Journal of Nutrition and Health (J Nutr Health) 2019; 52(3): 243 ~ 249 https://doi.org/10.4163/jnh.2019.52.3.243 eissn 2288-3959 Research Article 홍도라지추출물이마우스에서분리한비장세포에서 lipopolysaccharide 로유도된염증에미치는영향 * 박은정 1, 이유숙 1, 정현철 2, 이성현 3, 이해정 1 1 가천대학교식품영양학과, 2 SK 바이오랜드식품연구소, 3 농촌진흥청국립농업과학원 Mitigation effects of red Platycodon grandiflorum extract on lipopolysaccharide-induced inflammation in splenocytes isolated from mice* Eun-Jung Park 1, You-Suk Lee 1, Hyun Cheol Jeong 2, Sung-Hyen Lee 3 and Hae-Jeung Lee 1 1 Department of Food and Nutrition, Gachon University, Seongnam, Gyeonggi 13120, Korea 2 Food R&D Center, SK Bioland Co., Ltd, Ansan, Gyeonggi 15407, Korea 3 National Institute of Agricultural Sciences, Rural Department Administration, Wanju, Jeonbuk 55365, Korea ABSTRACT Purpose: Platycodon grandiflorum (PG) is known to have effective antimicrobial and anticancer activity. The main bioactive components of PG are saponins, and these could contribute to anti-inflammatory activity. However, little is known about the anti-inflammatory effect of PG. In this study, we aim to assess the anti-inflammatory response to Red PG Extract (RPGE) in splenocytes under ex vivo conditions. Methods: The cell viability of isolated splenocytes taken from mice was analyzed by performing a Cell Counting Kit-8 assay. The productions of nitric oxide (NO) and cytokines (specifically interleukin-6 (IL-6) and interleukin-10 (IL-10)) were measured utilizing Griess reagent and ELISA, respectively. Results: We found that co-treatment with RPGE and Lipopolysaccharide (LPS) decreased isolated splenocyte proliferation as compared with that of the LPS-stimulated control. We also observed that RPGE markedly suppressed NO synthesis and IL-6 production that was induced by LPS. There were no significant differences of IL-10 production between co-treatment with RPGE plus LPS and treatment with LPS alone. Conclusion: When taken together, our data has shown that RPGE mitigates LPS-induced inflammation in splenocytes isolated from mice. Further research is surely needed to confirm the anti-inflammation effects of RPGE in an in vivo model. KEY WORDS: Platycodon grandiflorum, anti-inflammatory, splenocytes 서론 면역은세균이나바이러스같은내부및외부병원성물질로부터신체를보호하는인체방어시스템이다 [1]. 최근전국적으로메르스, 독감등과같은전염성질병과천식, 크론병과같은알레르기성질환이증가추세에있어 [2] 연령과성별의구분없이면역에대한관심이증가하고있다. 염증은감염또는손상에대한숙주의초기면역반응이며 [3] 문제가된자극을제거하고, 회복을촉진하며면역계가기억하게하여추후같은감염이일어났을때숙주가 빠른대처를할수있게한다 [4]. 염증반응은대식세포, 림파구등면역세포의활성화를일으켜사이토카인및케모카인등을분비하는것으로시작된다 [5]. 염증은숙주의건강에필수적이나, 염증매개체의과잉생산은많은급성및만성염증성질환 ( 관절염, 암, 뇌졸중, 천식등 ) 의병인을유발할수있다 [6]. 