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Kor. J. Microbiol. Biotechnol. Vol. 38, No. 1, 77 83(2010) 항생제다제내성 Pseudomonas aeruginosa 및 Candida 균주에대한산사자의항균활성 류희영 안선미 김종식 1 정인창 손호용 * 안동대학교식품영양학과, 1 안동대학교생명과학과 Antimicrobial Activity of Fruit of Crataegus pinnatifida Bunge against Multidrug Resistant Pathogenic Pseudomonas aeruginosa and Candida sp. Ryu, Hee-Young, Seon-Mi Ahn, Jong-Sik Kim 1, In-Chang, Jung, and Ho-Yong Sohn*. Dept. of Food and Nutrition, Andong National University, Andong 760-749, Korea, 1 Dept. of Biological Science, School of Bioresource Science, Andong National University, Andong 760-749, Korea - The fructus of Crataegus pinnatifida Bunge (CBF) has been used as medicinal and food source in worldwide. In this study, antimicrobial activity of the methanol extract and its sequential organic solvent fractions of CBF against different pathogenic bacteria and fungi, including multidrug resistant Pseudomonas aeruginosa and Candida sp., were investigated. The methanol extract of CBF was active against various gram-positive and gram-negative bacteria, and the ethylacetate and butanol fractions of CBF showed strong antibacterial activity against Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus, Bacillus subtilis, Salmonella typhimurium, Proteus vulgaris, Escherichia coli and various multidrug resistant Pseudomonas aeruginosa with minimal inhibitory concentration of 1.0~7.5 mg/ml. Also the fractions showed anti-candida activity against C. albicans, C. kruseis and C. geochares. The methanol extract of CBF and its solvent fractions, except n-hexane fraction, did not show any hemolytic activity against human red blood cell up to 500 µg/ml, respectively. The hemolysis in n-hexane fraction at 500 µg/ml was less than 9.9%. Our results suggest that the CBF could be developed as a potent antibacterial agent, especially for multidrug resistant Pseudomonas aeruginosa Key words: Antibacterial, multidrug resistant Pseudomonas aeruginosa, anti-candida, fructus of Crataegus pinnatifida Bunge 서 산사자는장미과에속한낙엽교목인산사나무의성숙한과실로이가위, 이가배, 적과자, 산과자, 찔광이, 질구배등의다양한이름으로불리며, 주로식용및약용으로이용되고있다. 우리나라에서는주로지리산이북의전북, 경북, 강원등에서생산되며, 중국, 일본의각지에서도많이생산된다. 산사자는맛이좋으며 8% 의조단백질과 5% 의조지질을함유하여 [15] 떡, 잼, 청량음료및전통발효주의원료로이용되고있으며, 한방에서는음식을잘소화하게하며혈압을낮추는작용, 혈액순환을도와어혈을없애는작용및지질용해작용을한다고알려져있다 [4,24]. 한편산사자를이용한조충구제및세균성이질치료용제품들이개발되어국내에서도판매되고있으며, 최근에는줄기에가시가없는산사나무신품종도개발되어조경수로이용되고있다 [10]. 론 *Corresponding author Tel: 82-054-820-5491, Fax: 82-054-820-7804 E-mail: hysohn@andong.ac.kr 산사자의활성성분으로는 ursolic acid, corosolic acid, euscapic acid, oleanolic acid, cratagolic acid, hyperin, vitexin, caffeic acid, procatechuic acid 및 pyrogallol 등의다양한페놀성화합물이알려져있으며 [14, 15, 21], 산사나무잎에서도유사한약리성분과 quercetin 배당체, myricetin 배당체, apigenin 배당체와 epicatechin 등이확인되어있다 [6, 29, 30]. 