A Study on Antibacterial Activity of Daehwanggeonwoo-san(Dahwangqianniu-san) Ethanol Extract against Methicillin-Resistant Staphylococcus Aureus

대황견우산(大黃牽牛散) 에탄올 추출물의 Methicillin 내성 Staphylococcus aureus에 대한 항균활성 연구

  • Park, Ju-yeong (Department of third medicine of Korean Medicine, professional graduate school of Oriental medicine, Won-kwang University) ;
  • Na, Yong-su (Department of third medicine of Korean Medicine, professional graduate school of Oriental medicine, Won-kwang University) ;
  • Oh, Gong-cheon (Department of third medicine of Korean Medicine, professional graduate school of Oriental medicine, Won-kwang University) ;
  • Lee, Sang-mi (Department of third medicine of Korean Medicine, professional graduate school of Oriental medicine, Won-kwang University) ;
  • Choi, Byeong-kwon (Department of third medicine of Korean Medicine, professional graduate school of Oriental medicine, Won-kwang University) ;
  • Lee, Yoon-seung (Department of Rehabilitation Medicine of Korean Medicine, Colleage of Korean Medicine, Won-kwang University) ;
  • Song, Yung-sun (Department of Rehabilitation Medicine of Korean Medicine, Colleage of Korean Medicine, Won-kwang University)
  • 박주영 (원광대학교 한의학전문대학원 제3의학과) ;
  • 나용수 (원광대학교 한의학전문대학원 제3의학과) ;
  • 오공천 (원광대학교 한의학전문대학원 제3의학과) ;
  • 이상미 (원광대학교 한의학전문대학원 제3의학과) ;
  • 최병권 (원광대학교 한의학전문대학원 제3의학과) ;
  • 이윤승 (원광대학교 한의과대학 한방재활의학교실) ;
  • 송용선 (원광대학교 한의과대학 한방재활의학교실)
  • Received : 2018.03.18
  • Accepted : 2018.04.03
  • Published : 2018.04.30


Objectives The objective of this study is to determine the antimicrobial effect of Daehwanggyeonu-san(Dahwangqianniu-san,DGE) and synergistic effects with antibiotics oxacillin, ampicillin, and gentamicin against Methicillin-Resistant Staphylococcus aureus(MRSA). Methods The antibacterial activity of DGE extract was evaluated againest MRSA strains by using the Disc diffusion method, broth microdilution method(minimal inhibitory concentration; MIC), checkerboard dilution test. The checkerboard dilution test was used to examined synergetic effect of oxacillin, ampicillin, gentamicin, ciprofloxacin with DGE extract. Results DGE showed antimicrobial activity against MRSA with an MIC value of $125{\sim}250{\mu}g/mL$. In the checkerboard test, the interation of DGE with all tested antibiotics produced almost synergy or partial synergy against MRSA. Conclusions This study shows that DGE reduced the MICs of several antibiotics tested, and a remarkable antibacterial effect of DGE, with membrane permeability enhancers and ATP synthase inhibitors. This study can be a valuable source for the development of a new drug with low MRSA resistance.


