Research Article Synergistic Effect between Cryptotanshinone...

Research ArticleSynergistic Effect between Cryptotanshinone and Antibioticsagainst Clinic Methicillin and Vancomycin-ResistantStaphylococcus aureus

Jeong-Dan Cha1 Jeong-Ho Lee2 Kyung Min Choi1 Sung-Mi Choi3 and Jeong Hye Park4

1 Department of Research Development Institute of Jinan Red Ginseng Jinan Republic of Korea2Department of Oriental Medicine Resources College of Environmental and Bioresource SciencesChonbuk National University Republic of Korea

3 Department of Dental Hygiene Daegu Health College Daegu Republic of Korea4Department of Nursing Dong-eui University Republic of Korea

Correspondence should be addressed to Jeong Hye Park jhparkdeuackr

Received 25 November 2013 Revised 6 February 2014 Accepted 18 February 2014 Published 24 March 2014

Academic Editor Tai-Long Pan

Copyright copy 2014 Jeong-Dan Cha et alThis 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

Cryptotanshinone (CT) amajor tanshinone ofmedicinal plant SalviamiltiorrhizaBunge demonstrated strong antibacterial activityagainst clinic isolated methicillin and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA) in this experiment The CTwas determined against clinic isolatedMRSA 1ndash16 withMIC andMBC values ranging from 4 to 32 and 8 to 128120583gmL forMSSA 1-2from 16 to 32 120583gmL and 64 to 128 120583gmL for VRSA 1-2 from 2 to 4120583gmL and 4 to 16 120583gmL respectivelyThe range of MIC

50and

MIC90of CT was 05ndash8120583gmL and 4ndash64 120583gmL respectively The combination effects of CT with antibiotics were synergistic (FIC

index lt 05) against most of tested clinic isolated MRSA MSSA and VRSA except additive MRSA 4 and 16 in oxacillin MRSA 612 and 15 in ampicillin and MRSA 6 11 and 15 in vancomycin (FIC index lt 075ndash10) Furthermore a time-kill study showed thatthe growth of the tested bacteria was completely attenuated after 2ndash6 h of treatment with the 12 MIC of CT regardless of whetherit was administered alone or with ampicillin oxacillin or vancomycin The results suggest that CT could be employed as a naturalantibacterial agent against multidrug-resistant pathogens infection

1 Introduction

Staphylococcus aureus (S aureus) is one of themost importantpathogens in both hospitals and the community and cancause numerous syndromes in humans such as furuncle car-buncle abscess pneumonia meningitis bacterial arthritismyocarditis endocarditis osteomyelitis and septicemia [1ndash3] Methicillin-resistant Staphylococcus aureus (MRSA) is asignificant problem in hospitals and communities worldwideand awareness of MRSA in animals and reports of itszoonotic spread have increased in recent years [3 4] Theincreasing prevalence of methicillin-resistant Staphylococ-cus aureus (MRSA) has led to widespread increased useof vancomycin [5 6] Subsequently numerous reports ofelevated minimum inhibitory concentrations (MIC) to van-comycin among MRSA isolates have surfaced concomitant

with increased global vancomycin exposure to MRSA [7 8]Greater than 60 of S aureus isolates are now resistantto methicillin (oxacillin) and some strains have developedresistance to more than 20 different antimicrobial agentsnew agents are therefore needed for the treatment of Saureus [9 10] Plant medicines are used on a worldwidescale to prevent and treat infectious diseases They areof great demand both in the developed and developingcountries for the primary health care needs due to theirwide biological andmedicinal activities higher safetymarginand lesser costs [11 12] Plants are rich in a wide varietyof secondary metabolites such as tannins alkaloids ter-penoids and flavonoids having been found in vitro sincethey have antimicrobial properties and may serve as analternative effective cheap and safe antimicrobial for thetreatment of microbial infections [12ndash15] At the same time

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 450572 16 pageshttpdxdoiorg1011552014450572

2 Evidence-Based Complementary and Alternative Medicine

Table 1 Antibacterial activity of cryptotanshinone and antibiotics in isolated MRSA VRSA MSSA and some of reference bacteria

Samples Cryptotanshinone (120583gmL) Ampicillin Oxacillin VancomycinMIC50lt

MIC90lt

MICMBC MICMBC (120583gmL)MSSA ATCC 259231 4 64 64256 816 0251 052MRSA ATCC 335912 05 4 416 5122048 816 14VRSA 3A0633 05 2 24 5122048 5121024 1632VRSA 3A0664 1 4 416 128256 5121024 3264MSSA 15 2 16 1664 10242048 051 14MSSA 26 8 32 32128 256512 12 12MRSA 1 2 16 1632 128256 128256 12MRSA 2 8 64 64128 256256 832 24MRSA 3 1 4 416 128512 128512 24MRSA 4 05 4 48 128256 5122048 12MRSA 5 2 64 64128 128512 5121024 12MRSA 6 05 4 416 64128 64256 12MRSA 7 2 8 816 128256 5121024 14MRSA 8 4 32 3264 256256 5122048 14MRSA 9 2 8 816 64128 5121024 12MRSA 10 2 16 1664 128256 5121024 12MRSA 11 4 8 832 64128 64128 12MRSA 12 1 4 48 128256 128256 14MRSA 13 4 32 32128 128256 32128 12MRSA 14 05 8 816 64128 816 14MRSA 15 05 4 416 64128 128256 14MRSA 16 2 8 816 128512 128512 121MSSA (ATCC 25923) reference strain methicillin-sensitive Staphylococcus aureus2MRSA (ATCC 33591) reference strain methicillin-resistant Staphylococcus aureus3VRSA 3A063 vancomycin-resistant Staphylococcus aureus isolated clinically4VRSA 3A066 vancomycin-resistant Staphylococcus aureus isolated clinically5MSSA (1-2) methicillin-sensitive Staphylococcus aureus isolated clinically6MRSA (1ndash16) methicillin-resistant Staphylococcus aureus isolated clinically

because of the difficulty in developing chemical syntheticdrugs and because of their side effects scientists are makingmore efforts to search for new drugs from plant resourcesto combat clinical multidrug-resistant microbial infections[11 12 16]

The main components of S miltiorrhiza can be dividedinto two groups hydrophilic compounds such as salvianolicacids and lipophilic chemicals including diterpenoid andtanshinones [17] The second group of components labeledas tanshinone I tanshinone II and cryptotanshinone arethe major bioactive constituents and have various kinds ofpharmacological effects including antibacterial antioxidantand antitumor activities and prevention of angina pectorisand myocardial infarction [18ndash20] The cryptotanshinone(CT) exhibits antimicrobial activity against a broad rangeof Gram-positive bacteria including S aureus and Gram-negative bacteria as well as other microorganisms [18 21]The hexane and chloroform fractions of S miltiorrhizaevidenced profound antimicrobial activity and inhibitedresistant gene expression against Staphylococcus aureus andMRSA (methicillin-resistant Staphylococcus aureus) [21 22]

