<C7D1B9E6C0E7C8B0C7D0C8B B1C731C8A35F34C2F7C6EDC1FD E687770>

Transcription

1 Journal of Korean Medicine Rehabilitation Vol. 28 No. 2, April 2018 pissn eissn Original Article 大黃牽牛散에탄올추출물의 Methicillin 내성 Staphylococcus aureus 에대한항균활성연구 박주영 * 나용수 * 오공천 * 이상미 * 최병권 * 이윤승 송용선 원광대학교한의학전문대학원제 3 의학과 *, 원광대학교한의과대학한방재활의학교실 A Study on Antibacterial Activity of Daehwanggeonwoo-san(Dahwangqianniu-san) Ethanol Extract against Methicillin-Resistant Staphylococcus Aureus Ju-yeong Park, Ph.D.*, Yong-su Na*, Gong-cheon Oh*, Sang-mi Lee*, Byeong-kwon Choi*, Yoon-seung Lee. K.M.D., Yung-sun Song. K.M.D. Department of third medicine of Korean Medicine, professional graduate school of Oriental medicine, Won-kwang University*, Department of Rehabilitation Medicine of Korean Medicine, Colleage of Korean Medicine, Won-kwang University 본연구는 2017 년원광대학교교내연구비지원의결과입니다. RECEIVED Mar 18, 2018 ACCEPTED Apr 3, 2018 CORRESPONDING TO Yung-Sun SONG, Department of Rehabilitation Medicine of Korean Medicine, College of Korean Medicine, Won-Kwang University, 99, Garyeonsan-ro, Deokjin-gu, Jeonju , Korea TEL (063) FAX (063) yssong@wku.ac.kr Copyright 2018 The Society of Korean Medicine Rehabilitation Objectives The objective of this study is to determine the antimicrobial effect of Daehwanggyeonu-san(Dahwangqianniu-san,DGE) and synergistic effects with antibiotics oxacillin, ampicillin, and gentamicin against Methicillin-Resistant Staphylococcus aureus(mrsa). Methods The antibacterial activity of DGE extract was evaluated againest MRSA strains by using the Disc diffusion method, broth microdilution method(minimal inhibitory concentration; MIC), checkerboard dilution test. The checkerboard dilution test was used to examined synergetic effect of oxacillin, ampicillin, gentamicin, ciprofloxacin with DGE extract. Results DGE showed antimicrobial activity against MRSA with an MIC value of μg/ml. In the checkerboard test, the interation of DGE with all tested antibiotics produced almost synergy or partial synergy against MRSA. Conclusions This study shows that DGE reduced the MICs of several antibiotics tested, and a remarkable antibacterial effect of DGE, with membrane permeability enhancers and ATP synthase inhibitors. This study can be a valuable source for the development of a new drug with low MRSA resistance. (J Korean Med Rehabil 2018;28(2):21-35) Key words Daehwanggyeonu-san(Dahwangqianniu-san,DGE), methicillin-resistant Staphylococcus aureus(mrsa), antibacterial effect, MIC. 서론»»» 인간의거의모든포도상구균성질병은증식할때포도상세포군집을형성하는통성혐기성구균인 Staphylococcus aureus(s. aureus) 에의해서일어난다. S. aureus 는건강한사람또는가축의피부와비공에상재하여이중절반이상이식중독을일으키는내열성의장독소 (enterotoxin) 를생산한다 1-3). 1940년대초반이래로황색포도상구균감염 증의치료에항균력이우수한 penicillin, methicillin 등의항생제를사용하여왔으며, 1961년영국에서 MRSA(methicillin-resistant Staphylococcus aureus) 처음보고되었으며, 1981년국내에서처음보고된이래로대표적인병원감염균주인 MRSA는국내병원들의임상검체로부터분리한황색포도상구균의 70% 를차지하고, 중환자실분리균주중황색포도상구균의 80% 를차지하고있다 4). 항생제의과다사용은다제내성균의출현및제어실 21

