The Korean Journal of Microbiology (2010) Vol. 46, No. 2, pp. 200-206 Copyright c 2010, The Microbiological Society of Korea 구강질환세균에대한한약재의항균효과 유영은 1 박은영 1 정대화 1 변성희 2 김상찬 1,2 박성민 1 * 1 대구한의대학교한방생명자원연구센터, 2 대구한의대학교한의학과 Antibacterial Effect of Oriental Medicinal Herbs on Dental Pathogens Young-Eun Yu 1, Eun-Young Park 1, Dae-Hwa Jung 1, Sung Hui Byun 2, Sang Chan Kim 1,2, and Sung Min Park 1 * 1 The Research Center for Biomedical Resources of Oriental Medicine, Daegu Haany University, Daegu 706-828, Republic of Korea 2 College of Oriental Medicine, Daegu Haany University, Daegu 706-828, Republic of Korea (Received March 2, 2010/Accepted April 19, 2010) In this study, we investigated the antibacterial effect of 69 oriental medicinal herbs (OMHs) on Actinomyces viscocus, Streptococcus sobrinus, and two strains of Streptococcus mutans as oral bacteria. Methanol extracts of OMHs were used in the first antibacterial test, and then boiled water, ethanol, methanol, and ethyl acetate extracts of C. japonica, C. sappan, P. mume, and S. chinensis were used in the second test. Boiled water extract of C. japonica was shown to have the most superior effect on oral bacteria. The extract yield of boiled water extract of C. japonica was 22%, and the growth of oral bacteria was almost inhibited at over a 180 mg/ml concentration. Antibacterial effect compound analyzed by UPLC method was identified as berberine, and the content was 25.54%. In addition, the extract appeared to be stable at 121 C for 15 min. Keywords: antibacterial effect, dental pathogen, oriental medicinal herb 구강은외계와직접접하고있기때문에항상미생물의침입을받고있으며영양및생리적으로세균이증식하는데적합하여항상많은세균이정착하는상재세균총을이룬다. 보통사람의구강에는 30종이상의세균이분리되며구강세균총은각개인, 연령, 건강상태, 식이상태또는위생상태에따라그종류와비율이달라지는것으로알려져있다 (14). 대표적인구강질환인치아우식증과치주염은치면세균막내의세균에의하여발생하는감염성질환이며치면세균막질환이라고한다. 현재까지 Streptococcus mutans, Streptococcus sobrinus 그리고 Actinomyces sp. 가치아우식증과관련이있는것으로알려져있다 (18). 이러한균주들은 glucosyltransferase 를분비하여자당으로부터비수용성및수용성 glucan을생성하는데, 이중비수용성 glucan은치아표면에부착하게되고부착된 glucan에구강내여러세균이응집하여치면세균막을형성하게된다. 응집된치면세균막내의미생물에의하여대사산물의부산물로산을생성하게되며, 세균에의해생성된산 은치면세균막내에축적되고타액의완충작용의영향을받지않게되어초기치아우식증의원인이된다 (5). 따라서치아우식증을예방하기위해서는세균의성장을억제하거나치면세균막내에세균이부착하는것을억제할수있는항미생물제제가요구되고있다 (4). 현재구강질환을예방하기위한다양한종류의제품이시판되고있으며그중많이사용되고있는구강청결제의경우주된항미생물성분으로 chlorohexidine, cetylpyridinium chloride, benzetonium chloride와불소등을사용하고있다 (3). 그러나보다우수한제품을제조하기위하여구강세균에미치는각종화학물질의영향을조사하거나 (11) 팽이모자반 (4), 호장근 (13), 정향 (10), 오미자 (9), 카테킨 (21) 그리고다양한다류 (12) 를이용한항균효과등다양한연구가보고되었다. 이에본연구자들은한의학에서사용되고있는한약재를이용하여구강세균의성장을억제하여구강질환을예방또는치료할수있는연구를진행하기위한후보물질을선발하여조사하였으며결과를함께공유하고자한다. * For correspondence. E-mail: minshell@dhu.ac.kr; Tel: +82-53-770-2332; Fax: +82-53-770-2335
