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1 J Korean Med. 2016;37(3):39-46 pissn eissn Original Article 난소를절제한마우스에서갱년기증후군에대한오미자의효과 신다솜 1, 홍성빈 2, 금정호 2, 마진열 3, 정환석 3 1 경희대학교한의과대학생리학교실, 2 바이오믹스생명과학연구소, 3 한국한의학연구원한의기술응용센터 Effects of Schisandrae Fructus on menopause symptoms in ovariectomized mice Dasom Shin 1, Seong Bin Hong 2, Jeong Ho Geum 2, Jin Yeul Ma 3, Hwan-Suck Chung 3 1 Department of Physiology, College of Korean Medicine, Kyung Hee University, 2 BIOMIX Inc. 3 Korean Medicine (KM) Application Center, Korea Institute of Oriental Medicine (KIOM) Objectives: The present study aimed at investigating the effects of Schisandrae Fructus (SF) on menopause symptoms in ovariectomized mice. Methods: 6 weeks old female mice were ovariectomized (OVX), and the OVX mice were fed with a three doses of SF (25, 100, and 400 mg/kg, respectively) or 17β-estradiol (E2, 0.5 mg/kg) 3 times a week for 6 weeks. Body weight was measured at every week and uterus weight was also measured at the end of experiment. Lipids and osteocalcin levels in serum were analyzed. Results: The uterine weight was not changed in SF treated group, while E2 intake increased the reduced uterine weight by OVX. Although Body weight was increased since two weeks after ovariectomy compared with sham operated group, there was no change in SF or E2 treated group compared with OVX. But LDL was significantly reduced in SF (400 mg/kg) or E2 fed mice and SF or E2 treatment decreased enhanced osteocalcin level by OVX. Conclusion: These results indicated that SF would be effective in the improvement of LDL level and osteoporosis on menopause. Key Words : Menopause, Schisandrae fructus, Ovariectomized mice, Osteoporosis, Osteocalcin 서론최근우리나라는경제성장과더불어여성들의사회활동이급격이증가하는추세이다. 더욱이의료기술의발달로인하여사람의평균수명도길어져사회적으로갱년기에대한관심이점점높아지고있다. 여성의갱년기는에스트로겐의결핍으로인한안면홍조, 피로감, 불안감, 우울, 기억력장애, 수면장애, 질건조감, calcitonin 기능억제에의하여골교체율 (bone turnover) 증가에따른골다공증등이 대표적인증상이다 1,2). 이런갱년기증상을완화하기위해 estrogen이나 progestin을섭취하는호르몬보충요법이많이사용되며이는골다공증으로인한골절, 결장암을억제하는효과가있지만관상동맥질환, 중풍, 혈전색전증, 담낭염, 유방암, 자궁내막암등의부작용을유발한다는보고가있다 3-5). 반면, 우리주변에서접할수있는식물자원중아마씨, 칡, 석류등에함유된 isoflavone, lignan이 phytoestrogen으로작용하여여성갱년기에효과가있다는연구는이미보고되어있 Received:30 August 2016 Revised:20 September 2016 Accepted:20 September 2016 Correspondence to: 정환석 (Hwan-Suck Chung) 대구광역시첨단로 70 한국한의학연구원한의기술응용센터 Tel: , Fax: , hschung@kiom.re.kr 39