Supported by : 원광대학교


  1. Rao Q, Shang W, Hu X, Rao X. Staphylococcus aureus ST121: a globally disseminated hypervirulent clone. J Med Microbiol. 2015;64(12):1462-73. DOI:10.1099/jmm.0.000185.
  2. da Silva Sdos S, Cidral TA, Soares MJ, de Melo MC. Enterotoxin-encoding genes in staphylococcus spp. from food handlers in a university restaurant. Foodborne Pathog Dis. 2015;12(11):921-5.
  3. 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.
  4. Korea National Institute of Health. Investigation of Infection Patterns and carrier of MRSA in Newborn Infants [Internet]. 2011. Available from:
  5. Mihailescu R, Furustrand Tafin U, Corvec S, Oliva A, Betrisey B, Borens O, Trampuz A. High activity of fosfomycin and rifampin against methicillin-resistant Staphylococcus aureus biofilm In Vitro and in an experimental foreign-body infection model. Antimicrob Agents Chemother. 2014;58(5):2547-53.
  6. Pham MH, Delestre L, Dewitte A, Wattez N, Lepretre F, Lansiaux A. Synergistic effect of SN-38 in combination with cetuximab on angiogenesis and cancer cell invasion. Anticancer Res. 2015;35(11):5983-91.
  7. Arzuman L, Beale P, Proschogo N, Yu JQ, Huq F. Combination of genistein and cisplatin with two designed monofunctional platinum agents in human ovarian tumour models. Anticancer Res. 2015;35(11):6027-39.
  8. Son DJ, Hong JE, Ban JO, Park JH, Lee HL, Gu SM, Hwang JY, Jung MH, Lee DW, Han SB, Hong JT. Synergistic inhibitory effects of cetuximab and cisplatin on human colon cancer cell growth via inhibition of the erk-dependent egf receptor signaling pathway. Biomed Res Int. 2015;2015:397563.
  9. Yu WS. Byungigiyibomyungjip. Seoul:Mok and Toe. 2000:154-163.
  10. Heo J. Dongyibogam. Hadong:Dongyibogam Publisher. 2005:420-1.
  11. You X, Feng S, Luo S, Cong D, Yu Z, Yang Z, Zhang J. Studies on a rhein-producing endophytic fungus isolated from Rheum palmatum L. Fitoterapia. 2013;85:161-8.
  12. Ma YS, Hsiao YP, Lin JH, Hsu SC, Chueh FS, Weng SW, Lai KC, Lin JG, Chung JG. Crude extract of Rheum palmatum L inhibits migration and invasion of LS1034 human colon cancer cells acts through the inhibition of matrix metalloproteinase-2/-9 by MAPK signaling. Environ Toxicol. 2015;30(7):852-63.
  13. Campbell MJ, Hamilton B, Shoemaker M, Tagliaferri M, Cohen I, Tripathy D. Antiproliferative activity of Chinese medicinal herbs on breast cancer cells in vitro. Anticancer Res. 2002;22(6C):3843-52.
  14. Wang J, Zhao H, Kong W, Jin C, Zhao Y, Qu Y, Xiao X. Microcalorimetric assay on the antimicrobial property of five hydroxyanthraquinone derivativesin rhubarb (Rheum palmatum L.) to Bifidobacterium adolescentis. Phytomedicine. 2010;17(8-9):684-9.
  15. KO MJ, Park SY, Lee KJ, Yi TH, Lee WC, Han CK, Khil JH. Anti-inflammatory effects of Pharbitis nil Choisy in lipopolysaccharide-induceds RAW 264.7 cells. The Journal of Cosmetological Science. 2010;6(2):105-11.
  16. Keum SS, You KW. Effect of Wubangja on the serum lactate dehydrogenase and protein content of hepatic insufficiency. Journal of Koeran medicine. 1978;15(1):59-68.
  17. Lee SA. Antimicrobial activity of Papenfussiella kuromo against methicillin-resistant Staphylococcus aureus [master's thesis]. [Iksan]:wonkwang university;2014.
  18. 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.
  19. 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.
  20. Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standards. CLSI/ document M7-A5. Wayne, PA:CLS;2000.
  21. Mun SH, Kang OH, Joung DK, Kim SB, Seo YS, Choi JG, Lee YS, Cha SW, Ahn YS, Han SH, Kwon DY. Combination Therapy of Sophoraflavanone B against MRSA: In Vitro Synergy Testing. Foodborne Pathog Dis. 2014;11(3):234-9.
  22. Mun SH, Kang OH, Joung DK, Kim SB, Choi JG, Shin DW, Kwon DY. In vitro anti-MRSA activity of carvone with gentamicin. Exp Ther Med. 2014;7(4):891-6.
  23. Mun SH, Lee YS, Han SH, Lee SW, Cha SW, Kim SB, Seo YS, Kong R, Kang DH, Shin DW, Kang OH, Kwon DY. In vitro potential effect of morin in the combination with ${\beta}$-Lactam antibiotics against methicillin-resistant staphylococcus aureus. Foodborne Pathog Dis. 2015;12(6):545-50.
