J Korean Soc Food Sci Nutr 한국식품영양과학회지 37(12), 1679~1683(28) DOI: 1.3746/jkfn.28.37.12.1679 연구노트 포도씨추출물과분획물의 Tyrosinase 저해활성 한지영 성지혜 김대중 정헌상 이준수 충북대학교식품공학과 Inhibitory Effect of Methanol Extract and Its Fractions from Grape Seeds on Mushroom Tyrosinase Jiyoung Han, Jeehye Sung, Dae-Jung Kim, Heon-Sang Jeong, and Junsoo Lee Dept. of Food Science and Technology, Chungbuk National University, Chungbuk 361-763, Korea Abstract The objectives of this study were to determine inhibitory effect on tyrosinase activity and antioxidant activity of the methanolic extract from grape seeds and to investigate relationships between tyrosinase inhibitory activity and antioxidant activity of the extract. The 8% methanol extracts of grape seeds were fractionated subsequently with hexane, chloroform, ethyl acetate and water. The methanolic extract and fractions from grape seeds inhibited tyrosinase activity in a concentration dependent manner. The methanolic extracts showed the highest inhibitory effects on tyrosinase activity. The inhibitory effects of the ethyl acetate fraction from the methanolic extract on tyrosinase activity was higher than other fractions. The ethyl acetate fraction from methanolic extracts showed higher antioxidant activity and contained higher polyphenolic and flavonoid contents compared to other fractions. The correlation coefficients among the polyphenoilc content of methanolic extracts, ABTS radical cation scavenging activity and inhibitory effect of tyrosinase were relatively high. These results suggest that grape seeds may have potential as a depigmentation agent for cosmetics and functional food products. Key words: grape seeds, tyrosinase, antioxidant activity, antioxidant compound 서론급속한산업화에따른환경오염으로인해오존층이파괴되어자외선조사량이증가하고있다 (1). 이로인해기미, 주근깨, 검버섯등피부갈변또는피부스트레스가유발되며, 이러한색소침착은멜라닌색소의증가에기인한다. 멜라닌은동 식물과미생물계에널리존재하는페놀류의고분자천연색소로표피기저층에존재하는 melanocyte 내의 melanosome에서합성된다 (2). 멜라닌은노란색과빨간색을나타내는 pheomelanin과갈색과검은색을나타내는 eumelanin의두가지종류로체내에서합성되며피부, 머리카락, 눈동자등에분포되어있다 (3,4). 멜라닌은 tyrosinase (monophenol, dihydroxy-l-phenylalanine: oxygen oxidoreductase, EC 1.14.18.1) 효소의연속적산화반응으로생합성되며, tyrosinase는멜라닌생합성과정의 key enzyme 으로넓은범위의페놀화합물을기질로이용하는구리함유효소이다 (5). L-Tyrosine은 tyrosinase에의해 3,4-dihydroxyphenylalanine(L-DOPA) 로합성되고, L-DOPA는 phenylanine-3,4-quinone으로산화되며, 중간대사산물을 거쳐최종멜라닌으로합성된다 (6). 지금까지멜라닌색소의생합성을저해하는물질에관한연구는 tyrosinase 효소활성을저해하는수준에서주로이루어져왔으며현재까지알려진 tyrosinase 저해제로는 hydroquinone, ascorbic acid, 4-hydroxyanisole, kojic acid, azelaic acid, arbutin, corticosteroids 등이보고되고있으나, 안전성과경제성등의문제점으로사용이제한되고있다 (7,8). 