)joebxj1vcmjtijoh$psqpsbujpo &wjefodf … · 2019. 7. 31. · denture stomatitis (ds) is an...

Research ArticleThe Beneficial Effect of Equisetum giganteum Lagainst Candida Biofilm Formation New Approaches toDenture Stomatitis

Rafaela A S Alavarce1 Luiz L Saldanha2 Nara Ligia M Almeida1 Vinicius C Porto3

Anne L Dokkedal4 and Vanessa S Lara1

1Department of Stomatology (Pathology) Bauru School of Dentistry University of Sao Paulo (USP)Alameda Doutor Octavio Pinheiro Brisolla 9-75 17012-901 Bauru SP Brazil2Institute of Biosciences Sao Paulo State University (UNESP) Botucatu SP Brazil3Department of Prosthodontics Bauru School of Dentistry University of Sao Paulo (USP)Alameda Doutor Octavio Pinheiro Brisolla 9-75 17012-901 Bauru SP Brazil4Department of Biological Sciences Faculty of Sciences Sao Paulo State University (UNESP) Bauru SP Brazil

Correspondence should be addressed to Rafaela A S Alavarce rafaelafobuspbr

Received 25 March 2015 Accepted 9 July 2015

Academic Editor Youn C Kim

Copyright copy 2015 Rafaela A S Alavarce 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

Equisetum giganteum L (E giganteum) Equisetaceae commonly called ldquogiant horsetailrdquo is an endemic plant of Central and SouthAmerica and is used in traditional medicine as diuretic and hemostatic in urinary disorders and in inflammatory conditionsamong other applications The chemical composition of the extract EtOH 70 of E giganteum has shown a clear presence ofphenolic compounds derived from caffeic and ferulic acids and flavonoid heterosides derived from quercitin and kaempferol inaddition to styrylpyrones E giganteum mainly at the highest concentrations showed antimicrobial activity against the relevantmicroorganisms tested Escherichia coli Staphylococcus aureus and Candida albicans It also demonstrated antiadherent activityon C albicans biofilms in an experimental model that is similar to dentures Moreover all concentrations tested showed anti-inflammatory activity The extract did not show cytotoxicity in contact with human cells These properties might qualify Egiganteum extract to be a promising alternative for the topic treatment and prevention of oral candidiasis and denture stomatitis

1 Introduction

Denture stomatitis (DS) is an inflammatory disease thataffects the oral mucosa underlying a removable dentalprosthesis both partial and total DS is considered a verycommon disease affecting denture wearers especially thosewith maxillary complete dentures [1 2] Due to the strongrelationship between DS and Candida fungus the use ofproper hygiene aids by the denture wearer is fundamental byphysical means such as brushing and if necessary chemicaland immersion of dentures in disinfectant solutions Local orsystemic antifungal therapy may still be an option in somecases of DS [3] however one should know the predisposingfactors related to the user and eliminate them or change themwhenever possible There is no sense in using drug therapy if

it is not associated with awareness and change in the factorsthat contribute to DS

Currently treatments directed to DS include topicalantifungal therapy such as several formulations based onnystatin systemic antifungal medication such as azolesattention to oral hygiene denture disinfection proceduresremoval of the denture overnight and replacement of olderdentures [4]

Continued use of topical antifungal agents nystatin sus-pensions [5] and disinfectants such as sodium hypochlorite(NaOCl) glutaraldehyde and chlorhexidine can inducechanges in the properties of the resin surface such as rough-ness hardness andwettability which can contribute to fungaladhesion [5ndash7] The continued use of systemic antifungaltherapy can also lead to serious adverse effects such as

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 939625 9 pageshttpdxdoiorg1011552015939625

2 Evidence-Based Complementary and Alternative Medicine

hepatotoxicity and nephrotoxicity and microbial resistance[4] Moreover after completion of antifungal therapy thereis a rapid recurrence of the DS because the denture baseresin serves as a reservoir for the fungus and local orsystemic antifungal therapies are incapable of eliminating themicroorganisms present in denture bases leading to the needof a further treatment [8]

Following this reasoning the search for a better knowl-edge of antimicrobial action ofmedicinal plants has increasedexponentially and herbal products have proven to be analternative to synthetic chemicals and can play an impor-tant role in the treatment of DS [9] Studies using herbalmedicines to combat dental plaque microorganisms or oralbiofilmpresentingCandida have revealed the efficacy of theseagents as antimicrobial and antiadherent medications for theprevention of dental biofilmand treatment of candidiasis [10]

Equisetum giganteum L (E giganteum) is an endemicplant of Central and South America and is used as diureticand hemostatic in urinary disorders and in inflammatoryconditions in traditional medicine Besides E giganteum iscommonly used as a substitute for Equisetum arvense thisdrug is extensively commercialized for the purpose of ben-eficial role in the field of Medicine and Nutrition as a dietarysupplement [11] Although this plant has antimicrobial effect[12] until now there are no studies of E giganteum againstCandida albicans (C albicans)

Propolis is a resinous substance that has notable anti-fungal antibacterial antioxidant anti-inflammatory andimmunomodulatory properties In Odontology the propolishas been associated mainly with the process of healing[13ndash17] Considering their antifungal and anti-inflammatoryactivities the propolis could be an alternative for the localtreatment of DS [9 18 19]

Antiatherogenic [20] antitumor antioxidant [21] andvirucidal [22] effects have been reported for Punica granatum(P granatum) The findings of studies including some thathave related the inhibition of adherence suggest that oralbacteria and C albicans are sensitive to the extract of Pgranatum [23ndash25] In a clinical study negativity of yeastson lesions of DS in most subjects was observed after gelapplication of P granatum It could be concluded that Pgranatum extract may be used as a topical antifungal drug forthe treatment of this type of candidosis [9 26]

Therefore in the present study we evaluated the antimi-crobial and anti-inflammatory potential of E giganteumextract on C albicans as well as its influence on fungaladherence to the surface of heat-polymerized acrylic resinThe cytotoxic potential of the extract on human palatalepithelial cells and human monocytes was also assessed Thiswas done with a view to the future combination of the Egiganteum as a therapeuticpreventive alternative for topicalapplication or inclusion in soft denture lining materials oninner surface of dentures as a new alternative to the standardtreatment in DS

2 Materials and Methods

21 Plant Material and Extract Preparation The aerial partsofE giganteumwere collected inNovember 2011 at the Jardim

Botanico Municipal de Bauru SP Brazil (22∘2010158403010158401015840S and49∘0010158403010158401015840W) Voucher specimens were prepared identifiedand deposited at the Herbarium of the UNESP Sao PauloState University ldquoJulio de Mesquita Filhordquo UNBA (BauruSP Brazil) under number 5795 The fresh plants were driedat 40∘C for 48 h and the powdered raw material (13 kg)was extracted with EtOHH

2O (7 3 vv) by percolation at

room temperature The filtrate was concentrated to drynessunder reduced pressure at 40∘Cproviding the hydroethanolicextract (70EtOH) with a yield of 824 (364 g)