구강질환세균에대한한약재의항균효과조사 201 재료및방법재료구강세균에대하여항균효과를나타내는한약재를조사하기위하여본초학을바탕으로문헌조사를하였으며이를바탕으로한약재를선발하여수성구상동소재의대원약업사에서생약제제로포장되어판매하는제품을구입하여사용하였다 (Table 1). 공시균주및배양조건한약재의항균효과를조사하기위하여 S. sobrinus KCCM 11898, A. viscocus KCCM 12074, S. mutans KCCM 40105 및 S. mutans KCCM 41032를한국미생물보존센터로부터분양받아사용하였다. 공시균주의배양을위하여 Brain Heart Infusion (0.77% calf brains, 0.98% beef heart, 1.0% proteose peptone, 0.2% dextrose, 0.5% sodium chloride, 0.25% disodium phosphate, ph 7.4; Becton Dickinson, USA) 을사용하였다. 공시균주는 37 C에서 정치배양하였으며 S. mutans KCCM 41032의경우 37 C, 10% CO 2 조건의배양기 (Hera cell, Thermo, USA) 에서배양하였다. 실험을위해서공시균주를고체배지에접종하여배양한후 4 C에서보관하면서사용하였으며한주에한번씩계대배양하여실험 2일전다시계대배양하여균을활성화시킨후사용하였다. 추출물의제조본초학을바탕으로선발한 69종의한약재로부터항균효과를나타내는물질을추출하기위한용매로물, 에탄올, 메탄올및에틸아세테이트를사용하였다. 에탄올, 메탄올및에틸아세테이트를이용한추출물의제조는한약재에 10배량의용매를가하고상온에서 100 rpm, 24시간교반시킨후여과 (Filter paper No. 2, Whatman, Japan) 한후대형회전농축기 (Rotary evaporator, Buchi, Switzerland) 로감압농축하고각각의용매로다시현탁한후 0.2 μm filter (Minisart RC 25, Sartorius, Germany) 로제균하여준비하였다. 현탁용매에의한항균효과가나타날수있음으로실온에서무균적으로 30분이상건조 Table 1. List of oriental medicinal herbs for this study Species name (Part, recovery rate) Species name (Part, recovery rate) Areca catechu (Seed, 7) Lithospermum erythrorhizon (Root, 33) Artemisia apiacea (Aerial part, 9) Lonicera japonica (Flower, 45) Artemisia capillaries (Aerial part, 12) Lycium chinense (Fruit, 24) Artemisia princeps (Leaf, Stem, 11) Lycopus coreanus (Aerial part, 14) Caesalpina sappan (Stem, 8) Melia azedarach (Fruit, 21) Capsicum annuum (Fruit, green, 46) Mentha arvensis (Aerial part, 21) Capsicum annuum (Fruit, red, 59) Morus alba (Fruit, 64) Carthamus tinctorius (Flower, 6) Nelumbo nucifera (Leaf, 12) Chaenomeles sinensis (Fruit, 85) Oldenlandia diffusa (Leaf, 7) Chelidonium majus (Aerial part, 11) Paeonia suffruticosa (Root, 22) Chrysanthemum indicum (Flower, 49) Perilla frutescens (Leaf, Stem, 21) Cinnamomum cassia (Stem, 6) Persicaria tinctoria (Leaf, 2) Cirsium japonicum (All, 13) Phaseolus angularis (Seed, 0) Citrus unshiu (Fruit, 64) Phaseolus radiates (Seed, 0) Citrus unshiu pericarpium (Fruit, 35) Phellodendron amurense (Bark, 26) Clematis mandshurica (Root, 18) Polygonum