Antibacterial effects of Terminaliae chebula extract against major pathogens and methicillin-resistant Staphylococcus aureus (MRSA) from bovine mastitis milk

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(1)³¤¥Èó(2005) *45. *1a Korean J Vet Res(2005) 45(1) : 113~119. &¶ 5FSNJOBMJBFDIFCVMB ºÂb~<²FO""ºöÚ 5z~ ÖÚWï

(2) êç\ .34" ö&R]Î ;* Á^êÖÁË.RÁf«òÁÇ"ÿÁ·Ï ã>~"¦ö &v ¶"& (²Òß: 2005j 1ú 9¢) 1. Antibacterial effects of Terminaliae chebula extract against major pathogens and methicillin-resistant Staphylococcus aureus (MRSA) from bovine mastitis milk Hyun-Mi Kang*, Jin-San Moon, Gum-Chan Jang, Jong-Man Kim, Min-Dong Song15/ Si-Yong Yang1 National Veterinary Research and Quarantine Service, Anyang 430-824, Korea 1 College of Natural Science, Konkuk University, Chungju 380-701, Korea (Accepted=/ January 9, 2005) Abstract : Resistance to antibiotics is a problem all over the world, and this problem also is so extended in veterinary fields. Therefore, this study aimed to find out the antibacterial substances from natural medicinal herbs against bovine mastitis pathogens, especially methicillin-resistant Staphylococcus aureus (MRSA). Among seventy two medicinal herbs, Terminaliae chebula extract showed antimicrobial effect against MRSA isolated from mastitis milk in cow and patient in human. However, Terminaliae chebula extract didn’t show antibacterial effects on various strains including other contagious and environmental pathogens related with mastitis of dairy cows. Key words : Terminaliae chebula extract, antibacterial substances, medicinal herbs, mastitis pathogens, methicillin-resistant S. aureus (MRSA). *. . & öò jî¢ [10, 14], FO" B " Böò ~~, FV ~òB cloxacillinj ô Ò Ï~ ®º Ú * j 6n " r, 6" 7 z~. Ö ÚW ïêç Ò &ËW ® . $ , ïêç ~ FO" öÚê Bö & B ÚWj ¾æÚº B ÚW³ Ã& ®¢;rÒj Â* ~ 7' ^B 6b &v>B *'b n*~ Î"' < ² FO" ~òB BB . ; . ö V¢ "öº ^B~ B ÖJ& ìb B B¢ &Ú > ®º b $º ÿb F¾ W ª, ¯ Âbîj Ï Wbî~ BB B ® > ® . *Òræ ÚöB Â B~ B Bj * º £ ºÂböB~ W bîj. FO"f <² ¶Ú~ F*, Ò 5 ¦' º " ÏË Ò·&Ò¾ ÒG~ã ~ º B ' b ·Ï~ FO "æö ç~º b FOÚ ö «~ FFÚ "Ãj ;W~º ©bB FO" ~ öf 80« ç ·Ï~æ ß; &æ B ~ò~º ÚJæ ® [13, 20]. Ú~ ã Ö <² FO" æ³'b B~ Ú^> 50ò ç~ 4/ ÏË *Ú ÏË~ 30% *ê, 1993 j¦V Ú^>ö ~ F& Næ/B& > ®Ú FO"ö ~ ãB' ¶ f ï&~ . ß®, " º *"W FO" ö Staphylococcus aureusº. · ÷öW ¶¢ ªj~ ?"^~ >wö. *Corresponding author: Hyun-Mi, Kang National Veterinary Research and Quarantine Service, Anyang 430-824, Korea [Tel: +82-31-467-1775, Fax: +82-31-467-1766, E-mail: [email protected]]. 113.