22 Chemical Analysis by UHPLC-PAD-ESI-MSn The chem-ical profile of the 70 EtOH extract of E giganteum wasobtained by means of Accela High Speed LC (ThermoScientific San Jose CA USA) Thermo Scientific column(50 times 21mm 19 120583m) with PAD and coupled to an Accela(Thermo Scientific) LCQ Fleet with Ion Trap (IT) 3D andionization by electrospray (ESI) The mobile phase consistedof ultrapure water (eluent A) and methanol (eluent B) bothmixed in 01 of formic acid and we utilized a proportionof 5ndash100 of A in B during 20min The experimental protocolused the following parameters injection volume 100120583Lcolumn temperature 25∘C and flow ratio 04mLsdotminminus1and the chromatogram at 254 nm The effluent from theUHPLC was directed into the ESI probe The second scanevent was an MSMS experiment using a data dependentscan on deprotonatedmolecule [M-H]minus with collision energyto 10ndash25 After being dissolved in MeOH-H

2O (85 15)

the extract was infused in the ESI source by flow injectionanalysis (FIA) using a syringe pump at 5 120583gmL

23 Microorganisms For this study we used Staphylococcusaureus (ATCC 6536) Escherichia coli (O124) and C albicans(SC 5314) C albicans were grown in YEPD broth (DifcoSparks MD USA) and tested in Sabouraud broth (DifcoSparks MD USA) Bacteria were grown and tested in brain-heart infusion broth (BHI) (Difco Sparks MD USA)

24 Antimicrobial Assay In vitro antimicrobial actionwas analyzed by the broth microdilution method with theobjective of obtaining theminimum inhibitory concentration(MIC) of the E giganteum extract Bacterial and fungalsuspensions (1 times 105 cellsmL) were respectively inoculatedinto BHI and Sabouraud broth using 96-well plates Wellscontaining each inoculum with the 1 NaOCl solution(CTRLNaOCl) and culture medium (Sabouraud or BHIbroth) (CTRLMedium) served as the positive and negativecontrol respectively

25 Antiadherent Assay Ninety specimens of heat-polymer-ized acrylic resin (25 times 20 times 05 cm) were fabricated accord-ing to the manufacturersrsquo directions (Lucitone 550 DentsplyInternational Inc YorkUSA) Each surface of specimenswasabraded manually with high grade silicon carbide abrasivepaper (P80 Norton Abrasivos Sao Paulo Brazil) [27] topromote a rough surface of approximately 2 microns Afterthis the specimens were ultrasonically cleansed in sterilewater (Ultrasonic Cleaner Arotec Sao Paulo Brazil) [28] andsterilized with ethylene oxide (Acecil Campinas Brazil)

Evidence-Based Complementary and Alternative Medicine 3

According to the different treatments the resin specimenswere divided into five groups E50 E25 and E16 (50 25and 16mgmL) of E giganteum extract CTRLPBS sterilephosphate buffered saline (pH 72) or CTRLNaOCl-1NaOCl solution The treatments were performed in 6-wellplates at room temperature for 10min Sequentially thespecimens were incubated for 90min at 37∘C at 75 rpm(Fanem Guarulhos Brazil) with fungal suspension (1 times 107cellsmL) washed with PBS and incubated in RPMI-1640(GIBCO Grand Island NY) at 37∘C for 12 h at 75 rpmThreespecimens for group were stained by LIVEDEAD BacLightBacterial Viability Kit solution (Molecular Probes EugeneOR USA) [29] and incubated in the dark for 15min at roomtemperature

Subsequently the specimens were conditioned in theconfocal laser scanning microscopy (Leica MicrosystemsGmbH Mannheim Germany) from which digital imageswere obtainedThe viable cells were visualized in green whiledead cells were visualized in red With the use of BioImageSoftware program the biofilmmass was quantified includingboth viable and nonviable cells and expressed as 120583m3 Themore the biofilmmass on the specimens decreased the higherthe antiadherent activity was

26 Cell Culture HPEC were cultivated as previouslydescribed by Klingbeil et al [30] and humanmonocytes wereobtained from thirty-milliliter volumes of peripheral bloodwhich were drawn from apparently healthy five volunteersAfter obtaining peripheral blood mononuclear cells by cellseparation using Histopaque 1077 gradients according tothe manufacturerrsquos instructions (Sigma-Aldrich Company)monocytes were counted using neutral red (002) andincubated in 96-well culture plates by 2 h at 37∘C in 5 CO

2

Following nonadherent cells were removed by aspirationMonocytes were cultivated in RPMI-1640 (GIBCO GrandIsland NY) supplemented with 10 FBS (GIBCO) and 1penicillin-streptomycin (GIBCO)Themediumwas replacedevery other day until the cells grew to about 70 confluenceThe experimental protocol used in this study was approvedby the Committee on Ethics in Human Experimentation ofBauru School of Dentistry University of Sao Paulo (grantnumber 6706412200005417 November 8 2012) and writ-ten informed consent was obtained from all patients Thisstudy was performed in accordance with the Declaration ofHelsinki

261 Cell Viability Assay The different extracts were eval-uated regarding cytotoxicity by usingMTT (3-[45-dimethyl-thiazolyl-2]-25-diphenyltetrazolium bromide) colorimetricassay (Sigma Chemical Co St Louis USA) Briefly mono-cytes (2times 105 cellswell) suspended in 200 uL ofmediumwereplated into 96-well tissue culture plates for 24 h Thereafter200 uL of fresh medium containing various concentrationsof E giganteum extracts (50 25 16 8 and 4mgmL) wasadded to each well and incubated for time intervals of 1 12and 24 hours and compared with control cells (untreatedand water) After these periods the plates were washed twicein PBS and 200 uL of fresh medium mixed with 10 uL of5mgmL MTT dye in PBS was added to the wells for 4 h in

a humidified 5 CO2atmosphere at 37∘C After the removal

of MTT solution 150 uL of dimethyl sulphoxide (DMSO-LGCBiotecnologia Sao Paulo Brazil) was added and mixedto dissolve the dye crystals The absorbance was measuredat 500 nm by means of a multiplate reader (Synergy MxMonochromator-Based Biotek Washington DC USA) Thesame assay was performed with human palatal epithelial cells(HPEC) (104 cellswell) for 12 and 24 hours

27 Measurement of Intracellular Reactive Oxygen Species(ROS) Endogenous amounts of ROS in human mono-cytes were determined using the fluorescent probe 2101584071015840-dichlorofluorescin diacetate (Cell Rox Deep Red ReagentLife Technologies Grand Island NY USA) Monocytes (2times 105 cellswell) were in vitro incubated with E giganteumextract (50 25 16 8 and 4mgmL) for 1 hour at 37∘C in5 CO

2atmosphere After they were stimulated with LPS

at 5 120583gmL or C albicans SC5314 (1 times 107 cellsmL) for threehours and incubated with 5 120583M of Cell Rox at 37∘C in 5CO2atmosphere for 30minThefluorescence intensities (FIs)

of the cells were measured using the multiplate reader at640665 nm at 37∘C

28 Statistical Analysis Thedata are expressed as the mean plusmnstandard deviation of at least three independent experimentsin triplicate The data were analyzed where appropriateby means of the one-way ANOVA two-way ANOVA orKruskal-Wallis Test followed by the Tukey and DunnettTest using the STATISTICA software program (version 120StatSoft Inc Tulsa OK USA) We considered 119901 lt 005 as alevel of statistically significant difference

3 Results

31 Compounds Isolated from Equisetum giganteum L Theconstituents present in the hydroethanolic extract were iden-tified by 119877

