Research Article Evaluation of Mucoadhesive Gels with ...downloads.hindawi.com/journals/ecam/2013/641480.pdf · Vulvovaginal candidiasis is the second cause of vaginal infection in

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 641480 18 pageshttpdxdoiorg1011552013641480

Research ArticleEvaluation of Mucoadhesive Gels with Propolis (EPP-AF) inPreclinical Treatment of Candidiasis Vulvovaginal Infection

Andresa Aparecida Berretta12 Patriacutecia Alves de Castro1 Amanda Henriques Cavalheiro2

Vanessa Silveira Fortes2 Viniacutecius Pedro Bom1 Andresa Piacezzi Nascimento2

Franciane Marquele-Oliveira2 Viniacutecius Pedrazzi3 Leandra Naira Zambelli Ramalho4

and Gustavo Henrique Goldman15

1 Faculdade de Ciencias Farmaceuticas de Ribeirao Preto Universidade de Sao Paulo 14040-903 Ribeirao Preto SP Brazil2 Laboratorio de Pesquisa Desenvolvimento e Inovacao Apis Flora Indl Coml Ltda 14020-670 Ribeirao Preto SP Brazil3 Faculdade de Odontologia de Ribeirao Preto Universidade de Sao Paulo 14040-903 Ribeirao Preto SP Brazil4 Faculdade de Medicina de Ribeirao Preto Universidade de Sao Paulo 14040-040 Ribeirao Preto SP Brazil5 Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol-CTBE Caixa Postal 6170 13083-970 Campinas SP Brazil

Correspondence should be addressed to Andresa Aparecida Berretta andresaberrettahotmailcomand Gustavo Henrique Goldman ggoldmanuspbr

Received 29 March 2013 Revised 12 June 2013 Accepted 13 June 2013

Academic Editor Wojciech Krol

Copyright copy 2013 Andresa Aparecida Berretta et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Vulvovaginal candidiasis is the second cause of vaginal infection in the USA Clinical treatment of C albicans infections isroutinely performed with polyenes and azole derivatives However these drugs are responsible for undesirable side effects andtoxicity In addition C albicans azole and echinocandin resistance has been described Propolis is a bee product traditionallyused due to its antimicrobial anti-inflammatory and other properties Therefore the present work aimed to evaluate differentpropolis presentations in order to evaluate their in vitro and in vivo efficacy The methodologies involved antifungal evaluationchemical analysis and the effects of the rheological and mucoadhesive properties of propolis based gels The obtained resultsdemonstrated the fungicide action of propolis extracts against all three morphotypes (yeast pseudohyphae and hyphae) studiedThe highest level of fungal cytotoxicity was reached at 6ndash8 hours of propolis cell incubation Among the based gel formulationsdeveloped the rheological and mucoadhesive results suggest that propolis based carbopol (CP1) and chitosan gels were the mostpseudoplastic ones CP1 was the most mucoadhesive preparation and all of them presented low thixotropy Results of in vivoefficacy demonstrated that propolis based gels present antifungal action similar to clotrimazole cream suggesting that future clinicalstudies should be performed

1 Introduction

Vulvovaginal candidiasis is the second cause of vaginal infec-tion followed by bacterial vaginosis in the United States ofAmerica The costs involved with treatment diagnosis andthe loss of labor productivity are around $ 1 billion per yearAbout 13 million prescriptions for treating fungal infectionswere made in 1990 and these numbers were about doublethose from 1980 [1] Candida albicans is a fungal pathogenthat is found as a commensal in humans and is the most

common cause of mucosa and invasive fungal infections inhumans C albicans is a pleomorphic microorganism thatlives in the reproductive and gastrointestinal tracts in approx-imately half of the human population [2] The balancebetween normal microflora is essential for health since whenthis status is interrupted or immunological defenses are com-promised C albicans can be pathogenic due a transition inmorphological state from yeast to hyphae As a result C albi-cans infections are recognized as a serious challenge of publichealth with high social-economic and medical relevance [3]

2 Evidence-Based Complementary and Alternative Medicine

Clinical treatment of C albicans infections is routinely per-formed with polyenes azole derivatives allylamines thiocar-bamates fluoropyrimidines and echinocandins Howeverthese drugs are responsible for undesirable side effects andtoxicity In additionC albicans azole and echinocandin resis-tance has already been described [4ndash7] Thus consideringthe limited number of antifungal drugs and the continuousincrease of C albicans infection incidence it is important towork continuously in the development of new drugs to treatthis recurrent pathology especially new drugs with high ef-fectiveness and low adverse effects and costs

Propolis is a complex mixture produced by honey beesApis mellifera from the plant exudates consisting of resinousand balsamic materials The chemical composition includesflavonoids terpenoids phenylpropanoids and many othercompounds [8] It has been reported that the flavonoids andthe phenolic compounds are the main components respon-sible for the antibacterial antiviral and antifungal activitiesattributed to propolis extracts [9] Many different extractionprocesses have been reported for propolis extracts (eg alco-holic and aqueous) and currently the pharmaceutical tech-nology is focused on improving this field Many extracts thatfunction in different pharmaceutical applications such asmatrix microparticles and dry extracts have been reported[10] Therefore the characterization of chemical and antifun-gal properties of these new options of propolis extracts can bevaluable to the pharmacists who work with new products

Recent reports have suggested that the market formucoadhesive drug delivery systems is expanding rapidly[11] Various administration routes such as ocular nasal vag-inal rectal and others make mucoadhesive drug deliverysystems attractive and flexible in dosage form developmentThe advantages associated with the use of mucoadhesives indrug delivery systems include increased dosage form resi-dence time improved drug bioavailability reduced adminis-tration frequency simplified administration of a dosage formand termination of a therapy as well as the possibility of tar-geting particular body sites and tissues [11] In the presentwork we investigated different pharmaceutical forms ofpropolis extracts (alcoholic aqueous microparticles and dryextract) in order to choose one to composemucoadhesive gelsto treat vaginal candidiasis These semisolid pharmaceuticalpresentations were assessed both in vitro and in vivo andtheir efficacy as antifungal formulations against vulvovaginalcandidiasis was demonstrated

2 Materials and Methods21 Chemicals Purified water (Milli-Q) methanol HPLCgrade (J T Baker L9093-68) formic acid (Vetec L0804789)caffeic acid (Fluka L 43706045) 120588-coumaric acid (FlukaL3250759) trans-cinnamic acid (Fluka L21907066) isos-akuranetin (ChromaDex) artepillin C (Wako L 01619131)34-dicaffeoylquinic acid (34-DCQ) (Phytolab L13672938)35-dicaffeoylquinic acid (35-DCQ) (Phytolab L 13672946)45-dicaffeoylquinic acid (45-DCQ) (Phytolab L 13672903)trans-nerolidol (Sigma-Aldrich) gallic acid (Synth L109250)sodium bicarbonate (Vetec L0906112) and finally aroma-dendrin-41015840O-metil ether (previously isolated identified anddonated by Sousa et al [12]) were employed

22 Propolis Extracts Propolis ethanolic extract (PEE) prop-olis water extract (PWE) propolis matricial microparticles(PMM) and propolis soluble dry extract (PSDE) were sup-plied by Apis Flora Indl Coml Ltda (Ribeirao Preto SP Bra-zil) The extracts were obtained from a standardized propolisraw material (patent number PI0405483-0 Revista de Pro-priedade Industrial n 1778 2005) Since propolis is variableconsidering the plant vegetation of the area visited by honeybees propolis standardized raw material was prepared inorder to guarantee chemical reproducible composition of thepropolis in study For this the company employed a mixtureof propolis from different areas of Brazil for example MinasGerais Sao Paulo Parana and Santa Catarina states afterthe quality control of each one considering several micro-biological and physicochemical parameters and mainly theHPLC analysis according to what was previously publishedby Berretta et al [13] Propolis blend of standardized rawmaterial was previously cooled for 12 hours and pulverizedTo obtain PEE propolis raw material was macerated andpercolated with hydroalcoholic solution (7 3) and finallyfiltrated

PEE showed 11wv of extractable matter PWE PMMand PSDE were obtained from the same PEE batch

PMM was obtained by spray dryer employing PEE andexcipients such as modified corn starch (Capsul) and col-loidal silicon dioxide (1 1) The dryness condition involvedtemperature of 80∘C for sample exit sample flow of 120mLminute and system air output of 30m3min The spraydryer equipment used possessed a pneumatic atomizer with12mmof diameterThe ratio of drymatter and each excipientwas 1 05 05 resulting in approximately 50 of propolis drymatter in PMM

PSDE was produced after the dryness of PWE obtainedaccording to de Andrade et al [14] with modifications PEEwas submitted to complete evaporation of the solvent andto alkaline hydrolysis followed by purified water addition(PWE) Subsequently PWE was submitted to dryness withthe presence of maltodextrin (7 3 propolis excipient) byspray dryer process under the same condition used to PMM

23 HPLC Analysis Propolis extracts were evaluated forhigh pressure liquid chromatography (HPLC)with Shimadzuequipment with CBM-20A controller LC-20AT quaternarypump diode array detector SPD-M 20A and Shimadzu LCversion 121 SP1 software For analytical running ShimadzuShim-Pack CLC-ODS (M) column was used (46mm times 250mm with particle diameter of 5 120583m with porous diameter of100 A) To evaluate phenolic derivatives the eluent solutionconsisted of methanol and acidic water with formic acid(01ww) 20ndash95 77 minutes of running and 08mLminof flow [15] The detection was at 275 nm PEE PWE PMMand PSDE (119899 = 3) were individually diluted with metha-nolwater and homogenized with ultrasound bath After thatthe volume was acidified with formic acid to pH 270 Afterthe filtration (045120583m) 20120583L was injected in HPLC system

24 Strains Media and Culture Methods C albicans strainsused were SC5314 (wild type) [16] CAI4 (ura3imm434ura3imm434 iro1iro1imm434) [17] 3153A (wild type) (gener-ously donated by Paul Fidel Jr Department of Microbiology

Evidence-Based Complementary and Alternative Medicine 3

Immunology and Parasitology Louisiana State UniversityMedical Center New Orleans LA USA) Candida parapsilo-sisATCC 22019 and C glabrataATCC 90030 Saccharomycescerevisiae BY 4742 was also used The media used were thecomplete media YPD agar (2wv glucose 1wv yeastextract 2wv peptone and 2wv agar) and YPD liquidmedium with the same composition (but without agar)

25 Viability Determination and Kinetics Saccharomyces cer-evisiae BY 4742 Candida albicans SC5314 C albicans 3153AC parapsilosisATCC 22019C albicansCAI4 andC glabrataATCC 90030 were incubated in YPD liquid medium for 16hours (stationary phase growth) at 30∘C After this period1 times 106 cellsmL were inoculated into 30mL of YPD liquidin 125mL erlenmeyer flasks Cells were treated with 01250250 0500 075 and 100 of propolis dry matter fromPEE PWE PMM and PSDE For negative controls it wasemployed ethanol at the same amount present in each prop-olis concentration (PEE possess 55ww of ethanol) or phos-phate buffer solution (pH 74) The samples were maintainedin the medium for 1 2 4 6 8 12 and 24 hours The erlen-meyer flasks were kept in agitation of 180 rpm at 30∘C for24 hours After these periods of treatment in order to verifytheir viability tenfold serial dilution of these cells was platedon Petri dishes containing solid YPD medium They wereincubated at 30∘C for 24 hours Besides this procedure asample of treatments with 1 of propolis with 12 and 24 hsof incubation (119899 = 3) was dispersed in YPD medium incu-bated at 30∘C for 24 hours in order to plate and verify theviability of the strains in comparison with the viability of thestrains treated with the controls (alcoholic solution or PBS)All the colonies found in each plate were counted Controlscorrespond to 100 of viability and then the number ofcolonies found in the treatment groups was compared withthe controls in order to determine the percentage of viabilityin the presence of propolis extracts

In order to investigate the role of some phenolic com-pounds caffeoyl derivatives and the terpenoid trans-nerolidol present in propolis extracts in the antifungal actionthe isolated substances caffeic 119901-coumaric and cinnamicacids aromadendrin isossakuranetin Artepillin C and 3445 35 and 345 caffeoylquinic acids and finally trans-ner-olidol were studied in C albicans SC5314 For this purposeYPD liquid medium was used in 96 vessel plaque containing104 cellsvessel of C albicans and each standard was dilutedin DMSO and was evaluated at 1 2 3 4 5 125 50 75100 200 and 300 120583gvessel To control the experiment itwas also used (i) the inoculum group (ii) DMSO and (iii)fluconazole treatment The plaque was incubated at 30∘C for24 hours The evaluation involved visual inspection of ldquopel-letrdquo formation and the evaluation of viability For viabilityassessment 5 uL of each vessel content was put in YPD com-plete medium at 30∘C for 24 hs

