Journal of Life Science 2019 Vol. 29. No ~1392 ISSN (Print) ISSN (Online) DOI :

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1 Journal of Life Science 2019 Vol. 29. No ~1392 ISSN (Print) ISSN (Online) DOI : Antibacterial and Antibiofilm Activities of Alnus japonica Stem Extract against Porphyromonas gingivalis Hye Soo Kim and Soo Jeong Cho* Department of Pharmaceutical Engineering, Gyeongnam National University of Science and Technology, 33 Dongjinro, Jinju 52725, Korea Received November 12, 2019 /Revised November 29, 2019 /Accepted December 2, 2019 This study investigated the potential of dye plants as natural oral health products. The antibacterial activity of ethanol stem extracts of A. japonica, R. verniciflua Stokes, G. jasminoides, D. morbifera, P. amurense Rupr., and S. japonica against P. gingivalis KCTC 5352, S. mutans KCTC3065, S. downei KCTC3634, S. sanguinis KCTC3284, and S. gordonii KCTC 3286 was confirmed. Among the stem extracts from 6 dye plants grown in Korea, ethanol extract from A. japonica stem (1 mg/disc) showed the highest antibacterial activity against P. gingivalis KCTC5352. The A. japonica stem extracts showed antibacterial activity similar to chlorhexidine, which was used as a positive control. The MIC and MBC of P. gingivalis KCTC5352 were 0.4 mg/ml and 0.6 mg/ml, respectively. The biofilm production rate and cell growth of P. gingivalis KCTC5352 in the cultures treated with mg/ml of A. japonica extract were significantly decreased in a concentrationdependent manner. In addition, the mrna expression of the superoxide dismutase and fima associated with fimbriae formation in these cultures was suppressed, also in a concentrationdependent manner. Based on these results, it is concluded that A. japonica stem extracts can be used as an oral health product derived from natural materials, as demonstrated by its antibacterial action against and inhibition of biofilm formation of P. gingivalis KCTC5352. Key words : Alnus japonica stem, antibacterial activity, biofilm formation, fima, P. gingivalis KCTC5352 서 구강건강과관련된질환중치아우식증과치주질환은아동 에서부터성인, 노인에이르기까지다양한연령층에서발병되 는구강질환으로한번손상이발생되면영구손상으로남는 경향이높아생애전주기에걸쳐삶의질을떨어뜨리는원인 이되고있다 [19]. 치아우식증과치주질환은구강내존재하는 세균에의해발병되는세균감염성질환이며구강세균은음식 물섭취를통해영양분을공급받고세균밀도가높아지면치 아표면에치면세균막 (dental biofilm) 이라불리는세균막 (biofilm) 을형성하게된다. 