JNH 조윤희(K15-62).fm

Transcription

1 Journal of Nutrition and Health (J Nutr Health) 2016; 49(2): 63 ~ 71 pissn / eissn Research Article 자외선조사와병행된녹차식이공급이무모생쥐의표피산도관련인자함량및젖산탈수소효소의단백질발현과활성에미치는영향 * 이보민 1 김종례 1 황재성 2 조윤희 1 경희대학교동서의학대학원의학영양학과, 1 경희대학교생명자연과학연구원 2 Dietary effect of green tea extract on epidermal levels of skin ph related factors, lactate dehydrogenase protein expression and activity in UV-irradiated hairless mice* Lee, Bomin 1 Kim, Jongyei 1 Hwang, Jaesung 2 Cho, Yunhi 1 1 Department of Medical Nutrition, Graduate School of East-West Medical Science, Kyung Hee University, Yongin 17104, Korea 2 Institute of Life Sciences & Resources, Kyung Hee University, Yongin 17104, Korea ABSTRACT Purpose: Skin ph, an indicator of skin health, is maintained by various organic factors, which include lactate, free amino acid (FAA), and free fatty acid (FFA). As skin ages or with illness, skin ph becomes less acidic, and functional food has been developed to maintain the acidic ph of skin. In this study, we determined the dietary effect of green tea extract (GTE) on skin ph of photo-aged mice, as measured by epidermal levels of lactate, FAA, and FFA. The protein expression and activity of lactate dehydrogenase (LDH), an enzyme of pyruvate reduction for lactate generation, was further determined. Methods: Albino hairless mice were fed a control diet (group UV+) or a diet with 1% GTE (group GTE) in parallel with UV irradiation for 10 weeks. A normal control group was fed a control diet without UV irradiation for 10 weeks (group UV-). Results: Skin ph was higher (less acidic) in group UV+ than in group UV-. In parallel, epidermal levels of lactate and FFA, as well as of LDH protein expression and activity, were reduced in group UV+. Dietary supplementation of GTE (group GTE) reduced skin ph to similar to the level of group UV-, and inversely increased epidermal levels of lactate, LDH protein expression and activity, but not of FFA. Although epidermal levels of FAA were similar in groups UV- and UV+, it was increased in group GTE to a level higher than in group UV-. In further analysis of major FFA, epidermal levels of palmitic acid [16:0], oleic acid [18:1(n-9)], and linoleic acid [18:2(n-6), but not of stearic acid [18:0] in group GTE were similar to or lower than those in group UV+. Conclusion: Dietary GTE normalized skin ph with increased levels of lactate and FAA, as well as with increased protein expression and activity of LDH in the epidermis of UVB irradiated hairless mice. KEY WORDS: green tea extract, skin ph related factors, lactate dehydrogenase, ultraviolet irradiation, hairless mice 서 피부건강을나타내는주요지표인피부산도 (ph) 는피부의최외층인표피에서생성되는젖산 (lactate), 유리아미노산 (free amino acid, FAA), 유리지방산 (free fatty acid, FFA) 등여러인자들의전체함량에의해결정된다. 1-3 이들인자중젖산은피부 ph의주요결정인자로, 젖산생 론 성효소인젖산탈수소효소 (Lactate dehydrogenase, LDH) 는표피에서호기적인상태에서도강한활성을갖는것으로알려져있다. 4 표피의유리아미노산은약산성아미노산인 glutamate 및 histidine이 40% 이상을차지하는반면 methionine, phenylalanine, valine, isoleucine, leucine 및 cysteine은거의존재하지않는다. 1 표피에서 glutamate 및 histidine는 pyrrolidone carboxylic acid (PCA) 과 Received: December 17, 2015 / Revised: March 23, 2016 / Accepted: March 25, 2016 *This study was supported by a grant of the Korean Health Technology R & D Project, Ministry of Health & Welfare, Republic of Korea (Grant No. HN13C0076). To whom correspondence should be addressed. tel: , choyunhi@khu.ac.kr 2016 The Korean Nutrition Society 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 64 / 녹차추출물섭취에의한피부산도개선효능 urocanic acid (UCA) 로대사되기도한다. 1 또한표피의여러유리지방산에서초래되는약산성이피부산도유지에기여하는것으로알려져있다. 