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pissn 1229-1153 / eissn 2465-9223 J. Food Hyg. Saf. Vol. 30, No. 4, pp. 376~382 (2015) http://dx.doi.org/10.13103/jfhs.2015.30.4.376 Journal of Food Hygiene and Safety Available online at http://www.

1 pissn / eissn J. Food Hyg. Saf. Vol. 30, No. 4, pp. 376~382 (2015) Journal of Food Hygiene and Safety Available online at 낙지다리식물추출물의알레르기억제효과 조소영 김영미 * 덕성여자대학교약학대학 Inhibitory Effect of Penthorun chinense Extract on Allergic Responses in vitro and in vivo So young Jo and Young Mi Kim* College of Pharmacy, Duksung Women s University, Seoul 01369, Korea (Received November 26, 2015/Revised December 3, 2015/Accepted December 10, 2015) ABSTRACT - Allergic diseases have rapidly increased in recent years. Mast cells play a critical role in IgE-mediated allergy responses and, therefore, closely associated with rhinitis, asthma, and atopic dermatitis. We studied antiallergic effect of Penthorum chinense extract (PCE) in vitro and in vivo. PCE inhibited the degranulation of mast cells by antigen stimulation and its effect was dose-dependent and reversible in mast cells. PCE also suppressed the production of inflammatory cytokines such as TNF-α and IL-4 by antigen in mast cells. Mechanistically, PCE inhibited the activation of Syk/LAT pathway which is a key signaling pathway for the activation of mast cells by antigen. Notably, PCE suppressed IgE-mediated allergic responses by antigen in mice. These results strongly suggest that PCE is a potential candidate for anti-allergic treatment. Key words : Penthorum chinense extract (PCE), mast cell, syk kinases, allergy, anti-allergic effect 현대사회는기후변화에따른생태계변화, 대기오염, 황사, 실내주거환경, 서구화된식습관등에의하여천식, 알레르기비염, 아토피, 만성폐쇄성폐질환등알레르기질환이증가추세를보이고있다 1). 특히, 알레르기질환의대상이어린아이까지확대되면서사회적이슈가되고있으며다양한알레르기질환치료제들이개발되고있는상황이다. 외부알레르겐 (allergen) 이우리몸속으로침입하면, B 림프구는침입한알레르겐을인식하고, 형질세포로분화한다. 이때분화된형질세포는항원을특이적으로인식하는 Immunoglobulin E (IgE) 를분비하게된다. 이러한조건에의하여항원특이 IgE 항체가비정상적으로증가하게되며증가한 IgE는비만세포표면의 IgE 고친화성수용체 (FcεRI, high-affinity receptor for IgE) 에결합하며외부로부터알레르겐 (allergen) 이다시들어오면항원-항체반응이진행되면서비만세포를활성화시킨다 2). 