J KSM J Korean Soc Menopause 2012;18: ISSN J Korean Soc Menopause 2012;18: Origin

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J KSM ISSN 1226-8704 Original Article 혈관평활근세포증식에대한국내자생종승마의효과 강영희 1 이동윤 2 최두석 1,2 윤병구 1,2 이영주 3 류미라 4 삼성생명과학연구소 1, 성균관대학교의과대학산부인과학교실 2, 세종대학교생명공학과 3, 한국식품연구원대사기능연구본부 4 Effect of Korean Native

1 J KSM ISSN Original Article 혈관평활근세포증식에대한국내자생종승마의효과 강영희 1 이동윤 2 최두석 1,2 윤병구 1,2 이영주 3 류미라 4 삼성생명과학연구소 1, 성균관대학교의과대학산부인과학교실 2, 세종대학교생명공학과 3, 한국식품연구원대사기능연구본부 4 Effect of Korean Native Cimicifuga Species on Proliferation of Vascular Smooth Muscle Cells Young-Hee Kang, M.S. 1, Dong-Yun Lee, M.D. 2, Doo Seok Choi, M.D. 1,2, Byung-Koo Yoon, M.D. 1,2, Young Joo Lee, Ph.D. 3, Mee-Ra Rhyu, Ph.D. 4 Samsung Biomedical Research Institute 1, Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine 2, Department of Bioscience and Biotechnology, Sejong University 3, Seoul, Divisions of Metabolism and Functionality Research, Korea Food Research Institute 4, Seongnam, Korea Objectives: To examine the effect of extracts of Korean native Cimicifuge species on cell proliferation in vascular smooth muscle cells (VSMC). Methods: VSMC were isolated from rat aorta. Cell proliferation was assessed by measure of bromodeoxyuridine incorporation into the cells. Differences in Reactive oxygen species (ROS) levels were examined after exposure to the extracts of Korean native Cimicifuge species using the detection reagents dichlorofluorecin diacetate. The rhizomes/roots were air-dried and milled with a commercial food mixer. Milled rhizomes/roots of each Cimicifuga species were separately extracted by 80% ethanol, absolute methanol, and 40% 2-propanol using homogenizer and evaporated under reduced pressure at low temperatures. Effects of extracts dissolved in phosphate-buffered saline (0.3 mg/ml) were examined. Results: Ethanolic, methanolic or propanolic extracts of 4 Korean native Cimicifuge species (Cimicifuga [C] davurica, C. japonica, C. heracleifolia var. bifida Nakai, C. simplex) were screened. The addition of extracts of each Korean native Cimicifuge species to cells in the presence of 10% fetal bovine serum (FBS) potently inhibited cell proliferation. Significant decrease of 23%-30% was observed. Vitamin E, a potent antioxidant, inhibited 10% FBS-stimulated cell proliferation of VSMC. We also demonstrated that extracts of each Korean native Cimicifuge species decreased intracellular ROS generation induced with 10% FBS. The effect of Korean native Cimicifuge species was not species-specific and solvent-specific. Conclusion: TExtracts of Korean native Cimicifuge species inhibit VSMC proliferation via inhibition of intracellular ROS. These findings suggest that Cimicifuge species used for reducing menopause symptoms might be cardioprotective in women. Key Words: Cardiovascular diseases, Cimicifuga, Estrogens, Vascular smooth muscle cell 심혈관질환은폐경여성의가장중요한사망원인중하나이며, 여성호르몬의결핍으로혈중지질농도의변화와동맥혈관벽의변화로동맥경화증위험이증가한다. 1 동맥경화증은내피세포의기능장애, 염증, 기질변화등을포함하 는복잡한과정들이다. 주요원인으로혈관평활근세포증식및산화스트레스의증가가있다. 2~4 혈관벽에서 low-density lipoproteins (LDL) 의산화는산화스트레스를증가시킴으로써동맥경화를촉진시킨다. 산 Received: November 4, 2011 Revised: December 1, 2011 Accepted: December 1, 2011 Address for Correspondence: Byung-Koo Yoon, Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul , Korea Tel: , Fax: , bkyoon@skku.edu Copyright 2012 by The Korean Society of Meno pause This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (

2 강영희외. 혈관평활근세포증식에대한승마의효과 화 LDL 은또한내피세포와평활근세포에서기초섬유아세포성장인자의발현을유발하여평활근세포증식을자극한다. 5,6 호르몬대체요법 (hormone replacement therapy) 은에스트로겐감소로발생하는폐경증상을완화시킨다. 그러나호르몬투여로발생할수있는유방통, 비정상자궁출혈, 기분변화그리고체중증가와같은부작용과함께유방암위험증가에대한두려움으로호르몬치료를기피하는경향이있다. 따라서호르몬을대신할안전하고효과적인치료방법의필요성이요구되고있다. 최근식물성에스트로겐이갱년기증상, 골다공증, 악성종양, 그리고심장질환등에긍정적인효과를보이는것으로밝혀지고있어식물성에스트로겐에대한관심이높아지고있다. 7~12 승마 (black cohosh: Cimicifuga racemosa) 는북미에서자생하는식물로써오래전부터월경불순또는폐경과관련된부인과질병치료에이용되었다. 현재승마추출물은안면홍조, 발한, 발열등폐경증상의치료에흔히쓰이고있으며, 13~15 한국여성에서진행된승마와 St. John s Wort 의복합제에대한임상시험에서도중증열성홍조 16 또는갱년기증상완화 17 에효과적이었다. 그러나, 심혈관계에대한승마의영향연구는거의없는상태이다. 또한국내자생종승마에대한연구가전혀이루어지지않고있다. 따라서본연구에서는북미승마대체소재로서의활용성제고를위하여국내자생종승마를연구대상으로하였고, 천연식물자원유래의식물성에스트로겐인승마가갱년기증상완화이외에도심혈관계질환인동맥경화증예방효과를알아보고자하였다. 본연구목적은동맥경화증발생의대표적기전인혈관평활근세포증식에대한국내자생종승마의효과를밝히기위해시행되었다. 연구대상및방법 1. 혈관평활근세포의분리와배양 3개월된 Sprague-Dawley rat에서흉부대동맥을떼어내어혈관평활근세포를분리하였다. 18 세포는세포배양액 (Dulbecco s Modified Eagle Medium [DMEM]/F12 supplemented with 10% fetal bovine serum (FBS), mg/ ml penicillin, 0.1 mg/ml streptomycin) 에서 37 o C humidified 5% CO 2 /95% air 환경에서배양하였으며 5-10 번계대한후의세포를실험에사용하였다. 세포들이 70% confluence 상태에도달하면 defined serum-free medium (DMEM/F12 medium containing insulin M, transferrin 5 mg/ ml, ascorbic acid 0.2 mm) 로배양액을교환후 48시간배양하여 quiescent 상태의세포를사용하였다. 2. Bromodeoxyuridine (5-bromo-2 -deoxyuridine, BrdU) incorporation 세포증식을조사하기위하여 BrdU cell proliferation assay kit (Calbiochem, La Jolla, CA) 를사용하였다. 10% FBS 가첨가된배양액의세포에승마추출물을 0.3 mg/ml 농도로각각 24시간동안처리하였다. BrdU 는승마추출물처리시간 24시간중마지막 4시간전에첨가하였다. Excitation 320 nm (wavelength) 와 emission 460 nm (wavelength) 에서 fluorometer 를이용하여세포의 BrdU incorporation 정도를 relative fluorescence unit (RFU) 로측정하였다. 3. Reactive oxygen species (ROS) 측정 10% FBS 가첨가된배양액의세포에승마추출물을각각 0.3 mg/ml 농도로 24시간동안처리하였다. 10 um 2,7 -dichlorofluorecin diacetate (Calbiochem) 을첨가하여 30분간배양한후 fluorescence-activated cell sorter (FACs) 를이용하여세포내 ROS 생성을측정하였다. 4. 시료및추출강원도일대에서채취한자생종눈빛승마 (Cimicifuga [C] davurica), 왜승마 (C. japonica), 세잎승마 (C. heracleifolia var. bifida Nakai), 촛대승마 (C. simplex) 4종을한국자생식물원 ( 강원도평창군 ) 으로부터제공받아연구대상으로하였다. 각승마종건조분말에 20배가량의 80% ethanol, absolute methanol, 40% 2-propanol 을가하여 homogenizer (Ultra-Turrax T-25 basic, IKA Werke GmbH & Co. KG, Staufen, Germany) 로추출한후원심분리 (3,700 g, Sorvall RC50, GMI Inc., WA, USA), filtration 한여과액을 o C에서감압건조 (rotary evaporator RE121, Büchi, Switzerland), 동결건조하여추출물을제조하였다. 추출물을 phosphate-buffered saline 에녹인후 0.3 mg/ml 농도로처리하였다. 5. 통계분석결과는평균 ± 표준오차로표시하였고, n은반복실험한횟수를나타내었다. 실험한결과를 Wilcoxon s signed rank test 를사용하여통계분석하였다. P 값이 0.05 미만인경우통계적으로유의한차이로판정하였다. 101

