Research Article The Biological Activity of Propolis...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 704947 12 pageshttpdxdoiorg1011552013704947

Research ArticleThe Biological Activity of Propolis-Containing Toothpaste onOral Health Environment in Patients Who UnderwentImplant-Supported Prosthodontic Rehabilitation

Tadeusz Morawiec1 Arkadiusz Dziedzic2 Iwona Niedzielska1

Anna Mertas3 Marta Tanasiewicz2 Dariusz Skaba2 Jacek Kasperski4

Agnieszka Machorowska-Pienidhek5 Marek Kucharzewski6 Karolina Szaniawska7

WBodzimierz Wiwckiewicz8 and Mieszko Wiwckiewicz8

1 Department of Oral Surgery Medical University of Silesia Plac Akademicki 17 41-902 Bytom Poland2Department of Conservative Dentistry with Endodontics Medical University of Silesia Plac Akademicki 17 41-902 Bytom Poland3Department of Microbiology and Immunology Medical University of Silesia Jordana 19 41-808 Zabrze Poland4Department of Prosthetic Dentistry Medical University of Silesia Plac Akademicki 17 41-902 Bytom Poland5Department of Orthodontics Medical University of Silesia Plac Akademicki 17 41-902 Bytom Poland6Department of Descriptive and Topographic Anatomy Medical University of Silesia Jordana 19 41-808 Zabrze Poland7Division of Medicine and Dentistry Department of Oral Surgery Medical University of Warsaw Ulica Nowogrodzka 5902-006 Warszawa Poland

8Department of Prosthetic Dentistry Faculty of Dentistry Wrocław Medical University 50-425 Wrocław Poland

Correspondence should be addressed to Arkadiusz Dziedzic adziedzicsumedupl

Received 31 March 2013 Accepted 20 April 2013

Academic Editor Ewelina Szliszka

Copyright copy 2013 Tadeusz Morawiec et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The soft and periodontal tissues surrounding dental implants are particularly susceptible to bacteria invasion and inflammatoryreactions due to complex histological structures This study was carried out to investigate the influence of a propolis-containinghygienic agent on selected oral health parameters oral microflora and the condition of periodontal health Sixteen subjects whounderwent an oral rehabilitationwith dental implants were selected and randomly assigned into two groups which received a newlyformulated propolis-containing toothpaste (3 (CA)) or a negative control without an active ingredient (CC) Approximal plaqueindex (API) oral hygiene index (OHI debris component) and sulcus bleeding index (SBI) were assessed in three subsequent stagesDuring the first and last examinations the swabs were employed for microbiological inoculation Propolis-containing toothpastewas found to be distinctively effective in improving oral health and the occurrence of gingivitis triggered by dental plaque Thequalitative and quantitative changes in oral bacteria spectrum were observed Antibacterial measures containing propolis mightbe used as a natural adjuvant to other active substances in individuals with a high risk of periodontal problems against pathogenicoral microflora

1 Introduction

The bacterial plaque remaining over and under the gingivacontributes to the generation of inflammatory reaction intissues surrounding teeth and dental implants which leadsto the loss of collective tissue and alveolar bone attachment[1 2] Socransky et al divided the microorganisms located

on subgingival areas into five complexes One of them theso-called ldquored complexrdquo consisting of Tannerella forsythen-sis Porphyromonas gingivalis and Treponema denticola isstrongly associated with increased depth of periodontalandor peri-implant pockets as well as bleeding when aprobe is inserted [3] Results of clinical and microbiolog-ical investigations also indicate that Prevotella intermedia

2 Evidence-Based Complementary and Alternative Medicine

or Fusobacterium nucleatum among others are potentialetiological factors causing periodontitis and peri-implantitisproblems [4] Reduction of the number of those pathogenicmicro-organisms could potentially influence the epidemiol-ogy of periodontium diseases reducing their amount andintensification The hygiene regime related to oral healthmaintenance is a critical area of implant-supported casesof oral rehabilitation The periodontal tissues surroundingdental implants seem to be particularly susceptible to bacteriainvasion and local inflammatory reactions [5ndash7]

Effective oral hygiene around dental implants can bechallenging to achieve over the long term and the patientdentist and dental hygienistmust exercise considerable effortto achieve the desired results In more recent years implantmaintenance and effective patient home care have beenemphasized as critical factors needed for long-term successof dental implants [8] As the number of patients treated withdental implants continues to grow the patients must be fullyaware of a proper maintenance with the use of preventive andtherapeutic local measures [9 10] Among them propolismdasha resinous substance made by bees possessing biologicaltherapeutic activitiesmdashmay play an important role in oralhygiene and inmaintaining healthy soft tissueswithin the oralcavity It is known for its ability to nourish periodontal tissuesand prevent problems associated with gingiva and supportedhistological elements [8 11]

Humans began using propolis more than 2000 years agofor many purposes the foremost of which was applying it towounds to fight infection It promotes the healing processwith its mild anti-infective properties [12] Over time ithas been marketed in various forms such as toothpastesmouthrinses and lozenges Propolis extract is known topossess antimicrobial activity against Streptococcus mutans aGram-positive cocci facultative anaerobic strains commonlyfound in the human oral cavity and a significant contributorto plaque formation [13 14] Propolis has been also foundbeneficial in the treatment of gingivitis and oral mucosalesions in case studies and pilot clinical studies [15 16]

Over the last few decades a worldwide trend has beenobserved in the use of natural products for pharmacologicalpurposes due to their proven therapeutic effect Propoliswhich is a natural product widely consumed in the folkmedicine since ancient times and a substance produced bythe honeybees seems to be a promising agent to be addedto topical formulations due to its multidirectional properties[17] Besides antioxidant and anti-inflammatory activity [18ndash21] epidemiological studies have detected that propolis hasmany beneficial properties such as antibacterial antifungalantiviral and antitumor [22ndash26] Ethanolic extract of propo-lis (EEP) solutions has been used commercially on themarketas toothpaste mouth wash lozenges and so forth as aneffective antimicrobial and anti-inflammatory agent [27 28]However it is still an underestimated ingredient in academicmedicine and dentistry In general propolis is composed of50 resin and vegetable balsam 30 wax 10 essential andaromatic oils 5 pollen and 5 various other substancesincluding organic debris depending on the place and timeof collection [29] The constituents of propolis vary widelydue to climate season location and year and its chemical

formula is not stable [30 31] Flavonoidsmdashthe main propolisingredientmdashinhibit lipid peroxidation platelet aggregationcapillary permeability and fragility and the activity of enzymesystems including cyclo-oxygenase and lipoxygenase [28 31]

The study aimed at investigating the local effect of theorally administered extract of propolis in the form of tooth-paste on selected intraoral predictors representing oral healthcondition in the group of patients with dental implants-supported fixed and removable prosthodontic suprastruc-tures

2 Material and Methods

The clinical study was carried out to investigate the influ-ence of a propolis-containing toothpaste on gingival healthand the oral microbiota spectrum changes This researchwas conducted in Oral Surgery Department the AcademicCentre of Dentistry and Specialist Medicine (Bytom Poland)and Specialist Dental Clinic (Katowice Poland) which wereproviding comprehensive dental care for the patients whounderwent implant-prosthetic therapy All implants wereinstalled by the same operator a specialist in oral surgeryTheldquooldestrdquo installation was carried out 9 years ago and the mostrecent was 2 years ago The study was designed as a singleblind two-group parallel study The dental products and testsemployed in the study were legal merchandises commonlyavailable and used The study was not a clinical trial or aclinical experiment

