Antimicrobial Activity of Streptococcus mutans by Schizandrae fructus and Evodiae fructus extracts

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(1)Journal of Dental Hygiene Science Vol. 3, No. 1, pp. 39~ 44 (2003). , Streptococcus mutans

(2) . . †. . 1. Antimicrobial Activity of Streptococcus mutans by Schizandrae fructus and Evodiae fructus extracts †. Eun-Sook Jeon , Man-Deuk Han1 and Hyun-Day Kim 1. Dept. of Dental Hygiene, Dongpusan College, Pusan 612-715, Korea Dept. of Dental Hygiene, Kimcheon College, Gimcheon, Gyeongbuk 740-704, Korea. . This study was to investigate the antibacterial effect of Schizandrae fructus and Evodiae fructus extracts on S. mutans growth. The antibacterial effect of these extracts on S. mutans was good at Schizandrae fructus extract than the Evodiae fructus extract. The antibacterial activities of these extracts were measured 8.5 mm and 6.5 mm at 20 mg/ml concentration, respectively. The growth of S. mutans in control medium was the highest at 8 hrs, while the media of Schizandrae fructus and Evodiae fructus extracts added-medium(2 mg/ml) showed maximum growth at 16hrs. The pH values of the control medium was 5.08 at 8 hrs, but the media supplemented with Schizandrae fructus and Evodiae fructus extracts were 6.00, and 5.36 at 8 hrs, respectively. The amounts of total carbohydrate for the control media was 0.81 mg/ml at 8 hrs, but the media supplemented with Schizandrae and Evodiae fructus extracts were 1.32 mg/ml and 1.88 mg/ml at 8 hrs, respectively. In the change of culture medium proteins, the control culture broth and cultures supplemented with Schizandrae fructus and Evodiae fructus extracts were 8.39 mg/ml, 8.59 mg/ml and 9.97 mg/ml at 8 hrs, respectively. The S. mutans polysaccharide contents of the control medium and the media supplemented with Schizandrae and Evodiae fructus extracts were 300 mg/100 ml, 240 mg/100 ml and 280 mg/100 ml at 8 hrs, respectively..

(3) . Streptococcus mutans, Schizandrae fructus and Evodiae fructus extracts, Antimicrobial activity. . K noS l2p qrK no) stu . vQf XCY TUK@ wJf +, r xNKOu, y z{[4 +,) ghd | xNW } ~]N F . XC , xNp wKb <=)I, C), }~] I 3 5 F )t . <=N I HI xN4 V bK*, C) XC ,) aK lS . :C] &4 68C 3 C e XC , xNK@ w5 U). } ~]N XCY 4 t Z+,[ =, Z+,[ xN, S V #) +, " xN, 78 x N 3 #) t 5 . ] )I5 ~] xN4 5 V¡) ¢GZ f )4 V5 5 F )tG£ ¤, ¥ 79¦ 5§gm },o ¨85 7S =©4f 5 },o ) : D£Kª*, « 3 § ¬4f },o ¨8Kb Liriodendron tulipiferaF 5 },o FG£ : D£Kª. )%49 ] )IJ }, K 3).

(4) , . . !" #, $" , % &" '() *, +,, -)./, 0, 12 34 5. 67 89, :; <=)>?9, @A, B, :C, DEC 3 . ) + FG HI4 J KGL ) M 5FG L NOKPQ9 RS" TU, V$W . XCY Z+,[\ +,, :C], ^_ àHI4 Kb cd e Z+,[ \ +, M4 f9 Streptococcus mutansF ,)*, S. mutansF Z4 gh, YC i S8 Oj XC c K* , Z+,[ \ k +,D lgm i 1). 4). 2). 5). †. Corresponding author Tel: 051-540-3859 Fax: 051-540-3637 E-mail: [email protected]. 6). .