In this study the antimicrobial activities of cryptotan-shinone (CT) against methicillin and vancomycin-resistant

Staphylococcus aureus isolated in a clinic were assessed usingbrothmicrodilutionmethod and the checkerboard and time-kill methods for synergistic effect of the combination withantibiotics

2 Materials and Methods

21 Plant Extraction and Isolation The air-dried roots(20 kg) of S miltiorrhiza were crushed and extracted withMeOH (8 L times 3) at room temperature The MeOH extracts(210 g) were evaporated and suspended in distilled waterand partitioned sequentially with CH

2Cl2 EtOAc and n-

BuOH The CH2Cl2soluble fraction was concentrated in

vacuo and its crude extract (28 g) was subjected to silicagel column chromatography using a CHCl

3-MeOH gradient

solvent system to provide ten fractions (A1ndashA10) FractionA4was further subjected to silica gel column chromatographywith a CHCl

3-MeOH (10 1) to yield five fractions (B1ndashB5)

Fraction B3 was purified on recycling prep-HPLC (JAIGELGS column and 220 nm) eluted with MeOH (40mLmin) toyield cryptotanshinone (98mg) The structure of cryptotan-shinone was identified by comparing its spectral data withthose published [23 24]

Evidence-Based Complementary and Alternative Medicine 3

Time (hours)0 5 10 15 20 25

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VRSA 1

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Figure 1 Time-kill curves of MIC or 12 MIC of cryptotanshinone (CT) ampicillin (AMP) oxacillin (OXA) and vancomycin (VAN) aloneand its combination with MIC

50of AMP or OXA and VAN against VRSA 1 isolates and reference stains MSSA ATCC 25923 and MRSA

ATCC 33591 Bacteria were incubated with MIC of CT (e) AMP OXA and VAN and 12 MIC of CT + 12 MIC of AMP (I) 12 MIC of CT+ 12 MIC of OXA (998787) and 12 MIC of CT + 12 MIC of VAN (nabla) over time CFU colony-forming units


VRSA 2

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MSSA 1

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MSSA 1

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Figure 2 Time-kill curves of MIC or 12 MIC of cryptotanshinone (CT) ampicillin (AMP) oxacillin (OXA) and vancomycin (VAN) aloneand its combination with 12 MIC of AMP or OXA and VAN against VRSA 2 MSSA 1 and MSSA 2 isolates Bacteria were incubated withMIC of CT (e) AMP OXA and VAN and 12 MIC of CT + 12 MIC of AMP (I) 12 MIC of CT + 12 MIC of OXA (998787) and 12 MIC of CT+ 12 MIC of VAN (nabla) over time CFU colony-forming units


MRSA 1

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MRSA 2

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MRSA 2

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Figure 3 Time-kill curves of MIC or 12 MIC of cryptotanshinone (CT) ampicillin (AMP) oxacillin (OXA) and vancomycin (VAN) aloneand its combination with 12 MIC of AMP or OXA and VAN against MRSA 1 2 and 3 isolates Bacteria were incubated with MIC of CT (e)AMP OXA and VAN and 12 MIC of CT + 12 MIC of AMP (I) 12 MIC of CT + 12 MIC of OXA (998787) and 12 MIC of CT + 12 MIC ofVAN (nabla) over time CFU colony-forming units


MRSA 4

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MRSA 5

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Figure 4 Time-kill curves of MIC or 12 MIC of cryptotanshinone (CT) ampicillin (AMP) oxacillin (OXA) and vancomycin (VAN) aloneand its combination with 12 MIC of AMP or OXA and VAN against MRSA 4 5 and 6 isolates Bacteria were incubated with MIC of CT(e) AMP (I) OXA (998787) and VAN (nabla) and 12 MIC of CT + 12 MIC of AMP (e) 12 MIC of CT + 12 MIC of OXA (I) and 12 MIC of CT+ 12 MIC of VAN (998787) over time CFU colony-forming units


MRSA 7

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MRSA 8

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Figure 5 Time-kill curves of MIC or 12 MIC of cryptotanshinone (CT) ampicillin (AMP) oxacillin (OXA) and vancomycin (VAN) aloneand its combination with 12 MIC of AMP or OXA and VAN against MRSA 7 8 and 9 isolates Bacteria were incubated with MIC of CT (e)AMP (I) OXA (998787) and VAN (nabla) and 12 MIC of CT + 12 MIC of AMP (e) 12 MIC of CT + 12 MIC of OXA (I) and 12 MIC of CT +12 MIC of VAN (998787) over time CFU colony-forming units


MRSA 10

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MRSA 11

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MRSA 12

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MRSA 10

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MRSA 11

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MRSA 12

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MRSA 13

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Table 2 Synergistic effects of the cryptotanshinone with oxacillin in isolated MRSA VRSA MSSA and some of reference bacteria

Samples Agent MICMBC (120583gmL) FICFBC FICIFBCI2 OutcomeAlone Combination1

MSSA ATCC 259233 Cryptotanshinone 64256 832 01250125 03750375 SynergisticsynergisticOxacillin 0251 00625025 025025

MRSA ATCC 335914 Cryptotanshinone 416 14 025025 0505 SynergisticsynergisticOxacillin 816 24 025025

VRSA 3A0635 Cryptotanshinone 24 052 02505 03750375 SynergisticsynergisticOxacillin 5121024 64128 01250125


MSSA 17 Cryptotanshinone 1664 28 01250125 0375025 SynergisticsynergisticOxacillin 051 01250125 0250125


MRSA 18 Cryptotanshinone 1632 416 02505 05075 SynergisticadditiveOxacillin 128256 3264 025025

MRSA 2 Cryptotanshinone 64128 1616 0250125 050375 SynergisticsynergisticOxacillin 832 28 025025


MRSA 4 Cryptotanshinone 48 22 05025 0750375 AdditivesynergisticOxacillin 5122048 128256 0250125













1The MIC and MBC of cryptotanshinone with oxacillin2The FIC index3MSSA (ATCC 25923) reference strain methicillin-sensitive Staphylococcus aureus4MRSA (ATCC 33591) reference strain methicillin-resistant Staphylococcus aureus5VRSA 3A063 vancomycin-resistant Staphylococcus aureus isolated clinically6VRSA 3A066 vancomycin-resistant Staphylococcus aureus isolated clinically7MSSA (1-2) methicillin-sensitive Staphylococcus aureus isolated clinically8MRSA (1ndash16) methicillin-resistant Staphylococcus aureus isolated clinically


Table 3 Synergistic effects of cryptotanshinone with ampicillin in isolated MRSA VRSA MSSA and some of reference bacteria


MSSA ATCC 259233 Cryptotanshinone 64256 1664 025025 05075 SynergisticadditiveAmpicillin 816 28 02505

MRSA ATCC 335914 Cryptotanshinone 416 14 025025 0505 SynergisticsynergisticAmpicillin 5122048 128512 025025

VRSA 3A0635 Cryptotanshinone 24 052 02505 050375 SynergisticsynergisticAmpicillin 5122048 128256 0250125


