Research Article Antimicrobial Effects and Resistant ...

Research ArticleAntimicrobial Effects and Resistant Regulation of Magnolol andHonokiol on Methicillin-Resistant Staphylococcus aureus

Su Young Kim12 Ju Kim1 Seung-Il Jeong1 Kwang Yeop Jahng2 and Kang-Yeol Yu1

1 Jeonju Biomaterials Institute Jeonju Jeonbuk 561-360 Republic of Korea2Department of Life Science Chonbuk National University Jeonju Jeonbuk 561-756 Republic of Korea

Correspondence should be addressed to Kang-Yeol Yu kangyujbmirekr

Received 3 June 2015 Accepted 6 August 2015

Academic Editor James W Daily III

Copyright copy 2015 Su Young Kim et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The antimicrobial killing activity toward methicillin-resistant Staphylococcus aureus (MRSA) has been a serious emerging globalissue In a continuing search for compounds with antibacterial activity against several microorganisms including S aureus andMRSA an n-hexane extract ofMagnolia officinaliswas found to containmagnololThis compound exhibited potent activity againstS aureus standard methicillin-susceptible S aureus (MSSA) and MRSA as well as clinical MRSA isolates When combined withoxacillin the antibacterial activities ofmagnolol and honokiol against theMRSA strainwere increased compared to single treatmentwithout antibiotics at 10 120583gmL and 25 120583gmL respectively These activities of magnolol and honokiol were dose dependent Alsomagnolol showed synergistic effects with oxacillin against 13 clinical isolates of MRSA It was determined that magnolol andhonokiol had a synergistic effect with oxacillin against MRSA strain Furthermore the magnolol inhibited the expression of theresistant genes mecA mecI femA and femB in mRNA We concluded that the antibacterial activity of magnolol against MRSAstrain is more related to the mecIrsquos pathway and components of the cell wall than mecR1 Therefore the results obtained in thisstudy suggest that the combination of magnolol and antibiotics could lead to the development of new combination antibioticsagainst MRSA infection

1 Introduction

Staphylococcus aureus a Gram-positive coccus is an impor-tant human nosocomial pathogen which causes life-threat-ening systemic infections such as pneumonia osteomyelitisendocarditis and septicemia [1] Today the ongoing emer-gence of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and the infectiousdiseases caused by them are serious globally [2] Staphylo-coccal resistance to wide spectrum 120573-lactam antibiotics suchas methicillin cefotaxime and oxacillin emerged soon afterthe introduction of the first drug in this class and cannot betreated with conventional 120573-lactams making glycopeptides(vancomycin or teicoplanin) the only therapeutic solution[3 4] Particularly worrying is the increase in incidence ofmultidrug-resistant organisms such as MRSA and VRSAIn recent years emergence of MRSA strains with reducedsusceptibility to glycopeptides has emphasized the need

and new approaches for novel effective drugs to combatthese problems [5] Therefore combination therapy is oftenuseful for patients with serious infections caused by drug-resistant pathogens [6] In an attempt to overcome the seriousproblem of widespread multidrug MRSA it is essential tostudy the new antibacterial agents in combination with theexisting antibiotics [3] The use of combination therapy canbroaden the spectrum of antimicrobial activity canminimizethe emergence of resistant organisms and can sometimesresult in synergistic antibiotic interactions thereby exhibitingantimicrobial activity greater than would be expected fromeach antimicrobial drug individually [7ndash9]

The herb Magnolia officinalis has been used for thetreatment of diarrhea acute pain coughs diarrhea andurinary problems Magnolol and honokiol are main polyphe-nol compounds of the bark of this medicinal plant andhave a variety of pharmacological activities Several recentreports identified and proved that magnolol and honokiol

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 283630 9 pageshttpdxdoiorg1011552015283630

2 BioMed Research International

have several medicinal functions such as antioxidative anti-inflammatory and antitumor [10ndash12] In addition magnololand honokiol have been known to show high antimicrobialactivity against several microorganisms such as fungi Propi-onibacterium sp and S aureus [10 13ndash18] Recent researchreported that magnolol inhibited the transcription of hla (thegene encoding 120572-toxin) in both MSSA and MRSA resultingin a reduction of 120572-toxin production and hemolytic activities[19]

Methicillin a 120573-lactam antibiotic acts by inhibitingthe transpeptidation domain of penicillin-binding proteins(PBPs) that are involved in the synthesis of peptidoglycanWithout cross-linking reaction of the peptidoglycan thecell wall becomes mechanically weak some of the cytoplas-mic contents are released and the cell dies [20] Resistantstrains typically produced an enzyme called a 120573-lactamasewhich inactivated the 120573-lactam However S aureus canbecome resistant to methicillin and other 120573-lactam antibi-otics through the expression of a foreign PBP PBP2a (a low-affinity penicillin-binding protein) that is resistant to theaction of methicillin but which can perform the functions ofthe host PBPs [20]

The mechanism of resistance to methicillin is very com-plex and it is not completely characterized yet Howeverit may be due to the overproduction of 120573-lactamase [20]the expression of mecA producing PBP2a (also referred toas PBP21015840) showed a low affinity to 120573-lactam antibiotics [21]and the change of PBP type [22 23] detected factors otherthan the expression of the mecA gene that could controlthe induction of PBP2a production in S aureus E 67-0 andtermed it mecR this mecR is present in some S aureussuppresses the synthesis of PBP2a present in a certain geneticbackground and mediates the influence on the expressionof methicillin resistance This mecR consists of two genesmecI (the gene encoding a transcriptional regulator (MecI))and mecR1 (the gene encoding a membrane-bound signaltransduction protein (MecR1)) located in the upper streamofthe mecA gene [24] The mecR1 is activated upon the contactwith 120573-lactam that is equivalent to an inducing factor withMRSA and its signal binds to the promoter region of mecAand is transduced to mecI that suppresses transcription andthus the suppression is cleared

In addition previous studies [20 25] reported that thefemA and femB genes play important roles in building thepentaglycine crossbridges that link the glycan chains together

