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1 1) pissn J. Soc. Cosmet. Scientists Korea eissn Vol. 40, No. 1, March 2014, 국산생약추출물의항염증활성스크리닝 안상미 김형건 최은정 황형훈 이은석 백지훈 부용출 * 고재숙 ( 주 ) 더마프로피부과학연구소, * 경북대학교의학전문대학원분자의학교실세포기질연구소 (2013년 10월 11일접수, 2013년 10월 23일수정, 2013년 12월 3일채택 ) Screening for Anti-inflammatory Activities in Extracts from Korean Herb Medicines Sang Mi An, Hyoung Gun Kim, Eun Jung Choi, Hyoung Hoon Hwang, Eunseok Lee, Ji Hwoon Baek, Yong Chool Boo*, and Jae Sook Koh Dermapro Skin Research Center, DERMAPRO LTD., 4F Jiho B/D, Bangbaejoongang-ro 30, Seocho-gu, Seoul , Korea *Department of Molecular Medicine, Cell and Matrix Research Institute, BK21 Medical Education Program for Human Resources, Kyungpook National University School of Medicine (Received Octobet 11, 2013; Revised October 23, 2013; Accepted December 3, 2013) 요약 : 화장품은일반인이장기간연용하는제품으로인체피부에대한안전성이매우중요하다. 화장품에의한접촉피부염은자극성물질이피부에접촉후침투하여유발하는염증반응으로활성화된면역세포에서염증을매개하는다양한인자를분비함으로써시작된다. 본연구에서는국산생약추출물을이용하여 RAW264.7 대식세포에서염증관련인자에대한영향을통해항염증활성을스크리닝하였다. 51 종의국산생약추출물중측백, 측백엽 ( 초 ), 향부자, 형개, 동과자, 산약, 산약 ( 초 ), 상지, 송절, 택사의에탄올추출물 10 종이 lipopolysaccharide (LPS) 에의해유도된세포독성을감소시킴과동시에염증관련인자인 Nitric oxide (NO), interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α 의생성을억제하는것을확인하였다. 또한, 이들생약추출물은인체피부에서도자극을유발하지않음을인체첩포시험을통해검증하였다. 따라서, 항염증활성이확인된 10 종국산생약추출물은피부자극을예방할수있는화장품소재로서의활용이가능할것으로사료된다. Abstract: Cosmetics are products used over long periods by the public, and their safety is very important. Contact dermatitis induced by cosmetics is the result of an inflammatory response of the skin to direct irritancy. The initial event that this inflammatory response is observed is the release of pro-inflammatory cytokines. In this study, the anti-inflammatory activities of extracts from Korean herb medicines were investigated using RAW264.7 macrophage. Among the fifty one extracts tested, the ethanol extracts from Biotae Orientalis Folium, Biotae Orientalis Folium (roasted), Cyperi Rhizoma, Nepetae Spica, Benincasae Semen, Dioscoreae Rhizoma, Dioscoreae Rhizoma (roasted), Mori Ramulus, Pini Ramulus and Alismatis Rhizoma reduced the cytotoxicity and inhibited the productions of Nitric oxide (NO) and cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-αn lipopolysaccharide (LPS)-induced RAW264.7 macrophage. Additionally, they didn t induce the skin irritation when tested the human patch test. Overall, the result of this study suggests that the extracts of the ten Korean herb medicines are useful cosmetic agents for preventing the skin irritation. Keywords: Korean herb medicines, anti-inflammatory activities, Skin irritation, Cosmetics 주저자 ( dermapro@dermapro.co.kr) 95

2 96 안상미 김형건 최은정 황형훈 이은석 백지훈 부용출 고재숙 1. 서론 는화장품소재로서가능성을검토하고자하였다. 화장품과같은피부외용제는의약품과달리불특정다수가장기간연용하는제품으로사용방법이기본적으로사용자에게맡겨진다는특징을가지고있어모든가능성에대한안전성이확보되어야한다. 하지만화장품의소재나제형이급속도로다양화되고발전함에따라일반소비자들은다양한제품에노출되고있다. 때문에화장품에의한피부이상반응보고도증가하는추세이다 [1-4]. 화장품에의한피부이상반응중에는접촉피부염 (Contact Dermatitis) 이가장흔하게발생하는데접촉피부염은자극성물질이피부에접촉하고피부를통해침투하여유발하는염증반응으로활성화된면역세포에의해일어나는일련의반응이다. 염증반응은면역세포가생체의이물질등을인식하여활성화되고, 활성화된면역세포에서염증을매개하는많은인자를분비함으로써시작된다 [5-7]. Nitric oxide (NO) 는높은반응성을가진생체분자로서, NO synthase에의해 L-argine으로부터생성된다. NO는신경전달, 혈관의이완및세포매개성면역반응에관여하는데, 특히 lipopolysaccharide (LPS) 로자극하면 inducible NOS가발현되어 NO를생성하게된다 [8, 9]. 