Korean J. Plant Res. 31(5):423-432(2018) https://doi.org/10.7732/kjpr.2018.31.5.423 Print ISSN 1226-3591 Online ISSN 2287-8203 Original Research Article 여드름유발균 Propionibacterium acnes 에대한녹나무과 10 종의항균활성 조주성 1, 지래원 2, 장보국 3, 정헌상 4, 이철희 5 * 1 ( 주 ) 농우바이오, 박사연구원, 2 충북대학교원예과학과, 박사연구원, 3 충북대학교원예과학과, 박사과정생, 4 충북대학교식품생명공학과, 교수, 5 충북대학교원예과학과, 교수 Anti-microbial Activities of Ten Lauraceae Species against Propionibacterium acnes Ju Sung Cho 1, Lai Won Chi 2, Bo Kook Jang 3, Heon Sang Jeong 4 and Cheol Hee Lee 5 * 1 Ph.D. Researcher, Quality Control & Seed Tech Team, Nongwoo Bio CO., LTD., Yeoju 12655, Korea 2 Ph.D. Researcher and 3 Ph.D. Graduate Student, Major in Horticulture, Division of Animal, Horticultural, and Food Sciences, Chungbuk National University, Cheongju 28644, Korea 4 Professor, Department of Food Science and Biotechnology, Chungbuk National University, Cheongju 28644, Korea 5 Professor, Major in Horticulture, Division of Animal, Horticultural, and Food Sciences, Chungbuk National University, Cheongju 28644, Korea Abstract - This study was performed to develop a new natural antimicrobial materials by analyzing the effect of extracts obtained from Ten Lauraceae Species on the inhibitory activity against Propionibacterium acnes. The plant materials were collected from Wando and Jeju islands, and the antimicrobial activity of the crude extracts was examined by the agar diffusion method with different part (i.e., leaf and branch), solvents (i.e., distilled water, 80% ethanol, and 100% methanol) and at different ultrasonic extracting times (i.e., 15, 30, and 45 minutes). The control agents used were synthetic antimicrobials, methylparaben and phenoxyethanol, at concentrations of 0.4, 1, 2, and 4 mg /disc. Altogether, extracts of 10 species used in the study showed inhibitory activity, which confirmed their antimicrobial action against acnes. Among these, leaves of Laurus nobilis L. which was extracted in 80% ethanol for 45 min showed the largest clear zone (19.8 mm ). Leaves of L. nobilis L., showing highest antimicrobial activities among 10 species, were successively reextracted with n-hexane, chloroform, ethylacetate and n-butanol. As a results, in all fractions except butanol, clear zone above 10 mm were