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博士學位論文 苦參발효추출물이면역활성에 미치는영향 暻園大學校大學院 韓醫學科本草學專攻 金亨錫
博士學位論文 苦參발효추출물이면역활성에미치는영향 Effect of Fermented Sophorae Radix Extract on Immuno-modulating Activity 暻園大學校大學院 韓醫學科本草學專攻 金亨錫
博士學位請求論文 指導敎授李暎鍾 苦參발효추출물이면역활성에미치는영향 Effect of Fermented Sophorae Radix Extract on Immuno-modulating Activity 위論文을韓醫學博士學位論文으로提出함 2008 年 12 月日 暻園大學校大學院 韓醫學科本草學專攻 金亨錫
이論文을 金亨錫의 韓醫學博士學位論文으로認准함 2008 年 12 月日 審査委員長 安 德 均 ( 印 ) 審査委員 成 洛 戌 ( 印 ) 審査委員 朴 完 洙 ( 印 ) 審査委員 朴 雄 德 ( 印 ) 審査委員 李 暎 鍾 ( 印 )
국문초록 연구목적 : 苦參 (Sophorae Radix) 발효추출물이면역활성에미치는영향을 연구하고자하였다. 연구방법 : 苦參을 Saccharomyces cerevisiae STV89( 효모 ) 로발효추출한시료 SFS로마우스대식세포와인간 HepG2 cell을이용한 cell viability, 세포내 hydrogen peroxide 생성, NO 생성, 그리고 cytokines(tnf-α 와 IL-6 등 ) level 에미치는영향을측정하였다. 연구결과 : 1. 苦參발효추출물의세포독성을확인하기위해 MTT assay 를수행한결과처방발효추출물을농도별로처리했을때대식세포와 HepG2 cell 에독성을과도하게많이유발하지않았다. 2. 苦參발효추출물은대식세포와 HepG2 cell 의세포내 hydrogen peroxide 생성을증가시켰으며 LPS로유발된대식세포내의 hydrogen peroxide 생성억제를유의하게회복시켰다. 3. 苦參발효추출물은대식세포의 NO의생성을억제시켰으며 LPS로유발된대식세포의 NO 생성증가를유의하게억제시켰다. 4. 苦參발효추출물은 LPS에의해서유발된대식세포의 TNF-α 및 IL-6 생성량을 25 μg /ml 이상에서유의하게감소시켰다. 결론 : 苦參으로제조된발효추출물 SFS는대식세포와 HepG2 cell 에세포독성을과도하게많이유발하지않으면서대식세포와 HepG2 cell 의 hydrogen peroxide 생성을증가시키고대식세포의 NO 생성을감소시켰으며 LPS로유발된대식세포의 TNF-α와 IL-6 생성을억제하는등유의한 - v -
면역활성을가진것으로나타났다. 주제어 : 苦參, 발효, 세포독성, 면역활성, 활성산소종 - vi -
목 차 Ⅰ. 서론 1 Ⅱ. 실험 3 1. 재료 3 1) 약재 3 2) Cell line 3 3) 시약및기기 3 (1) 시약 3 (2) 기기 3 2. 방법 4 1) 시료의제조 4 (1) 苦參추출물제조 4 (2) 苦參발효추출물 (SFS) 제조 4 2) 세포배양 5 3) 세포독성검사 (cell viability assay) 5 4) Hydrogen peroxide(h 2 O 2 ) assay 6 5) Nitric oxide(no) 생성측정 6 6) 면역단백질 (cytokines) 에대한 Bio-Plex Cytokine Assay 7 3. 통계처리 7 Ⅲ. 결과 8 1. SFS가대식세포에미치는영향 8 1) Cytotoxicity 8 2) Hydrogen peroxide(h 2 O 2 ) 의생성 10 (1) Hydrogen peroxide(h 2O 2) 의생성 10 - vii -
(2) LPS로유발된 hydrogen peroxide(h 2 O 2 ) 생성감소에대한영향 12 3) Nitric oxide(no) 의생성 16 (1) Nitric oxide(no) 의생성 16 (2) LPS로유발된 nitric oxide(no) 생성증가에대한영향 18 4) LPS로유발된면역단백질생성증가에대한영향 20 (1) TNF-α 생성증가에대한영향 20 (2) IL-6 생성증가에대한영향 22 (3) IL-2 생성증가에대한영향 24 (4) IL-4 생성증가에대한영향 25 (5) IL-12 생성증가에대한영향 26 (6) MCP-1 생성증가에대한영향 27 (7) KC 생성증가에대한영향 28 (8) RANTES 생성증가에대한영향 29 2. SFS가 HepG2 cell 에미치는영향 30 1) Cytotoxicity 30 2) Hydrogen peroxide(h 2 O 2 ) 의생성 32 Ⅳ. 고찰 34 Ⅴ. 결론 39 참고문헌 40 ABSTRACT 44 - viii -
List of Tables Table 1. SFS on Cell Viability of Raw 264.7 Cell 8 Table 2. SFS on the Intracellular Production of Hydrogen Peroxide of Raw 264.7 Cell 10 Table 3-1. SFS on the Intracellular Production of Hydrogen Peroxide of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml) for 24 hr 12 Table 3-2. SFS on the Intracellular Production of Hydrogen Peroxide of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml) for 45 hr 14 Table 3-3. SFS on the Intracellular Production of Hydrogen Peroxide of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml) for 72 hr 15 Table 4. SFS on Nitric Oxide Production of Raw 264.7 Cell for 4, 24 hr 16 Table 5. SFS on Nitric Oxide Production of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml) for 24 hr 18 Table 6. SFS on the TNF-α Production of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml) 20 Table 7. SFS on the IL-6 Production of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml) 22 Table 8. SFS on the IL-2 Production of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml) 24 Table 9. SFS on the IL-4 Production of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml) 25 - ix -
Table 10. SFS on the IL-12 Production of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml) 26 Table 11. SFS on MCP-1 Production of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml) 27 Table 12. SFS on KC Production of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml) 28 Table 13. SFS on RANTES Production of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml) 29 Table 14. SFS on Cell Viability of HepG2 Cell 30 Table 15. SFS on the Intracellular Production of Hydrogen Peroxide of HepG2 Cell 32 - x -