이렇게생성된 NO는염증반응을매개하는역할을하게된다. 또한, 외부자극에의해활성화된대식세포는 interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α와같은 pro-inflammatory cytokine을생성하게된다 [10]. 생약 ( 生藥 Crude drugs) 은의약품의일종으로인체에유익한동식물의본체, 뿌리, 줄기등천연으로산출되는자연물을날로또는일차가공하여약으로사용하는것으로단일성분의화합물로된의약품과는달리많은종류의성분이혼재하고있어그효과가복합적이고상대적으로부작용이적다고알려져있고 [11], 그활성성분은항산화, 항암, 항균등다양한생리활성이보고되고있다 [12-15]. 특히, 인체의면역시스템중보체계 (complement system) 나면역관련 cytokine의조절을통해생체방어시스템을강화하는데많은영향을준다. 따라서, 본연구에서는국산생약추출물을통해염증매개물질인 NO, IL-1β, IL-6, TNFα의생성을억제하여피부자극반응을예방할수있 2. 재료및방법 2.1. 생약추출물본시험에사용된국산생약추출물은한국생명공학연구원 / 한국식물추출물은행에서구입하여사용하였다. 총 51종의식물추출물중 26종은 95.0% 에탄올로 45 에서 15 min간초음파처리후상온에서 2 h 정치시키는과정을하루에 10회반복하고이를 3일간실시하여추출되었다. 나머지 25종은증류수로전통적인생약추출방법인열탕추출 (100, 2.5 h) 을통해추출되었다. 모든생약시료는추출후여과, 농축, 건조과정을거쳐조제되었다 (Table 1) 시약및기기 Fetal bovine serum (FBS), Antibiotic-Antimycotic, RPMI 1640, Trypsin-EDTA 등의세포배양용시약들은 Life technologies (Gibco R, USA) 에서구입하여사용하였다. 실험에사용된시약중 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2h-tetrazolium bromide (MTT), Lipopolysaccharide (LPS), Dimethyl sulfoxide (DMSO), Squalane은 SIGMA (USA) 에서구입하였다. Cytokine 측정을위하여사용된 Mouse IL-1β ELISA Kit는 Life technologies (Invitrogen TM, USA) 에서구입하였고, OptEIA TM Mouse IL-6 ELISA Kit과 OptEIA TM Mouse TNF-α ELISA Kit 은 BD Bioscience (USA) 에서구입하였다 세포배양 RAW264.7 대식세포는 ATCC (CRL-2278 TM, USA) 에서구입하여 37, 5% CO 2 조건하에서 10% FBS와 1% Antibiotic-Antimycotic (10 units/ml penicillin, 100 µg/ml sterptomycin, 0.25 µg/ml amphotericin) 이첨가된 RPMI 1640 배지에서배양하였다 세포생존율측정세포생존율은살아있는세포의미토콘드리아탈수소효소에의해보라색 formazan으로환원되는 MTT의원리를이용하여분석하였다 [16]. RAW264.7 세포를 24-well plate에 cells/ml의농도로접종하여 24 h 배양한뒤배지를버리고 Phosphate Buffered Saline 대한화장품학회지, 제 40 권제 1 호, 2014

3 국산생약추출물의항염증활성스크리닝 97 Table 1. Lists of Korean Herb Medicines Used for This Study Scientific name Korean name Chinese name Solvent of extract Selaginellae Herba 권백 卷柏 Ethanol 95.0v/v% Perillae Folium 소엽 蘇葉 Ethanol 95.0v/v% Pruni Humilidis Semen 욱리인 郁李仁 Ethanol 95.0v/v% Arisaematis Rhizoma 남성 南星 Ethanol 95.0v/v% Massa Medicata Fermentata 신곡 神曲 Ethanol 95.0v/v% Peucedani Radix 전호 前胡 Ethanol 95.0v/v% Bambusae Folium 죽엽 竹葉 Ethanol 95.0v/v% Cnidii Rhizoma 천궁 川芎 Ethanol 95.0v/v% Biotae Orientalis Folium 측백 側柏 Ethanol 95.0v/v% Biotae Orientalis Folium 측백엽 ( 초 )* 側柏葉 Ethanol 95.0v/v% Gardeniae Fructus 치자 ( 초 )* 梔子 Ethanol 95.0v/v% Cyperi Rhizoma 향부자 香附子 Ethanol 95.0v/v% Nepetae Spica 형개 荊芥 Ethanol 95.0v/v% Benincasae Semen 동과자 冬瓜子 Ethanol 95.0v/v% Santali alba Lignum 백단향 白檀香 Ethanol 95.0v/v% Dioscoreae Rhizoma 산약 山藥 Ethanol 95.0v/v% Dioscoreae Rhizoma 산약 ( 초 )* 山藥 Ethanol 95.0v/v% Mori Ramulus 상지 桑枝 Ethanol 95.0v/v% Pini Ramulus 송절 松節 Ethanol 95.0v/v% Rehmaniae Radix crudus 지황 地黃 Ethanol 95.0v/v% Lycopi Herba 택란 澤蘭 Ethanol 95.0v/v% Alismatis Rhizoma 택사 澤瀉 Ethanol 95.0v/v% Polygoni Avicularis Herba 편축 萹蓄 Ethanol 95.0v/v% Vitidis Viniferae Caulis 포도등 葡萄藤 Ethanol 95.0v/v% Cartami Semen 홍화자 紅花子 Ethanol 95.0v/v% Glycine Semen nigra 흑두 黑豆 Ethanol 95.0v/v% Angelicae koreanae Radix 강활 羌活 Distilled Water Selaginellae Herba 권백 卷柏 Distilled Water Araliae Cordatae Radix 독활 獨活 Distilled Water Paeoniae Radix alba 백작약 白芍藥 Distilled Water Atractylodis Rhizoma alba 백출 白朮 Distilled Water Paeoniae Radix rubra 적작약 赤芍藥 Distilled Water Melonis