KOREAN J. FOOD SCI. TECHNOL. Vol. 42, No. 6, pp. 750~754 (2010) The Korean Society of Food Science and Technology 짝자래나무추출물의항산화및미백효과 서은종 홍은숙 최민희 김기선 이성준 * ( 주 ) 더페이스샵기술연구소 Antioxidant and Skin Whitening Effects of Rhamnus yoshinoi Extracts EunJong Seo, EunSuk Hong, MinHee Choi, KiSun Kim, and SungJun Lee* THEFACESHOP R&D Center Abstract The purpose of this study was to investigate the antioxidant and skin whitening effects of Rhamnus yoshinoi extracts. Rhamnus yoshinoi was extracted with 100% ethanol and water. The antioxidative and skin whitening effects of extracts were determined by in vitro assays using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method and inhibitory effects against tyrosinase activity and melanogenesis in B16F1 melanoma cells. The radical scavenging activities of the Rhamnus yoshinoi extracts were tested by DPPH assay and showed high DPPH radical scavenging activities (SC50; 21.6 ppm in EtOH, 40.5 ppm in water). As for tyrosinase inhibitory activity, the Rhamnus yoshinoi ethanol extract had the highest inhibition activity (IC50; 256.3 ppm). In B16F1 mouse melanoma cells, the Rhamnus yoshinoi ethanol extract significantly inhibited melanin synthesis by 53.36% at the concentration of 50 ppm. These results suggest that Rhamnus yoshinoi ethanol extract has significant antioxidant activity and whitening activity. Key words: Rhamnus yoshinoi, antioxidative, whitening, tyrosinase 서 유해산소라불리는활성산소란미토콘드리아, 식세포또는세포질에서정상적인대사과정중여러가지생물학적반응에의해생성되는일중항산소, 과산화수소 (H 2 O 2 ) 나 superoxide anion ( O 2 ) 및 hydroxyl 라디칼 ( OH) 등과같은짝짓지않은상태의전자를갖는유리라디칼들을말한다 (1). 이러한유리라디칼들은반응성과파괴성이매우높아생체내의세포막, 단백질, DNA 및효소등을손상시키는등세포와조직에해로운반응을일으켜암및동맥경화등의질병을유발하며노화와밀접한관련이있는것으로알려져있다 (2-8). 따라서이러한활성산소를제거하거나조절할수있는물질로알려진항산화제의개발에관한연구가활발히진행되고있으며여러항산화제에대한연구결과들이보고되어있다 (9-13). 연구결과에의하면식물에존재하는비타민 E, 비타민 C 등의비타민과플라보노이드류, 카로티노이드류, 폴리페놀과같은천연항산화제와 superoxide dismutase(sod), glutathione peroxidase 등과같은항산화효소및합성항산화제인 butylated hydroxytoluene(bht), butylated hydroxyanisole(bha) 등여러가지물질이항산화효과가있는것으로알려졌다 (9,13-16). 