건강한피부는 ph 4~6 범위의약산성 ph를유지하는반면, 1,3 아토피피부염, 여드름등의피부질환발병시피부 ph 증가와함께약산성의환경에서활성을나타내는피부장벽의주요지질인세라마이드생성관련효소인 glucocerebrosidase 및 sphingomyelinase 의활성이저하된다. 3,5,6 또한노화가진행되는피부에서는피부 ph 증가와함께히알루론산, 콜라겐등의보습및주름생체지표활성이저하된다. 7 피부노화는자연노화와광노화로나뉘어지는데과다한자외선노출에의해초래되는광노화는피부의주름형성, 건조, 표피과증식뿐아니라피부 ph의증가를야기한다 자연노화가시간경과에의해일어나는불가피한현상인반면, 광노화는자외선의노출정도를방지함에의해예방이가능할뿐아니라, 광노화의예방및개선을위해화장품뿐아니라다양한식이소재가개발되고있다. 국내에서는녹차, 소나무껍질추출물, 홍삼, 사상자, 산수유복합추출물등의다양한소재들이광노화보호를위한건강기능식품으로개발되고있으며이중녹차의피부노화억제효능은국내외적으로활발한연구가이루어지고있다 선별된차잎을고온에서가열하여산화와발효를억제한녹차는카테킨의일종인 epigallocatechin-3-gallate (EGCG) 가주요활성물질로알려져있으며, 콜라겐생성증가, 자외선에의한표피과증식방지등의피부노화개선효능이여러연구에서보고되었으나, 피부 ph 개선효능및 ph 관련여러인자들함량변화에대한연구는미흡한실정이다. 따라서본연구에서는무모생쥐에자외선조사와병행하여녹차를식이공급하고, 녹차섭취가표피의 ph을비롯하여젖산, 유리아미노산, 유리지방산의함량및 LDH의단백질발현과활성에미치는영향을알아보고자하였다. 연구방법 실험동물생후 6주령암컷무모생쥐 (Albino hairless mouse, SKH- 1) 를 ( 주 ) 오리엔트바이오에서공급받아 1주동안일반고형배합사료로적응시켰다. 실험군은체중에따라난괴법을이용해자외선조사를하지않은정상대조군 (UV-군, n = 5), 자외선조사군 (UV+ 군, n = 5), 자외선조사와함께녹차추출물 (green tea extract: GTE) 을전체식이의 1% 수준에서식이공급한실험군 (GTE군, n = 5) 으로나누었다. 사육기간중물과사료는자유롭게섭취하도록하였고 온도 24 ± 2 o C, 습도 55 ± 10% 로최적환경조건을유지시킨실험실에서 12 hr/day 명암주기로사육하였다. 본동물실험프로토콜은경기과학기술진흥원에서운영하는동물윤리위원회에서승인을득하였다 ( 승인번호 : ). 자외선조사 UVB lamp ( 약 30% UVA 함유 : TL 20W/12RS SLV, wave length 290 to 390 nm, Phillips, Netherlands) 를사용하여무모생쥐의등부위 ( 등중앙기준, 2 cm 3 cm) 를조사하였고자외선조사량은 UV meter (VARIOCONTROL, Waldmann ver. 2.03, Germany) 로측정하였다. 17 식이공급개시일로부터 1주일에 3회조사하였고 1주차에는 1 minimal erythema dose (MED; 75 mj/cm 2, 15분 ) 로시작하여 2 주차 2 MED (150 mj/cm 2, 30분 ), 3주차 3 MED (225 mj/ cm 2, 45분 ), 4주차부터 10주차까지는 4 MED (250 mj/cm 2, 50분 ) 로조사하였다. 실험식이 UV- 및 UV+ 군은 GTE가함유되지않은일반식이 (AIN- 93G 식이 ) 를공급하였고, GTE군은전체식이무게의 1.0% 에상응하는 GTE를식이에첨가하여 10주간공급하였다. 17 전체식이에서 GTE 추출물의 1.0% 첨가수준은섭취후독성및산화촉진등의부작용없이 18,19 피부산도개선의효능이나타난예비연구에기반하여결정하였다. GTE 시료첨가에의해발생하는건분식이무게차이는옥수수전분을감하여조정하였다. GTE 시료 ( 카테킨 50%, epigallocatechin-3-gallate (EGCG) 28% 함유 ) 는 ( 주 ) 아모레퍼시픽 (South Korea) 에서공급받았다. 각실험군의식이조성은 Table 1과같다. 피부 ph 측정실험동물의피부 ph는 ph meter (PH905, Courage- Khazaka, Germany) 를이용하여희생전날측정하였다. 측정시실험동물등부위의중앙부분을가볍게눌러총 5회측정하여평균값을기록하였다. 동물의희생과표피분리 10주간의실험식이공급후경추탈골방법으로희생시키고자외선조사가이루어진등부위 ( 등중앙기준, 2 cm 3 cm) 의피부조직을취하였다. 피부조직은 Hank s Balanced Salt Solution (HBSS, Gibco BRL, USA) 과 Dispase II (2.4 unit/ml, Roche, Germany) 가 1:1 (v/v) 로혼합된용액에담가 4 o C에서 16시간동안처리한후표피

3 Journal of Nutrition and Health (J Nutr Health) 2016; 49(2): 63 ~ 71 / 65 Table 1. Diet composition and UV irradiation of experimental groups (g/kg) Ingredients 를분리하였다. 17 Experimental group 1) UV- UV+ GTE 2) Corn starch Sucrose Cellulose Corn oil Vitamin mix 3) Mineral mix 4) L-Cystine Casein 5) GTE UV irradiation ) Groups UV- and UV+, hairless mice fed a control diet without (group UV-) or with (group UV+) UV irradiation for 10 weeks; Group GTE, hairless mice fed a diet containing 1.0% green tea extract (GTE) in parallel with UV irradiation for 10 weeks 2) GTE: green tea extract 3) AIN-93 vitamin mix # (Dyets Inc., Bethlehem, PA) 4) AIN-93G salt mix # (Dyets Inc., Bethlehem, PA) 5) Casein (nitrogen x 6.25), 870 g/kg 표피젖산함량측정표피에증류수를가하고 polytron (P-8340, Sigma Chemical Co., St. Louis, MO, USA) 을이용해 4 o C에서 20 분간초음파분쇄하였다. 분쇄한조직을 12,000g, 4 o C에서 15분간원심분리후상층액을취하였다. 젖산함량은단기간에대량분석이가능한 EnzyChrom TM L-lactate assay kit (ECLC-100, BioAssay Systems, Hayward, CA, USA) 을사용하여측정하였다 well plate에 LDH 효소와함께 NAD가함유된 buffer 및표피시료를 1 μl/well의농도로분주하고 30분간상온에서방치한후, LDH에의해젖산이피루브산으로전환시생성되는 NADH에비례하여환원되는 formazan의양을 595 nm에서측정하여간접적으로젖산의함량을파악하였다. 표피시료의단백질정량과함께젖산함량을 nmol/mg protein으로나타내었다. 표피유리아미노산총함량측정표피조직을 15% trichloroacetic acid (TCA) 용액과함께 polytron으로분쇄하고 4 o C에서 16시간동안방치하였다. 21 산침전단백질을제거하기위해 12,000 g에서 10분간원심분리후상층액을취하여 0.45 μm filter (Millipore, USA) 로여과하였다. 