21 Hygienic Compounds Preparation Two samples of tooth-pastes covered anonymously by a blank label and markedonly with the letters (A) or (C) were compared the testedtoothpaste containing the active ingredientmdash3 ethanolextract of Brazilian propolis (CA DentalPolisDX ldquogreenrdquopropolis) and a placebo as a control (CC) without activesubstance Raw propolis was collected from the beekeepingsection of the Seiri Alimentos Naturais Brazil Propolis sam-ples were obtained from colonies of Africanized honeybees(Apis mellifera) in Minas Gerais State Southeast Brazil andcollected over a period of 2008 year from the plant Baccharisdracunculifolia which is the main botanical source of theresin for the green propolis and determines the compositionof Brazilian EEP rich in artepillin C [32] The unprocessedpropolis was sent to the Nihon Natural Therapy Co Ltd(Tokyo Japan) for the preparation of the EEP ingredientThetoothpastes with 3 EEP and without of EEP (placebo) wereprepared in Nippon Zettoc Co Ltd Tokyo Japan

22 ResearchGroups InclusionCriteria Subjects qualificationfor the study was based on medical and dental historyinterview and review of clinical records The exclusioncriteria from the investigationwere as follows lack of patientrsquosvalid consent medically compromised patients inability tocomplywith the follow-up visit requirements patients receiv-ing concurrent antibiotic treatment for any other purposeindividuals with confirmed adverse reactions to bee productsnursing or pregnant women and recent postoperative oral

Evidence-Based Complementary and Alternative Medicine 3

surgery cases Inclusion criteria comprised the age group of22 to 65 years and patients free from systemic illness

Sixteen subjects were randomly assigned into two groupsof 8 subjects each which received an unrecognized propolis-containing toothpaste (group A) or a negative control tooth-paste (group C) Oral hygiene instructions were given in anattempt to improve their oral hygiene before entry into thestudy The toothpastes were allocated according to groupsto ensure balance All subjects received professional adviceregarding oral hygiene and were instructed to brush twotimes a day with the toothpaste recommended for at least twominutes and to refrain from all other oral hygiene measuresuntil the next examination

Targeted groups of 16 patients who received implant-supported oral rehabilitation were examined in three sub-sequent stages preliminary qualification at baseline (1stassessment) a follow-up after 7 days (2nd assessment) andafter 8 weeks since the initial examination (3rd assessment)During the first and last examination the swabs were usedfor microbiological examination of oral cavity microfloraThe microbial material for smear tests was collected fromthe oral mucosa around fixed prosthetic suprastructures andperi-implant areas Preliminary qualification of the subjectsincluded past and current health problems date of the lastdental appointment dental check-up intervals dietary habitscarbonate-rich products intake pattern and frequency ofteeth brushing and the use of additional domestic preventivemeasures (dental floss mouthrinses etc)

23 Clinical Examination Protocol The selected commonlyused indices of oral health condition APIOHI and SBI wereassessed and scored at baseline during follow-up visit andafter eight weeks since the initial examination Approximalplaque index (API Lange amp Ainamo 1988) scored thepresence of dental plaque in the interdental spaces and oralhygiene index-debris component (OHI-D Greene amp Vermil-lion 1960)was used for the assessment of amount of debris onall teeth and dental implants For the aproximal plaque indexonly the plaque (bacteria) in the spaces between the teeth wasevaluated in order to draw conclusions about the level of oralhygiene and individual risk of caries occurrenceOral hygieneindex determines the amount of soft debris or calculus on thefour buccal surfaces of the selected teeth (dental implants)upper right first molar upper right central incisor upperleft first molar and lower central incisor and on the lingualsurfaces of the lower left first molar and lower right firstmolar The periodontal status (gingival health) was evaluatedwith the use of the modified sulcus bleeding index (SBIMuhlemann-Son 1971) and the recording of only ldquobleedingpresencerdquo or ldquobleeding absencerdquo for all existing teeth or dentalimplants

In order to assess the oral hygiene related to the fixedprosthodontics themodified plaque index (MPI) was applied[33 34] including the protocol adequate to OHI scores MPIis valid for studies of peri-implant health in patients carryingmandibular implant overdentures on bars as long as thehygiene in abutments as in bars is evaluated asmuchThe lackof the barrsquos hygiene assessment would suppose an allocation

of plaque values lower to real ones so a poor hygienewould be undervalued These patients were included intoan oral hygiene protocol to avoid subsequent peri-implantcomplications as mucositis or peri-implantitis MPI scoresare as follows score 0mdashno plaque score 1mdashno plaque atfirst sight presence when slipping probe on abutment andoron bar score 2mdashmoderate plaque at first sight on abutmentandor on bar and score 3mdashplaque at first sight abundantthat occupies more than 13 of abutment andor bar

Certain mild degree of gingival recessions around dentalimplants was noted during examinations however theseobservations did not meet the criteria of periimplantitisor other substantial peri-implant pathology The mid-termand long-term success rates of dental implants were fullypredictable including good clinical prognosis None of thesigns indicating the possibilities of future implants failure wasobserved The initial clinical examination of stabilization ofdental implants and prosthodontic restorations was carriedout and was graded as ldquowithin acceptable limitsrdquo

Additionally the questionnaire containing the questionsabout organoleptic and rheological properties of toothpasteswas filled in by all patients in order to gather additionalinformation The questions taken into individual evaluationincluded color taste smell foaming ability and general opin-ion about tooth brushing effectiveness (tooth smoothnessfreshness feeling) and they were assessed in a 3-grade scaleThe data was then subjected to statistical analysis Patientswere assessed by two examiners using the same technique andprocedure in order to validate the objective clinical scores Allbacterial samples were taken in the same manner applyingthe standard protocol for oral microflora inoculums

24 Bacterial Strains Isolation and Microbiological Investiga-tion The estimation of bacterial spectrum was performed inquantitative and qualitative manner using standard methodsfor microbiological inoculation A number of 16 subjectswho participated in the study were scraped the gingivamargins surrounding dental implants with a sterile swabby the clinician Samples were collected by two examinersusing the same procedure The vials were delivered withina single working day and then immediately seeded in thelaboratory within 24 hours allowing the bacteria strainsisolation from clinical specimens which were subject forfurther inoculation

The biological material collected for microbiologicalinvestigation was cultured on a suitable medium (Columbiaagar Schaedler K3 agar Sabouraud agar) by bioMerieux(Marcy lrsquoEtoile France) Aerobic bacteria were multiplied onsolidmediumColumbia agar with 5 addition of ram bloodin the temperature of 37∘C Anaerobic bacteria were multi-plied on solidmedium Schaedler K3 with 5 addition of ramblood in the temperature of 37∘C in anaerobic conditionswith the use of GENbag anaer by bioMerieux (Marcy lrsquoEtoileFrance) Fungi of theCandida family on the other hand weremultiplied on selective solid medium Sabouraud agar in thetemperature of 35∘C in aerobic conditions

After they had been isolated and cultured further eachof the microorganisms was identified as regards its species


using the following set of reagents Api 20 E Api 20 NEand Api Candida by BioMerieux (Marcy lrsquoEtoile France)ENTEROtest 24 N andNEFERMtest 24 N STREPTOtest 24STAPHYtest 24 ANAEROtest 23 by Erba-Lachema (BrnoCzech Republic)

The data of individual patients were treated as confiden-tial and were not identifiable in any publication that emergedin relation to the examination The study represented aseparated part of the main research project of MedicalUniversity of Silesia supported byKNW-2-10210 SUMgrantThe research project was granted by the decision of BioethicsCommittee of the Medical University of Silesia (decision no62010)