그러나기존에개발된식물유래천연항산화제의경우항산화효과가비교적낮고원료수급의문제, 낮은수율및안정성등의문제점으로상업용추출물에대한효과가확인된보고 *Corresponding author: SeongJun Lee, THEFACESHOP R&D Center, Incheon 403-200, Korea Tel: 82-32-870-7401 Fax: 82-32-870-7400 E-mail: sjleec@thefaceshop.com Received August 23, 2010; revised October 4, 2010; accepted October 12, 2010 론 는없으며항산화효소는단백질로구성되어온도나 ph 에영향을많이받아산업적이용에어려움이있다 (15-17). 또한합성항산화제경우에는생체효소, 지방의변이및독성으로인해인체에암을유발할수있다는보고가있어보다안전하고활성이높은항산화제의연구가요구되고있다 (18-20). 멜라닌은자외선으로부터피부를보호하는역할을하나과도하게생성될경우피부에색소가침착되어기미, 주근깨를형성하며, 이와같은병변은나아가피부암의원인이되기도한다 (21). 따라서, 이러한색소침착현상을방지하기위해서는멜라닌생성과정의일부분을저해하여멜라닌의생성을감소시켜야한다. 멜라닌은피부에존재하는색소로서, 표피의기저층에존재하는멜라노사이트내의멜라노좀이라는소포체에서먼저 Tyrosinase, tyrosinase related protein-1(trp-1) 과 dopachrome tautomerase(dct) 등의효소에의해멜라닌이생성되게된다 (22). 특히 tyrosinase 는멜라닌생성첫단계를일으키는효소로, 티로신이도파 (DOPA) 를거쳐도파퀴논 (dopaquinone) 으로전환되고, 도파퀴논으로부터자동산화반응과효소반응으로도파크롬 (dopachrome) 을거쳐흑갈색의공중합체인멜라닌이생성되게된다. 현재미백제의개발에있어서, 생성된멜라닌색소를환원시켜탈색하는방법과멜라닌색소를형성하는효소인 tyrosinase 의활성을억제하는방법이알려져있다 (23,24). 기존화장품분야에서는미백성분으로서, 코지산 (kojic acid), 알부틴 (arbutin) 등과같은 tyrosinase 효소활성을억제하는물질, 하이드로퀴논 (hydroquinone), 비타민 C(L-ascorbic acid) 및이들의유도체와각종식물추출물이사용되어왔다 (25). 그러나, 이런물질들은불안정하여분해나착색, 이취, 효과의불분명및안전성문제등으로그사용이제한되고있는실정이다. 따라서기존미백제가갖고있는여러단점을극복하고자최근에는피부에안전한천연물을이용한미백제개발에많은연구가진행되고있다. 750
짝자래나무추출물의항산화및미백효과 751 따라서본연구는천연식물로서인체에안전하고항산화및미백효과를갖는소재를개발하고자수행되었다. 본연구에사용된짝자래나무 (Rhamnus yoshinoi) 는쌍떡잎식물갈매나무목갈매나무과의낙엽활엽관목이며한국의특산종으로경기, 충남을제외한전역에분포하나생리활성및항산화, 미백등과같은효능에대해서는알려진바가전혀없다. 이런짝자래나무추출물의신규미백소재로서의효능을확인하고자유효성분으로알려진폴리페놀및플라보노이드의함량측정및항산화효과, tyrosinase 저해효과, 멜라닌생합성저해효과를수행하였다. 재료및방법 재료본연구에서사용된짝자래나무 (Rhamnus yoshinoi) 는한국생명공학연구원한국식물추출물은행 (Daejeon, Korea) 에서건조분쇄되어있는것을구입하여 4 o C 에냉장보관하면서실험에사용하였다. 시약본연구의사용된 tannic acid, rutin hydrate, sodium metabisulfite, sodium carbonate, 1,1-diphenyl-2-picrylhydrazyl(DPPH), ascorbic acid, L-tyrosine, tyrosinase, 3-[4,5-Dimethylthiazol-2-yl]- 2,5-diphenyltetrazolium bromide (MTT), Folin-Ciocalteu s phenol reagent, diethylene glycol 는 Sigma사 (St. Louis, MO, USA) 에서구입하여사용하였다. 추출짝자래나무추출을위해용매로에탄올과물을이용하였으며추출은시료의 10 배의용매를가한후 30 o C 에서초음파 (Asia industry, Incheon, Korea) 를이용하여 3 회반복추출하였다. 100% 에탄올을이용하여추출한시료는 40 o C 이하에서감압농축 (rotary vacuum evaporator, N-N series, Eyela, Tokyo, Japan) 을하였으며물을이용하여추출한시료는동결건조 (FD8508, Ilshin Lab Co., Yangju, Korea) 하여중량법으로수율을계산하였다. 