02-두충과(노영창)

Size: px

Start display at page:

Download "02-두충과(노영창)"

현진 설
5 years ago
Views:

1 Journal of Radiation Industry 6 (1) : 11~15 (12) Review Paper 두충과느릅혼합추출물에의한염증억제효과 권희정 신영민 정성린 박종석 노영창 임윤묵 * 한국원자력연구원첨단방사선연구소공업환경연구부 In Vitro Anti-inflammatory Activities of Herbal Extracts with Eucommia ulimoides and Ulmus davidiana Hui-Jeong Gwon, Young Min Shin, Sung-In Jung, Jong-Seok Park, Young-Chang Nho and Youn-Mook Lim* Research Division for Industry & Environment, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 5-185, Korea Abstract - This study confirmed the cytotoxicity and anti-inflammatory activities of natural herbal extracts (HE) including Eucommia ulimoides and Ulmus davidiana against human mast cell (HMC-1). HE was extracted with distilled water (at 75 C) and then freeze-dried for 5 days. Finally, the HE was sterilized by gamma radiation with Co γ source at room temperature. Cytotoxicity of the HE against HMC-1 cell was measured using cell counting kit-8 (CCK-8) assay. In addition, inflammatory cytokines such as TNF-α, IL-6 and IL-8 were evaluated by ELISA kit on the HMC- 1 cells with calcimycin A23187 and phorbol 12-myristate 13-acetate (PMA). The results showed that HE had no toxicity and reduced TNF-α, IL-6, IL-8 response on HMC-1 cells. Key words : Ulmus davidiana, Eucommia ulimoides, Cytotoxicity, Cytokine 서 아토피성피부염은가장흔하게발생하는소아기만성피부질환으로생후몇년내에발병한다. 최근여러역학연구결과에따르면, 지난 3년간에걸쳐서산업화된국가에서아토피성피부염발병률이증가함을보이고있고난치성질환이라짧은기간내에완치하기어려운질병으로알려져있다 (Shultz and Hanifin 2). 아토피성피부염의발병원인은면역학적이상과생리적생화학적피부장벽구조의결함에의하여발병하여, 유전 론 * Corresponding author: Youn-Mook Lim, Tel , Fax , . ymlim71@kaeri.re.kr 적및환경적이상등이복잡하게연관되어작용한다. 이질환은피부건조, 소양, 찰상및태선, 홍반등의소견을보이며증상이심할수록혈청내 IgE가정상인에비해증가된다 (Leung and Bieber 3). 또한알레르기증상의대부분이비만세포나호산구가분비하는히스타민등의염증반응매개물질에의해서임상적증상이나타나며, 아토피성피부염환자의피부조직에서과도한각질화와염증세포의침윤등이관찰된다. 따라서피부장벽이제기능을하지못하게되고피부수분함량이줄어들어경피의수분손실이증가하게된다 (Kim et al. 6). 아토피성피부염에대한현재까지알려진치료방법은스테로이드제항히스타민제도포나복용및최근개발되고있는면역억제제가사용되고있으나장기간사용의부작용이문제점으로대두되고있다 (Furukawa et al. 11

