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1 Journal of Korean Academy of Oral Health 2016 March 40(1): Original Article 일부식물성오일의 Streptococcus mutans 와 Lactobacillus casei 성장에미치는영향 김세연 1, 김한나 3, 전은주 1, 김진범 1, 정승화 1,2 1 부산대학교치의학전문대학원예방치과학교실, 2 부산대학교중개치의학연구소, 3 청주대학교보건의료대학치위생학과 The growth inhibitory effect of some vegetable oils on Streptococcus mutans and Lactobacillus casei Se-Yeon Kim 1, Han-Na Kim 3, Eun-Joo Jun 1, Jin-Bom Kim 1, Seung-Hwa Jeong 1,2 1 Department of Preventive and Community Dentistry, 2 Institute of Translational Dental Sciences, Pusan National University School of Dentistry, Yangsan, 3 Department of Dental Hygiene, College of Health Sciences, Cheongju University, Cheongju, Korea Received: October 22, 2015 Revised: January 30, 2016 Accepted: February 18, 2016 Corresponding Author: Seung-Hwa Jeong Department of Preventive and Community Dentistry, Pusan National University School of Dentistry, Busandaehak-ro 49, Meulgeum-up, Yangsan 50612, Korea Tel: Fax: jsh0917@pusan.ac.kr *This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (No. 2014R1A1A ). Objectives: The purpose of this study was to evaluate the growth inhibitory effects of some vegetable oils on Streptococcus mutans (S. mutans) and Lactobacillus casei (L. casei). Methods: Two bacterial strains and 5 kinds of test solutions (3 experimental groups: orange essential oil, olive oil, soybean oil; 1 positive control group: chlorhexidine solution; 1 negative control group: broth medium) were used in this study. S. mutans and L. casei pellets were exposed to 1 ml of one of the test solutions for 1 minute. Then, the treated bacterial cells were incubated in fresh broth medium for 0, 4, 8, 16, and 24 hours. The optical density of the broth medium was measured using an ELISA reader at 620 nm. A nonparametric Kruskal-Wallis test (with Mann-Whitney U tests) was performed to compare the change in optical density between different groups at different time points. Results: Bacterial growth was significantly inhibited in all experimental groups compared to the negative control group. The growth of L. casei was less affected by experimental oils than that of S. mutans. Orange essential oil had the maximum growth inhibitory effect on S. mutans up to 8 hours, similar to that in the positive control group (P<0.01). Experimental oils had greater growth inhibitory effect on L. casei than chlorhexidine solution. Conclusions: This in vitro study confirmed the growth inhibitory effect of some vegetable oils on S. mutans and L. casei. Rising of the mouth using these vegetable oils is expected to have an anti-plaque effect, but additional clinical studies are needed to confirm this. Key Words: Essential oil, Growth inhibitory effect, Lactobacillus casei, Mouth rinse, Streptococcus mutans, Vegetable oil 서론 치면세균막은구강내에치아와같은경조직위의획득피막에다양한균이부착되어축적된세균복합체막이다 1). 