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 진경숙 1, 권현주 1,2, 김병우 1,2 * 1 동의대학교블루바이오소재개발및실용화지원센터 2 동의대학교생명응용학과 Received: April 28, 2014 / Revised: August 6, 2014 / Accepted: August 12, 2014 Anti-Oxidative and Anti-Inflammatory Effects of Malus huphensis, Ophiorrhiza cantonensis, and Psychotria rubra Ethanol Extracts 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, Dong-Eui University, Busan 614-714, Republic of Korea This study was orchestrated with the purpose of uncovering new nutraceutical resources possessing biological activities in the plant kingdom. To fulfill our objective, we analyzed several Chinese plants and selected three possessing powerful anti-oxidative activities. The anti-oxidative and anti-inflammatory effects these three Chinese plants, Malus hupehensis, Ophiorrhiza cantonensis, and Psychotria rubra ethanol extracts were then evaluated. First of all, they possessed potent scavenging activity against 1,1-diphenyl-2-picryl hydrazyl, similar with that of ascorbic acid, used as a positive control. Moreover, they inhibited lipopolysaccharide (LPS)- and hydrogen peroxide-induced reactive oxygen species, in a dose-dependent manner, in RAW 264.7 cells. Also, they induced the expression of an anti-oxidative enzyme, heme oxygenase 1, and its upstream transcription factor, nuclear factor-e2-related factor 2. Furthermore, they suppressed LPS-induced nitric oxide (NO) formation, without cytotoxicity. The inhibition of NO formation was the result of the down regulation of inducible NO synthase (inos). The suppression of NO and inos by the three extracts might be the result of modulation by the upstream transcription factors, nuclear factor κb and activator protein-1. Taken together, these results indicate that these three Chinese plants possess potent anti-oxidative and anti-inflammatory activities. Therefore, they might be utilized as promising materials in the field of nutraceuticals. Keywords: Three Chinese plants, anti-oxidative activity, anti-inflammatory activity, upstream transcription factors 서 론 활성산소종 (Reactive Oxygen Species, ROS) 은호흡을통해체내로유입된산소에의해생체대사과정에서끊임없이발생하게되며산화적스트레스 (oxidative stress) 를유발하여세포손상을일으킬뿐아니라, 염증유발인자를활성화시킴으로써세포및조직에염증을초래한다 [9, 24]. 이러한산화적스트레스는다양한질병의원인이되며, 노화를일으 *Corresponding author Tel: +82-51-890-2900, Fax: +82-51-890-2914 E-mail: bwkim@deu.ac.kr 2014, The Korean Society for Microbiology and Biotechnology 키는직 간접적원인물질로작용한다 [14, 24]. 그러므로세포는항상적정수준의항산화물질의보호를필요로하며, 인체에발생하는수많은질병에대응하기위해서는강한항산화능을보유한생리활성소재의개발이매우중요하다 [17, 18, 26]. 대표적인 cellular defensive phase 2 detoxifying antioxidant enzyme으로알려진 heme oxygenase (HO)-1의유도는산화적스트레스를방어하는중요한기전중하나로 carcinogen 등의외부자극으로부터세포를보호하는 chemoprevention에서중요한역할을한다 [5, 35]. 특히천연유래의다양한 dietary phytochemical은 nuclear factor E2- related factor 2 (Nrf2) 에의해조절되는 phase 2 detoxifying antioxidant enzyme의발현증가를통해 chemopreven- September 2014 Vol. 42 No. 3

