Antibacterial Effect of Haedokgeumhwa-san against Methicillin-Resistant Staphylococcus aureus

해독금화산(解毒金花散)의 Methicillin-Resistant Staphylococcus aureus에 대한 항균효과

  • Lee, Ha-Il (Department of Rehabilitation Medicine of Korean Medicine, College of Korean Medicine, Won-Kwang University) ;
  • Lee, Su-Kyung (Department of Rehabilitation Medicine of Korean Medicine, College of Korean Medicine, Won-Kwang University) ;
  • Kwon, Young-Mi (Department of Radiology, College of Korean Medicine, Won-Kwang University) ;
  • Song, Yung-Sun (Department of Rehabilitation Medicine of Korean Medicine, College of Korean Medicine, Won-Kwang University)
  • 이하일 (원광대학교 한의과대학 한방재활의학교실) ;
  • 이수경 (원광대학교 한의과대학 한방재활의학교실) ;
  • 권영미 (원광대학교 한의과대학 영상의학과) ;
  • 송용선 (원광대학교 한의과대학 한방재활의학교실)
  • Received : 2015.03.16
  • Accepted : 2015.03.26
  • Published : 2015.04.30


Objectives Methicillin-Resistant Staphylococcus aureus (MRSA) is a human pathogen and a major cause of hospital-acquired infections. New antibacterial agents that have not been compromised by bacterial resistance are needed to treat MRSA-related infections. In this study, we investigated the antimicrobial activity ofethanol extract of Haedokgeumhwa-san (HGH) which prescription is composed of korean medicine against MRSA. Methods The antibacterial activity of HGH extract was evaluated against MRSA strains by using the Disc diffusion method, broth microdilution method (minimal inhibitory concentration; MIC), checkerboard dilution test, and time-kill test; its mechanism of action was investigated by bacteriolysis, detergent or ATPase inhibitors. The checkerboard dilution test was used to examined synergistic effect of ampicillin, oxacillin, ciprofloxacin, vancomycin, gentamicin and norfloxacin in combination with HGH ethanol extract. A time-kill assay was performed a survival curve which was obtained by plotting viable colony counts depending on time on bacterial growth. Results The minimum inhibitory concentration (MIC) of ethanol extract (HGH) ranged from 1,000 to $2,000{\mu}g/mL$ against all the tested bacterial strains, respectively. We are able to confirm that HGH extract has potentially strong antibacterial activity. In the checkerboard dilution test, fractional inhibitory concentration index of HGH in combination with antibiotics indicated synergy or partial synergism against S. aureus. A time-kill study showed that the growth of the tested bacteria was considerably inhibited after 8 hr of treatment with the combination of HGH with selected antibiotics. For measurement of cell membrane permeability, HGH $250{\sim}1,000{\mu}g/mL$ along with concentration of Triton X-100 (TX) and Tris-(hydroxymethyl) aminomethane (Tris) were used. In the other hand, N,N-dicyclohexylcarbodimide (DCCD) and Sodium azide ($NaN_3$) was used as an inhibitor of ATPase. TX, Tris, DCCD and $NaN_3$ cooperation against S. aureus showed synergistic action. Accordingly, antimicrobial activity of HGH was affected by cell membrane and inhibitor of ATPase. Conclusions These results suggest that Haedokgeumhwa-san extract has antibacterial activity, and that HGH extract offers a potential as a natural antibiotic against MRSA.


Supported by : 원광대학교


  1. Kang MW, Kim YR. Infection of Methicillin resistant Staphylococcus aureus. J Korean So. Chemother. 1993; 11:17-26.
  2. You YO, Kim KJ, Min BM, Chung CP. Staphylococcus lugdunensis - a potential pathogen in oral infection. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1999;88(3):297-302.
  3. Choi OK, Noh YC, Hwang SY. Antimicrobial activity of grapefruit seed extracts and polylysine mixture against food-borne pathogens. Korean J Dietary Culture. 2000; 15(1):9-15.