Calyx 과체 瓜蒂 Distilled Water Cirsii Radix 대계근 大薊根 Distilled Water Cynanchi Wilfordii Radix 백하수오 白何首烏 Distilled Water Rubi Fructus 복분자 覆盆子 Distilled Water Polygonati Officinalis Rhizoma 옥죽 玉竹 Distilled Water Lonicerae Folium 인동 忍冬 Distilled Water Zingiberis Rhizoma 건강 乾薑 Distilled Water Angelicae tenuissimae Radix 고본 藁本 Distilled Water Fagopyri Semen 교맥 蕎麥 Distilled Water Cnidii Rhizoma 천궁 川芎 Distilled Water Aconiti Jaluencis Tuber 초오 草烏 Distilled Water Vitidis Vinferae Radix 포도근 葡萄根 Distilled Water Zizyphi Fructus 대추 大棗 Distilled Water Akebiae Caulis 목통 木通 Distilled Water Perillae Folium 소엽 蘇葉 Distilled Water Pini Pollen 송화 松花粉 Distilled Water Massa Medicata Fermentata 신곡 神曲 Distilled Water Astragali Radix 황기 黃耆 Distilled Water Siegesbeckiae Herba 희첨 豨僉 Distilled Water * The herb was roasted before the extraction. J. Soc. Cosmet. Scientists Korea, Vol. 40, No. 1, 2014

4 98 안상미 김형건 최은정 황형훈 이은석 백지훈 부용출 고재숙 (PBS) 로세척한다음 FBS를포함하지않는새로운배지로교환하였다. 세포는 LPS (200 ng/ml) 로자극시키거나혹은자극시키지않은상태에서최종 50 µg/ml의농도로각생약추출물을처리하고세포를다시 24 h 배양후, 0.5% MTT 용액을각 well에 100 µl씩첨가하여, 4 h 동안배양하였다. 배양액을제거한다음 DMSO를 200 µl씩을넣고 10 min간흔들어준다음 Microplate reader (Sunrise TM TECAN, Austria) 로 540 nm에서흡광도를측정하였다. 세포생존율은대조군에대한흡광도값을나누어백분율로나타내었다 NO 생성측정 NO 생성량은 Griess 시약을이용한 Nitrite/Nitrate Assay Kit (SIGMA, USA) 을통해세포배양액내존재하는 NO의안정된산화물인 NO - 2 를측정하여분석하였다. RAW264.7 세포를 24-well plate에 cells/ml 의농도로접종하여 24 h 배양한뒤배지를버리고 PBS로세척한다음 FBS를포함하지않는새로운배지로교환하였다. 생약추출물 (50 µg/ml) 과 LPS (200 ng/ml) 를처리하여다시 24 h 배양하였다. 세포배양상층액 100 µl와 Griess 시약 100 µl를혼합하여 96-well plate에서 10 min 동안반응시킨후 540 nm에서흡광도를측정하였다. 결과는 NaNO 2 를이용하여표준곡선을구하여정량하였다 Cytokine (IL-1β, IL-6, TNF-α) 측정위와동일한과정으로배양한 RAW264.7 세포는 24 h 배양후생약추출물 (50 µg/ml) 과 LPS (200 ng/ml) 를처리하여다시 24 h 배양하였다. 24 h 후세포배양상층액을회수하여 ELISA Kit을이용하여상층액에포함된 IL-1β, IL-6, TNF-α의함량을측정하였다. 측정법을간단하게요약하면, IL-1β, IL-6, TNF-α에특이적인단일항체로코팅된 96-well plate에세포배양상층액을첨가하여 2 h 반응시킨후 biotin이결합된 IL-1β, IL-6, TNF-α 단일항체와 streptavidin이결합된 peroxidase를차례로넣어검출한다. 발색기질은 TMB (3,3,5,5 -tetramethylbenzidine) 를사용하였고재조합된 IL-1β, IL-6, TNF-α를희석하여표준곡선을구하여정량하였다 인체첩포시험 인체첩포시험은헬싱키선언에근거한윤리규정 [17] 및식품의약품안전처화장품인체적용시험가이드라인 [18] 에따라실시되었으며, Frosch & Kligman이고안한방법 [19] 을응용하여특정피부질환및알레르기가없는 20세 ~47세의건강한여성피험자 33명 ( 평균연령 35.4 ± 9.3세 ) 을대상으로실시하였다. 시험시료로생약추출물을 5% 의농도로 80% 에탄올에녹인후최종농도는 0.5% 가되도록 squalane에희석하여적용하였다. 먼저, 첩포부위인등부위를 70% 에탄올로소독한후시험물질 20 µl가적용된 IQ chamber (Chemotechnique Diagnostics AB, Sweden) 를첩포하였다. 48 h 후첩포를제거하고 skin marker (Chemotechnique Diagnostics AB, Sweden) 로시험부위를표시하였으며, 30 min, 24 h 후에각시험부위를평가하였다. 평가기준은 Table 2에제시한 CTFA 가이드라인 [20] 의판정기준에따랐으며, 2회에걸쳐평가한피부반응은다음식을통해평균반응도를계산하였다 통계분석 인체첩포시험을제외한모든실험은최소 3회이상반복실험에대한평균 ± 표준편차 (mean ± SD) 로나타내었으며, 통계학적분석은 SPSS version (IBM, USA) 를이용하여 one-way analysis of variance (ANOVA) 를시행하여 p < 0.05인경우유의한것으로판정하였다. 3. 결과 3.1. RAW264.7 대식세포에서국산생약추출물의세포생존율분석 51종의국산생약추출물의세포독성을알아보기위하여 RAW264.7 대식세포에각생약추출물을 50 µg/ml의농도로처리하고 24 h 후세포생존율을측정하였다. 시험결과, 51종식물추출물중남성과건강의에탄올추출물이세포독성을보였고, 그외나머지 49종생약추출물은세포독성을보이지않았다 (Table 2). 한편, LPS를처리하여자극시킨 RAW264.7 대식세포는 LPS에의해세포생존율이감소한다고보 대한화장품학회지, 제 40 권제 1 호, 2014

