Action of Propolis on Microorganisms of the Oral Cavity : an Integrative Review revIew

Method: we opted for the integrative literature review, using the descriptors: oral cavity, propolis, anti-inflammatory activity, antimicrobial activity, endodontics, antimicrobial, propolis, saliva, metabolism, microbiology, antimicrobial activity and mouth; the consulted databases were PubMed, LILACS and SciELO, published between 1999 and 2014. The results were obtained through the selection of 09 articles, through close reading, critical and reflective texts, and then the organization of summary frames of data.


Introduction
In the mouth there are several microorganisms, including bacteria and fungi, which, under favorable conditions, can cause oral health problems.Therefore, the oral microbiota is one of the main objects of study in order to understand mechanisms that cause dental caries, mouth ulcers, gingivitis, periodontitis and alveolitis, as well as the treatment of these, as they are considered infectious diseases [1].
In combating these diseases there are several antibiotics and antifungals, synthetic drugs and also natural.with therapeutic purpose in vivo, the products of natural extracts base have been used.
Most of products of vegetable origin shows antibacterial substances, in its composition, capable of reducing the growth of bacteria in the mouth, and inhibit glucan synthesis from sucrose by the glycosyltransferase [2].
Among the natural compounds, propolis has been propagated in dentistry, for the treatment of the aforementioned diseases, due to its anti-inflammatory and efficiency in the removal of the oral microbial flora.
Propolis is a complex mixture, formed by resinous and balsamic material and is collected by bees in the branches, flowers, pollen, shoots and exudates of trees.This resin, mixed with salivary secretions of bees is used for the core protection against the proliferation of microorganisms, including fungi and bacteria [3].
In many countries, various components of propolis have been analyzed, and caffeic acid, phenethyl ester of caffeic acid (PECA) and flavonoids mainly responsible for the antibiotic power of this resin [4].
Thus, it has been demonstrated antibacterial activity of propolis, especially against gram-positive bacteria, and cariogenic involved in periodontal pathogenic process, highlighting the Streptococcus mutans, Lactobacillus casei, Prevotella intermedia/ Prevotella nigrescens, Porphyromonas gingivalis.Moreover, they highlight their potential antifungal and anti-inflammatory because it stimulates cell mediated immunity and enhances the phagocytic activity and the inhibition of prostaglandin synthesis [5].
Accordingly, the objective was to identify what is the action of propolis on the microorganisms present in the oral cavity, through the analysis of scientific production.