한편, 산사자의생리활성으로는항세균활성 [5], 내피세포의존성혈관이완활성 [4], 고지혈증개선및항우울증효과 [7, 16], 항염증활성 [12], 알콜성장해간보호효과 [24], 세포사멸활성 [1, 19], 항산화활성 [2, 3, 11], angiotensinconverting enzyme 저해효과 [28], chitin synthase II[9], HIV-I protease[18] 및 matrix metalloproteinase-1에대한저해활성 [17] 이알려져있으며, 최근본연구팀에서는산사자의다양한용매추출물및분획물들의트롬빈저해에따른강력한항혈전활성을보고한바있다 [23]. 그러나현재까지산사자추출물및유기용매분획물에대한광범위한항균활성탐색및항생제내성병원성미생물에대한항균활성을평가한보고는없다. 본연구에서는산사자의유용생리활성검토를위한연구

78 RYU et al. 의일환으로, 산사자의 methanol 추출물및이의다양한분획물을조제하여다양한병원성및식중독미생물에대한항균활성을평가하였으며, 그결과산사자추출물의 ethylacetate 및 butanol 분획에서우수한항미생물성활성을확인하였고, 특히이분획물들이항생제내성병원성 Pseudomonas aeruginosa 에대해강력한항균활성을나타냄을확인하였다. 본연구결과는산사자를이용한항생제내성병원성 P. aeruginosa 및 Candida sp. 의제어가능성을제시하고있다. 재료및방법 실험재료및시료의조제 2007 년경북안동에서구입한국내산산사자 ( 수분함량 18% w/w) 를사용하였다. 산사자 methanol 추출물의조제는시료 1kg 에 methanol 10 리터를가하여 24 시간상온추출하였으며 3 회반복추출하였다. 추출액은 50 에서감압농축하여분말로제조하고사용전까지저온밀봉보관하였다. 분획물제조의경우 methanol 추출물을물에현탁한후 n- hexane, ethylacetate, butanol 을이용하여순차적으로분획하고물잔류물을회수하였으며, filter paper(whatsman No. 2) 로거른후감압건조하여분말화하였다. 각각의시료는 dimethylsulfoxide(dmso) 에녹인후적당한농도로희석하여항균활성평가에사용하였다. 사용균주산사자 methanol 추출물및이의분획물들의항균활성평가에사용한세균및진균들은한국미생물자원센터 (KCTC), 한국농용미생물보존센터 (KACC) 및일본 Institute for Fermentation, Osaka(IFO) 에서분양받아사용하였다 (Table 1). P. aeruginosa CCARM 2010, 2020, 2030, 2200 및 2210 균주는한국항생제내성균주은행 (CCARM) 에서분양받아사용하였으며이들은 cefoperazone, gentamicin, norgloxacin, piperacillin, amikacin, aztreoman, ceftazidime, cefotaxime 및 imipenem의항생제중에서적어도 2종이상의항생제에대해내성을가지는다제내성균이며, 사람으로부터분리된균주이다. 한편 C. albicans CCARM 14020 및 14021, C. kruseis CCARM 14017, C. gillermordi CCARM 14018, C. tropicalis CCARM 14019는한국항생제내성균주은행 (CCARM) 에서분양받아사용하였으며이들은 amphotericin B, itaconazole, flucytosine 및 fluconazole에대해다제내성을나타내는균주이다 (http://www.ccarm.or.kr). 항균활성측정항세균및항진균활성평가는기존의보고한방법 [13, 26] 과동일하게이용하였으며, 각각 Nutrient agar(difco Co., USA) 및 Sabouraud dextrose(difco Co. USA) 배지상에서다양한시료 5 µl 를멸균 disc-paper( 지름 6.5 mm) 에가한 Table 1. The microorganisms used in this study. Gram negative bacteria Gram positive bacteria Fungi Escherichia coli KCTC 1682 Pseudomonas aeruginosa KACC 10186 Salmonella typhimurium KCTC 1926 Proteus vulgaris KCTC 2433 Pseudomonas aeruginosa KACC 10186 Pseudomonas aeruginosa KACC 10187 Pseudomonas aeruginosa KACC 10258 Pseudomonas aeruginosa KACC 10259 Pseudomonas aeruginosa KACC 10260 Pseudomonas aeruginosa CCARM 2010 Pseudomonas aeruginosa CCARM 2020 Pseudomonas aeruginosa CCARM 2030 Pseudomonas aeruginosa CCARM 2200 Pseudomonas aeruginosa CCARM 2210 Pseudomonas aeruginosa PAO-1 Staphylococcus aureus KCTC 1916 Listeria monocytogenes KACC 10550 