5 국산생약추출물의항염증활성스크리닝 99 Table 2. Evaluation Criteria of Skin Reactions by CTFA Gudeline Symbol Grade Clinical Description - 0 Negative reaction + 1 Slight erythema, either spotty or diffuse ++ 2 Moderate uniform erythema Intense erythema with edema Intense erythema with edema & vesicles 고되어있다 [21]. 이러한염증성유발모델에서생약추출물의세포생존율에대한영향을측정하였다. 시험결과, 죽엽, 측백, 측백엽 ( 초 ), 향부자, 백단향, 상지, 송절, 편축, 포도등, 흑두의에탄올추출물과초오, 대추, 송화분의증류수추출물은 LPS에의해유도된세포독성을억제하는효과를보였으며, 특히, 죽엽, 백단향, 상지, 포도등의에탄올추출물과초오, 대추, 송화분의증류수추출물은 LPS에의한자극유무에상관없이세포생존율을증가시키는것으로나타났다 (Table 3) 국산생약추출물이 NO 생성에미치는영향 RAW264.7 대식세포는 LPS에의해자극되고, 이렇게자극된대식세포는염증매개물질인 NO의생성이증가하게된다 [21]. 51종의생약추출물의 NO 생성에미치는영향을알아보기위하여 RAW264.7 대식세포에 LPS를 200 ng/ml 의농도로처리하여자극시킨후, 각생약추출물을 50 µg/ml의농도로처리하였다. 시험결과, 51종생약추출물모두 NO의생성을증가시키지않았다. 하지만, 죽엽, 천궁, 측백, 측백엽 ( 초 ), 치자 ( 초 ), 향부자, 형개, 동과자, 백단향, 산약, 산약 ( 초 ), 상지, 송절, 택란, 택사, 편축, 포도등, 홍화자, 흑두의에탄올추출물과강활, 권백, 백출의증류수추출물은 LPS에의해증가된 NO 생성량을효과적으로감소시켰다 (Table 4). 특히, 죽엽, 측백, 측백엽 ( 초 ), 향부자, 백단향, 상지, 송절, 편축, 포도등, 흑두의에탄올추출물은 RAW264.7 대식세포에서 LPS에의해유도된세포독성은억제하고동시에 NO의생성은효과적으로감소시켜항염증소재로서의가능성을보였다 (Tables 3, 4) 국산생약추출물이염증관련 cytokine 생성에미치는영향 LPS에의해자극된 RAW264.7 대식세포는 IL-1β, IL-6, TNF-α와같은 pro-inflammatory cytokine의분비를증가시킨다 [22,23]. 이세가지 cytokine에대한 51종생약추출물의영향을알아보기위하여 RAW264.7 대식세포에 LPS를 200 ng/ml의농도로처리하여자극시킨후, 각생약추출물을 50 µg/ml의농도로처리하였다. 시험결과, 염증성모델에서전호, 택란, 편축, 포도등을제외한모든에탄올추출물과옥죽, 초오, 대추, 목통의증류수추출물은 LPS에의해유도된 IL-1β의생성을유의적으로감소시켰다. 반면, 전호에탄올추출물은오히려 IL-1β의생성을증가시켰다. IL-6의경우, 욱리인, 전호, 치자 ( 초 ), 편축을제외한모든에탄올추출물은 LPS에의해유도된 IL-6의생성을유의적으로감소시켰고전호의에탄올추출물과백하수오, 복분자, 교맥, 포도근의증류수추출물은오히려 IL-6의생성을증가시켰다. TNF-α 의경우, 권백, 천궁, 측백, 측백엽 ( 초 ), 향부자, 형개, 동과자, 산약, 산약 ( 초 ) 상지, 송절, 지황, 택란, 택사의에탄올추출물이 LPS에의해유도된 TNF-α의생성을유의적으로감소시켰고전호의에탄올추출물과복분자, 교맥의증류수추출물은 TNF-α의생성을증가시켰다 (Table 4). 결과적으로, 국산생약추출물 51종을대상으로항염증활성을스크리닝한결과, 측백, 측백엽 ( 초 ), 향부자, 형개, 동과자, 산약, 산약 ( 초 ), 상지, 송절, 택사의에탄올추출물 10종이 RAW264.7 대식세포에서 LPS 에의해유도된세포독성을감소시킴과동시에염증매개물질인 NO, IL-1β, IL-6, TNF-α의생성을억제하는것으로나타났다 인체첩포시험에의한 10종국산생약추출물의피부자극평가 in vitro 항염증활성스크리닝결과효과적인항염증활성을보인 10종의생약추출물에대해인체피부에서의피부자극정도를평가하기위하여인체첩포시험을실시하였다. 0.5% 농도로적용한생약추출물중산약의에탄올추출물을제외한 9종의생약추출물은 33명의피험자에서모두음성반응을보였다. 산약의에탄올추출물은 1명의피험자에서경미한홍반반응인 grade 1의피부반응을유도하여반응도 0.4로 J. Soc. Cosmet. Scientists Korea, Vol. 40, No. 1, 2014

6 100 안상미 김형건 최은정 황형훈 이은석 백지훈 부용출 고재숙 Table 3. Effect of Korean Herb Medicines on Cell Viability of RAW264.7 Macrophage Materials Cell viability (%) without LPS with LPS Vehicle control (VC) ± ± 5.16* Selaginellae Herba ( 권백 ) a ± ± 8.48 Perillae Folium ( 소엽 ) a ± ± 1.16 Pruni Humilidis Semen ( 욱리인 ) a ± ± 2.93 Arisaematis Rhizoma ( 남성 ) a 76.1 ± 2.17* 65.4 ± 3.96 Massa Medicata Fermentata ( 신곡 ) a 97.9 ± ± Peucedani Radix ( 전호 ) a 89.8 ± ± 2.81 Bambusae Folium ( 죽엽 ) a ± 1.07* 85.5 ± 5.98 Cnidii Rhizoma ( 천궁 ) a ± ± 5.00 Biotae Orientalis Folium ( 측백 ) a ± ± 7.89 Biotae Orientalis Folium ( 측백엽 ( 초 )) a ± ± 5.56 Gardeniae Fructus ( 치자 ( 초 )) a ± ± 3.38 Cyperi Rhizoma ( 향부자 ) a ± ± 2.50 Nepetae Spica ( 형개 ) a 89.1 ± ± 0.21 Benincasae Semen ( 동과자 ) a ± ± Santali alba Lignum ( 백단향 ) a ± 5.77* ± 2.84 Dioscoreae Rhizoma ( 산약 ) a ± ± 3.37 Dioscoreae Rhizoma ( 산약 ( 초 )) a ± ± 1.05 Mori Ramulus ( 상지 ) a ± 3.27* ± 3.36 Pini Ramulus ( 송절 ) a ± ± 3.99 * Rehmaniae Radix crudus ( 지황 ) a ± ± 5.47 Lycopi Herba ( 택란 ) a ± ± 2.27 Alismatis Rhizoma ( 택사 ) a ± 3.80* 78.1 ± 5.07 Polygoni Avicularis Herba ( 편축 ) a 93.9 ± ± 5.55 Vitidis Viniferae Caulis ( 포도등 ) a ± 5.00* 97.4 ± 5.12 Cartami Semen ( 홍화자 ) a 99.3 ± ± 3.71 Glycine Semen nigra ( 흑두 ) a ± ± 3.91 Angelicae koreanae Radix ( 강활 ) b ± 6.22* 82.8 ± 4.28 Selaginellae Herba ( 권백 ) b ± 4.77* 76.9 ± 7.50 Araliae Cordatae Radix ( 독활 ) b ± ± 6.86 Paeoniae Radix alba ( 백작약 ) b ± 3.42* 63.2 ± 3.98 Atractylodis Rhizoma alba ( 백출 ) b ± 3.32* 65.3 ± 5.28 Paeoniae Radix rubra ( 적작약 ) b ± 3.39* 68.4 ± 2.90 Melonis Calyx ( 과체 ) b ± 3.65* 65.5 ± 8.26 Cirsii Radix ( 대계근 ) b ± 3.80* 60.2 ± Cynanchi Wilfordii Radix ( 백하수오 ) b ± ± 3.47 Rubi Fructus ( 복분자 ) b ± 1.60* 57.2 ± 6.34 Polygonati Officinalis Rhizoma ( 옥죽 ) b ± ± 4.46 대한화장품학회지, 제 40 권제 1 호, 2014

7 국산생약추출물의항염증활성스크리닝 101 Materials without LPS Cell viability (%) with LPS Lonicerae Folium ( 인동 ) b ± ± 4.55 Zingiberis Rhizoma ( 건강 ) b 79.5 ± 4.98* 44.4 ± 4.52 Angelicae tenuissimae Radix ( 고본 ) b ± 3.94* 58.8 ± 7.04 Fagopyri Semen ( 교맥 ) b 99.0 ± ± 7.48 Cnidii Rhizoma ( 천궁 ) b ± 2.24* 84.2 ± 9.25 Aconiti Jaluencis Tuber ( 초오 ) b ± 1.69* 90.0 ± Vitidis Vinferae Radix ( 포도근 ) b ± 3.00* 84.9 ± 4.06 Zizyphi Fructus ( 대추 ) b ± 4.57* 91.8 ± 6.79 Akebiae Caulis ( 목통 ) b ± ± Perillae Folium ( 소엽 ) b ± 2.41* 86.5 ± 8.69 Pini Pollen ( 송화분 ) b ± 3.52* 89.6 ± 9.67 Massa Medicata Fermentata ( 신곡 ) b ± 4.17* 62.8 ± 8.01 Astragali Radix ( 황기 ) b ± 1.15* 65.5 ± 3.09 Siegesbeckiae Herba ( 희첨 ) b ± 6.57* 69.2 ± 4.55 a Ethanol extracts, b Distilled water extracts Each value represents mean ± SD. Values are significantly different by one-way ANOVA test (p <0.05, * vs VC without LPS, vs VC with LPS). Table 4. Effect of Korean Herb Medicines on NO and Cytokines Production in RAW264.7 Macrophage Stimulated by LPS Materials NO (µm) IL-1β (pg/ml) IL-6 (pg/ml) TNF-α (pg/ml) Control 1.54 ± ± ± ± 1.76 VC with LPS ± 0.72* ± 3.05* ± 102.4* ± 14.62* Selaginellae Herba ( 권백 ) a ± ± ± ± Perillae Folium ( 소엽 ) a ± ± ± ± Pruni Humilidis Semen ( 욱리인 ) a ± ± ± ± Arisaematis Rhizoma ( 남성 ) a ± ± ± ± 4.16 Massa Medicata Fermentata ( 신곡 ) a ± ± ± ± 4.34 Peucedani Radix ( 전호 ) a ± ± ± ± Bambusae Folium ( 죽엽 ) a ± ± ± ± Cnidii Rhizoma ( 천궁 ) a ± ± ± ± Biotae Orientalis Folium ( 측백 ) a ± ± ± ± 7.37 Biotae Orientalis Folium ( 측백엽 ( 초 )) a ± ± ± ± Gardeniae Fructus ( 치자 ( 초 )) a ± ± ± ± 6.68 Cyperi Rhizoma ( 향부자 ) a ± ± ± ± Nepetae Spica ( 형개 ) a ± ± ± ± Benincasae Semen ( 동과자 ) a ± ± ± ± Santali alba Lignum ( 백단향 ) a ± ± ± ± Dioscoreae Rhizoma ( 산약 ) a ± ± ± ± Dioscoreae Rhizoma ( 산약 ( 초 )) a ± ± ± ± 9.05 J. Soc. Cosmet. Scientists Korea, Vol. 40, No. 1, 2014

8 102 안상미 김형건 최은정 황형훈 이은석 백지훈 부용출 고재숙 Materials NO (µm) IL-1β (pg/ml) IL-6 (pg/ml) TNF-α (pg/ml) Mori Ramulus ( 상지 ) a ± ± ± ± 6.68 Pini Ramulus ( 송절 ) a ± ± ± ± 9.82 Rehmaniae Radix crudus ( 지황 ) a ± ± ± ± Lycopi Herba ( 택란 ) a ± ± ± ± 7.81 Alismatis Rhizoma ( 택사 ) a ± ± ± ± 7.06 Polygoni Avicularis Herba ( 편축 ) a ± ± ± ± Vitidis Viniferae Caulis ( 포도등 ) a ± ± ± ± Cartami Semen ( 홍화자 ) a ± ± ± ± Glycine Semen nigra ( 흑두 ) a ± ± ± ± Angelicae koreanae Radix ( 강활 ) b ± ± ± ± Selaginellae Herba ( 권백 ) b ± ± ± ± Araliae Cordatae Radix ( 독활 ) b ± ± ± ± Paeoniae Radix alba ( 백작약 ) b ± ± ± ± Atractylodis Rhizoma alba ( 백출 ) b ± ± ± ± Paeoniae Radix rubra ( 적작약 ) b ± ± ± ± 3.79 Melonis Calyx ( 과체 ) b ± ± ± ± Cirsii Radix ( 대계근 ) b ± ± ± ± Cynanchi Wilfordii Radix ( 백하수오 ) b ± ± ± ± Rubi Fructus ( 복분자 ) b ± ± ± ± Polygonati Officinalis Rhizoma ( 옥죽 ) b ± ± ± ± 9.10 Lonicerae Folium ( 인동 ) b ± ± ± ± Zingiberis Rhizoma ( 건강 ) b ± ± ± ± 7.37 Angelicae tenuissimae Radix ( 고본 ) b ± ± ± ± 9.62 Fagopyri Semen ( 교맥 ) b ± ± ± ± Cnidii Rhizoma ( 천궁 ) b ± ± ± ± 4.68 Aconiti Jaluencis Tuber ( 초오 ) b ± ± ± ± Vitidis Vinferae Radix ( 포도근 ) b ± ± ± ± Zizyphi Fructus ( 대추 ) b ± ± ± ± Akebiae Caulis ( 목통 ) b ± ± ± ± Perillae Folium ( 소엽 ) b ± ± ± ± Pini Pollen ( 송화분 ) b ± ± ± ± Massa Medicata Fermentata ( 신곡 ) b ± ± ± ± 5.03 Astragali Radix ( 황기 ) b ± ± ± ± 9.34 Siegesbeckiae Herba ( 희첨 ) b ± ± ± ± a Ethanol extracts, b Distilled water extracts Each value represents mean ± SD. Values are significantly different by one-way ANOVA test (p < 0.05, * vs Control, vs VC with LPS). 대한화장품학회지, 제 40 권제 1 호, 2014

9 국산생약추출물의항염증활성스크리닝 103 Table 5. Results of human skin primary irritation test of Korean herb medicines (n = 33) Materials No. of responder Score Squalane (NC) Biotae Orientalis Folium ( 측백 ) Biotae Orientalis Folium ( 측백엽 ( 초 )) Cyperi Rhizoma ( 향부자 ) Nepetae Spica ( 형개 ) Benincasae Semen ( 동과자 ) Dioscoreae Rhizoma ( 산약 ) Dioscoreae Rhizoma ( 산약 ( 초 )) Mori Ramulus ( 상지 ) Pini Ramulus ( 송절 ) Alismatis Rhizoma ( 택사 ) NC: negative control 나타났지만, 이는전체피험자의 3% 이하로피부에대해저자극물질로판단하였다. 4. 고찰자극성접촉피부염은직접적인자극에의한피부의선천적면역반응의결과이다. 자극원에접촉된피부는외적인손상이유발되기전가장먼저 IL-1β와같은염증유발인자의분비가관찰된다. 이를시작으로혈관이확장되고피부의표피나진피로림프구, 대식세포와같은면역세포들의침투가일어나고, 그결과자극부위에국소적인통증과수포형성, 홍반, 경화, 부종등과같은표피의손상이나타난다. 또한, 자극의강도는자극원이피부의각질층을투과하는정도와살아있는세포에대한세포독성정도에따라결정된다. 따라서, 본시험에서는국산약용식물 51종을대상으로대식세포에서세포에대한독성과염증유발인자에대한억제효과를측정하여항염증효과를스크리닝하고항염증효과가나타난 10종에대해인체첩포시험을통해피부자극정도를검증하였다. 결과적으로, 측백, 측백엽 ( 초 ), 향부자, 형개, 동과자, 산약, 산약 ( 초 ), 상지, 송절, 택사의에탄올추출물은 RAW264.7 대식세포에서 LPS에의해유도된세포독성과염증매개물질인 NO, IL-1β, IL-6, TNF-α의생성을억제하는것으로나타나효과적인항염증활 성을보여주었고인체피부에서자극도일으키지않는것을확인하였다. 측백나무 (Biotae Orientalis) 는중국북부지방이원산지로, 한국, 일본, 인도등다른아시아지역에서도자생하고있으며 [24], 측백의항염증활성은이미많은연구에서보고되고있고 [25-28] Kim 등의연구에따르면측백의양모효과는이러한항염증활성에기인한것이라추측하고있다 [29]. 또한, 그잎이나열매를포함한측백의추출물은항균, 항산화활성에대해서도다양하게보고되고있다 [30-32]. 향부자 (Cyperi Rhizoma) 는강변이나해변모래사장에서서식하는식물로그뿌리는한방에서는통경, 진통제로서사용되어왔다 [24]. 본연구의결과와유사하게 Lee 등의연구에서도열수추출된향부자는대식세포에서세포생존율을증가시키고 LPS에의해증가된 NO 생성량을농도의존적으로감소시키는것으로나타났다 [33]. 이는향부자의알코올추출물에대한본연구의결과와유사하게나타났다. 한편, 최근에는 3-morpholinosydnonimine에의해자극된신경세포에서향부자추출물이 NO의생성을감소시켜신경세포의손상을억제한다는연구도보고되었다 [34]. 형개 (Nepetae Spica) 는한방에서전초를말려감기의발한, 해열목적이나볶아서지혈을목적으로사용되어왔으나 [24] 형개의약리효과에대한연구는부족한실정이다. 동과자 (Benincasae Semen) 는동과의 J. Soc. Cosmet. Scientists Korea, Vol. 40, No. 1, 2014

10 104 안상미 김형건 최은정 황형훈 이은석 백지훈 부용출 고재숙 씨로한방에서소염, 배농등의목적으로사용되었고차로도음용되고있다 [24]. 여러연구에서거담, 항산화, 항암효과등이보고되었으나 [35-37], 항염증활성은거의보고된바가거의없다. 산약 (Dioscoreae Rhizoma) 은일반적으로마라고불리는덩굴식물로뿌리를식용하고한방에서는기력을증진시켜주는자양강장제로활용되어왔다 [24]. 때문에산약의항산화, 항노화활성은다양한세포에서보고되고있다 [38-40]. 또한, 증류수추출물과발효추출물은대식세포에서세포독성없이 LPS에의해증가된 NO 생성량과염증관련 cytokine (IL-1β, IL-6, TNF-α) 의생성을농도의존적으로감소시켜본연구와유사한결과가보고되기도하였다 [41-43]. 송절 (Pini Ramulus) 은소나무줄기의마디로한방에서는풍증이나근육과골격의병을치료하는약재로사용되었고 [24], Choi 등의연구에의하면송절의메탄올추출물에서강력한항산화작용이보고되었다 [44]. 또한, 대식세포에서세포독성없이 LPS에의해증가된 NO 생성량과염증관련 cytokine (IL-1β, IL-6) 의생성을효과적으로감소시키는등본연구와유사한결과가보고되기도하였다 [45]. 상지 (Mori Ramulus) 는뽕나무의어린가지를말린것으로다양한약리작용이보고되고있다. 그중에서도미백활성이보고되어미백화장품의소재로활용되고있으나 [46] 항염증활성에대해서는보고된바가거의없다. 