The situation is that the action mechanism of the femAgene is not elucidated yet nevertheless it is thought that itdoes not influence PBP2a synthesis Significantly inactiva-tion of either femA or femB also results in a large reduction inmethicillin resistance by the interferencewith the crossbridgelength and the secretion of virulence factors which couldlimit the ability of the cell to cause infection [20] The femAgene is involved in the cell wall especially glycine content ofpeptidoglycan and peptidoglycan biosynthesis of S aureusand mediates the effect on drug sensitivity and thus it isinvolved in methicillin resistance [25 26]

In the present study we investigated antimicrobial effectsand resistant regulation of magnolol and honokiol againstMRSA strains for potential application as a natural product

Honokiol

Magnolol

Standard honokiol

Purified magnolol

Standard magnolol

(mAU

)

120

100

80

60

40

20

0

5 10 15

Honokiol

Magnolol

Standard honokiol

Purified magnolol

Standard magnolol

Figure 1 Comparison of the purified magnolol isolated fromMagnolia officinalis with standard compounds by HPLC

agent To the best of our knowledge this is the first workon the combined effect of magnolol and honokiol usingMICmethod and transcriptional regulation study of resistantgene to substantiate the synergistic antimicrobial activityof compoundantibiotics combination against MRSA andclinical MRSA isolates

2 Materials and Methods

21 Reagents Oxacillin honokiol (ge98) [3V5-di-2-pro-penyl-(11V-biphenyl)-24V-diol] and magnolol (ge95) [5V5-di-2-propenyl-(11V-biphenyl)-22V-diol] were purchasedfrom Sigma Magnolol and honokiol were dissolved in 100EtOH

22 Plant Materials Extraction and Isolation Magnolol waspurified from the bark ofM officinalis as described previously[27] In brief the methanol extracts were concentrated andlyophilized with a rotary evaporator and partitioned betweenwater and n-hexane The n-hexane fraction was then appliedto a silica gel column eluted with an n-hexane acetone(8 11 vv)mixtureThe n-hexane layerwas concentrated andmagnolol was directly obtained by prep-HPLC of n-hexanelayer The isolated magnolol was identified by thin-layerchromatograph and mixed melting point determination incomparison with authentic magnolol (Wako Osaka Japan)The purity (ge98) of magnolol was determined by high-performance thin-layer chromatograph (Figure 1)

23 Bacteria The bacteria used in this study were Bacillussubtilis (ATCC6633) Escherichia coli (ATCC8739) Propioni-bacterium acnes (ATCC 6919) Staphylococcus aureus (ATCC6538) standardmethicillin-susceptible Staphylococcus aureus(MSSA ATCC 25923) methicillin-resistant Staphylococcusaureus (MRSA ATCC 33591) and clinical MRSA isolatesused in [28 29] All strains were cultured at 34∘C for 24 hwith tryptic soy medium (Difco) under aerobic conditionsbefore the assay After culturing all strains onMueller-Hintonagar (Difco Detroit MI) the cells were resuspended in

BioMed Research International 3

Mueller-Hinton broth (Difco) to give 108 colony-formingunitsmL [29] The MRSA strains were defined on the basisof the occurrence of themecA gene and of their resistance toampicillin and methicillin according to the guidelines of theNational Committee for Clinical Laboratory Standards [29]

24 Antibacterial Activity Test and Determination of MICsAll strains were grown in tryptic soy broth for 24 hr at34∘C The S aureus suspensions were adjusted to the OD 03(approximately 1 times 108 CFUmL) and treated with samplesAfter 24 hr incubation 200120583L of MRSA suspension wastransferred to 96-well plate to measure OD by microplatereader (PerkinElmer multilabel counter) The data werereported as the minimum inhibitory concentrations (MICs)the lowest concentration ofmagnolol and honokiol inhibitingvisible growth after 24 hr of incubation 34∘C [30] The MICsof oxacillin magnolol and honokiol were also determinedand similarly defined as the lowest concentration at which novisible bacterial growth was observed

25 RT PCR and Real-Time PCR MRSA (ATCC 33591) strainwas grown in TSB at 34∘C with the graded subinhibitoryconcentrations of magnolol and honokiol to the postex-ponential growth phase Total RNA was isolated from thestrains with Trizol reagent (Molecular Research Center Inc)in accordance with the manufacturerrsquos specifications TheRNase-free DNase I (Takara) treatment was carried out toremove contaminating DNA First-strand cDNAwas synthe-sized using reverse transcription system (Promega) using 1120583gtotal RNA Ten percent of the RT product was added to aPCR reaction 27 PCR cycles were followed by denaturation at95∘C and extension at 72∘C Primer sequences are as follows[31] GAPDH F 51015840-TGACACTATGCAAGGTCGTTTCAC-31015840 R 51015840-TCAGAACCGTCTAACTCTTGGTGG-31015840 mecAF 51015840-GTAGAAATGACTGAACGTCCGATAA-31015840 R 51015840-CCAATTCCACATTGTTTCGGTCTAA-31015840 mecI F 51015840-CTGCAGAATGGGAAGTTATG-31015840 R 51015840-ACAAGTGAA-TTGAAACCGCC-31015840mecR1 F 51015840-AAGCACCGTTACTAT-CTGCACA-31015840 R 51015840-GAGTAATTTTGGTCGAATGCC-31015840femA F 51015840-CATGATGGCGAGATTACAGGCC-31015840 R 51015840-CGCTAAAGGTACTAACACACGG-31015840 femB F 51015840-TTA-CAGAGTTAACTGTTACC-31015840 R 51015840-ATACAAATCCAG-CACGCTCT-31015840 The real-time PCR reactions were per-formed in a 20120583L final volume and contained SYBR mastermix (Quantace) as recommended by the manufacturer Thereactions were carried out by using the Rotor-Gene 6000(Corbett Life Science) Cycling parameters were as follows95∘C for 10min 40 cycles at 95∘C for 10 s 60∘C for 15 s and72∘C for 20 s All samples were analyzed in triplicate and theGAPDH gene was used as an internal control housekeepinggene to normalize the levels of expression between samplesThe real-time PCR data were analyzed by the (ΔΔCt) methodusing the Rotor-Gene comparative concentration utility

