大韓本草學會誌제 29 권제 6 호 (2014 년 11 월 ) Kor. J. Herbology 2014;29(6):63-71 ISSN 1229-1765(Print), ISSN 2288-7199(Online) http://dx.doi.org/10.6116/kjh.2014.29.6.63. 금은화물추출물의항산화효과와 Ultraviolet(UV)B 로유도된사람각질형성세포손상에대한보호효과 서승희 1#, 배기상 2, 최선복 3,4, 조일주 3,4, 김동구 3,4, 신준연 3,4, 송호준 3,4, 박성주 2,3,4, 최미옥 5* 1 : 동신대학교뷰티미용학과, 2 : 원광대학교한방체액조절연구센터, 3 : 원광대학교한의과대학본초학교실, 4 : 원광대학교한의학전문대학원 BK21 플러스팀, 5 : 광주여자대학교미용과학과 The antioxidative and cytoprotective effect of Lonicerae japonicae Flos water extracts on the ultraviolet(uv)b-induced human HaCaT keratinocytes. Seung-Hee Seo 1,#, Gi-Sang Bae 2, Sun Bok Choi 3, Il-Joo Jo 3,4, Dong-Goo Kim 3,4, Joon-Yeon Shin 3,4, Ho-Joon Song 3,4, Sung-Joo Park 2,3,4, Mee-Ok Choi 5,* 1 : Department of Cosmetology, Dongshin University, Naju, Jeonnam, South Korea, 2 : Hanbang Body-Fluid Research Center, Wonkwang University, Iksan, Jeonbuk, South Korea, 3 : Department of Herbology, School of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk, South Korea, 4 : BK21 Plus Team, Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk, South Korea, 5 : Department of Beauty Science, Kwangju Women s University, Kwangju, South Korea ABSTRACT Objectives : Lonicerae japonicae Flos(LJF) has been reported to exhibit anti-oxidant, anti-inflammatory, anti-viral, anti-rheumatoid properties. However, it is still largely unknown whether LJF inhibits the ultraviolet(uv)b-induced oxidative damage in human HaCaT keratinocytes. Therefore in this paper, we investigated the anti-oxidative capacity and protective effect of LJF against UVB-induced oxidative demage in human HaCaT keratinocytes. Methods : To evaluate the anti-oxidative activity of LJF extracts, we measured total phenolic contents, total flavonoid contents, antioxidant capacity, and superoxide scavenging activity. To give an oxidative stress to HaCaT cells, UVB was irradiated with 200 mj/ cm2 to HaCaT cells. To detect the protective effect of LJF against UVB, we measured cell viability, DNA fragmentation and reactive oxygen species (ROS) production. In addition, we performed high-performance liquid chromatography (HPLC) analysis to find a major component of LJF. Results : LJF contained phenolic and flavonoid contents, and showed the anti-oxidant and superoxide scavenging activity. The UVB-induced oxidative conditions led to the cell death, DNA fragmentation and reactive oxygen species (ROS) production. However, pretreatment with LJF reduced oxidative conditions, including inhibition of cell death, DNA fragmentation and ROS production. In addition, we found out chlorogenic acid as major component of LJF. Conclusions : These results could suggest that LJF contained anti-oxidative contents and exhibited