Journal of Dental Hygiene Science Vol. 8, No. 4 pp. 367~373 (008) 황금 (Scutellaria baicalensis) 추출물에의한 Streptococcus mutans 의항균및부착억제효과 Á½ xá«x Á½ Á½ Áw w w w, 1 w e Effects of Antibacteria and Adhesive Inhibition of Scutellaria baicalensis Extract on Streptococcus mutans Jong-Yoon Paek, Young-Hyun Kim, Hyun-Jeoung Kwon 1, Eun-Nim Kim, Wan-Jong Kim and Man-Deuk Han Department of Biology, Soonchunhyang University, Asan, Chungnam, 336-745, Korea 1 Department of Dental hygiene, Shinheoung College, Uijeongbu, Gyeonggi, 480-701, Korea "CTUSBDU The natural products are used to be development of new antibacterial substances against human pathogenic bacteria. Adherence to the tooth surface by S. mutans is an important step in initiation of dental caries. This study was to examine antibacterial activity and anti-adhesive effect of Scutellaria baicalensis extract against S. mutans. Extracts of S. baicalensis were tested for antimicrobial activities by paper disc methods and radial diffusion assay methods, and bacterial adherence assay using 3 type of hydroxyapatite. The antibacterial level of ethyl acetate extract, IPK-3 on the growth of S. mutans was 15 mg/ml of minimum inhibitory concentration (MIC). The maximum growth of S. mutans in medium added with IPK-3 extract (50 mg/ml) was delayed to 30 hr, while the highest at 4 hr in control medium. The ph values of the control medium was 5.63 at 18 hr, but the media supplemented with IPK-3 extract was ph 6.50 at 1 hr. In adhesive inhibition assay, S. mutans was labelled with the fluorescent indicator DAPI and measured with fluorescence microscope. Adhesion of S. mutans on hydroxyapatite beads was inhibited by IPK-3 extracts. These results suggest that S. baicalensis extract can be used as an effective material for antibacterial activity and adhesive inhibition against S. mutans.,fz XPSET Streptococcus mutans, Antibacterial activity, Bacterial adhesion, Scutellaria baicalensis 서 론 w y y w ³ x e ƒ š 1). t e ³ Streptococcus mutans ) Lactobacillus casei, Actinomyces viscosus, S. sanguis, S. milleri, S. salivarius, S. rattus, S. cricetus 3,4). S. mutans Gram ³ m x» w e t w. e t ³ w S. mutans w s w» w, w glucosyltransferase (GTF) z w w jš glucan Corresponding author Tel: +81-41-530-470 Fax: +8-41-530-156 E-mail: mdhan@sch.ac.kr w w. glucan w e w w wš e j w. w w kz e w 3). e w w z ù w e w w w³ y š 5). w w alkaloid, flavonoid, phenolic compound, quinone 6). ù w w w³z ƒ û y. e w w tannin e 8) z ƒ š š7) Chinese black tea, Andrographis paniculata, Cassia alata w e w z ƒ š š 9). y (, Scutellaria baicalensis G.) w,,