치면세균막은구강내환경변화에도서로상호작용하며항상성을유지할수 있지만, 외인성병원균이더욱우세해지면치아우식증, 치주질환과같은구강질병이발생하게된다. 치아우식증과관련된대표적인균에는 Streptococcus mutans와 Lactobacilli species 2), 치주질환과관련된대표적인균에는 Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum 등 3) 이알려져있 Copyright 2016 by Journal of Korean Academy of Oral Health This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. JKAOH is available at pissn X / eissn

2 25 김세연외 일부식물성오일의 Streptococcus mutans와 Lactobacillus casei 성장에미치는영향 으며, 이러한구강질병은치면세균막의세균의양과비례한다 4,5). 따라서치아우식증과치주질환을예방하기위해서는치면세균막을물리, 화학적으로제거하거나치면세균막내의세균의성장을억제시켜줘야한다. 치면세균막을제거하는방법으로는물리적방법과화학적방법이있다. 화학적방법으로는클로르헥시딘 (Chlorhexidine) 이대표적으로, 구강내그람양성균, 그람음성균모두의활성을억제하여세균수를줄이는데효과적이다. 하지만맛이좋지못하고, 치아에착색및점막의통증을일으키는등의부작용이있으므로구강병예방이나구강미생물의활성억제를위한지속적인목적으로는장기간사용할수가없다 6). 따라서미생물의성장과치면세균막형성을억제시킬수있는천연물에서추출한항균물질활용에대한관심이높아지고있다 7). 대표적으로자일리톨과잔톨리졸은치면세균막구성균주의성장억제에효과적인것으로알려져있고 8,9). 이외에치면세균막구성균주의성장및억제를목적으로천연항균물질인에센셜오일을이용한항균에대한연구가활발히이루어지고있다 10-12). 에센셜오일 (Essential oil) 이란향기가나는식물에서물리적인방법 ( 증류법, 냉압착법, 용매추출법, 냉침법, SC-O2 추출법등 ) 으로추출한휘발성물질을말한다 13). 이러한에센셜오일은고대로부터오랫동안사용되어왔고, 주로방향제 화장품 향수 비누 세제 향신료등으로이용되고있다. 잎 나무 뿌리등의에센셜오일성분은식물에기생하는생물이나외부동물로부터자신을방어하는수단으로사용된다 14). Lee 등 11) 은구강내세균에대한에센셜오일의항균효과에관한연구로 5가지에센셜오일의농도를 1/2 씩희석하며항균능력을평가하였으며, Takarada 등 12) 은마누카오일 (manuka oil) 과티트리오일 (tea tree oil) 의강력한항균력을보고하였다. 최근에센셜오일과유사한식용가능한식물성오일을이용한건강유지방법으로오일풀링 (Oil pulling) 에대한대중의관심이높아진바있다 14). 오일풀링은인도의학서 아유르베다 (Ayurveda) 에도실린전통적인의술로써건강을위해고대에서부터전해져오는방법으로, 매일아침참깨또는코코넛과같은식물성오일을한스푼입에머금고가글을통해구강내에있는세균과입안의독소를오일과함께제거하여면역을강화시키는전통적인디톡스민간요법이다 14,15). 이때사용하는식물성오일은식물의씨나열매에서냉압착하여추출한오일이며, 이오일을사용했을때효과가극대화된다고보고하였다 15). Asokan 등 16) 은 Dentocult SM strip을이용하여식물성오일의구강내항균효과를확인한결과식물성오일은 Streptococcus mutans에대하여효과가있었으나클로르헥시딘보다항균효과가강하게나타나지않았다고보고하였다. 다양한에센셜오일의항균 11,12), 항염증 17), 항진균 18), 항암효과 19) 등에대해서는다수의연구에서보고되었으나, 식용가능한식물성오일에대한실험실상에서의연구에대한보고는많지않다. 따라서본연구의목적은구강내에서당을분해하고활발하게산을생산하여치아우식을유발하는균으로알려진 Streptococcus mutans (S. mutans) 와치아우식의 2차진행에관여하는 Lactobacillus casei (L. casei) 에대하여식물성오일이균성장에영향을미치는지를평가하고기존구강양치용액과비교하고자하였다. 1. 연구재료 연구대상및방법 1.1. 실험균주본실험에사용된균주는한국생명공학연구원생명자원센터에서분양받은 S. mutans KCTC3065와 L. casei KCTC3109이다. 균을배양하기위하여사용된배지는한국생명공학연구원생명자원센터에서추천하는배지로, S. mutans는 Brain Heart Infusion (Bacto BHI, BD, USA), L. casei는 Lactobacilli MRS (Difco MRS, BD, USA) 를사용하였다. 분양받은균주는동결건조상태로앰플에보관되어있었으며, l의액체배지를첨가하고골고루현탁하여고체평판배지에옮긴뒤, 활성확인을위해 37 o C에서 48시간배양하였다. 순수배양된균주의 colony를액체배지에서배양하여배양된균액과 100% glycerol의비율을 4:1로맞춰초저온냉동고 ( 76 o C) 에서동결보존하였다. 동결보존된균주의활성을위해초저온냉동고에서꺼낸후실온에서액체상태로녹인뒤, 1 ml의액체배지에넣어 24 시간배양하였으며, 활성된균주의용액을 4 ml의액체배지에넣어 24 시간, 2차계대배양하여실험에사용하였다 실험대상용액본연구에서는각실험균주의성장에미치는영향을평가하기위하여음성, 양성대조군을포함한총 5가지의실험용액을비교하였다. 