(5) . Antimicrobial Activity of Streptococcus mutans by Schizandrae fructus and Evodiae fructus extracts. F BK

(6) )tG£ . XC ,4 5 c 3 ) ®¯, ¯$( 3 °±]) 1, ² RS" xN5£ D£K ª£, « A³gm 1,4 J },o) 5 magnololS honokiol $Kª. ´5 °±]) c § )IJf Z+,[ S µ 4 5 ¶` " RS4 Jf9 D£ - . ¥ 3 5·i 5§ 63¦ ` ¸°±Kb ) p S. mutans4 5 },RSp ¹ºKª*, » 7S Gg ¼ ®½ °±]) X5 }, RSp u^\¾£ D£K ª. )¼ ) ]gm ¿¥K£ X5 @n ]4 5 5 F )tG£ u g cR ¹ º)u » @n4 V5 |) g)*, ) °±] S XC ,S VÀ4 5 ÁM" F d £ . )4 Â ·\4f cÃd Ä~, Äc 5§ gm ¸ °±] $5 A, S. mutans4 5 },RSp paper disc method ¹ºK£, },RSF uû 5§p )IKb S. mutans 4 ~ '( Å@ wKb rÆ, pH Ç, ¯ Ç, È É] Ç l 2 4 ~ RS 34 VKb ÊËK£ Kª. 7-9) 10). 11). 12,13). 14). 1.. . . Â ÌÍ4 I5 5§ fÎ ÏÐr4f E`! | ÑKb IKª.. 1) °±U ÒÒ 5§p "5 @ ÓKb 50 g Ô ÕÖ5 A ×ÒØQ/ 4 Ù£ Ð;F Ú@9Û Y2 1 l p F5 : 100 C4f 2ÜÒ@p )IKb Ý`4f 3ÐÞ Ï ¿ °±Kª. )p bSÐß bS_ à£ á Ð ° ± . bS S8 Oj b_ à A b_ âJ ¥?ã@ p IKb ä EÐå A Ð;p NKª. °± 5 § Id gw4 vQ Ð;æ ç, ¸è, é$, ¬ 3 ` Kª(Scheme 1). 2) Ð§ @@ ÌÍ4 I5 Ð§ BCA(Piece Co.), BSA(Piece Co.) Iê*, S. mutans « w5 «G brain heart infusion(Difco, U.S.A) IKª. @@ pH meter(Istek Co., Korea), UV/Vis spectrophotometer(Shimadzu Co., Japan), centrifuge(Hanil Co., Korea), ë@(BBL Co.), incubator 2.. o. (Dongyang Co., Korea), clean bench(Sangwoo Co., Korea), voltex(Dongyang Co., Korea), magnetic stirrer(Corning Co., U.S.A) . 3). 3 IKª , « ÌÍ4 I , 5ìíî ~]íS4f ï. S. mutans KCTC 3065p IKª*, DV À« . brain heart infusion(BHI) «G4 37 C4f 24ÐÞ Ï¿ ë o. Standard extract preparation from herbal medicines.. @4f ë@ « Kª. 4) },B ¨8 Ä~¼ Äc°±] },B ¹º paper disc method4 Kb ¨8Kª* , S. mutans , 1 loopp 10 ml BHI broth4 ð¦5 A 37 C4f 18~20ÐÞ ë@4 f ë@ « Kb )IKª. ñ, Müller hinton agar «Gp petri dish4 15 ml Ô Kb £ Ðå A, S. mutans « _ 50 µl p ð¦K b ,òK

(7) 9óKª. 5ô 5§ °±] 400 mg Ô ñ, 1 ml4 õ¥ö ÷b paper disc(6 mm)4 ÒÒ 50 µl Ô ø5 A EÐù. E disc ÐÍI Õú«G û Z4 ü ýhÐå :, 37 C4f 24ÐÞÏ¿ ë@ « K ª. °±]4 5 },B paper disc Ç clear zone þ(mm) ¨8Kb úKª. 5) =qJ?9 ¨8 =qJ?9(MIC, minimum inhibitory concentration) paper disc method4 Kb ¨8Kª* , S. mutans , 1 loopp 10 ml BHI broth4 ð¦5 A 37 C4f 18~20ÐÞ ë@" « Kb )IKª. ñ, Müller hinton agar «Gp petri dish4 15 ml Ô Kb ÿ£Ðå A, S. mutans « _ 50 µl p ð¦Kb ,òK

(8) 9óKª. 5§ °±] ?9p 10 mg/ml 2« 5 A 37 C4f 24ÐÞ Ï¿ ë@ « K b ²¿ VKb YC ?9¼ xN ?9 78Kª. 15) o. o. 15) o. o.

