280 Journal of Agriculture & Life Science 49(5) 서론 현대인들의건강에대한관심은과거다양한질병의원인규명과치료에서운동, 기능성식품의섭취를통한예방과개선을위한방안모색으로변화되고있다. 이러한차원에서 4,940여종에달하는우리나라의다양한자생식물소

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1 농업생명과학연구 49(5) pp Journal of Agriculture & Life Science 49(5) pp Print ISSN Online ISSN 생약재조성물의항산화및항염증활성 강민정 1 황초롱 1 이수정 2 신정혜 1* 1 ( 재 ) 남해마늘연구소, 2 경상대학교농업생명과학연구원 접수일 (2014 년 12 월 12 일 ), 수정일 (2015 년 10 월 12 일 ), 게재확정일 (2015 년 10 월 14 일 ) Antioxidant and Anti-inflammatory Activity of Medicinal Herbs Composites Min-Jung Kang 1 Cho-Rong Hwang 1 Soo-Jung Lee 2 Jung-Hye Shin 1* 1 Namhae Garilic Research Institute, Namhae 52430, Korea 2 Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Korea Received: DEC , Revised: OCT , Accepted: OCT 초록 생약재 5 종 ( 상황버섯, 황금, 단삼, 뽕잎및작약 ) 의물추출물을동량으로혼합한조성물 (MHE-1) 과동일한추출수율이되도록생약재를혼합한후물추출한조성물 (MHE-2) 의항산화및항염증활성을비교분석하였다. 조성물의총페놀및플라보노이드함량은 MHE-2 에서더많았다. DPPH, ABTS 및 superoxide anion 라디칼소거활성, Fe +2 킬레이팅활성, 환원력및 xanthine oxidase 저해활성등의항산화활성은 MHE-2 가더우수하였다. 항염증활성은 LPS 에의한염증유발군에비해조성물처리구에서유의적으로 NO 생성을억제하였으며, 100µg/mL 농도로처리시 PGE 2 와 cytokine 인 TNF-α, IL-2 및 IL-6 의생성도유의적으로감소시켰으나두조성물간에차이는적었다. 이상의결과를볼때여러종류의생약재를혼합한조성물의생리활성은원재료를혼합하여추출하는것이추출물의생리활성증대에효과적인것으로판단된다. 검색어 - 사이토카인, 생약재, 항산화, 항염증 ABSTRACT We studied the effects of mixing ratio on the antioxidants and anti-inflammatory activities in the 2 kinds of medicinal herbs composites by addition to Phellinus linteus, Scutellaria baicalensis Georgi, Salvia miltiorrhiza Bunge, Morus alba L. and Paeonia lactiflora Pall. The composite MHE-1 was the mixture by the same weight of each hot water extracts of 5 medicinal herbs, and MHE-2 was the hot water extract for the mixture of Phellinus linteus(100g), Scutellaria baicalensis(13.5g), Salvia miltiorrhiza(13.4g), Morus alba(21.2g) and Paeonia lactiflora(37.6g). The contents of total phenolic compounds and flavonoids were significantly higher in MHE-2 than MHE-1. DPPH, ABTS and superoxide radical scavenging activity, Fe +2 chelating activity, reducing power and xanthine oxidase inhibition activity were higher in MHE-2 than MHE-1. In anti-inflammatory activities, two composites were inhibited generation of the NO than LPS-induced inflammatory treatment in RAW264.7 cells. In 100µ g/ml concentration of composites treatment, PGE 2 and pro-inflammatory cytokines such as TNFα, IL-2 and IL-6 production were significantly reduced than LPS-induced inflammatory treatment but, no significant difference between MHE-1 and MHE-2. These result to the antioxidants and anti-inflammatory activities in the 2 kinds of medicinal herbs composites could be suggest that a extraction by mixing the raw materials is more effective than mixture of each extracts. Key words - Anti-inflammatory, Antioxidant, Cytokines, Medicinal herb * Corresponding author: Jung-Hye Shin Tel: whanbee@hanmail.net