비만세포는세포내에과립들을가지고있는데, 비만세포가활성화되면세포내의과립에저장되어있던히스타 *Correspondence to: Young Mi Kim, College of Pharmacy, Duksung Women's University 33, Samyangro 144-Gil, Dobong-Gu, Seoul 01369, Korea Tel: , kym123@duksung.ac.kr 민, 헤파린및단백분해효소등이세포밖으로분비되고류코트리엔, 프로스타글란딘등의화학매개체들을만들어낸다. 이러한화학매개체들은짧은시간내에혈관을확장시켜투과성을증가시키고우리몸의평활근수축및분비선의기능을증가시켜알레르기증상을유도한다 3). 비만세포표면에존재하는 FcεRI는 α, β, γ-subunit으로구성되어있다 4). 이중 β, γ-subunit는세포내신호전달에관여한다. 알레르겐이 IgE와교차결합하면 β-subunit에결합되어있던 Src-kinase에의해수용체가인산화되면서세포내신호전달이시작된다. 또한 β, γ-subunit은수용체에결합되어있던 Lyn kinase에의해 ITAM (Immunoreceptor tyrosine-based activation mitif) 이인산화된다 5). 인산화된 ITAM은세포활성화에중요한역할을하는 Syk kinase의결합부위를제공하여 Syk kinase를활성화시키고그후아래의하위신호전달물질의활성화를유도한다 6). 항원에의한비만세포활성화과정초기에가장중요한신호전달단백질인 Syk가활성화되면서하위신호전달 Linker for activation of T cells (LAT), phospholipase(pl)- Cγ, PKC (protein kinase C) 등다양한신호전달물질이활성화되고, 결과적으로비만세포가활성화되면서히스타민, 프로스타글라딘, 루코트리엔및과민증인자등을분비하여면역체계를활성화시키고, 염증및알레르기를발생 376

2 Inhibitory Effect of Penthorun chinense Extract on Allergic Responses in vitro and in vivo 377 시킨다 7). 이와같이세포내신호전달기전에대한연구는알레르기질환연구에서중요한부분을차지한다. 본논문에서도 FcεRI 매개한신호전달에중점을두어실험을진행하였다. 낙지다리 (Penthorum chinense Pursh) 는돌나물과 (Crassulaceae) 의다년생초본으로근경이나종자로번식한다. 전국적으로분포하며강가나풀밭의습기가많은곳에분포한다. 낙지다리추출물은간과신경세포의보호효과가있는것으로보고되어있다 8). 하지만알레르기질환에대한낙지다리의효능에대한보고는존재하지않는다. 본연구에서는낙지다리추출물의비만세포억제효과및알레르기유발동물모델에서알레르기억제효과등을규명하기위해수행하였다. Materials and Methods 재료 Dinitrophenol(DNP)-specific IgE와항원 (DNP-bovine serum albumin, DNP-BSA) 는 Sigma-Aldrich Co.(St. Louis, MO, USA) 에서구입하였다. 또한 PP2는 Calbiochem (La Jolla, CA, USA) 에서구입하였다. RPMI-1640 및 Minimal Essential Medium (MEM), 배양액에첨가되는 Glutamine과그외세포배양시약은 GIBCO Technologies Inc. (Rockville, MD, USA) 에서구입하였다. Western blot에사용되는 Phospho-Syk 및 Phospho-LAT 항체는 Cell signalling Technology (Danvers, MA, USA) 에서구입하였으며그외에 actin을검출하기위한항체는 Millipore (Billerica, MA, USA) 에서구입하였다. 기타모든시약은시중제품중특급시약을구입하여사용하였다. 실험동물실험에사용한동물은, ( 주 ) 오리엔탈바이오 ( 성남 ) 에서생후 5 주령수컷 Balb/c 마우스를구입하여사용하였다. 물과사료는자유롭게먹도록하였으며, 사육환경은온도 22 ±1 o C, 습도 55 ± 10% 로유지하였고, 낮과밤의주기는각각 12 시간으로하였다. 모든실험동물은 1 주일동안적응시킨후실험에사용하였다. 동물실험은덕성여자대학교동물실험윤리회의승인을받아, 동규정에따라실험을진행하였다. 낙지다리의메탄올추출물 (PCE) 제조낙지다리추출물 ( 분양번호, ) 은한국식물추출물은행으로부터분양받아실험을진행하였다. 낙지다리추출과정은건조된낙지다리전초 100 g을 50 o C 메탄올 1000 ml에서초음파추출기를이용하여하였으며스피드백을이용하여 40 o C에서 24시간건조과정을거쳤다. 이러한과정으로추출한최종회수율은약 15% 였다. 