음성대조군은액체배지 (NC) 를사용하였고, 양성대조군 Table 1. Test solution of this study Experimental Groups Negative control Positive control Experimental solution 1 Experimental solution 2 Experimental solution 3 Test solution Brain Heart Infusion broth (Bacto BHI, BD, USA) for S. mutans Lactobacilli MRS (Difco MRS, BD, USA) for L. casei Chlorhexidine solution (Hexamedine, Bukwang co., KOR) Olive oil (100% of Spanish Extra virgin olive oil, CJ co., KOR) Orange Essential oil (Sweet Orange, Euro Aroma, DEU) Soybean oil (Handmade in domestic soybean oil, CJ co., KOR)

3 26 J Korean Acad Oral Health 2016;40:24-30 은시판되는구강양치용액인클로르헥시딘용액 (CHx) 을사용하였다. 식용시판올리브오일 (OO), 에센셜오일중냉압착추출방법을사용하는오렌지오일 (OEO), 식용시판콩기름 (SO) 을사용하였다 (Table 1). 2. 연구방법 2.1. 실험대상용액의처리액체배지와균체분리를위해 S. mutans와 L. casei의배양액을 4분동안 8,000 rpm에서원심분리하여상층액을제거하였다. 상층액이제거된 S. mutans와 L. casei의 tube에각실험대상용액을 1 ml씩넣어 1분간처리하였다. 충분히현탁된실험대상용액에서균체분리를위해원심분리한뒤, 처리한실험대상용액을제거하였다. 마지막으로잔여처리용액을제거하기위해액체배지로 1분동안세척한후신선한배지로교체하기위해원심분리해주고신선한배지로교체하였다 실험대상용액별균의흡광도값평가각시간에서성장정도를평가하기위해처치군별샘플을준비하였다. 한샘플에는 990 l의액체배지에실험대상용액을처리한균체 10 l를접종하였으며, 각시간별 (0, 4, 8, 16, 24 시간 ) 3개의샘플을용액별로총 15개준비하였다. 37 o C의배양기에서배양하였다. 0, 4, 8, 16, 24 시간에배양기에서꺼내어충분히교반한후 96 well plate에 100 l씩분주한뒤, 실험대상용액별흡광도값평가를위해 96 well plate는흡광도측정기계 (ElISA Reader, Tecan, AUT) 로 620 nm의파장에서측정하였다. 각측정 시점에서의흡광도결과는동일실험군의 0시간에측정된흡광도값을뺀보정된값을분석에활용하였다 (Fig. 1). 3. 결과분석각실험군에서 5번의측정시점간의흡광도의통계적인차이여부를확인하기위하여비모수검정법인 Kruskal-Wallis test와사후분석으로유의수준이보정된 Mann-Whitney U test를적용하였다. 또한각측정시점에서실험군간의흡광도의차이를확인하기위해동일한비모수검정법을적용하였다. 본연구의통계분석은 IBM SPSS 21.0 (SPSS Inc., Chicago, IL, USA) 을사용하였으며, 유의수준은 5% 로설정하였다. 연구성적 1. S. mutans에대한실험용액처리후성장영향미처치군인음성대조군 (NC) 에서는시간이증가함에따라흡광도측정값이점점증가하여대수증식기를거쳐 8시간에흡광도측정값의최고값을나타냈다 (P<0.001). 양성대조군인클로르헥시딘용액 (CHx) 처치군에서는 Kruskal Wallis test 결과, 비록각시간간흡광도의미세한차이가관찰되었지만 (P=0.048), 사후분석에서는어떤집단간에도차이가관찰되지않은것으로보아 S. mutans의성장이지속적으로억제되었다. 오렌지에센셜오일 (OEO) 처치군에서는처치후 8시간까지도흡광도측정값 (0.00) 의변화가없다가 16시간후측정시점부터흡광도 (0.22) 가증가하기시작하여 24시간에서도계속성장하는양 Bacterial cells ( S. mutans, L. casei) were cultivated in each broth medium for 24 hours. Bacterial cells were separated from 1 ml of cultivated broth using centrifugation. The bacterial cells were treated with 1 ml of each test solution for 1 min. The treated bacterial cells were separated using centrifugation. The treated and separated bacterial cells were inoculated to 1 ml of new broth medium (A solution). 10 L of the A solution added to 990 L of new broth medium. Total 15 samples were prepared and cultivated for each time (0, 4, 8, 16, 24 hours). Three samples were selected at each time, and the optical density (OD) was measured three times using each 100 L of cultivated media to examine growth pattern. Total 9 measurements of OD were performed to examine the growth pattern at each time. Fig. 1. experimental procedure for measurement of optical density.

