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1 Korean Society for Biotechnology and Bioengineering Journal 30(1): (2015) ISSN / eissn 연구논문 Lactobacillus casei 로발효한톳추출물의항염증활성 문옥주 1, 권명숙 1, 배민주 1, 안별님 2, Fatih Karadeniz 1,3, 김미향 1, 이상현 4, 유기환 5, 김육용 5, 서영완 6, 공창숙 1 * Anti-inflammatory Activity of Hizikia fusiformis Extracts Fermented with Lactobacillus casei in LPS-stimulated RAW Macrophages Ok-Ju Mun 1, Myeong Sook Kwon 1, Min Joo Bae 1, Byul-Nim Ahn 2, Fatih Karadeniz 1,3, Mihyang Kim 1, Sang-Hyeon Lee 4, Ki Hwan Yu 5, Yuck Yong Kim 5, Youngwan Seo 6, and Chang-Suk Kong 1 * Received: 29 January 2015 / Accepted: 24 February The Korean Society for Biotechnology and Bioengineering 1 신라대학교의생명과학대학식품영양학과 1 Department of Food and Nutrition, College of Medical and Life Science, Silla University, Busan , Korea Tel: , Fax: cskong@silla.ac.kr 2 부산대학교공과대학유기소재시스템공학과 2 Department of Organic Material Science and Engineering, Pusan National University, Busan, Korea 3 신라대학교의생명과학대학해양신의약소재융합기술연구소 3 Marine Biotechnology Center for Pharmaceuticals and Foods, College of Medical and Life Science, Silla University, Busan , Korea 4 신라대학교의생명과학대학제약공학과 4 Department of Pharmaceutical Engineering, Silla University, Busan , Korea 5 ( 주 ) 아이에스푸드 5 IS Food CO., Marine Bio-industry Department Center, Busan , Korea 6 한국해양대학교해양생명과학부 6 Division of Marine Bioscience, Korea Maritime and Ocean University, Busan , Korea Abstract: In this study, we investigated the anti-inflammatory effects of fermented Hizikia fusiformis extracts in lipopolysaccharide (LPS)-stimulated RAW mouse macrophages. The fermentation was performed using Lactobacillus casei in mixture of carbon source at 30 o C for 30 days. The sample groups were prepared with/without L. casei group in order to demonstrate the anti-inflammatory activity of fermented H. fusiformis in regard to lactic acid bacteria. As a result, we confirmed the inhibitory effect of H. fusiformis extracts on LPS-stimulated NO production and expression of TNFα, while it had no regulatory effect on the expression of inos, COX-2, IL-1β and IL-6 as important inflammatory factors. However, L. casei fermented group significantly suppressed the expression of the above factors. In particular, the difference between the two groups in the matter of mrna expression of inos, which is directly associated with NO production, indicated that the fermentation with lactic acid bacteria effectively suppressed NO production by regulating inos expression. Also, effective suppression of pro-inflammatory cytokines showed that the fermentation using L. casei may provide an increment towards extraction of active ingredients that are effective anti-inflammatory agents. Keywords: Hizikia fusiformis, Lactobacillus casei, Inflammation, Fermentation 1. INTRODUCTION 염증반응이란외부로부터의병원균에의한감염, 조직의손상및물리화학적으로유해한자극에대항하는생체방어체계로, 체내에외부물질의유입이감지되었을때면역세포는염증매개물질들을분비하여체내면역반응을일으키는신호를전달하게된다 [1]. 염증반응은우리몸을보호하기위