2 박주영 나용수 오공천 이상미 최병권 이윤승 송용선 패의중요한원인이되는만큼주요의료관련감염원인균의항균제내성발현기전, 항균제내성전파방지를위한내성획득기전, 병원체의분자역학적분석을통한내성균전파규명을위해노력해야할것이다 5). 임상에서항균및항암치료효과를상승시키기위해교체내성 (cross-resistance) 이없고, 작용기전이서로다른약제를사용하는복합요법이주로이용되고있음에따라본연구에서는이러한항생제내성문제를낮추어주기위한천연물유래항균물질의가능성을알아보고자한다 6-8). 大黃牽牛散은 素問病機氣宜保命集 9) 卷中 熱論第十四 에최초로기재되었으며, 東醫寶鑑 10) 에수록된처방으로 治相火遊走臟腑, 大便秘結 이라하였다. 처방구성은大黃과牽牛子로되어있으며, 大黃은瀉下作用, 淸熱凉血의약리학적효능을가지고있어, 항균, 항염, 항바이러스, 항종양등의다양한효능이있다고알려져있다 11-14). 牽牛子는瀉下祛積, 逐水退腫, 殺蟲의약리학적효능을가지고있어, lipopolysaccharide 에의해유도된 RAW cell에서염증억제효과, 간기능개선효과등이보고된바있다 15,16). 이와같이大黃과牽牛子에단미에대한효과는보고되고있지만복합제제인大黃牽牛散에대한연구는아직까지보고된바가없다. 이에저자는大黃牽牛散의 Disc 확산실험을통해 MRSA 에대한항균활성을검증하였으며, 최소억제농도 (MIC) 를측정하여기존항생제와大黃牽牛散추출물을병행처리하였을때 synergy effect 및 partial synergy effect를조사하였고, 大黃牽牛散의항균활성이세포막투과성과 ATPase 억제에연관성이있는가를실험하여유의한결과를얻었기에보고하는바이다. Table I. The Composition of Daehwanggyeonu-san (Dahwangqianniu-san) Herbal name Pharmacognostic name Weight 大黃 Rhei Rhizoma 10 g 牽牛子 Pharbitidis Semen 5 g 2. 시약 Mueller-Hinton agar(mha) 와 Mueller-Hinton broth(mhb) 는 Difco (Baltimore, MD, USA) 에서, Tris-(hydroxymethyl) aminomethane은 AMRESCO(San Francisco, CA) 에서, Sodium azide(nan 3 ) 는 Fluka (Neu-Ulm, Germany) 에서, 항생제 oxacillin & ampicillin & gentamacin & ciprofloxacin과 Triton X-100, N,N-dicyclohexylcarbodimide(DCC) 는 Sigma-Aldrich(St. Louis, USA) 에서구입하여실험에사용하였다. 3. 균주및배양 본실험에사용한표준균주 (type strain) ATCC 33591은 American Type Culture Collection(Manassas, VA, USA) 에서구입하였고, CCARM 3090, 3091, 3095, 3102는서울여대항생제내성균주은행으로부터, DPS-1,2,3은원광대학교병원성형외과에서분양받아임상균주로사용하였다. 균주는 79 C 냉동고에서 30% glycerol 에보관하다가, Mueller-Hinton agar(mha) 에접종한후 37 C 항온기 (incubator) 에서 24시간배양하여실험에사용하였다. 재료및방법»»» 1. 大黃牽牛散의제조大黃牽牛散은 東醫寶鑑 10) 의처방을기준으로大黃 10 g, 牽牛子 5 g을 3첩분량 45 g을 70% ethanol 500 ml로추출하여감압농축, 동결건조의과정을거쳐 18.8% 의수득률을취했다 (Table I). 4. 항균활성측정 1) 디스크확산법 (Disc diffusion method) 大黃牽牛散에탄올추출물 (DGE) 의항균효과는디스크확산법을통해 MRSA에대한항균활성을측정하였다 17-19). 고압멸균된 MHA에 24시간배양된균 100 μl를골고루도말하여최종적으로 CFU/mL이되게하였다. 다음으로멸균된 paper disc(6 mm) 에 DGE(25 μg, 50 μg, 100 μg, 200 μg) 와 AP 10 μg을각각 20 μl씩주입하여완전히흡수시킨후 37 incubator 에서 24시간배양시켜 paper disc 주위에형성된억제직경을측정 22 J Korean Med Rehabil 2018;28(2):21-35

3 大黃牽牛散에탄올추출물의 Methicillin 내성 Staphylococcus aureus 에대한항균활성연구 하였다. 2) 최소억제농도 (Minimum inhibitory concentration, MIC) 최소억제농도 (MIC) 는 Clinical and Laboratory Standards Institute (CLSI 2000) 방법에따라액체배지희석법을통해측정하였다 20). DGE를액체배지에농도별로희석하여, 여기에탁도 0.5 McFarland CFU/mL을기준으로 10 μl씩접종하여최종적으로 CFU/well이되게하였다. 다음으로 37 C 항온기에서 24시간배양후미생물이증식되지않는가장낮은약물의농도를 MIC로측정하였다 21,22). 3) 약물병용효과 (Fractional inhibitory concentration index, FICI) DGE와항생제의병용효과를보기위해 checkerboard dilution test를실시하였다. 상용항생제는 oxacillin (OX), ampicillin (AP), gentamicin (GT). ciprofloxacin (CP) 이며상기액체배지희석법과동일한방법으로항생제를병합하였을때 MIC를측정하였다. FICI 산출기준은다음과같다 22,23). FICI = FIC A +FIC B = A/MIC A +B/MIC B (MIC A : 약물A의 MIC, MIC B : 약물B 의 MIC, A: 약물B 를병용한약물A의농도, B: 약물A를병용한약물B의농도 ) DGE 의항균활성이세포막투과성강화제와 ATP synthase 저해제를병합하였을때활성의변화를보기위해 detergent 인 Triton X-100(TX) 과 Tris-(hydroxymethyl) aminomethane (Tris) 및 ATP synthase inhibitor를사용하였다. TX는 non-ionic detergent로막가용화기능을높임으로써항생제감수성을증가시킬수있으며, TX와 Tris는막투과성증진제로사용하였다 26-27). N,N-Dicyclohexylcarbodiimide(DCCD) 와 sodium azide (NaN 3 ) 는 F 0 -F 1 -ATP synthase(f 0 F 1 ) 를저해함으로써 ATP 의대사를억제하는것으로 28), DGE의항균활성이 MRSA 의 ATP-dependent transport systems에관여하는지보기위해사용하였다. 5. 통계분석모든실험은 3회이상반복으로이루어졌으며, 실험결과는각항목에따라평균치 ± 표준편차 (SEM) 를구하여그유의성은 Student s t-test 분석법을이용하여신뢰수준 95% (p < 0.05) 에서통계적유의차를평가하였다. 결과»»» 1. 디스크확산법 (Disc diffusion method) FICI<0.5, synergy; 0.5 FICI<0.75, partial synergy; 0.75 FICI<1.0, additive effect; 1.0 FICI<4.0, no synergy; FICI>4.0, antagonism. 4) 생장곡선분석 (Growth curve assay) DGE와항생제 OX, AP, GT, CP를병용했을때 MRSA 증식변화를보기위해시간에따른 MRSA의생장곡선을흡광도법 (optical density, OD 600 ) 으로측정하였다 24,25). 5) 세포막투과성 (cytoplasmic membrane permeability) 과 Adenosine triphosphate(atp) synthase 억제 MRSA ATCC 33591, CCARM 3091, DPS-2에대한 DGE 의항균효과는디스크확산법을통해나타냈다. DGE의 100 μg/20 μl 및 200 μg/20 μl에서유효한항균활성을나타냈다 (Fig. 1-3). 2. 최소억제농도 (Minimum inhibitory concentration, MIC) DGE의항균활성은최소억제농도 (MIC) 를통해확인하였다. MRSA 8균주에대한 DGE의최소억제농도는 μg/ml이었다 (Table II). 23