Method
It is an integrative literature review, an approach that allows, from the analysis of the data, having a support helps in decision-making and improve clinical practice.The integrative review process includes six distinct stages, similar to conventional research stages of development [6]: 1 st ) identification of the theme and selecting the search issue.It begins with the definition of a problem and the formulation of a hypothesis of research that has relevance for health.In this sense, when you think of the mouth, their invasion by microbial agents and action of propolis on top of these, the central question of this study was: what is the action of propolis on microbial oral cavity?
2 nd ) Criteria for the selection of the sample.The survey was conducted on 2014 September 24 th , by searching in the databases of the Virtual Health Library (BVS): LILACS (Latin American and Caribbean Health Sciences) and SciELO (Scientific Electronic Library Online) and international database PubMed (Medical Published-serve of the US National Library of Medicine).
The descriptors were used: oral cavity, propolis, anti-inflammatory activity, antimicrobial activity, and endodontics, antimicrobial (85 articles in PubMed); In LILACS database, using the descriptors propolis, antibiotics and mouth was found only one article.In order to find more articles was decided to use two descriptors by search.Thus, the descriptors were used propolis and saliva (5 articles), propolis and metabolism (1 article), propolis and microbiology (3 articles), which referred to the oral cavity.Similarly it was proceeded in PubMed and SciELO, with no addition to the articles.
In Scielo, the descriptors with propolis, antimicrobial activity and mouth, totaled 4 articles, of which 2 contained in the LILACS database.Exclusion criteria were literature review articles of the narrative type and preliminary study.Articles published in Portuguese were included, English and Spanish, with abstracts and full text available on the selected databases; It referred to the action of propolis on microorganisms of the oral cavity, which method adopted allow to obtain strong evidence (levels 1, 2, 3 and 4), i.e., a systematic review and meta-analysis or not, randomized clinical trial (RCT) with 1000 patients, RCT with less than 1000 patients and cohort study [7].
These evidences were adopted considering the question that guided this review, because it is clinical question [7].However, given the lack of systematic review and cohort study, and the minimum publication of clinical trials, and that most of the studies found in the bases was accessed in vitro (evidence level 8), it was decided to include them.The strategies used to fetch the articles in the databases were adapted, because such bases present specific characteristics.
Thus, the research was guided by the question and inclusion and exclusion criteria, to maintain consistency in the search for articles and avoid possible biases.To refine your search, in addition to those descriptors were added to the filters: humans, free full text, languages (English, Spanish, and Portuguese).Thus, in PubMed and LILACS, the search amounted to ten articles in each; In SciELO, two articles.
3 rd ) Identification of pre-selected and selected studies.was held at first reading the summaries, being preselected five articles -PUBMED, ten -LI-LACS and two -SciELO.It was later performed in full, a close reading of the pre-selected articles and verified their suitability for inclusion criteria of the study.That done, there were obtained the bibliogra-phic material and the consolidation of the findings, to facilitating reflection and conclusion of the study.In reading found repeated articles in LILACS and SciELO databases.Finally, they selected nine articles, as follows: one -SciELO, two -PubMed, four -LI-LACS and two -LILACS and SciELO.
4 th ) Categorization of studies.An analogous phase to collecting the conventional survey data.For data collection of articles it was used a validated instrument containing the following items: identification of the original article, methodological characteristics of the study, assessment of the methodological rigor of measured interventions and results found [8].
Through data collection instrument it was possible an individual assessment of the studies included, both methodologically and in relation to the synthesis of the results, keeping in mind the issue problem, the findings were listed by reading and the inclusion criteria previously mentioned.
5 th ) Analysis and interpretation of results.For the analysis and subsequent synthesis of the articles that met the inclusion criteria was used a summary frame specially built for this purpose, which included the following aspects considered relevant: Name of the research, authors' names for article, level of evidence and the action of propolis on microbial oral cavity.Finally, there was analyzed descriptively in the light of scientific evidence.
6 th ) Presentation of synthesis of knowledge.The evidence was gathered and synthesized and conclusions of studies questioned because of their limitations.
The method of six articles [10-12, 14, 16-17] The in vitro study was considered weak evidence, one of them being combined with the experimentally [17]; and three clinical trials [9,13,15], with evidence level 3, as were a few patients.

Evidence about the action of propolis on oral cavity microorganisms
In response to the main question of this integrative review: "what is the action of propolis on the microorganisms of the oral cavity", table 2 presents a summary of the analyzed articles.

Discussion
The interest in propolis in Brazil happened in the 80s of the twentieth century, with record of pioneering work of Ernesto Ulrich Breyer.In his writing, "Bees and health," demonstrated the therapeutic properties of propolis and its use as a natural antibiotic [18].However, despite this elapsed time, are few and incipient research involving propolis, as evidenced by the quantitative research that this literature review and years of publication.
when the language of the reports analyzed, it is noticed that while English is considered a frontier language serving as a key point for the construction, reconstruction and improvement of research [19], it was found almost the same number of articles published in Portuguese.
The highlight in knowledge identified in the revised publications is due to the fact that propolis has different types of biological activity and applications in various therapies, especially in the dental field [2].
Although most studies have applied analytical statistics with reliable statistical tests, casts doubt on generalizations about the studied object, because besides the methodological designs were of weak evidence (level 8), the sample size was small, which does not reveal representation.In turn, in vitro studies are lower Evidence strength, because they mimic biological conditions in the laboratory and are used to test new materials or therapeutic or preventive methods.Such studies "do not establish guidelines for clinical management, nor do they provide priority information for clinical decision making" [20].
In addition, the above authors analyzed published research in Brazilian dental journals and concluded that the small number of research publications with higher level of evidence points to the importance of an expanded knowledge among Brazilian researchers on evidence-based clinical practice.
It is observed that the oral rinsing of alcohol-free propolis had a more effective action than chlorhexidine.However, statistical significance was the same for both products (p < 0.05) compared to Streptococcus mutans and lactobacilli [9].
A clinical study found that propolis reduced the plaque index by about 44.7% after treatment compared to placebo.In addition, the used mouthwash propolis decreased to 61.7%, the concentration of The extraction was performed by maceration, with ethyl alcohol 96 GL, for a period of fifteen days, then made the filtration and concentration of macerated in evaporator route, obtaining thus alcoholic extracts the 25%.

Paraná
Paraná -showed no bactericidal action on microorganisms tested, even at the highest concentrations tested.

Minas Gerais
Propolis from Minas Gerais was the one that proved most active of all, with the formation of inhibition halos slightly larger in relation to the propolis of São Paulo.