각각의추출물은밀봉하여 80 o C 에보관하며실험에사용하였다. 총폴리페놀함량측정총폴리페놀함량은 Gutfinger(26) 의방법을변형하여측정하였다. 각각의한약재추출물을 0.1-1 mg/ml 농도로희석하여준비한후실험에사용하였다. 시료 0.2 ml 와 Folin-ciocalteu reagent 0.2 ml 를혼합한후상온에서 3 분간반응하였다. 반응후 2M Na 2 CO 3 0.4 ml 와증류수 0.2 ml 를혼합한다음상온에서 30 분간반응후각각의시료를 96 well plate 에 0.2 ml 씩분주하여 725 nm 에서흡광도 (SpectraMAX Plus 384, Molecular devices Inc., Sunnyvale, CA, USA) 를측정하였다. 표준물질로 tannic acid 를이용하였으며표준물질의표준곡선으로부터총폴리페놀함량 (mg/ g) 을계산하였다. 총플라보노이드함량측정총플라보노이드함량은다음과같은방법으로측정하였다 (27). 각각의한약재추출물을 0.1-2 mg/ml 농도로희석한시료를조제한후시료 0.2 ml, diethylene glycol 2 ml, 1 N NaOH 0.2 ml 을첨가하여 37 o C 에서 1 시간반응시킨후 420 nm 에서흡광도를측정하였다. 표준물질로 rutin hydrate 를이용하였으며표준물질의표준곡선으로부터총플라보노이드함량 (mg/g) 을계산하였다. 전자공여능전자공여능은 DPPH 의환원력을이용하여측정하였다 (28). 96 well plate 에 400 µm 농도가되도록에탄올에용해한 DPPH 0.2 ml 와희석된추출물시료 0.2 ml 를분주한다음실온에서 30 분간반응시키고 525 nm 에서흡광도를측정하였다. 대조군은시료대신에탄올이첨가하였으며비교를위하여대표적인항산화제인 ascorbic acid 를사용하였다. Tyrosinase 활성저해효과 Bernard(29) 법을변형하여사용하였다. 96 well plate 에 0.1 M phosphate buffer(ph 6.5) 210 µl 를넣고여기에기질 (L-tyrosine) 40 µl 과 tyrosinase 20 µl, 추출물 30 µl 를넣고 37 o C 에서 10 분간반응시킨후 490 nm 에서흡광도를측정하였다. 이때대조군은시료대신완충액을넣은것으로하였으며비교를위하여 ascorbic acid 를사용하였다. 세포배양 Mouse immortalized fibroblast(nih3t3 cell) 와 mouse melanoma melanocyte(b16f1 cell) 를각각 10% 의 fetal bovine serum 이함유된 DMEM 배지에서 5% CO 2 에서 37 o C 로배양하여실험에사용하였다. 세포독성 MTT 시험은 Mosmann 법 (30) 이용하였다. Mouse immortalized fibroblast(nih3t3 cell) 를 96 well plate 에 well 당 1 10 4 cells 을접종하고 24 시간배양하였다. 여기에짝자래나무추출물을각 well 에첨가하여 24 시간배양후배지를버리고새로운배지와함께 MTT 용액을 10 µl 씩첨가하였다. 4 시간후배지를버리고 DMSO 를넣어 formazan 을용해시키고 570 nm 에서흡광도를측정하였다. 세포내멜라닌생합성억제효과 Tsuboi 의방법 (31) 을변형하여이용하였다. 사람의정상섬유아세포 mouse melanoma melanocyte(b16f1 cell) 를 6well plate 에접종하고 (1 10 5 cells/well), 24 시간배양하였다. 짝자래나무추출물을 DMEM 배지에첨가하여, 상기배지로교체한후 24 시간동안더배양하였다. 배지를모두제거하고 trypsin 을처리하여세포를회수한다. 회수한세포에 1 N NaOH(10% DMSO 포함 ) 를첨가하여 60 o C 항온조에서 1 시간방치하여멜라닌을녹여내었다. 490 nm 에서흡광도를측정하였다. 통계분석모든실험은 3 반복으로측정하여측정치를평균치 ± 표준편차로나타내었으며실험결과의통계적유의성은 Student s t-test 로하였으며 p 값이 0.05 미만일때통계적으로유의성이있다고판단하였다. 결과및고찰 한약재추출물의수율짝자래나무의추출수율은 Table 1 과같다. 추출수율은초기건조중량과추출후건조중량의백분비로계산하였다. 그결과물을이용한추출에서수율이 8% 로에탄올수율 6% 에비하여높게나타난것을확인하였으며이는기존의 Park(32) 의논문에서 8 종류의한약재를에탄올과물을이용하여열수추출을하였
