untitled

Journal of Korean Medicine Rehabilitation Vol. 27 No. 1, January 2017 pissn 1229-1854 eissn 2288-4114 https://doi.org/10.18325/jkmr.2017.27.1.19 Original Article 두충의건조법별지표성분및항염증활성비교 도호정 이진호 * 하인혁 * 정화진 * 이재웅 * 김민정 * 김은지 * 이인희 * 자생한방병원한방재활의학과, 자생의료재단척추관절연구소 * Comparison of Index Compound Content and Anti-inflammatory Activity of Eucommiae Cortex by Various Drying Methods Ho-Jeong Do, K.M.D., Jin-Ho Lee, K.M.D.*, In-Hyuk Ha, K.M.D.*, Hwa-Jin Chung, Ph.D.*, Jae-Woong Lee, M.S.*, Min-Jeong Kim, B.S.*, Eun-Jee Kim, M.S.*, In-Hee Lee, Ph.D.* Department of Korean Medicine Rehabilitation, Jaseng Korean Medicine Hospital, Jaseng Spine and Joint Research Institute, Jaseng Medical Foundation* RECEIVED December 20, 2016 ACCEPTED January 3, 2017 CORRESPONDING TO In-Hee Lee, Jaseng Spine and Joint Research Institute, Jaseng Medical Foundation, 858 Eonju-ro, Gangnam-gu, Seoul 06017, Korea TEL (02) 3218-2250 FAX (02) 3218-2244 E-mail ini3355@jaseng.co.kr Copyright 2017 The Society of Korean Medicine Rehabilitation Objectives Eucommiae cortex is one of the frequently used herbs for musculoskeletal disorders, with well-known anti-inflammatory effect. Meanwhile, powdered form of herbal drugs is more advantageous than decoction form, in that storing and taking is more convenient. Thus, the authors aimed to investigate whether the drying method affects the index compound level and anti-inflammatory effect of eucommiae cortex water extract. Methods Eucommiae cortex was extracted in distilled water at 100 o C for 3 hours, filtered, and then evaporated under vacuum. One half of the sample was freeze-dried at 80 o C. Another half of the sample was added with dextrin and then spray-dried at 180 o C. To assess the possible change in index compound content, pinoresinol diglucoside was selected as the index marker. The content level of the index compound in various extract sample was quantified through high performance liquid chromatography. In addition, inos assay in LPS-induced RAW 264.7 cells was adopted to determine the anti-inflammatory effect of various extract samples. Furthermore, MTT assay was performed to confirm that the result of inos assay was not due to cytotoxicity. Results There was no significance difference in index compound content between extract samples obtained through two different drying methods. Anti-inflammatory activity of extract samples were similar at the matching concentration, regardless of the drying methods. Extract samples did not show any significant cytotoxicity. Conclusions Extract samples of eucommiae cortex were obtained through freeze-drying and spray-drying. Neither change in index compound content nor difference in anti-inflammatory activity was observed between drying methods. (J Korean Med Rehabil 2017;27(1):19-25) Key words Eucommiae cortex, Pinoresinol diglucoside, Freeze-drying, Spray-drying, Anti-inflammatory 서론»»» 한약은한방치료수단중하나로변증, 약제의선택, 처방구성등의과정을통해처방된다. 통계청자료에따 르면탕약및한약제제를활용한한약치료가한방치료에서차지하는비율은 32.5% 로나타나한약이한방치료에서상당부분을차지하고있음을알수있다 1). 한편, 한약중탕제의경우제조과정의표준화및균일화가힘들다는 www.e-jkmr.org 19