3 J KSM 결과 고 찰 4 종의국내자생종승마 ( 왜, 세잎, 눈꽃, 촛대 ) 에서각각 추출한추출물이 FBS 자극에의해유도된혈관평활근세포 의증식에어떠한영향을미치는지알아보았다. Quiescent 상태인 cells 에 10% FBS 를첨가하고각각의승마추출물을 혈관평활근세포에처리하였다. Figure 1 은 FBS 의자극에 의해증가된세포증식이모든추출물에의해유의하게억 제되었음을보여준다. 억제효과가 % 까지차 이를보였다. 추출용매와종에따른차이는없었다. ROS 는혈관평활근세포증식에중요한역할을한다. 2~4 본 연구에서도 ROS inhibitor 인비타민 E 가 10% FBS 자극에의 해유도된혈관평활근세포증식을유의하게억제시킴을확 인하였다 (Fig. 2). Figure 3 은 10% FBS 자극하에서승마추출물이 ROS 생 성에미치는영향을조사한결과이다. 승마추출물의처리 는대조군에비해 ROS 생성을유의하게감소시켰다. 추출 용매와종에따른차이는볼수없었다. 이러한결과는국내 자생종승마에의한혈관평활근세포증식억제효과의기 전으로 ROS 감소가중요한역할을한다는것을보여준다. Percent of control DSF Control Fig. 1. The effects of extracts of Cimicifuga [C] species (0.3 mg/ ml) on 10% fetal bovine serum (FBS)-stimulated proliferation of vascular smooth muscle cells. The extracts were added to the medium containing 10% FBS and the incubation proceeded for 24 h. Cell proliferation was measured by bromodeoxyuridine assay. CJ: C. japonica, CH: C. heracleifolia var. bifida Nakai, CD: C. davurica, CS: C. simplex, DSF: defined serum-free medium, P: 40% 2-propanol, M: absolute methanol, E: 80% ethanol. P < 0.05 vs. control. P M E P M E P M E P M E CJ CH CD CS 10% FBS n=4 혈관평활근세포증식은고혈압, 죽상경화증, 재발협착증 과같은심혈관질환의잠재적주요원인이므로, 혈관평활 근세포증식억제는심혈관질환의중요한치료적전략이 다. 2,19 본연구에서는 10% FBS 자극에의해유도된혈관평 활근세포증식을국내자생종승마추출물이유의하게억 제시킴을보였다. 또한혈관평활근세포증식억제효과에 ROS 감소가중요한작용기전임을알수있었다. 이러한결 과는혈관평활근세포증식억제효과가있는국내자생종 승마가심혈관질환예방에효과적으로이용될수있는가 능성을보여준다. 심혈관질환, 특히관상동맥질환은여성의주요사망원인 중하나이며, 폐경후발병이증가한다. 여성호르몬감소가 가능한원인이며, 에스트로겐투여는발병을예방하는것으 로믿어져왔다. 1 호르몬대체요법은폐경증상에대한가장 우수한치료법이지만장기간투여시유방암위험이증가할 수있고노인에서시작한경우심혈관질환을오히려증가 시키므로최근들어식물성에스트로겐에대한관심이높아 지고있다. 20,21 승마 (C. racemosa) 는오래전부터미국, 중국, 그리고유 럽에서뇌하수체장애의치료, 그리고월경또는폐경과관 련된자율신경증상을치료하기위한천연생약물질로사용 되어왔다. 13~15 승마추출물은 triterpene glycosides, phenolic, Percent of control DSF Control Vitamin E 10% FBS n=3 Fig. 2. The effect of vitamin E on 10% fetal bovine serum (FBS)- stimulated proliferation of vascular smooth muscle cells, as assessed by bromodeoxyuridine assay. Vitamin E (40 mm) was added to the medium containing 10% FBS and the incubation proceeded for 24 h. DSF: defined serum-free medium. P < 0.05 vs. DSF; P < 0.05 vs. 10% FBS. 102