752 한국식품과학회지제 42 권제 6 호 (2010) Table 1. Yield of extraction from Rhamnus yoshinoi using 100% EtOH and water Yield (%) 100% EtOH Water Rhamnus yoshinoi 6±0.5 8±1.5 을때물에서추출수율이더높은것과동일한결과가얻어지는것을확인하였다. 총폴리페놀함량및총플라보노이드함량천연물에서얻어지는폴리페놀류와플라보노이드류의화합물은항산화효과를나타내는등생리활성기능을가지는것으로알려져있다 (33). 식물의 2 차대사산물중하나인페놀성화합물은분자내 phenolic hydroxyl 기가존재하여전자를수용하는기작으로항산화효과를가지며그외항돌연변이, 항암, 혈중콜레스테롤감소등의다양한생리활성을가지는것으로알려져있다 (34,35). 또한, 식물에널리분포하는노란색계통의색소인플라보노이드는항균, 항암, 항바이러스, 항알레르기, 지질저하작용, 면역증강작용, 모세혈관강화작용및항염증활성을지니며모든질병의원인이되는생체내산화작용을억제하는항산화효과를지니는것으로알려져있다 (36). 짝자래나무추출물에존재하는총폴리페놀과플라보노이드함량을측정한결과는 Table 2 와같다. 총폴리페놀과플라보노이드의함량은에탄올추출물에서 384.51, 120.39 mg/g 으로물추출물의 265.56, 80.72 mg/g 보다높게나타난것을확인하였으며짝자래나무의에탄올추출물과물추출물모두 200 mg/g 이상의높은폴리페놀을함유하여항산화효과및다른생리활성효과가우수할가능성이높을것으로생각된다. Fig. 1. DPPH radical scavenging effect of Rhamnus yoshinoi extracts. Results are expressed as mean±sd of data obtained from three independent experiments. 전자공여능전자공여능은항산화작용의지표로사용되고있으며식물추출물의항산화능측정에많이사용되고있다. 전자공여작용은인체내에서생성되는프리라디칼의전자를공여하여프리라디칼에의한노화와질병을억제하는작용으로이용되고있다 (37). 전자공여능측정은 DPPH 라디칼소거법을이용하여측정하였다. DPPH 는비교적안정한라디칼을가지고있는화합물로항산화능력을가지고있는물질과만나면라디칼이소거되어탈색이되는점을이용하여항산화효과를검정한다 (33). 짝자래나무추출물의라디칼소거능을비교한결과 Fig. 1 과같다. 짝자래나무추출물의 50% 라디칼소거능을가지는농도 (SC 50 ) 를비교한결과짝자래나무추출물중총폴리페놀함량이높은에탄올추출물이 21.6 ppm 으로물추출물의 40.5 ppm 보다약 2 배높은것을확인하였다. 이는폴리페놀의함량이증가하면 DPPH 라디칼소거능이비례적으로증가하는기존연구들과동일한결과를나타내었으며 (38,39) 대조구인 ascorbic acid 의 SC 50 의 7.20 ppm 과비교하여낮은전자공여능을나타내었으나항산화효과가좋다고잘알려진인삼, 갈근, 창출, 약쑥그리고느타리버섯의 70% 에탄올추출물의 100 µg/ml 를처리한결과와비교하 Fig. 2. Inhibitory effect of Rhamnus yoshinoi extracts against tyrosinase. Results are expressed as mean±sd of data obtained from three independent experiments. 였을때짝자래나무추출물의전자공여능이현저히높은것을확인하였다 (40). Tyrosinase 활성저해효과멜라닌생성에중요한역할을하는효소인 tyrosinase 의억제활성효과는기질인 L-tyrosine 과효소인 mushroom tyrosinase 에의해산화되어생성되는멜라닌의흡광도를측정하여 tyrosinase 억제효과를알아본결과는 Fig. 2 와같다. 실험결과짝자래나무의에탄올추출물의 tyrosinase 활성억제효과는농도의존적으로증가하고 50% tyrosinase 활성억제농도 (IC 50 ) 가 256.3 ppm 으로확인하였으나기존에미백효과가있다고알려진 ascorbic acid 의 IC 50 102.4 ppm 과비교하였을때낮은것으로확인되었다. 또한짝자래나무물추출물에서 tyrosinase 활성억제효과는실험최고 Table 2. Concentration of total polyphenol and total flavonoid in Rhamnus yoshinoi extracts Total polyphenol (mg/g) Total flavonoid (mg/g) 100% EtOH Water 100% EtOH Water Rhamnus yoshinoi 384.51±20.4 265.56±11.2 120.39±13.7 80.72±4.1