25 Statistical Analysis Nonparametric tests were appliedfor statistical verification of assumed research hypothesesand analysis of quantitative and qualitative data (OHI APISBI toothpaste features) The comparison of the two depen-dent groups was carried out using nonparametric Wilcoxonsigned-rank test (OHI SBI API) For the two independentsamples the nonparametric Mann-Whitney U-test (OHISBI API) was applied for comparing pairs of groups Threedependent groups were compared with the use of FriedmanANOVA test All tests applied were two tailed and a 119875-valueof le005 was considered statistically significant Statistica90 software (StatSoft USA) was used for statistical analysis(Medical University of Silesia licence Katowice Poland)

3 Results

From the total group of 21 patients 16 successfully completedthe study according to the research protocol (10 womenand 6 men) The restricted number of subjects with fitteddental implants supporting prosthetic restorations reflectedthe strict inclusion criteria for the study Table 1 presents theimplant-prosthetic profile of the examined groups in termsof the number of dental implants installed and the typeof prosthetic suprastructure (crown bridge overdenture)supported by endosseous implantsThe total number of singleendosseous titanium-made implants installed for the group(A) and (C) was 29 and 24 respectively One patient from thegroup (A) was using the bar-retained removable overdentureand one subject from the group (C) had an overdenturesupported by two-single implants and locators Vast majorityof examined patients were the users of single porcelain-to-metal fused crowns retained by to the single endosseousimplants with conical metal abutments and active threads

31 Oral Health Conditions The overall distribution of therange of Greene and Vermilionrsquos oral hygiene index (OHI-D debris component) scores in the evaluated groups isshown in Table 2 For one subject from groups (A) and (C)carrying mandibular implant-retained overdenture on barthemodified index of oral hygienewas applied [34]TheOHI-D index (median) for (A) and (C) groups was establishedas 02 and 008 respectively and the differences were notstatistically significant comparing both mentioned groupsafter 1st 2nd and 3rd examination (Figure 1) OHI-D value

Table 1 Total number of dental implants installed and the type ofprosthetic suprastructure (crown bridge overdenture) supportedby endosseous implants in each group (A) and (C)

Group (A) Group (C)Age (mean) 4950 4587Gender

Male 2 4Female 6 4

Number of single dental implants 29 24Mean number of dental implants per patient 362 3Number of other abutments (bars) 1 0Number of single crowns 16 13Number of fixed suprastructures(at least 3 pointic) 4 3

Number of implant-supported overdentures 1 1

OHI-D values (median min-max)

0

04

08

12

16O

HI

CA 1 CA 2 CA 3 CC 1 CC 2 CC 3

Max

Median

75Min

25

Figure 1 Oral hygiene index values (median min-max) for exam-ined groups A and C

(median) of the group (A) which consisted of the patientsusing propolis-containing toothpaste decreased significantlyafter 8 weeks of the study (119875 lt 005) On the other hand theinfluence of toothpaste CC was not statistically significantdespite of the OHI-D scores decreasing tendency at the endof the study and the ldquoPrdquo value close to 005 (119875 = 0061) Itmay be explained by the effect of patients participation inthe study the use of specific hygienic agents alone and theimproved toothbrushing following oral hygiene instructionswhich could influenced the obtained results irrespective ofthe presence or absence of a propolis additive

The decreasing tendency of the Lange API scores wasalso observed within the assessed groups (A) and (C) andthe significant differences were noted for group (A) onlyessentially between the first and the last clinical assessment(119875 lt 005) During the first visit the average oral hygieneprofile of patients from group (A)was classified as an ldquoaverageoral hygienerdquo (625) while at the end of the study themeanAPI scorewas determined as ldquooptimal hygienerdquo (875)Vast majority of the patients from group (C) represented


Table 2 OHI debris componentmdashassessment for the group (A) and group (C) ( of cases within the range and min-max value)

Oral hygiene assessment (OHI-debris) Friedman ANOVAtest (119875) Wilcoxon rank test (119875)

1st 2nd 3rd

Group (C)

00ndash05 625 00ndash05 750 00ndash05 8750061

(1) (2) na(2) (3) na(1) (3) na

06ndash10 125 06ndash10 125 06ndash10 12511ndash20 250 11ndash20 125 11ndash20 00

(016ndash16) (0ndash125) (0ndash075)

Group (A)

00ndash05 625 00ndash05 750 00ndash05 8750003lowast

(1) (2) = 0027lowast(2) (3) = 0027lowast(1) (3) = 0043lowast


(0ndash083) (0ndash075) (0ndash075)Mann-Whitney 119880 test (119875) 0958 0792 0494 mdash mdashlowastStatistically significant 119875 value lt 005

Table 3 API rangesmdashassessment for the group (A) and group (C) ( of cases)

Oral hygiene assessment (interproximal spaces) Friedman ANOVAtest (119875) Wilcoxon rank test (119875)

1st 2nd 3rd

Group (C)

Optimal 250 Optimal 125 Optimal 500

0149(1) (2) na(2) (3) na(1) (3) na

Quitegood 125 Quite

good 50 Quitegood 125

Average 50 Average 375 Average 375Bad 125 Bad 00 Bad 00

Group (A)


0005lowast(1) (2) = 0201(2) (3) = 0022lowast(1) (3) = 0456

Quitegood 125 Quite



Mann-Whitney 119880 test(119875) 0472 0109 0183 mdash mdash

lowastStatistically significant 119875 value lt 005

an rdquoaverage oral hygienerdquo and ldquoquite good oral hygienerdquoprofile during the first and second follow-up assessmentsrespectively (Table 3 Figure 2)

The third assessment of the periodontal tissues carriedout using Muhlemann-sonrsquos sulcus Bleeding index allowedto classify all the patients from group (A) as the subjectsdescribed as ldquonormal healthy gingiva no bleedingrdquo with SBIvalue lt10 (Table 4) However the differences between thegroups (A) and (C) were not statistically significant after 1st2nd and 3rd assessment (119875 gt 005 Mann-Whitney U-test)A significant decrease of SBI value during the last follow-upassessment was observed for the group (A) patients (119875 lt005) Patients belonging to the group (C) (50 119899 = 4)who used the CC toothpaste were initially qualified into thesubjects with slight initial gingivitis (bleeding on probingwithout shape and color changes of gingiva) which requiredoral hygiene instructions and hygienic regime

32 Patients Opinions Analysis Based on QuestionnaireThe questionnaire-based assessment of the rheological andorganoleptic properties of the investigated toothpastes as well

as the patients individual opinions regarding a comfort whenusing these preparations revealed differences between thegroupsA andCThe taste and smell were assessedwith higherscores while the main complaint was pointed out towardsthe foaming ability which was scored as ldquounsatisfactoryrdquoby 625 of patients (119899 = 5) from group A and as onlyldquosatisfactoryrdquo by 625 of patients from group C (119899 = 5) Thecolour of the toothpaste CA was also not graded well andonly one patient from group A rated it as ldquogoodrdquo Generallythe CC toothpaste did not evoke such subjective opinions andwas better accepted (Table 5)

33 Microbiological Identification of Peri-Implant Oral Micro-biota Total amount of isolatedmicroorganisms present fromperi-implant areas of the patients using toothpaste CA andCC is stated according to Table 6The results of microbiolog-ical examinations of the clinical specimens demonstrated asubstantial quantitative as well as qualitative differentiationof the oral cavity microflora composition in patients withdental implants (groupA)whowere using implant-supportedprosthetic appliances applying toothpaste with EEP (CA


Table 4 SBImdashassessment for the group (A) and group (C) ( of cases)

Periodontal assessment Friedman ANOVAtest (119875)

Wilcoxon rank test(119875)1st 2nd 3rd

Group (C)Normal gingiva

SBI lt 10 50 Normal gingivaSBI lt 10 625 Normal gingiva

SBI lt 10 625 0483(1) (2) na(2) (3) na(1) (3) naBleeding on

probing 50 Bleeding onprobing 375 Bleeding on

probing 375

Group (A)Normal gingiva


SBI lt 10 100 0015lowast(1) (2) = 0067(2) (3) = 0043lowast(1) (3) = 0874Bleeding on


probing 00

Mann-Whitney 119880test (119875)

