Korean J. Plant Res. 31(4):330-341(2018) https://doi.org/10.7732/kjpr.2018.31.4.330 Print ISSN 1226-3591 Online ISSN 2287-8203 Original Research Article 장보국 1, 지래원 1, 조주성 2, 이철희 1 * 1 충북대학교축산 원예 식품공학부생물건강소재산업화사업단, 2 농우바이오품질관리기술팀 Antimicrobial Activity Screening of Sixty-four Evergreen Woody Species According to Extraction Conditions against Trichophyton mentagrophytes Bo Kook Jang 1, Lai Won Chi 1, Ju Sung Cho 2 and Cheol Hee Lee 1 * 1 Brain Korea 21 Center for Bio-Resource Development, Division of Animal, Horticultural, and Food Sciences, Chungbuk National University, Cheongju 28644, Korea 2 Quality Control & Seed Tech Team, Nongwoo Bio CO., LTD., Yeoju 12655, Korea Abstract - This study was performed to investigate and measure the antimicrobial activity of evergreen woody species extracts on Trichophyton mentagrophytes. To do this, leaves and stems were collected from Wando and Jeju islands, and were used for the extraction with different solvents (i.e., distilled water, 80% ethanol, and 100% methanol), and at different ultrasonic extracting times (i.e., 15, 30, and 45 minutes). The experiment was conducted by using the agar diffusion method. The clear zone was measured after incubating the paper disc containing the plant extract in a bacterial culture medium. The controls were synthetic antimicrobials, methylparaben and phenoxyethanol, at concentrations of 0.4, 1, 2, and 4 mg/disc. Altogether, extracts of 56 out of 64 species used in this study had inhibitory activity, which confirmed their antimicrobial activity against Athlete s foot. Among them, the crude ethanolic extract of Elaeocarpus sylvestris in 45 min showed a zone of inhibition < 20.2 mm, while the clear zone of Actinodaphne lancifolia ethanolic extraction for 30 min was 23.5 mm. Also, Quercus acuta, Dendropanax morbiferus and Daphne odora showed clear zones of 28.0 mm (45 minutes ethanolic extraction), 20.5 mm (45 minutes crude methanolic extraction) and 19.7 mm (45 minutes methanolic extraction), respectively. Thus, these results confirm that the extracts of evergreen woody species have therapeutic potential against Athlete s foot, and suggest that in order to extract adequate amounts of antimicrobial substance from the plant sources, ideal extraction condition has to be considered. Key words - Athlete s foot, Methylparaben, Phenoxyethanol, Pruning product, Ultrasonic extraction 서언 진균 (fungi) 인피부사상균은피부, 머리카락, 손톱및발톱의각질층에침습하여피부진균감염증을유발하며 (Kwon-Chung and Bennett, 1992), Trichophyton mentagrophytes 는표재성진균증의대표적인원인균으로써 (Summerbell, 2011), 두피, 피부및손발톱을통해감염되어무좀을유발한다 (Shin and Lim, * 교신저자 : E-mail leech@chungbuk.ac.kr Tel. +82-43-261-2526 2004). 무좀치료에는항진균제가사용되며스테로이드, 면역억제제, 항생제및합성화학물질로구성되어치료에적합하다. 그러나강한자극성이나잔류독성등다양한부작용을동반하는것으로알려져있으며 (Cunha, 2001), 최근항생제의오남용으로내성균의발생도증가하고있어내성균치료제의개발도대두되고있다 (Shin and Lim, 2004; Lopes et al., 2017). 이러한문제의대안으로식물로부터유래한천연물질소재의탐색이시도되어왔으며, 망고, 월계수및양담쟁이등다양한식물군에서항진균의효과가조사되었다 (Leven et al., c 본학회지의저작권은 ( 사 ) 한국자원식물학회지에있으며, 이의무단전재나복제를금합니다. This is an Open-Access article distributed under the terms of the Creative Commons -330- 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.
