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1 Microbiol. Biotechnol. Lett. (2015), 43(3), pissn X eissn Microbiology and Biotechnology Letters Cotoneaster horizontalis Decne 추출물의항산화및항염증활성 이지영 1, 진경숙 1, 권현주 1,2, 김병우 1,2 * 1 동의대학교블루바이오소재개발및실용화지원센터 2 동의대학교생명응용학과 Received: July 31, 2015 / Revised: September 2, 2015 / Accepted: September 2, 2015 Anti-Oxidative and Anti-Inflammatory Activities of Cotoneaster horizontalis Decne Extract Ji Young Lee 1, Kyong-Suk Jin 1, Hyun Ju Kwon 1,2, and Byung Woo Kim 1,2* 1 Blue-Bio Industry Regional Innovation Center, 2 Department of Life Science and Biotechnology, College of Natural Science & Human Ecology, Dong-Eui University, Busan 47340, Republic of Korea Anti-oxidative and anti-inflammatory activities of Cotoneaster horizontalis Decne ethanol extract (CHEE) were evaluated. CHEE possessed a potent scavenging activity against 1,1-diphenyl-2-picryl hydrazyl, which was similar to the activity of ascorbic acid which was used as a positive control. CHEE also effectively suppressed hydrogen peroxide-induced reactive oxygen species on RAW cells. Furthermore, CHEE induced the expression of the anti-oxidative enzyme heme oxygenase 1, and its upstream transcription factor, nuclear factor-e2-related factor 2. CHEE inhibited LPS induced nitric oxide (NO) formation as a consequence of inducible NO synthase (inos) down regulation. Taken together, these results provide us with an important new insight; that C. horizontalis possesses anti-oxidative and anti-inflammatory activities. Therefore, C. horizontalis may be utilized as a promising material in the field of nutraceuticals. Keywords: Cotoneaster horizontalis, anti-oxidative, anti-inflammatory 생명체는산화촉진물질과산화억제물질이균형을이뤄생체내항상성을유지하고있으나, 다양한외부요인들로인해이러한균형상태를잃고산화가촉진되는방향으로기울게되면체내에산화적스트레스 (oxidative stress) 가유발되어세포손상및질병을일으키게된다. 이러한산화적스트레스의직접적원인이되는활성산소 (reactive oxygen species, 이하 ROS) 는화학적으로불안정하고반응성이높아 DNA, 단백질, 지질, 탄수화물과같은생체내고분자물질과쉽게반응할수있으며, 세포와조직에비가역적인손상을일으켜돌연변이, 세포독성, 암등을유발하게된다 [1, 5, 11, 21, 22]. 대표적인 cellular defensive phase 2 detoxifying antioxidant enzyme으로알려진 heme oxygenase (HO)-1의유도는산화적스트레스를방어하는중요한기전중하나로다양한 carcinogen으로부터세포를보호하는 chemoprevention에중요한역할을담당하는것으로알려져있으며, 특히천연에서 *Corresponding author Tel: , Fax: bwkim@deu.ac.kr 2015, The Korean Society for Microbiology and Biotechnology 유래한다양한 dietary phytochemical은 nuclear factor E2- related factor 2 (Nrf2) 에의해조절되는 phase 2 detoxifying antioxidant enzyme의발현증가를통해 chemopreventive function을나타낸다 [4, 31]. 