protective effects against UVB on human HaCaT keratinocytes. And the effective compound of LJF which could show protective activities against UVB is chlorogenic acid. Thus, LJF and chlorogenic acid would be useful for the development of drug or cosmetics treating skin troubles. Key words : HaCaT keratinocytes, Lonicerae japonicae Flos, UVB(ultravioletB), Reactive Oxygen Species (ROS), chlorogenic acid 서론 1) 피부는크게표피, 진피, 피하조직으로구성되어있으며체온조절, 보습, 감각작용, 방벽작용. 분비및배설작용, 호흡작용, 흡수작용, 영양소저장및비타민 D합성등다양한생리작용을함으로써정상적인피부를유지하는데주된작용을하고있다 1). 하지만피부표피에해당하는각질형성세포 (keratinocytes) 는태양광선 ( 자외선 ) 을비롯한외부자극에직접적으로노출되는
64 大韓本草學會誌 Vol. 29 No. 6, 2014 최외각층으로서반복적으로많은양의자외선에노출되면산화적스트레스 (oxidative stress) 를유도하여활성산소종 (reactive oxygen species, ROS) 이생성된다 2). 자외선으로유도된활성산소종은피부의광산화적손상을유도하여노화를가속화시킨다. 피부에직접적으로영향을주는자외선은표피와진피층까지투과하며다양한과정을거쳐서세포및조직에활성산소를생성시킨다 3). 생성된활성산소는피부의효소적, 비효소적항산화방어체계의불균형을발생시키고지질과산화로인한세포막의손상으로정상적인세포의기능을하지못하게된다. 그로인해피부는지속적인산화상태에회복되지못하게되면서표면이거칠고윤기가없어지면서탄력저하나주름발생과같은피부노화현상이표출된다 4). 따라서산화적스트레스 (oxidative stress) 로부터각질형성세포를보호하고노화를지연시키기위해서는체내항산화방어시스템을향상시키는것이매우중요하다 5). 이에따라자외선으로부터피부를보호하기위하여자외선의피부침투를차단시키거나, ROS를조절하여피부손상에따른염증을막고활성산소를제거하는자외선차단제, 항염증, 항산화제개발에대한관심이증가하고있다 6-8). 현재연구되고있는항산화제는크게합성항산화제와천연항산화제로구분할수있는데, 최근에몇몇합성항산화제와관련한위험성에대한보고로인하여효과적이면서도독성이없는천연에서얻어진약용식물을이용한항산화제개발에관한연구가활발히진행되고있다 9,10). 특히천연약용식물의추출물은여러약리적효능과더불어항산화제로작용할수있는생리활성물질이있어유용한연구의소재가되고있다 11). 금은화 ( 金銀花, Lonicerae japonicae Flos, LJF) 는인동과 (Caprifoliaceae) 에속하는인동덩굴 (Lonicera japonica Thunb.) 의꽃으로서한방에서는利尿, 淸熱解毒, 外感風熱, 熱毒瀉痢, 健胃, 涼散風熱, 擁腫精瘡, 喉痹, 丹毒, 熱血毒痢, 風血感冒, 溫炳發熱등에효능을갖고있는본초이다. 금은화에대한최근연구동향분석에따르면감염성질환, 외이도염, 화농증, 중이염, 바이러스성결막염, 인플루엔자, 폐렴등의염증성질병에사용되고있다 12,13). 뿐만아니라항균, 항바이러스효과 14), 항산화효과 15), 간보호효과 16), 항암효과 17), 면역증진효과 18) 등이있으며, 그외에혈소판활성및코카인중독, 망막허혈등에이르기까지폭넓은분야에걸쳐연구가이루어지고있다 19). 그동안진행된연구결과들에의하면다양한용매에추출된금은화의성분들은 chlorogenic acid, saponins, tannin계, flavonoid계의 luteolin, lonicerin, apiogenin, quercetin, ochnaflavone, astragalin등이배당체로존재하는것으로알려져있고, 이에대한다양한연구들이진행되고있다 20,21). 이와같이다양한효능을지니고있는금은화에대한약리학적작용과임상적이용가치에대해서는다수의정보가있으나, HaCaT Keratinocyte 사람각질형성세포에서금은화를이용한항산화효과에대한연구는본연구진의연구외에는미흡한실정이다 22). 따라서, 본연구는금은화물추출물에의한항산화능력조사와 Keratinocyte 에 서 UVB로유도된산화적손상에서의세포보호효과에대해서보고하는바이다. 재료및방법 1. 재료 1) 약재실험에사용한금은화는옴니허브 ( 경북영천, 대한민국 ) 에서구입하여정선해서사용하였다. 2) 세포주및배양본실험에사용된 HaCaT 세포는 Addexobio (San Diego, Califonia, USA) 로부터분양받아실험에사용하였다. 각질형성세포주인 HaCaT 세포주를 10% fetal bovine serum(fbs) 과 1% penicillin/streptomycine을첨가한 Dulbecco's modified Eagle's medium (DMEM) (GibcoBRL, Braunscheig, Germany) 배지를사용하여 5% CO 2, 37 세포배양기에서배양하면서실험에사용하였다. 2. 방법 1) 약물추출실험에사용된금은화 100g을 1L에약탕기 ( 대웅, 한국 ) 로 100 에서 3시간가열추출한다음여과한후, 동결건조하여금은화 17.4g의건조분말을얻었으며, 실험을위하여 4 에보관했다. 실험시에는증류수에녹여 stock solution (100 mg/ml) 을제조하였다. 제조된 stock solution 은 -2 0 에보관하면서분석방법에적합하도록희석하여사용하였다. 