Antibacteria and Adhesive Inhibition of S. baicalensis Extract on S. mutans sw my ãt w w t 0~60 cm wš, 10). š,»e, mx,, k k 11) baicalein, baicalin, wohonin, wogonoside, neobaicalein w, sitosterol sw 1). y w³ 13), w, w z,w k z 14). y w Cho 15) y w³p y œ ³ w³ šw š, Moon 16) e ³ wù S. mutans OMZ176 w y w³ š šw. š Lee 17) y Staphylococcus aureus Bacillus cereus w, Choi 18) Pseudomonas aeruginosa w w³ š š w. w w y w ³ w w³y (minimal inhibitory concentration, MIC) d wš, y S. mutans e w hydroxyapatite beads w z mw e t e ³ ƒ y w. 재료및방법 1. y w y k w w,» y w w. w y w z, 3 methanol ƒw 7 e g methanol aspirator (EYELA, A-3S, Tokyo Rikakikai Co.) w. methanol z»(rotavapor, Bchi RE-111, Switzerland) w 80 o C w (Oilbath, Bchi B-485, Switzerland) g. Methanol (IPK-6) 40 ml¾ w z» w hexane (IPK-1), chloroform (IPK- ), ethyl acetate (IPK-3), n-butanol (IPK-4), (IPK-5) ƒ z w. w w.. ³ w e³ Streptococcus mutans ATCC 5175 ³ w œw l (KCTC) w. ³ brain heart infusion (BHI, Difco, USA) 1~ z ³w 37 o C CO ) (Thermo electron corporation, USA) w. w ³y Meller hinton agar (MHA, Difco, USA) w. 3. w³y d y» z 1 w³y paper disc w. w S. mutans ³ BHI w 15~18 y y g w³ x MHA agar ³ 50 µl w. ³ filter paper disc (8 mm : 1.5 mm, Toyo Roshi Kaisha, Ltd., Japan) k z, y w. y» 1/ 4z w 1000 mg/ml, 500 mg/ml, 50 mg/ml, 15 mg/ml ƒ w, disc 50 µl g. Disc k 10 e g k z, S. mutans 37 o C CO incubator(5% CO ) 18~4 g. z disc inhibition zone diameter (mm) j» vernier caliper (Mitutoyo Co., Japan) d w ƒ w ³ y d w. 4. d Paper disc method 1 w³ y wš w y ethyl acetate (IPK-3) radial diffusion assay mw (MIC) d w. ³ tryptic soy borth (TSB, Bacto, USA) w y y k z, 37 o C CO incubator 18~4 w x tryptic soy agar w x w. x w, ³ O.D. 0.45 (4 x 10 6 cells/ml) underlay 10 ml ³ 45 µl w yww z, square petri dish (Falcon, USA) w 15 ew. 0.4 mm š y IPK-3 w. 1000 mg/ml w ƒ» 1/ 17 ¾ w. w z, 37 o C CO ) 18~4 w MIC d w. 5. S. mutans y y BHI y IPK-3 50 mg/ml ƒ w z ³ 1 10 cells/ml 7 w. z 37 o C CO ) 18~4 w BHI blank wš x IPK-3 ƒw BHI blank w Multi functional microplate reader (BMG Labtech, Germany) w 540 nm Ÿ d w. w ph y IPK-3 50 mg/ml ƒwš ph y 6 d w. phenol-sulfuric method, BCA protein assay kit(pierce Co.) w d w, w glucan z
Journal of Dental Hygiene Science Vol. 8, No. 4, pp. 367~373 (008). IPK-3 ƒ w d w. 6. Hydroxyapatite beads(ha) x S. mutans 19) ³ hydroxyapatite adhesion assay Gaines assay w xw. k w w ƒ conical tube w z, 4 C o 15,000 rpm 0 (Mega 17R, Hanil, Korea)w. k w 60 C 30 ƒ w z y y k z o - 0 C w x o w. ³ hydroxyapatite beads t w : (a) Macro-prep ceramic hydroxyapatite type II 80 µm (Bio-Rad Laboratories, Hercules, CA); (b) Fluka crude hydroxylapatite (Fluka cat. No. 13); (c) Hydroxyapatite (H-05, Sigma, USA). ƒ 30 mg wš 0. M NaOH 10 ml 10 w t y y jš w w. z wš bead 3 w. hydroxyapatite beads ph 7.0 y jš w w. hydroxyapatite beads k 1ml š 37 C 60 k gq g o (S-HA). gq S-HA 0.01 M potassium phosphate buffered saline (PBS, ph7.) z w z, BHI 1ml w. S-HA 96 well plate 10 µl wš, y IPK-3 15 mg/ml ƒw S. mutans ³ 1 10 cells/ml 7 w. 96 well plate CO incubator w 90, 1, 4 S-HA w. e k e-tube wš PBS 1z w z, š w S-HA DAPI (4',6-diamidino--phenylindole, Sigma, USA) ƒ w 37 C 1 o g. 1 k S-HA PBS 1z wš 0 µl w fluorescence microscope (Olympus DP71, Japan) w. 