Staphylococcus epidermidis KCTC 1917 Bacillus subtilis KCTC 1914 Candida albicans KCTC 7270 Candida albicans KCTC 7965 Candida albicans KACC 30003 Candida albicans KACC 30004 Candida albicans ATCC 10231 Candida albicans CCARM 14020 Candida albicans CCARM 14021 Candida kruseis CCARM 14017 Candida gillermordi CCARM 14018 Candida tropicalis CCARM 14019 Candida geochares KACC 30061 Candida saitoana KCTC 41238 Saccharomyces cerevisiae IF0 0233 후세균의경우 24 시간, 진균의경우 48 시간배양후, 생육저지환의크기를측정하여평가하였다 [26]. 대조구로는항세균제인 ampicillin 및 streptomycin sulfate 와항진균제인 miconazole 및 amphotericin B(Sigma Co., USA) 를각각 1 µg/disc 농도로사용하였으며, 육안으로생육저지환의지름을 mm 단위로측정하였고 3 회이상평가후대표결과로나타내었다 [13]. 항균활성이인정되는시료는최소생육억제농도 (MIC: minimal inhibitory concentration) 를측정하였으며, 0. 0.25, 0.5. 1.0, 1.5. 2.5, 5.0 및 7.5 mg/ml 농도의시료들을처리하여 24~48 시간배양한후생육억제를육안판정하여결정하였다. 인간적혈구용혈활성평가산사자추출물및분획물들의안전성평가의일환으로인간적혈구 (4%) 를이용하여용혈활성을평가하였다 [27]. PBS 로 3 회수세한인간적혈구 100 µl 를 96-well microplate 에가하고시료용액 100 µl 를가한다음 37 에서 30 분간반응시켰다. 이후, 반응액을 10 분간원심분리 (1,500 rpm) 하여상

ANTIMICROBIAL ACTIVITY OF CRATAEGUS PINNATIFIDA BUNGE 79 등액 100 µl 를새로운 microtiter plate 로옮긴후용혈에따른헤모글로빈유출정도를 414 nm 에서측정하였다. 시료의용매대조구로는 DMSO(2%), 실험대조구로는 triton X- 100(0.1%) 을사용하였다. 용혈활성은다음의수식을이용하여계산하였다. (%) hemolysis=[(abs. S Abs. C)/(Abs. T Abs. C)] 100. Abs. S: 시료첨가구의흡광도, Abs. C: DMSO 첨가구의흡광도, Abs. T: triton X-100 첨가구의흡광도 기타분석총 flavonoid 의함량측정은 Davis 방법의변법 [13] 에따라측정하였으며표준시약으로는 rutin 을사용하였다. 총 polyphenol 함량은 Singleton 등의방법 [25] 에따라측정하였으며, 표준시약으로는 tannic acid 를사용하였다. 모든결과는 3 회반복측정후평균값으로나타내었다. 결과및고찰 산사자의 methanol 추출물과이의순차적유기용매분획물을조제하였다. 산사자의 methanol 추출효율은 40.4±1.1% 이었으며, 추출물에대한 n-hexane, ethylacetate, butanol 및물잔류물의분획효율은각각 1.0, 5.1, 20.9 및 72.2% 로산사자추출물의대부분은 butanol 분획및물잔류물로이행되었다. 산사자추출물및분획물의총 polyphenol 함량은 methanol 추출물및 n-hexane, ethylacetate, butanol 및물잔류물에서각각 7.17, 1.33, 22.99, 14.62 및 0.78 mg/g 로나타나 ethylacetate 및 butanol 분획이대부분의 polyphenol 류들을포함하고있었다. 또한총 flavonoid 함량은추출물및분획물에서각각 8.02, 15.97, 52.43, 20.61 및 4.03 mg/ g 을포함하여 ethylacetate 분획, butanol 분획, n-hexane 분 획순으로 flavonoid 류들을포함하고있음을알수있었다. 다양한식중독및병원성세균들에대한항세균활성을평가하기위해준비된시료들을 500 µg/disc 농도로처리하였다. 그결과, 산사자 methanol 추출물은 P. aeruginosa 와 E. coli 를제외한모든균에대해전반적으로우수한활성을나타내었다 (Table 2). 특히알러지상식중독균인 P. vulgaris 에대해서는생육억제환의크기가 17 mm 로매우강력한항균활성을나타내었다. 분획물시료에서는 ethylacetate 분획물이실험에이용된세균모두에대해항균활성을나타내었으며, P. vulgaris, S. typhimurium 및 L. monocytogenes 에대해각각 20.5, 16.0 및 15.0 mm 의매우큰생육억제환을나타내었다. 한편 butanol 분획은 P. aeruginosa 를제외한세균들에서항균활성이나타났으며, 전반적으로 ethylacetate 분획물보다는약한활성을나타내었다. n-hexane 분획물과물잔류물은상대적으로한정된항균활성을나타내었으며, 대조구로사용된 ampicillin(1 µg/disc) 은사용균주모두에서 10.5~35.0 mm 의생육저지환을나타내어매우강력한항세균활성을가짐을보여주었다. 