총유리아미노산의농도는 high performance liquid chromatography (HPLC) 를이용하여분석하였으며, 분석조건은 Pico Tag column ( mm, 4 μm, WAT088131, Waters, USA) 에서 Acetonitrile:Methanol: Water (45:45:10, v/v/v) 용액을 mobile phase로유속 1.0 ml/min을유지하면서 254 nm에서흡광도를측정하였다. 표피시료의무게값과함께총유리아미노산함량을 μg/g wet weight로나타내었다. 표피유리지방산함량측정표피에증류수를가하고 polytron 을이용하여분쇄하고 12,000 g, 4 o C에서 15분간원심분리후상층액의일부를취하여단백질정량을하였다. 남은용액에 folch 용액 (CHCl 3 :MeOH=2:1, v/v) 을가하여지질을추출하고질소가스로건조시킨후 100 μl의 Folch 용액에다시녹였다. HPTLC (high performance thin-layered chromatography: silica gel, 60Å, 200 μm thickness, Whatman, USA) 에점적하고전개액 (haxane:diethyl ether:acetic acid = 14:16:0.2, v/v/v) 을이용하여유리지방산을분획하였다. 22,23 모든샘플은 automatic multiple development (AMD) apparatus (Linomat 5, CAMAG, Muttenz, Switzerland) 를이용하여분석하였고, 분획된유리지방산의함량은 cupric acetatephosphoric acid 처리및 160 o C에서 15분간가열후해당표준물질과함께 TLC III scanner (DigiStore2, CAMAG, Muttenz, Switzerland) 를이용하여정량하였다. 표피시료의단백질정량과함께유리지방산의총함량을 mg/μg protein으로나타내었다. 표피의주요개별유리지방산함량측정을위해, HPTLC 에의해분리된총유리지방산분획을박층으로부터긁어낸후 Folch 용액으로추출하고 methyl C17:0 (internal standard) 첨가후 6% HCl를함유한 methanol을이용하여지방산을 methylation하였다. 24 Fatty acid methyl ester는 320 Fused Silica Capillary column (30 cm 0.32 mm i.d., 0.25 μm film thickness, Supelco, USA) 을장착한 gas chromatograph (GC) (GC-17A, Shimadzu, Japan) 에주입하여각유리지방산을분석하고, 표피시료의단백질정량과함께 mg/μg protein으로나타내었다. GC 분석조건은 250 o C injector port와 260 o C detector (flameionization detector) port에서 oven 온도를 190 o C로 5분간유지시킨후 4 o C/min 비율로상승시켜 240 o C에서 10분간유지시켰다. 이때 Helium gas (carrier gas) 는 3 ml/min의유량으로흘려주었고 split ratio는 20:1의조건에서 1 μl의시료를주입하였다. 표피 LDH 단백질발현측정표피에 0.5% nonidet P-40 (TE-NP40 buffer extract), 10 mm EDTA, protease inhibitor, 40 mm Tris-HCL (ph 7.5)

4 66 / 녹차추출물섭취에의한피부산도개선효능 가함유된 Tris-EDTA Buffer (P-8340, Sigma Chemical Co., St. Louis, Mo, USA) 를가하고 polytron을이용하여분쇄하였다. 25 분쇄한표피조직을 12,000 g, 4 o C에서 15분간원심분리후얻어진상층액의일부를취하여단백질정량을하였다. 준비된단백질시료 (20 μg/well) 를 8% SDS/ PAGE gel에전기영동후 nitrocellulose membrane에흡착시켰다. Membrane을 1차항체 (anti-rabbit LDH antibody: sc-33781, Santa Cruz Biotech, Inc., USA) 와함께 4 o C에서 17시간동안반응시키고이어서 2차항체 (anti-rabbit IgG- HRP, Cell Signaling Technology Inc., USA) 와상온에서 1 시간동안반응시킨후 ECL hyperfilm을이용하여발색하였다. 25 해당 band의 intensity는 imaging densitometer (Labwork version 4,6 UVP., CA, USA) 를사용해정량하였다. 표피 LDH 활성측정표피 LDH 활성은 LDH Activity Colorimetric Assay Kit (K , BioVision, Milpitas, CA, USA) 를이용하여측정하였다. 26 표피에 LDH assay buffer를가하고 polytron 을이용해분쇄한후 12,000 g, 4 o C에서 15분간원심분리하여상층액을취하였다. 96 well plate에젖산과 NAD를함유한 buffer 및표피시료를 1 μl/well씩분주하고 30분간상온에서방치한후, 생성되는 NADH의양을 450 nm에서측정하여간접적으로 LDH 활성을파악하였다. 표피시료의단백질정량과함께 LDH 활성을 mu/mg protein으로나타내었다. 통계분석모든실험의결과는 SPSS 18.0 program (SPSS Inc., Chicago, IL, USA) 을이용하여통계처리하였다. 결과는각실험군별평균 (mean) 및표준오차 (standard error mean, SEM) 로나타냈으며, 각군간의비교는 one-way ANOVA 로분석후 p < 0.05 수준에서 Duncan s multiple range test로검증하였다. 또한자외선조사군 (UV+ 군 ) 을기준으로 GTE군의유리아미노산총함량의유의성을 p < 0.05 (*) 수준에서 independent student s t-test로재검증하였다. 결 자외선조사및녹차식이공급에의한피부 ph 변화 10주간자외선을조사한 UV+ 군의피부 ph는 UV-군보다유의적으로증가하였는데 (Fig. 1) 이는자외선조사가피부 ph의증가를초래하였음을의미한다. 반면자외선조 과 Fig. 1. Effect of UV irradiation and dietary green tea extract on skin ph. Groups UV- and UV, hairless mice fed a control diet without (group UV-) or with UV irradiation (group UV+) for 10 weeks; Group GTE, hairless mice fed a diet containing 1.0% green tea extract (GTE) in parallel with UV irradiation for 10 weeks. Values are mean ± SEM (n = 5). Values without a common letter are significantly different (p < 0.05) using one-way ANOVA and Duncan's multiple range test. 사와병행하여녹차추출물을 10주간식이공급한 GTE군의피부 ph는 UV+ 군에비해감소하였는데그감소정도는 UV-군과유사하였다. 이결과는자외선조사와병행된 GTE 식이공급이자외선조사에의해증가되는피부 ph 를정상화시켰음을의미한다. 자외선조사및녹차식이공급에의한피부 ph 주요인자의함량변화표피의젖산, 유리아미노산, 유리지방산은피부 ph를결정하는주요인자이다. 1-3 UV+ 군의젖산함량은 UV-에비해유의적으로감소한반면 GTE군의젖산함량은 UV+ 군에비해증가하였다 (Table 2). UV+ 군의총유리아미노산함량은 UV-군과유사하였으나 GTE군의총유리아미노산함량은 UV-군보다유의적으로증가하였다. UV+ 군의총유리지방산함량은 UV-군에비해감소하였으며, GTE군의총유리지방산의함량은 UV+ 군과유사하였다. 이결과는자외선조사는젖산및총유리지방산함량감소와함께피부 ph를증가시킨반면, 자외선조사와병행된 GTE의식이공급은젖산및총유리아미노산의함량증가와함께피부 ph를정상화시켰음을의미한다. 반면 GTE 식이공급은자외선조사에의해감소하는총유리지방산의함량을개선시키지않는것으로여겨진다. 자외선조사및녹차식이공급에의한표피 LDH 의발현및활성변화 LDH는피루브산과젖산의상호전환을촉매하는효소로써피부에서는호기적인상태에서도강한활성을나타

