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1 J Korean Soc Food Sci Nutr 한국식품영양과학회지 45(7), 1026~1034(2016) 식중독세균에대한소목추출물의항균활성 김이슬 심혜미 김광엽충북대학교식품생명공학과 Antimicrobial Effect of Caesalpinia sappan L. Extract on Foodborne Bacteria YiSeul Kim, HyeMee Shim, and KwangYup Kim Department of Food Science and Biotechnology, Chungbuk National University ABSTRACT To develop a natural antimicrobial agent, we investigated the antimicrobial activities of 13 species of edible herbal plant extracts against major Grampositive foodborne bacteria. Among the 13 screened edible herbal plants, Caesalpinia sappan L. showed the highest antimicrobial activity. In the paper disc agar diffusion assay, Caesalpinia sappan L. extracts had strong antibacterial activities against most Grampositive bacteria but did not have antibacterial activities against most Gramnegative bacteria. Minimum inhibitory concentrations of the ethanol extract were 0.06 mg/ml against Clostridium difficile and Listeria monocytogenes and 0.03 mg/ml against Staphylococcus aureus. Their inhibitory activities were not reduced by heat treatment or ph adjustment against C. difficile, L. monocytogenes, and S. aureus. Antimicrobial activities were higher in ethanol extract than in distilled water extract. These results support the potential use of Caesalpinia sappan L. ethanol extract as an antimicrobial agent or functional food components against Grampositive bacteria. Key words: Caesalpinia sappan L., antimicrobial effect, Clostridium difficile, Listeria monocytogenes, Staphylococcus aureus 서 평균수명의연장과더불어성인병발생이더욱증가하고있고, 생활환경의변화와함께건강에대한관심도더욱고조되고있다. 세계적인이상기후현상으로인해간단한먹거리에서부터각종식량자원의공급등이불안정해지고있고, 이로인해해외에서값싼식품재료등의수입을점차증가시키고있다. 축산물을수입하는경우이들의유통기한과저장등의과정중콜레라, 이질및각종식중독유발미생물등의오염이일어날확률이높아지고있으며, 기타수입식품의범람으로우리의식탁이여러가지위생학적인위험성에노출되어있다 (1). 우리나라식중독발생의경우주원인균으로살모넬라균이나포도상구균, 세균성이질균등을들수있는데 (2), 인간은이와같은식중독유발세균을통제하기위해수많은항생물질을개발했다. 그러나최근화학합성항생제에내성을가지는균주가증가함으로써새로운문제로대두하고있으며, 이를극복할수있는새로운천연항생물질의개발이요구되고있다. 즉기존의항생제와는다른작용메커니즘을통하여항생제내성균주에항균 Received 5 February 2016; Accepted 27 May 2016 Corresponding author: KwangYup Kim, Department of Food Science and Biotechnology, Chungbuk National University, Cheongju, Chungbuk 28644, Korea kimky@chungbuk.ac.kr, Phone: 론 효과를나타내야하며, 사용후분해가잘되어체내나자연계에서잔류성의위험이없는환경친화적항균제개발이필요하다 (35). 천연물질의경우일부미생물유래천연물질을제외하고대부분이그항균효과를검정하는데그치고있으며실제실생활에응용되어상품화된것은그리많지않다. 이는일부물질의경우인체에대한안정성을확보해야하는문제점이있을수있겠으며그유효물질을추출정제하는과정에많은시간과노력을해야하기때문으로생각된다. 한편다양한천연물질중에서도식물체, 특히식용으로이용되어온약용식물의경우항균효과뿐만아니라다양한생리활성을가지고있어이를굳이정제하거나순수분리하는과정을거치지않고도식품에직접첨가가가능할것으로생각되며, 그경우항균효과뿐만아니라인체에유용한다양한생리활성을동시에얻는효과가있을것으로생각된다 (6). 소목 (Caesalpinia sappan L.) 은인도, 말레이시아, 중국남부등의열대아시아에분포하는낙엽관목의콩과 (Leguminosae) 식물로밝은홍색을띠는심재를약용및홍색염료로사용한다. 소목추출물에관한생리활성연구로는항통증 (analgesic), 항염증 (antiinflammatory)(7), 조혈강장 (blood tonic), 거담 (expectorant), 통경 (emmenagogue)(8), 진정 (spasmolytic activity), 항경련 (anticonvulsant), 진경 (spasmo