각각의균주에대한 ethylacetate 및 butanol 분획에대한최소생육저해농도 (MIC) 를측정한결과는 Table 3 에나타내었다. Ethylacetate 분획의경우 L. monocytogenes, S. epidermidis, S. aureus 및 P. vulgaris 에대해서는 1.0~1.5 mg/ml 농도에서생육이완전히억제되었으며, S. typhimurium 에서는 2.5 mg/ml 농도에서, P. aeruginosa, E. coli 와 B. subtilis 에대해서는 5.0 mg/ml 농도에서생육이나타나지않았다. 반면 butanol 분획의경우 S. epidermidis 와 P. vulgaris 는 1.0~1.5 mg/ml, L. monocytogenes 와 S. aureus 는 2.5 mg/ml, S. typhimurium, E. coli 및 B. subtilis 는 5.0 mg/ml, P. aeruginosa 에대해서는 7.5 mg/ml 농도에서생육이나타나지않아 ethylacetate 분획이 butanol 분획보다전반적으로낮은 MIC 값을나타내었다. 한편산사자의 70% methanol 추출물이 S. Table 2. Antibacterial activity of the methanol extract of Crataegus pinnatifida Bunge and its organic solvent fractions against pathogenic bacteria. Extract/ fractions Growth inhibition zone (mm) Gram positive Gram negative L. m 3 S. e S. a B. s S. t P. a P.v E.c Ampicillin 25.0 21.5 24.0 13.0 22.0 10.5 35.0 10.5 Methanol ex 1. 9.5 8.0 7.5 10.0 8.0-4 17.0 - n-hexane fr 2. 10.0 11.0 8.5 12.0 - - 9.0 - Ethylacetate fr. 15.0 11.0 10.0 11.5 16.0 7.5 20.5 7.5 Butanol fr. 15.0 9.5 8.0 9.5 8.0-19.5 8.0 Water fr. 8.0 - - - 7.5-15.0-1 ex: extract, 2 fr: fraction, 3 L. m: Listeria monocytogenes, S. e: Staphylococcus epidermidis, S. a: Staphylococcus aureus, B. s: Bacillus subtilis, S. t: Salmonella typhimurium, P. a: Pseudomonas aeruginosa, P. v: Proteus vulgaris, E. c: Escherichia coli, 4 - : No activity The concentrations of the methanol extract, its organic solvent fractions, and ampicillin used were 500 µg/disc, 500 µg/disc, and 1 µg/disc, respectively. The growth inhibition zone expressed was included a size of disc-paper (6.5 mm of diameter). The data represent a representative result of three independent determinations.

80 RYU et al. Table 3. The minimal inhibitory concentration (MIC) values of ethylacetate and butanol fractions against different pathogenic bacteria. MIC (mg/ml) Fractions Gram positive Gram negative L. m 2 S. e S. a B. s S. t P. a P.v E.c Ethylacetate fr 1. 1.0 1.5 1.5 5.0 2.5 5.0 1.5 5.0 Butanol fr. 2.5 1.5 2.5 5.0 5.0 7.5 1.0 5.0 1 fr: fraction, 2 L. m: Listeria monocytogenes, S. e: Staphylococcus epidermidis, S. a: Staphylococcus aureus, B. s: Bacillus subtilis S. t: Salmonella typhimurium, P. a: Pseudomonas aeruginosa KACC 10186, P. v: Proteus vulgaris. E. c: Escherichia coli. aureus 에대해 9.1 mg/ml 의 MIC 를가진다는보고 [20] 와비교하면, 산사자의 ethylacetate 분획은 70% methanol 추출물보다우수한항균력을가지는것으로추측된다. 이러한결과는산사자추출물이다양한항균물질을함유하고있으며, ethylacetate 및 butanol 분획은다양한식중독및병원성세균의제어에이용가능함을제시하고있다. 한편산사자의 ethylacetate 분획은 P. aeruginosa 에대해 7.5 mm 의미약한항균활성을나타내었으며 (Table 2), 5.0 mg/ml 의 MIC 를나타내었다 (Table 3). 이는 P. aeruginosa 에대해항균활성을나타내는천연물이거의없다는기존보고 [8, 22] 로볼때의미있는결과로판단되었다. 