포도 (Vitis vinifera L.) 는갈대나무목 (Rhamnales) 포도과 (Vitaceae) 에속하는덩굴성과수로, 포도과에는 11속 7여종이있으며주로열대, 아열대지역에서자생하며일부는온대지방까지분포한다 (9). 포도는세계적으로광범위하게재배되고있으며세계과일생산량의약 3% 를차지하고, 우리나라에서는연간 4만톤의포도가생산된다 (1). 포도는세계적으로포도주생산에가장많이이용되고있으나국내에서는식용으로이용되며음료및주류의가공에이용되고있다 (11). 이런포도의가공공정에서약천톤정도의포도씨가부산물로배출되는것으로추정되지만포도씨에대한활용도는미비한실정이다 (12). 포도중량의 3~5% 를차지하고있는포도씨는지방 (9~12%), 단백질 (8~1%) 및 Corresponding author. E-mail: junsoo@chungbuk.ac.kr Phone: 82-43-261-2566, Fax: 82-43-271-4412
168 한지영 성지혜 김대중 정헌상 이준수 hemicellulose와같은식이섬유소 (45%) 를함유하고있을뿐만아니라 oleic acid 및 linoleic acid와같은불포화지방산을다량함유하고있다. 포도씨에는 (+)-catechins, (-)-epicatechin, (-)-epicatechin-3-o-gallate 등의 monomer 페놀화합물, polyhydroxy favan-3-ol 단위의 oligomer, 그리고 polymer인 proanthocyanidin이다량함유되어있어기능성신소재로각광받고있다. 포도씨에함유된유용물질들은심혈관계질환을예방하고항산화작용, 항암작용, 항균작용등여러가지생리활성을지니는것으로알려져있으나멜라닌생합성저해효과에대한연구는미비한실정이다. 따라서본연구에서는포도가공부산물인포도씨를이용하여피부노화및색소침착에관련된 tyrosinase 저해활성을알아보고자하였으며폴리페놀화합물과총항산화력과의상관관계를비교 분석하고자하였다. 재료및방법재료및시약본실험에사용한포도씨는삼랑진농산물가공공장에서제공받았으며, tyrosinase 저해활성실험에사용된 dimethysulfoxide(dmso), gallic acid, catechin, Folin-Ciocalteu (FC) reagent, mushroom tyrosinase, L-DOPA는 Sigma Chemical Co.(St. Louis, MO, USA) 에서구입하였다. 그밖에사용된추출용매및시약은 analytical 및 HPLC 등급을사용하였다. 시료추출및분획포도씨를분쇄기로일정하게마쇄하여시료 5 g에 8% methanol 5 ml을가하여실온에서 24시간추출후얻어진추출물은여과지 (Whatman No.2) 를사용하여고형분과분리하였고감압농축기 (EYELA, Tokyo, Japan) 로농축한후동결건조하여분말형태로사용하였다. 분말형태의포도씨추출물은 -74 C에보관하여사용하였다. 용매분획은농축된메탄올추출물을증류수로현탁시킨후 hexane, chloroform, ethyl acetate, water를이용하여순차분획하였으며감압농축하여분말형태로제조하였다. Tyrosinase 활성측정 Tyrosinase 활성은 Kubo 등 (13) 의방법을변형하여사용하였다. 반응액의총부피는 3 μl이며, 1/15 M sodium phosphate buffer(18 μl), 각추출액 (3 μl),.1 mm L-DOPA(4 μl), tyrosinase(5 μl) 를넣고 25 C에서 1분동안반응시킨후 475 nm에서흡광도를측정하였다. 총 polyphenol 함량측정총 polyphenol의함량측정은 Folin-Denis 방법을변형하여사용하였다 (14). 각추출액 (1 μl) 에 2% Na 2CO 3 용액 (2 ml) 를가하고 3분방치한후 5% FC reagent(1 μl) 를 가하였다. 3분후반응액의흡광도값을 75 nm에서측정하였고표준물질로.1% gallic acid를사용하여 mg gallic acid equivalent per g residue로나타내었다. 총 flavonoid 함량측정 총 flavonoid 의함량측정은발색방법을이용하여측정하였다 (15). 각추출액 (2 μl) 에 1.25 ml의증류수와 5% NaNO 2(75 μl) 를첨가하고 5분방치후 1% AlCl 3 H 2O(15 μl) 을넣고 5분간상온에서반응시킨후 1 M NaOH(5 μl) 을첨가한후잘혼합하여반응액의흡광도값을 51 nm 에서측정하였고표준물질로.2% (+)-cathechin 을사용하여 mg (+)-catechin equivalents per g residue로나타내었다. ABTS 라디칼을이용한총항산화력측정 총항산화력의측정은 ABTS radical cation을이용한방법 (16) 에의하여시행하였다. ABTS(7.4 mm) 와 potassium persulphate(2.6 mm) 를하루동안암소에방치하여 ABTS 양이온을형성시킨후이용액을 414 nm에서흡광도값이 1.5가되도록몰흡광계수 (ε=3.6 1 4 M -1 cm -1 ) 를이용하여증류수로희석하였다. 