MSSA 17 Cryptotanshinone 1664 48 0250125 050375 SynergisticsynergisticAmpicillin 64128 1632 025025


MRSA 18 Cryptotanshinone 1632 48 025025 0505 SynergisticsynergisticAmpicillin 128256 3264 025025





MRSA 6 Cryptotanshinone 416 12 0250125 0750625 AdditiveadditiveAmpicillin 64128 3264 0505





MRSA 11 Cryptotanshinone 832 28 025025 05075 SynergisticadditiveAmpicillin 64128 1664 02505

MRSA 12 Cryptotanshinone 48 12 025025 07505 AdditivesynergisticAmpicillin 128256 6464 05025





1The MIC and MBC of cryptotanshinone with ampicillin2The FIC index3MSSA (ATCC 25923) reference strain methicillin-sensitive Staphylococcus aureus4MRSA (ATCC 33591) reference strain methicillin-resistant Staphylococcus aureus5VRSA 3A063 vancomycin-resistant Staphylococcus aureus isolated clinically6VRSA 3A066 vancomycin-resistant Staphylococcus aureus isolated clinically7MSSA (1-2) methicillin-sensitive Staphylococcus aureus isolated clinically8MRSA (1ndash16) methicillin-resistant Staphylococcus aureus isolated clinically


Table 4 Synergistic effects of cryptotanshinone with vancomycin in isolated MRSA VRSA MSSA and some of reference bacteria


MSSA ATCC 259233 Cryptotanshinone 64256 1664 025025 0505 SynergisticsynergisticVancomycin 052 012505 025025

MRSA ATCC 335914 Cryptotanshinone 416 14 025025 0505 SynergisticsynergisticVancomycin 14 0251 025025

VRSA 3A0635 Cryptotanshinone 24 051 0125025 037505 SynergisticsynergisticVancomycin 1632 48 025025


MSSA 17 Cryptotanshinone 1664 416 025025 05075 SynergisticadditiveVancomycin 14 0252 02505


MRSA 18 Cryptotanshinone 1632 48 025025 0505 SynergisticsynergisticVancomycin 12 02505 025025

MRSA 2 Cryptotanshinone 64128 1632 025025 05075 SynergisticadditiveVancomycin 24 052 02505




MRSA 6 Cryptotanshinone 416 24 05025 07505 AdditivesynergisticVancomycin 12 02505 025025











1The MIC and MBC of cryptotanshinone with vancomycin2 The FIC index3MSSA (ATCC 25923) reference strain methicillin-sensitive Staphylococcus aureus4MRSA (ATCC 33591) reference strain methicillin-resistant Staphylococcus aureus5VRSA 3A063 vancomycin-resistant Staphylococcus aureus isolated clinically6VRSA 3A066 vancomycin-resistant Staphylococcus aureus isolated clinically7MSSA (1-2) methicillin-sensitive Staphylococcus aureus isolated clinically8MRSA (1ndash16) methicillin-resistant Staphylococcus aureus isolated clinically


22 Preparation of Bacterial Strains 16 isolates ofmethicillin-resistant Staphylococcus aureus 2 isolates of methicillin-sensitive S aureus (MSSA) and 2 isolates of vancomycin-resistant S aureus (VRSA) were purchased from the CultureCollection of Antimicrobial Resistant Microbes (CCARM)standard strains of methicillin-sensitive S aureus (MSSA)ATCC 25923 and methicillin-resistant S aureus (MRSA)ATCC 33591 were used as well (Table 1) Antibiotic sus-ceptibility was determined in testing the inhibition zones(inoculums 05 McFarland suspension 15 times 108 CFUmL)and MICMBC (inoculums 5 times 105 CFUmL) for strainsmeasured as described in the National Committee for Clini-cal Laboratory Standards (NCCLS 1999) Briefly the growthof bacteria was examined at 37∘C in 095mL of BHI brothcontaining various concentrations of CT These tubes wereinoculated with 5 times 105 colony-forming units (CFU)mL ofan overnight culture grown in BHI broth and incubated at37∘C After 24 h of incubation the optical density (OD) wasmeasured spectrophotometrically at 550 nmThree replicateswere measured for each concentration of tested drugs

23 Minimum Inhibitory ConcentrationMinimum Bacterici-dal Concentration Assay The antimicrobial activities of CTagainst clinical isolates MRSA 16 MSSA 2 VRSA 2 andreference strains were determined via the broth dilutionmethod [25] The minimum inhibitory concentration (MIC)was recorded as the lowest concentration of test samplesresulting in the complete inhibition of visible growth Forclinical strains MIC

50s and MIC

90s defined as MICs at

which 50 and 90 respectively of the isolates were inhibitedwere determined The minimum bactericidal concentration(MBC) was determined based on the lowest concentration ofthe extracts required to kill 999 of bacteria from the initialinoculum as determined by plating on agar

24 Checkerboard Dilution Test The synergistic combina-tions were investigated in the preliminary checkerboardmethod performed using the MRSA MSSA and VRSA ofclinical isolate strains via MIC andMBC determination [26]The fractional inhibitory concentration index (FICI) andfractional bactericidal concentration index (FBCI) are thesum of the FICs and FBCs of each of the drugs which weredefined as the MIC and MBC of each drug when used incombination divided by theMICandMBCof each drugwhenused aloneThe FIC and FBC index was calculated as followsFIC = (MIC of drug A in combinationMIC of drug A alone)+ (MIC of drug B in combinationMIC of drug B alone) andFBC = (MBC of drug A in combinationMBC of drug Aalone) + (MBC of drug B in combinationMBC of drug Balone) FIC and FBC indices were interpreted as follows theFIC index was interpreted as follows synergy lt05 partialsynergy 05ndash075 additive effect 076ndash10 indifference gt10ndash40 and antagonism gt40 [26]

25 Time-Kill Curves The bactericidal activities of the drugsevaluated in this study were also evaluated using time-killcurves constructed using the isolated and reference strainsCultures with an initial cell density of 1ndash5 times 106 CFUmL

were exposed to the MIC of CT alone or CT (12 MIC)plus oxacillin or ampicillin or vancomycin (12 MIC) Viablecounts were conducted at 0 05 1 2 3 4 5 6 12 and 24 hby plating aliquots of the samples on agar and subsequentincubation for 24 hours at 37∘C All experiments wererepeated several times and colony counts were conducted induplicate after which the means were determined

3 Results and Discussion

Many researchers are studying natural products that couldbe used as antibiotics against MRSA and are employingnovel dosing regimens and antimicrobials that would beadvantageous for combating the therapeutic problems asso-ciated with S aureus [10 13 14 16 27] The main bioactiveconstituents of S miltiorrhiza include water-soluble phe-nolic acids and lipophilic diterpenoid tanshinones [28ndash30]Cryptotanshinone was isolated from dried S miltiorrhizaroots and identified via comparison of their spectral datawith the data reported in the literature [23 24] lH-NMR(CDCl

3) 120575 764 (1H d J = 80Hz) 748 (1H d J = 80Hz)