  4. Sandel MK, McLillip JL. Virulence and recovery of Staphylococcus aureus relevant to the food industry using improvements on traditional approaches. Food Control. 2004;15:5-10.
  5. Karlsson-Kanth A, Tegmark-Wisell K, Arvidson S, Oscarsson J. Natural human isolates of Staphylococcus aureus selected for highproduction of proteases and $\alpha$- hemolysin are ${\sigma}B$ deficient. Int J Med Microbiol. 2006; 296(4-5):229-36.
  6. Marraro RV, Mitchell JL. Experiences and observations with the typing of Staphylococcus aureus phage 94. J Clin Microbiol. 1975;1(2):180-4.
  7. Lowy FD. Staphylococcus aureus infections. N Engl J Med. 1998;339:520-32.
  8. Linton AH, Hedges AJ, Bennet PM. Monitoring for the development of antimicrobial resistance during the use of olaquindox as a feed additive on commercial pig farms. J App. Bacteriol. 1988;64(4):311-27.
  9. Dancer SJ. The effect of antibiotics on methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother. 2008;61(2):246-53.
  10. Otto CC, Cunningham TM, Hansen MR, Haydel SE. Effects of antibacterial mineral leachates on the cellular ultrastructure, morphology, and membrane integrity of Escherichia coli and methicillin-resistant Staphylococcus aureus. Ann Clin Microbiol Antimicrob. 2010;16:9-26.
  11. Stephen HB. Herbal Antibiotics. 2nd ed. North Adams: Storey Publishing. 2012:77-86
  12. 朱震亨. 新編 丹溪心法附餘. 서울:大星文化社. 1993:256.
  13. 허준. 對譯 東醫寶鑑. 경남:동의보감출판사. 2005:401-2.
  14. 윤용갑. 新東醫方劑 2223 도해임상활용. 서울:정담출판사. 2006:2302.
  15. 서형식. 황련 전탕액이 Staphylococcus aureus와 Staphylococcus epidermidis에 미치는 항균효과에 대한 실험적 연구. 한방안이비인후피부과학회지. 2006;19(2):71-6.
  16. 성인화. 黃芩과 黃柏 추출물의 MRSA와 Candida에 대한 항균활성. 미생물학회지. 2004;40(1):17-22.
  17. 신선희, 성인화. 백작약 추출물의 Methicillin-Resistant Staphylococcus aureus에 대한 항균활성. 미생물학회지. 2006; 42(1):54-8.
  18. Choi EY, OH HJ, Park NK, Chun HJ, Ahn JW, Jeon BH, Han DS, Lee HO, Baek SH. Screening of Cytotoxicity and Antimicrobial Effects of Extracts from Atractylodes macrocephala Koidz. Kor J Oriental Physiol & Pathol. 2002;16(2):348-52.
  19. Lee KS, Lee MW, Lee JY. Studies on the Antibacterial Activity of Poria cocos. Kor J Mycol. 1982;10(1):27-31.
  20. Lee YS, Kang OH, Choi JG, Oh YC, Chae HS, Kim JH, Park H, Sohn DH, Wang ZT, Kwon DY. Synergistic effects of the combination of galangin with gentamicin against methicillin-resistant Staphylococcus aureus. J Microbiol. 2008;46(3):283-8.
  21. Dzink JL, Socransky SS. Comparative in vitro activity of sanguinarine against oral microbial isolates. Antimicrob Agents Chemother. 1985;27(4):663-5.
  22. Kwon DY, Kang OH, Choi JG, Lee YS, Oh YC, Chae HS, Lee GH, Park PS, Kim YC, Sohn DH, Park H, and Lee JH. Antibacterial effect of Dryopteris crassirhizoma against methicillin-resistant Staphylococcus aureus. Fitoterapia. 2007;78(6):430-3.
  23. Bala M, Ray K. Gupta SM. Comparison of disc diffusion results with minimum inhibitory concentration(MIC) values for antimicrobial susceptibility testing of Neisseria gonorrhoeae. Indian J Med Res. 2005;122(1):48-51.