택사 (Alismatis Rhizoma) 는습지에서자라는식물로그뿌리는한방에서이뇨제나부종, 습증등에약효가있다고알려져있다 [24]. 특히, 여러연구를통해항산화, 항균, 항당뇨활성등많은약리효과가보고되었으나 [47-49] 항염증활성에대해서는알려진바가없다. 그밖에도권백 (Selaginellae Herba)[50], 치자 ( 초 ) (Gardeniae Fructus)[51], 지황 (Rehmaniae Radix crudus) [52], 홍화자 (Cartami Semen)[53], 독활 (Araliae Cordatae Radix)[54,55], 적작약 (Paeoniae Radix rubra)[56], 복분자 (Rubi Fructus)[57], 건강 (Zingiberis Rhizoma)[58], 고본 (Angelicae tenuissimae Radix)[59], 천궁 (Cnidii Rhizoma) [60], 황기 (Astragali Radix)[61] 는이전의다른연구에서항염증효과가보고된바있고, 그중권백, 치자 ( 초 ), 지황, 홍화자, 복분자는본연구에서도 NO 혹은일부염증관련 cytokine에대해억제효과를보이기도하였다. 결론적으로본연구를통해항염증활성이확인된 10종의국산생약추출물은인체피부에대해피부자극을유발하지않아피부에안전한화장품소재임이입증되었고, 향후이러한항염증활성을활용하여피부장벽이손상된시험모델을이용하여피부장벽에대한효과를검증할예정이다. 감사의글본연구는보건복지부보건의료연구개발사업의지원에의하여이루어진것임 ( 과제고유번호 A103017). Reference 1. A. C. de Groot, Labelling cosmetics with their ingredients, BMJ (Clinical research ed.), 300(6740), 1636 (1990). 2. A. C. de Groot, E. G. Beverdam, C. T. Ayong, P. J. Coenraads, and J. P. Nater, The role of contact allergy in the spectrum of adverse effects caused by cosmetics and toiletries, Contact dermatitis, 19(3), 195 (1988). 3. E. Gendler, Adverse reactions to cosmetics, Cutis, 39(6), 525 (1987). 4. S. Wahie, J. J. Lloyd, and P. M. Farr, Sunscreen ingredients and labelling: a survey of products available in the UK, Clinical and experimental dermatology, 32(4), 359 (2007). 5. E. G. Lee, B. M. Mickle-Kawar, and R. M. Gallucci, IL-6 deficiency exacerbates skin inflammation in a murine model of irritant dermatitis, Journal of immunotoxicology, 10(2), 192 (2013). 6. M. Kamsteeg, P. A. Jansen, I. M. van Vlijmen- Willems, P. E. van Erp, D. Rodijk-Olthuis, P. G. van der Valk, T. Feuth, P. L. Zeeuwen, and J. Schalkwijk, Molecular diagnostics of psoriasis, atopic dermatitis, allergic contact dermatitis and irritant contact dermatitis, The British journal of dermatology, 162(3), 568 (2010). 7. J. Seneschal, E. Kubica, L. Boursault, J. Stokkermans, C. Labreze, B. Milpied, K. Ezzedine, 대한화장품학회지, 제 40 권제 1 호, 2014

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13 국산생약추출물의항염증활성스크리닝 107 Dioscoreae batatas Extract on Lipopolysaccharide-induced Macrophage Activation, Yakhak Hoeji, 55(5), 7 (2011). 44. J. H. Choi, H. S. Kim, M. J. Jung, and J. S. Choi, (+)-Catechin, an Antioxidant Principle from the Leaves of Pinus densiflora that Acts on 1,1- Diphenyl-2-picrylhydrazyl Radical, Natural Product Sciences, 7(1), 4 (2001). 45. K. P. Lee and J. Y. Moon, Pinus Densiflora Gnarl Extract for Pharmacopuncture Inhibits Inflammatory Responses through Heme Oxygenase-1 Induction in Lipopolysaccharide-Stimulated RAW264.7 Macrophages, Korean Journal of Acupuncture, 29(1), 10 (2012). 46. K. T. Lee, K. S. Lee, J. H. Jeong, B. K. Jo, M. Y. Heo, and H. P. Kim, Inhibitory effects of Ramulus mori extracts on melanogenesis, Journal of cosmetic science, 54(2), 133 (2003). 47. K. I. Seo, Y. S. Cho, J. R. Park, S. T. Yee, and C. K. Park, Antimicrobial and Antioxidative Effects of Alismatis Rhizoma Extract, Journal of Life Science, 10(5), 5 (2000). 48. J. W. Nam, S. H. Rhee, M. S. Kang, Y. K. Choi, C. Y. Jun, C. H. Park, and D. W. Kim, Effects of Different Lengths of Treatment with Rhizoma Alismatis on Diabetic Mellitus of Streptozotocin-Induced Hyperglycemic Rats, The Journal of Korean Oriental Internal Medicine, 27(4), 7 (2006). 