4 박주영 나용수 오공천 이상미 최병권 이윤승 송용선 Fig. 1. Diameter of inhibition zone(mm) of DGE against MRSA(ATCC 33591). Fig. 2. Diameter of inhibition zone(mm) of DGE against MRSA(CCARM 3091). Fig. 3. Diameter of inhibition zone(mm) of DGE against MRSA(DPS-2). 24 J Korean Med Rehabil 2018;28(2):21-35

5 大黃牽牛散에탄올추출물의 Methicillin 내성 Staphylococcus aureus 에대한항균활성연구 Table II. Antimicrobial Activity of DGE Against 8 Strains of MRSA. Strains MIC (μg/ml) DGE OX AP GT CP > CCARM , CCARM , CCARM CCARM DPS > DPS DPS > OX, oxacillin; AP, ampicillin; GT, gentamacin; CP, ciprofloxacin. Table III. Results of the Combination of DGE and OX Against MRSA. Strains agent MIC (μg/ml) Alone Combination FIC FICI Outcome ATCC DGE OX Partial S CCARM 3090 DGE OX 1, Partial S CCARM 3091 OX S CCARM 3095 OX S CCARM 3102 DGE OX AE DPS-1 DGE OX NS DPS-2 DGE OX NS DPS-3 DGE OX NS OX; oxacillin, S, synergy; AE, additive effect; NS, no synergy. 3. 약물병용효과 (Fractional inhibitory concentration index, FICI) 1) DGE 와 oxacillin(ox) 의병용효과 DGE와베타락탐계 (β-lactams) 항생제의상승효과를조사하기위해베타락탐계항생제인 OX를병용한결과, MRSA 4균주에서 synergy effect및 partial synergy effect 를나타냈다 (Table III) μg/ml DGE에서 OX 의농도를 2 32배감소시켰다. 2) DGE와 ampicillin(ap) 의병용효과 DGE와베타락탐계 (β-lactams) 항생제인 AP를병용한결과, MRSA 6균주에서 synergy effect및 partial synergy effect를나타냈다 (Table IV) μg/ml DGE에서 AP의농도를 2 64배감소시켰다. 25

6 박주영 나용수 오공천 이상미 최병권 이윤승 송용선 Table IV. Results of the Combination of DGE and AP Against MRSA. Strains agent MIC (μg/ml) Alone Combination FIC FICI Outcome ATCC DGE AP Partial S CCARM 3090 DGE AP Partial S CCARM 3091 DGE AP Partial S CCARM 3095 AP S CCARM 3102 AP Partial S DPS-1 DGE AP NS DPS-2 DGE AP S DPS-3 DGE AP NS AP; ampicillin, S, synergy; NS, no synergy. Table V. Results of the Combination of DGE and GT Against MRSA. Strains agent MIC (μg/ml) Alone Combination FIC FICI Outcome ATCC DGE GT NS CCARM 3090 DGE > GT > NS CCARM 3091 DGE GT 1, Partial S CCARM 3095 GT S CCARM 3102 GT Partial S DPS-1 DGE GT S DPS-2 GT S DPS-3 DGE GT 0.48> GT; gentamicin, S, synergy; NS, no synergy. 26 J Korean Med Rehabil 2018;28(2):21-35

7 大黃牽牛散에탄올추출물의 Methicillin 내성 Staphylococcus aureus 에대한항균활성연구 Table VI. Results of the Combination of DGE and CP Against MRSA. MIC (μg/ml) Strains agent Alone Combination ATCC CCARM 3090 CCARM 3091 CCARM 3095 CCARM 3102 DPS-1 DPS-2 DPS-3 CP; ciprofloxacin, S, synergy. DGE CP 0.48> - FIC FICI Outcome DGE > CP S DGE CP S DGE CP S CP Partial S DGE CP 0.48> DGE > CP S DGE CP Partial S 3) DGE와 gentamicin(gt) 의병용효과 DGE와아미노글리코사이드계 (aminoglysides) 항생제의상승효과를조사하기위해 GT를병용한결과, 실시한 MRSA 7균주중 5균주에서 synergy effect및 partial synergy effect를나타냈다 (Table V) μg/ml DGE 에서 GT의농도를 2 8배감소시켰다. 4) DGE와 ciprofloxacin(cp) 의병용효과 DGE와퀴놀론계 (quinolons) 항생제의상승효과를조사하기위해 CP를병용한결과, 실시한 MRSA 6균주에서 synergy effect및 partial synergy effect를나타냈다 (Table VI) μg/ml DGE에서 CP의농도를 2 32배감소시켰다. 4. Growth curve assay DGE(250 ug/ml) 와항생제 OX, AP, GT, CP를병용하였을때 MRSA의생장곡선은 Fig.4-7에나타냈다. 1) DGE + OX에서 MRSA(CCARM 3091) 의생장곡선 DGE와 OX의 1/2 MIC를단일및병용하여 24시간동안처리한결과, 약물을처리하지않은군에비해 OX군은비교적정상적인증식상태를나타냈으며 DGE군은약물활성에의해증식이억제되었으나 8시간이후부터 MRSA 증식이점차증가하는양상을나타냈다. 반면에 DGE와 OX를병용한군에서는확연하게억제된 MRSA의증식을보여주었다 (Fig. 4). 2) DGE + AP에서 MRSA(DPS-2) 의생장곡선 DGE와 AP의 1/2 MIC를단일및병용하여 24시간동안처리한결과, 약물을처리하지않은군에비해 AP와 DGE 군은약물활성에의해저해되었으나동일한양상을나타냈다. 반면에 DGE와 AP를병용한군에서는완전히억제된 MRSA의증식을보여주었다 (Fig. 5). 3) DGE + GT에서 MRSA(DPS-1) 의생장곡선 DGE와 GT의 1/2 MIC를단일및병용하여 24시간동안처리한결과, 약물을처리하지않은군과 GT군은비슷한증식양상을나타냈으나 DGE군과 DGE와 GT병용 27