이러한 chemoprevention은항산화활성을기초로하여암뿐만아니라염증, 뇌및심혈관계질환, 노화등의예방및치료기전과도상호작용하는것으로알려져있어그중요성이커지고있다 [2, 12, 25, 29]. 염증은외부자극에대한생체조직의주요방어기전이나지속적인염증반응은조직의손상을초래하여암을비롯한각종질병을유발한다 [3, 6]. 생체내염증반응은대식세포 (macrophage) 에서과량생산되는염증매개인자 (inflammatory mediators) 로부터유래되는데 inducible nitric oxide synthase (inos) 로부터생산되는 nitric oxide (NO) 와 cyclooxygenase 2 (COX-2) 로부터생산되는 prostaglandin E2 (PGE2) 등이대표적이다. 외부자극에의해과량생산된염증매개인자는 tumor necrosis factor α, interleukin 1β 등과같은사이토카인을생산하여염증반응을일으킨다 [12, 13]. 염증반응의대표적인세포실험계중하나인 RAW murine macrophage에 lipopolysaccharide (LPS) 와같은염증유발인자를처리하면 inos 및 COX-2의발현유도에의해 NO September 2015 Vol. 43 No. 3

2 281 Lee et al. 와 PGE2 등염증매개인자의생성및이를통한사이토카인분비량증가를확인할수있다 [17, 19, 26]. 그러므로이러한염증매개인자의생성을효과적으로제어할수있는물질들이염증의예방및치료를위한소재로서각광받고있으며이에따라최근많은연구들이항산화및항염증활성을바탕으로한생리활성보유신소재개발및그활성기전의규명에주력하고있다. 특히천연유래소재로부터유용성분을추출하고생리활성을규명하여기능성소재로서의가능성을타진하는연구가활발히이루어지고있다 [14, 15, 23]. Cotoneaster horizontalis Decne는장미과에속하는반상록또는낙엽성관목으로홍자단이라고도불린다. 아시아, 북아메리카, 유럽등의온대지역에약 50여종이분포하며, 보통 1 m 내외로자라는데, 가지는낮은포복으로덩굴모양으로늘어지며, 윤기있는잎이달린다. 5 6월에연분홍의꽃을피우며, 9 10월에열매를맺는다. C. horizontalis의현재까지보고된생리활성으로는 Mohamed 등이이집트에서재배된 C. horizontalis 지상부의주요성분과점액질의항당뇨및항이상지질혈증효과를보고하였고 [18], Sokkar 등이이집트에서수집한 C. horizontalis 가지에탄올추출물의항산화, 항암및간보호효과를보고하였으며 [24], Khan 등은 C. horizontalis 전초의메탄올추출물의분획으로부터분리한콜린에스터라아제억제제에대해보고한바있다 [10]. 현재까지 C. horizontalis의규명된생리활성은상기의연구결과뿐이며그구체적인작용기작에대한연구, 특히항산화및항염증효과에대한연구는매우미흡하다. 이에본연구에서는천연에서유래한생리활성보유신소재개발의일환으로 C. horizontalis 95% 에탄올추출물 (CHEE) 이보유한항산화및항염증활성과그작용기작을분석함으로써기능성소재로서의활용가능성을확인해보고자하였다. 본연구에사용한시료는한국생명공학연구원, 해외생물소재허브센터에서구입 ( 분양번호 FBM ) 하여사용하였다. 건조및분쇄한시료를 95% 에탄올을용매로하여 45 o C에서 3일간초음파추출을수행하였다. 추출이끝난시료를 filter로여과하여고형물을없애고감압농축 (N-1000SW, EYELA, Japan) 및동결건조 (FDU2100, EYELA, Japan) 하여사용전까지 4 o C에보관하였다. 항산화작용의주요기전중하나인전자공여능은인체내에서생성되는 free radical의전자를공여함으로써 free radical에의한노화와질병을억제한다. 항산화작용의주요지표로서의전자공여능은특히천연물이보유한항산화능의측정에많이사용되고있으며 [5], 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical 소거능분석을이용하여측정하였다. DPPH는비교적안정한 free radical로써, ascorbic acid, tocopherol, polyhydroxy 방향족화합물, 방향족아민류에의해환원되어짙은자색이탈색되는원리를이용하여항산화 활성을간단히측정할수있는동시에식물체의항산화활성과도연관성이매우높기때문에많이이용되고있는방법이다 [9]. DPPH radical scavenging activity 측정을위해 CHEE를농도별 ( μg/ml) 로메탄올에녹여준비하고 96 well plate에메탄올에용해된 M DPPH 40 μl와각시료 160 μl를분주한혼합액을실온에서 30분간반응시킨후, multi-plate reader (Paradigm, Beckman, CA, USA) 를이용하여 520 nm에서흡광도를측정하였다. 