2 식중독세균에대한소목추출물의항균활성 1027 Table 1. List of herbal plants used for experiments Scientific name (abbreviation) Used part Therminalia chebula Retz (Tc) Rosa laevigata Michx (Rl) Platycodon grandiflorum ADC (Pg) Ledebouriella seseloides Wolff (Ls) Rubus coreanus Miquel (Rc) Caesalpinia sappan L. (Cs) Rhus javanica (Rj) Forsythia koreana Nakai (Fk) Achyranthes japonica Nakai (Aj) Myristica fragrans Houtt (Mf) Citrus aurantium L. (Ca) Phellodendron amurense Ruprecht (Pa) Siegesbeckia glabrescens Makino (Sg) Root Root Wood Root Root Whole lytic activity)(9), 혈관이완 (vasorelaxation)(10), 항동맥경화 (antiatherogenic), 항보체활성 (anticomplementary) (11), 면역조절 (immunomodulation)(12), 면역작용의유도 (induction of immmunomodulation)(13), 항균 (antimicrobial)(14,15), 세포독성 (cytotoxic)(16,17), 항바이러스 (antiviral)(18), 혈당저하 (hypoglycemic)(19,20), 항산화효과 (antioxidation)(21) 등이보고되었다. 소목의주성분가운데약 2% 를차지하는염료성화합물은무색의 flavonoid 구조를갖는 brazilin이며, 이는공기중에산화되어 brazilein이된다. Brazilin은 Benz(b)indeno(2,1d)pyran 유도체로주로염료나산및알칼리지시약또는식품첨가물등으로사용해왔다. 또한, 한방에서 brazilin은타박손상에의한어혈, 월경통, 월경폐색, 현훈, 출산후각종증상에사용해왔다 (2224). Moon 등 (25) 은 brazilin이고혈압에효과가있음을보고하였고, Hwang 등 (26) 은 brazilin이혈소판에서칼슘농도를조절함을보고하였으며, Kim 등 (27) 은혈액에서 brazilin의혈당저하작용을보고하였다. 또한, Kwon 등 (28) 은 brazilin이식중독의주된원인균일뿐만아니라화농성질환의원인균인 Staphylococcus aureus와치아우식증을유발하는 Streptococcus mutans에대해항균효과가있다고보고하였다. 본연구에서는식용가능한약용식물중문헌조사를통해항균효과가알려진 13가지의약용식물을선정하여미생물에대해생육을억제하는효과를알아보고, 가장높은활성을갖는소목추출물의미생물에대한기초자료확보와이러한소목추출물의효능을이용한천연항균제제또는기능성식품을상품화하기위한기초자료로사용하고자한다. 재료및방법실험재료본연구에사용된 13종의약용식물은서울경동시장약재상가에서구입하여사용하였다. 항생제로는 vancomycin HCl(SigmaAldrich Co., St. Louis, MO, USA) 을구입하여대조군으로사용하였다. 실험에사용된 13종의시료들은 Table 1에나타내었다. 시료의용매는 75% 에탄올을사용하였으며 25 C에서 24시간 200 rpm으로교반추출하였다. 추출된시료들은 paper disc agar diffusion method 와 broth microdilution method를통해항균활성을검색하였고, 13종의시료중소목의 75% 에탄올추출물이강한항균활성을나타내어소목을선발하여실험에사용하였다. 사용균주및배지본연구에서는한국생물자원센터 (Jeongeup, Korea) 에서분양받은, Clostridium perfringens KCTC 3269, Eubacterium limosum KCTC 3266,, Bifidobacterium bifidum KCTC 3472, Lactobacillus acidophilus KCTC 3164,, Streptococcus thermophilus KCTC 2190, Enterobacter aerogenes KCTC 2190,,, Salmonella Typhimurium KCTC 2515, Salmonella Enteritidis KCTC 12021, 과한국미생물보존센터 (Seoul, Korea) 에서분양받은 Leuconostoc mesenteroides ATCC 8293, Listeria monocytogenes ATCC 19115와충북대학교식품공학과식품미생물학실험실에서자체보유하고있는 Bifidobacterium adolescentis, Lactobacillus fermentum YL3을실험에사용하였다. 