2 Lactobacillus casei 로발효한톳추출물의항염증활성 39 한면역체계반응이지만지속적인염증반응의진행은세포조직의손상을초래하여암, 뇌질환, 알츠하이머병, 관절염, 천식, 염증성장질환및동맥경화등여러질병뿐만아니라노화의직 간접적원인이되는것으로알려져있다 [2-4]. 대식세포는염증반응에관여하는주요세포로손상부위로의면역세포의이동과 Tumor necrosis factor (TNF)-α, Interleukin (IL)-1β 및 IL-6 등의 pro-inflammatory cytokine 분비에관여하는등매우중요한역할을한다 [1,3,5-7]. 즉, 대식세포의사이토카인에의한활성화는 pro-inflammatory cytokine, Prostaglandin E2 (PGE2) 및 Nitric oxide (NO) 등을생성함으로써염증성반응의유발과염증부위로의면역세포의이동을촉진하게된다 [1]. 이중 NO 는염증반응의대표적인지표물질로체내에서 NO synthase (NOS) 촉매반응을통하여 L- arginine 으로부터생성되며 [7], inducible NOS (inos) 및 Cyclooxygenase-2 (COX-2) 가과도하게발현될경우다량의 NO 를생성하게된다. 대식세포는그람음성균의세포벽을구성하는내독소작용물질인 Lipopolysaccharide (LPS) 의자극에의해 inos 의발현이급격하게증가하는것으로알려져있다 [5,6]. 따라서항염증활성및염증매개인자들의발현을조절하는물질에대한연구는 LPS 를자극한대식세포를이용하여진행되고있다. 해조류는독특한화학적구조의화합물을함유하고있으며풍부한영양성분을가지고있어생리활성에대한연구가많이진행되고있으며 [8], 해조류의유용성분을추출하기위한방법으로주로열수및에탄올추출법을이용하고있다 [9]. 그러나해조류에포함되어있는탄수화물의대부분이비소화성복합다당류로서이러한기존추출방법으로는유용성분을다량추출해내는데한계점이있다 [10]. 이러한단점을극복하기위한방법으로효소처리법과유용미생물을이용한발효법으로유용성분을추출하고자하는시도가행해지고있다 [9,11-13]. 특히, 발효는미생물의대사활동에의해유용성분의추출효율성을극대화시키는동시에다양한효능을가지는물질을생성할수있는것으로알려져있다 [11]. Lactobacillus casei 는주로치즈생산에이용되는유산균으로탄수화물을분해시키는효소인아밀라아제 (amylase) 를생산하며다당성분의처리에의해기능성해조올리고당이생성된다고알려져있어탄소원으로포도당을첨가하여유용성분의추출률을높이는데이용되고있다 [9]. 따라서본연구에서는 L. casei 로발효시킨톳추출물을이용하여 LPS 로염증을유도한 RAW 대식세포에서 NO 생성억제와염증관련인자의발현에미치는영향을검토하여발효톳의항염증소재로서의이용가능성을알아보고자하였다. 2. MATERIALS AND METHOD (Jeju, Korea) 에서구입하여사용하였으며, 탈염된톳건조물을초미세기류식분쇄기 (KMS-200, Korea) 로분쇄한다음밀봉하여 4 o C 에보관하면서사용하였다 발효균주발효균주로는 Lactobacillus casei (( 주 ) 메디오젠, Jecheon, Korea) 를실험에사용하였다. L. casei 는유산균선택배지인 MRS 액체배지 (Difco, USA) 에배양하였고, 멸균된 80% glycerol (Duchefa Biochemie, Netherlands) 에배양액을현탁하여 -80 o C deep freezer (NF-140SF, HFC, Japan) 에서장기보관하였다 발효균주를이용한톳발효액의제조발효균인 L. casei 는 MRS 액체배지를이용하여 30 o C 에서정치배양하였다. 발효액은이전연구방법에따라제조하였다 [9]. 톳분말 10 g 에 5% (w/v) 로되게 ddh 2 O 를첨가한후, 탄소원으로 1% glucose 의농도로첨가하여 121 o C 의가압조건에서 30 분간멸균하였다. 여기에발효균을 0.05% 의농도로접종한후 30 o C 배양기에서 30 일간배양하여톳발효액을제조하였다 발효톳추출물의제조발효가완료된톳발효액을진공농축기 (R-200, Buchi, Switzerland) 를이용하여농축시킨후, 동결건조 (FD8518, Ilshin BioBae, Korea) 하여발효톳건조시료를얻었다. 건조시료에 10 배량 (w/v) 의 70% 에탄올을첨가한후, 80 o C 에서 4 시간씩 2 회추출을수행하였으며진공농축기를이용하여농축시켜추출물을얻었다 세포배양및세포독성실험마우스대식세포인 RAW 264.7세포는 10% fetal bovine serum (FBS, Atlas, Fort Collins, CO, USA) 및 1% penicillin (Sigma- Aldrich, St. Lousi, MO, USA) 을포함한 phenol red-free Dulbecco's modified Eagle medium (DMEM, Gibco-BRL, Gaithersburg, MD, USA) 배지를사용하여 37 o C, 5% CO 2 조건에서배양하였다. 세포독성은 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay를이용하여측정하였다. RAW cells을 cells/well이되도록 96-well plates에분주하고 24시간동안배양한후, 농도별로시료를처리하여 24시간동안배양하였다. MTT (1 mg/ml) 용액을가하고동일한배양조건에서 4시간더배양하였다. 이때생성된 formazan crystal은 DMSO에녹여서 ELISA Reader (Multiskan GO, Thermo Scientific, Finland) 를이용하여 540 nm에서흡광도를측정하여세포생존율 (%) 을다음과같이계산하였다 실험재료본실험에사용한톳 (Hizikia fusiformis) 은 2013 년파라제주 Cytotoxicity (%) = 대조군의흡광도 시료처리군의흡광도 100 대조군의흡광도