시료를첨가하지않은음성대조군의흡광도와비교하여 free radical 소거정도를백분율로나타내고, 50% 저해농도 (Inhibitory Concentration, IC 50 ) 를계산하였다. 대표적인항산화제로 DPPH radical 소거활성측정시양성대조군으로주로사용되는 ascorbic acid를함께비교분석하였다. 항산화및항염증활성의세포실험은 murine macrophage cell line인 RAW 264.7을 American Type Culture Collection (ATCC, VA, USA) 로부터구입하여 10% fetal bovine serum (FBS, Invitrogen Corporation, CA, USA) 및 penicillin/ streptomycin (Invitrogen) 이포함된 DMEM 배지 (Invitrogen) 에서배양하여수행하였다 [20]. 활성분석수행전먼저시료가세포생존율에미치는영향을확인함과동시에세포독성을유발하지않는시료의처리농도를결정하기위해 CHEE 에의한세포독성유발유무를 WST assay를통해수행하였다 cell을 24-well tissue culture plate에분주하여 24시간동안부착시키고, EPEE 처리 24시간후 WST 시약 (Daeil Lab Service, South Korea) 이든배지로교체하여한시간동안반응시킨다음 multi-plate reader를이용하여 450 nm에서흡광도를측정하였다. ROS는과량생산시 DNA, 단백질, 지질을포함한생체내분자에산화적인변형을유발하여다양한질병의원인이되므로 ROS 소거능은항산화능의중요한지표로활용된다 [16]. Hydrogen peroxide (H 2 O 2 ) 는대표적인 ROS 중하나로항산화능분석을위한많은연구에서산화적스트레스유발제로사용되고있다 [27, 28, 30]. 본연구에서는 CHEE가보유한항산화능을 H 2 O 2 로유도한 ROS 생성에미치는영향을통해분석하였다. 이를위해 RAW cell에 cell permeable fluorescent dye인 50 μm의 dichlorofluorescin diacetate (DCFH-DA, Sigma-aldrich, MO, USA) 를 2시간동안전처리한후제거하고 500 μm의 H 2 O 2 를농도별시료와함께처리한후시료에의한 ROS 생성억제능을 multiplate reader 를이용한 fluorescence 측정을통해분석하였다. CHEE의항산화활성기전을알아보기위해대표적인항산화효소인 HO-1과그전사인자인 Nrf2의시료처리에의한단백질발현변화를 Western blot hybridization으로분석하였다. HO-1의일차항체는 Cell Signaling Technology (MA, USA) 로부터구입하였고, Nrf2와 Actin의일차항체와

3 Biological Activities of C. horizontalis 282 anti-goat와 anti-rabbit 등의이차항체는 Santa Cruz Biotechnology (CA, USA) 에서구입하여사용하였다. 시료처리가끝난배양세포에서단백질을추출하여 Bradford assay로단백질농도를결정한후 50 μg의단백질을 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 로전기영동하고 nitrocellulose membrane에 blotting한후 1:1,000 5,000으로희석한대상단백질의일차항체와 hybridization하였다. Membrane washing 후 horse radish peroxidase (HRP) 가부착된이차항체 (1:1,000) 로한시간동안반응시키고 chemiluminescence detection system (FluoChem FC2, AlphaInnotech, USA) 을이용하여단백질발현을분석하였다. 대표적인 free radical 중하나인 NO는생체내에서중요한세포신호전달물질로서작용하나과잉생산시산화적스트레스의유발을통해염증및세포손상의원인이된다 [8]. 이러한 NO 생성억제능의분석은 Park 등 [20] 의방법을변형하여수행하였다. RAW cell을 24-well tissue culture plate에 well 당 cell을 seeding하여부착시킨후 1 μg/ml의 LPS를처리하여 NO 생성을유도하고시료에의한 NO 생성저해능을 Griess reaction을통해분석하였다. 