2 (340) 다 6). 이런이유로부작용이적은생약재를이용한건강기능식품및천연물신약개발이꾸준히진행되고있다. 오미자는구기자, 사상자, 토사자, 복분자와함께五子로불리며전통적으로많이알려진생약재로서오미자과 (Schisandraceae) 과에속한낙엽목질등본으로열매가붉게맺히며주로열매를사용한다. 오미자는신맛, 쓴맛, 매운맛, 짠맛, 단맛등다섯가지의맛을가지고있다고하여五味子로불리며, 性은溫하고, 味는酸甘하며肺, 心, 腎經에들어가上으로는肺氣를斂하고, 下로는腎陰을滋하며澀精止瀉, 生津, 止汗등의작용을가지고있다 7). 예로부터기침등의호흡기증상과간질환, 만성간질환등에효과가있다고하여차로많이사용되어왔으며현재에는식품원료로사용이허가되어있을정도로無毒하다. 오미자는 schizandrin, gomisin N, gomisin A, deoxyschizandrin 등의리그난을주요성분으로함유하고있다고보고되어있으며 8), 이런 lignan이약물의주된효과를발휘하는화합물로알려져있다. 오미자추출물이 in vitro 실험을통해파골세포의분화를억제하고, estrogen receptor 를활성화한다는결과가보고되어있지만 9,10). in vivo에서갱년기증상에미치는효능은아직알려져있지않다. 난소절제후오미자추출물 100 mg/kg 투여군 (OVX-SCB100), 난소절제후오미자추출물 400 mg/kg 투여군 (OVX-SCB400)] 으로분류하여, 주 3 회오미자혹은대조물질을존데를이용하여일정한시간에경구투여로 6주동안진행하였다. 실험동물의체중은동일한시간에측정하였으며동물실험실의환경조건은온도 23±3, 상대습도 55±15%, 환기횟수 10-20회 /hr, 조명시간 12시간및조도 Lux를유지하였으며물과식이는자유공급하였고, 실험시료는증류수에용해하여 1마리당 200 μl씩경구투여하였으며, 대조군은동일용량의증류수를투여하였다. 본연구는동물보호법 ( 제정 1991년 05월 31일법률제 4379호, 일부개정 2008년 2월 29일법률제 8852호 ) 에근거한경희대동물윤리위원회에의해승인되었다. 2. 시료의제조본실험에사용한오미자 (Schisandra chinensis Baillon) 추출물은경북문경에서구입하여수세, 정선및탈수과정을거쳐건조된오미자를 30% 주정으로 3시간추출후, 여과하여분말화공정을거쳐제조되었다. 본실험에사용된시료는 ( 주 ) 바이오믹스에서제공받아사용하였다. 재료및방법 1. 실험동물실험동물은 g인 6주령암컷 (C57BL/6J) mice의난소절제모델 (OVX model mice) 과비난소절제군 (sham-operated mice) 를 오리엔트바이오 ( 성남 ) 에서구입해 1주일간무균실에서순응시켰다. 순응기간종료후난괴법 (Randomized Complete Block Design) 에의하여 8마리씩 6군 [ 비난소절제정상대조군 (Sham) 즉, 난소절제는실시하지않았으나수술의스트레스를동일하게받은군, 난소를절제한대조군 (OVX-Vehicle), 난소절제후 17-beta -estradiol 투여한양성대조군 (OVX-E2), 난소절제후오미자추출물 25 mg/kg 투여군 (OVX-SCB25), 3. HPLC 에의한지표성분분석오미자의지표물질인 Gomisin N 은한약진흥재단으로부터구입하였으며화학구조는그림 1A와같다. 제조한오미자추출물을 20 ml의 methanol에녹인후 30분간 sonication을시행하였다. 이용액을 0.45 µm 필터로여과후 10 µl의검액을 HPLC system에주입하였다. HPLC는 Waters 2695 Alliance HPLC system과 Waters 996 PDA가검출기로구성되어있으며, 컬럼은 Fortis C18 (250 X 4.6 mm, 5µm) 을사용하였다. 이동계는 acetonitrile 과 0.1% formic acid (70/30, v/v) 에서 40분간, 유속은 1 ml/min로하였다. 40