119905in RP-UHPLC UV-vis and MSMSn spectra

analysis and comparison with data from the literature [11] Intotal 13 constituents were identified in the 70 EtOH extract(Table 1) Figure 1 shows the structures of styrylpyrones andflavonoids identified

The UHPLC-PAD-ESI-MSn analysis of the hydroethano-lic extract highlighted UV spectrum characteristic of styryl-pyrones with bands at 253ndash258 and 355ndash373 nm [31] andflavonoids at 240ndash290 nm related to A-ring and at 300ndash390 nm related to B-ring [32] Diagnostics precursor ionsat mz 379 301 and 285 confirm the presence of styrylpy-rones and flavonoid glucosides derivatives of quercetin andkaempferol respectivelyThe signals ofmass fragments atmz178 and 192 were related to presence of caffeic and ferulic acidderivatives The neutral losses of 162 mass units allowed theidentification of hexoses (sophoroside or glucoside)

In Figure 2 the UPLC-PAD chromatogram of the Egiganteum 70 EtOH extract is presented

32 Antimicrobial Activity of E giganteum The E giganteumextract (E50 E25 E16 E8 and E4) showed antimicrobialactivity against all strains studied (Figure 3) The strainmost sensitive to the E giganteum extract was S aureus


Table 1 UHPLC-PAD-ESI-MS data (UV-vis spectra and detected ions) and MS119899 (product ions) of compounds detected in EtOH 70 of theaerial parts of E giganteum

Peak 119877119905

(min) UV (120582max) UHPLC-MS ions [M-H]minus ESI-IT-MS119899 Compound1 999 266 350 787 463 301 Quercetin-tri-O-hexoside2 1056 265 344 771 609 428 285 Kaempferol-3-O-sophoroside-7-O-glu3 1122 254 366 585 422 379 259 3-Hydroxyhispidin-341015840-di-O-glucoside4 1161 218 295sh 327 62507 463 301 Quercetin-37-di-O-glucoside5 1169 278 305sh 327 62501 463 301 255 178 Quercetin-3-O-(caffeoyl)-glucoside6 1208 264 341 609 447 327 285 255 Kaempferol-37-di-O-glucoside7 1309 270 361 625 462 301 Flavonol-di-O-hexoside8 1344 254 331 369 423 379 287 261 217 Equisetumpyrone9 1353 216 291 328 mdash 215 178 160 142 Caffeic acid derivative10 1404 265 287sh 341 609 285 Kaempferol-3-O-sophoroside11 1455 270 347 463 301 Quercetin-hexoside12 1482 275 297sh 331 463 309 192 177 133 Ferulic acid derivative13 11568 265 344 447 285 Kaempferol-3-O-glucoside

O

O O

OH

HO

HO

O

OH

R1

R1 R1

R1

R2

R2 R2

R2

R3

R3Compound

Kaempferol-3-O-sophoroside-7-O-glu

Kaempferol-37-di-O-glucoside

Kaempferol-3-O-sophoroside

Kaempferol-3-O-glucoside

Quercetin-tri-O-hexoside

Quercetin-37-di-O-glucoside

Quercetin-3-O-(caffeoyl)-glucoside

Quercetin-hexoside

Flavonol-di-O-hexoside

O-Soph

O-Glu

O-Soph

O-Glu

O-Glu-Glu-Glu

O-Glu

O-Glu-Caff

O-Hex

O-Hex-Hex

O-Glu

O-Glu

H

H

OH

O-Glu

OH

OH

OH

Compound

Equisetumpyrone

3-Hydroxyhispidin-34998400-di-O-glucoside

H

H

H

H

OH

OH

OH

OH

OH

O-Glu OH

O-Glu O-Glu

Figure 1 Structure of the flavonoids and styrylpyrones identified by UHPLC-PAD-ESI-MSn in the hydroethanolic extract of E giganteum

At the highest concentration of the extract (50mgmL) theresults were similar to 1 NaOCl for all strains testedEven at its lowest concentrations the extract still showed amicrobicidal action

33 Antibiofilm Effect of E giganteum against C albicansSC5314 The specimens pretreated with sterile phosphatebuffered saline (CTRLPBS) showed an expressive presenceof biofilm mass of C albicans on the acrylic resin specimensurface thereby revealing biofilms at the stage of proliferationand filamentation (Figure 4(d)) As expected this biofilmwas

mainly formed of viable (green) cells (Table 2) In contrastwith PBS 1 sodium hypochlorite pretreatments removedthemajority of biofilm (Figure 4(e)) leaving only a few viablecells but most of the cells were nonviable (Table 2)

The surface of resin specimens pretreated with thedifferent concentrations of E giganteum extract (50 25and 16mgmL) revealed that increasing the concentrationof the extract improved its antiadherent activity becausebiofilm formation decreased with an increase in amount ofherbal extract used (Figures 4(a) 4(b) and 4(c)) As shownfor 1 NaOCl E50 significantly reduced the biofilm mass


7 8 9 10 11 12 13 14 15 16 17 18 19

Time (min)

1056

1169

1161

1208

13441122 1404

1482999 15681309965 1599 1653 1763 1888831796

1

2

3

4

5 6

7

810

911

12 13

200

406080

100120140160180200220times103

(120583AU

)

RT 700ndash1900

Figure 2 UHPLC-PAD analytical chromatogram of the 70 EtOHextract of the aerial parts of E giganteum at 120582 = 254 nm Mobilephase was water ultrapure (eluent A) and methanol (eluent B)both containing 01 of formic acid The ratio was 5ndash100 ofA in B in 20min Injection volume 100120583L Thermo Scientificcolumn (50times 21mm 19 120583m) column temperature 25∘C flow ratio04mLsdotminminus1

0102030405060708090

100110

Candida albicans Staphylococcus Escherichia coli

Killi

ng (

)

lowast lowast lowast

CTRL

NaO

ClE5

0

E25

E16

E8 E4 CTRL

NaO

ClE5

0

E25

E16

E8 E4 CTRL

NaO

ClE5

0

E25

E16

E8 E4

aureus

Figure 3 Median plusmn SD of percentage of killing against E coliO124 S aureus ATCC 6538 and C albicans SC 5314 after 24 hoursin contact with extract at different concentrations (mgmL) 50(E50) 25 (E25) 16 (E16) 8 (E8) and 4 (E4) The respective controlswere incubated with 1 sodium hypochlorite (CTRLNaOCl) orwith culture medium (CTRLMedium) which showed no killingof microorganisms (0) lowast119901 lt 005 represents a significant changecompared with the CTRLMedium Five independent experimentswere performed in triplicate (119899 = 15)

on the specimen surfaces although scarce viable cells canstill be noted (Figure 4(a)) All groups of the E giganteumextract presented lower numbers of remaining fungal cellsin comparison with control (PBS) showing marked anti-adherent activity of the E giganteum extracts against Calbicans biofilms At 25 and 16mgmL the values of biofilmmass were similar to those obtained for PBS however theydiffered statistically from both control (NaOCl) and E50No statistical differences were observed between E50 andCTRLNaOCl with respect to the total number of remainingnonviable cells

34 ROS Production in Monocytes Treated with E giganteumExtract The results revealed that all the extract concentra-tions (50 25 16 8 and 4mgmL) were able to decrease thelevels of ROS production by cells stimulated with LPS or Calbicans returning to baseline levels Values for cells treated