96-well plate에세포배양액상층 100 μl를분주하고 50 μl의 sulphanilamide (1% in 5% phosphoric acid) 와 50 μl의 naphthynaphthylene dihydrochloride (0.1%) 를섞은후실온에서 10분간반응시켰다. Multiplate reader를사용하여 550 nm에서흡광도를측정하고 sodium nitrite (NaNO 2 ) 를단계희석한표준액으로표준정량선을그린후 NO 생성량을계산하였다. 실험에사용한시약은모두 Sigma 에서구입하여사용하였다. 또한 CHEE 가보유한 NO 생성억제능의기전을밝히기위해 NO 생성의핵심단백질인 inos 의단백질발현을분석하였다. Western blot hybridization 을위한 inos 의일차항체는 Cell Signaling Technology (MA, USA) 로부터구입하여사용하였으며분석과정은상기 HO-1 및 Nrf2 의경우와같다. 모든실험결과는최소 3 회이상의반복실험을통하여얻은데이터를평균 (mean) ± 표준편차 (standard deviation, SD) 로나타내었고, 필요시대표적인그림이나데이터를제시하였다. 각데이터의통계분석은 SPSS 20.0 software 를이용한 unpaired Student s t-test 를통해 p 값이 0.05 미만 (p < 0.05) 인경우유의성이있는것으로판단하였다. 이상의실험을통한 CHEE 의항산화능및항염증생리활성의분석결과는다음과같다. 먼저 CHEE 의항산화능보유유무및그정도를알아보기위해항산화능의주요지표중하나인 DPPH radical scavenging activity 를분석한결과 CHEE 의농도증가에따른강한 radical 소거능을보여 , 0.512, 2.56, 12.8 μg/ml 의시료처리에의해 DPPH Table 1. DPPH radical scavenging activity of CHEE. Reagent Concentration (µg/ml) CHEE Ascorbic acid (Positive control) Inhibition rate (%) ± ± ± ± ± ± ± 0.16 radical 소거능이각각 26.82, 34.25, 66.21, 96.99% 로나타나 50% 소거농도를나타내는 IC 50 값이 1.52 μg/ml 로양성대조군으로사용한 ascrobic acid, 즉 vitamin C 의 IC 50 값인 1.53 μg/ml 와유사한정도의높은활성을보여매우강한항산화능을보유함을확인하였다 (Table 1). 이는앞서 Sokkar 등이보고한결과에서와유사하게농도의존적인활성의증가를보였으나더낮은농도에서더높은활성을나타내었다 [24]. 이와같이 DPPH radical scavenging activity 분석을통해 CHEE 가보유한높은항산화능이확인됨에따라그작용기전을좀더자세히알아보기위해 CHEE 가보유한항산화능의정도및기전을세포수준에서확인하고자하였다. CHEE 의세포수준에서의생리활성을분석하기전먼저시료가세포생존율에미치는영향을 RAW cell 에서분석한결과 μg/ml 의 CHEE 처리에의해세포독성이일어나지않음을확인하였다 (Fig. 1A). 또한 RAW cell 에대표적인산화적스트레스유도인자인 H 2 O 2 를처리하여 CHEE 에의한 ROS scavenging activity 를분석한결과 H 2 O 2 단독처리에의해유도되는과량의 ROS 생성이 CHEE 의병용처리에의해효과적으로저해되는것으로나타나 CHEE 가 DPPH radical 뿐만아니라세포실험계에서 H 2 O 2 에의해유도된산화적스트레스또한효과적으로감소시킴을확인하였다 (Fig. 1B). 강한항산화능을보유한천연유래소재들이 Nrf2 에의한항산화효소계의발현유도를통해활성을나타낸다는것이여러연구를통해밝혀짐에따라 CHEE 가보유한항산화능의작용기작을알아보고자하였다 [7, 25]. 이를위해대표적인항산화효소로천연물에의한항산화활성에의해주로발현이유도되는 HO-1 과그상위전사인자인 Nrf2 의단백질발현에 CHEE 가미치는영향을분석한결과 6 시간동안 μg/ml 의시료처리에의해 HO-1 과 Nrf2 의단백질발현이증가되는것으로나타났다 (Fig. 1C). CHEE 가항산화능뿐만아니라항염증활성또한보유하는지를알아보기위해 NO 생성에미치는효과를알아보았 September 2015 Vol. 43 No. 3

283 Lee et al. Fig. 2. Modulation of CHEE on LPS-induced NO formation (A) and inos protein expression (B) in RAW 264.7 cells. (A) Values are represented as the mean ± SD (n = 3).