8 박주영 나용수 오공천 이상미 최병권 이윤승 송용선 Fig. 4. Growth Curves for MRSA(CCARM 3091) in the Presence or Absence of DGE and Oxacillin(OX). Bacterial Cell Growth was Measured by Using OD at 600 nm at Indicated Time Points. Fig. 5. Growth Curves for MRSA(DPS-2) in the Presence or Absence of DGE and Ampicillin(AP). Bacterial Cell Growth was Measured by Using OD at 600 nm at Indicated Time Points. Fig. 6. Growth Curves for MRSA(DPS-1) in the Presence or Absence of DGE and Gentamacin(GT). Bacterial Cell Growth was Measured by Using OD at 600 nm at Indicated Time Points. 28 J Korean Med Rehabil 2018;28(2):21-35

9 大黃牽牛散에탄올추출물의 Methicillin 내성 Staphylococcus aureus 에대한항균활성연구 Fig. 7. Growth Curves for MRSA(CCARM 3095) in the Presence or Absence of DGE and Ciprofloxacin(CP). Bacterial Cell Growth was Measured by Using OD at 600 nm at Indicated Time Points. Fig. 8. Effect of the Membrane-Permeabilizing Agent TX on the Susceptibility of MRSA(ATCC 33591) to DGE. The Viability of Bacteria was Determined Spectrophotometrically(Optical Density at 600 nm, OD 600 ) After Incubation for 24 hr with 7.8 μg/ml DGE and % TX. The Data are Average of Triple-Independent Experiments. *p < 0.01 Compared to Control(CON). Fig. 9. Effect of the Membrane-Permeabilizing Agent TRIS on the Susceptibility of MRSA(ATCC 33591) to DGE. The Viability of Bacteria was Determined Spectrophotometrically(Optical Density at 600 nm, OD 600 ) After Incubation for 24Hr with 7.8 μg/ml DGE and 62.5 μg/ml Tris. The Data are Average of Triple-Independent Experiments. *p < 0.01 Compared to Control(CON). 29

10 박주영 나용수 오공천 이상미 최병권 이윤승 송용선 Fig. 10. Effect of the ATP Synthase Inhibitor DCCD on the Susceptibility of MRSA(ATCC 33591) to DGE. The Viability of Bacteria was Determined Spectrophotometrically(OD 600 ) after Incubation for 24 Hr with 7.8 μg/ml DGE and 62.5 μg/ml DCCD. The Data are Average of Triple-Independent Experiments. *p < 0.01 Compared to Control(CON). Fig. 11. Effect of the ATP Synthase Inhibitor NaN 3 on the Susceptibility of MRSA(ATCC 33591) to DGE. The Viability of Bacteria was Determined Pectrophotometrically(OD 600 ) after Incubation for 24Hr with 7.8 μg/ml DGE and 50 μg/ml NaN 3. The Data are Average of Triple-Independent Experiments. *p < 0.01 Compared to Control(CON). 군은약물활성에의해확연하게억제된 MRSA의증식을보여주었다 (Fig. 6). 4) DGE + CP에서 MRSA(CCARM 3095) 의생장곡선 DGE와 CP의 1/2 MIC를단일및병용하여 24시간동안처리한결과, 약물을처리하지않은군에비해 CP군은비교적정상적인증식상태를나타냈으며 DGE군은약물활성에의해증식이억제되었으나 8시간이후부터 MRSA 증식이점차증가하는양상을나타냈다. DGE와 CP를병용한군에서는확연하게억제되다가 8시간이후점차증가하는 MRSA의증식을보여주었다 (Fig. 7). 5. DGE 와막투과성증진제및 ATP synthase 억제제 1) DGE 와막투과성증진제의효과 DGE의항균활성과막투과성증진제의효과를조사하기위해, 7.8 μg/ml DGE와 % TX 및 62.5 μ g/ml Tris를각각병용하였다. 그결과, 병용처리한약물에서 MRSA(ATCC 33591) 의생존력을각각 70% 와 85% 로감소시켰다 (Fig. 8, 9). 2) DGE 와 ATP synthase 억제제의효과 ATP synthase 를감소시키는대사억제제로써, DCCD 와 30 J Korean Med Rehabil 2018;28(2):21-35