4 27 김세연외 일부식물성오일의 Streptococcus mutans와 Lactobacillus casei 성장에미치는영향 상을나타냈다 (P<0.001). 콩기름 (SO) 처치군에서는처치후 4시간까지흡광도의변화가없다가, 8시간후측정시점부터흡광도 (0.06) 가증가하기시작하여 16시간에최대흡광도 (0.26) 를나타낸후, 24시간후에는흡광도 (0.23) 가감소하였다 (P<0.001). 올리브오일 (OO) 처치군에서는처치직후 (0.00) 와비교하여 4시간측정시점 (0.01) 부터흡광도의통계적인차이가 4시간부터관찰되고 8시간후 (0.18) 에는흡광도가급격히증가하였다 (P<0.001). 각실험용액처치후 4시간시점에서흡광도비교결과, 오렌지에센셜오일처치군과콩기름처치군은양성대조군인클로르헥시딘처치군과동일한성장억제양상을나타냈다 (P<0.001). 처치후 8시간시점에서는오렌지에센셜오일처치군만이클로르헥시딘처치군과동일한성장억제양상을나타냈으며, 콩기름과올리브오일은비록클로르헥시딘보다는못하지만, 미처치한군보다는성장을억제하는것으로나타났다.(P<0.001). 처치후 16시간시점에서는오렌지에센션오일처치군의성장억제양상은클로르헥시딘에미치지못하였으나, 미처치군보다는성장이억제되었다. 이를통해오렌지에센셜오일과콩기름은각각 8시간, 4시간까지클로르헥시딘의효과에상응하는 S. mutans의성장억제능력을확인할수있었으며, 성장시간이지속될수록식물성오일의성장에미치는영향은사라지는것을확인할수있었다 (Table 2, Fig. 2). 2. L. casei에대한실험용액처리후성장영향 L. casei에각실험용액을 1분간처리후 4시간후의흡광도를관찰한결과, 미처치대조군에비해모든실험용액처치군이통계적으로유의하게더낮게나타났으며 (P<0.001), 흡광도는미처치대조군 (0.26) 에비해오렌지에센셜오일 (0.12), 콩기름 (0.15), 올리브오일 (0.17), 클로르헥시딘 (0.21) 이각각 46%, 58%, 65%, 81% 로나타나오렌지에센셜오일이 L. casei의성장을가장억제하는것으로나타났다. 각실험용액처리후 8시간후흡광도를관찰한결과, 흡광도는큰변화를보이며증가하였으며, 대조군 (0.59) 에비해오렌지에센셜오일 (0.49) 과콩기름 (0.49) 만이통계적으로유의하게더낮게나타났으며, 미처치대조군에비해 83% 만이성장하였다. 16시간후에는직전측정시점 (4, 8시간 ) 의각실험군간비교양상과는다른양상으로대조군에비해콩기름, 올리브오일이각각 82%, 88% 로나타나고오렌지에센셜오일은그차이가나타나지않았고, 24시간에는대조군과식물성오일의성장의차이는나타나지않았다. 이러한결과를통해식물성오일은처치후초기시간동안대조군에비해성장을억제하고, 이후시간이지남에따라그효과가빠르게사라짐을알수있었다. 또한클로르헥시딘은식물성오 Table 2. Optical density (620 nm) of S. mutans according to the test solution per time (Mean±SD) N 0 h 4 h 8 h 16 h 24 h P-value NC ±0.00 a 0.02±0.01 ba 0.26±0.02 ca 0.25±0.01 ca 0.23±0.01 da P<0.001 CHx ±0.00 a 0.00±0.00 ab 0.00±0.00 ab 0.00±0.00 ab 0.00±0.01 ab P = OEO ±0.00 a 0.00±0.01 abc 0.00±0.01 ab 0.22±0.01 bc 0.24±0.01 ba P<0.001 OO ±0.00 a 0.01±0.00 bad 0.18±0.01 cc 0.25±0.01 da 0.23±0.01 ea P<0.001 SO ±0.00 a 0.01±0.01 abd 0.06±0.01 bd 0.26±0.01 ca 0.23±0.01 da P<0.001 P-value P=1.000 P<0.001 P<0.001 P<0.001 P<0.001 NC: Negative control, CHx: hexamedine solution, OO: olive oil, EO: essential oil, SO: soybean oil. P-value was determined by nonparametric Kruskal-Wallis test. abcde Indicates difference between measurement time at each group by post hoc Mann-Whitney test. ABCD Indicates difference between groups at each time by post hoc Mann-Whitney test S. mutans 1.2 L. casei nm OD NC CHx OED 0.05 OO SO Time (h) 620 nm OD NC CHx OED 0.2 OO SO Time (h) Fig. 2. The result of growth curve on S. mutans and L. casie for 0, 4, 8, 16, 24 hours. NC: Negative control, CHx: hexamedine solution, LS: listerine zero, OO: olive oil, OEO: essential oil, SO: soybean oil.