486 (1H t J = 92Hz) 436 (1H dd J = 60 and 60Hz)360 (1H m) 321 (2H br t) 169 (4H m) 136 (3H d J= 64Hz) 131 (6H s) 13C-NMR (CDCl

3) 120575 2967 (C-1)

1908 (C-2) 3782 (C-3) 3486 (C-4) 14370 (C-5) 13256(C-6) 12250 (C-7) 12842 (C-8) 12627 (C-9) 15237 (C-10) 18427 (C-11) 17572 (C-12) 11830 (C-13) 17075 (C-14)8146 (C-15) 3462 (C-16) 1885 (C-17) 3194 (C-18) and3189 (C-19) Among the lipophilic diterpenoid tanshinonescryptotanshinone dihydrotanshinone I tanshinone IIA andtanshinone I exhibited strong antimicrobial activity against( Agrobacterium tumefaciens ATCC 11158 Escherichia coliATCC 29425 Pseudomonas lachrymans ATCC 11921 Ralsto-nia solanacearum ATCC 11696 and Xanthomonas vesicatoriaATCC 11633) and three Gram-positive bacteria (Bacillussubtilis ATCC 11562 Staphylococcus aureus ATCC 6538 andStaphylococcus haemolyticus ATCC 29970) [18] Our resultsof the antibacterial activity showed that the CT exhibitedinhibitory activities against isolates MSSA MRSA VRSAand reference stains The MICs and MBCs values of CTand antibiotics ampicillin oxacillin and vancomycin againstMSSA ATCC 25923 and MRSA ATCC 33591 and isolatesMSSA 1-2 MRSA 1ndash16 and VRSA 1-2 are shown in Table 1The MICs and MBCs values of CT against isolates MSSA1-2 were in the range of 16 and 32 120583gmL and 64 and128 120583gmL isolates MRSA 1ndash16 in the range of 4ndash64 120583gmLand 8ndash128120583gmL isolates VRSA 1-2 in the range of 2 and4 120583gmL and 4 and 16 120583gmL and reference stains in rangeof 4 and 64 120583gmL and 16 and 256 120583gmL respectively TheMICsMBCs for ampicillin were determined to be either816 or 10242048120583gmL for oxacillin either 0251 or5122048120583gmL for vancomycin either 052 or 3264120583gmLagainst reference strains and MSSA 1-2 MRSA 1ndash16 andVRSA 1-2 isolates The MIC

50and MIC

90values of CT for

reference strains were 05 and 4 120583gmL and 4 and 64 120583gmLwhile for MSSA 1-2 and VRSA 1-2 isolates were 05ndash8120583gmLand 2ndash32 120583gmL and for MRSA 1ndash16 isolates were 05ndash8 120583gmL and 4ndash64120583gmL respectively (Table 1)


MRSA 16

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Time (hours)


VAN (MIC)

Figure 8 Time-kill curves of MIC or 12 MIC of cryptotanshinone (CT) ampicillin (AMP) oxacillin (OXA) and vancomycin (VAN) aloneand its combination with 12 MIC of AMP or OXA and VAN against MRSA 16 isolate Bacteria were incubated with MIC of CT (e) AMP(I) OXA (998787) and VAN (nabla) and 12 MIC of CT + 12 MIC of AMP (e) 12 MIC of CT + 12 MIC of OXA (I) and 12 MIC of CT + 12 MICof VAN (998787) over time CFU colony-forming units

Evaluation of in vivo effectiveness of the antimicrobialcombinations is necessary to generate data that can be extrap-olated to the clinical situation as well as predicting relevantconcentration of optimal dosing regimens for both agentsof the combinations [31 32] Combination antibiotic therapyhas been studied to promote the effective use of antibioticsin increasing in vivo activity of antibiotics in preventing thespread of drug-resistant strains and in minimizing toxicity[32 33] The combination of oxacillin and CT resulted ina reduction in the MICsMBCs for isolates VRSA 1-2 andMSSA 1-2 with the MICsMBCs of 216 or 8128120583gmL foroxacillin becoming 01250125ndash025 120583gmL and reduced byge4-fold evidencing a synergistic effect as defined by a FICIand FBCI of le05 (Table 2) The combination of oxacillinand CT resulted in a reduction against isolates MRSA 1ndash16 with the MICsMBCs values of 05ndash162ndash32 120583gmL foroxacillin becoming 1ndash1284ndash256120583gmL and reduced by ge4-fold evidencing a synergistic effect as defined by a FICI andFBCI of le05 except MRSA 4 and 16 of additive (FICI ge05) and MRSA 1 7 and 15 of additive (FBCI ge 05) Thecombination of ampicillin and CT resulted in a reductionin the MICsMBCs for isolates VRSA 1-2 and MSSA 1-2 with the MICsMBCs of 051 or 24 120583gmL and 48 or816 for ampicillin becoming 32128 or 64256 120583gmL and1664 or 32128 120583gmL and reduced by ge4-fold evidencing asynergistic effect as defined by a FICI and FBCI of le05 and inmost of MRSA tested were reduced by ge4-fold evidencing asynergistic effect as defined by a FICI and FBCI ofle05 exceptMRSA 6 12 and 15 (FICI ge 05) andMRSA 6 11 and 13 (FBCIge 05) respectively (Table 3)Thecombination of vancomycinand CT resulted in a reduction against isolates MRSA 1ndash16 with the MICsMBCs values of 1ndash162ndash32 120583gmL for

vancomycin becoming 0125ndash0505ndash2120583gmL and reducedbyge4-fold evidencing a synergistic effect as defined by a FICIand FBCI of le 05 except MRSA 6 11 and 15 of additive (FICIge 05) andMRSA 1 and 9 of additive (FBCI ge 05) and for iso-lates VRSA 1-2 and MSSA 1-2 with the MICsMBCs of 051or 14 120583gmL and 48 or 1616 for vancomycin becoming 48or 816 120583gmL and 0252 or 0251 120583gmL and reduced by ge4-fold evidencing a synergistic effect as defined by a FICI andFBCI of le05 except MSSA 1 and 2 of additive (FBCI ge 05)(Table 4)

The effects of CT administered in combination withoxacillin andor ampicillin andor vancomycin against stan-dard (MSSA and MRSA) and clinical isolates of MSSA (12) VRSA (12) and MRSA (MRSA 1ndash16) were confirmedby time-kill curve experiments (Figures 1 2 3 4 5 67 and 8) Cultures of each strain of bacteria with a celldensity of 106 CFUmL were exposed to the MIC of CTand antibiotics alone or CT (12 MIC) with oxacillin (12MIC) ampicillin (12 MIC) or vancomycin (12 MIC) Weobserved that 30 minutes of CT treatment with ampicillinoxacillin or vancomycin resulted in an increased rate ofkilling as compared to that observed with CT (MIC) alone Aprofound bactericidal effect was exerted when a combinationof drugs was utilized The growth of the tested bacteria wascompletely attenuated after 2ndash5 h of treatment with the 12MICofCT regardless of whether it was administered alone orwith oxacillin (12MIC) ampicillin (12MIC) or vancomycin(12 MIC) (Figures 1ndash8)