4 283 Lee et al. Fig. 2. Modulation of CHEE on LPS-induced NO formation (A) and inos protein expression (B) in RAW cells. (A) Values are represented as the mean ± SD (n = 3). *, # Significantly different from the vehicle control [0, LPS ( )] and LPS-induced control [0, LPS (+)], respectively (p < 0.05). (B) Actin was used as an internal control. Fig. 1. Effect of CHEE on cell viability (A), H 2 O 2 -induced ROS scavenging activity (B), modulation of an anti-oxidative enzyme, HO-1 and its upstream transcription factor, Nrf2 protein expression (C) in RAW cells. (A, B) Values are represented as the mean ± SD (n = 3). (B) *, # Significantly different from the vehicle control [0, H 2 O 2 ( )] and H 2 O 2 - induced control [0, H 2 O 2 (+)], respectively (p < 0.05). (C) Actin was used as an internal control. 다. LPS 로자극을유도한쥐대식세포주 RAW cell 에서농도별 CHEE 의처리에따른 NO 생성양의변화를분석한결과 μg/ml 의시료처리에의해농도의존적인 NO 생성저해능을보였으며이는 NO 생성단백질인 inos 의발현저해에서기인하는것으로나타났다 (Fig. 2). 이러한결과를통해 CHEE 가산화적스트레스뿐만아니라염증성자극에대한방어능또한보유함을확인하였다. 한편 CHEE 의처리는 inos 의단백질발현은유의적으로감소시켰으나 COX-2 의발현에는영향을주지않았다 (data not shown). 이상의결과를통해 C. horizontalis 가항산화능과항염증활성을보유함을세포수준에서처음으로확인하였으며, 이 러한결과는신규소재에대한새로운기능성데이터를구축함과동시에향후생리활성보유기능성소재로서의활용을위한근거자료로이용될수있을것이다. 후속연구를통해 C. horizontalis 의활성성분을분리함과동시에각생리활성의상위신호전달경로의규명이필요할것으로판단된다. 요 약 Cotoneaster horizontalis Decne 에탄올추출물 (CHEE) 의항산화능과항염증생리활성을분석하였다. CHEE의항산화능을 DPPH radical scavenging activity로분석한결과 radical 소거능의정도가양성대조군으로사용한 ascorbic acid와유사한정도의높은활성을보여매우강한항산화능을보유함을확인하였다. 또한 RAW 세포주를이용하여 H 2 O 2 유도에의해생성된 ROS에대한소거능을분석한결과에서도강한소거능을보였다. 뿐만아니라항산화효소 HO-1 및그전사인자인 Nrf2의단백질발현이 CHEE의처리에의해증가되었다. 한편 CHEE가 LPS에의해유도된 NO 생성에미치는영향을분석한결과농도의존적인 NO 생성저해능을보였으며이는 NO 생성단백질인 inos의발

5 Biological Activities of C. horizontalis 284 현저해에서기인함을확인하였다. 이러한결과를통해 C. horizontalis 의항산화능과항염증활성을세포수준에서처음으로확인하였으며향후기능성소재로서유용하게활용될수있을것으로판단된다. Acknowledgments This work was supported by Blue-Bio Industry Regional Innovation Center (RIC ) at Dong-Eui University as a RIC program under Ministry of Trade, Industry and Energy (MOTIE) and Busan city. References 1. Cencioni C, Spallotta F, Martelli F, Valente S, Mai A, Zeiher AM, Gaetano C Oxidative stress and epigenetic regulation in ageing and age-related diseases. Int. J. Mol. Sci. 14: Chapple SJ, Siow RC, Mann GE Crosstalk between Nrf2 and the proteasome: therapeutic potential of Nrf2 inducers in vascular disease and aging. Int. J. Biochem. Cell Biol. 44: Chawla A, Nguyen KD, Goh YP Macrophage-mediated inflammation in metabolic disease. Nat. Rev. Immunol. 11: Giudice A, Arra C, Turco MC Review of molecular mechanisms involved in the activation of the Nrf2-ARE signaling pathway by chemopreventive agents. Methods Mol. Biol. 647: Gonzalez-Burgos E, Gomez-Serranillos MP Terpene compounds in nature: a review of their potential antioxidant activity. Curr. Med. Chem. 19: Grivennikov SI, Greten FR, Karin M Immunity, inflammation, and cancer. Cell 140: Hu R, Saw CL, Yu R, Kong AN Regulation of NF-E2- related factor 2 signaling for cancer chemoprevention: antioxidant coupled with antiinflammatory. Antioxid. Redox Signal. 13: Kalyanaraman B Teaching the basics of redox biology to medical and graduate students: Oxidants, antioxidants and disease mechanisms. Redox Biol. 1: Kedare SB, Singh RP Genesis and development of DPPH method of antioxidant assay. J. Food Sci. Technol. 