결과및고찰 1. y w» y hexane, chloroform, ethyl acetate, n-butanol» z w, Table 1 butanol (IPK-4) 4.15%, ethyl acetate (IPK-3) 1.19%, hexane (IPK-1) 0.65%, chloroform (IPK-) 0.39%. (IPK-5) ƒ 4.45% ùkû.. w³y d y» z ³ ƒ w³y» w MHA ³ wš paper disc 1,000 mg/ml, 100 mg/ml, 10 mg/ml ƒw 37 o C CO ) w w. x Table S. mutans³ 1,000 mg/ml w³y ùk ù butanol (IPK-4) (IPK-5) w³ y ùk. p, IPK-3» w w³y ƒ j ùkû, IPK- ³ w w³y. 100 mg/ml IPK-3 ƒ. 5BCMF Average yield of organic solvent fractions extracted from S. baicalensis Dried herbal weight (g) Solvent fractions Extract weight ± S.D 1) Yield (%) ) ± S.D IPK-1 0.65 ± 0.54 0.65 ± 0.54 IPK- 0.39 ± 0. 0.39 ± 0. 100 IPK-3 1.19 ± 0.45 1.19 ± 0.45 IPK-4 4.15 ± 0.75 4.15 ± 0.75 IPK-5 4.45 ± 0.79 4.45 ± 0.79 1) S.D = Standard Deviation ) Yield(%) = Dried weight of extract fraction (g) 100 Dried weight of S. baicalensis (g) 5BCMF Antibacterial effects of organic solvent fractions from S. baicalensis on pathogenic bacteria. Extract of S. baicalensis 1) Bacterial Strain 1000 mg/ml 100 mg/ml 10 mg/ml H ) C E B W H C E B W H C E B W Streptococcus mutans ATCC 5175 + + ++ - - - - + - - - - - - - 1) Concentration of organic solvent extract from S. baicalensis. ) Fractionated procedure of organic solvent from S. baicalensis : H (hexane extract), C (chloroform extract), E (ethyl acetate extract), B (n-butanol extract), W (water extract)
Antibacteria and Adhesive Inhibition of S. baicalensis Extract on S. mutans 3. d Paper disc w³y ùk y IPK-3 w d w» w radial diffusion assay xw. S. mutans w d w, IPK-3 15 mg/ml (Fig. 1). 4. y S. mutans e w 1) e w y IPK-3 S. mutansƒ w e z d w» w IPK-3 50 mg/ml ƒw z ³ Multi functional microplate reader (BMG Labtech, Germany) d w Fig.. 4 z Ÿ 1.17 'JH Minimal inhibitory concentrations(mic) of the ethyl acetate extract (IPK-3) from S. baicalensis on S. mutans. Concentrations of IPK-3 extract were A) 1000 mg/ml, B) 500 mg/ml, C) 50 mg/ml, D) 15 mg/ml, E) 6.5 mg/ml, F) 31.5 mg/ml, G) 15.63 mg/ml, H) 7.81 mg/ml, I)3.90 mg/ml, J) 1.95 mg/ml, K) 0.97 mg/ml, L) 0.48 mg/ml, M) 0.4 mg/ml, N) 0.1 mg/ml, O) 0.06 mg/ml, P) 0.03 mg/ml, and Q) 0.015 mg/ml. y IPK-3 50 mg/ml n w 30 Ÿ ƒ 0.49 ùk. mw y IPK-3 e ³ S. mutans w š j z ƒ. ) ph y e w y IPK-3 50mg/ml S. mutans ƒw ph y e z Fig. 3.» ph 6.8 w, 18 ph 5.63 w y z w ph w. IPK-3 n 1 ¾ w ph 6.5 w ù z w. y IPK-3 S. mutans³ w ph y. 3) k y y e w y IPK-3 S. mutans ƒw z k y y» w 6 d w.» k y ml 0.56 mg k y 18 ù z 0.4 mg/ ml ù 50 mg ƒ ƒ y IPK-3 n 0.5 mg/ml w 4 ù z k y w y w (Fig. 4). 4) y e w w w, x, k, w (p ) w y. y BHI š ³ g, y y w ³ y y w. y IPK-3 ƒ S. mutans wš yw d w, 0.5 mg/ml. ù ³ 4.31 mg/ml š, y IPK-3 'JH The growth curve of S. mutans in culture medium by addition of ethyl acetate fraction (IPK-3) extracted from S. baicalensis. ; control, ; 50 mg/ml of IPK-3. 'JH The changes of ph in S. mutans culture medium by addition of ethyl acetate fraction (IPK-3) extracted from S. baicalensis. ; control, ; 50 mg/ml of IPK-3.
