J Physiol & Pathol Korean Med 29(1):33~38, ,4-Dinitrochlorobenzene 으로유도된 BALB/c 마우스에서 Black currant seed oil 의아토피성피부염억제효과 이예서 박교현 김배환 * 계명대학교자연과

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1 J Physiol & Pathol Korean Med 29(1):33~38, ,4-Dinitrochlorobenzene 으로유도된 BALB/c 마우스에서 Black currant seed oil 의아토피성피부염억제효과 이예서 박교현 김배환 * 계명대학교자연과학대학공중보건학과 I nhibitory Effects of Black currant seed oil on 2,4-Dinitrochlorobenzene I nduced Atopic Dermatitis in BALB/c M ice M odel Ye Seo Lee, Kyo Hyun Park, Bae Hwan Kim* Department of Public Health, College of Natural Sciences, Keimyung University This study investigated the clinical parameters of atopic dermatitis and evaluated the inhibitory effects of Black currant seed oil for atopic dermatitis by using a Dinitrochlorobenzene(DNCB) induced BALB/c mice model. Five-week-old BALB/c mice were divided into four groups: normal group (N, non-treated), control group (C, atopic dermatitis-induced), positive control group (PC, Tacrolimus ointment-treated against induced atopic dermatitis, PC), experiment group (E, Black currant seed oil-treated against induced atopic dermatitis). After induction of atopic dermatitis by DNCB, the erythema, edema, eschar, and scratching were severely observed. The symptoms of atopic dermatitis were improved after 2 weeks, and almost disappeared after 4 weeks in PC and E group. The increased frequencies of scratching in C group were decreased in PC and E group. Transepidermal water loss, erythema index and serum IgE level were significantly decreased in E and PC compared to that in C after 4 weeks of the treatment. The results indicated that Black currant seed oil can relieve atopic dermatitis symptoms effectively, and may be possibly used as a functional material for suppression of atopic dermatitis. keywords : Black currant seed oil, atopic dermatitis, BALB/c mice, DNCB 서론 아토피성피부염은홍반성구진, 강한소양감등의주요증상을특징으로하는만성염증성피부질환이다 1). 주로아동기에높은발병률을보이며전세계적으로꾸준히증가하는추세로산업이발달한선진국일수록유병률은지속적으로증가하고있는모습을보인다 2). 아토피성피부염의원인은명확하게밝혀져있지않지만가족력, 식생활의변화, 면역조절장애등의요인이복합적으로작용하여발병하는것으로보여지며, 최근에는피부표면장벽의이상이병인에중요하게작용한다는근거들이제시되면서각질층의구성인자에대한연구들이보고되고있다 3). 아토피성피부염은혈청 immunoglobulin E(IgE) 가비정상적으로항진하는외인성과 IgE의수준은정상이나병리학적으로아토피성피부염과유사한증상이나타나는내인성으로나누어진다 4). 항원제시세포는항원과결합하여항원의정보를 T cell에전달하고항원정보를받은 T cell은세포매개반응을일으켜병변유발및지속적 으로면역반응을유지하는데큰역할을하게된다 5). T cell은생성하는 cytokine에의해 Th1, Th2 유형으로분화되며, Th1 유형에서생성되는 cytokine은 interleukin-2(il-2), interferon-gamma (INF-γ), tumor necrosis factor-alpha(tnf-α) 등을분비하여대식세포를활성화시켜지연형면역반응을유도하고, Th2 유형의 cytokine인 interleukin-4(il-4), interleukin-5(il-5) 및 interleukin-13(il-13) 등은 IgE의합성과비만세포의분화를유도함으로써염증반응과면역병리학적증상을유발하게된다 6,7). 