(9) . Journal of Dental Hygiene Science Vol. 3, No. 1, pp. 39~44 (2003). 6) rÆ ¨8 rÆ S. mutans , 1 loopp 10 ml BHI broth4 ð¦5 A 37 C4f 18~20ÐÞ ë@" « Kb B Ðå _ 50 µl¼ 5§ °±] 50 µlp BHI broth 10 ml4 ÑKª. 37 C4f ë@ « KZf ÐÞæ Kb , ² spectrophotometerp )IJf ø9(600 nm)p ¨8Kª. 7) pH ¨8 pH Ç :S ) ¨8Kª. S. mutans , 1 loopp 10 ml BHI broth4 ð¦5 A 37 C4f 18~20ÐÞ ë@ « Kb B Ðå _ 50 µl¼ 5§ °±] 50 µl p BHI broth 10 ml4 ÑKª. 37 C4f ë@ « KZ f ÐÞæ Kb pH meterp )IJf ¨8Kª. 8) É] 8Ö ¨8 «G\ É] Ç phenol sulfuric acid U )I Kª. û_ N ¹ÖÆ »$@ wKb û] D-glucosep IKª*, glucosep 100 µl/ml stock solution L£, Y2 Kb Àæ û_ L¾ . ¹Ö H 20 ml I ÐÍV4 Àæ û_ 1 ml FKb, b@4 80% phenol 25 µl¼ 05 ®i 2.5 ml FK£, _4 û_ & Y2 1 ml Fê. ´ 5 sample 1 ml S b@4 80% phenol 25 µl ¼ 05 ®i 2.5 ml Fê. ÐÍV â5 A 30 C4f 20Þ

(10) ÿÐß, Ì> ÜÒ5 :, û_ _ ø9 p 490 nm4f ¨8ê. 9) ¯ 8Ö ¨8 «G\ ¯ Ç BCA kitp )IKª. û_ N ¹ÖÆ »$@ wKb û] BSA(Bovine Serum albumine) )IKª. BSAp 1 mg/ml stock solution L£, Y2 Kb Àæ û_ L ¾. ¹Ö H 20 ml I ÐÍV4 BCA â_ (100:2) 4 ml FKb, b@4 Àæ û_ ÒÒ 0.1 ml Ô FK£, _4 û_ & Y2 0.2 ml Fê . ´5 sample 1 ml 4 BCA reagent â_ Fê. ÐÍV â5 A 37 C4f 30Þ

(11) ÿÐß Ì>4f ÜÒ5 :, û_ _ ø9p 562 nm4f ¨8 ê. 10) S. mutans l2 $ 5·i 5§ °±]) S. mutans glucan 4 ~ '( ÅD@ wJ « _ \ l2 5 3 U Ç Kb Ì Kª. Ð; p wKb BHI «G 4 5§ °±] F(2 mg/ml)Kb Ò ÐÞæ yK b «Gp 4 C Ür£4 DVKª. DV «G\ l2 Ö :S ) ¨8Kª. l2p à@ wKb «G4 2.5N ?9F d9Û NaOHp FK£ 12ÐÞ Ï¿ UÐù. U « _4 acetic acidp FKb M 5 : cell debrisp ¡$Kb NO5 A, `_4 3 «Ö(v/v) 4É FKb K Ï¿ UÐù. 4É4 J ¥ l2 £¡$@(Beckman XL-90) ¥Ðß(15,000g, 10 min) l2 $Kª. $ l2 60 C4f EÐå A EMÖ(mg/ml ) ¨8K ª. o. o. 1..

(12). . Ä~, Äc 5§gm °±] 20 mg/ml ?9 paper disc U S. mutans4 5 },RSp ¨85 7 S, Ä~ 8.5 mm, Äc 6.5 mm ,YC xNo D ª(Fig. 1).. o. o. o. o. Antimicrobial activity by paper disc method of the DH47 Schizandrae fructus and DH-48 Evodiae fructus extracts on the growth of S. mutans KCTC 3065..

(13) 4 J },B u^ Ä~, Äc °±] =qJ?9p ¨85 7S Table 1S *, S. mutans4 J Ä~, Äc °±] =qJ?9p ¨8 5 7S 2.5 mg/ml FÐ ²) VdG . vQf S. mutans4 5 Ä~, Äc °±] =qJ?9 5 mg/ml u^(Figs. 2, 3). 2.. S. mutans.

(14) Minimal inhibitory concentrations of the herbal medicines extracts on the growth of S. mutans KCTC 3065 Minimal growth inhibitory concentrations mg/ml of the medicinal herb extracts. The herbal medicines extracts Schizandrae fructus Evodiae fructus. 5 5. 16). o. o. Minimal inhibitory concentrations of the Schizandrae fructus extracts on the growth of S. mutans KCTC 3065. [ 10 mg/ml 5 mg/ml 2.5 mg/ml 1.25 mg/ml ]. . . . .