49. C. A. Toh, Antimicrobial and Antifungal Studies on Alismae Rhizoma, Korean Journal of Pharmacognosy, 27(4), 5 (1996). 50. B. T. Ahn, S. A. Shin, J. G. Kim, and W. H. Park, Inhibitory Effect of Amentoflavone of Selaginella Tamariscina on MMP-9 Expression through NF-B and AP-1 in Macrophage Raw cells, Korean Journal of Oriental Physiology and Pathology, 21(1), 7 (2007). 51. D. H. Shon, D. W. Choi, and M. H. Kim, Improvement of Anti-Inflammation Activity of Gardeniae fructus Extract by the Treatment of β -Glucosidase, Korean journal of food science and technology, 44(3), 6 (2012). 52. B. S. Chae and T. Y. Shin, Effect of Fresh Rehmanniae Radix Methanol Extracts on the Production of Cytokines, Journal of the Pharmaceutical Society of Korea, 50(3), 7 (2006). 53. D. H. Kim, E. Y. Hwang, and J. H. Son, Anti-Inflammatory Activity of Carthamus tinctorious Seed Extracts in Raw cell, Journal of Life Science, 23(1), 8 (2013). 54. C. H. Kang, J. R. Koo, and J. S. So, Inhibitory Effects of Aralia cordata Thunb Extracts on Nitric Oxide Synthesis in RAW Macrophage Cells, Korean journal of food science and technology, 44(5), 7 (2012). 55. T. D. Kim, J. Y. Lee, B. J. Cho, T. W. Park, and C. J. Kim, The analgesic and anti-inflammatory effects of 7-oxosandaracopimaric acid isolated from the roots of Aralia cordata, Archives of pharmacal research, 33(4), 509 (2010). 56. S. Y. Han and E. Yy. Lee, Effect of Paeoniae Radix Rubra Extract on the Production of NO and Prostaglandin E 2 in LPS-stimulated RAW264.7 Marcrophages, The Journal of Korean Acupuncture & Moxibustion Society, 28(1), 8 (2011). 57. S. Y. Choi, K. C. Lee, Y. J. Jeoung, and B. O. Lim, In vitro Anti-inflammatory Activity of Rubus coreanus Miq. on Nitric Oxide, Interferon-γ, Cycloxygenase-2, and Tumor Necrosis Factor-α Production in the Macrophage like Cell Line RAW Activated by Lipopolysccharide, Korean Journal of Medicinal Crop Science, 15(5), 5 (2007). 58. H. W. Jung, C. H. Yoon, K. M. Park, H. S. Han, and Y. K. Park, Hexane fraction of Zingiberis Rhizoma Crudus extract inhibits the production of nitric oxide and proinflammatory cytokines in LPS-stimulated BV2 microglial cells via the NF-kappaB pathway, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association, 47(6), 1190 (2009). 59. S. Y. Lee, H. J. Park, D. S. Cha, T. Y. Shin, H. J. J. Soc. Cosmet. Scientists Korea, Vol. 40, No. 1, 2014

14 108 안상미 김형건 최은정 황형훈 이은석 백지훈 부용출 고재숙 Na, W. S. Moon, Y. G. Kang, and H. Jeon, Antioxidant and Anti-inflammatory Effect of Angelica Tenuissima in IFN-γ/LPS-stimulated Peritoneal Macrophage, Korean J. Oriental Physiology & Pathology, 22(6), 8 (2008). 60. S. W. Choi, E. O. Kim, H.-H. Leem, and J.-K. Kim, Anti-Inflammatory Effects of Volatile Flavor Extracts from Cnidium officinale and Angelica giga, J. Korean Soc. Food Sci. Nutr., 41(8), 9 (2012). 61. M. Ryu, E. H. Kim, M. Chun, S. Kang, B. Shim, Y. B. Yu, G. Jeong, and J. S. Lee, Astragali Radix elicits anti-inflammation via activation of MKP-1, concomitant with attenuation of p38 and Erk, Journal of ethnopharmacology, 115(2), 184 (2008). 대한화장품학회지, 제 40 권제 1 호, 2014