26 Minimum Fungicidal Concentration (MFC) The brothdilution method recommended by the Clinical and Labo-ratory Standards Institute (document M27-A3 CLSI 2008)[18] was used in this study with some modifications inorder to determineminimum fungicidal concentration of the

samples In this study the following microorganisms wereused C albicans SC5314 C parapsilosis ATCC 22019 and Cglabrata ATCC 90030

Because of the turbidity that occurred in test brothwhen propolis extracts were diluted in the culture mediumit was not possible to determine the minimum inhibitoryconcentration (MIC)Therefore the antifungal activity of thesamples was assessed by means of the minimum fungicidalconcentration (MFC)whichwas determined by subculturing20120583L aliquots from each tube of the broth dilution seriesonto Potato Dextrose Agar (Difco Detroit MI USA) Theplates were incubated at 35∘C aerobically for 48 h After theincubation period the MFC was determined It was definedas the lowest concentration of the sample required to kill themicroorganism being tested

27 Propolis Effects during Morphologic Transition Aimingto evaluate C albicans sensitivity to propolis (PMM) (05subinhibitory concentration) in different morphologic typesSC5314 strain was inoculated in YPD medium for 16 hoursat 30∘C under agitation After that cells were counted and2 times 107 cellsmL were incubated for 4 hours under the sameprevious conditions (yeast) in YPD at 37∘C (to inducepseudo-hyphae) and in YPD liquid with bovine fetal serum(20) at 37∘C To control the experiment phosphate bufferwas used in substitution of PMM for the same time 6 hoursTo evaluate the efficiency of morphologic conditions thesample was evaluated by microscopy After treatments a ten-fold dilution was done from 106 cellsmL and ldquodrop outrdquounder YPD complete medium Plates were incubated for 24hours at 30∘C

28 Gel Preparation

281 Propolis Based Carbopol 940 Gel (CP1) Carbopol(1ww)was dispersed inwater previously conserved (potas-sium sorbate 01 and EDTA 001) and the polymer wasmaintained under hydration for 24 hs Then the mixtureof propolis extract (1) Melaleuca (Melaleuca alternifolia)sweet birch (Betula lenta)Mentha (Mentha spicata) and rose-mary (Rosmarinus officinalis) essential oils and propyleneglycol was added into the dispersion previously obtainedwithstirring Under stirring triethanolamine was used to adjustpH of the preparation

282 Propolis Based Poloxamer 407 Gel with Carbopol 940(PP1) Initially the co-polymer Poloxamer 407 (LutrolF127) (13ww) was dispersed in water and the polymer wasmaintained under hydration for 24 hours at 5∘C MeanwhileCarbopol (1ww) was dispersed in water previously con-served (potassium sorbate 01 and EDTA 001) and wasmaintained under hydration for 24 hours (phase B) Thenunder stirring the mixture of propolis extract (1) Mela-leuca (M alternifolia) sweet birch (B lenta) M spicata androsemary (R officinalis) essential oils and propylene glycolwas added gradually into the poloxamer dispersion previousobtained (phase A) Afterwards carbopol 940 dispersion wasmixed in phase A Under stirring triethanolamine was usedto adjust pH and confer the jellification of the preparationaround pH 60


283 Propolis Alginate with Pectin (AlP1) Sodium alginate(4) was dispersed in water andmaintained under hydrationfor 2 hoursThen themixture of pectin (2) propolis extract(1) Melaleuca (M alternifolia) sweet birch (B lenta) Mspicata and rosemary (R officinalis) essential oils and propy-lene glycol was added gradually into the dispersion previouslyobtained with stirring to complete homogenization

284 Propolis Based Chitosan Gel with Natrosol (ChP1) Todissolve and jellify natrosol (40) the sample was dispersedin water under warming at about 70∘C After that the prep-aration was cooled (A) Meanwhile chitosan (15) propolisextract (1) propylene glycol potassium sorbate EDTAMelaleuca (M alternifolia) sweet birch (B lenta) andM spi-cata and rosemary (R officinalis) essential oils were weightedand blended (B) Phase B was included in phase A under stir-ring Acetic acid was dripped and gel formation could beobserved

For all previous preparations controls were preparedusing alcoholic solution (55ww) in substitution of propolisand were identified as the controls CC-(carbopol) PC-(poloxamer) AlC-(alginate) and ChC- (chitosan) Moreovernone of the controls possess essential oils

29 Flow Rheology For this study a RS Plus RheometerBrookfield v90 was used coupled with a Peltier system withSoftware Brookfield RHEO 2000 version 28 The conditionof study was 375∘C to resemble body temperature and plateP25 was used (P25 module) because it is the analysis systemfor semisolids As parameter of analysis a race of 120 secondswas done with upward curve from 1199050 to 11990560 s and descendantfrom 11990560 s to 119905120 s obtaining values of shear stress shear rateand viscosity every 2 seconds Assays were done in triplicateand statistical analysis using software Brookfield based oncalculations by Ostwald Law and Prism 50 software

210 Mucoadhesion Tests To mucoadhesion studies thereproductive system of the cow was removed immediatelyafter sacrificing the animal in the slaughterhouse Olhos Drsquoagua (Ribeirao Preto SP Brazil) and vaginalmucosa removedwith scalpel and surgical scissors and immediately frozenat minus10∘C On the day of the experiment the mucosa wasthawed and cleaned using 09 NaCl solution at 25∘C cutinto disks of 1 cm diameter and gluedwith cyanoacrylate glue(SuperBonder) in a holder made of Plexiglass holderThe testwas done by lowering the load cell to contact with themucosafor a time of 30 seconds and tensile strength of 05N at arate of 05mmmin Triplicates were performed for each gelevaluated and statistical analysis with Prism 50 software Inthis study the machine Emic DL 2000 was used with a loadcell 19 Trd with the program Tesc version 301

211 Candidiasis Vaginal ldquoIn Vivordquo Model This experimentwas approved by the Animal Experimentation Ethics Com-mittee of the Faculty of Pharmaceutical Sciences Universityof Sao Paulo (FCFRPUSP) Ribeirao Preto SP Brazil To thisstudy female Balbc mice with 7-8 weeks of age weighingapproximately from 20 to 22 g were used in this study

This protocol was performed as described by Yano andFidel Jr [19] with some modifications Thus to induce

vaginal candidiasis in mice Balbc animals it was necessaryto simulate pseudoestrus condition for optimal Candidacolonization For that 03mg of estradiol valerate (17 120573-estra-diol valerate) previously dissolved in 100 uL of castor oil(Sigma-Aldrich) was injected in dorsal area of each animalfollowed by a massage to disperse the suspension 72 hoursbefore infection Pseudoestrus condition was maintainedweekly with the application of the hormone a condition nec-essary to guarantee the results of the protocol For the infec-tion 20120583L of theC albicans strain suspensionwas inoculatedby inserting the pipette tip about 5mm deep into the vaginallumen

2111 Candida albicans Suspension C albicans strain wasincubated for 24 hours in YPD complete medium at 30∘CThen one colony was dispersed in liquid YPD mediumunder agitation at 30∘C for 16 hours The suspension waswashed with PBS until a cleaner suspension was obtainedthen after centrifugation the pellet was suspended in PBScounted with Neubauer chamber and diluted to 25 times 106cellmL The suspension of Candida albicans was intravagi-nally inoculated (20120583L) [19]

2112 Standardization of the Protocol In order to define thebest strain and time after inoculation of C albicans to starttreatments the fungal burden and histological slices of theanimals after 48 and 72 hours after inoculation of C albicanswas investigated After that the protocol with the time prede-fined in the last protocol was used to compare between Calbicans SC5314 and 3153A

2113 Treatments Protocol The groups of animals weredivided into six (119899 = 10) (i) control (animals infectedwithouttreatment) (ii) treated with CPb (carbopol gel base) (iii)treatedwith PPb (poloxamer gel base) (iv) treatedwith CP1(propolis 1 carbopol gel) (v) treated with PP1 (propolis1 poloxamer gel) and finally (vi) treated with clotrimazolcream (Neo Quımica) Each group was treated every 12hours with 60 uL of each product (except for group onemdashwithout treatment) during 7 (seven) and 10 (ten) days Aftertreatments the evaluation of the animals was done with theculture of intravaginal lavage (100 uL of PBS) in each timeand with the histological evaluation of vaginal mucosa of oneanimal of each groupThen the animals were supervised dur-ing the 10 days after stopping of the treatment and re-evaluated with the culture of intravaginal lavage and the his-tological slices of the vaginal mucosa

One or two animals per group was euthanized (cervicaldislocation) with 7 and 10 days of treatment and in the con-clusion of the protocol (10 days after the last treatment) forvaginal mucous removal and histological slices preparationThe specimens were removed and fixed for 24 hours in 37formaldehydemdashPBS Samples were washed several times in70 alcohol before dehydration in a series of alcohol solu-tions of increasing concentrations Finally the samples werediaphonized in xylol and embedded in paraffin For eachsample sequential 5120583m thick sections were collected on glassslides and stained with Gomori methenamine silver (GMS)or hematoxylin and eosin (HampE) stain Briefly sections were


deparaffinized oxidized with 4 chromic acid stained withmethenamine silver solution and counterstained with picricacid For HampE staining sections were deparaffinized andstained first with hematoxylin and then with eosin Allstained slides were immediately washed preserved withmounting medium and sealed with a coverslip Next theywere analyzed and photographed under a microscope (Jen-aval-Zeiss) coupled to a digital camera (Leica DFC425) Theparameters analyzed were descriptive and examined by asingle researcher who was blinded to the analysis of thegroupsThe parameters analyzedwere inflammatory reactionand C albicans infection

212 Statistical Analysis The analysis of variance ANOVA(one way) and Bonferroni multiple comparison test was per-formed with a level of significance of 5 or by Studentrsquos 119905-test(120572 = 005) according to the protocol used Statistical analysiswas done using Prism 4 (Graph Pad)

3 Results

31 Chemical Characterization Although the standardiza-tion of propolis extract (EPP-AF) was previously shown byBerretta et al [13] the HPLC fingerprints of PMM and PSDEextracts were also performed (Figure 1) since these extrac-tion processes involve several steps such as temperaturefiltrationpurification dryness and others These steps couldprovide different chemical composition for each extract dif-fering from the same EEP standardized propolis batch Theresults of each extract are presented in Table 1 and the resultsnormalized to the same propolis concentration (1) areshown in Figure 2 It is possible to detect that the preparationprocess led to the absence of cinnamic acid and aroma-dendrin in PSDE and the absence of caffeic acid in PMM(lower than quantification limit) It was observed that PEEand PMM presented few differences considering most ofthe standards evaluated (normalized by propolis dry mat-ter) however artepillin C presented a reduction of about25 of the content present in PEE Interestingly PSDEshowed expressive values to artepillin C 0816mgg Caffeoylquinic acid derivatives were also evaluated since a recentwork attributed antiviral activity to these compounds [20]Results are presented in Figure 1 and Table 1 It is possibleto observe similarities in PEE and PMM corroborating theresults obtained with phenolic derivatives It is known thatpropolis aqueous extract usually presents caffeoyl quinic acidderivatives in their composition [20ndash22] however this is notthe case in the present work since PWE was obtained fromPEE and the extraction solvent was not water

Taken together the chemical characterization resultsshowed that PEE and PMM are similar (119875 gt 005) consider-ing the standards investigated while PSDE is different fromPEE and PMM (119875 lt 005)

32 Antifungal Comparison of Propolis Extracts in ldquoIn VitrordquoModels In order to compare the antifungal activities of eachextract PWE PEE PMMandPSDE (0125 025 and 050ofpropolis) were evaluated against Saccharomyces cerevisiae andC albicans (data not shown) The experimental conditions

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70(min)

800

50100150200250300350400

(mAU

)

1 2

3

45

6

7 8

9

10

275 nm 4 nm (100)

(a)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70(min)

80(m

AU)

0255075

100125150175200225250275

2

3

4

56

7 8

10275 nm 4 nm (100)

(b)

5 10 15 20 25 30 35 40 45 50 55 60 65 70 80(min)

(mAU

)

0255075

100125150175200225250275

3

2 45 6

7 8

10

275 nm 4 nm (100)

(c)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70(min)

80

(mAU

)

0255075

100125150175200225250275

32 45

6

10

275 nm 4 nm (100)

(d)

Figure 1 Chemical fingerprint of (a) standards used (1) internalstandard gallic acid (2) caffeic acid (3) p-coumaric acid (4)34-dicaffeoylquinic acid (5) 35-dicaffeoylquinic acid (6) 45-dicaffeoylquinic acid (7) cinnamic acid (8) aromadendrin (9)isosakuranetin and (10) Artepillin C and of the extracts evaluatedPEE (b) PMM (c) and PSDE (d) (119899 = 3) The chromatograms wereplotted at 275 nm using the RP-HPLC C18 (Shim-pack CLC-ODS(M) 25 cm times 46) column and gradient elution with methanol andacidic water (formic acid pH = 27)


