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1 Korean J. Plant Res. 26(5): (2013) Print ISSN Online ISSN Data 한약재추출물의항산화및사구체혈관간세포증식억제활성탐색 손은화, 장선아 1, 우한구 1, 구현정 1, 한효상 2 *, 강세찬 1 * 강원대학교생약자원개발학과, 1 가천대학교생명과학과, 2 중부대학교보건행정학과 Screening of Traditional Medicines for Antioxidative and Anti-proliferative Effects on Rat Mesangial Cells Eun-Hwa Sohn, Seon-A Jang 1, Han Goo Woo 1, Hyun Jung Koo 1, Hyo-Sang Han 2 * and Se Chan Kang 1 * Department of Herbal Medicine Resources, Kangwon National University, Samcheok , Korea 1 Department of Life Science, Gachon University, Seongnam , Korea 2 Department of Health Administration, Joongbu University, Geumsan , Korea Abstract - In the present study, anti-oxidative and the RMC proliferation inhibitory propeties of 80% ethanol extracts from 63 kinds of traditional medicines were investigated. Inhibitory effects of RMC proliferation were showed that Dalbergia odorifera T. Chen., Melia azedarach Linné and Hydnocarpus anthelmintica Pierre. Among them Hydnocarpus anthelmintica Pierre had the highest anti-oxidative activity (ORAC PE value = 1.6, DPPH = 81.1), but Dalbergia odorifera T. Chen. and Melia azedarach Linné had no effects. These results suggest that the Hydnocarpus anthelmintica Pierre could prevent or protect from kidney disease as antioxidant and anti-proliferative agent for RMC. Key words - Oxygen radical absorbance capacity (ORAC), Hydnocarpus anthelmintica Pierre, Rat mesangial cell (RMC), Renal failure 서언 활성산소종 (reactive oxygen species, ROS) 은산화적스트레스에의해생성되며, superoxide anion, hydrogen peroxide, hydroxyl radical, hydroperoxyl radical, single oxygen 등이이에속한다. ROS 가과도하게생성되면체내세포와조직에산화적세포손상을일으켜노화를촉진시키고암, 동맥경화, 파킨슨병, 알츠하이머병, 2형당뇨병등의각종질병을유발한다고알려져있다 (Arai et al., 2007; Farooqui and Farooqui., 2011; Garcia-Bailo et al., 2011; Hong et al., 2011; Jomova et al., 2010; Kim et al., 2010; Matés, 2000; Park et al., 2007). 또한, 신장에서산화적스트레스로인해생성되는 ROS는만성신부전과같은신장질환및신장질환에서의합병증을야기시키고, 항암제로사용되는 cisplatin 에의한신장독성에도관련이 * 교신저자 ( ) : sckang73@gachon.ac.kr; hanhs@joongbu.ac.kr 있다고보고된바있다 (Feng et al., 2013; Hong et al., 2011; Modlinger et al., 2004; Rodrigo and Rivera, 2002; Sung et al., 2008). 한편, ROS 에대한방어기구로서사용되는항산화제는생체이물, 약물, 발암물질, 유해한라디칼반응의부작용에대해직접또는간접적으로세포를보호하는물질로서약용식물에포함된여러성분들은높은항산화활성을나타내며, 많은항산화제들이자유라디칼과 superoxide 를제거하거나해독및방어하는단백질을활성화시키는능력을지니고있다고알려져있다 (Halliwell, 1995; Kocic et al., 2002; Shin et al., 2012). 사구체혈관간세포 (rat mesangial cells) 의증식은신부전의특징중하나로서, 혈관간세포의증식과이에따른세포외기질의과도한축적은사구체간질의확장을야기시켜신부전증에서나타나는사구체경화에큰영향을미치는것으로알려져있다 (Catherwood et al., 2002). 활성산소에의한신부전증유발및사구체혈관간세포의증 c 본학회지의저작권은 ( 사 ) 한국자원식물학회지에 있으며, 이의무단전재나복제를금합니다.