도호정 이진호 하인혁 정화진 이재웅 김민정 김은지 이인희 점, 약효의재현성확보가어려운점, 보관및저장으로인한치료의질적저하가초래될우려가있다는점등의이유로문제가되고있다 2). 한약의제형은과학기술의발전과함께새로운제제및포장재료의도입, 품질및공정관리의발달, 의료의진보및사회의변화를통해종류의다양화, 효과의극대화, 그리고복용의편리성을높이는방향으로나아가고있다 3). 또한한방의료기관에내원한환자 217명을대상으로한한방제형에대한설문조사에서정제나캅셀제등복용이나휴대가편리한제형의선호도가 45.1% 로나타나복용이편리한한약제형에대한소비자들의요구가높음을알수있다 4). 복용이나소지가간편한한방제형인과립제나정제, 캅셀제등을사용하기위해서기존탕약처방에따라배합한약재를기존조제과정과동일한공정을거쳐여액 ( 濾液 ) 을추출한뒤이를건조시켜서만든엑스분말을원료로이용한다. 이렇게한약재를추출하여건조하여보관하는방법은오염을방지하는효과가있어한약을장기간보존하고자할때쓰인다. 두충 (Eucommiae Cortex) 은식물분류학상두충과 (Eucommiaceae) 에속하는낙엽교목인 Eucommia ulmoides Olive의껍질을건조시킨것이다. 한의학적관점으로그氣味는辛溫無毒하고神農本草經에서不老長壽, 輕身益氣의효과가있다고밝히고있고本草綱目에서는腎과肝에작용하여肝腎을윤택하고강하게하며허리, 무릎통증을치료하고正氣를보태고筋骨을튼튼하게하고잔뇨를없애며몸을가볍게하고노화를방지한다고하였다 5,6). 최근에는다양한연구를통해두충의약리적인효과에대해서도밝혀지고있다. 두충은혈압강하, 심장수축력억제, 혈관확장, 축동작용뿐만아니라이뇨, 이담작용, 혈당강하, 피로해소작용, 진통소염작용, 단백질합성및지질대사촉진작용이있고류마티스및신경통치료효과가있는것으로나타났고 7-11) 항돌연변이효과도있는것으로알려져있다 12). 이에본연구에서는한방에서널리사용되는두충을활용하여두충물추출물에대하여건조법을달리하였을때, 지표성분의변화가있는지확인하고항염증의지표가되는 inos assay를통하여생리활성의차이를비교하고자하였다. 재료및방법»»» 1. 실험재료본실험에서사용한두충은그린명품제약 ( 남양주, 한국 ) 자체적으로감별위원회를거쳐검증된것을제공받아사용하였다. 2. 기기및시약지표물질의정량분석실험에사용된 HPLC는 Shimadzu Co. (Kyoto, Japan) 의 LC-20AD, SIL-20AC, CBM-20A, SPD-20A, CTO-20AC 을사용하였으며, HPLC 용매인 Acetonitrile (ACN) 과증류수는 J.T.Baker Co. (Phillipsburg, USA) 의제품을사용하였다. 두충의지표물질인 Pinoresinol diglucoside 의순도는 98.00% 였으며 Sigma-Aldrich Co. (Saint Louis, USA) 에서구입하여사용하였다. inos assay 측정을위한 micro plate reader 는 Tecan Co. (Chapel Hill, USA) 의 Sunrise 모델을사용하였고, 세포배양에사용된 DMEM, fetal bovine serum (FBS), antibiotic-antimycotic은 Gibco Co. (Waltham, USA) 의제품을사용하였다. 3-(4,5-di-methylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) 시약과 Lipopolysaccharide (LPS) 는 Sigma-Aldrich Co. (Saint Louis, USA) 의제품을사용하였다. 3. 통계모든실험은 3회이상반복하였고실험값은평균 ± 표준오차로표시하였으며, 통계분석은 SPSS (Statistical Package for the Social Science, Ver. 12.0) program을이용하여 ANOVA test를실시한후, Duncan s multiple range test로분석하였다. 4. 두충의추출두충 200 g을 2 L의증류수로 100 o C에서 3시간동안추출한후 pore size 1 μm 필터로여과하였다. 여과후감압농축하여 15 brix로보정하고남은용액을반씩나누어절반은 80 o C의 deep freezer ( 일신바이오베이스, 한 20 J Korean Med Rehabil 2017;27(1):19-25

두충의건조법별지표성분및항염증활성비교 국 ) 로동결건조하였으며, 나머지절반은분무건조를위한 dextrin 을 50% 첨가한후 180 o C로분무건조하였다 (EYELA, Japan). 각각의방법으로건조후분말의무게를측정하여수율을계산하였다. 5. 지표성분함량분석두충의지표물질인 pinoresinol diglucoside 는대한약전의두충정량법으로분석하였다. C18 (4.6 250 mm, 5 μm, Agilent) 컬럼을사용하였고, 컬럼온도는 35 o C, 검출기는 UV 230 nm, 이동상으로는 0.1% formic acid의물 (A) 과 acetonitrile(b) 을사용하여 gradient profile 로최초 5% B로시작하여 8 15 min까지 20% B, 30 35 min까지 5% B로하였고유속은 1 ml/min 을사용하였다. 6. 산화질소 (NO) 의측정마우스의대식세포인 RAW 264.7 세포주는한국세포주은행 (No.40071, 서울, 한국 ) 에서구입하여사용하였다. 