2) UVB 조사세포의자외선조사는 300~315 nm를가장강하게방출하는 UVB lamp 306 nm ( 동서과학, 한국 ) 를사용하였으며, UV 광량은 UVA/UVB light meter 850009(Sper scientific, Scottsdale, AZ, USA) 로측정하였다. UVB의조건을잡기위해서 DMEM 배지를제거한다음, Phosphate buffered saline ph 7.4 (PBS) 로 2번씻어낸후자외선조사동안세포가건조되지않도록 PBS 1 ml을세포배양접시에넣은후 UVB를선량별로 50, 100, 200, 300 mj/ cm2를조사하였다. UVB 200 mj/ cm2에서조건이잡힌후실험에서는금은화추출물및천연추출물을 0.1, 0.25, 0.5 mg/ml 농도로 1시간처리한후 UVB 200 mj/ cm2조사하였다. PBS 1 ml로 1 번씻어낸다음금은화추출물및천연추출물이 0.1, 0.25, 0.5 mg/ml 농도로첨가된배양액으로갈아주었다. 세포는 UVB 200 mj/ cm2를조사하고 4시간이지난후에실험에사용하였다. *Corresponding author : Mee-Ok Choi. Department of Beauty Science, Kwangju Women s University, Kwangju, South Korea Tel : +82-61-330-3297 E-mail : cmo0323@hanmail.net #First author : Seung-Hee Seo. Department of Cosmetology, Dongshin University, Naju, Jeonnam, South Korea Tel : +82-61-330-3297 E-mail : ssh@dsu.ac.kr Received:14 October 2014 Revised:3 November 2014 Accepted:11 November 2014
금은화물추출물의항산화효과와 Ultraviolet(UV)B 로유도된사람각질형성세포손상에대한보호효과 65 3) 세포독성분석 HaCaT cell의생존율은밀집세포의미토콘드리아탈수소효소에의해자줏빛 formazan 생성물로변하는 MTT 환원을바탕으로 MTT 분석법으로측정했다. 세포들은 DMEM 배지에서 5 10 4 cells/well 의밀도로현탁하여 0.1, 0.25, 0.5 mg/ml 의농도로금은화추출물및천연추출물을처리하였다. 24시간배양후 5 mg/ml 의농도로 MTT 용액을첨가후 30분동안배양하였다. MTT-formazan 생성물은 DMSO 200 µl를첨가함으로써용해했다. formazan 의양은용해액을 96well plate에 loading 한후, 생성물을 DMSO로녹여생성된크리스탈의양을 spectrometer (molecular devices, CA, USA) 기기를사용하여, 최대세포생존률을반영하는파장인흡광도 540 nm에흡수되는양을측정함으로서결정하였다. 4) 추출물의성분분석 (1) 총페놀함량측정금은화추출물 100 µl(1 mg/ml) 에 Folin-ciocalteau reagent 50 µl를가하고 5분간상온에서안정화시킨후 20% sodium carbonate 300 µl를가하고, 상온에서 15분동안안정화시킨후증류수 1 ml를넣어혼합한후에 725 nm 에서흡광도를측정하였다. 페놀화합물함량은표준물질 gallic acid를이용하여검량선을작성한다음정량하여 GAE(gallic acid equivalents) 로나타내었다. (2) 총플라보노이드함량측정금은화추출물 100 µl(1 mg/ml) 에 10% aluminum nitrate 20 µl, 1 M potassium acetate 20 µl 그리고 80% 에탄올 860 µl를차례로가하여혼합하고실온에서 40분동안안정화시킨다음 415 nm에서흡광도를측정하였다. Quercetin 을표준물질로이용하여검량선을작성한다음 QE(quercetin equivalents) 로타나내었다. 5) 추출물의항산화활성측정 (1) 총항산화능측정금은화추출물에 0.35 M acetate 완충용액과 0.89 mm 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) 용액및 0.44 mm hydrogen peroxide 용액등을첨가하고, 혼합한뒤 5분후에 655 nm 에서흡광도를측정하였다. Trolox를표준시약으로사용하여표준곡선을작성하였고, 총항산화능은 mm Trolox equivalent 로표기하였다. (2) Superoxide 소거활성측정금은화추출물에 62 µm nitroblue tetrazolium(nbt) 와 98 µm β-nicotinamide adenine dinucleotide (NADH) 를함유한 20 mm Tris 용액 (ph 8.0) 을혼합한다음, 20 mm Tris 용액과 33 µm phenazine methosulfate(pms) 를각각첨가하였다. 즉, 비효소적으로 PMS/NADH 로유발된 superoxide 는 NBT 를자주색의 formazan 으로환원시키며, 생성된 formazan 을측정하기위해 540 nm에서 10 분동안반응물의흡광도를측정하였다. 추출물의 superoxide 소거활성 (%) 은다음과 같은공식으로구하였다. 시료첨가흡광도 Superoxide scavenging activity (%) = ( ) X 100 시료무첨가흡광도 6) 형광유세포분석 (Fluorescence-activated cell sorting, FACS) (1) ROS 생성도분석 (dichlorofluorescein diacetate, DCF-DA) 세포내활성산소의생성을측정하기위하여형광 prob는 DCF-DA(Sigma, MO, USA) 를이용하였다. HaCaT 세포에금은화추출물을 0.1, 0.25, 0.5 mg/ml 농도로 1시간전처리한후 UVB 200 mj/ cm2를조사한후 4시간동안배양하였다. 