formed. The ethyl acetate fraction showed the highest inhibitory activity (13.3 mm ) and the inhibitory activity was significantly higher than that of crude extract (10.2 mm ) and phenoxyethanol as a control (12.5 mm ). Key words - Laurel, Methylparaben, Phenoxyethanol, Pruning product, Skin disease pathogens 서언 현대사회에서피부미용에투자하는시간과비용은점차증가되고있다. 하지만잘못된세정습관, 합성화학성분제나오일제품의장기간사용및신체리듬과생리적인변화에따른 testosterone 의불균형등은피지선을비대시키고피지분비를왕성하게한다. 과다분비된피지에의해모낭벽세포에이상각 * 교신저자 : leech@chungbuk.ac.kr Tel. +82-43-261-2526 화가일어나모낭구가막히면피지가배출되지못하고정체하게된다 (Suh, 2010). 이에피부는항상성이깨지고, 모공내미생물들이성장하기좋은조건으로조성됨으로써혐기성균, 호기성구균, 진균등이번식하게된다 (Brown and Shalita, 1998; Ahn et al., 2002). 여드름유발균인 Propionibacterium acnes (P. acnes) 는피지과다분비부위에서번식하면서 lipase를분비하여피지성분의중성지방을유리지방산으로변화시킨다. 이후모낭주위에밀집된백혈구가모낭벽을파괴하고모낭내용물이진피내로 c 본학회지의저작권은 ( 사 ) 한국자원식물학회지에있으며, 이의무단전재나복제를금합니다. This is an Open-Access article distributed under the terms of the Creative Commons -423- Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Korean J. Plant Res. 31(5) : 423~432(2018) 유입되어염증성여드름을야기하며 (Kim and Byeom, 2007), 추후오목흉터 (potted scar) 나비대흉터 (hypertrophicscar) 와같은후유증을동반하기도한다 (Brown and Shalita, 1998). 염증성여드름치료에사용되는스테로이드제는피부위축, 구순주위피부염, 자반증및간손상등의부작용을일으킬수있다 (Kim et al., 1995). 또한광범위하게사용되는화학방부제인 paraben 의과용은알레르기성접촉피부염을유발하며, 체내세포조직에축적되어유방암세포에서고농도의파라벤이검출되는등의부작용이보고된바있다 (Lee et al., 2006). 염증성질환치료제인레티노이드는비타민 A의천연또는합성유도체이며생체내에서는 retinoic acid 와같은활성형태로각질형성세포의증식과분화조절, 피지선억제, 면역조절및항염증작용, 여드름및건선등의피부질환에사용된다 (Kim, 2008). 또한 retinoic acid 는광노화와내인성노화피부의주름살및피부탄력을호전시킬수도있으나레티노이드피부염 (retinoid dermatitis) 이라불리는일종의자극성접촉피부염으로홍반, 인설, 건조, 작열감, 소양감등을호소하는부작용을유발할수있다 (Kim, 2008). 최근들어화학합성항생제에내성을가지는균주가발견되고관련된부작용들이표출됨에따라이를해결하고자천연물유래의항균 항산화물질탐색이활발하다 (Otvos, 2000). 국내에서편백, 나한백, 유칼립투스, 소목등의수목류는항산화효과와세포독성효과, 항염효과, 항알러지효과등이연구되었으며 (Kang et al., 2002; Jeon et al., 2010; Jung, 2010), 그중소목 (Caesalpinia sappan L.) 에서분리한 brazilin은항산화활성과구강상피암증식억제효과가보고된바있다. 또한수목류의정유성분에대한항균 항진균및모발기능성물질의이용기술개발 (RDA, 2007), 한국산자생식물 125종에대한항균력탐색및대황의 Staphylococcus aureus 에대한항균활성 (Choi et al., 2002) 및치자의항산화활성및활성성분의분리 (Yang et al., 2011) 등의연구가진행되었다. 최근에는자원식물의메탄올추출물을이용한식물병원성곰팡이의포자발아억제활성연구도보고되었다 (Park et al., 2017). 이처럼국내에서도식물유래항균물질탐색에관한연구가꾸준히진행되고있으나대부분초본의약용식물에국한되어있어, 상록성목본수종에대한관심과연구는부족한실정이다. 