1979). 식물에함유된다양한천연물과생리활성물질은합성화학물질에비해부작용이나잔류독성이적은편으로이를약용하고자하는연구가현재까지도활발히진행되고있다 (Nychas, 1995; Iwu et al., 1999; Chandra et al., 2017; Kim et al., 2017). 국내에자생하는상록성목본식물들은다양한페놀화합물과플라보노이드성분을함유하고있으며, 항산화와항균작용을유도하는것으로알려져개발가치가높다 (Kim and Han, 1997; Rim et al., 2000; Yang et al., 2011; Cha et al., 2015). 이와같은상록목본류는지리적환경에적응하여생장하기때문에원산지와다른기능성을나타내며, 채취시기와환경에따른효능도달라지는것으로알려져있다. 또한재료자체의추출방법 (Nostro et al., 2000), 용매의종류및농도에따라서도성분의차이가발생한다 (Sultana et al., 2009). 남부지방상록수종은 1970 년대후반부터정원, 공원, 도시녹지, 가로수, 조경및방풍림등에대규모로식재하여관상수및원예 조경수로많이이용해왔다 (Lee and Kim, 2017). 그러나최근겨울철전정으로버려지는부산물의규모가증가하고있고, 부산물은대부분가지와잎부위이므로이를활용할수있는방안이요구되고있다. 본연구는연중수시로채취가가능하여재료수급이용이하고일부우수한기능성이보고된바있는상록목본식물들의부위별추출물을소재로무좀원인균 (Trichophyton mentagrophytes) 피부질환치료를목적으로하는천연의약소재를개발하기위해수행되었다. 재료및방법식물재료전라남도완도및제주도일대에자생하는 64종상록목본식물의잎과가지를 2013 년 1월에채취하였다 (Table 1). 채취한재료는동결건조기 (Freeze dryer FD8512, Ilshin Bio Base, Korea) 를이용하여 48시간동안동결건조하였다. 건조시료는분쇄기 (Hood mixer FM-681C, Hanil, Korea) 로곱게분쇄한후 -70±2 의 deep freezer (MDF-U73V, Sanyo Electric Co., Ltd., Japan) 에저장하면서실험에사용하였다. 추출방법추출은 ultrasonic cleaner (300 240 145 mm, 5510-DTH, Bransonic, USA) 를이용하여초음파추출하였다. 건조시료 1 g 이충분히잠길정도의용매를유리병에넣어혼합한후아크릴판에부착하였으며, 초음파수조내부에 5 l의증류수를넣어하단으로부터약 9 cm높이로채운후 30분동안추출하였다 (20± 2 ). 모든추출은 1회 2반복으로추출하였다. 추출이끝난후바로여과지 (Advantec No. 2, Toyo Roshi Kaisha Ltd., Japan) 를사용하여 vacuum pump 로감압여과하였으며, 여과된추출물은용매를보충하여최종농도를 50배로정량하였다. 이후추출물을감압농축하여 DMSO 로녹여농도를 50 mg ml -1 로조절하였다. 추출물은 deep freezer에보관하면서실험에사용하였다. 공시균의배양추출물의항균활성을측정하기위해사용된공시균은피부에무좀을유발할수있는 Trichophyton mentagrophytes (KCTC 6316) 균으로한국유전자은행에서분양받았다. 무좀균배양에사용한배지는 sabouraud agar (M11S2103YJ, MB Cell, Korea) 였으며, 균의활성을유지하고자 3 5 주간격으로계대배양하였으며, 실험 2주전, 3 5 일간격으로 10 ml agar broth 에무좀균배양액 100 μl를활성화시켜사용하였다. 한천확산법 (agar diffusion assay) Shin and Lee (2010) 의 agar diffusion assay을응용하여실험을수행하였다. 실험전 visible spectrophotometer (Novaspec Plus, Biochrom, England) 를이용하여무좀균배양액의 O.D. 값을 1.0으로조정하였으며, 접종을위해 0.7% agar가첨가된 soft agar broth 배지를멸균후항온수조에서 60 로유지하였다. 준비된 soft agar broth에무좀균배양액을 1% v/v 수준으로첨가후교반하였으며, 멸균된페트리디쉬에 12 ml씩분주후무균작업대에서굳혔다. 그후페이퍼디스크 (8 mm, Advantec Toyo Roshi International Inc., Japan) 에추출물을 2 mg /disc (40 μl ) 농도로주입하였으며, 추출물내용매가충분히휘발되도록건조하였다. 배지내추출물의침투가용이하도록 4 냉장고에서 1시간동안배양한후, 37 의생장상으로옮겨 24시간동안배양하였다. 배양후페이퍼디스크의직경을포함한생육억제환을측정하였으며, 추출물의항균활성을비교하기위하여합성항균제로사용되는 phenoxyethanol (0H2108, Junsei Chemical Co., Ltd., Japan) 과 methylparaben (8K5015, Junsei Chemical Co., Ltd., Japan) 을 0.4, 1, 2, 4 mg /disc 농도로희석하여무좀원인균의생육억제정도를비교하였다. -331-
Korean J. Plant Res. 31(4) : 330~341(2018) Table 1. List of plants used in this study and antimicrobial activities of extracts obtained from leaves of 64 species against Trichophyton mentagrophytes Family name Scientific name Clear zone ( mm ) Family name Scientific name Clear zone ( mm ) Control (mg/disc) y Phenoxyethanol 0.2 - x Gramineae Phyllostachys pubescens 12.67 ± 0.23 f 0.4 - Sasa palmata - 2.0 13.57 ± 0.76 ef w Hamamelidaceae Distylium racemosum 12.60 ± 1.14 g 4.0 14.17 ± 0.73 e Illiciaceae