3 40 Korean Society for Biotechnology and Bioengineering Journal 30(1): (2015) 2.6. Nitric oxide (NO) 생성억제능 RAW 세포를 96-well plates 에 cells/well 로분주하여 24 시간동안배양한후, 시료를농도별로처리하였으며 NO 생성을유도하기위해 LPS (1 µg/ml) 를처리하여 24 시간동안배양하였다. 세포배양액을 Griess reagent (Sigma- Aldrich, St. Lousi, MO, USA) 와 1:1 로혼합하여 15 분동안실온에방치한후, ELISA Reader (Multiskan GO, Thermo Scientific, Finland) 를이용하여 540 nm 에서흡광도를측정하였다. 생성된 NO 양은표준물질인 sodium nitrate (NaNO 2 ) 를표준용액으로사용하여계산하였다 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 상기와동일한조건에서배양한 RAW 세포를 Trizol reagent (Invitrogen Co., CA, USA) 를이용하여 total RNA를추출, 정량하였다. 분리된 RNA (2 µg) 는 37 o C 에서 1시간동안역전사반응을실시하여 cdna를합성하였다. 합성된 cdna에 COX-2, inos, IL-1β, IL-6와 TNFα primer를첨가 Table 1. Sequences of primers used for RT-PCR Gene Direction Sequence COX-2 Forward 5'-AGA-AGG-AAA-TGG-CTG-CAG-AA-3' Reverse 5'-GCT-CGG-CTT-CCA-GTA-TTG-AG-3' inos Forward 5'-TTC-CAG-AAT-CCC-TGG-ACA-AG-3' Reverse 5'-TGG-TCA-AAC-TCT-TGG-GGT-TC-3' IL-1β Forward 5'-GGG-CCT-CAA-AGG-AAA-GAA-TC-3' Reverse 5'-TAC-CAG-TTG-GGG-AAC-TCT-GC-3' IL-6 Forward 5'-AGT-TGC-CTT-CTT-GGG-ACT-GA-3' Reverse 5'-CAG-AAT-TGC-CAT-TGC-ACA-AC-3' TNFα Forward 5'-AGC-CCC-CAG-TCT-GTA-TCC-TT-3' Reverse 5'-CAT-TCG-AGG-CTC-CAG-TGA-AT-3' β-actin Forward 5'-CCA-CAG-CTG-AGA-GGG-AAA-TC-3' Reverse 5'-AAG-GAA-GGC-TGG-AAA-AGA-GC-3' 하였으며 internal control 로 β-actin 을사용하였다 (Table 1). T100 Thermal Cycler (Bio-Rad, CA, USA) 을이용하여 95 o C 에서 2 분간 denaturation 시킨뒤, 95 o C 에서 30 초간 Denaturation, 60 o C 에서 45 초간 Annealing, 72 o C 에서 1 분간 Extension 과정을 30 번반복함으로써증폭시킨후마지막으로 72 o C 에서 5 분간 Extension 한후에 4 o C 에서종결하였다. PCR 산물은 TAE buffer (Biosesang, Korea) 를전해질로사용한 1.5% agarose gel 을이용하여 10 분간전기영동한후, Ethidium bromide (EtBr, AMRESCO, USA) 용액에담궈 30 분동안염색한후 UV (Davinch-Chemi imager, CAS-400SM, Wako Co., Osaka, Japan) 하에서확인하였다 통계처리실험결과는평균 표준편차 (Mean Standard deviation, SD) 로표시하였으며, SPSS + /WIN 12.0 (Statistical Package for Social Science, version 12.0) 통계프로그램을이용하여통계적유의성을검토하였다. 집단간의유의성을검정하기위해일원배치분산분석 (Oneway Analysis of Variance: ANOVA) 을통해분석하였고, 사후검증은 Duncan's multiple range test 를실시하여 p<0.05 수준에서검증하였다. 3. RESULTS AND DISCUSSION 3.1. 발효톳추출물의 RAW264.7 세포에대한독성측정발효톳에탄올추출물의 RAW 세포에대한세포독성은 MTT assay 를통하여확인하였다 (Fig. 1(a)). RAW 세포에발효톳에탄올추출물을농도별 (10, 100, 500 µg/ml) 로처리한결과, L. casei 접종군및 L. casei 무접종군모두 90% 이상의생존율을보여 RAW 세포에대한독성이나타나지않음을확인하였으며, 이농도에서발효톳추출물의염 Fig. 1. Effect of ethanol extracts from fermented H. fusiformis with/without L. casei on cell viability (a) and NO level (b) in LPSstimulated RAW macrophages. The cells were pretreated with fermented H. fusiformis for 1 h and following by treating with LPS (1 µg/ml) for 24 h. The nitrite content of culture media was analyzed. a-d Means with the different letters in the each sample are significantly different (p<0.05) by Duncan's multiple range test. *p<0.01.