따라서아토피성피부염에서혈청 IgE 생성과관련된 IL-4와 IgE 농도변화에대한기전은치료에중요한지표라할수있다 8). 현재피부염의치료방법으로사용되어지는스테로이드제는 1950년에도입되어수년동안사용되어왔지만사용횟수와농도, 기간등에따른피부의안전성과내성의문제점으로인해장기간의사용이제한되고있다 9-11). 특히아토피성피부염의경우면역력이약한유 소아에서유병률이높기때문에안전성이보장된치료제의확보가무엇보다시급한것으로보여진다. 리놀렌산, 감마리놀렌산, * Corresponding author Bae-Hwan Kim, Department of Public Health, College of Natural Sciences, Keimyung University, Daegu , Korea kim9399@kmu.ac.kr Tel : Received : 2014/11/12 Revised : 2015/01/20 Accepted : 2015/01/23 c The Korean Society of Oriental Pathology, The Physiological Society of Korean Medicine pissn eissn Available online at

2 34 Y. S. Lee et al 알파리놀렌산등은대표적인불포화지방산으로동물실험과임상실험을통해염증성질환의증상완화와표피의장벽보호에효과적이라인정되었으며, 임신기간동안불포화지방산을임신부가섭취할경우태반에서 Th2 cytokine의감소로인하여출생후아토피성피부염의발병률이낮은것으로보고되었다 12-14). 또한비만세포주 RBL-2H3 cell과마우스의골수에서분화된 BMMCs cell에서비만세포의특이적전사인자를조절함으로 IL-4, IL-13의생성을억제시켰으며, 마우스의대식세포주 RAW cell에서염증작용을억제하였다고보고된바있다 15,16). 오메가-3 지방산인알파리놀렌산과오메가-6 지방산인리놀렌산이풍부한 Black currant seed oil의경우, 경구섭취로인한염증억제및면역증가등이보고되어있으나 17), 경피흡수를통한효과연구는아직미비한실정이다. 본연구에서는 BALB/c 마우스와 2,4-dinitrochlorobenzene(DNCB) 를이용하여아토피성피부염과면역학적특성이유사한실험모형으로 Black currant seed oil를경피에처치하였을때아토피성피부염의일반적인지표변화와혈청내 IgE 레벨등면역학적지표의변화를관찰하여아토피성피부염의억제원료로개발가능성의여부를판단하고자하였다. 재료및방법 1. 실험재료실험에사용된 Black currant seed oil은 Douglas Laboratories (USA, Lot. #99857) 에서제조되었다. 실험물질은물로세척하고건조후압착하여추출한것을실험에사용하였으며, 본실험에사용한 Black currant seed oil의성분은 Table 1과같다. 성피부염을유발한뒤 Black currant seed oil 을처치한실험군 (E) 으로구성하였다. Table 2. Experimental group characteristics Normal (N) Control (C) Positive Control (PC) Experimental (E) Atopic dermatitis induction 3. 아토피성피부염유발및시료도포 Test compound No. of mice % Tacrolimus 6 + Black currant seed oil 아토피성피부염의유발및시료도포과정은다음과같다 (Fig. 1). DNCB 를이용하여아토피성피부염을유발하였다. 마우스의제 모된등부위피부에 DNCB 를용매물질 (acetone:olive oil = 1:3) 에녹인 1% DNCB 용액을 3 일간격으로 100 μl씩 2 회처치하여 아토피성피부염을감작 (sensitization) 시킨후, 7 일째되는다음일 주일은 0.2% DNCB 용액을 3 일간격으로 2 회도포하여아토피성 피부염을유발 (challenge) 하였다. 아토피성피부염의발현후 0.1% tacrolimus 150 μl, Black currant seed oil 150 μl씩주 5 회, 4 주간환부에처치하였다. 지속적인아토피성피부염의유지를위해 0.2% DNCB(sigma, USA) 용액을시료처치기간중주 1 회마우 스의등부위에처치하였다. 6 Table 1. Composition of Black currant seed oil Ingredients % Linoleic acid 44.8 ɤ-Linoleic acid 16.1 α-linoleic acid 13.6 Oleic acid 10.1 Stearic acid 1.5 Palmitic acid 7.8 Others 6.1 Source : Douglas Laboratories 2. 