Table 1 Fingerprint of chemical compounds of different propolis extracts evaluated (119899 = 3) (mgg)

Samples PEE PMM PSDEStandards Mean SD Mean SD Mean SDCaffeic acid 0345 0011 ND mdash 2699 0398119901-Coumaric acid 1712 0069 6312 0061 5979 068634-DCQ acid 0488 0006 1138 0014 5842 013235-DCQ acid 1614 0012 6147 0129 2667 009245-DCQ acid 3442 0023 13308 0203 7430 0045Cinnamic acid 0167 0006 0494 0006 ND mdashAromadendrin 0808 0024 2628 0024 ND mdashArtepillin C 6621 0306 6593 0348 51384 6036ND not detected

Chemical compounds

1 2 3 4 5 6 7 800

02

04

06

08

10

PEEPMMPSDE

(mg

g)

Figure 2 Chemical results obtained for PEE PMM and PSDE after normalization of the extracts to 1 of propolis dry matter (mgg) Theresults originated from the medium previously presented in Table 1 however in order to pollute the figure less the standard deviations weresuppressed (1) Caffeic acid (2) p-coumaric acid (3) 34-DCQ acid (4) 35-DCQ acid (5) 45-DCQ acid (6) cinnamic acid (7) aromadendrinand (8) artepillin C

such as propolis subinhibitory concentration appropriategrowth phase of microorganism and others were previouslydefined by de Castro et al [23] S cerevisiae was the lesstolerant strain to all tested extracts The results showed thatPEE was the most potent for both microorganisms followedby PWE PMM and PSDE The MFC was determined sinceit was not possible to establish the MIC determination dueto propolis turbidity The MFC value found was 70mgmLfor PEE considering the microorganisms C albicans and Cparapsilosis and 140mgmL forC glabrataThe other resultscan be seen in Table 2 where it is evident the better efficacyof PEE compared to the other extracts In this assay theMFCvalue of PWEwas very low and consequently this extract wasnot investigated in other steps including chemical character-ization After the first trial extracts were evaluated in propolisconcentrations from 025 to 10 against S cerevisiae Calbicans SC5314 and CAI4 strains C parapsilosis and Cglabrata (Figure 3) The results demonstrated that PEE wasmore effective than PMM and PSDE considering all strainstested (Figure 3) S cerevisiae followed by C albicans were

more sensitive than the other strains whileC parapsilosiswasthemost resistant strain studied (Figure 3)These results werecorroborated with the MFC values presented in Table 2 forPEE PMM and PSDE

To evaluate C albicans cell viability upon propolis expo-sure PMM was incubated for 1 2 4 6 8 12 and 24 hourswith 106 cellsmL The results showed that the best growthinhibition occurred at 6 and 8 hours of propolis exposure(Figure 4(a)) Unexpectedly at 24 hs incubation with PMMC albicans grew at the same density as the control(Figure 4(a)) Thus C albicans viability was assessed for alldifferent propolis extracts incubated for 12 and 24 hs andaccordingly fungicide and fungistatic actions of the extractswere evaluated considering the results of samples platedand incubated for 24 hs at 30∘C Viability results with 1 ofpropolis showed that PEE has fungicide effects while PMMand PSDE have fungistatic action at this concentration thatis 10mgmL and also considering the incubation time of12 and 24 hs (Figure 4(b)) Taken into consideration theresults observed the chemical changes among the extracts


Negative control PEE 0125 PEE 0250 PEE 0500 PEE 0750 PEE 100

106 105 104 103

(A)

(B)

(C)

(D)

(E)

(a)

PMM 0125 PMM 0250 PMM 0500 PMM 0750 PMM 100

106 105 104 103

Negative control

(A)

(B)

(C)

(D)

(E)

(b)

Negative control PSDE 0125 PSDE 0250 PSDE 0500 PSDE 0750 PSDE 100

106 105 104 103

(A)

(B)

(C)

(D)

(E)

(c)

Figure 3 Antimicrobial activity of different propolis extracts evaluated PEE PMM and PSDE using C albicans SC5314 (A) CAI4 (B) Scerevisiae (C) C parapsilosis (D) and C glabrata (E) strains with 0125 0250 050 075 and 10 The controls of the experimentwere the strain in YPD medium alcoholic solution in the same concentration of propolis extract and phosphate buffer The strains wereevaluated in YPD complete medium with propolis at 30∘C 24 hs

emerge as the possible responsible for these growth differen-ces Thus volatile substances were researched by Gas Chro-matography (GC) since both PMM and PSDE were obtainedwith high pressure and relatively high temperatures (spraydrying process) and consequently volatile substances couldbe different The results demonstrated that PEE presentedinnumerous volatile compounds specially trans-nerolidoland spathulenol (J P De Sousa amp J K Bastos data not pub-lished) while PMM and PSDE did not show any volatilesubstances in the analytical running used (data not shown)Although these volatile substances were different these iso-lated compounds were investigated and the results showedthat none of them demonstrated inhibitory action up to100 120583gvessel except for fluconazole that exhibited this effectupon 10 120583gvessel

The dimorphic transition is an important step for C albi-cans pathogenicity and the growth inhibition of differentmorphological forms like hyphae pseudohyphae and yeast(Figures 5(a) and 5(c)) was studied in the presence of propolis

Table 2 Minimum fungicidal concentration (MFC) of PEE PWEPMM and PSDE for distinct Candida strains (119899 = 3)

Sample MFC plusmn SD (mgmL)C albicans SC5314 C parapsilosis C glabrata

PEE 70 plusmn 00 70 plusmn 00 140 plusmn 00PWE 245 plusmn 00 245 plusmn 00 245 plusmn 00PMM 137 plusmn 00 275 plusmn 00 137 plusmn 00PSDE 1173 plusmn 00 1173 plusmn 00 586 plusmn 00

(PMM) Figure 5(a) shows that the treatment of C albicansduring 6 hours with PMM (05 propolis) is effective for alltransition stages and this fact is dependent on PMM con-centrations (Figure 5(b))

33 Propolis Gels Evaluation Antifungal Rheology andMucoadhesion in In Vitro Models Among all available poly-mers we have chosen for the present study chitosan sodium


5 120583L from theculture medium

10-fold serial dilution 5 120583L

C-

1 h of treatment 2 h 4 h

6 h 8 h 12 h 24 h

025

050

075

100

C-

025

050

075

100

(a)

Cellular viability

0

50

100

150

lowast

lowast

PEE PMM PSDEC-

Surv

ival

()

12 hs24 hs

(b)

Figure 4 (a) Antimicrobial activity of PMM using C albicans SC5314 with 0250 050 075 and 10 of propolis with 1 2 4 6 8 12and 24 hours of treatments (30∘C 24 hs under agitation) The controls of the experiment were the strain in YPD medium and PBS Afterthe treatments 5120583L and subsequently tenfold dilutions were applied in YPD complete medium with propolis at 30∘C 24 hs (b) graphicpresentation of cellular viability of C albicans treated during 12 and 24 hours with PEE PMM and PSDE 1 (119899 = 3)

alginate carbopol 940 and poloxamer 407 Scheme 1 Chi-tosan a cationic chain known to be an excellent material fordrug preparation is a plentiful natural biopolymer nontoxicbiocompatible and biodegradable [24] Alginic acids or thesalt forms sodium alginate are food ingredients and theyhave been used as additives for drug preparation due to theirsafety to oral administration and for controlling drugrelease and biocompatibility [25] Poloxamer 407 a non-ionic copolymer of poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) has been studied as a potential base forthermosensitive hydrogels It can carry sufficient drug andshows good water solubility tolerability biodegradabilitynontoxicity and controlled release [26] Carbopol 940 is across-linked polyacrylate polymer of anionic character It isan extremely efficient rheology modifier able to provide highviscosity and forms sparkling clear water or hydroalcoholic

gels and creams All formulations were evaluated for in vitroeffectiveness dissolving each one in YPD top agar mediumwith propolis at 0125 025 050 075 and 10 Sub-sequently tenfold serial dilution from S cerevisiae C albi-cans C parapsilosis and C glabrata cells was plated in eachpropolis concentration It was observed that all formulationsat concentrations higher than 05 propolis demonstratedsimilar results to every tested strain (data not shown) At con-centrations lower than 05 of propolis S cerevisiae was theleast propolis-tolerant microorganism After demonstratingthe in vitro effectiveness formulations were physicochemi-cally characterized aiming to perform the in vivo evaluation

Rheological studies are important to develop semisolidpreparations especially when certain characteristics shouldbe present such as easiness in product removal from packag-ing and application adequate spreading and smooth texture


Yeast

Hyphae

Yeast

HyphaePMM

-pro

polis

PMM treatment 05 propolis for 6 hours

Pseudo-hyphae

Pseudo-hyphae

0

20

40

60

80

100

120

Control 025 05 075 10Propolis concentration

Cel

lula

r via

bilit

y (

)

Leveduriform C albicans SC3514 viability with propolis

12 hs24 hs

Yeast

Hyphae

Morphology

Pseudohyphae

(a)

(b) (c)

1∘ 2∘ 3∘ 4∘ 5∘ 6∘ 7∘ 8∘

Figure 5 (a) Antimicrobial activity of PMMusingC albicans SC5314 (yeast pseudohyphae and hyphae) with 050 of propolis with 6 hoursof treatments (30∘C 24 hs under agitation) The controls of the experiment were the strain in YPD medium and PBS After the treatments5120583L and subsequently tenfold dilutions were applied in YPD complete medium with propolis at 30∘C 24 hs (b) graphic presentation ofcellular viability of C albicans treated during 12 and 24 hours with 025 050 075 and 10 of propolis (PMM) (119899 = 3) (c) microscopy ofthe strains used during protocol yeasts pseudohyphae and hyphae

on the application site [27] Some important features asspreadability and retention characteristics are essential toclinical outcomeof vaginal semisolids particularly thosewithcontraceptives and microbicidal activity [28]

Flow properties of vaginal formulations determine theease of product administration into the mucosa membraneand the (time-dependent) recovery of the product follow-ing administration In this context Newtonian and non-Newtonian behaviors can be seen In continuous shear rhe-ometry study all formulations exhibited shear-thinningbehavior (pseudoplastic flow) with low degrees of thixotropy(Figure 6(a)) The decreases in the non-Newtonian viscosityas a function of increasing shear rate were most appropriatelymathematically modelled using the Power law (Oswald-deWaele) model where the flow behavior index (119899) was deter-mined [29] Although Figure 6(a) clearly shows that all prep-arations presented pseudoplastic behavior its magnitude canonly be clearly observed by the results shown in Table 3 It isknown that the Newtonian characteristics are as higher as thevalue found for the material is closer to 1 and consequentlyas the value is lower than 1 the more pseudoplastic the mate-rial will be [28] Pseudoplasticity of poloxamer and chitosanbased gels was modified by propolis presence (Table 3) while

no changes were observed to sodium alginate and carbopolgels (119875 gt 005)The flow indexes values were 04455plusmn0001702554 plusmn 00095 02607 plusmn 00073 and 03674 plusmn 0007 forPP1 AlP1 CP1 and ChP1 respectively (Table 3) Asthe flow index values were all below 1 graphic representa-tion was confirmed showing that all formulations presentedpseudoplastic behavior [30] which is typical of the polymericsystem [28] There were significant differences (119875 lt 005)among all formulations except when comparing alginate andcarbopol (119875 gt 005)

Considering viscosity results Figure 6(b) shows that car-bopol and chitosan hydrogels are less viscous preparationswhile poloxamer is themost viscous oneThis is an interestingproperty that can help to retain the preparation into vaginalmucosa Although poloxamer presented the worst pseudo-plastic results it is important to consider that this gel base hasproperties such as thermoreversible behavior with increasedviscosity in corporeal temperature [31 32] and usually thiscomposition offers a delivery system information that can beinteresting in the pathology in study

An essential property that governs the clinical perfor-mance of mucosal gels is the ability to adhere to the host epi-thelium and provide residency during the therapeutic period


OO

OHO

HO

HO

HO

HO

HO HO

OH

OH

OH

OH

OH

OHCH

C C

C

2 CH2

NH2

NH2

NH2

CH2CH2 CH2O O

O

O

O O

O O

O

O OO

O

CH3

CH H

H H

H

Ominus

Ominus

a b c

m

n

n

n

ChitosanCarbopol

AlginatePoloxamer 407

Scheme 1

Hence mucoadhesive formulations of limited viscosity havebeen employed for this purpose [33]Themucoadhesive bondstrength was examined using a previously reported test inwhich cow vaginal mucosa was employed and the forcerequired to partially separate the mucosa disc from the sur-face of the formulation is determined The ability of a vagi-nally applied formulation to adhere to the vaginal epitheliumis essential to maximize its residency and thereby clinicalperformance [34] It has been reported that the establishmentof amucoadhesive bond between polymeric components anda biological substrate may be influenced by the surface ofthe biological substrate surface of the bioadhesive layer andinterfacial layer between the two [35] Assuming that the sur-face of the mucosa in each experiment is similar slight dif-ferences occurring in the mucoadhesive ability of the gelformulations may be attributed to the formulation surfaceeffects The results showed that propolis inclusion increasedmucoadhesion of CP1 and PP1 a fact not observed in algi-nate and chitosan vehicles (data not shown) It was observedthat the CP1 based gel demonstrated higher strength(024N) followed by ChP1 (018N) and PP1 (017N) Thedifference that favors poloxamer is the little time to reach themucoadhesion stabilization in comparison to chitosan basedgel This could be related to the differences in the polymerchain flexibility ability to form hydrogen bonds andor theextent of swelling of polymers