0599 0344 0127 mdash mdash


Table 5 Descriptive scale of tested toothpastes properties (CA andCC)

Subjects number ()Toothpaste features

Group A (CA) Group C (CC)

TasteUnsatisfactory 1 (125) 0 (00)Satisfactory 1 (125) 0 (00)Good 6 (750) 8 (100)

SmellUnsatisfactory 2 (250) 0 (00)Satisfactory 1 (125) 2 (25)Good 5 (625) 6 (750)

ColourUnsatisfactory 5 (625) 0 (00)Satisfactory 2 (250) 2 (250)Good 1 (125) 6 (75)

FoamingUnsatisfactory 5 (625) 1 (125)Satisfactory 0 (000) 5 (625)Good 3 (375) 2 (250)

Cleaning abilityUnsatisfactory 2 (250) 0 (00)Satisfactory 6 (750) 0 (00)Good 0 (00) 8 (100)

preparation) for oral hygiene maintenance a few times perday The microbiological material collected before the use ofpropolis-containing toothpaste (first assessment) allowed toidentify a total of 16 isolates of microorganismsrsquo representing10 speciesstrains in the group of patients applying CAtoothpaste with active organic ingredient

Second microbiological assessment performed aftereight weeks since the study commencement revealed a totalof 32 isolates of microorganisms representing 15 species(Table 7) In the patients of group (A) four species ofmicroorganisms were eliminated (Klebsiella oxytoca Serratialiquefaciens Staphylococcus epidermidisMSCNS Sarcina sp)at the same time the physiological and transient micro-floraof oral cavity has been enriched by the addition of 9 new

API values for groups A and C

01020304050607080

API

Max

Median


75Min

25

Figure 2 Approximal plaque index values (median min-max) forexamined groups (A) and (C)

species of microorganisms Streptococcus sanguinis Strepto-coccus vestibularis Streptococcus acidominimus Ruminococ-cus productus Veillonella parvula Bifidobacterium adoles-centis Bifidobacterium dentium Actinomyces naeslundii andCitrobacter freundii The second assessment of the (A)group of patients also revealed a less numerous presence ofEscherichia coli isolates than the first examination did At thesame time the second examination revealed the presence ofmore isolates of the bacteria Streptococcus mitis Streptococcussalivarius Neisseria species and Candida fungi During thefirst examination Candida albicans has been isolated in oneperson while Candida famata in another person whereas inthe case of the second examination three isolates of Candidaalbicans have been found (subjects different than the case of1st examination) as well as one isolate of Candida famata(the same subject as in 1st examination) These findingscan be related to the improved and excessive daily oralhygiene following the thorough instruction given during firstexamination


Table 6 Total amount of isolated microorganisms isolated from periimplant areas in the patients using toothpastes CA and CC

Micrococci Gram (+) Micrococci Gram (minus) Rods Gram (+) Rods and bacilli Gram (minus) Fungi Total

Group (A) Test 1 6 2 0 6 2 16Test 2 14 9 3 2 4 32

Group (C) Test 1 9 5 0 7 2 23Test 2 12 7 1 4 4 28

Table 7 Microorganisms isolated from oral cavity samples the investigated group (A) of patients with dental implants (119899 = 8) and implant-retained prosthetic suprastructures who were using propolis-containing toothpaste (CA)

Isolated microorganisms (strains) Baseline examination Final examination1 2 3 4 5 6 7 8 all 1 2 3 4 5 6 7 8 all

Gram-positive cocciStreptococcus mitis x x x 3 x x x x x 5Streptococcus sanguinis mdash x 1Streptococcus salivarius x 1 x x x x 4Streptococcus vestibularis mdash x 1Streptococcus acidominimus mdash x x 2Staphylococcus epidermidisMSCNS x 1 mdashRuminococcus productus mdash x 1Sarcina sp x 1 mdash

Gram-negative cocciNeisseria spp x x 2 x x x x x x x 7Veillonella parvula mdash x x 2

Gram-positive ActinobacteriaBifidobacterium adolescentis mdash x 1Bifidobacterium dentium mdash x 1Actinomyces naeslundii mdash x 1

Gram-negative bacilli (rod-shaped)Citrobacter freundii mdash x 1Escherichia coli x x x 3 x 1Klebsiella oxytoca x 1 mdashSerratia liquefaciens x x 2 mdash

FungiCandida albicans x 1 x x x 3Candida famata x 1 x 1

Total isolated strains 1 1 3 1 3 4 2 1 16 3 3 6 4 5 4 3 4 32x the presence of bacterial strain in investigated material

Considering the microbiological profile in the group ofeight patients who were using toothpaste without EEP (groupC) for oral cavity hygiene after eight weeks of application ofCC preparation a similar count of microorganisms isolateswas observed (Table 8) The material collected before thetoothpaste CC application commencement (1st examination)was represented by a total of 23 isolates of microorganismsbelonging to 13 species As a result of the second exam-ination carried out after eight weeks since CC applica-tion commenced a total of 28 isolates of microorganismsallocated to 16 species have been obtained The secondmicrobiological examination of group (C) patients revealed

the elimination of 7 species of microorganisms Streptococcussanguinis Escherichia coli Enterobacter aerogenes Enterobac-ter amnigenus Mitsuokella multiacidus Serratia liquefaciensand Serratia grimes After the period of 8 weeks the oralcavity micro-flora got enriched with 10 new species ofphysiological flora microorganism and potential pathogensStreptococcus vestibularis Streptococcus 120573 haemolyticus Fgroup Staphylococcus epidermidis MSCNS Sarcina sp Veil-lonella parvula Bifidobacterium adolescentis Klebsiella oxy-toca Klebsiella pneumoniae Pseudomonas aeruginosa andSerratia marcescensThe findings of the second examinationconcerning the patients marked as a group (C) also showed


Table 8 Microorganisms isolated from oral cavity samples the investigated group (C) of patients with dental implants (119899 = 8) who wereusing toothpaste (CC) without EEP


Gram-positive cocciStreptococcus mitis x x 2 x x x x 4Streptococcus sanguinis x x x 3 mdashStreptococcus salivarius x x 2 x x 2Streptococcus vestibularis mdash x 1Streptococcus haemolyticus F group mdash x 1Staphylococcus aureus MSSA x x 2 x x 2Staphylococcus epidermidis MSCNS mdash x 1Sarcina sp mdash x 1

Gram-negative cocciNeisseria spp x x x x x 5 x x x x x x 6Veillonella parvula mdash x 1

Gram-positive ActinobacteriaBifidobacterium adolescentis mdash x 1

Gram-negative bacilli (rod-shaped)Escherichia coli x 1 mdashEnterobacter aerogenes x 1 mdashEnterobacter amnigenus x 1 mdashKlebsiella oxytoca mdash x 1Klebsiella pneumoniae mdash x 1Mitsuokella multiacidus x x 2 mdashPseudomonas aeruginosa mdash x 1Serratia liquefaciens x 1 mdashSerratia grimesii x 1 mdashSerratia marcescens mdash x 1

FungiCandida albicans x 1 x x 2Candida glabrata x 1 x x 2

Total isolated strains 4 2 3 2 3 4 4 1 23 4 5 2 2 6 3 2 4 28

a higher count of isolated Streptococcus mitis and isolates ofthe Neisseria species as well as fungi of the Candida family(Candida albicans Candida glabrata)

4 Discussion

Individual home care and consistent professional mainte-nance have proven to be critical to the success and longevityof endosseous dental implants This is especially true in anenvironment with adjacent natural teeth which if affected byperiodontal disease could act as a reservoir for pathogenicbacteria and seed the peri-implant sulcus [35 36] An implantpatientrsquos home care regimen should be individually tailoredaccording to each patientrsquos needs These needs are basedon the location and angulation of the dental implants theposition and length of transmucosal abutments the type ofprosthesis and the rate of plaque and calculus accumulation[37] Proper monitoring and maintenance are essential to

ensure the longevity of the dental implant through a com-bination of appropriate professional care evaluation andeffective patient oral hygiene Oral rinses with antimicrobialproperties containing essential oils or chlorhexidine havebeen advocated for use in patients with implants [38] In thisarea the routine use of propolis-containing toothpaste seemsto have a beneficial effect on peri-implant tissues and plaqueaccumulation In a recent study authors Tanasiewicz et al[16] demonstrated that hygienic experimental preparations(toothpaste and gel) containing 3 ethanol propolis extractefficiently support reduction of dental plaque and have thetherapeutic local effect on marginal periodontium Theseresults are coherent to our findings and observations basedon the SBI values constituting the conclusion that domesticproducts for oral hygiene support the antiplaque action andhave the anti-inflammatory effect onmarginal periodontiumThe proven anti-inflammatory action of propolis seems tobe particularly advantageous for prophylactic proceduresof the patients with dental implants and increased risk of


periodontal inflammatory problems that is gingivitis andchronic periodontitis [39ndash41]

Current trend reveals a return to complementary med-icine and alternative treatment methods due to the devel-oping resistance to modern medications and antibioticsOnly few studies have investigated the activity of propolisethanolic extract towards oral pathogens particularly peri-odontopathic bacteria [36 38 39 42] Propolis samples werefound to be active mainly against Gram-positive bacteriaand some fungi They presented also a weak activity againstGram-negative bacteria [14 27 41 42] The pharmacolog-ically active constituents against oral bacteria in Brazilianpropolis are flavonoids (flavones flavonols flavanones) phe-nolics and aromatics including p-coumaric acid ferulicacid cinnamic acid and its derivativemdashdrupanin baccha-rin and artepillin C chrysinmdashtectochrysin pinocembrinpinobanksin isosakuranetin kaempferol kaempferide andquercetin [43ndash45]

In our study the analysis of the influence of toothpastecontaining 3 ethanol extract of propolis upon the mouthcavity micro-flora revealed beneficial quality changes in itsspecies composition consisting mainly of elimination ofpotential bacteria pathogens particularly Enterobacteriaceaefamily rods To summarize the results of microbiologicalexaminations it can be stated that the propolis includedin toothpaste thanks to its antimicrobial properties hasbeneficial influence upon the modification of oral cavitybacterial micro-flora whereas it hardly influences fungi ofthe Candida family The increase of the Candida strainsduring the second assessment may be caused by intensiveoral hygiene regime and hygienic habits improvement Theapplication of propolis in preparations used for routine oralcavity hygiene allows to eliminate microorganisms that arepathogens as well as microorganisms of physiological floralisted among opportunistic pathogens The European PatentEP 1738781 A2 describes the method which involves usingpropolis as coating material for medical implants [46]

Clinical researches have examined the associationbetween oralmicroorganismswhich are found in the saliva asnon-adhering populations and as plaque amicrobial biofilmand specific oral conditions such as dental caries periodontaldisease and oral mucosa diseases [5 6 36 39 41 47] Kooet al stated that mouthrinses containing propolis showedsignificant reduction of dental plaque and also inhibition ofbacterial polysaccharides formation [47] Similar to thesefindings we assumed that the choice of specific therapeuticproduct including propolis-containing toothpaste may havea direct influence on oral hygiene regime improvement andelimination of hygiene negligence in case of healthy patientsor with minor periodontal problems Propolis constitutes anatural alternative which helps to maintain oral health andhealthy periodontium

Study accomplished by Dodwad and Jha Kukreja [48]evaluated the effect of propolis mouthwash on plaque accu-mulation and gingivitis of a group of subjects who completelystopped the oral hygiene regime that is toothbrushingPlaque index increased on the 5th day with the estimatedvalue for propolis 68 and 16 for chlorhexidine Moreoverpropolis revealed 7 and chlorhexidine 9 increase in

gingival index These results suggested that propolis is notbetter than chlorhexidine in reducing plaque formation butmay be more efficient in reduction of gingival inflamma-tion This is in accordance with studies by Murray et al1997 [49] The research reported by Koo et al indicatedsubstantial effectiveness of propolis extract in inhibiting thegrowth of bacteria that belong to the ldquored complexrdquo [50]Santos et al Feres et al and Koru et al also confirmedthe antibacterial effectiveness of propolis extract towards thepathogens causing periodontitis [5 6 26 51 52] Differentgroups of organic compounds have been identified frompropolismainly flavonoids and phenolic acids (esters) whichare responsible for many of the biological activities attributedto European Brazilian and Asian propolis [53ndash55]

Due to the inhibition of the development of pathogenscausing parodontitisperi-implantitis extracts of propolis formouth rinsing or toothpastes based on propolis extractseems to be a promising agent not only for prophylaxis butalso for the treatment [5 6] Bruschi et al demonstratedthat the therapeutic propolis-containing gel applied locallyto gingival pockets may be effective for the treatment ofperiodontal diseases [5 6 56] The findings of Coutinhorsquosmicrobiological study revealed that subgingival irrigationswith the propolis extract applied in periodontal course oftreatment improved the treatment outcomes more signifi-cantly than scaling and root planning alone [57] The pro-phylactic action of propolis towards periodontal tissues as anadditive to mouths rinses or toothpastes allows to reduce thedental plaque formation and the initial signs of gingivitis [58]Numerous studies have also proven immunomodulatoryaction of propolis extract [59] among the other pharmaco-logical activities such as inhibitory action towards bacterialbiofilm formation [60]

The general improvement of oral hygiene which wasobserved during the study can be partially related to ldquosubjectof investigation effectrdquo Patients taking part in any clinicalresearch focusing on oral hygiene adjust and improve theirbrushing habits and carry them out more efficiently thanroutinely Our findings indicate that propolis andor itscompounds are promising antibacterial agents for preventionof oral diseases The effective biological action observed forthe propolis extract suggests its usage as an adjuvant to forexample therapy of periodontal problems The results of thepresented clinical study may suggest a positive influence ofpropolis-containing toothpaste (3 EEP) in patients with theoccurrence of gingivitis caused by dental plaque The routinedaily use of propolis-containing hygienic measures seems tohave a beneficial effect on peri-implant tissues by reducing aplaque accumulation and preventing development of chronicperiodontal disease

A further step should be given to verify if a dosesufficient to eradicate the target microorganisms can bereached within the subgingival environment without causingadverse effects over a long-term period of use Howeverthe representative group containing more individuals withdental implants is needed for relevant evaluation The localhygienic and domestic agents (eg toothpastes) containingnatural products need to have the appropriate and improvedorganoleptic features including more accepted colour and


foaming action It needs to be emphasised that when usingpropolis-based hygienic preparations the patient needs to beaware and needs to be informed about potential occurrenceof side effects particularly possible allergic reactions [16]

5 Conclusion

This study indicates a positive biological activity of propolis-containing toothpaste with respect to the oral microbiotaspectrum The results obtained suggest that propolis mightbe used as a natural alternative or additive to chemicalmouthwashes for example chlorhexidine in individuals suf-fering from periodontal problems associated with implantsusage Although further and long-term trials are required formore conclusive evidence antibacterial measures containingpropolis would be promising local agents acting againstpathogenic oral microflora