2 280 Journal of Agriculture & Life Science 49(5) 서론 현대인들의건강에대한관심은과거다양한질병의원인규명과치료에서운동, 기능성식품의섭취를통한예방과개선을위한방안모색으로변화되고있다. 이러한차원에서 4,940여종에달하는우리나라의다양한자생식물소재 (Kim et al., 2012) b 는기능성연구를위한우선적인연구대상이되고있는데, 이는오래전부터식용또는약용으로이용되어왔기때문에안전성이확보되어있고, 민간에서장기간사용되므로써다소간의효능이검증되어있기때문이다. 이러한차원에서식물류는용이하게현대인의건강유지에도움을줄수있는소재로인식되고있으며, 생리활성을과학적으로규명하기위한연구와더불어이들을이용한음료및가공식품의개발도지속적으로증가되고있는추세에있다. 천연의식물소재는미량의유효물질을포함하여비타민 C, 토코페롤, 카로티노이드, 플라보노이드및페놀산등식물체의모든부위에존재하는페놀성화합물이주요활성물질이며 (Lee et al., 2012) a, 이들성분은대부분항산화활성을가지는것으로확인되고있다. 특히플라보노이드화합물은유리기를소거하거나산화의연쇄반응을종결시키는강력한항산화활성을가지고있으며 (Jia et al., 1999), 페놀성화합물은 phenolic hydroxyl 기가단백질등과결합하고, 강한환원력을지님으로써라디칼을소거하여항산화활성을나타낸다 (Droge, 2001). 이들의항산화활성은시료에함유된항산화성분과활성의측정방법에따라서도차이를나타내는데, DPPH 라디칼소거활성은비타민 C와상관성이높고, ABTs 라디칼소거활성은총페놀및플라보노이드함량과높은상관관계를가지며 (Lee et al., 2012) a, 환원력은페놀화합물보다는플라보노이드화합물의함량과더큰상관관계를가지는것으로보고되어있다 (Jang et al., 2012). 이와같이천연소재의다양한생리활성은시료가보유한항산화활성을기본으로하는데 (Kundu & Surh, 2012), 식물체의항산화와관련한연구는생체내에서일어나는산화적스트레스를효 과적으로제어할수있는소재탐색에도유용하게이용된다. 인체는생명유지를위해끊임없이체내로산소를유입시켜야하고, 이러한과정에서일부는활성산소로전환되어산화적스트레스를유발한다 (Droge, 2001; Lee et al., 2013). 지속적이고과도한산화적스트레스는세포막, DNA, 단백질및지단백의손상을초래하고, 염증반응의신호와연결되어전신적인만성적염증반응을유도하게된다 (Lodovici et al., 2001). 활성산소이외에산화질소도체내산화적스트레스를증가시키며, 염증반응의매개체로작용한다 (Jung et al., 2013). 즉, 지속적인산화와염증반응은혈관이나조직의손상, 동맥경화와같은심혈관계질환, 생활습관병, 치매, 암발생의위험을증가시키고, 노화를촉진하는원인이되기때문에 (Kim et al., 2008) b, 생체내항산화기작의균형이파괴될경우외부식품으로부터항산화성물질의공급은필수적이라고볼수있다 (Kwak & Lee, 2014). 따라서항산화활성이우수한소재는생체내에서의과산화작용에의한염증유발및면역체계의활성화조절에도관여하는것으로추정되고있다. 단일원료보다는여러원료가혼합될경우그효과가상승되므로 (Cho et al., 2007) 최근, 성공적인기능성식의약품개발을위하여과학적인근거에의한복합소재의기능성연구가활발히진행되고있다. 본연구에서는항산화및항염증활성을가지는소재를탐색하고, 그활성을검증하는연구의일환으로선행연구 (Lee et al., 2013) 를통하여선정된 5종의생약재열수추출물을각각동량으로혼합한조성물과생약재원재료의추출수율을고려하여혼합한조성물로구분하였을때, 각조성물의생리활성을 in vitro에서비교함으로써기능성향상을위한식물체복합물개발에활용코져하였다. 재료및방법 1 생약재추출물및조성물의제조문헌조사를통하여선발전생약재 16종을대상으

3 Kang et al: Antioxidant and Anti-inflammatory Activity of Medicinal Herbs Composites 281 Table 1. The combined ratio for the MHE-2 as medicinal herbs composites Sample name Phellinus linteus Scutellaria baicalensis Georgi Salvia miltiorrhiza Bunge Morus alba L. Paeonia lactiflora Pall. Yield(%) Yield ratio to P. linteus Weight(g) 로항산화및항염증활성을스크리닝한선행연구결과 (Lee et al., 2013) 활성이우수한상황버섯 (Phellinus linteus), 황금 (Scutellaria baicalensis Georgi), 단삼 (Salvia miltiorrhiza Bunge), 뽕잎 (Morus alba L.) 및작약 (Paeonia lactiflora Pall.) 이선별되었으며, 이들 5종생약재로구성된조성물의생리활성을검정하고자다음과같이 2가지조성물을제조하였다. 각시료를 90 의진탕배양기 (JSSB-50T, JSR, Gongju, Korea) 에서 60rpm으로회전시키면서 12시간씩 3회반복하여열수추출하였으며, 이를모두모아여과한후동결건조시킨각각의추출건조물을동량으로혼합한것을조성물 MHE-1 로하였다. 또한생약재원재료를추출수율 (Lee et al., 2013) 을고려하여혼합한후추출 건조한것을조성물 MHE-2 로하였다. 즉, 수율이가장낮은상황버섯을 100g으로하여황금, 단삼, 뽕잎및작약의첨가량을각각 13.5g, 13.4g, 21.2g 및 37.6g으로하여 (Table 1) 상기와동일한방법으로추출하고, 여액을동결건조한후분말화하였다. 이때생약재조성물 MHE-2 의추출수율은 20.03±0.87% 였으며, 제조된조성물은 -40 에서보관하면서실험에사용하였다. 2 총페놀화합물및플라보노이드정량총페놀화합물함량은각조성물을 1mg/mL의농도로증류수에용해하여 Foline-Ciocalteu 시약및 10% Na 2CO 3 용액을각각 1mL씩차례로가하여실온에서 1시간반응시킨후 760nm에서흡광도를 측정하였다 (Gutfinger, 1981). 총플라보노이드정량은상기시료액 1mL에 10% aluminum nitrate 및 1M potassium acetate 각 0.1mL와 ethanol 4.3mL를차례로가하여혼합하고실온에서 40분간반응시킨다음 415nm에서흡광도를측정하였다 (Moreno et al., 2000). 총페놀화합물및플라보노이드함량은표준물질로각각 caffeic acid 및 quercetin(sigma- Aldrich Co., St. Louis, MO, USA) 을이용한검량선에의해계산하였다. 3 라디칼소거활성측정 DPPH 라디칼소거능은 1,1-diphenyl-2-picrylhydrazyl(DPPH) 에대한전자공여효과로측정하였다. 96well plate에 10mg/100mL 농도로메탄올에용해한 DPPH 용액 100µL 에농도별시료액 50 µl 를가한후상온에서 10초간 plate shaker(mx2, FINEPCR, Seoul, Korea) 로혼합한후 30분간반응시켜 ELISA reader를이용하여 520nm에서흡광도를측정하였다. DPPH 라디칼소거능은시료첨가구와무첨가구의흡광도비 (%) 로나타내었다 (Blois, 1958). ABTS 라디칼소거능은 7mM ABTS 용액에 potassium persulfate 를 2.4mM이되도록용해시킨다음암실에서 12~16시간동안반응시킨후이를 415nm에서흡광도가 1.5가되도록증류수로조정하여사용하였다. 96well plate에 ABTS 용액 200µL 와시료액 50µL 를혼합하여 ELISA reader로 415nm에서흡광도를측정하였으며, DPPH 및 ABTS 라디칼소거능은시료무첨가구에대한시료첨가구의흡광도비 (%) 로나타내었다 (Re et al.,