비장은초기면역반응을담당하는주요말초면역기관으로, 가로막아래복부의왼쪽윗부분에위치하고있다 [7]. 비장은혈액유래항원에대한면역반응을하며, 노화된적혈구및손상된세포를제거하는역할을수행한다 Received: May 13, 2019 / Revised: June 3, 2019 / Accepted: June 3, 2019 * This work was carried out with the support of Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01381002) Rural Development Administration, Republic of Korea. To whom correspondence should be addressed. tel: +82-31-750-5968, e-mail: skysea@gachon.ac.kr 2019 The Korean Nutrition Society This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons. org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
244 / 홍도라지추출물염증완화 [8]. 비장세포는주로 T림프구, B림프구, 대식세포와같은다양한면역세포로구성되어있어 [8] 항염증연구에적합하게이용되고있다. Lipopolysaccharide (LPS) 는그람음성박테리아의외막의주요구성요소로포유류의면역계에중대한영향을미친다 [9]. LPS는선천면역반응을활성화시켜 interleukin-6 (IL-6) 와같은 pro-inflammatory cytokine을방출시키는데, 이는염증을촉진하여숙주에게큰이익을준다 [10]. 하지만앞서설명하였듯이사이토카인의과다분비는천식, 아토피피부염, 호산구식도염과같은다양한질병의주요원인이될수있으므로 [11,12] LPS는면역반응의증폭메커니즘연구뿐만아니라, 다양한항염증반응연구에사용되어져왔다 [13]. LPS로유도된과도한염증반응을조절하기위하여기능성을가진다양한천연물들이연구되고있다 [5]. 현재식품의약품안전처인정을받은천연물유래과민면역반응개선에도움을주는건강기능식품은구아바잎추출물등복합물, 다래추출물, 피카오프레토분말등복합물, 소엽추출물의 4종이다. 도라지는인삼과마찬가지로사포닌함량이풍부해예로부터항균, 항암등에효과가있는것으로알려져있으며, 중국, 일본, 우리나라에서만유일하게자생하는식물로전국각지에서야생하여다년생약용으로사용되어왔다 [14,15]. 도라지의항염증효과에대한기능성연구는미흡한실정이나, 도라지의유효성분인 platycodin D는 LPS와 interferon-γ에의하여활성화된 RAW 264.7 대식세포에서 nitric oxide (NO) 의생성과 tumor necrosis factor alpha (TNF-α) 의분비를억제하여항염증효과의가능성을보였다 [16]. 하지만, 현재까지의연구에서비장세포를활용한도라지의항염증효과를평가한연구는진행된바없다. 기능성식품산업에서는원물소재의기능성분을증가시키기위한다양한가공방법을시도하고있다 [17]. 홍도라지는고려도경의고문헌에서언급된바와같이인삼을말리고찌는과정을반복한홍삼의가공방법과유사하게제조하며붉은색이특징이다 [18]. 따라서본연구에서는홍도라지추출물 (red Platycodon grandiflorum extract, RPGE) 의항염증효능을알아보기위하여 LPS로활성화된마우스비장세포에서 NO 생성및염증관련사이토카인을측정하였다. 연구방법 실험재료실험에사용된홍도라지추출물은생산관리기준에따 라엄격히관리및생산하고있는 SK바이오랜드 (Ansan, Gyeonggi, Korea) 에서제공받았다. 국내산생도라지뿌리를 2회세척하여 120분동안증숙하고, 이를 24시간건조하여다시 90분증숙하는것을 9번반복한뒤, 72시간건조하여홍도라지를제조하였다. 홍도라지원료 10 kg과 50% 주정을원료대비 15배수첨가하여 80 C에서 8시간동안추출하였다. 1차추출액을회수하고 50% 주정으로 80 C에서 8시간동안 2차추출하였다. 추출액을모두혼합하여필터프레스여과하였다. 이를고형분 60% 이상감압농축하고살균하였다. 추출물은 dimethyl sulfoxide를용매로 100 mg/ml stock으로제조하여각실험방법에제시한농도를최종농도 (working concentration) 로사용하였다. HPLC 분석홍도라지추출물의유효성분인 platycodin D를 HPLC (High Performance Liquid Chromatography)-ELSD (Evaporative Light Scattering Detector, Waters 2424 ELSD, Waters, Milford, MA, USA) 로분석하였다. 컬럼은 Sunfire C18 (3.5 μm, 4.6 mm 150 mm, Waters, Milford, MA, USA) 을사용하였고, 컬럼온도는 25 C로설정하였으며, 이동상으로는 0.0015% formic acid (Solvent A, Sigma Aldrich, St. Louis, MO, USA) 와아세토니트릴 (Solvent B, Burdick & Jackson, Morristown, NJ, USA), 메탄올 (Solvent C, Burdick & Jackson, Morristown, NJ, USA) 을사용하여 0.6 ml/min 유속으로시료 20 μl를주입하여 Table 1에나타낸 gradient 조건으로분석하였다. 