It has been reported that some plant-derived compoundscan improve the in vitro activity of some cell-wall inhibit-ing antibiotics by directly attacking the same target sitethat is peptidoglycan [7 9 34] CT exhibits antimicrobial


activity against a broad range of Gram-positive bacteriaincluding S aureus and Gram-negative bacteria as well asother microorganisms [21 22] Despite the pharmacologi-cal activities potential risks regarding combination use ofS miltiorrhiza and drugs have been observed [28 35 36]Scientific findings concluded S miltiorrhiza fraction andits components (cryptotanshinone and dihydrotanshinoneI) showed antibacterial activity against a broad range ofbacteria including the broad range of Gram-positive bacteriaand Gram-negative bacteria and superoxide radicals areconsidered important in the antibacterial actions of theagents [18]The Gram-positive bacteria-specific properties ofCT are caused by the inhibition of RNAand protein synthesisrather than by attacking the bacterial membrane [21 22]

In conclusion CT of S miltiorrhiza is expected to berecognized as natural sources for the development of newfunctional drugs against multiresistant S aureus MRSA andVRSA

Conflict of Interests

The authors have declared no conflict of interests

Authorsrsquo Contribution

Jeong-Dan Cha and Jeong-Ho Lee contributed equally to thiswork

Acknowledgment

This research was supported by Dong-eui University Grant(2011AA106)

References

[1] G R Corey ldquoStaphylococcus aureusbloodstream infectionsdefinitions and treatmentrdquo Clinical Infectious Diseases vol 48no 4 pp S254ndashS259 2009

[2] C A Petti and V G Fowler Jr ldquoStaphylococcus aureus bac-teremia and endocarditisrdquo Cardiology Clinics vol 21 no 2 pp219ndash233 2003

[3] A Malm A Biernasiuk R Łos et al ldquoSlime productionand cell surface hydrophobicity of nasopharyngeal and skinstaphylococci isolated from healthy peoplerdquo Polish Journal ofMicrobiology vol 54 no 2 pp 117ndash121 2005

[4] S Defres C Marwick and D Nathwani ldquoMRSA as a cause oflung infection including airway infection community-acquiredpneumonia and hospital-acquired pneumoniardquo European Res-piratory Journal vol 34 no 6 pp 1470ndash1476 2009

[5] D P Levine ldquoVancomycin understanding its past and preserv-ing its futurerdquo SouthernMedical Journal vol 101 no 3 pp 284ndash291 2008

[6] Z Moravvej F Estaji E Askari K Solhjou M N Nasab and SSaadat ldquoUpdate on the global number of vancomycin-resistantStaphylococcus aureus (VRSA) strainsrdquo International Journal ofAntimicrobial Agents vol 42 no 4 pp 370ndash371 2013

[7] C-H Huang and Y-H Chen ldquoThe detection and clinicalimpact of vancomycin MIC among patients with methicillin-resistant Staphylococcus aureus bacteremiardquo Journal of Micro-biology Immunology and Infection vol 46 no 4 pp 315ndash3162013

[8] D I Hsu L KHidayat R Quist et al ldquoComparison ofmethod-specific vancomycin minimum inhibitory concentration valuesand their predictability for treatment outcome of meticillin-resistant Staphylococcus aureus (MRSA) infectionsrdquo Interna-tional Journal of Antimicrobial Agents vol 32 no 5 pp 378ndash3852008

[9] H C Maltezou and H Giamarellou ldquoCommunity-acquiredmethicillin-resistant Staphylococcus aureus infectionsrdquo Interna-tional Journal of Antimicrobial Agents vol 27 no 2 pp 87ndash962006

[10] F Aqil M S A Khan M Owais and I Ahmad ldquoEffect of cer-tain bioactive plant extracts on clinical isolates of 120573-lactamaseproducing methicillin resistant Staphylococcus aureusrdquo Journalof Basic Microbiology vol 45 no 2 pp 106ndash114 2005

[11] J N Eloff ldquoWhich extractant should be used for the screeningand isolation of antimicrobial components from plantsrdquo Jour-nal of Ethnopharmacology vol 60 no 1 pp 1ndash8 1998

[12] J Parekh and S V Chanda ldquoIn vitro antimicrobial activityand phytochemical analysis of some Indian medicinal plantsrdquoTurkish Journal of Biology vol 31 no 1 pp 53ndash58 2007

[13] T Hatano Y Shintani Y Aga S Shiota T Tsuchiya and TYoshida ldquoPhenolic constituents of licorice VIII Structures ofglicophenone and glicoisoflavanone and effects of licorice phe-nolics onmethicillin-resistant Staphylococcus aureusrdquoChemicaland Pharmaceutical Bulletin vol 48 no 9 pp 1286ndash1292 2000

[14] T Fukai A Marumo K Kaitou T Kanda S Terada and TNomura ldquoAntimicrobial activity of licorice flavonoids againstmethicillin-resistant Staphylococcus aureusrdquo Fitoterapia vol 73no 6 pp 536ndash539 2002

[15] E Tripoli M L Guardia S Giammanco D D Majo and MGiammanco ldquoCitrus flavonoids molecular structure biologicalactivity and nutritional properties a reviewrdquo Food Chemistryvol 104 no 2 pp 466ndash479 2007

[16] G B Mahady ldquoMedicinal plants for the prevention and treat-ment of bacterial infectionsrdquo Current Pharmaceutical Designvol 11 no 19 pp 2405ndash2427 2005

[17] J Cao Y-J Wei L-W Qi et al ldquoDetermination of fifteen bioac-tive components in Radix et Rhizoma Salviae Miltiorrhizae byhigh-performance liquid chromatography with ultraviolet andmass spectrometric detectionrdquo Biomedical Chromatographyvol 22 no 2 pp 164ndash172 2008

[18] D-S Lee S-H Lee J-G Noh and S-D Hong ldquoAntibacterialactivities of cryptotanshinone and dihydrotanshinone I from amedicinal herb Salvia miltiorrhiza bungerdquo Bioscience Biotech-nology and Biochemistry vol 63 no 12 pp 2236ndash2239 1999

[19] Y-M Ahn S K Kim S-H Lee et al ldquoRenoprotective effectof Tanshinone IIA an active component of Salvia miltiorrhizaon rats with chronic kidney diseaserdquoPhytotherapy Research vol24 no 12 pp 1886ndash1892 2010

[20] W Chen L Liu Y Luo et al ldquoCryptotanshinone activatesp38JNK and inhibits Erk12 leading to caspase-independentcell death in tumor cellsrdquoCancer Prevention Research vol 5 no5 pp 778ndash787 2012

[21] H Feng H Xiang J Zhang et al ldquoGenome-wide transcrip-tional profiling of the response of Staphylococcus aureus to


cryptotanshinonerdquo Journal of Biomedicine and Biotechnologyvol 2009 Article ID 617509 8 pages 2009