3 난소를절제한마우스에서갱년기증후군에대한오미자의효과 (341) Table 1. Experimental Design of Animal Group Operation Treatment Sham sham-operated OVX-Vehicle ovariectomized OVX-E2 ovariectomized Estradiol 0.5 mg/kg bw/day OVX-SCB25 ovariectomized Schisandra chinensis Baillon 25 mg/kg bw/day OVX-SCB100 ovariectomized Schisandra chinensis Baillon 100 mg/kg bw/day OVX-SCB400 ovariectomized Schisandra chinensis Baillon 400 mg/kg bw/day 4. 혈중지질농도측정혈청의생화학적분석을위해, 응고된혈액표본을 1,500 g, 10 분간원심분리하여혈청을얻었다. 혈청은 Automated analyser (7080, Hitachi Ltd., Tokyo, Japan) 를사용하여 Total cholesterol(tc), Triglyceride(TG), Low density lipoprotein(ldl), High density lipoprotein(hdl) 을측정하였다. 5. 혈중 Osteocalcin 측정 Osteocalcin 측정을위해혈청을수집하였다. 혈중 Osteocalcin은 Takara Bio In. 의효소면역측정세트를이용하여제조회사의사용방법에따라측정하였다. 96 well microtiter plate는 Anti-mouse Osteocalcin monoclonal antibody가코팅되어져있다. 표준물질과표본을코팅된 96 well microtiter plate에넣어준후, 실온에서 1시간반응시킨후 2차 peroxidase-labeled anti-gla-osteocalcin monoclonal antibody를넣어준후, 실온에서 1시간반응시켰다. 마지막으로, plate에 Substrate Solution (TMBZ) 를넣어 30분동안반응시킨후, TMBZ stop solution 을첨가하여반응을종결시켰다. Optical density는 microplate reader (SoftMax PRO, version 3.1 software, CA, USA) 를사용하여 450 nm에서측정하였다. 6. 통계처리모든실험결과는통계프로그램 Graphpad Prism software (version 5, CA, USA) 를이용하여분석하 였으며, 평균 ± 표준편차로표시하였다. 정상대조군 (Sham) 대비오미자투여군 (OVX-SCB) 과난소절제군 (OVX-Vehicle) 대비오미자투여군 (OVX-SCB) 과의차이를분석하였으며, one-way ANOVA를이용하여유의확률 (p-value) 이 5% 미만 (p<0.05) 일때유의한차이를나타낸다고판단하였다. 결과 1. 지표물질정량오미자의주성분인 Gomisin N을 HPLC 분석으로정량하여오미자를확인하고자하였다. 표준품 Gomisin N을이용해 ~250 µg/ml (10.417, , 31.25, 62.5, 125 and 250 µg/ml) 까지검량선을작성하였으며 (Fig. 1B), 오미자의 Gomisin N 은 분의 retention time에서피크를확인할수있었다. peak area를계산한결과 1g의오미자추출물에는 µg의 Gomisin N을함유하였다. 2. 자궁무게난소를절제하면자궁내막을자라게하는에스트로겐이감소해자궁무게가감소하며, 자궁무게감소는난소절제술의성공여부를판단하는잣대가되기도한다 11). 자궁무게비교분석결과, 정상대조군에비해난소절제군에서통계적유의성있는자궁무게감소를나타내었다 (***p<0.001, one-way ANOVA). 또한, 난소절제군에비해양성대조물질투여군 (OVX-E2) 은유의성있는증가를나타내었 41

4 (342) Fig. 1. HPLC analysis of SCB. The structure of Gomisin N(A), Calibration curve of Gomisin N (B), Chromatogram of SCB (C) and Gomisin N (D) using HPLC systems. 다 (###p<0.001, one-way ANOVA). 그러나정상대조군대비오미자추출물투여군 (OVX-SCB), 난소절제군대비오미자투여군에서모두유의성있는자궁무게의차이를보이지않았다 (Fig. 2). Fig. 2. Effects of SCB on uterus weight in ovariectomized mice (OVX). Data are shown as mean ± S.E.M. Statistical analyses were conducted by one-way ANOVA (***p<0.001 vs Sham and, ***p<0.001 vs OVX+Vehicle). 3. 체중및혈중지질수준체중증가는폐경기에나타나는특징으로, 폐경후체내콜레스테롤이에스트로겐으로전환율이감소하면서혈중콜레스테롤이상승하고, 지방조직의증가로인해내장지방이증가하면서복부지방이늘어나게된다 12). 몸무게비교분석결과, 식이 2주후부터정상대조군에비해난소절제군에서통계적으로유의성있는몸무게증가를나타내었다 (***p<0.001, **p<0.01, *p<0.05 one-way ANOVA). 그러나정상대조군및난소절제군대비오미자투여군에서는모두유의성있는몸무게의차이를보이지않았다 (Fig. 3). Serum 을이용하여 Total cholesterol (TC), Triglyceride (TG), Low density lipoprotein (LDL), High density lipoprotein (HDL) 을측정하였다. TC와 LDL 분석결과, 정상대조군대비난소절제군에서유의성있는증가를나타내었다 (*p<0.01, *p<0.05 one-way ANOVA). LDL은난소절제군대비양성대조물질투여군 42

난소를절제한마우스에서갱년기증후군에대한오미자의효과 (343) Fig. 3. Effects of SCB on body weight in ovariectomized mice (OVX). Data are shown as mean ± S.E.M. Statistical analyses were conducted by one-way ANOVA (***p<0.