세포는 10% fetal bovine serum (Gibco, Waltham, USA) 과 1% antibiotic-antimycotic (Gibco, Waltham, USA) 이함유된 DMEM (Gibco, Waltham, USA) 을사용하였다. 37 o C와 5% CO 2 조건하에서배양하였다. RAW 264.7 세포가 LPS에의해활성화되면산화질소 (NO) 를생성하고생성된산화질소는세포배양배지에서 NO 2 의형태로존재한다. 그양을 griess 시약과의반응을통해측정하였다. RAW 264.7 세포를 96 well plate에 1 10 6 cell/ml의농도로분주하고 37 o C와 5% CO 2 incubator에서 24시간동안배양하였다. 1 μg/ml LPS로자극시킨후각각의건조된두충물추출분말을 (800, 400, 200, 100, 50 μg/ml) 로처리한후 24시간동안배양하였다. 상층액을옮겨담고 griess reagent (0.1%N-(1-naphthyl) ethylene diamide dihydrochloride, 과 1% sulfanilamide in 5% phosphoric acid) 을동량첨가한후 540 nm의 microplate reader 로흡광도를측정하였다. NO 2 의농도는 sodium nitrite 의표준곡선에대입하여계산하였다. 7. 세포생존율측정 Griess 활성시험후, 각 well에 MTT solution (final 500 g/ml) 을첨가하고 4시간동안 37 o C에서배양하였다. 배지제거하고 dimethyl sulfoxide (DMSO) 를첨가하여생성된 formazan 을용해시켰다. 570 nm에서흡광도를측정하였고 control group (LPS+) 과비교하여세포생존율 (%) 을측정하였다. 결과»»» 1. 지표성분함량분석동결건조와분무건조의건조법별로유효성분의변화를확인하기위하여두충의지표성분인 pinoresinol diglucoside의표준품을농도별로분석하여표준곡선을그린후각각의샘플을분석하여대응하는함량으로정량하였다. 표준곡선의 R 2 값은 1로매우정확한직선을형성하였다 (Fig. 1A, B). 이때의기울기와편차에대입하여계산한결과, pinoresinol diglucoside 의함량은동결건조분말 1 g에 27.51 mg, 분무건조분말 1 g에는 13.89 mg이었다 (Fig. 1C). 2. 산화질소의측정건조법에따른두충물추출물의생리활성을비교하기위하여 inos assay 를진행하였다. 분무건조의경우 50% 의 dextrin 을첨가하기때문에활성비교시두배의농도를사용하였다. 동결건조는 400, 200, 100, 50 μg/ml의농도로처리하였으며, 분무건조는 800, 400, 200, 100 μg/ml 의농도를처리하였다. NaNO 2 를이용하여본실험의표준곡선을그린결과 R 2 값은 0.9999 로유효한직선을형성하였다. 이때의기울기값과표준편차에대입하여계산한결과, 형성된산화질소의함량은 blank 로사용한 RAW 264.7 세포만배양한군은 0.177±0.138 μm, 대조군으로 blank 에 LPS를 1 μg/ml 처리한군은 38.104±0.918 μm, 두충물추출물동결건조분말을 400, 200, 100, 50 μg/ml 처리한군은각각 15.155±0.473, 23.356±0.200, 32.333± 0.936, 33.564±1.531 μm이었으며분무건조분말을 800, 400, 200, 100, 50 μg/ml 처리한군은각각 15.061± 0.200, 23.527±0.798, 32.826±0.988, 36.027±1.270 μm 의값을형성하였다 (Fig. 2A, B). www.e-jkmr.org 21

도호정 이진호 하인혁 정화진 이재웅 김민정 김은지 이인희 (A) (A) (B) (B) (C) Fig. 2. Quantification of anti-inflammatory effect of eucommiae cortex samples from different drying methods. (A) Standard curve of NaNO 2. (B) inos assay of eucommaie cortex samples from different drying methods; in order to compare the effect of equivalent mass of eucommiae cortex, the concentration of final sample was adjusted accordingly. *p<0.001, p<0.05, p>0.05 (=insignificant). Fig. 1. HPLC analysis of pinoresinol diglucoside amount in eucommiae cortex samples from different drying methods. (A) Chromatogram of pinoresinol diglucoside standard at various concentrations (500, 100, 50, 10, 5, 1 μg/ml). (B) Standard curve of pinoresinol diglucoside. (C) Chromatogram of eucommiae cortex samples from different drying methods; 10 μl of spray-dried samples and 5 μl of freeze-dried samples were injected to be analyzed. Fig. 3. Cytotoxicity assay of eucommiae cortex samples from different drying methods. Cell viability >85% was regarded as no significant cytotoxicity. 3. 세포생존율 산화질소를측정한실험에서사용된두충물추출건조분말의독성에의한것이아닌항염증효능임을입증하기 22 J Korean Med Rehabil 2017;27(1):19-25