(15) . Antimicrobial Activity of Streptococcus mutans by Schizandrae fructus and Evodiae fructus extracts. Minimal inhibitory concentrations of the Evodiae fructus extracts on the growth of S. mutans KCTC 3065. [ 10 mg/ml 5 mg/ml 2.5 mg/ml 1.25 mg/ml ]. . . . . , S. mutans !" Ä~ Äc °±]) S. mutans ²4 ~ R Sp ¨8K@ wJ 2 mg/ml ?9 «G4 F5 A , ² Spectrophotometer ¨85 7S Fig. 4~Fig. 7S . 1) rÆ4 ~ '( S. mutans rÆ ) « 8ÐÞ A4 ø9 1.05 r D

(16) Z, Ä~°±](2 mg/ml) b5 «G4f r) Gdt 16ÐÞ A4 ø9 0.90 r u^(Fig. 4). ´5 Äc °±] Ð r) Gdt 16ÐÞ A4 ø9 0.80 r u^(Fig. 4). vQf Ä~, Äc °±] XC , S. mutans r xNK | u^. 2) pH Ç4 ~ '( Ä~ °±] S. mutans «G4 FKb 8ÐÞ Ï¿ « 5 A pHp ¨85 7S pH 5.08 5 Ç p DªGL, Ä~ °±]) b «G pH 6 î" õL5 Çp Db 4 J pH ÇF O ò 3.. Growth curve of S. mutans by addition of Schizandrae fructus and Evodiae fructus extracts. − − Schizandrae fructus, − − Evodiae fructus, − − Control . . . . pH changes in S. mutans culture medium by addition of Schizandrae fructus and Evodiae fructus extracts. − − Schizandrae fructus, − − Evodiae fructus, − − Control . . . . Changes of total carbohydrate in S. mutans culture medium by addition of Schizandrae fructus and Evodiae fructus extracts. − − Schizandrae fructus, − − Evodiae fructus, − − Control. . . . . Protein changes in S. mutans culture medium by addition of Schizandrae fructus Evodiae fructus extracts. − − Schizandrae fructus, − − Evodiae fructus, − − Control. . . . .

(17) . Journal of Dental Hygiene Science Vol. 3, No. 1, pp. 39~44 (2003). tuG (Fig. 5). Â 7S Ä~ °±] FÐ S. mutans «G @ pH qK4 '( G u, « 8ÐÞL4 5 =¼ },RSp u^ | D c@i L Q ´ k ]) },RS4 VbW | ;. Ä~ #)5 U(S & ) 5 |) #)*, ÞrDaHI , w! xNHI 3) *, º=] Å"0 anthocyanin X }in) | Å"G£ . ³ 3 Ä~ °±] M c@ i) pHp qKÐß },RSp u^£ Kª£, ¶ 3. Ä~ °±] «G4 F5 A 0.1N HCl )IKb ci, ²4 ~ Ä~ °±] ²xN RSp ¨ 8Kª*, Ä~ °±] M c@i fumaric acid¼ citric acid, malic acid, itaconic acidp «G4 FKb ¼ ²xNn î5 - . Äc °±] X S. mutansp 8ÐÞ Ï¿ « 5 A «G\ pHp ¨85 7S pH 5.08 5 Ç p DªGL, pH 5.36 î" õL5 Çp Dª(Fig. 5). )`S ) S. mutans «G\ pH Çp Ä~, Äc °±] b5 A RSp Å Â 7S S. mutans , Y C xNK | uû }XC RSp @W | ;. 3) «G\ É] Ç4 ~ '( Ä~, Äc °±] S. mutans4 F5 A ÐÞæ « Ð «G\ É] Çp ÅD. )u Ä~ , Äc °±]) F «G @ È É] . ml l 2.5~3 mg É]) 8Öd¾. « 8ÐÞ A 0.81 mg/ml )¾u, Ä~ °±]) b «G 1.32 mg/ml É] ) O ÇKG (Fig. 6). Äc °±]) b «G X « 8ÐÞ L4 1.88 mg/ml u^\t É] ) O ÇKG (Fig. 6). Äc alkaloid¼ limomoid ) $ D£ - * » §$HI >cGHI , ¡ HI , }#i=YHI , $ãHI , %&2 YHI , §5 &äKHI ¡r³Ï' =HI 3) Å"0 - . 4) «G\ ¯ Ç4 ~ '( Ä~ Äc °±]) F BHI «G4 S. mutans KCTC 3065p « K£ ÐÞæ ÇK ¯Ö ¨8K ª. « 0ÐÞ4f () «G4f 10 mg/ml ¯) 8Öd¾. , rÖ D 8ÐÞ4f ) 8.39 mg/ml )¾£, Ä~ °±]) b «G 8.59 mg/ml ¯) 8Öd¾* Äc °±]) b «G 9.97 mg/ml )¾(Fig. 7). ) 7S @Â «G I5 BHI \ È ¯) 4f +, @ r4 vQ =Ìdu, Äc4f , r xN4 vQ ¯ =(F

