Effects of Lignum sappan Extract on the Growth of Streptococcus mutans KCTC 3065

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(1)Journal of Dental Hygiene Science Vol. 4, No. 3, pp. 127~ 131 (2004). ²Ï ª³b Streptococcus mutans KCTC 3065~ Ëö ~º Î. *B? ÁRò{ †.

(2). ÿ¦Ö& ~* BÂ& ~* 1. Effects of Lignum sappan Extract on the Growth of Streptococcus mutans KCTC 3065 †. Eun-Sook Jeon and Man-Deuk Han1 Dept. of Dental Hygiene, Dongpusan College, Busan 612-715, Korea Dept. of Dental Hygiene, Gimcheon College, Gimcheon, Gyeongbuk 740-704, Korea. 1. " "#! #!#. This study was to study the antibacterial effect of Lignum sappan extract on S. mutans growth. The antibacterial activities of Lignum sappan's crude extract was measured 17.3 mm at 20 mg/ml concentration. The growth of S. mutans in control medium was the highest at 8hr, while the media of Lignum sappan extract added-medium (2 mg/ml) showed maximum growth at 16hr. The pH values of the control media was 5.08 at 8hr, but the media supplemented with Lignum sappan extract was 6.69 at 8hr. The amounts of total carbohydrate of the control media was 0.81 mg/ml at 8hr, but the media supplemented with Lignum sappan extract was 2.06 mg/ml at 8hr. In the protein change of culture medium, the control culture broth and the cultures supplemented with Lignum sappan extract was 8.39 mg/ml and 12.3 mg/ml at 8hr, respectively. The S. mutans polysaccharide contents of the control media and the media supplemented with Lignum sappan extract was 300 mg/100ml and 60 mg/100 ml at 8hr, respectively. ,FZ XP XPSE SETT Streptococcus mutans, antibacterial effect, Lignum sappan extract. B. > ®j © . " Â>îö & S. mutans~ Î"ö & · Úæ ® . * f J¶, J>F ºÂ>j >ºÂ ê S. mutansö & Î"¢ ¦ï~&b, FJ; f ¶ãN, Ö«¶ 5 æö ~ S. mutans~ Ã ÛBÎ"ö & : ®, öê flavonoid , £Ò 5 Ëò , &7 , . >î B : ® . ²Ï(Lignum sappan)f "ö ³~º Ï~ £ÒB 7 ¦ªò ~ ÒÏ~ .*Ã~ ~ò, êÛB, úã /ê 5 æ;ç ö ® . FB &N >îº brazilin, homoisoflavonoids, dibenz[b,d]oxocins (protosappanins A, B and C, protosappanin E-1 and E-2) rJ^ ® . ÚöBº S. mutansö & ²Ï~ º ~ >Ú ®æ pf ;, ²Ï ºÂ>~ Öz Î" , ²Ï ºÂ > ®¦N >~ WË" ªêG ö ~º 'Ë B : ® . ö º ÚöB FÛ>º ²Ïb¦V > ºÂ >j ªÒ ê, S. mutansö & Î"¢ paper disc method ¦ï~, S. mutans~ &Òö ~º 'Ëj ï& ~V *~ ËF, pHæz, Wî æz, C ê>z>~. . ~jÖf ~^ï(plaque)Ú ^ö ~ Wª ª >ÚB, r ¾Jº Öb ~j î²> ~î Ú FVWª Ï>º ";j ö . ~jÖ &N^bº Streptococci(S. mutans), Lactobacilli(L. casei), Actinomyces (A. biscosus) S. mutansº ~j ïÖ 5 .VÖö &~, L. caseiº ²f¾ Ö 5 ê¯Ö÷²ö A. viscosusº ~"Öö &~º ©b rJ^ ® . ß® S. mutansº ¶j &Ò~ ®ÏW ^ ~¢ W~º º ~jö ¦O~ ôf ·~ ~^ïj ;W~² B . $ S. mutansº ~^ï ;W ^ ¶j Ö b *~Êº ËK" ~¢ j» vº ËK Ú¾. . f ?f F S. mutansº ~jÖ ö 7öBê &Ë 7º~² *æ ® . V¢B ;ÚöB L. caseif S. mutans¢ ;^~ WËj Ò ÛB > ®. ~jÖÃj ² 6²Ê¾ z ç~ ê¯j ïj. 2). 3). 4). 5). 6). 7). 8). 9). 10-12). 1). 13). 14). †. Corresponding author Tel: 051-540-3859 Fax: 051-540-3643 E-mail: [email protected].