치면세균막은치아표면에부착된 당단백질 (glycoprotein) 성분의얇은막에약 700 여종이상의 세균들이부착하여형성된점착성세균막이다 [16]. 구강내치 면세균막은물리적으로두꺼운층을형성하기때문에외부 물질의침투가어렵고, 세포간상호작용을통해유전적변이 가일어나기때문에부유세균에비해항균물질에대한저항 *Corresponding author *Tel : , Fax : * sjcho@gntech.ac.kr This is an OpenAccess article distributed under the terms of the Creative Commons Attribution NonCommercial License ( which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 론 성이높다 [24, 25]. 치면세균막이적절하게제거되지못하면치면에침착하게되고시간이지나면서치석으로변하게된다. 치석은계속방치하게되면치은출혈, 부종, 치조골흡수로인한치아흔들림이발생될수있고치아우식증과치주질환의원인이된다 [10]. 치주질환은치아주변조직인치은, 치조골, 치주인대및백악질에염증이발생하는질환으로치아소실의주요한원인이며동맥경화증과같은심혈관질환뿐만아니라폐혈증, 유산, 조산, 폐렴및당뇨병과도밀접한관련이있는것으로보고되었다. 치주질환과관련된세균들은주로치은연하치면세균막에존재하는그람음성혐기성세균으로 P. gingivalis가대표적이다 [5, 8]. P. gingivalis는콜라겐을분해하고암모니아, 황화수소, 아민, 리포폴리사카라이드등과같은독소를분비하여치주조직에직접적인영향을미친다 [9, 18, 23]. 또한, P. gingivalis의대사산물들은생체면역계를자극하고자극된체액성및세포성면역계는여러작용에의해활성산소, 인터루킨과같은사이토카인을분비하여잇몸염증을유발하기도한다 [9]. 그러므로치주질환을예방하고치료하는데있어치면세균막을제거하는것은필수적이다. 치면세균막을제거하는가장좋은방법은올바른잇솔질이지만잇솔질로는치아교합면에존재하는세균막이나치아인접면에있는세균막까지는제거하기어렵다. 이러한단점을보완하기위해치실이나항균제가첨가된구강케어제품을사용하고있다. 현재구강용항균제로는 bisbiguanides 제제 (chlorhexidine), 4급암모늄제제 (cetylpyridiym, benzalkonium chloride), 페놀화

2 Journal of Life Science 2019, Vol. 29. No 합물 (triclosan) 이많이사용되고있으나이들화합물유래항균제는장기간사용시치아나연조직에착색이유발되고미각변화가능성등이보고되고있어서이를대체할수있는천연물유래구강건강소재개발에관한연구가주목을받고있다 [9, 11, 13]. 최근들어오배자추출물 [29], 편백 [14], 몰약 [2], 라타니아 [2], 카모밀레 [2], 인도감나무줄기추출물 [13] 등이 P. gingivalis에대해항균작용이있음이보고되면서천연물로부터인체에무해하고구강병원미생물에대해서는항균활성이우수한구강건강소재개발에관한연구가활발히이루어지고있다 [4]. 오리나무 (Alnus japonica) 는자작나무과 (Betulaceae) 에속하는낙엽교목으로우리나라중부이북의해발 m 지역에주로분포하고있으며꽃은취산화서이다. 한방에서는오리나무수피를적양이라고부르며해열, 지혈, 수렴등의효능이있어오래전부터장염, 설사, 외상출혈, 혈변등의치료에사용해왔고민간에서는숙취해소에사용하였다 [1]. 오리나무에는 lupenon, βamylin, glutenol, taraxerol, betulinic acid 등여러종류의 triterpenoid 외에 βsitosterol, heptacosane, 지방족 alcohol, pyrocatechol 계열 tannin과 pillioin, salvigenin 및 5hydroxy4, 7dimethoxyflavone 등의 flavonoids 화합물이포함되어있으며 [3, 12, 17, 21, 26, 32] 오리나무의항암효과 [30], 항산화효과 [15, 22], 항염효과 [20], 간보호효과 [19] 등에관한생리활성연구가보고되었지만구강미생물에대한오리나무줄기추출물의항균활성에관한연구는미비한실정이다. 본연구에서는구강미생물인 P. gingivalis에대한오리나무줄기추출물의항균활성과바이오필름생성억제효과를조사하여천연물유래구강건강소재로써오리나무줄기추출물의이용가능성을알아보고자한다. 