São Paulo
Minas Gerais and São Paulo -showed antimicrobial activity for the microorganism S. mutans from concentration equivalent to 0.6 µg/L.
[15] The propolis extract was sprayed and macerated in hydroalcoholic, obtaining a propolis solution to 60% and determined the antimicrobial activity of strain of S. mutans in solid medium for determining the Minimum Inhibitory Concentration (MIC).

Southern Region of the State of São Paulo
Propolis solution to 6.25% interfered significantly on growth of microorganisms (p < 0.001), being observed similar reduction when the use of chlorhexidine solution to 0.12% (p < 0.01).
[ insoluble polysaccharides, plate, compared to placebo suggesting that had an effect on the oral microflora.However, the study did not specify what concentration or solute treatment was performed [21].
The glycolic extract of propolis reduced C. albicans and E. coli completely eliminated, while the calcium hydroxide reduced growth, demonstrating resistance against this bacterium used product [11].However, the actual mechanism of the antimicrobial properties of propolis appears to be complex and not fully understood.In this some authors attribute to the highest antimicrobial activity flavonoids present in propolis extract [22].Using alcoholic extract of propolis, a study obtained better results in relation to the strains of S. mutans, S. salivarius, S. mitis and L. casei, compared to aqueous extract [11].In vitro research, developed using as antimicrobial agent propolis extracts (not specified where concentration nor solute), isolated fractions and purified compounds showed reductions in Streptococcus mutans count, as well as interference with its adhesion capacity and activity glycosyltransferase, which are considered large properties in the establishment of cariogenic process [23].
Research In vitro followed by ex-alive, it used dry extract of propolis at low concentrations (11%, 20% and 30%), comparing the results to those of industrialized products Listerine®, Malvatricin®, Pa-rodontax® and Periogard® and found that there was inhibition of glucose consumption, similarly to those products [12].In this regard, a survey showed antibacterial inhibitory effect of propolis in concentration reduced front of Staphylococcus aureus, a gram-positive species with broad-spectrum oral pathogenicity [24].
In an experiment with 41 young people who did not alter the oral hygiene, or eating habits during the experiment, it was used propolis extract mouthwash (without solute information or concentration) three times daily for seven days.Saliva was collected from participants before the first use and after an hour and seven days after the first rinse.The results showed that in 49% of all samples collected after the use of the extract, a reduction of the concentration of S. mutans.However, 26% of the samples there was no change, and 25% noted an increase of S. mutans [13].This phenomenon can happen because of caries be a mixed infection and oral microflora is highly diversified.Furthermore, S. mutans may be present in the oral cavity in three different serotypes C, E and F [25].
Using the propolis of three distinct regions, they observed that the Paraná (even in higher concentrations) showed no bactericidal activity on the tested microorganisms, unlike the propolis of Minas Gerais, which showed antimicrobial property to the S. mutans, from equivalent concentration to 0.6 µg/L, and introduced more active regarding that of Paraná and São Paulo.[14] Referring to the propolis by regions of Brazil, points out research in the Japanese market, the propolis produced in Minas Gerais State is considered the world's best, according to the Beekeepers Federation of Minas Gerais [2].
The fact of the biological properties of propolis are directly related to its chemical composition, which varies with the type of vegetation in the area, the time of collection, the technique employed, the species of bee and the level of Africanization of Apis mellifera in Brazil suggests that the antimicrobial action is closely related to the concentration of flavonoids and caffeic acid [2,26].
In crossover trial (15 children) analyzing the effects of mouthwash with propolis and chlorhexidine, showed that, similar to the 0.12% chlorhexidine mouthwash, the hydroalcoholic extract of propolis to 6.25% significantly reduced levels of S. mutans, bacteria that act on the conditions of gum disease and dental plaque accumulation, suggesting that propolis mouthwash may be indicated as therapeutic chemical agent [15].
Another study showed that the ethanol extract of propolis has antimicrobial activity against all tes-ted periodontal bacteria, and also capable of causing about superinfection (S. aureus, P. aeruginosa, Escherichia coli); and antifungal activity against C. albicans [16].Moreover, other research inhibitory results obtained with gram-negative anaerobes, such as Prevotella intermedia, Prevotella nigrescens and Porphyromonas gingivalis, which have been associated with periodontal disease, confirmed the antibacterial activity of the compound of propolis [27].
Authors tested the minimum inhibitory dilution of propolis and Periogard® against yeasts oral cavity, lesions and healthy, and found that most strains were sensitive to both antimicrobials.However, they observed that propolis 50% inhibited 77.1% of the strains, most of C. albicans (96.5%).Noting that C. albicans and C. tropicalis were sensitive to the same concentrations (01:20 ou 01:40), while the other species are sensitive to the concentration of 01:20.Interestingly, the isolated species of oral injury, C. albicans, C. tropicalis and C. glabrata were resistant to all Propolis dilutions tested [17].
The resistance of C. albicans may be related to its ability to adapt to different environmental conditions, since this fungus is able to survive as a dinner in adaptation to acidic or basic pH.Furthermore, the C. albicans has the ability to undergo morphological transition, hyphae yeast change for representing a change of state pathogenicity for dinner, being able to invade host tissues and escaping phagocytosis by macrophages; this yeast also form biofilms on different surfaces [28].
In the analysis of all studies we noticed the use of propolis in different concentrations and solutes, resulting in some regions of Brazil, once identified.In the early twenty-first century, Brazil was responsible for 10-15% of world production of propolis, and that Japan has preference for Brazilian propolis, due to its pharmacological properties, its organoleptic characteristics and the lowest content of heavy metals and other pollutants environmental [2].
These facts have been confirmed, since the studies analyzed showed the antimicrobial activi-ty of propolis in the oral cavity, enhancing their inhibitory character on gram-negative and grampositive, anaerobic and aerobic, as well as antifungal activity on Candida albicans; excepting to the fact that most research revised result from in vitro studies and some did not specify what type of extract, if alcoholic, aqueous or glycolic, either concentrations.