34 Propolis Gels Preclinical Efficacy Evaluation The in vivovulvovaginal candidiasismodelwas established 48 hours afterinoculation (Figure 7(a)) In this model C albicans 3153Astrain was more virulent than SC5314 (Figure 7(b)) a fact

corroborated by Figures 8(a)ndash8(d) The histological analysisdemonstrated that the control group (Figure 8(a)) displayednormal tissue while vaginal mucous infected with SC5314strain showed keratin deposition and decreased thickness abehavior more characteristic of chronic infection and com-mon in less virulent strains (Figure 8(b)) Finally Figures 8(c)and 8(d) showed superficial erosion and neutrophil influxrespectively features characteristic of acute infection

To evaluate efficacy two formulations were chosen con-sidering rheology and mucoadhesion results carbopol andpoloxamer gel with propolis extract the CP1 and PP1respectively Either preparation was applied every 12 hoursfor 7 and 10 days As a control a group did not receive anytreatment and two other groups were treated with carbopoland poloxamer gel base To compare propolis effects clotri-mazole (10mgg) cream (Neo Quımica Brazil) a conven-tional medicine used in this kind of pathology was used likea positive control

Our results showed that 7 days treatments reduced fungalburden in 602 848 and 972 for CP1 PP1 and clotri-mazole respectively while with 10 days treatment fungalburden reduction was 842 894 and 979 respectivelyStatistical analysis did not show any difference between 7 and10 days treatment (119875 gt 005) either using ANOVA one-wayor Studentrsquos 119905-test with 95 and 99 of confidence intervalAll treatments used demonstrated difference with negativecontrol groups (119875 lt 005) while CP1 andPP1were similarto clotrimazole cream (119875 gt 005) for 7 and 10 days of treat-ment respectively Figures 9(a) and 9(b)

The results presented are corroborated by histologicalanalysis upon 10 days treatment (Figures 10(a) and 10(b) the


0 15 30 45 600

250

500

750

1000

1250

1500

1750

AlP1PP1

CP1ChP1

D (1s)

120591(P

a)

(a)

0 10 20 30 40 50 600

40

80

120

160

200

AlP1PP1

CP1ChP1

D (1s)

Visc

osity

(Pa s

)

(b)

00 05 10 15 20000

005

010

015

020

025

PP1AlP1

ChP1CP1

Deformation (mm)

Stre

ngth

(N)

(c)

Figure 6 Rheograms of formulations were (a) presents shear stress (b) viscosity and (c) mucoadhesive results

Table 3 Yield exponent values to gel base and propolis gel (119899 = 3)

Samples Gel base Propolis gelPolymers Mean plusmn SD Mean plusmn SDPoloxamer 407 (Pluronic) 03845 plusmn 00098 04455 plusmn 00049Sodium alginate 02425 plusmn 00041 02554 plusmn 00126Carbopol 940 02750 plusmn 00060 02608 plusmn 00143Chitosan 03326 plusmn 00193 03674 plusmn 00212

control without any treatment) and Figures 10(c) and 10(d)(clotrimazole group) The results showed that estradiol min-istration maintained the pseudoestrus condition and conse-quent tissue infection considering that C albicans(Figure 10(b)) and inflammation (Figure 10(a)) persisted Incontrast the clotrimazole treatment presented normal epi-thelium (Figures 10(c) and 10(d)) The histological analysesof these experiments corroborate fungal burden analysisand in addition demonstrate the absence of irritation or

inflammation induced by CP1 and PP1 (Figures 11(a) and11(c)) Figures 11(a) and 11(c) show the morphological aspectsof tissue after 10 days of treatmentwithCP1andPP1whereboth groups displayed normal architecture and thickness inpseudoestrus phase with keratin deposition indicating theabsence of C albicans infection (Figures 11(b) and 11(d))

4 Discussion

C albicans is a common vaginal inhabitant in humans and ispresent in their vaginal mucosa during their lives normallynot displaying any symptoms or signs of vaginitis and usuallywith little concentration of yeasts C albicans can be a com-mensal or a pathogen into the vagina depending on changesin the host vaginal environment that induce the pathogenicstate [1] Vaginal candidiasis incidence caused by Candidanon-albicans strains has increased in function of unique anti-fungal dosage forms low dosage maintenance of the azoleposology and by indiscriminate use of antimicotics [1]


Groups

15 times 105

10 times 105

50 times 104

0C- 48 hs 72 hs

C a

lbica

ns S

C531

4 (c

ells

mL)

lowast

lowast

(a)

Control SC 5314 3153A

10 times 108

10 times 107

10 times 106

10 times 105

10 times 104

10 times 103

10 times 102

10 times 101

10 times 100

Groups n = 10

C a

lbica

ns (c

ells

mL)

(b)

Figure 7 (a) Fungal burden of vaginal mucosa lavage after 48 and 72 hours of inoculation of C albicans SC5314 (20 120583L 25 times 106 cellsmL)(YPD complete medium 30∘C 24 hs) (b) fungal burden of vaginal mucosa lavage after inoculation of C albicans SC5314 and 3153A with 48hours after inoculation (YPD complete medium 30∘C 24 hs) (lowast119875 lt 005mdashStudentrsquos 119905-test)

C albicans species represent 85ndash95 of yeasts isolated fromvagina and considering non-albicans strains the most com-mon is C glabrata present in 10ndash20 of the infected womenfollowed byC parapsilosis Azoles do not present good resultswith C glabrata strain [1]

Propolis ethanolic extracts alone or incorporated inpharmaceutical presentations are commonly used therapeu-tically [36] Considering some disadvantages of this presen-tation the present work tried to study other options to usepropolis in medicine such as aqueous microparticles andsoluble dry extract Rocha et al [37] have demonstrated thatit is possible to achieve aqueous propolis extract similar inchemical composition to the alcoholic ones and Bruschi et al[38 39] obtained gelatin microparticles with propolis with-out taste strong odour and without the presence of alcoholHere we evaluated PWE PMM and PSDE that were devel-oped with higher concentrations of propolis dry matter thatis around 11 50 and 70 of genuine propolis extractThe antifungal results obtained for PWE were different whencompared with the antibacterial results obtained by Rocha etal [37] and since undesirable results were found PWE wasnot considered in the mucoadhesive preparations

Antifungal effects of propolis in vitro have been demon-strated to C albicans by Fernandes Jr et al [40] results cor-roborated by Longhini et al [41] and the present workSawaya et al [42] who also studied the inhibition of C albi-cans C tropicalis C krusei C parapsilosis and others found20mgmL as the MFC for propolis alcoholic extract Thisconcentration is higher than the values found for PEE whichwas 140mgmL forC glabrata themost PEE resistant strainand for PSDE which was 1173mgmL to C albicans andC parapsilosis Berretta et al [13] have shown that differentbatches of propolis extracts were chemically reproducibleconsidering phenolic derivatives and also presented anti-inflammatory effects [13 15 21 31] However the extractsproposed in this study (PWE PMM and PSDE) can show

different chemical fingerprints since these kinds of prepara-tions involve several other steps including high pressure andtemperature This fact was confirmed once a few compoundsare absent in some extracts especially cinnamic acid andaromadendrin in PSDE and caffeic acid in PMM This pos-sibly occurred due to the temperature or pressure for PMMand PSDE necessary steps for the preparation of the pharma-ceutics presentations

Propolis like other natural products exert a biologicaction through synergic effect between numerous constit-uents and the results presented corroborate this informationsince none of the constituents studied presented anti-Can-dida action up to 100120583gvessel Based on the results obtainedhere for PEE (fungicide) PMM and PSDE (fungistatic) itis important to consider the volatile compounds of propolisfor the better action of propolis extracts since the differencein PEE and the other extracts by GC analysis was so out-standing Considering the inherent lipophilic characteristicof terpenoids they show affinity and partition with biologicalmembranes where its presence can substantiallymodify theirstructural and functional properties [43] trans-Nerolidol isa known compound that increases bacterial plasmatic mem-brane permeability (S aureus and E coli) [44] and actsin inhibiting the dimorphic transition of C albicans [45]Brehm-Stecher and Johnson [44] showed that sesquiter-penoids can break the normal barrier of cellular membranesof bacteria increasing the uptake of exogenous compounds tointracellular medium such as ethidium bromide and antibi-otics This effect was more pronounced in Gram-positivebacteria possibly because they lack additional barriers to theexternal membrane for example Gram-negative bacteriaBrehm-Stecher and Johnson [44] evaluated the ability ofnerolidol farnesol bisabolol and apritone sesquiterpenoidsto increase the bacterial permeability and the susceptibility ofexogenous antimicrobial compounds uptake These authorsdemonstrated that sesquiterpenoids promoted intracellular


(a) (b)

(c) (d)

Figure 8 (a) Control group (without infection) vaginal mucosa with normal architecture and normal thickness showing discrete keratindeposition (HampE-40x) (b) C albicans SC5314 infection group normal architecture with decreased thickness with signs of epithelialregeneration (mitosis) and increased of keratin deposition (HampE-40x) (c) C albicans 3153A infection normal thickness with precursorcells increase superficial erosion and neutrophils influx without keratin (HampE-40x) (d) neutrophils presence in lavage microscopy (ASD-Naphtol-20x)

7 days treatment

0

20

60

40

C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo

lowast

lowastlowast

times105

(a)

10 days treatment

0

50

40

30

20

10C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo

lowast lowast lowast

times105

(b)

Figure 9 Fungal burden of vaginal mucosa lavage after 7 (a) and 10 days (b) of treatment with CP1 PP1 and clotrimazol cream (NeoQuımica) after infection of C albicans 3153A (20120583L 25 times 106 cellsmL) (YPD complete medium 30∘C 24 hs) (lowast119875 lt 005mdashANOVA one-waywith Bonferronirsquos posttest)

accumulation of ethidium bromide a nucleic acid markerimpermeable to cellular membrane in Lactobacillus fermen-tum living cells suggesting that the increase of permeabilitywas a result of cytoplasmatic membrane disorder Hornby etal [45] showed that nerolidol and farnesol prevented dimor-phic transition of C albicans from yeast to mycelial form

Considering that hyphae are associated with a pathogenicsituation the inhibition of transition can be a nonlethal wayto control this pathogen

Due to their low cost ease of manufacture and prece-dence of use in the topical administration of drugs conven-tional gel systems are commonly employed to administer


(a) (b)

(c) (d)

Figure 10 Histological slices of vaginal mucosa with 10 days of infection showing ((a)-(b)) negative control group and ((c)-(d)) clotrimazolecream treatment (10 days of treatment) Tissular inflammation can be seen in ldquoardquo and ldquocrdquo with the presence of neutrophils influx (HampEcoloration) GMS coloration (b and d) shows C albicans presence in Control group (b) showing the efficacy of the model implementationand absence of C albicans in clotrimazole at this time (d) (times40 magnification)

drugs via the vaginal route mainly for the treatment of vagi-nal infection contraception and hormone replacement ther-apy More recently gel-based formulations are being widelydeveloped for sustained delivery of HIV microbicides andmucosal vaccines The retention of vaginal gel formulationsis fundamental to the improvement of clinical performancePoor vaginal retention of conventional gel formulations rep-resents a significant challenge for those clinical indicationswhere sustained delivery would enhance efficacy [11] Flowrheological characterization offered important informationconsidering stability and spreadability Newtonian and non-Newtonian behavior thixotropy and viscosity and importantpoints to consider when developing a topical medicine Itis known that a pseudoplastic material can break down foreasy spreading and the applied film can gain viscosity instan-taneously to resist running a fact not observed with Newto-nian fluids independent of the time Pseudoplasticity is com-monly observed in polymeric semisolid preparations suchas skin moisturizing vaginal microbicides sunscreens forexample [28 29] Moreover Newtonian fluids that is thosewhose viscosity is similar independently of shear rate whereviscosity is measured are interesting in developing spermi-cides [28] or massage fluids such as mineral oil Besidestherapeutical application rheological information possiblypredicts flowing easiness of the package intravaginal applica-tion and retention therein and associated with the viscosity

and mucoadhesion results it is possible to suggest in situbehavior Considering rheological data our results indicatethat CP1 and PP1 were the most promising formulationsbecause of their pseudoplasticity and the viscosity respec-tively The nonlinear responses to shear stresses exhibited bythe formulations under study were probably a result of struc-tural changes caused by shearingThe formulations consistedprimarily of high-molecular weight components organizedinmicelles with the water Following exposure to shear stressthe dispersion could flow and the chains of the polymerscould align along the direction of shear releasing water orwater and propolis As a result subsequent shearing occurredmore readily and the apparent viscosity was decreased favor-ing the flow a fact that was reversed with the absence of shearstress Furthermore the low degree of thixotropy indicatesthat the restoration of the original configuration would re-quire only a short time after removal of the shear stress (afterthe administration) therefore enhancing retention therein