세포를수확하기전에 10 µm DCF-DA를처리하여 37 에서 20분동안배양하였다. 배양한세포는 PBS로세척하여 1% Trypsin-EDTA(Sigma, MO, USA) 용액을처리하여세포를수확하고다시 PBS로세척하여 flow cytometry (FACS CAlibur, BD Biosciences) 로형광을측정하였다. 정보의분석은 Cell Quest software (Becton Dickinson) 를이용하였다. 7) 형광현미경분석실험 (Flourscence microscopy) (1) DNA 염색분석 (4',6-diamidino-2-phenylimdole, DAPI) HaCaT 세포에금은화추출물을 0.1, 0.25, 0.5 mg/ml로전처리한후 UVB 200 mj/ cm2조사후 4시간동안배양하였다. PBS로 2회세척후 4% paraformaldehyde 용액으로 30분동안실온에서고정하고 PBS로 3회세척하였다. 세포의핵은 DAPI (Sigma, MO, USA) (10 mg/ml) 사용하여 5분간염색하고 PBS로 3회세척하였고, prolong gold anti-fading mount solution (invitrogen, CA, USA) 를이용하여마운팅하였다. 그후 Flourscence microscopy (Olympus X 70, Tokyo, Japan) 으로관찰하였다. 8) DNA 분절측정 (1) 세포 DNA분절샘플준비 Cellular DNA fragmentation Kit (Millipore, MA, USA) 를이용하여 DNA의분절정도를측정하였다. 먼저 HaCaT 세포를 2 10 5 cells/ml 로현탁하고, bradu labeling solution을 10 µm을첨가하여 5% CO 2, 37 조건하에서 2시간배양하였다. 그후 250 g에서 10분간원심분리하여상층액을제거한다음, 세포에 DMEM 배지를각 200 µl씩첨가하여 96well plate에넣어주었다. HaCaT 세포에금은화추출물을 0.1, 0.25, 0.5 mg/ml 농도로처리한후 1시간뒤 UVB 200 mj/ cm2조사후 5% CO 2, 37 조건하에서 1시간배양하였다. 1시간후에 96well plate를 250 g에서 10분간원심분리후, 상층액을제거하였다. 96well plate 각 well에 1 incubation solution 200 µl를첨가한후, 상온에서 30분간배양하였다. 그후 250 g에서 10분간원심분리하여나온상층액을 100 µl 취하여실험에사용하였다.
66 大韓本草學會誌 Vol. 29 No. 6, 2014 (2) 세포 DNA 분절분석 96 well plate에 DNA coating solution 100 µl를첨가한뒤, 4 에서 overnight 하였다. 현탁액을털어내어버린뒤, 1 incubation solution 200 µl를첨가하여상온에서 30분간 blocking 하였다. 그후, washing buffer 200 µl를첨가하여 3회씻어낸다음, exonuclease Ⅲ solution 100 µl를첨가하여 37 에서 30분간 denaturing 하였다. washing buffer 200 µl를첨가하여 3회씻어낸다음, anti-brad-pod conjugate solution을각 well에 100 µl씩첨가하여 4 에서 overnight 하였다. 그후, washing buffer 200 µl를첨가하여 3회씻어낸다음, substrate solution 100 µl를첨가해 DNA가분절된정도를 370 nm에서 spectrometer 를이용하여흡광도를측정함으로서결정하였다. 9) High-Performance Liquid Chromatography (HPLC) 본연구에서사용한금은화추출물과화합물은 HPLC 를사용하여함유성분의패턴을분석하였다. 사용한 HPLC 장치는 YL9100 system (Younglin, Korea) 이었으며, 컬럼은 YMC-Triart C18 (4.6 150 mm, 5 µm) 을사용하였다. 이동상은 MeOH, (JT Baker, 영국 ), 0.1% formic acid in water 를사용하였으며, 온도는실온이었으며, 유속은분당 0.7ml 이었다. 크로마토그램은 YL9100 UV/BIS Detector (Sykam, 독일 ) 를이용하여 254nm에서검출하였다. 이동상용매로는 MeOH과 water의비율을달리하여처음 10분간 10%~20% MeOH 조성으로, 다음 10분 ~55분간 20%~70% MeOH 조성으로, 다음 55분 ~60분간 70%~100% MeOH 조성으로총 60분간변화시켰다. 실험에 chlorogenic acid는 sigma(st. Louis, MO, USA) 에서구입하여사용하였다. 며, 이는금은화물추출물에플라보노이드가함유되어있음을알수있다. 2. 금은화물추출물의총항산화능및 superoxide 소거활성효과금은화추출물 0.1 mg/ml 농도의총항산화능은 0.70 mm Trolox equivalent 이었으며, 추출물농도가증가함에따라총항산화능도비례적으로증가하여, 0.1, 0.25 및 0.5 mg/ml 농도에서는각각 0.70, 1.56 및 2.43 mm Trolox equivalent를나타내었다. 반면에, 양성대조군으로사용한 α -tocopherol 의총항산화능은 0.1, 0.25 및 0.5 mg/ml 농도에서각각 0.56, 1.22, 1.89 mm Trolox equivalent 로측정되었다 (Fig. 1C). 