5 난소를절제한마우스에서갱년기증후군에대한오미자의효과 (343) Fig. 3. Effects of SCB on body weight in ovariectomized mice (OVX). Data are shown as mean ± S.E.M. Statistical analyses were conducted by one-way ANOVA (***p<0.001, **p<0.01, *p<0.05 vs Sham). Fig. 4. Effects of SCB on serum lipids in ovariectomized mice (OVX). Data are shown as mean ± S.E.M. Statistical analyses were conducted by one-way ANOVA (**p<0.01, *p<0.05 vs Sham and, ###p<0.001, #p<0.05 vs OVX+Vehicle). (OVX-E2) 과오미자투여군 (OVX-SCB400) 에서통계적유의성있는감소를나타내었다 (###p<0.001, one-way ANOVA). 반면, TG와 HDL 분석결과, 정상대조군에비해난소절제군에서통계적유의성있는차이를나타내지않았다 (Fig. 4). 4. 혈중 Osteocalcin Osteocalcin은폐경기에증가하여폐경후골다공증의조기진단의마커로사용된다 13). Serum 내 Osteocalcin의농도분석결과, 정상대조군대비난소절제군에서유의성있는증가를나타내었다 (**p<0.01, one-way ANOVA). 반면, 양성대조물질투여군과오미자투여군 (OVX-SCB25) 은난소절제군대비유의성있는감소를나타내었다 (Fig. 5, 43

(344) Fig. 5. Effects of SCB on osteocalcin in ovariectomized mice (OVX). Data are shown as mean ± S.E.M. Statistical analyses were conducted by one-way ANOVA (**p<0.01 vs Sham and, ***p<0.001, **p<0.

6 (344) Fig. 5. Effects of SCB on osteocalcin in ovariectomized mice (OVX). Data are shown as mean ± S.E.M. Statistical analyses were conducted by one-way ANOVA (**p<0.01 vs Sham and, ***p<0.001, **p<0.01 vs OVX+Vehicle). ###p<0.001, ##p<0.01, one-way ANOVA). 고찰 五味子는收斂固澁, 益氣生津, 補腎寧心등의효과가있어임상에서久嗽虛喘, 夢遺滑精, 遺尿頻尿, 久瀉不止, 自汗, 盜汗, 津傷口渴, 心悸失眠등의증상에응용되어왔다 14). 여성의 50% 는안면홍조, 빈맥, 발한과같은갱년기증상을보이며일부는피로감, 불안감, 우울, 기억력장애를동반하기도한다. 이러한갱년기증상과관련된오미자의효능을보면 醫方類聚 에서는腎을보하고열을내리며渴症을멈추고몸을든든하게하고성기능도높이며, 진액을생겨나게하며元氣가모자랄때쓰면크게보한다고기록되어있다. 醫寶鑑 에서는몹시여윈것을보하며消渴과煩熱을낫게한다고 15) 한다. 최근연구결과에의하면, 갱년기모델인난소절제군은 oestrogen 부족으로인해인지능력이감소하게되는데, 오미자의주성분인 Schizandrin이스냅스전달과항산화활성이보고되었다 16). 본연구는오미자추출물이난소절제술로갱년기를유도한마우스에서여성갱년기증상개선정도를평가하고자실시하였다. 체중증가는갱년기의증상중하나로호르몬불균형에의한현상이며 17) 일반적인에너지섭취과량혹은소비저하로인한대사성질환과는성격이다르다. 난소절제술을시행하여갱 년기를유도한후 6주간정상식이한결과정상대조군에비하여체중이증가하는전형적인갱년기증상을보였고, 오미자추출물투여군역시정상대조군에비하여체중이증가하였다. 하지만, 혈중지질이정상대조군에비하여난소절제술을시행한마우스는 Total cholesterol이상승하고, LDL도증가하였다. 하지만오미자추출물을투여한결과, 혈중 LDL 수치를감소시켜혈중지질대사에유익한변화를보였음을확인하였다. 난소를절제하면여성호르몬부족에의한자궁위축현상이발생하여자궁의무게가줄어들며, 정상대조군에대비하여난소절제군과오미자투여군에서의자궁무게가감소되는경향을보였다. 반면, 호르몬치료요법의하나인 17-beta-Estradiol를투여받은양성대조물질투약군은자궁무게증가현상을보였다. 갱년기증상의하나인골다공증은지속적인뼈재형성과정중뼈를생성시키는조골세포와흡수시키는파골세포의불균형에의하여발생되는질환이다. 정상적인뼈는재형성 (bone remodeling) 과정으로인한골손실이없지만, 폐경기여성의경우불균형에의한골교체율 (bone turnover) 이증가하면서혈청내 osteocalcin 의농도가증가한다. Osteocalcin 은골조직에서발견되는단백질이며파골세포의작용으로인하여혈중으로방출된다. 따라서체내 estrogen이부족해지면골교체율이증가하여혈청내 osteocalcin이증가됨이보고된바있다 12). 본실험에서난소절제로갱년기를유도시킨동물모델에서도정상대조군에비하여혈청내높아진 osteocalcin 농도가확인되었으며, 오미자를투여한군에서난소절제군대비통계적으로유의성있게감소된결과를확인할수있었다. 즉폐경기모델에서의골세포불균형의예방을의미하며나아가골다공증예방에효과를지닌다고말할수있다. 결론난소절제로유도된갱년기마우스모델에서오미 44