희석된 ABTS 용액 1 ml에추출액 3 μl를가하여흡광도의변화를정확히 9분후에측정하였으며표준물질로서 Trolox를동량첨가하여표준곡선을작성한후계산하였다. 결과및고찰 포도씨추출물과분획물의 tyrosinase 활성과항산화성분과의상관성포도씨 methanol 추출물과분획물의수율은 Table 1에나타내었다. Methanol 추출물과각분획물의수율은 methanol 추출물의경우 5.58% 이며 hexane 분획층은 methanol 추출물의 4.67, chloroform 분획층은 2.2, ethyl acetate 분획층은 2.41, water 분획층은 72.72% 로나타났다. 여러연구결과에의하면추출에사용된용매의극성이증가할수록유용성분의추출률이높아지며물추출물보다는 aqueous methanol 과 ethanol 을사용하였을경우추출물의활성이증가하는것으로보고되었다 (17,18). 따라서본연구에서는 Table 1. Polyphenol and flavonoid contents of methanolic extracts and its' fraction obtained from grape seeds and extraction yields 8% methanol extract Hexane fraction Chloroform fraction Ethyl acetate fraction Water fraction Polyphenol 1) Flavonoid 2) Yield (%) 687.17±8.75 192.96±5.89 199.95±5.62 865.41±16.55 535.94±18.59 26.27±6.25 58.44±3.79 61.1±1.57 477.29±9.3 166.58±1.3 5.58 4.67 2.2 2.41 72.72 1) Mean of triplicate determinations expressed as mg gallic acid equivalents per g residue. 2) Mean of triplicate determinations expressed as mg catechin equivalents per g residue.
포도씨추출물과분획물의 Tyrosinase 저해활성 1681 8% methanol을추출용매로선택하여여러가지활성성분을추출하고포도씨에존재하는생리활성물질의손실을최소화하고자하였다. 포도씨 methanol 추출물과그분획물의 tyrosinase 저해효과는.1,.1,.25,.5, 1. mg/ml 의농도로측정하였다. 포도씨 methanol 추출물은 1 mg/ml 의농도에서약 18% 의 tyrosinase 활성을나타냈으며농도의존적인경향을나타내었다 (Fig. 1). 포도씨 methanol 추출분획물의 tyrosinase 활성은 Fig. 2에나타내었다. Tyrosinase 저해활성은 1 mg/ ml의농도에서 hexane, chloroform, ethyl acetate 및 water 분획층은각각 64.18, 13.52, 24.18, 47.25% 의 tyrosinase 활성을나타내었고.1 mg/ml의농도에서는 4가지층모두 tyrosinase 억제효과를보이지않았다. Chloroform 분획층은실험에사용한모든농도에서거의효과가나타나지않았 Tyrosinase activity (%) 12 1 8 6 4 2.1.1.25.5 1. Concentration (mg/ml) Fig. 1. Inhibitory effect of 8% methanol extracts from grape seeds against tyrosinase activity. Data values are expressed as mean±sd of triplicate determination. Tyrosinase activity(%) 14 12 1 8 6 4 2 Hexane fraction Chloroform fraction Ethyl acetate fraction Water fraction..1.1.25.5 1. Concentration (mg/ml) Fig. 2. Inhibitory effects of fraction from grape seeds 8% methanol extracts against tyrosinase activity. Data values are expressed as mean±sd of triplicate determination. 으나 ethyl acetate 분획층은농도의존적으로매우우수한저해활성을나타냈다. Woo와 Yang(19) 의연구결과에의하면송이에서분리된균사배양액의분획물또한 ethyl acetate 층에서매우놓은활성을나타내었고본연구결과와유사한경향을보였다. 포도씨의 methanol 추출물과분획물은 tyrosinase 저해활성에서모두농도의존적인경향을나타내었으며, 이결과로포도씨에서 tyrosinase 저해활성을나타내는물질은 hexane, chloroform 정도의비극성유기용매보다 ethyl acetate, water 정도의용매에잘추출되는비교적극성이강한물질이라고여겨진다. 포도씨 methanol 추출물은그분획물보다같은농도에서 (1 mg/ml) 더높은 tyrosinase 저해활성을나타내었다. 