4 282 Journal of Agriculture & Life Science 49(5) 1999). Superoxide anion 라디칼소거활성은시료액에 0.1M Tris-HCl 완충용액 (ph 8.5) 및 100µM phenazine methosulfate 용액을잘혼합하여 560nm에서흡광도를측정하였다. 여기에 500µM nitroblue tetrazolium(nbt) 및 β-nicotinamide adenine dinucleotide- reduced disodium salt (NADH) 용액을혼합한후다시흡광도를측정하였다. 시료대신증류수를사용하여시료무첨가구를얻었으며, NBT와 NADH 시약의첨가전 후의흡광도차이를구해백분율로산출하였다 (Nishikimi et al., 1972). 4 Fe +2 킬레이팅활성측정 Fe +2 킬레이팅활성은농도별시료액 0.2mL, methanol 0.8mL, 2mM FeCl 2 4H 2O용액 0.05mL 및 5mM ferrozine[3-(2-pyridyl)-5,6-diphenyl- 1,2,4-triazine-4,4'-disulfonic acid] 용액 0.2mL 를혼합하여실온에서 10분간반응시킨후 562nm 에서흡광도를측정하였다. 시료의 Fe +2 킬레이팅활성은시료무첨가구에대한시료첨가구의흡광도비로나타내었다 (Yen et al., 2002). 5 환원력측정농도별시료액 1mL에 200mM의인산완충액 (ph 6.6) 및 1% 의 potassium ferricyanide 1mL를차례로가한다음 50 의수욕상에서 20분간반응시켰다. 여기에 10% trichloroacetic acid(tca) 용액 1mL를가하여반응을정지시키고 5,000 rpm에서 5 분간원심분리하여얻은상징액에동량의증류수및 ferric chloride 용액을혼합한후 700nm에서흡광도를측정하였으며, 시료의환원력은흡광도의값으로나타내었다 (Oyaizu, 1986). 6 Xanthine oxidase(xo) 저해활성측정 XO 저해활성측정은일정농도로희석한시료액 0.3mL에 0.1M potassium phosphate buffer(ph 7.5) 와 xanthine 2mM을녹인기질액 3mL를첨가하였다. 여기에 0.2U/mL(Sigma-Aldrich Co.) 농 도의 XO 0.3mL를가하여 37 에서 30분간반응시킨다음 20% TCA 1mL를가하여반응을정지시킨후, 반응액중에생성된 uric acid를 292nm에서측정하였다. 시료에대한 XO 저해활성은시료첨가구와무첨가구의흡광도감소율을백분율 (%) 로나타내었다. 7 대식세포를이용한항염증활성측정마우스대식세포에대한독성측정은 CCK-8 assay(cell Counting Kit 8, Dojindo Molecular Technologies, Inc. Gaithersburg, MD, USA) 를이용한발색분석법을사용하였다. RAW264.7 세포는한국세포주은행 (KCLB, Korea Cell Line Bank, Seoul, Korea) 에서분양받았으며, 세포배양을위해 10% FBS와 1% penicillin-streptomycin 을포함하는 DMEM 배지에서 37, 5% CO 2 조건으로배양하였다. 세포농도를 cells/ml로분산시킨다음세포배양용 96well plate에 80µL 씩분주하여 37 및 5% CO 2 incubator 에서 24시간배양한후, 시료액을 20µL 씩접종하였다. 이를다시 24시간배양한후 CCK-8 용액을 10µL 씩분주하여 37 에서 3시간배양하고 ELISA reader(epoch, BioTeck Instrument, Winooski, VT, USA) 로 450nm에서흡광도를측정하였다. 세포로부터생성된 nitric oxide(no) 의양은세포배양액중에존재하는 NO - 2 의형태로 Griess reagent system(promega Corp., Madison, WI, USA) 을이용하여측정하였다. RAW264.7 세포를 cells/well 농도로 24well plate에분주하여 24시간배양한후농도별조성물을각각 20µL 씩처리한다음, 1시간후에 lipopolysaccharide(lps) 를 1µg/mL 씩처리하여 24시간동안배양하였다. 배양액을회수해원심분리 (3,000rpm, 5min, 4 ) 하여세포배양상층액을얻었다. 세포배양액 50µL 와 sulfanilamide solution 50µL 를혼합하여상온에서 10분간반응시켰다. 여기에 NED 용액을 50µL 혼합하여상온에서다시 10분간반응시킨후 ELISA reader를이용하여 540nm에서흡광도를측정하였