Table 2 Mean plusmn SD of biofilm cells (biofilm mass) includingthose that are viable and nonviable (mean) remaining on thesurface of the specimens after different pretreatments with extract50mg (E50) 25mg (E25) or 16mg (E16) and PBS (CTRLPBS)or 1 sodium hypochlorite (CTRLNaOCl) for 10 minutes Thedata were obtained by using the confocal laser scanning microscopyand BioImage software program Six independent experiments wereperformed in triplicate (119899 = 18)

Groups Biofilm mass (120583m3) (Viable + nonviable)E50 426303 plusmn 403727 (423174 + 3129)E25 3140834 plusmn 555214 (2882529 + 258305)E16 3544857 plusmn 495006 (3406322 + 138535)CTRLPBS 9548884 plusmn 4038917 (9406039 + 142845)CTRLNaOCl 48936 plusmn 24411 (38617 + 10319)

with E giganteum extract which were not stimulated withLPS or C albicans remained at baseline levels (Figure 5)

35 Cytotoxicity Test Both monocytes and HPEC remainedviable in the presence of E giganteum extract but at the low-est concentration (E4) for 12 hours on monocytes there weresignificant differences between the cells treated with extractand untreated cells In addition at highest concentrationsof E giganteum the percentage of cell viability was higherthan it was for untreated cells suggesting an increased cellmetabolism (Figures 6(a) and 6(b))

4 Discussion

C albicans is the predominant yeast species isolated frompatients with DS due to its greater capacity to form biofilmson surfaces [33ndash36] including denture acrylic surfacesTherefore decontamination of dentures is a fundamentalaspect of effective oral hygiene in order to prevent thedenture stomatitis Typically research on Candida biofilmshas evaluated the efficacy of different disinfectant solutionssuch as 1 and 2 NaOCl and 2 glutaraldehyde on heat-polymerized acrylic resin [37]

Considering that various disinfectants affect the physicalproperties of resin surface of denture [37ndash44] alternativetherapies such as phytotherapeutic agents with antimi-crobial potential and ability to eliminate or prevent theadhesion of C albicans to the inner surface of the denturewithout damaging themucosa or denturesmay be of extremeinterest to removable denture wearers particularly those withmaxillary complete dentures [45 46]

In the present study the crude extract of E giganteumshowed a potent antimicrobial effect on C albicans and onfungus adherence to heat-polymerized acrylic resin Further-more the plant extract demonstrated a potential in vitro anti-inflammatory effect on monocytes while it had no cytotoxiceffect in vitro on monocytes and HPEC Our study is the firstto verify the beneficial effect of E giganteum against Candidabiofilm formation on surfaces and its biocompatibility

Although E giganteum does not have bacteriostaticpotential the antimicrobial action was evident in our find-ings even at the lowest concentration tested (4mgmL)This


(a) (b) (c)

(d) (e)

Figure 4 Confocalmicroscopy images ofCandida albicans biofilms after each treatment with extract (a) E50 (b) E25 (c) E16 (d) CTRLPBSand (e) CTRLNaOCl Six independent experiments were performed in triplicate

0

2000

4000

6000

8000

Relat

ive fl

uore

scen

ce u

nit (

rfu)

Medium LPS C albicans

Med

ium

E50

E25

E16

E8 E4 LPS

E50

E25

E16

E8 E4 E50

E25

E16

E8 E4C a

lbica

ns

120575

lowast

Figure 5 Production of ROS by human monocyte cells culturedand treated with LPS and C albicans in the presence or absenceof extracts as described in Materials and Methods Results areexpressed as mean plusmn SD of at least four experiments Symbolsindicate significant difference (119901 lt 005) lowastin comparison with LPS-treated cells and 120575 compared with C albicans-treated cells in thepresence of extract in all concentrations tested

was shown not only against C albicans but also against the Saureus and E coli bacteria used as parameters for comparisonin our study At a concentration of 50mgmL by meansof antimicrobial assay based on the broth microdilutionmethod the herbal extract showed antimicrobial action

similar to 1 sodium hypochlorite an agent widely appliedfor immersion of dentures during treatment of DS

We emphasize the values of antimicrobial action obtainedin our study which were higher than those reported in a pre-vious study [12] that assessed the same herbal extract againstGram-negative and Gram-positive bacteria and obtainedno activity against Escherichia coli (E coli) In our studyanother aspect that highlights the significant antimicrobialactivity of E giganteum is that this phytotherapeutic agentwas effective against a clinical strain of E coli (O1124) whichusually presents more resistance and higher pathogenicitythan standard strains when they are isolated from patients[47] However even at low concentrations of the extract (8and 4mgmL) we observed that its antimicrobial activityagainst this strain of E coli ranges from 65 to 54 of death

The chemical composition of the extract EtOH 70 of Egiganteumhas shown a clear presence of phenolic compoundsderived from caffeic and ferulic acids and flavonoid hetero-sides derived from quercitin and kaempferol in addition tostyrylpyrones (Table 1 Figure 1)This herbal extract producesbioactive compounds such as flavonoids that are responsiblefor its anti-inflammatory and antioxidant effects in additionto having analgesic antimicrobial and immunomodulatoryproperties [48 49] Therefore the flavonoids present inthe composition of E giganteum probably are the con-stituents responsible for the microbicidal effects observed


E50 E25 E4Control E8E16

00

02

04

06

08

10

1h 24 h12 h

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

OD

500

nm

(a)

00

02

04

06

08

10

E50 E25 E16 E8 E4Control

lowast

24 h12 h

OD

500

nm

(b)

Figure 6 (a) Effects of E giganteum on cell viability of humanmonocytesThe viability of cells under treatments with different concentrationsof E giganteumwas investigated byMTT assays Data shown are the mean plusmn SD of at least four independent experiments Asterisks indicatesignificant difference compared with the control (119901 lt 005) (b) The effects of E giganteum on cell viability of HEPCs The viability of cellsunder treatments with different concentrations of E giganteum was investigated by MTT assays Data shown are the mean plusmn SD of at leastthree independent experiments lowast119901 lt 005 represents a significant change compared with the control

here because they have the ability to inactivate microbialadhesion proteins and transport proteins and to rupture themicrobial membrane

Furthermore in order to simulate the resinous portionof removable dentures that have been contaminated byC albicans as frequently it occurs with dentures underthe conditions of the intraoral environment we developedcontaminated specimens made of denture base material Theresin surface pretreated with extract of E giganteum at allconcentrations showed a significant reduction in the biofilmmass of viable C albicans cells in comparison with theuntreated resin In other words we can assert that a brief10-minute treatment of the resin surface with the extractswas able to significantly reduce the capacity of C albicansto adhere to the surface We emphasize that at 50mgmLthe treatment with E giganteum resulted in the lowestbiofilm mass found when compared with any of the otherconcentrations Interestingly after these pretreatments theresin surface showed dispersed fungus in both its filamentousand yeast forms especially at the highest concentration ofthe extract As observed in our control samples confluentbiofilms presenting predominantly filamentous forms aredeveloped on the resin surface Taken together we believethat the herbal extract was able to interfere in the expressionof fungal virulence factors in addition to having an antiad-herent effect on the microorganism