List of Figures Fig. 1. Effect of SFS on cell viability of Raw 264.7 cell. Cells were incubated for 24 hr 9 Fig. 2. Effect of SFS on the intracellular production of hydrogen peroxide of Raw 264.7 cell. Cells were incubated for 24 hr 11 Fig. 3. Effect of SFS on the intracellular production of hydrogen peroxide of Raw 264.7 cell treated with lipopolysaccharide(2 μg /ml). Cells were incubated for 24 hr 13 Fig. 4. Effect of SFS on nitric oxide production of Raw 264.7 cell. Cells were incubated for 4, 24 hr 17 Fig. 5. Effect of SFS on nitric oxide production of Raw 264.7 cell treated with lipopolysaccharide(2 μg /ml) for 24 hr 19 Fig. 6. Effect of SFS on the TNF-α production of Raw 264.7 cell treated with lipopolysaccharide(2 μg /ml) for 3 hr 21 Fig. 7. Effect of SFS on the IL-6 production of Raw 264.7 cell treated with lipopolysaccharide(2 μg /ml)for 3 hr 23 Fig. 8. Effect of SFS on cell viability of HepG2 cell. Cells were incubated for 24hr 31 Fig. 9. Effect of SFS on the intracellular production of hydrogen peroxide of HepG2 cell. Cells were incubated for 24 hr 33 - xi -
I. 서론 1) 苦參은神農本草經 中品에 苦參, 味苦寒. 主心腹結氣, 癥痂積聚, 黃疸, 溺 有餘瀝, 逐水, 除癰腫, 補中, 明目, 止淚. 一名水槐, 一名苦識, 生山谷及田野. 라 고처음收載되었으며, 임상에서淸熱燥濕, 祛風殺蟲의효능으로濕熱瀉痢, 腸風便血, 黃疸, 小便不利, 水腫, 帶下, 陰痒, 疥癬, 麻風, 皮膚瘙痒, 濕毒瘡瘍 등을치료하는약물로사용되고있다 2). 苦參의기원으로대한약전 3) 에 고삼 Sophora flavescens Solander ex Aiton( 콩과 Leguminosae) 의뿌리로서그대로또는주피를제거한것 이라 고수재되어있으며, 중국약전 4), 일본약전 5), 북한약전 6), 대만약전 7) 모두동일 하게고삼 S. flavescens 단일종으로되어있다. 苦參의성분으로는여러종류의 alkaloid 가함유되어있는데, 그주성분은 matrine, oxymatrine, N-oxy-sophocarpine, sophoranol N-oxide, supanine, anagyrine 등이다. 8) 또한 flavonoid 로서 kushenol H, kushenol K, kurarinol, sophoflavescenol 및 kuraridine 등이함유되어있다. 기타 triterpene 계의 saponin 으로서 soyasapogenol B 가함유되어있다. 9) 약리작용으로는심혈관계에대한작용 10-13), 당뇨병개선효능 14), 항위염작용 15), 항염증작용 16), 항균작용 15,17-19), 항암활성작용 20), 발모촉진작용 21) 등이보고 되었다. 발효한약은전통발효공법을통해한약재를미생물이잘이용할수있게찌거나삶은다음, 공기중의미생물또는유산균과같은순수분리미생물을이용하여발효한한약재를말한다. 이는한약재약효성분의체내흡수율과생체이용률을모두극대화시킨일종의가공방법으로약리적기능성뿐만아니라한약의제형개량과포제방법을향상시킬수있고이를통해한약의새로운수요를창출하고고부가가치의새로운한약제품을개발할수있다는데그의의를두고있다. 22) 발효한약에대한선행연구논문으로윤등 23) 은유산균으로발효된한국 - 1 -
산겨우살이추출물의항종양활성및면역자극효과를보고하였고, 손 24) 은버섯균사체로발효시킨복령과후박의항산화및항암효과를보고하였고, 정등 22) 은인진호, 금은화, 지구자등의한방소재를주원료로제조된발효한약추출물로숙취해소효능을보고하였고, 차등 25) 은차가버섯과어성초함유발효조성물로암예방및항암식 의약품의소재개발가능성을제시하였다. 이를토대로苦參발효추출물을통한세포독성과면역활성에대한연구를하게되었다. 이에著者는본연구에서苦參을 Saccharomyces cerevisiae STV89( 효모 ) 로발효추출하여제조한시료 SFS로마우스 Raw 264.7 cell과인간 HepG2 cell 을이용한 cell viability, 세포내 hydrogen peroxide 생성, NO 생성, 그리고 cytokines(tnf-α, IL-6 등 ) level 에미치는영향을측정한결과 SFS는대식세포와 HepG2 cell 에세포독성을과도히유발하지않으면서대식세포와 HepG2 cell의 hydrogen peroxide 생성을증가시키고대식세포의 NO 생성을감소시켰으며특히 LPS(lipopolysaccharide) 로유발된대식세포의 TNF-α 와 IL-6 생성을억제하는등유의한면역활성을나타내었기에보고하는바이다. - 2 -
Ⅱ. 실험 1. 재료 1) 약재실험에사용된苦參 (Sphorae Radix) 은서울의경동시장에서 2007 년 10월에구입하였으며, 약재는경원대학교한의과대학본초학교실에서감정하였고모든약재는쓰기전에초음파세척기 (Branson, USA) 를이용하여불순물을제거하고실험에사용하였다. 苦參의발효에사용한균주로는 Saccharomyces cerevisiae STV89( 효모 ) 를사용하였다. 2) Cell line 실험에사용된대식세포는 mouse 대식세포 (Raw 264.7 cell line) 이며간조직세포는 human hepatocytes(hepg2 cell line) 로서한국세포주은행 (KCLB, Korea) 에서구입하였다. 3) 시약및기기 (1) 시약본실험을위해서 ethyl alcohol(samchun Chemical, Korea), DMSO(Sigma, USA), DMEM(Sigma, USA), 1 PBS(Sigma, USA), EDTA(Sigma, USA), isopropanol(sigma, USA), trypsin-edta(sigma, USA), Bio-Plex cytokine assay kit(panomics, USA) 등이사용되었다. (2) 기기본실험에사용된기기는 CO 2 incuvator (NUAIRE, USA), rotary vacuum evaporator(eyela, Japan), air compressor(tamiya, Japan), homogenizer (Omni, USA), research microscope(becton dickinson, USA), centrifuge (Hanil, Korea), fume hood(hanil, Korea), clean bench(jeio thec, - 3 -
Korea), ultrasonic cleaner(branson, USA), microplate reader(bio-rad, U SA), vortex m ixer(v ision S cientific C o, K orea), w ater b ath (intron biotech, Korea), ice-maker(vision Scientific Co, Korea) 등이다. 2. 방법 1) 시료의제조 (1) 苦參추출물제조苦參 50 g을정확하게중량을측정한뒤환류추출기에 1차증류수 2,000 ml와함께넣은뒤탕액이끓는시점으로부터 2시간동안가열하여추출한다음추출액을 filter paper(advantec No.2, Japan) 로감압여과한여과액을 rotary vacuum evaporator 를이용하여농축액을얻었다. 이농축액을동결건조기를이용하여건조한분말을시료로사용하였다. 동결건조추출물은 9.5g을얻었으며, 수율은 19 % 였다. (2) 苦參발효추출물 (SFS) 제조위에서제조된苦參추출물을이용하여다음과같이苦參발효추출물을제조하였다. 1 조효소조제 : 조효소제인 α-herbzyme( 한국효소 ) 3 g에증류수 100 ml를가하고 37 에서 30분간침출하여여과시킨후그여액을조효소액으로사용하였다. 2 열수출하여건조한苦參 (3.0 g, ph:4.67) 을 screw cap tube에담고미리추출된조효소액 2.2 ml를첨가하여 37 에서 2시간효소반응하였다. 3 효소반응후 95 에서 10분간살균하였다. 4 Saccharomyces cerevisiae STV89를苦參에 4 % 씩접종하여 Saccharomyces cerevisiae STV89는 30 에서 4일간배양하였다. - 4 -