4 강영희외. 혈관평활근세포증식에대한승마의효과 Percent of control Control P M E P M E P M E P M E CJ CH CD CS 10% FBS n=4 소를통하여혈관평활근세포증식을억제함을확인하였다. 앞으로승마가폐경증상의조절과함께심혈관질환을예방할수있는가능성이기대된다. 감사의글 본연구는삼성생명과학연구소 (#SBRI C-A ), 성균관대학교산학협력단 (S ), 그리고농림수산식품부농림기술개발사업 ( SB010) 의지원으로시행되었음. Fig. 3. The effects of extracts of Cimicifuge species (0.3 mg/ ml) on reactive oxygen species (ROS) generation of vascular smooth muscle cells, as assessed by fluorescence-activated cell sorter (FACs) analysis. The extracts were added to the medium containing 10% fetal bovine serum (FBS). P: 40% 2-propanol, M: absolute methanol, E: 80% ethanol. P < 0.05 vs. control. CJ: C. japonica, CH: C. heracleifolia var. bifi da Nakai, CD: C. davurica, CS: C. simplex. flavonoids, volatile oils, tannins 과다른 pharmacologically 활성성분들을함유하고있다. 15,22 또한 selective estrogen receptor modulator 로서잘알려져있다. 13 폐경여성에게승 마추출물의투여가조골세포의활성을증가시킴으로써골 다공증에효과가있을것이라생각되고, 최근에는승마에서 추출된 triterpenoid glycoside 가 in vitro 에서 cytokines 에의 한파골세포생성을억제시키고 tumor necrosis factor 에의 해유도된골손실을완화시킨다는연구결과가있다. 23 또한 질점막에서는에스트로겐과비슷한활성을나타내었으나, 1 년간승마추출물을투여한여성에게서자궁내막증식증이 발견되지않았다. 24,25 현재까지승마는폐경증상개선에있어효과적이며안전 한물질로판단된다. 그러나심혈관계에대한승마의영향 연구는매우미흡한상황이다. 따라서본연구는동맥경화 증발생의대표적기전인혈관평활근세포증식및산화스 트레스에대한국내자생종승마의효과를밝히기위해시 행되었다. 그결과국내자생종승마추출물에의해혈관평 활근세포증식이억제되었다. 또한 ROS 생성을크게감소 시켰다. 추출방법과승마종에따른증식억제나 ROS 생성 의차이는없었다. 결과로서국내자생종승마가동맥경화 증억제효과가있음을확인하였다. 본연구를바탕으로향 후다양한심혈관계영향에대한추가연구가필요할것으 로사료된다. 결론적으로국내자생종승마추출물이산화스트레스감 참고문헌 1. Barrett-Connor E, Grady D. Hormone replacement therapy, heart disease, and other considerations. Annu Rev Public Health 1998; 19: Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993; 362: Schwartz SM, Virmani R, Rosenfeld ME. The good smooth muscle cells in atherosclerosis. Curr Atheroscler Rep 2000; 2: Stocker R, Keaney JF Jr. Role of oxidative modifications in atherosclerosis. Physiol Rev 2004; 84: Catapano AL, Maggi FM, Tragni E. Low density lipoprotein oxidation, antioxidants, and atherosclerosis. Curr Opin Cardiol 2000; 15: Sherer Y, Shoenfeld Y. Mechanisms of disease: atherosclerosis in autoimmune diseases. Nat Clin Pract Rheumatol 2006; 2: Hurd WW. Menopause. In: Berek JS, Adashi EY, Hillard PA, Rinehart RD, Novak HW, Hengst TC editors. Novak s gynecology. Baltimore, MD: Lippincott Williams & Wilkins, p Gennari C, Adami S, Agnusdei D, Bufalíno L, Cervetti R, Crepaldi G, et al. Effect of chronic treatment with ipriflavone in postmenopausal women with low bone mass. Calcif Tissue Int 1997; 61 Suppl 1: S Ingram D, Sanders K, Kolybaba M, Lopez D. Case-control study of phyto-oestrogens and breast cancer. Lancet 1997; 350: Anthony MS, Clarkson TB, Hughes CL Jr, Morgan TM, Burke GL. Soybean isoflavones improve cardiovascular risk factors without affecting the reproductive system of peripubertal rhesus monkeys. J Nutr 1996; 126: Washburn S, Burke GL, Morgan T, Anthony M. Effect of 103

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6 강영희외. 혈관평활근세포증식에대한승마의효과 국문초록 연구목적 : 혈관평활근세포증식에대한국내자생종승마의효과를밝히기위해시행되었다. 연구재료및방법 : 3개월된암컷 Sprague-Dawley 쥐의대동맥으로부터혈관평활근세포를얻었다. 눈빛승마, 왜승마, 세잎승마, 촛대승마등 4종의자생승마를 80% ethanol, absolute methanol 그리고 40% 2-propanol 을사용하여추출하였다. 10% fetal bovine serum (FBS) 에서배양한혈관평활근세포를승마추출물 (0.3 mg/ml) 로 24시간동안처리한후 bromodeoxyuridine incorporation assay 를사용하여세포증식을조사하였다. dichlorofluorecin diacetate 을첨가한후 flow cytometry 를이용하여세포내 ROS 생성을정량화하였다. 결과 : 추출법이다른국내자생종승마추출물을처리한결과, 혈관평활근세포의증식이유의하게 23-30% 억제되었다. 또한강력한항산화제인비타민 E는 10% FBS 로유도된세포증식을감소시켰다. 승마추출물의작용기전을밝히기위한추가연구상 10% FBS 에의한 ROS 생성이승마에의해감소되었다. 세포증식이나 ROS 생성에대한효과는추출방법과승마종에따라차이가없었다. 결론 : 국내자생종승마는산화스트레스감소를통하여혈관평활근세포증식을억제한다. 향후다양한심혈관계영향에대한추가연구가필요할것으로사료된다. 중심단어 : 심혈관질환, 승마, 에스트로겐, 혈관평활근세포 105

Treatment and Role of Hormaonal Replaement Therapy

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