그이유는유기용매를사용하여극성별로유용성분을추출하여 tyrosinase 활성을측정하는것보다극성, 비극성물질의유용성분들이모두추출되어나온 crude extract 인 methanol 추출물에서서로상호간의상승효과가나타난것으로사료된다. Jo 등 (2) 은대장암세포주에대한만형자추출물의항암효과를측정하였고만형자 acetone 추출물이 acetone 추출물의분획물보다효과적인암세포성장억제활성을나타낸다는것을보고하였다. 포도씨는다량의 phenolic compound 를함유하며이들이체내에서여러생리활성을나타내는것은널리알려진사실이다. 따라서포도씨의 methanol 추출물의 polyphenol 함량과 flavonoid 함량을분석하여 tyrosinase 저해활성과의상관성을분석하고자하였다. 포도씨 methanol 추출물의총 polyphenol 함량은 687.17 mg/g residue의함량을나타내었고 hexane, chloroform, ethyl acetate, water 분획층은각각 192.96, 199.95, 865.41, 535.94 mg/g residue의함량을나타냈다. 총 flavonoid 함량은 methanol 추출물이 26.27 mg/g residue, 분획층별로는 58.44, 61.1, 477.29, 166.58 mg/g residue 의함량을나타내었고 polyphenol 함량과 flavonoid 함량모두 ethyl acetate 층에서가장높게나타났다. Makris 등 (21) 의연구결과에의하면포도씨의 total polyphenol 함량이 6465 mg/1 g sample로나타났고본연구결과와비슷한결과를나타내었다. 포도씨의 polyphenol 및 flavonoid 함량과 tyrosinase 저해활성과의경향을비교해보면 phenolic compound 의함량이가장높은 ethyl acetate 분획층이가장높은 tyrosinase 저해활성을보였고 phenolic compound 함량이높은 water 분획층도비교적높은 tyrosinase 저해활성을보였다. Phenolic compound의함량이비교적낮은 hexane 및 chloroform 분획층은 tyrosinase 저해효과가미비하였다. 따라서 tyrosinase 저해활성은 phenolic compound 의함량과매우높은상관성이있는것으로생각한다. 포도씨추출물의총항산화력과 tyrosinase 저해활성과의상관성본연구에서는표준물질로 Trolox를사용하여총항산화
1682 한지영 성지혜 김대중 정헌상 이준수 ABTS radical scanvenging activity (%) (mg TEAC/1g residue) 5 4 3 2 1 포도씨분획물중 ethyl acetate 층이 polyphenol과 flavonoid 함량이가장높았고 ABTS 라디칼소거능또한가장높은결과를나타내었으며 water 분획층도비교적높은활성을나타내었다. 본연구결과 tyrosinase 활성과항산화성분, 항산화활성경향이일치하는것을볼수있었으며포도씨는미백, 항노화효과로화장품산업에서천연물질로잠재적인기능성을가지고있을것으로사료된다. 감사의글 A B C D E Fig. 3. Total antioxidant activity of the 8% methanolic extracts from grape seed on ABTS radical. Each value represents the mean of triplicate measurements of analyzed samples (TEAC: mg trolox equivalent antioxidant capacity). (A) 8% methanol extract, (B) hexane fraction, (C) chloroform fraction, (D) ethyl acetate fraction, (E) water fraction. 력을 Trolox equivalent antioxidant capacity(teac mg/g residue) 로산출하여 Fig. 3에나타내었다. 포도씨 methanol 추출물은 TEAC값이 1794. mg Trolox/g residue로표현되는데이것은포도씨 methanol 추출물 1 g의잔사량당 Trolox 1794. mg에해당하는항산화력을지니는것으로해석할수있다. 포도씨 methanol 추출물과분획물의 TEAC 값은 ethyl acetate 분획층이가장높은 391.67 mg Trolox/g residue를나타내었고 water, chloroform, hexane 분획층순으로 1566.67, 546., 386.67 mg Trolox/g residue 의결과를나타내었다. 포도씨추출물은 catechin, epicatechin, quercetin, anthocyanin, resveratrol 등의 polyphenol류성분들이다량함유되어있어항산화효과가높은것으로알려져있으며포도씨분획물중 ethyl acetate 층에서가장높은항산화력을나타내었고이는 tyrosinase 활성과도매우높은상관성을보였다 (22). 천연물에서유래된 tyrosinase 활성저해제로알려진물질인 quercetin, resveratrol 등이포도에다량함유되어있는것으로예상되어기능성을나타내는물질에대한추가적인연구가진행되어야할것으로사료된다 (23). 