짝자래나무추출물의항산화및미백효과 753 Fig. 3. Effect of Rhamnus yoshinoi extracts on cell viability of mouse immortalized fibroblast. Results are expressed as mean ±SD of data obtained from three independent experiments. Table 3. Inhibitory activity of Rhamnus yoshinoi extracts (50 ppm) on melanin biosynthesis Inhibitory effect of melanin biosynthesis (%) 100% EtOH Water Ascorbic acid Rhamnus yoshinoi 53.36±2.81-17.40±3.19 농도인 1000 ppm 까지유의한결과를확인할수없었다. 이는에탄올과물을이용하여짝자래나무를추출하였을때서로다른성분이추출되어물추출물에서는효과가나타나지않은것으로생각된다. 세포독성 MTT assay 는살아있는세포에서탈수소효소작용에의하여노란색의수용성기질은 MTT tetrazolium 을청자색을띄는비수용성의 MTT formazan 으로환원시키는미토콘드리아의능력을이용하는검사법으로짝자래나무추출물의세포생존율에미치는영향을알아보았다. 세포독성실험결과짝자래나무추출물은추출용매에상관없이 500 ppm 까지독성이없는것으로나타났다 (Fig. 3). 세포내멜라닌생합성억제효과멜라닌생성세포인멜라노사이트를배양하여시료를처리함으로서멜라닌생성에어떠한영향을미치는가에대하여알아보고자하였다. 마우스유래의멜라노사이트세포 (B16F1 cell) 에짝자래나무에탄올및물추출물 50 ppm 을첨가하여배양한후세포내멜라닌양을측정하였다. 대조구로 ascorbic acid 를사용하였다. 실험결과짝자래나무에탄올추출물에서멜라닌생합성억제효과는 53.36% 로대조구인 ascorbic acid 50 ppm 의 17.4% 보다높게나타났으나물추출물에서는효과가없는것을확인하였다 (Table 3). 이는 tyrosinase 억제효과와결과가같았으며 tyrosinase 가멜라닌생성에중요한역할을하는것으로확인되었다. 요 본연구는짝자래나무의항산화및미백제로서의유효성을알아보기위해 100% 에탄올과물을이용한추출물의폴리페놀함량, 플라보노이드함량, 전자공여능, tyrosinase 활성저해효과및세포내멜라닌생합성억제효과를조사하였다. 유용한생리활성 약 을가질것으로예상되는폴리페놀및플라보노이드함량을실험한결과총폴리페놀과플라보노이드의함량은에탄올추출물에서 384.51, 120.39 mg/g 으로물추출물의 265.56, 80.72 mg/g 보다높게나타난것을확인하였으며짝자래나무의에탄올추출물과물추출물모두 200 mg/g 이상의높은폴리페놀을함유하는것을확인하였다. 항산화능을평가하기위해 DPPH 를이용한전자공여능실험결과짝자래나무의추출물중총폴리페놀함량이높은에탄올추출물이 21.6 ppm 으로물추출물의 40.5 ppm 보다약 2 배높은것을확인하였다. 미백제로의효능을알아보고자실행한 tyrosinase 활성저해및세포내멜라닌생합성억제에관한결과짝자래나무의에탄올추출물에서 tyrosinase 활성억제농도 (IC 50 ) 는 256.3 ppm, 멜라노사이트에 50 ppm 농도로처리하였을때멜라닌생성억제효과가 53.36% 로높은효과를가지는것을확인하였다. 이상의결과에따르면짝자래나무의에탄올추출물의경우항산화제및미백제로서큰가능성을가지는것으로나타났다. 감사의글 본연구는보건복지부보건의료연구개발사업 ( 과제고유번호 : A092055) 의지원에의해이루어진연구의일부이며지원에감사드립니다. 문 1. Papa S, Skulachev VP. Reactive oxygen species, mitochondria, apoptosis and aging. Mol. Cell Biochem. 