전배양및본배양을위한생육배지로장내유해균은 Reinforced Clostridial Medium(RCM) broth(difco, Detroit, MI, USA), 유산균은 Lactobacilli MRS broth(difco), 그외의균은 Tryptic soy broth(difco) 를사용하였다. 이들균주는 50% 글리세롤이포함된배지에넣어 80 C에보관하여사용하였으며, 실험에사용하기전 3회이상계대배양하여활성화한후사용하였다. 시료추출및분획시료 25 g에 20배의 75% 에탄올과증류수를각각가하여교반추출기 (VS8480, Vision Scientific Co., Ltd., Seoul, Korea) 에서 25 C로 24시간동안 200 rpm으로추출하여여과지 (Whatman No. 3, GE Healthcare, Buckinghamshire, UK) 로여과한다음회전진공농축기 (EYELA CCA 1110, Tokyo Rikakikai Co., Tokyo, Japan) 를이용하여 40 C에서용매를완전히제거한후추출물로사용하였다. 제조된각각의에탄올, 열수추출물을 50 ml로정용하여분획깔때기에넣고헥산, 클로로포름, 에틸아세테이트및부탄올을 150 ml씩 3회순차적으로가하여분획한후각각의용매분획물을얻은다음최종남은용액을물분획물로하였다. 각각의용매분획물들은질소농축및감압농축한다음실험에사용하였다.

3 1028 김이슬 심혜미 김광엽 Paper disc agar diffusion method 항균활성검색은 paper disc agar diffusion 법 (29) 을응용하였다 CFU/mL로배양된시험균주들을멸균된각각의 agar 배지에 2~3% 접종하여배지를조성하였다. 여기에 paper disc를배지위에떨어지지않도록부착시킨후농도가 50, 100, 200 mg/ml인시료의추출물을 30 μl 주입하여배양장치 (BF250IN, BioFree, Seoul, Korea) 에넣고 37 C 에서 24시간배양한다음생육억제환생성유무를확인하였다. 추출물을녹인용매에대한영향을알아보기위해대조군으로는 30% DMSO를사용하였다. 최소저해농도 (minimum inhibitory concentration, MIC) 측정미생물의최소저해농도는 broth microdilution method (30) 를응용하여다음과같이측정하였다. 18~24시간배양된세균배양액을 650 nm에서흡광도가 0.3이되게조절한후 96 well plate에각시험균배양액을 100 μl씩분주하고, 시료추출물을최고농도 20 mg/ml에서부터 2배씩희석하여최저농도로 100 μl씩처리하여 24시간배양한후시료추출물이세균의증식에미치는영향을 650 nm에서 ELISA Reader를통해측정하여비교분석하였다. 열및 ph 안정성측정소목추출물의항균활성물질에대한열안정성측정은시료를 60, 80, 100 C에서 30분동안열처리하고 121 C에서는 15분열처리한후대조구와같이 paper disc agar diffusion 법으로생육억제환의지름을측정하여비교하였고, 대조구로는열처리하지않은소목추출물을사용하였다. ph 안정성은 ph meter를사용하여시료를 0.1 N HCl과 0.1 N NaOH로 ph 2, 4, 8로조절한후대조구와같이 paper disc agar diffusion 법으로생육억제환의지름을측정하여비교하였고, 대조구로는 0.1 N HCl, 0.1 N NaOH와 ph를조절하지않은소목추출물을사용하였다. 결과및고찰 Paper disc agar diffusion method 에따른항균활성소목의 75% 에탄올추출물과열수추출물을각각 200 mg/ml, 100 mg/ml, 50 mg/ml의농도로하여 paper disc agar diffusion assay로항균활성을검색한결과는 Table 2에나타내었다. 그람양성균인 C. difficile KCTC 5009, L. monocytogenes ATCC 19115와 S. aureus KCTC 1916 은소목의 75% 에탄올추출물의처리구에서열수추출물보다강한항균활성효과를나타내었고추출물의농도가높아질수록높은항균활성을보였다. 소목의열수추출물의처리구에서는항균활성효과가나타났으나 75% 에탄올추출물보다는그항균활성이적었다. 특히그람양성균인 C. difficile KCTC 5009, L. monocytogenes ATCC 19115와 S. aureus KCTC 1916은소목의 75% 에탄올추출물 50 mg/ml의처리구에서도다른식중독세균과는달리억제환의크기가 20 mm 이상의항균활성효과를나타내었다 (Fig. 1). Shin 등 (31) 은소목의에탄올추출물에대해 S. aureus 와 L. monocytogenes에서가장뛰어난항균활성을나타내었고에탄올추출물의첨가농도에관계없이생육이정지 Table 2. Antimicrobial activity of Caesalpinia sappan L. 75% ethanol extract and distilled water extract by the paper disc agar diffusion method Strains Bifidobacterium adolescentis Caesalpinia sappan L. 75% ethanol extract Caesalpinia sappan L. distilled water extract 50 mg/ml 100 mg/ml 200 mg/ml 50 mg/ml 100 mg/ml 200 mg/ml 1) 1) Growth inhibition size of clear zone:, not detected;, smaller than 8 12 mm;, mm;, mm;, mm;, large than 25 mm.