26 Statistical Analysis of the Results Experimental data wereanalyzed using SigmaPlot 100 statistical software Data areexpressed as the mean plusmn SD Statistical differences were

examined using the independent Student 119905-test A119901 value lessthan 005 was considered statistically significant

3 Results and Discussion

Infection caused by MRSA poses a serious global problembecause MRSA strains are resistant to many antibioticsin the hospital environment Particularly worrying is theincrease in the incidence of multidrug-resistant organismssuch as MRSA and VRSAThus novel antimicrobials for newapproaches to resolve these problems are urgently neededIn a continuing search for the compounds with antibacterialactivity against MRSA Magnolia officinalis was investigatedThe MeOH extract of M officinalis was fractionated intohexane chloroform ethyl acetate and n-butanol solublefractions Of these only the hexane extract showed notableantibacterial activity against the MRSA strain Thereforeaiming to identify the active substances the hexane fractionwas submitted to column chromatography in silica gel A nee-dle white compound was purified by repeated prep-HPLCas the active constituent against the bacteria On the basisof the foregoing findings the compound was determinedto be named as magnolol Comparative chromatograms ofthe purified magnolol the standard magnolol and honokiolshowed that the purity of the purified magnolol the standardmagnolol and honokiol was 96 99 and 98 respectivelyThe result also showed that the HPLC retention time ofmagnolol was different from honokiol (Figure 1) Also thedifferent HPLC retention time of magnolol from honokiolrespectively showed that they are different compounds

In a continuing search for the compounds with antibac-terial activity against several microorganisms including Saureus MRSA (ATCC 33591) B subtilis and E coli ann-hexane extract of M officinalis showed antimicrobialactivities Magnolol showed antibacterial activities against Saureus MRSA and 13 clinical MRSA isolates (Figure 2(a)and Table 1) According to previous studies [13 14] magnololand honokiol showed antibacterial activities against P acnes(data not shown) and B subtilis except E coli (Figure 2)Moreover the growth of MRSA cultured with magnololwas significantly decreased in the range of 10ndash25 120583gmLsimilar to S aureus As shown in Figure 2(b) the growth ofMRSA cultured with honokiol was significantly decreased inthe range of 10ndash50120583gmL However there was no obviousantimicrobial effect of honokiol against S aureus B subtilisand E coli at 50120583gmLMagnolol showed better antibacterialactivity against S aureus and MRSA than honokiol Thesedata suggest that magnolol may be useful to treat S aureusand MRSA infections

The antibacterial activities ofmagnolol and honokiol withantibiotics against MRSA were shown in Figure 3 Whencombinedwith 50120583gmL oxacillin the antibacterial activitiesof magnolol and honokiol against the MRSA standard strainwere increased compared to single treatment without antibi-otics at 10 120583gmL and 25120583gmL respectively These activi-ties of magnolol and honokiol were dose dependent Alsomagnolol exhibited potent activities against the standardMSSA and MRSA as well as the clinical isolates of MRSA As


Table 1 Antibacterial activity tests of magnolol against methicillin-resistant S aureus (MRSA)

S aureus strain Class Magnolol (120583gmL)lowast Oxacillin 25 120583gmL + magnolol (120583gmL)lowast

0 1 10 25 50 0 1 10 25 50ATCC25923 MSSA +++ +++ +++ +minus mdash mdash mdash mdash mdash mdashATCC33591 MRSA +++ +++ +++ +minus mdash +++ +++ +++ mdash mdashClinical isolates

S aureus 78 MRSA +++ +++ +++ mdash mdash +++ +++ mdash mdash mdashS aureusM11 MRSA +++ +++ +++ +minus mdash +++ +++ mdash mdash mdashS aureus 21-8 MRSA +++ +++ +++ mdash mdash +++ +++ +minus mdash mdashS aureus 6-2 MRSA +++ +++ +++ mdash mdash +++ +++ mdash mdash mdashS aureus 7-3 MRSA +++ +++ +++ +minus mdash +++ +++ mdash mdash mdashS aureus 8-4-1 MRSA +++ +++ +++ +minus mdash +++ +++ mdash mdash mdashS aureus 8-4-2 MRSA +++ +++ +++ +minus mdash +++ +++ mdash mdash mdashS aureus 9-5 MRSA +++ +++ +++ +minus mdash +++ +++ mdash mdash mdashS aureus 13-7 MRSA +++ +++ +++ mdash mdash +++ +++ +minus mdash mdashS aureus 27-9 MRSA +++ +++ +++ mdash mdash +++ +++ mdash mdash mdashS aureus 47-10 MRSA +++ +++ +++ mdash mdash +minus +minus mdash mdash mdashS aureus 105-13 MRSA +++ +++ +++ +minus mdash +++ +++ mdash mdash mdashS aureus 106-14 MRSA +++ +++ +++ +minus mdash +++ +++ +minus mdash mdash

lowast+++ growing very well +minus growing slightly mdash no growth

00

20

40

60

80

100

0 1 10 25 50 100

log 1

0(C

FUm

L)

Magnolol (120583gmL)

MRSAE coliB subtilis

S aureus

(a)

00

20

40

60

80

100

0 1 10 25 50 100

log 1

0(C

FUm

L)

Honokiol (120583gmL)


S aureus

(b)

Figure 2 Antimicrobial activities of magnolol (a) and honokiol (b) against Staphylococcus aureus (ATCC 6538) methicillin-resistantStaphylococcus aureus (MRSA ATCC 33591) Bacillus subtilis (ATCC 6633) and Escherichia coli (ATCC 8739) All strains were culturedat 34∘C for 24 h with tryptic soy medium (Difco) under aerobic conditions before the assay For antibacterial activity test Mueller-Hintonmedium (Difco Detroit MI) was used

shown in Table 1 magnolol showed more significant antibac-terial activity with oxacillin than single treatment withoutantibiotics against 13 clinical isolates of MRSA at 10 120583gmLThese results determined that magnolol and honokiol had asynergistic effect with oxacillin against MRSA strain These

data suggest that magnolol and honokiol are potentiallyuseful for the treatment of drug-resistant S aureus infectionswhen used in combination with lower concentration ofantibiotics Against the MRSA standard strain the MICsof magnolol and oxacillin were 20 120583gmL and 177 120583gmL