One possible hypothesis for explaining the effect ofbiofilm removal by E giganteum extract could be based onbiofilm pH Since the most of biofilms are formed at neutralpH the conditions having very high or very low pH valuescan provide changes on microbial metabolism and surfaceproperties interfering in their adhesion process to surfaces[50] Hypochlorite solutions for example having alkaline pH(pHgt 11) can increase the electrostatic repulsion between theyeast cells and the surface of the resin material dissolvingthe biofilm cells [51] Thus the acidic pH (pH = 548) of

the hydroalcoholic extract of E giganteum associated withantimicrobial activity especially in higher concentrationscould explain their important antiadherent effect on Calbicans biofilms however further studies are required aboutthe mechanisms involved

In addition to the beneficial results with respect to theantimicrobial and antiadherent activities all concentrationsof E giganteum extract demonstrated suppression of reactiveoxygen species in human monocytes stimulated in vitroby LPS or C albicans Previous studies have also shownreduced levels of ROS resulting from the action of E arvenseand have attributed this effect to the presence of flavonoids[52] In fact flavonoids are able to eliminate ROS andRNS by donation of electron and hydrogen (H+) from thehydroxyl groups present in their composition stabilizingthese free radicals [49 53] or suppressing the formation ofROS by inhibiting the enzymes involved in generating them[49 54] The presence of phenolic acids was also detectedin the E giganteum extract This group of substances ischaracterized by their antioxidant properties since they wereable to reduce the levels of reactive oxygen species due totrapping free radicals directly or scavenging them througha series of combined reactions with antioxidant enzymes[49 52 53 55] Other inflammatory mediators should beevaluated for a more accurate conclusion and to imply thatE giganteum extract could negatively modulate monocyte-mediated inflammatory responses

Another interesting finding was that the extract did notalter the viability of monocytes and HPEC for up to 24 hoursThis has important clinical repercussions since these cells areclosely related to the inner surface of the denture in denturewearers The oral epithelial cells are the first cells to interactwith C albicans during the establishment of DS

In summary our results suggest that the phytotherapeuticE giganteum extract has the following properties antimicro-bial against C albicans S aureus and E coli antiadherence


of C albicans to heat-cured acrylic resin specimens and anti-inflammatory effects on human monocytes activated by Calbicans In addition the phytotherapeutic extract did notcompromise the viability of human monocytes or humanpalatal epithelial cells These properties might qualify Egiganteum extract to be a promising alternative for the topictreatment and prevention of oral candidiasis and denturestomatitis

Conflict of Interests

The authors declare that they have no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors wish to acknowledge the financial support fromSao Paulo Research Foundation (FAPESP) (no 201212458-9)

References

[1] L Gendreau and Z G Loewy ldquoEpidemiology and etiology ofdenture stomatitisrdquo Journal of Prosthodontics vol 20 no 4 pp251ndash260 2011

[2] S Sharma and V Hegde ldquoComparative evaluation of antifungalactivity of melaleuca oil and fluconazole when incorporated intissue conditioner an in vitro studyrdquo Journal of Prosthodonticsvol 23 no 5 pp 367ndash373 2014

[3] L F Perezous C M Flaitz M E Goldschmidt and RL Engelmeier ldquoColonization of Candida species in denturewearers with emphasis on HIV infection a literature reviewrdquoJournal of Prosthetic Dentistry vol 93 supplement 3 pp 288ndash293 2005

[4] T Lombardi and E Budtz-Jorgensen ldquoTreatment of denture-induced stomatitis a reviewrdquo The European Journal ofProsthodontics and Restorative Dentistry vol 2 supplement 1pp 17ndash22 1993

[5] K H Neppelenbroek A C Pavarina C E Vergani and E TGiampaolo ldquoHardness of heat-polymerized acrylic resins afterdisinfection and long-term water immersionrdquo The Journal ofProsthetic Dentistry vol 93 no 2 pp 171ndash176 2005

[6] Z N AL-Dwairi F A AL-Quran and O Y AL-Omari ldquoTheeffect of antifungal agents on surface properties of poly(methylmethacrylate) and its relation to adherence of Candida albi-cansrdquo Journal of Prosthodontic Research vol 56 no 4 pp 272ndash280 2012

[7] C F Carvalho A D Vanderlei S M S Marocho S MB Pereira L Nogueira and T J A Paes-Junior ldquoEffect ofdisinfectant solutions on a denture base acrylic resinrdquo ActaOdontologica Latinoamericana vol 25 no 3 pp 255ndash260 2012

[8] J Chandra P K Mukherjee and M A Ghannoum ldquoCandidabiofilms associated with CVC and medical devicesrdquo Mycosesvol 55 supplement 1 pp 46ndash57 2012

[9] A R Casaroto and V S Lara ldquoPhytomedicines for Candida-associated denture stomatitisrdquoFitoterapia vol 81 no 5 pp 323ndash328 2010

[10] P M Alves L M G Queiroz J V Pereira and M S VPereira ldquoAtividade antimicrobiana antiaderente e antifungicain vitro de plantas medicinais brasileiras sobre microrganismosdo biofilme dental e cepas do genero Candidardquo Revista da

Sociedade Brasileira de Medicina Tropical vol 42 supplement2 pp 222ndash224 2009

[11] L N Francescato S L Debenedetti T G Schwanz V LBassani and A T Henriques ldquoIdentification of phenolic com-pounds in Equisetum giganteum by LCndashESI-MSMS and a newapproach to total flavonoid quantificationrdquo Talanta vol 105 pp192ndash203 2013

[12] P Kloucek Z Polesny B Svobodova EVlkova and LKokoskaldquoAntibacterial screening of some Peruvian medicinal plantsused inCallerıa Districtrdquo Journal of Ethnopharmacology vol 99supplement 2 pp 309ndash312 2005

[13] F Borrelli P Maffia L Pinto et al ldquoPhytochemical compoundsinvolved in the anti-inflammatory effect of propolis extractrdquoFitoterapia vol 73 supplement 1 pp S53ndashS63 2002

[14] T P T Cushnie and A J Lamb ldquoDetection of galangin-inducedcytoplasmic membrane damage in Staphylococcus aureus bymeasuring potassium lossrdquo Journal of Ethnopharmacology vol101 no 1ndash3 pp 243ndash248 2005

[15] J E Johnston H A Sepe C L Miano R G Brannan andA L Alderton ldquoHoney inhibits lipid oxidation in ready-to-eatground beef pattiesrdquo Meat Science vol 70 supplement 4 pp627ndash631 2005

[16] F Mani H C R Damasceno E L B Novelli E A M Martinsand J M Sforcin ldquoPropolis effect of different concentrationsextracts and intake period on seric biochemical variablesrdquoJournal of Ethnopharmacology vol 105 no 1-2 pp 95ndash98 2006

[17] F R Victorino S L Franco T I E Svidzinski et al ldquoPharma-cological evaluation of propolis solutions for endodontic userdquoPharmaceutical Biology vol 45 no 9 pp 721ndash727 2007

[18] V R Santos F J G S Pimenta M C F Aguiar M A V doCarmo M D Naves and R A Mesquita ldquoOral candidiasistreatment with Brazilian ethanol propolis extractrdquo PhytotherapyResearch vol 19 supplement 7 pp 652ndash654 2005

[19] V R Santos R T Gomes R A de Mesquita et al ldquoEfficacy ofBrazilian propolis gel for the management of denture stomatitisa pilot studyrdquo Phytotherapy Research vol 22 supplement 11 pp1544ndash1547 2008