요 본연구에서는포도가공부산물인포도씨를이용하여피부노화및색소침착에관련된 tyrosinase 저해활성을알아보고자하였으며폴리페놀화합물과총항산화력과의상관관계를비교 분석하고자하였다. 포도씨 8% 메탄올추출물은 hexane, chloroform, ethyl acetate, water 층의순서로분획되었다. Tyrosinase 저해활성은각분획물보다 crude한형태인 methanol 추출물의효과가더높았으며분획물중 ethyl acetate 층이가장높은 tyrosinase 저해활성을나타내었고추출물과분획물모두농도의존적인경향을나타내었다. 약 이논문은농림부 / 농림기술관리센터지정포도연구사업단의연구비지원에의해연구되었으며이에감사드립니다. 문 1. Yoo YG, Joung MS, Choi JW, Kim JH. 25. The study on the whitening effect of Ephedra sinica extract. J Soc Cosmet Scientists Korea 31: 153-159. 2. Seo SY. 21. Screening of tyrosinase inhibitors from oriental herbs. Korean J Plant Res 14: 32-37. 3. Wakamatsu K, Shosuke I. 22. Advanced chemical methods in melanin determination. Pigment Cell Res 15: 174-183. 4. Chin JE, Kim KC. 25. Effects of chestnet bark extracts on tyrosinase gene expression. Korean J Sanitation 2: 1-16. 5. Slominski A, Tobin DJ, Shibahara S, Wortsman J. 24. Melanin pigmentation in mammalian skin and its hormonal regulation. Rhysiol Rev 84: 1155-1228. 6. Solano F, Briganti S, Picardo M, Ghanem G. 26. Hypopigmenting agents: an updated review on biological, chemical and clinical aspects. Pigment Cell Res 19: 55-571. 7. Lee HB, Bai S, Chin JE. 25. Inhibitory effect of Lithospermum erythrorhizon extracts on melanin biosynthesis. J Korean Soc Food Sci Nutr 34: 1325-1329. 8. Kwak JH, Kim YH, Chang HR, Park CW, Han YH. 24. Inhibitory effect of gardenia fruit extracts on tyrosinase activity and melanogenesis. Korean J Biotechnol Bioeng 19: 437-44. 9. Choi SW, Chung US, Lee KT. 25. Preparation of high quality grape seed oil by solvent extraction and chemical refining process. Korean J Food Preserv 12: 6-67. 1. Lee EJ, Kwon JH. 26. Characteristics of microwave-assisted extraction for grape seed components with different solvents. Korean J Food Preserv 13: 216-222. 11. Jeong SM, Kim SY, Ha JU, Lee SC. 25. Effect of far-infrared irradiation on the antioxidant activity of extracts from grape seed. J Korean Soc Food Sci Nutr 34: 1619-1624. 12. Woo MJ, Seo JW, Byun SY. 25. Extraction of resveratrol containing grape seed oil with supercritical carbon dioxide. Korean J Biotechnol Bioeng 2: 383-386. 13. Kubo I, Hori IK, Yokokawa Y. 1994. Tyrosinase inhibitors from Anacardium occidentale fruits. J Nat Prod 57: 545-551. 14. Choi Y, Jeong HS, Lee J. 27. Antioxidant activity of methanolic extracts from some grain consumed in Korea. Food Chem 13: 13-138. 헌
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