llicium anisatum 14.50 ± 0.46 e Methylparaben 0.2 - Lardizabalaceae Stauntonia hexaphylla 8.63 ± 4.32 h 0.4 - Lauraceae Actinodaphne lancifolia 22.33 ± 0.62 a 2.0 14.23 ± 0.32 e Cinnamomum loureirii 14.67 ± 1.12 e 4.0 21.40 ± 1.45 ab Cinnamomum yabunikkei 11.37 ± 0.38 fg Apocynaceae Nerium oleander - Cinnamomum camphora 12.13 ± 0.88 f Trachelospermum asiaticum var. - Laurus nobilis 14.63 ± 0.38 e Trachelospermum asiaticum - Litsea japonica 11.70 ± 0.38 f Aquifoliaceae Ilex cornuta 11.43 ± 0.44 fg Machilus japonica 18.27 ± 0.13 bc Ilexⅹwandoensis 12.43 ± 0.75 f Machilus thunbergii 11.00 ± 0.25 g Ilex crenata 12.03 ± 0.43 f Neolitsea aciculata 13.87 ± 0.28 ef Ilex rotunda - Neolitsea sericea 17.13 ± 0.03 c Araliaceae Dendropanax morbiferus 22.60 ± 0.57 a Loganiaceae Gardneria insularis - Fatsia japonica 15.80 ± 1.21 d Magnoliaceae Magnolia grandiflora 20.10 ± 0.49 b Hedera rhombea 12.77 ± 0.61 f Moraceae Ficus oxyphylla 11.47 ± 0.32 fg Caprifoliaceae Viburnum odoratissimum 15.37 ± 0.15 d Myricaceae Myrica rubra 14.10 ± 2.00 e Cephalotaxaceae Cephalotaxus koreana - Myrsinaceae Ardisia crenata 10.00 ± 5.72 h Cupressaceae Chamaecyparis obtusa 17.73 ± 3.38 c Oleaceae Ligustrum lucidum 14.40 ± 0.76 e Juniperus rigida 15.77 ± 0.37 d Osmanthus fragrans - Daphniphyllaceae Daphniphyllum macropodum 10.63 ± 0.79 g Osmanthus fragrans var. 9.50 ± 0.30 h Elaeagnaceae Elaeagnus glabra 10.10 ± 0.55 h Pinaceae Pinus thunbergii 13.77 ± 0.74 ef Elaeagnus umbellata 9.30 ± 0.26 h Rosaceae Eriobotrya japonica 13.93 ± 0.47 e Elaeocarpaceae Elaeocarpus sylvestris 20.20 ± 1.01 b Raphiolepis indica 12.87 ± 0.29 f Ericaceae Rhododendron brachycarpum 13.47 ± 0.99 ef Rutaceae Citrus junos - Vaccinium bracteatum - Schisandraceae Kadsura japonica 10.33 ± 0.29 g Vaccinium oxycoccus - Taxaceae Torreya nucifera 13.97 ± 0.24 e Fagaceae Castanopsis sieboldii 17.83 ± 1.19 c Taxodiaceae Cryptomeria japonica 16.47 ± 0.87 cd Quercus acuta 22.27 ± 0.49 a Theaceae Camellia japonica 12.27 ± 0.27 f Quercus gilva 15.13 ± 2.02 d Cleyera japonica 19.27 ± 1.83 bc Quercus glauca 10.50 ± 0.40 g Eurya emarginata - Quercus myrsinifolia 11.33 ± 0.91 g Eurya japonica - Quercus salicina 10.50 ± 0.90 g Ternstroemia gymnanthera 13.80 ± 0.65 ef Flacourtiaceae Xylosma congesta. 8.93 ± 4.47 h Thymelaeaceae Daphne odora 22.13 ± 1.05 a z EN; Evergreen Needle-leaved, EB; Evergreen Broad-leaved, EC; Evergreen Climber. y Positive control as a synthetic antiseptic. x Not detected. w Means ± S.E. (n=10) and separation. within columns by Duncan s multiple range test, p<0.05. 상록성목본 64종의항균활성스크리닝상록성목본 64종의항균활성정도를확인하고자, 상록성목본 64종의잎동결건조시료 1 g과 100% 메탄올 (Merck, Germany) 을용매로 30분동안초음파추출하였다. 64종의잎조추출물은 agar diffusion assay 를이용하여 2 mg /disc 농도로 항균활성을조사하였다. 추출조건에따른항균활성측정잎조추출물의스크리닝에서우수한항균활성이확인된시료 5종을선발하여실험에사용하였다 (Table 2-6). 추출부위, -332-