[20] M Kaplan T Hayek A Raz et al ldquoPomegranate juice sup-plementation to atherosclerotic mice reduces macrophage lipidperoxidation cellular cholesterol accumulation and develop-ment of atherosclerosisrdquo The Journal of Nutrition vol 131 no8 pp 2082ndash2089 2001

[21] J-D Su T Osawa S Kawakishi and M Namiki ldquoTanninantioxidants from Osbeckia chinensisrdquo Phytochemistry vol 27no 5 pp 1315ndash1319 1988

[22] M Haidari M Ali S W Casscells III and M MadjidldquoPomegranate (Punica granatum) purified polyphenol extractinhibits influenza virus and has a synergistic effect withoseltamivirrdquo Phytomedicine vol 16 no 12 pp 1127ndash1136 2009

[23] C Anesini and C Perez ldquoScreening of plants used in Argentinefolk medicine for antimicrobial activityrdquo Journal of Ethnophar-macology vol 39 no 2 pp 119ndash128 1993

[24] M S Ali-Shtayeh and S I Abu Ghdeib ldquoAntifungal activity ofplant extracts against dermatophytesrdquoMycoses vol 42 no 11-12pp 665ndash672 1999

[25] L C D S Vasconcelos F C Sampaio M C C Sampaio MD S V Pereira J S Higino and M H P Peixoto ldquoMinimuminhibitory concentration of adherence of Punica granatum Linn(pomegranate) gel against S mutans S mitis and C albicansrdquoBrazilian Dental Journal vol 17 no 3 pp 223ndash227 2006

[26] M A L Vasconcelos D D S Ferreira M L Andrade e SilvaR C S Veneziani andW R Cunha ldquoAnalgesic effects of crudeextracts of Miconia albicans (Melastomataceae)rdquo Bollettino


Chimico Farmaceutico vol 142 supplement 8 pp 333ndash3352003

[27] J Verran and C J Maryan ldquoRetention of Candida albicans onacrylic resin and silicone of different surface topographyrdquo TheJournal of Prosthetic Dentistry vol 77 no 5 pp 535ndash539 1997

[28] T Pereira A A del Bel Cury M S Cenci and R C MRodrigues-Garcia ldquoIn vitro Candida colonization on acrylicresins and denture liners Influence of surface free energyroughness saliva and adhering bacteriardquo International Journalof Prosthodontics vol 20 no 3 pp 308ndash310 2007

[29] Y Jin T Zhang Y H Samaranayake H H P Fang H KYip and L P Samaranayake ldquoThe use of new probes andstains for improved assessment of cell viability and extra-cellular polymeric substances in Candida albicans biofilmsrdquoMycopathologia vol 159 no 3 pp 353ndash360 2005

[30] M F G Klingbeil M B Mathor F S Giudice D Yoshito andD dos Santos Pinto Jr ldquoIs it safe to utilize in vitro reconstitutedhuman oral epithelium An oncogenetic pathway studyrdquo Celland Tissue Banking vol 13 supplement 1 pp 27ndash35 2012

[31] C Beckert Biosynthese Akkumulation und Strukturen vonStyrylpyronen in gametophytischen und sporophytischenGeweben von Equisetum [PhD dissertation] University ofWurzburg Wurzburg Germany 2002

[32] O M Andersen and K R Markham Flavonoides ChemistryBiochemistry and Applications Taylor amp Francis New York NYUSA 2006

[33] X Y He J H Meurman K Kari R Rautemaa and L PSamaranayake ldquoIn vitro adhesion ofCandida species to denturebase materialsrdquoMycoses vol 49 no 2 pp 80ndash84 2006

[34] G Ramage J P Martınez and J L Lopez-Ribot ldquoCandidabiofilms on implanted biomaterials a clinically significantproblemrdquo FEMS Yeast Research vol 6 supplement 7 pp 979ndash986 2006

[35] L Coulthwaite and J Verran ldquoPotential pathogenic aspects ofdenture plaquerdquo British Journal of Biomedical Science vol 64supplement 4 pp 180ndash189 2007

[36] R B Shinde J S Raut andM SKaruppayil ldquoBiofilm formationby Candida albicans on various prosthetic materials and itsfluconazole sensitivity a kinetic studyrdquoMycoscience vol 53 no3 pp 220ndash226 2012

[37] I A Orsi A G Junior C A Villabona F H C N Fernandesand I Y Ito ldquoEvaluation of the efficacy of chemical disinfectantsfor disinfection of heat-polymerised acrylic resinrdquo Gerodontol-ogy vol 28 no 4 pp 253ndash257 2011

[38] C Shen N S Javid and F A Colaizzi ldquoThe effect of glutaralde-hyde base disinfectants on denture base resinsrdquo The Journal ofProsthetic Dentistry vol 61 no 5 pp 583ndash589 1989

[39] T Asad A C Watkinson and R Huggett ldquoThe effects ofvarious disinfectant solutions on the surface hardness of anacrylic resin denture base materialrdquo The International Journalof Prosthodontics vol 6 no 1 pp 9ndash12 1993

[40] T Ma G H Johnson and G E Gordon ldquoEffects of chemicaldisinfectants on the surface characteristics and color of dentureresinsrdquo Journal of Prosthetic Dentistry vol 77 no 2 pp 197ndash2041997

[41] R C M M Rodrigues Garcia J A de Souza Jr R N Rachedand A A Del Bel Cury ldquoEffect of denture cleansers on thesurface roughness and hardness of a microwave-cured acrylicresin and dental alloysrdquo Journal of Prosthodontics vol 13 no 3pp 173ndash178 2004

[42] H D F O Paranhos L R Davi A Peracini R B Soares C HD S Lovato and R F de Souza ldquoComparison of physical andmechanical properties of microwave-polymerized acrylic resin

after disinfection in sodium hypochlorite solutionsrdquo BrazilianDental Journal vol 20 no 4 pp 331ndash335 2009

[43] A L Machado E T Giampaolo C E Vergani J F de Souzaand J H Jorge ldquoChanges in roughness of denture base andreline materials by chemical disinfection or microwave irradi-ation Surface roughness of denture base and reline materialsrdquoJournal of Applied Oral Science vol 19 no 5 pp 521ndash528 2011

[44] M C Goiato D M D Santos A Moreno M G Iyda MC R A Rezende and M F Haddad ldquoEffect of disinfectionand storage on the flexural strength of ocular prosthetic acrylicresinsrdquo Gerodontology vol 29 no 2 pp e838ndashe844 2012

[45] N Radulovic G Stojanovic and R Palic ldquoComposition andantimicrobial activity of Equisetum arvense L essential oilrdquoPhytotherapy Research vol 20 no 1 pp 85ndash88 2006

[46] A Catalan J G Pacheco A Martınez and M A MondacaldquoIn vitro and in vivo activity of Melaleuca alternifolia mixedwith tissue conditioner onCandida albicansrdquoOral Surgery OralMedicine Oral Pathology Oral Radiology and Endodontologyvol 105 no 3 pp 327ndash332 2008

[47] S H Gillespie ldquoAntibiotic resistance in the absence of selectivepressurerdquo International Journal of Antimicrobial Agents vol 17no 3 pp 171ndash176 2001

[48] JM Calderon-Montano E Burgos-Moron C Perez-Guerreroand M Lopez-Lazaro ldquoA review on the dietary flavonoidkaempferolrdquo Mini-Reviews in Medicinal Chemistry vol 11supplement 4 pp 298ndash344 2011

