Characteristics of Baicalin Synergy with Penicillin or Notopterygium Ethanol Extracts Against Staphy

TSINGHUA SCIENCEISSN1007-0214A31TECHNOLOGY8pp459-461Volume 11, Number 4, August 2006Characteristics of Baicalin Synergy with Penicillin or NotopterygiumEthanol Extracts Against Staphylococcus aureusMENG Zhen(孟甄, Chongjin Sop(郑进燮), YoU Xuefu(游雪甫),Ⅺ NG Dongming(邢东明)WANG Wei(王伟), DU Lijun(杜力军)Laboratory of Pharmaceutical Sciences, Department of Biological Sciences and BiotechnologyTsinghua University, Beijing 100084, China;t Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and PekingUnion Medical College, Beijing 100050, ChinaAbstract: Baicalin facilitates the effectiveness of both penicillin and Notopterygium ethanol extracts(NEE)on Staphylococcus aureus. Two Staphylococcus aureus strains, ATCC29213 and 03-21(a clinical isolate)were tested. When combined with 128 ug mL of baicalin, the minimum inhibitory concentration(MIC) ofpenicillin was reduced from 0.25 ug mL-1to 0.06 ughile that of NEE was reduced from 32 ugto 8 ug mL against ATCC29213. Against the 03-21 strain, the MIc of penicillin was reduced from 4ug mL to 1 ug mL while that of NEe was reduced from 32 ug mL- to 4 ug mL. Viable number countingresults show that combination of baicalin and penicillin acts on the killing mechanismthe baicalin andNEE acts on the inhibition. These results show that baicalin acts as a synergistic allyoth penicillin andnee but has different modes of actionKey words: baicalin synergy; minimum inhibitory concentration; Staphylococcus aureuscumarin-like compounds), and penicillin(a B-lactameIntroductionantibiotic) on the in vitro antibacterial activity of theStaphylococcus aureus (s. aureus) resistance to moscombinations of baicalin plus penicillin and baicalinplus nEe against two strains of Staphylococcus aureusonce powerful p-lactame antibiotics is now a severeand compared the different characteristics of theirproblem world wide, so agents are urgently needed toactionsreduce or moderate resistance to existing antibioticsTraditional medicinal herbs with low toxicity and 1 Materials and Methodsminimal possibility of inducing resistance have at-tracted significant interest. Traditional herbs and com- 1.1 Materials and strainsbined application of medicinal herbs and antibioticshave been used to treat infections This study analyzedBaicalin was extracted from Radix scutellariathe effects of baicalin(a flavone compound), Notocalensis (purchased from Beijing Songlan Pharmaterygium ethanol extracts (NEE, mainly includingtical Herbal Company). The stock solution was dissolved in distilled water with the ph adjusted to 7.2Received: 2005-09-21: revised: 2006-01-17with I mol/L NaOH. Notopterygium ethanol extractsSupported by the Basic Research Foundation of Tsinghuawere extracted in ethanol from rhisoma et radixUniversitynotopterygii (bairintore). The col* To whom correspondence should be addressedlected distillate中国煤化工E-mail:pharm@mail.tsinghua.edu.cn;Tel:86-10-62773630CNMHG460Tsinghua Science and Technology, August 2006, 11(4): 459-461coccus aureus strains ATCC29213 and the checkerboard was performed to estimate the synergis-clinical isolate 03-21 were from the pharmacology lab tic effect of the two combinations as described aboveof Chinese Academy of Medical Sciences and Peking except with each plate well containing 50 HL of eachUnion Medicine College. Mh broth was obtained from drugthe National Institute for the Control of pharmaceuticaland Biological Products(Beijing, China). Penicillin1.3 Determinations of growth curveswas obtained from Sigma. Test organisms were incu- Inoculum of 1x10 CFU. mL-I was added to 4 mLbated in 4 mL of MH broth for 18 h at 37C. The cell of mh broth medium containing the selected drugsconcentrations were adjusted with MH broth to about The mixtures were incubated at 37 C. After contact2X10 CFU mL-(CFU is colony-forming units) by times of 0, 2, 4, 6, 8, and 24 h, a 100-uL sample ofmeasuring the absorbency at 600 nm. Over-dried nutri- each mixture was aseptically transferred to 900 HL MHtion agar plates were used for determining the CFUbroth and serially diluted. Growing colonies were1. 