재료및방법사용균주및추출물구강미생물에대한국내자생염료식물의항균활성을알아보기위해사용된균주는그람음성균인 Porphyromonas gingivalis KCTC5352와그람양성균인 Streptococcus mutans KCTC 3065, Streptococcus downei KCTC3634, Streptococcus sanguinis KCTC3284, Streptococcus gordonii KCTC3286 등으로한국생명공학연구원생물자원센터 (KCTC) 에서분양받아사용하였다. P. gingivalis는 tryptic soy agar hemin menadione 배지에계대한다음 37 에서혐기배양하였고 S. mutans는 brainheart infusion agar (BHIA; BD, Franklin Lakes, USA) 에계대한다음 37 에서혐기배양하였다. S. downei, S. sanguinis, S. gordonii는 tryptic soy yeast extract medium 배지에계대한다음 37 에서혐기배양하였다. 국내자생염료식물인오리나무 (A. japonica), 옻나무 (Rhus verniciflua Stokes), 치자나무 (Gardenia jasminoides), 황칠나무 (Dendropanax morbifera), 황벽나무 (Phellodendron amurense Rupr.), 회화나무 (Sophora japonica) 등의줄기는서울경동시장도매상가에서건조된것을구매한다음분쇄하여사용하였다. 분쇄한오리나무, 옻나무, 치자나무, 황칠나무, 황벽나무, 회화나무등의줄기는각각 80% 에탄올에침지한다음상온에서 2시간동안 3회반복추출하였다. 추출물은 Whatman filter paper (No. 2) 로여과한후회전감압농축기 (Eyela, Tokyo Rikakikai Co., Tokyo, Japan) 를이용하여용매를완전히제거한다음 DMSO에용해하여사용하였다. 조추출물의추출수율은각각약 37.5%, 30.3%, 32.1%, 27.8%, 18.9%, 22.6% 였다. 추출물의항균활성추출물의구강미생물에대한항균활성은 agar diffusion method [6] 에따라 P. gingivalis, S. mutans, S. downei, S. sanguinis, S. gordonii가각각도말된고체배지에추출물의농도가 1 mg/disc인 paper disc (8 mm diameter, ADVANTEC, Tokyo, Japan) 를얹어배양한다음 paper disc 주위에나타난 clear zone의크기를측정하여조사하였다. 양성대조구로는구강케어제품에사용되고있는항균제인 chlorhexidine (Sigma Aldrich, St. Louis, USA), sodium lauryl sulfate (Sigma Aldrich, St. Louis, USA), triclosan (Sigma Aldrich, St. Louis, USA) 등을사용하였다. 추출물의최소성장억제농도 (MIC) 와최소살균농도 (MBC) 측정추출물의최소성장억제농도 (minimal inhibitory concentration, MIC) 는액체배지희석법을이용하여조사하였다 [14]. 구강미생물은 37 에서 24시간동안혐기배양한다음배양액을액체배지에희석하였다 (O.D 600=0.40.6). 희석된배양액에추출물을 mg/ml 농도로처리한다음 37 에서 24시간동안혐기배양한후 600 nm에서흡광도를측정하여구강미생물의증식이나타나지않는최소농도를최소성장억제농도로정하였다. 추출물의최소살균농도 (minimal bactericidal concentration, MBC) 는최소성장억제농도로확인된추출물농도로부터그이상의농도에해당하는배양액을고체배지에도말하여 37 에서 24시간동안혐기배양한다음균수의 99.9% 가사멸된추출물의농도로정하였다. 추출물에의한구강미생물의바이오필름 (biofilm) 생성억제추출물이구강미생물의바이오필름형성에미치는영향은 Zhou 등 [34] 의방법을이용하여조사하였다. 구강미생물은고체배지에도말하여 24시간동안혐기배양한다음액체배지에현탁하여 (O.D 600=0.40.6) 96well plate에분주하였다 mg/ml 농도의추출물을구강미생물이분주된 96well plate 에처리한다음 37 에서혐기배양하면서바이오필름생성을

3 1388 생명과학회지 2019, Vol. 29. No. 12 유도하였다. 96well plate에형성된바이오필름은멸균증류수로 2회세척한다음건조한후 0.5% crystal violet으로 20분동안염색하였다. 바이오필름생성억제능은 crystal violet에염색된바이오필름을 70% 에탄올에용해한후 575 nm에서 spectrophotometer (Softmax M5, Molecular Devices, USA) 로흡광도를측정하여확인하였다. 