11 大黃牽牛散에탄올추출물의 Methicillin 내성 Staphylococcus aureus 에대한항균활성연구 NaN 3 을 7.8 μg/ml DGE와각각병용한결과, 각각 82% 와 47% 의억제율을나타냈다 (Fig. 10, 11). 고찰»»» 황색포도상구균 (Staphylococcus aureus, S. aureus) 는농가진, 종기, 상처의농양등화농성의피부감염뿐아니라결핵, 폐렴및독성쇼크증후군등의중증감염을포함한광범위한감염을일으킬수있다 29,30). 병원성 S. aureus 는높은 2차감염률의원인균으로혈액응고효소 (coagulase), 백혈구사멸소 (leucocidin) 와용혈소 (hemolysin) 등의전염병발생에중요한세포외단백질을생성해낸다 31-33). 이중반합성 penicillin인 methicillin 에내성을보이는것을 methicillin 내성황색포도상구균 (methicillin-resistant Staphylococcus aureus ; MRSA) 이라한다 34). S. aureus 는 β-lactam 계열항생제의표적인새로운 penicillin binding protein(pbp 2a) 을생성하여 mec 유전자에의해 β-lactam 항생제와의친화도가낮은새로운페니실린결합단백 (penicillin binding protein, PBP 2a) 을생성하여 methicillin에내성을가지게된다 35-37). 세계곳곳에서항생제내성으로인해다제내성균이발생하고있다. 항생제의과다사용은다제내성균의출현및제어실패의중요한원인이되는만큼적절한대책이시급하다 38,39). 최근국내병원중환자실에서분리되는 S. aureus에서메티실린 (methicilllin) 내성이 90% 로보고되는등여러계열의항생제에다제내성을보이고있어이러한다제내성균의증가와확산은기존의항생제로는더이상치료하기어려운상황에직면하게했다 40,41). 최근연구동향은기존의항균제들과는다른차별화된새로운작용기전을갖거나기존항균제의구조를변형시켜내성균에효과를보이는광범위스펙트럼항생제를개발하는것이다. 또한항생제내성발생으로인한항생제의시장판매주기가짧은점으로개발비율이낮은점을고려하여야한다. 항균제병용요법은 1차단독요법의실패를극복, 효과적인항균범위를확보하기위한 2차항생제에있어다제내성균에유효한항생효과를발휘하여야하며병용약제와의상호작용등임상적특성도고려되어야한다 42-44). 大黃牽牛散은 東醫寶鑑 에수록된처방으로大黃牽牛散을이루는개별약재의본초학적인효능을살펴보면, 大 黃은蓼科 ( 여뀌과 ; Polygonaceae) 에속한多年生高大草本인장군풀및同屬近緣植物의根및根莖이다. 苦, 寒, 無毒하고脾, 胃, 大腸, 肝, 心五經에작용하며攻積導滯, 瀉火解毒行瘀通經, 淸利濕熱등의효능이있어實熱便秘, 積滯腹痛, 腸癰, 瀉利不爽, 瘀血經閉, 濕熱黃疸등의병증을치료한다. 牽牛子는旋花科 ( 메꽃과 ; Convolvulaceae) 에속한 1 年生攀援草本인나팔꽃및둥근잎나팔꽃의成熟한種子이다. 苦, 辛, 寒, 大毒하고肺, 腎, 大腸三經에작용하며瀉下祛積, 逐水退腫, 殺蟲, 瀉肺逐痰등의효능이있어水腫脹滿, 二便不通, 諸蟲病, 痰飮積聚, 氣逆咳喘등의병증을치료한다 45). 개별약재의성분및이전연구를살펴보면, 大黃의주요성분은 glucosides rhein-8monoglucoside, physcion monoglucoside, aloeemodin monoglucoside, sennoside tannin, gallic acid, calechin 등으로보고되었다 46). 大黃에관련된이전연구로는최등 47) 이지방조직의염증및인슐린저항성에미치는영향을알아보았으며, 장등 48), 조등 49), 이등 50) 이大黃의혈당및혈청지질개선에효과가있다는발표가있었으며, 윤등 51) 은 HepG2 Cell에대한항암효과, 박등 52) 의항산화효과와 MMP-1의활성저해효과에대한실험등다양한실험적연구가있었다. 또한大黃에대한항균활성에대한연구로는이 53) 의大黃에서분리된플로로탄닌의여드름균에대한항균및시너지효과를보고하였으며, 엄 54) 의 MRSA에대한항균물질의화학구조구명및작용기작을해석한연구가있으며, 최 55) 의大黃에서추출한 emodin의 MRSA에대한항균작용이보고되고있고, 이와같이항균효과에대해많은보고가있었다. 牽牛子의대표성분은 Gibberel- lenic acid, Pharbitin, Nilic acid, Pharbitic acid 등이함유되어있다고보고되어져있으며 56), 牽牛子의성분중 Pharbitin은腸道, 腎腸, 중추신경계통등에손상을일으킨다 57). 牽牛子에대한실험적연구로는고등 15), 윤등 58) 이소염및해열작용에미치는영향을보고하였고, 김등 59) 이조골세포분화에미치는영향을연구하였고, MRSA에대한항균작용에대한보고는없었다. 현재까지大黃牽牛散에대한연구는보고되지않았으며, 항균활성연구가보고된大黃에瀉下祛積, 殺蟲의효능을가진牽牛子가처방되어보다높은항균효과를높일수있기를기대하는大黃牽牛散에대한연구가필요하리라사료된다. 본실험은 DGE의 MRSA에대한항균효과와기존항생 31