276 Jin et al. tive function 을나타낸다 [19]. 이러한 chemoprevention 은항산화활성을기초로하여암, 염증, 뇌및심혈관계질환, 노화등의예방및치료기전과도상호작용하는것으로알려지고있어그중요성이매우크다 [3, 15, 27, 33]. 염증은외부자극에대한생체조직의방어기전중하나이나지속적인염증반응의발생은조직의손상을일으켜암을비롯한각종질병을유발한다 [4, 7]. 생체내염증반응은대식세포 (macrophage) 에서과량생산되는염증매개인자 (inflammatory mediators) 로부터유래되며, 이러한염증매개인자로는 inducible nitric oxide synthase (inos) 에의해생산되는 nitric oxide (NO) 와 cyclooxygenase 2 (COX-2) 로부터생산되는 prostaglandin E2 (PGE2) 등이알려져있다. 이러한염증매개인자는 tumor necrosis factor α, interleukin 1β 등과같은사이토카인의생산을유도하여염증반응을일으킨다 [15, 16]. 염증반응의대표적인세포실험계중하나인 RAW 264.7 murine macrophage 에염증유발인자인 lipopolysaccharide (LPS) 를처리하면 inos 및 COX-2 의발현유도에의해 NO 와 PGE2 등의염증매개인자가과량생성되며이는사이토카인분비량증가를유도한다. 이러한일련의반응은염증상위신호전달기전인 nuclear factor (NF)-κB 와 activator protein (AP)-1 에의해조절되는것으로알려져있다 [21, 28]. 그러므로염증매개인자와그상위신호전달기전을효과적으로제어할수있는물질의개발에많은연구가집중되고있다. 이에본연구에서는천연에서유래한항산화및항염증생리활성보유신소재개발의일환으로중국자생식물백종의생리활성을분석하여그중높은항산화능을보이는 3 종의식물, 즉 Malus hupehensis, Ophiorrhiza cantonensis, 그리고 Psychotria rubra 를선별하였다. M. hupehensis 는장미과 (Rosaceae) 에속하는낙엽수로일본및타이완을원산지로하며, 동아시아지역에널리분포한다. 일반적인높이는약 7m 이고, 개화시기는 4-5 월이며, 10 월에씨를맺는다. 열매는식용가능하며, 일반적으로는차의형태로복용한다. 열매가약용으로알려져있으나구체적인효능에대해서는알려진바가없다. O. cantonensis 는꼭두서니과 (Rubiaceae) 에속하는아관목으로일반적으로 Rubiaceae 과의식물이아시아전역에널리분포하는데반해 O. cantonensis 는중국에만분포하는것으로알려져있다. 일반적인높이는 1-2 m 이고, 겨울과봄사이에개화하며, 봄에서여름사이에씨를맺는다. 잎이식용가능한것으로알려져있으나구체적인효능에대해서는알려진바가없다. P. rubra 또한 Rubiaceae 에속하는하부층관목으로동아시아지역, 특히중국, 일본및타이완에분포한다. 일반적인높이는 0.5-3 m 이고, 개화시기는 4-11 월이며, 7-12 월사이에열매를맺는다. P. rubra 의식용 및약용에대해서는알려진바가없으며, 특히상기세소재의항산화및항염증효과에대해서는전혀알려진바없다. 이에본연구에서는 M. hupehensis, O. cantonensis, 그리고 P. rubra의 95% 에탄올추출물 ( 이하 MHEE, OCEE, PREE) 이보유한항산화및항염증활성을분석함으로써각소재의기능성자원으로서의활용가능성을확인해보고자하였다. 재료및방법 추출물의제조본연구에서사용한 M. hupehensis, O. cantonensis, 그리고 P. rubra의 95% 에탄올추출물 ( 이하 MHEE, OCEE, PREE) 은한국생명공학연구원, 해외생물소재허브센터에서구입 ( 분양번호 FBM123-027, 077, 100) 하여사용하였으며추출과정은다음과같다. MHEE와 PREE는줄기부위를, OCEE는줄기를포함한전초를건조하여분쇄하고, 95% 에탄올을용매로하여 45 o C에서 3일간추출을수행하였다. 추출이끝난시료를 filter로여과하여고형물을없애고감압농축 (N-1000SW, EYELA, Japan) 및동결건조 (FDU2100, EYELA, Japan) 하여사용전까지 4 o C에보관하였다. DPPH radical 소거능측정을통한항산화능분석각추출물의항산화능보유유무및그정도를알아보기위해항산화능의주요지표로활용되고있는 1,1-diphenyl- 2-picryl hydrazyl (DPPH) radical 소거능분석을수행하였다 [6]. DPPH는비교적안정한 free radical로써, ascorbic acid, tocopherol, polyhydroxy 방향족화합물, 방향족아민류등에의해환원되어짙은자색이탈색되며이러한원리를이용하여항산화활성을간단히측정할수있는동시에식물체의항산화활성과도연관성이매우높기때문에많이이용되고있다 [13]. DPPH radical 소거능측정을위해각시료를농도별 (0.512-12.8 μg/ml) 로메탄올에녹여준비하고 96 well plate 에메탄올에용해된 1.5 10 4 M DPPH 40 μl와각시료 160 μl를분주한혼합액을실온에서 30분간반응시킨후, multi-plate reader (Paradigm, Beckman, CA, USA) 를이용하여 520 nm에서흡광도를측정하였다. 시료를첨가하지않은음성대조군과대비하여 free radical 소거정도를백분율로나타내고, 50% 저해농도 (Inhibitory Concentration, IC 50 ) 를계산하였다. 대표적인항산화제로 DPPH radical 소거능측정시양성대조군으로주로사용되는 ascorbic acid 를함께비교분석하였으며측정값은 3회반복실험의평균값으로나타내었다.