비장세포분리 5주령의마우스는 Orient Bio (Seongnam, Gyeonggi, Korea) 에서구입하여무균적으로비장을적출하여 RPMI 1640 (Gibco, Grand Island, NY, USA) 배지로씻어준뒤분 Table 1. Gradient mobile phase conditions Time (min) Solvent A (%) Solvent B (%) Solvent C (%) 0 75 20 5 10 75 20 5 17 72.5 22.5 5 25 72.5 22.5 5 34 71 24 5 42 71 24 5 52 70 25 5 60 70 25 5 67.5 0 50 50 77.5 0 50 50 82.5 75 20 5 90 75 20 5
Journal of Nutrition and Health (J Nutr Health) 2019; 52(3): 243 ~ 249 / 245 쇄한세포를 200 μm cell strainer (BD Biosciences, San Jose, CA, USA) 로여과하고원심분리 (4 C, 3,000 rpm, 10 min) 하였다. 상층액을제거한세포에적혈구제거를위하여 lysing buffer를넣고 5분후원심분리 (4 C, 3,000 rpm, 10 min) 하였다. RPMI 1640 배지로세척하여원심분리 (4 C, 3,000 rpm, 10 min) 하여비장세포를얻었다. 본연구에사용된동물실험은국립농업과학원동물실험윤리위원회의승인 (NAS-201807) 후수행하였다. 비장세포증식능측정마우스비장세포를 10% FBS RPMI 1640 (Gibco, Grand Island, NY, USA) 배지에 5.0 10 6 cells/ml의농도로 96-well plate에 well 당 100 μl 씩분주한뒤, 홍도라지추출물 0.8, 4, 20, 100, 500 μg/ml 단독또는 LPS (1 μg/ml, Sigma-Aldrich, St. Louis, MO, USA) 과홍도라지추출물을함께처리하였다. 24시간동안 37 C, 5% CO 2 조건에서배양후에각 well에 Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Tokyo, Japan) 용액 10 μl 가하여 2시간동안다시배양하고 Microplate reader (Epoch Microplate Spectrophotometer, Biotek Inc., Winooski, VT, USA) 로 450 nm의파장에서흡광도를측정하였다. 통계분석통계분석은 Graphpad Prism 5 (Graphpad software, San Diego, CA, USA) 를이용하였다. 실험결과는평균 ± 표준오차 (mean ± SEM) 로표시하였으며, One-way ANOVA 를수행하여사후검증은 Tukey test로하였고, p < 0.05인경우를유의한것으로인정하였다. 결과 홍도라지추출물의유효성분분석홍도라지추출물의유효성분인 platycodin D의함유여부를확인하기위하여 HPLC분석을수행하였다. 표준품과비교하였을때, 본실험에사용된홍도라지추출물에서 platycodin D의 peak가뚜렷하게나타났다 (Fig. 1). 홍도라지추출물의 LPS로유도된비장림프구증식능억제효과홍도라지추출물에의한비장림프구증식능억제정도를측정하기위하여, 마우스비장세포를 LPS로활성화시키고, Fig. 2A에서나타낸것과같이세포독성이없는최 Nitric Oxide 측정마우스비장세포를 1.6 10 6 cells/ml의농도로 12-well plate에 well 당 1mL 씩분주한뒤, LPS (1 μg/ml) 과홍도라지추출물을 20, 100, 500 μg/ml 농도로처리하였다. 24 시간동안 37 C, 5% CO 2 조건에서배양후에상층액을수거하여 3,000 rpm에서 5분간원심분리하였다. 시료를 Griess reagent (Promega, Madison, WI, USA) 와섞어상온에서 10분간반응시킨뒤 coloring solution을섞고상온에서차광하고 10분간반응시켜 540 nm에서흡광도를측정하였다. nitrite 표준액을이용하여 0 ~ 1,000 μm 농도범위에서표준곡선을작성한후시료의 nitrite를계산하였다. Cytokine 측정 IL-6와 interleukin-10 (IL-10) 은마우스비장세포에서 Mouse Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) 이용하여제조사의권고방법대로측정하였다. 비장세포를 1.6 10 6 cells/ml의농도로 12-well plate에 well 당 1 ml 씩분주한뒤, LPS (1 μg/ml) 과홍도라지추출물을 20, 100, 500 μg/ml 농도로처리하였다. 24시간동안 37 C, 5% CO 2 조건에서배양후에상층액을수거하여 3,000 rpm에서 5분간원심분리하여시료로사용하였다. Fig. 1. Chromatograms of active ingredients in Red Platycodon grandiflorum Extract (RPGE)