Mucoadhesive polymers have been exploited for severaldecades by pharmaceutical scientists to formulate novel dos-age forms for various routes of drug administration (buccaloral nasal ocular and vaginal)The research in this area con-tinues to develop very quickly with more than a hundrednew papers being published each year (for revision see [46])The current efforts in this area are focused on the designof mucoadhesive polymers since the characteristic of most


(a) (b)

(c) (d)

Figure 11 Histological slices of vaginal mucosa after 10 days of infection showing CP1 group ((a)-(b)) and PP1 ((c)-(d)) treatmentduring 10 days Normal tissue can be seen in all pictures (HampE and GMS coloration) and the absence of inflammation and C albicans(times40 magnification)

mucins is carrying a negative charge net due to the presenceof carboxylic groups (sialic acid) and ester sulfates at the ter-minus of some sugar units Additionally the approximatep119870119886 of these acidic groups is 10ndash26 resulting in their com-plete ionization under physiological conditions [46] which isan interesting interface to the adherence of some polymers

Regarding the mucoadhesive strengths presented hereit is possible to observe that the results obtained that ismucoadhesion for carbopol poloxamer sodium alginate andchitosan polymers are in accordance with other authors [1027 47] Although in some cases mucoadhesive strength waslarger than 030N such as nonsteroidal anti-inflammatorygels evaluated by Barry [48] Perioli et al [49] evaluated chi-tosan gels to vaginal application (05 and 20) with 0043and 0093N of mucoadhesion Moreover Cid et al [27]found values of 009ndash020N for chitosan gels (1 and 3)Considering that ChC- and ChP1 offered 033 and 037Nrespectively the results obtained were very interesting andcoherent since although ChC- and ChP1 possess 15 ofchitosan the effect was complemented with the presence ofnatrosol (cellulose derivate)

The results showed that carbopol was the most effectivemucoadhesive polymer Initially it was expected that chi-tosan due to its positive charges from amino groups couldbe the most interesting formulation to electrically adhere tovaginal mucous and epithelium (negatively charged) how-ever it was not successful Moreover concerning the longpolymeric chain of carbopol rich in carboxyl and hydroxyl

groups it was possible to foresee the numerous hydrogenbonds and Van der Waals forces that can be done betweenpolymeric chain and charges of mucous and epitheliumThis observation justifies the results obtained in which thenumber of interactions of carbopol is higher than the inter-actions done by amino groups of chitosan therefore theeffect observed is coherent

Results of physicochemical and in vitro biological charac-terization showed that all formulations were effective againstallCandida strainsThe rheological andmucoadhesive resultssuggest that CP1 and ChP1 were the most pseudoplasticformulations CP1 and ChP1 were less viscous prepara-tions while poloxamer was the most viscous gel CP1 wasthe most mucoadhesive preparation followed by both PP1and ChP1 All preparations possess low thixotropy Takentogether it is possible to suggest that all formulations can flowunder shear stress a fact that can be restored after the stop-ping of strength application (pseudoplastic behavior with lit-tle thixotropy) Considering this data it is interesting to usea deformable packing to all preparations obtained The vis-cosity and mucoadhesion can suggest that carbopol andpoloxamer are interesting preparations to proceed biologicalefficacy tests

Vaginally applied imidazoles are the first line treatmentof vulvovaginal candidiasisThe need to develop a less incon-venient andmore patient acceptable regimen led to the devel-opment of a treatment schedule from the original 3 weeks to7 days protocols [50] Currently some topical preparations


like clotrimazole 500mg vaginal tablet 2 65 tioconazole(Vagistat-1) and 2 miconazole ovule (Monistat1) amongothers are currently available in USA This class of com-pounds is fungicidal only at high concentrations and afterprolonged incubation [51] Gonzalez et al [52] evaluated vor-iconazole and fluconazole therapies by oral or topical route(05 1 and 5mgkg once daily) where results showed thatall therapy regimens administered topically for the treatmentof vaginal infection significantly reduced the fungal loadwith respect to the negative control group (excipient) Con-sidering topical treatments at analogous doses voriconazolewas as effective as fluconazole nevertheless neither drugadministered orally nor topically was able to eradicate thismicroorganism from the vagina [52] These results were cor-roborated by Stevens et al [53] in topical treatments withclotrimazole and zeamantine treatments Scott et al [51]evaluated in systemically and topical murine models a newclass of antifungal agents jasplakinolide a structure derivedfrom marine sponge The topical administration of jasplaki-nolide 2 was very effective in reducing infection with re-sults showing 53of negative cultures in this group 47withmiconazole nitrate and 7 of the control (4 days after infec-tion) however the toxicity of this structure compromised itsutilization

The analysis of the results presented here can suggest thatsome improvements in propolis formulations possibly canincrease the response in animal treatments Besides propolisconcentration maybe some changes in the thixotropy of theformulations or the presence of an oil phase can improve thedelivery frompropolis extracts ofmore lipophilic compoundsfrom pharmaceutical systems Actually this was the wayBachhav and Patravale [54] improved fluconazole vaginaleffects offering completely eradication of themicroorganism

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

The authors are grateful to Conselho Nacional de Desen-volvimento Cientıfico e Tecnologico (CNPq) Fundacao deAmparo a Pesquisa do Estado de Sao Paulo (FAPESP) Finan-ciadora de Estudos e Projetos (FINEP) and Apis Flora IndlComl Ltda all from Brazil for financial support and forgranting research fellowships Also the authors thank Raul deBarros Ferreira FelipeMiguel Galetti and VinıciusMatos fordiscussing protocols and results and for technical assistancerespectively

References

[1] J D Sobel ldquoVulvovaginal candidosisrdquo Lancet vol 369 no 9577pp 1961ndash1971 2007

[2] P L Fidel Jr ldquoVaginal candidiasis review and role of local mu-cosal immunityrdquo AIDS Patient Care and STDs vol 12 no 5 pp359ndash366 1998

[3] J R Naglik S J Challacombe and B Hube ldquoCandida albicanssecreted aspartyl proteinases in virulence and pathogenesisrdquo

Microbiology and Molecular Biology Reviews vol 67 no 3 pp400ndash428 2003

[4] P L Fidel Jr J L Cutright and J D Sobel ldquoEfficacy of D0870treatment of experimental Candida vaginitisrdquo AntimicrobialAgents and Chemotherapy vol 41 no 7 pp 1455ndash1459 1997

[5] H Lotz H Brunner and S Rupp ldquoHyphae-specific cell wallproteins of Candidardquo US20070196378A1 2007

[6] S Perea J L Lopez-Ribot W R Kirkpatrick et al ldquoPrevalenceof molecular mechanisms of resistance to azole antifungalagents inCandida albicans strains displaying high-level flucona-zole resistance isolated from human immunodeficiency virus-infected patientsrdquo Antimicrobial Agents and Chemotherapy vol45 no 10 pp 2676ndash2684 2001

[7] M A Pfaller and D J Diekema ldquoRare and emerging oppor-tunistic fungal pathogens concern for resistance beyond Can-dida albicans and Aspergillus fumigatusrdquo Journal of ClinicalMicrobiology vol 42 no 10 pp 4419ndash4431 2004

[8] B A Rocha M R Rodrigues P C P Bueno et al ldquoPreparationand thermal characterization of inclusion complex of Braziliangreen propolis and hydroxypropyl-120573-cyclodextrin increasedwater solubility of the chemical constituents and antioxidantactivityrdquo Journal of Thermal Analysis and Calorimetry vol 108no 1 pp 87ndash94 2012

[9] N Volpi and G Bergonzini ldquoAnalysis of flavonoids from prop-olis by on-line HPLC-electrospray mass spectrometryrdquo Journalof Pharmaceutical and Biomedical Analysis vol 42 no 3 pp354ndash361 2006

[10] M L Bruschi D S Jones H Panzeri M P D Gremiao ODe Freitas and E H G Lara ldquoSemisolid systems containingpropolis for the treatment of periodontal disease in vitro releasekinetics syringeability rheological textural andmucoadhesivepropertiesrdquo Journal of Pharmaceutical Sciences vol 96 no 8 pp2074ndash2089 2007

[11] G P Andrews T P Laverty and D S Jones ldquoMucoadhesivepolymeric platforms for controlled drug deliveryrdquo EuropeanJournal of Pharmaceutics and Biopharmaceutics vol 71 no 3pp 505ndash518 2009

[12] J P B de Sousa P C P Bueno L E Gregorio et al ldquoA reliablequantitative method for the analysis of phenolic compoundsin Brazilian propolis by reverse phase high performance liquidchromatographyrdquo Journal of Separation Science vol 30 no 16pp 2656ndash2665 2007

[13] A A Berretta A P Nascimento P C P Bueno M M D O LLeite Vaz and J M Marchetti ldquoPropolis standardized extract(EPP-AF) an innovative chemically and biologically repro-ducible pharmaceutical compound for treating woundsrdquo Inter-national Journal of Biological Sciences vol 8 no 4 pp 512ndash5212012

[14] U V C de AndradeWHartmann S Funayama SM de Alen-car and M L Masson ldquoPropolis obtained by means of alka-line hydrolysis and action on Staphylococcus aureusrdquo Ars Vet-erinaria vol 25 no 3 pp 151ndash154 2009

[15] J L Machado A K M Assuncao M C P da Silva et al ldquoBra-zilian green propolis anti-inflammatory property by an im-munomodulatory activityrdquo Evidence-Based Complementary andAlternativeMedicine vol 2010 Article ID 157652 10 pages 2012

[16] A M Gillum E Y H Tsay and D R Kirsch ldquoIsolation of theCandida albicans gene for orotidine-51015840-phosphate decarboxy-lase by complementation of S cerevisiae ura3 and E coli pyrFmutationsrdquo Molecular and General Genetics vol 198 no 1 pp179ndash182 1984


[17] W A Fonzi and M Y Irwin ldquoInvestigationsmdashisogenic strainconstruction and gene mapping in Candida albicansrdquo Geneticsvol 134 no 3 pp 717ndash728 1993

[18] Clinical and Laboratory Standards Institute (CLSI) ReferenceMethod for Broth Dilution Antifungal Susceptibility Testing ofYeasts Approved Standard CLSI document M27-A3 3rd edi-tion 2008

[19] J Yano and P L Fidel Jr ldquoProtocols for vaginal inoculation andsample collection in the experimental mouse model of Candidavaginitisrdquo Journal of Visualized Experiments no 58 article 33822011

[20] T Urushisaki T Takemura S Tazawa et al ldquoCaffeoylquinicacids aremajor constituentswith potent anti-influenza effects inbrazilian green propolis water extractrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2011 Article ID 2549147 pages 2011

[21] S P Andrade S A LDMoura GNegri et al ldquoAqueous extractof Brazilian green propolis primary components evaluationof inflammation and wound healing by using subcutaneousimplanted spongesrdquo Evidence-Based Complementary and Alter-native Medicine vol 2011 Article ID 748283 8 pages 2011

[22] T Matsui S Ebuchi T Fujise et al ldquoStrong antihyperglycemiceffects of water-soluble fraction of Brazilian propolis and itsbioactive constituent 345-tri-O-caffeoylquinic acidrdquo Biologi-cal and Pharmaceutical Bulletin vol 27 no 11 pp 1797ndash18032004

[23] P A de Castro M Savoldi D Bonatto et al ldquoMolecular char-acterization of propolis-induced cell death in Saccharomycescerevisiaerdquo Eukaryotic Cell vol 10 no 3 pp 398ndash411 2011

[24] K Kofuji T Ito Y Murata and S Kawashima ldquoBiodegradationand drug release of chitosan gel beads in subcutaneous airpouches of micerdquo Biological and Pharmaceutical Bulletin vol24 no 2 pp 205ndash208 2001

[25] Y Murata S Toniwa E Miyamoto and S Kawashima ldquoPrepa-ration of alginate gel beads containing chitosan salt and theirfunctionrdquo International Journal of Pharmaceutics vol 176 no2 pp 265ndash268 1999

[26] J J Xuan P Balakrishnan D H Oh et al ldquoRheological char-acterization and in vivo evaluation of thermosensitive polox-amer-based hydrogel for intramuscular injection of piroxicamrdquoInternational Journal of Pharmaceutics vol 395 no 1-2 pp 317ndash323 2010