다양한상록성목본수종에서유래한 polyphenol 과 flavonoid 는항산화및항균효과를나타내기때문에, 잎과가지등의재료자급율이높은천연소재로서경제적가치가풍부하여개발가능성이매우높다 (Kim and Han, 1997; Bae and Moon, 2001). 그러나동일종이라도지리적인환경및채취시기에따라구성성분이변하고, 용매종류및농도에따라항균력에차이가있기에국내수목연구가무엇보다도중요하다 (Park et al., 1992; Kim et al., 2004). 우리나라남부에자생하는상록성목본의기능성연구로는솔잎추출물의항균활성 (Park et al., 2017), 황칠나무의부위별항산화활성분석 (Yu and Moon, 2018) 및구실잣밤나무의항산화및항균활성 (Kim et al., 2011) 등꾸준히진행되고있어활용성은점차증가될것으로기대된다. 또한상록성목본은전정으로인해버려지는부산물을이용하여 1년내내재료의수급이수월하고더불어식물의부가가치를상승시킬수있다는장점이있다. 본연구는우리나라남부지방에자생하는상록성목본식물인녹나무과 10종의피부상재균주 P. acnes 증식억제효과를검증함으로써염증성여드름질환의치료를목적으로하는새로운천연추출물을활용한기능성화장품용소재를개발하고자시행되었다. 재료및방법식물재료남부지방에자생하는녹나무과 6속 10종상록목본의잎과가지를 2013 년 1월중순부터 2월초까지전라남도완도및제주도일대에서채취하였다 (Table 1). 식물재료는생체중을측정한후 48시간동안동결건조 (FD8512, IlShin Lab. Co. Ltd., Seoul, Korea) 하였으며, 이후건물중을측정하여각재료의수분함량을계산하였다 (Table 1). 건조된시료는분쇄기 (Hood mixer FM-681C, Hanil, Gwangju, Korea) 로곱게분쇄한후, -70±2 의 deep freezer (Ultra-low temperature freezer SW-U5-200, Samwon, Goyang, Korea) 에저장하면서실험에사용하였다. 실험균주및배지실험에사용된균주 P. acnes (KCTC 3320) 는그람양성균이며한국유전자은행에서분양받았다. 배지는 RCM broth (Reinforced Clostridial Medium, Difco, Detroit, MI, USA) 를사용하였으며, 3 5 주간격으로계대배양하였다. 실험 2주전에는 3 5 일간격으로 broth 10 ml에미생물배양액 100 μl를접종하여활성도를높였다. 혐기성세균인 P. acnes 는배양시 gas pak (AnaeroPack, Mitsubishi Gas Chemical Co., Inc, Tokyo, Japan) 을넣은밀폐용기에넣어 37±0.5 의 CO 2 incubator (VS-9108MS, Vision, Yeoju, Korea) 에서관리되었다. -424-
여드름유발균 Propionibacterium acnes 에대한녹나무과 10 종의항균활성 Table 1. List of ten Lauraceae evergreen broad-leaved species used in this study Scientific name Korean name Collection site Moisture content (%) Leaf Stem Actinodaphne lanecifolia (Sieb. et Zucc.) Meisn. 육박나무 Wando, Jeollanam-do 71.5±1.11 52.6±2.15 Cinnamomum loureirii Ness. 육계나무 Jeju-si, Jeju-do 58.0±1.13 40.7±1.18 Cinnamomum yabunikkei H.Ohba 생달나무 Wando, Jeollanam-do 47.4±2.80 50.3±2.12 Cinnamomum camphora (L.) J.Presl 녹나무 Jeju-si, Jeju-do 53.2±0.99 51.8±3.34 Laurus nobilis L. 월계수 Wando, Jeollanam-do 58.3±3.50 49.4±3.32 Litsea japonica (Thunb.) Juss. 까마귀쪽나무 Wando, Jeollanam-do 57.0±2.97 57.1±1.68 Machilus japonica Siebold & Zucc. 센달나무 Wando, Jeollanam-do 55.2±4.41 52.7±1.13 Machilus thunbergii Siebold & Zucc. 후박나무 Wando, Jeollanam-do 50.2±5.08 52.5±1.97 Neolitsea aciculata (Blume) Koidz. 새덕이 Jeju-si, Jeju-do 51.2±1.01 53.5±2.31 Neolitsea sericea (Blume) Koidz. 