12 박주영 나용수 오공천 이상미 최병권 이윤승 송용선 제와의상승작용및세포의구조적특성과에너지대사에기인하여항균활성의작용기작을조사하였다. 大黃과牽牛子의두약물로이루어진 DGE는디스크확산법을통해 MRSA ATCC 33591, CCARM 3091, DPS-2에유효한항균활성을나타냈다. 또한 MRSA 표준균주와임상균주를포함한 8균주에서우수한항균효과를나타냈다. DGE를항생제와병용처리하였을때 MRSA에대한상승효과를확인하기위해베타락탐계 (OX & AP), 아미노글리코사이드계 (GT) 및퀴놀론계 (CP) 항생제를병합한결과, 대부분상승효과를나타내었으며, DGE와항생제 OX, AP, GT, CP를병용하였을때 MRSA의생장곡선을분석한결과, 병용한군에서는확연하게억제된 MRSA의증식을보여주었다. 이는 DGE가다제내성을갖는 MRSA 의항생제내성을낮추어줄수있는천연물항균제로써의가능성을유추할수있었다 21,60,61). DGE의항균활성과막투과성증진제및 ATP synthase 억제제효과를조사하기위해, 7.8 μg/ml DGE와 % TX 및 62.5 μg/ml Tris를각각병용하였다. 그결과, 병용처리한약물에서 MRSA(ATCC 33591) 의생존력을감소시켰으며, ATP synthase를감소시키는대사억제제로써, DCCD와 NaN 3 을 7.8 μg/ml DGE와각각병용한결과, 억제율을나타냈다. DGE의항균활성은세포막의구조적특성과에너지대사기능에있어서침투력이상승했다. 세포벽안쪽에서세포질을둘러싸고있는세포막 (cytoplasmic membrane) 은세포내외로물질의선택적투과성을부여한다. 이는어떤분자나이온들은쉽게막을통과할수있지만어떤것들은제한을받는다 62). 大黃牽牛散과병용투여하였을때 TX와 Tris가 MRSA의막표면구조를이완시켜세포내로침투하는항생제의이동을증가시키는데기인하여大黃牽牛散항균활성의침투력을높여주었다고사료된다. 세포막은 cytochrome oxidase, dehydrogenase, ATPase, protein synthetase 등에너지생산및물질생산에필요한다양한효소와전자운반체를갖고있다. ATP synthase는 ADP와인산을합성하여 ATP를만들어내는반응촉매효소로써 DCCD와 NaN 3 는이러한 ATP 대사를저해하는데기인하여大黃牽牛散의항균활성을증진시켰을것으로사료된다. 위실험들로보아大黃牽牛散에탄올추출물에는 MRSA 를저해할수있는강한항균효과가있음을확인하였고, 또한기존의항생제와병용하였을때약물상승효과를볼 수있어 MRSA에대한항생제후보물질중하나로大黃牽牛散또는大黃牽牛散과항생제의병용요법이충분한가치가있음을, 더나아가기존항생제의사용을감소시킬수있는후보물질임을기대할수있을것으로사료된다. 그러나임상에서실제치료에응용하기위하여향후임상시험을통한항균효과및항생제병용효과에대한추가적인연구가필요할것이라사료된다. 결론»»» 大黃牽牛散에탄올추출물 (DGE) 의 Methicillin 내성 Staphylococcus aureus(mrsa) 에대한항균활성을측정하여다음과같은결과를얻었다. 1. DGE는디스크확산법을통해 MRSA 세균주에대해 100 μg/20 μl 및 200 μg/20 μl에서항균효과를나타냈다. 2. DGE의 MRSA 최소억제농도는 (MIC) 는 μ g/ml 이었다. 3. DGE와항생제 OX, AP, GT, CP의병용결과, 大黃牽牛散 μg/ml에서상승효과를나타냈으며, 항생제의농도를 2배 64배낮추어주었다. 4. DGE는 TX와 Tris가막투과성을증가시켜항균활성의침투력을증가시켰다. 5. DGE는 DCCD와 NaN 3 가 ATP 대사를저해하여항균활성이증진되었다. 이상실험결과, 大黃牽牛散에탄올추출물이 MRSA에유효함과, 기존항생제와의상승효과로항생제농도를낮추어줄수있어다제내성 MRSA에대한항생제후보물질로써가능성을시사하는바이다. References»»» 1. Rao Q, Shang W, Hu X, Rao X. Staphylococcus aureus ST121: a globally disseminated hypervirulent clone. J Med Microbiol. 2015;64(12): DOI: /jmm da Silva Sdos S, Cidral TA, Soares MJ, de Melo MC. Enterotoxin-encoding genes in staphylococcus spp. from food handlers in a university restaurant. Foodborne 32 J Korean Med Rehabil 2018;28(2):21-35