  24. Santiago C, Pang EL, Lim KH, Loh HS, Ting KN. Reversal of ampicillin resistance in MRSA via inhibition of penicillin-binding protein 2a by Acalypha wilkesiana. Biomed Res Int. 2014;2014:965348.
  25. Santiago C, Pang EL, Lim KH, Loh HS, Ting KN. Inhibition of penicillin-binding protein 2a (PBP2a) in methicillin resistant Staphylococcus aureus(MRSA) by combination of ampicillin and a bioactive fraction from Duabanga grandiflora. BMC Complement Altern Med. 2015;15:178.
  26. Komatsuzawa H, Suzuki J, Sugai M, Miyake Y, Suginaka H. The effect of Triton X-100 on the in vitro susceptibility of methicillin-resistant Staphylococcus aureus to oxacillin. J Antimicrob Chemother. 1994;34(6):885-97.
  27. Irvin RT, MacAlister TJ, Costerton JW. Tris (hydroxymethyl) aminomethane buffer modification of Escherichia coli outer membrane permeability. J Bacteriol. 1981;145(3):1397-403. PMid:7009585 PMCid:PMC217144
  28. Toei M, Noji H. Single-molecule analysis of F0F1-ATP synthase inhibited by N,N-dicyclohexylcarbodiimide. J Biol Chem. 2013;288(36):25717-25726.
  29. Rudkin JK, Edwards AM, Bowden MG, Brown EL, Pozzi C, Waters EM, Chan WC, Williams P, O'Gara JP, Massey RC. Methicillin resistance reduces the virulence of healthcare-associated methicillin-resistant Staphylococcus aureus by interfering with the agr quorum sensing system. J Infect Dis. 2012;205(5):798-806.
  30. Takahashi N, Imanishi K, Uchiyama T. Overall picture of an emerging neonatal infectious disease induced by a superantigenic exotoxin mainly produced by methicillin-resistant Staphylococcus aureus. Microbiol Immunol. 2013;57(11):737-45.
  31. Stanier RY, John L. Ingraham, Mark L. Wheelis, Page R. Palnter. Basic and applied microbiology. Bae M, translator. Seoul:Academy company;1998:544.
  32. Moellering RC Jr. MRSA: the first half century. J Antimicrob Chemother. 2012;67(1):4-11.
  33. Khosravi AD, Hoveizavi H, Farshadzadeh Z. The prevalence of genes encoding leukocidins in Staphylococcus aureus strains resistant and sensitive to methicillin isolated from burn patients in Taleghani Hospital, Ahvaz, Iran. Burns. 2012;38(2):247-51.
  34. Ba X, Harrison EM, Lovering AL, Gleadall N, Zadoks R, Parkhill J, Peacock SJ, Holden MT, Paterson GK, Holmes MA. Old drugs to treat resistant bugs: methicillin- resistant Staphylococcus aureus isolates with mecC are susceptible to a combination of penicillin and clavulanic acid. Antimicrob Agents Chemother. 2015;59(12):7396-404.
  35. Mun SH, Joung DK, Kim YS, Kang OH, Kim SB, Seo YS, Kim YC, Lee DS, Shin DW, Kweon KT, Kwon DY. Synergistic antibacterial effect of curcumin against methicillin-resistant Staphylococcus aureus. Phytomedicine. 2013;20(8-9):714-8.
  36. Hiramatsu K, Cui L, Kuroda M, Ito T. The emergence and evolution of methicillin-resistant Staphylococcus aureus. Trends Microbiol 2001;9:486-93.
  37. Hiramatsu K, Katayama Y, Yuzawa H, Ito T. Molecular genetics of methicillin-resistant Staphylococcus aureus. Int J Med Microbiol. 2002;292(2):67-74.
  38. Klitgaard JK, Skov MN, Kallipolitis BH, Kolmos HJ. Reversal of methicillin resistance in Staphylococcus aureus by thioridazine. J Antimicrob Chemother. 2008;62(6):1215-21.
  39. Ohlsen K, Lorenz U. Novel targets for antibiotics in Staphylococcus aureus. Future Microbiol. 2007;2(6):655-6.
  40. Amaral L, Viveiros M, Kristiansen JE. 'Non-antibiotics': alternative therapy for the management of MDRTB and MRSA in economically disadvantaged countries. Curr Drug Targets. 2006;7(7):887-91.
  41. Viswanathan K, Frey KM, Scocchera EW, Martin BD, Swain Iii PW, Alverson JB, Priestley ND, Anderson AC, Wright DL. Toward new therapeutics for skin and soft tissue infections: propargyl-linked antifolates are potent inhibitors of MRSA and Streptococcus pyogenes. PLoS One. 2012;7(2):e29434.
  42. Poulsen MO, Jacobsen K, Thorsing M, Kristensen NR, Clasen J, Lillebaek EM, Skov MN, Kallipolitis BH, Kolmos HJ, Klitgaard JK. Thioridazine potentiates the effect of a beta-lactam antibiotic against Staphylococcus aureus independently of mecA expression. Res Microbiol. 2013;164(2):181-8.