5 Journal of Nutrition and Health (J Nutr Health) 2016; 49(2): 63 ~ 71 / 67 Table 2. Effect of UV irradiation and dietary green tea extract on epidermal levels of lactate, total free amino acids and total free fatty acids Skin ph related factors Experimental group 1) UV- UV+ GTE Lactate (nmol/mg protein) ± )a 91.5 ± 6.6 b ± 5.9 a Total free amino acids (µg/g wet weight) 55.1 ± 4.0 b 69.2 ± 2.3 ab 84.6 ± 1.2 a* Total free fatty acids (mg/µg protein) 16.5 ± 0.6 a 9.8 ± 0.9 b 11.0 ± 1.0 b 1) Groups UV- and UV+, hairless mice fed a control diet without (group UV-) or with UV irradiation (group UV+) for 10 weeks; Group GTE, hairless mice fed a diet containing 1.0% green tea extract (GTE) in parallel with UV irradiation for 10 weeks. 2) Data are mean ± SEM (n = 5). Values without a common letter are significantly different (p < 0.05) using one-way ANOVA and Duncan's multiple range test. *p < 0.05 versus group UV+ by non-paired student's t-test Fig. 2. Effect of UV irradiation and dietary green tea extract on lactate dehydrogenase (LDH) protein expression and activity in the epidermis of mice. Groups UV- and UV+, hairless mice fed a control diet without (group UV-) or with UV irradiation (group UV+) for 10 weeks; Group GTE, hairless mice fed a diet containing 1.0% green tea extract (GTE) in parallel with UV irradiation for 10 weeks. (A) Representative expressions of LDH and actin in the epidermis of groups. (B) Signal intensities from multiple experiments of A were quantified and the integrated areas were normalized to the corresponding value of actin (the loading control). Values are mean ± SEM (n = 5). (C) The activity of LDH was determined in the epidermis of group by LDH activity colorimetric assay kit based on NADH generation in epidermis of groups. Values are mean ± SEM (n = 5). Values without a common letter are significantly different (p < 0.05) using one-way ANOVA and Duncan s multiple range test. 낸다. 4 UV+ 군의 LDH 단백질발현은정상대조군인 UV- 군에비해유의적으로감소하였으나, GTE군에서는 UV+ 군보다증가하였다 (Fig. 2A, 2B). 이는자외선조사에의해 LDH 단백질발현이감소하는반면자외선조사와병행된녹차추출물의식이공급은 LDH 단백질발현을증가시켰음을의미한다. LDH의활성또한 UV+ 군에서감소한반면 GTE군에서는 UV-군과유사한수준으로증가하였다 (Fig. 2C). 이는 10주간의자외선조사가표피의 LDH 단백질발현과활성을감소시키는반면자외선조사와병행된 GTE 식이공급은 LDH의단백질발현및활성을증가시켜궁극적으로젖산함량을증가시킴을의미한다. 자외선조사및녹차식이공급에의한표피주요유리지방산의함량변화팔미트산 (palmitic acid, PA, 16:0), 스테아르산 (stearic acid, SA, 18:0), 올레산 (oleic acid, OA, 18:1n-9) 및리놀레산 (linoleic acid, LA, 18:2n-6) 은표피의주요유리지방산이다. 27 체내에서생성이불가능한 LA는식이공급이절대적으로요구되는필수불포화지방산인반면 PA 및 SA 는체내에서생성이가능한불필수포화지방산이다. 28 한편체내에서생성된 PA는장쇄효소 (elongase) 에의해 SA 로전환될수있고, SA는 Δ9 탈포화효소 (desaturase) 에의해 OA로대사될수있으므로, 28 SA 및 OA은개별지방산보다는 PA에서대사가능한지방산의개념에서군간함량

6 68 / 녹차추출물섭취에의한피부산도개선효능 Table 3. Effect of UV irradiation and dietary green tea extract on epidermal levels of major free fatty acids Major FFA 1) Experimental group2) (mg/µg protein) UV- UV+ GTE 16:0 3) 3.2 ± 0.1 4)a 2.1 ± 0.1 b 2.0 ± 0.2 b 18:0 1.7 ± 0.1 a 1.1 ± 0.1 b 1.7 ± 0.2 a 18:1n ± 0.3 a 2.3 ± 0.2 b 0.9 ± 0.0 c 18:2n ± 0.3 a 2.1 ± 0.1 b 1.7 ± 0.2 b 1) FFA: free fatty acid 2) Groups UV- and UV+, hairless mice fed a control diet without (group UV-) or with UV irradiation (group UV+) for 10 weeks; Group GTE, hairless mice fed a diet containing 1.0% green tea extract (GTE) in parallel with UV irradiation for 10 weeks 3) Only major free fatty acids (FFA) are listed. 