용매및시간에따른항균활성정도를구명하고자, 잎과가지부위를시료, 증류수 (Deionized water; nano pure grade), 80% 에탄올 (Ethanol, Jin Chemical Parmaceut., Korea), 100% 메탄올을용매로각 15, 30 및 45분동안초음파추출하였다. 스크리닝실험과동일하게 agar diffusion assay를이용하여 2 mg / disc 농도로항균활성을조사하였다. 조사및통계분석 Agar diffusion assay 를이용한스크리닝은 3반복수행하였으며, 추출물의농도및배양시간에따른추가실험은 6반복을 1회로 2회반복하였다. 모든결과값은평균과표준오차범위를구하고 SAS version 9.3 (SAS Institute Inc., USA) 의 Duncan s multiple range test 를이용하여 p<0.05 수준에서유의성을검정하였으며, 추출조건의요인간유의성검정을실시하였다. 결과및고찰항생제는균을포함하는미생물의생장을억제하거나, 사멸하는역할을하며, 20 세기처음발견된이래로전인류의건강과의학에혁신을불러왔다. 그러나무분별한항생제의오남용과미생물이항생물질에적응하기위해내성을가지게되면서일부균이나미생물에는효과가나타내지않아문제가발생되고있다. 이와같은문제를해결하기위한대안으로많은연구자들은식물의천연물질에관심을가지게되었으며, 지속적으로연구가진행되고있다. 우리의연구는상록목본식물로부터유래된추출물을이용하여균의생장을억제하거나, 사멸하는작용을하는식물대상을탐색하고동일분야의기초자료로제공하고자한다. 이연구에사용된 64종의잎추출물중황칠나무등 56종의처리구에서생육억제환이형성되어공시균으로사용된 T. mentagrophytes ( 무좀원인균 ) 에대한항균효과를확인할수있었다 (Table 1). 그중황칠나무 (22.60 mm ), 육박나무 (22.33 mm ), 붉가시나무 (22.27 mm ), 서향 (22.13 mm ) 등 4종의잎추출물은대조구로사용된 methylparaben(21.40 mm ) 보다우수한생육억제활성을보였으며, 담팔수 (20.20 mm ) 등 15종의잎추출물의경우, phenoxyethanol (14.17 mm ) 에비해높은억제활성을나타냈다. 상록성목본식물은종에따라무좀원인균의생육억제정도가달랐지만, 다수의종에서항균활성여부를확인할수있었으며, 천연항균제로써이용가능성을제시한결과라고생각되었다. 식물체에존재하는생리활성물질을활용하기위해서는추출물의형태로사용하는것이효율적이다. 물, 에탄올, 메탄올은식물체에서유효성분을추출하기위한용매로가장많이사용되며, 물은수용성생리활성물질인페놀성화합물을용출시킨다 (Cha et al., 2009). Cowan (1999) 에따르면, 식물로부터추출된물질중미생물에대한활성이확인된물질들은방향족화합물또는유기화합물로에탄올과메탄올에쉽게용출된다. 또한추출용매의비율에따라미생물의억제활성도다르다고알려져있다. 차조기의경우, 에탄올비율 (0, 30, 50, 70, 95%) 에따라 Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa 균주의억제활성이달랐으며 (Kim et al., 2007), 80% 에탄올을용매로복분자, 가자육, 오배자등 62종의약용식물을추출한결과에서다양한미생물의억제활성을보고한바있다 (Lee et al., 2002). 선행연구에서무좀원인균의억제활성이우수하였던, 황칠나무 (Dendropanax morbiferus), 육박나무 (Actinodaphne lancifolia), 붉가시나무 (Quercus acuta), 서향 (Daphne odora), 담팔수 (Elaeocarpus sylvestris) 5종을선발하여, 부위 ( 잎, 가지 ), 추출용매 (100% 메탄올, 80% 에탄올, 증류수 ) 및초음파추출시간 (15, 30, 45분 ) 을달리하여추출조건의영향을조사하고자하였다. 담팔수의 T. mentagrophytes 억제는부위에따라항균활성이달랐으며, 가지보다잎부위에서비교적우수한결과를나타냈다 (Table 2). 대조구로사용된 phenoxyethanol (14.90 mm ) 과의비교에서도가지보다잎부위가대조구와유사한효과를나타내는것으로조사되었다. 그중, 에탄올을용매로 45분동안초음파추출했을때, 생육억제환이 20.2 mm로가장많은억제율을나타냈으며, 높은통계적유의성을보인부위조건을제외한용매, 시간별조건에서는뚜렷한차이를확인할수없었다. 담팔수잎추출물에는 PGG, geraniin, elaeocarpusin, ellagitannin 등의물질들이존재하며 (Tanaka et al., 1986), 항종양, 항염증, 항균, 항산화활성및혈관확장에강력한효과를보인다 (Piao et al., 2009; Perera et al., 2015). Lee et al. (2004) 에따르면, 담팔수추출물은유해미생물 Helicobacter pylori를억제하는생육억제환 (20.0 mm ) 을형성하는것으로보고되었다. 연구에사용된 T. mentagrophytes도유사한억제력을나타냈으며, 담팔수잎에함유된화합물들에의한시너지효과로추정되었다. 각물질별효과에대해서는추가적인연구가요구된다. 서향의추출물도가지보다잎추출물에서높은생육억제활성이조사되었다. 메탄올 (19.7 mm ) 과에탄올 (19.2 mm ) 을 45분간추출한조건이가장효과적이었다 (Table 3). 가지추출물의경 -333-
Korean J. Plant Res. 31(4) : 330~341(2018) Table 2. Antimicrobial activities of extracts obtained from leaves and stems of Elaeocarpus sylvestris var. ellipticus (Thunb.) H. Hara against Trichophyton mentagrophytes according to extraction methods 0.4 Part Solvent Extraction time (min.) Clear zone ( mm ) Control Methylparaben - y (mg/disc) z 1.0-2.0 14.1 ± 0.24 c-e x 4.0 21.1 ± 0.19 a Phenoxyethanol 0.4-1.0-2.0 14.3 ± 0.53 c-e 4.0 14.9 ± 0.55 c-e Leaf MeOH 100% 15 15.9 ± 0.77 b-d 30 