[27] Y P Cid V Pedrazzi V P de Sousa andM B R Pierre ldquoIn vitrocharacterization of chitosan gels for buccal delivery of celecoxibinfluence of a penetration enhancerrdquo Amercian Association ofPharmaceutical Scientists vol 13 no 1 pp 101ndash111 2012

[28] J Das Neves M V Da Silva M P Goncalves M H Amaraland M F Bahia ldquoRheological properties of vaginal hydrophilicpolymer gelsrdquo Current Drug Delivery vol 6 no 1 pp 83ndash922009

[29] L R Gaspar and P M B G Maia Campos ldquoRheological behav-ior and the SPF of sunscreensrdquo International Journal of Pharma-ceutics vol 250 no 1 pp 35ndash44 2003

[30] A Martin Physical Pharmacy Lea amp Febiger Philadelphia PaUSA 4th edition 1993

[31] A A and Berretta Desenvolvimento e avaliacao de uma for-ma farmaceutica de liberacao sustentada contendo extrato pa-dronizado de propolis para o tratamento de queimaduras [disser-tacao de mestrado] Faculdade de Ciencias Farmaceuticas deRibeirao Preto Universidade de Sao Paulo Sao Paulo Brazil2003

[32] E J Ricci M V L B Bentley M Farah R E S Bretas and JM Marchetti ldquoRheological characterization of Poloxamer 407lidocaine hydrochloride gelsrdquo European Journal of Pharmaceu-tical Sciences vol 17 no 3 pp 161ndash167 2002

[33] G P Andrews and D S Jones ldquoRheological characterizationof bioadhesive binary polymeric systems designed as platformsfor drug delivery implantsrdquo Biomacromolecules vol 7 no 3 pp899ndash906 2006

[34] J das Neves and M F Bahia ldquoReviewmdashgels as vaginal drugdelivery systemsrdquo International Journal of Pharmaceutics vol318 no 1-2 pp 1ndash14 2006

[35] AGMikos andNA Peppas ldquoMeasurement of the surface ten-sion ofmucin solutionsrdquo International Journal of Pharmaceuticsvol 53 no 1 pp 1ndash5 1989

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

[37] B A Rocha P C P Bueno M M O L L Vaz et alldquoEvaluation of a propolis water extract using a reliable RP-HPLC methodology and in vitro and in vivo efficacy and safetycharacterisationrdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2013 Article ID 670451 11 pages 2013

[38] M L Bruschi M L C Cardoso M B Lucchesi and M P DGremiao ldquoGelatin microparticles containing propolis obtainedby spray-drying technique preparation and characterizationrdquoInternational Journal of Pharmaceutics vol 264 no 1-2 pp 45ndash55 2003

[39] M L Bruschi EHG Lara CHGMartins et al ldquoPreparationand antimicrobial activity of gelatin microparticles containingpropolis against oral pathogensrdquo Drug Development and Indus-trial Pharmacy vol 32 no 2 pp 229ndash238 2006

[40] A Fernandes Jr E C Balestrin J E C Betoni R De OliveiraOrsi M D L R De Souza Da Cunha and A C MontellildquoPropolis anti-Staphylococcus aureus activity and synergismwith antimicrobial drugsrdquoMemorias do Instituto Oswaldo Cruzvol 100 no 5 pp 563ndash566 2005

[41] R Longhini S M Raksa A C P Oliveira T I Svidzinski andS L Franco ldquoObtencao de extratos de propolis sob diferentescondicoes e avaliacao de sua atividade antifungicardquo RevistaBrasileira Farmacognosia vol 17 no 3 pp 388ndash395 2007

[42] A C H F Sawaya A M Palma F M Caetano et al ldquoCom-parative study of in vitro methods used to analyse the activityof propolis extracts with different compositions against speciesof Candidardquo Letters in Applied Microbiology vol 35 no 3 pp203ndash207 2002

[43] J Sikkema J A M De Bont and B Poolman ldquoMechanisms ofmembrane toxicity of hydrocarbonsrdquo Microbiological Reviewsvol 59 no 2 pp 201ndash222 1995

[44] B F Brehm-Stecher and E A Johnson ldquoSensitization of Staph-ylococcus aureus and Escherichia coli to antibiotics by the ses-quiterpenoids nerolidol farnesol bisabolol and apritonerdquoAnti-microbial Agents and Chemotherapy vol 47 no 10 pp 3357ndash3360 2003

[45] J M Hornby E C Jensen A D Lisec et al ldquoQuorum sensingin the dimorphic fungus Candida albicans is mediated by far-nesolrdquo Applied and Environmental Microbiology vol 67 no 7pp 2982ndash2992 2001

[46] V V Khutoryanskiy ldquoAdvances in mucoadhesion and mucoad-hesive polymersrdquo Macromolecular Bioscience vol 11 no 6 pp748ndash764 2011

[47] S A Mortazavi and J D Smart ldquoAn investigation of some fac-tors influencing the in vitro assessment of mucoadhesionrdquo


International Journal of Pharmaceutics vol 116 no 2 pp 223ndash230 1995

[48] B W Barry ldquoRecent advanced in pharmaceutical rheologyrdquoJournal of Texture Studies vol 1 pp 405ndash430 2007

[49] L Perioli V Ambrogi L Venezia C Pagano M Ricci and CRossi ldquoChitosan and a modified chitosan as agents to improveperformances of mucoadhesive vaginal gelsrdquo Colloids and Sur-faces B vol 66 no 1 pp 141ndash145 2008

[50] M R Henzl ldquoProperties of sustained-release single-dose for-mulations for vulvovaginal candidiasis the example of buto-conazole vaginal emulsion as a drug-sparing formulationrdquoAmerican Journal of Drug Delivery vol 3 no 4 pp 227ndash2372005

[51] V R Scott R Boehme and T R Matthews ldquoNew class ofantifungal agents jasplakinolide a cyclodepsipeptide from themarine sponge Jaspis speciesrdquoAntimicrobial Agents and Chem-otherapy vol 32 no 8 pp 1154ndash1157 1988

[52] G M Gonzalez O J Portillo G I Uscanga et al ldquoTherapeuticefficacy of voriconazole against a fluconazole-resistant Candidaalbicans isolate in a vaginal modelrdquo Journal of AntimicrobialChemotherapy vol 64 no 3 pp 571ndash573 2009

[53] D A Stevens L Calderon M Martinez K V Clemons S JWilson and C P Selitrennikoff ldquoZeamatin clotrimazole andnikkomycin Z in therapy of a Candida vaginitis modelrdquo Journalof Antimicrobial Chemotherapy vol 50 no 3 pp 361ndash364 2002

[54] Y G Bachhav and V B Patravale ldquoPharmaceutical nano-technologymdashmicroemulsion based vaginal gel of fluconazoleformulation in vitro and in vivo evaluationrdquo International Jour-nal of Pharmaceutics vol 365 no 1-2 pp 175ndash179 2009

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


Clinical treatment of C albicans infections is routinely per-formed with polyenes azole derivatives allylamines thiocar-bamates fluoropyrimidines and echinocandins Howeverthese drugs are responsible for undesirable side effects andtoxicity In additionC albicans azole and echinocandin resis-tance has already been described [4ndash7] Thus consideringthe limited number of antifungal drugs and the continuousincrease of C albicans infection incidence it is important towork continuously in the development of new drugs to treatthis recurrent pathology especially new drugs with high ef-fectiveness and low adverse effects and costs

Propolis is a complex mixture produced by honey beesApis mellifera from the plant exudates consisting of resinousand balsamic materials The chemical composition includesflavonoids terpenoids phenylpropanoids and many othercompounds [8] It has been reported that the flavonoids andthe phenolic compounds are the main components respon-sible for the antibacterial antiviral and antifungal activitiesattributed to propolis extracts [9] Many different extractionprocesses have been reported for propolis extracts (eg alco-holic and aqueous) and currently the pharmaceutical tech-nology is focused on improving this field Many extracts thatfunction in different pharmaceutical applications such asmatrix microparticles and dry extracts have been reported[10] Therefore the characterization of chemical and antifun-gal properties of these new options of propolis extracts can bevaluable to the pharmacists who work with new products

Recent reports have suggested that the market formucoadhesive drug delivery systems is expanding rapidly[11] Various administration routes such as ocular nasal vag-inal rectal and others make mucoadhesive drug deliverysystems attractive and flexible in dosage form developmentThe advantages associated with the use of mucoadhesives indrug delivery systems include increased dosage form resi-dence time improved drug bioavailability reduced adminis-tration frequency simplified administration of a dosage formand termination of a therapy as well as the possibility of tar-geting particular body sites and tissues [11] In the presentwork we investigated different pharmaceutical forms ofpropolis extracts (alcoholic aqueous microparticles and dryextract) in order to choose one to composemucoadhesive gelsto treat vaginal candidiasis These semisolid pharmaceuticalpresentations were assessed both in vitro and in vivo andtheir efficacy as antifungal formulations against vulvovaginalcandidiasis was demonstrated

2 Materials and Methods21 Chemicals Purified water (Milli-Q) methanol HPLCgrade (J T Baker L9093-68) formic acid (Vetec L0804789)caffeic acid (Fluka L 43706045) 120588-coumaric acid (FlukaL3250759) trans-cinnamic acid (Fluka L21907066) isos-akuranetin (ChromaDex) artepillin C (Wako L 01619131)34-dicaffeoylquinic acid (34-DCQ) (Phytolab L13672938)35-dicaffeoylquinic acid (35-DCQ) (Phytolab L 13672946)45-dicaffeoylquinic acid (45-DCQ) (Phytolab L 13672903)trans-nerolidol (Sigma-Aldrich) gallic acid (Synth L109250)sodium bicarbonate (Vetec L0906112) and finally aroma-dendrin-41015840O-metil ether (previously isolated identified anddonated by Sousa et al [12]) were employed

22 Propolis Extracts Propolis ethanolic extract (PEE) prop-olis water extract (PWE) propolis matricial microparticles(PMM) and propolis soluble dry extract (PSDE) were sup-plied by Apis Flora Indl Coml Ltda (Ribeirao Preto SP Bra-zil) The extracts were obtained from a standardized propolisraw material (patent number PI0405483-0 Revista de Pro-priedade Industrial n 1778 2005) Since propolis is variableconsidering the plant vegetation of the area visited by honeybees propolis standardized raw material was prepared inorder to guarantee chemical reproducible composition of thepropolis in study For this the company employed a mixtureof propolis from different areas of Brazil for example MinasGerais Sao Paulo Parana and Santa Catarina states afterthe quality control of each one considering several micro-biological and physicochemical parameters and mainly theHPLC analysis according to what was previously publishedby Berretta et al [13] Propolis blend of standardized rawmaterial was previously cooled for 12 hours and pulverizedTo obtain PEE propolis raw material was macerated andpercolated with hydroalcoholic solution (7 3) and finallyfiltrated

PEE showed 11wv of extractable matter PWE PMMand PSDE were obtained from the same PEE batch

PMM was obtained by spray dryer employing PEE andexcipients such as modified corn starch (Capsul) and col-loidal silicon dioxide (1 1) The dryness condition involvedtemperature of 80∘C for sample exit sample flow of 120mLminute and system air output of 30m3min The spraydryer equipment used possessed a pneumatic atomizer with12mmof diameterThe ratio of drymatter and each excipientwas 1 05 05 resulting in approximately 50 of propolis drymatter in PMM

PSDE was produced after the dryness of PWE obtainedaccording to de Andrade et al [14] with modifications PEEwas submitted to complete evaporation of the solvent andto alkaline hydrolysis followed by purified water addition(PWE) Subsequently PWE was submitted to dryness withthe presence of maltodextrin (7 3 propolis excipient) byspray dryer process under the same condition used to PMM

23 HPLC Analysis Propolis extracts were evaluated forhigh pressure liquid chromatography (HPLC)with Shimadzuequipment with CBM-20A controller LC-20AT quaternarypump diode array detector SPD-M 20A and Shimadzu LCversion 121 SP1 software For analytical running ShimadzuShim-Pack CLC-ODS (M) column was used (46mm times 250mm with particle diameter of 5 120583m with porous diameter of100 A) To evaluate phenolic derivatives the eluent solutionconsisted of methanol and acidic water with formic acid(01ww) 20ndash95 77 minutes of running and 08mLminof flow [15] The detection was at 275 nm PEE PWE PMMand PSDE (119899 = 3) were individually diluted with metha-nolwater and homogenized with ultrasound bath After thatthe volume was acidified with formic acid to pH 270 Afterthe filtration (045120583m) 20120583L was injected in HPLC system

24 Strains Media and Culture Methods C albicans strainsused were SC5314 (wild type) [16] CAI4 (ura3imm434ura3imm434 iro1iro1imm434) [17] 3153A (wild type) (gener-ously donated by Paul Fidel Jr Department of Microbiology









28 Gel Preparation



















3 Results




0 5 10 15 20 25 30 35 40 45 50 55 60 65 70(min)

800

50100150200250300350400

(mAU

)