5 Kang et al: Antioxidant and Anti-inflammatory Activity of Medicinal Herbs Composites 283 Table 2. Total phenol and flavonoid contents in the medicinal herbs composites (mg/g dried extract) Sample code Total phenol Flavonoids MHE ± ±1.34 MHE ±9.64 * 28.77±1.52 * Each value represents mean±sd(n=3). * This superscripts are significantly different among the different sample by Student t-test at P<0.05. MHE-1; the mixture by the same weight of each hot water extracts as Phellinus linteus, Scutellaria baicalensis, Salvia miltiorrhiza, Morus alba and Paeonia lactiflora. MHE-2; the hot water extract for the mixture of Phellinus linteus(100g), Scutellaria baicalensis(13.5g), Salvia miltiorrhiza(13.4g), Morus alba(21.2g) and Paeonia lactiflora(37.6g). 고, sodium nitrate로표준곡선을작성하여 NO 함량을산출하였다. PGE 2 및 cytokine 생성량측정으로 Prostaglandin E 2(PGE 2) 는 commercial competitive enzyme immunoassay kit(r&d system, Minneapolis, MS, USA) 로 cytokine 인 TNF-α, IL-1ß 및 IL-6는각각의해당 ELISA kit(invitrogen, Carlsbad, CA, USA) 를이용하여시료액과 LPS를순차적으로처리하여얻은세포배양액중의함량을측정하였다. PGE 2 생성양은 anti-mouse PGE 2 로 pre-coating된 well에세포배양액 150µL 를넣은다음 primary antibody solution 을 150µL 첨가하여실온에서 1시간반응시켰다. PGE 2 conjugate 를 50µL 씩첨가한후실온에서 2시간반응시키고, washing buffer로 3회세척하여 substrate solution 을 200 µl 첨가하여실온에서 30분간빛을차단한조건에서반응시켰다. 이어반응정지용액을 100µL 씩처리한다음 ELISA reader를이용하여 450nm에서흡광도를측정하였다. TNF-α, IL-1β및 IL-6는각각의단일클론항체가코팅된 96well plate에세포배양액 50µL 와반응액을넣은후실온에서 1시간 30분간반응시켰다. 반응후 washing buffer로 4회세척한다음 streptavidin-hrp buffer를 100µL 첨가하여실온에서 30분간반응시킨후 4회세척하였다. 이어 stabilized chromogen 을 100µL 첨가하여실온에서 30분간빛을차단한조건에서반응시킨후, 반응정지액을 100µL 처리하여 450nm에서 ELISA reader 로흡광도를측정하였다. 8 통계분석각실험은 3~5회이상반복실험한결과에대하여 SPSS 12.0(SPSS, Inc., Chicago, IL, USA) 을사용하여통계처리하였으며, 각각의시료에대해평균 ± 표준편차로나타내었다. 각시료군에대한유의차검정은 p<0.05 수준에서 Student t-test 또는분산분석을한후 p<0.05 수준에서 Duncan's multiple test에따라분석하였다. 결과및고찰 1 총페놀화합물및플라보노이드함량생약재 5종의혼합조건을달리한조성물에서총페놀및플라보노이드함량을측정한결과는 Table 2와같다. MHE-2 의총페놀및플라보노이드함량은 mg/g 및 28.77mg/g 으로 MHE-1 에비해유의적으로높았다. 조성물을구성하는각시료의총페놀화합물과플라보노이드함량의평균값은각각 64.66mg/g 과 20.53mg/g(Lee et al., 2013) 으로추출물을동량으로혼합한조성물인 MHE-1 과비교하였을때플라보노이드함량은유사하였으나총페놀화합물의함량은조성물에서더많았으며, MHE-2 와같이원재료를혼합하여추출한경우이들성분의함량은더높게정량되었다. 항산화활성이있는순수페놀성물질을일정비율

6 284 Journal of Agriculture & Life Science 49(5) Table 3. Radical scavenging activity of the medicinal herbs composites Scavenging activity Concentration(μg/mL) (%) DPPH radical ABTS radical MHE ±2.69 a 68.07±4.08 b 87.64±1.42 c 89.71±1.91 c 90.36±0.89 c MHE ±2.44 a* 76.18±3.63 b* 91.18±0.64 c* 91.55±1.21 c 92.50±0.62 c* MHE ±1.54 a 40.08±1.30 b 82.18±0.88 c 97.64±0.68 d 98.35±0.86 e MHE ±2.53 a* 50.60±1.27 b* 95.26±2.04 c* 97.87±1.14 cd 99.55±0.29 d Superoxide anion radical MHE ±0.46 a 11.09±1.59 b 28.53±3.40 c 52.10±3.06 d 68.91±3.32 e MHE ±1.60 a* 13.03±1.22 b 48.90±4.21 c* 70.04±3.26 d* 94.61±4.79 e* Sample code refer to the foot note of Table 2. Each value represents mean±sd(n=3). a-e Means with different superscripts in the same row are significantly different at P<0.05. * This superscripts are significantly different between MHE-1 and MHE-2 for each radical by Student t-test at P<0.05. 로혼합한혼합물의항산화활성은각물질의항산화활성에대한합과유사하여시너지효과보다는오히려첨가효과라는보고가있다 (Heo et al., 2007). 반면에대부분의식물체혼합물에서항산화활성은식물체간의상호작용에의한시너지효과가주요인으로보고되어있으며 (Kim et al., 2004) a, 한약재는여러성분과공존할경우시너지작용이나타나생체방어시스템의증강및체내항상성유지에효과적인것으로알려져있다 (Wasser & Ewis, 1999). 특히식물류에따라페놀성화합물의종류및결합정도가상이하고, 열처리에의한추출과정에서결합형폴리페놀이유리형폴리페놀로전환되기때문에추출물중유효물질의함량이증가한다는보고도있다 (Jang et al., 2012). 본연구에서생약재조성물 MHE-2 의총페놀화합물이나플라보노이드함량이 MHE-1 에비해높게정량되었는데, 이는조성물 MHE-2 가각생약재의추출시수율과동일하도록각시료가혼합되었으나, 추출과정에서공존하는시료에의해상대적인 용출정도에차이를보였기때문으로추정된다. 