참식나무 Jeju-si, Jeju-do 55.3±2.30 54.9±1.19 녹나무과 10종의항균활성탐색각종의동결건조한잎시료 1.0 g을 100% 메탄올 (methanol, Merck, Darmstadt, Hesse, Germany) 에잠기도록유리병에넣어혼합한후 Ultrasonic cleaner (30 24 14.5 cm, 5510-DTH, Branson, Danbury, CT, USA) 로 30분동안초음파추출을시행하였다. 모든추출물은잔사를재추출하지않고 1회, 2반복추출하였다. 추출직후 vacuum pump (GAST) 와여과지 (Advantec No. 2, Toyo Roshi Kaisha Ltd., Tokyo, Japan) 를이용하여감압여과하였으며, 여과된추출물은용매를사용하여최종농도를 50배로정량하였다. 이후추출물은질소충전하여 -70± 2 deep freezer (Samwon Engineering Co.) 에보관하면서항균활성평가에사용하였으며, 5 ml를취하여증발접시에담고 60±2 의 dry oven (KPI-507L, Korea Power Ind. Co. Ltd, Seoul, Korea) 에 48시간건조시켜추출물의가용성고형분함량 (g/g db) 을구하였다 (Table 1). Agar diffusion assay 는다음의순서로진행하였다. 50 배추출물을감압농축 (N-1000, EYELA, Tokyo, Japan) 후 DMSO 로녹여 50 mg / ml로농축한다음클린벤치내에서멸균한 8 mm의 paper disc (Advantec No. 2, Toyo Roshi Kaisha Ltd., Tokyo, Japan) 에 2 mg /disc 가되도록하였으며, 용매를충분히건조시켰다. 0.7% 의 agar를첨가한 soft agar broth는멸균한다음미리활성화시킨미생물배양액 (O.D. value = 1.0) 을 60 의 soft agar broth 에 1% v/v 로접종하였다. 그리고 magnetic stirrer 로혼합한다음 90 15 mm의페트리디쉬에 12 ml씩분주하였으며, 15분이내에추출물을주입한 disc 를치상하였다. 이후 37 의 incubator 에서 24시간동안배양하였으며, 배양후 disc 의직경 을포함한 clear zone 을측정하였다. 추출물의항균활성을화장품제조시사용되는합성항균제인 phenoxyethanol (0H2108, Junsei Chemical Co., Ltd., Tokyo, Japan) 과 methylparaben (8K5015, Junsei Chemical Co., Ltd., Tokyo, Japan) 을 0.4, 1, 2, 4 mg /disc 의농도로희석하여추출물과같은조건으로배양하였다. 추출조건에따른항균활성잎추출물의항균활성이가장우수한종 ( 월계수 ) 을선발하여적정추출부위, 용매및시간을구명하고자하였다. 동결건조한가지와잎시료를사전연구를통해선정된물 (deionized water; nano pure grade), 80% 에탄올 (Jin chemical pharmaceutical Co., LTD, Siheung, Korea) 및 100% 메탄올 (Merck) 을용매로하여 15, 30 및 45분동안각각초음파추출하였다. 추출당시초음파수조내수온은 20±2 였으며, 추출 15분후에는 1.0±0.5, 30분후에는 2.5±0.8, 45분후에는 3.6±0.7 가상승하였다. 이후동일한방법으로 agar diffusion assay 를수행하여항균활성을분석하였다. 용매분획에따른항균활성추출조건에따른항균활성이가장우수하였던종 ( 월계수 ) 의잎시료를 100% 메탄올을용매로 30분간초음파추출하여조추출물을제조하였다. 조추출물을감압농축 (EYELA) 후동결건조하여고형분시료 1.0 g을얻었다. 이후고형분시료, 증류수및 n-hexane을 1:9:10 의비율로혼합하여분획하였으며 n-hexane 층을농축하여분획물을얻었다. n-hexane 분획후수층은다 -425-
Korean J. Plant Res. 31(5) : 423~432(2018) Fig. 1. Schematic diagram for solvent fractionation of whole extract using n-hexane, chloroform, ethyl acetate, and n-butanol. 시 chloroform, ethyl acetate, n-butanol 순으로순차적으로용매분획하였으며 (Fig. 1), 각각의분획층으로부터동일한방법으로고형분시료를얻은후증류수에녹여항균실험에사용하였다. 통계분석녹나무과 10종의잎추출물에대한항균활성은 3반복, 그외항균활성은 6반복을 1회로 2회반복실험하였다. 또한추출조건에따른항균활성은부위, 용매및시간간의요인별유의성을검정하였다. 