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(5) . Effects of Lignum sappan Extract on the Growth of Streptococcus mutans KCTC 3065. æz 5 ~ Wö ~º Î" ö &~ «~¶ ~& . Òò5O» 1.. þÒò. þö ÒÏ ²Ïf BÞ ãÿËöB ç~ © j \«~ ÒÏ~& .. þO» 1) ºÂO» ²Ïj '. V ªê~ 50 g O ïï ê â'2 ¢Êö I ò& ÆVê Ã~> 1 l¢ & r 100 CöB ~~ï'V¢ Ï~ >»çöB 3* ÿn ºÂ~ & . ¢ * j á ºÒº ºÂ . ;j ö j áf ê j ²*³»V¢ ÒÏ ~ 6{ Î ê ò¢ B~& (Scheme 1). 2.. o. j ÒÏ~& . " 5 V·. þö ÒÏB "º Î&L >öB ª· Af S. mutans KCTC 3065¢ ÒÏ~&b, 5 ê&V·f brain heart infusion(BHI) Væö 37 CöB 24* ÿn 6 V¶öB 6V V·~& . 4) W G; ²Ï~ W ¦ïf paper disc methodö ~~ G; ~&b , S. mutans " 1 loop¢ 10 ml BHI brothö 7« ê 37 CöB 18~20* 6V¶öB 6V V·~ Ï~& . B Müller hinton agar Væ¢ petri dishö 15 mlO ª"~ ;z Î ê, S. mutans V· 50 µl¢ 7«~ ¢~² êö~& . Þ ²Ï ºÂ> 400 mgj > 1 mlö j*® paper disc(6 mm)ö 50 µl O W> ê 8 . B discº þÏ ï6Væ Rö ¹j &O Î r, 37 CöB 24*ÿn 6V V·~& . ºÂ>ö & Wf paper disc "æ~ clear zone çã(mm)j G;~ 6~& . 5) ²&³ê G; ²&³ê(MIC, minimum inhibitory concentration)º paper disc methodö ~~ G;~&b , S. mutans " 1 loop¢ 10 ml BHI brothö 7« ê 37 CöB 18~20 * 6V'b V·~ Ï~& . B Müller hinton agar Væ¢ petri dishö 15 ml O ª"~ wÎ ê, S. mutans V· 50 µl¢ 7«~ ¢~² êö~& . ²Ï ºÂ>f . ³ê¢ 10 mg/ml 2V ³ \C ê 37 CöB 24* ÿn 6V V·~ G nb V~ ÃB ³êf ÛBB ³ê Ö;~& . 6) ËF G; ËFf S. mutans " 1 loop¢ 10 ml BHI brothö 7« ê 37 CöB 18~20* 6V'b V·~ Wz Î 50 µlf ²Ï ºÂ> 50 µl¢ BHI broth 10 mlö "«~& . 37 CöB 6V V·~B *ê j~ ~ Gj spectrophotometer¢ ÏB W%ê(600 nm)¢ G;~& . 7) pH G; pH æzº S. mutans " 1 loop¢ 10 ml BHI brothö 7« ê 37 CöB 18~20* 6V V·~ Wz Î 50 µlf ²Ï ºÂ> 50 µl¢ BHI broth 10 mlö "« ê 37 CöB 6V V·~B *ê j~ pH meter¢ ÏB G;~& . 8) ê>z> ;ï G; VæÚ ê>z> æzº phenol sulfuric acid O»j Ï ~& . R&~ Bº ¦ïFj ÒV *~ R&>î D-glucose¢ ÒÏ~&b, glucose¢ 100 µl/ml stock solutionj ò Ã~> \C~ êê R&j ò î. . ¦ïF~ ·Wf 20 mlÏ þö êê R& 1 mlj &~, Vö 80% phenol 25 µlf ê Ö 2.5 mlj Î &~, &öº R& & Ã~> 1 mlj &® . $ sample 1 mlf Vö 80% phenol 25 µl, ê Ö 2.5 ml j Î&® . þj ¾ b ê 30 CöB 20ª* >w (Corning Co., U.S.A) 3). o. 15). o. o. 15). o. o. o. o. o. o. 4DIFNF Standard extract preparation from Lignum sappan.. 2) £ 5 VV. þö ÒÏ £bº BCA(Piece Co.), BSA(Piece Co.)j ÒÏ®b, S. mutans V·j * Væº brain heart infusion(Difco, U.S.A)j ÒÏ~& . VVº pH meter (Istek Co., Korea), UV/Vis spectrophotometer(Shimadzu Co., Japan), centrifuge(Hanil Co., Korea), 6V¶(BBL Co.), incubator(Dongyang Co., Korea), clean bench(Sangwoo Co., Korea), voltex(Dongyang Co., Korea), magnetic stirrer. o.