7 난소를절제한마우스에서갱년기증후군에대한오미자의효과 (345) 자추출물의효능을평가한결과혈중 LDL 수치를낮춰주고, 골교체율을저하시켜골다공증을경감시킬수있지만, 자궁에는특정효과를나타내지않는선택적인 phytoestrogen effect를가지는것으로평가되었다. 이러한효능은 genistein나 daidzein이자궁에는영향을미치지않으며, 골교체율을감소시키는선택적인 phytoestrogens으로작용한다는이전의보고와유사하다 11). 결론적으로오미자는호르몬치료제인 Estradiol처럼자궁에직접적인영향을미치지않으면서 LDL 수치를낮추며골다공증을개선하는효과가있을것으로기대된다. 감사의글 This work was supported by a grant (K16281) awarded to the KIOM from the Ministry of Science, ICT and Future Planning (MISP), Republic of Korea. 참고문헌 1. Archer DF, Sturdee DW, Baber R, de Villiers TJ, Pines A, Freedman RR, et al. Menopausal hot flushes and night sweats: where are we now? Climacteric. 2011: 14: Body JJ. Calcitonin for the long-term prevention and treatment of postmenopausal osteoporosis. Bone. 2002: 30(5 Suppl):75S-79S. 3. Ross RK, Paganini-Hill A, Wan PC, Pike MC. Effect of hormone replacement therapy on breast cancer risk: estrogen versus estrogen plus progestin. J Natl Cancer Inst. 2000:92(4): Ziel HK. Estrogen's role in endometrial cancer. Obstet Gynecol 1982;60: Nelson HD, Humphrey LL, Nygren P, Teutsch SM, Allan JD. Postmenopausal hormone replacement therapy: scientific review. JAMA. 2002:288(7): Ahn KH, Kim SM, Yi KW, Park HT, Shin JH, Kim YT, et al. The Effect of Pomegranate on Postmenopausal Syndrome: A Randomized, Double-blind, Placebo-controlled Trial. J Korean Soc Menopause. 2010:16(2): Herbology Association of Korea's Colleges of Oriental Medicine. Textbook of Herbology. Younglimsa. 2011: Koo DC, Suh WS, Baek SY, Shim SH. Quantitative Determination of Lignans from Schizandra chinensis by HPLC. Kor. J. Pharmacogn. 2011:42(3): Lee Y, Lee HS, Jang SJ, Song JH. Effect of Water Extract of Schisandra Chinensis on Osteoclast Differentiation. Korean J. Oriental Physiology & Pathology 2010: 24(5): Lee YJ, Cho JY, Kim JH, Park WK, Kim DK, Rhyu MR. Extracts from Schizandra chinensis Fruit Activate Estrogen Receptors: A Possible Clue to Its Effects on Nitric Oxide-Mediated Vasorelaxation. Biol Pharm Bull. 2004:27(7): Picherit C, Coxam V, Bennetau-Pelissero C, Kati-Coulibaly S, Davicco MJ, Lebecque P, et al. Daidzein Is More Efficient than Genistein in Preventing Ovariectomy-Induced Bone Loss in Rats. J Nutr. 2000:130(7): So H, Ahn S, Song R, Kim H. Relationships among Obesity, Bone Mineral Density, and Cardiovascular Risks in Post-menopausal Women. Korean J Women Health Nurs. 2010: 16(3): Garnero P, Sornay-Rendu E, Claustrat B, Delmas PD. Biochemical Markers of Bone Turnover, Endogenous Hormones and the Risk of Fractures in Postmenopausal Women: The OFELY Study. J Bone Miner Res. 2000:15(8): 45

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Treatment and Role of Hormaonal Replaement Therapy

Treatment and Role of Hormaonal Replaement Therapy Treatment and Role of Hormone Replacement Therapy Chung-Ang University Hospital Chee Jeong Kim 7.2 5.3 6.4 5.6 7.3 5.5 1 1 2 3 4 10 11 7 34 49 122 123 1.0 - - 10. 2001 1998 1998 Improvement of postmenopausal