2 12 권희정 신영민 정성린 박종석 노영창 임윤묵 4). 일부동양권에서는천연약용식물에서추출한몇몇성분들을이용하여아토피성피부염을치료하는데사용하여왔다. 두충 (Eucommia ulimoides) 은두충나무과에속하는 1 과 1속 1종의낙엽교목으로서높이 15 m, 지름 cm까지자란다. 원산지는중국의호북, 사천성등지로알려져있으며우리나라중부이남지역어디에서나잘서식하며특히계곡이나산녹지대또는토질이좋고수분이적절한곳에서잘자라며약용가치가높아전국적으로많이재배되고있다. 두충및두충차의약리활성에관한연구로는활성산소종의소거작용 (Hsieh et al. ) 및항산화효과 (Yen et al. ) 가보고되고있으며콜라겐합성을촉진시키는효과 (Li et al. 1998), 알파글루코시다아제를억제하는기전에의거한항당뇨효과 (Watanabe et al. 1997), 암발생저해효과 (Nakamura et al. 1997), 항보체효과 (Oshima et al. 1998) 등이보고되고있다. 느릅나무 (Ulmus davidiana var. japonica) 는이른봄이나가을에뿌리껍질을벗겨서약으로사용되어왔다. 분포지역으로는우리나라를비롯하여일본, 사할린, 쿠릴열도, 중국북부및동시베리아등이다. 한방에서는껍질을유피라는약재로사용하며, 치습, 이뇨소종독의처방에사용하고있다. 유피의기원식물에는크게개느릅나무인비술나무를포함한느릅나무와시무나무가있으며약용부위로근피와수피가함께사용되고있다. 유피의기원식물중느릅나무의효능에관한연구로는항암및면역활성 (Yang and Kim 1), 항균효과 (Kim et al. 3), 항산화효과 (Lee and Han ), 소염진통작용 (Cho et al. 1996), 약효 (Hong et al. 199), 위염및위궤양관련연구 (Lim and Cui 2) 와성분연구 (Bae and Kim ) 가보고되었으며시무나무에있어서는성분과항산화활성 (Chang et al. 4) 이보고되었다. 이처럼천연약용식물의효능이입증되고있으나각약용식물의혼합물에대한세포독성평가및아토피피부염완화및치료에관한자세한보고는없는실정이다. 따라서본연구에서는몇가지천연약용식물들을선별하고각각을열수추출한뒤, Cell Counting Kit-8 (CCK -8) assay를통해추출액의세포독성을비교측정하고 ELISA kit를이용하여항염증반응을확인하여, 향후아토피성피부염완화및치료에활용해보고자하였다. 재료및방법 1. 시약및재료 Cell counting kit-8 (CCK-8) 은 Dojindo사 (Kumamoto, Japan) 제품을이용하였다. Cell culture -dish, 96-well plate, 24-well plate는 NUNC사 (Naperville, IL, USA) 제품을이용하였고, Iscove s Modified Dulbecco s Medium (IMDM), Dulbecco s phosphate Bufferd saline (DPBS), Penicillin Streptomycin, Fetal Bovine Serum (FBS), Pen Strep- Penicillin Streptomycin은 GIBCO BRL (Grand Island, NY, USA) 제품을이용하였다. Calcimycin A23187, PMA (Phorbol 12-myristate 13-acetate) 은 Sigma-Aldrich co. (St. Louis, Mo, USA) 제품을이용하였다. Human TNF-α, IL-6, IL-8 ELISA kit는 R&D system (Minneapolis, MN USA) 제품을이용하였다. 두충과느릅은 ShinHeung science (Daejeon, Korea) 를통해서구입하여사용하였다. 2. 추출물제조방법 추출용매로는 3차증류수를이용하였다. 두충, 느릅을각각 1 :, 8 : 2, 5 : 5, 2 : 8, : 1의조성비율로 3차증류수 1L를가하여 75 C 에서 24시간동안열수추출하였다 (Table 1). 여기서얻은추출물을냉동건조 (-55 C, 5 일 ) 시켜분말로만든후 25 kgy (1 kgy hr -1 ) 로방사선멸균하였다. 3. 세포배양 비만세포 (HMC-1) 는한국세포주은행 (Seoul, Korea) 으로부터구입하였고, 세포배양액은 HMC-1 cell을 IMDM 에 1% FBS, unit ml -1 의 penicillin과 unit ml -1 의 streptomycin을첨가한것을사용하였다. 배양은배양기를이용하여 37 C, 5% CO 2 상태를유지하였다. 4. 세포독성실험 추출물의세포독성여부확인은 CCK-8 assay를이용하여측정하였다. HMC-1 cell을 96-well plate에 cells well -1 이되도록분주하고 37 C, 5% CO 2 상태로 24 시간배양하였다. 배양후 96-well plate에분주된 HMC- 1 cell은추출물을 mg ml -1,.6 mg ml -1, 1.2 mg ml -1, 1.8 mg ml -1, 2.1 mg ml -1, 2.4 mg ml -1, 2.7 mg ml -1, 또는 3mg ml -1 의농도로처리하여 37 C, 5% CO 2 상태로 24시간 Table 1. Composition of herbal extracts mixtures Group Eucommia ulmoides Ulmus davidiana I 1 II 8 2 III 5 5 IV 2 8 V 1

두충과느릅혼합추출물에의한염증억제효과 13 동안배양한후세포독성을측정하였다. 5. 항염증반응실험비만세포는 24-well plate에 1 1 6 cells well -1 이되도록분주하고 PMA 5 nm ml -1 와 A23187 1 μm ml -1 을처리한후 7시간동안배양후, 세포배양액을취하여사이토카인을측정하였다.

특히활성화된비만세포로부터후천적으로생성되는 leukotriene, platelet activating factor prostaglandin (PG) 등은혈관투과성과점액분비물을증가시키고, 부종을유발하고, 기도내염증세포의침윤등을유발함으로써실제알레르기천식과직접연관되는것으로알려져있다 (Brown et al. 8).

추출혼합물의세포독성평가비만세포는알레르기반응의유발에필수적인세포로알려져있다. 비만세포는세포질내과립을풍부하게가진면역세포로서결체조직과점막에존재하며알레르기염증반응에관여한다. 비만세포는 IgE가매개하는면역반응과관계되어있으며, 탈과립을유도하는물질인 PMA와 A23187에의해비만세포가활성화된다.

