h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /
Clinical
Microbiology
Antibacterial
activity
of
3,3
,4
-Trihydroxyflavone
from
Justicia
wynaadensis
against
diabetic
wound
and
urinary
tract
infection
Dorin
Dsouza,
Lakshmidevi
Nanjaiah
∗UniversityofMysore,DepartmentofStudiesinMicrobiology,Manasagangotri,Mysore,India
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received24January2017 Accepted9May2017
Availableonline10August2017 AssociateEditor:LuisHenrique Guimarães Keywords: 3,3,4-Trihydroxyflavone Justiciawynaadensis Antibacterialactivity UTI Wound
a
b
s
t
r
a
c
t
Thepresentinvestigation wasdesigned tostudytheeffectofanactivecompound iso-latedfromJusticiawynaadensisagainstmultidrugresistantorganisms(MDRO’s)associated withdiabetic patients.The drug resistantpathogensimplicatedin woundandurinary tractinfectionofdiabeticpatientswereisolatedandidentifiedbymolecularsequencing. Solvent–solventfractionationofcrudemethanolextractproducedhexane,chloroform,ethyl acetateandmethanol–waterfraction,amongwhichchloroformfractionwasfoundtobe potentwhencomparedwithotherthreefractions.Further,chloroformfractionwas sub-jectedtopreparatoryHPLC(High-PerformanceLiquidChromatography),thatproducedfour sub-fractions;chloroformHPLCfraction1(CHF1)throughCHF4.Amongthesub-fractions, CHF1inhibitedthepathogenseffectivelyincomparisontootherthreesub-fractions.The purityofCHF1wasfoundtobe>95%.Therefore,CHF1wasfurthercharacterizedbyNMRand FTIRanalysisandbasedonthestructureelucidated,thecompoundwasfoundtobe3,3,4 -Trihydroxyflavone.Theeffectivedoseofthisbioactivecompoundrangedfrom32g/mL to1.2mg/mL.Thus,thepresentstudyshowsthat3,3,4-TrihydroxyflavoneisolatedfromJ. wynaadensisisaninterestingbiopharmaceuticalagentandcouldbeconsideredasasourceof antimicrobialagentforthetreatmentofvariousinfectionsandusedasatemplatemolecule forfuturedrugdevelopment.
©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Introduction
Infectious diseases are amongthe leading causes ofdeath globallyandaremorefrequentinpatientswithdiabetes mel-litus.Therecurrenceofmicrobialcomplicationsindiabetic
∗ Correspondingauthor.
E-mail:nlakshmidevi7@gmail.com(L.Nanjaiah).
patientsisbroughtaboutbythehyperglycaemicenvironment that favours immune dysfunction, neuropathy, reduced antibacterialactionofurine,urinarydysmotilityand promi-nent usage of various medications.1 Common pathologies
amongdiabetesarewoundandurinarytractinfection(UTI). Wound in diabetic patients is associated with peripheral
https://doi.org/10.1016/j.bjm.2017.05.002
1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
vascular disease, foot ulceration and gangrene that lead to limb amputation.2 While, UTI is allied with acute
pyelonephritis(upperUTI)andemphysematous pyelonephri-tis necessitating hospital admission.3 The first line of
treatmentinthesepatientsistheusageofantibiotics.The choiceofantibioticdependsonseveralfactorssuchasseverity ofinfection,antibioticsusedpreviouslyandtheresistanceto antibioticsbytheinfectingmicroorganism. However, exces-siveuseofantibioticsisofconcernastheiroveralleffecton thepatientisunclear.Theincreasingincidenceofantibiotic resistance among human pathogens against conventional antibioticsdemandssearchforalternatemodesoftreatment.4 Medicinalplantshavebeenfoundtobeeffectiveintreating microbialinfectionsassociatedwithdiabetes.Bioassaywith activity-guidedfractionationhasprovideddirectionsto iso-latenewbioactivecompoundswithnoveltargetstoovercome drugresistanceamongthepathogenicmicroorganisms.5The
searchformorepotentandsaferbiomoleculeshascontinued tobeanimportantareaofactiveresearch.Therefore,inthis study,weselectedJusticiawynaadensis,whichisknown tradi-tionallytopossess vitalbioactivemoleculeshavingvarious medicinal properties.6 Justicia, the largest genus of
Acan-thaceae, has approximately 600 species that are found in pantropicalandtropicalregions.7J.wynaadensisisendemicto
therainforestregionoftheWesternGhats.8Tothebestofour
knowledge,therearenoreportsavailable,thatdescribesthe isolationandcharacterizationofanyantimicrobialcompound fromthisplant.
Inthepresentstudy,anattempthasbeenmadetoisolatea bioactivecompoundwithpotentantibacterialactivityagainst drugresistantmicroorganismsisolatedfromwoundandUTI ofpatientswithdiabetes.
Materials
and
methods
MultidrugresistanceorganismsIsolationandidentificationofMDRO’sfrom diabeticwound and UTI were performed as follows: briefly, one hundred woundandurinesampleseachwerecollectedfromdiabetic patients with random blood glucose levels >140mg/dl and HbA1c>7.5percentadoptingasepticconditions.Theisolates were culturedon variousselectivemedia andidentified by biochemicalmethods.9Further,theisolateswereculturedon
Mueller-Hintonmedium, and tested againstknown antibi-otics (Hi Media Laboratories, Pvt Ltd., Mumbai). Penicillin-G, oxacillin, erythromycin, linezolid, co-trimoxazole, van-comycin,cefotaxime, ciprofloxacin,tetracycline, imipenem, amoxyclav,ampicillin,gentamycin,cefuroxime,levofloxacin, norfloxacin, doxycycline HCl, amikacin chloramphenicol, cefoxitin,amphotericin-B,and clotrimazole.Theorganisms thatofferedresistancetothreeormoreclassesofantibiotics wereselectedandidentifiedbymolecularsequencing. Molecularidentificationoftheisolates
Bacteria
Genomic DNA was isolated by employing the conven-tional phenol-chloroform method.10 Amplification of
16S rDNA was performed using the universal primers 27f (5-AGAGTTTGATCCTGGCTCA-3) and 1492r (5 -TACGGCTACCTTGTTACGACTT-3).11 The optimal cycling
conditions were the following:95◦Cfor5min; 35cycles of 94◦Cfor15s,56◦Cfor30s,and72◦Cfor90s;andafinal exten-sionat72◦Cfor7min.EachPCRmixtureof50Lcontained 1×Taqbuffer(Fermentas,USA),200MeachdNTPs,1.5mM MgCl2, 0.4M of primers, template DNA (100–250ng) and
1.25UofTaqpolymerase(Fermentas,USA).ThePCRproducts wereloadedin1.5%agarosegelin1×TAE(TrisacetateEDTA) bufferandwereallowedtorunat50Vfor45min.Thegelwas stained withethidiumbromidebath(10g/mL)and thegel imagewascapturedinageldoc.DNAsequencingwascarried outatAmnionBiosciencesPvt.Ltd.,Bangalore.
Collectionofplantmaterial
J. wynaadensis was collected in the month of August 2014 fromWesternGhatsofKarnataka,Indiaandauthenticatedby ataxonomist.Avoucher specimenofthisplantmaterialis depositedattheherbariumlibrary,JSScollegeof Pharmacol-ogy,Mysore,India(AccessionNo.:Jsscp-Pcog-16).
Extractpreparation
Theleaveswere separatedfromtheplantmaterial,washed thoroughlyunderrunningtapwaterandshade-driedon ster-ileblotters.Thegroundmaterial(100g)wassoakedin500mL of methanol and was placed on a water bath ina sealed container for propersteam effectat 50◦C for4h with fre-quentstirring. Theextract wasfiltered throughWhattman filterpaperandthefiltratewasevaporatedtodrynessunder reduced pressureusingarotavapor(Buchi,RotavapourR-3, Switzerland).
Bioassayguidedfractionation
Fractionationofcrudemethanolextract
The methanol extract was weighed and reconstituted in methanol:water1:1(v/v),subjectedforsolvent–solvent par-titioningwithsequentialadditionofhexane,chloroformand ethylacetateintheratio1:1(v/v),theextractionwasrepeated thrice.Thesolventfractionswereconcentratedtodrynessin rotavaportoaffordfoursolventfractions:hexane(HF), chlo-roform(CF),ethylacetate(EF)andmethanol–water(MF).
Antibacterialactivityoffoursolventfractions
Thepathogens isolatedfrom diabeticwoundandUTIwere inoculatedinto10mLofsterilenutrientbrothandincubated at37◦Cfor24h. Thecultureswere asepticallyswabbedon sterileMueller-Hintonagarplatesusingsterilecottonswab. Twenty-fivemicroliterofeachofthefourfractions(0.5mg)was loadedontothesterilediscplacedonthemicrobiallawnand antibacterialactivitywasevaluatedbymeasuringthezoneof inhibitionagainstthetestorganisms.
AnalyticalHPLCanalysis
AnalyticalRPHPLCwascarriedoutinaShimadzuHPLCsystem usingaC18column(100 ˚A,5m,4.6mm×250mm) contain-ing LC-10AT vp pump, SCL-10A vp system controller, and
SPD-10AV vp UV/vis detector. Chromatographic separation wasachievedusingmethanol,aceticacid,andwaterinthe ratio18:2:80 (v/v)asthemobile phaseunder isocratic con-dition.Thecolumnwaswashedwithmethanol,equilibrated withthefilteredanddegassedmobilephasefor30min,and 20Lof 1mg/mL standard mixtureor 2mg/mLchloroform fraction(CF)wasinjected.Thesampleorstandardrunwas car-riedoutfor1hat1mL/minflowrateandthedetectionsystem wassetat280nm.
PreparativeHPLCanalysis
Preparative HPLC analysis of the chloroform fraction (CF) wascarriedusingShimadzu(LC-8A)seriesHPLCsystem,DI diodearraydetector(SP-M10AVP),reversephaseC18column (20mm×250mm). Samples(2mL, 30mg/mL) were injected into the column repeatedly and the reference molecule, quercetinwas usedas astandard (1mL,1mg/mL).An Iso-craticelutionmethodwasemployedusingamobilephaseof methanol,aceticacidandwaterintheratio18:2:80.Theflow ratewas10mL/minand theabsorptionwas measuredata wavelengthof280and320nm.Fromthegradientelution,four majorpeakswereobtainedatretentiontime20.5,30.1,37.5 and57.1minandwerecollected,andlabelledCHF1through CHF4.Thecollectedisolatedfractionsweredriedbyremoving thesolventusingrotavapourat40◦C.Thesampleswerekept inavacuumdesiccatorforremovaloftracesofwaterandthen storedat4◦Cuntilfurtheranalysis.
Antibacterialactivityoffourfractionsobtainedfrom preparatoryHPLC
Twenty-five microliter of each of the four fractions CHF1-CHF4(0.25mg)obtainedfrompreparativeHPLCwereloaded ontothesterilediscand placedonthe microbiallawnand antibacterialactivity wasevaluated bymeasuringthezone ofinhibitionagainstthetestorganisms.Eachorganismwas testedintriplicates.
Characterizationofisolatedactiveprinciple
Amongthe isolatedfractions,CHF1elutingat20.5minwas againre-chromatographedundersameconditiontoverifyits purityandthencharacterizedbyspectroscopicmethodsas follows.
LC/MS
Theanalytical LC/MSexperimentwasperformedinWaters AcquityUPLCsystemcoupledwithSynaptG2SiHDMSmass spectrometer. Waters MassLynx and TargetLynx softwares wereusedfordataacquisitionand dataprocessing respec-tively.
LCconditions
Twomicrolitreof2mg/mLCHF1wasinjectedintoAcquityBEH C18 RPcolumn(130 ˚A,1.7m, 10mm×50mm). Binary gra-dientcontainingmobilephaseA(0.1%formicacidinwater, v/v)andmobilephaseB(acetonitrile)wasusedfor chromato-graphicseparationat15,000psiand0.3mL/minflowrate.The gradientusedwasasfollows:98%Aand2%Bfrom0to4min, 2%Aand98%Bfrom4to6min,and98%Aand2%Bfrom6 to8min.Columnandsampletemperaturesweremaintained
at50◦Cand24◦Crespectively;andtheruntimewassetfor 8min.
MSconditions
TimeofflightMS(TOF-MS)wasusedinnegativeelectronspray ionizationmode.Capillary,samplingconeandextractioncone voltagesweresetat1.8kV,40kVand4kVrespectively,witha sourcetemperatureof100◦Candpressureof3.5mbar. Nitro-gen was usedas the desolvationgasat200◦Cwith aflow rateof500L/handpressureof1.4mbar.Helium(dampinggas, 180mL/min,2mbar),Argon(trapgas,2mL/min,7.6mbar)and nitrogen(IMSgas,90mL/min,2.5mbar)werethegasesused inTri-wave.Collision induceddissociationwasusedasthe fragmentationmethodatatrapcollisionenergyof4eV.Ion mobilityseparation(IMS)wassetonalinearrampwithIMS gasvelocityascentfrom300to600m/s,andwaveheight(V) ascentfrom8to20.Transferwavevelocitywassetat247m/s withawaveheightof0.2.TOFwasoperatedbetween50and 1500m/zwithlowmassresolutionof4.7andhighmass reso-lutionof15.Datawasobtainedincontinuummodewithfull scanacquisitiontimeof8min,scantimeof1sandinterscan delayof0.024s.
Minimuminhibitoryconcentrationoftheactiveprinciple Minimum inhibitory concentration of the active com-pound was determined by micro broth dilution tech-nique as per CLSI guidelines. Briefly, the cell suspensions were prepared from bacterial cultures grown on Trypti-cose soya broth and was adjusted to 1–2×105cells/mL as
per McFarland’s standards. The standard reference drug, chloramphenicol (4–246g/mL) and the bioactivemolecule, 3,3,4-Trihydroxyflavone (16–1024g/mL) were prepared in Mueller-Hinton broth. Each individual drug concentrations (90L)weremixedwith10Linoculumin96wellplate. Chlor-amphenicolwasusedasapositivecontrol,whilethebroth without active compound served as negative control. The above treated bacterial plates were incubated at37◦C and observedafter24–48handopticaldensitywasmeasuredat 600nminTecanplatereader.Allthetestswereconductedin triplicates.
Statisticalanalyses
Statistical analysis was performed with Graph Pad Prism (GraphPadSoftware,Inc.).One-wayANOVAfollowedby stu-dent’s‘t’testwasusedfortestingpotentfractionwithother fractions.*p<0.05;**p<0.01;***p<0.001wereconsidered sta-tisticallysignificant.Dataareexpressedasmean±SEM.
Results
Speciesidentificationoftheisolates
Theclinicalisolatesusedinthisstudywereidentifiedby16S rDNAsequenceanalysis.Fig.1showsthesinglesharp1500bp amplifiedregionofthe16SrDNA(Fig.1AandB).Thesizeof theamplifiedDNAwasconfirmedbyusingastandardmarker DNAladder.Thepartial16SrDNAsequencesthusobtained
M 1 2 3 4 5 6 7 8 M 1 2 3 4 5 6 7 8
1.1 1.2
1500 bp
1500 bp
Fig.1–Clinicalisolatesidentifiedby16SrDNAanalysis.(1.1and1.2):1500bpamplifiedregionofthe16SrDNAofthe
bacterialisolatesfromdiabeticwoundandUTI,respectively.
wereretrievedinFASTAformatandsubjectedtoBLASTsearch anddepositedinGenbankundertheaccessionnumbersas listedin(Table1).Intotal,16organismswereidentifiedwith 8organismseachfromwoundandurinesamples.
Bioassayguidedfractionation
Fractionationofmethanolcrudeextractandphytochemical analysis
The total yield of crude methanol extract was found to be 5.25g/100g of the dry powder. Fractionation of the crude methanol extract (Fig. 2) by solvent–solvent parti-tioninggavefourdifferentfractions:hexane(yield=47.23%), chloroform (yield=24.57%), ethyl acetate (yield=15.23%) and methanol–water (yield=7.04%). Phytochemical analy-sis of these four fractions revealed the presence and absence of medicinally important phytoconstituents such asalkaloids, tannins, saponins,terpenoids, flavonoids, gly-cosides, phlobatannins and steroids. Chloroform fraction had alkaoids,tannins, saponins, flavonoids,glycosides and
steroids whereas, methanol–water fraction had alkaloids, tannins and glycosides. However, the hexane fraction and ethylacetatefractionwererichinatleastoneof phlobatan-nins,terpenoids,glycosides,saponinsandsteroids.
Antibacterialactivity
The four solventfractions (HF, CF,EFand MF) were tested fortheir efficacy againstthe isolated MDROs. The antibac-terial activity of the four fractions against a total of 16 isolatesisshowninFig.3AandB.Chloroformfractionwas foundto bemorepotentwhencomparedwithother three fractions. It showed maximum activity against Enterococ-cusfaecalis,Staphylococcus aureus,Escherichia coli,Enterobacter aerogenes,StaphylococcusepidermidisandKlebsiellapneumoniae and less inhibition against Staphylococcus haemolyticus and Pseudomonas aeruginosa.Hexane fractionshowed maximum activityagainstS.haemolyticusandE.aerogenesbutaminimum effectwasfoundonotherorganismswhencomparedto chlo-roformfraction.While,ethylacetatefractionwaslesseffective Leaf powder 100 g Methanolic extract 5.25 g Hexane Fraction (HF) 2.48 g Chloroform Fraction (CF) 1.29 g
Ethyl acetate Fraction (CF) 0.80 g Methanol-water Fraction (MF) 0.37 g Fraction 1 (CHF1) 0.46 g Fraction 2 (CHF2) 0.26 g Fraction 3 (CHF3) 0.31 g Fraction 4 (CHF4) 0.18 g FT-IR LC-MS NMR
Fig.2–Flowchartofbioassayguidedfractionation.Crudemethanolextractwasfractionatedbysolvent–solvent
fractionationfollowedwithseparationusingpreparativeHPLCtoobtainsub-fractions.Thepotentfractionischaracterized
Table1–Listofdrugresistantorganismsusedinthestudy.
Typeofinfection Gramnegative Grampositive
Diabeticwound
Pseudomonasaeruginosa–KY069054 Staphylococcusaureus–KX611101
Klebsiellapneumoniae–KY069048 Enterococcusfaecalis–KY069051
Enterobacteraerogenes–KY069049 Staphylococcusepidermidis–KY069052
Escherichiacoli–KY069050 Staphylococcushaemolyticus–KY069053
Urinarytract
infection
Escherichiacoli–KX816955 Staphylococcusepidermidis–KX816954
Klebsiellapneumoniae–KY021157 Staphylococcusaureus–KY021156
Pseudomonasaeruginosa–KX816953 Enterococcusfaecalis–KY021157
Enterobacteraerogenes–KX878983
Proteusmirabilis–KX878984 whencomparedwithothertwofractionsandmethanol–water fractionhadtheleastactivityagainstorganismstestedfrom woundsamples.
Interestingly,incaseofUTIisolates,chloroform fraction showed maximum antibacterial activity.Maximum activity wasobservedagainstS.aureus,P. aeruginosa,K.pneumoniae, E.faecalisandE.coli.HexanefractionwaseffectiveagainstE. aerogenes,ProteusmirabilisandS.epidermidisbutshowedleast
activityagainstK.pneumoniaeandE.coli.Ethylacetatefraction wasfoundtobeeffectiveagainstE.aerogenesandE.faecalisbut amoderateactivitywasobservedforP.aeruginosa,P.mirabilis
andS.aureus.However,themethanol-waterfractionwasnot foundtobeeffectiveagainstanyorganisms.
Sincechloroform fractionshowedpotentialantibacterial effect,itwassub-fractionatedusingpreparativeHPLCwhich resulted infour Sub-fractions(Fig.4). Thetotalpercentage
25 25 25 25 25 25 20 20 20 20 20 20 15 15 15 15 15 15 10 10 10 10 10 10 5 5 5 5 5 5 0 0 25 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 5 0 25 20 20 15 15 10 10 5 5 0 0 25 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 5 0 0 25 20 15 10 5 0 25 20 15 10 5 0 0 0 0 HF HF HF HF HF CF CF CF CF CF EF EF EF EF EF MF MF MF MF MF C C HF CF EF MF C HF CF EF MF C HF CF EF MF C HF CF EF MF C HF CF EF MF C HF CF EF MF C HF CF EF MF C HF CF EF MF C HF CF EF MF C HF CF EF MF C HF CF EF MF C C C C Enterococcus faecalis
Enterobacter aerogenes Enterococcus faecalis Staphylococcus aureus Staphylococcus aureus
Staphylococcus epidermidis Staphylococcus epidermidis
Staphylococcus haemolyticus
Klebsiella pneumoniae
Klebsiella pneumoniae pseudomonas aeruginosa
pseudomonas aeruginosa Enterobacter aerogenes Proteus mirabilis Escherichia coli Escherichia coli Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm) (mm) * * *** *** ** ** *** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * **
A
B
Fig.3–(A)Antibacterialactivityoffourextractsagainstwoundisolates:HF,hexanefraction;CF,chloroformfraction;EF,
ethyl-acetatefraction;MF,methanol–waterfraction;C,chloramphenicol.CFshowedmaximumactivityagainstE.faecalis,S.
aureus,E.coli,E.aerogenes,S.epidermidisandK.pneumoniaeandlessinhibitionagainstS.haemolyticusandP.aeruginosa.
Dataarerepresentedasmean±SD.*p<0.05;**p<0.01;***p<0.001,CFcomparedwithHF,EFandMF.(B)Antibacterialactivity
offourextractsagainstUTIisolates:HF,hexanefraction;CF,chloroformfraction;EF,ethyl-acetatefraction;MF,
methanol–waterfraction;C,chloramphenicol.Chloroformfraction(CF)showedmaximumactivityagainstS.aureus,P.
aeruginosa,K.pneumoniae,E.faecalisandE.coliandlessinhibitiononP.mirabilis,S.epidermidisandE.aerogenes.Dataare
CHF2 30.1000 CHF3 57.1000 40.3833 CHF4 37.5333 CHF1 20.5000 10.00 8.00 6.00 4.00 2.00 -0.00 60.00 50.00 40.00 Time[min] V oltage[mV] 30.00 20.00 10.00 10.5167 0.00 12.00 14.00
Fig.4–Sub-fractionationofchloroformfractionusingpreparativeHPLC.Fourdifferentsub-fractionswereobtainedfrom
chloroformfraction(CF)ataretentiontimeof20.5min(chloroformHPLCfraction1,CHF1),30.1min(CHF2),37.53min(CHF3)
and57.1min(CHF4). Staphylococcus aureus 25 20 15 10 5 0 0 CHF1 CHF2 CHF3 CHF4 c Zone of inhibition (mm) 25 20 15 10 5 0 CHF1 CHF2 CHF3 CHF4 c Zone of inhibition (mm) 25 20 15 10 5 0 CHF1 CHF2 CHF3 CHF4 c Zone of inhibition (mm) 25 20 15 10 5 0 CHF1 CHF2 CHF3 CHF4 c Zone of inhibition (mm) 25 20 15 10 5 0 CHF1 CHF2CHF3 CHF4 c Zone of inhibition (mm) 25 20 15 10 5 0 CHF1 CHF2CHF3 CHF4 c Zone of inhibition (mm) 25 20 15 10 5 0 CHF1 CHF2 CHF3 CHF4 c Zone of inhibition (mm) 25 20 15 10 5 0 CHF1 CHF2 CHF3 CHF4 c Zone of inhibition (mm) 25 20 15 10 5 0 CHF1 CHF2 CHF3 CHF4 c Zone of inhibition (mm) 20 15 10 5 0 CHF1 CHF2 CHF3 CHF4 c Zone of inhibition (mm) 25 20 15 10 5 CHF1 CHF2 CHF3 CHF4 c Zone of inhibition (mm) 0 25 20 15 10 5 CHF1 CHF2 CHF3 CHF4 c Zone of inhibition (mm) 0 25 20 15 10 5 CHF1 CHF2 CHF3 CHF4 c Zone of inhibition (mm) 0 25 20 15 10 5 CHF1 CHF2 CHF3 CHF4 c Zone of inhibition (mm) 25 20 15 10 5 0 CHF1 CHF2 CHF3 CHF4 c Zone of inhibition (mm) 0 25 20 15 10 5 CHF1 CHF2 CHF3 CHF4 c Zone of inhibition (mm)
Enterococcus faecalis Klebsiella pneumoniae
Klebsiella pneumoniae
Staphylococcus aureus
Staphylococcus epidermidis Enterobacter aerogenes Escherichia coli
Escherichia coli
Pseudomonas aeruginosa Enterococcus faecalis
Staphylococcus epidermidis
Staphylococcus haemolyticus Pseudomonas aeruginosa Enterobacter aerogenes Proteus mirabilis ** ** * * *** * *** *** *** * ** ** ** ** ** ** ** * * * *** * * *** * ** **
A
B
Fig.5–(A)Antibacterialactivityagainstdiabeticwoundisolates(HPLCfraction):CHF1,chloroformHPLCfraction1;CHF2,
chloroformHPLCfraction2;CHF3,chloroformHPLCfraction3;CHF4,chloroformHPLCfraction4;C,chloramphenicol.
MaximumactivitywasofferedbyCHF1againstE.faecalis,E.aerogenes,S.aureus,E.coli,andK.pneumoniaeandless
inhibitiononS.epidermidisandS.haemolyticus.Dataarerepresentedasmean±SD.*p<0.05;**p<0.01;***p<0.001,CHF1
comparedwithCHF2,CHF3andCHF4.(B)AntibacterialactivityagainstUTIisolatesindiabeticpatients(HPLCfraction):
CHF1showedmaximuminhibitoryactivityagainstP.aeruginosa,E.faecalis,S.aureus,K.pneumonaie,andE.coliand
minimuminhibitionagainstE.aerogenes,P.mirabilisandS.epidermidis.Dataarerepresentedasmean±SD.*p<0.05;
recoveryofthesesub-fractions;CHF1,CHF2,CHF3,andCHF4 were35.65,20.15,24.03and13.95%respectively.
Sub-fractionationofchloroformfraction
Amongthefoursub-fractionsscreenedforpotential antibac-terialactivityagainstorganismsisolatedfromdiabeticwound (Fig.5A),CHF1showedmaximumactivity againstE.coli,K. pneumoniaeandE.aerogenesamonggramnegativeisolatesand alsoforE.faecalisandS.aureusamonggrampositiveisolates. AmoderateinhibitionwasobservedforS.epidermidisandS. haemolyticus.CHF2waseffectiveagainstE.faecalis,E.aerogenes andE.coli.However,CHF3andCHF4werelesseffectivewhen comparedwithCHF1andCHF2.
Wefoundthat,theresultsobtainedfromchloroform frac-tionagainstmicroorganismofdiabeticwoundweresimilarto thatofUTIsamples(Fig.5B).Consistently,CHF1showed max-imuminhibitoryactivityforP.aeruginosa,E.faecalis,S.aureus, K.pneumonaie,andE.coli.While,minimuminhibitoryeffect wasobservedforE. aerogenes,P. mirabilisand S.epidermidis. CHF2was effective againstE. faecalis,K. pneumoniae and S. aureusandamoderate zoneofinhibitionwasobservedfor P.mirabilis,S.epidermidisandE.colibuttheactivitywasless whencompared toCHF1.ChloroformHPLC fraction(CHF3) wasfoundto bemoreeffectiveagainstGram negative iso-latesthanGrampositiveisolates.ChloroformHPLCfraction 4(CHF4)hadleastantibacterialeffectwhencomparedwith otherthreesub-fractions.Itwasfurthersubjectedtocheck thepurityoftheCHF1throughanalyticalHPLC(Fig.6A)and thestructureoftheactivecompoundinCHF1wasidentifiedas 3,3,4-Trihydroxyflavone(Fig.6B)basedontheNMRandother spectraldata(Fig.7).
Minimalinhibitoryconcentrationof 3,3,4-Trihydroxyflavone
Theactivecompound, 3,3,4-Trihydroxyflavonewas signifi-cantlyeffectiveagainstdiabeticwoundisolatescomparedto UTIisolates.ItshowedanMICvalueof32g/mLagainstE. faecalisandS.aureus,and64and128g/mLK.pneumoniae,E. aerogenesandE.coli,respectively.While,theconcentrationof referenceantibioticforthecorrespondingorganismsranged between8and32g/mL.However,wefoundthe MICvalue againstS.epidermidisandS.haemolyticustobe1024g/mL.In comparisontopathogensfromwoundsamples,theMICvalue of3,3,4-Trihydroxyflavone againstK.pneumoniae, S.aureus, E.faecalisandE.coliwasalsofoundtobe64and128g/mL, respectively.TheMICofthereferenceantibioticforthese iso-latesrangedbetween4and32g/mL.However,theMIC of P.aeruginosawasfoundtobe32g/mL,and appearedtobe relativelygreaterthanthatofreferenceantibiotic.
Discussion
Opportunisticorganismstakeadvantageoflowresistanceof thediabetichosttoinfection.Thecommensals ofthebody become pathogenic in conditions such as diabetic wound and UTI. Diabetics have severe neutropenia and impaired humoral as well as cellular immunity which may be the
0.00 -0.00 2.00 4.00 6.00 20.8333 8.00 10.00 12.00 14.00 5.00 10.00 15.0020.00 25.00 30.00 35.00 40.00 45.00 V oltage [m v] Time [min] O O OH OH OH
A
B
Fig.6–(A)HPLCfractionofCHF1;Asinglepeakshowingat
RT20.5min.(B)Structureof3,3,4-Trihydroxyflavone;IR:
3336cm−1OHstretch,1588cm−1,1471cm−1aromaticring
stretching,1416cm−1C Ostretch,1195cm−1C–O–Cstretch,
1007cm−1,781cm−1,637cm−1aromaticCHstretch,.1H
NMR(400MHz,DMSO-d6,ppm):ı5.35(s2protons,phenolic
OH)ı6.87–8.07(m,7H,phenylrings),9.24–9.51(s,1H,OH
proton).13CNMR(100MHz,DMSO-d
6,ppm):ı115.73,116.0,
118.57,120.44,121.75,122.74,124.79,125.1,133.77,138.29,
145.52,146.57,148.06,154.81,172.9.LC–MSm/z272(M+).
majorcontributingfactortothecauseofinfection.12Although
opportunisticfungiarerarecomparedwithbacteria,theycan bethecauseoflifethreateninginfectionsinimmune compro-misedindividuals.Thus,themajorinfectiveorganismsmay dependonseveralfactorssuchasage,genderandseverityof infection.
Weidentified eightdifferentbacterialisolatesinwound and UTI ofpatientswith diabetes. Polymicrobialinfections werealsoobservedwithasmanyasfiveisolatesinasingle individual. While, S. aureus was the most common orga-nisms in wound; E. coli was most common in UTI. Gram negative bacteria were predominant in both wound and UTI whencomparedto Grampositive bacteria. This obser-vation isin agreement with studies reported from several investigators.13–16 The presence ofE. coli as the most
pre-dominantpathogen indiabetic UTIissupportedbyseveral studies.17–20 The major mechanism of drug resistance in
UTI among patients with diabetes is the ESBL production by the infecting organisms. In our study, 30.8% of E. coli and 44.4% of K. pnemoniae were ESBL producers. This was
101 95 90 85 65 64 3500 2500 1500 1000 600 4000 3000 2000 75 80 70 cm-1 %T
A
C
D
B
Fig.7–Spectraldataof3,3,4-Trihydroxyflavone.(A)LC–MS,(B)FTIR,(C)CNMRand(D)HNMRofchloroformHPLCfraction
1.
higherthan26.6%reportedbyKhurana.21Thepresentstudy
also confirms the colonizationof MDRO notably by MRSA predominancewith over22% ofS. aureusbeingmethicillin resistant.ThisobservationagreeswiththestudyofCaputo,22
whoreported20%MRSAindiabeticwoundinfections. Inhibi-tionzoneamongassociatedbacteriaarethegreatinterestto searchforantibacterialsubstances.Isolationandscreeningfor secondarymetabolite-producingbacteriahavebeenstrongly investigated.Therearenumerousreportsontheantimicrobial activityofcrudeplantextractsanditsbioactivecompounds.23
Inthe present study,we haveshown the potential ofJ. wynaadensisasasourceofnaturalantimicrobials.The chloro-formfractionobtainedthroughsolvent–solventfractionation showed strong antimicrobial activity against most of the tested drug resistant organisms. While the other fractions showedmoderateantimicrobialactivity.Thisislikelydueto thepresenceofbioactivemoleculeinchloroformfraction.The antibacterialactivityofthemethanolicextractofJ. wynaaden-sisagainstK.pneumoniaehasalsobeenreportedbyPonnamma andManjunath.24InarecentstudyreportedbySubbu,25the
methanolextractofTragiainvolucratawasfoundtobeeffective againstE.aerogenes,P.aeruginosa,S.aureus,Proteusvulgarisand E.coliofwhichwereisolatedfromdiabeticfootulcersandUTI, whichisinagreementwithourresults.
Weperformedfurtherfractionationofthechloroform frac-tion to obtain four sub-fractions and upon screening for antibacterialproperties,wefoundCHF1tobehighlypotent whencomparedwithotherthreesub-fractions(CHF2,CHF3 andCHF4).ThisledustoknowthatCHF1indeedpossesses thebioactivemoleculeresponsibleforitspotenteffects.The maximuminhibitoryactivityagainstE.coli,K.pneumonaie,P. aeruginosa,S.aureusandE.faecaliscouldbeduetoacommon mechanismofaction.However,theminimuminhibitoryeffect
ofallthesub-fractionsobservedagainstP.mirabilis, E. aero-genesandS.haemolyticussuggeststhatthesepathogensmight adoptadifferentkind ofresistant pattern inorderto mul-tiplyingreatnumber.Identifyingsuchresistantpatternwill providenewinsightstocombatsuchMDROs.Therefore,we subjectedCHF1foritspuritythroughHPLCandfound>95% pure. Oncethe purity wasconfirmed, characterization and structureelucidationoftheisolatedmoleculewascarriedout byusingdifferentspectroscopictechniques:1H(400MHz)and 13CNMR(100MHz).Comparisonofthe1Hand13CNMRalong
withtheFTIR spectrumindicatedthepresenceofhydroxyl groupandpresenceofhydrogenandcarbonatoms.The iden-tifiedcompound,3,3,4-Trihydroxyflavoneisaflavonoid.The antimicrobialactivityofflavonoidshasbeenreportedearlier.26
Wespeculatethattheantimicrobialactivityobservedinthis studyisduetothepresenceofflavonoid.Aflavonoidrichplant extractsfromdifferentspecieshavebeenreportedtopossess antibacterial activity.27,28 Several flavonoids including
api-genin,galangin,flavoneandflavonolglycosides,isoflavones, flavanones,andchalconeshavebeenshowntopossesspotent antibacterialactivity.29Antibacterialflavonoidsmayhave
var-iouscelltargets,insteadofoneparticularsiteofactivity.One oftheirmolecularactivityistoformcomplexwithproteins throughnonspecificstrengthssuchashydrogenbondingand hydrophobicimpacts,andalsobycovalentbondformation. Hence,theirmethodofantimicrobialactivitymightbe identi-fiedwiththeircapacitytoinactivatemicrobialadhesins, com-poundsandcellenvelopetransportproteins.30,31The
antimi-crobialactivity of3,3,4-Trihydroxyflavonemight bedueto oneormoreofthemechanismsofactionasmentionedabove. The minimal inhibitory concentration (MIC) of 3,3,4 -Trihydroxyflavone,theisolatedcompoundfromJ.wynaadensis extractagainstthetestedbacteriaarepromising.According
toRiosandRecio,32MICvalueranginglessthan1mg/mLfor
crudeextractsor0.1mg/mLforisolatedcompoundsshould beconsideredeffectiveandalsoproposedthatbioactive com-poundsthatshow activityinthe rangebetween0.1mg/mL (extract) and 0.01mg/mL (isolated compound) would be of veryinteresting.While,Gibbons33suggeststhatisolated
phy-tochemicalsshouldhaveMICvalues<1mg/mL.TheMICof thiscompoundrangingfrom32to1024g/mLsuggeststhat the3,3,4-Trihydroxyflavoneholdalotofpromisesincethe organismstestedweremultidrugresistant.Andhence,canbe derivatizedtoincreaseitsspecificityandpotency.The3,3,4 -Trihydroxyflavonestructurecanserveasausefultemplatefor thesynthesisofnovelanti-microbialagents.
Fromourstudyandfromotherstudies,itisevidentthat emergenceofdrugresistanceisbecomingamajorobstacleto effectivetreatmentofinfectionsindiabeticindividuals.The lackofeffectiveandsafe antibioticstotreatserious bacte-rialinfectionsandlackofnewantibioticdrugdevelopmentby pharmaindustryisalarming.Consequently,botanicalsaswell ascomplementaryandalternativemedicineareseriously con-sideredtoaddresstheissueofemergingdrugresistance.The useoftraditionalmedicinalplantsforprimaryhealthcarehas steadilyincreasedworldwide.Plantsarearichsourceof phyto-chemicalsthatcouldbeusedasantimicrobialsanddeveloped asnewdrugs.
Conclusion
Prevention and cure of infectious diseases using phyto-chemicalsespeciallyflavonoidsarewellknown.Thepresent study shows that 3,3,4-Trihydroxyflavone isolated from J. wynaadensisisaninterestingbiopharmaceuticalagentagainst the multi drug resistant organisms isolated from diabetic woundandUTIinfections.Itcouldbeconsideredasasource ofantimicrobialagentforthetreatmentofvariousinfections andusedasatemplatemoleculeforfuturedrugdevelopment.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
r
e
f
e
r
e
n
c
e
s
1. CasqueiroJ,CasqueiroJ,AlvesC.Infectionsinpatientswith diabetesmellitus:areviewofpathogenesis.IndianJ EndocrinolMetab.2012;16(Suppl.1):S27–S36.
2. ShakilS,KhanAU.Infectedfootulcersinmaleandfemale diabeticpatients:aclinico-bioinformativestudy.AnnClin MicrobiolAntimicrob.2010;9:2.
3. NicolleLE.Urinarytractinfectionindiabetes.CurrOpinInfect Dis.2005;18:49–53.
4. JasmineR,SelvakumarBN,AishwaryaS.Roleofanovel terpenoidaseffluxinhibitorintargetingtheeffluxprotein (mexa)ofmultidrugresistant,Pseudomonasaeruginosa.IntJ PharmSciRes.2012;3:1647e–1651e.
5. HostettmannK,LeaPJ.Biologicallyactivenaturalproducts. In:ProcPhytochemSocEur,vol.27.Oxford:ClarendonPress; 1987:56–57.
6.PonnammaSU,ManjunathU.GC–MSanalysisof phytocomponentsinthemethanolicextractofJusticia Wynaadensis(Nees)T.Anders.IntJPharmBiosci. 2012;3:570–576.
7.DurkeeLH.FloraCostaricensis:Acanthaceae.Fieldiana Botany.vol.18;1986:1–87.
8.GambleJS.FloraofthePresidencyofMadras.AdlardandSons Ltd.;1967:755–756.
9.MurrayPR,BaronEJ,PfallerMA,TenoverFC,YolkenRH. ManualofClinicalMicrobiology.6thed.Washington,DC: AmericanSocietyofMicrobiologyPress;1995:1482. 10.SambrookJ,RussellDW.MolecularCloning–ALaboratory
Manual.3rded.ColdSpringHarbor:ColdSpringHarbor LaboratoryPress;2001.
11.LaneDJ.16S/23SrRNAsequencing.In:StackebrandtE,Good fellowM,eds.NucleicAcidsTechniquesinBacterialSystematics. NewYork:JohnWiley&sons;1991:115–175.
12.PelegAY,WeerarathnaT,McCarthyJS,DavisTM.Common infectionsindiabetes:pathogenesis,managementand relationshiptoglycaemiccontrol.DiabetesMetabResRev. 2007;23:3–13.
13.ShankarEM,MohanV,PremalathaG,SrinivasanRS,Usha AR.BacterialetiologyofdiabeticfootinfectionsinSouth India.EurJInternMed.2005;16:567–570.
14.GadepalliR,DhawanB,SreenivasV,KapilA,AmminiAC, ChaudhryR.Aclinico-microbiologicalstudyofdiabeticfoot ulcersinanIndiantertiarycarehospital.DiabetesCare. 2006;29:1727–1732.
15.RajaNS.Microbiologyofdiabeticfootinfectionsina teachinghospitalinMalaysia:aretrospectivestudyof194 cases.JMicrobiolImmunolInfect=Weimianyuganranzazhi. 2007;40:39–44.
16.RamakantP,VermaAK,MisraR,etal.Changing
microbiologicalprofileofpathogenicbacteriaindiabeticfoot infections:timeforarethinkonwhichempiricaltherapyto choose?Diabetologia.2011;54:58–64.
17.GuptaK,HootonTM,WobbeCL,StammWE.Theprevalence ofantimicrobialresistanceamonguropathogenscausing acuteuncomplicatedcystitisinyoungwomen.IntJ AntimicrobAgents.1999;11:305–308.
18.BarrettSP,SavageMA,RebecMP,GuyotA,AndrewsN, ShrimptonSB.Antibioticsensitivityofbacteriaassociated withcommunity-acquiredurinarytractinfectioninBritain.J AntimicrobChemother.1999;44:359–365.
19.JonesRN,KuglerKC,PfallerMA,WinokurPL.Characteristics ofpathogenscausingurinarytractinfectionsinhospitalsin NorthAmerica:resultsfromtheSENTRYAntimicrobial SurveillanceProgram,1997.DiagnMicrobiolInfectDis. 1999;35:55–63.
20.VromenM,vanderVenAJ,KnolsA,StobberinghEE. Antimicrobialresistancepatternsinurinaryisolatesfrom nursinghomeresidents.Fifteenyearsofdatareviewed.J AntimicrobChemother.1999;44:113–116.
21.KhuranaS,TanejaN,SharmaM.Extendedspectrum beta-lactamasemediatedresistanceinurinarytractisolates offamilyEnterobacteriaceae.IndianJMedRes.
2002;116:145–149.
22.CaputoGM,CavanaghPR,UlbrechtJS,GibbonsGW,
KarchmerAW.Assessmentandmanagementoffootdisease inpatientswithdiabetes.NEnglJMed.1994;331:
854–860.
23.Puupponen-PimiaR,NohynekL,AlakomiHL, Oksman-CaldenteyKM.Theactionofberryphenolics againsthumanintestinalpathogens.BioFactors(Oxford, England).2005;23:243–251.
24.PonnammaSU,ManjunathK.TLC-bioautographyguided screeningforcompoundsinhibitorytoKlebsiellapneumoniae fromJusticiaWynaadensis(Nees)T.Anders.IndianJApplRes. 2015;5:629–630.
25.SubbuLakshmiS,ChelladuraiG,SureshB.Invitrostudieson medicinalplantsusedagainstbacterialdiabeticfootulcer (BDFU)andurinarytractinfected(UTI)causingpathogens.J ParasitDis.2016;40:667–673.
26.TaguriT,TanakaT,KounoI.Antimicrobialactivityof10 differentplantpolyphenolsagainstbacteriacausing food-bornedisease.BiolPharmBull.2004;27:1965–1969. 27.MishraA,KumarS,BhargavaA,SharmaB,PandeyAK. Studiesoninvitroantioxidantandantistaphylococcal activitiesofsomeimportantmedicinalplants.CellMolBiol (Noisy-le-Grand).2011;57:16–25.
28.MishraA,KumarS,PandeyAK.Scientificvalidationofthe medicinalefficacyofTinosporacordifolia.ScientificWorldJournal. 2013;2013:292934.
29.CushnieTP,LambAJ.Antimicrobialactivityofflavonoids.Int JAntimicrobAgents.2005;26:343–356.
30.CowanMM.Plantproductsasantimicrobialagents.Clin MicrobiolRev.1999;12:564–582.
31.MishraAK,MishraA,KehriHK,SharmaB,PandeyAK. InhibitoryactivityofIndianspiceplantCinnamomum zeylanicumextractsagainstAlternariasolaniandCurvularia lunata,thepathogenicdematiaceousmoulds.AnnClin MicrobiolAntimicrob.2009;8:9.
32.RiosJL,RecioMC.Medicinalplantsandantimicrobial activity.JEthnopharmacol.2005;100:80–84.
33.GibbonsS.Plantsasasourceofbacterialresistance modulatorsandanti-infectiveagents.PhytochemRev. 2005;4:63–78.