따라서, 금은화추출물의총항산화능효과는 α-tocopherol 보다우수함을확인하였다. 또한, 금은화추출물 0.1 mg/ml 농도의 superoxide 소거활성은 11.4% 이었으며, 추출물농도가증가함에따라소거활성도증가하여, 0.1, 0.25, 0.5 mg/ml 농도의금은화추출물의 superoxide 소거활성은각각 11.4, 31.4, 44.5 % 로나타났다. 양성대조군으로사용한 catechin의 superoxide 소거활성은 0.1, 0.25, 0.5 mg/ml 농도에서각각 16.0, 46.6, 70.9 % 로측정되었다 (Fig. 1D). 10) 통계처리실험결과에대한통계처리는 SPSS V10.0을이용하여 one way ANOVA로검정하여 P값이 0.05 미만일때통계적으로유의한차이가있는것으로판정하였다. 결과 1. 금은화물추출물의총페놀및총플라보노이드함량측정금은화추출물 0.1 mg/ml 농도의총페놀함량은 1.10 mm gallic acid equivalent 이었으며, 추출물농도가증가함에따라총페놀함량도비례적으로증가하여, 0.25, 0.5 mg/ml 농도에서각각 1.38, 2.09 mm gallic acid equivalent로나타났다 (Fig. 1A). 이는금은화물추출물에총페놀함량이우수하게포함되어있음을의미한다. 또한, 금은화물추출물 0.1 mg/ml 농도의총플라보노이드함량은 0.1 mm quercetin equivalents 이었으며, 추출물농도가증가함에따라플라보노이드함량도비례적으로증가하여, 0.25, 0.5 mg/ml 농도에서각각 0.83, 1.2 mm quercetin equivalent로나타났다 (Fig. 1B). 금은화물추출물의농도가증가할수록 quercetin equivalents 의 mm이증가하는경향이나타났으 Fig. 1. The effects of LJF extracts on total phenolic content (A), total flavonoid contents (B), total antioxidant capacity (C), and superoxide radical scavenging activity (D). Data results were expressed as in terms of mm gallic acid equivalent (A). Data results were expressed as in terms of mm quercetin equivalent (B). Data results were expressed as in terms of mm and trolox equivalent (C). Data results were expressed as % inhibition of the activity (D). The data represent the mean±s.d. of 3 experiments. 3. 금은화물추출물이사람각질형성세포에서 UVB 로유도된독성에대한세포보호효과및 DNA 손상에미치는영향 다음으로금은화물추출물의 UVB로유도된세포손상에대한세포보호효과를확인하고자하였다. HaCaT 세포에금은화물추출물을 0.1, 0.25, 0.5 mg/ml 농도별로처리하
금은화물추출물의항산화효과와 Ultraviolet(UV)B 로유도된사람각질형성세포손상에대한보호효과 67 여 1시간동안배양한후 UVB 200 mj/ cm2을조사후 4시간처리한뒤세포생존률을살펴보았다. 그결과 UVB 200 mj/ cm2을조사하였을때감소하던세포생존률이금은화물추출물처리군에서농도의존적으로증가한것을확인하였다. 아무것도처리하지않은그룹의생존률 100% 에비해 UVB 200 mj/ cm2조사시 52% 의세포생존율을보였으며, 금은화물추출물을 0.1, 0.25, 0.5 mg/ml 로전처리시 72.22%, 76.31%, 77.01% 로생존율이증가하는것을확인하였다 (Fig. 2). UVB 조사후나타나는세포 DNA 손상에금은화물추출물이주는영향을알아보기위하여, 금은화물추출물을농도별 (0.1, 0.25, 0.5 mg/ml) 로 1시간전처리하고, UVB 200 mj/ cm2조사후인큐베이터에서 4시간배양하였다. 그결과정상군에비하여대조군인 UVB 200 mj/ cm2처리군에서 DAPI의응집, 즉핵의응축이일어나는것을확인할수있었다. 하지만금은화를 0.1, 0.25, 0.5 mg/ml을처리하였을때 UVB 200 mj/ cm2처리로인한핵의응축이유의하게감소되는것을확인하였다 (Fig. 3). 4. 금은화물추출물이사람각질형성세포에서 UVB 로유도된 DNA 분절및 ROS 생성에미치는영향 UVB 조사후나타나는세포 DNA 분절에금은화물추출물이주는영향을알아보기위하여, 금은화물추출물을농도별로 (0.1, 0.25, 0.5 mg/ml) 을 UVB 조사 1시간전에전처리하고, 1시간후 UVB 200 mj/ cm2을조사후인큐베이터에서 4시간배양하였다. 그결과정상군에비하여대조군인 UVB 200 mj/ cm2자극군에서 Brad U positive 흡광도가증가하여, DNA분절이일어나는것을확인할수있었다. 하지만금은화물추출물을 0.1, 0.25, 0.5 mg/ml 을처리하였을때 UVB 200 mj/ cm2자극으로인한 DNA 분절을유의하게감소시킬수있었다 (Fig. 4). 금은화물추출물이 ROS의생성에어떤영향을주는지알아보기위하여, 금은화물추출물을농도별 (0.1, 0.25, 0.5 mg/ml) 로 1시간전에전처리하고, UVB 200 mj/ cm2을조사하였다. UVB 200 mj/ cm2조사 1 시간후세포에 DCF-DA를염색하여, ROS의생성을관찰하였다. 