5 배양후 60 에서 20 분간열처리하였다. 6 Saccharomyces cerevisiae STV89 로배양한후 ph 는 4.66 였다. 2) 세포배양 Raw 264.7 cells 과 HepG2 cells 은 37, 5 % CO 2 조건에서 10 % FBS, penicillin(100 U/mL), streptomycin(100 μg /ml) 이첨가된 DMEM 배지로배 양되었다. HepG2 cells 은 75 cm 2 flask(falcon, USA) 에서충분히증식된후 배양 3일간격으로배양세포표면을 phosphate buffered saline(pbs) 용액으로씻어준후 50 ml flask 당 1 ml의 0.25 % trypsin-edta 용액을넣고실온에서 1분간처리한다음 trypsin 용액을버리고 37 에서 5분간보관하여세포를탈착하여계대배양하였다. 탈착된세포는 10 % FBS가첨가된 DMEM 배양액 10 ml에부유시킨다음새로운배양용기 (50 ml culture flask) 에옮겨 1 : 2의 split ratio 로 CO 2 배양기 (37, 5 % CO 2 ) 에서배양하였다. 3) 세포독성검사 (cytotoxicity assay) 준비된시료가 Raw 264.7 cells 과 HepG2 cells 에나타내는세포독성유발효과를알아보기위하여 MTT assay를실시하였다. 96 well plate 에 1 10 4 cells/well 의 cell 을 100 μl씩넣고 37, 5 % CO 2 incubator 에서 24시간동안배양한후배지를버리고배양세포표면을 phosphate buffered saline(1 PBS) 용액으로씻어주었다. 같은양의배지와 PBS에녹인시료 (25, 50, 100, 200 μg /ml) 를각 well에처리하고 24시간동안배양하였다. 배 양이끝난후 PBS 에녹인 1 mg /ml MTT(Sigma, USA) 를 100 μl씩각 well에처리하여알루미늄호일로차광시킨후 2시간동안같은조건에서배양하였다. 배양액을모두제거한후 DMSO를 100 μl처리하고 37 에서 2 시간방치후 microplate reader(molecular Devices, USA) 를이용하여 540 nm에서흡광도를측정하였다. Cell viability 는다음공식으로계산하였다. Cell viability(%) = 100 AT / AC - 5 -
AC : absorbance of control AT : absorbance of tested extract solution. 4) Hydrogen peroxide(h 2 O 2 ) assay 세포내의 hydrogen peroxide(h 2O 2) 는 dihydrorhodamine 123(DHR) assay 를이용하여측정하였다. 96 well plate 1 10 4 cells/well 의 cell 을 100 μl씩넣고 37, 5 % CO 2 incubator 에서 24시간동안배양한후배지를버리고배양세포표면을 phosphate buffered saline(1 PBS) 용액으로씻어주었다. LPS와시료를처리하기전에우선 DHR(10 um) 이담긴배지를 30분간각 well 에처리한뒤배지를제거하였다. LPS를단독처리 (2 μg /ml) 하거나혹은다양한농도의시료 (25, 50, 100, 200 μg /ml) 와함께배지에담아각 well 에처리하고 24시간동안 37, 5 % CO 2 incubator 에서배양한후 microplate reader (Bio-Rad, USA) 를이용하여 490 nm 에서흡광도를측정하였다. 세포내 H 2 O 2 생성은다음공식으로계산하였다. Intracellular Productions of H 2O 2(%) = 100 AT / AC AC : absorbance of control AT : absorbance of tested extract solution. 5) Nitric oxide(no) 생성측정 NO의기질인 L-알기닌은 L-시트룰린과일산화질소로변하는데, 이는빠르게안정된이산화질소, 아질산염, 질산염으로변한다. 그리스시약 (griess reagent : 0.5 % 의설파닐아미드, 2.5 % 의인산및 0.5 % 의나프틸에틸렌아민 ) 은아질산염과화학반응하여보라색의아조염을형성하고이것은일산화질소의농도와일치하기때문에, 아조염의농도로부터아질산염의농도를추정하기위해 microplate reader 를이용, 540 nm에서흡광도를측정하여 NO의생성정도를비교하였다. 이를위해다음과같이실험하였다. LPS를단독처리 (2 μg /ml) 하거나혹은다양한농도의시료 (25, 50, 100, 200 μg /ml) 와함께배지에담아각 well 에처리하고 24시간동안 37, 5 % CO 2 incubator 에서 - 6 -
배양한후세포배양상등액 100 μl을채취하여여기에그리스시약 100 μl을혼합하여 15분동안반응시킨후 microplate reader (Bio-Rad, USA) 를이용하여 540 nm에서흡광도를측정하였다. 세포의 nitric oxide 생성은다음공식으로계산하였다. Productions of Nitric oxide(%) = 100 AT / AC AC : absorbance of control AT : absorbance of tested extract solution. 6) 면역단백질 (cytokines) 에대한 Bio-Plex Cytokine Assay 면역단백질분비와관련된시료의영향을알아보기위해 Anderson 등 26) 의방법을응용하여다음과같이실험을시행하였다. 96 well plate 에 1 10 5 cells/ml 의 cell 을 100 μl씩넣고 37, 5 % CO 2 incubator 에서 24 시간동안 배양한후배지를버리고배양세포표면을 1 PBS 용액으로씻어준뒤각 well 에 LPS를단독처리 (2 μg /ml) 하거나혹은다양한농도의시료 (25, 50, 100 μg /ml) 와함께배지에담아처리하고 3시간동안배양하였다. 배양이끝나면상층액을채취하여 Bio-Plex Suspension Array System 의 Bio-Plex Cytokine Assays 를실시하여면역단백질들 (TNF-α, IL-6, IL-2, IL-4, IL-12, MCP-1, KC, RANTES) 의발현에대한시료의영향을계산, 비교하였다. 3. 통계처리본실험에서얻은결과에대해서는평균치 ± 표준오차 (mean ± SD) 로나타내었으며, 대조군과각실험군과의평균의차이는 Student's t-test 와 ANOVA test 로분석하여 p-value 값이 0.05 미만일때통계적으로유의한차이가있는것으로판정하였다. - 7 -
Ⅲ. 결과 1. SFS가대식세포에미치는영향 1) Cytotoxicity SFS가대식세포의증식에미치는영향을비교하였다. 24시간처리한결과 25 μg /ml 이상의모든농도에서유의한감소는없었다 (Table 1, Fig. 1). Table 1. SFS on Cell Viability of Raw 264.7 Cell. Samples( μg /ml) Cell Viability(% of normal; mean±sd) Normal 100.0 ± 17.4 SFS 25 163.5 ± 43.3 * SFS 50 177.5 ± 54.1 * SFS 100 177.1 ± 50.8 * SFS 200 165.7 ± 49.0 * Cells were incubated for 24 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with SFS. * represents P < 0.05 compared to the normal. - 8 -
Cell viability (% of normal) 250 225 200 175 150 125 100 75 50 25 0 * * * * Normal 25 50 100 200 Concentration (ug/ml) Fig. 1. Effect of SFS on cell viability of Raw 264.7 cell. Cells were incubated for 24 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with SFS. * represents P < 0.05 compared to the normal. - 9 -
2) Hydrogen peroxide(h 2 O 2 ) 의생성 (1) Hydrogen peroxide(h 2O 2) 의생성 SFS가대식세포의 hydrogen peroxide(h 2O 2) 의생성에미치는영향을비교하였다. 24시간에서 25 μg /ml 이상일때유의한증가를나타내었다 (Table 2, Fig. 2). Table 2. SFS on the Intracellular Production of Hydrogen Peroxide of Raw 264.7 Cell. Samples( μg /ml) H 2O 2 Production(% of normal; mean±sd) Normal 100.0 ± 7.0 SFS 25 123.6 ± 13.3 * SFS 50 129.9 ± 17.4 * SFS 100 136.6 ± 34.1 * SFS 200 110.1 ± 8.3 * Cells were incubated for 24 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with SFS. * represents P < 0.05 compared to the normal. - 10 -