비만세 포억제실험에사용하기위해서 DMSO (dimethyl sulfoxide) 에용해시켜 100 mg/ml 농도로제조한후, 최종처리농도로희석하여실험에적용하였다. 비만세포 (BMMC, RBL-2H3) 의분리및배양 RBL-2H3 (Rat Basophil Leukemia) cell은 ATCC (American Type Culture Collection) 에서분양받아 15% FBS가포함된 MEM (penicillin /streptomycin 및 L-glutamine 포함 ) 을배양액으로하여 5% CO 2 incubator에서, 2-3일간격으로계대배양하였다. 생쥐의골수에서유래한비만세포 (BMMCs, Bone marrowderived mast cells) 는 5주령의수컷 Balb/c 마우스의골수로부터채취하였다. 마우스로부터분리한골수의세포는 RPMI배지 (RPMI 1640, 10% FBS, 0.1 mm non-essesntial amino acid 및 2 mm L-glutamine) 에 IL-3(10 ng/ml) 이포함된배양액을사용하여 4주이상배양후 BMMC로의분화가 98% 이상되었음을 FACS분석을통해확인한후실험에사용하였다. 비만세포의탈과립 (β-hexosaminidase) 측정 BMMCs( /well) 혹은 RBL-2H3 cells( / well) 두종류의비만세포를 24-flask에분주하고 20 ng/ml DNP-특이 IgE를포함한배지에서 12시간이상감작하여비만세포 FcεRI에 IgE가결합하도록하였다. β-hexosaminidase 분비측정은 Tyrode (135 mm NaCl, 5 mm KCl, 20 mm HEPES, 5.6 mm glucose, 1.8 mm CaCl 2, 1 mm MgCl 2, 0.5% fatty acid-free fraction V from bovine serum, ph 7.4) 완충용액과 PIPES (25 mm PIPES, 159 mm NaCl, 5 mm KCl, 0.4 mm MgCl 2, 1 mm CaCl mm glucose, 0.5% fatty acid-free fraction V from bovine serum, ph 7.4) 완충용액을이용하여실험을진행하였다. 우선 37 o C 완충용액으로각각의세포를 2번세척후 well당각농도별로완충용액에희석된추출물을첨가하고 30분전처리를하였다. 그후 20 ng/ml의항원을각 well에첨가하여 10분동안세포자극을진행하였다. 10분 incubation 후약 7분간얼음위에서자극을정지시켰다. 탈과립에의해세포밖으로분비된 β-hexosaminidase을측정하기위해위의과정을거친각 well의상층액과 Triton X-100으로세포를파쇄하여얻은파쇄액을각각의 96 well plate에 30 ul씩분주하고 1 mm p-nitrophenyl-n-acetyl-β-d-gluco saminide를넣고 1시간동안반응시킨후 250 ul의 0.1 M carbonate buffer 를넣어반응을종료한뒤 405 nm에서흡광도측정하여정량하였다. Cell viability 96well plate에 well 당 개의세포를분주하고 5% CO 2 incubator에서 12시간배양하였다. 그후 PCE를

3 378 So young Jo and Young Mi Kim 10 ug/ml, 30 ug/ml, 100 ug/ml 농도별로처리한후 CO 2 incubator에서 1시간동안반응시켰다. 1시간후각 well에 CCK-8 (cell counting kit-8) 을첨가한후 1시간동안incu- bation을시킨다음 ELISA reader를이용하여 450 nm에서흡광도를측정하였다. Immunoblotting 분석 IgE로감작된 BMMCs, RBL-2H3 cells에 PIPES (or Tyrod buffer) 로희석한 PCE를각각농도별로첨가한다음, 20 ng/ml의항원을넣어 10분동안자극하고얼음위에서반응을정지시켰다. 항원에의해자극된세포는약 4 o C PBS 완충용액으로 2번세척한후세포파쇄용액 (20 mm HEPES, ph 7.5, 150 mm NaCl, 1% nonidet P-40, 60 mm octyl β-glucoside, 10% glycerol, 10 mm NaF, 1 mm phenylmethyl-sulfonyl fluoride (PMSF), 1 mm Na3VO4, 2.5 mm nitrophenyl-phosphate, 0.7 mg/ml pepstatin, and a proteaseinhibior cocktail tablet) 으로 10분간세포를파쇄후 15,000rpm, 10분간 4 o C에서원심분리기한다음상등액을얻었다. 