(3) ;*Á^êÖÁË.RÁf«òÁÇ"ÿÁ·Ï. 114. ¦ï~& [16, 17, 21], ¢böBº Yang [24] ãÎ¢&~ ¶b 80«öB 4«~ "ö & Wj ¦Æ : ® . öê N [25], 9 [15], º´ [12], ¶. [22], â [19] öB K ®r B>î . f ? Âbî~ ·Ïö & º &¦ª W rJê &æ& > ®. [9]. $, £j Ï ®~ Î&BB~ ÒÏ &ËWj rj¶ ÷öW bö & W ¢¦ > [1, 9] ®b, " >~ª¢ö ®Ú £Ò ºÂbj Ï~ "æ~ &ËÃj j ^W î÷ö & .O $º ~ò &ËWj ~&b¾ [8], <² FO"ö & £Ò~ 'Ïö & º ~ *ææ p ® . V¢B, öBº W ® rJê 72. «~ £j Ï~ <² FO" "º öÚö & Kj ÎîVç~, ß®, *"W öÚ S. aureus 5 z~ Ö ÚW S. aureus "ö & K ¦Ò¢. b B &Ú £ Âbîj <² FO " .O $º, ~òö wÏ~V * V.¶ò¢ á¶ >¯~& .. Òò 5 O» ÒÏ "º ã>~"¦ö ^"ö & 7 FO" F¾ 17"~ Methicillin resistance Staphylococcus aureus(MRSA) ¢ªÒ"¢ j~ Staphylococcus aureus 305, vancomycin-resistant enterococci. Table 1. List of the medicinal herbs used for antibacterial activity test Scientific Name. Korean. Morus bombycis var. caudatifolia Angelica gigas Nakai Cinnamomum cassia PRESL Crataegus pinnatifida Bunge Sinomenium acutum Granati Pericarpium Prunus mume Artemisia princeps PAMPAN Dianthus superbus Schizandra chinensis Ginkgo biloba Acorus calamus var. angustatus Zanthoxylum coreanum Epimedium koreanum Reynoutria elliptica Thuja orientalis Punica granatum Ampelopsis brevipedunculata var. heterophylla Juniperus rigida Agrimonia pilosa Inula helenium Allium fistulosum LINNE Sanguisorba officinalis Houttuynia cordata Eugenia aromatica (L.) Merr Commelina communis Viscum alvum L. var. coloratum Asperagus chchinchinensis Forsythia koreana NAKAI Prunella vulgaris Ledebouriella seseloides Scutellaria baicalensis GEORGE Imperata cylindrica (LINNE) BEAUV.var koenigii (RETZ.)DURAND et SCHINZ Lonicera japonica THUNB Xanthium strumarium LINNE Foeniculum vulgare Gaertner. {~4Ò æ êæ ÖÒ¾Z OV CFb. z¾Z # FNÓE J¶ f¯¾Z Wc {.b¾Z r·# ^Ë" GW¾Z CF B^¾Z *>¾Z Ïj. ÏË CW J¦ ÚW. ;Ë Bj ÎÖÚ Â^ÿ v ~. O³ . WÎ" .fz c¶ ²Ë. Scientific Name Curcuma Zedoaria Japonica Miq Akebia quinata Decaisne Coptis chinensis FRANCH Crategus oxyacantha Lythrum anceps Patrinia villosa JUSSIEU Impatiens balsamina Morus alba LINNE Paeonia suffruticosa Andr Taraxacum platycarpum Pulsatilla koreana Paeonia lactiflora Polygonatum stenophyllum Portulaca oleracea Anemarrhena asphodeloides Ulmus davidiana var. japonica Pleuropterus multflorus Angelica dahurica Belamcanda chinensis Zingiber officinale Asplenium sarlii Allium sativum for. pekinense Lilium concolor var. parthneion Allium cepa Rumex crispus Stemona japonica Miquel Aster tataricus Rheum officinale BAILLON Machilus thunbergii Liriope platyphylla Evodia officinalis Phellodendron amurense Paeonia lactiflora PALL. var. hortensis MAKINO Cornus officinalis Piper longum Terminaliae chebula. Korean /jÂ Úª N ÖÒ Âj NË" /Fz çWb Ïb " . E '·£ ; î~* æÎ ¶«¾Z ~>J å& Ò* ; òÒÒ î¾ ~¾¾Ò ·2 ²Òç W¦ ¶j & ê; ^ÿ J>F W W·£ Ö>F jB &¶.