H2O2 production (% of normal) 180 160 140 120 100 80 60 40 20 0 * * * * Normal 25 50 100 200 Concentration (ug/ml) Fig. 2. Effect of SFS on the intracellular production of hydrogen peroxide of Raw 264.7 cell. Cells were incubated for 24 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with SFS. * represents P < 0.05 compared to the normal. - 11 -
(2) LPS 로유발된 Hydrogen peroxide(h 2 O 2 ) 생성감소에대한영향 SFS 가 LPS 로유발된대식세포의 hydrogen peroxide(h 2O 2) 의생성감소에 미치는영향을비교하였다. 24 시간에서모두 LPS 단독으로배양한경우 Normal 군보다유의하게 H 2 O 2 생성이감소하였으며 LPS 와 SFS 을함께배 양한경우 SFS 의농도가 50 μg /ml 이상일때 LPS 에의한감소를유의하게 회복하였다 (Table 3-1, Fig. 3). Table 3-1. SFS on the Intracellular Production of Hydrogen Peroxide of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml) for 24 hr. Samples( μg /ml) H 2 O 2 Production(% of control; mean±sd) Normal 208.5 ± 2.5 Control 100.0 ± 1.6 # SFS 25 100.8 ± 1.9 SFS 50 102.7 ± 1.5 * SFS 100 105.4 ±2.0 * SFS 200 108.3 ± 3.8 * Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with LPS. Control : Treated with Lipopolysaccharide(2.0 μg / ml ) only. # represents P < 0.05 compared to the normal. * represents P < 0.05 compared to the control. - 12 -
H2O2 production (% of control) 250 200 150 100 50 0 * # * * Normal Control 25 50 100 200 Concentration (ug/ml) Fig. 3. Effect of SFS on the intracellular production of hydrogen peroxide of Raw 264.7 cell treated with lipopolysaccharide(2 μg /ml). Cells were incubated for 24 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with LPS. Control : Treated with Lipopolysaccharide(2.0 μg / ml ) only. # represents P < 0.05 compared to the normal. * represents P < 0.05 compared to the control. - 13 -
45시간의배양에서는 LPS 단독으로배양한경우 Normal 군보다유의하게 hydrogen peroxide 생성이감소하였으며 LPS와 SFS을함께배양한경우 SFS의농도가 100 μg /ml이상일때 LPS에의한감소를유의하게회복하였다 (Table 3-2). Table 3-2. SFS on the Intracellular Production of Hydrogen Peroxide of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml) for 45 hr. Samples( μg /ml) H 2O 2 Production(% of control; mean±sd) Normal 219.6 ± 3.8 Control 100.0 ± 3.6 # SFS 25 99.7 ± 3.0 SFS 50 102.3 ± 4.1 SFS 100 105.7 ± 3.5 * SFS 200 108.7 ± 4.9 * Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with LPS. Control : Treated with Lipopolysaccharide(2.0 μg / ml ) only. # represents P < 0.05 compared to the normal. * represents P < 0.05 compared to the control. - 14 -
72시간에서모두 LPS 단독으로배양한경우 Normal 군보다유의하게 hydrogen peroxide 생성이감소하였으며 LPS와 SFS을함께배양한경우 SFS의농도가 200 μg /ml일때만 LPS에의한감소를유의하게회복하였다 (Table 3-3). Table 3-3. SFS on the Intracellular Production of Hydrogen Peroxide of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml) for 72 hr. Samples( μg /ml) H 2O 2 Production(% of control; mean±sd) Normal 214.8 ± 7.9 Control 100.0 ± 7.4 # SFS 25 100.2 ± 3.7 SFS 50 101.1 ± 1.9 SFS 100 104.3 ± 3.2 SFS 200 107.3 ± 5.7 * Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with LPS. Control : Treated with Lipopolysaccharide(2.0 μg / ml ) only. # represents P < 0.05 compared to the normal. * represents P < 0.05 compared to the control. - 15 -
3) Nitric oxide(no) 의생성 (1) Nitric oxide(no) 의생성 SFS가대식세포의 nitric oxide(no) 의생성에미치는영향을비교하였다. 4시간의배양에서는 25, 50, 200 μg /ml일때유의한감소를나타내었으며, 24시간의배양에서는 25 μg /ml이상일때모두유의한감소를나타내었다 (Table 4, Fig. 4). Table 4. SFS on Nitric Oxide Production of Raw 264.7 Cell for 4, 24 hr. Samples( μg /ml) NO Production(% of normal; mean±sd) 4 hr 24 hr Normal 100.0 ± 25.5 100.0 ± 42.8 SFS 25 63.1 ± 17.0 * 50.1 ± 23.9 * SFS 50 70.8 ± 32.7 * 50.9 ± 21.2 * SFS 100 75.3 ± 40.3 53.0 ± 24.4 * SFS 200 70.8 ± 17.8 * 57.3 ± 27.8 * Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with SFS. * represents P < 0.05 compared to the normal. - 16 -
NO production (% of normal) 160 140 120 100 80 60 40 20 0 4 hr 24 hr * * * * * * * Normal 25 50 100 200 Concentration (ug/ml) Fig. 4. Effect of SFS on the nitric oxide production of Raw 264.7 cell. Cells were incubated for 4, 24 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with SFS. * represents P < 0.05 compared to the normal. - 17 -