식중독세균에대한소목추출물의항균활성 1029 A C 1 2 3 B C 1 2 3 Fig. 1. Antimicrobial activity of Caesalpinia sappan L. 75% ethanol extract on L. monocytogenes ATCC 19115 (A) and S. aureus KCTC 1916 (B).

aureus KCTC 1916이소목에탄올추출물에서우수한항균효과를보인것과유사한결과를보였다. 반대로그람음성균인 E. coli KCTC 1039, Sal. Enteritidis KCTC 12021, Sal. Typhimurium KCTC 2515에서는소목의 75% 에탄올추출물과열수추출물처리구모두에서항균활성효과가나타나지않았다.

4 식중독세균에대한소목추출물의항균활성 1029 A C B C Fig. 1. Antimicrobial activity of Caesalpinia sappan L. 75% ethanol extract on L. monocytogenes ATCC (A) and S. aureus KCTC 1916 (B). C, Control (30% DMSO); 1, 50 mg/ml extract; 2, 100 mg/ml extract; 3, 200 mg/ml extract. 된상태를보이다가 10 ppm 첨가후배양 48시간이되어서야비로소증식한다는것을보고했다. 이것은본실험에서 L. monocytogenes ATCC 19115와 S. aureus KCTC 1916이소목에탄올추출물에서우수한항균효과를보인것과유사한결과를보였다. 반대로그람음성균인 E. coli KCTC 1039, Sal. Enteritidis KCTC 12021, Sal. Typhimurium KCTC 2515에서는소목의 75% 에탄올추출물과열수추출물처리구모두에서항균활성효과가나타나지않았다. Kwon과 Park(32) 의실험에서그람음성균인 E. coli와 Sal. Typhimurium에대해오미자추출물의항균활성이나타나지않았고, Kwon 등 (33) 의실험에서도그람음성균보다그람양성균에대해높은활성을보였다는결과와일치하였다. 하지만본실험의결과와는달리 Chang과 Choi (34) 는소목에탄올추출물에서 E. coli에대해 5 mg/ml 수준에서항균활성을나타내었는데이는사용한균주의차이로생각되며, 또한 Park 등 (35) 은항균성물질을추출할때추출온도, 용매농도및용매의종류에따라항균력이차이가난다고하였다. 이를통하여소목에탄올추출물이특히그람양성균인 C. difficile KCTC 5009, L. monocytogenes ATCC 19115와 S. aureus KCTC 1916에대해우수한항균활성을나타내항균제제로활용할가능성이있다고판단된다. 이에더하여소목추출물의헥산, 클로로포름, 에틸아세테이트, 부탄올및물분획물들에대하여항균활성을검색하였다. 각분획물을 50 mg/ml의농도로하여 paper disc agar diffusion assay로항균활성을검색한결과 75% 에탄올추출물의분획물의경우그람양성균인 C. difficile KCTC 5009는헥산, 클로로포름, 에틸아세테이트, 부탄올층에서, L. monocytogenes ATCC 19115는클로로포름, 에틸아세테이트, 부탄올층에서, S. aureus KCTC 1916은헥산, 클로로포름, 에틸아세테이트, 부탄올층에서억제환이생성되어항균활성을나타내었고, 열수추출물의분획물의경우 C. difficile KCTC 5009는부탄올층에서, L. monocytogenes ATCC 19115는에틸아세테이트, 부탄올층에서, S. aureus KCTC 1916은에틸아세테이트, 부탄올층에서억 제환이생성되어항균활성을나타내었다. 추출물의결과와같이 75% 에탄올추출물의분획물이열수추출물의분획물보다높은항균활성을나타내었다. 그람음성균인 E. coli KCTC 1039, Sal. Enteritidis KCTC 12021, Sal. Typhimurium KCTC 2515는소목의 75% 에탄올추출물과열수추출물처리구모두에서항균활성효과가나타나지않았다. 이에대한결과는 Table 3, 4에나타내었다. 