5 28 J Korean Acad Oral Health 2016;40:24-30 Table 3. Optical density (620 nm) of L. casei according to the test solution per time (Mean±SD) N 0 h 4 h 8 h 16 h 24 h P-value NC ±0.00 a 0.26±0.01 ba 0.59±0.06 ca 0.94±0.06 da 1.03±0.03 eab P<0.001 CHx ±0.00 a 0.21±0.01 bb 0.54±0.08 cac 0.91±0.08 dab 1.00±0.01 da P<0.001 OEO ±0.00 a 0.12±0.02 bc 0.49±0.09 cbc 0.85±0.09 dacd 1.01±0.02 eab P<0.001 OO ±0.00 a 0.17±0.03 bd 0.56±0.06 cab 0.83±0.06 dbc 1.03±0.02 eb P<0.001 SO ±0.00 a 0.15±0.01 bcd 0.49±0.04 cb 0.77±0.04 dcd 1.01±0.01 eab P<0.001 P-value P=1.000 P<0.001 P<0.001 P<0.001 P<0.05 NC: Negative control, CHx: hexamedine solution, OO: olive oil, EO: essential oil, SO: soybean oil. P-value were determined from nonparametric Kruskal-Wallis test. was evaluated to growth on L casei, abcde Denoted by Mann-Whitney U test. was performed to compare the optical density between hours, ABCD Denoted by Mann-Whitney U test. 일보다 L. casei의성장억제에영향을덜미치는것을확인하였다 (Table 3, Fig. 2). 고안 소득과교육, 생활수준이향상되면서최근건강에대한국민들의관심은점점더높아지고있고, 이러한관심과욕구가 TV 및인터넷, 책등과같은다양한매체를통하여충족되고있다. 더나아가건강에대한관심은구강질병예방및구강건강증진에대한관심에까지이르고있다 20). 구강은다양한미생물이서식하는곳으로구강질환및전신질환이많이발생하는신체부위중하나이며, 구강건강을향상시키기위해미생물의생육조건을조절하는방법이다양하게연구되고개발되고있다 11). 최근약물의오남용으로내성균에대한문제가대두되면서치면세균막형성을억제시킬수있는천연물에서추출한항균물질활용에대한관심이높아지고있다. 따라서, 본연구에서대표적인치아우식원인균인 Streptococcus mutans (S. mutans) 와 Lactobacillus casei (L. casei) 에대한식물성오일의성장에미치는영향을평가하고기존구강양치용액과비교하고자하였다. 본연구에서는오일풀링요법에서주로사용되는식물성오일인올리브오일, 시중에널리유통되고있는대표적인식물성오일인콩기름, 그리고시중에유통되는에센션오일중임의로오렌지에센셜오일을실험군으로선정하였으며, 대표적인항균구강양치액인클로르헥시딘용액과미처치군 ( 배지용액 ) 과비교하였다. S. mutans, L. casei에대한각실험용액의 1분간처치후성장양상은실험용액에따라다르게나타났다. 본연구결과, S. mutans에대한성장억제효과는오렌지에센셜오일, 콩기름, 올리브오일순으로나타났으며, 처치후 8시간에서미처치군의 S. mutans 성장 (100%) 과대비하여오렌지에센셜오일은 0%, 콩기름은 23%, 올리브오일은 69% 성장하였다. 하지만, 처치후 16시간에서는콩기름과올리브오일의성장억제효과는없었으며오렌지에센셜오일만이미약한성장억제효과 (88%) 를나타냈고, 이후시간에서는관찰할수없었다. L. casei에대한성장억제효과는 S. mutans와동일한오렌지에센션오일, 콩기름, 올리브오일순으로나타났으나, 처치후 4시간에서미처치군의성장 (100%) 와비교하여오렌지 에센셜오일은 46%, 콩기름은 58%, 올리브오일은 65% 성장하여, L.casei에대한식물성오일의성장영향은 S. mutans보다적은것을알수있었다. 본연구에서식물성오일은 S. mutans, L. casei의성장에영향을미치는것을확인할수있었다. 오일고유의친유성 (lipophilicity) 은세포벽과세포막에쉽게결합하여세포막의다당류, 지방산, 인지질구조를와해시켜세포의투과성을높여막손상을야기하고세포내이온의소실과세포막전위의소실을야기해세포독성을야기할수있다 21). 이러한오일의성질이본실험균주의초기성장에영향을미쳤으며, 오일의종류와실험균주의세포막특성에따라성장양상이다르게나타났을것으로여겨진다. 