246 / 홍도라지추출물염증완화 대농도의홍도라지추출물을농도별로첨가하여 CCK-8 assay로세포생존율을측정하였다. LPS를처리하였을경우 vehicle control에비하여비장세포가약 3배이상증식 하였으며, 홍도라지추출물 100, 500 μg/ml을 LPS와함께처리하였을때, 유의적으로증식이감소한것을확인하였다 (Fig. 2B). (A) 홍도라지추출물의 LPS로유도된 NO 생성억제효과홍도라지의항염증활성을 NO 생성억제여부로측정하기위하여마우스비장세포에 LPS를처리하여 NO 생성을유도하였다. 20, 100, 500 μg/ml의홍도라지추출물을 LPS와함께처리하였을때, LPS에의해유도된 NO 생성량이농도의존적으로감소하였다 (Fig. 3). 홍도라지추출물 100 μg/ml은 LPS에의한비장세포의 NO 생성을 100% 로잡았을때 62% 감소시켰으며, 홍도라지추출물 500 μg/ml은 74% 감소시키는것으로나타났다. (B) 홍도라지추출물의마우스비장세포사이토카인생성에미치는영향홍도라지추출물이비장세포에서 LPS로유도된 IL-6 생성에미치는효과를알아보기위하여 20, 100, 500 μg/ml (A) Fig. 2. RPGE reduces LPS-induced cell proliferation in splenocytes. The cells were treated with various concentrations of RPGE for 24 hours in the absence (A) or presence (B) LPS (1 μg/ml). Cell viability was measured by CCK-8 assay. LPS, Lipopolysaccharide; VC, vehicle control. The data represents the mean ± SEM. ** p < 0.01, *** p < 0.005 (one-way ANOVA followed by Tukey s post hoc test). (B) Fig. 3. RPGE inhibits LPS-induced NO synthesis in splenocytes. The cells were treated with LPS (1 μg/ml) and the various concentrations of RPGE for 24 hours. The culture media was collected to measure NO synthesis using Griess reagent. LPS, Lipopolysaccharide; NO, Nitric oxide; VC, vehicle control. The data represents the mean ± SEM. ** p < 0.01, *** p < 0.005 (one-way ANOVA followed by Tukey s post hoc test). Fig. 4. RPGE modulates LPS-induced cytokine levels in splenocytes. The cells were treated with LPS (1 μg/ml) and the various concentrations of RPGE for 24 hours. The culture media was collected to assess cytokine levels for (A) IL-6 and (B) IL-10. LPS, Lipopolysaccharide; IL, interleukin; ND, not detected; VC, vehicle control. The data represents the mean ± SEM. ** p < 0.01, *** p< 0.005 (one-way ANOVA followed by Tukey s post hoc test).
Journal of Nutrition and Health (J Nutr Health) 2019; 52(3): 243 ~ 249 / 247 의홍도라지추출물을 LPS와함께 24시간동안처리한결과 100 μg/ml 이상농도에서 LPS에의해증가한 IL-6 생성이억제되었다 (Fig. 4A). 같은방법으로 LPS로활성화된비장세포에서 IL-10의생성증가에대한홍도라지추출물의효과를알아본결과, LPS에의한 IL-10 의생성에는유의적인변화가없었다 (Fig. 4B). 고찰 본연구의목적은 ex vivo 조건에서비장세포를이용하여홍도라지추출물의항염증효과를평가하는것이다. 도라지 (Platycodon grandiflorum) 는초롱꽃과 (Campanulaceae) 의다년생작물로섬유질이풍부하고칼륨및마그네슙등무기질에많이함유되어오래전부터식용및약용으로널리이용되어왔다 [19]. 항균, 항암및감기예방효과가있는소재로알려져있으나 [20] 비장세포에서항염증효과를평가한연구는진행된바없다. 도라지의유효성분은대표적으로 platycodin D가있으며, 그이외에도약 30여종의사포닌을함유하고있다 [21]. 홍삼이인삼에비하여제조과정에서유효성분이증가하는것과같이 [22], 홍도라지역시도라지를아홉번증숙하고건조시켜유효성분함량을증가시켰고도라지특유의아린맛을완화시킴으로써, 홍도라지추출물은기능뿐만아니라관능적으로도움이될것으로사료된다. 조혈모세포는백혈구, 적혈구, 혈소판등의림프구를생산하고, 림프구는비장으로들어가분화되고성숙하여방출된다 [23]. 이러한림프구중하나인 T세포는선천면역과적응면역에서매우중요한역할을하는데, 특히사이토카인분비를통해서면역반응을활성화시키는보조 (Helper) T 세포는주변자극에의해서 Th1 세포와 Th2 세포로나누어진다 [24]. Th1 세포는세포매개성 (cell-mediated) 면역반응활성화시키고, Th2 세포는주로체액성 (humoral) 면역반응에관여한다 [25]. 