일반적으로식물의에틸아세테이트분획층에서는사포닌성분, 유기산류, 탄닌당, 배당체및기타알칼로이드류가주로용출되는것으로알려져있고, phenol류및 flavonoid 등의성분이용출되는클로로포름분획물에도상당한항균력이확인되었다고하였다 (36). Shin 등 (31) 은소목의에틸아세테이트분획층에서 10종의유해균에대해뛰어난항균력을보였다고하였다. Kwon 등 (28) 역시소목의에틸아세테이트분획층에서식중독유발세균인 S. aureus와치아우식증의주요원인균인 Str. mutans에대하여강한항균력을보여주었고그항균물질로 brazilin을분리동정하였다. 본연구결과에서는소목의클로로포름, 에틸아세테이트, 부탄올층에서강한항균활성을나타낸바항균성물질은특정용매에만국한되어용출되는것이아니라여러가지성분이복합적으로작용하여여러용매에도용출될수있음을암시해주었다. Vancomycin 과의항균활성비교그람양성균과관련된경험적치료에사용되는현재까지가장강력한항생제로알려진 vancomycin과의항균활성을비교한결과 50 mg/ml 농도에서소목 75% 에탄올추출물의항균활성은 vancomycin 2.5 mg/ml를사용하였을때와유사한항균활성을나타냈고, 소목열수추출물의항균활성은 C. difficile KCTC 5009, L. monocytogenes ATCC 19115에서는 vancomycin 0.04 mg/ml, S. aureus KCTC 1916에서는 vancomycin 1.3 mg/ml를사용하였을때와항균활성이유사하게나타났다 (Table 5). 이러한결과로소목에탄올추출물은 vancomycin보다낮은항균활성을보였지만그람양성균에대한항균활성이우수하다고할수있으며 vancomycin과같은항생제에내성을가진식중독원인균에대해서도천연항균제로서의적용가능성을기대한다. 최소저해농도소목의에탄올추출물을가지고미생물에대한최소저해농도를측정한결과는 Table 6에나타내었다. C. difficile KCTC 5009, L. monocytogenes ATCC 19115의최소저해농도는 0.06 mg/ml였고, S. aureus KCTC 1916에대한최소저해농도는 0.03 mg/ml였다. Jung 등 (37) 의연구결과에따르면 vancomycin의유효혈청농도는 0.005~0.04 mg/ml로보고하였는데, 본실험결과정제되지않은소목에탄올추출물이 vancomycin에견줄만한항균활성을나타

5 1030 김이슬 심혜미 김광엽 Table 3. Antimicrobial activity of solvent s of Caesalpinia sappan L. 75% ethanol extract by the paper disc agar diffusion method Strains Bifidobacterium. adolescentis Hexane 1) Chloroform Ethyl acetate Butanol Water 1) Growth inhibition size of clear zone:, not detected;, smaller than 8 12 mm;, mm;, mm;, Table 4. Antimicrobial activity of solvent s of Caesalpinia sappan L. distilled water extract by the paper disc agar diffusion method Strains Bifidobacterium adolescentis Hexane 1) Chloroform Ethyl acetate Butanol Water 1) Growth inhibition size of clear zone:, not detected;, smaller than 8 12 mm;, mm;, mm;, 내는것을알수있었다. 이러한결과는 Choi 등 (38) 의소목에탄올추출물의항균활성에대한연구에서 S. aureus에대한 MIC를측정한결과 1.2 mg/ml, Lee와 Min(39) 이소목메탄올추출물에서 S. aureus, L. monocytogenes에대한 MIC를측정한결과각각 mg/ml, mg/ml보다낮은농도에서높은항균활성을나타냄을알수있었다. 소목의열수추출물을가지고미생물에대한최소저해농도를측정한결과는 Table 7에나타내었다. C. difficile KCTC 5009, L. monocytogenes ATCC 19115의최소저해농도는 mg/ml였고, S. aureus KCTC 1916에대한최소저해농도는 mg/ml였다.