Biological Activities of Three Chinese Plants 277 RAW 264.7 murine macrophage의배양항산화및항염증활성의세포실험모델계로 murine macrophage cell line인 RAW 264.7을 American Type Tissue Collection (ATCC, TIB-71 TM, Manassas, VA, USA) 로부터구입하여 10% fetal bovine serum (FBS) 및 penicillin/streptomycin (Pen/Strep) 이포함된 DMEM 배지에서배양하였다 [23]. 세포생존율분석활성분석수행전시료가세포생존율에미치는영향을확인하고, 세포독성을유발하지않는시료의처리농도를결정하기위해각추출물에의한세포독성유발유무를 WST assay를통해수행하였다. 1.0 10 5 cell을 24-well tissue culture plate에분주하여 24시간동안부착시키고, 농도별시료처리 24시간경과후 WST 시약이든배지로교체하여한시간동안반응시킨다음 multi-plate reader를이용하여 450 nm에서흡광도를측정하였다. 측정값은 3회반복실험의평균값으로나타내었으며독성을유발하지않는농도범위에서이후실험을수행하였다. Reactive oxygen species (ROS) 소거능분석 ROS는과량생산시핵산, 단백질, 지질등의생체내고분자에산화적스트레스를유발하여다양한질병의원인이되므로이러한 ROS의소거능은항산화력의중요한지표가된다 [20]. Hydrogen peroxide (H 2 O 2 ) 는대표적인 ROS 중하나로소재의항산화능을규명하기위한많은연구에서 ROS 유도제로사용되고있다 [29, 31, 34]. 또한그람음성박테리아의세포외벽구성인자인 lipopolysaccharide (LPS) 는대표적인염증유발인자로산화적스트레스또한유발하는것으로알려져있어항산화능및항염증활성을규명하기위한많은연구에서스트레스유도제로사용되고있다 [1, 22]. 본연구에서는각소재가보유한항산화능을 H 2 O 2 및 LPS 로유도한 ROS 생성에시료가미치는영향을통해분석하였다. 이를위해 RAW 264.7 cell에 cell permeable fluorescent dye인 50 μm의 dichlorofluorescin diacetate (DCFH-DA) 를 2시간동안전처리한후제거하고 500 μm의 H 2 O 2 혹은 1 μg/ml의 LPS를농도별시료와함께처리한후시료에의한 ROS 생성억제능을 multiplate reader 를이용한 fluorescence 측정을통해분석하였다. 측정값은 3 회반복실험의평균값으로나타내었다. 항산화효소 HO-1 및상위전사인자 Nrf2 의발현조절능분석각소재의항산화활성기전을알아보기위해대표적인항산화효소인 HO-1 과그상위전사인자인 Nrf2 의시료처리에의한단백질발현변화를 Western blot hybridization 으로분 석하였다. HO-1의일차항체는 Cell Signaling Technology (MA, USA) 로부터구입하였고, Nrf2 그리고 Actin의일차항체와 anti-goat, 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 1:1,000으로희석한대상단백질의일차항체와 hybridization하였다. Membrane 수세후 horse radish peroxidase (HRP) 가부착된이차항체 (1:1,000) 로한시간동안반응시키고 chemiluminescence detection system (Fluo- Chem FC2, AlphaInnotech, USA) 을이용하여단백질발현을분석하였다. 실험의결과는 3회반복실험을통해유의적인단백질발현변화를확인한후대표적인데이터를제시하였다. NO 생성억제능분석대표적인 free radical 중하나인 NO는중요한세포신호전달물질로서작용하나과잉생산시산화적스트레스의유발을통해염증및세포손상의원인이된다 [12]. 이러한 NO 생성억제능의분석은 Park 등 [23] 의방법을변형하여다음과같이수행하였다. RAW 264.7 cell을 24-well tissue culture plate에 well 당 1.0 10 5 cell을분주하여부착시킨후 1 μg/ ml의 LPS를처리하여 NO 생성을유도하고식물추출물에의한 NO 생성저해능을 Griess reaction을통해분석하였으며측정값은 3회반복실험의평균값으로나타내었다. 항염증활성기전분석각소재가보유한 NO 생성억제능의기전을밝히기위해 NO 생성유전자인 inos의단백질발현을분석하였다. 