3 두충과느릅혼합추출물에의한염증억제효과 13 동안배양한후세포독성을측정하였다. 5. 항염증반응실험비만세포는 24-well plate에 cells well -1 이되도록분주하고 PMA 5 nm ml -1 와 A μm ml -1 을처리한후 7시간동안배양후, 세포배양액을취하여사이토카인을측정하였다. 수거된배지는측정전까지 4 C 에서보관하고, TNF-α, IL-6, IL-8 함량은 R&D Systems ELISA kit를사용하여측정하였으며, 각사이토카인의함량은표준물질의반응으로부터얻어진표준곡선을이용하여환산하였다. 요한역할을한다 (Galli et al. 8). 특히활성화된비만세포로부터후천적으로생성되는 leukotriene, platelet activating factor prostaglandin (PG) 등은혈관투과성과점액분비물을증가시키고, 부종을유발하고, 기도내염증세포의침윤등을유발함으로써실제알레르기천식과직접연관되는것으로알려져있다 (Brown et al. 8). 각추출혼합물이비만세포에미치는세포독성을알아보기위하여실시한실험결과모든그룹에서 3. mg ml -1 의농도에서 75.% 이상으로독성이없음을나타내었다 (Fig. 1). 따라서그룹 I, II, III, IV, V은 3. mg ml -1 의농도에서항염증반응실험을진행해야할것으로판단되었다. 2. 추출혼합물의항염증반응 결과및논의 1. 추출혼합물의세포독성평가비만세포는알레르기반응의유발에필수적인세포로알려져있다. 비만세포는세포질내과립을풍부하게가진면역세포로서결체조직과점막에존재하며알레르기염증반응에관여한다. 비만세포는 IgE가매개하는면역반응과관계되어있으며, 탈과립을유도하는물질인 PMA와 A23187에의해비만세포가활성화된다. 특히외부자극으로부터활성화된비만세포는다양한히스타민과프로테아제를분비하여혈관을확장하고, 염증유발물질인다양한사이토카인의분비를자극한다. 비만세포로부터분비되는사이토카인들은알레르기염증의초기와후기반응을유발하고만성염증반응을지속시키는데중 본연구에서열수추출한천연약용식물에의해염증매개물질인사이토카인의생성이억제되는것을확인하였다. TNF-α의경우그룹 I, II, III, IV, V의사이토카인저감률은각각 61.1%, 7.1%, 79.2%, 94.8%, 97.4% 로느릅의함량비율이높아질수록사이토카인의생성이억제되는것을확인하였다 (Fig. 2). IL-8의경우도마찬가지 TNF-α inhibition (%) Fig. 2. Effects of extracts on the production of TNF-α in A23187 and PMA stimulated HMC-1 cells. HMC-1 viability (%) Concentration (mg ml -1 ) IL-8 inhibition (%) Fig. 1. Cytotoxicity test of herbal extracts mixtures on HMC-1 cell line. Fig. 3. Effects of extracts on the production of IL-8 in A23187 and PMA stimulated HMC-1 cells.