  24. CLSI. Performance standards for antimicrobial disk susceptibility tests. Approved standards. CLSI document M2-A7. Wayne, PA. 2001.
  25. Veljic M, Tarbuk M, Marin PD, Ciric A, Sokovic M, Marin M. Antimicrobial activity of methanol extracts of mosses from Serbia. Pharm Biol. 2008;46(12):871-5.
  26. Witte W, Pasemann B, Cuny C. Detection of low-level oxacillin resistance in mecA-positive Staphylococcus aureus. Clin Microbiol Infect. 2007;13(4):408-12.
  27. Shahverdi AR, Fakhimi A, Zarrini G, Dehghan A, Iranshahi M. Galbanic acid from Ferula szowitsiana enhanced the antibacterial activity of penicillin G and cephalexin against Staphylococcus aureus. Biol Pharm Bull. 2007;30(9):1805-7.
  28. Noble WC, Virani Z, Cree RG. Co-transfer of vancomycin and other resistance genes from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus. FEMS Microbiol Lett. 1992;72(2):195-8.
  29. Guadalupe MN, Blanca EL, Mariano VP, Fortino SS. In vitro activity effects of combinations of cephalothin, dicloxacillin, imipenem, vancomycin and amikacin against methicillin-resistant Staphylococcus spp. strains. Ann Clin Microbiol Antimicrob. 2006;5:25.
  30. Mazumdar K, Dutta NK, Kumar KA, Dastidar SG. In vitro and in vivo synergism between tetracycline and the cardiovascular agent oxyfedrine HCl against common bacterial strains. Biol Pharm Bull. 2005;28(4):713-7.
  31. Timurkaynak F, Can F, Azap OK, Demirbilek M, Arslan H, Karaman SO. In vitro activities of non-traditional antimicrobials alone or in combination against multidrug- resistant strains of Pseudomonas aeruginosa and Acinetobacter baumannii isolated from intensive care units. Int J Antimicrob Agents. 2006;27(3): 224-8.
  32. Nascimento AM, Brandao MG, Oliveira GB, Fortes IC, Chartone SE. Synergistic bactericidal activity of Eremanthus erythropappus oil or $\beta$-bisabolene with ampicillin against Staphylococcus aureus. Antonie Van Leeuwenheok. 2007; 92(1): 95-100.
  33. Choi JG, Kang OH, Obiang-Obounou B, Lee YS, Chae HS, Oh YC, Sohn DH, Park H, Choi HG, Kim SG, Shin DW, and Kwon DY. Antibacterial activity of Ecklonia cava against methicillin-resistant Staphylococcus aureus and Salmonella spp. Foodborne Pathog Dis. 2010;7(4): 435-41.
  34. Linnett PE and Beechey RB. Inhibitors of the ATP synthethase system. Methods Enzymol. 1979;55:472-518.
  35. Zhao WH, Hu ZQ, Okubo S, Hara Y, Shimamura T. Mechanism of synergy between epigallocatechin gallate and $\beta$-lactams against methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2001;45(6):1737-42.
  36. Irvin RT, MacAlister TJ and Costerton JW. Tris(hydroxymethyl) aminomethane buffer modification of Escherichia coli outer membrane permeability. J Bacteriol. 1981; 145(3):1397-403.
  37. Jung HJ, Lee DG. Synergistic antibacterial effect between silybin and N,N'-dicyclohexylcarbodiimide in clinical Pseudomonas aeruginosa isolates. J Microbiol. 2008; 46(4):462-7.
  38. 辛民敎. 臨床本草學 -改正增補 修訂版-. 서울:永林社出版 1997:179-80, 242, 400-7, 650.
  39. 강소신의학원 편. 中藥大辭典 서울:정담출판사. 1988:2215-28, 2319-27, 4839-45, 6448-59, 6478-99, 6512-25.
  40. 杜鎬京, 朴憲在. 黃連解毒湯의 藥理學的 硏究. 慶熙韓醫大論文集. 1982;5:103-14.