실험후모든결과값은 SAS version 9.3 (SAS instritute Inc., Cary, NC, USA) 를이용하여평균과표준오차를구하였으며, P<0.05의유의수준에서 Duncan's multiple range test 를이용하여통계분석하였다. 결과및고찰 P. acnes 에대한녹나무과 10종의항균활성연구에사용된 10종의모든잎추출물에서우수한 P. acnes 억제활성이확인되었다 (Table 2). 여드름균생육억제환을측 정한결과, 모든추출물에서지름 20 mm이상으로조사되었으며, 특히월계수 (30.00 mm ) 가가장우수한활성을보였다. 다음으로육박나무 (28.05 mm ), 센달나무 (27.70 mm ), 육계나무 (27.65 mm ) 및생달나무 (25.75 mm ) 순으로높은활성을나타내었다. 이상의 5종의잎추출물은대조구인 phenoxyethanol (4 mg /disc, 15.20 mm ) 과 methylparaben (2 mg /disc, 24.70 mm ) 에비해서도높은항균활성을기록하였다. 실험에사용된녹나무과식물재료들은예전부터국내 외에서다양한민간요법치료제로사용되었으며, 최근에그효능들이검증되었다. 월계수는상록활엽교목으로우리나라경남과전남지방에분포되어있다. 월계수추출물은신경통, 류마티스진통제, 발진, 구풍제, 소화제, 수렵제등의사용된다고알려져있으며 (Kivcakb and Mert, 2002; Simic, 2003), glutamic acid 과 leucin 및 vitamin A, C가다량함유되어있다 (Lee et al., 2005). 제주도에자생하는녹나무의약재로사용되는장뇌에는정유성분인 camphor 외에 cineol, 1-camphene 등이함유되어있고, 제주도에서는녹나무잎다린물을당뇨병치료에민간요법으로사용해왔다 (Kim et al., 2007). 또한녹나무의잎추출물은플라보노이드가풍부하여, 항산화활성과항염증효과가높 -426-
여드름유발균 Propionibacterium acnes 에대한녹나무과 10 종의항균활성 Table 2. Antimicrobial activities of extracts obtained from leaves of ten Lauraceae species against Propionibacterium acnes Korean name Contents of soluble solids ( mg /g) Concentration ( mg /disc) Clear zone ( mm ) Scientific name Leaf Stem Phenoxyethanol z 0.40 - y 1.00 8.20±1.20g x 2.00 13.40±0.75f 4.00 15.20±1.00ef Methylparaben 0.40-1.00 8.50±2.30g 2.00 24.70±1.20bc 4.00 15.20±1.45ef Actinodaphne lanecifolia (Sieb. et Zucc.) Meisn. 육박나무 0.16 0.18 2.00 28.05±1.13ab Cinnamomum loureirii Ness. 육계나무 0.12 0.27 2.00 27.65±2.68ab Cinnamomum yabunikkei H.Ohba 생달나무 0.27 0.32 2.00 25.75±1.41bc Cinnamomum camphora (L.) J.Presl 녹나무 0.20 0.26 2.00 24.35±0.09c Laurus nobilis L. 월계수 0.16 0.15 2.00 30.00±2.02a Litsea japonica (Thunb.) Juss. 까마귀쪽나무 0.32 0.21 2.00 21.95±1.24d Machilus japonica Siebold & Zucc. 센달나무 0.26 0.31 2.00 27.70±1.44ab Machilus thunbergii Siebold & Zucc. 후박나무 0.15 0.31 2.00 22.80±1.10cd Neolitsea aciculata (Blume) Koidz. 새덕이 0.20 0.34 2.00 24.00±0.58c Neolitsea sericea (Blume) Koidz. 참식나무 0.21 0.22 2.00 23.90±0.23c z Positive control as a synthetic antioxidant. y Not detected. x Means ± S.E. (n=10) and separation within columns by Duncan s multiple range test, P<0.05. 은것으로보고되었다 (Lee and Balick, 2005). 생달나무는전라남도및제주도에분포한다. 강한향을내는녹나무에비해향에대한만족도가높아향기가인체에미치는생리적측면의영향에대한연구 (Kim et al., 2007) 가이뤄진바있다. 