두충의건조법별지표성분및항염증활성비교 위하여 MTT assay를진행하였다. 아무것도처리하지않은 blank를 100% 생존한것으로하였을때모든실험결과치는 85% 이상으로유의적인독성이없었음을확인하였다 (Fig. 3). 고찰»»» 최근한약에대한수요가지속적으로증가하고이에따라한약의효능과안전성, 안정성과관련된연구들이요구되고있는상황이다 13). 다양한물질들이액상형태로추출된탕약은한약의제형중가장많이사용되고있으나시간의흐름에따라그품질이변하고빛, 산소, 수분등여러요인에의해영향을받는다. 그중온도는탕약의품질변화에가장큰요인으로작용한다고알려져있다 14,15). 하지만모든탕약에이러한내용을일반화하기에는과학적근거가부족한상황이며효능이나안정성이어떤조건에서얼마의기간동안유지되는지에대해서는관련연구가부족한실정이므로한약제제의종류에따른보관기간및유통기한설정등다양한연구와그에따른평가방법을모색하는것이필요하다 16,17). 이에본연구에서는탕약에비해저장과복용이편리하다고알려진엑스분말에있어서제조공법중분무건조법과동결건조법을적용하였을때지표성분함량변화및생리활성변화가나타나는지를살펴보고자하였다. 추출물등을건조하는방법은열풍건조, 분무건조, 드럼건조, 동결건조등다양한방법이있다 18). 이중산업적으로는분무건조와동결건조가가장많이사용되어왔다. 분무건조는산업적건조법중가장오래된방법이며경제적이라는장점을가지고있다 19). 물추출된용액이분무시스템으로부터미립화되어면적이증대되면서약 180 o C 정도의열풍공기와의접촉으로빠르게증발되어미세캡슐을형성시키는분무건조는다른건조방식에비해용해성, 유동성이좋으며미립화에서분말제품에이르기까지하나의공정으로이루어지므로식품, 의약품등다양한응용이가능하다 20,21). 그러나공정에이용되는 180 o C 정도의고온과급속건조로인하여성분이변성될수있다는단점을가지고있다 20). 동결건조는영하의온도에서승화를통해건조가진행되기때문에성분의변성면에서가 장선호되는방법이다 22). 건조전특성을잘보존하며건조물의재수화율이높은반면 23), 공정에서에너지소모가높으며시간이오래걸리는단점을가지고있다 24,25). 이와같은장단점이있는두가지건조법을적용하여두충을추출하고건조하였을때, 건조법별지표성분및활성의차이를보기위하여본연구를진행하였다. 결과적으로두충은온도가올라가지않는동결건조와고온의건조과정이포함되는분무건조에서유의한차이를보이지않았다. 두충에함유되어있는유효성분으로는 geniposidic acid, chlorogenic acid, geniposide, pinoresinol diglucoside, liriodendrin, genipin 등이있다 26). 이중대한약전에등재되어있는 pinoresinol diglucoside 를지표물질로설정하였는데 pinoresinol diglucoside 는온도와시간이증가하면함량이감소한다는보고도있었지만 27), 반면반대의결과를보인보고도있었다 28,29). 본연구에서도고온의처리과정을보이는분무건조방식을사용하여도함량에유의한변화가없음을확인할수있었다. 항염증활성을통하여비교한생리활성면에서도 p-value 를 0.05로하였을때동결건조 50 μg/ml 과분무건조 100 μg/ml 사이에는유의한차이를보였으나, 그밖의농도에서는모두유의한차이를보이지않아활성면에서도유의한차이가없음을확인할수있었다. 두충의효과는실험적연구를통해서도많이밝혀진바있다. 두충이뼈의흡수및손실방지, 난소절제에따른배뇨량증가의방지효과가있고골신생에유의한효과가있을뿐만아니라 30,31), 골모세포증식의촉진, 파골세포증식의억제의효과가있고 32) 난소를적출한흰쥐를대상으로한실험에서두충의투여가골조직형태에유의한변화를초래한것으로나타났다 33). 또한콜라겐합성의촉진 34), 면역기능증가 35), 혈청지질함량감소 36), 뇌조직및혈장내 catecholamine 함량저하에대해서보고된바있고 28) 암세포의증식을저해하는것으로나타났다 37). 이외에두충은신경절단에의한근육위축시탈신경지배를통한근육무게와근섬유굵기의감소를억제하는것으로알려져있다 38). 향후연구에서는 pinoresinol diglucoside 이외의지표물질도다양한건조법에서효능과안정성이유지되는지확인할필요가있으며, 두충이외의다양한한약재를이용한연구가필요할것으로사료된다. www.e-jkmr.org 23

도호정 이진호 하인혁 정화진 이재웅 김민정 김은지 이인희 결론»»» 두충물추출물의동결건조와분무건조에서지표성분인 pinoresinol diglucoside 의함량에차이가없었으며항염증작용을통해살펴본생리활성에있어서도큰차이를보이지않았다. 동결건조나분모건조등의건조법을이용한한약제형의개발은한방은물론이거니와식품및제약업등다양한분야에서활용할수있을것이다. References»»» 1. Choi H, Choi J, Lee CH. The chart of herbal medicine in Korean. 1st ed. Paju:Jipmoondang. 2010:191-2. 2. Jeong YS, Moon BK, Nam HW, Park JS, Eum SY, Choe BM. The Survey on Using Alternative Medicine in General Population with Medical Problems : A Pilot Study. Korean J Psychosomatic Medicine. 1998;6(1):70-8. 3. Research institute of east oriental medicine. Introduction to east oriental medicine. Seoul:Rao-gang. 1994:333. 4. Choi HJ, Bang NY, Song BW, Kim NJ, Ryu BH. Survey on the preference for the dosage forms of oriental herbal medicine. J Kyung Hee Univ Med Cent. 2004;20(1): 356-67. 5. Ahn DG. Mysterious treatment of eucommia ulmoides, Paju:Openbooks, 1993. 