4 14 권희정 신영민 정성린 박종석 노영창 임윤묵 IL-6 inhibition (%) Fig. 4. Effects of extracts on the production of IL-6 in A23187 and PMA stimulated HMC-1 cells. 로, 그룹 I, II, III, IV, V의사이토카인저감률은각각 27.9%, 41.9%, 52.8%, 84.7%, 85.4% 로느릅의함량비율이높아질수록사이토카인의생성이억제되는것을확인하였다 (Fig. 3). 반면 IL-6의경우에는두충과느릅의혼합비율이 5:5일때가장적은저감률을보였다 (Fig. 4). 두충과느릅의조성의변화에따라 TNF-α, IL-8, IL-6의저감률이다양한결과를보이는데이러한추출물간의차이는추출물에유리된성분의차이에의한것으로판단된다. 성분의분석은추후실험이더필요할것으로보인다. 결 본연구에서는열수추출한두충과느릅의천연약용식물성분을방사선멸균한후세포독성과항염증작용을측정하였다. 각각의천연약용식물의추출물은효능이입증되었으나각약용식물의혼합물에대해서는독성평가및아토피성피부염에미치는영향에관한보고가없기에본연구를실시하였다. 그결과, 비만세포에서는 3. mg ml -1 이하의농도에서독성이없는것으로나타났고, 항염증반응에서는유의적인값을확인할수가있었다. 따라서이번실험에서얻어진결과를바탕으로독성이없는농도범위하에서천연약용식물의혼합물을이용하여아토피피부염을억제할수있는연구에관한새로운발판을마련할수있을것으로판단된다. 사 이논문은교육과학기술부의재원으로시행하는한국과학재단의원자력기술개발사업으로수해되었습니다. 론 사 참고문헌 Bae YS and Kim JK.. Extractives of the bark of ash and elm as medicinal hardwood tree species. MokchaeKonghak. 28: Brown JM, Wilson TM and Metcalfe DD. 8. The mast cell and implications for therapy. Clin. Exp. Allergy 38:4-18. Chang BS, Kwon YS and Kim CM. 4. The chemical structures and their antioxidant activity of the components isolated from the heartwood of Hemiptelea davidii. Korean J. Pharmacogn. 35:-87. Cho SK, Lee SG and Kim CJ Anti-inflammatory and analgesic activities of water extract of root bark of Ulmus parvifolia. Korean J. Pharmacogn. 27: Furukawa H, Nakamure K, Zheng X, Tojo M, Oyama N, Akiba H, Nishibu A, Kaneko F, Tsunemi Y, Sseki H and Tamaki K. 4. Enhanced TARC production by dust-mite allergens and its modulation by immunosuppressive drugs in PBMCs from patients with atopic dermatitis. J. Dermatol Sci. 35: Galli SJ, Tsai M and Piliponsky AM. 8. The development of allergic inflammation. Nature 454: Hong ND, Rho YS, Kim NJ and Kim JS A study on efficacy of Ulmi cortex. Korean J. Pharmacogn. 21: Hsieh CL and Yen GC.. Antioxidant actions of duzhong (Eucommia ulmiodes oliv.) toward oxidative damage in biomolecules. Life Sci. 66: Kim CS, Lee JM, Choi CO, Park SB and Eom TJ. 3. Chemical analysis and isolation of antibacterial compound from Ulmus specied (I): chemical analysis and antibacterial activity of extractives. MokchacKonghak. 3: Kim DW, Park JY, Na GY, Lee SJ and Lee WJ. 6. Correlation of clinical features and skin barrier function in adolescent and adult patients with atopic dermatitis. Int J. Dermatol. 45: Lee YJ and Han JP.. Antioxidative activities and nitric scavenging abilities of extracts from Ulmus devidiana. J. Korean Soc. Food Sci. Nutr. 29: Leung DY and Bieber T. 3. Atopic dermatitis. Lancet. 361: Li Y, Sato T, Metori K, Koike K, Che QM and Takahashi S The promoting effects of geniposidic acid and aucubin in Eucommia ulmiodes oliver leaves on collagen synthesis. Biol. Pharm. Bull. 21: Lim JP and Cui X. 2. Effect of water extract of Ulmi pumilae cortex on gastric ulcer in rats. Korean J. Medicinal Crop Sci. 1: Nakamura T, Nakazawa Y, Onizuka S, Satoh S, Chiba A, Sekihashi K, Miura A, Yasugahira N and Sasaki YF

5 두충과느릅혼합추출물에의한염증억제효과 15 Antimutagenicity of Tochu tea (an aqueous extract of Eucommia ulmoides leaves): 1. The clastogen-suppressing effects of Tochu tea in CHO cells and mice. Mutat. Res. 388:7-. Oshima T, Takata S, Kikino H, Deyama T and Kinoshita G Anticomplementary activity of the constituents of Eucommia ulmoides bark. J. Ethnopharmacol. 23: Shultz LF and Hanifin JM. 2. Epidemiology of atopic dermatitis. Immunol Allergy Clin North Am. 22:1-24. Watanabe J, Kawabata J, Kurihara H and Niki R Isolation and identification of alpha-glucosidase inhibitors from tochu-cha (Eucommia ulmiodes). Biosci Biotech. Bioch. 61: Yang YL and Kim YJ. 1. Immunostimulating exopolysaccharide with anticancer activity from Enterobacter sp. SSYL (KCTC 687BP) screened from Ulmus parvifolia. Korean J. Biotechnol. Bioeng. 16: Yen GC and Hsieh CL.. Reactive oxygen species scavenging activity of Du-zhong (Eucommia ulmiodes oliv.) and its active compounds. J. Agric. Food Chem. 48: Manuscript Received: December 14, 11 Revised: January 1, 12 Revision Accepted: January 3, 12

14-TNF-a(dh)nn

14-TNF-a(dh)nn Journal of Radiation Industry 5 (3) : 273~277 (11) Review Paper TNF-α, IL-6, IL-8 저해를통한천연약용식물추출물의항염증특성 김용수 권희정 박종석 임윤묵 노영창 * 한국원자력연구원정읍방사선과학연구소방사선공업환경연구부 Anti-inflammatory Activity of Herbal Extracts through