육박나무는남쪽섬지방에분포하며관절염, 복통, 과로등의치료에사용되었다 (KBIS, 2018). 본연구에서월계수잎추출물의 P. acnes 에대한항균활성은 Choi et al. (1998) 이연구한 97종의천연물, 새삼및실새삼 (Suk et al., 2004), 감초등한약복합추출물 (Kim et al., 2006), 대황, 황백및황금의복합추출물 (Son et al., 2005) 및자몽종자추출물과법제유황수 (Ha et al., 2009), 강활, 백작약, 물푸레피, 현삼및황금추출물 (Kim, 2008) 에비해서도우수하였다. 시판합성치료제인 methylparaben 은쉽게분해되지않으므로신체조직에축적되거나환경오염물질로남아다양한부작 용을유발하며, 그외에도내분비계교란 (Foran et al., 2000) 및내성균주출현 (Eady et al., 2003) 등다양한부작용을유발하는것으로알려져있으므로사용에제한이있다. 본연구에서월계수등 5종은 methylparaben 뿐만아니라 phenoxyethanol 에비해서도우수한활성을보여, P. acnes 에대한합성항균제를대체할수있는천연물유래여드름예방및치료소재로개발이가능할것으로생각된다. 추출조건에따른월계수의항균활성녹나무과 10종의잎추출물중 P. acnes 억제활성이가장우수하였던월계수를부위, 용매및시간을달리하여추출한다음항균활성분석하였다. 연구의결과, 월계수잎을 80% 에탄올을용매로 45분간추출하였을때억제활성이가장우수하였으며 (19.8 mm ), 대조구의모든농도에비해서도높았다 (Table 3). 또 -427-
Korean J. Plant Res. 31(5) : 423~432(2018) Table 3. Antimicrobial activities of extracts obtained from leaves and stems of Laurus nobilis L. against Propionibacterium acnes according to extraction methods Part Solvent Extraction time (min.) Clear zone ( mm ) Methylparaben z 0.4-1.0 8.6±0.65i y 2.0 14.2±0.27de Control 4.0 14.8±0.38c- e ( mg /disc) Phenoxyethanol 0.4-1.0-2.0 13.1±0.80d-g 4.0 18.7±0.27ab 15 10.9±0.79g-i MeOH 100% 30 10.2±0.76hi 45 18.6±0.20ab 15 14.9±1.18cd Leaf EtOH 80% 30 13.9±0.60d-f 45 19.8±0.68a 15 14.7±0.90c-e Distilled water 30 17.1±0.05bc 45 18.8±0.06ab 15 14.6±0.95de MeOH 100% 30 14.3±0.55de 45 17.7±0.03ab 15 12.2±0.84e-h Stem EtOH 80% 30 13.3±0.10e-h 45 11.6±0.61f-g 15 - Distilled water 30-45 - Significance x P (Part) *** S (Solvent) *** T (Time) *** P S *** P T *** T S *** P T S ns z Positive control as a synthetic antioxidant. y Means ± S.E. (n=10) and separation within columns by Duncan s multiple range test, P<0.05. x ns, *, **, *** : nonsignificant or significant at P<0.05, 0.01 or 0.001, respectively. -428-
여드름유발균 Propionibacterium acnes 에대한녹나무과 10 종의항균활성 한월계수는가지부위의물추출물을제외한모든조건에서항균활성을나타내었으며, 추출부위, 용매및시간에따른유의적인활성도의차이를보였다 (P<0.001). 본연구에서월계수가지의물추출물은항균활성이전혀없었는데, 이는 Park (1998) 의솔잎추출물이식중독세균의생존에미치는영향에관한연구에서에탄올추출물이물추출물에비해월등히높은항균활성을가진다는것과유사하다. Park et al. (2017) 의연구에서는에탄올을용매로한소나무의부위별추출물은 P. acnes 에대해항균활성을나타내었으나열수추출물에서는부위에관계없이활성이나타나지않았다. 또한동일실험에서송엽, 송절및송화등추출부위에따른활성도는다른것으로보고된바있다. 