동물시험구성 생후 5 주령의암컷 BALB/c mouse 를 ( 주 ) 샘타코 ( 오산, 한국 ) 로 부터분양받아 1 주간특정병원체부재무균사육실에서온도는 22±3, 습도는 50±5%, 명암주기 12 시간밤과낮단위로조절되 는표준사육환경에서적응과정을거친후실험에사용하였다. 실험 기간동안사료 (Purina, 한국 ) 와물의양은제한없이공급하였다. 실험의모든과정은계명대학교동물실험윤리위원회의승인 ( 승인번 호 KM ) 을얻은후가이드라인에의거하여실험을진행하 였다. 본연구의실험군구성은 Table 2 와같다. 각군당 6 마리씩난 괴법에따라분류하고, 아무런처치를하지않은정상군 (N), DNCB 를도포하여아 ; 토피성피부염을유발한대조군 (C), 아토피성피부 염을유발한뒤 0.1% tacrolimus 를처치한양성대조군 (PC), 아토피 Fig. 1. Scheme of experimental design. 4. Itching behavior 관찰시료에따른가려움증의정도를관찰하기위해마우스가뒷다리를이용하여안면, 목, 등부위의피부를긁는횟수를 micro-camera를이용하여 30분간녹화하여군별로비교하였다. 5. 피부병변육안적관찰제모된등부위의피부병변정도를보기위해주 1회사진촬영하였다. 육안으로관찰하였을때각군의아토피성피부염상태는홍반, 부종, 가피, 찰과상, 태선화, 건조등의증상에따라 scorad index 평가방법으로없음 (0), 경증 (1), 중간 (2), 중증 (3) 으로판정하였으며, 각군별로평균치를산정하여분석하였다. 6. 경피수분손실량 (Transepidermal water loss, TEWL) 측정

3 Y. S. Lee et al 35 시료처치전과실험종료후의마우스의경피수분손실량을측정하기위해 tewameter를이용하여동일한등부위에 10회반복하여비침습방법으로측정후평균값을구한다음군간비교, 분석하였다. 7. 홍반도측정시료처치전과실험종료후의홍반도를측정하기위해 mexameter를이용하여마우스의동일한등부위에 5회반복비침습방법으로측정후평균값을구한다음군간비교, 분석하였다. 8. 혈액채취시료처치전안와정맥총 (periorbital plexus) 에서 capillary tube를이용하여약 100 μl의혈액을채취하였고, 실험종료후희생부검시심장채혈을실시하여혈액을채취하였다. 안와채혈혹은심장채혈을통해얻은혈액을원심분리하여혈청을분리한다음분석시까지 80 에서냉동보관하였다. 9. 혈액 IgE 측정혈청내 IgE 수준은 ELISA(enzyme-linked immunosorbent assay, 효소면역측정법 ) 방법에의해정량되었다. 원심분리후냉동보관중인혈청을측정전에해동하여, IgE ELISA Kit(Sibayagi, Japan) 를이용하여 IgE 농도측정을하였다. 항체가코팅된 well에 50 μl씩 sample과 IgE standard solution을각각넣어 20~25 조건에서 2시간배양시킨후 biotin-conjugated anti-ige 항체를 50 μl씩넣어 20~25 조건에서 2시간배양시켰다. 이후, HRP(horse radish peroxidase)-conjugated avidin을 50 μl씩넣어 20~25 조건에서 1시간배양시킨후, 마지막으로 chromogenic substrate reagent를 50 μl씩넣어 20~25 조건에서 20분간배양시키고 ELISA reader를이용해 450nm 파장으로흡광도를측정하였다. 제모한등부위피부병변을육안으로관찰한결과는 Table 4, Fig. 2 와같다. 정상군에서는실험기간동안피부병변이관찰되지 않았고아토피성피부염을유발시킨대조군, 양성대조군, 실험군에 서는홍반, 부종, 가피형성이관찰된것으로보아아토피성피부염 의유발이잘되었음을보여준다. 양성대조군과실험군에서시료처 치 2 주차부터서서히회복되는양상을보이며시료처치 4 주차에는 거의정상상태에가까울정도로육안적변화를보였다. Table 3. Scratching frequencies during the experiment period ± ± ± ± ±4.18 a 13.29±4.61 b 5.17±3.43 a 3.83±1.72 a * Unit: number. Values are mean±sd of 6 mice. Test compounds were topically DNCB + tacrolimus, E : DNCB + Black currant seed oil. Values with different Table 4. Comparison in scorad index during experimental period in mice 0 1.2±0.8 a 5.5±0.5 ab 5.7±1.6 d 5.3±0.8 c 1 1±0.6 a 6.2±0.8 b 4±1.5 c 4.5±0.5 b 2 0.8±0.8 a 5.7±0.5 ab 3.2±1 bc 3.3±0.8 a 3 1.2±0.4 a 5.3±0.5 ab 2.5±0.5 ab 3±0.6 a 4 0.8±0.4 a 5±0.9 a 1.5±0.8 a 2.8±0.4 a Unit: number. Values are mean±sd of 6 mice. Test compounds were topically DNCB + tacrolimus, E : DNCB + Black currant seed oil. Values with different 0W 10. 