Lactobacillus casei 로발효한톳추출물의항염증활성 41 Fig. 2. Effect of ethanol extracts from fermented H. fusiformis with/without L. casei on COX-2 and inos in LPS-stimulated RAW 264.7 macrophages.

4 Lactobacillus casei 로발효한톳추출물의항염증활성 41 Fig. 2. Effect of ethanol extracts from fermented H. fusiformis with/without L. casei on COX-2 and inos in LPS-stimulated RAW macrophages. The cells were pre-treated with different concentration of the fermented H. fusiformis for 1 h followed by stimulated with LPS (1 µg/ml) for 24 h. a-e Means with the different letters are significantly different in each gene (p<0.05) by Duncan's multiple range test. 증억제활성을확인하기위한실험을진행하였다 발효톳추출물이 LPS 에의해유도된 NO 생성에미치는영향 NO 는 L-arginine 을기질로하여 NOS 에의해서합성되며 NO 가과량으로생성되면염증반응을촉진시키고조직및신경을손상시키는것으로알려져있다 [14]. 발효톳추출물의 NO 생성억제활성을알아보기위해 RAW 세포에서 LPS 로자극을유도하고시료를농도별 (10, 100, 500 µg/ml) 로처리하여 24 시간후 NO 생성량을측정하였다 (Fig. 1(b)). LPS 단독처리군에비하여 L. casei 접종군과 L. casei 무접종군모두 NO 생성을저해하였다. 특히, L. casei 접종군은 L. casei 무접종군에비해 10, 100, 500 ìg/ml 의처리농도에서각각 1.4 배, 1.3 배, 2.1 배의 NO 생성저해효과를보였다 발효톳추출물의 inos 와 COX-2 발현에미치는영향 inos 와 COX-2 는염증유발에관여하는유전자로대식세포에서 LPS 자극에의해유도된염증이 NO 를포함한다량의염증매개체를생합성하는것으로알려져있다. 세종류의 NOS 에서 neuronla NOS (nnos) 와 endothelial NOS (enos) 는세포내에서항상발현이유지되지만 inos 는 LPS 와 proinflammatory cytokine 에의해발현이증가한다. inos 에의해 NO 가과량증가되어염증성질환을유발한다. COX 는염증매개물질인 prostaglandins 분비를자극하는효소이다. COX-1 은정상적인생체기능에적용되어인체에유익한 prostaglandins 형성에관여하는효소인반면, COX-2 는염증유발자극인자들에의해활성화되어통증과염증을유발시키는 prostaglandins 의분비에작용한다 [14-16]. 발효톳에탄올추출물의 NO 생성억제효과가염증관련유전자의발현 조절과관련이있는지확인하기위하여 RT-PCR 을수행하였다 (Fig. 2). 유전자발현에큰변화를보이지않은 L. casei 무접종군에비하여 L. casei 접종군은 COX-2 의발현을농도의존적으로저해하였으며, 특히 500 µg/ml 의농도에서 inos 와 COX-2 의발현을현저히억제하였다 발효톳추출물이 pro-inflammatory cytokine 발현에미치는영향 TNFα, IL-1β 및 IL-6 는대표적인염증성 cytokine 으로 LPS 로유도된대식세포에서과도하게생성되어발열, 조직을손상시키고염증반응을촉진한다 [17]. 이들중 TNFα 는 endotoxin shock 에관련된중요한인자로과발현되면치명적인독성을유발하여패혈증의원인이되는것으로알려지고있다 [18]. 발효톳에탄올추출물이 pro-inflammatory cytokine 의유전자발현에미치는효과를 RT-PCR 을통하여확인하였다 (Fig. 3). L. casei 무접종군은 IL-1β 와 IL-6 발현은억제하지못하였지만 TNFα 발현은유의적으로감소시켰다. 반면, L. casei 접종군은 TNFα, IL-1β 와 IL-6 발현모두농도의존적으로저해하였으며, 500 µg/ml 농도에서는 L. casei 무접종군에비해 L. casei 접종군의 TNFα, IL-1β 와 IL-6 발현이현저히억제되어발효톳에탄올추출물이 pro-inflammatory cytokine 생성을저해하는것을확인하였다. 4. CONCLUSION 톳의분해성을높여유용성분의활성을증진시키고자유산균 L. casei 을이용한발효공정을도입하여유산균발효에따른항염증활성을 L. casei 접종군과 L. casei 무접종군으로나

42 Korean Society for Biotechnology and Bioengineering Journal 30(1): 38-43 (2015) Fig. 3. Effect of ethanol extracts from fermented H. fusiformis with/without L.