따라서산사자시료들을대상으로다양한 P. aeruginosa 균주에대한항균력을평가하였다. 먼저대조구로사용된 ampicillin 의경우 P. aeruginosa KACC 10186 에대해서는 10.5 mm 의생육저지환을형성하여양호한항균활성이나타났으나다른 10 종의 P. aeruginosa 균주에대해서는전혀항균활성이나타나지않아 (Table 4), 대부분의 P. aeruginosa 균주들이 ampicillin 에내성을가지고있음을확인하였다. 또한 streptomycin sulfate처리시에도 P. aeruginosa CCARM 2020, CCARM 2030, CCARM 2200, CCARM 2210 및P. aeruginosa PAO-1는전혀생육저해가나타나지않아, 상당수의 P. aeruginosa 균주들이 streptomycin sulfate 내성상태였다. 그러나, 산사자 methanol 추출물은항생제내성 P. aeruginosa CCARM 2010, CCARM 2020, CCARM 2030 및 KACC 10187에대해 8.0~8.5 mm 정도의생육억제활성을나타내었다. 가장우수한항균활성은 ethylacetate 및 butanol 분획에서확인되었으며, 이분획물들은항생제다제내성병원성 P. aeruginosa 균주모두에대해우수한항균력을나타내었다. 각각의분획물들의최저생육억제농도평가결과, P. aeruginosa CCARM 2010을제외한항생제내성균주와 PAO-1 균주는 MIC값이 2.5~5.0 mg/ml 농도로확인되었으며, 기타의 P. aeruginosa 균주들은 MIC값이 5.0~7.5 mg/ ml 농도로나타났다 (Table 5). 이는산사자의 ethylacetate 및 butanol 분획물은기존내성항생제와는다른기작으로 P. aeruginosa생육을억제하며, 활성물질의정제가이루어진다면산사자가 P. aeruginosa 생육억제제로개발가능함을암시하고있다. 한편, 산사자 methanol 추출물및이의분획물에대한항진균활성을평가한결과는 Table 6에나타내었다. 대조구로사용된 miconazole(1 µg/disc) 은 C. albicans KACC 30003 균주와항생제내성균주인 C. albicans CCARM 14020, CCARM 14021, C. kruseis CCARM 14017 및 C. tropicalis CCARM 14019에대해항균력을나타내지못하였으며, amphotericin B(1 µg/disc) 경우는 C. kruseis CCARM 14017을제외한균주에대해서 11.0~28.0 mm의생육억제환을나타내었다. 따라서 miconazole에대한 Candida sp. 에대한내성율은 40% 를상회하며, amphotericin B에대해서는 10% 이하의내성율을나타내는것으로추측되었다. 산사자시료들은다양한활성패턴을나타내었으며, 특히 ethylacetate 및 Table 4. Antibacterial activity of the methanol extract of Crataegus pinnatifida Bunge and its organic solvent fractions against multidrug-resistant Pseudomonas aeruginosa Extract/Fractions Growth inhibition zone (mm) 1 3 2 3 4 5 6 7 8 9 10 11 Ampicillin 10.5 - - - - - - - - - - Streptomycin sulfate 14.0 8.0 7.5 7.5 8.0 9.0 - - - - - Methanol ex 1. - 8.5 - - - 8.0 8.0 8.0 - - - n-hexane fr 2. - - - - - - - - - - - Ethylacetate fr. 7.5 11.0 9.0 8.5 11.0 9.0 9.0 8.5 9.0 10.0 9.0 Butanol fr. - 4 9.0 9.0 8.0 9.0 9.5 10.0 9.0 8.5 8.0 8.5 Water fr. - - 8.0 - - - - - - - - 1 ex: extract, 2 fr: fraction, 3 1: Pseudomonas aeruginosa KACC 10186, 2: P. aeruginosa KACC 10187, 3: P. aeruginosa KACC 10258, 4: P. aeruginosa KACC 10259, 5: P. aeruginosa KACC 10260, 6: P. aeruginosa CCARM 2010, 7: P. aeruginosa CCARM 2020, 8: P. aeruginosa CCARM 2030, 9: P. aeruginosa CCARM 2200, 10: P. aeruginosa CCARM 2210, 11: P. aeruginosa PAO-1, 4 -: No activity. The concentrations of the methanol extract, its organic solvent fractions, and ampicillin used were 500 µg/disc, 500 µg/disc, and 1 µg/disc, respectively. The growth inhibition zone expressed was included a size of disc-paper (6.5 mm of diameter). The data represent a representative result of three independent determinations.

