의 HaCaT 세포에서의독성정도를확인해보기위해 HaCaT 세포에 kinetin을각각 0, 50, 100, 200, 400 μm 농도로 24 h 처리한뒤세포독성을확인한결과 100 μm까지는큰변화가없었으나 200 μm kinetin 처리시에는 8.88%, 400 μm 처리시에는 18.99% 세포생존율이감소하는것으로나타났다 (Figure 2). 200 μm 이상의농도에서는세포독성이점차강해져향후 HaCaT 세포모델실험에서는 200 μm 까지농도에서만 kinetin을사용하였다. 2. Kinetin의 H 2 O 2 에의한세포손상억제효과 HaCaT 세포에 H 2 O 2 를각 40, 60, 80, 100, 200, 400, 600 μm로처리할경우 100 μm의농도에서는생존율이 77.13% 였으며, 그이상의농도에서는생존율이 70% 이하로나타났다. H 2 O 2 의적용농도는 100 μm로설정하였다 (Figure 3). Kinetin에 H 2 O 2 100 μm를함께처리하여세포생존율의변화를확인한결과농도의존적으로세포생존율이변화하고있음을알수있었다. Kinetin 50 μm은 81.94%, 100 μm www.kosac.or.kr 61
Figure 4. The effect of kinetin on cell viability in H 2 O 2 -treated HaCaT keratinocytes. Figure 5. The effect of kinetin on the intracellular ROS. 은 91.29%, 200 μm은 97.40% 의생존율을보였다. 이를통해 H 2 O 2 100 μm 처리시 24.49% 감소했던세포생존율이 kinetin에의해점차농도의존적으로회복됨을확인할수있다 (Figure 4). 향후실험은 kinetin 100, 200 μm 농도를사용하였다. 3. Kinetin의활성산소제거능각질형성세포에 H 2 O 2 를처리하여산화적스트레스를유발한후형광 probe DCF-DA를이용하여 ROS 제거능을확인하였다. H 2 O 2 100 μm를처리하면 ROS가 3배이상증가했으나 kinetin 100 μm을전처리하면 2.13배, 200 μm 전처리시에는 1.36배로농도의존적으로감소하였다 (Figure 5). 실험결과 kinetin이 HaCaT 각질형성세포에서 H 2 O 2 에의한 ROS를효과적으로제거하는것을확인할수있었다. 본연구에서는세포효력시험을통해 HaCaT 각질형성세포에서 kinetin의항산화능을확인하였다. 먼저 kinetin의세포독성을확인한결과, 인간각질형성세포주 HaCaT에서 200 μm까지는세포독성이 10% 미만이었으나 200 μm이상에서는세포독성이 10% 를넘어 600 μm에서 70% 의세포생존율을기록하였다. 따라서세포생존율이 90% 이상을기록하는 200 μm을최고농도로하여이후실험을진행하였다. H 2 O 2 를사용하여활성산소로인한세포생존율변화를확인한결과, 100 μm의 H 2 O 2 농도에서는생존율이 77.13% 였으며, 그이상의농도에서는생존율이 70% 이하로나타나, 이후실험에서사용할 H 2 O 2 의적용농도는 100 μm로하였다. H 2 O 2 처리로인해손상된각질형성세포에 Kinetin을처리 함으로써 kinetin 이손상된각질형성세포의세포생존율에미치는영향을확인해보았다. Kinetin을농도의존적으로처리하여 H 2 O 2 로인해손상된각질형성세포가회복되는것을세포생존력증가를통해확인하였고그농도에따라 kinetin 50 μm, 100 μm, 200 μm에서각각 81.94%, 91.29%, 97.40% 의생존율을보여 H 2 O 2 처리로인해 75% 까지감소한세포생존율이다시증가하는것을확인하였다. 이를통해 kinetin이활성산소로인한세포의사멸을저해하는것을확인하였다. 다음으로 DCF-DA를이용하여 kinetin의 ROS 제거능을확인한결과 H 2 O 2 처리에의해 3.44배증가하였던활성산소가 kinetin 100 μm에서 2.13배로감소하였고 kinetin 200 μm에서는 1.36배로 kinetin 농도의존적으로감소한것을확인하였다. DCF-DA를이용한 ROS 제거능확인으로 kinetin의항산화효과를확인할수있었다. 각질형성세포에서의 kinetin 실험을통하여, H 2 O 2 로인해유도되는각질형성세포의손상에대한 kinetin 의항산화효과를확인하였으며이로써 kinetin이손상된피부장벽을개선시키는효과를유추해볼수있다. 본연구는 kinetin이표피에미치는영향과효능에대한연구결과를통해 kinetin의항노화성분및기능성화장품원료로서의가능성을제시한다. 감사의글본논문은보건복지부보건의료연구개발사업의지원 ( 과제번호 : HN13C0075) 에의한것이며, 이에감사드립니다. Beckman K, Ames BN. The free radical theory of aging natures. Physiol., 78: 547-581, 1988. Blanken R, Vilsteren MJ, Tupker RA, Coenraads PJ. 62 www.kosac.or.kr
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oxidant Activity of Oil Extracted from Korean Red Ginseng and Its Moisturizing Function. Kor. Soc. Aesthet. Cosmetol., 11: 489-494, 2013. Song YA. Influence in application of sesame seed oil to the skin on a change in the aging and dry skin condition. Master s Thesis, Sungshin Women s Univ., 2011. Won CH. Kinetin enhances in vitro development of porcine parthenogenetic and nuclear transfer embryos. Master s Thesis, Seoul nat. Univ., 2007. Woo SJ. A Study on Make-up and Nail Art in College Women (Centering on Seoul and Jeonbuk Areas). Master s Thesis, Sookmyung Woomen s Univ., 2005. 64 www.kosac.or.kr