2 Minimum inhibitory concentration (MIC)counted after plating in triplicate and incubating at 37determinationC overnight. Positive controls contained similar cellsand solvent concentrationsStock solutions of the drugs were diluted with mhbroth to the required test concentrations. MICs were 2 Resultsdetermined using microtitre methods of twofold serialdilution. The first concentrations of each drug were2.1 miC determinatidoubled. Each well of the culture plates( costar) con- The fractional inhibitory concentration(FIC) index fortained 100 uL of the drugs with the same volume of the combination was derived as the ratio of the concen-bacteria prepared as described to give a final concentration of each antibacterial drug present in a combinatration of 10 CFU mL The plates were coveretion of antibacterial drugs at the mic for the combinaand incubated at 37C for 16-18 h. The MiC was tion to the MIC concentrations for the antibacterialtaken as the lowest concentration at which the test or- drug used alone. An FIC index of 0. 8 or below wasganisms did not show visible growth. The culture ab- presumed to a synergistic interaction I!. Table 1 listedsorbency in each well was determined at 600 nm. An 8 the synergistic indication for the two combinationsTable 1 MICs of NEE and penicillin with and without baicalin(128 ug. mL )against S aureus strainsMC/(g·mL-)s aureus strainsMC/(pg·mL)NEE NEE +baicalin NEE Penicillin Penicillin +baicalin penicilinATCC292130.362.2 Growth curve determinationsBaicalin, a flavone compound, is one of the mostimportant active constituents in the Chinese herbAlthough baicalin showed obvious synergistic activity Scutellaria baicalensis, whose antibacterial activitywith NEE and penicillin, the variation of the number of has been ascertained by modern pharmacological reviable colonies with time demonstrated different searches 2-4. In this study, baicalin showed synergisticmodes of action, The inhibition of theffect with both NEE and penicillin at 128 ug.mLaureus by baicalin and nee was not obvious within 24(87.5% lower than its MICh, while the combination of baicalin and penicillin The counting of the viable number for the clinicalcompletely killed the bacteria in a few hoursisolate 03-21 indicated different modes of action for3 Discussionthe two comb中国煤化工ng curve wasobserved withTHCNMHGMENG Zhen(n h)et al: Characteristics of Baicalin Synergy with Penicillin461lin, with most bacteria killed within 4 h, the use of only not kill the bacteria but only inhibited growth withinbaicalin or only penicillin had no bactericidal effect 24 h of application with insufficient bacteria growth to(Fig. 1b). The combination of baicalin and nee did the degree to be visible(Fig. la)icillin◆128μ g mL baicalinX 128 ugmL baicalin +lug mL penicillin68 ug.mL NEE◆128 ug- mL baicalinx 128 ug mL baicalin+8 ugmL NEE12162024Time(h)at37℃812162at37℃(a)with NEE(b)With penicillinFig. 1 Effect of baicalin(128Hg. mL-)combined with NEE (a)or penicillin (b)on the viable counts of 03-21.The values are the means of three observationsThe combination of baicalin and penicillin restoredto treatment of burn wound infections, international jourpenicillins bactericidal effect at low dose through bai-l of Pharmaceutics, 1991, 75: 81-85calin's three distinct types of activities: weak antibacte- [2] Wang S. Research of TCM on anti-bacteria infectionrial action, inhibition of B-lactamase hydrolysis, andJournal of Beijing TCM, 2002, 21: 249-251.(in Chinese)inhibitory interactions between B-lactamase and peni- [3] Yan W D, Wang R J, Yan H E, Yang Y XDevelopmentcillin-binding proteins. The mechanism for the actionon the pharmacology of baicalin. Journal of shanxi TCM,of baicalin and nee needs further research. The cura2002,23:1127-1129.( in Chinese)tive effect of the combination of the traditional herbs [4] Yang D, Hu H, Huang S, Chaumont J P, Millet J. Studyseems to be an integrated effect of their multiple func-on the inhibitory activity, in vitro, of baicalein and bations in vivo. For example, baicalin at low dose markcalin against skin fungi and bacteria. Journal of chinesedly enhanced the ingestion function of mouse peneal macrophages in vivo. This study shows that bai- [5] Liu I X, Durham D G, Richards R M. Baicalin synerg.alin acts as a synergistic ally with both penicillin andwith B-lactam antibiotics against methicillin-resistantnee but has different modes of action that needs to betaphylococcus aureusβ actam-resistantfurther investigatedstrains of S. aureus. Journal of pharmacy and Pharma-cology,2000,52:361-36Acknowledgement[6 Cai X D, Tan JP, Wang L X Effect of baicalin on the celWe thank Dr ZHANG Lujun for kindly providing baicalinlular immunity of mice. Jounal of Nanjing Railway Medical College, 1994, 13: 65-68.(in ChineseReferences[1 Richards R M e, Xing D K L. Evaluation of synergisticeffects of combinations of antibacterials having relevance中国煤化工CNMHG