추출물처리에따른 P. gingivalis 의 superoxide dismutase (SOD) 와섬모 (fimbriae) 에대한 mrna 발현변화추출물이구강미생물의 SOD와섬모에대한 mrna 발현에미치는영향은 sod와 fima유전자를 primer로사용한 realtime PCR로조사하였다. Total RNA는 37 에서 24시간동안혐기배양한후액체배지에현탁한구강미생물 (O.D 600=0.40.6) 에 mg/ml 농도의추출물을처리한다음 37 에서배양한배양액을원심분리하여얻은 pellet으로부터 RNeasy Kit (Qiagen, Hillden, Germany) 를이용하여분리하였다. cdna 는 superscript VILO cdna synthesis (Thermo Fisher Scientific, USA) 를이용하여합성하였고 realtime PCR은 CFX96 touch TM realtimepcrdetectionsystem (BioRad,USA) 을이용하여수행하였으며실험에사용된 primer는 Table 1과같다 [14, 31]. PCR 반응은 ssoadvanced TM universalsybr R green supermix (BioRad, USA) 를사용하여 95 에서 30초, 95 에서 10초, 60 에서 20초씩 35 cycle을반복하여수행하였다. PCR 반응종결후 meltcurve analysis를수행하여유전자증폭의정확성을재확인하였으며유전자의상대적발현량은 endogenous control로사용한 P. gingivalis의 housekeeping gene 인 gyra에대한 sod와 fima의상대적유전자발현값으로나타내었다. 통계처리모든실험은 5회이상반복실험을수행하여얻어진결과이며실험결과의평균값과표준오차는 SAS (Statistical analysis system, USA) program을사용하여구하였고 Duncan s 다중검정법으로 p<0.05 수준에서통계적유의성검정을실시하였다. 결과및고찰오리나무줄기추출물의항균활성국내자생염료식물인오리나무, 옻나무, 치자나무, 황벽나 Fig. 1. Antibacterial activity of ethanol extracts (1 mg/disc) from A. japonica stem against P. gingivalis KCTC5352. a: DMSO as a negative control, b: ethanol as a negative control, c: sodium lauryl sulfate (1 mg/ml), d: triclosan (1 mg/ ml), e: chlorhexidine (1 mg/ml), f: ethanol extracts from A. japonica stem. 무, 황칠나무, 회화나무등의줄기를추출한에탄올추출물의구강미생물에대한항균활성을측정한결과, 오리나무줄기추출물은 P. gingivalis와 S. sanguinis에대해서, 옻나무줄기추출물은 P. gingivalis와 S. mutans, S. downei, S. sanguinis, S. gordonii에대해서, 치자나무줄기추출물은 P. gingivalis에대해서, 황벽나무와황칠나무줄기추출물은 P. gingivalis와 S. sanguinis에대해서, 회화나무줄기추출물은 P. gingivalis, S. downei, S. sanguinis, S. gordonii에대해서항균활성을나타내었으며이중오리나무줄기추출물의 P. gingivalis에대한항균활성이가장우수하였다 (Table 2). P. gingivalis에대한오리나무줄기추출물의항균활성을구강케어제품에사용되고있는 chlorhexidine, sodium lauryl sulfate, triclosan과비교한결과는 Fig. 1과같다. 시판되고있는구강케어제품에서 chlorhexidine, sodium lauryl sulfate, triclosan은각각 0.1%, 0.2%, 0.3% 의농도로사용되고있으며본실험에서는오리나무줄기추출물과 chlorhexidine, sodium lauryl sulfate, triclosan의항균활성을 1 mg/disc의농도에서비교하였다. 그결과, 오리나무줄기추출물은 P. gingivalis에대해 chlorhexidine과유사한항균활성을나타내었다. 양이온성항균물질인 chlorhexidine 은구강내에존재하는세균들을광범위하게사멸시킬수있는대표적인항균제제이지만장기간사용시치아나연조직에착색이유발되고미각변화가능성이보고되고있기때문에이를대체할항균제의개발이필요한실정이다 [11]. 따라서오리나무줄기추출물은 P. gingivalis에대해항균활성이있는 Table 1. Oligonucleotides used for realtime PCR in this study Gene Forward primer (5 3 ) Reverse primer (5 3 ) gyra sod fima 5 AAT ATC ATC CCC TCT TCC ACC 3 5 CCT ATG TGG ACA ACC TCA AT3 5 CTG TGT GTT TAT GGC AAA CTT C3 5 GTT GGA GTA TCC GAT TTC GTT3 5 GGC TTC CTT ATG TAT TGG TG3 5 AAC CCC GCT CCC TGT ATT CCG A 3