aviculare (All, 10) Cnidium monieri (Fruit, 7) Poncirus trifoliate (Fruit, 32) Commelina communis (Aerial part, 6) Prunus mume (Fruit, 73) Coptis japonica (Root, 12) Rheumtanguticum (Root, 89) Cornus officinalis (Fruit, 123) Rubus coreanus (Fruit, 8) Corydalis ternate (Stem, 2) Salvia miltiorrhiza (Root, 15) Crataegus pinnatifida (Fruit, 129) Santalum album (Stem, 9) Curcuma longa radix (Root, 29) Sasa japonica (Leaf, 11) Curcuma longa Rhizoma (Root, 14) Saururus chinensis (Aerial part, 38) Curcuma zedoaria (Root, 7) Schisandra chinensis (Fruit, 99) Dianthus chinensis (Aerial part, 21) Schizonepeta tenuifolia (Flower, 5) Eclipta prostrate (All, 21) Scrophularia buergeriana(root, 22) Elsholtzia ciliate (All, 15) Scutellaria baicalensis (Root, 25) Foeniculum vulgare (Fruit, 7) Sophora japonica (Flower, 24) Forsythia viridissima (Fruit, 31) Spatholobus suberectus (Stem, 16) Gardenia jasminoides (Fruit, 44) Spirodela polyrhiza (Leaf, 16) Gentiana scabra (Root, 52) Taraxacum platycarpum (All, 18) Isatis tinctoria (Root, 27) Thuja orientalis (Branch, 35) Leonurus sibiricus (Aerial part, 9) Xanthium strumarium (Fruit, 5) Ligustrum lucidum (Fruit, 26)
202 Yu et al. 시켜용매에의한영향을배제시킨후연구에사용하였으며별도의대조구는처리하지않았다. 물추출물의경우에는 10배량의 1차정제수를가하여 85 C 에서 2시간정도끓여추출한후용매를이용한추출물과동일한방법으로준비하여사용하였다. 추출물의회수율은회수된추출물의중량을추출시사용한한약재의중량으로나눈후백분율로계산하였다. 항균효과조사 항균효과를조사하기위하여미리배양해둔공시균주를원심분리 (7,085 g, 2 min) 하여회수한후상등액을제거하고잔존배지성분등의제거를위하여멸균수로현탁시키고동일한방법으로 3번반복한후희석하여준비하였으며 1.5±0.5 10 8 cells/ml로균수를조정하여사용하였다. 항균효과를나타내는한약재를선별하기위하여메탄올추출물을이용하여선행조사하였으며 (1차) 그결과를바탕으로한약재를선별하고용매별추출물을제조하여항균효과를조사하였다 (2차). 한약재의경우추출물을제조할때마다구입하여사용하였다. 1차와 2차조사는추출물을 6 mm paper disc (Advantec, Japan) 에 20 μl 점적한후한천평판확산법 (agar diffusion method) 으로조사하였으며공시균주의생육이억제되면서형성되는생육저지환 (clear zone) 의크기로항균효과의유ㆍ무와정도를확인하였다. 2차조사의결과를바탕으로가장우수한항균효과를나타내는한약재를최종선발하고공시균주의생육에미치는영향을조사하였다. 전배양한공시균주의수를조정한후 100 μl 접종하고추출물 20 μl를첨가하여 24시간배양하면서조사하였다. 분광광도계 (Spectrophotometer, Shimadzu, Japan) 를이용하여 660 nm에서생육도를조사하였으며추출물의첨가에따라배지의흡광도가변함으로배지에추출물만첨가한후동일한조건에서배양하고흡광도측정시대조구로사용하였다. 실험에서얻어진결과의유의성은 SPSS 11.0 (Statistical package for social sciences) program을이용하여실험군당평균 ± 표준편차로표시하였다. 열안정성 한약재중가장우수한항균효과를나타내는추출물을 121 C 에서 15분간열처리를한후실온에방치하여식힌후열처리를하지않은대조구와함께한천평판확산법을이용하여항균효과를비교하였다. 항균효과물질의확인 가장우수한항균효과를나타내는한약재의추출물을 UPLC (ultra high performance liquid chromatography) 를이용하여표준품과비교하였으며이때의조건은 Table 2와같이하였다. 사용용매는모두특급을사용하였으며증류수는탈이온처리하였으며, 사용전 0.45 μm filter로여과하여사용하였다. 