(6) Journal of Dental Hygiene Science Vol. 4, No. 3, pp. 127~131 (2004). * Nb ï' r R& 5 &~ W%ê¢ 490 nmöB G;® . 9) Wî ;ï G; VæÚ Wî æzº BCA kit¢ Ï~ G;~& . R &~ Bº ¦ïFj ÒV *~ R&>î BSA (Bovine Serum albumine)j Ï~& . BSA¢ 1 mg/ml stock solutionj ò , Ã~> \C~ êê R&j ò î . ¦ïF~ ·Wf 20 mlÏ þö BCA b (100:2)j 4 ml &~, Vö êê R&j '' 0.1 ml O Î&~, &öº R& & Ã~> 0.2 mlj &®. . $ sample 1 mlö BCA reagent bj &® . þj ¾ b ê 37 CöB 30ª* >w* NöB ï ' r, R& 5 &~ W%ê¢ 562 nmöB G;® . 10) S. mutans~ ~ ªÒ ²Ï ºÂ> S. mutans~ glucan Wö ~º 'Ëj rjV * V· Ú ~~ ·f ~ O» j æ ;~ ¯~& . ò j¢ *~ BHI Væö ²Ï º Â>j Î&(2 mg/ml)~ ' *ê >³ ê Væ¢ 4 C ïËö ~& . B VæÚ~ ~ ïj G;~ V *~ jB Væö 2.5N~ ³ê& >ê NaOH¢ & ~ 12*ÿn O~8 . O~B V·ö acetic acid¢ &~ 7z r cell debris¢ öªÒ ~ B ê, ç[ö 3Vï(v/v)~ öêRj &~ ~ ÿn O~8 . öêRö ~ *B ~º ³öªÒV(Beckman XL90) **(15,000Üg, 10 min) ~ Wªj ªÒ~&. . ªÒB ~º 60 CöB Î ê 7ï(mg/ml)j G;~& .. . >, ²Ï~ ÚÊ Wª S. mutansö & ·Ïj ¾æ Úºæ V*ö & \& z jº ©b .. ²³ê S. mutans KCTC 3065ö & ²Ï ºÂ>~ ²&³ ê¢ G; Ö 2.5 mg/ml Î& G V>æ p~b ¾, 1.25 mg/mlöBº ~ Ë ¢ÚÒ . V¢B S. mutans ö & ²Ï ºÂ>~ ²&³êº 2.5 mg/mlb ¾æÒ. (Fig. 2). 2.. o. 16). o. 'JH Minimal inhibitory concentrations of the Lignum sappan extract on the growth of S. mutans KCTC 3065. (1) 10 mg/ml (2) 5 mg/ml (3) 2.5 mg/ml (4) 1.25 mg/ml. o. Ö5V. ö & Î. 1. S. mutans KCTC 3065 20 mg/ml mutans 17) (Fig. 1). aureus Salmonella typhimurium. ²Ï ºÂ>j ~ ³ê paper disc O»b S. ö & Î¢ G; Ö, 17.3 mm~ Ã ÛBKj &. *öã Staphylococcus f ö & Ã&¢ Öfº ² N& ®b¾ &~j W º º ¢ ~~& . ©f ²Ï ºÂ> \;Ú~ ~jÖÃj FB ~º öö & Î'b 'ÏF > ®j ©b Òò. 'JH . Antimicrobial activity by paper disc method of the Lignum sappan extract(DH-39) on the growth of S. mutans KCTC 3065.. ²Ï ºÂ> Îö 8 S. mutans KCTC 3065 Ëö ~º Ë ²Ï ºÂ> S. mutans KCTC 3065~ Gö ~º Î¢ G;~V * ²Ï ºÂ>j 2 mg/ml~ ³ê Væ ö Î& ê ~ Gj Spectrophotometer G; Ö º Fig. 3 - 6 ? . 3.. 'JH The growth curve of S. mutans KCTC 3065 by addition of Lignum sappan extract. - - Lignum sappan, - - Control. ø.