4 Journal of Life Science 2019, Vol. 29. No Table 2. Antimicrobial activities of ethanol extracts (1 mg/ml) from stem of dye plants against oral microorganisms Oral microorganisms P. gingivalis KCTC5352 S. mutans KCTC3065 S. downei KCTC3634 S. sanguinis KCTC3284 S. gordonii KCTC3286 A. japonica * Ethanol extracts from stem of dye plants (1 mg/ml) R. verniciflua Stokes G. jasminoides D. morbifera P. amurense Rupr. * Growth inhibition size of clear zone:, not detected;, less than 4 mm;, 48 mm;, more than 8 mm. S. japonica 천연물유래구강건강소재로써개발가능성이있다고판단되어오리나무줄기추출물을선발하여추가적인실험을수행하였다. P. gingivalis KCTC5352 에대한오리나무줄기추출물의최소성장억제농도및최소살균농도오리나무줄기추출물은구강미생물중 P. gingivalis에대해우수한항균활성을나타내었으며오리나무줄기추출물처리에따른 P. gingivalis 의 MIC는 0.4 mg/ml였다. P. gingivalis 의 MBC는 MIC인 0.4 mg/ml 및그이상의농도에해당하는 P. gingivalis 배양액을고체배지에배양하였을때 0.6 mg/ml 및그이상의농도에서 colony를관찰할수없었으므로 0.6 mg/ml로결정하였다 (Table 3). 생육이감소한다는 Zhang [33] 등의보고와도일치하는결과이다. 따라서오리나무추출물은 P. gingivalis의생육을억제할수있을뿐만아니라생물막생성도억제할수있는천연물유래구강건강소재로써개발가능성이있다고판단된다. 오리나무줄기추출물이 P. gingivalis의 superoxide dismutase (SOD) 에대한 mrna 발현에미치는영향오리나무줄기추출물이 P. gingivalis의 SOD에대한 mrna 발현에미치는영향은 sod 유전자를 primer로이용한 real timepcr로확인하였으며그결과는 Fig. 3에나타내었다. Real timepcr을이용하여추출물 ( mg/ml) 처리농도에따른 sod 유전자의발현변화를확인한결과 P. gingivalis의 오리나무줄기추출물이바이오필름생성에미치는영향오리나무줄기추출물이 P. gingivalis의생물막형성에미치는영향은 Zhou 등 [34] 의양적분석방법을이용하여조사하였다. P. gingivalis 배양액에추출물을 mg/ml 농도로처리하였을때, P. gingivalis 배양액에서생물막생성이억제되는것을확인할수있었다. 추출물이 mg/ml 농도로처리된배양액에서 P. gingivalis의생물막생성율은각각 80.9±0.09%, 65.4±0.08%, 31.2±0.06%, 27.1±0.02%, 24.8±0.01%, 25.3±0.02% 로추출물의농도가높아질수록생물막생성이억제되는것을확인할수있었고균주의생육도추출물의농도 ( mg/ml) 가높아질수록억제되는것을확인할수있었다 (Fig. 2). 이는에센셜오일에서추출한유제놀의 P. gingivalis 에대한항균활성및생물막생성억제에관한연구에서유제놀 (eugenol) 의농도가높아질수록생물막생성율및균주의 Fig. 2. Bacterial growth and biofilm formation of P. gingivalis KCTC5352 in brainheart infusion broth (BHIB) treated with ethanol extracts of A. japonica stem ( mg/ml). Growth and biofilm formation were measured under anaerobic condition. All assays were performed in triplicate, and mean values and standard deviations are shown. Table 3. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ethanol extract of A. japonica stem against P. gingivalis Concentration (mg/ml) Minimum inhibitory concentration a / c b Minimum bactericidal concentration / a, growth on test medium; b,nogrowthontestmedium; c,slowgrowthontestmedium; d,nondetection.