이상등액을이용하여단백질정량후 100 o C에서 3x Laenmmli buffer로약 5분간끓였다. 정량된단백질은 SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) 로분리후 nitrocellulose membrane으로옮겨 5% BSA를포함한 TBS-T (10 mm Tris-HCl, ph 7.5, 150 mm NaCl, 0.05% Tween 20) 완충액으로 1시간동안 blocking을시켰다. 이후분석하고자하는단백질에대한 1차항체를 1000배로희석하여 4 o C에서 overnight로반응시킨후 TBS-T로세척한뒤 2000배로희석한 HRP-conjugation된 2차항체로 1시간상온에서반응시켰다. 이후 10분씩 5번 TBS-T 완충액으로세척하고 chemiluminescence analyzer로단백질밴드를확인하였다. 의비교는 one-way analysis of variance (ANOVA) 에이은 Student s t-test 분석을실시하였다. 대조군과비교하여 p 값이 0.05 미만일경우에통계적으로유의성이있다고판정하였다. 모든실험은 3회반복으로실시하였다. Results 비만세포 (RBL-2H3 세포 ) 의탈과립 (β-hexosaminidase) 억제효과 IgE 감작된비만세포는항원으로부터자극을받으면비만세포과립안에있던 histamine, β-hexosaminidase, 다양한 proteases 등을세포밖으로방출하여알레르기반응을유도한다. 본실험에서는탈과립의지표로사용되는 β- hexosaminidase를측정하여낙지다리추출물의억제효과를확인하였다 9). 항원자극에의한비만세포에서의 β-hexosaminidase 분비가농도의존적으로억제되었으며, 처리농도 100 μg/ml에서비만세포의탈과립현상이약 80% 정도가억제되는우수한효능이관찰되었다 (Fig. 1). 이러한억제효과는대조약물로사용된 PP2의억제효과와유사하게나타났다. PCE의비만세포탈과립억제효과의가역성실험 RBL-2H3 세포에한그룹은 PCE를 100 μg/ml으로 30분동안전처리를진행하였고다른한그룹은 PCE를동일한농도로 30분전처리한후 PIPES 완충용액으로 5회세척하여탈과립의현상이회복되는것을관찰하였다. PCE 100 μg/ml 농도로전처리한군에서 β-hexosaminidase 분비는대조군과비교하여 70% 이상억제되었으나 PIPES 완 국소적수동피부알레르기마우스동물모델 DNP (dinitrophenol)-specific IgE에의한국소알레르기피부반응 (passive cutaneous anaphylaxis, PCA) 을유도하기위해마우스 (BALB/c, 5주령수컷 ) 의오른쪽귀에 DNPspecific IgE를피내로 0.5 μg를주사하였다. 다음날 5% 아라비아검용액에녹인낙지다리추출물을 40 mg/kg, 200 mg/kg, 1000 mg/kg 경구투여하였다. 1 시간후항원 (DNP- BSA, 1 mg/ml) 을에반스블루용액을이용하여 5mg/ml 농도로제조하여 250 μl를꼬리로정맥투여하였다. 1 시간후마우스를안락사시키고귀를적출하여 700 μl의포름아마이드로 63 o C에서 12 시간동안에반스블루를추출후상등액을 620 nm의파장에서흡광도를측정하였다. 에반스블루의정량은표준정량곡선을이용하여측정하였다. 통계처리모든실험결과는 mean ± S.E. 로나타내었으며, 각군간 Fig. 1. Effect of Penthorum chinense extract (PCE) on antigen (Ag)-stimulated degranulation in RBL-2H3 mast cells. After incubating RBL-2H3 cells with 50 ng/ml DNP-specific IgE, the cells were stimulated with 25 ng/ml antigen for 10 min. The degranulation was determined by measurement of the activity of β-hexosaminidase in culture media. The values indicate mean ± S.E.M. from three independent experiments. *p < 0.05 and **p < 0.01, significant differences with the Ag-stimulated control without PCE. PP2 is a general Src-family kinase inhibitor.