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(8) . Effects of Lignum sappan Extract on the Growth of Streptococcus mutans KCTC 3065. 'JH . The pH changes in S. mutans KCTC 3065 culture medium by addition of Lignum sappan extract. - - Lignum sappan, - - Control. ø. 'JH . The changes of total carbohydrate in S. mutans KCTC 3065 culture medium by addition of Lignum sappan extract. - - Lignum sappan, - - Control. ø. 'JH The protein changes in S. mutans KCTC 3065 culture medium by addition of Lignum sappan extract. - - Lignum sappan, - - Control. ø. 'JH The effect of Lignum sappan extract on S. mutans KCTC 3065 polysaccharide production. - - Lignum sappan, - - Control. ø. 1) ²Ï ºÂ> ËFö ~º 'Ë ²Ï ºÂ>~ S. mutans Ëö & Îº &` V · 8* êö W%ê 1.05 &~ Ëj &b¾, ²Ï ºÂ>(2 mg/ml)j 4 VæöBº Ë æ>Ú 16* êö W%ê 0.36b &~ Ëj ¾æî (Fig. 3). ? f Ö j ²Ï ºÂ>f ÖW ö S. mutansö &~ 5 G æÎ& ®º ©b Îê . 2) ²Ï ºÂ> pH æzö ~º 'Ë ²Ï ºÂ>j S. mutans KCTC 3065ö Î&~ 8* ÿn V· ê VæÚ pH¢ G; Ö &`f pH 5.08 /Ï æz¢ &æò, ²Ï ºÂ> 4B Væº pH 6.69 jL' jò æz¢ &`ö j pH æz& ~ ¢Ú¾æ p~ (Fig. 4). \;~ pHº ;ç' ãÖº 7Wæò, j ô S~º ãÖöº ~^ï Ú ^ ö ~ ª ;b Ö W>Ú ~^ïÚ pH& ÎÚæ, ~^ï Ú pH& 5.5 ~ ÎÚæ² > ~ î î²>Ú ~jÖÃj FB . þ~ Ö ²Ï ºÂ>f &`ö j pH~ /Ï æz¢ ^~ Ã Û Bf ?f O»b ÛB~º ©b ¾æÒbæ '. O »j Û~ \;Úö 'Ï>Úæ ~jÖ6² Î¢ V& > ®j ©b ÒòB . 3) ²Ï ºÂb ê>zb æzö ~º 'Ë ²Ï ºÂbj S. mutans KCTC 3065 ö Î& ê Væ Ú~ ê>zb æz¢ rjV * *ê G;~& . & `¾ ²Ï ºÂb Î&B Væ~ .V C ê>zb~ ·f ml 2.5~3 mg~ ê>zb ;ï >î . V· 8* ê &`f 0.81 mg/ml îb¾, ²Ï ºÂb 4B V æº 2.06 mg/ml ê>zb~ · ~ æ~æ p~. (Fig. 5). 4) ²Ï ºÂb Wî æzö ~º 'Ë ²Ï ºÂb Î&B BHI Væö S. mutans KCTC 3065 ¢ V·~ *ê æz~º Wîïj G;~& . V· 0 *öBº Î VæöB 10 mg/ml~ Wî ;ï>î . Ú~ & Ëïj 8*öBº &` 8.39 mg/ml î, ²Ï ºÂb 4B Væº 12.3 mg/ml~ Wî 18).

(9) Journal of Dental Hygiene Science Vol. 4, No. 3, pp. 127~131 (2004). ;ï>î (Fig. 6). ?f Öº V Væ ÒÏ BHI Ú C Wî &`öBº ^~ .V Ëö V¢ ². >¾, ²ÏöBº ~ Ë ÛBö V¢ Wî~ ²Î& ² 6²~æ pf Ö Îê . ¾ * ãö V¢ Wî~ Ã&º ~ Ëö V¢ Ú Wî Ã&~ ¾æÂ Ö ÒòB .. ²Ï ºÂ> S. mutans KCTC 3065~ ~ Wö ~º Î S. mutans KCTC 3065~ ~º ~jÖÃö ~º ~^ï(dental plaque)j ;W~º &Ë 7º j ~º ~ . V¢B \öBº ²Ï ºÂb S. mutans~ ~ Wj Ú¶ ;ê ÛB~ºæ {~V *~ ÚVæöB *ê ~~ ·' æz¢ rj~ . Fig. 7öB º :f ? &`öBº V· 8*òö 300 mg/100 ml~ ~¢ áîb¾, ²Ï ºÂb Î&B VæöBº 60 mg/100 ml~ ~¢ >³~ £ 1/5;ê Ô ² W~& (Fig. 7). ç ?f Ö " r ²Ï ºÂbf ~jÖÃj F B~º S. mutansö & Î, Ëæ, pH 6²ÛB ?f Î¢ ¾æÚæ Ëê ~jÖj .O Ï'b ~£, \;Ó;B ~ Wªb Î& > ®j ©b Îê . 4.. º. £. \º ²Ï ºÂb~ S. mutans KCTC 3065ö & Î¢ paper disc method ¦ï~, ²Ï~ S. mutans KCTC 3065 Ëö 'Ëj ï&~& . 1. S. mutans KCTC 3065ö & ²Ï ºÂb~ ²Ë ÛB³êº 2.5 mg/mlî . 2. ²Ï ºÂb~ S. mutans KCTC 3065Ëö & Î º &` 8*öB 1.05 & Ëj &b¾, ²Ï ºÂbj 2 mg/mlj 4 VæöBº 16* ò ö 0.36b &Ëj & . 3. S. mutans KCTC 3065¢ 8* ÿn V· ê Væ Ú pH¢ G; Ö &`f pH 5.08îb, ²Ï ºÂb 4B Væº pH 6.69î . 4. S. mutans KCTC 3065¢ 8* ÿn V· ê Væ Ú ê>zb æzïj G; Ö &`f 0.81 mg/ml îb, ²Ï ºÂb 4B Væº 2.06 mg/ml î . 5. S. mutans KCTC 3065¢ 8* ÿn V· ê Væ Ú Wî æzïj G; Ö &`f 8.39 mg/ml îb, ²Ï ºÂb 4B Væº 12.3 mg/ml .