6. Research institute of east oriental medicine. Herbology. Seoul:Ryo-gang, 1994. 7. Chien TH. Pharmacological action of Eucommia ulmoides. Japanese Journal of Pharmacology. 1957;6(2):122-37. 8. Kin KC, Ting KS. Studies on drugs for treatment of hypertension: II. Toxicity and experimental therapy of Eucommia ulmoides Oliv. Acta Physiologica Sinica. 1956;20:247-54. 9. Au C, Yang CP, Luang CJ, Leu HJ. The diuretic and cardiovascular effects of cortex eucommiae. Bull Taipei Med Coll. 1986. 10. Sih CJ, Ravikumar PR, Huang FC, Buckner C, Whitlock H. Isolation and synthesis of pinoresinol diglucoside a major antihypertensive principle of Tu-Chung(Eucommia ulmoides). Journal of the American Chemical Society. 1976;98(17):5412-5. 11. Xu SL, Zeng QZ, Huang WG, Yin Q. Effects of Duzhong on plasma camp and cgmp levels in mice. Chinese Traditional and Herbal Drugs. 1986;17:204. 12. Nakamura T, Nakazawa Y, Onizuka S. Antimutagenicity of Tochu tea(an aqueous of Eucomia ulmoides leaves): 1. The clastogen- suppressing effects of Tochu tea in CHO cell and mice. Mutation Research. 1997;388(1);7-20. 13. Man SC, Durairajan SSK, Kum WF, Lu JH, Huang JD, Cheng CF, Chung V, Xu M, Li M. Systematic review on the efficacy and safety of herbal medicines for Alzheimer s disease. Journal of Alzheimer s disease. 2008;14(2):209-23. 14. Park IH, Kim YH, Choi SH, Yu SN, Kim SH, Ahn SC, Cho SI, Lee I. Effect of Preservation Conditions on the Stability of Samul-tang Decoctions. Journal of Life Science. 2015;25(10):1124-31. 15. Yoon SJ, Lee SI. Study of Jagyakgamchotang s effect change by time. J of Herbology. 1991;6(1):29-34. 16. Jin SE, Kim OS, Seo CS, Shin HK, Jeong SJ. Comparative study on stability and efficacy of Banhasasim-tang decoction depending on the preservation temperature and period. Journal of Korean Medicine. 2016;37(1):21-33. 17. Jin SE, Kim OS, Shin HK, Jeong SJ. Comparative Study on Biological Activities of Gwakhyangjeonggi-san Decoction According to the Preservation Periods. Journal of Korean Medicine. 2014;35(3):60-9. 18. Tsami E, Krokida MK, Drouzas AE. Effect of drying method on the sorption chracteristics of medel fruit powder. Journal of food engineering. 1998;38(4):381-92. 19. Kailasapathy K. Microencapsulation of probiotic bacteria: technology and potential applications. Curr Issues Intest Microbiol. 2002;3(2):39-48. 20. Cho YH, Shin DS, Park JY. Microencapsulation technology in food industry. Food Science and Industry. 1997; 30:98-111. 21. Sano Y. Gas flow behaviour in spray dryer. Drying Technol. 1993;11(4):697-718. 22. Frank F. Freezing-drying of bioproducts; putting princicples into practice. European Journal of Pharmaceutics and Biopharmaceutics. 1997;45:221-9. 23. Jafar F, Farid M. Analysis of Heat and Mass transfer in freeze drying. Drying technology. 