00

20

40

60

80

100

0 1 10 25 50 100

log 1

0(C

FUm

L)


Oxacillin200120583gmL

minusoxacillin+oxacillin 50120583gmL

(a)

00

20

40

60

80

100

0 1 10 25 50 100

log 1

0(C

FUm

L)




(b)

Figure 3 Synergistic effect of magnolol and honokiol with oxacillin againstMRSA Oxacillin 50 120583gmLwas used for synergistic effect MRSAstrain was grown in tryptic soy broth for 24 hr at 34∘C

respectively (Table 2) The MIC of magnolol in combinationwith 50 120583gmL oxacillin was 16 120583gmL against the MRSAstandard strain AlsoMIC of honokiol was 33 120583gmL and theMIC of honokiol in combination with 50 120583gmL oxacillinwas 23 120583gmL against the MRSA standard strain There wasno significant synergistic effect between single treatment ofmagnolol and the combination treatment with antibioticswhen compared to MICs

In the present study five antibiotics were chosen accord-ing to their different mechanism of action against MRSABoth oxacillin and ampicillin from penicillin class andcefoxitin from cephalosporin class are the inhibitors of cellwall biosynthesis while chloramphenicol and tetracycline areprotein inhibitors [3 32] The combination of antimicrobialagent acts on the different target site of bacteria that couldlead to synergistic effect [33] Antibacterial activities ofmagnolol and honokiol with the combination of five repre-sentative antibiotics oxacillin (Ox) ampicillin (Amp) chlo-ramphenicol [33] tetracycline (Tet) and cefoxitin (Cef) wereevaluated against standard MRSA strain (Table 2) Whencombined with 50120583gmL oxacillin the magnolol showed thebest synergistic effect (77) compared to other antibioticsagainst MRSA strain On the other hand when combinedwith 25120583gmL chloramphenicol and 10 120583gmL cefoxitin thehonokiol showed the best synergistic effect (93) comparedto other antibiotics It was shown from these results thatmagnolol and honokiol had a synergistic effect with severalrepresentative antibiotics as well as oxacillin against MRSAstrain These data suggest that magnolol and honokiol arepotentially useful for the treatment of multidrug-resistantS aureus infections when used in combination with lowerconcentration of antibiotics

To investigate the resistant regulation of magnolol andhonokiol againstMRSA strains we tried the RT-PCR (reversetranscriptase-polymerase chain reaction) analysis of resistantgenes involved in MRSA resistance to 120573-lactam antibi-otics Figure 4 shows the results of resistant gene expres-sion for MRSA and clinical MRSA isolates using RT-PCR

mecA

mecRI

mecI

GAPDH

femA

femB

MRSA clinical isolates

MSS

AM

RSA 78

M11

21-8 6-2

7-3

8-4-1

8-4-2

9-5

13-7

27-9

47-10

105-13

106-14

Figure 4 RT-PCR analysis of the level ofmecAmecImecR1 femAand femB mRNA in S aureus strains All strains were grown intryptic soy broth for 24 hr at 34∘C

The standard MRSA (ATCC 33591) was mecA-positive andthe standard MSSA (ATCC 25923) was mecA-negative Wecould not confirm the gene expression of mecA mecI andmecR1 in S aureus (ATCC 6538) as well as MSSA (ATCC25923) but we could see these genes appear clearly inMRSA (ATCC 33591) The mecA gene expressed all MRSAstrains including standard MRSA (ATCC 33591) and 13clinical MRSA isolates For further study about the resistantgene regulation of magnolol and honokiol we selected thestandard MRSA (ATCC 33591) expressing all resistant genes

As shown in Figure 5 the expression of the mecA genewas inhibited in a dose-dependent manner by the magnololand was weekly decreased by honokiol Also femA and femBgenes known to be related with the composition of cell wallalso exhibited that magnolol inhibited gene expression Real-time PCRwas carried out to assess the transcriptional level ofthe resistant genesmecA mecI mecR1 femA and femB aftertreatment with subinhibitory concentrations of magnololand honokiol As shown in Figure 6 magnolol significantlyinhibited the transcription ofmecA mecI femA and femB inMRSA (ATCC 33591)When culturedwith 1120583gmL 5 120583gmL


Table 2 The MICs and synergistic effects of the magnolol and honokiol against standard MRSA strain

Antibacterial agent MIC (120583gmL) Antibioticslowast

minus Oxacillin + Oxacillin 50 120583gmL Ox Amp Cam Tet CefOxacillin 177Magnolol 20 16 syn (77) mdash syn (15) syn (22) syn (54)Honokiol 33 23 syn (38) mdash syn (93) mdash syn (93)lowastOx oxacillin 50120583gmL Amp ampicillin 100120583gmL Cam chloramphenicol 25 120583gmL Tet tetracycline 15 120583gmL Cef cefoxitin 10 120583gmL

mecA

mecRI

mecI

GAPDH

femA

femB

Magnolol (120583gmL)0 1 5 10 OX 50Control

(a)

mecA

mecRI

mecI

GAPDH

femA

femB

0 1 5 10 OX 50Honokiol (120583gmL)

Control

(b)

Figure 5 RT-PCR analysis of the level ofmecAmecImecR1 femA and femBmRNA inMRSA (ATCC 33591) strain treated with the indicatedconcentrations of the magnolol (a) and honokiol (b) GAPDHwas employed as an internal control and oxacillin 50120583gmL (OX 50) was usedin this study MRSA strain was grown in tryptic soy broth for 24 hr at 34∘C

and 10 120583gmL of magnolol the transcriptional levels ofmecAin MRSA strain were decreased by 040- 030- and 019-fold respectively Also the transcriptional levels ofmecI weredecreased at 5 120583gmL and 10 120583gmL by 070- and 032-foldrespectively The transcriptional levels of femA and femBwere decreased at 10 120583gmL magnolol by 063- and 059-foldrespectively When cultured with 10 120583gmL of honokiol thetranscriptional level of mecA in MRSA strain was decreasedby 054-foldThemecA genewas affected in a dose-dependentmanner by magnolol and honokiol at the transcriptionallevel in a dose-dependent manner This was due to theregulation ofmethicillin resistance bymagnolol and honokiolthereby inhibiting mecA gene expression that could controlthe induction of PBP2a production However mecR1 genedid not show the effectiveness against MRSA strain so weconcluded that the antibacterial activity of magnolol againstMRSA strain is more related tomecIrsquos pathway thanmecR1