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(11). î . 6. S. mutans KCTC 3065~ ~ Wö ~º Îº &`~ ãÖ V· 8*ö 300 mg/100 mlj W~& b¾, ²Ï ºÂb 4B VæöBº 60 mg/100 mlj W~& . ^^ò 1. Norman PW, Robert RW, Rosen S: Essential Dental Microbiology. Appleton & Lange 341-355, 1991. 2. , , : , Streptococcus . 3(1): 39-44, 2003. mutans 3. , , , , , , , : , Streptococcus mutans . 20(1): 107-120, 1996. 4. , , : Flavonoids . 20(2): 189-202, 1996. 5. , , : Streptococcus mutans . 21(2): 187-191, 1993. 6. , , : . 40(4): 478-481, 1996. 7. , , , : , Streptococcus mutans OMZ 176 . 28(3): 99-103, 1997. 8. , : . , 1996. 9. : ( ) . , 1990. 10. Nagai M, Nagumo S, Eguchi I, Lee SM, Suzuki T: Sappanchalcone from Caesalpinia sappan L., the proposed biosynthetic precursor of brazilin. Yakugaku zasshi 104: 935, 1984. 11. Namikoshi M, Nakata H, Saitoh T: Homoisoflavonoids from Caesalpinia sappan. Phytochemistry 26: 1831, 1987. 12. Nagai M, Nagumo S: Protosappanins E-1 and E-2, stereoisomeric dibenzoxocins combined with brazilin from sappan Lignim. Chem Pharm Bull 38: 1490, 1990. 13. , , : . 28(1): 77-82, 1996. 14. : (Caesalpinia sappan L.) . 1-24, 1999. 15. Marvin T, Robert I, Lindemeyer, Robert G. Petersdorf: Comparison of single-disc and tube-dilution techniques in determining antibiotic sensitivities of gram-negative pathogens. Annals of Internal Medicine 58(1): 56-65, 1962. 16. Han MD, Jeong H, Lee JW, Back SJ, Kim SW, Yoon KH: The composition and bioactivities of ganoderan by mycelial fractionation of Ganoderma lucidum IY009. Kor J mycol 23(4): 285-297, 1995. 17. : , Topoisomerase I , . 1-43, 1998. 18. Loesche WJ: Role of Streptococcus mutans in human dental decay. Microbiol Rev 50: 353-380, 1990. (Received November 17, 2004; Accepted December 15, 2004). *B? Rò{ B*& J¶ J>F ºÂb~ ö &R Î" ~*"²æ FJ; #új ËV Ë\B ²^" ß¢ ;ÏC ;Ò ¶ãN Ö«¶ 5 æö ~ ~ Ã ÛB Î" &;²æ ;& Òß ËVj >« ~ Ööö & ^ Î" &;²æ F'F ;V^ f'V £Ò 5 Ëò~ Ã ÛB Î" Öëb²æ êÿF "÷F VV~ &7~ Wbî ªÒ 5 Ï~ö & K ï& £²æ ^Ï7 F\ "÷F VV~ Ï~ ö & .~ WWª £²æ Ëç^ fÿö £¶öb ^Â6Ò "v êË£ £ &Ò* 'âÒ. ª&& ë ÿz ²Ï ºÂb~ Öz Î" ®"² æ ^öC ²Ï ºÂb ®¦N b~ WË" ªêG ö ~º 'Ë &ÎW&ÎÓ&v ~ ã"&ö ®"" CÒ*¢^. *öã ²Ï ºÂb~ W & ^ëW Î"f îÖÊ¢ Ï /WëW þö & &v &ö b" CÒ*¢^.

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