Authorsrsquo Contribution

Tadeusz Morawiec and Arkadiusz Dziedzic contributedequally to this work

Acknowledgments

The authors would like to thank Mr Rindai Yamamoto thePresident of Nihon Natural Food Co Ltd (Tokyo Japan)for the samples of toothpaste-containing ethanol extractof Brazilian green propolis The clinical study project wassupported by Medical University of Silesia Grant (KNW-2-10210 SUM) The paper is original and the authors takethe responsibility for the submission The authors declare noconflict of interests They confirm that all their affiliationswith or financial involvements in or any financial conflictwith the subject matter discussed in the paper are completelydisclosed (eg employment consultancies honoraria experttestimony grants and royalties)

References

[1] R T Demmer and P N Papapanou ldquoEpidemiologic patterns ofchronic and aggressive periodontitisrdquo Periodontology 2000 vol53 no 1 pp 28ndash44 2010

[2] S Dhir ldquoBiofilm and dental implant themicrobial linkrdquo Journalof Indian Society of Periodontology vol 17 no 1 pp 5ndash11 2013

[3] S S Socransky A D Haffajee M A Cugini C Smith and R LKent Jr ldquoMicrobial complexes in subgingival plaquerdquo Journal ofClinical Periodontology vol 25 no 2 pp 134ndash144 1998

[4] M Feres S C Cortellii L C Figueiredo A D Haffajee andS S Socransky ldquoMicrobiological basis for periodontal therapyrdquoJournal of Applied Oral Science vol 12 no 4 pp 256ndash266 2004

[5] WWięckiewiczMMiernikMWięckiewicz andTMorawiecldquoDoes propolis help to maintain oral healthrdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID351062 8 pages 2013

[6] G Kurtzman ldquoDental implants oral hygiene andmaintenancerdquoImplant Dentistry Today vol 1 no 3 2007

[7] A Mombelli and N P Lang ldquoAntimicrobial treatment of peri-implant infectionsrdquo Clinical Oral Implants Research vol 3 no4 pp 162ndash168 1992

[8] A K Garg F Duarte and K Funari ldquoHygienic maintenance ofdental implants the key to successrdquo Journal of Practical Hygienevol 6 no 2 pp 13ndash20 1997

[9] G R BaumanMMills JW Rapley andWHHallmon ldquoClin-ical parameters of evaluation during implant maintenancerdquoTheInternational Journal of Oral ampMaxillofacial Implants vol 7 no2 pp 220ndash227 1992

[10] S G Ciancio F Lauciello O Shibly M Vitello and MMather ldquoThe effect of an antiseptic mouthrinse on implantmaintenance plaque and peri-implant gingival tissuesrdquo Journalof Periodontology vol 66 no 11 pp 962ndash965 1995

[11] S Ciancio ldquoExpanded and future uses of mouthrinsesrdquo TheJournal of the AmericanDental Association vol 125 supplement2 pp 29ndash32 1994

[12] G P Rezende F C Pimenta and L R Costa ldquoAntimicrobialactivity of two Brazilian commercial propolis extractsrdquo Brazil-ian Journal of Oral Sciences vol 5 pp 967ndash970 2006

[13] G A Elbaz and I I Elsayad ldquoComparison of the antimicrobialeffect of Egyptian propolis versus New Zealand propolis onStreptococcus mutans and Lactobacilli in salivardquo Oral HealthPreventive Dentistry vol 10 no 2 pp 155ndash160 2012

[14] A Dziedzic R Kubina R D Wojtyczka A Kabała-DzikM Tanasiewicz and T Morawiec ldquoThe antibacterial effect ofethanol extract of Polish propolis on mutans steptococci andLactobacilli isolated from salivardquo Evidence-Based Complemen-tary and Alternative Medicine vol 2013 Article ID 681891 12pages 2013

[15] G Martınez Silveira A Gou Godoy R Ona Torriente M CPalmer Ortiz and M A Falcon Cuellar ldquoPreliminary study ofthe effects of propolis in the treatment of chronic gingivitis andoral ulcerationrdquo Revista Cubana de Estomatologıa vol 25 no 3pp 36ndash44 1988

[16] M Tanasiewicz M Skucha-Nowak M Dawiec W Krol DSkaba and H Twardawa ldquoInfluence of hygienic preparationswith a 3 content of ethanol extract of Brazilian propolis on thestate of the oral cavityrdquo Advanced in Clinical and ExperimentalMedicine vol 21 no 1 pp 81ndash92 2012

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

[18] Z P Czuba and W Krol ldquoThe importance of hydroxyl sub-stituent in position 41015840 in flavonoids for modulation of chemi-luminescence generated by an enzymatic system (horseradishperoxidase-luminol-hydrogen peroxide)rdquo Current Topics inBiophysics vol 20 pp 38ndash41 1996

[19] S Kumazawa T Hamasaka and T Nakayama ldquoAntioxidantactivity of propolis of various geographic originsrdquo Food Chem-istry vol 84 no 3 pp 329ndash339 2004

[20] W Krol S Scheller Z Czuba et al ldquoInhibition of neutrophilsrsquochemiluminescence by ethanol extract of propolis (EEP) and itsphenolic componentsrdquo Journal of Ethnopharmacology vol 55no 1 pp 19ndash25 1996

[21] W Krol Z Czuba S Scheller Z Paradowski and J ShanildquoStructure-activity relationship in the ability of flavonols toinhibit chemiluminescencerdquo Journal of Ethnopharmacology vol41 no 1-2 pp 121ndash126 1994

[22] A Kujumgiev I Tsvetkova Y Serkedjieva V Bankova RChristov and S Popov ldquoAntibacterial antifungal and antiviral


activity of propolis of different geographic originrdquo Journal ofEthnopharmacology vol 64 no 3 pp 235ndash240 1999

[23] L Drago B Mombelli E de Vecchi M C Fassina L TocalliandM R Gismondo ldquoIn vitro antimicrobial activity of propolisdry extractrdquo Journal of Chemotherapy vol 12 no 5 pp 390ndash3952000

[24] E Szliszka G Zydowicz B Janoszka C Dobosz G Kowalczyk-Ziomek and W Krol ldquoEthanolic extract of Brazilian greenpropolis sensitizes prostate cancer cells to TRAIL-inducedapoptosisrdquo International Journal of Oncology vol 38 no 4 pp941ndash953 2011

[25] E Szliszka and W Krol ldquoThe role of dietary polyphe-nols in tumor necrosis factor-related apoptosis inducing lig-and (TRAIL)-induced apoptosis for cancer chemopreventionrdquoEuropean Journal of Cancer Prevention vol 20 no 1 pp 63ndash692011

[26] F A Santos E M A Bastos M Uzeda et al ldquoAntibacterialactivity of Brazilian propolis and fractions against oral anaer-obic bacteriardquo Journal of Ethnopharmacology vol 80 no 1 pp1ndash7 2002

[27] M I Nieva Moreno M I Isla N G Cudmani M A Vattuoneand A R Sampietro ldquoScreening of antibacterial activity ofAmaicha del Valle (Tucuman Argentina) propolisrdquo Journal ofEthnopharmacology vol 68 no 1ndash3 pp 97ndash102 1999

[28] M Viuda-Martos Y Ruiz-Navajas J Fernandez-Lopez and JA Perez-Alvarez ldquoFunctional properties of honey propolis androyal jellyrdquo Journal of Food Science vol 73 no 9 pp 117ndash1242008

[29] V Bankova R Christov A Kujumgiev M C Marcucci andS Popov ldquoChemical composition and antibacterial activity ofBrazilian propolisrdquo Zeitschrift fur Naturforschung C vol 50 no3-4 pp 167ndash172 1995

[30] V Seidel E Peyfoon D G Watson and J Fearnley ldquoCompara-tive study of the antibacterial activity of propolis from differentgeographical and climatic zonesrdquo Phytotherapy Research vol22 no 9 pp 1256ndash1263 2008