그결과정상군보다 UVB 200 mj/ cm2조사군에서 DCFDA의발현이증가하였고, 이는 ROS 생성이크게증가함을보여준다. 하지만금은화물추출물을처리하였을때 UVB 조사로인한 ROS 생성이농도의존적으로유의하게억제됨을확인할수있었다 (Fig. 5). Fig. 2. The effects of LJF extracts on the UVB-induced cytotoxicity in human keratinocyte HaCaT cells. HaCaT cells were pre-treated with or without LJF (0.1, 0.25, or 0.5 mg/ml) for 1h, then stimulated with UVB (200mJ/ cm2 ). After 4 h, the cell viability was measured by MTT assay. The results were similar in 3 additional experiments. * p < 0.05 significant as compared to untreated normal, p < 0.05 : significant as compared to UVB alone. Fig. 4. The effects of LJF on the UVB-induced DNA fragmentation in human keratinocyte HaCaT cells. The cells were pre-treated with or without LJF (0.1, 0.25, or 0.5 mg/ml) for 1h, and then stimulated with or without UVB (200mJ/ cm2 ). After 4h, the cells were harvested for detection of DNA fragmentation. The cells were visualized by DNA fragmentation assay. The results were normalized by control absorbance to 1. The results were similar in 3 additional experiments. * p < 0.05 significant as compared to untreated normal, p < 0.05 : significant as compared to UVB alone. Fig. 3. The effects of LJF on the UVB-induced cell death in human keratinocyte HaCaT cells. The cells were pre-treated with or without LJF (0.1, 0.25, or 0.5 mg/ml) for 1h, and then stimluated with or without UVB (200mJ/ cm2 ). After 4h, the cells were harvested for detection of DAPI. To find our the DNA fragment, the cells were stained with DAPI. The condensed DNA was indicated with arrow. The relative condensed intensity per field was calculated by Olympus software. The results were similar in 3 additional experiments. * p < 0.05 significant as compared to untreated normal, p < 0.05 : significant as compared to UVB alone. Fig. 5. The effects of LJF on the UVB-induced ROS production in human keratinocyte HaCaT cells. The cells were pre-treated with or without LJF (0.1, 0.25, or 0.5 mg/ml) for 1h, and then stimulated with or without UVB (200mJ/ cm2 ). After 1h, the cells were harvested for detection of ROS. Then the ROS production were measured by flourscence microscopy and relative DCFDA intensity. The percentage of shifted area was indicated by graph below. The results were similar in 3 additional experiments. * p < 0.05 significant as compared to untreated normal, p < 0.05 : significant as compared to UVB alone.