통계처리 SPSS 21.0 for windows(spss Inc., USA) 통계프로그램을이용하여일원배치분산분석 (one-way ANOVA) 을실시하였다. 각그룹간의차이를검정하기위하여 Duncan's multiple range test를이용하여사후분석을실시하였다. 본연구의통계학적인유의성검증수준은 p<0.05로하였다. 2W 결 과 4W 1. Itching behavior 관찰결과시료처치전과실험종료후에 30분간동영상촬영하여마우스가뒷다리를이용해안면, 목, 등부위의피부를긁는횟수를측정한결과는 Table 3과같다. 아토피성피부염을유도하지않은정상군에서는긁는횟수가증가되지않았고, 양성대조군과실험군에서시료처치 4주후대조군에비해유의하게감소하였다 (p<0.05). 2. 피부병변육안적관찰결과 Fig. 2. Clinical skin features and severity of DNCB-induced atopic dermatitis in balb/c mice. N : Non treat, C : DNCB, PC : DNCB + tacrolimus, E : DNCB + Black currant seed oil 3. 경피수분손실량 (Transepidermal water loss, TEWL) 측정결과 시료처치전과종료후의경피수분손실량을측정한결과는 Table 5 와같다. 정상군에비해아토피성피부염을유발한군에서높 은수치를보였고, 시료처치종료후모든군에서감소하였으며대조 군에비해양성대조군과실험군에서유의하게감소하였다 (p<0.05).

4 36 Y. S. Lee et al Table 5. Changes in transepidermal water loss of BALB/c mouse skin in 4 weeks experiment of Black currant seed oil ±3.67 a * 65.94±7.98 b 76.43±11.53 b 70.36±11.19 b ±3.68 a * 28.09±5.62 c 14.97±4.4a* 20.8±4.98 b Unit: g/h m2, Values are mean±sd of 6 mice. Test compounds were topically DNCB + tacrolimus, E : DNCB + Black currant seed oil, Values with different 4. 홍반도측정 시료처치전과종료후의홍반도를측정한결과는 Table 6 과 같다. 시료처치전정상군에비해아토피성피부염을유발한군에 서높은수치를보였고대조군, 양성대조군, 실험군간에유의한차 이는없었으며, 시료처치종료후대조군에비해양성대조군, 실험 군은유의하게감소하였다 (p<0.001). Table 6. Changes in erythema index of BALB/c mouse skin in 4weeks experiment of Black currant seed oil ±55.06 a * ±35.07 b ±15.81 b ±14.73 b ±17.09 a * ±10.65 d ±24.71 b * ±32.24 c * Unit: AU(arbitrary unit), Values are mean±sd of 6 mice. Test compounds were topically applied to the backs of BALB/c mice for 4weeks. N : Non treat, C : DNCB, PC : DNCB + tacrolimus, E : DNCB + Black currant seed oil, Values with different superscripts are significantly different (p<0.05) by ANOVA and Duncan's multiple range tests. The asterisk (*) indicates significantly different (p<0.001) from the 5. 혈액 IgE 측정결과 혈청내 IgE 수준의변화를측정한결과 Table 7 과같다. 아토 피성피부염을유발후정상군에비해 IgE 수준이약 10 배정도급 격하게상승하였고, 군간의차이는크게없었다. 시험 4 주후대조 군과실험군의 IgE 수준이증가하였으나, 대조군에비해실험군의 IgE 수준이경미하게증가하였다. 양성대조군의 IgE 수준은감소하 였으며, 정상군은큰변화가없었다. Table 7. Changes in IgE level change of mouse serum ± a * ± b ± b ± b ± a * ± d ± b * ± c * Unit: ng/ml, Values are mean±sd of 6 mice. Test compounds were topically DNCB + tacrolimus, E : DNCB + Black currant seed oil, Values with different 고 최근아토피성피부염의발생원인으로피부표피의구성성분 인필라그린 (filaggrin) 단백질의구조적이상, 세라마이드지질의 이상등이만성염증성피부염을유발한다는근거가꾸준히제시되 고있다 18,19). 