5 1390 생명과학회지 2019, Vol. 29. No. 12 Fig. 3. Effects of A. japonica stem extract on expression of sod and fima genes in P. gingivalis KCTC5352 by realtime PCR analysis. The expression was normalized to gyra gene used as a reference gene. Results are shown as the ± SD of five replicates. *p<0.05, as compared with the control. sod 유전자는 0.8 mg/ml 까지는추출물의농도가높아질수록감소하는경향을나타내었다. SOD는초과산화이온을산소와과산화수소로바꿔주는불균등화반응을촉매하는효소로써산소에노출되는거의모든세포의항산화방어기전으로중요하다. 초과산화이온이가지고있는자유라디칼음이온은세포손상을초래하기때문에 SOD에의해초과산화이온이산소와과산화수소로바뀜으로서독성으로부터세포를방어하는역할을한다. P. gingivalis의초과산화이온을약화시키는 SOD에대한 mrna 발현은 0.4 mg/ml의오리나무줄기추출물에노출되었을때현저히감소되었다. 이결과는산화물질이축적될때축적된산화물질의독성을중화시켜줄수있는 P. gingivalis의능력이감소한다는것을나타내며 Kim 등 [14] 의보고에서도 0.005% 피톤치드에노출시켰을때 P. gingivalis의 sod 발현은현저히감소되었다. Nakayama 등 [28] 은 P. gingivalis 는 SOD를생성하기때문에편성혐기성균임에도불구하고산소가미량존재하는환경에서도어느정도저항성이있다고보고하였다. 따라서오리나무줄기추출물의처리농도에따라 SOD 에대한 mrna 발현이감소하였다는것은오리나무줄기추출물에노출되면산화물질의중화능력이감소하기때문에 P. gingivalis의생존력도감소됨을나타낸다. 오리나무줄기추출물이 P. gingivalis 의섬모에대한 mrna 발현에미치는영향오리나무줄기추출물이 P. gingivalis의섬모에대한 mrna 발현에미치는영향은 fima 유전자를 primer로이용한 real timepcr로확인하였으며그결과는 Fig. 3에나타내었다. Real timepcr을이용하여추출물 ( mg/ml) 처리농도에따른 fima 유전자의발현변화를확인한결과, P. gingivalis 의 fima 유전자는추출물의농도가높아질수록감소하는경향을나타내었다. 섬모 (fimbriae) 는세균의외막에부착된단백질성부속기 (proteinaceous appendages) 로서생물막형성과정중초기부착단계에관여하며 P. gingivalis 표면에는 FimA subunit으로구성된긴섬모와 Mfa1 subunit으로구성된짧은섬모가존재하고있다. 긴섬모는생물막형성과정에서 P. gingivalis 의치은섬유모세포및상피세포부착에필수적일뿐만아니라 P. gingivalis와 Actinomyces viscosus, Treponema denticola, Streptococcus gordonii, Streptococcus oralis의 aggregation 및 P. gingivalis 의 autoaggregation에도관여한다 [7]. 섬모관련유전자인 fimx, pgma 및 fimabcde 유전자는 fim 클러스터 (cluster) 를이루고있으며 fima유전자결손돌연변이체는인간치은섬유모세포및상피세포에대한부착을감소시켜 P. gingivalis가치은연하 (subgingival) 에서생물막을형성할수없다 [27]. 따라서오리나무줄기추출물의처리농도에따라 fima 유전자발현이감소하였다는것은생물막형성단계에서 P. gingivalis의초기치은섬유모세포및상피세포부착이저해됨을나타낸다. 감사의글 본연구는 2018년경남과학기술대학교대학회계연구비지원에의하여수행된결과의일부이며이에감사드립니다. References 1. An, S. W., Kim, Y. G., Kim, M. H., Lee, B. I., Lee, S. H., Kwon, H. I., Hwang, B. and Lee, H. Y Comparison of hepatic detoxification activity and reducing serum alcohol concentration of Hovenia dulsis Thumb and Alnus japonica Steud. Kor. J. Med. Crop Sci. 7, Back, H. S., Kang, S. K., Auh, K. S., Chun, Y. S. and Hong, J. P Effect of antibacterial effects of myrrh, rhatany, chamomomilla against to oral microorganisms. J. Oral Med. Pain 38, Buitelaar, R. M., Langenhoff, A. A. M., Heidstra, R. and Tramper, J Growth and thiophene production by hairy root culture in various two liquid phase bioreactiors. Enz. Microbiol. Technol. 13, Choi, H. J., Heo, N. S., Choi, Y. W., Lee, Y. G., Jeong, Y. K. and Joo, W. H Antimicrobial and antihalitosis effects of Alnus firma extracts. J. Life Sci. 22, Darveau, R. P., Tanner, A. and Page, R. C The microbial challenge in periodontitis. Periodontol , Davidson, P. M. and Parish, M. E Methods for testing the efficacy of food antimicrobials. Food Technol. 43, Gerits, E., Verstraeten, N. and Michiels, J New approaches to combat Porphyromonas gingivalis biofilms. J. Oral Microbiol. 9, Haffajee, A. D. and Socransky, S. S Microbial etiological agents of destructive periodontal diseases. Periodontol , Han, S. M., Hong, I. P., Woo, S. O., Park, K. K. and Chang,