(2) LPS로유발된 nitric oxide(no) 생성증가에대한영향 SFS가 LPS로유발된대식세포의 nitric oxide(no) 의생성증가에미치는영향을비교하였다. 24시간동안 LPS 단독으로배양한경우 Normal 군보다유의하게 NO생성이증가하였으며 LPS와 SFS을함께배양한경우 SFS의농도가 50, 100 μg /ml일때 LPS에의한증가를유의하게억제시켰다 (Table 5, Fig. 5). Table 5. SFS on the Nitric Oxide Production of Raw 264.7 Cell treated with Lipopolysaccharide(LPS 2 μg /ml) for 24 hr. Samples( μg /ml) No Production(% of control; mean±sd) Normal 83.5 ± 5.7 Control 100.0 ± 5.0 # SFS 25 97.1 ± 11.2 SFS 50 85.9 ± 13.7 * SFS 100 83.1 ± 13.8 * SFS 200 96.2 ± 18.8 Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with LPS. Control : Treated with Lipopolysaccharide(2.0 μg / ml ) only. # represents P < 0.05 compared to the normal. * represents P < 0.05 compared to the control. - 18 -
140 NO production (% of control) 120 100 80 60 40 20 # * * 0 Normal Control 25 50 100 200 Concentration (ug/ml) Fig. 5. Effect of SFS on the nitric oxide production of Raw 264.7 cell treated with lipopolysaccharide(2 μg /ml). Cells were incubated for 24 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with LPS. Control : Treated with Lipopolysaccharide(2.0 μg / ml ) only. # represents P < 0.05 compared to the normal. * represents P < 0.05 compared to the control. - 19 -
4) LPS로유발된면역단백질생성증가에대한영향 (1) TNF-α 생성증가에대한영향 SFS가 LPS로유발된대식세포의 TNF-α 생성증가에미치는영향을비교하였다. 3시간에서 LPS 단독으로배양한경우 Normal 군보다유의하게 TNF-α 생성이증가하였으며 LPS와 SFS을함께배양한경우 SFS의농도가 25 μg /ml이상일때모두 LPS에의한증가를유의하게감소시켰다 (Table 6, Fig. 6). Table 6. SFS on the TNF-α Production of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml). Samples( μg /ml) TNF-α Production(pg/mL) Normal 47.9 ± 21.2 Control 2175.9 ± 306.4 # SFS 25 1518.7 ± 473.2 * SFS 50 1346.0 ± 637.3 * SFS 100 1283.1 ± 523.5 * Cells were incubated for 3 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with LPS. Control : Treated with Lipopolysaccharide(2.0 μg /ml) only. # represents P < 0.05 compared to the normal. * represents P < 0.05 compared to the control. - 20 -
3000 TNF-α production (pg/ml) 2500 2000 1500 1000 500 # * * * 0 Normal Control 25 50 100 Concentration (ug/ml) Fig. 6. Effect of SFS on TNF-α production of Raw 264.7 cell treated with lipopolysaccharide(2 μg /ml). Cells were incubated for 3 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with LPS. Control : Treated with Lipopolysaccharide(2.0 μg / ml ) only. # represents P < 0.05 compared to the normal. * represents P < 0.05 compared to the control. - 21 -
(2) IL-6 생성증가에대한영향 SFS가 LPS로유발된대식세포의 IL-6 생성증가에미치는영향을비교하였다. 3시간에서 LPS 단독으로배양한경우 Normal 군보다유의하게 IL-6 생성이증가하였으며 LPS와 SFS을함께배양한경우 SFS의농도가 25 μg /ml 이상일때모두 LPS에의한증가를유의하게감소시켰다 (Table 7, Fig. 7). Table 7. SFS on the IL-6 Production of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml). Samples( μg /ml) IL-6 Production(pg/mL) Normal 5.3 ± 11.2 Control 16.7± 5.8 # SFS 25 9.7 ± 5.1 * SFS 50 9.0 ± 5.9 * SFS 100 8.8 ± 4.5 * Cells were incubated for 3 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Lactobacillus pentosus K34. Normal : Not treated with LPS. Control : Lipopolysaccharide(2.0 μg / ml ) #, P < 0.05 compared to the normal. *, P < 0.05 compared to the control. - 22 -
25 # IL-6 production (pg/ml) 20 15 10 5 * * * 0 Normal Control 25 50 100 Concentration (ug/ml) Fig. 7. Effect of SFS on the IL-6 production of Raw 264.7 cell treated with lipopolysaccharide(2 μg /ml). Cells were incubated for 3 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with LPS. Control : Treated with Lipopolysaccharide(2.0 μg /ml) only. # represents P < 0.05 compared to the normal. * represents P < 0.05 compared to the control. - 23 -
(3) IL-2 생성증가에대한영향 SFS가 LPS로유발된대식세포의 IL-2 생성증가에미치는영향을비교하였다. 24시간에서 LPS 단독으로배양한경우 Normal 군보다유의하게 IL-2 생성이증가하였으며 LPS와 SFS을함께배양한경우 50, 100 μg /ml 일때만유의한감소가나타났다 (Table 8). Table 8. SFS on IL-2 Production of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml). Samples( μg /ml) IL-2 Production(pg/mL) Normal 6.3 ± 9.5 Control 37.9 ± 25.0 # SFS 25 20.4 ± 16.0 SFS 50 18.4 ± 12.1 * SFS 100 17.0 ± 8.4 * Cells were incubated for 24 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with LPS. Control : Lipopolysaccharide(2.0 μg / ml ) #, P < 0.05 compared to the normal. *, P < 0.05 compared to the control. - 24 -