Journal of Dental Hygiene Science Vol. 8, No. 4, pp. 367~373 (008) 'JH The changes of total carbohydrate in S. mutans culture medium by addition of ethyl acetate fraction (IPK-3) extracted from S. baicalensis. ; control, ; 50 mg/ml of IPK-3. 'JH The changes of total protein in S. mutans culture medium by addition of ethyl acetate fraction (IPK-3) extracted from S. baicalensis. ; control, ; 50 mg/ml of IPK-3. n.51 mg/ml y (Fig. 5). 5. S. mutans z S. mutans e w e ³ x w ƒ w w w. y IPK-3 50 mg/ml S. mutans n w w y w» w y Fig. 6. 18 500 mg/100 ml ù, y n 80 mg/100 ml ƒ y. w19) w w S. mutans ³ glucan š. y e ³ x w w S. mutans w y w w ƒ. 'JH The polysaccharide contents in S. mutans culture medium by addition of ethyl acetate fraction (IPK-3) extracted from S. baicalensis. ; control, ; 50 mg/ml of IPK-3. 6. Hydroxyapatite(HA) S. mutans z S-HA w x Macro-prep ceramic hydroxyapatite type II 80 (Bio-Rad Laboratories, Hercules, CA), Fluka crude hydroxylapatite (Fluka cat. No. 13), Hydroxyapatite (Sigma, USA) 3 hydroxyapatite beads w. y ethyl acetate S-HA w S. mutans ³ z ƒ» w 15 mg/ ml xw Fig. 7~Fig. 9. Fig. 7 Macro-prep ceramic hydroxyapatite type II 80 µm (Bio-Rad Laboratories, Hercules, CA) t w xw Fig. 8 Fluka crude hydroxylapatite (Fluka cat. No. 13), Fig. 9 Hydroxyapatite (Sigma, USA) t w w. ³ S-HA l z¾» w 0, 90, 1, 4 S-HA wš DAPI w w. y IPK-3 ƒ S- 'JH Fluorescent microscope image ( 400 magnification) illustrating S. mutans labelled with DAPI, hydroxyapatite under transmission light and an image overlay illustrating S. mutans adhering to hydroxylapatite (Bio-Rad Co.). (A) control. (B) HA beads treated with 15mg/ml of IPK-3 extracted from S. baicalensis.
Antibacteria and Adhesive Inhibition of S. baicalensis Extract on S. mutans 'JH Fluorescent microscope image ( 400 magnification) illustrating S. mutans labelled with DAPI, hydroxyapatite under transmission light and an image overlay illustrating S. mutans adhering to hydroxylapatite (Fluka Co. product). (A) control. (B) HA beads treated with 15 mg/ml of IPK-3 extracted from S. baicalensis. w y ethyl acetate (IPK-3) 15 mg/ml.. y IPK-3 50 mg/ml S. mutans n w ³. 3. y IPK-3 50 mg/ml S. mutans n w, ph y 18 ph 5.63 w y y IPK-3 50 mg/ml 6.50 w phƒ yƒ. 4. y IPK-3 ƒw k y, ³ y w û. 5. ³ xÿ (DAPI) w S-HA y w, w S. mutans hydroxyapatite(ha) ƒ ƒ ù, y IPK-3 ƒ x S-HA ³. y IPK-3 (ethyl acetate ) e ³ S. mutans w w³y z hydroxyapatite z. 감사의글 'JH Fluorescent microscope image ( 400 magnification) illustrating S. mutans labelled with DAPI, hydroxyapatite under transmission light and an image overlay illustrating S. mutans adhering to hydroxylapatite (Sigma Co. product). (A) control. (B) HA beads treated with 15 mg/ml of IPK-3 extracted from S. baicalensis HA w S. mutans w ³ w. 0)ƒ Gaines xw w y w. w S. mutans HA k DMSO ³ w. y IPK-3 0% DMSO w x y w ù, DMSO w DMSO w. 요 약 y l» e ³ S. mutans w w³y, y y, š w. 1. e t ³ S. mutans x l (MOCIE- RIC060605),. 참고문헌 1. Allaker RP, Ian Douglas CW: Novel anti-microbial therapies for dental plaque-related diseases. Int J Antimicrob Agents 33: 8-13, 009.. Koga T, Askawa H, Okahashi N, Hamada S: Sucrosedependent cell adherence and cariogenicity of serotype c Streptococcus mutans. J Gen Microbiol 10: 873-883, 1986. 