15.4 ± 0.13 c-e 45 15.6 ± 0.67 b-d EtOH 80% 15 18.5 ± 0.50 a-c 30 17.3 ± 0.40 a-d 45 20.2 ± 0.10 ab Distilled water 15-30 - 45 - Stem MeOH 100% 15 14.2 ± 0.12 c-e 30 13.0 ± 0.38 de 45 10.7 ± 2.00 e EtOH 80% 15 14.7 ± 0.47 c-e 30 12.6 ± 0.28 de 45 13.0 ± 0.65 de Distilled water 15-30 - 45 - P (Part) *** w S (Solvent) T (Time) P S P T T S P T S z Positive control as a synthetic antiseptic. y Not detected. x Means ± S.E. (n=10) and separation. within columns by Duncan s multiple range test, p<0.05. w, *, **, ***; nonsignificant or significant at p<0.05, 0.01 or 0.001, respectively. -334-
Table 3. Antimicrobial activities of extracts obtained from leaves and stems of Daphne odora Thunb. against Trichophyton mentagrophytes according to extraction methods 0.4 Part Solvent Extraction time (min.) Clear zone ( mm ) Control Methylparaben - y (mg/disc) z 1.0-2.0 14.1 ± 0.24 cd x 4.0 21.1 ± 0.19 a Phenoxyethanol 0.4-1.0-2.0 14.3 ± 0.53 cd 4.0 14.9 ± 0.55 cd Leaf MeOH 100% 15 12.5 ± 0.17 d 30 12.2 ± 0.99 d 45 19.7 ± 0.75 ab EtOH 80% 15 16.7 ± 0.60 bc 30 15.0 ± 0.58 cd 45 19.2 ± 0.23 ab Distilled water 15-30 - 45 - Stem MeOH 100% 15 13.3 ± 0.95 cd 30 12.2 ± 0.50 d 45 16.2 ± 0.45 b-d EtOH 80% 15 14.0 ± 0.85 cd 30 15.0 ± 0.35 cd 45 14.9 ± 0.55 cd Distilled water 15-30 - 45 - P (Part) w S (Solvent) T (Time) * P S P T T S P T S z Positive control as a synthetic antiseptic. y Not detected. x Means ± S.E. (n=10) and separation. within columns by Duncan s multiple range test, p<0.05. w, *, **, ***; nonsignificant or significant at p<0.05, 0.01 or 0.001, respectively. -335-
Korean J. Plant Res. 31(4) : 330~341(2018) Table 4. Antimicrobial activities of extracts obtained from leaves and stems of Actinodaphne lancifolia (Sibold & Zucc.) Meisn. against Trichophyton mentagrophytes according to extraction methods 0.4 Part Solvent Extraction time (min.) Clear zone ( mm ) Control Methylparaben - y (mg/disc) z 1.0-2.0 14.1 ± 0.24 cd x 4.0 21.1 ± 0.19 b Phenoxyethanol 0.4-1.0-2.0 14.3 ± 0.53 cd 4.0 14.9 ± 0.55 c Leaf MeOH 100% 15 20.0 ± 0.29 b 30 21.9 ± 0.01 ab 45 20.8 ± 0.72 b EtOH 80% 15 12.2 ± 0.23 d 30 23.5 ± 1.99 a 45 22.2 ± 0.72 ab Distilled water 15-30 - 45 - Stem MeOH 100% 15 13.3 ± 0.03 cd 30 14.0 ± 0.06 cd 45 12.2 ± 0.29 d EtOH 80% 15 14.0 ± 0.62 cd 30 15.0 ± 1.28 c 45 15.0 ± 0.31 c Distilled water 15-30 - 45 - P (Part) *** w S (Solvent) T (Time) *** P S ** P T *** T S *** P T S ** z Positive control as a synthetic antiseptic. y Not detected. x Means ± S.E. (n=10) and separation. within columns by Duncan s multiple range test, p<0.05. w, *, **, ***; nonsignificant or significant at p<0.05, 0.01 or 0.001, respectively. -336-