2 한약재추출물의항산화및사구체혈관간세포증식억제활성탐색 식은급 만성신부전증을초래한다. 따라서, 본연구에서는 63 종의한약재추출물에서항산화활성및사구체혈관간세포의증식억제능을탐색함으로써신장질환의예방및치료제로서개발가능한한약재를선별하고자하였다. 재료및방법재료본실험에서는경동시장 ( 서울, 한국 ) 에서 63종의한약재를구입하였으며, 증거표본및추출물을가천대학교생명과학과천연물신약연구실에보관하였다 (voucher specimen No. GNMR1 ~ GNMR63). 추출및분획 63종의한약재를세절하고음건한후 80% EtOH 을이용하여추출교반기로 24시간동안추출및여과하였다. 모든시료추출물은감압농축하고동결건조하여 -80 에보관하였다. 이중대풍자 1.8 kg 에서얻은 MeOH 추출물 598 g을증류수 1 L에현탁하고 n-hexane(1 L 3), CH 2 Cl 2 (1 L 3), EtOAc(1 L 3) 및 n-buoh(1 L 3) 순으로 1 : 1 비율로용매분획하여각각 g, 9.0 g, 13.1 g, 17.2 g과 208 g의 n-hexane, CH 2 Cl 2, EtOAc, n-buoh 및 H2O 분획물을얻었다 (Fig. 1). 세포배양 Rat mesangial cells(rmc) 는 American Type Culture Collection(ATCC, USA) 로부터분양받았으며, 10% fetal bovine serume(fbs, Invitrogen), 100 U/ ml penicillin, 100 μg / ml streptomycin 이포함된 Dulbeccos Modified Eagles Medium(DMEM, Invitrogen) 을사용하였다. 세포는 37, 5% CO 2 에서배양하였으며, 세포의과밀도현상을해소하기위하여계대배양하였다. RMC proliferation assay 세포에 63종의추출물 (50 μg /ml) 을 24시간처리한후배지를제거하고 200 μl의 MTT(0.5 mg/ml in media) 시약을각 well 에첨가하여 37 에서 3시간배양하였다. 상층액을제거하고 100 μl의 dimethyl sulfoxide(dmso) 를각 well 에첨가하여비수용성의보라색 formazan 을용해시켜 540 nm 에서흡광도를측정하였다. ORAC(oxygen radical absorbance capacity) assay 각시료를 DMSO 에희석하여 96-well 에넣은후양성대조군으로 trolox(fluka) 를시료의농도와동일하게넣어주었다. 이후 75 mm PE buffer 와 β-phycoerythrin(sigma) 을섞어서넣어준후, AAPH(Sigma) 를첨가하여 fluorometer(victor 3, Perkin Elmer) 를이용하여형광의변화과정을 excitation 535 nm, emission 590 nm에서 60분동안 2분간격으로측정하였다. AUC 및 Value PE 의산출방법은다음과같다. AUC(Area under he curve) = 1 + f1/f0 + f2/f0 + f2/f0 + + f19/f0 + f20/f0 <Relative ORAC value> = [(AUC sample - AUC black )/(AUC Trolox - AUC blank )] (molarity of Trolox/molarity of sample) DPPH(1,1-diphenyl-2-picryl hydrazil) assay ORAC법과비교하기위하여각시료의항산화활성은 DPPH free radical 소거법에의한전자공여능 (EDA) 방법으로측정하였다. 시료 0.5 ml 에 DPPH(1,1-diphenyl-2-picryl hydrazil) 를 3 ml 를가하고, 실온에서 30분간반응시킨후 UV-visible spectrophotometer(phanrmaca Biotech Ultraspec 3000, England) 를이용하여 517 nm 에서측정하였다. 결과및고찰본연구에서는경동시장에서구입한 63종의한약재를세절하고음건하여 80% EtOH 에서 24시간동안추출및여과하고감압농축한뒤동결건조하여 -80 에서보관하면서실험에사용하기전에실온에서조제하여항산화활성및생쥐의사구체혈관간세포주인 RMC 세포의증식억제능을확인하였다. 그결과, 강진향, 고련피, 대풍자, 목향이 50 μg /ml 의농도에서 RMC 세포의증식을 50% 이상억제하는것으로나타났으며, ORAC 와 DPPH assay를통한항산화활성을확인한결과, 백단향, 백렴은양성대조군 trolox(vitamin E, ORAC value = 1) 보다 2배이상증가하여가장우수한 ORAC 활성효능을보였고, 계혈등, 귀전우, 반대해는 1.5배이상의 ORAC 활성효능을보였으나, 반대로갈화, 감국, 감송, 건율, 고련피, 곡기생, 곤포, 구자, 구절초, 귀판, 백반은양성대조군인 trolox(vitamin E, ORAC value =1) 보다못미치는 ORAC 활성을나타냈다. 이중계혈등, -653-