(18) =KG 7S *H. ».u ÐÞ) S4 vQ ¯ YF , r4 vQ ,% ¯ 3) YFKb u^+ 7S ;. 17). 19). 18). 20). 21). 2). vQf Â 4f 5§ °±]) S. mutans l2 t, 89 xNKG K@ wKb _« G4f ÐÞæ l2 " Çp ÅD. Table 24f D -¼ ) 4f « 8ÐÞL4 300 mg/100 ml l2p à¾u, Ä~ X 240 mg/100 ml, Äc F 280 mg/100 ml l2p yKb 4 J )p u^\G | u^(Fig. 8). ) @ b Ö) =xN?9D " ml l 2 mg bê@ ./ | ;d*, 5 ) 5 l2 y í" # 5 - glucan l2ª. 30).

(19) Effects of herbal medicines on S. mutans KCTC 3065 polysaccharide production Polysaccharide (mg/100 ml cultured medium). Time(hr.) Sample Names Schizandrae fructus Evodiae fructus Control. 0. 8. 12. 16. 24. 48. 18 15 20. 240 280 300. 225 240 280. 180 190 220. 165 170 220. 170 170 210. 22). 23). 25). 26). 24). 27). 28). 29). # $%& ' () l2 XCY4 VbK Z+,[ K¤ Fr M5 W K l. 4. S. mutans S. mutans (dental plaque). Effect of Schizandrae fructus and Evodiae fructus extracts on S. mutans KCTC 3065 polysaccharide production. − − Schizandrae fructus, − − Evodiae fructus, − − Control. . . . )` 7S Ä~, Äc °±] Z+,[ 4 M K

(20) HIK S. mutans l2 xNW | b0G*, ) °±] wN FKb B IW | ;.. . Â Ä~, Äc °±] S. mutans4 5 } ,RSp paper disc method ¹ºKb, S. mutans r4 V5 '( ÕFKª. 1. Ä~, Äc ¸ °±] },RSp 1º5 7S S. mutans4 5 },o) X5 2f Ä~, Ä c 2)¾*, Paper disc method4 5 },B ¨ 8(20 mg/ml b) 7S Ä~F 8.5 mm, ÄcF.

(21) . Antimicrobial Activity of Streptococcus mutans by Schizandrae fructus and Evodiae fructus extracts. )¾. 2. 5§ °±] S. mutans r4 5 RS ) 8ÐÞ4f 1.05 r3 Dªu, Ä~, Äc °±] 2 mg/ml b5 «G4f 16ÐÞL 4 0.90, 0.80 r Dª. 3. S. mutans p 8ÐÞ Ï¿ « 5 A «G\ pHp ¨8 5 7S 5.08 )¾*, Ä~, Äc °±] ) b «G 6.00, 5.36 )¾. 4. S. mutansp 8ÐÞ Ï¿ « 5 A «G\ É] Ç Ö ¨85 7S 0.81 mg/ml )¾*, Ä ~, Äc °±]) b «G 1.32 mg/ml, 1.88 mg/ml )¾. 5. S. mutansp 8ÐÞ Ï¿ « 5 A «G\ ¯ Ç Ö ¨85 7S 8.39 mg/ml )¾*, Ä~ , Äc °±]) b «G 8.59 mg/ml 9.97 mg/ml )¾. 6. S. mutans l2 4 ~ RS X « 8ÐÞ4 300 mg/100 ml Kªu, Ä~, Äc °±]) b «G4f 240 mg /100 ml, 280 mg/100 ml Kª. 6.5 mm. . 1.. , , , ,

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(23) @EF G HIJ$ KL MN ./ 6=OPQ@#RS

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(26)