단핵구는골수에서성숙되는데조직에서는대식세포로분화하게된다 [26]. 대식세포는환경신호에따라전통적방법으로활성화되는 M1, 대체방법으로활성화되는 M2 등으로나뉘는데, M1 대식세포는 IL-6 등의사이토카인, 케모카인, NO등을분비하여 Th1 세포나자연살해 (NK) 세포를끌어들여감염된세포를공격하게하는반면 M2 대식세포는항염증반응으로 Th2 세포를통해세포독성 T세포의기능억제및신생혈관촉진등의작용으로알레르기반응이나암세포의성장을돕게된다 [26,27]. IL-10은수지상세포와대식세포에작용하여 Th1 세포의염증유발사이토카인인 IL-6 등의생성을억제하는면역억제사이토카인으로알려져있다 [28], 또한 IL-10은 IL-10을생산하는 Treg 세포의발달을촉진한다 [29]. 이로인해 IL-10은만성염증질환의치료제로이용할수있다. 예를들어, in vivo에서증식시킨 FOXP3-expressing CD4+ Treg 세포는효과 (effector) T 세포의빈도를감소시켜알레르기성염증으로부터보호기작을유도한다 [30]. 본연구에서 LPS로유도된마우스비장세포에서 NO의생성및 IL-6의분비에대한홍도라지추출물의효과를확인한결과, 홍도라지추출물 100 μg/ml 이상농도에서 LPS에의해증가한 NO 생성및 IL-6 분비가억제되었다. 이는홍도라지추출물이염증성인자및사이토카인의억제를통해항염효과를나타내는것으로생각된다. 또한같은조건에서홍도라지추출물에의한 IL-10의분비에는변화가없었는데, 이로인하여염증성사이토카인인 IL-6 의생성이억제되었을것으로생각된다. 이러한결과는비장세포에서 red cabbage juice의항염증의효과로측정된 IL-6, IL-10의분비양상과비슷하였다 [31]. 이상의결과는 LPS로활성화된비장세포에서홍도라지추출물의항염증효과를처음으로확인한것이며, 도라지의항염증관련사이토카인조절기작은 IL-6는억제시키고 IL-10은변화시키지않는것과관련이있는것으로추정되어진다. 이를통해홍도라지추출물의과민면역반응완화를위한건강기능성물질로서의잠재성이확인된것으로사료되며, 나아가자세한기전을밝히기위한심도있는연구가진행될필요성이있다. 요약 본연구에서는 LPS로활성화된마우스비장세포에서 Platycodin D가함유된홍도라지추출물의항염증효능을알아보기위하여비장세포증식능과 NO 생성및염증관련사이토카인을측정하였다. 결과를요약하면다음과같다. 1. 마우스비장세포에 1 μg/ml 농도의 LPS를처리하였을때비장세포의증식능이 3배이상증가하였으며홍도라지추출물처리시증가된증식능이유의하게감소되었다. 2. 마우스비장세포에 1 μg/ml 농도의 LPS를처리하였을때비장세포의 NO생성이증가하였으며, 홍도라지추출물처리시농도의존적으로증가된 NO생성이줄어들었다. 3. 마우스비장세포에 1 μg/ml 농도의 LPS를처리하였을때염증관련사이토카인 IL-6와항염증사이토카인 IL-10 분비가증가되었으며, 홍도라지추출물처리시농도의존적으로증가된 IL-6의분비가감소되었다. IL-10 분비에는유의적인차이가없었다. 위의결과를종합하여볼때, 본연구는홍도라지추출물이 ex vivo 실험
248 / 홍도라지추출물염증완화 을통해항염증관련인자들의조절을통하여과민면역반응을효과적으로억제한다는근거를확인하였다. 이에동물실험과인체적용시험을통해홍도라지추출물의과민면역반응억제효능에관한후속연구가필요할것으로사료된다. ORCID 박은정 : https://orcid.org/0000-0002-2778-1834 이유숙 : https://orcid.org/0000-0001-8360-1976 정현철 : https://orcid.org/0000-0001-5940-0621 이성현 : https://orcid.org/0000-0002-7886-4752 이해정 : https://orcid.org/0000-0001-8353-3619 References 1. Kovarik J. From immunosuppression to immunomodulation: current principles and future strategies. Pathobiology 2013; 80(6): 275-281. 2. Song MR, Kang MH, Park JS, Jo HK. A comparative study of the prevalence of allergic disease between rural and urban elementary school students. Child Health Nurs Res 2012; 18(1): 29-35. 3. Chen L, Deng H, Cui H, Fang J, Zuo Z, Deng J, et al. Inflammatory responses and inflammation-associated diseases in organs. Oncotarget 2017; 9(6): 7204-7218. 4. Park SO, Han YW, Aleyas AG, George JA, Yoon HA, Eo SK. The kinetics of secondary response of antigen-specific CD4+ T cells primed in vitro with antigen. 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