The RBL-2H3 cells were washed out five times with PIPES buffer after pre-treating with 100 μg/ml PCE for 1 h and then stimulated for 10 min with antigen. The values indicate mean ± S.E. from three independent experiments.

4 Inhibitory Effect of Penthorun chinense Extract on Allergic Responses in vitro and in vivo 379 Fig. 2. Reversibility of Penthorum chinense extract (PCE) on antigen (Ag)-stimulated degranulation in RBL-2H3 mast cells. The RBL-2H3 cells were washed out five times with PIPES buffer after pre-treating with 100 μg/ml PCE for 1 h and then stimulated for 10 min with antigen. The values indicate mean ± S.E. from three independent experiments. **p < 0.01, significant differences with control. Fig. 4. Effect of Penthorum chinense extract (PCE) on ionomycin or thasigargin-stimulated degranulation in mast cells. The RBL- 2H3 cells were stimulated with 1 μm ionomycin (Ion) or 300 nm thapsigargin (Tg), and degranulation was measured by assessing the activity of β-hexosaminidase in culture media. The values indicate mean ± SEM from three independent experiments. PP2 is a general Src-family kinase inhibitor. *p < 0.05 and **p < 0.01, significant differences with Ag-stimulated controls. PCE의비만세포에서염증성사이토카인분비억제효과본연구에서는 PCE 전처리에의한비만세포에서의사이토카인생산억제효과를 RT-PCR 기법으로측정하였다. PCE는농도의존적으로 TNF-α 와 IL-4의 mrna 발현을현저히억제하였다 (Fig. 3A). IL-4의 mrna 발현은 PCE 30 μg/ml 농도에서부터대조군과비교하여유의적으로억제되었으며 (p < 0.05), TNF-α의 mrna 발현은 PCE 100 μg/ml 농도에서대조군과비교하여유의적으로억제되었다 (p < 0.05), PCE 100 μg/ml에서는대조약물로사용된 PP2 와유사한정도의억제효과가관찰되었다 (Fig. 3B). Fig. 3. Effect of Penthorum chinense extract (PCE) on the expression of tumor necrosis factor (TNF)-α and interleukin (IL)-4 in RBL-2H3 mast cells. RBL-2H3 cells were incubated with IgE and then stimulated with 25 ng/ml Ag. Total RNA was isolated and then reversely transcribed. Polymerase chain reaction using IL-4- or TNF-α specific primers was performed as described in the section for Materials and Methods. (A) The representative images and (B) band density from three independent experiments are shown. *p < 0.05 and **p < 0.01, significant differences with Ag-stimulated controls. PP2 is a general Src-family kinase inhibitor. PCE의세포내칼슘농도증가에따른탈과립의억제효과본연구에서 PCE의비만세포탈과립억제효과의기전을연구하기위해우선이억제효과가칼슘이온증가의상위신호전달물질인지하위신호경로를억제하는것인지를알아보기위해본실험을진행하였다. 비만세포를이오노마이신과탭시가긴을처리시비만세포에서탈과립이증가하는것을관찰할수있었다. 하지만이러한물질을처리하기전 PCE을전처리하였을때 PP2 와는달리 PCE의전처리에의해서탈과립의억제효과는보이지않았다 (Fig. 4). 이러한결과는 PCE에의한비만세포에서의탈과립억제효과는칼슘에의한신호전달경로상위에서일어나는것으로추정된다. 충용액으로 5회세척해준군은대조군의약 90% 까지회복되었다 (Fig. 2). 이러한결과로부터, PCE의비만세포탈과립억제효과는가역성을가짐을알수있었다. PCE의비만세포에서의 Syk 및 LAT의인산화억제효과 Fig. 5에서, PCE는농도의존적으로 Syk kinase의인산화를억제하여활성을감소시키는것을확인할수있었다. 또한 Syk kinsae의인산화활성이억제됨으로 LAT 인

380 So young Jo and Young Mi Kim Fig. 5. Effect of Penthorum chinense extract (PCE) on the activating phosphorylation of Syk and LAT in Ag-stimulated mast cells.