본연구에서사용한식물성오일중, 오렌지에센셜오일이실험균주에대한가장큰성장억제효과를확인할수있었다. 에센션오일은오래전부터각종세균, 바이러스, 곰팡이균, 기생충, 곤충등의감염방지와성장억제를목적으로사용되어왔으며, 에센셜오일에포함된터펜 (Terpenes), 터페노이드 (terpenoid), 페놀성방향물질등은항산화제로알려져있다 21). 치면세균막구성균주의에센셜오일의다수의항균효과연구가수행되었으며 11,12,22), 세균의세포벽을파괴하여세포의효소활성을방해하고, 치면세균막구성균주중그람양성균들의군집을막아치면세균막의양을감소시킨다고알려져있다 23). 리스테린은페놀구조의 4가지에센셜오일을주성분으로하는대표적인항균구강양치액이다 24). 본연구에서에센셜오일의고유의항균력과오일특유의친유성이다른식물성오일보다실험균주의성장억제에더큰영향을미쳤을것으로생각된다. 본연구에서양성대조군이었던클로르헥시딘은 S. mutans에대해서는강력한성장억제효과를보인반면, L. casei에대해서는성장억제효과를확인할수없었다. 클로르헥시딘은친수성과소수성을모두갖는 bisbiguanide 계열의양이온성의광범위항균제로음이온성의구강세균의세포벽에부착하여세포막을파괴한다 25). 고농도에서는세포의비가역적손상을일으켜살균 (bactericidal) 효과를나타내며, 저농도에서는세포기능을저해하여정균 (bacteriostatic) 효과를나타낸다고알려져있다 26). 그람음성균보다그람양성균에보다큰효과를보는광범위항균제로, 실험실상에서항균효과보다임상에서치면세균막에대한항균, 성장저해

6 29 김세연외 일부식물성오일의 Streptococcus mutans와 Lactobacillus casei 성장에미치는영향 효과에대한많은연구가보고되었다 27,28). 실험실상에서는주로 Mutans steptococci (MS) 에대한연구가주로수행되었으며, 다른균에비해클로르헥시딘이더특이적으로반응하는것으로알려져있으며, S. mutans에대한최소억제농도는 1-4 g/ml이다 27). 하지만, MS 중에서도 S. sanguinis와같은일부종에서항균력의변이가존재하며, MS 이외의균주에대해서는클로르헥시딘의항균효과는찾아보기힘들다. 방사선조사전후의대상자에게클로르헥시딘사용후, 치면세균막의 MS와 lactobacilli의변화를비교한임상연구에서는 MS는낮은수준을유지한반면, lactobacilii는양이지속증가하여높은수준을유지하였다고보고하였다 29). 이러한선행연구와본연구결과를통해클로르헥시딘은 Lactobacillus casei 에대한항균효과는없다고판단되며, 클로르헥시딘보다식물성오일이처치초기에 L. casei의성장에더영향을미치는것으로사료된다. 하지만 Cleghorn 등 30) 은 ph와클로르헥시딘의농도에따라성장에영향을미친다고보고하였기때문에클로르헥시딘의 L. casei에대한영향에관한추가적인연구가필요할것이다. 본연구에서는치아우식증의대표적인원인균인 S. mutans와 L. casei에대한식물성오일의성장억제효과를실험적으로평가하였지만, 식물성오일을이용한오일풀링의구강내주효과는치은염완화로알려져있다 17). 향후연구에서는치주질환원인균에대한영향을평가하고, 식물성오일의세균에대한직접적인영향을확인하기위하여투과전자현미경 (Transmission Electron Microscope) 등을통한세포벽변화를관찰하는연구가필요할것이다. 또한이러한실험실연구결과를바탕으로실제식물성오일양치후, 세균조성의변화와치은염완화효과를평가하는임상연구가수행될필요가있다. 결론 본연구는식물성오일의구강양치가구강세균에미치는영향을알아보기위하여, 대표적인치아우식유발균주인 Streptococcus mutans와 Lactobacillus casei를실험실상에서배양한후, 오렌지에센셜오일, 올리브오일, 콩기름을 1분동안노출시킨후, 0, 4, 8, 16, 24시간후에흡광도평가를통해성장을비교하여다음과같은결과를얻었다. 1. Streptococcus mutans에대하여식물성오일은성장억제효과를나타냈으며, 오렌지에센셜오일 > 콩기름 > 올리브오일순으로성장억제효과가있었다. 2. 오렌지에센셜오일과콩기름은각각 8시간, 4시간까지클로르헥시딘의효과에상응하는 S. mutans의성장억제능력을나타냈다. 3. Lactobacillus casei에대하여식물성오일은 4시간까지대조군에비해유의한성장억제효과를나타냈으며, 오렌지에센셜오일이가장큰효과를나타냈다. 4. 식물성오일은클로르헥시딘보다 Lactobacillus casei에대해처치후 4시간까지성장억제효과가크게나타났다. 본실험실연구를통해식물성오일이 Streptococcus mutans 와 Lactobacillus casei에대한성장억제효과가있음을확인하였으며, 올리브오일, 콩기름보다오렌지에센셜오일이가장큰성장억제효과가있음을확인하였다. 