[22] J-W Lee Y-J Ji S-O Lee and I-S Lee ldquoEffect of Salivamiltiorrhiza Bunge on antimicrobial activity and resistant generegulation against Methicillin-Resistant Staphylococcus aureus(MRSA)rdquo Journal of Microbiology vol 45 no 4 pp 350ndash3572007

[23] M Gu G Zhang Z Su and F Ouyang ldquoIdentification of majoractive constituents in the fingerprint of Salvia miltiorrhizaBunge developed by high-speed counter-current chromatogra-phyrdquo Journal of Chromatography A vol 1041 no 1-2 pp 239ndash243 2004

[24] E J Park H Y Ji N J Kim et al ldquoSimultaneous determinationof tanshinone I dihydrotanshinone I tanshinone IIA andcryptotanshinone in rat plasma by liquid chromatographymdashtandem mass spectrometry application to a pharmacokineticstudy of a standardized fraction of Salvia miltiorrhiza PF2401-SFrdquo Biomedical Chromatography vol 22 no 5 pp 548ndash5552008

[25] R D Wojtyczka A Dziedzic D Idzik et al ldquoSusceptibility ofStaphylococcus aureus clinical isolates to propolis extract aloneor in combination with antimicrobial drugsrdquoMolecules vol 18no 8 pp 9623ndash9640 2013

[26] W Seesom A Jaratrungtawee S Suksumran C Mekseep-ralard P Ratananukul and W Sukhumsirichart ldquoAntileptospi-ral activity of xanthones fromGarciniamangostana and synergyof gamma-mangostin with penicillin Grdquo BMC Complementaryand Alternative Medicine vol 13 article 1 2013

[27] A Ribeiro A Z Coronado M C Silva-Carvalho et alldquoDetection and characterization of international community-acquired infections by methicillin-resistant Staphylococcusaureus clones in Rio de Janeiro and Porto Alegre cities causingboth community- and hospital-associated diseasesrdquo DiagnosticMicrobiology and Infectious Disease vol 59 no 3 pp 339ndash3452007

[28] D Yuan Y-N Pan W-W Fu T Makino and Y KanoldquoQuantitative analysis of the marker compounds in Salvia mil-tiorrihiza root and its phytomedicinal preparationsrdquo Chemicaland Pharmaceutical Bulletin vol 53 no 5 pp 508ndash514 2005

[29] Y-G Li L Song M Liu Z-B Zhi-Bi-Hu and Z-T WangldquoAdvancement in analysis of Salviae miltiorrhizae Radix etRhizoma (Danshen)rdquo Journal of Chromatography A vol 1216no 11 pp 1941ndash1953 2009

[30] G Zeng H Xiao J Liu and X Liang ldquoIdentification ofphenolic constituents in Radix Salvia miltiorrhizae by liquidchromatographyelectrospray ionization mass spectrometryrdquoRapid Communications in Mass Spectrometry vol 20 no 3 pp499ndash506 2006

[31] C Jacqueline J Caillon V Le Mabecque et al ldquoIn vitroactivity of linezolid alone and in combination with gentam-icin vancomycin or rifampicin against methicillin-resistantStaphylococcus aureus by time-kill curve methodsrdquo Journal ofAntimicrobial Chemotherapy vol 51 no 4 pp 857ndash864 2003

[32] O Aiyegoro A Adewusi S Oyedemi D Akinpelu andA Okoh ldquoInteractions of antibiotics and methanolic crudeextracts of Afzelia Africana (Smith) against drug resistancebacterial isolatesrdquo International Journal of Molecular Sciencesvol 12 no 7 pp 4477ndash4487 2011

[33] J-Y Lee S O Won S K Kwan et al ldquoSynergy of arbekacin-based combinations against vancomycin hetero-intermediate

Staphylococcus aureusrdquo Journal of Korean Medical Science vol21 no 2 pp 188ndash192 2006

[34] M N Alekshun and S B Levy ldquoMolecular mechanisms ofantibacterialmultidrug resistancerdquoCell vol 128 no 6 pp 1037ndash1050 2007

[35] T-H Chen Y-T Hsu C-H Chen S-H Kao and H-M Lee ldquoTanshinone IIA from Salvia miltiorrhiza inducesheme oxygenase-1 expression and inhibits lipopolysaccharide-induced nitric oxide expression in RAW 2647 cellsrdquoMitochon-drion vol 7 no 1-2 pp 101ndash105 2007

[36] J D Adams R Wang J Yang and E J Lien ldquoPreclinical andclinical examinations of Salvia miltiorrhiza and its tanshinonesin ischemic conditionsrdquo Chinese Medicine vol 1 article 3 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Antibacterial activity of cryptotanshinone and antibiotics in isolated MRSA VRSA MSSA and some of reference bacteria

Samples Cryptotanshinone (120583gmL) Ampicillin Oxacillin VancomycinMIC50lt

MIC90lt

MICMBC MICMBC (120583gmL)MSSA ATCC 259231 4 64 64256 816 0251 052MRSA ATCC 335912 05 4 416 5122048 816 14VRSA 3A0633 05 2 24 5122048 5121024 1632VRSA 3A0664 1 4 416 128256 5121024 3264MSSA 15 2 16 1664 10242048 051 14MSSA 26 8 32 32128 256512 12 12MRSA 1 2 16 1632 128256 128256 12MRSA 2 8 64 64128 256256 832 24MRSA 3 1 4 416 128512 128512 24MRSA 4 05 4 48 128256 5122048 12MRSA 5 2 64 64128 128512 5121024 12MRSA 6 05 4 416 64128 64256 12MRSA 7 2 8 816 128256 5121024 14MRSA 8 4 32 3264 256256 5122048 14MRSA 9 2 8 816 64128 5121024 12MRSA 10 2 16 1664 128256 5121024 12MRSA 11 4 8 832 64128 64128 12MRSA 12 1 4 48 128256 128256 14MRSA 13 4 32 32128 128256 32128 12MRSA 14 05 8 816 64128 816 14MRSA 15 05 4 416 64128 128256 14MRSA 16 2 8 816 128512 128512 121MSSA (ATCC 25923) reference strain methicillin-sensitive Staphylococcus aureus2MRSA (ATCC 33591) reference strain methicillin-resistant Staphylococcus aureus3VRSA 3A063 vancomycin-resistant Staphylococcus aureus isolated clinically4VRSA 3A066 vancomycin-resistant Staphylococcus aureus isolated clinically5MSSA (1-2) methicillin-sensitive Staphylococcus aureus isolated clinically6MRSA (1ndash16) methicillin-resistant Staphylococcus aureus isolated clinically

because of the difficulty in developing chemical syntheticdrugs and because of their side effects scientists are makingmore efforts to search for new drugs from plant resourcesto combat clinical multidrug-resistant microbial infections[11 12 16]