[49] S Kumar andA K Pandey ldquoChemistry and biological activitiesof flavonoids an overviewrdquo The Scientific World Journal vol2013 Article ID 162750 16 pages 2013

[50] T R Bott ldquoBiological growth on heat exchanger surfacesrdquoin Fouling of Heat Exchangers pp 1ndash515 Elsevier ScienceAmsterdam The Netherlands 1995

[51] S Fukuzaki ldquoMechanisms of actions of sodium hypochlorite incleaning and disinfection processesrdquo Biocontrol Science vol 11no 4 pp 147ndash157 2006

[52] H Oh D-H Kim J-H Cho and Y-C Kim ldquoHepatoprotectiveand free radical scavenging activities of phenolic petrosinsand flavonoids isolated from Equisetum arvenserdquo Journal ofEthnopharmacology vol 95 supplement 2-3 pp 421ndash424 2004

[53] C-P Lin F-Y Lin P-H Huang et al ldquoEndothelial progenitorcell dysfunction in cardiovascular diseases role of reactive oxy-gen species and inflammationrdquo BioMed Research Internationalvol 2013 Article ID 845037 10 pages 2013

[54] F C Vila Identification of flavonoids with antioxidant activity ofsugar cane (Saccharum officinarum L) [dissertation] Univer-sity of Sao Paulo Sao Paulo Brazil 2006

[55] M V Rao G Paliyath and D P Ormrod ldquoUltraviolet-B- andozone-induced biochemical changes in antioxidant enzymes ofArabidopsis thalianardquo Plant Physiology vol 110 no 1 pp 125ndash136 1996

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


hepatotoxicity and nephrotoxicity and microbial resistance[4] Moreover after completion of antifungal therapy thereis a rapid recurrence of the DS because the denture baseresin serves as a reservoir for the fungus and local orsystemic antifungal therapies are incapable of eliminating themicroorganisms present in denture bases leading to the needof a further treatment [8]

Following this reasoning the search for a better knowl-edge of antimicrobial action ofmedicinal plants has increasedexponentially and herbal products have proven to be analternative to synthetic chemicals and can play an impor-tant role in the treatment of DS [9] Studies using herbalmedicines to combat dental plaque microorganisms or oralbiofilmpresentingCandida have revealed the efficacy of theseagents as antimicrobial and antiadherent medications for theprevention of dental biofilmand treatment of candidiasis [10]

Equisetum giganteum L (E giganteum) is an endemicplant of Central and South America and is used as diureticand hemostatic in urinary disorders and in inflammatoryconditions in traditional medicine Besides E giganteum iscommonly used as a substitute for Equisetum arvense thisdrug is extensively commercialized for the purpose of ben-eficial role in the field of Medicine and Nutrition as a dietarysupplement [11] Although this plant has antimicrobial effect[12] until now there are no studies of E giganteum againstCandida albicans (C albicans)

Propolis is a resinous substance that has notable anti-fungal antibacterial antioxidant anti-inflammatory andimmunomodulatory properties In Odontology the propolishas been associated mainly with the process of healing[13ndash17] Considering their antifungal and anti-inflammatoryactivities the propolis could be an alternative for the localtreatment of DS [9 18 19]

Antiatherogenic [20] antitumor antioxidant [21] andvirucidal [22] effects have been reported for Punica granatum(P granatum) The findings of studies including some thathave related the inhibition of adherence suggest that oralbacteria and C albicans are sensitive to the extract of Pgranatum [23ndash25] In a clinical study negativity of yeastson lesions of DS in most subjects was observed after gelapplication of P granatum It could be concluded that Pgranatum extract may be used as a topical antifungal drug forthe treatment of this type of candidosis [9 26]

Therefore in the present study we evaluated the antimi-crobial and anti-inflammatory potential of E giganteumextract on C albicans as well as its influence on fungaladherence to the surface of heat-polymerized acrylic resinThe cytotoxic potential of the extract on human palatalepithelial cells and human monocytes was also assessed Thiswas done with a view to the future combination of the Egiganteum as a therapeuticpreventive alternative for topicalapplication or inclusion in soft denture lining materials oninner surface of dentures as a new alternative to the standardtreatment in DS

2 Materials and Methods

21 Plant Material and Extract Preparation The aerial partsofE giganteumwere collected inNovember 2011 at the Jardim

Botanico Municipal de Bauru SP Brazil (22∘2010158403010158401015840S and49∘0010158403010158401015840W) Voucher specimens were prepared identifiedand deposited at the Herbarium of the UNESP Sao PauloState University ldquoJulio de Mesquita Filhordquo UNBA (BauruSP Brazil) under number 5795 The fresh plants were driedat 40∘C for 48 h and the powdered raw material (13 kg)was extracted with EtOHH

2O (7 3 vv) by percolation at

room temperature The filtrate was concentrated to drynessunder reduced pressure at 40∘Cproviding the hydroethanolicextract (70EtOH) with a yield of 824 (364 g)

22 Chemical Analysis by UHPLC-PAD-ESI-MSn The chem-ical profile of the 70 EtOH extract of E giganteum wasobtained by means of Accela High Speed LC (ThermoScientific San Jose CA USA) Thermo Scientific column(50 times 21mm 19 120583m) with PAD and coupled to an Accela(Thermo Scientific) LCQ Fleet with Ion Trap (IT) 3D andionization by electrospray (ESI) The mobile phase consistedof ultrapure water (eluent A) and methanol (eluent B) bothmixed in 01 of formic acid and we utilized a proportionof 5ndash100 of A in B during 20min The experimental protocolused the following parameters injection volume 100120583Lcolumn temperature 25∘C and flow ratio 04mLsdotminminus1and the chromatogram at 254 nm The effluent from theUHPLC was directed into the ESI probe The second scanevent was an MSMS experiment using a data dependentscan on deprotonatedmolecule [M-H]minus with collision energyto 10ndash25 After being dissolved in MeOH-H

2O (85 15)

the extract was infused in the ESI source by flow injectionanalysis (FIA) using a syringe pump at 5 120583gmL

23 Microorganisms For this study we used Staphylococcusaureus (ATCC 6536) Escherichia coli (O124) and C albicans(SC 5314) C albicans were grown in YEPD broth (DifcoSparks MD USA) and tested in Sabouraud broth (DifcoSparks MD USA) Bacteria were grown and tested in brain-heart infusion broth (BHI) (Difco Sparks MD USA)

24 Antimicrobial Assay In vitro antimicrobial actionwas analyzed by the broth microdilution method with theobjective of obtaining theminimum inhibitory concentration(MIC) of the E giganteum extract Bacterial and fungalsuspensions (1 times 105 cellsmL) were respectively inoculatedinto BHI and Sabouraud broth using 96-well plates Wellscontaining each inoculum with the 1 NaOCl solution(CTRLNaOCl) and culture medium (Sabouraud or BHIbroth) (CTRLMedium) served as the positive and negativecontrol respectively

25 Antiadherent Assay Ninety specimens of heat-polymer-ized acrylic resin (25 times 20 times 05 cm) were fabricated accord-ing to the manufacturersrsquo directions (Lucitone 550 DentsplyInternational Inc YorkUSA) Each surface of specimenswasabraded manually with high grade silicon carbide abrasivepaper (P80 Norton Abrasivos Sao Paulo Brazil) [27] topromote a rough surface of approximately 2 microns Afterthis the specimens were ultrasonically cleansed in sterilewater (Ultrasonic Cleaner Arotec Sao Paulo Brazil) [28] andsterilized with ethylene oxide (Acecil Campinas Brazil)