13 大黃牽牛散에탄올추출물의 Methicillin 내성 Staphylococcus aureus 에대한항균활성연구 Pathog Dis. 2015;12(11): Zhao WH, Hu ZQ, Okubo S, Hara Y, Shimamura T. Mechanism of synergy between epigallocatechin gallate and beta-lactams against methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2001;45(6): Korea National Institute of Health. Investigation of Infection Patterns and carrier of MRSA in Newborn Infants [Internet] Available from: 5. Mihailescu R, Furustrand Tafin U, Corvec S, Oliva A, Betrisey B, Borens O, Trampuz A. High activity of fosfomycin and rifampin against methicillin-resistant Staphylococcus aureus biofilm In Vitro and in an experimental foreign-body infection model. Antimicrob Agents Chemother. 2014;58(5): Pham MH, Delestre L, Dewitte A, Wattez N, Lepretre F, Lansiaux A. Synergistic effect of SN-38 in combination with cetuximab on angiogenesis and cancer cell invasion. Anticancer Res. 2015;35(11): Arzuman L, Beale P, Proschogo N, Yu JQ, Huq F. Combination of genistein and cisplatin with two designed monofunctional platinum agents in human ovarian tumour models. Anticancer Res. 2015;35(11): Son DJ, Hong JE, Ban JO, Park JH, Lee HL, Gu SM, Hwang JY, Jung MH, Lee DW, Han SB, Hong JT. Synergistic inhibitory effects of cetuximab and cisplatin on human colon cancer cell growth via inhibition of the erk-dependent egf receptor signaling pathway. Biomed Res Int. 2015;2015: Yu WS. Byungigiyibomyungjip. Seoul:Mok and Toe. 2000: Heo J. Dongyibogam. Hadong:Dongyibogam Publisher. 2005: You X, Feng S, Luo S, Cong D, Yu Z, Yang Z, Zhang J. Studies on a rhein-producing endophytic fungus isolated from Rheum palmatum L. Fitoterapia. 2013;85: Ma YS, Hsiao YP, Lin JH, Hsu SC, Chueh FS, Weng SW, Lai KC, Lin JG, Chung JG. Crude extract of Rheum palmatum L inhibits migration and invasion of LS1034 human colon cancer cells acts through the inhibition of matrix metalloproteinase-2/-9 by MAPK signaling. Environ Toxicol. 2015;30(7): Campbell MJ, Hamilton B, Shoemaker M, Tagliaferri M, Cohen I, Tripathy D. Antiproliferative activity of Chinese medicinal herbs on breast cancer cells in vitro. Anticancer Res. 2002;22(6C): Wang J, Zhao H, Kong W, Jin C, Zhao Y, Qu Y, Xiao X. Microcalorimetric assay on the antimicrobial property of five hydroxyanthraquinone derivativesin rhubarb (Rheum palmatum L.) to Bifidobacterium adolescentis. Phytomedicine. 2010;17(8-9): KO MJ, Park SY, Lee KJ, Yi TH, Lee WC, Han CK, Khil JH. Anti-inflammatory effects of Pharbitis nil Choisy in lipopolysaccharide-induceds RAW cells. The Journal of Cosmetological Science. 2010;6(2): Keum SS, You KW. Effect of Wubangja on the serum lactate dehydrogenase and protein content of hepatic insufficiency. Journal of Koeran medicine. 1978;15(1): Lee SA. Antimicrobial activity of Papenfussiella kuromo against methicillin-resistant Staphylococcus aureus [master's thesis]. [Iksan]:wonkwang university; Veljić M, Tarbuk M, Marin PD, Ćirić A, Soković M, Marin M. Antimicrobial activity of methanol extracts of mosses from Serbia. Pharm Biol. 2008;46(12): Bala M, Ray K, Gupta SM. Comparison of disc diffusion results with minimum inhibitory concentration (MIC) values for antimicrobial susceptibility testing of Neisseria gonorrhoeae. Indian J Med Res. 2005;122(1): Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standards. CLSI/ document M7-A5. Wayne, PA:CLS; Mun SH, Kang OH, Joung DK, Kim SB, Seo YS, Choi JG, Lee YS, Cha SW, Ahn YS, Han SH, Kwon DY. Combination Therapy of Sophoraflavanone B against MRSA: In Vitro Synergy Testing. Foodborne Pathog Dis. 2014;11(3): Mun SH, Kang OH, Joung DK, Kim SB, Choi JG, Shin DW, Kwon DY. In vitro anti-mrsa activity of carvone with gentamicin. Exp Ther Med. 2014;7(4): Mun SH, Lee YS, Han SH, Lee SW, Cha SW, Kim SB, Seo YS, Kong R, Kang DH, Shin DW, Kang OH, Kwon DY. In vitro potential effect of morin in the combination with β-lactam antibiotics against methicillin-resistant staphylococcus aureus. Foodborne Pathog Dis. 2015;12(6): Santiago C, Pang EL, Lim KH, Loh HS, Ting KN. Reversal of ampicillin resistance in MRSA via inhibition of penicillin-binding protein 2a by Acalypha wilkesiana. Biomed Res Int. 2014;2014: Santiago C, Pang EL, Lim KH, Loh HS, Ting KN. Inhibition of penicillin-binding protein 2a (PBP2a) in methicillin resistant Staphylococcus aureus(mrsa) by combination of ampicillin and a bioactive fraction from Duabanga grandiflora. BMC Complement Altern Med. 2015;15: Komatsuzawa H, Suzuki J, Sugai M, Miyake Y, Suginaka H. The effect of Triton X-100 on the in vitro susceptibility of methicillin-resistant Staphylococcus aureus to oxacillin. J Antimicrob Chemother. 1994;34(6): Irvin RT, MacAlister TJ, Costerton JW. Tris (hydroxymethyl) aminomethane buffer modification of Escherichia coli outer membrane permeability. J Bacteriol. 1981;145(3):