68 大韓本草學會誌 Vol. 29 No. 6, 2014 6. High Performance Liquid Chromatography (HPLC) 를이용한금은화물추출물과 chlorogenic acid 의성분분석연구 금은화물추출물의유효활성성분을알아보고자 HPLC 성분패턴분석을진행하였다. 먼저, 금은화물추출물의 HPLC 성분패턴을분석하여다음과같은결과를얻었다 (Fig 6A). 다음으로금은화의주요성분으로알려져있는 chlorogenic acid를이용하여금은화물추출물과같은조건에서패턴분석을진행하였다 (Fig 6B). 그결과 chlorogenic acid는 28.35분에서피크가나타났다. 따라서금은화물추출물의 28.35분에나타나는피크가 chlorogenic acid 임을추측할수있었고, 이러한결과를바탕으로실험에사용된금은화물추출물의주요성분중의하나로 chlorogenic acid가포함되어있었음을확인할수있었다. Fig.6. HPLC chromatogram of LJF extract (A) and chlorogenic acid (B). 고찰 의학의발달로평균수명이연장되고고령화시대가초래됨에따라웰빙, 안티에이징, 웰에이징등의키워드가부각되면서스파와헬스케어등과같은뷰티산업에대한관심이집중되고있다. 특히탄력적이고건강한피부를유지하고자하는소비자층의증가로노화방지를위한항산화, 항노화관련기능성화장품에대한연구가활발히진행중이다 23). 피부는인체의최외각에존재함으로서자외선에직접적으로노출되기쉬우며, 노출된피부는지속적으로영향을받게되면, 활성산소종에의한광산화적노화가발생된다 24). 자외선중피부손상의주된원인은 280-380 nm의중파장을갖고있는 UVB 이다. UVB는진피의상층부까지도달하여피부내 ROS의생성을촉진시켜세포에돌연변이를유발하고 aspartate 나 glutamate 와같은흥분성아미노산의분비를촉진시켜단백질기능에손상을유도한다 25-27). 또한 nitric oxide(no) 와의상호작용에의한 peroxinitrate 의독성물질생성 28,29), 세포내칼슘의과잉축적되어세포손상을유도한다 25,30). 특히표피에존재 하는각질형성세포 (keratinocyte) 와색소세포 (melanocyte), 면역세포 (langerhans cell) 에직접적인영향을미침으로서세포의기능적, 피부의구조적문제를야기시키게된다. 그로인해피부에비정상적인각화주기로인한각질의들뜸이나예민함, 색소침착, 피부염증등이발생하게된다. 이렇듯건강한피부는다양한 ROS에대항하여세포보호작용을나타내지만활성산소가과하게발생되거나지속적인산화상태에서회복되지못하게되면산화적손상에대한방어체계의불균형으로인해활성산소종유래의다양한질환을유발시킨다 31,32). 따라서활성산소종은피부세포및조직의손상과항산화방어체계의균형을파괴시키며, 지질과산화, 단백질산화, 콜라겐과엘라스틴등의결함섬유사슬절단및멜라닌생성반응의촉진 DNA손상으로인한핵분절등과같은각종효소와단백질의비정상적활성화를야기시킴으로써피부노화를가속화시킨다 33,34). 이에본연구에서는선행연구에서 H 2O 2 로유도된산화적손상에대한금은화물추출물의세포보호효과를확인하였고 22), 그결과를바탕으로 UVB로인한피부각질형성세포손상을금은화가 ROS를억제함으로서, 산화적손상에대한세포사멸을억제할수있는지에대하여접근하였다. 최근연구에서는광노화를지연시키고억제하기위해서자외선에의해발생하는활성산소종을억제하여젊고건강한피부를유지하고자하는목적에따라항노화, 항산화기능성원료및화장품에대한연구가활발히진행중이다 23). 따라서, 금은화물추출물의항산화능력을평가하기위하여성분분석, 항산화력측정에미치는영향에대해서분석해보았다. 그결과, 금은화물추출물에총페놀함량이우수하게포함되어있으며, 총플라보노이드함량도금은화물추출물의농도가증가함에따라비례적으로증가하는것을확인하였다. 이를바탕으로 UVB로유도한세포손상에대한세포보호효과에대해서알아보고자하였다. 각질형성세포에 UVB를 (50, 100, 200, 300 mj/ cm2 ) 선량별로조사한결과각질형성세포 HaCaT에서 UVB로유도된산화적스트레스에따른 ROS를과생성하고있음을알수있었다. 이에금은화추출물을 (0.1, 0.25, 0.5 mg/ml) 농도별로전처리한후, UVB 200mJ/ cm2의과도한산화적스트레스를유도하였을때, ROS의과생성이억제되고, 세포사멸및손상을농도의존적으로억제함을알수있었다. UVB에의한세포사멸은세포가위축되면서사멸체 (apoptotic body) 라는포낭을형성한다. 세포사이에틈새가생기고, 핵응축 (chromatin) 이일어나세포내 DNA 가규칙적으로절단되면서인접해있는세포의식작용에의해먹혀버림으로서죽음에이르게된다 35-39). 그러므로자극후세포내핵응축진행정도를관찰하고, 세포내 DNA 분절을측정하는것은세포사멸분석에서중요한지표가된다. 본연구에서는 HaCaT 세포에 UVB를조사하여자극에따른세포손상정도를핵염색을통해그형태와응축정도를파악하고, 세포의 DNA를추출하여 DNA 분절정도를살펴보았다. 그결과 HaCaT 세포에 UVB 조사시증가하던핵응축과 DNA 분절현상이금은화추출물을전처리하였을때, 농도의존적으로감소시킴을확인하였다. 이러한결과를바탕으로, 금은화물추출물이함유하고있는주요유효성분이무엇인지알아보고자 HPLC를이용하여지표성분분석을수행하였다. 이에, 금은화의주성분으로
금은화물추출물의항산화효과와 Ultraviolet(UV)B 로유도된사람각질형성세포손상에대한보호효과 69 알려진 Loganin, Lupeol, chlorogenic acid 등을같은조건에서분석해보았다. 그중에서, Fig. 6과같이 chlorogenic acid만이본실험에사용된금은화물추출물에서확인되었다. Chlorogenic acid는폴리페놀화합물의일종이며, 커피콩특유의착색원인물질로감자, 고구마의껍질에도많이함유되어있다. 생체내에서과산화지질의생성억제효과, 콜레스테롤생합성억제및항산화와함암작용을한다고알려져있다 38,39). 본연구에서는금은화물추출물에서나오는피크와 chlorogenic acid의 HPLC 피크의 retention time이 28.35분에서동일하게나타났고, 이는금은화물추출물은 chlorogenic acid를주요한성분중의하나로함유하고있는것을나타낸다. 또한, Cha et al 40) 의 chlorogenic acid가 UVB에의한 HacaT손상을억제한다는연구를참고해보면, 금은화의주요성분중에하나인 chlorogenic acid가금은화물추출물의보호작용에부분적으로는기여하지않았을가추측해볼수있다. 