3. Hamada S, Slade HD: Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol Rev 35: 331-384, 1980. 4. Slots J, Taubman MA: Contemporary oral microbiology and immunology. Missouri. Mosby-Year Book pp. 377-44, 199. 5. Cohen SH, Morita MM, Bradford M: A-seven year experience with methicillin-resistant Staphylococcus aureus. Am J Med 91: 33S-37S, 1991. 6. Choi I, Cho JY, Lim SC: Antimicrobial activity of medicinal herbs against Staphylococcus aureus. J Kor Plant Res 19(4): 491-496, 006 7. Otake S, Makimura M, Kuroki T, Nishibara Y, Hirasawa M: Anticaries effects of polyphenolic compounds from Japanese green tea. Caries Res 5: 438-443, 1991. 8. Nakahara K, Kawabata S, Ono H, Ogura K, Tanaka T, Ooshima T, Hamada S: Inhibitory effect of oolong tea polyphenols on glycosyltransferases of mutans Streptococci.
Journal of Dental Hygiene Science Vol. 8, No. 4, pp. 367~373 (008) Appl and environ microbiol 6: 09-13, 1997. 9. Limsong J, Benjavongkulchai E, Kuvatanasuchati J: Inhibitory effect of some herbal extracts on adherence of Streptococcus mutans. J Ethnopharmacol 9: 81-89, 004. 10. Choi MR, Lee JS, Lim HS: Changes in physiological activities of Scutellariae baicalensis by heating. J Life Sci 17(10): 1381-1386, 007. 11. Kim YH, Park YS: Effect of Scutellaria baicalensis water extract on antioxidative activity and epidermal thickness in DNCB-induced allergic contact dermatitis animal model. J Korean Soc Food Sci Nutr 35(5): 543-548, 006. 1. Rhee JS, Woo ER, Kim NH, Lee EJ, An DK, Lee JH, Park SK, Park HK: A study on qualitative and quantitative analysis of major ingredients in Scutellariae radix. Anal Sci Technol 10(): 91-104, 1997. 13. Bae JH, Lee MJ, Lee SM: Antimicrobial effect of Cutellaria Baicalensis george extracts on Food-Borne pathogens. J Kor Micribiol Biotechnol 33(1): 35-40, 005. 14. Woo IT, Park KN, Lee SH: Antimicrobial activities of Scutellaria baicalensis Georgi against various pathogens and spoilage bacterial isolated from Tofu. J Kor Soc Food Sci Nutr 36(4): 470-475, 007. 15. Cho SH, Kim YR: Antimicrobial characteristics of Scutellariae Radix extract. J Kor Soc Food Sci Nutr 30(5): 964-968, 001. 16. Moon YH, Lee YH, Min BS, Bae KH: Antibacterial constituents from Scutellariae Radix against Streptococcous mutans OMZ176. J Kor Phar 8(3): 99-103, 1997 17. Lee CK, Seo JJ: Antimicrobial activity of Whangkumtang extract and Scutellariae Radix extract on the Food-Borne pathogens. J Kor Soc Food Sci Nutr 34(10): 1606-1610, 005. 18. Choi OK, Kim YS, Cho GS, Sung CK: Screening for antimicrobial activity from korean plants. J Kor Food Sci Nutr 5(4): 300-306, 00. 19. Han MD: Lee JW, Ra SJ, Lee ES, Jeon ES. Chemical properties of Streptococcus mutans KCTC 306 polysaccharide purified by fractions. J Kor Acad Dent Health 4(3): 59-70, 000. 0. Gaines S, James TC, Folan M, Baird AW, O Farrelly C: A novel spectrofluorometric microassay for Streptococcus mutans adherence to hydroxyapatite. J Microbiol Meth 54: 315-33, 003. (Received October 3, 008; Accepted December, 008)