Magnolol and honokiol main compounds isolated fromthe bark ofM officinalis increase the susceptibility of MRSAisolates to 120573-lactams either by affecting the expression of themecA gene or by interfering with either the signalling domainofMecI or some other protein involved in signal transductionand components of the cell wall By targeting and inhibitingthe proteins such as PBP2a FemA and FemB involvingthe formation of these substrates methicillin resistance can

be modulated Compounds such as magnolol and honokiolare attractive compounds to develop into therapeutic agentssince these compounds already exist and are known tomodulate methicillin resistance

4 Conclusion

The present study investigated the antimicrobial effects andthe resistant regulationmechanismofmagnolol and honokiolagainst MRSA strains for potential application as a naturalproduct agent These compounds exhibited potent activityagainst MSSA as well as clinical isolates of MRSA Further-more magnolol and honokiol showed the synergistic effectwith oxacillin againstMRSA strain and reduced expression ofthe resistant genes mecA mecI femA and femB in mRNAFrom the study it was concluded that magnolol has thepotential to restore the effectiveness of 120573-lactam antibioticsagainst MRSA and other strains of 120573-lactam-resistant Saureus In a view of its limited toxicity magnolol offersthe potential for the development of a valuable adjunct to120573-lactam treatments against otherwise resistant strains ofmicroorganismsTherefore the results obtained in this studysuggest that the combination of magnolol and antibioticscould lead to the development of new combination antibioticsagainst MRSA infection


00

05

10

15

20

25

Control 0 1 5 10 OX 50Magnolol (120583gmL)

Relat

ive g

ene e

xpre

ssio

nm

ecA

lowastlowastlowast

(a)

00

10

20

30

40

50

0 1 5 10 OX 50ControlMagnolol (120583gmL)

Relat

ive g

ene e

xpre

ssio

nm

ecI

lowast

(b)

00

10

20

30

40

50

0 1 5 10 OX 50Magnolol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nm

ecRI

(c)

00

05

10

15

20

25

0 1 5 10 OX 50Magnolol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nfem

Alowast

(d)

00

05

10

15

20

25

0 1 5 10 OX 50

Relat

ive g

ene e

xpre

ssio

nfem

B

lowastlowast

Magnolol (120583gmL)Control

(e)

00

05

10

15

20

25

0 1 5 10 OX 50Honokiol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nm

ecA

lowast

(f)

Figure 6 Relative gene expression of mecA mecI mecR1 femA and femB gene in MRSA (ATCC 33591) strain treated with the indicatedconcentrations of the magnolol ((a)ndash(e)) and honokiol (f) Oxacillin 50 120583gmL (OX 50) was used in this study MRSA strain was grown intryptic soy broth for 24 hr at 34∘C Values represent the mean and standard error of three independent experiments Statistical differenceswere examined using the independent Student 119905-test A 119901 value less than 005 was considered statistically significant lowast represents 119901 lt 005

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by the Regional Specialized Tech-nology Convergence RampD Program funded by the Ministryof Trade Industry and Energy

References

[1] X-S Pan P J Hamlyn R Talens-Visconti F L AloveroR H Manzo and L M Fisher ldquoSmall-colony mutants ofStaphylococcus aureus allow selection of gyrase-mediated resis-tance to dual-target fluoroquinolonesrdquo Antimicrobial Agentsand Chemotherapy vol 46 no 8 pp 2498ndash2506 2002

[2] M Li B A Diep A E Villaruz et al ldquoEvolution of virulence inepidemic community-associatedmethicillin-resistant Staphylo-coccus aureusrdquo Proceedings of the National Academy of Sciences


of the United States of America vol 106 no 14 pp 5883ndash58882009

[3] D F Basri L W Xian N I Abdul Shukor and J Latip ldquoBac-teriostatic antimicrobial combination antagonistic interactionbetween epsilon-viniferin and vancomycin against methicillin-resistant Staphylococcus aureusrdquoBioMed Research Internationalvol 2014 Article ID 461756 8 pages 2014

[4] G Lin L M Ednie and P C Appelbaum ldquoAntistaphylococcalactivity of ACHN-490 tested alone and in combination withother agents by time-kill assayrdquo Antimicrobial Agents andChemotherapy vol 54 no 5 pp 2258ndash2261 2010

[5] C E Udobi A F Obajuluwa and J A Onaolapo ldquoPreva-lence and antibiotic resistance pattern of methicillin-resistantStaphylococcus aureus from an orthopaedic hospital in nigeriardquoBioMed Research International vol 2013 Article ID 860467 4pages 2013

[6] M A Dawis H D Isenberg K A France and S G Jenk-ins ldquoIn vitro activity of gatifloxacin alone and in combina-tion with cefepime meropenem piperacillin and gentamicinagainstmultidrug-resistant organismsrdquo Journal of AntimicrobialChemotherapy vol 51 no 5 pp 1203ndash1211 2003

[7] G M Eliopoulos and C T Eliopoulos ldquoAntibiotic combina-tions should they be testedrdquoClinicalMicrobiology Reviews vol1 no 2 pp 139ndash156 1988

[8] A Belley E Neesham-Grenon F F Arhin G A McKay T RParr Jr andGMoeck ldquoAssessment by time-killmethodology ofthe synergistic effects of oritavancin in combination with otherantimicrobial agents against Staphylococcus aureusrdquo Antimicro-bial Agents and Chemotherapy vol 52 no 10 pp 3820ndash38222008

[9] D F Basri and R Khairon ldquoPharmacodynamic interactionof Quercus infectoria galls extract in combination with van-comycin against MRSA using microdilution checkerboard andtime-kill assayrdquoEvidence-Based Complementary andAlternativeMedicine vol 2012 Article ID 493156 6 pages 2012