아토피성피부염환자의피부표면장벽의기능적이상 은병변부위뿐아니라정상적인곳에서도관찰되며, 경피수분손실 면적또한정상적인곳에서도보이고있다 20). 이러한건조증상은체 찰 내불포화지방산이부족할경우나타나며합성되는양이미량이기때문에인체외부에서공급되어야한다. 리놀렌산의대사과정을통하여감마리놀레산이생성되며, 생성된감마리놀레산은 TNF-α, nitric oxide(no), cyclooxygenase-2 (COX-2) 의생성과배출을억제하고, 손상된피부표면장벽을회복하여수분보유량을증가시킨다 21-23). 리놀렌산의최종대사산물인아라키돈산 (arachidonic acids) 이 COX 효소에의해 prostaglandin E(PGE) 가생산되고 PGE는 T cell의증식과세포사멸억제, B cell의증식과분화를조절하여면역반응을향상시키는것으로알려져있다 24,25). 음식으로섭취할경우대사과정에서충분한 PGE의생성이어렵고수명이짧기때문에고순도의불포화지방산을환부에직접바르는것은매우효과적일것이라생각되어다량의불포화지방산이함유된 Black currant seed oil의아토피성피부염억제에관한연구를실시하였다. 아토피성피부염은대표적증상인소양감으로인해병변부위를긁어 2차피부질환을유발하게되는데본실험에서도아토피성피부염을유발한군에서정상군에비해긁는횟수가증가된것이관찰되었다. 시료처치 4주후대조군에서는시료처치전보다긁는횟수가증가하였고양성대조군과실험군에서는감소하였다. 항원에감작될경우항원제시세포와항원이결합하여 T cell에항원을제공하게되면 T cell이활성화하여 IL-4와같은 cytokine에의해 B cell이형질세포로분화되어 Ig E를분비하게되는데, 이때생성된 IgE가비만세포의표면에부착하여히스타민 (histamine) 의분비로가려움증, 홍반등의알르레기반응이일어나게된다. 본실험결과시료의처치로인하여 Ig E의분비가감소되어비만세포에서분비되는히스타민을억제시켜소양감을감소시킨것으로보여진다. 이러한소양감은피부가건조할경우더욱심해지며아토피성피부염환자의경우표피의수분보유량이일반인에비해감소되어있는데본연구에서도앞서언급한바와같이아토피성피부염을유발한마우스에서경피수분손실이정상군에비해 3배이상증가되었고, 4 주간시료를처치한결과대조군에비해양성대조군과실험군에서유의하게감소하였다 (p<0.05). 홍반도를측정한결과아토피성피부염유도후정상군을제외한모든군에서높은수치를보였고, 시료처치종료후에는정상군에서는큰변화가관찰되지않았으며대조군에서는처치전보다증가하였다. 양성대조군과실험군에서는시료처치 4주후대조군에비해유의하게감소하였으며 (p<0.001), 시료처치전과후의차이를검증한결과대조군에비해양성대조군과실험군에서유의하게감소하였다 (p<0.05). 이같은결과는 Black currant seed oil이표피의수분보유량을높여표면장벽을보호하는데기인한것으로사료된다. 아토피성피부염의중증도를임상적으로평가하는방법으로 scorad index를사용하는데홍반, 부종, 가피, 찰과상, 태선화, 건조등과같은증상을점수화한것으로 15 18점일때가장심한정도로보고있다. 본연구결과아토피성피부염유도직후에는위와같은증상들이관찰되었고, 시료처치 2주부터모든군에서호전되는양상을보이며 4주차에양성대조군과실험군에서는정상과가까운육안적변화를보였다. 대조군에서도회복되긴했으나약간의흉터가관찰되었다. 아토피성피부염은 IgE 매개성으로혈청내 IgE가증가되어있

5 Y. S. Lee et al 37 는것으로보고되고있으며 IgE의수준은아토피성피부염의염증반응에매우중요한지표로여겨진다. 항원이인체에들어오면항원제시세포 (antigen presenting cell), 랑게르한스세포 (Langerhans cell) 가활성화되어항원의정보가제시되고 T cell은 Th2로분화되어 IL-4, IL-13 등의 cytokine의생성을유도한다 27). 이러한 cytokine은 immunoglobulin isotype 전환으로 B cell을자극하여 IgE가증가하게된다. 이러한기전은본연구에서도 DNCB의처치로혈중 IgE가정상군에비해 10배이상증가한것을알수있었으며시료처치 4주후정상군에서는큰변화가관찰되지않았고대조군과실험군에서는시료처치전보다증가하는양상을보였다. 이는시료처치중에도자연치유의오차를피하기위해주 1회 DNCB 를처치한것이아토피성피부염을유지시킨것으로보인다. 그러나대조군과비교했을때실험군과양성대조군은유의한감소를보였다 (p<0.001). 