(4) ¶(Terminaliae chebula) ºÂ>~ <² FO" º öÚ 5 z~ ÖÚW ï

(5) êç\(MRSA)ö &R ]Î 115. (VRE) &" Enterococcus faecium BM7641, E. faecalis V583, E. gallinarum GS 5 E. casseliflavus ATCC 25788j Ï~& . $, Streptococcus uberis ATCC 27958, Streptococcus dysgalactiae ATCC 27957" Streptococcus agalactiae ATCC 13813j Ï~&b, Escherichia coli, Citrobacter freundii, Enterobacter cloacae, Pseudomonas aeruginosa ~ ²rW" Bacillus cereus ¢ªÒ"¢ £~ &ç " Ï~& . ÒÏ £Ò 5 ºÂO» Â bîº ÖÒ ¾¢öB £Ò &Ë Þ'b ¾ Ï> ®b, $, ^òç [7]öBê K ® rJê 72«~ £j «~ Ò Ï~& (Table 1). Ò £~ ºÂ ¦*º óî, . , òÒ, ´ j &çb ~& . W bîf b" zêR ºÂ~&º, b ºÂ ãÖ ï'w»V & J~B â'2¢Êö òf Ã~>¢ 1:9~ jN I 100oCöB 1* ÿn & .ºÂB áf j "æ(Whatman, No. 1) "~ B~ & . $, zêR ºÂf >ºÂ" ÿ¢ Ë~ö 9 Vï~ 95% zêRj &~ çNöB 24* ÿn º Â r, ÿ¢ O»b "B áf j ²*ê ÃBV .ï~ 1/9 ³»~ ÒÏ~& . Î £ ºÂb~ FO" "º öö & Kf :Ê {Ö» [11]ö ~ G;~& . ¯, þ 0.1 ml¢ Ò BÛÎ plate*ö ¢~² êö ê. Table 2. Antibacterial effects of medicinal herbs against bovine mastitis causing pathogens Antibacterial effect of medicinal herbs* (Inhibition zone, mm). Pathogens Methicillin-resistant S. aureus 132 Methicillin-resistant S. aureus 425 Methicillin-resistant S. aureus 136 Methicillin-resistant S. aureus 428 Methicillin-resistant S. aureus 133 Methicillin-resistant S. aureus 357 Methicillin-resistant S. aureus 345 Methicillin-resistant S. aureus 355 Methicillin-resistant S. aureus 290 Methicillin-resistant S. aureus 369 Methicillin-resistant S. aureus 342 Methicillin-resistant S. aureus 370 Methicillin-resistant S. aureus 372 Methicillin-resistant S. aureus 363 Methicillin-resistant S. aureus 164 Methicillin-resistant S. aureus 256 Methicillin-resistant S. aureus 292 S. aureus 305 Enterococcus faecium BM7641 Enterococcus faecalis V583 Enterococcus gallinarum GS Enterococcus casseliflavus ATCC 25788 Streptococcus uberis ATCC 27958 Streptococcus dysgalactiae ATCC 27957 Streptococcus agalactiae ATCC 13812 Escherichia coli Citrobacter freundii Enterobacter cloacae Pseudomonas aeruginosa Bacillus cereus *. a. b. c. d. e. f. 8 NI 8 8 10I NI NI 1NI NI 8 NI 8 8 8 8 8 NI NI NI NI NI NI NI NI NI NI NI NI NI NI. NI** NI NI NI NI NI NI NI NI NI NI NI NI NI 8 NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI. 8 8 10 8 14 14 14 14 8 8 14 10 8 14 NI 10 10 NI NI NI NI NI NI NI NI NI NI NI NI NI. NI NI NI NI NI NI NI NI NI NI 14 NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI. NI NI NI NI 12 NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI. NI NI NI 14 10 14 10 10 NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI. a, Eugenia aromatica; b, Cornus officinalis; c, Terminaliae chebula; d, Punica granatum; e, Artemisia princeps PAMPAN; f, Schizandra chinensis ** NI, no inhibition.