또한각추출물에의한 NO 생성및 inos의발현저해능이 NFκB 및 AP-1에의해조절될가능성을알아보기위해 LPS로유도된 NF-κB p65와 inhibitory κbα (IκBα), 그리고 AP-1 의 subunit인 c-jun의인산화에각추출물이미치는영향을분석하였다. Western blot hybridization을위한 inos, p- p65, p-iκbα, 그리고 p-c-jun의일차항체는 Cell Signaling Technology (MA, USA) 로부터구입하여사용하였다. 시료처리가끝난배양세포에서단백질을추출하여 Bradford assay로단백질농도를결정한후 50 μg의단백질을 10% SDS-PAGE로전기영동하고 nitrocellulose membrane에 blotting한후대상단백질의항체와 hybridization하였다. Membrane 수세후 HRP가 tagging된이차항체로한시간동안반응시키고 chemiluminescence detection system을이용하여단백질발현을분석하였다. 실험의결과는 3회반 September 2014 Vol. 42 No. 3

278 Jin et al. 복실험을통해유의적인단백질발현변화를확인한후대표적인데이터를제시하였다. 통계분석실험의결과는평균 (mean) ± 표준편차 (standard deviation, SD) 로나타내었고, 각데이터의통계분석은 SPSS 20.0 software를이용한 unpaired Student s t-test를통해 p 값이 0.05 미만 (p < 0.05) 인경우유의성이있는것으로판단하였다. 결과및고찰 추출물의 ROS 소거능분석 DPPH radical 소거능분석에의해각소재가보유한높은항산화능이확인됨에따라그작용기전을좀더자세히알아보기위해먼저 RAW 264.7 cell 에대표적인산화적스트레스유도인자인 H 2 O 2 와 LPS 를각각처리하여각소재의 ROS 소거능을분석하였다. 그결과 Fig. 1 과 Fig. 2 에제시한바와같이 H 2 O 2 와 LPS 에의해각각유도된 ROS 생성이소재의농도별처리에의해효과적으로저해되는것으로나타나세소재가 DPPH radical 뿐만아니라세포수준에서 H 2 O 2 와 LPS 에의해유도된산화적스트레스또한효과적으로감소시킴을확인하였다. 추출물의항산화능분석 3종소재의항산화능보유유무및그정도를알아보기위해먼저항산화능의주요지표중하나인 DPPH radical 소거능을분석하였다. 그결과 Table 1에제시된바와같이 MHEE, OCEE, 그리고 PREE 모두농도증가에따른강한 radical 소거능을보여 12.8 μg/ml의시료처리에의해 DPPH radical 소거능이각각 97.32, 95.42, 95.01% 로나타나양성대조군으로사용한 ascrobic acid, 즉 vitamin C의 97.49% 와유사한정도의활성을보여매우강한항산화능을보유함을확인하였다. 각소재의 DPPH radical에대한 50% 소거농도를나타내는 IC 50 값은 MHEE, OCEE, PREE가각각 1.10, 1.49, 1.38 μg/ml으로나타났다. 이에각소재가보유한항산화능의정도및기전을세포수준에서확인하고자하였다. Table 1. DPPH radical scavenging activity of MHEE, OCEE, and PREE. Reagent Concentration (µg/ml) Inhibition rate (%) MHEE 10.512 38.10 ± 0.19 12.56 79.86 ± 0.37 * OCEE 10.512 12.56 12.8 PREE 10.512 12.56 12.8 Ascorbic acid (Positive control) 12.8 97.32 ± 0.29 * 33.58 ± 0.27 67.84 ± 0.46 * 95.42 ± 0.25 * 33.14 ± 0.23 73.13 ± 0.62 * 95.01 ± 0.10 * 10.512 30.10 ± 0.15 12.56 12.8 96.68 ± 0.18 * 97.49 ± 0.06 * *Significantly different from the inhibition rate of each reagent s first concentration, 0.512 µg/ml (p < 0.05). Fig. 1. Effect of MHEE (A), OCEE (B), and PREE (C) on H 2 O 2 - induced ROS scavenging activity in RAW 264.7 cells. Values are represented as the mean ± SD (n = 6). *, #Significantly different from the vehicle control [Con ( )] and H 2 O 2 -induced control [Con (+)], respectively (p < 0.05).