6 Journal of Life Science 2019, Vol. 29. No Y. C Anticariogenic activity purified bee venom (Apis mellifera L.) against four cariogenic bacteria. Kor. J. Pharmacogn. 47, Hwang, Y. J., Cho, Y. S. and Lee, S. Y Awareness and satisfaction of health insurance coverage of dental scaling. J. Dent. Hyg. Sci. 15, James, P., Worthington, H. V., Parnell, C., Harding, M., Lamont, T., Cheung, A., Whelton, H. and Riley, P Chlorhexidine mouthrinse as an adjunctive treatment for gingival health. Cochrane Database Syst. Rev. 3, CD Kim, H. J., Yeom, S. H., Kim, M. K., Shim, J. G., Paek, I. N. and Lee, M. W Nitric oxide and prostaglandin E2 synthesis inhibitory activities of diarylheptanoids from the barks of Alnus japonica Steudel. Arch. Pharm. Res. 28, Kim, H. S., Kwon, H. S., Kim, C. H., Lee, S. W., Sydara, K. and Cho, S. J Effects of methanol extracts from Diospyros malabarica stems on growth and biofilm formation of oral bacteria. J. Life Sci. 28, Kim, S. Q., Shin, M. K., Auh, Q. S., Lee, J. Y., Hong, J. P. and Chun, Y. H Effect of phytoncide on Porphyromonas gingivalis. J. Oral Med. Pain 32, Kim, S. T., Kim, J. D., Ahn, S. H., Ahn, G. S., Lee, Y. I. and Jeong, Y. S Hepatoprotective and antioxidant effects of Alnus japonica extracts on acetaminopheninduced hepatotoxicity in rats. Phytother. Res. 18, Kim, S. Y., Woo, D. H., Lee, M. A., Kim, J. S. and Lee, J. H Red fluorescence of oral bacteria interacting with Porphyromonas gingivalis. J. Kor. Acad. Oral Health 41, Kuroyanagi, M., Shimomae, M., Nagashima, Y., Muto, N., Okuda, T., Kawahara, N., Nakane, T. and Sano, T New diarylheptanoids from Alnus japonica and their antioxidative activity. Chem. Pharm. Bull. 53, Kwon, H. J., Parkm J. W., Yoon, M. S., Chung, S. K. and Han, M. D Dental hygiene and dental education: Factors associated with selfreported halitosis in korean patients. J. Kor. Acad. Oral Health 32, Lee, J. Y. and Lee, M. O Influential factors for the knowledge and awareness of adults on periodontal diseases and their belief. J. Kor. Cont. Associ. 16, Lee, M. W., Kim, J. H., Jeong, D. W., Ahn, K. H., Toh, S. H. and Surh, Y. J Inhibition of cyclooxygenase2 expression by diarylheptanoids from the bark of Alnus hirsuta var. Sibrica. Biol. Pharm. Bull. 23, Lee, M. W., Tanaka, T., Nonaka, G. I. and Nishioka, I Dimeric ellagitannins from Alnus japonica. Phytochemistry 31, Lee, Y. A., Kim, K. H., Kim, J. S., Cho, S. M., Kim, S. W. and Lee, M. W Antioxidative effects of diarylhepatonoids from Alnus hirsuta. Arch. Pharm. Res. 44, Lopez, N. J Occurrence of actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia in progressive adult periodontitis. J. Periodontol. 