또한추출수율은낮으나, 총페놀화합물의함량이가장많은상황버섯 (Lee et al., 2013) 의함량이가장높았던것도 MHE-2에서총페놀화합물및플라보노이드함량이높게정량된것과관련이있는것으로여겨진다. 2 라디칼소거활성생약재 5종의혼합조건을달리한조성물의라디칼소거활성은 Table 3과같다. 50~1000µg/mL 의농도범위에서시료의첨가농도가많아짐에따라라디칼소거활성은상승하였는데, DPPH 라디칼소거활성은두조성물모두 250~1000µg/mL 범위에서농도증가에따른소거활성의유의적인차이는없었으나, 모든농도에서 MHE-1 보다 MHE-2 에서더높은활성을보였다. ABTS 및 superoxide anion 라디칼의소거활성은시료의첨가농도에의존하여유의적으로상승하였다. ABTS 라디칼소거활성은 MHE-1 에서 27.53~98.35%, MHE-2 는 28.18~

7 Kang et al: Antioxidant and Anti-inflammatory Activity of Medicinal Herbs Composites 285 Table 4. Fe +2 chelating activity of the medicinal herbs composites Concentration(µg/mL) Sample code (%) MHE ±1.74 a 21.10±1.61 b 53.45±1.63 c 76.98±1.87 d 89.97±1.22 e MHE ±1.24 a 24.52±1.84 b* 62.89±1.78 c* 85.99±1.01 d* 95.75±1.13 e* Sample code refer to the foot note of Table 2. Each value represents mean±sd(n=3). a-e Means with different superscripts in the same row are significantly different at P<0.05. * Superscripts in the same column are significantly different among the different at each concentration by Student t-test at P< % 였으며, 특히 50~250µg/mL 농도에서 MHE-2 의활성이유의적으로높았으나, 500µg/mL 이상의농도에서는조성물간에유의차를보이지않았다. Superoxide anion 라디칼소거활성은모든농도범위에서 MHE-2 조성물이유의적으로활성이높았다. 약용식물류추출물의 superoxide anion 라디칼소거활성에서 DPPH 및 ABTS 라디칼소거활성과의상관관계가각각 및 0.851로높은관련성이있었다는보고 (Cho et al., 2007) 는본연구와도유사한결과였다. Lee et al.(2012) b 은와송과한약재복합조성물의 in vitro에서항산화활성차이가각구성물질의페놀화합물및플라보노이드함량에의존적인것으로보고한바있다. 또한 8종의한약재복합물에서금은화의첨가유무에따라항산화활성에차이를보인다는보고 (Kim et al., 2004) b 로볼때, 여러시료의복합물에서항산화활성은시료의혼합이나추출조건에따라달라지며, 시료중의특정물질은복합물의활성증가를위한보조제로써이용가능성이높은것으로추정되고있다. 본연구에서생약재의각추출물을혼합한조성물 (MHE-1) 보다는생약재의원재료를모두혼합하여추출한조성물 (MHE-2) 에서라디칼소거활성이더우수한것으로나타난것도식물류의유리라디칼소거활성이폴리페놀및플라보노이드화합물의함유량에의존적이며, 대부분의식물류는이들물질의함량에따라항산화활성이증가하는경향을보인다는보고 (Jung et al., 2004) 와잘일치한다. 3 Fe +2 킬레이팅활성조성물 MHE-1 과 MHE-2 의 Fe +2 킬레이팅활성을나타낸결과는 Table 4와같다. 시료의첨가농도가증가함에따라 Fe +2 킬레이팅활성은의존적으로높아졌으며, 50µg/mL 의농도에서는조성물간에유의적인차이가없었으나 100µg/mL 이상의농도에서는조성물간에유의적인차이로 MHE-2 의활성이더높아시료의농도가생리활성의발현에영향을주는요인임을알수있었다. 식물체의페놀화합물이나플라보노이드는만성질환의발병과병의진행을지연시키는데도움이되는데, 이들은생체내에서자유기와반응하여새로운자유기의생성을억제하거나, 지질산화를촉진하는 Fe +2 과같은금속이온과의결합을통해산화스트레스를억제한다는보고 (Ebrahimzadeh et al., 2008) 와함께식물체의 Fe +2 킬레이팅활성은생체내산화스트레스억제와관련이있는것으로알려져있다. 하지만 Fe +2 이존재하는반응계에서항산화활성은다소간감소되는것으로알려져있는데 (Kang, 2009), 본연구에서조성물의 Fe +2 킬레이팅활성이두드러지게나타난것으로보아본연구에사용된생약재조성물은생체내항산화작용에도효과적일것으로예상된다. 4 환원력조성물 MHE-1, MHE-2 의환원력은 Table 5와같이흡광도값으로나타낸결과시료의농도가높아질수록증가하는경향이었다. 50µg/mL 의농도에

8 286 Journal of Agriculture & Life Science 49(5) Table 5. Reducing power of the medicinal herbs composites Sample code Concentration(μg/mL) (Absorbance value at 700nm) MHE ±0.006 a 0.162±0.002 b 0.325±0.004 c 0.581±0.007 d 1.038±0.014 e MHE ±0.001 a 0.179±0.005 b* 0.363±0.006 c* 0.599±0.004 d* 1.107±0.022 e* Sample code refer to the foot note of Table 2. Each value represents mean±sd(n=3). a-e Means with different superscripts in the same row are significantly different at P<0.05. * Superscripts in the same column are significantly different among the different at each concentration by Student t-test at P<0.05. 서는두조성물간에유의차가없었으나 100µg/mL 이상의농도에서는 MHE-2 의환원력이유의적으로높았다. 