Biological Activities of Three Chinese Plants 279 Fig. 2. Effect of MHEE (A), OCEE (B), and PREE (C) on LPSinduced ROS scavenging activity in RAW 264.7 cells. Values are represented as the mean ± SD (n = 6). *, #Significantly different from the vehicle control [Con ( )] and LPS-induced control [Con (+)], respectively (p < 0.05). 각추출물이항산화효소 HO-1 및상위전사인자 Nrf2 의발현에미치는영향강한항산화능을보유한천연소재들이 Nrf2 에의한항산화효소계의발현유도를통해활성을나타낸다는것이여러연구를통해밝혀짐에따라각소재가보유한항산화능의작용기작을알아보고자하였다 [11, 27]. 이를위해각소재가대표적인항산화효소인 HO-1 과그상위전사인자인 Nrf2 의단백질발현에미치는영향을분석하였다. 그결과 Fig. 3 에제시된바와같이 10-50 μg/ml 의시료처리에의해 HO-1 및상위전사인자인 Nrf2 의단백질발현이증가되는 Fig. 3. Modulation of an anti-oxidative enzyme, HO-1, and its upstream transcription factor, Nrf2 protein expression in RAW 264.7 cells by MHEE (A), OCEE (B), and PREE (C). Actin was used as an internal control. *Significantly different from the vehicle control (0) (p < 0.05). September 2014 Vol. 42 No. 3

280 Jin et al. Fig. 4. Effect of MHEE (A), OCEE (B), and PREE (C) on the viability of RAW 264.7 cells. Values are represented as the mean ± SD (n = 3). *Significantly different from the LPS-induced control [Con (+)] (p < 0.05). 것으로나타나각소재에의한 HO-1 의발현유도가 Nrf2 의발현증가에서기인할것으로판단되었다 [2, 5]. 추출물의세포생존율및항염증활성분석각소재가보유한항염증활성을알아보기전추출물이 RAW 264.7 세포생존율에미치는영향을살펴보았다. 그결과 Fig. 4 에제시한바와같이세추출물모두 100 μg/ml 까지의처리에서심한세포독성을유발하지않는것을확인하였으며이후실험의농도를 100 μg/ml 까지로결정하여수행하였다. Fig. 5. Modulation of LPS-induced NO formation in RAW 264.7 cells by MHEE (A), OCEE (B), and PREE (C). Values are represented as the mean ± SD (n = 3). *, #Significantly different from the vehicle control [Con, LPS ( )] and LPS-induced control [Con, LPS (+)], respectively (p < 0.05). 항염증활성의확인을위해각소재의 NO 생성억제능을분석하였다. LPS로자극을유도한쥐대식세포주 RAW 264.7 cell에서농도별각시료의처리에따른 NO 생성양의변화를분석한결과 Fig. 5에제시된바와같이세시료모두 10-100 μg/ml의시료처리에의해농도의존적인 NO 생성저해능을보였으며이는 NO 생성단백질인 inos의발현저해에서기인하는것으로나타났다 (Fig. 6). 이러한결과를통해세소재가항산화능뿐만아니라항염증활성또한보유함을확인하였다.