Table 5. Antimicrobial activities of extracts obtained from leaves and stems of Quercus acuta Thunb. against Trichophyton mentagrophytes according to extraction methods 0.4 Part Solvent Extraction time (min.) Clear zone ( mm ) Control Methylparaben - y (mg/disc) z 1.0-2.0 14.1 ± 0.24 fg x 4.0 21.1 ± 0.19 bc Phenoxyethanol 0.4-1.0-2.0 14.3 ± 0.53 fg 4.0 14.9 ± 0.55 fg Leaf MeOH 100% 15 18.5 ± 0.32 c-f 30 23.7 ± 0.27 ab 45 20.5 ± 0.10 b-d EtOH 80% 15 20.5 ± 0.63 b-e 30 26.2 ± 0.83 a 45 28.0 ± 0.87 a Distilled water 15 17.4 ± 0.90 c-g 30 26.0 ± 0.27 a 45 21.4 ± 0.10 bc Stem MeOH 100% 15 14.5 ± 0.33 fg 30 16.2 ± 0.50 d-g 45 14.0 ± 0.50 fg EtOH 80% 15 15.6 ± 0.79 fg 30 14.9 ± 0.90 fg 45 15.0 ± 0.40 fg Distilled water 15 13.6 ± 0.43 g 30 17.2 ± 0.38 c-g 45 15.9 ± 0.32 e-g P (Part) *** w S (Solvent) T (Time) *** P S P T T S P T S z Positive control as a synthetic antiseptic. y Not detected. x Means ± S.E. (n=10) and separation. within columns by Duncan s multiple range test, p<0.05. w, *, **, ***; nonsignificant or significant at p<0.05, 0.01 or 0.001, respectively. -337-
Korean J. Plant Res. 31(4) : 330~341(2018) Table 6. Antimicrobial activities of extracts obtained from leaves and stems of Dendropanax morbiferus H.Lév. against Trichophyton mentagrophytes according to extraction methods 0.4 Part Solvent Extraction time (min.) Clear zone ( mm ) Control Methylparaben - y (mg/disc) z 1.0-2.0 14.1 ± 0.24 f-h x 4.0 21.1 ± 0.19 a Phenoxyethanol 0.4-1.0-2.0 14.3 ± 0.53 cd 4.0 14.9 ± 0.55 c Leaf MeOH 100% 15 18.2 ± 0.00 a-f 30 16.2 ± 0.00 c-h 45 20.5 ± 0.00 ab EtOH 80% 15 15.0 ± 0.00 f-h 30 14.9 ± 0.00 f-h 45 19.7 ± 0.00 a-d Distilled water 15 15.9 ± 0.00 d-g 30 17.2 ± 0.00 a-g 45 19.3 ± 0.00 a-e Stem MeOH 100% 15 14.0 ± 0.00 gh 30 15.6 ± 0.00 e-h 45 12.5 ± 0.00 h EtOH 80% 15 15.0 ± 0.00 f-h 30 20.2 ± 0.00 a-c 45 16.7 ± 0.00 b-g Distilled water 15 15.9 ± 0.00 d-h 30 16.4 ± 0.00 c-h 45 20.1 ± 0.00 a-c P (Part) w S (Solvent) T (Time) ** P S ** P T * T S P T S z Positive control as a synthetic antiseptic. y Not detected. x Means ± S.E. (n=10) and separation. within columns by Duncan s multiple range test, p<0.05. w, *, **, ***; nonsignificant or significant at p<0.05, 0.01 or 0.001, respectively. -338-
우, 메탄올 45분추출조건 (16.2 mm ) 에서비교적우수하였다. 육박나무의무좀원인균억제활성은부위와시간에따라항균력이달랐으며, 특히부위별처리조건에서뚜렷한차이를확인할수있었다. 잎시료를메탄올 (21.9 mm ) 과에탄올 (23.5 mm ) 로 30분간추출하였을때, 가장높은생육억제환이형성되었다 (Table 4). 가지는잎보다활성이저조하였지만, 에탄올을용매로 30분이상추출시대조구로사용된 phenoxyethanol 과유사한억제력을나타냈다. 한편담팔수, 서향및육박나무는증류수를용매로추출하였을때, 시간과부위에관계없이생육억제환이형성되지않아항균활성을확인할수없었다. 식물의추출물은종별로도성분의차이가크지만, 추출용매및방법에따라용출되는성분이달라지고용출된물질도다양하다고알려져있다 (Wang and Weller, 2006; Dai and Mumper, 2010). 최근보고에따르면, 수용성용매보다유기용매 (alcoholic, chloroformic acid and hexane) 를포함한추출물이더강력한피부사상균억제활성을나타냈다 (Soares et al., 2013; Iqbal et al., 2015; Lopes et al., 2017). 이는무좀원인균에대한항균활성을가진물질이담팔수, 서향및육박나무내에서비극성으로존재한다고추정할수있었다. 붉가시나무는부위, 용매및추출시간에관계없이모든처리구에서무좀원인균에대한생육억제활성을확인할수있었으며, 대조구인 phenoxyethanol과의비교에서도높은활성을나타냈다 (Table 5). 잎추출물중메탄올 30분 (23.70 mm ), 에탄올 30, 45분 (26.2, 28,00 mm ) 및증류수 30분 (26.00 mm ) 처리는다른대조구인 methylparaben (21.10 mm ) 보다높은생육억제환이조사되었다. 또한추출시간에따라생육억제활성이상이하였는데, 15분과 30분추출조건에서의차이가가장컸다. 통계적으로도부위와시간별처리조건이뚜렷한영향을미치는것으로조사되었다. 그러나 45분추출조건에서는감소하거나비슷한수준으로유지되어추출효율이극대화되는시점이존재함을추정할수있었다. 