6 식중독세균에대한소목추출물의항균활성 1031 Table 5. Antimicrobial activity of vancomycin by paper disc agar diffusion method 2.5 mg/ml 1.3 mg/ml 0.65 mg/ml 0.33 mg/ml 0.16 mg/ml 0.08 mg/ml 0.04 mg/ml Clostridium difficile KCTC ) Listeria monocytogenes ATCC Staphylococcus aureus KCTC 1916 EtOH extract Water extract 1) Growth inhibition size of clear zone:, not detected;, smaller than 8 12 mm;, mm;, mm;, Table 6. Minimum inhibitory concentration (MIC) of 75% ethanol extract of Caesalpinia sappan L. Strain Bifidobacterium adolescentis 1) No growth. 2) Growth. Growth of various concentration (mg/ml) MIC 1) 2) 열및 ph 안정성식품등의제조시에는일련의공정에서열처리와 ph의변화를수반하기도한다. 때로는이러한열처리및 ph의변화로인하여유효성분들이분해되는문제가발생하며공정상의제약요인이된다. 따라서소목의 75% 에탄올추출물과열수추출물의열및 ph에대한안정성을조사하였다 (40). 소목 75% 에탄올추출물과소목열수추출물을가지고열안정성을측정한결과는 Table 8과같다. 시료를 60 C, 80 C, 100 C에서 30분, 121 C에서 15분간열처리한후항균력을살펴본결과 C. difficile KCTC 5009, L. monocytogenes ATCC 19115, S. aureus KCTC 1916에대한항균력은 60 C, 80 C, 100 C, 121 C에서모두대조구와비슷하게억제환이생성되어소목추출물의항균성물질은열에대체로안정한결과를보여주었다. 소목 75% 에탄올추출물과소목열수추출물을가지고 ph 안정성을측정한결과는 Table 9와같다. 시료를 ph 2, 4, 8로조정한후항균력을살펴본결과 0.1 N HCl(pH 1.06) 과 0.1 N NaOH(pH 12.93) 에대해서는영향을받지않았고, 항균력은 C. difficile KCTC 5009, L. monocytogenes ATCC 19115, S. aureus KCTC 1916에서모두 ph 를조절하지않은소목추출물의결과와비슷하게억제환이생성되었다. 소목추출물의항균성물질은 ph에대체로안정한결과를보여주었으며본결과로열및 ph의변화에안정적인소목추출물에함유된항균물질을이용하여기능성식품의소재및식품의제조공정에적합한첨가물로서도유용하게이용될수있다고기대된다. 요약본실험은식용가능한약용식물을대상으로하여항균활성을검색하였다. 미생물에대하여약용식물을 50 mg/ml, 100 mg/ml, 200 mg/ml의농도로하여 paper disc agar

7 1032 김이슬 심혜미 김광엽 Table 7. Minimum inhibitory concentration (MIC) of distilled water extract of Caesalpinia sappan L. Strain Bifidobacterium adolescentis 1) No growth. 2) Growth. Growth of various concentration (mg/ml) MIC 1) 2) 0.25 Table 8. Heated stability of 75% ethanol extract and distilled water extract of Caesalpinia sappan L. Strain C. difficile L. monocytogenes S. aureus Caesalpinia sappan L. 75% ethanol extract Caesalpinia sappan L. distilled water extract 60 C 80 C 100 C 121 C Control 2) 60 C 80 C 100 C 121 C Control 3) 1) 1) Growth inhibition size of clear zone:, not detected;, smaller than 8 12 mm;, mm;, mm;, 2) Control: Not heated treatment on Caesalpinia sappan L. 75% ethanol extract. 3) Control: Not heated treatment on Caesalpinia sappan L. distilled water extract. Table 9. ph stability of 75% ethanol extract and distilled water extract of Caesalpinia sappan L. Strain C. difficile L. monocytogenes S. aureus Caesalpinia sappan L. 75% ethanol extract 2) 0.1 N 0.1 N ph 2 ph 4 ph 8 Control NaOH HCl 1) Caesalpinia sappan L. distilled water extract 3) 0.1 N 0.1 N ph 2 ph 4 ph 8 Control NaOH HCl 1) Growth inhibition size of clear zone:, not detected;, smaller than 8 12 mm;, mm;, mm;, 2) Control: not ph variation on Caesalpinia sappan L. 75% ethanol extract. 