3 韓資植誌 Korean J. Plant Res. 26(5) : 652~657(2013) Table 1. Effects of RMC proliferation inhibitory and anti-oxidant from natural plant library Voucher Name Korean names Herb medicine names Scientific names RMC inhibition(%) ORAC PE value DPPH inhibition (ug/ml) 1 갈화 Puerariae Flos Pueraria lobata Ohwi NE 2 감국 Chrysanthemi Flos Chrysanthemum indicum Linné NE 3 감송향 Nardostachyos Rhizoma Nardostachys chinensis Batal NE 4 감수 Euphorbiae Kansui Radix Euphorbia kansui Liou ex Wang NE 5 강진향 Dalbergiae Odoriferae Lignum Dalbergia odorifera T. Chen NE 6 건율 Castaneae Semen Castanea crenata Siebold et Zuccarini NE 0.75 NE 7 건칠 Lacca Sinica Exsiccata Rhus verniciflua Stokes NE 1.17 NE 8 계내금 Galli Stomachichum Corium Gallus domesticus Brisson NE 1.08 NE 9 계지 Cinnamomi Ramulus Cinnamomum cassia Presl NE 10 계혈등 Spatholobi Caulis Spatholobus suberectus Dunn NE 고련피 Meliae Cortex Melia azedarach Linné NE 12 고본 Angelicae Tenuissimae Radix Ligusticum tenuissimum Kitagawa NE 1.04 NE 13 곡기생 Visci Herba Viscum album L. var. coloratum Ohwi NE 0.97 NE 14 곤포 Laminariae Japonicae Thallus Laminaria japonica Areschoung NE 15 골담초근 Caraganae Radix Caragana sinica (Buchoz) Rehder NE 1.08 NE 16 곽향 Agastachis Herba Agastache rugosa (Fischer et Meyer) O. Kuntze NE 1.07 NE 17 괴각 Sophorae Fructus Sophora japonica Linné NE 1.01 NE 18 구맥 Dianthi Herba Dianthus chinensis Linné NE 1.05 NE 19 구자 AlliTuberosi Semen Allium tuberosum Rottler NE 0.47 NE 20 구절초 Chrysanthemi Zawadskii Herba ChrysanthemumzawadskiiHerbichvar. latilobum(maxim.) Kitamura NE 0.85 NE 21 권백 Selaginellae Herba Selaginella tamariscina Spring NE 22 귀전우 EuonymiLignum Suberalatum Euonymus alatus Siebold 귀판 Testudinis Plastrum Chinemys reevesii Gray NE 24 금전초 Lysimachiae Herba Lysimachia christinae Hance NE 1.42 NE 25 나도근 Oryzae Radix Oryza sativa L. var. glutinosa Matsumura NE 26 낙석등 Trachelospermi Caulis Trachelospermum jasminoides (Lindl.) Lem NE 27 낭독 Euphorbiae Fischerianae Radix Euphorbia fischeriana Steudel NE 1.43 NE 28 노감석 - Calamina NE 1.33 NE 29 노근 Phragmitis Rhizoma Phragmites communis Trinius NE 1.38 NE 30 노로통 Liquidambaris Fructus Liquidambar formosana Hance NE 1.35 NE 31 노봉방 Vespae Nidus Polistes mandarinus Saussure NE 1.36 NE 32 노회 Aloe Aloe barbadensis Linné, NE 1.35 NE 33 누로 Echinopsis Radix Echinopssetifer Linné NE 1.39 NE 34 능소화 Campsitis Flos Campsis grandiflora Schumann NE 1.07 NE 35 대극 Euphorbiae Pekinensis Radix Euphorbia pekinensis Ruprecht NE 1.15 NE 36 대청엽 Isatidis Folium Isatis indigotica Fort. NE 1.40 NE 37 대풍자 Hydnocarpi Semen Hydnocarpus anthelmintica Pierre NE, No effect