The main components of S miltiorrhiza can be dividedinto two groups hydrophilic compounds such as salvianolicacids and lipophilic chemicals including diterpenoid andtanshinones [17] The second group of components labeledas tanshinone I tanshinone II and cryptotanshinone arethe major bioactive constituents and have various kinds ofpharmacological effects including antibacterial antioxidantand antitumor activities and prevention of angina pectorisand myocardial infarction [18ndash20] The cryptotanshinone(CT) exhibits antimicrobial activity against a broad rangeof Gram-positive bacteria including S aureus and Gram-negative bacteria as well as other microorganisms [18 21]The hexane and chloroform fractions of S miltiorrhizaevidenced profound antimicrobial activity and inhibitedresistant gene expression against Staphylococcus aureus andMRSA (methicillin-resistant Staphylococcus aureus) [21 22]

In this study the antimicrobial activities of cryptotan-shinone (CT) against methicillin and vancomycin-resistant

Staphylococcus aureus isolated in a clinic were assessed usingbrothmicrodilutionmethod and the checkerboard and time-kill methods for synergistic effect of the combination withantibiotics

2 Materials and Methods

21 Plant Extraction and Isolation The air-dried roots(20 kg) of S miltiorrhiza were crushed and extracted withMeOH (8 L times 3) at room temperature The MeOH extracts(210 g) were evaporated and suspended in distilled waterand partitioned sequentially with CH

2Cl2 EtOAc and n-

BuOH The CH2Cl2soluble fraction was concentrated in

vacuo and its crude extract (28 g) was subjected to silicagel column chromatography using a CHCl

3-MeOH gradient

solvent system to provide ten fractions (A1ndashA10) FractionA4was further subjected to silica gel column chromatographywith a CHCl

3-MeOH (10 1) to yield five fractions (B1ndashB5)

Fraction B3 was purified on recycling prep-HPLC (JAIGELGS column and 220 nm) eluted with MeOH (40mLmin) toyield cryptotanshinone (98mg) The structure of cryptotan-shinone was identified by comparing its spectral data withthose published [23 24]


Time (hours)0 5 10 15 20 25

0

20

40

60

80MSSA 25923

Bact

eria

(times105

CFU

mL)

MRSA 33591

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


VRSA 1

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)0 5 10 15 20 25

0

20

40

60

80MSSA 25923

Bact

eria

(times105

CFU

mL)

MRSA 33591

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

VRSA 1

0 5 10 15 20 25

0

20

40

60

80

Time (hours)


VAN (MIC)

Bact

eria

(times105

CFU

mL)





VRSA 2

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

MSSA 1

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MSSA 2

0 5 10 15 20 25

0

20

40

60

80


Time (hours)

Bact

eria

(times105

CFU

mL)

VRSA 2

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MSSA 1

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MSSA 2

0 5 10 15 20 25

0

20

40

60

80

Time (hours)


VAN (MIC)

Bact

eria

(times105

CFU

mL)



MRSA 1

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 2

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 3

0 5 10 15 20 25

0

10

20

30

40

50

60

70


Time (hours)

Bact

eria

(times105

CFU

mL)

MRSA 1

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 2

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 3

0 5 10 15 20 25

0

20

40

60

80

Time (hours)


VAN (MIC)

Bact

eria

(times105

CFU

mL)



MRSA 4

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)0 5 10 15 20 25

MRSA 5

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

0 5 10 15 20 25

0

10

20

30

40

50

60

70MRSA 6


Time (hours)

Bact

eria

(times105

CFU

mL)

MRSA 4

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 5

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 6

0 5 10 15 20 25

0

20

40

60

80

Time (hours)


VAN (MIC)

Bact

eria

(times105

CFU

mL)



MRSA 7

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

MRSA 8

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

( times105

CFU

mL)

MRSA 9

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

( times105

CFU

mL)


Time (hours)

MRSA 7

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 8

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 9

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



MRSA 10

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 11

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

MRSA 12

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


Time (hours)

MRSA 10

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 11

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 12

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



MRSA 13

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 14

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 15

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


MRSA 13

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 14

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 15

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



















































































50s and MIC








3) 120575 764 (1H d J = 80Hz) 748 (1H d J = 80Hz)


3) 120575 2967 (C-1)


50and MIC

90values of CT for



MRSA 16

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


Time (hours)

MRSA 16

0 5 10 15 20 25

0

20

40

60

80Ba

cter

ia (times

105

CFU

mL)

Time (hours)


VAN (MIC)













Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Time (hours)0 5 10 15 20 25

0

20

40

60

80MSSA 25923

Bact

eria

(times105

CFU

mL)

MRSA 33591

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


VRSA 1

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)0 5 10 15 20 25

0

20

40

60

80MSSA 25923

Bact

eria

(times105

CFU

mL)

MRSA 33591

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

VRSA 1

0 5 10 15 20 25

0

20

40

60

80

Time (hours)


VAN (MIC)

Bact

eria

(times105

CFU

mL)





VRSA 2

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

MSSA 1

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MSSA 2

0 5 10 15 20 25

0

20

40

60

80


Time (hours)

Bact

eria

(times105

CFU

mL)

VRSA 2

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MSSA 1

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MSSA 2

0 5 10 15 20 25

0

20

40

60

80

Time (hours)


VAN (MIC)

Bact

eria

(times105

CFU

mL)



MRSA 1

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 2

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 3

0 5 10 15 20 25

0

10

20

30

40

50

60

70


Time (hours)

Bact

eria

(times105

CFU

mL)

MRSA 1

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 2

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 3

0 5 10 15 20 25

0

20

40

60

80

Time (hours)


VAN (MIC)

Bact

eria

(times105

CFU

mL)



MRSA 4

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)0 5 10 15 20 25

MRSA 5

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

0 5 10 15 20 25

0

10

20

30

40

50

60

70MRSA 6


Time (hours)

Bact

eria

(times105

CFU

mL)

MRSA 4

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 5

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 6

0 5 10 15 20 25

0

20

40

60

80

Time (hours)


VAN (MIC)

Bact

eria

(times105

CFU

mL)



MRSA 7

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

MRSA 8

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

( times105

CFU

mL)

MRSA 9

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

( times105

CFU

mL)


Time (hours)

MRSA 7

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 8

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 9

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



MRSA 10

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 11

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

MRSA 12

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


Time (hours)

MRSA 10

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 11

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 12

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



MRSA 13

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 14

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 15

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


MRSA 13

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 14

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 15

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



















































































50s and MIC








3) 120575 764 (1H d J = 80Hz) 748 (1H d J = 80Hz)


3) 120575 2967 (C-1)


50and MIC

90values of CT for



MRSA 16

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


Time (hours)

MRSA 16

0 5 10 15 20 25

0

20

40

60

80Ba

cter

ia (times

105

CFU

mL)

Time (hours)


VAN (MIC)













Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















VRSA 2

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

MSSA 1

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MSSA 2

0 5 10 15 20 25

0

20

40

60

80


Time (hours)

Bact

eria

(times105

CFU

mL)

VRSA 2

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MSSA 1

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MSSA 2

0 5 10 15 20 25

0

20

40

60

80

Time (hours)


VAN (MIC)

Bact

eria

(times105

CFU

mL)