4) Data are mean ± SEM (n = 5). Values without a common letter are significantly different (p < 0.05) using one-way ANOVA and Duncan's multiple range test. 을비교함이타당한것으로여겨진다. UV+ 군에서 PA, SA 및 OA의개별함량은 UV-군에비해유의적으로감소하였으며 (Table 3), GTE군에서 PA 함량은 UV+ 군과유사한반면, SA 함량은 UV-군과유사한수준으로증가하였고 OA 함량은 UV+ 군보다더욱현저히감소하였는데이는자외선조사와병행된 GTE 공급이 Δ9 탈포화효소 (desaturase) 의억제를초래하여 SA의축적및 OA 함량의현저한감소를초래한것으로여겨진다. UV+ 군에서 LA의함량은 UV-군에비해유의적으로감소하였으며, GTE군의 LA 함량은 UV+ 군과유사하였다. 이결과는총유리지방산의함량변화결과와유사하게, 자외선조사와병행된 GTE 식이공급은자외선조사에의해감소하는표피의주요유리지방산의개별함량을개선시키지않았음을제안한다. 흥미롭게도자외선조사와병행된 GTE 식이공급은 Δ9 탈포화효소 (desaturase) 의발현이나활성을억제하여지방산대사의변화를초래하는것으로여겨지나, 이는추후연구에서심도있게다루어질필요가있다. 고 찰 본연구는무모생쥐에 10 주간자외선조사와병행하여 1.0% 수준의녹차추출물을식이공급후피부산도및관련인자의함량과 LDH 효소의단백질발현및활성변화를자외선비조사군 (UV-군 ) 및자외선조사군 (UV+ 군 ) 과비교분석하였다. 식이에첨가된녹차추출물의첨가수준은섭취후독성및산화촉진등의부작용없이 18,19 피부산도개선효능이예상되는 0.3%, 0.5%, 1.0% 및 1.25% 수준에서행해진예비연구에기반하여결정하였다. 신체의최외각에서자외선에항시노출되어있는피부에서녹차섭취에의한항산화효능은간, 폐, 및소장의내부장기 보다고용량을필요로하는데, 29 4주간 2% 수준의녹차카테킨섭취가피부에서산화촉진없이항산화관련여러효소의활성증가와함께광노화를억제하는반면, 18 90일간의섭취기간동안백서의체중, 간기능및혈액지표이상등의독성을유발하지않는녹차추출물의식이첨가수준은 0.3~1.25% 수준으로파악되었다. 19 이를근거로 0.3~ 1.25% 수준에서예비연구를시행한결과 0.3% 및 0.5% 에서는효능이나타나지않은반면 1.0% 및 1.25% 의수준에서피부산도개선의효능이나타났으며, 이중저용량인 1.0% 를본연구의식이첨가수준으로결정하였다. 10주간자외선조사와함께병행된 1% 수준의녹차추출물의식이공급은자외선조사에의해증가된피부산도를정상대조군과유사한수준으로감소시켰다. 병행하여, 피부산도관련인자중젖산및총유리아미노산의함량이표피에서증가된반면총유리지방산의함량은자외선조사군과유사한수준으로긍정적인효과를나타내지않았다. 이결과는녹차추출물이젖산의생성및총유리아미노산함량을증가시켜궁극적으로피부산도를정상화시켰음을의미한다. 자연노화와광노화로나뉘어지는피부노화는건조증, 가려움증, 피부장벽지질의생성저하뿐아니라피부의 ph 증가를초래한다. 3,6,30 특히과다한자외선노출에의한피부의광노화는색소침착, 굵은주름형성, 세포사멸, 표피과증식과더불어피부 ph의증가를일으켜피부장벽항상성의손상을초래하는것으로알려져있다. 31,32 광노화의예방및개선을위해화장품뿐아니라다양한식이소재가개발되고있으며, 특히녹차, 소나무껍질추출물복합물, 홍삼, 사상자, 산수유복합추출물등의여러식품소재들이광노화보호를위한건강기능식품으로제품화되고있다 이중녹차추출물및녹차의주요성분인카테킨은자외선노출에의한피부의 DNA 손상, 멜라닌생성억제등의구체적인피부노화억제효능이국내외적으로알려져있다 무모생쥐에 10주간의자외선조사는피부 ph를증가시켰는데이는 60명의사람을대상으로 2 주동안의지속적인자외선조사가피부 ph 증가시킴을보고한선행연구와 35 일치하는결과이다. 반면 10주간의자외선조사와병행된 1.0% 수준의녹차추출물의식이공급은피부 ph를 UV-군과유사한수준으로감소시켰다. 건강한피부는 ph 4~6 범위의약산성 ph를유지하고있으며이는피부의항상성유지의지표로여겨지고있다. 5 광노화에의한피부 ph 증가는피부의여러효소들의활성변화를초래하는것으로보고되어있으며녹차식이공급에의한피부 ph 감소는녹차의공급이피부의여러효소들의활성을정상화시켰음을의미한다. 피부의 ph는표피에서생성되는젖산, 유리아미노산,

7 Journal of Nutrition and Health (J Nutr Health) 2016; 49(2): 63 ~ 71 / 69 유리지방산등여러인자들의전체함량에의해결정된다. 1-3 땀에포함되어있는암모니아 (NH 3 ) 및중탄산이온 (HCO 3 ) 또한피부 ph에영향을미치나이들의함량은극소량으로표피의젖산, 유리아미노산및유리지방산이피부 ph의주요결정인자로알려져있다. 표피에서는호기적인상태에서도젖산생성이활발히이루어지며, 4 유리아미노산조성또한대부분의아미노산이일정한범위에서큰변화없이유지되는혈액의아미노산조성과상이한데, 36 이는산성아미노산인 glutamate 및 histidine이 40% 이상을차지하고있는반면 methionine, phenylalanine, valine, isoleucine, leucine 및 cysteine은거의존재하지않는것으로설명된다. 1 표피에서 glutamate 및 histidine는 pyrrolidone carboxylic acid (PCA) 과 urocanic acid (UCA) 로대사되기도한다. 1 표피의여러유리지방산또한피부산도유지에기여하는데, 젖산과유리지방산뿐아니라, glutamate 등의산성유리아미노산및 PCA와 UCA에함유되어있는카르복실기의수소이온이 pka 2~6 범위에서해리되어궁극적으로 ph 4~6 범위약산성의건강한피부산도를유지한다. 1,3 또한표피의유리아미노산은피부산도증가시젖산이나유리지방산에비해완충력 (buffering capacity) 이뛰어나피부산도를약산성으로신속하게정상화시킨다고알려져있다. 37 피부에서 lactate dehydrogenase (LDH) 에의한피루브산의젖산으로의전환은호기적인상태에서도활발히이루어지는데 4 표피의 LDH 단백질발현은진피에비해현저히높은것으로파악되었다. 38,39 GTE군의표피에서 LDH 단백질발현및활성이 UV+ 군에비해증가되었는데, 이는자외선조사에의해감소된 LDH 단백질발현과활성이녹차추출물의식이공급에의해증가됨을의미한다. LDH의 mrna 전사는 protein kinase C (PKC) 경로를통해활성화되는데, 40 녹차의카테킨이 PKC 경로를활성화하는것이선행연구에의해보고되었다. 41 본연구에서는 LDH의 mrna 발현수준을파악하지않았으나이결과들은녹차의카테킨성분들이 PKC 경로의활성화를초래하여간접적으로 LDH의 mrna 전사및단백질발현을증가시키고활성증가를초래하여궁극적으로젖산생성을증가시킴을제안한다. 표피의주에너지로사용되는젖산은유리지방산, 탄산및인산결합의총량보다피부 ph 의약산성유지에크게기여할뿐만아니라피부건조증상을완화시켜주는인자이며, 1 젖산이포함되지않은환경에서표피세포의성장이현저히감소한다는선행연구에 4 의해젖산이피부표면을산성화시킬뿐만아니라피부건강의주요요소임이확인되었다. UV-군에비해 UV+ 군의젖산함량이현저히감소하였으며 GTE군에서는젖산함량 이 UV-군과유사한수준으로증가하였는데, 이결과는젖산분비가감소된신부전환자의피부 ph 증가가젖산함량감소와병행된다는사전보고와 42 함께녹차식이공급이자외선노출로인해감소된 LDH의발현과활성증가를초래하여결과적으로젖산함량을증가시켰음을의미한다. 