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

[32] E Szliszka G Zydowicz E Mizgala and W Krol ldquoArtepillinC (35-diprenyl-4-hydroxycinnamic acid) sensitizes LNCaPprostate cancer cells to TRAIL-induced apoptosisrdquo Interna-tional Journal of Oncology vol 41 no 3 pp 818ndash828 2012

[33] C Gatti and M Chiapasco ldquoImmediate loading of Branemarkimplants a 24-month follow-up of a comparative prospectivepilot study between mandibular overdentures supported byConical transmucosal and standard MK II implantsrdquo ClinicalImplant Dentistry and Related Research vol 4 no 4 pp 190ndash199 2002

[34] J Faus-Lopez E Canet and J P Firmino ldquoModification ofthe index of oral hygiene in Patients carrying mandibularimplant overdentures on barsrdquo Universidad Cardenal Herrera-CEU Valenci Spain httpwwwivioesMODIFICATION20OF20THE20INDEX20OF20ORAL20HYGIENE202pdf

[35] W Papaioannou M Quirynen M Nys and D van Steen-berghe ldquoThe effect of periodontal parameters on the sub-gingival microbiota around implantsrdquo Clinical Oral ImplantsResearch vol 6 no 4 pp 197ndash204 1995

[36] E C E Gebara L A Lima and M P A Mayer ldquoPropo-lis antimicrobial activity against periodontopathic bacteriardquo

Brazilian Journal of Microbiology vol 33 no 4 pp 365ndash3692002

[37] R M Meffert ldquoIn the spotlight implantology and the dentalhygienistrsquos rolerdquo Journal of Practical Hygiene vol 12ndash14 1995

[38] K Schwach-Abdellaoui N Vivien-Castioni and R GurnyldquoLocal delivery of antimicrobial agents for the treatment ofperiodontal diseasesrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 50 no 1 pp 83ndash99 2000

[39] Y K Park M H Koo J A S Abreu M Ikegaki J A Curyand P L Rosalen ldquoAntimicrobial activity of propolis on oralmicroorganismsrdquo Current Microbiology vol 36 no 1 pp 24ndash28 1998

[40] S Sonmez L Kirilmaz M Yucesoy B Yucel and B YilmazldquoThe effects of bee propolis on oral pathogens and humangingival fibroblastsrdquo Journal of Ethnopharmacology vol 102 pp71ndash76 2005

[41] F Ozan Z Sumer Z A Polat K Er U Ozan and H DegerldquoEffect of propolis containing mouthrinse on oral microor-ganisms and human gingival fibroblastsrdquo European Jounal ofDentistry vol 1 pp 195ndash201 2007

[42] D Steinberg G Kaine and I Gedalia ldquoAntibacterial effectof propolis and honey on oral bacteriardquo American Journal ofDentistry vol 9 no 6 pp 236ndash239 1996

[43] J M Sforcin A Fernandes C A M Lopes V Bankova and SR C Funari ldquoSeasonal effect on Brazilian propolis antibacterialactivityrdquo Journal of Ethnopharmacology vol 73 no 1-2 pp 243ndash249 2000

[44] M C Marcucci ldquoPropolis chemical composition biologicalproperties and therapeutic activityrdquo Apidologie vol 26 pp 83ndash99 1995

[45] A Uzel K Sorkun O Oncag D Cogulu O Gencay andB Salih ldquoChemical compositions and antimicrobial activitiesof four different Anatolian propolis samplesrdquo MicrobiologicalResearch vol 160 no 2 pp 189ndash195 2005

[46] httpwwwgooglecompatentsEP1738781B1cl=en[47] H Koo J A Cury P L Rosalen G M B Ambrosano M

Ikegaki and Y K Park ldquoEffect of a mouthrinse containingselected propolis on 3-day dental plaque accumulation andpolysaccharide formationrdquo Caries Research vol 36 no 6 pp445ndash448 2002

[48] V Dodwad and B Jha Kukreja ldquoPropolis mouthwash a newbeginningrdquo Journal of Indian Society of Periodontology vol 15no 2 pp 121ndash125 2001

[49] M CMurray H VWorthington and A S Blinkhorn ldquoA studyto investigate the effect of a propolis-containing mouthrinse onthe inhibition of de novo plaque formationrdquo Journal of ClinicalPeriodontology vol 24 no 11 pp 796ndash798 1997

[50] H Koo B P F A Gomes P L Rosalen G M B AmbrosanoY K Park and J A Cury ldquoIn vitro antimicrobial activity ofpropolis and Arnica montana against oral pathogensrdquo Archivesof Oral Biology vol 45 no 2 pp 141ndash148 2000

[51] M Feres L C Figueiredo I M Barreto M H Coelho MW Araujo and S C Cortelli ldquoIn vitro antimicrobial activityof plant extracts and propolis in saliva samples of healthy andperiodontally-involved subjectsrdquo Journal of the InternationalAcademy of Periodontology vol 7 no 3 pp 90ndash96 2005

[52] O Koru F Toksoy C H Acikel et al ldquoIn vitro antimicrobialactivity of propolis samples from different geographical originsagainst certain oral pathogensrdquo Anaerobe vol 13 no 3-4 pp140ndash145 2007


[53] Z Czuba W Krol S Scheller and J Shani ldquoEffect of cinnamicand acrylic acidsrsquo derivatives on luminol-enhanced chemilumi-nescence of neutrophilsrdquo Zeitschrift fur Naturforschung C vol47 no 9-10 pp 753ndash756 1992

[54] W Krol Z P Czuba M D Threadgill B D M Cunninghamand J Shani ldquoModulation of luminol-dependent chemilumi-nescence of murine macrophages by flavone and its syntheticderivativesrdquo Arzneimittel-Forschung vol 45 no 7 pp 815ndash8181995

[55] W Krol J Shani Z Czuba and S Scheller ldquoModulat-ing luminol-dependent chemiluminescence of neutrophils byflavonesrdquo Zeitschrift fur Naturforschung C vol 47 no 11-12 pp889ndash892 1992

[56] 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

[57] A Coutinho ldquoHoneybee propolis extract in periodontal treat-ment A clinical and microbiological study of propolis inperiodontal treatmentrdquo Indian Journal of Dental Research vol23 no 2 p 294 2012

[58] S Hidaka Y Okamoto K Ishiyama and K Hashimoto ldquoInhi-bition of the formation of oral calcium phosphate precipitatesthe possible effects of certain honeybee productsrdquo Journal ofPeriodontal Research vol 43 no 4 pp 450ndash458 2008

[59] J M Sforcin ldquoPropolis and the immune system a reviewrdquoJournal of Ethnopharmacology vol 113 no 1 pp 1ndash14 2007

[60] R DWojtyczka M Kępa D Idzik et al ldquoIn vitro antimicrobialactivity of ethanolic extract of Polish propolis against biofilmforming Staphylococcus epidermidis strainsrdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID590703 11 pages 2013

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


or Fusobacterium nucleatum among others are potentialetiological factors causing periodontitis and peri-implantitisproblems [4] Reduction of the number of those pathogenicmicro-organisms could potentially influence the epidemiol-ogy of periodontium diseases reducing their amount andintensification The hygiene regime related to oral healthmaintenance is a critical area of implant-supported casesof oral rehabilitation The periodontal tissues surroundingdental implants seem to be particularly susceptible to bacteriainvasion and local inflammatory reactions [5ndash7]