[10] J-L Shen K-M Man P-H Huang et al ldquoHonokiol andmagnolol as multifunctional antioxidative molecules for der-matologic disordersrdquo Molecules vol 15 no 9 pp 6452ndash64652010

[11] K L E Hon T F Leung P C Ng et al ldquoEfficacy and tolerabilityof a Chinese herbalmedicine concoction for treatment of atopicdermatitis a randomized double-blind placebo-controlledstudyrdquo British Journal of Dermatology vol 157 no 2 pp 357ndash363 2007

[12] L E Fried and J L Arbiser ldquoHonokiol a multifunctionalantiangiogenic and antitumor agentrdquo Antioxidants amp RedoxSignaling vol 11 no 5 pp 1139ndash1148 2009

[13] J Park J Lee E Jung et al ldquoIn vitro antibacterial andanti-inflammatory effects of honokiol and magnolol againstPropionibacterium sprdquo European Journal of Pharmacology vol496 no 1 pp 189ndash195 2004

[14] C P Bopaiah and N Pradhan ldquoAntimicrobial activity ofhonokiol and magnolol isolated from Magnolia officinalisrdquoPhytotherapy Research vol 15 no 2 pp 139ndash141 2001

[15] K H Bang Y K Kim B S Min et al ldquoAntifungal activity ofmagnolol and honokiolrdquo Archives of Pharmacal Research vol23 no 1 pp 46ndash49 2000

[16] N H Choi G J Choi B-S Min et al ldquoEffects of neolignansfrom the stem bark of Magnolia obovata on plant pathogenicfungirdquo Journal of AppliedMicrobiology vol 106 no 6 pp 2057ndash2063 2009

[17] W-J Syu Jr C-C Shen J-J Lu G-H Lee and C-M SunldquoAntimicrobial and cytotoxic activities of neolignans fromMagnolia officinalisrdquo Chemistry amp Biodiversity vol 1 no 3 pp530ndash537 2004

[18] Y-S Kim J-Y Lee J ParkW Hwang J Lee and D Park ldquoSyn-thesis andmicrobiological evaluation of honokiol derivatives asnew antimicrobial agentsrdquo Archives of Pharmacal Research vol33 no 1 pp 61ndash65 2010

[19] H Xiang J-Z Qiu D-C Wang Y-S Jiang L-J Xia and X-M Deng ldquoInfluence of magnolol on the secretion of 120572-toxin byStaphylococcus aureusrdquo Molecules vol 15 no 3 pp 1679ndash16892010

[20] P D Stapleton and P W Taylor ldquoMethicillin resistance inStaphylococcus aureus mechanisms and modulationrdquo ScienceProgress vol 85 no 1 pp 57ndash72 2002

[21] K Ubukata R Nonoguchi M Matsuhashi and M KonnoldquoExpression and inducibility in Staphylococcus aureus of themecA gene which encodes a methicillin-resistant S aureus-specific penicillin-binding proteinrdquo Journal of Bacteriology vol171 no 5 pp 2882ndash2885 1989

[22] W Tesch C Ryffel A Strasle F H Kayser and B Berger-BachildquoEvidence of a novel staphylococcal mec-encoded element(mecR) controlling expression of penicillin-binding protein 2rsquordquoAntimicrobial Agents and Chemotherapy vol 34 no 9 pp 1703ndash1706 1990

[23] B Guignard JM Entenza and PMoreillon ldquo120573-lactams againstmethicillin-resistant Staphylococcus aureusrdquoCurrent Opinion inPharmacology vol 5 no 5 pp 479ndash489 2005

[24] H Z Zhang C J Hackbarth K M Chansky and H FChambers ldquoA proteolytic transmembrane signaling pathwayand resistance to 120573-lactams in staphylococcirdquo Science vol 291no 5510 pp 1962ndash1965 2001

[25] H Maidhof B Reinicke P Blumel B Berger-Bachi andH Labischinski ldquofemA which encodes a factor essentialfor expression of methicillin resistance affects glycine con-tent of peptidoglycan in methicillin-resistant and methicillin-susceptible Staphylococcus aureus strainsrdquo Journal of Bacteriol-ogy vol 173 no 11 pp 3507ndash3513 1991

[26] N Koyama J Inokoshi andH Tomoda ldquoAnti-infectious agentsagainst MRSArdquoMolecules vol 18 no 1 pp 204ndash224 2012

[27] S I Jeong Y S Kim M Y Lee et al ldquoRegulation of contractileactivity by magnolol in the rat isolated gastrointestinal tractsrdquoPharmacological Research vol 59 no 3 pp 183ndash188 2009

[28] S-I Jeong S-Y Kim S-J Kim et al ldquoAntibacterial activityof phytochemicals isolated from Atractylodes japonica againstmethicillin-resistant Staphylococcus aureusrdquo Molecules vol 15no 10 pp 7395ndash7402 2010

[29] S-I JeongW-S Han Y-H Yun and K-J Kim ldquoContinentalicacid from Aralia continentalis shows activity against meth-icillin-resistant Staphylococcus aureusrdquo Phytotherapy Researchvol 20 no 6 pp 511ndash514 2006

[30] A R Shahverdi A Fakhimi G Zarrini G Dehghan and MIranshahi ldquoGalbanic acid from Ferula szowitsiana enhancedthe antibacterial activity of penicillin G and cephalexin againstStaphylococcus aureusrdquo Biological and Pharmaceutical Bulletinvol 30 no 9 pp 1805ndash1807 2007

[31] J-W Lee Y-J Ji M-H Yu et al ldquoAntimicrobial effect andresistant regulation of Glycyrrhiza uralensis on methicillin-resistant Staphylococcus aureusrdquo Natural Product Research vol23 no 2 pp 101ndash111 2009


[32] S T Micek ldquoAlternatives to vancomycin for the treatment ofmethicillin-resistant Staphylococcus aureus infectionsrdquo ClinicalInfectious Diseases vol 45 supplement 3 pp S184ndashS190 2007