(6) ;*Á^êÖÁË.RÁf«òÁÇ"ÿÁ·Ï. 116. ''~ £ ºÂb 50 µl¢ >Î paper disc (TOYO 8 mm; Advantec, USA)¢ ¹, &ç ^ j 37oCöB 24* V· ê Ã~æ á clear zone~ çãj G;~ ;ê¢ jv~& . Ò*¶*ãj Ï K { FO" öj Tryptic soy broth(Difco, USA) V æö 7« r, Kj £j Î&~ V ·Î r, PBS 3² ö^¿~ "Ò*¶*ã (Hitachi, Japan)b ^~ ¶ç¦¢ {~& .. . Ö. £ºÂb~ K {~ 4Ò, æ, êæ C 72«~~ £Ò¢ > ºÂ»j Ï~ <² FO" "º öÚö & Wj Ò Ö" ;Ë, Ö>F, &¶, CF, # 5. J¶öB ¢¦ Wj {~& . ¯, 17"~ FO " F¾ MRSAö & :Ê {Ö»j Ï~ Î"¢ Ò :, Ö>F, CFbf #f 1"~ MRSA ö &Bò Î"& ®îæò, ;Ë" J¶º 11 "~ MRSAf 5"~ MRSAö & '' Î"¢ ¾æîb, &¶ ºÂbf 16"~ MRSAö &B Î"¢ ¾æÚî (Table 2). ¾, MRSA ~ ²·W" ²rW~ FO" "º öÚöº Î"& ìº ©b ¾æÒ . &¶ºÂb~ ºÂO»ö 8 Î jL þöB z~ Ö ÚW ïêçö & K &Ë ÚÂ £ &¶ ºÂbj <² FO" "º ö S. aureus 5 E. coli ²·W" ²rWö & zêRºÂO»" >ºÂO»*~ Î"ö &B jv~& . Ö", &¶ ºÂbf 17"~ MRSA ¢ªÒ"ö & v &æ ºÂ O»*. Table 3. Comparison of methanol extract and water extract methods of Terminaliae chebula against MRSA from bovine mastitis milk Antibacterial effects (Inhibition size, mm). MRSA from bovine mastitis milk Methicillin-resistant Methicillin-resistant Methicillin-resistant Methicillin-resistant Methicillin-resistant Methicillin-resistant Methicillin-resistant Methicillin-resistant Methicillin-resistant Methicillin-resistant Methicillin-resistant Methicillin-resistant Methicillin-resistant Methicillin-resistant Methicillin-resistant Methicillin-resistant Methicillin-resistant *. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S.. aureus aureus aureus aureus aureus aureus aureus aureus aureus aureus aureus aureus aureus aureus aureus aureus aureus. 132 425 136 428 133 357 345 355 290 369 342 370 372 363 164 256 292. MeOH extraction. Water extraction. 14 14 16 14 14 14 15 15 15 14 14 14 13 14 NI 14 14. 13 13 16 13 14 11 11 18 16 13 11 12 14 14 NI 13 12. NI, no inhibition. Table 4. Comparison of methanol extract and water extract methods of Terminaliae chebula extract against MRSA from patient in human Antibacterial effects (Inhibition size, mm). MRSA from human Methicillin-resistant Methicillin-resistant Methicillin-resistant Methicillin-resistant. S. S. S. S.. aureus aureus aureus aureus. ATCC ATCC ATCC ATCC. 3287 3299 43300 2246. MeOH extraction. Water extraction. 12 14 12 13. 11 13 13 12.


(8) êç\(MRSA)ö &R ]Î 117. FÒ Kj ¾æÚî (Table 3). $, &¶ ºÂ bf Ò² ~¶ F¾ MRSAö & Î"öBê . þ 4"~ MRSA& zêRºÂb" >ºÂb Îv öB Î"¢ ¾æÚî (Table 4). Fig. 1f >ºÂ»j Ï~ Muller-Hinton agar çöB MRSA 425 ªÒ"f Ò² F¾ &" MRSA 43300ö & &¶ºÂb~ Î"¢ ¾æÞ ©, Fig. 2º "Ò*¶*ãj Ï~ ;' æz ¦¢ &V Ö" MRSA 425 "~ ãÖ &¶ ºÂbö ~ ^>& *Ú ^& Ï>Ú ¶ ç>îrj { > ®î .. Fig. 1. Antibacterial effects of Terminaliae chebula extract on Muller-Hinton agar against methicillin-resistant S. aureus 425 isolate and methicillin-resistant S. aureus ATCC 43300.. V. MRSAö Î"¢ &æ ®º &¶(Terminaliae chebula)º Ò¶"(combretaceae) Ò¶>(Terminaliae. Fig. 2. Scanning electron microscopy of methicillin-resistant S. aureus 425 isolate normal cells (A) and cell grown on Tryptic soy broth contained sublethal dose of Terminaliae chebula extract (B)..