2003;21(2):249-63. 24. Hammami C, Rene F. Determination of freezing-drying process variables for strawberries. Journal of Food engineering. 1997;32(2)133-54. 25. Erbay Z, Icier F. Optimization of hot air drying of olive leaves using response surface methodology. Journal of food engineering. 2009;91(4):533-41. 26. Seo CS, Kim JH, Shin HK, Kim BS. Quantitative Determination of the Six Marker Compounds in Eucommiae Cortex by Processing Method. Korean Journal of Pharmacognosy. 2015;46(2):123-32. 27. Chen XY, Luo LL, Ren GC, Qu GY, Dong LS. Determination of pinoresinol diglucoside in processed Eucommia ulmoides and crude Eucommia ulmoides. West China Journal of Pharmaceutical Sciences. 2008; 23(5):592-3. 28. Park SD, Kim GW. Experimental studies of Eucommiae 24 J Korean Med Rehabil 2017;27(1):19-25

두충의건조법별지표성분및항염증활성비교 Cortex according to processing. The Journal of Dong Guk Oriental Medicine. 1992;1(1):81-107. 29. Kim HK. Studies for standardization of processed herbal medicines(i). Seoul:Ministry of Food and Drug Safety. 2003;31-56. 30. Oh HS, Kim HC, Lee SI, Ahn DK. Effects of Eucommiae Cortex and Folium on the Ovariectomized Rat as the Model of Postmenopaual Osteoporosis. Journal of Herbology. 1995;10(1):59-68. 31. Lee JA, Noh SH, Ahn DK, Choi HY. Effects of the Eucommiae Cortex and Chanenomelis Fructus on the Aged Ovariectomized Rat of Postmenopausal Osteoporosis. Journal of Herbology. 2001;16(1):201-6. 32. Ha HY, Ho JY, Shin SM, Kim HJ, Koo SJ, Kim IH, Kim CS. Effects of Eucommiae Cortex on osteoblast-like cell proliferation and osteoclast inhibition. Archives of Pharmacal Research. 2003;26(11):929-36. 33. Jang JB, Kim KJ, Choi GY, Cho JH, Park YK, Lee KS. The Effects of Eucommiae Cortex on Bone Histomorphologic Change in the Ovariectomized Rats. The Journal of oriental obstetrics and gynecology. 2003;16(4):1-14. 34. Li Y, Kamo S, Metori K, Koike K, Che QM, Takahashi S. The promoting effect of eucommiol from Eucommiae cortex on collagen synthesis. Biological and Pharmaceutical Bulletin. 2000;23(1):54-9. 35. Kim UJ, Kim SH. Effect of Eucommia Cortex and Eucommia Cortex Leaf on Immune Function. Journal of Herbology. 1994;9(1):99-113. 36. Lee CG, Lee BK. Effect of Eucommia Cortex and Eucommia Cortex Leaf on the Development of Experimental Hypercholesterolemia in the Rats. The Journal of East-West Medicines. 1987;12(2):17-31. 37. Kim JB, Park JR, Jeon JR, Cha MH. Isolation and Identification of Anticancer Compounds from Eucommia ulmoides Leaves. Journal of the Korean Society of Food Science and Nutrition. 2001;30(4):732-8. 38. Cho JH, Kim KS, Cha JD, Lee HS, Choi H, Jung HS, Sohn NW, Sohn YJ. Effect of Eucommiae Cortex on Hind Limb Muscle Atrophy of Sciatic Nerve Transectioned Rats. Korean J. Oriental Physiology & Pathology. 2008;22(6):1454-61. www.e-jkmr.org 25