Research Article Synergistic Effect and Antiquorum Sensing...

Research ArticleSynergistic Effect and Antiquorum Sensing Activity ofNymphaea tetragona (Water Lily) Extract

Md Akil Hossain1 Ji-Yong Park12 Jin-Yoon Kim1 Joo-Won Suh3 and Seung-Chun Park1

1 Laboratory of Clinical Pharmacokinetics and Pharmacodynamics Department of Pharmacology College of Veterinary MedicineKyungpook National University Daegu 702-701 Republic of Korea

2 Institute of Clean Bio Daejeon 301-212 Republic of Korea3 Division of Bioscience and Bioinformatics Myongji University Science Campus Gyeonggi 449-728 Republic of Korea

Correspondence should be addressed to Seung-Chun Park parkschknuackr

Received 26 February 2014 Accepted 1 April 2014 Published 8 May 2014

Academic Editor Kota V Ramana

Copyright copy 2014 Md Akil Hossain et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Salmonellosis is a common and widely distributed food borne disease where Salmonella typhimurium is one of the most importantetiologic agents The purpose of this study was to investigate the antimicrobial activity of Nymphaea tetragona alone and incombination with antibiotics against S typhimurium It also aimed to assess the plant for quorum sensing inhibition (QSI) activityand to identify the bioactive compoundsThe antibacterial activities of the extract were assessed using brothmicrodilutionmethodDisk agar diffusionmethodwas employed to determine theQSI and bioactive compounds were identified byGC-MS analysis Ethylacetate fraction of N tetragona extract (EFNTE) demonstrated good antimicrobial activity (MIC 781120583gmL) against 4 strains outof 5 FIC index ranged from 0375 to 1031 between EFNTEtylosin and 0515 to 1250 between EFNTEstreptomycin against Styphimurium Among all extracts EFNTE and butanol fraction more significantly inhibited pigment production of C violaceumPolyphenols were identified as major compound of EFNTE and butanol fractionThese results indicate that combination amongNtetragona extract and antibiotics could be useful to combat drug-resistance Salmonella infections and polyphenols are promisingnew components from N tetragona that warrant further investigation as a candidate anti-Salmonella agent and quorum sensinginhibitor

1 Introduction

Salmonella species are the leading cause of bacterial gastroen-teritis in humans and animals all over the world [1 2] Foodanimals and water are the most important reservoirs of thebacteria [2] where the outbreaks of Salmonella infectionshave increasingly been associated with processed foods [3 4]There are 1300 million cases of gastroenteritis 16 millioncases of typhoid fever and 3 million cases of deaths world-wide each year due to Salmonella infections [5] Salmonellatyphimurium is one of the most common serovars associatedwith clinically reported salmonellosis in humans in mostparts of the world accounting for at least 15 of infections[2 6]

S typhimurium infects a wide range of animal hostsincluding poultry cattle and pigs and is termed ubiqui-tous which usually causes a self-limited gastroenteritis in

humans [7] The use of antibiotics is a major strategy andthey are commonly used therapeutically and prophylacticallyto treat S typhimurium infections in human and animalHowever increased antimicrobial resistance is exacerbatingimpact on public health worldwide which leads to increasedmorbidity mortality and treatment costs [8 9] Scientificstudies showed that tylosin has low or no inhibitory effects onexperimentally inoculated S typhimurium in pigs [10 11] Itwas also reported that S typhimurium illustrated resistance toampicillin chloramphenicol streptomycin sulphonamidesand tetracycline in 1980s in the UK and later distributedextensively through Europe and North America Additionalresistance had attained to trimethoprim-sulfamethoxazoleciprofloxacin and extended-spectrum cephalosporins withinthe 1990s [12] Development of alternative antibacterial ther-apies is necessary to overcome this outbreak Approximately80of theworldrsquos inhabitants rely on traditionalmedicine for

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 562173 10 pageshttpdxdoiorg1011552014562173

2 BioMed Research International

their primary health care and plants also play an importantrole in the health care system [13] The possible therapeuticuse ofNymphaeaceaemay be a good alternative of traditionalantibacterial

The Nymphaeaceae also called water lilies have a broadrange of flower colors and are living on the banks of lakesand rivers distributed in tropical areas around the world [14ndash16] A number of species of Nymphaea in Nepal India andChina are thought to act as functional drug plants [17] Manybioactive and pharmacologically important compounds havebeen obtained from Nymphaea species and used in medicineand pharmacy [18] Flower extract of N nouchali whichpossess compounds with high antibacterial and cytotoxicproperties [19 20] and ethyl acetate extract of N nouchalileaf extract have antibacterial activity against a wide rangeof strains [21] Nymphaea lotus extract was reported to havebioactive compounds such as tannins flavonoids alkaloidsanthraquinones saponins cardiac glycosides and phenolicswhere methicillin and vancomycin resistant S aureus Spyogenes and E coli were highly susceptible to N lotus[13 22 23] The pygmy water lily Nymphaea tetragona (Ait)Georgi (Nymphaceae) is one of the widely distributed plantsranging globally from Asia-temperate Asia-tropical Europeand northern America [24] It has ethnomedical uses as therhizome is used to cure acute diarrhea and dysentery by tribalherbal practitioners in Indian region [24]

Although there are many reports in the ethnomedicinalvalues of Nymphaea information on their antibacterial effi-cacy is scarce and with low scientific caliber for further com-mercial use Hence it is important to determine the antibac-terial activity very clearly and to identify the antibacterialactive compounds of this plant Furthermore the potentialsof water lily in combating antimicrobial resistance alone andin combination with antibiotics and the inhibition of quorumsensing controlled virulent factors of microbial pathogenswere not explored previously Thus the current study wasdesignated to evaluate the antibacterial activity of Nymphaeatetragona extract alone and in combination with commercialantibiotics against Salmonella typhimuriumQuorum sensinginhibition activity of the extract against biomonitor strainChromobacterium violaceum was also aimed at assessingin this study Finally a GC-MS analysis was performedto identify and quantify the major compounds of the Ntetragona

2 Materials and Methods

21 Bacterial Strains and Culture Medium Salmonellatyphimurium QC strain KTCC2515 and clinical isolatesST171 ST482 ST688 and ST21A were used for experimentsin this study (Table 1) which were collected from differentfarms in Republic of Korea Bacterial strains were suspendedin Mueller Hinton broth (MHB Difco USA) and thenincubated at 37∘C with 200 rpm for 20 h Mueller Hintonagar (MHA Difco) was used for the agar diffusion method

22 Plant Extraction and Fractionation N tetragona powderof body and root mixture was purchased from Chamsamgol

Lotus Farm (Chungju Republic of Korea) 100 g of thepowderedmaterial was boiled with 1000mL of 50methanolin a 2000mL three-neck round bottom boiling flask (SchottDuran NY USA) at 100∘C setting temperature on nonas-bestos surface for 3 h when the Brix and absorbance of theextract became the highest The supernatant of N tetragona50 methanol extract (NTME) was collected by filtration(70mm Advantec Toyo Roshi Kaisha Ltd Tokyo Japan)and solid particles retained on the filter were discardedThe solvent was then removed under reduced pressure inBuchi Rotavapor R-114 (BUCHI Labortechnik AG in FlawilSwitzerland) at 10 rpm and Eyela CCA-1111 (Tokyo RikakikaiCo Ltd made in China) and solidified by freeze-drying priorto use The yield of extract was 1071

10 g of lyophilized N tetragona extract was suspended in50mL of water and fractionated with equivalent amount ofdichloromethane ethyl acetate and butanol correspondinglyby separating funnelThe solvent fractionswere solidified andthe distribution of extract was 043 in dichloromethane401 in ethyl acetate and 4674 in butanol 4736 wasobtained as residue in water and 146 was process loss

23 Antibacterial Resistance Testing The disk-agar diffusionmethod validated by the Clinical and Laboratory StandardsInstitute [25] was used to verify the resistance pattern of fourS typhimurium clinical isolates against different commercialantibiotics S typhimurium KTCC 2515 was used as qualitycontrol strain in this experiment Antibiotic sensitivity wasconsidered according to the zone diameter interpretativestandards of CLSI [26]

24 Determination of MIC and MBC MIC was determinedby the standard broth microdilution method according tothe CLSI guidelines [27] in MHB using sim5 times 105 CFUmLof inoculums concentration The MHB was supplementedwith serial dilutions ranging from 244ndash25000 49ndash2500122ndash6250 and 49ndash25000120583gmL respectively for NTMEDFNTE EFNTE and BFNTE in 100 120583L volumes in 96-wellplates Bacterial suspensions were adjusted to 01 OD at600 nm diluted 1100 and dispensed in 100 120583L aliquots toall the wells including drug-free controls Initial CFUmL ofthe bacterial suspensions was determined by plating 10-folddilutions onMHA plates After overnight incubation at 37∘Cinoculums from each well were diluted 10-fold and plated onMHA plates to determine the CFUmL of each well MIC wasdetermined by comparing the final CFUwith initial CFUThelowest concentration of the extract inhibiting the increase ofCFUwas considered asMIC 100 120583Ldrug dilutions fromwellsof 96-well plates were cultured on MHA plates to determinethe existing bacterial number MBC was considered as thelowest concentrationwhich can eradicate 999bacteria [28]

25 Killing Rate of Salmonella typhimurium Killing rate wasevaluated as previously described by Tia Dubuisson et al[28] with some modification 025 times MIC 05 times MIC 1 timesMIC and 2 times MIC of EFNTE were prepared in 10mL ofMHB Four-hour old S typhimurium (KTCC 2515) cultureswere adjusted to 01 OD in 600 nm diluted to make suitable

BioMed Research International 3

Table 1 List of Salmonella strains used in this study along with their sources and sensitivities against commercial antibiotics

Antimicrobials Concentration (120583gdisk) KCTC2515 IsolatesST171 (chicken) ST482 (pig) ST688 (cattle) ST21A (pig)

Chloramphenicol 30 S S S R SCiprofloxacin 5 S S S S SErythromycin 15 R R R R RGentamycin 10 S R S R RKanamycin 30 S S R R INorfloxacin 10 S S S S SStreptomycin 10 I R R R RTetracycline 30 S R R R SAntibiotic sensitivity is considered according to the zone diameter interpretative standards of CLSI [26] R resistant I intermediate and S sensitive

concentration which will be 106 CFUmL after inoculatingto each tube Incubation of tubes was done at 200 rpm ona shaker incubator at 37∘C In 0 1 2 3 4 6 8 12 and 24hours the CFU of the cultures was determined by culturing20120583L aliquots of 10-fold dilutions onMHAplates Plates wereincubated at 37∘C overnight before counting CFUThe meanlog10CFUmL for each extract was plotted against different

times

26 Nymphaea tetragona Extract and Antimicrobial Com-bination Activities In Vitro Combination effects of EFNTEwith ampicillin marbofloxacin norfloxacin Streptomycintrimethoprim and tylosin (Sigma-Aldrich Co St Louis MOUSA) against S typhimurium were screened out since theseantimicrobials have reported for becoming resistant to Styphimurium [10ndash12] Previously described double disk agardiffusion method [29] was used with slight modification Azone diameter of individual antimicrobial was determined byCLSI guideline [25]

Disks were placed separately with a distance equal to thesum of the zone radii for each disk tested separately Theinterface of inhibition zones was observed after incubationGenerally synergism shows an improved zone indifferenceshows no change and antagonism shows a reduced zonecompared to the zones of individual test [29] Further test ofsynergism was done only for tylosin and streptomycin withthe extract fraction as those 2 antibiotics showed synergismin disk diffusion test

Combination interactions of the extract fraction withtylosin and streptomycin were determined in 96-well platesby previously described chequerboardmicrodilutionmethodwith slight modifications [30] Antibiotic was vertically andthe extract was horizontally diluted to get amatrix of differentcombinations of 2 antibacterials Similar dilutions of individ-ual drugs and the drug-free medium control were includedin each test plate Plates were incubated at 35∘C for 16 to 20hours after the addition of sim5 times 105 CFUmL of inoculumsFrom the MIC of the drugs alone and in combination wecalculated the fractional inhibitory concentration (FIC) andthe FIC index (FICI) FIC is the MIC in combination dividedby the MIC of the individual drug and FICI is the sum of

the FICs of individual drugs An FICI of le05 is consideredsynergistic an FICI of 40 is considered antagonistic and anFICI of 05ndash4 is considered to indicate no interaction [31]

27 Quorum Sensing Inhibition Quorum sensing inhibitionofNTME and its solvent fractionswere verified in accordancewith themethod described byAlvarez et al [32]C violaceumCV12472 was employed to find out the pigment inhibitionof extracts for attaining a qualitative screening 100 120583L offresh culture diluted as 25times 106 CFUmL was poured onmedia for the preparation of LB agar plates 60120583L of eachextract with specific concentrations was applied to saturatethe sterile paper disks (8mm) Normal saline (60120583L) wasused as negative control and purified furanone (100 120583g) wasapplied as positive control whereas tetracycline (10 120583g) wasemployed to compare antibacterial and antiquorum sensingactivity Inhibition of pigment production around the discwas checked after 18ndash24 h incubation at 30∘C The sensitivityto different agents was classified by the diameter of theinhibition zones as follows ldquonot sensitiverdquo for diameter lessthan 8mm ldquosensitiverdquo for diameter between 9 and 14mmldquovery sensitiverdquo for diameter between 15 and 19mm andldquoextremely sensitiverdquo for diameter larger than 20mm [33 34]

28 Gas Liquid Chromatography Coupled Mass Spectropho-tometric (GC-MS) Analysis GC-MS analysis of DFNTEEFNTE and BFNTE was performed by ldquoCenter for ScientificInstrumentsrdquo of Kyungpook National University and carriedout using a HP 6890 Plus GC gas chromatograph with a(MSD)mdashHP 5973 MSD mass selective detector (Hewlett-Packard) Samples were diluted 1 1000 (v v) with HPLCgrade dichloromethane Aliquots of the sample (1 120583L) wereinjected into an HP-5 columnThe GC oven temperature wasset at 50∘C for 4min increased to 280∘C at a rate of 4∘Cminand held at the final temperature for 2min Velocity of the Hecarrier gas (9999) was 07mLmin Quantitative analysiswas performed using the area normalization method

29 Statistical Analysis The mean values and the standarddeviation were calculated from the data obtained from tripli-cate trials Analysis of variance (ANOVA) was used to verify


Table 2 Antimicrobial activity of NTME and its solvent fractions against five strains of Salmonella typhimurium

KCTC 2515 ST171 ST482 ST688 ST21AMIC (120583gmL)

NORa 0063 0063 0125 0063 0125NTME 6250 6250 6250 6250 6250DFNTE gt2500 gt2500 gt2500 gt2500 gt2500EFNTE 781 781 gt781 781 781BFNTE 6250 6250 6250 6250 6250

MBC (120583gmL)NORa 1 1 1 1 1NTME 12500 12500 25000 12500 12500DFNTE gt2500 gt2500 gt2500 gt2500 gt2500EFNTE 1562 1562 1562 1562 1562BFNTE 25000 25000 25000 25000 25000

NOR norfloxacin NTMEN tetragona 50methanol extract EFNTE ethyl acetate fraction ofN tetragona 50methanol extract DFNTE dichloromethanefraction of N tetragona 50 methanol extract BFNTE butanol fraction of N tetragona 50 methanol extract aPositive control

differences and F-test was applied for the determination ofstatistical significance between groups

3 Results

31 MIC and MBC of Nymphaea tetragona 50 MethanolExtract The antimicrobial activity of NTME and its solventfractions were confirmed by determining the minimuminhibitory concentration (MIC) and minimum bacterici-dal concentration (MBC) against different strains of StyphimuriumThe results indicated that the EFNTEpossessedthe strongest antibacterial activity among all fractions MBCof EFNTE against all tested S typhimurium strains wasle1562120583gmL whereas theMIC values were within 781 120583gmLfor all strains except one clinical isolate (Table 2)

32 Killing Rate of Salmonella typhimurium Time-kill curvesof S typhimurium after treatment with EFNTE are demon-strated in Figure 1 The EFNTE of both the 1 times MIC and2 timesMIC concentrations showed complete inhibition up to 8hours and started their log phase from this time point At 24hours the growth level was 2-fold lower in 1 times MIC and 3-fold lower in 2 timesMIC of EFNTE than the growth control At025 times MIC and 05 times MIC bacteria reached log phase after1 hour and stationary phase after 8 hours whereas the controlstarted stationary phase after the 4th hour None of the testedconcentrations showed complete killing effect within 24 h

33 In Vitro Synergy with Commercial Antimicrobials Toinvestigate whether there is any synergy between EFNTEand commercial antibiotics we examined six antibioticswith EFNTE by disc diffusion method EFNTE exhibitedsynergism only with tylosin and streptomycin and has addi-tiveindifferent effects with other antibiotics There was noantagonistic effect observed in EFNTE with those antibiotics(Figure 2)

To reconfirm the synergistic activity of EFNTE withtylosin and streptomycin we further performed checker-board microdilution assay The results of the checkerboard

0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14 16 18 20 22 24

Control

Time (H)

log 1

0C

FUm

L

2 times MIC1 times MIC


Figure 1 Time-kill curves of S typhimurium KCTC 2515 aftertreatment with EFNTE at 025 timesMIC (n) 050 timesMIC (times) 1 timesMIC(998771) and 2 times MIC (◼) Ca-MHB was used as a control (X) Theresults are presented as mean plusmn standard deviations (119899 = 3) andthe coefficients of variation for all concentrations are le5

analysis are summarized in Tables 3 and 4 and Figure 3An increased sensitivity against tylosin was observed incombination with EFNTE The corresponding FICIs werele05 in tested strain demonstrating a synergistic effectHowever EFNTE in combination with streptomycin showedadditive and indifferent instead of synergistic interaction

34 Quorum Sensing Inhibition of NTME The inhibitiondiameter of C violaceum pigment in presence of NTMEand solvent fractions of NTME is presented in Table 5 Cviolaceum showed sensitivity to all fractions including thecrude extract Although the crude extract and all fractionsof extract significantly prevented the pigment ethyl acetateand butanol fractions showed highest inhibition among all


(a) (b)

Figure 2 Double disk synergy of EFNTE with commercial antibiotics against S typhimurium KCTC 2515 EAF ethyl acetate fraction of Ntetragona 50 methanol extract TMR trimethoprim NOR norfloxacin MAR marbofloxacin AMP ampicillin STR streptomycin andTYL tylosin Red arrows indicate synergistic interactions

EFNTE EFNTE EFNTE EFNTE EFNTE EFNTE EFNTE EFNTE EFNTE EFNTE EFNTE

MIC

ADI

ADI

ADI

SYN SYN

TYL

TYL

TYL

TYL

TYL

TYL

TYL

IND IND IND

EFNTE (120583gmL)

Tylo

sin (120583

gm

L)

6250 3125 1563 781 391 195 98 49 24 12 6

MIC

4096

2048

1024

512

256

128

64

0

(a)

Stre

ptom

ycin

(120583g

mL)

STR

STR

STR

STR

STR

STR

STR128

64

32

16

8

4

2

0MIC

MIC

IND

IND

IND

IND

IND ADI ADI ADI ADI ADI


EFNTE (120583gmL)

6250 3125 1563 781 391 195 98 49 24 6

(b)

Figure 3 Combination interaction of EFNTE with (a) tylosin and (b) streptomycin against S typhimurium KCTC 2515 by checkerboardmicrodilution method Ash colors indicate bacterial growth and without color zones are free of bacteria EFNTE ethyl acetate fraction of Ntetragona 50 methanol extract TYL tylosin STR streptomycin SYN synergistic effect ADI additive effect IND indifferent effect MICminimum inhibitory concentration

Tetracycline demonstrated bactericidal furanone confirmedpigment inhibitory effect and the negative control had noactivity

35 Gas Liquid Chromatography Coupled Mass Spectrophoto-metric (GC-MS) Analysis The major identified compoundswith their biological activities are illustrated in Table 6according to their elusion order The major chemicalcompounds in DFNTE were mainly hydrocarbons (about4646) and EFNTE contains methyl gallate (7044)1 2 3-benzenetriol or pyrogallol (2061) and 6 8-dimethylbenzocyclooctene (590) The major compounds

found in BFNTE were 2-hydrazinoquinoline (5761) pyro-gallol (2009) and methyl gallate (1277) GC-MS chro-matogram of EFNTE and chemical structures of methylgallate and pyrogallol are presented in Figure 4 We haveshown only the chromatogram of EFNTE and structures ofthose compounds as they are expected for desired effects andare abundant in EFNTE

4 Discussion

There are recent reports indicating the resistance of severalbacterial strains against different antibiotics that have been


1600000

1400000

1200000

1000000

800000

600000

400000

200000

12 14 16 18 20 22 24 26 28 30 32

Time (min)

Abun

danc

e

OH

OH

HO

Pyrogallol(RT 15787min)

OO

Methyl gallate(RT 27649min)

OH

OH

HO

Figure 4 Gas chromatography coupled with mass spectroscopy (GC-MS) chromatogram of EFNTE and chemical structure of majorcompounds

Table 3 Fractional inhibitory concentration (FIC) and FIC index (FICI) of combination between tylosin and EFNTE

Tylosin EFNTE FICI InterpretationConc (120583gmL) FIC of drug A Conc (120583gmL) FIC of drug B (FIC of A + FIC of B)1024 MIC of A 0 MIC of A MIC of A MIC of A1024 1 6 00077 1008 IND1024 1 12 00154 1015 IND1024 1 24 00307 1031 IND512 05 49 0063 0563 ADI256 025 98 0125 0375 SYN256 025 195 025 0499 SYN128 0125 391 05 0626 ADI64 00625 391 05 0563 ADI0 MIC of B 781 MIC of B MIC of B MIC of BEFNTE ethyl acetate fraction ofN tetragona 50methanol extract Conc concentration drugA tylosin drug B EFNTE SYN synergistic effect ADI additiveeffect IND indifferent effect MIC minimum inhibitory concentration The FICI was interpreted as follows synergistic effect (0 lt FICI le 05) additive effect(05 lt FICI le 1) and indifferent effect (1 lt FICI le 4)

Table 4 Fractional inhibitory concentration (FIC) and FIC index (FICI) of combination between streptomycin and EFNTE

Streptomycin EFNTE FICI InterpretationConc (120583gmL) FIC of drug A Conc (120583gmL) FIC of drug B (FIC of A + FIC of B)32 MIC of A 0 MIC of A MIC of A MIC of A32 1 6 00077 1008 IND16 05 12 00154 0515 ADI16 05 24 00307 0531 ADI16 05 49 0063 0563 ADI16 05 98 0125 0625 ADI16 05 195 025 075 ADI16 05 391 05 1 IND8 025 781 1 125 IND4 0125 781 1 1125 IND2 00625 781 1 1063 IND0 MIC of B 781 MIC of B MIC of B MIC of BEFNTE ethyl acetate fraction of N tetragona 50 methanol extract Conc concentration drug A streptomycin drug B EFNTE ADI additive effect INDindifferent effect MIC minimum inhibitory concentration The FICI was interpreted as follows synergistic effect (0 lt FICI le 05) additive effect (05 lt FICIle 1) and indifferent effect (1 lt FICI le 4)


Table 5Quorum sensing inhibition activity (as pigment inhibition zone diameters) ofNymphaea tetragona 50methanol extract and solventfractions of the crude extract against C violaceum

Solvent fractionantibiotic Concentration Pigment inhibition diameter (mm)(120583gdisk) Mean plusmn SD

Tetracycline 10 1633 plusmn 058lowast

Furanone 100 1667 plusmn 058a

Normal Saline mdash NDNTME 600 1367 plusmn 115

c

DFNTE 600 1333 plusmn 058c

EFNTE 600 1467 plusmn 115b

BFNTE 600 1433 plusmn 153b

NTME N tetragona 50 methanol extract DFNTE dichloromethane fraction of N tetragona 50 methanol extract EFNTE ethyl acetate fraction of Ntetragona 50 methanol extract BFNTE butanol fraction of N tetragona 50 methanol extract lowastDiameter of clear zone ND no activity detected Datashown represent the mean plusmn SD of three replicates Different alphabets indicate significant difference

used in the treatment of infectious diseases of human and ani-mals [45]Thus to combat infectious diseases associated withresistant pathogens development of alternative antimicrobialdrugs is urgent [46 47]The in vitro activity of EFNTE againstresistance strains of Salmonella typhimurium (Table 1) reflectsthat the plant could be a good candidate as a source of activephytoconstituents to minimize the development of bacterialresistance and to ensure clinical cure of bacterial infection

In the present study MIC results of EFNTE exhibitedantibacterial activity against all strains of S typhimuriumtested which have been shown to be resistant in one to six outof eight antibiotics (Table 1) The time-kill assay also exposedthat the extract fraction effectively inhibited the growth of Styphimurium Utilities of EFNTE are again explored by thecombination interactions with commercial antibiotics whereit possessed synergistic effect with tylosin and additive andorindifferent effects with other tested antibiotics Quorumsensing inhibition activity of NTME and solvent fractions ofthe extract demonstrated varied level of effects statisticallywherever EFNTE and BFNTE showed the greatest activityamong all fractions and crude extract Furthermore the GC-MS analysis was performed to investigate possible compo-nents from theNTME for its antibacterial potentialThis GC-MS analysis efficiently confirmed the existence of somemajorphenolic compounds (methyl gallate and pyrogallol) alongwith several other minor constituents

MIC and MBC of NTME and its solvent fractions werestudied against 5 strains of S typhimurium The results inTable 2 indicated that the EFNTE possessed the strongestantibacterial activity among all the fractions and crudeextract Considering the antibacterial activity EFNTE wasselected for further investigation Extract having MIC valuesless than 8mgmL was believed as active crude extract [48]Again it was suggested to avoid the MIC greater than1mgmL for crude extract and 01mgmL for isolated com-pounds 01mgmL and 001mgmL MICs correspondinglyfor crude extract and isolated compounds would be veryinteresting activity [49] Including all MIC values less than1mgmL were considered as good activity in the currentstudy

Time-kill curves have been used to determine whetherthe effects of EFNTE are either bacteriostatic or bactericidal

and are useful for the evaluation of the pharmacodynamiccharacteristics of new antimicrobial agents [50] According toour results EFNTE shows bacteriostatic activity against thetested bacteria since a reduction ge999 of the inoculumswas not observed compared to the growth control (Figure 1)At its 1 times MIC and 2 times MIC EFNTE was able to inhibit Styphimurium in the first 8 h of incubation Then 1 times MICand 2 times MIC of EFNTE treated groups started their logphase At the end of the incubation period (24 h) 3-foldreduced inoculums concentration in 2 timesMIC of EFNTE and2-fold lower inoculums concentration in 1 timesMIC of EFNTEwere achieved in contrast to control indicating that EFNTEdisplays a bacteriostatic effect

It is always suggested to treat bacterial infections witha combination of antimicrobial agents for the prevention ofdrug resistance development and to improve efficacy Drugcombinations having synergistic interactions are generallyconsidered as more effective and therefore preferable [28]Incidentally EFNTE is proved to have synergistic effectswith tylosin against S typhimurium The EFNTE also hasadditive and indifferent effects with streptomycin and otherantibiotics tested against the same bacterial strain and hasno antagonistic interactions with those antibiotics Excel-lent in vitro activity combined with synergistic effects withother antibacterial drugs underscores the potential utility ofEFNTE for the treatment of Salmonella associated infections

In this study NTME and its solvent fractions alsopossessed quorum sensing inhibition activity EFNTE andBFNTE are considered as very sensitive or sensitive amongthem whereas the crude extract and the rest of the fractionsare sensitive according to the previous report [33] Everyfraction of extract may have many major and minor com-pounds with QS-inhibition activity which is reflected in theresult Furthermore many compounds having antimicrobialactivity were identified by the GC-MS analysis of DFNTEEFNTE and BFNTE Methyl gallate pyrogallol and somehydrocarbons (hexacosan heptacosan octacosan etc) areimportant in response to desired type of effects and theproportion of those compounds in extracts Pyrogallol isreported to have quorum sensing inhibition activity [36 37]and antimicrobial activity [38] that is present in both EFNTE(2061) and BFNTE (2209) Methyl gallate is identified


Table6Major

compo

undlisto

fthree

solventfractions

accordingto

theirc

ontributionin

respectiv

esolvent

andcompo

unds

werelisted

follo

wed

bytheire

lutio

norderincluding

repo

rted

activ

ity

RTarea

Com

poun

dAc

tivity

Reference

Dichlorom

ethane

fractio

nof

Nymphaeatetra

gona

50methano

lextract

3546

318

Heneicosan

mdash3712

544

Tetracosan

mdash3871

79Pentacosan

mdash40

24

1118

Hexacosan

Antim

icrobialeffects

[35]

4171

1057

Heptacosan

Antifu

ngalactiv

ity[35]

4314

819

Octacosan

Antim

icrobialeffects

[35]

Ethylacetatefractio

nof

Nymphaeatetra

gona

50methano

lextract

1437

59

68-Dim

ethylbenzocyclo

octene

mdash1579

2061

Pyrogallo

lAntibacteria

lQSinhibitio

nandpo

tent

tyrosin

aseinh

ibito

r[36ndash

39]

1679

131

14-C

yclohexanedicarboxylicacid25-dioxo-diethylester

Anticolon

cancer

[40]

2690

139

1-[71015840-M

ethylbenzofuran-21015840-carbo

nyl]-3-ethylazulene

Derivatives

have

antib

acteria

lantifun

galanti-inflammatory

analgesic

antidepressantantic

onvulsa

ntantitu

mor

anti-HIVantidiabetic

antitu

bercular

activ

ity

[4142]

2765

7044

Methylgallate

Antim

icrobial

[43]

Butano

lfractionof

Nymphaeatetra

gona

50methano

lextract

1286

5761

2-Hydrazino

quinoline

mdash158

2209

Pyrogallo

lAntibacteria

lQSinhibitio

nandpo

tent

tyrosin

aseinh

ibito

r[36ndash

39]

2739

1277

Methylgallate

Antibacteria

l[4344

]


from EFNTE (7044) and BFNTE (1277) which is anantibacterial agent [43]The scenario is now clear that methylgallate and pyrogallol were contributing the vital role ofthe activity of EFNTE where they occupy about 91 of thetotal The similar level of QS inhibition activity of EFNTEand BFNTE may be because of the presence of pyrogallolalmost in equivalent amount in both fractionsThe combinedactivity of those hydrocarbons or minor compounds presentin DFNTE may possess the anti-QS activity

This is the first study describing the combination inter-action with commercial antibiotics and quorum sensinginhibition activity of N tetragona extract This study is alsoreporting the existence of methyl gallate and pyrogallol forthe first time from this species Together with all the promis-ing in vitro assay findings we believe that N tetragona 50methanol extract is expected to become a novel antimicrobialtreatment for Salmonella infection of animals and humanFurther study is needed to explore themechanism of quorumsensing inhibition antibacterial and synergistic activity ofthe extract In vivo activity and cytotoxic effects of the extractare also necessary to expose

Abbreviations

NTME Nymphaea tetragona 50 methanol extractDFNTE Dichloromethane fraction of Nymphaea

tetragona 50 methanol extractEFNTE Ethyl acetate fraction of Nymphaea tetragona

50 methanol extractBFNTE Butanol fraction of Nymphaea tetragona 50

methanol extractGC-MS Gas chromatography-mass spectrometry

Conflict of Interests

None of the authors have any conflict of interests to declare

Acknowledgments

This research was supported in part by the Next GenerationBioGreen 21 Program (no PJ009007) Republic of Koreaand in part by the Ministry of Trade Industry and Energy(MOTIE) and Korea Institute for Advancement of Tech-nology (KIAT) through the Promoting Regional specializedIndustry

References

[1] T J Humphrey ldquoPublic health aspects of Salmonella infectionsrdquoin Salmonella in Domestic Animals C Wray and A Wray EdsCABI Wallingford UK 2000

[2] E Scallan RMHoekstra F J Angulo et al ldquoFoodborne illnessacquired in the United Statesmdashmajor pathogensrdquo EmergingInfectious Diseases vol 17 no 1 pp 7ndash15 2011

[3] M J Sotir G Ewald A C Kimura et al ldquoOutbreak ofSalmonella wandsworth and Typhimurium infections in infantsand toddlers traced to a commercial vegetable-coated snackfoodrdquo The Pediatric Infectious Disease Journal vol 28 no 12pp 1041ndash1046 2009

[4] Foodborne Outbreaks due to Confirmed Bacteria EtiologiesCenters for Disease Control and Prevention Atlanta Ga USA2007

[5] A K Bhunia Foodborne Microbial Pathogens Mechanisms andPathogenesis Food Science Text Series Springer NewYork NYUSA 2008

[6] G A Grassl Y Valdez K S B Bergstrom B A Vallance and BB Finlay ldquoChronic enteric Salmonella infection inmice leads tosevere and persistent intestinal fibrosisrdquo Gastroenterology vol134 no 3 pp 768ndash780 2008

[7] M E Ohl and S I Miller ldquoSalmonella a model for bacterialpathogenesisrdquo Annual Review of Medicine vol 52 pp 259ndash2742001

[8] A E Mather S W J Reid D J Maskell et al ldquoDistinguish-able epidemics of multidrug-resistant Salmonella typhimuriumDT104 in different hostsrdquo Science vol 341 no 6153 pp 1514ndash1517 2013

[9] M-H Lee H A Kwon D-Y Kwon et al ldquoAntibacterial activityof medicinal herb extracts against Salmonellardquo InternationalJournal of Food Microbiology vol 111 no 3 pp 270ndash275 2006

[10] D L Baggesen A Wingstrand B Carstensen B Nielsenand F M Aarestrup ldquoEffects of the antimicrobial growth pro-moter tylosin on subclinical infection of pigs with Salmonellaenterica serotype typhimuriumrdquoAmerican Journal of VeterinaryResearch vol 60 no 10 pp 1201ndash1206 1999

[11] T R Shryock R A Elliott T H Bennett R P Basson andR E Bowen ldquoEffect of tylosin on an experimental Salmonellainfection in pigsrdquo Journal of Swine Health and Production vol6 no 5 pp 211ndash216 1998

[12] M B Zaidi V Leon C Canche et al ldquoRapid and widespreaddissemination of multidrug-resistant 119887119897119886

119862119872119884minus2

Salmonellatyphimurium in Mexicordquo Journal of AntimicrobialChemotherapy vol 60 no 2 pp 398ndash401 2007

[13] O J Akinjogunla C S Yah N O Eghafona and F O Ogbe-mudia ldquoAntibacterial activity of leave extracts of Nymphaealotus (Nymphaeaceae) on Methicillin resistant Staphylococ-cus aureus (MRSA) and Vancomycin resistant Staphylococcusaureus (VRSA) isolated from clinical samplesrdquo Annals of Bio-logical Research vol 1 no 2 pp 174ndash184 2010

[14] V K Agnihotri H N ElSohly S I Khan T J Smillie I AKhan and L AWalker ldquoAntioxidant constituents ofNymphaeacaerulea flowersrdquo Phytochemistry vol 69 no 10 pp 2061ndash20662008

[15] J Zhao T Liu L Ma et al ldquoAntioxidant and preventiveeffects of extract from Nymphaea candida flower on in vitroimmunological liver injury of rat primary hepatocyte culturesrdquoEvidence-Based Complementary and Alternative Medicine vol2011 Article ID 497673 8 pages 2011

[16] K A BeckettTheConcise Encyclopedia of Garden Plants OrbisLondon UK 1984

[17] J-H Cheng S-Y Lee Y-Y Lien M-S Lee and S-CSheu ldquoImmunomodulating activity of Nymphaea rubra Roxbextracts activation of rat dendritic cells and improvement ofthe TH1 immune responserdquo International Journal of MolecularSciences vol 13 no 9 pp 10722ndash10735 2012

[18] A K Siddhanta KHMody B K Ramavat et al ldquoBioactivity ofmarine organisms partVIIImdashscreening of somemarine flora ofwestern coast of Indiardquo Indian Journal of Experimental Biologyvol 35 no 6 pp 638ndash643 1997

[19] B K Dash M K Sen K Alam et al ldquoAntibacterial activityof Nymphaea nouchali (Burm f) flowerrdquo Annals of ClinicalMicrobiology and Antimicrobials vol 12 article 27 2013


[20] M A A Sikder H R Jisha Md R Kuddus F RumiM A Kaisar and M A Rashid ldquoEvaluation of bioactivi-ties of Nymphaea nouchali (Burm f)mdashthe national flower ofBangladeshrdquo Bangladesh Pharmaceutical Journal vol 15 no 1pp 1ndash5 2012

[21] A K Kumar S babu and K Ammani ldquoAntimicrobial andphytochemical analysis of Nymphaea nauchali leaf extractsrdquoInternational Journal of Research and Reviews in Pharmacy andApplied Science vol 2 no 2 pp 142ndash151 2012

[22] O J Akinjogunla A A Adegoke I P Udokang and BC Adebayo-Tayo ldquoAntimicrobial potential of Nymphaea lotus(Nymphaeaceae) against wound pathogensrdquo Journal of Medici-nal Plant Research vol 3 no 3 pp 138ndash141 2009

[23] J Yisa ldquoPhytochemical analysis and antimicrobial activity ofScoparia dulcis andNymphaea lotusrdquoAustralian Journal of Basicand Applied Sciences vol 3 no 4 pp 3975ndash3979 2009

[24] 2014 httpwwwplant-talkorgnymphaea-tetragona-indiahtm

[25] Clinical and Laboratory Standards Institute ldquoPerformancestandards for antimicrobial disk susceptibility testsrdquo CLSIApproved StandardsM2-A7 Clinical and Laboratory StandardsInstitute Wayne Pa USA 2001

[26] Clinical and Laboratory Standards Institute ldquoPerformancestandard for antimicrobial susceptibility testingrdquo CLSI SeventhInformational Supplement M100-S17 Clinical and LaboratoryStandards Institute Wayne Pa USA 2007

[27] Clinical and Laboratory Standards Institute ldquoMethods fordilution antimicrobial susceptibility tests for bacteria that growaerobicallyrdquo CLSI Approved Standards M7-A5 Clinical andLaboratory Standards Institute Wayne Pa USA 2000

[28] T Dubuisson E Bogatcheva M Y Krishnan et al ldquoIn vitroantimicrobial activities of capuramycin analogues againstnon-tuberculous mycobacteriardquo Journal of AntimicrobialChemotherapy vol 65 no 12 Article ID dkq372 pp 2590ndash2597 2010

[29] G M Eliopoulos and R C Moellering ldquoAntimicrobial combi-nationsrdquo in Antibiotics in Laboratory Medicine V Lorian Edpp 330ndash396 Williams amp Wilkins Baltimore Md USA 4thedition 1996

[30] V M Reddy L Einck and C A Nacy ldquoIn vitro antimycobacte-rial activities of capuramycin analoguesrdquo Antimicrobial Agentsand Chemotherapy vol 52 no 2 pp 719ndash721 2008

[31] F C Odds ldquoSynergy antagonism and what the chequerboardputs between themrdquo Journal of Antimicrobial Chemotherapyvol 52 no 1 article 1 2003

[32] M V Alvarez M R Moreira and A Ponce ldquoAntiquorum sens-ing and antimicrobial activity of natural agents with potentialuse in foodrdquo Journal of Food Safety vol 32 no 3 pp 379ndash3872012

[33] A G Ponce R Fritz C Del Valle and S I Roura ldquoAntimicro-bial activity of essential oils on the native microflora of organicSwiss chardrdquo LWTmdashFood Science and Technology vol 36 no 7pp 679ndash684 2003

[34] M R Moreira A G Ponce C E Del Valle and S I RouraldquoInhibitory parameters of essential oils to reduce a foodbornepathogenrdquo LWTmdashFood Science and Technology vol 38 no 5pp 565ndash570 2005

[35] C Gaspar-Marques M F Simoes M L Valdeira and BRodrıguez ldquoTerpenoids and phenolics from Plectranthus strigo-sus bioactivity screeningrdquoNatural Product Research vol 22 no2 pp 167ndash177 2008

[36] N Ni G Choudhary M Li and B Wang ldquoPyrogallol andits analogs can antagonize bacterial quorum sensing in Vibrioharveyirdquo Bioorganic amp Medicinal Chemistry Letters vol 18 no5 pp 1567ndash1572 2008

[37] T Defoirdt G S Pande K Baruah and P Bossier ldquoTheapparent quorum-sensing inhibitory activity of pyrogallol is aside effect of peroxide productionrdquo Antimicrobial Agents andChemotherapy vol 57 no 6 pp 2870ndash2873 2013

[38] I Kocacaliskan I Talan and I Terzi ldquoAntimicrobial activityof catechol and pyrogallol as allelochemicalsrdquo Zeitschrift furNaturforschung C Journal of Biosciences vol 61 no 9-10 pp639ndash642 2006

[39] J Choi S J Bae Y M Ha et al ldquoA newly synthesizedpotent tyrosinase inhibitor 5-(6-Hydroxy-2-naphthyl)- 123-benzenetriolrdquoBioorganicampMedicinal Chemistry Letters vol 20no 16 pp 4882ndash4884 2010

[40] W Wichitnithad U Nimmannit S Wacharasindhu and PRojsitthisak ldquoSynthesis characterization and biological evalu-ation of succinate prodrugs of curcuminoids for colon cancertreatmentrdquoMolecules vol 16 no 2 pp 1888ndash1900 2011

[41] H A Abdel-Aziz A A I Mekawey and K M DawoodldquoConvenient synthesis and antimicrobial evaluation of somenovel 2-substituted-3-methylbenzofuran derivativesrdquo EuropeanJournal of Medicinal Chemistry vol 44 no 9 pp 3637ndash36442009

[42] M Kamal A K Shakya and T Jawaid ldquoBenzofurans a newprofile of biological activitiesrdquo International Journal of Medicaland Pharmaceutical Sciences vol 1 no 3 pp 1ndash15 2011

[43] J-G Choi O-H Kang Y-S Lee et al ldquoAntibacterial activity ofmethyl gallate isolated from galla rhois or carvacrol combinedwith nalidixic acid against nalidixic acid resistant bacteriardquoMolecules vol 14 no 5 pp 1773ndash1780 2009

[44] J-G Choi O-H Kang Y-S Lee et al ldquoIn vitro activity ofmethyl gallate isolated from Galla Rhois alone and in combina-tion with ciprofloxacin against clinical isolates of SalmonellardquoJournal of Microbiology and Biotechnology vol 18 no 11 pp1848ndash1852 2008

[45] J Davies and D Davies ldquoOrigins and evolution of antibioticresistancerdquoMicrobiology andMolecular Biology Reviews vol 74no 3 pp 417ndash433 2010

[46] A Berahou A Auhmani N Fdil A Benharref M Jana and CA Gadhi ldquoAntibacterial activity ofQuercus ilex barkrsquos extractsrdquoJournal of Ethnopharmacology vol 112 no 3 pp 426ndash429 2007

[47] K Salomao P R S Pereira L C Campos et al ldquoBrazil-ian propolis correlation between chemical composition andantimicrobial activityrdquo Evidence-Based Complementary andAlternative Medicine vol 5 no 3 pp 317ndash324 2008

[48] B Ncube J F Finnie and J van Staden ldquoIn vitro antimicrobialsynergism within plant extract combinations from three SouthAfrican medicinal bulbsrdquo Journal of Ethnopharmacology vol139 no 1 pp 81ndash89 2012

[49] J L Rıos and M C Recio ldquoMedicinal plants and antimicrobialactivityrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 80ndash84 2005

[50] M A Pfaller D J Sheehan and J H Rex ldquoDetermination offungicidal activities against yeasts and molds lessons learnedfrom bactericidal testing and the need for standardizationrdquoClinical Microbiology Reviews vol 17 no 2 pp 268ndash280 2004

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


their primary health care and plants also play an importantrole in the health care system [13] The possible therapeuticuse ofNymphaeaceaemay be a good alternative of traditionalantibacterial

The Nymphaeaceae also called water lilies have a broadrange of flower colors and are living on the banks of lakesand rivers distributed in tropical areas around the world [14ndash16] A number of species of Nymphaea in Nepal India andChina are thought to act as functional drug plants [17] Manybioactive and pharmacologically important compounds havebeen obtained from Nymphaea species and used in medicineand pharmacy [18] Flower extract of N nouchali whichpossess compounds with high antibacterial and cytotoxicproperties [19 20] and ethyl acetate extract of N nouchalileaf extract have antibacterial activity against a wide rangeof strains [21] Nymphaea lotus extract was reported to havebioactive compounds such as tannins flavonoids alkaloidsanthraquinones saponins cardiac glycosides and phenolicswhere methicillin and vancomycin resistant S aureus Spyogenes and E coli were highly susceptible to N lotus[13 22 23] The pygmy water lily Nymphaea tetragona (Ait)Georgi (Nymphaceae) is one of the widely distributed plantsranging globally from Asia-temperate Asia-tropical Europeand northern America [24] It has ethnomedical uses as therhizome is used to cure acute diarrhea and dysentery by tribalherbal practitioners in Indian region [24]

Although there are many reports in the ethnomedicinalvalues of Nymphaea information on their antibacterial effi-cacy is scarce and with low scientific caliber for further com-mercial use Hence it is important to determine the antibac-terial activity very clearly and to identify the antibacterialactive compounds of this plant Furthermore the potentialsof water lily in combating antimicrobial resistance alone andin combination with antibiotics and the inhibition of quorumsensing controlled virulent factors of microbial pathogenswere not explored previously Thus the current study wasdesignated to evaluate the antibacterial activity of Nymphaeatetragona extract alone and in combination with commercialantibiotics against Salmonella typhimuriumQuorum sensinginhibition activity of the extract against biomonitor strainChromobacterium violaceum was also aimed at assessingin this study Finally a GC-MS analysis was performedto identify and quantify the major compounds of the Ntetragona

2 Materials and Methods

21 Bacterial Strains and Culture Medium Salmonellatyphimurium QC strain KTCC2515 and clinical isolatesST171 ST482 ST688 and ST21A were used for experimentsin this study (Table 1) which were collected from differentfarms in Republic of Korea Bacterial strains were suspendedin Mueller Hinton broth (MHB Difco USA) and thenincubated at 37∘C with 200 rpm for 20 h Mueller Hintonagar (MHA Difco) was used for the agar diffusion method

22 Plant Extraction and Fractionation N tetragona powderof body and root mixture was purchased from Chamsamgol

Lotus Farm (Chungju Republic of Korea) 100 g of thepowderedmaterial was boiled with 1000mL of 50methanolin a 2000mL three-neck round bottom boiling flask (SchottDuran NY USA) at 100∘C setting temperature on nonas-bestos surface for 3 h when the Brix and absorbance of theextract became the highest The supernatant of N tetragona50 methanol extract (NTME) was collected by filtration(70mm Advantec Toyo Roshi Kaisha Ltd Tokyo Japan)and solid particles retained on the filter were discardedThe solvent was then removed under reduced pressure inBuchi Rotavapor R-114 (BUCHI Labortechnik AG in FlawilSwitzerland) at 10 rpm and Eyela CCA-1111 (Tokyo RikakikaiCo Ltd made in China) and solidified by freeze-drying priorto use The yield of extract was 1071

10 g of lyophilized N tetragona extract was suspended in50mL of water and fractionated with equivalent amount ofdichloromethane ethyl acetate and butanol correspondinglyby separating funnelThe solvent fractionswere solidified andthe distribution of extract was 043 in dichloromethane401 in ethyl acetate and 4674 in butanol 4736 wasobtained as residue in water and 146 was process loss

23 Antibacterial Resistance Testing The disk-agar diffusionmethod validated by the Clinical and Laboratory StandardsInstitute [25] was used to verify the resistance pattern of fourS typhimurium clinical isolates against different commercialantibiotics S typhimurium KTCC 2515 was used as qualitycontrol strain in this experiment Antibiotic sensitivity wasconsidered according to the zone diameter interpretativestandards of CLSI [26]

24 Determination of MIC and MBC MIC was determinedby the standard broth microdilution method according tothe CLSI guidelines [27] in MHB using sim5 times 105 CFUmLof inoculums concentration The MHB was supplementedwith serial dilutions ranging from 244ndash25000 49ndash2500122ndash6250 and 49ndash25000120583gmL respectively for NTMEDFNTE EFNTE and BFNTE in 100 120583L volumes in 96-wellplates Bacterial suspensions were adjusted to 01 OD at600 nm diluted 1100 and dispensed in 100 120583L aliquots toall the wells including drug-free controls Initial CFUmL ofthe bacterial suspensions was determined by plating 10-folddilutions onMHA plates After overnight incubation at 37∘Cinoculums from each well were diluted 10-fold and plated onMHA plates to determine the CFUmL of each well MIC wasdetermined by comparing the final CFUwith initial CFUThelowest concentration of the extract inhibiting the increase ofCFUwas considered asMIC 100 120583Ldrug dilutions fromwellsof 96-well plates were cultured on MHA plates to determinethe existing bacterial number MBC was considered as thelowest concentrationwhich can eradicate 999bacteria [28]

25 Killing Rate of Salmonella typhimurium Killing rate wasevaluated as previously described by Tia Dubuisson et al[28] with some modification 025 times MIC 05 times MIC 1 timesMIC and 2 times MIC of EFNTE were prepared in 10mL ofMHB Four-hour old S typhimurium (KTCC 2515) cultureswere adjusted to 01 OD in 600 nm diluted to make suitable






times















3 Results





0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14 16 18 20 22 24

Control

Time (H)

log 1

0C

FUm

L







(a) (b)



MIC

ADI

ADI

ADI

SYN SYN

TYL

TYL

TYL

TYL

TYL

TYL

TYL

IND IND IND

EFNTE (120583gmL)

Tylo

sin (120583

gm

L)

6250 3125 1563 781 391 195 98 49 24 12 6

MIC

4096

2048

1024

512

256

128

64

0

(a)

Stre

ptom

ycin

(120583g

mL)

STR

STR

STR

STR

STR

STR

STR128

64

32

16

8

4

2

0MIC

MIC

IND

IND

IND

IND



EFNTE (120583gmL)

6250 3125 1563 781 391 195 98 49 24 6

(b)





4 Discussion



1600000

1400000

1200000

1000000

800000

600000

400000

200000

12 14 16 18 20 22 24 26 28 30 32

Time (min)

Abun

danc

e

OH

OH

HO


OO


OH

OH

HO












c













Table6Major

compo

undlisto

fthree

solventfractions

accordingto

theirc

ontributionin

respectiv

esolvent

andcompo

unds

werelisted

follo

wed

bytheire

lutio

norderincluding

repo

rted

activ

ity

RTarea

Com

poun

dAc

tivity

Reference

Dichlorom

ethane

fractio

nof

Nymphaeatetra

gona

50methano

lextract

3546

318

Heneicosan

mdash3712

544

Tetracosan

mdash3871

79Pentacosan

mdash40

24

1118

Hexacosan

Antim

icrobialeffects

[35]

4171

1057

Heptacosan

Antifu

ngalactiv

ity[35]

4314

819

Octacosan

Antim

icrobialeffects

[35]

Ethylacetatefractio

nof

Nymphaeatetra

gona

50methano

lextract

1437

59

68-Dim

ethylbenzocyclo

octene

mdash1579

2061

Pyrogallo

lAntibacteria

lQSinhibitio

nandpo

tent

tyrosin

aseinh

ibito

r[36ndash

39]

1679

131

14-C


Anticolon

cancer

[40]

2690

139

1-[71015840-M


nyl]-3-ethylazulene

Derivatives

have

antib

acteria

lantifun


analgesic

antidepressantantic

onvulsa

ntantitu

mor


antitu

bercular

activ

ity

[4142]

2765

7044

Methylgallate

Antim

icrobial

[43]

Butano

lfractionof

Nymphaeatetra

gona

50methano

lextract

1286

5761

2-Hydrazino

quinoline

mdash158

2209

Pyrogallo

lAntibacteria

lQSinhibitio

nandpo

tent

tyrosin

aseinh

ibito

r[36ndash

39]

2739

1277

Methylgallate

Antibacteria

l[4344

]




Abbreviations







Acknowledgments


References













119862119872119884minus2













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






times















3 Results





0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14 16 18 20 22 24

Control

Time (H)

log 1

0C

FUm

L







(a) (b)



MIC

ADI

ADI

ADI

SYN SYN

TYL

TYL

TYL

TYL

TYL

TYL

TYL

IND IND IND

EFNTE (120583gmL)

Tylo

sin (120583

gm

L)

6250 3125 1563 781 391 195 98 49 24 12 6

MIC

4096

2048

1024

512

256

128

64

0

(a)

Stre

ptom

ycin

(120583g

mL)

STR

STR

STR

STR

STR

STR

STR128

64

32

16

8

4

2

0MIC

MIC

IND

IND

IND

IND



EFNTE (120583gmL)

6250 3125 1563 781 391 195 98 49 24 6

(b)





4 Discussion



1600000

1400000

1200000

1000000

800000

600000

400000

200000

12 14 16 18 20 22 24 26 28 30 32

Time (min)

Abun

danc

e

OH

OH

HO


OO


OH

OH

HO












c













Table6Major

compo

undlisto

fthree

solventfractions

accordingto

theirc

ontributionin

respectiv

esolvent

andcompo

unds

werelisted

follo

wed

bytheire

lutio

norderincluding

repo

rted

activ

ity

RTarea

Com

poun

dAc

tivity

Reference

Dichlorom

ethane

fractio

nof

Nymphaeatetra

gona

50methano

lextract

3546

318

Heneicosan

mdash3712

544

Tetracosan

mdash3871

79Pentacosan

mdash40

24

1118

Hexacosan

Antim

icrobialeffects

[35]

4171

1057

Heptacosan

Antifu

ngalactiv

ity[35]

4314

819

Octacosan

Antim

icrobialeffects

[35]

Ethylacetatefractio

nof

Nymphaeatetra

gona

50methano

lextract

1437

59

68-Dim

ethylbenzocyclo

octene

mdash1579

2061

Pyrogallo

lAntibacteria

lQSinhibitio

nandpo

tent

tyrosin

aseinh

ibito

r[36ndash

39]

1679

131

14-C


Anticolon

cancer

[40]

2690

139

1-[71015840-M


nyl]-3-ethylazulene

Derivatives

have

antib

acteria

lantifun


analgesic

antidepressantantic

onvulsa

ntantitu

mor


antitu

bercular

activ

ity

[4142]

2765

7044

Methylgallate

Antim

icrobial

[43]

Butano

lfractionof

Nymphaeatetra

gona

50methano

lextract

1286

5761

2-Hydrazino

quinoline

mdash158

2209

Pyrogallo

lAntibacteria

lQSinhibitio

nandpo

tent

tyrosin

aseinh

ibito

r[36ndash

39]

2739

1277

Methylgallate

Antibacteria

l[4344

]




Abbreviations







Acknowledgments


References













119862119872119884minus2













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of








3 Results





0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14 16 18 20 22 24

Control

Time (H)

log 1

0C

FUm

L







(a) (b)



MIC

ADI

ADI

ADI

SYN SYN

TYL

TYL

TYL

TYL

TYL

TYL

TYL

IND IND IND

EFNTE (120583gmL)

Tylo

sin (120583

gm

L)

6250 3125 1563 781 391 195 98 49 24 12 6

MIC

4096

2048

1024

512

256

128

64

0

(a)

Stre

ptom

ycin

(120583g

mL)

STR

STR

STR

STR

STR

STR

STR128

64

32

16

8

4

2

0MIC

MIC

IND

IND

IND

IND



EFNTE (120583gmL)

6250 3125 1563 781 391 195 98 49 24 6

(b)





4 Discussion



1600000

1400000

1200000

1000000

800000

600000

400000

200000

12 14 16 18 20 22 24 26 28 30 32

Time (min)

Abun

danc

e

OH

OH

HO


OO


OH

OH

HO












c













Table6Major

compo

undlisto

fthree

solventfractions

accordingto

theirc

ontributionin

respectiv

esolvent

andcompo

unds

werelisted

follo

wed

bytheire

lutio

norderincluding

repo

rted

activ

ity

RTarea

Com

poun

dAc

tivity

Reference

Dichlorom

ethane

fractio

nof

Nymphaeatetra

gona

50methano

lextract

3546

318

Heneicosan

mdash3712

544

Tetracosan

mdash3871

79Pentacosan

mdash40

24

1118

Hexacosan

Antim

icrobialeffects

[35]

4171

1057

Heptacosan

Antifu

ngalactiv

ity[35]

4314

819

Octacosan

Antim

icrobialeffects

[35]

Ethylacetatefractio

nof

Nymphaeatetra

gona

50methano

lextract

1437

59

68-Dim

ethylbenzocyclo

octene

mdash1579

2061

Pyrogallo

lAntibacteria

lQSinhibitio

nandpo

tent

tyrosin

aseinh

ibito

r[36ndash

39]

1679

131

14-C


Anticolon

cancer

[40]

2690

139

1-[71015840-M


nyl]-3-ethylazulene

Derivatives

have

antib

acteria

lantifun


analgesic

antidepressantantic

onvulsa

ntantitu

mor


antitu

bercular

activ

ity

[4142]

2765

7044

Methylgallate

Antim

icrobial

[43]

Butano

lfractionof

Nymphaeatetra

gona

50methano

lextract

1286

5761

2-Hydrazino

quinoline

mdash158

2209

Pyrogallo

lAntibacteria

lQSinhibitio

nandpo

tent

tyrosin

aseinh

ibito

r[36ndash

39]

2739

1277

Methylgallate

Antibacteria

l[4344

]




Abbreviations







Acknowledgments


References













119862119872119884minus2













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(a) (b)



MIC

ADI

ADI

ADI

SYN SYN

TYL

TYL

TYL

TYL

TYL

TYL

TYL

IND IND IND

EFNTE (120583gmL)

Tylo

sin (120583

gm

L)

6250 3125 1563 781 391 195 98 49 24 12 6

MIC

4096

2048

1024

512

256

128

64

0

(a)

Stre

ptom

ycin

(120583g

mL)

STR

STR

STR

STR

STR

STR

STR128

64

32

16

8

4

2

0MIC

MIC

IND

IND

IND

IND



EFNTE (120583gmL)

6250 3125 1563 781 391 195 98 49 24 6

(b)





4 Discussion



1600000

1400000

1200000

1000000

800000

600000

400000

200000

12 14 16 18 20 22 24 26 28 30 32

Time (min)

Abun

danc

e

OH

OH

HO


OO


OH

OH

HO












c













Table6Major

compo

undlisto

fthree

solventfractions

accordingto

theirc

ontributionin

respectiv

esolvent

andcompo

unds

werelisted

follo

wed

bytheire

lutio

norderincluding

repo

rted

activ

ity

RTarea

Com

poun

dAc

tivity

Reference

Dichlorom

ethane

fractio

nof

Nymphaeatetra

gona

50methano

lextract

3546

318

Heneicosan

mdash3712

544

Tetracosan

mdash3871

79Pentacosan

mdash40

24

1118

Hexacosan

Antim

icrobialeffects

[35]

4171

1057

Heptacosan

Antifu

ngalactiv

ity[35]

4314

819

Octacosan

Antim

icrobialeffects

[35]

Ethylacetatefractio

nof

Nymphaeatetra

gona

50methano

lextract

1437

59

68-Dim

ethylbenzocyclo

octene

mdash1579

2061

Pyrogallo

lAntibacteria

lQSinhibitio

nandpo

tent

tyrosin

aseinh

ibito

r[36ndash

39]

1679

131

14-C


Anticolon

cancer

[40]

2690

139

1-[71015840-M


nyl]-3-ethylazulene

Derivatives

have

antib

acteria

lantifun


analgesic

antidepressantantic

onvulsa

ntantitu

mor


antitu

bercular

activ

ity

[4142]

2765

7044

Methylgallate

Antim

icrobial

[43]

Butano

lfractionof

Nymphaeatetra

gona

50methano

lextract

1286

5761

2-Hydrazino

quinoline

mdash158

2209

Pyrogallo

lAntibacteria

lQSinhibitio

nandpo

tent

tyrosin

aseinh

ibito

r[36ndash

39]

2739

1277

Methylgallate

Antibacteria

l[4344

]




Abbreviations







Acknowledgments


References













119862119872119884minus2













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


1600000

1400000

1200000

1000000

800000

600000

400000

200000

12 14 16 18 20 22 24 26 28 30 32

Time (min)

Abun

danc

e

OH

OH

HO


OO


OH

OH

HO












c













Table6Major

compo

undlisto

fthree

solventfractions

accordingto

theirc

ontributionin

respectiv

esolvent

andcompo

unds

werelisted

follo

wed

bytheire

lutio

norderincluding

repo

rted

activ

ity

RTarea

Com

poun

dAc

tivity

Reference

Dichlorom

ethane

fractio

nof

Nymphaeatetra

gona

50methano

lextract

3546

318

Heneicosan

mdash3712

544

Tetracosan

mdash3871

79Pentacosan

mdash40

24

1118

Hexacosan

Antim

icrobialeffects

[35]

4171

1057

Heptacosan

Antifu

ngalactiv

ity[35]

4314

819

Octacosan

Antim

icrobialeffects

[35]

Ethylacetatefractio

nof

Nymphaeatetra

gona

50methano

lextract

1437

59

68-Dim

ethylbenzocyclo

octene

mdash1579

2061

Pyrogallo

lAntibacteria

lQSinhibitio

nandpo

tent

tyrosin

aseinh

ibito

r[36ndash

39]

1679

131

14-C


Anticolon

cancer

[40]

2690

139

1-[71015840-M


nyl]-3-ethylazulene

Derivatives

have

antib

acteria

lantifun


analgesic

antidepressantantic

onvulsa

ntantitu

mor


antitu

bercular

activ

ity

[4142]

2765

7044

Methylgallate

Antim

icrobial

[43]

Butano

lfractionof

Nymphaeatetra

gona

50methano

lextract

1286

5761

2-Hydrazino

quinoline

mdash158

2209

Pyrogallo

lAntibacteria

lQSinhibitio

nandpo

tent

tyrosin

aseinh

ibito

r[36ndash

39]

2739

1277

Methylgallate

Antibacteria

l[4344

]




Abbreviations







Acknowledgments


References













119862119872119884minus2













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







c













Table6Major

compo

undlisto

fthree

solventfractions

accordingto

theirc

ontributionin

respectiv

esolvent

andcompo

unds

werelisted

follo

wed

bytheire

lutio

norderincluding

repo

rted

activ

ity

RTarea

Com

poun

dAc

tivity

Reference

Dichlorom

ethane

fractio

nof

Nymphaeatetra

gona

50methano

lextract

3546

318

Heneicosan

mdash3712

544

Tetracosan

mdash3871

79Pentacosan

mdash40

24

1118

Hexacosan

Antim

icrobialeffects

[35]

4171

1057

Heptacosan

Antifu

ngalactiv

ity[35]

4314

819

Octacosan

Antim

icrobialeffects

[35]

Ethylacetatefractio

nof

Nymphaeatetra

gona

50methano

lextract

1437

59

68-Dim

ethylbenzocyclo

octene

mdash1579

2061

Pyrogallo

lAntibacteria

lQSinhibitio

nandpo

tent

tyrosin

aseinh

ibito

r[36ndash

39]

1679

131

14-C


Anticolon

cancer

[40]

2690

139

1-[71015840-M


nyl]-3-ethylazulene

Derivatives

have

antib

acteria

lantifun


analgesic

antidepressantantic

onvulsa

ntantitu

mor


antitu

bercular

activ

ity

[4142]

2765

7044

Methylgallate

Antim

icrobial

[43]

Butano

lfractionof

Nymphaeatetra

gona

50methano

lextract

1286

5761

2-Hydrazino

quinoline

mdash158

2209

Pyrogallo

lAntibacteria

lQSinhibitio

nandpo

tent

tyrosin

aseinh

ibito

r[36ndash

39]

2739

1277

Methylgallate

Antibacteria

l[4344

]




Abbreviations







Acknowledgments


References













119862119872119884minus2













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Table6Major

compo

undlisto

fthree

solventfractions

accordingto

theirc

ontributionin

respectiv

esolvent

andcompo

unds

werelisted

follo

wed

bytheire

lutio

norderincluding

repo

rted

activ

ity

RTarea

Com

poun

dAc

tivity

Reference

Dichlorom

ethane

fractio

nof

Nymphaeatetra

gona

50methano

lextract

3546

318

Heneicosan

mdash3712

544

Tetracosan

mdash3871

79Pentacosan

mdash40

24

1118

Hexacosan

Antim

icrobialeffects

[35]

4171

1057

Heptacosan

Antifu

ngalactiv

ity[35]

4314

819

Octacosan

Antim

icrobialeffects

[35]

Ethylacetatefractio

nof

Nymphaeatetra

gona

50methano

lextract

1437

59

68-Dim

ethylbenzocyclo

octene

mdash1579

2061

Pyrogallo

lAntibacteria

lQSinhibitio

nandpo

tent

tyrosin

aseinh

ibito

r[36ndash

39]

1679

131

14-C


Anticolon

cancer

[40]

2690

139

1-[71015840-M


nyl]-3-ethylazulene

Derivatives

have

antib

acteria

lantifun


analgesic

antidepressantantic

onvulsa

ntantitu

mor


antitu

bercular

activ

ity

[4142]

2765

7044

Methylgallate

Antim

icrobial

[43]

Butano

lfractionof

Nymphaeatetra

gona

50methano

lextract

1286

5761

2-Hydrazino

quinoline

mdash158

2209

Pyrogallo

lAntibacteria

lQSinhibitio

nandpo

tent

tyrosin

aseinh

ibito

r[36ndash

39]

2739

1277

Methylgallate

Antibacteria

l[4344

]




Abbreviations







Acknowledgments


References













119862119872119884minus2













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




Abbreviations







Acknowledgments


References













119862119872119884minus2













































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 Synergistic Effect and Antiquorum Sensing...

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Transcript of Research Article Synergistic Effect and Antiquorum Sensing...