1 2

3

45

6

7 8

9

10

275 nm 4 nm (100)

(a)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70(min)

80(m

AU)

0255075

100125150175200225250275

2

3

4

56

7 8

10275 nm 4 nm (100)

(b)

5 10 15 20 25 30 35 40 45 50 55 60 65 70 80(min)

(mAU

)

0255075

100125150175200225250275

3

2 45 6

7 8

10

275 nm 4 nm (100)

(c)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70(min)

80

(mAU

)

0255075

100125150175200225250275

32 45

6

10

275 nm 4 nm (100)

(d)





Chemical compounds

1 2 3 4 5 6 7 800

02

04

06

08

10

PEEPMMPSDE

(mg

g)







106 105 104 103

(A)

(B)

(C)

(D)

(E)

(a)


106 105 104 103

Negative control

(A)

(B)

(C)

(D)

(E)

(b)


106 105 104 103

(A)

(B)

(C)

(D)

(E)

(c)












C-


6 h 8 h 12 h 24 h

025

050

075

100

C-

025

050

075

100

(a)

Cellular viability

0

50

100

150

lowast

lowast

PEE PMM PSDEC-

Surv

ival

()

12 hs24 hs

(b)






Yeast

Hyphae

Yeast

HyphaePMM

-pro

polis


Pseudo-hyphae

Pseudo-hyphae

0

20

40

60

80

100

120


Cel

lula

r via

bilit

y (

)


12 hs24 hs

Yeast

Hyphae

Morphology

Pseudohyphae

(a)

(b) (c)

1∘ 2∘ 3∘ 4∘ 5∘ 6∘ 7∘ 8∘








OO

OHO

HO

HO

HO

HO

HO HO

OH

OH

OH

OH

OH

OHCH

C C

C

2 CH2

NH2

NH2

NH2

CH2CH2 CH2O O

O

O

O O

O O

O

O OO

O

CH3

CH H

H H

H

Ominus

Ominus

a b c

m

n

n

n

ChitosanCarbopol


Scheme 1








0 15 30 45 600

250

500

750

1000

1250

1500

1750

AlP1PP1

CP1ChP1

D (1s)

120591(P

a)

(a)

0 10 20 30 40 50 600

40

80

120

160

200

AlP1PP1

CP1ChP1

D (1s)

Visc

osity

(Pa s

)

(b)

00 05 10 15 20000

005

010

015

020

025

PP1AlP1

ChP1CP1

Deformation (mm)

Stre

ngth

(N)

(c)






4 Discussion



Groups

15 times 105

10 times 105

50 times 104

0C- 48 hs 72 hs

C a

lbica

ns S

C531

4 (c

ells

mL)

lowast

lowast

(a)


10 times 108

10 times 107

10 times 106

10 times 105

10 times 104

10 times 103

10 times 102

10 times 101

10 times 100

Groups n = 10

C a

lbica

ns (c

ells

mL)

(b)








(a) (b)

(c) (d)


7 days treatment

0

20

60

40

C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo

lowast

lowastlowast

times105

(a)

10 days treatment

0

50

40

30

20

10C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo


times105

(b)






(a) (b)

(c) (d)






(a) (b)

(c) (d)













Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























28 Gel Preparation



















3 Results




0 5 10 15 20 25 30 35 40 45 50 55 60 65 70(min)

800

50100150200250300350400

(mAU

)

1 2

3

45

6

7 8

9

10

275 nm 4 nm (100)

(a)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70(min)

80(m

AU)

0255075

100125150175200225250275

2

3

4

56

7 8

10275 nm 4 nm (100)

(b)

5 10 15 20 25 30 35 40 45 50 55 60 65 70 80(min)

(mAU

)

0255075

100125150175200225250275

3

2 45 6

7 8

10

275 nm 4 nm (100)

(c)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70(min)

80

(mAU

)

0255075

100125150175200225250275

32 45

6

10

275 nm 4 nm (100)

(d)





Chemical compounds

1 2 3 4 5 6 7 800

02

04

06

08

10

PEEPMMPSDE

(mg

g)







106 105 104 103

(A)

(B)

(C)

(D)

(E)

(a)


106 105 104 103

Negative control

(A)

(B)

(C)

(D)

(E)

(b)


106 105 104 103

(A)

(B)

(C)

(D)

(E)

(c)












C-


6 h 8 h 12 h 24 h

025

050

075

100

C-

025

050

075

100

(a)

Cellular viability

0

50

100

150

lowast

lowast

PEE PMM PSDEC-

Surv

ival

()

12 hs24 hs

(b)






Yeast

Hyphae

Yeast

HyphaePMM

-pro

polis


Pseudo-hyphae

Pseudo-hyphae

0

20

40

60

80

100

120


Cel

lula

r via

bilit

y (

)


12 hs24 hs

Yeast

Hyphae

Morphology

Pseudohyphae

(a)

(b) (c)

1∘ 2∘ 3∘ 4∘ 5∘ 6∘ 7∘ 8∘








OO

OHO

HO

HO

HO

HO

HO HO

OH

OH

OH

OH

OH

OHCH

C C

C

2 CH2

NH2

NH2

NH2

CH2CH2 CH2O O

O

O

O O

O O

O

O OO

O

CH3

CH H

H H

H

Ominus

Ominus

a b c

m

n

n

n

ChitosanCarbopol


Scheme 1








0 15 30 45 600

250

500

750

1000

1250

1500

1750

AlP1PP1

CP1ChP1

D (1s)

120591(P

a)

(a)

0 10 20 30 40 50 600

40

80

120

160

200

AlP1PP1

CP1ChP1

D (1s)

Visc

osity

(Pa s

)

(b)

00 05 10 15 20000

005

010

015

020

025

PP1AlP1

ChP1CP1

Deformation (mm)

Stre

ngth

(N)

(c)






4 Discussion



Groups

15 times 105

10 times 105

50 times 104

0C- 48 hs 72 hs

C a

lbica

ns S

C531

4 (c

ells

mL)

lowast

lowast

(a)


10 times 108

10 times 107

10 times 106

10 times 105

10 times 104

10 times 103

10 times 102

10 times 101

10 times 100

Groups n = 10

C a

lbica

ns (c

ells

mL)

(b)








(a) (b)

(c) (d)


7 days treatment

0

20

60

40

C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo

lowast

lowastlowast

times105

(a)

10 days treatment

0

50

40

30

20

10C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo


times105

(b)






(a) (b)

(c) (d)






(a) (b)

(c) (d)













Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































3 Results




0 5 10 15 20 25 30 35 40 45 50 55 60 65 70(min)

800

50100150200250300350400

(mAU

)

1 2

3

45

6

7 8

9

10

275 nm 4 nm (100)

(a)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70(min)

80(m

AU)

0255075

100125150175200225250275

2

3

4

56

7 8

10275 nm 4 nm (100)

(b)

5 10 15 20 25 30 35 40 45 50 55 60 65 70 80(min)

(mAU

)

0255075

100125150175200225250275

3

2 45 6

7 8

10

275 nm 4 nm (100)

(c)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70(min)

80

(mAU

)

0255075

100125150175200225250275

32 45

6

10

275 nm 4 nm (100)

(d)





Chemical compounds

1 2 3 4 5 6 7 800

02

04

06

08

10

PEEPMMPSDE

(mg

g)







106 105 104 103

(A)

(B)

(C)

(D)

(E)

(a)


106 105 104 103

Negative control

(A)

(B)

(C)

(D)

(E)

(b)


106 105 104 103

(A)

(B)

(C)

(D)

(E)

(c)












C-


6 h 8 h 12 h 24 h

025

050

075

100

C-

025

050

075

100

(a)

Cellular viability

0

50

100

150

lowast

lowast

PEE PMM PSDEC-

Surv

ival

()

12 hs24 hs

(b)






Yeast

Hyphae

Yeast

HyphaePMM

-pro

polis


Pseudo-hyphae

Pseudo-hyphae

0

20

40

60

80

100

120


Cel

lula

r via

bilit

y (

)


12 hs24 hs

Yeast

Hyphae

Morphology

Pseudohyphae

(a)

(b) (c)

1∘ 2∘ 3∘ 4∘ 5∘ 6∘ 7∘ 8∘








OO

OHO

HO

HO

HO

HO

HO HO

OH

OH

OH

OH

OH

OHCH

C C

C

2 CH2

NH2

NH2

NH2

CH2CH2 CH2O O

O

O

O O

O O

O

O OO

O

CH3

CH H

H H

H

Ominus

Ominus

a b c

m

n

n

n

ChitosanCarbopol


Scheme 1








0 15 30 45 600

250

500

750

1000

1250

1500

1750

AlP1PP1

CP1ChP1

D (1s)

120591(P

a)

(a)

0 10 20 30 40 50 600

40

80

120

160

200

AlP1PP1

CP1ChP1

D (1s)

Visc

osity

(Pa s

)

(b)

00 05 10 15 20000

005

010

015

020

025

PP1AlP1

ChP1CP1

Deformation (mm)

Stre

ngth

(N)

(c)






4 Discussion



Groups

15 times 105

10 times 105

50 times 104

0C- 48 hs 72 hs

C a

lbica

ns S

C531

4 (c

ells

mL)

lowast

lowast

(a)


10 times 108

10 times 107

10 times 106

10 times 105

10 times 104

10 times 103

10 times 102

10 times 101

10 times 100

Groups n = 10

C a

lbica

ns (c

ells

mL)

(b)








(a) (b)

(c) (d)


7 days treatment

0

20

60

40

C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo

lowast

lowastlowast

times105

(a)

10 days treatment

0

50

40

30

20

10C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo


times105

(b)






(a) (b)

(c) (d)






(a) (b)

(c) (d)













Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















3 Results




0 5 10 15 20 25 30 35 40 45 50 55 60 65 70(min)

800

50100150200250300350400

(mAU

)

1 2

3

45

6

7 8

9

10

275 nm 4 nm (100)

(a)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70(min)

80(m

AU)

0255075

100125150175200225250275

2

3

4

56

7 8

10275 nm 4 nm (100)

(b)

5 10 15 20 25 30 35 40 45 50 55 60 65 70 80(min)

(mAU

)

0255075

100125150175200225250275

3

2 45 6

7 8

10

275 nm 4 nm (100)

(c)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70(min)

80

(mAU

)

0255075

100125150175200225250275

32 45

6

10

275 nm 4 nm (100)

(d)





Chemical compounds

1 2 3 4 5 6 7 800

02

04

06

08

10

PEEPMMPSDE

(mg

g)







106 105 104 103

(A)

(B)

(C)

(D)

(E)

(a)


106 105 104 103

Negative control

(A)

(B)

(C)

(D)

(E)

(b)


106 105 104 103

(A)

(B)

(C)

(D)

(E)

(c)












C-


6 h 8 h 12 h 24 h

025

050

075

100

C-

025

050

075

100

(a)

Cellular viability

0

50

100

150

lowast

lowast

PEE PMM PSDEC-

Surv

ival

()

12 hs24 hs

(b)






Yeast

Hyphae

Yeast

HyphaePMM

-pro

polis


Pseudo-hyphae

Pseudo-hyphae

0

20

40

60

80

100

120


Cel

lula

r via

bilit

y (

)


12 hs24 hs

Yeast

Hyphae

Morphology

Pseudohyphae

(a)

(b) (c)

1∘ 2∘ 3∘ 4∘ 5∘ 6∘ 7∘ 8∘








OO

OHO

HO

HO

HO

HO

HO HO

OH

OH

OH

OH

OH

OHCH

C C

C

2 CH2

NH2

NH2

NH2

CH2CH2 CH2O O

O

O

O O

O O

O

O OO

O

CH3

CH H

H H

H

Ominus

Ominus

a b c

m

n

n

n

ChitosanCarbopol


Scheme 1








0 15 30 45 600

250

500

750

1000

1250

1500

1750

AlP1PP1

CP1ChP1

D (1s)

120591(P

a)

(a)

0 10 20 30 40 50 600

40

80

120

160

200

AlP1PP1

CP1ChP1

D (1s)

Visc

osity

(Pa s

)

(b)

00 05 10 15 20000

005

010

015

020

025

PP1AlP1

ChP1CP1

Deformation (mm)

Stre

ngth

(N)

(c)






4 Discussion



Groups

15 times 105

10 times 105

50 times 104

0C- 48 hs 72 hs

C a

lbica

ns S

C531

4 (c

ells

mL)

lowast

lowast

(a)


10 times 108

10 times 107

10 times 106

10 times 105

10 times 104

10 times 103

10 times 102

10 times 101

10 times 100

Groups n = 10

C a

lbica

ns (c

ells

mL)

(b)








(a) (b)

(c) (d)


7 days treatment

0

20

60

40

C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo

lowast

lowastlowast

times105

(a)

10 days treatment

0

50

40

30

20

10C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo


times105

(b)






(a) (b)

(c) (d)






(a) (b)

(c) (d)













Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Chemical compounds

1 2 3 4 5 6 7 800

02

04

06

08

10

PEEPMMPSDE

(mg

g)