식물성추출물의약리활성은식물종과추출부위별로다르게나타나며, 동일추출부위에서도추출용매에따라용출되는성분이달라지기때문에차이를보인다 (Lee et al., 2006). 식물소재의에탄올추출물은 polyphenol, flavonoid, polypeptide 등의성분으로이루어져있으며, 그중 phenolic compound 와 flavonoid 가미생물의세포막에침투하여세포막을파괴시킴으로써항균활성을나타낸다 (Paniwnyk et al., 2001; Lambert et al., 2011) 의연구에서는식물원료를물로추출할경우추출물내에수산기 radical 이형성되어생리활성이저하되는결과가보고된바있다. 한편 Lee and Yoon (2010) 의연구에서는월계수잎의메탄올및열수추출물에서피부의 AGEs 형성을억제하는항주름활성과최종당화산물생성억제활성이높았으므로월계수는추출의목적에따라추출조건을달리할필요하다. 따라서 P. acnes 에대한항균활성을목적으로월계수를추출할때잎은 80% 에탄올을용매로 45분간추출하고, 가지부위는물추출을피하는것이효과적일것으로생각된다. 월계수추출물의용매분획별항균활성 P. acnes 억제활성이우수하였던월계수잎을 100% 메탄올을용매로초음파 30분추출한다음 n-hexane, chloroform, ethyl acetate, n-butanol 그리고 water 순으로용매분획하여항균활성을비교분석하였다. 각분획물에서얻어진가용성고형분의함량은조추출물에서 161.7 mg /g 수준이었으며, Water 분획물에서 544.0 mg /g의고형분을얻어내가장높은수율을보였다 (Table 4). 반면 n-hexane 분획물에서는 89.3 mg /g으로수율이가장낮아, 분획물별가용성고형분함량은차이가있었다. Table 4. Antimicrobial activities of different solvent fractions obtained from leaf of Laurus nobilis L. against Propionibacterium acnes No. Treatment ( mg /disc) Soluble solids contents ( mg /g) Clear zone ( mm ) Methylparaben z 0.4 - y 1.0 12.4±0.34c x 2.0 14.4±1.00b 4.0 18.2±1.44a 0.4 - Phenoxyethanol 1.0-2.0-4.0 12.5±0.85c 1 n-hexane 2.0 208.1 12.8±0.23c 2 Chloroform 2.0 89.3 10.9±0.17d 3 Ethyl acetate 2.0 107.9 13.3±0.17bc 4 n-butanol 2.0 289.8-5 Water 2.0 544.0 10.0±0.25d Crude extract 2.0 161.7 10.2±0.12d z Positive control as a synthetic antioxidant. y Not detected. x Means ± S.E. (n=10) and separation within columns by Duncan s multiple range test, P<0.05. -429-
Korean J. Plant Res. 31(5) : 423~432(2018) 월계수잎용매분획물의항균활성을분석한결과, ethyl acetate 분획물에서억제환이 13.3 mm로가장컸으며, 조추출물 (10.2 mm ) 과대조구인 phenoxyethanol (12.5 mm ) 에비해서도유의적으로우수한억제활성을나타내었다. 또한 butanol 분획물을제외한모든분획물에서 10.0 mm이상의억제환이형성되었다. 자생상록활엽수인두릅나무과의황칠나무는잎의 ethyl acetate 분획물에서 tyrosinase 저해활성과 melanin 생성억제활성이높다는연구결과가있다 (Mo and Oh, 2013). 또한입과줄기의 ethyl acetate 분획물에서 DPPH 라디컬소거능이가장높았다. 그러나황칠나무의 Acetylcholinesterase 저해활성은 dichloromethane 분획물에서가장높게조사되었다. Kim et al. (2011) 의연구에서우리나라남해안에자생하는상록활엽수구실잣밤나무의 DPPH 라디컬소거능과 xanthine oxidase 저해활성은 butanol 분획물에서, superoxide 라디컬소거활성은 ethyl acetate 분획물에서우수하였다. 또한 Staphylococcus aureus 외 5종의균억제활성을측정하였을때 ethyl acetate 분획물에서높은항균활성을나타내었다. 양치식물인관중과더부살이고사리는 butanol 분획물에서, 참지네고사리는 ethyl acetate 분획물에서총폴리페놀함량이많았으며, 총플라보노이드함량은관중이 chloroform 분획물에서, 참지네고사리와더부살이고사리는 hexane 분획물에서많았다. 