(4) IL-4 생성증가에대한영향 SFS가 LPS로유발된대식세포의 IL-4 생성증가에미치는영향을비교하였다. 24시간에서 LPS 단독으로배양한경우 Normal 군보다유의하게 IL-4 생성이증가하였으며 LPS와 SFS을함께배양한경우유의한변화는나타나지않았다 (Table 9). Table 9. SFS on IL-4 Production of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml). Samples( μg /ml) IL-4 Production(pg/mL) Normal 6.3 ± 2.5 Control 10.7 ± 4.1 # SFS 25 8.2 ± 1.0 SFS 50 9.0 ± 1.1 SFS 100 11.3 ± 0.9 Cells were incubated for 24 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with LPS. Control : Lipopolysaccharide(2.0 μg / ml ) #, P < 0.05 compared to the normal. *, P < 0.05 compared to the control. - 25 -
(5) IL-12 생성증가에대한영향 SFS가 LPS로유발된대식세포의 IL-12 생성증가에미치는영향을비교하였다. 24시간에서 LPS 단독으로배양한경우 Normal 군보다유의하게 IL-12 생성이증가하였으며 LPS와 SFS을함께배양한경우 SFS의농도가 25 μg /ml 일때는유의한감소를, 50 μg /ml 일때는유의한증가를나타내었다 (Table 10). Table 10. SFS on the IL-12 Production of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml). Samples( μg /ml) IL-12 Production(pg/mL) Normal 12.0 ± 4.3 Control 137.1 ± 11.2 # SFS 25 92.5 ± 7.5 * SFS 50 143.0 ± 27.0 * SFS 100 153.4 ± 35.5 Cells were incubated for 24 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with LPS. Control : Lipopolysaccharide(2.0 μg / ml ) #, P < 0.05 compared to the normal. *, P < 0.05 compared to the control. - 26 -
(6) MCP-1 생성증가에대한영향 SFS가 LPS로유발된대식세포의 monocyte chemoattractant protein-1 (MCP-1) 생성증가에미치는영향을비교하였다. 24시간에서 LPS 단독으로배양한경우 Normal 군보다유의하게 MCP-1 생성이증가하였으며 LPS와 SFS을함께배양한경우 SFS의농도가 25 μg /ml일때는유의한감소를, 100 μg /ml일때는유의한증가를나타내었다 (Table 11). Table 11. SFS on MCP-1 Production of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml). Samples( μg /ml) MCP-1 Production(pg/mL) Normal 80.5 ± 41.0 Control 11473.2 ± 435.3 # SFS 25 8471.5 ± 788.6 * SFS 50 7861.9 ± 561.3 SFS 100 12993.6 ± 580.0 * Cells were incubated for 24 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with LPS. Control : Lipopolysaccharide(2.0 μg / ml ) #, P < 0.05 compared to the normal. *, P < 0.05 compared to the control. - 27 -
(7) KC 생성증가에대한영향 SFS가 LPS로유발된대식세포의 KC 생성증가에미치는영향을비교하였 다. 24시간에서 LPS 단독으로배양한경우 Normal 군보다유의하게 KC 생 성이증가하였으며 LPS와 SFS을함께배양한경우 SFS의농도가 25 μg /ml일때는 유의한 감소를, 50 μg /ml일때는 유의한 증가를 나타내었다 (Table 12). Table 12. SFS on KC Production of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml). Samples( μg /ml) KC Production(pg/mL) Normal 6.7 ± 2.9 Control 10.5 ± 1.0 # SFS 25 8.4 ± 0.7 * SFS 50 10.6 ± 1.3 * SFS 100 11.4 ± 2.0 Cells were incubated for 24 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with LPS. Control : Lipopolysaccharide(2.0 μg / ml ) #, P < 0.05 compared to the normal. *, P < 0.05 compared to the control. - 28 -
(8) RANTES 생성증가에대한영향 SFS 가 LPS 로유발된대식세포의 RANTES 생성증가에미치는영향을비 교하였다. 24 시간에서 LPS 단독으로배양한경우 Normal 군보다유의하게 RANTES 생성이증가하였으며 LPS 와 SFS 을함께배양한경우 SFS 의농 도가 25 μg /ml 일때는유의한감소를, 100 μg /ml 일때는유의한증가를나 타내었다 (Table 13). Table 13. SFS on RANTES Production of Raw 264.7 Cell treated with Lipopolysaccharide(2 μg /ml). Samples( μg /ml) RANTES Production(pg/mL) Normal 161.1 ± 21.0 Control 11146.7 ± 457.4 # SFS 25 10454.1 ± 410.2 * SFS 50 10614.1 ± 396.3 SFS 100 12434.9 ± 434.1 * Cells were incubated for 24 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with LPS. Control : Lipopolysaccharide(2.0 μg / ml ) #, P < 0.05 compared to the normal. *, P < 0.05 compared to the control. - 29 -
2.SFS가 HepG2 cell에미치는영향 1) Cytotoxicity SFS가 HepG2 cell의증식에미치는영향을비교하였다. 24시간에서 25 μg /ml 이상일때세포증식률증가를나타내었다 (Table 14, Fig. 8). Table 14. SFS on Cell Viability of HepG2 Cell. Samples( μg /ml) Cell Viability(% of normal; mean±sd) Normal 100.0 ± 69.6 SFS 25 159.1 ± 33.4 * SFS 50 155.8 ± 33.7 * SFS 100 150.6 ± 22.2 * SFS 200 145.2 ± 19.0 * Cells were incubated for 24 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with SFS. * represents P < 0.05 compared to the normal. - 30 -
200 175 * * * * Cell viability (% of normal) 150 125 100 75 50 25 0 Normal 25 50 100 200 Concentration (ug/ml) Fig. 8. Effect of SFS on cell viability of HepG2 cell. Cells were incubated for 24 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with SFS. * represents P < 0.05 compared to the normal. - 31 -
2) Hydrogen peroxide(h 2 O 2 ) 의생성 SFS가 HepG2 cell 의 hydrogen peroxide(h 2O 2) 의생성에미치는영향을비교하였다. 24시간에서 25, 50, 100 μg /ml일때유의한증가를나타내었다 (Table 15, Fig. 9). Table 15. SFS on the Intracellular Production of Hydrogen Peroxide of HepG2 Cell. Samples( μg /ml) H 2O 2 Production(% of normal; mean±sd) Normal 100.0 ± 1.3 SFS 25 107.2 ± 7.1 * SFS 50 111.4 ± 12.2 * SFS 100 109.4 ± 9.9 * SFS 200 102.0 ± 4.7 Cells were incubated for 24 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with SFS. * represents P < 0.05 compared to the normal. - 32 -