이러한연구결과를바탕으로추후연구를통해식물성오일의구강세균성장억제기전및임상에서의치면세균막의성장억제에관한연구가필요할것이다. References 1. Paster BJ, Olsen I, Aas JA, Dewhirst FE. The breadth of bacterial diversity in the human periodontal pocket and other oral sites. Periodontol ;42: Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev : Song YJ. Classification of Periodontal Pathogens Based on Genetic Specificity[master s thesis]. Seoul: Seoul National University;2014. [Korean] 4. Listgarten MA. The structure of dental plaque. Periodontol 2000, 1994;5: Kim EJ. Study on the reduction effects on oral microorganisms through the different methods of controlling dental plaque[master s thesis]seoul:dankook University;2003. [Korean] 6. Filoche SK, Soma K, Sissons CH. Antimicrobial effects of essential oils in combination with chlorhexidine digluconate. Oral Microbiol Immunol 2005;20: Palombo EA. Traditional medicinal plant extracts and natural products with activity against oral bacteria: potential application in the prevention and treatment of oral diseases. Evid Based Complement Alternat Med 2011;2011: Trahan L. Xylitol: a review of its action on mutans streptococci and dental plaque-its clinical significance. Int Dent J 1995;45: Hwang JK, Shim JS, Baek NI, Pyun YR. Xanthorrhizol: a potential antibacterial agent from Curcuma xanthorrhiza against Streptococcus mutans. Planta Med 2000;66: Baratta MT, Dorman HJ, Deans SG, Figueiredo AC, Barroso JG, Ruberto G. Antimicrobial and antioxidant properties of some commercial essential oils. Flavour Fragr J 1998;13: Lee SY, Kim JK, Baek BJ, Yang YM, Lee KY, Lee YH, et al. Antibacterial effect of essential oils on oral bacteria. J Korean Acad Pediatr Dent 2009;36: Takarada K, Kimizuka R, Takahashi N, Honma K, Okuda K, Kato T. A comparison of the antibacterial efficacies of essential oils against oral pathogens. Oral Microbiol Immunol 2004;19: Lim DJ. Extraction of natural citrus by SC-CO2 and their pharmacological effect on aroma therapy[docter of philosophy] Busan:Pukyong National University;2014.[Korean] 14. Fife B. Oil pulling therapy : detoxifying and healing the body through oral cleansing. Seoul:Piccadilly Books;2013: Tomar P, Hongal S, Jain M, Rana K, Saxena V. Oil Pulling and Oral Health: A Review. IJSS 2014;1: Asokan S, Rathan J, Muthu MS, Rathna PV, Emmadi P, Raghuraman, et al. Effect of oil pulling on Streptococcus mutans count in plaque and saliva using Dentocult SM Strip mutans test: a randomized, controlled, triple-blind study. J Indian Soc Pedod Prev Dent 2008;26: Amith