Biological Activities of Three Chinese Plants 281 Fig. 6. Modulation of LPS-induced inos protein expression in RAW 264.7 cells by MHEE (A), OCEE (B), and PREE (C). Actin was used as an internal control. *, #Significantly different from the vehicle control [Con ( )] and LPS-induced control [Con (+)], respectively (p < 0.05). 추출물의항염증활성상위신호전달기전분석각추출물이 inos 의발현저해를통한 NO 생성억제능을보임에따라항염증활성의상위신호전달기전인 NF-κB 와 AP-1 의연관성을알아보기위해추출물의처리가 LPS 에의해유도된 NF-κB p65 와 IκBα, 그리고 AP-1 의 subunit 중하나인 c-jun 의인산화에미치는영향을분석하였다. 그결과 Fig. 7 에제시된바와같이 2 시간동안의 LPS 처리에의해유도된세전사인자의인산화가시료농도의증가에따라유의적으로억제되는것으로나타났다. 이러한결과는각소재가보유한항염증활성이 NF-κB 및 AP-1 과같은상위신호전달인자들의일련의조절기작을통해이루어질가능성을시사하였다. 이러한결과를통해중국자생식물인 M. hupehensis, O. cantonensis, 그리고 P. rubra 의에탄올추출물이항산화능과항염증활성을보유함을확인하였다. 이러한결과는기능성에대한보고가거의없는상기의소재에대한생리활성을처음으로규명한것으로신규소재에대한새로운기능성데이터를구축함과동시에향후생리활성보유기능성소재로서의활용을위한근거자료로사용될수있을것으로판단된다. M. hupehensis 호북꽃사과나무로불리는데열매인꽃사과는과당, 포도당, 주석산, 비타민 C 등의함유하고, 한방에서는소화불량, 식욕부진, 위산분비조절등위장관계관련질환에이용되며, 민간에서는고기를부드럽게하는연육제로, 과즙은숙취해소에사용된다. 보고된바로는 M. hupehensis 잎의 polyphenol 과 flavonoid 가 doxorubicin 으로유도한심장세포사멸에대한보호효과를나타내며, 동물모델에서기생충으로유도한간섬유증의억제효과를보유하는것으로밝혀졌다 [30, 32]. 규명된 M. hupehensis 의활성성분으로는잎으로부터분리한 12 종의 flavonoid 와 3 종의 biflavonoid glycoside 가있으며이중에는강한항산화능을보유하는것으로알려진 quercetin 을포함하였다 [30]. 또한 M. hupehensis 의 flavon 조성을분석한결과, 사과나무껍질등에서주로채취하는배당체인 phlorizin 이약 1.6% 함유되어있는것으로나타났다 [8]. Phlorizin 은포도당수용체인 SGLT-2 의억제제로작용하여당뇨병치료제개발의기초물질로활용되고있다 [25]. 본연구에서사용한 M. hupehensis 줄기부위에대해서는기능성및활성성분에대한보고는전무하다. 다만상기의연구결과에서보고된활성성분이줄기부위에도함유되어있을가능성이있다고판단되며, 그중대부분이항산화와함염증활성을보유할것으로생각되어이에대한분석이필요할것으로사료된다. O. cantonensis 의기능성에대해보고된문헌은현재까지없으며, P. rubra 의기능성또한에탄올추출물에서분리된신종 naphthoquinone 인 psychorubrin 의항암활성에대한보고가유일하다 [10]. 이에추후계속적인연구를통해상기세소재가보유한항산 September 2014 Vol. 42 No. 3

282 Jin et al. Fig. 7. Modulation of upstream signaling pathways for the anti-inflammatory activity by MHEE (A), OCEE (B), and PREE (C) in RAW 264.7 cells. Actin was used as an internal control. *, #Significantly different from the vehicle control [Con ( )] and LPS-induced control [Con (+)], respectively (p < 0.05). 화및항염증활성의작용물질및그작용기작의세부적인규명이필요할것으로판단된다. 요 약 본연구에서는식물자원으로부터생리활성을보유한새로운기능성소재를찾고자하였다. 이를위해수종의중국자생식물을분석하여강한항산화능을보유한 3종 (Malus hupehensis, Ophiorrhiza cantonensis, 그리고 Psychotria rubra) 을선별하고, 각추출물의항산화능과항염증생리활성을분석하였다. 먼저각추출물의항산화능을 DPPH radical 소거능을통해분석한결과모두양성대조군으로사용한 ascorbic acid와유사한정도의높은활성을보여각소재가매우강한항산화능을보유함을확인하였다. 또한 RAW 264.7 세포주에서 H 2 O 2 및 LPS 에의해유도된 ROS 에대한각추출물의소거능을분석한결과에서도농도의존적인강한소거능을보였다. 뿐만아니라대표적인항산화효소중하나로항산화능보유천연물에의해발현이유도되는 HO- 1 및그전사인자인 Nrf-2 의단백질발현이각추출물의처리에의해증가됨을보였다. 한편각소재가 LPS 에의해유도된 NO 생성에미치는영향을분석한결과모두농도의존적인 NO 생성저해능을보였으며이는 NO 생성단백질인 inos 의발현저해에서기인함을확인하였다. 이와같은각소재의 NO 생성및 inos 발현억제효과는염증상위신호전달계인 NF-κB 및 AP-1 의조절을통해일어날가능성을보였다. 이러한결과를통해중국자생식물 3 종의높은항산화능과항염증활성을처음으로확인하였으며향후기능성소재로서유용하게활용될수있을것으로판단된다.