2










3 Results









Peak 119877119905


O

O O

OH

HO

HO

O

OH

R1

R1 R1

R1

R2

R2 R2

R2

R3

R3Compound








Quercetin-hexoside


O-Soph

O-Glu

O-Soph

O-Glu

O-Glu-Glu-Glu

O-Glu

O-Glu-Caff

O-Hex

O-Hex-Hex

O-Glu

O-Glu

H

H

OH

O-Glu

OH

OH

OH

Compound

Equisetumpyrone


H

H

H

H

OH

OH

OH

OH

OH

O-Glu OH

O-Glu O-Glu







7 8 9 10 11 12 13 14 15 16 17 18 19

Time (min)

1056

1169

1161

1208

13441122 1404

1482999 15681309965 1599 1653 1763 1888831796

1

2

3

4

5 6

7

810

911

12 13

200

406080

100120140160180200220times103

(120583AU

)

RT 700ndash1900


0102030405060708090

100110


Killi

ng (

)


CTRL

NaO

ClE5

0

E25

E16

E8 E4 CTRL

NaO

ClE5

0

E25

E16

E8 E4 CTRL

NaO

ClE5

0

E25

E16

E8 E4

aureus








4 Discussion






(a) (b) (c)

(d) (e)


0

2000

4000

6000

8000

Relat

ive fl

uore

scen

ce u

nit (

rfu)


Med

ium

E50

E25

E16

E8 E4 LPS

E50

E25

E16

E8 E4 E50

E25

E16

E8 E4C a

lbica

ns

120575

lowast








00

02

04

06

08

10

1h 24 h12 h

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

OD

500

nm

(a)

00

02

04

06

08

10


lowast

24 h12 h

OD

500

nm

(b)













Acknowledgment


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2










3 Results









Peak 119877119905


O

O O

OH

HO

HO

O

OH

R1

R1 R1

R1

R2

R2 R2

R2

R3

R3Compound








Quercetin-hexoside


O-Soph

O-Glu

O-Soph

O-Glu

O-Glu-Glu-Glu

O-Glu

O-Glu-Caff

O-Hex

O-Hex-Hex

O-Glu

O-Glu

H

H

OH

O-Glu

OH

OH

OH

Compound

Equisetumpyrone


H

H

H

H

OH

OH

OH

OH

OH

O-Glu OH

O-Glu O-Glu







7 8 9 10 11 12 13 14 15 16 17 18 19

Time (min)

1056

1169

1161

1208

13441122 1404

1482999 15681309965 1599 1653 1763 1888831796

1

2

3

4

5 6

7

810

911

12 13

200

406080

100120140160180200220times103

(120583AU

)

RT 700ndash1900


0102030405060708090

100110


Killi

ng (

)


CTRL

NaO

ClE5

0

E25

E16

E8 E4 CTRL

NaO

ClE5

0

E25

E16

E8 E4 CTRL

NaO

ClE5

0

E25

E16

E8 E4

aureus








4 Discussion






(a) (b) (c)

(d) (e)


0

2000

4000

6000

8000

Relat

ive fl

uore

scen

ce u

nit (

rfu)


Med

ium

E50

E25

E16

E8 E4 LPS

E50

E25

E16

E8 E4 E50

E25

E16

E8 E4C a

lbica

ns

120575

lowast








00

02

04

06

08

10

1h 24 h12 h

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

OD

500

nm

(a)

00

02

04

06

08

10


lowast

24 h12 h

OD

500

nm

(b)













Acknowledgment


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Peak 119877119905


O

O O

OH

HO

HO

O

OH

R1

R1 R1

R1

R2

R2 R2

R2

R3

R3Compound








Quercetin-hexoside


O-Soph

O-Glu

O-Soph

O-Glu

O-Glu-Glu-Glu

O-Glu

O-Glu-Caff

O-Hex

O-Hex-Hex

O-Glu

O-Glu

H

H

OH

O-Glu

OH

OH

OH

Compound

Equisetumpyrone


H

H

H

H

OH

OH

OH

OH

OH

O-Glu OH

O-Glu O-Glu







7 8 9 10 11 12 13 14 15 16 17 18 19

Time (min)

1056

1169

1161

1208

13441122 1404

1482999 15681309965 1599 1653 1763 1888831796

1

2

3

4

5 6

7

810

911

12 13

200

406080

100120140160180200220times103

(120583AU

)

RT 700ndash1900


0102030405060708090

100110


Killi

ng (

)


CTRL

NaO

ClE5

0

E25

E16

E8 E4 CTRL

NaO

ClE5

0

E25

E16

E8 E4 CTRL

NaO

ClE5

0

E25

E16

E8 E4

aureus








4 Discussion






(a) (b) (c)

(d) (e)


0

2000

4000

6000

8000

Relat

ive fl

uore

scen

ce u

nit (

rfu)


Med

ium

E50

E25

E16

E8 E4 LPS

E50

E25

E16

E8 E4 E50

E25

E16

E8 E4C a

lbica

ns

120575

lowast








00

02

04

06

08

10

1h 24 h12 h

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

OD

500

nm

(a)

00

02

04

06

08

10


lowast

24 h12 h

OD

500

nm

(b)













Acknowledgment


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















7 8 9 10 11 12 13 14 15 16 17 18 19

Time (min)

1056

1169

1161

1208

13441122 1404

1482999 15681309965 1599 1653 1763 1888831796

1

2

3

4

5 6

7

810

911

12 13

200

406080

100120140160180200220times103

(120583AU

)

RT 700ndash1900


0102030405060708090

100110


Killi

ng (

)


CTRL

NaO

ClE5

0

E25

E16

E8 E4 CTRL

NaO

ClE5

0

E25

E16

E8 E4 CTRL

NaO

ClE5

0

E25

E16

E8 E4

aureus








4 Discussion






(a) (b) (c)

(d) (e)


0

2000

4000

6000

8000

Relat

ive fl

uore

scen

ce u

nit (

rfu)


Med

ium

E50

E25

E16

E8 E4 LPS

E50

E25

E16

E8 E4 E50

E25

E16

E8 E4C a

lbica

ns

120575

lowast








00

02

04

06

08

10

1h 24 h12 h

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

OD

500

nm

(a)

00

02

04

06

08

10


lowast

24 h12 h

OD

500

nm

(b)













Acknowledgment


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c)

(d) (e)


0

2000

4000

6000

8000

Relat

ive fl

uore

scen

ce u

nit (

rfu)


Med

ium

E50

E25

E16

E8 E4 LPS

E50

E25

E16

E8 E4 E50

E25

E16

E8 E4C a

lbica

ns

120575

lowast








00

02

04

06

08

10

1h 24 h12 h

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

OD

500

nm

(a)

00

02

04

06

08

10


lowast

24 h12 h

OD

500

nm

(b)













Acknowledgment


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















00

02

04

06

08

10

1h 24 h12 h

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

OD

500

nm

(a)

00

02

04

06

08

10


lowast

24 h12 h

OD

500

nm

(b)













Acknowledgment


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgment


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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