유리아미노산의유일한근원단백질인필라그린 (filaggrin) 은 43,44 peptidylarginine deiminase-3 (PAD3) 에의해분해가유도되고표피분화최종단계에서 caspase-14, calpain-1 및 bleomycin hydrolase (BH) 등의다양한단백질분해효소에의해표피의유리아미노산으로최종분해되는것으로여겨지고있는데이과정에관여하는효소는아직명확하게파악되지않았다. 45,46 필라그린의분해산물인유리아미노산의조성은필라그린의구성아미노산조성과유사한반면, 45,46 대부분의아미노산이일정한범위에서큰변화없이유지되는혈액의아미노산조성과상이한데, 36 이는산성아미노산인 glutamate 및 histidine이 40% 이상을차지하고있는반면 methionine, phenylalanine, valine, isoleucine, leucine 및 cysteine은거의존재하지않는것으로설명된다. 1 표피에서 glutamate 및 histidine는 pyrrolidone carboxylic acid (PCA) 과 urocanic acid (UCA) 로대사되기도한다. 1 표피에서 glutamate 등의산성유리아미노산및 PCA와 UCA에함유되어있는카르복실기의수소이온이 pka 2~6 범위에서해리되어궁극적으로 ph 4~6 범위의약산성의건강한피부산도를유지할뿐아니라, 1,3 한편으로표피의유리아미노산은피부산도증가시젖산이나유리지방산에비해완충력 (buffering capacity) 이뛰어나피부산도를약산성으로신속하게정상화시킨다고알려져있다. 37 UV+ 군과 UV-군의총유리아미노산함량이유사한반면 GTE군에서현저히증가한총유리아미노산함량은녹차식이공급이표피의총아미노산함량을증가시켰음을의미한다. 녹차섭취에의한총유리아미노산의함량증가가 glutamate, histidine 등의주요개별아미노산및, PCA, UCA의함량증가를직접적으로의미하지는않으나, 이들이표피의주요유리아미노산및대사체임이사전연구에서보고되어있고, 1,3 피부산도증가시표피유리아미노산은전체적으로젖산이나유리지방산에비해완충력이뛰어나다는점에서, 37 총유리아미노산의함량증가는피부산도개선에기여하였음을의미한다. 추후녹차추출물의공급에의한필라그린을비롯한개별유리아미노산함량, 관련효소들의발현및활성변화등에대한심도있는연구가필요할것으로사료된다. 표피의과립층과각질층사이에서분비되는 phospholipase A 2 (PLA 2 ) 에의해생성되는유리지방산은각질층의피부장벽을이루는주요구성성분일뿐아니라피부

8 70 / 녹차추출물섭취에의한피부산도개선효능 ph의약산성유지에기여한다. 47 UV+ 군의총유리지방산함량이 UV-군보다감소하였는데이는광노화로인한유리지방산합성효소들의 mrna 발현및 PLA 2 의활성이변화하였다는사전보고와 48 함께자외선조사에의해피부지방산함량의감소가초래되었음을의미한다. 녹차카테킨은광노화에의한지방의과산화반응을감소시키는반면, 유리지방산의생성을저해하는것으로알려져있다. 49 즉자외선조사와병행된녹차식이공급은지방산함량에는긍정적인효과를나타내지않는것으로여겨진다. 결론적으로자외선조사와병행된녹차추출물식이공급은 LDH 발현과활성증가와함께젖산과유리아미노산함량을증가시켰으며궁극적으로자외선조사에의해증가된피부 ph를정상화시켰다. 요약 본연구에서는자외선조사와함께녹차추출물식이를 10주간공급한무모생쥐 (GTE군) 의표피 ph 관련인자의함량과 LDH 효소의단백질발현및활성변화를자외선비조사군 (UV-군 ) 및자외선조사군 (UV+ 군 ) 과비교분석하였으며결과는다음과같다. UV+ 군의피부 ph는 UV-군보다유의적으로증가한반면 GTE군의피부 ph는 UV+ 군에비해감소하였으며그감소정도는 UV-군과유사하였다. UV+ 군의젖산함량은 UV-군에비해현저히감소한반면 GTE군의젖산함량은 UV+ 군에비해증가하였다. UV+ 군과 UV-군의총유리아미노산함량은군간유사하였으나 GTE군의총유리아미노산함량은 UV-군에비해현저히증가하였다. UV+ 군및 GTE군의총유리지방산함량은군간차이없이모두 UV-군에비해감소하였으며, GTE군에서주요유리지방산인 PA, OA 및 LA의개별함량은모두 UV-군과유사하거나감소하였다. UV+ 군의 LDH 단백질발현및활성은 UV-군에비해유의적으로감소한반면, GTE군의 LDH 단백질발현및활성은모두 UV+ 군보다증가하였으며특히 LDH 활성의증가정도는 UV-군과유사하였다. 결론적으로자외선조사와병행된녹차추출물식이공급은 LDH 발현및활성증가와함께젖산과총유리아미노산함량을증가시켰으며궁극적으로자외선조사에의해증가된피부 ph를정상화시켰다. References 1. Rippke F, Schreiner V, Schwanitz HJ. The acidic milieu of the horny layer: new findings on the physiology and pathophysiology of skin ph. Am J Clin Dermatol 2002; 3(4): Nakagawa N, Sakai S, Matsumoto M, Yamada K, Nagano M, Yuki T, Sumida Y, Uchiwa H. Relationship between NMF (lactate and potassium) content and the physical properties of the stratum corneum in healthy subjects. J Invest Dermatol 2004; 122(3): Elias PM. Stratum corneum acidification: how and why? Exp Dermatol 2015; 24(3): Lambers H, Piessens S, Bloem A, Pronk H, Finkel P. Natural skin surface ph is on average below 5, which is beneficial for its resident flora. Int J Cosmet Sci 2006; 28(5): Ali SM, Yosipovitch G. Skin ph: from basic science to basic skin care. Acta Derm Venereol 2013; 93(3): Choi EH, Man MQ, Xu P, Xin S, Liu Z, Crumrine DA, Jiang YJ, Fluhr JW, Feingold KR, Elias PM, Mauro TM. Stratum corneum acidification is impaired in moderately aged human and murine skin. J Invest Dermatol 2007; 127(12): Rabe JH, Mamelak AJ, McElgunn PJ, Morison WL, Sauder DN. Photoaging: mechanisms and repair. J Am Acad Dermatol 2006; 55(1): El-Domyati M, Attia S, Saleh F, Brown D, Birk DE, Gasparro F, Ahmad H, Uitto J. Intrinsic aging vs. photoaging: a comparative histopathological, immunohistochemical, and ultrastructural study of skin. Exp Dermatol 2002; 11(5): Bissett DL, Hannon DP, Orr TV. An animal model of solar-aged skin: histological, physical, and visible changes in UV-irradiated hairless mouse skin. Photochem Photobiol 