또한 DPPH 라디컬소거활성은관중과더부살이고사리가 butanol 분획물에서, 참지네고사리가 water 분획물에서가장높았다 (Kim et al., 2012). 따라서추출물의활용목적에따라식물종별적정분획용매는다르며, 본실험에서월계수잎의 ethyl acetate 분획물은 P. acnes 의생육억제에가장효과적인것으로판단되었다. 본연구의결과, 연구에사용된녹나무과 10종은모두 P. acnes 에대해유의적인생육억제활성이증명되었으므로합성항균제를대체할천연물유래항균소재로써개발가치가높은것으로생각된다. 특히월계수잎추출물은기존에 P. acnes 억제활성이인정된천연또는합성항균소재에비해서도효과가우수하였기때문에저비용으로높은효과를낼수있는경제적인여드름예방및치료용천연소재로다양하게활용될수있을것으로기대된다. 적요본연구는녹나무과 10종의추출물이 Propionibacterium acnes 의억제활성에미치는영향을분석하여새로운천연항균 소재를개발하기위해수행되었다. 실험재료는완도와제주도에서채집하였으며, 부위 ( 잎, 가지 ), 용매 ( 증류수, 80% 에탄올, 100% 메탄올 ) 및초음파추출시간 (15, 30, 45 분 ) 에따른조추출물의항균활성을 agar diffusion method 로조사하였다. 대조구로는합성항균제인 methylparaben과 phenoxyethanol 0.4, 1, 2, 4 mg /disc 를사용하였다. 연구에사용된 10종의모든추출물에서억제활성을보였으며, 여드름균에대한항균작용을확인할수있었다. 이중월계수는 80% 에탄올에 45분간추출한조건에서 20.2 mm의생육억제환을나타내어가장우수한여드름균에대한항균작용을확인할수있었다. 월계수잎을 100% 메탄올을용매로초음파 30분추출한다음 n-hexane, chloroform, ethyl acetate, n-butanol 그리고 water 순으로용매분획하여항균활성을비교분석하였다. 결과, butanol 분획물을제외한모든분획물에서 10.0 mm이상의억제환이형성되었다. 그중 ethyl acetate 분획물에서억제환이 13.3 mm로가장컸으며, 조추출물 (10.2 mm ) 과대조구인 phenoxyethanol (12.5 mm ) 에비해서도유의적으로우수한억제활성을나타내었다. References Ahn, B.K., E.H. Choi and S. Lee. 2002. The pathogenesis of acne. J.S.B.R. 4:62-70. Bae, H.G. and S.P. Moon. 2001. Structure of evergreen broadleaved forest community at Baegunbong area in Wando. Forest Sci. Technol. 90:756-766. Brown, S.K. and A.R. Shalita. 1998. Acne vulgaris. The Lancet 351:1871-1876. Choi, S.M., M.J. Kim, Y.H. Choi, H.J. Ahn and Y.P. Yun. 1998. Screening of the antibacterial activity of natural products against Propionibacterium acnes. Yakhak Hoeji 42:89-94. Choi, Y.G., Y.S. Kim, G.S. Cho and C.K. Sung. 2002. Screening for antimicrobial activity from Korean plants. J. Korean Soc. Food Nutr. 15:300-306. Eady, E.A., M. Gloor and J.J. Leyden. 2003 Propionibacterium acnes resistance a worldwide problem. Dermatology 206: 54-56. Foran, C.M., E.R. Bennett and W.H. Bebson. 2000. Developmental evaluation of a potential non-steroidal estrogen: triclosan. Mar. Environ. Res. 50:153-156. Ha, Y.M., B.B. Lee, H.J. Bae, K.M. Je, S.R. Kim, J.S. Choi and I.S. Choi. 2009. Anti-microbial activity of grapefruit seed extract and processed sulfur solution against human skin -430-
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