[33] M A Campos P Morey and J A Bengoechea ldquoQuinolonessensitize gram-negative bacteria to antimicrobial peptidesrdquoAntimicrobial Agents and Chemotherapy vol 50 no 7 pp 2361ndash2367 2006

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


have several medicinal functions such as antioxidative anti-inflammatory and antitumor [10ndash12] In addition magnololand honokiol have been known to show high antimicrobialactivity against several microorganisms such as fungi Propi-onibacterium sp and S aureus [10 13ndash18] Recent researchreported that magnolol inhibited the transcription of hla (thegene encoding 120572-toxin) in both MSSA and MRSA resultingin a reduction of 120572-toxin production and hemolytic activities[19]

Methicillin a 120573-lactam antibiotic acts by inhibitingthe transpeptidation domain of penicillin-binding proteins(PBPs) that are involved in the synthesis of peptidoglycanWithout cross-linking reaction of the peptidoglycan thecell wall becomes mechanically weak some of the cytoplas-mic contents are released and the cell dies [20] Resistantstrains typically produced an enzyme called a 120573-lactamasewhich inactivated the 120573-lactam However S aureus canbecome resistant to methicillin and other 120573-lactam antibi-otics through the expression of a foreign PBP PBP2a (a low-affinity penicillin-binding protein) that is resistant to theaction of methicillin but which can perform the functions ofthe host PBPs [20]

The mechanism of resistance to methicillin is very com-plex and it is not completely characterized yet Howeverit may be due to the overproduction of 120573-lactamase [20]the expression of mecA producing PBP2a (also referred toas PBP21015840) showed a low affinity to 120573-lactam antibiotics [21]and the change of PBP type [22 23] detected factors otherthan the expression of the mecA gene that could controlthe induction of PBP2a production in S aureus E 67-0 andtermed it mecR this mecR is present in some S aureussuppresses the synthesis of PBP2a present in a certain geneticbackground and mediates the influence on the expressionof methicillin resistance This mecR consists of two genesmecI (the gene encoding a transcriptional regulator (MecI))and mecR1 (the gene encoding a membrane-bound signaltransduction protein (MecR1)) located in the upper streamofthe mecA gene [24] The mecR1 is activated upon the contactwith 120573-lactam that is equivalent to an inducing factor withMRSA and its signal binds to the promoter region of mecAand is transduced to mecI that suppresses transcription andthus the suppression is cleared

In addition previous studies [20 25] reported that thefemA and femB genes play important roles in building thepentaglycine crossbridges that link the glycan chains together

The situation is that the action mechanism of the femAgene is not elucidated yet nevertheless it is thought that itdoes not influence PBP2a synthesis Significantly inactiva-tion of either femA or femB also results in a large reduction inmethicillin resistance by the interferencewith the crossbridgelength and the secretion of virulence factors which couldlimit the ability of the cell to cause infection [20] The femAgene is involved in the cell wall especially glycine content ofpeptidoglycan and peptidoglycan biosynthesis of S aureusand mediates the effect on drug sensitivity and thus it isinvolved in methicillin resistance [25 26]

In the present study we investigated antimicrobial effectsand resistant regulation of magnolol and honokiol againstMRSA strains for potential application as a natural product

Honokiol

Magnolol

Standard honokiol

Purified magnolol

Standard magnolol

(mAU

)

120

100

80

60

40

20

0

5 10 15

Honokiol

Magnolol

Standard honokiol

Purified magnolol

Standard magnolol

Figure 1 Comparison of the purified magnolol isolated fromMagnolia officinalis with standard compounds by HPLC

agent To the best of our knowledge this is the first workon the combined effect of magnolol and honokiol usingMICmethod and transcriptional regulation study of resistantgene to substantiate the synergistic antimicrobial activityof compoundantibiotics combination against MRSA andclinical MRSA isolates

2 Materials and Methods

21 Reagents Oxacillin honokiol (ge98) [3V5-di-2-pro-penyl-(11V-biphenyl)-24V-diol] and magnolol (ge95) [5V5-di-2-propenyl-(11V-biphenyl)-22V-diol] were purchasedfrom Sigma Magnolol and honokiol were dissolved in 100EtOH

22 Plant Materials Extraction and Isolation Magnolol waspurified from the bark ofM officinalis as described previously[27] In brief the methanol extracts were concentrated andlyophilized with a rotary evaporator and partitioned betweenwater and n-hexane The n-hexane fraction was then appliedto a silica gel column eluted with an n-hexane acetone(8 11 vv)mixtureThe n-hexane layerwas concentrated andmagnolol was directly obtained by prep-HPLC of n-hexanelayer The isolated magnolol was identified by thin-layerchromatograph and mixed melting point determination incomparison with authentic magnolol (Wako Osaka Japan)The purity (ge98) of magnolol was determined by high-performance thin-layer chromatograph (Figure 1)

23 Bacteria The bacteria used in this study were Bacillussubtilis (ATCC6633) Escherichia coli (ATCC8739) Propioni-bacterium acnes (ATCC 6919) Staphylococcus aureus (ATCC6538) standardmethicillin-susceptible Staphylococcus aureus(MSSA ATCC 25923) methicillin-resistant Staphylococcusaureus (MRSA ATCC 33591) and clinical MRSA isolatesused in [28 29] All strains were cultured at 34∘C for 24 hwith tryptic soy medium (Difco) under aerobic conditionsbefore the assay After culturing all strains onMueller-Hintonagar (Difco Detroit MI) the cells were resuspended in

















00

20

40

60

80

100

0 1 10 25 50 100

log 1

0(C

FUm

L)



S aureus

(a)

00

20

40

60

80

100

0 1 10 25 50 100

log 1

0(C

FUm

L)



S aureus

(b)





00

20

40

60

80

100

0 1 10 25 50 100

log 1

0(C

FUm

L)




(a)

00

20

40

60

80

100

0 1 10 25 50 100

log 1

0(C

FUm

L)