HV, Ankola AV, Nagesh L. Effect of oil pulling on plaque and gingivitis. J Oral Health Comm Dent 2007;1: Kim JH. Antiocidant and antimicrobial effects of lemon and eucalyptus essential oils[master s thesis]. Seoul: Konkuk University;2011. [Korean] 19. Kang SM. Effect of essential oil mouthrinse on oral squamous epithelial cells (KB Cells)[master s thesis]. Seoul: Chung-ang Univer-

7 30 J Korean Acad Oral Health 2016;40:24-30 sity;2010. [Korean] 20. Kim SH, Lim SA, Park SJ, Kim DK. Assessment oral Health-related quality of life using the Oral Health Impact Profile(OHIP). J Korean Acad Oral Health 2004;28: Bakkali F, Averbeck S, Averbeck D, Idaomar M. Biological effects of essential oils-a review. Food Chem Toxicol 2008;46: Shapiro S, Meier A, Guggenheim B. The antimicrobial activity of essential oils and essential oil components towards oral bacteria. Oral Microbiol Immunol 1994;9: Ouhayoun JP. Penetrating the plaque biofilm: impact of essential oil mouthwash. J Clin Periodontol 2003;30: Charles CH, Mostler KM, Bartels LL, Mankodi SM. Comparative antiplaque and antigingivitis effectiveness of a chlorhexidine and an essential oil mouthrinse: 6-month clinical trial. J Clin Periodontol 2004 Oct;31(10): Scheie AA, Rukke HV, Perterson FC. Are antibacterials necessary in caries prophylaxis? In:Fejerskov O, Nyvad B, Kidd E. Dental caries: the disease and its clinical management. 3rd ed. Oxford: Wiley Blackwell; 2015: Hugo WB, Longworth AR. Some aspects of the mode of action of chlorhexidine. J Pharm Phamacol 1964;16: Emilson CG. Susceptibility of various microorganisms to chlorhexidine. Scand J Dent Res 1977;85: Emilson CG. Outlook for Hibitane in dental caries. J Clin Periodontol 1977 Dec;4: Joyston-Bechal S, Hayes K. Davenport ES, Hardie JM. Caries incidence, mutans streptococci and lactobacilli in irradiated patients during a 12-month preventive programme using chlorhexidine and fluoride. Caries Res 1992;26: Cleghorn B, Bowden GH. The effect of ph on the sensitivity of species of Lactobacillus to chlorhexidine and the antibiotics minocycline and spiramycin. J Dent Res 1989;68:

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012임수진 Received : 2012. 11. 27 Reviewed : 2012. 12. 10 Accepted : 2012. 12. 12 A Clinical Study on Effect of Electro-acupuncture Treatment for Low Back Pain and Radicular Pain in Patients Diagnosed with Lumbar