Effective oral hygiene around dental implants can bechallenging to achieve over the long term and the patientdentist and dental hygienistmust exercise considerable effortto achieve the desired results In more recent years implantmaintenance and effective patient home care have beenemphasized as critical factors needed for long-term successof dental implants [8] As the number of patients treated withdental implants continues to grow the patients must be fullyaware of a proper maintenance with the use of preventive andtherapeutic local measures [9 10] Among them propolismdasha resinous substance made by bees possessing biologicaltherapeutic activitiesmdashmay play an important role in oralhygiene and inmaintaining healthy soft tissueswithin the oralcavity It is known for its ability to nourish periodontal tissuesand prevent problems associated with gingiva and supportedhistological elements [8 11]

Humans began using propolis more than 2000 years agofor many purposes the foremost of which was applying it towounds to fight infection It promotes the healing processwith its mild anti-infective properties [12] Over time ithas been marketed in various forms such as toothpastesmouthrinses and lozenges Propolis extract is known topossess antimicrobial activity against Streptococcus mutans aGram-positive cocci facultative anaerobic strains commonlyfound in the human oral cavity and a significant contributorto plaque formation [13 14] Propolis has been also foundbeneficial in the treatment of gingivitis and oral mucosalesions in case studies and pilot clinical studies [15 16]

Over the last few decades a worldwide trend has beenobserved in the use of natural products for pharmacologicalpurposes due to their proven therapeutic effect Propoliswhich is a natural product widely consumed in the folkmedicine since ancient times and a substance produced bythe honeybees seems to be a promising agent to be addedto topical formulations due to its multidirectional properties[17] Besides antioxidant and anti-inflammatory activity [18ndash21] epidemiological studies have detected that propolis hasmany beneficial properties such as antibacterial antifungalantiviral and antitumor [22ndash26] Ethanolic extract of propo-lis (EEP) solutions has been used commercially on themarketas toothpaste mouth wash lozenges and so forth as aneffective antimicrobial and anti-inflammatory agent [27 28]However it is still an underestimated ingredient in academicmedicine and dentistry In general propolis is composed of50 resin and vegetable balsam 30 wax 10 essential andaromatic oils 5 pollen and 5 various other substancesincluding organic debris depending on the place and timeof collection [29] The constituents of propolis vary widelydue to climate season location and year and its chemical

formula is not stable [30 31] Flavonoidsmdashthe main propolisingredientmdashinhibit lipid peroxidation platelet aggregationcapillary permeability and fragility and the activity of enzymesystems including cyclo-oxygenase and lipoxygenase [28 31]

The study aimed at investigating the local effect of theorally administered extract of propolis in the form of tooth-paste on selected intraoral predictors representing oral healthcondition in the group of patients with dental implants-supported fixed and removable prosthodontic suprastruc-tures

2 Material and Methods

The clinical study was carried out to investigate the influ-ence of a propolis-containing toothpaste on gingival healthand the oral microbiota spectrum changes This researchwas conducted in Oral Surgery Department the AcademicCentre of Dentistry and Specialist Medicine (Bytom Poland)and Specialist Dental Clinic (Katowice Poland) which wereproviding comprehensive dental care for the patients whounderwent implant-prosthetic therapy All implants wereinstalled by the same operator a specialist in oral surgeryTheldquooldestrdquo installation was carried out 9 years ago and the mostrecent was 2 years ago The study was designed as a singleblind two-group parallel study The dental products and testsemployed in the study were legal merchandises commonlyavailable and used The study was not a clinical trial or aclinical experiment

21 Hygienic Compounds Preparation Two samples of tooth-pastes covered anonymously by a blank label and markedonly with the letters (A) or (C) were compared the testedtoothpaste containing the active ingredientmdash3 ethanolextract of Brazilian propolis (CA DentalPolisDX ldquogreenrdquopropolis) and a placebo as a control (CC) without activesubstance Raw propolis was collected from the beekeepingsection of the Seiri Alimentos Naturais Brazil Propolis sam-ples were obtained from colonies of Africanized honeybees(Apis mellifera) in Minas Gerais State Southeast Brazil andcollected over a period of 2008 year from the plant Baccharisdracunculifolia which is the main botanical source of theresin for the green propolis and determines the compositionof Brazilian EEP rich in artepillin C [32] The unprocessedpropolis was sent to the Nihon Natural Therapy Co Ltd(Tokyo Japan) for the preparation of the EEP ingredientThetoothpastes with 3 EEP and without of EEP (placebo) wereprepared in Nippon Zettoc Co Ltd Tokyo Japan

22 ResearchGroups InclusionCriteria Subjects qualificationfor the study was based on medical and dental historyinterview and review of clinical records The exclusioncriteria from the investigationwere as follows lack of patientrsquosvalid consent medically compromised patients inability tocomplywith the follow-up visit requirements patients receiv-ing concurrent antibiotic treatment for any other purposeindividuals with confirmed adverse reactions to bee productsnursing or pregnant women and recent postoperative oral

















3 Results





Male 2 4Female 6 4




0

04

08

12

16O

HI


Max

Median

75Min

25







1st 2nd 3rd

Group (C)


(1) (2) na(2) (3) na(1) (3) na



Group (A)







1st 2nd 3rd

Group (C)


0149(1) (2) na(2) (3) na(1) (3) na

Quitegood 125 Quite



Group (A)


0005lowast(1) (2) = 0201(2) (3) = 0022lowast(1) (3) = 0456

Quitegood 125 Quite


















probing 375





probing 00


0599 0344 0127 mdash mdash













01020304050607080

API

Max

Median


75Min

25






Group (A) Test 1 6 2 0 6 2 16Test 2 14 9 3 2 4 32

Group (C) Test 1 9 5 0 7 2 23Test 2 12 7 1 4 4 28





















4 Discussion















5 Conclusion




Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































3 Results





Male 2 4Female 6 4




0

04

08

12

16O

HI


Max

Median

75Min

25







1st 2nd 3rd

Group (C)


(1) (2) na(2) (3) na(1) (3) na



Group (A)







1st 2nd 3rd

Group (C)


0149(1) (2) na(2) (3) na(1) (3) na

Quitegood 125 Quite



Group (A)


0005lowast(1) (2) = 0201(2) (3) = 0022lowast(1) (3) = 0456

Quitegood 125 Quite


















probing 375





probing 00


0599 0344 0127 mdash mdash













01020304050607080

API

Max

Median


75Min

25






Group (A) Test 1 6 2 0 6 2 16Test 2 14 9 3 2 4 32

Group (C) Test 1 9 5 0 7 2 23Test 2 12 7 1 4 4 28





















4 Discussion















5 Conclusion




Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















1st 2nd 3rd

Group (C)


(1) (2) na(2) (3) na(1) (3) na



Group (A)







1st 2nd 3rd

Group (C)


0149(1) (2) na(2) (3) na(1) (3) na

Quitegood 125 Quite



Group (A)


0005lowast(1) (2) = 0201(2) (3) = 0022lowast(1) (3) = 0456

Quitegood 125 Quite


















probing 375





probing 00


0599 0344 0127 mdash mdash













01020304050607080

API

Max

Median


75Min

25






Group (A) Test 1 6 2 0 6 2 16Test 2 14 9 3 2 4 32

Group (C) Test 1 9 5 0 7 2 23Test 2 12 7 1 4 4 28





















4 Discussion















5 Conclusion




Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























probing 375





probing 00


0599 0344 0127 mdash mdash













01020304050607080

API

Max

Median


75Min

25






Group (A) Test 1 6 2 0 6 2 16Test 2 14 9 3 2 4 32

Group (C) Test 1 9 5 0 7 2 23Test 2 12 7 1 4 4 28





















4 Discussion















5 Conclusion




Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Group (A) Test 1 6 2 0 6 2 16Test 2 14 9 3 2 4 32

Group (C) Test 1 9 5 0 7 2 23Test 2 12 7 1 4 4 28





















4 Discussion















5 Conclusion




Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























4 Discussion















5 Conclusion




Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























5 Conclusion




Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article The Biological Activity of Propolis...

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