3) Control: not ph variation on Caesalpinia sappan L. distilled water extract. diffusion assay로항균활성을검색한결과소목추출물에서가장높은항균활성을나타내었다. 이에따라소목의에탄올추출물과열수추출물을각각 200 mg/ml, 100 mg/ ml, 50 mg/ml의농도로하여 paper disc agar diffusion assay로항균활성을검색한결과소목추출물은그람양성균에서높은항균활성을나타내었고, 소목의에탄올추출물이소목의열수추출물보다높은항균활성을나타내었다. 소목에탄올추출물과열수추출물의각분획물을 50 mg/ ml의농도로하여 paper disc agar diffusion assay로항균 활성을검색한결과그람양성균은클로로포름, 에틸아세테이트, 부탄올층에서항균활성을나타내었고, 그람음성균은항균활성을나타내지않았다. 소목의항균물질로추정되는 brazilin은주로에틸아세테이트분획층에존재하므로소목에탄올추출물에서클로로포름, 에틸아세테이트, 부탄올의항균활성이유사한점으로미루어 brazilin 외에도다양한항균물질이존재하여이들이복합적으로작용하여항균활성을나타내는것으로생각한다. 현재까지가장강력한항생제로알려진 vancomycin과의항균활성을비교한결과

8 식중독세균에대한소목추출물의항균활성 mg/ml 농도에서소목 75% 에탄올추출물의항균활성은 vancomycin 2.5 mg/ml를사용하였을때와유사한항균활성을나타내었다. 소목의추출물을가지고미생물에대한최소저해농도를측정한결과 C. difficile KCTC 5009, L. monocytogens ATCC 19115의최소저해농도는 0.06 mg/ml였고, S. aureus KCTC 1916에대한최소저해농도는 0.03 mg/ml였다. Vancomycin의유효혈청농도는 ~0.04 mg/ml로견줄만한항균활성을나타내었다. 소목추출물을가지고열및 ph 안정성을측정한결과는항균력은 C. difficile KCTC 5009, L. monocytogens ATCC 19115, S. aureus KCTC 1916에서모두대조구와비슷하게억제환이생성되어소목추출물의항균성물질은열및 ph에대체로안정한결과를보여주었다. 상기결과들을종합해볼때소목에탄올추출물이그람양성균에대해항균활성이뛰어난것으로보아소목을그람양성의병원성미생물을억제하기위한항균제제및기능성식품의소재로써활용할수있다고판단된다. 감사의글 이논문은 2013년도충북대학교학술연구지원사업의연구비지원에의하여연구되었음. REFERENCES 1. Bae JH Effect of extracts from Paeonia japonica on the growth of foodborne pathogens. J East Asian Soc Diet Life 21: Bae JH Antimicrobial effect of Hedyotis diffusa extracts on foodborne pathogens. J Korean Soc Food Sci Nutr 34: Otvos L Jr Antibacterial peptides isolated from insects. J Pept Sci 6: Sitaram N, Nagaraj R Hostdefense antimicrobial peptides: importance of structure for activity. Curr Pharm Des 8: Wu M, Maier E, Benz R, Hancock RE Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli. Biochemistry 38: Lee YC, Oh SW, Hong HD Antimicrobial characteristics of edible medicinal herbs extracts. Korean J Food Sci Technol 34: Hikino H, Taguchi T, Fujimura H, Hiramatsu Y Antiinflammatory principles of Caesalpinia sappan wood and of Haematoxylon campechianum wood. Planta Med 31: Badami S, Moorkoth S, Rai SR, Kannan E, Bhojraj S Antioxidant activity of Caesalpinia sappan heartwood. Biol Pharm Bull 26: RodríguezLópez V, Salazar L, Estrada S Spasmolytic activity of several extracts obtained from some Mexican medicinal plants. Fitoterapia 74: Hu CM, Kang JJ, Lee CC, Li CH, Liao JW, Cheng YW Induction of vasorelaxation through activation of nitric oxide synthase in endothelial cells by brazilin. Eur J Pharmacol 468: Oh SR, Kim DS, Lee IS, Jung KY, Lee JJ, Lee HK Anticomplementary activity of constituents from the heartwood of Caesalpinia sappan. Planta Med 64: Choi SY, Yang KM, Jeon SD, Kim JH, Khil LY, Chang TS, Moon CK Brazilin modulates immune function mainly by augmenting T cell activity in halothane administered mice. Planta Med 63: Mok MS, Jeon SD, Yang KM, So