사용기기는 UPLC (ACQUITY TM, Waters, USA) 를이용하였으며검량선작성은표준품 (25, 50, 100 ppm) 을이용하여검량 Table 2. Operation conditions for UPLC analysis Parameter Column Conditions ACQUITY UPLC BEN C18 Column (2.1 100 mm) Solvent system 0.1% formic acid in 100% ACN, 0.1% formic acid in H 2O Flow rate 0.4 ml/min Detector PDA 345 nm Injector volume 2 μl 선을작성하여사용하였다. 한약재의항균효과 결과및고찰 선발한한약재의메탄올추출을통한회수율은 Table 1과같이조사되었다. 산사와산수유의경우 120% 이상의회수율을나타내어가장높은회수율을나타내었으며계지외 16종의한약재는 10% 미만의낮은회수율을, 녹두와적소두의경우에는회수되지않는것으로조사되었다. 미리배양해둔공시균주의수를조정한후 100 μl를첨가하여고체배지를제조한후한약재의메탄올추출물 20 μl를 paper disc에점적하여상온에서건조시키고배지에얹어 37 C 에서 24시간배양하여 1차조사한항균효과의결과는다음과같았다. S. sobrinus의경우에는 16종의한약재 ( 단삼, 대황, 박하, 백굴채, 삼백초, 상심자, 소목, 오매, 오미자, 측백엽, 치자, 판람근, 현호색, 황금, 황련, 황백 ) 에서, A. viscocus의경우에는 11 종의한약재 ( 단삼, 대황, 박하, 삼백초, 상심자, 소목, 오매, 측 백엽, 황금, 황련, 황백 ) 에서, S. mutans KCCM 40105의경우에는 14종의한약재 ( 단삼, 단향, 대황, 산수유, 소목, 오매, 오미자, 측백엽, 하엽, 현호색, 홍화, 황금, 황련, 황백 ) 에서그리고 S. mutans KCCM 41032의경우에는 20종의한약재 ( 단삼, 단향, 대계, 대황, 박하, 사상자, 산수유, 삼백초, 상심자, 소목, 소회향, 아출, 오매, 오미자, 지실, 측백엽, 판람근, 황금, 황련, 황백 ) 에서항균효과를나태내는것으로조사되었다. 메탄올추출물의항균효과를바탕으로공시균주에대하여양호한항균효과를나타낸소목, 오매, 오미자그리고황련을항균효과조사를위한한약재로선별하고각각의용매로추출하여조사한항균효과와회수율은 Table 3과같았다. 선별된한약재중소목의경우메탄올추출물에서, 오매, 오미자그리고황련은열수추출물에서가장우수한항균효과를나타내었으며선별된한약재중황련이가장우수한항균효과를나타내는것으로조사되었으며 (Fig. 1) 에틸아세테이트추출물의경우대부분의결과에서항균성이나타나지않는것으로미루어추출물을현탁한유기용매가나타낼수있는항균성은배제할수있을것으로판단되었다. 이결과는안등 (1), 배 (2) 와장등 (8) 의연구결과, 특히장등이보고한황련의 berberine에의한항균효과에대한연구결과와유사한것으로
구강질환세균에대한한약재의항균효과조사 203 Table 3. Antibacterial effect of selected oriental medicinal herbs by different kinds of solvent extracts Selected herb Solvent (recovery rate) Clear zone (mm) 11898 12074 40105 41032 C. sappan Water (9) 11.5 12.0 10.5 12.0 Ethanol (3) 16.0 15.0 14.0 17.0 Methanol (10) 16.0 17.0 15.5 19.0 Ethyl acetate (2) 10.0 8.5 9.0 10.5 P. mume Water (79) 14.0 10.5 14.0 15.5 Ethanol (21) 10.5 8.0 11.0 13.0 Methanol (70) 9.0 10.0 9.5 10.5 Ethyl acetate (4) - - - - S. chinensis Water (112) 11.8 11.0 13.0 15.0 Ethanol (64) 7.0 - - 8.0 Methanol (110) 10.0 9.0 11.0 12.5 Ethyl acetate (6) - - - - C. japonica Water (15) 22.0 22.0 20.0 18.0 Ethanol (1) 10.0 10.5 11.0 - Methanol (10) 21.0 20.0 19.0 17.0 Ethyl acetate (0.1) - - - - 판단되었다 (5). 이에본연구자들은추출한황련열수추출물에서 berberine 을확인하기위하여 UPLC로분석한결과표준품과동일한머무름시간에서 (6분) peak를확인할수있었으며 (Fig. 2) 그함량은 25.54% (25.54 mg/l) 로확인되었다. 그러나조정제수준의시료에서 berberine에의한항균효과라고단정짓기는어려 2 1 1 2 4 3 3 4 S. sobrinus KCCM 11898 A. viscocus KCCM 12074 울것으로판단되었으나기존의보고 (5) 와비교할때가능성은확인할수있었으며황련열수추출물로부터 berberine 성분을분리ㆍ정제과정을진행한후이부분에대한확인연구를진행할예정이다. 