The whole cell lysates were subjected to immunoblot analysis to detect the phosphorylated forms of Syk and LAT. Representative immunoblot images are shown from three independent experiments.

5 380 So young Jo and Young Mi Kim Fig. 5. Effect of Penthorum chinense extract (PCE) on the activating phosphorylation of Syk and LAT in Ag-stimulated mast cells. The IgE-primed RBL-2H3 cells and BMMCs were stimulated with 25 ng/ml Ag (DNP-BSA), with or without PCE at the indicated doses, for 7 min. The whole cell lysates were subjected to immunoblot analysis to detect the phosphorylated forms of Syk and LAT. Representative immunoblot images are shown from three independent experiments. PP2 is a general Src-family kinase inhibitor. 산화활성역시감소하는것을확인하였다. 따라서낙지다리추출물은 Syk kinase의인산화를억제하므로서항원에의해유도되는비만세포의탈과립을억제할수있음을알수있었다 (Fig. 5). 이러한결과는 RBL-2H3 cell 뿐만아니라골수유래의세포에서분화시킨비만세포 (BMMC) 에서도항원자극에의한 Syk/LAT 신호전달경로가유사한정도로억제된다는것을확인할수있었다 (Fig. 5). PCE의국소피부자극알레르기반응의억제효과알레르기동물모델에서 PCE의알레르기반응억제효과를측정한결과, 항원자극에의한알레르기반응은잘유도되었으며 PCE 처리에의해억제되었다 (Fig. 6A). 이러한억제결과는 PCE mg/kg 농도범위내에서농도의존적으로억제되었으며최고농도인 1000 mg/kg 투여군에서는대조약물로사용된세트리진과동일한효과를보였다. Discussion 질병관리본부가실시한 천식및알레르기질환조사 결과에따르면, 최근 15년간 ( 95년 ~ 10년 ) 알레르기비염은어린이 1.3배, 청소년은 1.4배증가했고, 알레르기결막염은어린이 1.9배, 청소년 1.9배증가했다고발표했다. 이와같이알레르기발병률은어린아이로부터성인까지계 Fig. 6. Effect of Penthorum chinense extract (PCE) on IgE-mediated passive cutaneous anaphylaxis (PCA). (A) The anti-allergic effect was assessed using PCA mouse model. Balb/c mice were intravenously injected with a 250-mg antigen (DNP-BSA) containing 4% Evans blue 24 h after intradermal administration of a dinitrophenol (DNP)-specific IgE antibody (0.5 mg) into a mouse ear. PCE ( mg/kg) was orally administrated at 1 h before treatment of antigen. (A) The representative photographs of ears are shown. B) The dye extravasated by antigen was extracted from the ear and the amount was measured by absorbance from three independent experiments, each with 5 mice. *p < 0.05; **p < 0.01, significant differences with Ag-stimulated controls. Cetrizine (CZ, 50 mg/kg) was used as a typical anti-histamine reference drug. 속해서증가하는추세이다 10). 현재알레르기치료제로는알레르기를일으키는알레르겐 (allergen) 으로부터보호하는물리적방법, 항히스타민제, 류코트리엔수용체길항제등이있는데, 이중가장널리쓰이는항히스타민제는알레르기반응시, 체내에과다하게분비되는히스타민을억제함으로알레르기반응을감소시키는효과를가지고있지만, 과량으로사용할경우중추신경계억제및녹내장, 전립선비대등다양한부작용을동반하고있다 11). 이와같이현재시판되고있는약물중알레르기를근본적으로치료할수있는치료법은미비한상태이다. 따라서알레르기질환을치료할근본적인연구가필요하다. 알레르기질환에서증상발현에중요한역할을하는비만세포 (mast cell) 의활성의조절은알레르기질환치료제개발에서많은연구의대상이되고있다. 호염기구와비만세포에는알레르기반응을유도하는화학매개체들이포함된과립들이세포질내에존재한다. 알레르겐이외부로부터항원에의해자극되어지면알레르기반응을일으키는물질을생산, 분비한다 12). 이러한화학매개체들은과립안에이미생성되어저장되어있던물질 (preformed mediators)