(9) ;*Á^êÖÁË.RÁf«òÁÇ"ÿÁ·Ï. 118. chebula Retzss)~ W?" j ©b .*¦V ^W ËÚî~ö ¶" ÒÏ>º £b " *Ë £~ VËj~ cc, î, JÒ " ËÚ bö & ·Ï 5 :Êö & Î"& Ö > ©b rJ^ ® [6]. ÂbîöB º Â Wª~ W bîf ¢ Wª jò & æ zb 'b ·Ï ¢Ú¾º ©b > ®b, 7öBê phenolW zb" alkaloid~ & " &~, ·Ïê &æ &ÒãöB ¢Ú¾º ©b º;> ® [4]. #" ;Ëê bî rJ^ ®º, ö Bê ¢¦ MRSA "ö &B Î"¢ ¾æÚî æò £ö & & j ; . Þ, # [1]f Ö>F& E. coliö & Kj &b¾ S. aureusö &Bº Kj ¾æÚæ p~ ~ &b¾, öBº Ö>F& 1B MRSA "ö Wj & . $, öB &¶ ºÂb. rb MRSA "ö Î"¢ ¾æÞ J¶ (Schizandra chinensis)º ÏN"ö ³~º ¶ÏbB OöB ê;, ê 5 ~ 7ºÛB ·Ï" * ^ 5 .{;~, rzRö & ë·Ï b ÒÏ > ® [2, 3, 5]. J¶~ >ºÂb" ö êRºÂb~ Kf ºÂb 7~ FVÖ Væ~ pH¢ &~B ¾æÞ ~&b¾ [23], Leef Im [18] f FVÖ Wª ~ W bî G ÛBö · Ï ~º *Òræ J¶~ ÚÆ Wª Kj <ºæö &Bº «{® «>æ p ® . æ, Ö"¢ Æ& Wj ¾æÚ º £ 7 ÚÆ Wª ÷öW bö Î"¢ ¾æÚºæf Î"~ ·ÏV*ö & . >¯>Ú¢^¢ © . Þ, Î" ¢ º £Ò¢ <² FO" .O 5 ~ò¢ * Ò Ï~V *Bº þÿb 5 Ï'ÿbö & n*W ï&& ê¯>Ú¢ ©b ÒòB .. Ö. . W ® rJê 72«~ £j Ï~ <² FO" "º öÚö & K ¦Ò¢ :, &¶, ;Ë, Ö>F, J¶, # 5 CF ºÂb z~ ÖÚW ïêç(MRSA)ö &B ; Î"¢ ¾æÚî . 7öBê &¶ ºÂbf <² 5 Ò²F¾ MRSA " &¦ªö &B ¸f Î"¢ ¾æÚîb¾ FO" öÚ ²· W" ²rWö &Bº Wj ¾æÚæ p. º ßWj ¾æî .. ^^ò 1. #9, ·Òö, 7ß. ¢¦ ÷öW bö & Wj º £~ öï. ®*²æ 1993, 8, 141-145. 2. Bãª, Î;, ²j. J¶ ¢>Wª, FVÖ 5 anthocyanin ï²ö &~. ®"²æ 1973, 5, 178-182. 3. BÆ, 8ÿ, J;. JV¶(Rhus japonica Linne) methanol ºÂb~ ÖzÎ". ®*²æ 1992, 7, 107-112. 4. ÎÏK, B.?, ËF;. Streptococcus mutans JC-2~ ÖWö ~º &¶~ Î". &;²æ 1995, 19, 495-503. 5. Jç, B9>, "÷Ï, ;ÿÎ. V¶(Lycium chinensis Miller), æ(Angelica acutiloba Kitag), J ¶(Schizandra chinensis Bailon), J.b(Acanthopanax sessiliflorum Seeman) ºÂb~ FÒ, FÒjÖ, FVÖ 5 æò~ W. ®"²æ 1990, 22, 76-81. 6. ç. £ªçwÏ, pp. 392-393, WÒ, BÞ 1990. 7. ;"5, "v. Ë££ &Ò* (bÞ). 'âÒ 1990. 8. ÒÏ, R¢, ~ã. Escherichia coliö & £Ò ºÂb~ W. &>~²æ 2003, 43, 625631. 9. Rö, ÏÆ, 9. ¶ã«¶ºÂb" ÒÒ bb~ ®¦Nö & Î". Korean J Dietary Culture 2000, 15, 9-15. 10. Aarestrup FM, Dangler CA, Sordillo LM. Prevalence of coagulase gene polymorphism in Staphylococcus aureus isolates causing mastitis. Can J Vet Res 1995, 59, 124-128. 11. Bauer AW, Kirby M, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966, 45, 493. 12. Choi MY, Choi EJ, Lee E, Rhim TJ, Cha BC, Park HJ. Antimicrobial activities of pine needle (Pinus densiflora Seib et Zucc.) extract. Korean J Appl Microbial Biotechnol 1997, 25, 293-297. 13. Gilmour A, Harvey J. Staphylococci in milk and milk products. Soc Appl Bacteriol Symp Ser 1990, 19, 147s166s. 14. Gruet P, Maincent P, Berthelot X, Kaltasatos V. Bovine mastitis and intramammary drug delivery. review and perspectives. Adv Drug Deliv Rev 2001, 50, 245-259. 15. Kang SK, Kim D, Park SK. Effects of antimicrobial of Leaf Mustard (Brassica juncea) extract on.


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