  41. 임대환, 윤지연, 장선일, 윤용갑. 黃連解毒湯이 Dextran Sulfate Sodium 유도 마우스 대장염에 미치는 영향. 대한한의학방제학회지. 2011;19(2):11-22 .
  42. 孫昌鳳. 抗菌活性에 작용에 대한 黃連解毒湯의 效能에 관한 연구. 원광대학교. 1986. 석사논문.
  43. 백상인, 배종훈, 김진영, 심성흠, 강경화, 이승연, 감철우, 박동일. PMA와 A23187로 자극된 RBL-2H3 세포에 미치는 黃連解毒湯의 영향. 동의생리병리학회지 2008;22(3):600-6.
  44. 鞠潤範. 黃連解毒湯이 自發的 高血壓 白鼠의 血壓 및 腎臟機能에 미치는 영향. 대한한의학방제학회지. 2002;10(1): 113-129.
  45. 서형식. 소염약침액, 황련해독탕, 황련이 Staphylococcus aureus에 미치는 항균효과에 대한 실험적 연구. 대한약침학회지. 2006;9(2):87-92.
  46. Oyaizu M, Fujimoto Y, Ogihara H, Sekimoto K, Naruse A, Naruse U. Antioxidative and antimicrobial activities of extracts from several utility plants. Food Preserv Sci. 2003;29(1):33-8.
  47. Bycroft BW, Shute RE. The Molecular Basis for the Mode of Action of Beta-Lactam Antibiotics and Mechanisms of Resistance. Pharm Res. 1985;2(1):3-14.
  48. Al-Habib A, Al-Saleh E, Safer A., Afzal M. Bactericidal effect of grape seed extract on methicillin-resistant Staphylococcus aureus (MRSA). J Toxicol. 2010;35(3):357-64.
  49. Ifesan BO, Joycharat N, Voravuthikunchai SP. The mode of antistaphylococcal action of Eleutherine americana. FEMS Immunol Med. Microbial. 2009;57(2):193-201.
  50. Drago L, De Vecchi E, Nicola L, Gismondo MR. In vitro evaluation of antibiotics'combinations for empirical therapy of suspected methicillin resistant Staphylococcus aureus severe respiratory infections. BMC Infect Dis. 2007;7:111.
  51. Darwish R.M, Aburjai T, Al-Khalil S, Mahafzah A. Screening of antibiotic resistant inhibitors from local plant materials against two different strains of Staphylococcus aureus. J Ethnopharmacol. 2002;79(3):359-64.
  52. Nascimento GGF, Locatelli J, Freitas PC, Silva GL. Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistance bacteria. Braz J Microbiol. 2000; 31(4):247-56.
  53. Yam TS, Hamilton-Mueller JM, Shah S. The effect of a component of tea Camellia sinensis on methicillin resistance, PBP2'synthesis, and $\beta$-lactamase production in Staphylococcus aureus. J Antimicrob Chemother. 1998; 42(2):211-16.
  54. Cha JD, Jeong MR, Jeong SI, Lee KY. Antibacterial activity of sophoraflavanone G isolated from the roots of Sophora flavescens. J Med Microbiol. 2007;17(5):858-64.
  55. Cha JD, Jeong MR, Jeong SI, Moon SE, Kim JY, Kil BS, Song YH. Chemical composition and antimicrobial activity of the essential oils of Artemisia scoparia and A.capillaris. Planta Medica. 2005;71(2):186-190.
  56. Jung KJ, Cha JD, Lee SH, Woo WH, Lim DS, Choi BK, Kim KJ. Involvement of staphylococcal protein A and cytoskeletal actin in Staphylococcus aureus invasion of cultured human oral epithelial cells. J Med Microbiol. 2001;50(1)35-41.
  57. Kim KJ, Yu HH, Cha JD, Seo SJ, Choi NY, You YO. Antibacterial activity of Curcuma longa L. against methicillin- resistant Staphylococcus aureus. Phytotherapy Research. 2005;19(7)599-604.