1987; 46(3): Holleran WM, Uchida Y, Halkier-Sorensen L, Haratake A, Hara M, Epstein JH, Elias PM. Structural and biochemical basis for the UVB-induced alterations in epidermal barrier function. Photodermatol Photoimmunol Photomed 1997; 13(4): Sterenborg HJ, de Gruijl FR, van der Leun JC. UV-induced epidermal hyperplasia in hairless mice. Photodermatol 1986; 3(4): Sime S, Reeve VE. Protection from inflammation, immunosuppression and carcinogenesis induced by UV radiation in mice by topical Pycnogenol. Photochem Photobiol 2004; 79(2): Kang TH, Park HM, Kim YB, Kim H, Kim N, Do JH, Kang C, Cho Y, Kim SY. Effects of red ginseng extract on UVB irradiationinduced skin aging in hairless mice. J Ethnopharmacol 2009; 123(3): Pazyar N, Feily A, Kazerouni A. Green tea in dermatology. Skinmed 2012; 10(6): Vayalil PK, Mittal A, Hara Y, Elmets CA, Katiyar SK. Green tea polyphenols prevent ultraviolet light-induced oxidative damage and matrix metalloproteinases expression in mouse skin. J Invest Dermatol 2004; 122(6): Li YH, Wu Y, Wei HC, Xu YY, Jia LL, Chen J, Yang XS, Dong GH, Gao XH, Chen HD. Protective effects of green tea extracts on photoaging and photommunosuppression. Skin Res Technol 2009; 15(3): Min J, Lee Y, Han SM, Choi Y. Dietary effect of royal jelly supplementation on epidermal levels of hydration, filaggrins, free amino acids and the related enzyme expression in UV irradiated hairless mice. Korean J Nutr 2013; 46(2): Jeon SE, Choi-Kwon S, Park KA, Lee HJ, Park MS, Lee JH, Kwon

9 Journal of Nutrition and Health (J Nutr Health) 2016; 49(2): 63 ~ 71 / 71 SB, Park KC. Dietary supplementation of (+)-catechin protects against UVB-induced skin damage by modulating antioxidant enzyme activities. Photodermatol Photoimmunol Photomed 2003; 19(5): Takami S, Imai T, Hasumura M, Cho YM, Onose J, Hirose M. Evaluation of toxicity of green tea catechins with 90-day dietary administration to F344 rats. Food Chem Toxicol 2008; 46(6): Senadheera D, Krastel K, Mair R, Persadmehr A, Abranches J, Burne RA, Cvitkovitch DG. Inactivation of VicK affects acid production and acid survival of Streptococcus mutans. J Bacteriol 2009; 191(20): Nachat R, Méchin MC, Takahara H, Chavanas S, Charveron M, Serre G, Simon M. Peptidylarginine deiminase isoforms 1-3 are expressed in the epidermis and involved in the deimination of K1 and filaggrin. J Invest Dermatol 2005; 124(2): Hamanaka S, Hara M, Nishio H, Otsuka F, Suzuki A, Uchida Y. Human epidermal glucosylceramides are major precursors of stratum corneum ceramides. J Invest Dermatol 2002; 119(2): Uchida Y, Hara M, Nishio H, Sidransky E, Inoue S, Otsuka F, Suzuki A, Elias PM, Holleran WM, Hamanaka S. Epidermal sphingomyelins are precursors for selected stratum corneum ceramides. J Lipid Res 2000; 41(12): Tang W, Ziboh VA. Reversal of epidermal hyperproliferation in essential fatty acid deficient guinea pigs is accompanied by rapid generation of inositol triphosphate. Arch Dermatol Res 1988; 280(5): Takagi Y, Nakagawa H, Yaginuma T, Takema Y, Imokawa G. An accumulation of glucosylceramide in the stratum corneum due to attenuated activity of beta-glucocerebrosidase is associated with the early phase of UVB-induced alteration in cutaneous barrier function. Arch Dermatol Res 2005; 297(1): Beamer CA, Holian A. Silica suppresses Toll-like receptor ligandinduced dendritic cell activation. FASEB J 2008; 22(6): Lampe MA, Burlingame AL, Whitney J, Williams ML, Brown BE, Roitman E, Elias PM. Human stratum corneum lipids: characterization and regional variations. J Lipid Res 1983; 24(2): Cook HW. Fatty acid desaturation and chain elongation in eukaryotes. In: Vance