6 Inhibitory Effect of Penthorun chinense Extract on Allergic Responses in vitro and in vivo 381 과, 자극후새로생성되는물질 (newly synthesized mediators) 로구성된다. 비만세포활성화시분비되는물질로는히스타민과세로토닌이있으며, 그외에도 IL-4, IL-13, TNF-α 등의사이토카인을분비하여알레르기성염증및림프구의증식과이동을유도한다 13). 이러한과정은알레르겐과비만세포표면의고친화성 IgE 수용체 (FcεRI) 가결합한후신호전달체계가활성화되는 Fyn-Gab2-PI3K 경로와 Lyn-Syk-LAT 경로에따라진행된다 14). 알레르겐과 FcεRI 간의집합이이루어지면수용체의 β, γ-subunit의 ITAM이 Src-kinase에의해인산화되고 Syk kinase와인산화된 ITAM이결합하여 Syk의구조가변형되면서신호가활성화된다. 그후하위신호전달분자및어댑터단백질이순차적으로활성되어비만세포에서알레르기유도물질을분비하고알레르기반응을유도하게된다 15). PCE은세포독성이없는 μg/ml에서비만세포초기활성신호전달경로인 Syk/LAT 의신호전달경로활성을농도의존적으로억제함을확인하였다 (Fig. 5). 이러한신호전달을바탕으로활성화된비만세포는세포질내칼슘농도가증가되는데이러한증가는비만세포내의과립의탈과립을유도한다. PCE가비만세포탈과립억제의효과가있음을확인하였다 (Fig. 1) 새로운의약품이개발될때그물질의체내가역성은매우중요하다. 일반적으로비가역적인물질은체내투여시체내단백질에결합하여그단백질이제거될때까지약효를진속하게되어부작용가능성을증가시킨다 16). 이러한비가역성에의한부작용가능성을관찰하기위해우리는낙지다리추출물의가역성실험을진행하였다. 실험결과낙지다리추출물은비만세포활성억제효과가가역적임을관찰하였다 (Fig. 2). 이러한가역성은낙지다리추추물의부작용가능성을다소낮추어준것으로평가되었다. 최근에비만세포억제를통한알레르기반응억제효능이있는식물추출물에대한보고가다양하게진행되어지고있다. 그중에서갈매나무잎추출물은항원자극에의한비만세포의 Fyn kinase 활성화억제를통해알레르기반응을억제하는것으로보고되었다 17). 꾸지뽕나무추출물은항원에의한비만세포의 Lyn/Syk 경로의활성화억제를통해알레르기반응억제효능을가지는것으로보고되어갈매나무추출물의억제기전과는다소차이가있었다 18). 이러한식물추출물의마우스에서의알레르기반응억제 ED 50 는대부분 mg/kg 이며본연구의낙지다리추출물의경우에도유사한 ED 50 를보였다. 또한, 낙지다리추출물은비만세포의신호전달과정중 Syk kinase 의활성화억제를통해알레르기반응을억제하였다. 하지만이러한억제가어떤상위 kinase의활성조절에의한것인지는추후보완연구가더필요하다. 종합적으로우리나라에서자생하는낙지다리식물추출물은항원자극에의한비만세포의탈과립, 사이토카인분 비및동물모델에서알레르기반응을세포내 Syk/LAT 신호전달경로를억제하여나타남을확인할수있었다. 이러한억제현상은가역성을가져추후사람에게의적용가능성이높아환자에적용하기위해독성및성분연구에대한추가연구가필요한것으로생각된다. Acknowledgments 본연구는 2014년도덕성여자대학교연구지원을받아수행되었으며이에감사드립니다. References 1. Holgate, S.T. and Polosa, R.: Treatment strategies for allergy and asthma. Nat. Rev. Immunol., 8, (2008). 2. Siraganian RP, Zhang J, Suzuki K and Sada K: Protein tyrosine kinase Syk in mast cell signaling. Mol Immunol, 38, (2002). 3. Beaven MA: Our perception of the mast cell from Paul Ehrlich to now. Eur J Immunol, 39, (2009). 4. Kang Mo Ahn: Role of Mast Cells in Allergic Inflammation and Innate Immunity. Korean Journal of Pediatrics, 47, (2004). 5. Xiao W, Nishimoto H, Hong H, Kitaura J, Nunomura S, Maeda-Yamamoto M, Kawakami Y, Lowell CA, Ra C and Kawakami T : Positive and Negative Regulation of Mast Cell Activation by Lyn via the FcεRI. J Immunol, 175, (2005). 