48: Khan S WZ, Wang R, Zhang L Horizontoates A-C: New cholinesterase inhibitors from Cotoneaster horizontalis. Phytochemistry Letters 10: Khansari N, Shakiba Y, Mahmoudi M Chronic inflammation and oxidative stress as a major cause of age-related diseases and cancer. Recent Pat. Inflamm. Allergy Drug Discov. 3: Kundu JK, Surh YJ Inflammation: gearing the journey to cancer. Mutat. Res. 659: Kundu JK, Surh YJ Emerging avenues linking inflammation and cancer. Free Radic. Biol. Med. 52: Lee JC, Hou MF, Huang HW, Chang FR, Yeh CC, Tang JY, et al Marine algal natural products with anti-oxidative, anti-inflammatory, and anti-cancer properties. Cancer Cell Int. 13: Li J, Zhang H, Huang W, Qian H, Li Y TNF-alpha inhibitors with anti-oxidative stress activity from natural products. Curr. Top Med. Chem. 12: Liochev SI Reactive oxygen species and the free radical theory of aging. Free Radic. Biol. Med. 60: Lu Y, Suh SJ, Kwak CH, Kwon KM, Seo CS, Li Y, et al Saucerneol F, a new lignan, inhibits inos expression via MAPKs, NF-kappaB and AP-1 inactivation in LPS-induced RAW264.7 cells. Int. Immunopharmacol. 12: Mohamed SA SN, El-Gindi O, Ali ZY, Alfishawy IM Phytoconstituents Investigation, Anti-diabetic and Anti-dyslipidemic Activities of Cotoneaster horizontalis Decne Cultivated in Egypt. Life Sci. J. 9: Park CM, Jin KS, Lee YW, Song YS Luteolin and chicoric acid synergistically inhibited inflammatory responses via inactivation of PI3K-Akt pathway and impairment of NF-kappaB translocation in LPS stimulated RAW cells. Eur. J. Pharmacol. 660: Park CM, Park JY, Noh KH, Shin JH, Song YS Taraxacum officinale Weber extracts inhibit LPS-induced oxidative stress and nitric oxide production via the NF-kappaB modulation in RAW cells. J. Ethnopharmacol. 133: Pillai S, Oresajo C, Hayward J Ultraviolet radiation and skin aging: roles of reactive oxygen species, inflammation and protease activation, and strategies for prevention of inflammation-induced matrix degradation - a review. Int. J. Cosmet. Sci. 27: Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB Oxidative stress, inflammation, and cancer: how are they linked? Free Radic. Biol. Med. 49: Saw CL, Wu Q, Su ZY, Wang H, Yang Y, Xu X, et al Effects of natural phytochemicals in Angelica sinensis (Danggui) on Nrf2-mediated gene expression of phase II drug metabolizing enzymes and anti-inflammation. Biopharm. Drug Dispos. 34: Sokkar N E-GO, Sayed S, Mohamed S, Ali Z, Alfishawy I Antioxidant, andticancer and hepatoprotective activities of Cotoneaster horizontalis Decne extract as well as a-tocopherol and amygdalin production from in vitro culture. Acta Physiol. Plant. 35: Su ZY, Shu L, Khor TO, Lee JH, Fuentes F, Kong AN A perspective on dietary phytochemicals and cancer chemoprevention: oxidative stress, nrf2, and epigenomics. Top Curr. Chem. 329: Tsai HH, Lee WR, Wang PH, Cheng KT, Chen YC, Shen SC Propionibacterium acnes-induced inos and COX-2 September 2015 Vol. 43 No. 3

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KJMB fm Korean J. Microbiol. Biotechnol. (2014), 42(3), 275 284 pissn 1598-642X eissn 2234-7305 Korean Journal of Microbiology and Biotechnology Malus huphensis, Ophiorrhiza cantonensis, Psychotria rubra 에탄올추출물의항산화및항염증활성