4 한약재추출물의항산화및사구체혈관간세포증식억제활성탐색 Table 1. Continued Voucher Name Korean names Herb medicine names Scientific names RMC inhibition(%) ORAC PE value DPPH inhibition (ug/ml) 38 동충하초 Cordyceps Cordyceps sinensis Sacc NE 1.41 NE 39 두엽 ( 두충엽 ) Eucommlae Folium Eucommia ulmoides Oliv. NE 1.23 NE 40 마치현 Portulacae Herba Portulaca oleracea Linné NE 1.37 NE 41 마편초 Verbenae Herba Verbena officinalis Linné NE 1.14 NE 42 마황근 Ephedrae Radix Ephedra sinica Stapf NE 43 망초 Natrii Sulfas Glauber's salt NE 1.33 NE 44 맥아 HordeiFructus Germinatus Hordeum vulgare Linné NE 1.33 NE 45 목별자 Momordicae Semen Momordica cochinchinensis Sprenger NE 1.09 NE 46 목적 Equiseti Herba Equisetum hyemale Linné NE 1.43 NE 47 목향 Aucklandiae Radix Aucklandia lappa Decne NE 48 밀몽화 Buddlejae Flos Buddleja officinalis Maximowicz NE 49 반대해 Sterculiae Scaphigerae Semen Sterculia scaphigera Wall. NE 반지련 Scutellariae Barbatae Herba Scutellaria barbata D. Don NE 51 방기 Sinomenium stem and Rhizome Sinomenium acutum Rehder et Wilson NE 1.29 NE 52 백강잠 Batryticatus Bombyx Bombyx mori (Linné) NE 53 백계화 Althaea Flower NE 54 백굴채 Chelidonii Herba Chelidonium majus Linné NE 55 백급 Bletillae Rhizoma Bletilla striata (Thunberg) Reichenbach fil NE 56 백단향 Santali albi Lignum Santalum album Linné NE 백두옹 Pulsatillae Radix Pulsatilla koreana Nakai NE 58 백렴 Ampelopsis Radix Ampelopsis japonica Makino NE 백미 Cynanchi Radix Cynanchum atratum Bunge NE 1.29 NE 60 백반 Alum Potassium aluminium sulfate NE 0.39 NE 61 시엽 Persimmon Diospyros kaki NE 62 애엽 Artemisiae Arhyi Folium Artemisiaprinceps Pamp. var. orientlishara NE 63 육계 Cinnamon Bark Cinnamomum cassia Presl NE 1.24 NE 귀전우, 대풍자, 반대해, 백단향, 백렴은 DPPH 라디칼소거활성또한높은것으로나타났다 (Table 1). 특히, 대풍자추출물과목향추출물은항산화활성과사구체혈관간세포증식억제능모두뛰어난효능을나타냈다. 대풍자는산유자나무과에속하는대풍수의씨앗으로서, 항산화및항균, 항염증효능이보고된바있으며주성분은 Hydnocarpic acid, Chaulmoogric acid, Gorlic acid로알려져있다 (Lee et al., 2012a). 대풍자유는전통적으로한센병을치료하는데사용되어져왔으며, 여러피부질환을치료하는한약재로사용되어져왔으나현재까지대풍자의항산화활성및신부전증억제효능에대해보고된바가없다 (Kibbi et al., 1998; Oommen et al., 1999; Oommen, 2000; Lee et al., 2012b). 목향은국화과에속하는다년생식물의뿌리로서, sesquiterpene 및 sesquiterpene lactone 계화합물을함유하고있으며, 주성분은 esequiterpene lactone 계화합물들인 costunolide 와 dehdrocostus lactone으로알려져있고, 항균작용, 항염증작용및혈관생성억제효능등이알려져있다 (Park et al., 1996; Kang et al., 1999; Wedge et al., 2000; Jeong et al., 2002). 그러나목향이함유하고있는 aristolochic acid는임상적으로신장에독성을일으킴으로신장질환치료제에서제외되는한약재로알려져있다 (Shum et al., 2007). 본연구결과목향의사구체혈관간세포증식억제능은세포독성에의한결과인것으로 -655-

5 韓資植誌 Korean J. Plant Res. 26(5) : 652~657(2013) 위한한약재후보물질로서분획별항산화활성과유효성분규명의연구가요구된다. 인용문헌 Fig. 1. Fractionation scheme and their inhibitory effects for RMC proliferation. NE, no effect. 사료된다. 따라서, 본연구에서는항산화활성이우수하면서 RMC 세포증식억제능이가장우수한대풍자로부터신부전증예방및치료효능에대한기초연구를위하여용매분획을통한항산화활성및 RMC 세포에대한증식억제능을확인하고자하였으며, 그결과대풍자의 n-hexane 분획에서가장높은항산화활성과함께 RMC 세포증식억제능을나타내었다 (Fig. 1). 향후실험에서는가장우수한효능을보인대풍자추출물의분획별항산화활성과유효성분이규명되어야할것이며, 신장질환예방및치료의관점에서보았을때대풍자추출물의연구개발가능성을제시하는바이다. 