14 박주영 나용수 오공천 이상미 최병권 이윤승 송용선 PMid: PMCid:PMC Toei M, Noji H. Single-molecule analysis of F0F1-ATP synthase inhibited by N,N-dicyclohexylcarbodiimide. J Biol Chem. 2013;288(36): Rudkin JK, Edwards AM, Bowden MG, Brown EL, Pozzi C, Waters EM, Chan WC, Williams P, O'Gara JP, Massey RC. Methicillin resistance reduces the virulence of healthcare-associated methicillin-resistant Staphylococcus aureus by interfering with the agr quorum sensing system. J Infect Dis. 2012;205(5): Takahashi N, Imanishi K, Uchiyama T. Overall picture of an emerging neonatal infectious disease induced by a superantigenic exotoxin mainly produced by methicillin-resistant Staphylococcus aureus. Microbiol Immunol. 2013;57(11): Stanier RY, John L. Ingraham, Mark L. Wheelis, Page R. Palnter. Basic and applied microbiology. Bae M, translator. Seoul:Academy company;1998: Moellering RC Jr. MRSA: the first half century. J Antimicrob Chemother. 2012;67(1): Khosravi AD, Hoveizavi H, Farshadzadeh Z. The prevalence of genes encoding leukocidins in Staphylococcus aureus strains resistant and sensitive to methicillin isolated from burn patients in Taleghani Hospital, Ahvaz, Iran. Burns. 2012;38(2): Ba X, Harrison EM, Lovering AL, Gleadall N, Zadoks R, Parkhill J, Peacock SJ, Holden MT, Paterson GK, Holmes MA. Old drugs to treat resistant bugs: methicillin-resistant Staphylococcus aureus isolates with mecc are susceptible to a combination of penicillin and clavulanic acid. Antimicrob Agents Chemother. 2015;59(12): Mun SH, Joung DK, Kim YS, Kang OH, Kim SB, Seo YS, Kim YC, Lee DS, Shin DW, Kweon KT, Kwon DY. Synergistic antibacterial effect of curcumin against methicillin-resistant Staphylococcus aureus. Phytomedicine. 2013;20(8-9): Hiramatsu K, Cui L, Kuroda M, Ito T. The emergence and evolution of methicillin-resistant Staphylococcus aureus. Trends Microbiol 2001;9: Hiramatsu K, Katayama Y, Yuzawa H, Ito T. Molecular genetics of methicillin-resistant Staphylococcus aureus. Int J Med Microbiol. 2002;292(2): Klitgaard JK, Skov MN, Kallipolitis BH, Kolmos HJ. Reversal of methicillin resistance in Staphylococcus aureus by thioridazine. J Antimicrob Chemother. 2008;62(6): Ohlsen K, Lorenz U. Novel targets for antibiotics in Staphylococcus aureus. Future Microbiol. 2007;2(6): Amaral L, Viveiros M, Kristiansen JE. Non-antibiotics : alternative therapy for the management of MDRTB and MRSA in economically disadvantaged countries. Curr Drug Targets. 2006;7(7): Viswanathan K, Frey KM, Scocchera EW, Martin BD, Swain Iii PW, Alverson JB, Priestley ND, Anderson AC, Wright DL. Toward new therapeutics for skin and soft tissue infections: propargyl-linked antifolates are potent inhibitors of MRSA and Streptococcus pyogenes. PLoS One. 2012;7(2):e Poulsen MØ, Jacobsen K, Thorsing M, Kristensen NR, Clasen J, Lillebæk EM, Skov MN, Kallipolitis BH, Kolmos HJ, Klitgaard JK. Thioridazine potentiates the effect of a beta-lactam antibiotic against Staphylococcus aureus independently of meca expression. Res Microbiol. 2013;164(2): Segatore B, Bellio P, Setacci D, Brisdelli F, Piovano M, Garbarino JA, Nicoletti M, Amicosante G, Perilli M, Celenza G. In vitro interaction of usnic acid in combination with antimicrobial agents against methicillin-resistant Staphylococcus ureus clinical isolates determined by FICI and ΔE model methods. Phytomedicine. 2012;19(3-4): Barber KE, Werth BJ, Ireland CE, Stone NE, Nonejuie P, Sakoulas G, Pogliano J, Rybak MJ. Potent synergy of ceftobiprole plus daptomycin against multiple strains of Staphylococcus aureus with various resistance phenotypes. J Antimicrob Chemother. 2014;69(11): Sin MK. Yimsangbonchohak. Seoul:YounglimSa. 2006:785-7, Shah CS, Qadry JS, Bhatt JG. Qualitative and quantitative evaluation of anthraquinone derivatives in Indian rhubarb. Planta Med. 1972;22(1): Choi SB, Ma YH, Han YH, Jeong SJ, Jo HS. Effects of Rhei Radix et Rhizoma on insulin resistance and adipose tissue inflammatory response in high fat diet induced obese C57BL/6 mice. J Int Korean Med. 2014;35(2): Jang YH, Choi SW, Cho SH. Effect of Eisenia Bicyclis and its pill on serum lipid status in rats fed high fat diet. Korean J Nutr. 2008;41(1): Cho SH, Park SY, Choi SW. Effects of Eisenia Bicyclis extracts and pill on blood glucose and lipid profile in streptozotocin-induced diabetic mice. Korean J Nutr. 2008;41(6): Lee YJ. The effects of fraction obtained from rhei rhizoma on the blood lipids in hypercholesterol rats. Kor J Herbology. 2000;15(2): Yun HJ, Hwang SG, Yun HJ, Kim CH, Seo GS, Park WH, Park SD. Anticancer effect of Rheum Rhizoma on human liver cancer HepG2 cells. Kor J Herbology. 2006;21(4): Park SM, Lee GW, Cho YH. Effect of Rheum undulatum extract on antioxidant activity and activity of matrix metalloproteinase-1 in human skin fibroblasts. Journal 34 J Korean Med Rehabil 2018;28(2):21-35

15 大黃牽牛散에탄올추출물의 Methicillin 내성 Staphylococcus aureus 에대한항균활성연구 of Life Science. 2008;18(12): Lee JH. Antibacterial and synergistic effect of phlorotannins isolated from Eisenia bicyclis against acne-related bacteria [dissertation]. [Busan]:Pukyong National University; Eom SH. Anti-MRSA (methicillin-resistant Staphylococcus aureus) substance isolated from Eisenia bicyclis and its action mechanism [dissertation]. [Busan]:Pukyong National University; Choi MS. The antibacterial effects of emodin from rhei radix et rhizoma against methicillin-resistant Staphylococcus aureus [master's thesis]. [Iksan]:Wonkwang University; Lyu KW, Yun ES. A study on the influence of pharbitidis semen on the activity of serum transaminase in carbon tetrachloride poisoned rabbits. The Journal of The Yellow-Emperor`s Medicine. 1977;2(3): Seo BI, Lee YJ. A philological study on poisoning of pharbitidis semen. The Journal of East-West Medicine. 2008;33(2): Yoon SG, Park YH, Lee MJ. Effects of PS (Pharbitidis Semen) on anti-inflamatory and anti-febrile activities in arthritic rats. J Oriental Rehab Med. 2003;13(3): Kim JH, Seong JM, Moon HJ, Hwang JM, Hwang KS, Kwon YD, Kwon IK, Park YD. Effects of pharbitidis semen extract on osteoblast differentiation in MC3T3-E1 cells. Tissue Engineering and Regenerative Medicine. 2010;7(2): Hu Y, Liu A, Vaudrey J, Vaiciunaite B, Moigboi C, McTavish SM, Kearns A, Coates A. Combinations of β -lactam or aminoglycoside antibiotics with plectasin are synergistic against methicillin-sensitive and methicillin-resistant Staphylococcus aureus. PLoS One. 2015;10(2):e Dosler S, Gerceker AA. In vitro activities of nisin alone or in combination with vancomycin and ciprofloxacin against methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains. Chemotherapy. 2011;57(6): Nester EW, Roberts CE, Pearsall NN, Anderson DG, Nester MT. Functional Anatomy of Prokaryotes and Eukaryotes. In Kane KT, Ronald E, Terrance S, Editors. Microbiology. 2nd ed. New York:WCB/McGraw-Hill;1998: