Research Article Chemical Composition and Disruption of ...downloads.hindawi.com/journals/ecam/2015/472593.pdf · Research Article Chemical Composition and Disruption of Quorum Sensing

Research ArticleChemical Composition and Disruption of Quorum SensingSignaling in Geographically Diverse United States Propolis

Michael A Savka1 Lucas Dailey1 Milena Popova2 Ralitsa Mihaylova2

Benjamin Merritt1 Marissa Masek1 Phuong Le1 Sharifah Radziah Mat Nor1

Muhammad Ahmad1 Andreacute O Hudson1 and Vassya Bankova2

1TheThomas H Gosnell School of Life Sciences College of Science Rochester Institute of Technology 85 Lomb Memorial DrA350 Gosnell Hall Rochester NY 14623 USA2Institute of Organic Chemistry with Centre of Phytochemistry Bulgarian Academy of Sciences Academic G Bonchev StreetBuilding 9 1113 Sofia Bulgaria

Correspondence should be addressed to Michael A Savka massbiritedu and Vassya Bankova bankovaorgchmbasbg

Received 7 December 2014 Revised 12 March 2015 Accepted 25 March 2015

Academic Editor Veronique Seidel

Copyright copy 2015 Michael A Savka 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

Propolis or bee glue has been used for centuries for various purposes and is especially important in human health due tomany of its biological and pharmacological properties In this work we showed quorum sensing inhibitory (QSI) activity often geographically distinct propolis samples from the United States using the acyl-homoserine lactone- (AHL-) dependentChromobacterium violaceum strain CV026 Based on GC-MS chemical profiling the propolis samples can be classified into severalgroups that are as follows (1) rich in cinnamic acid derivatives (2) rich in flavonoids and (3) rich in triterpenes An in-depthanalysis of the propolis from North Carolina led to the isolation and identification of a triterpenic acid that was recently isolatedfrom Hondurian propolis (Central America) and ethyl ether of p-coumaric alcohol not previously identified in bee propolis QSIactivity was also observed in the second group US propolis samples which contained the flavonoid pinocembrin in addition toother flavonoid compoundsThe discovery of compounds that are involved in QSI activity has the potential to facilitate studies thatmay lead to the development of antivirulence therapies that can be complementary andor alternative treatments against antibioticresistant bacterial pathogens andor emerging pathogens that have yet to be identified

1 Introduction

Propolis is a chemically complex substance collected byhoneybees from regional macroflora [1] Bees use propolis tostrengthen the hive to block holes and cracks in the hive andto protect the hive from invading insects and microorgan-ismsThe chemical constituents of propolis are related to budbark and wound exudates collected by bees from accessibleflora [2 3] As a result the chemical composition of propolisdepends on the local flora froma specific geographical regionPropolis is composed of resin and balsam wax essentialand aromatic oils pollen and other organic materials [45] Over 300 compounds have been identified in differentpropolis samples and it is accepted that the flavonoidsaromatic acids diterpenic acids and phenolic compounds act

as bioactive principles in different chemical types of propolis[6ndash8] Propolis has been used in human medicine sincethe 17th century due to its biological properties pertainingto antibacterial anticavity antitumor antioxidant antivi-ral anti-inflammatory and immunomodulatory effects inaddition to other beneficial properties [8 9] Propolis isincreasingly recognized as a factor in social immunity traitsof the honeybee and these traits are important for increasingadult longevity decreasing brood mortality and increasinghive productivity [10] A constituent of some propolis typespinocembrin has recently been shown to regulate immunegenes in the honey bee Apis mellifera [11] Thus propolis isa diverse and rich natural product source to search for noveltherapeutic compounds

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 472593 10 pageshttpdxdoiorg1011552015472593

2 Evidence-Based Complementary and Alternative Medicine

In bacteria the expression of certain bacterial genesincluding the virulome or the whole set of genes requiredfor virulence frequently depends on the cell density of thepopulation In one system this phenomenon termed quo-rum sensing (QS) is mediated by specific molecules calledN-acyl-homoserine lactone (AHL) QS signals also known asautoinducers [12ndash14] The specificity of AHLs is determinedby the acyl side chain length degree of its saturation and thepresence or lack of an oxo- or hydroxy-group in C3 position[15 16] AHLs are characterized as long- or short-chain AHLsdepending on whether their acyl moieties consist of gt8 orle8 carbon atoms respectively [16] Examples of bacterialphenotypes controlled by QS include conjugal transfer ofplasmids biofilm formation swarming motility virulencefactor expression bioluminescence pigment production andother traits [12 13] The QS mechanism in bacteria enablesthe regulation of gene expression and coordinated functionsbeneficial only when carried out by a large number ofbacterial cells for example high cell density

QS is important in host-microbial interactions inhumans marine systems and some phytopathogens ManyGram-negative bacterial species that use AHL-based QScontain a complete AHL QS regulatory circuit This includesthe QS core proteins known as LuxI-type protein (the AHLsynthase) and a LuxR-type protein (the response regulatoror receptor) [12 17] The manipulation of the regulationof QS may provide alternative and novel therapeuticapproaches against bacterial pathogens [18 19] Detailson the biochemical and molecular mechanism underlyingQS regulation and developing approaches to manipulateQS regulated behaviors in bacteria have recently beeninvestigated [18 20 21]

One approach tomitigate treatment towards bacteria thatare resistant to clinically relevant antibiotic is to develop newmethods of antipathogenic treatments that act to attenuatethe expression of disease progression (virulence) whichmay be less likely to impose a selection pressure for thedevelopment of bacterial resistance [19 22] A strategy todevelop novel antipathogenic treatments is by blocking thecell-to-cell communication mediated by QS systems Recentstudies have identified many natural and synthetic com-pounds as QS inhibitors (QSI) [18 22 23] The majorityof the QSI compounds have been shown to inhibit QSsignaling in screens using AHL-dependent biosensor strainsA preliminary screening for QS inhibitors revealed thatpropolis displayed a qualitative inhibitor activity based on asingle LuxR-based AHL-dependent biosensor strain [24]

In our previous work we investigated the effectsof commercially prepared propolis tinctures to affectQS-regulated responses using five different AHL-signal-dependent reporter strains each containing a differentLuxR-homolog receptor [25] We showed that propolissamples that differ in their region of origin chemical profileand absorption spectrum exhibit different QSI responses andthat these responses depend on the AHL-dependent receptorprotein

Studies investigating the chemical constituents and phy-totherapeutic compounds of propolis from hives in theUnited States are underrepresented in the literature [26 27]

Table 1 United States propolis samples

Sample ID Origin Geographic coordinatesLA-1 Baton Rouge LA 30∘22101584041410158401015840N 91∘09101584056210158401015840WNY-2 Beaver Dam NY 41∘2510158405010158401015840N 74∘0710158400910158401015840WNY-3 Dundee NY 42∘3110158402810158401015840N 76∘5810158402910158401015840WNE-4 Lincoln NE 41∘09101584051410158401015840N 96∘28101584057910158401015840WNV-5 Fallon Nevada 39∘2810158402210158401015840N 118∘4610158404410158401015840WPA-6 Millerton PA 41∘5110158405010158401015840N 76∘5710158401810158401015840WNC-7 Raleigh NC 35∘43101584028810158401015840N 78∘40101584033410158401015840WNY-8 Bloomfield NY 42∘5310158405710158401015840N 77∘2510158404710158401015840WMN-9 St Paul MN 44∘59101584027810158401015840N 93∘11101584017510158401015840WNY-10 Yates Co NY 42∘3910158403610158401015840N 77∘310158402010158401015840W

In this work we report antiquorum sensing activity inten propolis samples harvested from geographically diverseregions in theUnited StatesThe sampling locations representdistinct botanical characteristics and include samples fromthe cold North the wet Southeast and the dry Southwestregions of the United States Furthermore we determine thechemical profiles of each propolis provenience by GC-MSanalysis and characterized the propolis into three groups byprincipal component analysis Lastly we identified pinocem-brin a flavonoid from propolis which we show disruptingAHL-dependent QS in bacteria

2 Materials and Methods

21 Chemicals All common chemicals and solvents areanalytical reagent grade from worldwide recognized brandsBis(trimethylsilyl)-trifluoroacetamide (BSTFA) and Lobarprepacked column size A [LiChroprep Si 60 (40ndash63 120583m)]were purchased from Merck Polyamide 6 was purchasedfromFluka Pinocembrinwas isolated fromBulgarian propo-lis as described in our earlier work [28] Purified standardsof N-acyl-homoserine lactones (AHLs) QS signals werepurchased from Cayman Chemical Co (Ann Arbor MIUSA) Abbreviations forN-acyl homoserine lactones (AHLs)include the following C4-HSL N-butanoyl-homoserine lac-tone and 3-oxo-C12-HSL N-3-oxo-dodecanoyl homoserinelactone Bacto agar was purchased from VWR InternationalRadnor Pennsylvania The antibiotic gentamycin and otherchemicals were purchased from Sigma-Aldrich (St Louis)

22 Propolis Samples The sample identification geographicorigin and coordinates of the rawUnited States propolis usedin this study are provided (Table 1)

23 Propolis Sample Preparation forQuorumSensing BioassayFrozen propolis samples were ground to a fine powderand extracted with 70 ethanol by shaking for 24 hoursThe insoluble materials were removed by centrifugation at12000 g for 10 minutes at 4∘C The extracts were dilutedto 5 based on dry weight for AHL-based QS biosensorinvestigations as previously described by our group [25]

Evidence-Based Complementary and Alternative Medicine 3

A translucent zone around the cellulose discs indicate quo-rum sensing inhibition due to the reduction of the AHL-dependent violacein synthesis and this is in clear contrast toa transparent zone which indicates death via cell lysis in thewhole-cell biosensor strain CV026 as previously described inour work [16 25]

Raw Hungarian propolis (prepared in 70 EtOH extractand based on 5dryweight) andHungarian propolis tincture(30 commercial) as previously studied in our laboratorywere used as internal propolis standards [25] and bothpossess antiquorum sensing activity against CV026 as wellas four additional whole-cell biosensors that monitor AHL-regulated QS activity

24 CV026 Biosensor Strain in Reverse Bioassay The poten-tial of United States propolis to inhibit QS was tested in areverse bioassay using theChromobacterium violaceum strainCV026 In C violaceum strain CV026 the LuxR homologCviR regulates the production of a purple pigment violaceinwith exogenous short-chain (C4 to C8) alkanoyl or 3-oxo-alkanoyl side chain AHL [16 29] Violacein production inCV026 in the presence of short-chain AHLs is inhibited bythe presence of long-chain AHLs (C10 to C14) thus inhibitingviolacein production in the presence of the stimulator AHLin reverse bioassays (C6-HSL and C4-HSL) [15 24] Thisphenomenon allows the use of CV026 in reverse bioassayto identify compounds that disrupt AHL-mediated QS sig-naling In this bioassay the long-chain AHL 3-oxo-C12-HSL was used as a positive control and impregnated intodisc (2120583L of 1mM stock) to inhibit violacein production inthe presence of inducing concentrations of short-chain C4-HSL AHL as previously shown in our laboratory [16 25]The extinction coefficient of CV026 AHL produced violaceinhas been determined to be 005601mL 120583gminus1 cmminus1 [30] Theantibiotic gentamycin (disc impregnated with 10 120583g was usedto visualize biosensor death as a transparent zone of growthinhibition

25 Inhibition of Bacterial QS by Raw Propolis Collectedin the United States To test the effect of propolis extractson the AHL-dependent phenotype (pigment production) ofbiosensor CV026 cellulose discs of 6mm in diameter wereimpregnatedwith ethanol extracts of the ten propolis samplesfrom the United States placed on the surface of a soft agarplate seeded with strain CV026 induced with AHL C4-HSLand incubated at 28∘Covernight Eightmicroliters of propolisat a 5 preparation in 70 EtOHwas applied to the cellulosediscs Short-chain signal C4-HSL (final concentration of242 120583M) combined with CV026 was added to a soft agarplate as previously described by our group [25 29] Weused Hungarian propolis previously studied by our groupas internal standards in the bioassays since this propolissample has been characterized for the inhibition of bacterialQS responses using five different whole-cell biosensors thatcontained different LuxR AHL signal receptor proteins [25]The zone of inhibition was observed and measured as atranslucent zone directly adjacent to the cellulose disc [25]

26 Quantification of Violacein Pigmentation A 20mm discof the CV026 seeded soft agar medium below the discscontaining the propolis was harvested and the violaceinpigmentation was extracted andmeasured spectrophotomet-rically at 119860

585 nm as described by Blosser and Gray [31] Allexperiments were repeated at least three times

27 Extraction and Sample Preparation for GC-MS AnalysisSilylation was performed according to [32] In brief propolisgrated after cooling was extracted twice with 70 ethanol(1 10 w v) at room temperature for 24 h A part of theethanol extract (5mL) was evaporated to dryness About5mg of the extract was mixed with 50120583L of dry (water-free)pyridine and 75 120583L of bis(trimethylsilyl)-trifluoroacetamide(BSTFA) and heated at 80∘C for 20minThe silylated extractswere analysed by GC-MS

28 GC-MS Analysis and Identification of Compounds TheGC-MS analysis was performed with a Hewlett-Packard gaschromatograph 5890 series II Plus linked to a Hewlett-Packard 5972 mass spectrometer system (single quadrupole)equipped with a 30m long 025mm id and 05 120583m filmthickness HP5-MS capillary column (Hewlett-Packard) Thetemperature was programmed from 60 to 300∘C at a rateof 5∘Cmin and a 10min hold at 300∘C Helium was usedas a carrier gas at a flow rate of 08mLmin The splitratio was 1 10 the injector temperature 280∘C the interfacetemperature 300∘C and the ionization voltage (EI) 70 eV119898119911range 50ndash700

The identification of individual compounds by GC-MSwas performed using computer searches on commerciallibraries comparison with spectra of authentic samplesand literature data If no reference spectra were availableidentification was performed based on the mass-spectralfragmentation in such cases for some compounds only ten-tative structures were proposed Some constituents remainedunidentified because of the lack of relevant references andinformation (all of them constituted minor percentage ofTIC)

29 NMR Experiments One- and two-dimensional NMRspectra (1H- 13C- DEPT HSQC and HMBC) were taken onBruker AV 600 in CDCl

3

210 Isolation of Individual Compounds Individual com-pounds were isolated from the sample NC-7 (North Car-olina) The total 70 ethanol extract was concentratedand extracted successively with petrol ether (3x) and ethylacetate (3x) The ethyl acetate extract was evaporated to yield804 g dry extract which was subjected to vacuum liquidchromatography on polyamide 6 eluted with chloroform-methanol-ethyl methyl ketone (20 2 1 to 20 12 6) Sixteenfractions were obtained Fraction 3 (299 g) eluted withchloroform-methanol-ethyl methyl ketone (20 4 2) wasrechromatographed on a column with polyamide 6 usingchloroform-ethyl acetate (1 to 100) as a mobile phaseand 19 fractions were obtained Fraction 3 (1874mg) wassubjected to column chromatography on silica gel (Lobar)


NY-3 NE-4 NV-5 PA-6 NY-8NC-7 EtOH Gm Hg 3OC12

(a)

Propolis samples

70

E

tOH

3-O

-C12 3 4 6 8 5 7

120140

100806040200

25

20

15

10

5

0

Hun

garia

n

Solid bars violacein inhibition in millimeters (left axis)Stripped bars violacein inhibition as percentage ofHungarian (right axis)

(b)

Propolis samples

08

06

04

02

00

Back

grou

nd

70

E

tOH

3-O

-C12 3 4 6 8 5 7

120100806040200

Solid bar violacein absorbance at OD at 585nanometer (left axis)Stripped bar percent of background pigmentation (right axis)

(c)

Figure 1 Inhibition of AHL-dependent violacein synthesis in C violaceum strain CV026 in the presence of United States propolis (a)Inhibition of AHL-regulated violacein synthesis in CV026 by the selected propolis in the disc diffusion assay Abbreviations include thefollowing 3OC12 positive control disc impregnatedwith long-chain 3-oxo-C12-HSLAHL signalOthers include the followingHg an internalstandard the Hungarian raw propolis to visualize violacein synthesis inhibition as a translucent zone adjacent to the disc and as previouslyreported by our laboratory [25] EtOH pure solvent of 70 ethyl alcohol (control) andGm antibiotic gentamycin to visualize biosensor deathas a transparent zone adjacent to the disc All experiments were performed in triplicate and (a) are a representative result of one replication (b)Quorum sensing inhibition (QSI) by propolis samples inmillimeters indicated by a translucent zone of violacein synthesis across the cellulosedisc Data presented is mean plusmn standard deviation Data is also presented as percent of the Hungarian raw propolis (internal standard) (Hg)(c) Quantification of violacein pigment synthesis inhibition in CV026 after treatment with propolis This data is presented as percent ofviolacein present in the soft agar plate background (positive control full induction of violacein synthesis) Using the propolis samples noCV026 biosensor growth inhibition (only observed in the antibiotic gentimycin containing disc) was observed as identified by transparentzone around the cellulose disc All experiments were repeated at least three times with representative data of one experiment shown

and eluted with chloroform-ethyl acetate (1 to 100) and20 fractions were obtained Fraction 5 yielded 22mg ofcinnamyl-p-coumarate [33] and 69mg benzyl-p-coumarate[33] Fraction 7 yielded 55mg of (E)-4-(31015840-ethoxyprop-11015840-enyl) phenol (ethyl ether of p-coumaric alcohol) [34] andfraction 15 gave 166mg of 3-oxo-6120573-hydroxy-lup-20(29)-en-28-oic acid [33]

211 Statistical Analysis Multivariate analysis of propolischemical profiles was performed by PCA using the GCMSdata for groups of identified compounds expressed as apercentage of the TIC respectively Statistica Version 80was used for the analysis For biological tests data werestatistically analyzed using SAS (version 91 SAS InstituteInc Cary NC USA) All experiments were repeated at leastthree times

3 Results and Discussion

31 QS Inhibitory (QSI) Activity of US Propolis Samples Weassessed whether propolis collected from different regionsof the United States can disrupt the AHL-dependent QSresponse in biosensor CV026 All ten proveniences were

initially screened for QS inhibitory (QSI) activity usingthe CviR-based (LuxR-receptor) AHL-dependent biosensorstrain in reverse bioassays We then selected six proveniencesbased on initial zone of inhibition responses in the reversebioassay with CV026 and availability of sample (Table 2)

The selected proveniences include the following NY-3NE-4 NV-5 PA-6 NC-7 and NY-8 (Figure 1) We quanti-fied these six samples for their potential to disrupt AHL-dependentQS communication using theC violaceumCV026and propolis-containing cellulose discs (1) to determine thesize of the diffusion zone of inhibition (Figure 1(b)) and (2)to measure the amount of inhibition of the synthesis of theQS-regulated trait in CV026 violacein pigment production(Figure 1(c)) The differences between selected propolis sam-ples are highly significant (119875 lt 001) When compared tonegative control (70EtOH) all six samples had significantlylarger zone of pigment inhibition and when compared topositive control (pure long-chain AHL 3-O-C12) all treat-ments had significantly smaller zones of pigment inhibitionThe NE-4 PA-6 and NY-8 propolis samples showed thelargest zones of pigment inhibition adjacent to the propolisimpregnated discs (Figure 1(a)) which were between 70 and80 of the zone of inhibition observed with the pure long-chain 3-oxo-C12-HSL signal (positive control) Compared to


Table 2 Quorum sensing inhibitory (QSI) activity of United States propolis samples

Propolis origin sample ID QSI activityZOI (mm)a

Used in additionalQSI bioassays

Baton Rouge LA (LA-1) 7 NoBeaver Dam NY (NY-2) 7 NoDundee NY (NY-3) 10 YesLincoln NE (NE-4) 11 YesFallon NV (NV-5) 10 YesMillerton PA (PA-6) 13 YesRaleigh NC (NC-7) 14 YesBloomfield NY (NY-8) 14 YesSt Paul MN (MN-9) 10 NoYates Co NY (NY-10) 7 NoInternal standardHungarian (raw prepared by our group and previously reported [24]) 12 Yes

Internal standardHungarian (tincture commercial and reported in [24]) 14 No

Positive controllong-chain AHL signal 3-oxo-C12-HSL (1 uL of 1mM)

17 QSI zone(translucent)b Yes

AntibioticGentamycin (2 uL of 50mgmL)

14 death zone(transparent)b Yes

aZOI zone of violacein synthesis inhibition as identified by a translucent halo adjacent to the 6 mm cellulose discbA transparent zone of inhibition around the disc indicates death of the biosensor strain CV026 due to cell lysis whereas a translucent zone indicates inhibitionof violacein synthesis in the AHL-dependent QS response in whole-cell biosensor CV026

Table 3 Compound types identified in ethanolic extracts by GC-MS ( of TIC)

Compound type LA-1 NY-2 NY-3 NE-4 NV-5 PA-6 NC-7 NY-8 MN-9 NY-10Simple phenols and benzoic acid derivatives 19 157 124 04 02 25 05 19 131 166Cinnamic acid derivatives 92 372 286 32 13 89 62 92 525 366Fatty acids 03 11 03 02 02 07 0 18 0 17Chalcones 52 68 45 182 82 72 99 60 25 34Flavanones and dihydroflavonols 130 84 118 366 230 275 229 183 32 89Flavones and flavonols 58 84 129 246 164 247 158 138 46 78Phenolic glycerides 0 01 01 0 0 0 0 0 36 02Triterpenes 209 0 0 0 69 06 115 04 0 07Unknowns 0 0 0 0 80 0 92 14 0 0

the internal propolis standard the Hungarian raw propolisthese three US samples were between 94 and 106 of theinternal standardrsquos pigment inhibition zone (Figure 1(b))

The violacein was extracted from the soft agar samplesof the zones of inhibition and quantified (Figure 1(b)) Theamounts of pigment differed significantly (119875 lt 001) forall propolis extracts when compared to the backgroundpigmentation of randomly selected regions of the CV026-seeded soft agar distal to cellulose discs When violaceinamounts were compared to the background pigmentationall extracts except the 70 EtOH (control) were significantlylower indicating disruption of violacein synthesis in the zoneunder and adjacent to the disc containing propolis Thesix propolis samples ranged from 18 to 43 in comparisonto the background pigmentation (100) and 16 for thepositive control pure long-chain 3-oxo-C12-HSL AHL signal(Figure 1(c))

32 Chemical Profiles of US Propolis Samples The chemi-cal composition of all ten propolis samples from differentregions of the United States (the States New York NevadaPennsylvania North Carolina and Louisiana) was analyzedby GC-MS after silylation Over 60 individual compoundswere identified Distinct chemical profiles were observed forsamples fromdifferent location in accordancewith the recentfindings of Wilson et al [10] The chemical composition ispresented bymeans of themain type of compounds identified(Table 3) and is obviously qualitatively and quantitativelyvariable Detailed data about the percentage of the totalion current (TIC) in the mass chromatogram for individualconstituents can be found in Table S1 (supplementary data)(see Table S1 in Supplementary Material available onlineat httpdxdoiorg1011552015472593) In all samples ana-lyzed aromatic acids and their esters flavonoids and chal-cones were found while the presence of triterpenes was

6 Evidence-Based Complementary and Alternative MedicineFa

ctor

219

41

30252015100500

minus05minus10minus15minus20minus25

Factor 1 5250minus6 minus5 minus4 minus3 minus2 minus1 0 1 2 3 4 5

Projection of the cases on the factor-plane (1 times 2)

9

3

210

17

5

8 6 4

Cases with sum of cosine square ge 000

Figure 2 Principal component analysis (PCA) of the main classesof compounds identified The numbers correspond to the samplenumbers in Table 1

detected only in six samples The triterpenes are compoundsfound in propolis from tropical and subtropical regions andhave not been found in North American propolis samples

Because of the complex chemical composition of thesamples the chemometric approach principal componentanalysis (PCA) was applied using the relative amounts of themain classes of compounds (percentage of TIC) from theGC-MS analysis The obtained two-dimensional plot covers 72of the total variation (Figure 2) Based on the PCA resultsthree groups of propolis samples were distinguished one richof cinnamic acid derivatives (group I samples NY-2 NY-3MN-9 and NY-10) a second group with high concentrationof flavonoids (group II samples NE-4 PA-6 and NY-8) anda third group characterized by relatively high concentrationof triterpenes (group III samples LA-1 NV-5 and NC-7)It is obvious that the samples from the first two groupsoriginate predominately from Northern American statesSuch differentiation is not surprising because in differentclimatic and geographical regions the bees collect resins fromdifferent plants

The phenolic compounds identified in all samples (phe-nolic acids esters and flavonoids) are known propolis con-stituents found in poplar type propolis the most widelydistributed propolis type in the temperate regions [35ndash37]This indicates that one of the propolis plant sources isrepresentatives of genus Populus In North America poplarsfrom section Aigeiros Tacamahaca and Leuce are widely dis-tributed The major constituents of group I propolis samples(NY-2 NY-3MN-9 andNY-10) are aromatic acids and esterssuch as benzoic cinnamic p-coumaric and ferulic acids andbenzyl-p-coumarate The high percent of aromatic acids andlow concentration of flavonoids are characteristic for thebud exudates of Populus spp of section Leuce [38] such asPopulus tremula (aspen) in Europe and Populus tremuloidesMichx (American aspen) in North America P tremuloidesis native to cooler areas of North America regions where

the samples NY-2 NY-3 MN-9 and NY-10 were collectedThe American aspen is shown as a source of the Canadianpropolis [32] and is the possible botanical source of thesamples from Minnesota and New York The sample fromMinnesota is the only one containing significant amountsof phenolic glycerides These compounds have been foundin propolis from Russia and Switzerland [38 39] They aretypical constituents of bud exudates of poplars belonging tothe section Leuce [40] Their mass spectra (of the silylatedcompounds) are highly characteristic and can be successfullyused for positive identification [39 40]

The propolis samples from group II (NE-4 PA-6 andNY-8) were characterized by poplar bud flavonoids suchas pinocembrin pinobanksin pinobanksin-3-O-acetatechrysin galangin and pinocembrin chalcone The aromaticacids and esters were minor constituents This chemicalprofile is typical for resins of poplar buds from sectionAigeiros [41] and Populus fremontii is the most probableplant source Recently the American poplars from boththe Tacamahaca (P trichocarpa and P balsamifera) andthe Aigeiros sections (P fremontii P deltoides and P alba)were identified as sources of propolis from some Americanstates (Oregon and California Minnesota) [27 42] Thisgroup (NE-4 PA-6 and NY-8) contained the samples thatdemonstrated the most pronounced effect on the QS asseen in Figure 1 They displayed high concentrations offlavonoid aglycones (flavones flavanones and chalcones)This fact supports recent findings flavonoids and especiallyflavanones have been shown to interfere with quorumsensing and reduce the expression of several QS-controlledgenes [43]

Group III was characterized by significant amounts oftriterpenes Samples NV-5 and NC-7 in the third group arerich in flavonoids as well However the high amounts oftriterpenesmight interfere with the effect of the flavonoids onQS Poplars from Aigeiros section are the main source of thepropolis from the third group Nevertheless the considerableamounts of triterpenes found in these samples indicatedparticipation of other source plants Among the triterpenesoleanolic and oleanonic acids were the major constituentsin the samples from coastal regions whereas in the samplefrom Nevada (NV-5) a series of lupane type triterpenes weredetected

The samples from Pennsylvania (PA-6) and New York(NY-8 and NY-10) also contained triterpenes but theiramount was low (less than 1 of TIC) and there the ldquotriter-penerdquo source is obviously minor

33 Isolation of Individual Compounds from Triterpene Con-taining Propolis Among the samples analyzed the one fromNorth Carolina was a typical representative of a mixed typepropolis with triterpenes as important compounds Thissample was chosen for further analysis in order to isolate andidentify some of its bioactive constituents From the ethylacetate extract after using vacuum liquid and column chro-matography four known natural compounds were isolatedThey were identified by comparison of their spectra withliterature data By comparison of 1H-NMR and mass spectra


Figure 3The flavonoid pinocembrin which is present in the second groupUnited States propolis confers QSI activity Abbreviations includethe following 3OC12 pure 3-oxo-C12 acyl-homoserine lactone (positive control) Pinocembrin ldquoEuropean type propolisrdquo fraction containingpinocembrin EtOH ethyl alcohol EtOAc ethyl acetate

with published spectra cinnamyl-p-coumarate [32] benzyl-p-coumarate [32] and ethyl ether of p-coumaric alcohol [(E)-4-(31015840-ethoxyprop-11015840-enyl)phenol] [33] were identified Thetriterpene 3-oxo-6120573-hydroxy-lup-20(29)-en-28-oic acid wasidentified by analysis of its 1H- 13C DEPT HMBC andHSQC NMR spectra and comparison of 1H- and 3C-NMRspectra with literature data [44] as shown in Figure 4 Theethyl p-coumaryl ether is a new propolis constituent and thetriterpenic acid has been isolated from Hondurian propolis(Central America) [32] as shown in Figure 4

It is interesting to note that the chemical compositionof propolis from group III is somewhat similar to propolisfrom Honduras Lotti et al [32] isolated a series of cinnamicacid derivatives from Hondurian propolis as well as fla-vanones and triterpenes The tree Liquidambar styraciflua L(Honduras styrax) is suggested as a botanical source of thesepropolis constituents Among the isolated compounds 3-oxo-6120573-hydroxy-lup-20(29)-en-28-oic acid benzyl p-coumarateand cinnamyl cinnamatewere identified asmajor compoundsin Hondurian propolis On the other hand the balsam ofL styraciflua is characterized by predomination of cinnamylcinnamate and the above-mentioned triterpenic acid is oneof themajor triterpenes in the cones of L styraciflua All theseconstituents were detected in high relative concentrations inthe samples fromTheUnited States LA andNC As L styraci-flua is distributed in south and east regions ofNorthAmericait is possible that it plays a role as a secondary plant sourceof the samples analyzed The sample from NV although itcontains triterpenes has different triterpenic profile and lacksthe cinnamyl derivatives typical for Liquidambar Perhapssome other plants are the source of their triterpenes Ingeneral the search for the source plants in the USA requiresfurther studies

34 Quorum Sensing Inhibitory (QSI) Activity of PinocembrinThe United States group II propolis displayed QSI activity(Figure 1) and also contained poplar flavonoids including

pinocembrinHaving previously purified a pinocembrin frac-tion from ldquoEuropean typerdquo propolis we tested the pinocem-brin fraction for QSI activity in disc diffusion reverse bioas-say using AHL-dependent biosensor CV026 as previouslydescribedThepinocembrin fraction from the European-typepoplar propolis displayed a translucent zone of inhibitionaround the disc indicating inhibition of the AHL-dependentQS violacein synthesis consistent with pinocembrin exhibit-ing QSI activity (Figure 3) The translucent zone is similarin extent of response when compared to the positive controldisc containing pure long-chain AHL 3-oxo-C12 homoserinelactone (3OC12) This is in contrast to the two solvents usedethyl alcohol (EtOH) and ethyl acetate (EtOAc) that did notdisplay QSI activity (Figure 3) Pinocembrin has been testedin mice and rats and appears not to be toxic up to levels of 40and 100mg kgminus1 dayminus1 [45ndash47]

4 Conclusions

In this study we analyzed 10 propolis samples from differentgeographic regions of the United States for QSI activitythat disrupts QS AHL bacterial communication mechanismcorrelated the QSI activity of propolis with its chemicalcomposition and identified the poplar flavonoid pinocem-brin as a potential propolis active principle that disruptsAHL-dependent QS in bacteria There are indications thatadditional flavonoids are important propolis constituents inthis respect It is obvious that propolis from theUSA deservesfurther studies as a promising source of compounds andcompound mixtures in the search for new approaches forantipathogenic treatments of bacterial pathogens based onnatural products

Conflict of Interests

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


HO

OO

COOH

OH

(E)-4-(3998400-Ethoxyprop-1998400-enyl)phenol 3-Oxo-6120573-hydroxy-lup-20(29)-en-28-oic acid

Figure 4

Authorsrsquo Contribution

This study was conceived directed and coordinated byVassya Bankova and Michael A Savka Vassya BankovaMuhammad Ahmad Milena Popova and Andre O Hudsonwrote and revised the paper Lucas Dailey Benjamin MerrittandMarissa Masek participated as an undergraduate studentin the Biotechnology program at Rochester Institute ofTechnology during the study by carrying out the experimentsunder the direction ofMichael A Savka Chemical separationand identification studies were performed by Milena Popovaand Ralitsa Mihaylova Vassya Bankova and Michael ASavka assisted in the overall data interpretation PhuongLe performed statistical analyses on the data sets underthe direction of Michael A Savka All the authors readand approved the final paper Michael A Savka and VassyaBankova contributed equally to the work and should beconsidered as cocorresponding authors

Acknowledgments

The authors thankMichael Simone-Finstrom then in the Spi-vak Lab at the University ofMinnesota for providing theMNNE LA NV and NC raw propolis samples Lucas Dailey andBenjaminMerritt are currently enrolled in the Biotechnologyand Molecular Bioscience program at Rochester Institute ofTechnology (RIT) and were supported by the College ofScience at RIT Sharifah Radziah Mat Nor and MuhammadAhmad graduated in the Biotechnology Program at RITMarissa Masek graduated from the College of Science at RITin 2014The rawHungarian propolis and tincture preparationwas kindly provided by Erno Szegedi Research Institute forViticulture and Enology Kecskemet PO Box 25 H-6001Hungary Michael A Savka and Andre O Hudson wish tothank theThomasH Gosnell School of Life Sciences Collegeof Science at RIT for the support of this work throughFaculty Evaluation and Development (FEAD) 2013 and 2014awards

References

[1] J M Sforcin and V Bankova ldquoPropolis is there a potential forthe development of new drugsrdquo Journal of Ethnopharmacologyvol 133 no 2 pp 253ndash260 2011

[2] G A Burdock ldquoReview of the biological properties and toxicityof bee propolis (Propolis)rdquo Food and Chemical Toxicology vol36 no 4 pp 347ndash363 1998

[3] E L Ghisalberti ldquoPropolis a reviewrdquo BeeWorld vol 60 pp 59ndash84 1979

[4] V S Bankova S L de Castro and M C Marcucci ldquoPropolisrecent advances in chemistry and plant originrdquo Apidologie vol31 no 1 pp 3ndash15 2000

[5] V Bankova ldquoChemical diversity of propolis and the problem ofstandardizationrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 114ndash117 2005

[6] S Castaldo and F Capasso ldquoPropolis an old remedy used inmodern medicinerdquo Fitoterapia vol 73 supplement 1 pp S1ndashS62002

[7] S Silici and S Kutluca ldquoChemical composition and antibac-terial activity of propolis collected by three different races ofhoneybees in the same regionrdquo Journal of Ethnopharmacologyvol 99 no 1 pp 69ndash73 2005

[8] M Viuda-Martos Y Ruiz-Navajas J Fernandez-Lopez andJ A Perez-Alvarez ldquoFunctional properties of honey propolisand royal jellyrdquo Journal of Food Science vol 73 no 9 pp R117ndashR124 2008

[9] V D Wagh ldquoPropolis a wonder bees product and its pharma-cological potentialsrdquo Advances in Pharmacological Sciences vol2013 Article ID 308249 11 pages 2013

[10] M Wilson D Brinkman M Spivak G Gardner and J CohenldquoRegional variation in composition and antimicrobial activity ofUS propolis against Paenibacillus larvae and Ascosphaera apisrdquoJournal of Invertebrate Pathology vol 124 pp 44ndash50 2015

[11] W Mao M A Schuler and M R Berenbaum ldquoHoney con-stituents up-regulate detoxification and immunity genes in thewestern honey bee Apis melliferardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no22 pp 8842ndash8846 2013


[12] S Atkinson and P Williams ldquoQuorum sensing and socialnetworking in the microbial worldrdquo Journal of the Royal SocietyInterface vol 6 no 40 pp 959ndash978 2009

[13] C A Lowery T J Dickerson and K D Janda ldquoInterspecies andinterkingdom communication mediated by bacterial quorumsensingrdquo Chemical Society Reviews vol 37 no 7 pp 1337ndash13462008

[14] C E White and S C Winans ldquoCell-cell communication inthe plant pathogen Agrobacterium tumefaciensrdquo PhilosophicalTransactions of the Royal Society B Biological Sciences vol 362no 1483 pp 1135ndash1148 2007

[15] A Camilli and B L Bassler ldquoBacterial small-molecule signalingpathwaysrdquo Science vol 311 no 5764 pp 1113ndash1116 2006

[16] R A Scott J Weil P T le et al ldquoLong- and short-chainplant-produced bacterial N-acyl-homoserine lactones becomecomponents of phyllosphere rhizophere and soilrdquo MolecularPlant-Microbe Interactions vol 19 no 3 pp 227ndash239 2006

[17] R J Case M Labbate and S Kjelleberg ldquoAHL-driven quorum-sensing circuits their frequency and function among theProteobacteriardquo The ISME Journal vol 2 no 4 pp 345ndash3492008

[18] S Dobretsov M Teplitski and V Paul ldquoMini-review quorumsensing in the marine environment and its relationship tobiofoulingrdquo Biofouling The Journal of Bioadhesion and BiofilmResearch vol 25 no 5 pp 413ndash427 2009

[19] T B Rasmussen and M Givskov ldquoQuorum-sensing inhibitorsas anti-pathogenic drugsrdquo International Journal of MedicalMicrobiology vol 296 no 2-3 pp 149ndash161 2006

[20] D McDougald S A Rice and S Kjelleberg ldquoBacterial quorumsensing and interference by naturally occurring biomimicsrdquoAnalytical and Bioanalytical Chemistry vol 387 no 2 pp 445ndash453 2007

[21] S A Rice D McDougald N Kumar and S Kjelleberg ldquoTheuse of quorum-sensing blockers as therapeutic agents for thecontrol of biofilm-associated infectionsrdquo Current Opinion inInvestigational Drugs vol 6 no 2 pp 178ndash184 2005

[22] DA Rasko andV Sperandio ldquoAnti-virulence strategies to com-bat bacteria-mediated diseaserdquoNature Reviews Drug Discoveryvol 9 no 2 pp 117ndash128 2010

[23] T B Rasmussen and M Givskov ldquoQuorum sensing inhibitorsa bargain of effectsrdquo Microbiology vol 152 no 4 pp 895ndash9042006

[24] T B Rasmussen T Bjarnsholt M E Skindersoe et al ldquoScreen-ing for quorum-sensing inhibitors (QSI) by use of a novelgenetic system the QSI selectorrdquo Journal of Bacteriology vol187 no 5 pp 1799ndash1814 2005

[25] Z Bulman P Le A O Hudson and M A Savka ldquoA novelproperty of propolis (bee glue) anti-pathogenic activity byinhibition of N-acyl-homoserine lactone mediated signaling inbacteriardquo Journal of Ethnopharmacology vol 138 no 3 pp 788ndash797 2011

[26] K S Johnson F A Eischen and D E Giannasi ldquoChemicalcomposition of North American bee propolis and biologicalactivity towards larvae of greater wax moth (LepidopteraPyralidae)rdquo Journal of Chemical Ecology vol 20 no 7 pp 1783ndash1791 1994

[27] A Aliboni ldquoPropolis from Northern California and Oregonchemical composition botanical origin and content of aller-gensrdquoZeitschrift fur Naturforschung C vol 69 no 1-2 pp 10ndash202014

[28] K Bilikova M Popova B Trusheva and V Bankova ldquoNewanti-Paenibacillus larvae substances purified from propolisrdquoApidologie vol 44 no 3 pp 278ndash285 2013

[29] K H McClean M K Winson L Fish et al ldquoQuorum sensingand Chromobacterium violaceum exploitation of violacein pro-duction and inhibition for the detection of N-acylhomoserinelactonesrdquoMicrobiology vol 143 no 12 pp 3703ndash3711 1997

[30] A S Mendes J E de Carvalho M C T Duarte N Duranand R E Bruns ldquoFactorial design and response surface opti-mization of crude violacein for Chromobacterium violaceumproductionrdquoBiotechnology Letters vol 23 no 23 pp 1963ndash19692001

[31] R S Blosser and K M Gray ldquoExtraction of violacein fromChromobacterium violaceum provides a new quantitative bioas-say for N-acyl-homoserine lactone autoiducersrdquo Journal ofMicrobiological Methods vol 40 no 1 pp 47ndash55 2000

[32] R Christov B Trusheva M Popova V Bankova and MBertrand ldquoChemical composition of propolis from Canada itsantiradical activity and plant originrdquo Natural Product Researchvol 20 no 6 pp 531ndash536 2006

[33] C Lotti A L Piccinelli C Arevalo et al ldquoConstituents ofHondurian propolis with inhibitory effects on Saccharomycescerevisiae multidrug resistance protein Pdr5prdquo Journal of Agri-cultural and Food Chemistry vol 60 no 42 pp 10540ndash105452012

[34] N Sukhirun W Pluempanupat V Bullangpoti and OKoul ldquoBioefficacy of Alpinia galanga (Zingiberaceae) rhizomeextracts (E)-p- acetoxycinnamyl alcohol and (E)-p-coumarylalcohol thyl ether against Bactrocera dorsalis (Diptera Tephri-tidae) and the impact on detoxification enzyme activitiesrdquoJournal of Economic Entomology vol 104 no 5 pp 1534ndash15402011

[35] V S Bankova S S Popov and N L Marekov ldquoHigh-performance liquid chromatographic analysis of flavonoidsfrom propolisrdquo Journal of Chromatography A vol 242 no 1 pp135ndash143 1982

[36] V Bankova B Trusheva and M Popova ldquoNew developmentsin propolis chemical diversity studies (since 2000)rdquo in ScientificEvidence of Use of Propolis in Ethnomedicine N Orsolichand I Basic Eds pp 1ndash13 Transworld Research NetworkTrivandrum India 2008

[37] V Bankova and L Kuleva ldquoPhenolic compounds in propolisfrom different regions in Bulgariardquo Animal Sciences vol 1989no 2 pp 94ndash98 1989 (Bulgarian)

[38] S A Popravko I V Sokolov and I V Torgov ldquoNew naturalphenolic triglyceridesrdquo Khimiya Prirodnykh Soedinenii vol1982 no 2 pp 169ndash173 1982 (Russian)

[39] V Bankova M Popova S Bogdanov and A G SabatinildquoChemical composition of European propolis expected andunexpected resultsrdquo Zeitschrift fur Naturforschung C vol 57 no5-6 pp 530ndash533 2002

[40] V A Isidorov M Brzozowska U Czyzewska and L GlinkaldquoGas chromatographic investigation of phenylpropenoid glyc-erides from aspen (Populus tremula L) budsrdquo Journal ofChromatography A vol 1198-1199 no 1-2 pp 196ndash201 2008

[41] S English W Greenaway and F R Whatley ldquoAnalysis ofphenolics in the bud exudates of Populus deltoides P fremontiiP sargentii and P wislizenii by GC-MSrdquo Phytochemistry vol 31no 4 pp 1255ndash1260 1992

[42] M B Wilson M Spivak A D Hegeman A Rendahl and JD Cohen ldquoMetabolomics reveals the origins of antimicrobial


plant resins collected by honey beesrdquo PLoS ONE vol 8 no 10Article ID e77512 2013

[43] F Nazzaro F Fratianni and R Coppola ldquoQuorum sensing andphytochemicalsrdquo International Journal of Molecular Sciencesvol 14 no 6 pp 12607ndash12619 2013

[44] M Kuroyanagi M Shiotsu T Ebihara H Kawai A Uenoand S Fukushima ldquoChemical studies on Viburnum awabuki KKOCHrdquoChemical and Pharmaceutical Bulleti vol 34 no 10 pp4012ndash4017 1986

[45] R Liu J-Z Li J-K Song et al ldquoPinocembrin protects humanbrain microvascular endothelial cells against fibrillar amyloid-1205731minus40

injury by suppressing the MAPKNF-120581B inflammatorypathwaysrdquo BioMed Research International vol 2014 Article ID470393 14 pages 2014

[46] R Liu C-X Wu D Zhou et al ldquoPinocembrin protectsagainst 120573-amyloid-induced toxicity in neurons throughinhibiting receptor for advanced glycation end products(RAGE)-independent signaling pathways and regulatingmitochondrion-mediated apoptosisrdquo BMC Medicine vol 10article 105 21 pages 2012

[47] S Charoensin C Punvittayagul W Pompimon U Mevateeand R Wongpoomchai ldquoToxicological and clastogenic evalu-ation of pinocembrin and pinostrobin isolated from Boesenber-gia pandurata inWistar ratsrdquoThai Journal of Toxicology vol 25no 1 pp 29ndash40 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



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OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


In bacteria the expression of certain bacterial genesincluding the virulome or the whole set of genes requiredfor virulence frequently depends on the cell density of thepopulation In one system this phenomenon termed quo-rum sensing (QS) is mediated by specific molecules calledN-acyl-homoserine lactone (AHL) QS signals also known asautoinducers [12ndash14] The specificity of AHLs is determinedby the acyl side chain length degree of its saturation and thepresence or lack of an oxo- or hydroxy-group in C3 position[15 16] AHLs are characterized as long- or short-chain AHLsdepending on whether their acyl moieties consist of gt8 orle8 carbon atoms respectively [16] Examples of bacterialphenotypes controlled by QS include conjugal transfer ofplasmids biofilm formation swarming motility virulencefactor expression bioluminescence pigment production andother traits [12 13] The QS mechanism in bacteria enablesthe regulation of gene expression and coordinated functionsbeneficial only when carried out by a large number ofbacterial cells for example high cell density

QS is important in host-microbial interactions inhumans marine systems and some phytopathogens ManyGram-negative bacterial species that use AHL-based QScontain a complete AHL QS regulatory circuit This includesthe QS core proteins known as LuxI-type protein (the AHLsynthase) and a LuxR-type protein (the response regulatoror receptor) [12 17] The manipulation of the regulationof QS may provide alternative and novel therapeuticapproaches against bacterial pathogens [18 19] Detailson the biochemical and molecular mechanism underlyingQS regulation and developing approaches to manipulateQS regulated behaviors in bacteria have recently beeninvestigated [18 20 21]

One approach tomitigate treatment towards bacteria thatare resistant to clinically relevant antibiotic is to develop newmethods of antipathogenic treatments that act to attenuatethe expression of disease progression (virulence) whichmay be less likely to impose a selection pressure for thedevelopment of bacterial resistance [19 22] A strategy todevelop novel antipathogenic treatments is by blocking thecell-to-cell communication mediated by QS systems Recentstudies have identified many natural and synthetic com-pounds as QS inhibitors (QSI) [18 22 23] The majorityof the QSI compounds have been shown to inhibit QSsignaling in screens using AHL-dependent biosensor strainsA preliminary screening for QS inhibitors revealed thatpropolis displayed a qualitative inhibitor activity based on asingle LuxR-based AHL-dependent biosensor strain [24]

In our previous work we investigated the effectsof commercially prepared propolis tinctures to affectQS-regulated responses using five different AHL-signal-dependent reporter strains each containing a differentLuxR-homolog receptor [25] We showed that propolissamples that differ in their region of origin chemical profileand absorption spectrum exhibit different QSI responses andthat these responses depend on the AHL-dependent receptorprotein

Studies investigating the chemical constituents and phy-totherapeutic compounds of propolis from hives in theUnited States are underrepresented in the literature [26 27]

Table 1 United States propolis samples

Sample ID Origin Geographic coordinatesLA-1 Baton Rouge LA 30∘22101584041410158401015840N 91∘09101584056210158401015840WNY-2 Beaver Dam NY 41∘2510158405010158401015840N 74∘0710158400910158401015840WNY-3 Dundee NY 42∘3110158402810158401015840N 76∘5810158402910158401015840WNE-4 Lincoln NE 41∘09101584051410158401015840N 96∘28101584057910158401015840WNV-5 Fallon Nevada 39∘2810158402210158401015840N 118∘4610158404410158401015840WPA-6 Millerton PA 41∘5110158405010158401015840N 76∘5710158401810158401015840WNC-7 Raleigh NC 35∘43101584028810158401015840N 78∘40101584033410158401015840WNY-8 Bloomfield NY 42∘5310158405710158401015840N 77∘2510158404710158401015840WMN-9 St Paul MN 44∘59101584027810158401015840N 93∘11101584017510158401015840WNY-10 Yates Co NY 42∘3910158403610158401015840N 77∘310158402010158401015840W

In this work we report antiquorum sensing activity inten propolis samples harvested from geographically diverseregions in theUnited StatesThe sampling locations representdistinct botanical characteristics and include samples fromthe cold North the wet Southeast and the dry Southwestregions of the United States Furthermore we determine thechemical profiles of each propolis provenience by GC-MSanalysis and characterized the propolis into three groups byprincipal component analysis Lastly we identified pinocem-brin a flavonoid from propolis which we show disruptingAHL-dependent QS in bacteria

2 Materials and Methods

21 Chemicals All common chemicals and solvents areanalytical reagent grade from worldwide recognized brandsBis(trimethylsilyl)-trifluoroacetamide (BSTFA) and Lobarprepacked column size A [LiChroprep Si 60 (40ndash63 120583m)]were purchased from Merck Polyamide 6 was purchasedfromFluka Pinocembrinwas isolated fromBulgarian propo-lis as described in our earlier work [28] Purified standardsof N-acyl-homoserine lactones (AHLs) QS signals werepurchased from Cayman Chemical Co (Ann Arbor MIUSA) Abbreviations forN-acyl homoserine lactones (AHLs)include the following C4-HSL N-butanoyl-homoserine lac-tone and 3-oxo-C12-HSL N-3-oxo-dodecanoyl homoserinelactone Bacto agar was purchased from VWR InternationalRadnor Pennsylvania The antibiotic gentamycin and otherchemicals were purchased from Sigma-Aldrich (St Louis)

22 Propolis Samples The sample identification geographicorigin and coordinates of the rawUnited States propolis usedin this study are provided (Table 1)

23 Propolis Sample Preparation forQuorumSensing BioassayFrozen propolis samples were ground to a fine powderand extracted with 70 ethanol by shaking for 24 hoursThe insoluble materials were removed by centrifugation at12000 g for 10 minutes at 4∘C The extracts were dilutedto 5 based on dry weight for AHL-based QS biosensorinvestigations as previously described by our group [25]












3




(a)

Propolis samples

70

E

tOH

3-O

-C12 3 4 6 8 5 7

120140

100806040200

25

20

15

10

5

0

Hun

garia

n


(b)

Propolis samples

08

06

04

02

00

Back

grou

nd

70

E

tOH

3-O

-C12 3 4 6 8 5 7

120100806040200


(c)

























ctor

219

41

30252015100500




9

3

210

17

5

8 6 4

















4 Conclusions





HO

OO

COOH

OH


Figure 4



Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























3




(a)

Propolis samples

70

E

tOH

3-O

-C12 3 4 6 8 5 7

120140

100806040200

25

20

15

10

5

0

Hun

garia

n


(b)

Propolis samples

08

06

04

02

00

Back

grou

nd

70

E

tOH

3-O

-C12 3 4 6 8 5 7

120100806040200


(c)

























ctor

219

41

30252015100500




9

3

210

17

5

8 6 4

















4 Conclusions





HO

OO

COOH

OH


Figure 4



Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)

Propolis samples

70

E

tOH

3-O

-C12 3 4 6 8 5 7

120140

100806040200

25

20

15

10

5

0

Hun

garia

n


(b)

Propolis samples

08

06

04

02

00

Back

grou

nd

70

E

tOH

3-O

-C12 3 4 6 8 5 7

120100806040200


(c)

























ctor

219

41

30252015100500




9

3

210

17

5

8 6 4

















4 Conclusions





HO

OO

COOH

OH


Figure 4



Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































ctor

219

41

30252015100500




9

3

210

17

5

8 6 4

















4 Conclusions





HO

OO

COOH

OH


Figure 4



Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ctor

219

41

30252015100500




9

3

210

17

5

8 6 4

















4 Conclusions





HO

OO

COOH

OH


Figure 4



Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















4 Conclusions





HO

OO

COOH

OH


Figure 4



Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HO

OO

COOH

OH


Figure 4



Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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Research Article Chemical Composition and Disruption of ...downloads.hindawi.com/journals/ecam/2015/472593.pdf · Research Article Chemical Composition and Disruption of Quorum Sensing