적요 국내유통되는한약재를이용한신장질환의예방및치료제개발을위한기초자료를제시하고자경동시장에서구입한 63 종의한약재추출물을이용하여항산화활성및사구체혈관간세포의증식억제능탐색활성을확인하였다. 그결과강진향, 고련피, 대풍자, 목향이 RMC 세포의증식을 50% 이상억제하는것으로나타났고, ORAC 와 DPPH assay를통한항산화활성을확인한결과백단향, 백렴이가장우수한 ORAC 활성효능을보였으며, DPPH 라디칼소거활성에서는계혈등, 귀전우, 대풍자, 반대해, 백단향, 백렴이우수한효능을나타냈다. 이중대풍자추출물과목향추출물은항산화활성과사구체혈관간세포증식억제능모두뛰어난효능을나타냈으나, 목향이함유하고있는 aristolochic acid는임상적으로신장에독성을일으켜신장질환치료제에서제외되는한약재로알려져있다. 따라서, 가장뛰어난효능을보인대풍자추출물은신장질환치료및예방을 Arai, M., Y. Shibata, K. Pugdee, Y. Abiko and Y. Ogata Effects of reactive oxygen species (ROS) on antioxidant system and osteoblastic differentiation in MC3T3-E1 cells. IUBMB Life 59: Catherwood, M.A., L.A. Powell, P. Anderson, D. McMaster, P.C. Sharpe and E.R. Trimble Glucose-induced oxidative stress in mesangial cells. Kidney Int. 61: Farooqui, T. and A.A. Farooqui Lipid-mediated oxidative stress and inflammation in the pathogenesis of Parkinson's disease. Parkinson Dis. 2011:1-9. Feng, Y., Y. Liu, L. Wang, X. Cai, D. Wang, K. Wu, H. Chen, J. Li and W. Lei Sustained oxidative stress causes late acute renal failure via duplex regulation on p38 MAPK and Akt phosphorylation in severely burned rats. PLoS One 8:1-11. Garcia-Bailo, B., A. El-Sohemy, P.S. Haddad, P. Arora, F. Benzaied, M. Karmali and A. Badawi Vitamins D, C, and E in the prevention of type 2 diabetes mellitus: modulation of inflammation and oxidative stress. Biologics 5:7-19. Halliwell, B Antioxidant characterization. Methodology and mechanism. Biochem. Pharmacol. 49: Hong, C.O., S.T. Hong, Y.C. Koo, S.Y. Yang, J.Y. Lee, Y.H. Lee, Y.M. Ha and K.W. Lee Protective effect of Plantago asiatica L. extract against ferric nitrilotriacetate (Fe-NTA) induced renal oxidative stress in wistar rats. J. Fd Hyg. Safety 26: Jeong, S.J., T. Itokawa, M. Shibuya, M. Kuwano, M. Ono, R. Higuchi and T. Miyamoto Costunolide, a sesquiterpene lactone from Saussurea lappa, inhibits the VEGFR KDR/Flk-1 signaling pathway. Cancer Lett. 187: Jomova, K., D. Vondrakova, M. Lawson and M. Valko Metals, oxidative stress and neurodegenerative disorders. Cell. Biochem. 345: Kang, S.S., J.S. Kim, H.J. Chi, S.Y. Chang and K.W. Ha Isolation and quantitative determination of costunolide from saussurea root. Korean J. Pharmacogn. 30:48-53 (in Korean). Kibbi, A.G. and Z. Tannous Skin diseases caused by heat and cold. Clin. Dermatol. 16:

6 한약재추출물의항산화및사구체혈관간세포증식억제활성탐색 Kim, H.H., G.H. Park, K.S. Park, J.Y. Lee and B.J. An Anti-oxidant and anti-inflammation activity of fractions from aster glehni Fr. Schm. Korean J. Microbiol. Biotechnol. 38: (in Korean). Kocic, G., V. Djordjevic, P. Vlahovic, R. Kocic, D. Pavlovic and T. Jevtovic Antioxidants modulate adenosine metabolism in rat mesangial cells cultured under high glucose conditions. Ren. Fail. 24: Lee, G.S., D. Yim, J.H. Cheong and T.J. Kang. 2012a. The n-hexane, ethylacetate, and butanol fractions from Hydnocarpi Semen enhanced wound healing in a mice ulcer model. Immunopharmacol Immunotoxicol. 34: Lee, G.S., H. Shim, K.M. Lee, S.H. Kim, D. Yim, J.H. Cheong and T.J. Kang. 2012b. The role of the ethylacetate fraction from hydnocarpi semen in acute inflammation in vitro model. Immune Netw. 12: Matés, J.M Effects of antioxidant enzymes in the molecular control of reactive oxygen species toxicology. Toxicology 153: Modlinger P.S., C.S. Wilcox and S. Aslam Nitric oxide, oxidative stress, and progression of chronic renal failure. Semin. Nephrol. 24: Oommen, S.T., M. Rao and C.V. Raju Effect of oil of hydnocarpus on wound healing. Int. J. Lepr. Other Mycobact. Dis. 67: Oommen, S.T The effect of oil of hydnocarpus on excision wounds. Int. J. Lepr. Other Mycobact. Dis. 68: Park, H.J., W.T. Jung, P. Basnet, S. Kadota and T. Namba Syringin 4-O-beta-glucoside, a new phenylpropanoid glycoside, and costunolide, a nitric oxide synthase inhibitor, from the stem bark of Magnolia sieboldii. J. Nat. Prod. 59: Park, Y.K., S.H. Choi, S.H. Kim, J.K. Han and H.G. Chung Changes in antioxidant activity, total phenolics and vitamin C content during fruit ripening in Rubus occidentalis. Korean J. Plant Res. 20: Rodrigo, R. and G. Rivera Renal damage mediated by oxidative stress: a hypothesis of protective effects of red wine. Free Radic. Biol. Med. 33: Shin, S.W., J.H. Lee and K.S. Bang Antioxidant and antimicrobial activities of Xanthium sibiricum. Korean J. Plant Res. 25: (in Korean). Shum, K.C., F. Chen, S.L. Li, J. Wang, P.P. But and P.C. Shaw Authentication of radix aucklandiae and its substitutes by GC-MS and hierarchical clustering analysis. J. Sep. Sci. 30: Sung, M.J., D.H. Kim, Y.J. Jung, K.P. Kang, A.S. Lee, S. Lee, W. Kim, M. Davaatseren, J.T. Hwang, H.J. Kim, M.S. Kim, D.Y. Kwon and S.K. Park Genistein protects the kidney from cisplatin-induced injury. Kidney Int. 74: Wedge, D.E., J.C. Galindo and F.A. Macías Fungicidal activity of natural and synthetic sesquiterpene lactone analogs. Phytochemistry 53: (Received 14 August 2013 ; Revised 8 October 2013 ; Accepted 10 October 2013) -657-

Journal of Life Science 2011, Vol. 21. No μ μ

Journal of Life Science 2011 Vol. 21. No. 8. 1120~1126 ISSN : 1225-9918 DOI : http://dx.doi.org/10.5352/jls.2011.21.8.1120 μ μ μ α β Journal of Life Science 2011, Vol. 21. No. 8 1121 μ μ 1122 생명과학회지 2011,