(b)





mecA

mecRI

mecI

GAPDH

femA

femB


MSS

AM

RSA 78

M11

21-8 6-2

7-3

8-4-1

8-4-2

9-5

13-7

27-9

47-10

105-13

106-14








mecA

mecRI

mecI

GAPDH

femA

femB


(a)

mecA

mecRI

mecI

GAPDH

femA

femB

0 1 5 10 OX 50Honokiol (120583gmL)

Control

(b)





4 Conclusion



00

05

10

15

20

25


Relat

ive g

ene e

xpre

ssio

nm

ecA

lowastlowastlowast

(a)

00

10

20

30

40

50


Relat

ive g

ene e

xpre

ssio

nm

ecI

lowast

(b)

00

10

20

30

40

50

0 1 5 10 OX 50Magnolol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nm

ecRI

(c)

00

05

10

15

20

25

0 1 5 10 OX 50Magnolol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nfem

Alowast

(d)

00

05

10

15

20

25

0 1 5 10 OX 50

Relat

ive g

ene e

xpre

ssio

nfem

B

lowastlowast


(e)

00

05

10

15

20

25

0 1 5 10 OX 50Honokiol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nm

ecA

lowast

(f)




Acknowledgment


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

















00

20

40

60

80

100

0 1 10 25 50 100

log 1

0(C

FUm

L)



S aureus

(a)

00

20

40

60

80

100

0 1 10 25 50 100

log 1

0(C

FUm

L)



S aureus

(b)





00

20

40

60

80

100

0 1 10 25 50 100

log 1

0(C

FUm

L)




(a)

00

20

40

60

80

100

0 1 10 25 50 100

log 1

0(C

FUm

L)




(b)





mecA

mecRI

mecI

GAPDH

femA

femB


MSS

AM

RSA 78

M11

21-8 6-2

7-3

8-4-1

8-4-2

9-5

13-7

27-9

47-10

105-13

106-14








mecA

mecRI

mecI

GAPDH

femA

femB


(a)

mecA

mecRI

mecI

GAPDH

femA

femB

0 1 5 10 OX 50Honokiol (120583gmL)

Control

(b)





4 Conclusion



00

05

10

15

20

25


Relat

ive g

ene e

xpre

ssio

nm

ecA

lowastlowastlowast

(a)

00

10

20

30

40

50


Relat

ive g

ene e

xpre

ssio

nm

ecI

lowast

(b)

00

10

20

30

40

50

0 1 5 10 OX 50Magnolol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nm

ecRI

(c)

00

05

10

15

20

25

0 1 5 10 OX 50Magnolol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nfem

Alowast

(d)

00

05

10

15

20

25

0 1 5 10 OX 50

Relat

ive g

ene e

xpre

ssio

nfem

B

lowastlowast


(e)

00

05

10

15

20

25

0 1 5 10 OX 50Honokiol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nm

ecA

lowast

(f)




Acknowledgment


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


00

20

40

60

80

100

0 1 10 25 50 100

log 1

0(C

FUm

L)




(a)

00

20

40

60

80

100

0 1 10 25 50 100

log 1

0(C

FUm

L)




(b)





mecA

mecRI

mecI

GAPDH

femA

femB


MSS

AM

RSA 78

M11

21-8 6-2

7-3

8-4-1

8-4-2

9-5

13-7

27-9

47-10

105-13

106-14








mecA

mecRI

mecI

GAPDH

femA

femB


(a)

mecA

mecRI

mecI

GAPDH

femA

femB

0 1 5 10 OX 50Honokiol (120583gmL)

Control

(b)





4 Conclusion



00

05

10

15

20

25


Relat

ive g

ene e

xpre

ssio

nm

ecA

lowastlowastlowast

(a)

00

10

20

30

40

50


Relat

ive g

ene e

xpre

ssio

nm

ecI

lowast

(b)

00

10

20

30

40

50

0 1 5 10 OX 50Magnolol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nm

ecRI

(c)

00

05

10

15

20

25

0 1 5 10 OX 50Magnolol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nfem

Alowast

(d)

00

05

10

15

20

25

0 1 5 10 OX 50

Relat

ive g

ene e

xpre

ssio

nfem

B

lowastlowast


(e)

00

05

10

15

20

25

0 1 5 10 OX 50Honokiol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nm

ecA

lowast

(f)




Acknowledgment


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





mecA

mecRI

mecI

GAPDH

femA

femB


(a)

mecA

mecRI

mecI

GAPDH

femA

femB

0 1 5 10 OX 50Honokiol (120583gmL)

Control

(b)





4 Conclusion



00

05

10

15

20

25


Relat

ive g

ene e

xpre

ssio

nm

ecA

lowastlowastlowast

(a)

00

10

20

30

40

50


Relat

ive g

ene e

xpre

ssio

nm

ecI

lowast

(b)

00

10

20

30

40

50

0 1 5 10 OX 50Magnolol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nm

ecRI

(c)

00

05

10

15

20

25

0 1 5 10 OX 50Magnolol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nfem

Alowast

(d)

00

05

10

15

20

25

0 1 5 10 OX 50

Relat

ive g

ene e

xpre

ssio

nfem

B

lowastlowast


(e)

00

05

10

15

20

25

0 1 5 10 OX 50Honokiol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nm

ecA

lowast

(f)




Acknowledgment


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


00

05

10

15

20

25


Relat

ive g

ene e

xpre

ssio

nm

ecA

lowastlowastlowast

(a)

00

10

20

30

40

50


Relat

ive g

ene e

xpre

ssio

nm

ecI

lowast

(b)

00

10

20

30

40

50

0 1 5 10 OX 50Magnolol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nm

ecRI

(c)

00

05

10

15

20

25

0 1 5 10 OX 50Magnolol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nfem

Alowast

(d)

00

05

10

15

20

25

0 1 5 10 OX 50

Relat

ive g

ene e

xpre

ssio

nfem

B

lowastlowast


(e)

00

05

10

15

20

25

0 1 5 10 OX 50Honokiol (120583gmL)

Control

Relat

ive g

ene e

xpre

ssio

nm

ecA

lowast

(f)




Acknowledgment


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Research Article Antimicrobial Effects and Resistant ...

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Transcript of Research Article Antimicrobial Effects and Resistant ...