71, Marsh, P. D Dental plaque as a microbial biofilm. Caries Res. 38, Marsh, P. D Dental plaque as a biofilm and a microbial communityimplications for health and disease. BMC Oral Health 6, S Na, C. S., Lee, S. B., Kim, J. B., Chung, H. S. and Dong, M. S Effect of hot water extract of Alnus japonica Steud on the experimentallyinduced acute gastritis and peptic ulcers in rats. Kor. J. Pharmacogn. 43, Nagano, K., Abiko, Y., Yoshida, Y. and Yoshimura, F Porphyromonas gingivalis fima fimbriae: roles of the fim gene cluster in the fimbrial assembly and antigenic heterogeneity among fima genotypes. J. Oral Biosci. 54, Nakayama, K. J Rapid viability loss on exposure to air in a superoxide dismutasedeficient mutant of Porphyromonas gingivalis. J. Bacteriol. 176, Park, H. R. and Hong, S. J Research on natural medicine for wellness and oral health J. Digital Convergence 13, Stevic, T., Savikin, K., Zdunic, G., Stanojkovic, T., Juranic, Z., Jankovic, T. and Menkovic, N Antioxidant, cytotoxic, and antimicrobial activity of Alnus incana (L.) ssp. incana Moench and A. viridis (Chaix) DC ssp. viridis extracts. J. Med. Food 13, Van der Ploeg, J. R., Giertsen, E., Lüdin, B., Mörgeli, C., Zinkernagel, A. S. and Gmür, R Quantitative detection of Porphyromonas gingivalis fima genotypes in dental plaque. FEMS Microbiol. Lett. 232, Wada, H., Tachibana, H., Fuchino, H. and Tanaka, N Three new diarylheptanoid glycosides from Alnus japonica. Chem. Pharm. Bull. 46, Zhang, Y., Wang, Y., Zhu, X., Cao, P., Wei, S. and Lu, Y Antibacterial and antibiofilm activities of eugenol from essential oil of Syzygium aromaticum (L.) Merr. & L. M. perry (clove) leaf against periodontal pathogen Porphyromonas gingivalis. Microb. Pathog. 113, Zhou, L., Ding, Y., Chen, W., Zhang, P., Chen, Y. and Lv, X The in vitro study of ursolic acid and oleanolic acid inhibiting cariogenic microorganisms as well as biofilm. Oral Dis. 19,

7 1392 생명과학회지 2019, Vol. 29. No. 12 초록 :Porphyromonas gingivalis 에대한오리나무줄기추출물의항균활성및생물막형성억제효과김혜수 조수정 * ( 경남과학기술대학교제약공학과 ) 본연구에서는천연물유래구강건강소재로써염료식물의이용가능성을알아보기위해오리나무, 옻나무, 치자나무, 황칠나무, 황벽나무, 회화나무줄기추출물의구강미생물에대한항균활성을조사하였다. 6 종의국내자생염료식물의줄기에서추출한에탄올추출물중오리나무줄기추출물 (1 mg/disc) 이치주질환원인균인 P. gingivalis KCTC5352에대해가장우수한항균활성을나타내었다. 오리나무줄기추출물은 P. gingivalis KCTC5352에대해양성대조구로사용한 chlorhexidine과유사한항균활성을나타내었으며 P. gingivalis KCTC5352의 MIC는 0.4 mg/ml였고 MBC는 0.6 mg/ml였다. 오리나무줄기추출물이 mg/ml 농도로처리된배양액에서 P. gingivalis KCTC5352의바이오필름생성율과세균생육은추출물의농도가증가할수록저해되는경향을보였다. 또한 P. gingivalis KCTC5352의 superoxide dismutase (SOD) 와섬모에대한 mrna 발현도추출물의농도가높아질수록감소하는것을확인할수있었다. 이상의결과를종합하면오리나무줄기추출물은치주질환원인균인 P. gingivalis KCTC5352에대한항균활성과생물막생성억제능이우수하기때문에천연물유래구강건강소재로써이용가능성이높을것으로판단된다.

Journal of Life Science 2011, Vol. 21. No μ μ

Journal of Life Science 2011 Vol. 21. No. 8. 1120~1126 ISSN : 1225-9918 DOI : http://dx.doi.org/10.5352/jls.2011.21.8.1120 μ μ μ α β Journal of Life Science 2011, Vol. 21. No. 8 1121 μ μ 1122 생명과학회지 2011,