ANTIMICROBIAL ACTIVITY OF CRATAEGUS PINNATIFIDA BUNGE 81 Table 5. The minimal inhibitory concentration (MIC) values of ethylacetate and butanol fractions against pathogenic and multidrug resistant Pseudomonas aeruginosa. Extract/Fractions MIC (mg/ml) 1 2 2 3 4 5 6 7 8 9 10 11 Ethylacetate fr. 1 5.0 7.5 >7.5 >7.5 7.5 >7.5 5.0 5.0 2.5 2.5 2.5 Butanol fr. 7.5 5.0 5.0 5.0 2.5 7.5 2.5 2.5 2.5 2.5 5.0 1 fr: fraction, 2 1: Pseudomonas aeruginosa KACC 10186, 2: P. aeruginosa KACC 10187, 3: P. aeruginosa KACC 10258, 4: P. aeruginosa KACC 10259, 5: P. aeruginosa KACC 10260, 6: P. aeruginosa CCARM 2010, 7: P. aeruginosa CCARM 2020, 8: P. aeruginosa CCARM 2030, 9: P. aeruginosa CCARM 2200, 10: P. aeruginosa CCARM 2210, 11: P. aeruginosa PAO-1. Table 6. Antifungal activity of the methanol extract of Crataegus pinnatifida Bunge and its organic solvent fractions against pathogenic Candida sp. Growth inhibition zone (mm) Extract/ Candida albicans (mm) Candida sp. (mm) fractions 1 4 2 3 4 5 6 7 8 9 10 11 12 S. c 5 Miconazole 28.0 6-20.0 8.0 19.0 - - - 11.0-30.0 7.0 8.5 Amp B 1 16.0 14.0 13.0 19.0 15.0 14.0 14.0-11.0 11.0 28.0 15.0 13.5 Methanol ex 2. - 6 - - 7.5 7.0 - - 7.0-8.0 - - - n-hexane fr 3. - - - - - - - - - - 8.0 8.0 - Ethylacetate fr. - 7.5 8.0 8.0 8.0 - - - - - 10.0 - - Butanol fr. - 7.5-7.5 9.5 - - 8.0 - - 7.5 7.0 7.5 Water fr. 7.0 - - 7.5 - - - - - 7.0 7.5-7.0 1 Amp B: Amphotericin B, 2 ex: extract, 3 fr: fraction, 4 1: Candida albicans KCTC 7270, 2: C. albicans KCTC 7965, 3: C. albicans KACC 30003, 4: C. albicans KACC 30004, 5: C. albicans ATCC 10231, 6: C. albicans CCARM 14020, 7: C. albicans CCARM 14021, 8: C. kruseis CCARM 14017, 9: C. gillermordi CCARM 14018, 10: C. tropicalis CCARM 14019, 11: C. geochares KACC 30061, 12: C. saitoana KCTC 41238, 5 S. c: Saccharomyces cerevisiae IF0 0233, 6 - : No activity. The concentrations of the methanol extract, and its organic solvent fractions used were 500 µg/disc, respectively. The concentrations of miconazole and amphotericin B as positive controls used were 1 µg/disc, respectively. The growth inhibition zone expressed was included a size of disc-paper (6.5 mm of diameter). The data represent a representative result of three independent determinations. butanol 분획물에서의미있는항 Candida sp. 활성이나타났다. 그러나산사자의 methanol 추출물이 5 종의항생제내성 Candida sp. 중 C. kruseis CCARM 14017 및 C. tropicalis CCARM 14019 균주에대해항균력을나타낸것과는달리 ethylacetate 분획은항생제내성 Candida sp. 에대해항균력을나타나지않았으며, butanol 분획은 C. kruseis CCARM 14017 에대해서만항균력을나타내었다. Ethylacetate 및 butanol 분획을대상으로 MIC 를평가한결과 ethylacetate 분획은 5 mg/ml 의 MIC 를, butanol 분획은 5.0~7.5 mg/ml 의 MIC 를나타내어 (result not shown), 산사자가 Candida sp. 제어에도이용가능함을알수있었다. 산사자 methanol 추출물및분획물의독성평가의일환으로인간적혈구용혈활성을평가한결과는 Fig. 1 에나타내었다. 