H2O2 production (% of normal) 140 120 100 80 60 40 20 0 * * * Normal 25 50 100 200 Concentration (ug/ml) Fig. 9. Effect of SFS on the intracellular production of hydrogen peroxide of HepG2 cell. Cells were incubated for 24 hr. Results are represented as mean±sd. SFS : Water extract of Sophorae Radix Fermented by Saccharomyces cerevisiae STV89. Normal : Not treated with SFS. * represents P < 0.05 compared to the normal. - 33 -
Ⅳ. 고찰 苦參의기원으로대한약전 3) 에 고삼 Sophora flavescens Solander ex Aiton( 콩과 Leguminosae) 의뿌리로서그대로또는주피를제거한것 이라 고수재되어있으며, 중국약전 4), 일본약전 5), 북한약전 6), 대만약전 7) 모두동일 하게고삼 S. flavescens 단일종으로되어있다. 1) 苦參은神農本草經 中品에 苦參, 味苦寒. 主心腹結氣, 癥痂積聚, 黃疸, 溺有 餘瀝, 逐水, 除癰腫, 補中, 明目, 止淚. 一名水槐, 一名苦識, 生山谷及田野. 라고 처음收載되었으며, 名醫別錄 27) 에 治惡瘡, 下部慝, 平胃氣, 令人嗜食, 輕身. 이 라고하였고李時珍 28) 은 苦以味名, 參以功名, 槐似葉形名也 라고한이래味 苦性寒 28) 하고, 淸熱燥濕, 祛風殺蟲의효능으로濕熱瀉痢, 腸風便血, 黃疸, 小 便不利, 水腫, 帶下, 陰痒, 疥癬, 麻風, 皮膚瘙痒, 濕毒瘡瘍등을치료는약물 로사용되고있다 2). 이약의성상은원주형을이루고길이 5 ~ 20 cm, 지름 2 ~ 3 cm 이다. 바깥면은어두운갈색 ~ 황갈색이며세로주름이뚜렷하고가로로긴피목 이있다. 주피를벗긴것은황백색이며꺾인면은약간섬유성이다. 이약의 횡단면은연한황갈색이고피층의두께가 1 ~ 2 mm 이며형성층부근은 약간어두운색을띠고목부와의사이에세포간극이생긴것이있다. 이약 은특유한냄새가있고맛은매우쓰며오래남는다. 3) 산지는우리나라各 地에분포한다. 중국에서는주로山西, 河南, 河北등에서생산되며기타대 부분의성에서도역시생산된다 29). 苦參의성분으로는여러종류의 alkaloid 가함유되어있는데, 그주성분은 matrine, oxymatrine, N-oxy-sophocarpine, sophoranol N-oxide, supanine, anagyrine 등이다. 8) 최근의연구에의하면고삼에서분리한 flavonoid 성분인 8-lavandulylkaempferol 가 free radical 과 ONOO - 를제거하는능력이있는것으 로보고된바있으며, lavanduly flavanones 성분인 (2S)-2 -methoxykurarinone, - 34 -
sophra-flavanone G, leachianone A 및 (-)-kurarinone 등이항말라리아활성을가진다는보고도있다. 15) 약리에대한연구로는 Yamahara 등 10), 권등 11), 김등 12), 송등 13) 은심혈관계에대한작용을, 김 14) 은당뇨병개선효능을, 강등 15) 은항위염작용을, Yamaguchi-Miyamoto 등 16) 은항염증작용을, 강등 15), 안등 17), 조등 18), 이등 19) 은항균작용을, 조등 20) 은항암활성작용을, Roh 등 21) 은발모촉진작용을보고하였다. 발효 (fermentation) 는넓은의미에서미생물을이용하여그효소작용으로유기물을전환시키는것을뜻한다. 발효현상은유사이래인류에의해서관찰되고이용되어왔으나, 명확하게발효현상을인식하게된시기는확실하지않다. 지구상에생명체의탄생과더불어발생하여인류의역사만큼오랫동안이용되어온발효현상도그본체가밝혀지기시작한것은 200년이되지않는다. 발효란뜻의 fermentation 은라틴어의 ferverve( 끓는다 ) 로부터유래되었으며, 이것은알콜발효시에발생하는탄산가스에의해서거품이일게되는현상을나타낸것으로추측된다. 30) 최근한의학계에서도발효한약이라는제제를도입하여임상에서사용하고있으나아직탕약이주된제형으로사용되는추세여서발효한약은다소생소한이름이다. 하지만환자들을치료하는데좀더나은방법이있다면새로운제제로의접근도의미있을것으로생각된다 31). 이에著者는발효한약의유의성을적용하여苦參을발효추출하여만들어진시료 SFS로마우스대식세포와인간 HepG2 cell을이용한 cell viability, 세포내 hydrogen peroxide 생성, NO 생성, 그리고 cytokines(tnf-α 와 IL-6 등 ) level 에미치는영향을측정하였다. Saccharomyces속의대표균종인 Saccharomyces cerevisiae는영국의맥주공장에서분리된맥주효모로맥주주발효가지나면액표면에거품과함께떠오르는상면발효효모 ( 上面醱酵酵母, top fermentation yeast) 이다. 맥주, 포도주등의각종주류, 알코올, 빵등의제조에이용되는등이용범위가넓은 - 35 -
효모로 glucose, galactose, maltose, sucrose 및일부 raffinose 를발효하고 lactose 는발효하지못한다. 32) 세포는구형, 난형또는긴것도있다. 세포내에 thiamine( 비타민 B1) 을비교적많이생성하므로약용효모로도이용된다. 33) 발효미생물 Saccharomyces계통은생화학적성질이일정하고, 물에잘분산되며, 자기소화에대한내성이있어서보존성이좋고, 당밀배지에서증식속도가빠르고수득률이높다는등의성질이있다 34). 이에본실험에서는 Saccharomyces cerevisiae STV89( 효모 ) 를균주로이용하여발효하였다. 본연구에서는한국세포주은행 (KCLB, Korea) 으로부터분양받은 Raw 264.7 cells 과 HepG2 cells 에발효苦參추출물을 25, 50, 100, 200 μg /ml의농도로처리한뒤에 24시간동안 37 에서배양한후, 세포의증식을 MTT assay을이용하여확인한결과대조군에비하여苦參발효추출물이 Raw 264.7 cells과 HepG2 cell에유의한세포증식률감소를일으키지않았으며이는발효苦參추출물이세포독성을과도히유발하지않는다는것으로볼수있다. Hydroperoxide(H 2 O 2 ) 는세포내에서발생하는 reactive oxygen species (ROS) 의일종이며일반적으로세포의산화적 stress 를유발하는인자로알려져있다. 또한인체의면역기능과도중요한관계에있다. 즉인체내의염증반응이커지면 neutrophils 등의세포는 hydrogen peroxide 생성을많이하고이는 immunologic reaction 을유발하는것으로알려져있다. 최근의연구에서는대식세포의 hydrogen peroxide 생성증가가 T-cell 과관련된 arthritis 를억제하는등자가면역질환발생을방어하는작용이있음이보고되었다 35-37). SFS가대식세포의 hydrogen peroxide(h 2 O 2 ) 의생성에미치는영향을비교하였다. 24시간에서 25 μg /ml 이상일때유의한증가를나타내었다. 그리고 24시간, 45시간, 그리고 72시간동안 LPS 단독으로배양한경우 Normal 군보다유의하게 H 2O 2 생성이감소하였으며 LPS 와 SFS 을함께배양한경우 SFS 의농도가 각각 50 μg /ml, 100 μg /ml, 200 μg /ml 이상일때 LPS 에의한감소를유의 - 36 -