DS, Moon CK Effects of brazilin on induction of immunological tolerance by sheep red blood cells in C57BL/6 female mice. Arch Pharm Res 21: Lim MY, Jeon JH, Jeong EY, Lee CH, Lee HS Antimicrobial activity of 5hydroxy1,4naphthoquinone isolated from Caesalpinia sappan toward intestinal bacteria. Food Chem 100: Woldemichael GM, Singh MP, Maiese WM, Timmermann BN Constituents of antibacterial extract of Caesalpinia paraguariensis Burk. Z Naturforsch C 58: Ueda JY, Tezuka Y, Banskota AH, Le Tran Q, Tran QK, Harimaya Y, Saiki I, Kadota S Antiproliferative activity of Vietnamese medicinal plants. Biol Pharm Bull 25: Park KJ, Yang S, Eun YA, Kim SY, Lee HH, Kang H Cytotoxic effects of Korean medicinal herbs determined with hepatocellular carcinoma cell lines. Pharm Biol 40: Chiang LC, Chiang W, Liu MC, Lin CC In vitro antiviral activities of Caesalpinia pulcherrima and its related flavonoids. J Antimicrob Chemother 52: Parameshwara S, Srinivasanb KK, Rao CM Oral antidiabetic activities of different extracts of Caesalpinia bonducella seed kernels. Pharm Biol 40: You EJ, Khil LY, Kwak WJ, Won HS, Chae SH, Lee BH, Moon CK Effects of brazilin on the production of fructose2,6bisphosphate in rat hepatocytes. J Ethnopharmacol 102: Nguyen MT, Awale S, Tezuka Y, Ueda JY, Tran QL, Kadota S Xanthine oxidase inhibitors from the flowers of Chrysanthemum sinense. Planta Med 72: de Oliveira LFC, Edwards HGM, Velozo ES, Nesbitt M Vibrational spectroscopic study of brazilin and brazilein, the main constituents of brazilwood from Brazil. Vib Spectrosc 28: Kee CH The pharmacology of Chinese herbs. CRC Press, Boca Raton, FL, USA. p Puchtler H, Meloan SN, Waldrop FS Application of current chemical concepts to metalhematein and brazilein stains. Histochemisty 85: Moon CK, Park KS, Kim SG, Won HS, Chung JH Brazilin protects cultured rat hepatocytes from BrCCl 3induced toxicity. Drug Chem Toxicol 15: Hwang GS, Kim JY, Chang TS, Jeon SD, So DS, Moon CK Effects of brazilin on the phospholipase A2 activity and changes of intracellular free calcium concentration in rat platelets. Arch Pharm Res 21: Kim SG, Kim YM, Khil LY, Jeon SD, So DS, Moon CH, Moon CK Brazilin inhibits activities of protein kinase C and insulin receptor serine kinase in rat liver. Arch Pharm Res 21: Kwon HJ, Kim YH, Han KI, Jeon MA, Han MD Antimicrobial activities and adherence inhibition on Streptococcus mutans by ethyl acetate extract from Caesalpinia sappan L.. J Dent Hyg Sci 12: Ha MH, Park WP, Lee SC, Heo HJ, Cho SH Antimi

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386-390.hwp 386 HANYANG MEDICAL REVIEWS Vol. 29 No. 4, 2009 우리나라 미숙아의 통계와 의료비용 Statistics and Medical Cost of Preterm in Korea 윤혜선 을지대학교 노원을지병원 소아청소년과학교실 Hye Sun Yoon, M.D., Ph.D., Department of Pediatrics, Nowon