소목의경우 4종의용매추출물에서모두항균효과를나타내었으나소목을제외한나머지한약재의경우추출용매에따라선택적으로항균효과를나타내는것으로조사되었다. 소목의경우이미암세포주에대한증식억제 (6) 와가축의질병원인균 (7, 17) 에대한항균성이있다고보고되었으며, 특히 S. mutans에대한성장억제 (20) 및소목의성분중 brazilin에의한항균효과 (15) 와비교할때유사한결과를나타내는것으로판단되었다. 회수율의경우오미자열수추출물이 112% 로가장높이조사되었고황련에틸아세테이트추출물이 0.1% 로가장낮은것으로조사되었으며회수율과항균효과의상관관계는확인할수없었다. 또한회수율의경우추출물을제조한경우마다약간의차이를나타내었다. 황련열수추출물이공시균주의생육에미치는영향 1 3 4 2 S. mutans KCCM 40105 S. mutans KCCM 41032 Fig. 1. Antibacterial effect of different kinds of C. japonica extracts on oral bacteria. (1) water extract, (2) ethanol extract, (3) methanol extract, (4) ethylacetate extract 3 1 4 2 선별된한약재중가장우수한항균효과를나타내는것으로조사된황련열수추출물을이용하여공시균주의생육에미치는영향을조사한결과반복된실험에따라큰차이는나타나지않았으며 Fig. 3과같이조사되었다. 추출물을첨가하지않은대조구와비교할때 S. mutans KCCM 40105의경우 30 mg/ml의농도에서 76% 의생육이저해되는것으로조사되었으며 60 mg/ml 이상의농도에서는거의생육하지않는것으로조사되어공시균주중가장낮은농도에서가장높은저해를나타내었다. S. sobrinus의경우 30 mg/ml에서 64%, 60 mg/ml 에서 97% 의생육이저해되는것으로조사되었으나 120 mg/ml 에서생육도가다시증가하다가다시감소하여 180 mg/ml에
204 Yu et al. 0.070 0.060 0.050 0.040 0.030 0.020 0.010 0.000 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 Fig. 2. Chromatogram of water extract of C. japonica by UPLC. Time (min) 서는생육을하지않는것으로조사되었다. A. viscocus 경우에도 30-90 mg/ml 구간에서생육이저해되었으나 150 mg/ml에서다시증가하다가 180 mg/ml에서는생육하지않는것으로조사되었다. S. mutans KCCM 41032의경우 30 mg/ml의농도에서 13% 이하의저해를받는것으로조사되었으며다른공시균과달리농도에따른생육도의일시적인상승없이농도에따라저해를받는것으로조사되었으며황련열수추출물에대한저항성이가장높은것으로조사되었다. S. mutans KCCM 40105와 S. mutans KCCM 41032는동일균주이기는하나다른저해양상을나타내었으며이는동종이라고하여도주위환경에대한반응에서다양성을나타내는미생물의일반적인특성으로판단되었다. 150 mg/ml 전, 후의농도에서생육도의일시적인상승이반복실험에서지속적으로확인되는것으로미루어박등 (19) 의연구에서와같이분광광도계를이용한조사결과첨가물에의하여생육이저해되다가다시증가하는것과같은양상을나타내었으나그이유는불명확하여이부분에대한연구가필요한것으로판단되었다. 때황련열수추출물은다양한온도분포에서안정성을가진다고판단할수있었다. 적요본연구에서는 69가지의한약재를이용하여구강세균인 Actinomyces viscocus, Streptococcus sobrinus, 그리고 Streptococcus mutans 2종에대한항균효과를조사하였다. 우선선발된한약재를메탄올로추출하여조사하였으며그결과를바탕으로소목, 오매, 오미자, 그리고황련을선별하여열수, 에 황련열수추출물의열안정성열수추출물을 121 C에서 15분간열처리후한천평판확산법을이용하여대조구와항균효과를비교한결과 Fig. 4와같이공시균주에대하여거의유사한수준의항균효과를나타내는것으로조사되었으나실험구에서약간더우수한항균효과를나타내는것으로조사되었다. 이는황련열수추출물의경우온도에대한안정성이높다는것을확인할수있었으며이등 (16) 이황련의지표물질을추출한후실온과냉장조건에서 5개월간비교하였을때크게차이가없었다는결과를적용할 Fig. 3. Growth inhibition of different concentrations of C. japonica water extract on oral bacteria. ( ) S. sobrinus KCCM 11898, ( ) A. viscocus KCCM 12074, ( ) S. mutans KCCM 40105, ( ) S. mutans KCCM 41032. Results are expressed as Means±SD (n=3).