하게회복하였다. SFS가 HepG2 cell 의 hydrogen peroxide(h 2 O 2 ) 의생성에미치는영향을알아보기위해서 24시간동안배양한결과 25, 50, 100 μg /ml 일때유의한증가를나타내었다. 이와같이 SFS 에의해대식세포의 H 2O 2 이증가함은 SFS 가자가면역질환발생억제제로서의가능성이있음을의미하는것이다. Nitric oxide 는 L-arginine 으로부터 nitric oxide synthase(nos) 에의해만들어지며, NOS는항상성유지에필요한 NO를생성하는 endothelial NOS(eNOS) 및 neuronal NOS(nNOS) 와염증성인자등에의해유도되는 inducible NOS(iNOS) 로분류할수있다. 38) SFS가대식세포의 nitric oxide(no) 의생성에미치는영향을비교하였는데 4시간의배양에서는 25, 50, 200 μg /ml일때유의한감소를나타내었고, 24시간의배양에서는 25 μg /ml 이상일때모두유의한감소를나타내었다. 24 시간동안 LPS 단독으로배양 한경우 Normal 군보다유의하게 NO생성이증가하였으며 LPS와 SFS을함께배양한경우 SFS의농도가 50, 100 μg /ml일때 LPS에의한증가를유의하게억제시켰다. 이와같이 SFS에의해서 LPS에의해유발된대식세포의 NO 배출증가를억제함은 SFS 가염증반응억제의효능이있음을의미한다. 싸이토카인은한세포에서생산되어다른세포의형태에영향을미치는단백으로림프구에서생산되는싸이토카인을림포카인 (lymphokine) 또는인터루킨 (interleukin, IL) 이라고한다. 싸이토카인은표적세포의특이수용체와결합한다. 39) TNF-α는활성화된대식세포에의하여주로생성되며, 세균감염이나악성종양발생시숙주의반응에있어서중요한역할을한다. 40,41) SFS 가 LPS로유발된대식세포의 TNF-α 생성증가에미치는영향을비교했을때 3시간동안 LPS 단독으로배양한경우 Normal 군보다유의하게 TNF-α 생성이증가하였으며 LPS와 SFS을함께배양한경우 SFS의농도가 25 μg /ml이상일때모두 LPS에의한증가를유의하게감소시켰다. IL-6 는단핵대식세포, 혈관내피세포, 섬유세포및 T-cell 에서만들어지며, - 37 -
많은기능과효과를나타낸다. 42) SFS가 LPS로유발된대식세포의 IL-6 생성증가에미치는영향을비교하였는데 3시간동안 LPS 단독으로배양한경우 Normal 군보다유의하게 IL-6 생성이증가하였으며 LPS와 SFS을함께배양한경우 SFS의농도가 25 μg /ml 이상일때모두 LPS에의한증가를유의하게감소시켰다. 이밖에 LPS에의한 IL-2, IL-4, IL-12, MCP-1, KC, RANTES 등의면역단백질생성증가에대해서는 SFS 가별다른유의성을나타내지않았다. SFS에의해서 LPS에의해유발된대식세포의 TNF-α와 IL-6 생성증가가억제됨은 SFS 가 cytokine 발현조절을통한염증반응조절의효능이있음을의미한다. 이상의결과, 苦參발효추출물 SFS는대식세포와 HepG2 cell 에세포독성을과도히유발하지않으면서대식세포와 HepG2 cell 의 hydrogen peroxide 생성을증가시키고대식세포의 NO 생성을감소시켰으며특히 LPS로유발된대식세포의 TNF-α와 IL-6 생성을억제하는등유의한면역활성이있음을알수있었다. 앞으로苦參발효추출물을이용한대식세포연관면역질환치료제로의개발을위하여더욱많은연구가필요할것으로사료된다. - 38 -
Ⅴ. 결론 苦參을 Saccharomyces cerevisiae STV89( 효모 ) 로발효추출한후얻은추출시료 SFS 로마우스 Raw 264.7 cell 과인간 HepG2 cell 을이용한 cell viability, 세포내 hydrogen peroxide 생성, NO 생성, 그리고 cytokines(tnf-α 와 IL-6 등 ) level 에미치는영향을측정하여다음과같은결론을얻었다. 1. 苦參발효추출물의세포독성을확인하기위해 MTT assay 를수행한결과 苦參발효추출물을농도별로처리했을때대식세포와 HepG2 cell 에독성 을과도히유발하지않았다. 2. 苦參발효추출물은대식세포와 HepG2 cell 의세포내 hydrogen peroxide 생성을증가시켰으며 LPS 로유발된대식세포내의 hydrogen peroxide 생성 억제를유의하게회복시켰다. 3. 苦參발효추출물은대식세포의 NO 의생성을억제하였으며 LPS 로유발된 대식세포의 NO 생성증가를유의하게억제하였다. 4. 苦參발효추출물은 LPS 에의해서유발된대식세포의 TNF-α 및 IL-6 생성량을 25 μg /ml 이상에서유의하게감소시켰다. 이상의결과, 苦參으로제조된발효추출물 SFS는대식세포와 HepG2 cell 에세포독성을과도히유발하지않으면서대식세포와 HepG2 cell 의 hydrogen peroxide 생성을증가시키고대식세포의 NO 생성을감소시켰으며특히 LPS 로유발된대식세포의 TNF-α와 IL-6 생성을억제하는등유의한면역활성을가진것으로나타났다. - 39 -
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ABSTRACT Effect of Fermented Sophorae Radix Extract on Immuno-modulating Activity Kim, Hyung Seok Supervised by Lee, Young Jong Dept. of Oriental Medicine, Graduate School. Kyungwon University Objective: This study aims at examining the effect of the fermentative extract of root of Sophorae Radix on the immuno-modulating activity. Method: Measurements were done for the influences on the cell viability, the generation of hydrogen peroxide in cells and of NO and the level of cytokines (TNF-α, IL-6, etc.), using the macrophage of mouse and human HepG2 cell with the specimen SFS of the fermentative extract with Saccharomyces cerevisiae STV89 (a yeast fungus). Results: 1. As a result of carrying out MTT assay to check the cellular toxicity of the fermentative extract of Sophorae Radix, There was not any excessive toxicity to the macrophage and HepG2 cell when the fermentative extract of root of Sophorae Radix was treated in - 44 -
different concentrations. 2. The fermentative extract of Sophorae Radix increased the generation of hydrogen peroxide in the macrophage and HepG2 cell and significantly restored the suppression of the generation of the hydrogen peroxide in the macrophage induced by LPS. 3. The fermentative extract of Sophorae Radixt reduced the generation of NO in the macrophage and significantly suppressed the increase of the generation of NO in the macrophage induced by LPS. 4. The fermentative extract of Sophorae Radix significantly decreased the amount of TNF-α generated in the macrophage induced by LPS when it was 25 μg /ml or higher. Conclusions: SFS, the fermentative extract of Sophorae Radix, did not excessively cause the cellular toxicity in the macrophage and HepG2 cell, but increased the generation of hydrogen peroxide in the macrophage and HepG2 cell, decreased the generation of NO in the macrophage, and especially it was found that there was a significant immuno-modulating activity such as suppressing the generation of TNF-α, IL-6, in the macrophage led by LPS. Keywords: Sophorae Radix, fermentation, cellular toxicity, immuno-modulating activity, reactive oxygen species(ros) - 45 -