참나무과에속하는붉가시나무는강력한항산화및항염증활성을보이는페놀화합물을가지고있으며 (Oh et al., 2014), Bacillus brevis, B. coagulans, B. stearothermophilus, Escherichia coli B, Pseudomonas aeruginosa 등의미생물에대한억제활성도조사되었다 (Serti et al., 1991). 또한동일속에 Quercus acutissima Carruth., Q. salicina Blume, Q. serrata Roxb. 는항알러지에효과를나타내며 (Bak et al., 2011), 붉가시나무의추출물은미생물억제에영향을미치는물질이다수함유되어있는것으로추정된다. 황칠나무시료는부위, 용매및시간별처리조건에관계없이 모두무좀원인균에대한생육억제환이형성되어항균력이우수한종으로조사되었다. 부위및용매별항균력의차이는없었지만, 추출시간에따른차이는존재하는것으로확인되었다. 잎시료를세가지용매로 45분간초음파추출하였을때, 각각메탄올 (20.50 mm ), 에탄올 (19.70 mm ), 증류수 (19.30 mm ) 로가장높은생육억제환이조사되었으며, 가지의경우, 에탄올 30분 (20.20 mm ) 과증류수 45분 (20.10 mm ) 추출조건에서항균력이우수하였다 (Table 6). 대조구인 phenoxyethanol (14.90 mm ) 보다높은항균효과를나타냈으며, 또다른대조구인 methylparaben (21.10 mm ) 과도유사한억제력을보이는것으로조사되었다. 황칠나무는다양한페놀및플라보노이드화합물들이함유되어있으며, 항암, 항산화, 미백, 당뇨및간세포재생에효과를보인다 (Park and Han 2016). Lee et al. (2015a) 에따르면, 황칠나무잎의에탄올추출물은식중독, 비듬, 충치원인균의증식을억제하는것으로조사되었으며, n-hexane 분획물에서분리한 1-tetradecanol 과 B-sitosterol 은피부개선및탈모방지에효과를나타내는것으로보고되었다 (Lee et al., 2015b). 황칠나무도무좀원인균에억제효과를보이는다양한화합물이존재하는것으로생각된다. 이러한결과로미루어볼때, 상록목본식물의잎과가지를메탄올, 에탄올및증류수로초음파추출한조추출물은 T. mentagrophytes 에대한억제활성을가지는것으로조사되었다. 특히담팔수, 서향, 육박나무, 붉가시나무및황칠나무는가장높은항균활성을나타내어피부질환개선및치료제로의개발가능성을제시하였다. 차후실제치료제개발을위해서는무좀원인균을억제하는물질의분리및동정이필요할것으로생각된다. 적요본연구는무좀균 (Trichophyton mentagrophytes) 에대한상록성목본추출물의항균활성을조사하고측정하기위해수행되었다. 이를위해완도와제주도에서채집한잎과줄기를용매 ( 증류수, 80% 에탄올, 100% 메탄올 ) 와초음파처리시간 (15, 30, 45분 ) 을달리하여추출하여실험에사용하였다. 실험은한천확산법을사용하여, 박테리아배지에식물추출물이함유된종이디스크를배양한뒤클리어존 ( 생육억제환 ) 을측정하였다. 대조군은합성항균제인 methylparaben 과 phenoxyethanol 0.4, 1, 2, 4 mg /disc 에농도로사용하였다. 연구에사용된 64종중 56 종의추출물에서클리어존이보여, 무좀균에대한항균작용을 -339-
Korean J. Plant Res. 31(4) : 330~341(2018) 확인할수있었다. 그중담팔수는에탄올에 45분간추출한처리구에서 20.2 mm, 육박나무는 80% 에탄올로 30분간추출한처리구에서 23.5 mm의클리어존을나타냈다. 또한붉가시나무, 황칠나무및서향의잎추출물은각각 28.0 mm (80% 에탄올 45분추출 ), 20.5 mm (100% 메탄올 45분추출 ) 및 19.7 mm (100% 메탄올 45분추출 ) 의클리어존이조사되었다. 따라서, 이러한결과는상록성목본추출물의무좀균에대한치료가능성을확인할수있었으며, 항균물질이많이함유한식물소재를얻기위해서는식물의적정추출조건을고려해야할것으로생각된다. References Bak, J.P., J.B. Kim, J.H. Park, Y.J. Yang, I.S. Kim, E.S. Choung and S.C. Kang. 2011. Screening and compound isolation from natural plants for anti-allergic activity. Appl. Biol. Chem. 54:367-375. Cha, J.D., M.R. Choi, E.S. Ko, S.M. Hwang, J.R. Kang, J.S. Oh, Y.J. Park, Y.H. Jung, A.L. Jeon and K.M. Choi. 2015. Synergistic effects of Cinnamomum camphora leaves extract against clinical isolated methicillin-resistant Staphylococcus aureus. Korean J. Plant Res. 28:9-15 (in Korean). Cha, W.S., I.S. Ju, D.H. Yun, S.S. Chun, J.H. Kim and Y.J. Cho. 2009. Biological activity of extracts from cherry sage (Salvia officinalis L.). Korean J. Life Sci. 19:390-396 (in Korean). Chandra, H., P. Bishnoi, A. Yadav, B. Patni, A.P. Mishra and A.R. Nautiyal. 2017. Antimicrobial resistance and the alternative resources with special emphasis on plant-based antimicrobials-a review. Plants 6:16. Cowan, M.M. 1999. Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12:564-582. Cunha, B.A. 2001. Antibiotic side effects. Medical Clinics 85:149-185. Dai, J. and R.J. Mumper. 2010. Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules 15:7313-7352. Iqbal, J., R.P. Mishra and A.H. Allie. 2015. Antidermatophytic activity of angiospermic plants: A review. Asian J. Pharm. Clin. Res. 8:75-80. Iwu, M.W., A.R. Duncan and C.O. Okunji. 1999. New antimicrobials of plant origin: In Janick, J (ed.), Perspectives on New Crops and New Uses, ASHS Press, Alexandria, VA (USA). pp. 457-462. Kim, M.H., N.H. Lee, M.H. Lee, D.J. Kwon and U.K. Choi. 2007. Antimicrobial activity of aqueous ethanol extracts of Perilla frutescens var. acuta leaf. Korean J. Food Culture. 22:266-273 (in Korean). Kim, S.I. and Y.S. Han. 1997. Isolation and identification of antimicrobial compound from Sancho (Zanthoxylum schinifolium). Korean J. Soc. Food. Sci. 13:56-63 (in Korean). Kim, S.M., J.H. Park, H.O. Boo, S.G. Song and H.Y. Park. 2017. In vitro comparision of biological activities of solvent fraction extracts from Orostachys japonicus. Korean J. Plant Res. 30:133-143 (in Korean). Kwon-Chung, K.J. and J.E. Bennett. 1992. Medical Mycology. Lea and Febiger. Philadelphia, Pennsylvania (USA). pp. 866-866. Lee, H.K., H.B. Lee, C.S. Kim and Y.J. Ahn. 2004. Anti- Helicobacter pylori activity of methanol extracts from Korean native plant species in Jeju island. J. Korean Soc. Appl. Biol. Chem. 47:91-96. Lee, K.W. and D.G. Kim. 2017. Freezing injury of evergreen broad-leaved trees in warm-temperature in the southern region in Korea. J. Korean Env. Res. Tech. 20:77-96 (in Korean). Lee, S.G., S.H. Lee and E.J. Park. 2015a. Antimicrobial and antioxidant activities of ethanol leaf extract of Dendropanax morbiferus Lev. Korean J. Food Cook. Sci. 31:515-523 (in Korean). Lee, S.Y., E.J. Choi, D.H. Bae, D.W. Lee and S.O. Kim. 2015b. Effects of 1-tetradecanol and β-sitosterol isolated from Dendropanax morbifera Lev. on skin whitening, moisturizing and preventing hair loss. J. Soc. Cosmet. Sci. Korea 41: 73-83 (in Korean). Lee, Y.C., S.W. Oh and H.D. Hong. 2002. Antimicrobial characteristics of edible medicinal herbs extracts. Korean J. Food Sci. Technol. 34:700-709 (in Korean). Leven, M., D.A.V. Berghe, F. Mertens, A. Vlietinck and E. Lammens. 1979. Screening of higher plants for biological activities I. antimicrobial activity. Planta Medica 36:311-321. Lopes, G., E. Pinto and L. Salgueiro. 2017. Natural products: an alternative to conventional therapy for dermatophytosis? Mycopathologia 182:143-167. Nostro, A., M.P. Germanò, V. D Angelo, A. Marino and M.A. Cannatelli. 2000. Extraction methods and bioautography for evaluation of medicinal plant antimicrobial activity. Lett. Appl. Microbiol. 30:379-384. Nychas, G.J.E. 1995. Natural antimicrobials from plants: In Gould, G.W. (ed.), New Methods of Food Preservation, Springer, Boston, MA (USA). pp. 58-89. -340-
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