MRSA 1

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 2

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 3

0 5 10 15 20 25

0

10

20

30

40

50

60

70


Time (hours)

Bact

eria

(times105

CFU

mL)

MRSA 1

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 2

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 3

0 5 10 15 20 25

0

20

40

60

80

Time (hours)


VAN (MIC)

Bact

eria

(times105

CFU

mL)



MRSA 4

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)0 5 10 15 20 25

MRSA 5

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

0 5 10 15 20 25

0

10

20

30

40

50

60

70MRSA 6


Time (hours)

Bact

eria

(times105

CFU

mL)

MRSA 4

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 5

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 6

0 5 10 15 20 25

0

20

40

60

80

Time (hours)


VAN (MIC)

Bact

eria

(times105

CFU

mL)



MRSA 7

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

MRSA 8

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

( times105

CFU

mL)

MRSA 9

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

( times105

CFU

mL)


Time (hours)

MRSA 7

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 8

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 9

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



MRSA 10

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 11

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

MRSA 12

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


Time (hours)

MRSA 10

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 11

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 12

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



MRSA 13

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 14

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 15

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


MRSA 13

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 14

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 15

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



















































































50s and MIC








3) 120575 764 (1H d J = 80Hz) 748 (1H d J = 80Hz)


3) 120575 2967 (C-1)


50and MIC

90values of CT for



MRSA 16

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


Time (hours)

MRSA 16

0 5 10 15 20 25

0

20

40

60

80Ba

cter

ia (times

105

CFU

mL)

Time (hours)


VAN (MIC)













Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MRSA 1

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 2

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 3

0 5 10 15 20 25

0

10

20

30

40

50

60

70


Time (hours)

Bact

eria

(times105

CFU

mL)

MRSA 1

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 2

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 3

0 5 10 15 20 25

0

20

40

60

80

Time (hours)


VAN (MIC)

Bact

eria

(times105

CFU

mL)



MRSA 4

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)0 5 10 15 20 25

MRSA 5

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

0 5 10 15 20 25

0

10

20

30

40

50

60

70MRSA 6


Time (hours)

Bact

eria

(times105

CFU

mL)

MRSA 4

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 5

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 6

0 5 10 15 20 25

0

20

40

60

80

Time (hours)


VAN (MIC)

Bact

eria

(times105

CFU

mL)



MRSA 7

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

MRSA 8

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

( times105

CFU

mL)

MRSA 9

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

( times105

CFU

mL)


Time (hours)

MRSA 7

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 8

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 9

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



MRSA 10

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 11

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

MRSA 12

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


Time (hours)

MRSA 10

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 11

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 12

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



MRSA 13

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 14

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 15

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


MRSA 13

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 14

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 15

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



















































































50s and MIC








3) 120575 764 (1H d J = 80Hz) 748 (1H d J = 80Hz)


3) 120575 2967 (C-1)


50and MIC

90values of CT for



MRSA 16

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


Time (hours)

MRSA 16

0 5 10 15 20 25

0

20

40

60

80Ba

cter

ia (times

105

CFU

mL)

Time (hours)


VAN (MIC)













Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MRSA 4

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)0 5 10 15 20 25

MRSA 5

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

0 5 10 15 20 25

0

10

20

30

40

50

60

70MRSA 6


Time (hours)

Bact

eria

(times105

CFU

mL)

MRSA 4

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 5

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 6

0 5 10 15 20 25

0

20

40

60

80

Time (hours)


VAN (MIC)

Bact

eria

(times105

CFU

mL)



MRSA 7

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

MRSA 8

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

( times105

CFU

mL)

MRSA 9

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

( times105

CFU

mL)


Time (hours)

MRSA 7

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 8

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 9

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



MRSA 10

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 11

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

MRSA 12

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


Time (hours)

MRSA 10

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 11

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 12

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



MRSA 13

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 14

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 15

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


MRSA 13

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 14

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 15

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



















































































50s and MIC








3) 120575 764 (1H d J = 80Hz) 748 (1H d J = 80Hz)


3) 120575 2967 (C-1)


50and MIC

90values of CT for



MRSA 16

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


Time (hours)

MRSA 16

0 5 10 15 20 25

0

20

40

60

80Ba

cter

ia (times

105

CFU

mL)

Time (hours)


VAN (MIC)













Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MRSA 7

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

MRSA 8

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

( times105

CFU

mL)

MRSA 9

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

( times105

CFU

mL)


Time (hours)

MRSA 7

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 8

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 9

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



MRSA 10

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 11

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

MRSA 12

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


Time (hours)

MRSA 10

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 11

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 12

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



MRSA 13

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 14

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 15

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


MRSA 13

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 14

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 15

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



















































































50s and MIC








3) 120575 764 (1H d J = 80Hz) 748 (1H d J = 80Hz)


3) 120575 2967 (C-1)


50and MIC

90values of CT for



MRSA 16

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


Time (hours)

MRSA 16

0 5 10 15 20 25

0

20

40

60

80Ba

cter

ia (times

105

CFU

mL)

Time (hours)


VAN (MIC)













Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MRSA 10

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 11

Time (hours)0 5 10 15 20 25

0

10

20

30

40

50

60

70

Bact

eria

(times105

CFU

mL)

MRSA 12

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


Time (hours)

MRSA 10

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 11

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 12

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



MRSA 13

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 14

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 15

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


MRSA 13

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 14

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 15

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



















































































50s and MIC








3) 120575 764 (1H d J = 80Hz) 748 (1H d J = 80Hz)


3) 120575 2967 (C-1)


50and MIC

90values of CT for



MRSA 16

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


Time (hours)

MRSA 16

0 5 10 15 20 25

0

20

40

60

80Ba

cter

ia (times

105

CFU

mL)

Time (hours)


VAN (MIC)













Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MRSA 13

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 14

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 15

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


MRSA 13

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 14

Time (hours)0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

MRSA 15

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)

Time (hours)


VAN (MIC)



















































































50s and MIC








3) 120575 764 (1H d J = 80Hz) 748 (1H d J = 80Hz)


3) 120575 2967 (C-1)


50and MIC

90values of CT for



MRSA 16

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


Time (hours)

MRSA 16

0 5 10 15 20 25

0

20

40

60

80Ba

cter

ia (times

105

CFU

mL)

Time (hours)


VAN (MIC)













Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































































































50s and MIC








3) 120575 764 (1H d J = 80Hz) 748 (1H d J = 80Hz)


3) 120575 2967 (C-1)


50and MIC

90values of CT for



MRSA 16

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


Time (hours)

MRSA 16

0 5 10 15 20 25

0

20

40

60

80Ba

cter

ia (times

105

CFU

mL)

Time (hours)


VAN (MIC)













Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MRSA 16

0 5 10 15 20 25

0

20

40

60

80

Bact

eria

(times105

CFU

mL)


Time (hours)

MRSA 16

0 5 10 15 20 25

0

20

40

60

80Ba

cter

ia (times

105

CFU

mL)

Time (hours)


VAN (MIC)













Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Synergistic Effect between Cryptotanshinone...

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