결론 본연구에서는 Black currant seed oil의아토피성피부염억제효과를입증하기위하여 DNCB으로유도된 BALB/c 마우스에 Itching behavior 관찰, 피부병변의육안적관찰, 경피수분손실량측정, 홍반도측정, 혈액내 IgE 측정을하였다. Black currant seed oil은이기능성소재로서의적용가능성을확인하기위하여아토피성피부염억제효과를시험하였다. Black currant seed oil 은 DNCB으로유도한아토피성피부염에서경피수분손실량과홍반도를감소시켰고, 육안적피부병변의회복및긁는횟수가줄어들어피부표면장벽을강화하여물리적인자극을완화하였으며혈청내 IgE를효과적으로감소시키는결과를보이므로아토피성피부염억제원료로활용가능하리라사료된다. References 1. Boguniewicz, M., Leung, D.Y.M. Atopic dermatitis. J Allergy Clin Immunol 117: , Leung, D.Y.M., Bieber, T. Atopic dermatitis. Lancet 361: , Su, R.C., Becker, A.B., Kozyrskyj, A.L., HayGlass, K.T. Epigenetic regulation of established human type 1 versus type 2 cytokine responses. J Allergy Clin Immunol 121: 57-63, Novak, N., Bieber, T. Allergic and nonallergic forms of atopic diseases. J Allergy Clin Immunol 112: , Kim, M.A., Son, H.U., Nam, D.Y., Cha, Y.S., Shin, Y.K. Inhibitory Effect of Angelica keiskei Extract in an Atopic Dermatitis Animal Model. Korean J Food Preserv 19: , Jartti, T., Burmeister, KA., Seroogy, C.M., Jennens-Clough, M.L., Tisler, C.J., Salazar, L.P., DaSilva, D.F., Evans, M.D., Vrtis, R.F., Wallace, P.K., Ruiz-Perez, B., Gangnon, R.E., Lemanske, R.F.Jr., Gern, J.E. Association between CD4+CD25high T cells and atopy in children. J Allergy Clin Immunol 120: , Leung, D.Y.M., Soter, N.A. Cellular and immunologic mechanisms in atopic dermatitis. J Am Acad Dermatol 44: 1-12, Wills-Karp, M. Interleukin-13 in asthma pathogenesis. Curr Allergy Asthma Rep 4: , Ainley-Walker, P.F., Patel, L., David, T.J. Side to side comparison of topical treatment in atopic dermatitis. Arch Dis Child 79: , Hanifin, J.M., Cooper, K.D., Ho, V.C., Kang, S., Krafchik, B.R., Margolis, D.J., Schachner, L.A., Sidbury, R., Whitmore, S.E., Sieck, C.K., Van Voorhees, A.S. Guidelines of care for atopic dermatitis. J Am Acad Dermatol 50: , Hoare, C., Li Wan, P.A., Williams, H. Systematic review of treatments for atopic eczema. Health Technol Assess 4: 1-191, Artemis, P.S. Omega-3 fatty acids and cardiovascular disease: the epidemiological evidence. Environ Health Prev Med 6: , Chung. S., Kong. S., Seong. K., Cho, Y. Gamma-linolenic acid in borage oil reverses epidermal hyperproliferation in guinea pigs. J Nutr 132: , Olsen, S.F., Østerdal, M.L., Salvig, J.D., Mortensen, L.M., Ryttrer, D., Secher, N.J., Henriksen, T.B. Fish oil intake compared with olive oil intake in late pregnancy and asthma in the offspring: y of registry-based follow-up from a randomized controlled trial. Am J Clin Nutr 88: , Noh, K.H., Jang, J.H., Min, K.H., Chinzorig, R., Lee, M.O., Song, Y.S. Suppressive Effect of Green Tea Seed Coat Ethyl Acetate Fraction on Inflammation and Its Mechanism in RAW264.7 Macrophage Cell. J Korean Soc Food Sci Nutr 40: , Chang, C.S., Sun, H.L., Lii, C.K., Chen, H.W., Chen, P.Y., Liu