Biological Activities of Three Chinese Plants 283 Acknowledgments This work was supported by Blue-Bio Industry Regional Innovation Center (RIC08-06-07) at Dong-Eui University as a RIC program under Ministry of Trade, Industry and Energy (MOTIE) and Busan city. References 1. Awad N, Khatib N, Ginsberg Y, Weiner Z, Maravi N, Thaler I, et al. 2011. N-acetyl-cysteine (NAC) attenuates LPS-induced maternal and amniotic fluid oxidative stress and inflammatory responses in the preterm gestation. Am. J. Obstet. Gynecol. 204: 450.e15-20. 2. Bryan HK, Olayanju A, Goldring CE, Park BK. 2013. The Nrf2 cell defence pathway: Keap1-dependent and -independent mechanisms of regulation. Biochem. Pharmacol. 85: 705-717. 3. Chapple SJ, Siow RC, Mann GE. 2012. Crosstalk between Nrf2 and the proteasome: therapeutic potential of Nrf2 inducers in vascular disease and aging. Int. J. Biochem. Cell Biol. 44: 1315-1320. 4. Chawla A, Nguyen KD, Goh YP. 2011. Macrophage-mediated inflammation in metabolic disease. Nat. Rev. Immunol. 11: 738-749. 5. Giudice A, Arra C, Turco MC. 2010. Review of molecular mechanisms involved in the activation of the Nrf2-ARE signaling pathway by chemopreventive agents. Meth. Mol. Biol. 647: 37-74. 6. Gonzalez-Burgos E, Gomez-Serranillos MP. 2012. Terpene compounds in nature: a review of their potential antioxidant activity. Curr. Med. Chem. 19: 5319-5341. 7. Grivennikov SI, Greten FR, Karin M. 2010. Immunity, inflammation, and cancer. Cell 140: 883-899. 8. Guo DY, Li J, Shi YQ, Wang JZ. 2011. Study on the flavon ingredients of Malus hupehensis. Zhong Yao Cai. 34: 1026-1029. 9. Harman D. 2009. Origin and evolution of the free radical theory of aging: a brief personal history, 1954-2009. Biogerontology 10: 773-781. 10. Hayashi T, Smith FT, Lee KH. 1987. Antitumor agents. 89. Psychorubrin, a new cytotoxic naphthoquinone from Psychotria rubra and its structure-activity relationships. J. Med. Chem. 30: 2005-2008. 11. Hu R, Saw CL, Yu R, Kong AN. 2010. Regulation of NF-E2- related factor 2 signaling for cancer chemoprevention: antioxidant coupled with antiinflammatory. Antioxid. Redox. Signal. 13: 1679-1698. 12. Kalyanaraman B. 2013. Teaching the basics of redox biology to medical and graduate students: Oxidants, antioxidants and disease mechanisms. Redox. Biol. 1: 244-257. 13. Kedare SB, Singh RP. 2011. Genesis and development of DPPH method of antioxidant assay. J. Food Sci. Technol. 48: 412-422. 14. Khansari N, Shakiba Y, Mahmoudi M. 2009. Chronic inflammation and oxidative stress as a major cause of age-related diseases and cancer. Recent. Pat. Inflamm. Allergy Drug. Discov. 3: 73-80. 15. Kundu JK, Surh YJ. 2008. Inflammation: gearing the journey to cancer. Mutat. Res. 659: 15-30. 16. Kundu JK, Surh YJ. 2012. Emerging avenues linking inflammation and cancer. Free Radic. Biol. Med. 52: 2013-2037. 17. Lee JC, Hou MF, Huang HW, Chang FR, Yeh CC, Tang JY, et al. 2013. Marine algal natural products with anti-oxidative, anti-inflammatory, and anti-cancer properties. Cancer Cell Int. 13: 55. 18. Li J, Zhang H, Huang W, Qian H, Li Y. 2012. TNF-alpha inhibitors with anti-oxidative stress activity from natural products. Curr. Top Med. Chem. 12: 1408-1421. 