DE, Vance JE, editors. Biochemistry of Lipids and Membranes. Menlo Park, CA: Benjamin/Cummings Pub. Co.; p Khan SG, Katiyar SK, Agarwal R, Mukhtar H. Enhancement of antioxidant and phase II enzymes by oral feeding of green tea polyphenols in drinking water to SKH-1 hairless mice: possible role in cancer chemoprevention. Cancer Res 1992; 52(14): Kim EJ, Jin XJ, Kim YK, Oh IK, Kim JE, Park CH, Chung JH. UV decreases the synthesis of free fatty acids and triglycerides in the epidermis of human skin in vivo, contributing to development of skin photoaging. J Dermatol Sci 2010; 57(1): Bhawan J, Andersen W, Lee J, Labadie R, Solares G. Photoaging versus intrinsic aging: a morphologic assessment of facial skin. J Cutan Pathol 1995; 22(2): Hsu S. Green tea and the skin. J Am Acad Dermatol 2005; 52(6): Kim DS, Park SH, Kwon SB, Li K, Youn SW, Park KC. (-)-Epigallocatechin-3-gallate and hinokitiol reduce melanin synthesis via decreased MITF production. Arch Pharm Res 2004; 27(3): Katiyar SK, Elmets CA. Green tea polyphenolic antioxidants and skin photoprotection (Review). Int J Oncol 2001; 18(6): Di Cerbo A, Laurino C, Palmieri B, Iannitti T. A dietary supplement improves facial photoaging and skin sebum, hydration and tonicity modulating serum fibronectin, hyaluronic acid and carbonylated proteins. J Photochem Photobiol B 2015; 144: Kim H, Park KH, Yeo JH, Lee KG, Jeong DH, Kim SH, Cho Y. Dietary effect of silk protein sericin or fibroin on plasma and epidermal amino acid concentration of NC/Nga mice. Korean J Nutr 2006; 39(6): Levin J, Maibach H. Human skin buffering capacity: an overview. Skin Res Technol 2008; 14(2): Ronquist G, Andersson A, Bendsoe N, Falck B. Human epidermal energy metabolism is functionally anaerobic. Exp Dermatol 2003; 12(5): Lewis C Jr, Schmitt M, Hershey FB. Heterogeneity of lactic dehydrogenase of human skin. J Invest Dermatol 1967; 48(3): Huang D, Jungmann RA. Transcriptional regulation of the lactate dehydrogenase A subunit gene by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Mol Cell Endocrinol 1995; 108(1-2): Levites Y, Amit T, Mandel S, Youdim MB. Neuroprotection and neurorescue against Aβ toxicity and PKC-dependent release of nonamyloidogenic soluble precursor protein by green tea polyphenol (-)-epigallocatechin-3-gallate. FASEB J 2003; 17(8): Yosipovitch G, Tur E, Morduchowicz G, Boner G. Skin surface ph, moisture, and pruritus in haemodialysis patients. Nephrol Dial Transplant 1993; 8(10): Scott IR, Harding CR, Barrett JG. Histidine-rich protein of the keratohyalin granules: source of the free amino acids, urocanic acid and pyrrolidone carboxylic acid in the stratum corneum. Biochim Biophys Acta 1982; 719(1): Rawlings AV, Scott IR, Harding CR, Bowser PA. Stratum corneum moisturization at the molecular level. J Invest Dermatol 1994; 103(5): Thulin CD, Walsh KA. Identification of the amino terminus of human filaggrin using differential LC/MS techniques: implications for profilaggrin processing. Biochemistry 1995; 34(27): McGrath JA, Uitto J. The filaggrin story: novel insights into skinbarrier function and disease. Trends Mol Med 2008; 14(1): Hachem JP, Crumrine D, Fluhr J, Brown BE, Feingold KR, Elias PM. ph directly regulates epidermal permeability barrier homeostasis, and stratum corneum integrity/cohesion. J Invest Dermatol 2003; 121(2): Tyrrell RM. Activation of mammalian gene expression by the UV component of sunlight--from models to reality. BioEssays 1996; 18(2): Wang X, Song KS, Guo QX, Tian WX. The galloyl moiety of green tea catechins is the critical structural feature to inhibit fattyacid synthase. Biochem Pharmacol 2003; 66(10):