KL. Gamma-linolenic acid inhibits inflammatory responses by regulating Nf-κB and AP-1 activation in lipopolysaccharide -induced RAW macrophages. Inflammation 33: 46-57, Linnamaa, P., Savolainen, J., Koulu, L., Tuomasjukka, S., Kallio, H., Yang, B., Vahlberg, T., Tahvonen, R. Blackcurrant seed oil for prevention of atopic dermatitis in newborns: a randomized, double-blind, placebo-controlled trial. Clin Exp Allergy 40: , 2010.

6 38 Y. S. Lee et al 18. Akdis, CA., Akdis, M., Bieber, T., Bindslev-Jensen, C., Boguniewicz, M., Eigenmann, P., Hamid, Q., Kapp, A., Leung, D.Y.M., Lipozencic, J., Luger, T.A., Muraro, A., Novak, N., Platts-Mills, T.A.E., Rosenwasser, L., Scheynius, A., Simons, F.E.R., Spergel, J., Turjanmaa, K., Wahn, U., Weidinger, S., Werfel, T., Zuberbier, T. Diagnosis and treatment of atopic dermatitis in children and adults: european academy of allergology and clinical immunology/american academy of allergy, asthma and immunology/practall consensus report. J Allergy Clin Immunol 118: , Weidinger, S., Rodrĺguez, E., Stahl, C., Wagenpfeil, S., Klopp, N., Illig, T., Novak, N. Filaggrin mutations strongly predispose to early-onset and extrinsic atopic dermatitis. J Invest Dermatol 127: , Kim, D.H., Lee, K.J., Qi, X.F., Lee, Y.M., Yoon, Y.S., Kim, J.L., Chang, B.S., Ryang, Y.S. The Effects of Magnesium Rich Sea Mineral Water on Atopic Dermatitis-like Skin Lesions in Hairless Mice. Korean J Microscopy 38: , Dooper, M.M.B.W., Riel, B.V., Graus, Y.M.F., M'Rabet, L. Dihomo-γ-linolenic acid inhibits tumour necrosis factorα production by human leucocytes independently of cyclooxygenase activity. Immunology 110: , Furse, R.K., Rossetti, R.G., Seiler, C.M., Zurier, R.B. Oral administration of gammalinolenic acid, an unsaturated fatty acid with anti-inflammatory properties, modulates interleukin-1[beta] production by human monocytes. J Clin Immunol 22: 83-91, Yen, C.H., Dai, Y.S., Yang, Y.H., Wang, L.C., Lee, J.H., Chiang, B.L. Linoleic acid metabolite levels and transepidermal water loss in children with atopic dermatitis. Ann Allergy Asthma Immunol 100: 66-73, Kim, K.B., Park, J.S., Jeong, J.M. Effects of Phyto-Extract Mixture (PEM381) in Type I Allergic Reaction-Induced Mice. J Korean Soc Food Sci Nutr 36: , Calder, P.C., Yaqoob, P., Thies, F., Wallace, F.A., Miles, E.A. Fatty acids and lymphocyte functions. Br J Nutr 87: 31-48, 2002.

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