19. Li L, Dong H, Song E, Xu X, Liu L, Song Y. 2014. Nrf2/ARE pathway activation, HO-1 and NQO1 induction by polychlorinated biphenyl quinone is associated with reactive oxygen species and PI3K/AKT signaling. Chem. Biol. Interact. 209: 56-67. 20. Liochev SI. 2013. Reactive oxygen species and the free radical theory of aging. Free Radic. Biol. Med. 60: 1-4. 21. Lu Y, Suh SJ, Kwak CH, Kwon KM, Seo CS, Li Y, et al. 2012. 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: 175-181. 22. Noworyta-Sokolowska K, Gorska A, Golembiowska K. 2013. LPS-induced oxidative stress and inflammatory reaction in the rat striatum. Pharmacol. Rep. 65: 863-869. 23. Park CM, Park JY, Noh KH, Shin JH, Song YS. 2011. Taraxacum officinale Weber extracts inhibit LPS-induced oxidative stress and nitric oxide production via the NF-kappaB modulation in RAW 264.7 cells. J. Ethnopharmacol. 133: 834-842. 24. Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. 2010. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic. Biol. Med. 49: 1603-1616. 25. Rosenwasser RF, Sultan S, Sutton D, Choksi R, Epstein BJ. 2013. SGLT-2 inhibitors and their potential in the treatment of diabetes. Diabetes Metab. Syndr. Obes. 6: 453-467. 26. Saw CL, Wu Q, Su ZY, Wang H, Yang Y, Xu X, et al. 2013. 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: 303-311. 27. Su ZY, Shu L, Khor TO, Lee JH, Fuentes F, Kong AN. 2013. A perspective on dietary phytochemicals and cancer chemoprevention: oxidative stress, nrf2, and epigenomics. Top Curr. Chem. 329: 133-162. 28. Tsai HH, Lee WR, Wang PH, Cheng KT, Chen YC, Shen SC. 2013. Propionibacterium acnes-induced inos and COX-2 protein expression via ROS-dependent NF-kappaB and AP-1 activation in macrophages. J. Dermatol. Sci. 69: 122-131. 29. Wang FW, Wang Z, Zhang YM, Du ZX, Zhang XL, Liu Q, et al. September 2014 Vol. 42 No. 3

284 Jin et al. 2013. Protective effect of melatonin on bone marrow mesenchymal stem cells against hydrogen peroxide-induced apoptosis in vitro. J. Cell Biochem. 114: 2346-2355. 30. Wang SQ, Zhu XF, Wang XN, Shen T, Xiang F, Lou HX. 2013. Flavonoids from Malus hupehensis and their cardioprotective effects against doxorubicin-induced toxicity in H9c2 cells. Phytochemistry 87: 119-125. 31. Yagi H, Tan J, Tuan RS. 2013. Polyphenols suppress hydrogen peroxide-induced oxidative stress in human bone-marrow derived mesenchymal stem cells. J. Cell Biochem. 114: 1163-1173. 32. You-Qin D, Tian-Yan F, Gai-Gai D, Ying L, Jun-Zhi W. 2011. [Inhibitory effect of total flavonoids of Malus hupehensis on hepatic fibrosis induced by Schistosoma japonicum in mice]. Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi. 23: 551-554. 33. Zhang M, An C, Gao Y, Leak RK, Chen J, Zhang F. 2013. Emerging roles of Nrf2 and phase II antioxidant enzymes in neuroprotection. Prog. Neurobiol. 100: 30-47. 34. Zhang R, Kang KA, Piao MJ, Maeng YH, Lee KH, Chang WY, et al. 2009. Cellular protection of morin against the oxidative stress induced by hydrogen peroxide. Chem. Biol. Interact. 177: 21-27. 35. Zhao CR, Gao ZH, Qu XJ. 2010. Nrf2-ARE signaling pathway and natural products for cancer chemoprevention. Cancer. Epidemiol. 34: 523-533.