더욱이조성물의농도가 50~100µg/mL 일때시료농도증가에따른환원력은약 1.4배증가한반면 500~1000µg/mL 에서환원력은약 1.8배증가하여시료의농도가높을경우환원력의증가폭은더높아지는경향이었다. Joo(2013) 는 1mg/mL 의농도에서황금추출물의환원력은 1.69정도로동시에실험된 10종의시료중높은수준이었으며, 시료의환원력은총페놀화합물및플라보노이드함량과높은상관관계인것으로보고한바있다. 3종의이소플라본화합물에마늘메탄올추출물을농도별로첨가하였을때환원력은이소플라본자체의환원력보다더증가하는경향으로채소류와마늘의병행섭취가항산화계에서더효과적이라는보고 (Kang, 2013) 로볼때본연구에사용된조성물은각각의단일물질에비해높은항산화활성을보였으며, 이는시료의혼합에따른시 너지효과로추정된다. 따라서본연구결과조성물 MHE-2 의항산화활성이높았던것은원료의혼합추출시상대적인수율의변화에따른유효물질의함량차이에의존적인것으로생각된다. 즉, 식물류혼합에의한조성물의항산화활성증가경향은조성물내에여러활성물질이공존됨에따른반응조건의상호작용에기인된다는보고 (Kim et al., 2004) a 와유사한결과라사료된다. 5 Xanthine oxidase 저해활성생약재조성물 MHE-1 및 MHE-2의 XO 저해활성을 250~5000µg/mL 농도범위에서측정한결과는 Table 6과같다. 시료의농도가증가할수록활성이유의적으로증가하는경향이었으나, 200~ 1000µg/mL 농도에서는 50% 미만의활성이었으며, 5000µg/mL 농도에서만 MHE-1과 MHE-2간에유의적인차이를보였다. Table 6. Xanthine oxidase inhibition activity of the medicinal herbs composites Concentration(µg/mL) Sample code (%) MHE ±0.40 a 17.38±1.42 b 29.79±0.21 c 63.87±1.03 d 87.45±0.95 e MHE ±1.17 a 20.07±0.65 b 30.38±0.70 c 64.89±0.84 d 93.43±0.46 e* Sample code refer to the foot note of Table 2. Each value represents mean±sd(n=5). a-e Means with different superscripts in the same row are significantly different at p<0.05. * Superscripts in the same column are significantly different among the different at each concentration by Student t-test at P<0.05.

9 Kang et al: Antioxidant and Anti-inflammatory Activity of Medicinal Herbs Composites 287 XO는 purine 대사에관여하는효소로서 xanthine 또는 hypoxanthine 의산소를떼어내면서과산화수소를생성하게되고, 나머지골격부분이 uric acid 를형성하여혈장내에과잉으로존재할때뼈에축적되어심한통증을유발하는통풍과신장에침착되어신장질환을일으키는효소로알려져있으며일종의항산화활성을측정하는방법으로도사용되고있다 (Cho et al., 2010). XO 저해활성은다양한탄닌류및페놀성물질이기여하는바가큰데 (Cho et al., 1993), 그중플라보노이드류는 hydroxy 기의수와위치에따라각종효소의저해효과가다르게나타나는것으로보고되어있다 (An et al., 1998). 본연구에서조성물을구성하는황금추출물의총페놀및플라보노이드함량은각각 mg/g 및 mg/g 으로보고된바있으며 (Joo, 2013), 단삼물추출물의페놀함량은 41.4mg/g 으로 16종의약용식물중가장높았으며 (Ju et al., 2006), 상황버섯에탄올추출물의페놀및플라보노이드함량은각각 mg/g 및 mg/g이었다는보고가있다 (Kim et al., 2008) a. 또한전보 (Lee et al., 2013) 에서 5종생약재의총페놀화합물함량에서상황버섯이 78.53mg/g, 황금은 68.82mg/g, 단삼은 75.42mg/g, 뽕잎은 53.16mg/g, 작약은 47.40mg/g 으로, 각추출물을동량으로혼합한조성물인 MHE-1 은각추출물에비해총페놀화합물및항산화활성이높게측정되었으며, 이러한결과는 XO 저해활성과도관련성이있는것으로사료된다. 6 세포생존율 CCK assay를이용하여 RAW264.7 대식세포에서생약재조성물 MHE-1 및 MHE-2 를농도별 (12.5, 25, 50, 100 및 200µg/mL) 로처리한결과 (Fig. 1), 100µg/mL 이하의농도에서세포생존율은 101.2~ 103.7% 의범위로대조군과비교하여 100% 이상의생존율을보여어떠한독성도없는것으로확인되었다. 200µg/mL 농도에서세포생존율은 MHE-1 처리시 86.7% 였고, MHE-2 처리시는 88.4% 로이후의실험에서는세포독성을나타내지않는 100µ Fig. 1. Effect of the medicinal herbs composites on cell viability in RAW264.7 cells. Sample codes refer to the foot note of Table 2. MHE-1 and MHE-2 were treated with various concentrations in RAW264.7 cells for 24hr. Each value represents the mean±sd of the determinations made in triplicate experiments. g/ml 이하의농도에서실험을수행하였다. 7 Nitric oxide 함량 NO 는 NO 합성효소에의해생성되는무기유리체 로면역반응, 세포독성, 신경전달계및혈관이완등 여러생물학적인과정에관여하며농도에따라세포 기능유지에중요한작용을하며세포독성및염증 유발에도중요한역할을하는것으로알려져있다 (Tang et al., 2004). RAW264.7 세포에 1µg/mL 의 LPS 단독처리구에 서 NO 생성량은 69.33µM 이었는데, 조성물의농도 가 25µg/mL 일때 MHE-1 처리구는 LPS 단독처리 구에비해유의적인감소를보였으나, MHE-2 처리 구는유의차를보이지않았다. 50µg/mL 일때 NO 생성량은 및 63.94µM 이었으며, 조성물의처 리농도가 100µg/mL 일때 MHE-1 과 MHE-2 처리 구의 NO 생성량은각각 46.24µM 과 47.51µM 로 LPS 단독처리구에비해유의적으로감소하였으나 두조성물간에유의차는보이지않았다 (Fig. 2). RAW264.7 세포에대한황금추출물의 NO 생성 저해효과는농도의존적으로 NO 의생성이감소되었