6. Roth K, Chen WM and Lin TJ: Positive and negative regulatory mechanisms in high-affinity IgE receptor-mediated mast cell activation. Arch, Immunol Ther Exp, 56, (2008). 7. D G Raible, E S Schulman, J DiMuzio, R Cardillo and T J Post: Mast cell mediators prostaglandin-d2 and histamine activate human eosinophils., J Immunol, 148, (1992). 8. Wang A, Lin L and Wang Y: Traditional Chinese Herbal Medicine Penthorum chinense Pursh: A Phytochemical and Pharmacological Review. Am J Chin Med, 43, (2015). 9. Schwartz LB and Austen KF: Enzymes of the Mast cell Granule. J Inv Der, 74, (1980). 10. Korea Centers for Disease Control and Prevention: Trend increase in allergic diseases in children youth. 13, 1-13 (2010). 11. Kyu Eon Kim: Allergic rhinitis diagnosis and treatment guidelines in Korea. AARD, 19, (1999). 12. Gilllan AM and Rivera J: The tyrosine kinase network regulating mast cell activation. Immunol Rev, 228, (2009). 13. Sang Yup Lee and Gwang Ho In: Immunopathogenesis of Asthma. Tuberculosis and Respiratory Diseases, 60, 1-12 (2006). 14. Gifillan AM and Tkaczyk C: Integrated signalling pathways for mast-cell activation. Nat Rev Immunol, 6, (2006). 15. Giorgio Bertona, Attila Mócsaib and Clifford A. Lowellc: Src and Syk kinases: key regulators of phagocytic cell acti-

7 382 So young Jo and Young Mi Kim vation. cell press, 26, (2005). 16. Eun Lee, Byung Wook Park, Kum Jeong Huh and Heung Go: A Study on The Side Effects and Toxicity of Herbal Medicine. Journal of Korean Oriental Internal Medicine, 23, (2002). 17. Kim JH, Kim AR, Kim HS, Kim HW, Park YH, You JS, Park YM, Her E, Kim HS, Kim YM and Choi WS: Rhamnus davurica leaf extract inhibits Fyn activation by antigen in mast cells for anti-allergic activity. BMC Complement Altern Med (2015). 18. Kim JD, Kim do K, Kim HS, Kim AR, Kim B, Her E, Park KH, Kim HS, Kim YM and Choi WS: Morus bombycis extract suppresses mast cell activation and IgE-mediated allergic reaction in mice. J Ethnopharmacol, 146, (2013).

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ÀÇÇÐ°�ÁÂc00Ì»óÀÏ˘ Common Allergic Diseases in Children Sang - Il Lee, M.D. Department of pediatrics Sungkyunkwan University School of Medicine, Samsung Medical Center E - mail : silee@smc.samsung.co.kr Abstract Allergy