본연구결과에서금은화물추출물은 UVB 로유도된산화적손상으로부터농도의존적으로세포를보호하였고, ROS를억제하고 DNA 분절을억제함으로서세포보호효과가있음을제시하였다. 하지만이연구는본저자의논문을부분인용한것으로 41), 추가적으로 HaCaT 세포에서금은화추출물이보호효과를갖는세부기전에관한연구가이루어진다면, 금은화물추출물이자외선에의한피부질환개선제품을개발하는데유용한소재로이용될수있을것으로사료된다. 결론 본연구에서는금은화물추출물을이용하여사람각질형성세포인 HaCaT keratinocyte 에서 UVB로유도된산화적인손상에대한효과를관찰한결과다음과같은결론을얻었다. 1. 금은화물추출물의총페놀및총플라보노이드함량측정결과우수한것을확인하였다. 2. 금은화물추출물의총항산화능및 superoxide 소거활성효과가우수한것을확인하였다. 3. 금은화물추출물은 HaCaT keratinocyte 에서 UVB (200 mj/ cm2 ) 유도된산화적손상에대해농도의존적으로세포생존률을증가시켰다. 4. 금은화물추출물은 HaCaT keratinocyte 에서 UVB로유도된 DNA chromatin의응축및 DNA 분절을감소시켰다. 5. 금은화물추출물은 HaCaT keratinocyte 에서 UVB로유도된 ROS의생성을농도의존적으로억제하였다. 6. 금은화물추출물의주요성분을 HPLC로분석한결과 chlorogenic acid가주요함유성분으로나타났다. 이상의결과, 금은화물추출물은사람각질형성세포인 HaCaT keratinocyte 에서 UVB로유도된산화적인스트레스에서세포보호및 ROS 생성억제및 DNA 분절억제효과가우수하게작용하는것으로나타났다. 이와같은결과를바탕으로금은화물추출물이피부의광산화적손상을개선하는데주요하게작용함으로서항산화, 항노화의기능성화장품으로의활용가능성을시사한다. 추후에금은화물추출물의세포보호효과를나타내는주요성분연구및 chlorogenic acid 에대한세부적인후속연구, 피부에서산화적인손상을억제하는구체적인기전연구와 in vivo 동물실험을통한임상적용의기초연구가추가적으로필요할것으로사료된다. 감사의글 References 1. Halliwell B, Gutteridge JM. The importance of free radicals and catalytic ions in human diseases. Mol Aspects Med. 1985 ; 8(2) : 89-193. 2. Park SN. Antioxidative Properties of Baicalein, Component from Scutellaria baicalensis Georgi and Its Application to Cosmetics (I). J Korean Ind Eng Chem. 2003 ; 14(5) : 657-65. 3. Park KJ, Park SH, Kim JK. Anti-wrinkle Activity of Acanthopanax senticosus Extract in Ultraviolet B (UVB)-induced Photoaging. J Korean Soc Food Sci Nutr. 2010 ; 39(1) : 42-6. 4. Azizi E, Lusky A, Kushelevsky AP, Schewach MM. Skin type, hair color, and freckles are predictors of decreased minimal erythema ultraviolet readiation does. J Am Acad Dermatol. 1988 ; 19(1 pt 1) : 32-8. 5. Applegate LA, Noel A, Vile G, Frenk E, Tyrrell RM. Two genes contribute to different extents to the heme oxygenase enzyme activity measured in cultured human skin fibroblasts and keratinocytes: implications for protection against oxidant stress. Photochem Photobiol. 1995 ; 61(3) : 285-91. 6. Jo HR, Lee HJ, Kang MH. Antioxidative Status, DNA Damage and Lipid Profiles in Korean Young Adults by Glutathione S-Transferase Polymorphisms. J Nutr Health. 2011 ; 44(1) : 16-28. 7. Lee EH, Lee JK, Hong JT, Jung KM, Kim YK, Lee SH, Chung SY, Lee YW. Protective effect of green tea extract, cat-echin on UVB-induced skin damage. J Foof Hygine saf. 2001 ; 16(2) ; 117-24. 8. Kim JS, Kim JB. The Effects of Extracts from Mulberry (Morus alba) Leaves and Branches on UVB-induced Skin Damage. J Kor Soc Cosm. 2011 ; 17(6) : 993-9. 9. Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: Impact on human
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