10 288 Journal of Agriculture & Life Science 49(5) Fig. 2. Effect of the medicinal herbs composites on NO production in RAW264.7 cells. Sample codes refer to the foot note of Table 2. RAW264.7( cells/ml) cells were pre-treated for 2hr with the MHE-1 and MHE-2(25, 50, 100µg/mL) before being stimulated with LPS(1 µg/ml) for 24hr. Each value represents the mean±sd of determinations made in triplicate experiments. Fig. 3. Effect of the medicinal herbs composites on PGE 2 production in RAW264.7 cells. Sample codes refer to the foot note of Table 2. RAW264.7( cells/ml) cells were pre-treated for 2hr with the MHE-1 and MHE-2(100µg/mL) before being stimulated with LPS(1µg/mL) for 24hr. Each value represents the mean±sd of determinations made in triplicate experiments. 으며 (Yoon et al., 2011), 작약에틸아세테이트추출물의경우 100µg/mL 의농도에서 LPS 처리구에비해 50% 이상의 NO 생성저해효과를보였으며 (Im & Lee, 2012), 단삼메탄올추출물은 300µg/mL 에서 95% 수준의저해활성이보고된바있다 (Yoon et al., 2007). 이러한연구결과로미루어볼때이들이모두혼합된조성물에서도높은 NO 생성저해활성이있을것으로기대되었으나, 50µg/mL 이상의농도에서만유의적인활성이확인되어전체반응계에일정이상의시료농도가유지되어야조성물의생리활성이발휘되는것으로여겨진다. 8 PGE 2 함량 RAW264.7 세포에생약재조성물 MHE-1 과 MHE-2 를 LPS와병행처리하였을때 PGE 2 의함량을측정한결과는 Fig. 3과같다. LPS 단독처리구의 PGE 2 생성량은 pg/mL 수준으로무처리구에비해월등히증가하였으며, MHE-1 과 MHE-2 를각각 100µg/mL 농도로처리하였을때 pg/mL 및 pg/mL 로유의적인감소를보였다. PGE 2 는 NO와더불어세포의분열이나증식을조 절하고, 염증성질환의병리기전에서가장중요한염증촉진물질이며, NO는백혈구가염증부위로이동하는초기단계에서주된역할을하는반면 PGE 2 는발열과통증이나타나는염증의후반부에주로작용하는것으로알려져있다 (Kwak & Lee, 2014). PGE 2 생성억제는 COX-2 효소의발현억제및 NO 의생성억제를통해조절되는데, 배암차즈기잎추출물의 PGE 2 저해효과에서 NO의생성량이감소함에따라동일한경향으로감소되었다는보고 (Jeong et al., 2012) 는본연구와도일치하는결과였다. 반면에본연구에서 100µg/mL 농도의조성물 MHE-1 과 MHE-2 처리시 LPS 단독처리구에비해 NO 생성억제정도가 33.3% 및 31.5% 였으나, PGE 2 생성억제는 14.5% 및 10.8% 에불과하였다. 9 Cytokine 함량 Cytokine 은염증을나타내는중요한지표로서 LPS 자극에의해염증반응이활성화된세포는 TNF-α, IL-1β 및 IL-6와같은전염증성 cytokine을생산하게된다 (Kim et al., 2011; Kim & Son, 2012). 혼합조건이다른생약재조성물 MHE-1과

11 Kang et al: Antioxidant and Anti-inflammatory Activity of Medicinal Herbs Composites 289 같다. LPS 처리로 RAW264.7 대식세포의 TNFα, IL-2 및 IL-6의생성량은무처리구에비해큰폭으로증가였는데, TNF-α 생성량은 LPS 처리구 (213.79pg/mL) 에비해 100µg/mL 의 MHE-1 과 MHE-2 처리시 56.67pg/mL 과 59.87pg/mL 로유의적으로감소하였으나, 두조성물간의유의차는없었다 (Fig. 4A). IL-6는림프계세포와골수성림프계세포에서생성되며, 감염이나손상등에급성반응을보이고, 면역반응에관여하는 cytokine 으로서 B와 T 림프구기능을조절한다 (Kim et al., 2009). RAW264.7 세포에 LPS를처리하였을때, IL-6의생성량은 pg/mL 로증가하였으나 100µg/mL 의농도로 MHE-1 과 MHE-2 를처리한결과각각 pg/mL 과 pg/mL 로유의적으로 IL-6의생성이억제되었다 (Fig. 4B). 또한 100µg/mL 농도로 MHE-1 과 MHE-2 처리시 IL-2 생성량은각각 34.83pg/mL 과 38.91pg/mL 으로 LPS 처리시 (76.01pg/mL) 에비해각각 54.2% 와 48.8% 의억제를보였다 (Fig. 4C). IL-6의생성억제는조성물간에유의차를보였으나, TNF-α 및 IL-2의경우에는조성물간에유의차를보이지않았다. 황금과작약으로구성된황금작약탕은 LPS로처 Fig. 4. Effect of the medicinal herbs composites on TNF-α(A), IL-2(B) and IL-6(C) production in RAW264.7 cells. Sample codes refer to the foot note of Table 2. RAW264.7( cells/ml) cells were pre-treated for 2hr with the MHE-1 and MHE-2(100µg/mL) before being stimulated with LPS(1µg/mL) for 24hr. Each value represents the mean±sd of determinations made in triplicate experiments. MHE-2 를 LPS 와병행하여 RAW264.7 세포에처 리후 cytokine 농도를측정한결과는 Fig. 4 와 리된 RAW264.7 세포에처리시 NO 생성량, PGE 2 및 IL-6의발현을강하게억제시켜염증억제에효과적인것으로보고된바있다 (Kim et al., 2013). 