174: 305-319 (1997) 2. Block G. Vitamin C, cancer, and aging. Age 16: 55-58 (1993) 3. Feskanich D, Ziegler RG, Michaud DS, Giovannucci EL, Speizer FE, Willett WC, Colditz GA. Prospective study of fruit and vegetable consumption and risk of lung cancer among men and women. J. Natl. Cancer I. 92: 1812-1823 (2000) 4. Halliwell B, Gutteridge JM. Role of free radicals and catalytic metal ions in human disease: An overview. Method Enzymol. 186: 1-85 (1990) 5. Regnstrm J, Nilsson J, Tornvall P, Landou C, Hamsten A. Susceptibility to low-density lipoprotein oxidation and coronary atherosclerosis in man. Lancet 339: 1183-1186 (1992) 6. Gey KF, Puska P, Jordan P, Moser UK. Inverse correlation between plasma vitamin E and mortality from ischemic heart disease in cross-cultural epidemiology. Am. J. Clin. Nutr. 53: 326-334 (1991) 7. Abuja PM, Albertini R. Methods for monitoring oxidative stress, lipid peroxidation and oxidation resistance of lipoproteins. Clin. Chim. Acta 306: 1-17 (2001) 8. Ames BN. Endogenous DNA damage as related to cancer and aging. Mutat. Res. 214: 41-46 (1989) 9. Nho JW, Hwang IG, Joung EM, Kim HY, Chang SJ, Jeong HS. Biological activities of Magnolia denudata Desr. flower extracts. J. Korean Soc. Food Sci. Nutr. 38: 1478-1484 (2009) 10. Lee WC, Kim AJ, Kim SY. The study on the functional materials and effects of mulberry leaf. Food Sci. Ind. 36: 2-14 (2003) 11. Cerutti PA. Oxy-radicals and cancer. Lancet 344: 862-863. (1994) 12. Cort WM. Antioxidant activity of tocopherols, ascorbyl palmitate, and ascorbic acid and their mode of action. J. Am. Oil Chem. Soc. 51: 321-325 (1974) 13. Coleman MD, Fernandes S, Khanderia L. A preliminary evaluation of a novel method to monitor a triple antioxidant combination (vitamins E, C, and α-lipoic acid) in diabetic volunteers using in vitro methaemoglobin formation. Environ. Toxicol. Phar. 14: 69-75 (2003) 14. Namiki MO. Antioxidants and antimutagens in food. Crit. Rev. Food Sci. 29: 273-300 (1990) 15. Cort WM. Antioxidant activity of tocopherols, ascorbyl palmitate, 헌
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