진핵세포의 sterol 성분과결합하여세포막파괴를나타내는 amphotericin B 의경우, 6.25 µg/ml 농도에서 53%, 25 µg/ml 농도에서 90% 이상의용혈활성을나타내었으며 (Fig. 1a), triton X-100 은 0.1% 농도에서 100% 용혈현상이나타났다. n-hexane 분획물을제외한산사자의추출물및분 획물들은 250 µg/ml 농도에서용혈현상이나타나지않았으며, n-hexane 분획물은약 3.2% 의용혈이나타났다 (Fig. 1b). 이러한용혈현상은농도의존적이었으며, 500 µg/ml 농도에서는약 9.9% 의용혈을나타내었다 (Fig. 1c). 그러나 n-hexane 분획물을제외한시료의경우, 500 µg/ml 농도에서도용혈현상이전혀나타나지않으며산사자추출물의 n- hexnae 분획물의분획율이 1% 수준이므로일반적인사용에있어서산사자의독성은없을것으로판단되었다. 본연구결과는산사자의 ethylacetate 및 butanol 분획물이항생제내성균주들을포함한다양한세균및 Candida sp. 에우수한항균활성을가짐을나타내며, 이들로부터항생제내성균주에대한항균제개발가능성을제시하고있다. 현재기존항생제와산사자 ethylacetate 분획물의동시처리에의한항생제내성균주제어가능성을검토중에있으며, ethylacetate 및 butanol 분획물로부터항균활성물질의정제가필요하다. 요 산사자는전세계적으로이용되는있는식용 / 약용생물자 약

82 RYU et al. 원중하나이다. 본연구에서는산사자의유용생리활성검토를위한연구의일환으로, 산사자의 methanol 추출물및이의 n-hexane, ethylacetate, butanol 분획물및물잔류물을조제하여항생제다제내성 Pseudomonas aeruginosa 및 Candida sp. 를포함하는다양한병원성및식중독미생물에대한항균활성을평가하였다. 산사자의 methanol 추출물은그람양성및음성의다양한세균에대해항균활성을나타내었고, 이의분획물중 ethylacetate 및 butanol 분획물은 Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus, Bacillus subtilis, Salmonella typhimurium, Proteus vulgaris, Escherichia coli는물론 10종의항생제내성병원성 Pseudomonas aeruginosa에대해서도우수한항세균활성을나타내었다 ( 최소생육억제농도 1.0~7.5 mg/ ml). 또한 ethylacetate 및 butanol 분획물은일부의 Candida sp. 에대해서도항균활성을나타내었다. 한편 n-hexane 분획물을제외한산사자 methanol 추출물및분획물들은 500 µg/ml 농도까지인간적혈구에대한용혈현상을보이지않았으며, n-hexane 분획물은 500 µg/ml 농도에서약 9.9% 의미미한용혈활성을나타내었다. 이러한결과는산사자가다양한세균의제어는물론항생제내성 Pseudomonas aeruginosa 제어를위한생물자원으로개발가능함을제시하고있다. REFERENCES 1. Ahn, K. S., M. S. Hahm, E. J. Park, H. K. Lee, and I. H. Kim. 1998. Corosolic acid isolated from the fruit of Crataegus pinnatifida var. psilosa is a protein kinase C inhibitor as well as a cytotoxic agent. Planta Med. 64: 468-470. 2. An, B. J., B. Y. Kang, and J. T. Lee. 2002. Development of cosmetic material from Korean Crataegi fructus extract. Kor. J. Herbology 17: 39-50. 3. An, B. J. and J. T. Lee. 2002. Studies on biological activity from extract of Crataegi fructus. Kor. J. Herbology 17: 29-38. 4. Bae, M. H. and H. H. Kim. 2003. Mechanism of Crataegi fructus extract induced endothelium-dependent vasorelaxation in rabbit carotid artery. Kor. J. Herbology 18: 169-180. 5. Choi, O.-K., Y. Kim, G.-S. Cho, and C.-K. Sung. 2002. Screening for antimicrobial activity from korean plants. Kor. J. Food. Nutr. 15: 300-306. 6. El-Mousallamy, A. M. D., 1998. Chemical investigation of the constitutive flavonoid glycosides of the leaves of Crataegus sinaica. Natural Prod. Sci. 4: 53-57. 7. Hong, S. S., J. S. Hwang, S. A. Lee, X. H. Han, J. S. Ro, and K. S. Lee. 2002. Inhibitors of monoamine oxidase activity from the fruits of Crataegus pinnatifida Bunge. Kor. J. Pharmacogn. 33: 285-290. 8. Hooi, D. S. W., B. W. Bycroft, S. R. Chhabra, P. Williams, and D. I. Pritchard. 2004. Differential immune modulatory activity of Pseudomonas aeruginosa quorum sensing signal molecules. Infect. Immun. 72: 6463-6470. 