106 105 104 103

(A)

(B)

(C)

(D)

(E)

(a)


106 105 104 103

Negative control

(A)

(B)

(C)

(D)

(E)

(b)


106 105 104 103

(A)

(B)

(C)

(D)

(E)

(c)












C-


6 h 8 h 12 h 24 h

025

050

075

100

C-

025

050

075

100

(a)

Cellular viability

0

50

100

150

lowast

lowast

PEE PMM PSDEC-

Surv

ival

()

12 hs24 hs

(b)






Yeast

Hyphae

Yeast

HyphaePMM

-pro

polis


Pseudo-hyphae

Pseudo-hyphae

0

20

40

60

80

100

120


Cel

lula

r via

bilit

y (

)


12 hs24 hs

Yeast

Hyphae

Morphology

Pseudohyphae

(a)

(b) (c)

1∘ 2∘ 3∘ 4∘ 5∘ 6∘ 7∘ 8∘








OO

OHO

HO

HO

HO

HO

HO HO

OH

OH

OH

OH

OH

OHCH

C C

C

2 CH2

NH2

NH2

NH2

CH2CH2 CH2O O

O

O

O O

O O

O

O OO

O

CH3

CH H

H H

H

Ominus

Ominus

a b c

m

n

n

n

ChitosanCarbopol


Scheme 1








0 15 30 45 600

250

500

750

1000

1250

1500

1750

AlP1PP1

CP1ChP1

D (1s)

120591(P

a)

(a)

0 10 20 30 40 50 600

40

80

120

160

200

AlP1PP1

CP1ChP1

D (1s)

Visc

osity

(Pa s

)

(b)

00 05 10 15 20000

005

010

015

020

025

PP1AlP1

ChP1CP1

Deformation (mm)

Stre

ngth

(N)

(c)






4 Discussion



Groups

15 times 105

10 times 105

50 times 104

0C- 48 hs 72 hs

C a

lbica

ns S

C531

4 (c

ells

mL)

lowast

lowast

(a)


10 times 108

10 times 107

10 times 106

10 times 105

10 times 104

10 times 103

10 times 102

10 times 101

10 times 100

Groups n = 10

C a

lbica

ns (c

ells

mL)

(b)








(a) (b)

(c) (d)


7 days treatment

0

20

60

40

C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo

lowast

lowastlowast

times105

(a)

10 days treatment

0

50

40

30

20

10C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo


times105

(b)






(a) (b)

(c) (d)






(a) (b)

(c) (d)













Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















106 105 104 103

(A)

(B)

(C)

(D)

(E)

(a)


106 105 104 103

Negative control

(A)

(B)

(C)

(D)

(E)

(b)


106 105 104 103

(A)

(B)

(C)

(D)

(E)

(c)












C-


6 h 8 h 12 h 24 h

025

050

075

100

C-

025

050

075

100

(a)

Cellular viability

0

50

100

150

lowast

lowast

PEE PMM PSDEC-

Surv

ival

()

12 hs24 hs

(b)






Yeast

Hyphae

Yeast

HyphaePMM

-pro

polis


Pseudo-hyphae

Pseudo-hyphae

0

20

40

60

80

100

120


Cel

lula

r via

bilit

y (

)


12 hs24 hs

Yeast

Hyphae

Morphology

Pseudohyphae

(a)

(b) (c)

1∘ 2∘ 3∘ 4∘ 5∘ 6∘ 7∘ 8∘








OO

OHO

HO

HO

HO

HO

HO HO

OH

OH

OH

OH

OH

OHCH

C C

C

2 CH2

NH2

NH2

NH2

CH2CH2 CH2O O

O

O

O O

O O

O

O OO

O

CH3

CH H

H H

H

Ominus

Ominus

a b c

m

n

n

n

ChitosanCarbopol


Scheme 1








0 15 30 45 600

250

500

750

1000

1250

1500

1750

AlP1PP1

CP1ChP1

D (1s)

120591(P

a)

(a)

0 10 20 30 40 50 600

40

80

120

160

200

AlP1PP1

CP1ChP1

D (1s)

Visc

osity

(Pa s

)

(b)

00 05 10 15 20000

005

010

015

020

025

PP1AlP1

ChP1CP1

Deformation (mm)

Stre

ngth

(N)

(c)






4 Discussion



Groups

15 times 105

10 times 105

50 times 104

0C- 48 hs 72 hs

C a

lbica

ns S

C531

4 (c

ells

mL)

lowast

lowast

(a)


10 times 108

10 times 107

10 times 106

10 times 105

10 times 104

10 times 103

10 times 102

10 times 101

10 times 100

Groups n = 10

C a

lbica

ns (c

ells

mL)

(b)








(a) (b)

(c) (d)


7 days treatment

0

20

60

40

C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo

lowast

lowastlowast

times105

(a)

10 days treatment

0

50

40

30

20

10C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo


times105

(b)






(a) (b)

(c) (d)






(a) (b)

(c) (d)













Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















C-


6 h 8 h 12 h 24 h

025

050

075

100

C-

025

050

075

100

(a)

Cellular viability

0

50

100

150

lowast

lowast

PEE PMM PSDEC-

Surv

ival

()

12 hs24 hs

(b)






Yeast

Hyphae

Yeast

HyphaePMM

-pro

polis


Pseudo-hyphae

Pseudo-hyphae

0

20

40

60

80

100

120


Cel

lula

r via

bilit

y (

)


12 hs24 hs

Yeast

Hyphae

Morphology

Pseudohyphae

(a)

(b) (c)

1∘ 2∘ 3∘ 4∘ 5∘ 6∘ 7∘ 8∘








OO

OHO

HO

HO

HO

HO

HO HO

OH

OH

OH

OH

OH

OHCH

C C

C

2 CH2

NH2

NH2

NH2

CH2CH2 CH2O O

O

O

O O

O O

O

O OO

O

CH3

CH H

H H

H

Ominus

Ominus

a b c

m

n

n

n

ChitosanCarbopol


Scheme 1








0 15 30 45 600

250

500

750

1000

1250

1500

1750

AlP1PP1

CP1ChP1

D (1s)

120591(P

a)

(a)

0 10 20 30 40 50 600

40

80

120

160

200

AlP1PP1

CP1ChP1

D (1s)

Visc

osity

(Pa s

)

(b)

00 05 10 15 20000

005

010

015

020

025

PP1AlP1

ChP1CP1

Deformation (mm)

Stre

ngth

(N)

(c)






4 Discussion



Groups

15 times 105

10 times 105

50 times 104

0C- 48 hs 72 hs

C a

lbica

ns S

C531

4 (c

ells

mL)

lowast

lowast

(a)


10 times 108

10 times 107

10 times 106

10 times 105

10 times 104

10 times 103

10 times 102

10 times 101

10 times 100

Groups n = 10

C a

lbica

ns (c

ells

mL)

(b)








(a) (b)

(c) (d)


7 days treatment

0

20

60

40

C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo

lowast

lowastlowast

times105

(a)

10 days treatment

0

50

40

30

20

10C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo


times105

(b)






(a) (b)

(c) (d)






(a) (b)

(c) (d)













Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Yeast

Hyphae

Yeast

HyphaePMM

-pro

polis


Pseudo-hyphae

Pseudo-hyphae

0

20

40

60

80

100

120


Cel

lula

r via

bilit

y (

)


12 hs24 hs

Yeast

Hyphae

Morphology

Pseudohyphae

(a)

(b) (c)

1∘ 2∘ 3∘ 4∘ 5∘ 6∘ 7∘ 8∘








OO

OHO

HO

HO

HO

HO

HO HO

OH

OH

OH

OH

OH

OHCH

C C

C

2 CH2

NH2

NH2

NH2

CH2CH2 CH2O O

O

O

O O

O O

O

O OO

O

CH3

CH H

H H

H

Ominus

Ominus

a b c

m

n

n

n

ChitosanCarbopol


Scheme 1








0 15 30 45 600

250

500

750

1000

1250

1500

1750

AlP1PP1

CP1ChP1

D (1s)

120591(P

a)

(a)

0 10 20 30 40 50 600

40

80

120

160

200

AlP1PP1

CP1ChP1

D (1s)

Visc

osity

(Pa s

)

(b)

00 05 10 15 20000

005

010

015

020

025

PP1AlP1

ChP1CP1

Deformation (mm)

Stre

ngth

(N)

(c)






4 Discussion



Groups

15 times 105

10 times 105

50 times 104

0C- 48 hs 72 hs

C a

lbica

ns S

C531

4 (c

ells

mL)

lowast

lowast

(a)


10 times 108

10 times 107

10 times 106

10 times 105

10 times 104

10 times 103

10 times 102

10 times 101

10 times 100

Groups n = 10

C a

lbica

ns (c

ells

mL)

(b)








(a) (b)

(c) (d)


7 days treatment

0

20

60

40

C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo

lowast

lowastlowast

times105

(a)

10 days treatment

0

50

40

30

20

10C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo


times105

(b)






(a) (b)

(c) (d)






(a) (b)

(c) (d)













Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















OO

OHO

HO

HO

HO

HO

HO HO

OH

OH

OH

OH

OH

OHCH

C C

C

2 CH2

NH2

NH2

NH2

CH2CH2 CH2O O

O

O

O O

O O

O

O OO

O

CH3

CH H

H H

H

Ominus

Ominus

a b c

m

n

n

n

ChitosanCarbopol


Scheme 1








0 15 30 45 600

250

500

750

1000

1250

1500

1750

AlP1PP1

CP1ChP1

D (1s)

120591(P

a)

(a)

0 10 20 30 40 50 600

40

80

120

160

200

AlP1PP1

CP1ChP1

D (1s)

Visc

osity

(Pa s

)

(b)

00 05 10 15 20000

005

010

015

020

025

PP1AlP1

ChP1CP1

Deformation (mm)

Stre

ngth

(N)

(c)






4 Discussion



Groups

15 times 105

10 times 105

50 times 104

0C- 48 hs 72 hs

C a

lbica

ns S

C531

4 (c

ells

mL)

lowast

lowast

(a)


10 times 108

10 times 107

10 times 106

10 times 105

10 times 104

10 times 103

10 times 102

10 times 101

10 times 100

Groups n = 10

C a

lbica

ns (c

ells

mL)

(b)








(a) (b)

(c) (d)


7 days treatment

0

20

60

40

C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo

lowast

lowastlowast

times105

(a)

10 days treatment

0

50

40

30

20

10C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo


times105

(b)






(a) (b)

(c) (d)






(a) (b)

(c) (d)













Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 15 30 45 600

250

500

750

1000

1250

1500

1750

AlP1PP1

CP1ChP1

D (1s)

120591(P

a)

(a)

0 10 20 30 40 50 600

40

80

120

160

200

AlP1PP1

CP1ChP1

D (1s)

Visc

osity

(Pa s

)

(b)

00 05 10 15 20000

005

010

015

020

025

PP1AlP1

ChP1CP1

Deformation (mm)

Stre

ngth

(N)

(c)






4 Discussion



Groups

15 times 105

10 times 105

50 times 104

0C- 48 hs 72 hs

C a

lbica

ns S

C531

4 (c

ells

mL)

lowast

lowast

(a)


10 times 108

10 times 107

10 times 106

10 times 105

10 times 104

10 times 103

10 times 102

10 times 101

10 times 100

Groups n = 10

C a

lbica

ns (c

ells

mL)

(b)








(a) (b)

(c) (d)


7 days treatment

0

20

60

40

C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo

lowast

lowastlowast

times105

(a)

10 days treatment

0

50

40

30

20

10C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo


times105

(b)






(a) (b)

(c) (d)






(a) (b)

(c) (d)













Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Groups

15 times 105

10 times 105

50 times 104

0C- 48 hs 72 hs

C a

lbica

ns S

C531

4 (c

ells

mL)

lowast

lowast

(a)


10 times 108

10 times 107

10 times 106

10 times 105

10 times 104

10 times 103

10 times 102

10 times 101

10 times 100

Groups n = 10

C a

lbica

ns (c

ells

mL)

(b)








(a) (b)

(c) (d)


7 days treatment

0

20

60

40

C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo

lowast

lowastlowast

times105

(a)

10 days treatment

0

50

40

30

20

10C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo


times105

(b)






(a) (b)

(c) (d)






(a) (b)

(c) (d)













Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)


7 days treatment

0

20

60

40

C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo

lowast

lowastlowast

times105

(a)

10 days treatment

0

50

40

30

20

10C a

lbica

ns 3

153A

(cel

lsm

L)

C- CP1 PP1 Clo


times105

(b)






(a) (b)

(c) (d)






(a) (b)

(c) (d)













Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)






(a) (b)

(c) (d)













Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)













Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















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
















Documents

Research Article Evaluation of Mucoadhesive Gels with ...downloads.hindawi.com/journals/ecam/2013/641480.pdf · Vulvovaginal candidiasis is the second cause of vaginal infection in