  43. Segatore B, Bellio P, Setacci D, Brisdelli F, Piovano M, Garbarino JA, Nicoletti M, Amicosante G, Perilli M, Celenza G. In vitro interaction of usnic acid in combination with antimicrobial agents against methicillin-resistant Staphylococcus ureus clinical isolates determined by FICI and ΔE model methods. Phytomedicine. 2012;19(3-4):341-7.
  44. Barber KE, Werth BJ, Ireland CE, Stone NE, Nonejuie P, Sakoulas G, Pogliano J, Rybak MJ. Potent synergy of ceftobiprole plus daptomycin against multiple strains of Staphylococcus aureus with various resistance phenotypes. J Antimicrob Chemother. 2014;69(11):3006-10.
  45. Sin MK. Yimsangbonchohak. Seoul:YounglimSa. 2006:785-7, 791-3.
  46. Shah CS, Qadry JS, Bhatt JG. Qualitative and quantitative evaluation of anthraquinone derivatives in Indian rhubarb. Planta Med. 1972;22(1):103-8.
  47. Choi SB, Ma YH, Han YH, Jeong SJ, Jo HS. Effects of Rhei Radix et Rhizoma on insulin resistance and adipose tissue inflammatory response in high fat diet induced obese C57BL/6 mice. J Int Korean Med. 2014;35(2):195-207.
  48. Jang YH, Choi SW, Cho SH. Effect of Eisenia Bicyclis and its pill on serum lipid status in rats fed high fat diet. Korean J Nutr. 2008;41(1):5-12.
  49. Cho SH, Park SY, Choi SW. Effects of Eisenia Bicyclis extracts and pill on blood glucose and lipid profile in streptozotocin-induced diabetic mice. Korean J Nutr. 2008;41(6):493-501.
  50. Lee YJ. The effects of fraction obtained from rhei rhizoma on the blood lipids in hypercholesterol rats. Kor J Herbology. 2000;15(2):87-93.
  51. Yun HJ, Hwang SG, Yun HJ, Kim CH, Seo GS, Park WH, Park SD. Anticancer effect of Rheum Rhizoma on human liver cancer HepG2 cells. Kor J Herbology. 2006;21(4):27-36.
  52. Park SM, Lee GW, Cho YH. Effect of Rheum undulatum extract on antioxidant activity and activity of matrix metalloproteinase-1 in human skin fibroblasts. Journal of Life Science. 2008;18(12):1700-4.
  53. Lee JH. Antibacterial and synergistic effect of phlorotannins isolated from Eisenia bicyclis against acne-related bacteria [dissertation]. [Busan]:Pukyong National University;2014.
  54. Eom SH. Anti-MRSA (methicillin-resistant Staphylococcus aureus) substance isolated from Eisenia bicyclis and its action mechanism [dissertation]. [Busan]:Pukyong National University;2012.
  55. Choi MS. The antibacterial effects of emodin from rhei radix et rhizoma against methicillin-resistant Staphylococcus aureus [master's thesis]. [Iksan]:Wonkwang University;2007.
  56. Lyu KW, Yun ES. A study on the influence of pharbitidis semen on the activity of serum transaminase in carbon tetrachloride poisoned rabbits. The Journal of The Yellow-Emperor's Medicine. 1977;2(3):14-20.
  57. Seo BI, Lee YJ. A philological study on poisoning of pharbitidis semen. The Journal of East-West Medicine. 2008;33(2):1-13.
  58. Yoon SG, Park YH, Lee MJ. Effects of PS (Pharbitidis Semen) on anti-inflamatory and anti-febrile activities in arthritic rats. J Oriental Rehab Med. 2003;13(3):63-72.
  59. Kim JH, Seong JM, Moon HJ, Hwang JM, Hwang KS, Kwon YD, Kwon IK, Park YD. Effects of pharbitidis semen extract on osteoblast differentiation in MC3T3-E1 cells. Tissue Engineering and Regenerative Medicine. 2010;7(2):230-6.
  60. Hu Y, Liu A, Vaudrey J, Vaiciunaite B, Moigboi C, McTavish SM, Kearns A, Coates A. Combinations of ${\beta}$ -lactam or aminoglycoside antibiotics with plectasin are synergistic against methicillin-sensitive and methicillin-resistant Staphylococcus aureus. PLoS One. 2015;10(2):e0117664.
  61. Dosler S, Gerceker AA. In vitro activities of nisin alone or in combination with vancomycin and ciprofloxacin against methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains. Chemotherapy. 2011;57(6):511-6.
  62. Nester EW, Roberts CE, Pearsall NN, Anderson DG, Nester MT. Functional Anatomy of Prokaryotes and Eukaryotes. In Kane KT, Ronald E, Terrance S, Editors. Microbiology. 2nd ed. New York:WCB/McGraw-Hill;1998:63-6.

Cited by

  1. Synergistic effect of natural antifungal agents for postharvest diseases of blackberry fruits pp.00225142, 2019,