처방이유사한한약재복합물 2종에대한항산화및항염증활성의비교에서특정시료에의해두복합물모두항산화와항염증활성은효과적이었으나, 두활성이일치하지는않았다는보고 (Kim et al., 2012) a 는본연구결과와도유사한경향이었다. 본연구에서사용된조성물 MHE-2 는 MHE-1 에비해생리활성이높은것으로확인되었다. 식생활에이용되는대부분의식품은혼합형태이며, 특히한약재의경우단일원료의사용보다는여러원료의혼합후추출과정이수반되는데, 이때원료의종류나배합비율등의반응조건에따라유효성분의용출이상이한것으로보고되어있다 (Kim et al., 2004). 따라서복합조성물의제조시추출편이성

12 290 Journal of Agriculture & Life Science 49(5) 과기능성증대를위해원료의혼합후추출이시료내유효성분의상호작용에의한용출증대로생리활성증가에더효과적일것으로판단된다. 감사의글 본논문은농림수산식품부농림기술개발사업 ( ) 의수행에따른연구성과의일부이며이에감사드립니다. 참고문헌 An BJ, Lee JT and Bae MJ Isolation of a novel polyphenol from oolong tea and its effective prevention of the gout. Korean J. Food Sci. Technol. 30: Blois MS Antioxidant determination by the use of a stable free radical. Nature 181: Cho HE, Choi YJ and Cho EK Antioxidant and nitrite scavenging activity and α- glucosidase inhibitory effect of water extract from Schzandra chinensis Baillon. J. Korean Soc. Food Sci. Nutr. 39: Cho HS, Lee SJ, Shin JH, Kang MJ, Cho HS, Lee HJ and Sung NJ Antioxidative activity and nitrite scavenging effect of the composites containing medicinal plant extracts. J. Life Sci. 17: Cho YC, An BJ and Choi C Isolation and enzyme inhibition of tannins from Korean green tea. J. Korean Biochem. 26: Droge W Free radicals in the physiological control of cell function. Physiol. Rev. 82: Ebrahimzadeh MA, Pourmorad F and Bekhradnia AR Iron chelating activity, phenol and flavonoid content of some medicinal plants from Iran. Afr. J. Biotechnol. 7: Gutfinger T Polyphenols in olive oil. J. Am. Oil Chem. Soc. 58: Heo HJ, Kim YJ, Chung D and Kim DO Antioxidant capacities of individual and combined phenolics in a model system. Food Chem. 104: Im DY and Lee KI Nitric oxide production inhibitory effect and antibacterial activity of the extract and fractions from Paeoniae Radix. Kor. J. Pharmacogn. 43: Jang GY, Kim HY, Lee SH, Kang YK, Hwang IG, Woo KS, Kang TS, Lee JS and Jeong HS Effects of heat treatment and extraction method on antioxidant activity of several medicinal plants. J. Korean Soc. Food Sci. Nutr. 41: Jeong HR, Sung MS, Kim YH, Ham HM, Choi YM and Lee JS Anti-inflammatory activity of Salvia plebeia R. Br. leaf through heme oxygenase-1 induction in LPS-stimulated RAW264.7 macrophages. J. Korean Soc. Food Sci. Nutr. 41: Jia Z, Tang M and Wu J The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 64: Joo SY Antioxidant activities of medicinal plant extracts. J. Korean Soc. Food Sci. Nutr. 42: Ju JC, Shin JH, Lee SJ, Cho HS and Sung NJ Antioxidative activity of hot water extracts from medicinal plants. J. Korean Soc. Food Sci. Nutr. 35: Jung SH, Kim SJ, Jun BG, Lee KT, Hong SP, Oh MS, Jang DS and Choi JH α- Cyperone, isolated from the rhizomes of Cyperus rotundus, inhibits LPS-induced COX-2 expression and PGE 2 production through the negative regulation of NFκ

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