5 42 Korean Society for Biotechnology and Bioengineering Journal 30(1): (2015) Fig. 3. Effect of ethanol extracts from fermented H. fusiformis with/without L. casei on TNFα, IL-1β and IL-6 in LPS-stimulated RAW macrophages. The cells were pre-treated with different concentration of the fermented H. fusiformis for 1 h followed by stimulated with LPS (1 µg/ml) for 24 h. a-d Means with the different letters are significantly different in each gene (p<0.05) by Duncan's multiple range test. 누어비교하였다. 그결과, L. casei 무접종군은 LPS 에의해유도된 NO 생성과 TNFα 의발현을억제하여톳발효추출물그자체로도염증억제효과를보임을확인하였다. 하지만중요한염증유발인자인 inos 와 COX-2 및 IL-1β, IL-6 의발현조절에는효과가없었던반면, L. casei 접종군은위인자들의발현을유의적으로저해시켰다. 특히, NO 생성과직접적으로연관된 inos 의발현이두그룹간큰차이를보인점은유산균발효가전사수준에서 inos 유전자를조절하여 NO 생성을효과적으로억제한다는것을시사한다. 또한, proinflammatory cytokine 에보인뛰어난억제효과는 L. casei 를이용한발효가염증억제에효과가있는유효성분의추출률을높여줄수있음을보여준다. 본연구를바탕으로향후개선된발효조건을확립하여항염증활성물질개발연구에기초자료로활용할수있을것으로기대되며, 톳발효추출물을기능성소재로개발하기위해서항염증억제기전에대한추가적인연구가필요할것으로사료된다. Acknowledgements 본연구는교육과학기술부와한국연구재단의지역혁신인력양성사업으로수행된연구결과이며이에감사드립니다 (No. NRF-2013H1B8A ). REFERENCES 1. Kim, Y. S., S. J. Lee, J. W. Hwang, E. H. Kim, P. J. Park, and J. H. Jeong (2012) Anti-inflammatory effects of extracts from Ligustrum ovalifolium H. leaves on RAW macrophages. J. Korean Soc. Food Sci. Nutr. 41: Park, S. H., J. I. Kim, Y. K. Jeong, and Y. H. Choi (2011) Extracts of Allium fistulosum attenuate pro-inflammatory action in the lipopolysaccharide-stimulated BV2 microglia cells. Korean J. Life Sci. 21: Kim, Y. J. (2011) Anti-inflammatory effect and mechanism of an extract from Anethum graveloens flowers. MS Thesis. Yon-Sei University, Seoul, South Korea. 4. Jin, K. S., J. Y. Lee, H. J. Kwon, and B. W. Kim (2014) Antioxidative and anti-inflammatory activities of Ardisia arborescens ethanol extract. Korean J. Life Sci. 24: Cho, W. I. (2010) Anti-inflammatory effect of Houttuynia cordata extract in lipopolysaccharide-induced Raw Cells. MS Thesis. Chung-Ang University, Seoul, South Korea 6. Jung, Y. S., C. S. Eun, Y. T. Jung, H. J. Kim, and M. H. Yu (2013) Anti-inflammatory effects of Picrasma quassioides (D.DON) BENN leaves extracts. Korean J. Life Sci. 23: Kang, C. H., S. H. Han, and J. S. So (2013) Anti-inflammatory effect of chloroform extract from Potentilla chinensis. Korean Soc. Biotechnol. Bioeng. J. 28: Jeon, M. R. and S. H. Choi (2012) Quality characteristics of pork patties added with seaweed powder. Korean J. Food Sci. Ani. Resour. 32: Park, S. H., S. J. Lee, M. J. Jeon, S. Y. Kim, O. J. Mun, M. H. Kim, C. S. Kong, D. G. Lee, K. H. Yu, Y. Y. Kim, and S. H. Lee (2014) Evaluation of biological activities of fermented Hizikia fusiformis extracts. Korean J. Life Sci. 24: Song, H. S., H. K. Kim, H. O. Min, J. D. Choi, and Y. M. Kim (2011) Changes in physicochemical and sensory properties of Hizikia fusiforme water extract by fermentation of lactic acid bacteria. Korean J. Fish Aquat. Sci. 44:

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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,