9. Jeong T. S., E. I. Hwang, H. B. Lee, E. S. Lee, Y. K. Kim, B. S. Min, K. H. Bae, S. H. Bok, and S. U. Kim. 1999. Chitin synthase II inhibitory activity of ursolic acid, isolated from Crataegus pinnatifida. Planta Med. 65: 261-263. 10. Kang, H. C., K. K. Shim, Y. M. Ha, and W. H. Lee. 2002. New varieties with thronless branches of Crataegus pinnatifida Bunge. Kor. J. Hor. Sci. Technol. 20: 252-256. 11. Kang, I.-H., J.-H. Cha, S.-W. Lee, H.-J. Kim, S.-H. Kwon, I.-H. Ham, B.-S. Hwang, and W.-K. Whang. 2005. Isolation of anti-oxidant from domestic Crataegus pinnatifida Bunge leaves. Kor. J. Pharmacogn. 36: 121-128. 12. Kao, E. S., C. J. Wang, W. L. Lin, Y. F. Yin, C. P. Wang, and T. H. Tseng. 2005. Anti-inflammatory potential of flavonoid contents from dried fruit of Crataegus pinnatifida in vitro and in vivo. J. Agric. Food. Chem. 53: 430-436. 13. Kim J. I., H. S. Jang, J. S. Kim, and H. Y. Sohn. 2009. Evaluation of antimicrobial, antithrombin, and antioxidant activity of Dioscorea batatas Decne. Kor. J. Microbiol. Biotechnol. 37:133-139. 14. Kim, J. S., G. D. Lee, J. H. Kwon, and H. S. Yeon. 1993. Antioxidative effectiveness of ether extract in Crataegus pinnatifida Bunge and Terminalia chebula Rets. J. Kor. Agric. Chem. Soc. 36: 203-207. 15. Kim, J. S., G. D. Lee, J. H. Kwon, and H. S. Yeon. 1993. Identification of phenolic antioxidative components in Crataegus pinnatifida Bunge. J. Kor. Agric. Chem. Soc. 36: 154-157. 16. Lee, H. J., and M. S. Choi. 1998. Changes of plasma and hepatic lipids, hydroxy-methyl-glutaryl CoA reductase activity and acyl-coa: cholesterol acyltransferase activity by supplementation of hot water extracts from Rosa rugosa, Crataegus pinnatifida and Polygon. J. Food. Sci. Nutr. 3: 344-350. 17. Lee, S. Y., J. H. An, H. Chun, and H. Y. Cho. 2003. Isolation and characterization of MMP-1 inhibitor peptide from Crataegus pinnatifida Bunge in fibroblast cell line HS68 cells. J. Kor. Sco. Agric. Chem. Biotechnol. 46: 60-65. 18. Min, B. S., H. J. Jung, J. S. Lee, Y. H. Kim, S. H. Bok, C. M. Ma, N, Nakamura, M. Hattori, and K. Bae. 1999. Inhibitory effect of triterpenes from Crataegus pinatifida on HIV-I protease. Planta Med. 65: 374-375. 19. Min, B. S., Y. H. Kim, S. M. Lee, H. J. Jung, J. S. Lee, M. K. Ma, C. O. Lee, J. P. Lee, and K. Bae. 2000. Cytotoxic triterpenes from Crataegus pinnatifida. Arch. Pharm. Res. 23: 155-158. 20. Park, C. G., K. H. Bang, S. E. Lee, M. S. Cha, J. S. Sung, H. W. Park, and N. S. Seong. 2001. Antibacterial activity from medicinal plant extracts on the Staphylococcus aureus. Kor. J. Med. Crop Sci. 9: 251-258. 21. Park, S. W., C. S. Yook, and H. K. Lee. 1994. Chemical components from the fruits of Crataegus pinnatifida var psilosa. Kor. J. Pharmacogn. 25: 328-335.

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