구강질환세균에대한한약재의항균효과조사 205 C H C H (A) (B) C H C H (C) (D) Fig. 4. Antibacterial effect of C. japonica water extract (4.4 mg/20 μl) C (untreated) and H (heat treated) on oral bacteria. (A) S. sobrinus KCCM 11898, (B) A. viscocus KCCM 12074, (C) S. mutans KCCM 40105, (D) S. mutans KCCM 41032 탄올, 메탄올, 그리고에틸아세테이트를이용하여항균효과를조사하였다. 조사결과황련열수추출물이가장우수한효과를나타내는것으로조사되었다. 황련열수추출물을이용하여공시균주에대한저해양상을조사한결과 180 mg/ml 이상의농도에서공시균주의생육이대부분저해되는것을확인할수있었으며이때황련열수추출물의회수율은 22% 였다. 황련열수추출물을 UPLC를이용하여 berberine 함량을조사한결과 25.54% 로조사되었다. 또한 121 C에서 15분간열처리후황련열수추출물의열안정성을조사한결과열에대하여안정하다는것을확인할수있었다. 감사의말 본연구는지식경제부지역혁신센터사업을수행하고있는대구한의대학교한방생명자원연구센터 (B0009008) 의지원에의하여이루어졌으며이에감사드립니다. 참고문헌 1. Ahn, S.M., D.S. Lee, M.S. Kim, S.J. Choi, C.S. Choi, J.B. Lee, H.S. Jang, and H.Y. Sohn. 2009. Bioactivity of the extract of Coptis chinensis: In-vitro antifungal against Phytophthora capsici and growth-promotion effect in Red-pepper. Kor. J. Microbiol. Biotechnol. 37, 280-286. 2. Bae, J.H. 2005. Antimicrobial effect of Plagiorhegama dubium extracts on food-borne pathogen. Korean J. Food Nutr. 18, 81-87. 3. Baek, D.H. 2007. Screening of the natural plant extracts for the antimicrobial activity on dental pathogens. Kor. J. Microbiol. 43, 27-231. 4. Chang, K.W., H.G. Kim, and C.H. Cho. 1997. Antibacterial effects of Sargassum horneri extract to the Streptococcus mutans and Streptococcus sobrinus strains. J. Korean Acad. Dent. Health 21, 379-388. 5. Chang, K.W., K.J. Koh, and Y.G. Yoo. 1997. Antibacterial effects of berberine to the mutans streptococci. J. Korean Acad. Dent. Health 21, 537-544. 6. Han, M.D. and E.K. Kim. 2007. Antiproliferative effects of Caesalpinia sappan extract on human epithelial cell line HaCaT and cencer cell lines. J. Dental Hygiene Science 7, 31-35. 7. Hur, T.Y., S.J. Kang, and G.H. Suh. 2006. Antibacterial effect of Caesalpinia sappan extract against mastitis pathogens from dairy cows. J. Vet. Clin. 23, 286-290. 8. Jang, G.H., B.Y. Ahn, S.H. Oh, D.S. Choi, and Y.J. Kown. 2000. Anticariogenic effects of Coptis chinensis franch extract. Korean J. Food Sci. Technol. 32, 1396-1402. 9. Jeong, H.J., Y.A. Lee, and W.D. Ji. 2002. Effect of the extract of Schizandra chinensis baill on bacteria isolated from oral cavity. J. Dental Hygiene Science 2, 85-88. 10. Jeong, H.J., H.S. Lee, and W.D. Ji. 2003. Effect of the extract of Syzygium aromaticum merr. et perry on bacteria isolated from oral cavity. J. Korean Soc. Hygienic Sciences 9, 69-75. 11. Ji, W.D., S.G. Seo, D.J. Kwak, S.Y. Kim, and Y.G. Chung. 1997. Isolation and identification of oral bacteria and growth inhibition of chemical substances on these strains. J. Korean Soc. Hygienic Sciences 3, 69-76. 12. Kim, M.S., H.Y. Lee, and Y.S. Kim. 1999. Inhibitory effect of tea extracts on the growth of oral bacteria. J. Korean Soc. Hygienic Sciences 5, 111-119. 13. Kim, S.K., J.H. Song, J.B. Kim, K.W. Chang, and J.G. Jeon. 2005. In vitro anticariogenic activity of Polygoni radix. J. Korean Acad. Dent. Health 29, 80-90. 14. Lee, B.B., Y.M. Ha, S.H. Shin, K.M. Je, S.R. Kim, J.S. Choi,
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