Synthesis of knowledge about the action of propolis on the microorganisms of the oral cavity
Several species, from bacteria and fungi naturally colonize the oral cavity, which, in favorable conditions, can cause diseases, highlighting caries, periodontitis and others.In this sense, propolis is a natural alternative substance to be used in the control and prevention of such diseases.
Thus, this review showed that researchers testing of low concentrations propolis, with prevalence used alcohol as a solvent, being effective against gram-positive bacteria Staphylococcus mutans, S. salivarius and S. mitis; and gram-negative (periodontal pathogens), as follows: Actinobacillus actinomycetemcomitans, pseudomonas intermedia, Pseudomonas melaningenic, Pseudomonas gingivalis and Fusobacterium nucleatum, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus (particularly those last three species are also capable of causing superinfection).
Used in typed form, free of alcohol, propolis was able to reduce the salivary levels of S. mutans, demonstrating preventive against caries treatment effect and residual compared to chlorhexidine, widely used as a mouthwash.
However, the aqueous propolis, in pure form and in dilutions 1:1 to 1:4, showed an effect only on S. mitis.It was found also that glycolic extract of propolis and 12%, and propolis containing 5.63 mg/mL flavonoids, associated with calcium hydroxide, were able to completely eliminate E. coli and E. faecalis reduce and endotoxins and C. albicans root canal.
The antifungal activity of propolis in the oral cavity has been demonstrated when used glycolic extract of propolis 12% and the ethanol extract of propolis (70% ethanol) causing efficacy in reducing C. albicans.In addition, it was found that the C. albicans and C. tropicalis, in clinically healthy mouths, were sensitive to the alcoholic propolis extract concentrations at 01:20 or 01:40.
Moreover, to safely say the effectiveness of propolis on the microorganism in the oral cavity would require evidence from studies of systematic reviews and randomized clinical trials with humans, because they are considered major evidence force; since the results presented in this study derived in most part from in vitro studies, evidence of which strength is weak.In addition, some studies do not make clear which the solvent of the propolis extract or concentration is.
Otherwise, this literature indicates the need for therapeutic parameters are defined and the minimum chemical composition, the different types of propolis, so that they have the appropriate/desired therapeutic properties.Where Doctors exchange clinical experiences, review their cases and share clinical knowledge.You can also access lots of medical publications for free.Join Now! http://medicalia.org/

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Table 1 .
Characterization of the analyzed publications.
3 [10] Effect of Zingiber officinale and propolis on microorganisms and endotoxins in root canals English/J Appl Oral Sci Evaluating the action of glycolic acid Propolis (PRO) and Ginger extracts (GIN), calcium hydroxide (CH), chlorhexidine gel (CLX) and their combinations used as irrigation intracanal against C. albicans, E. faecalis and Escherichia coli endotoxin.In vitro study -fill intracanal 8 [11] Antimicrobial activity of natural products in front of the dental biofilm-forming bacteria Portuguese/ Odontoped Clin Integr.Assessing the sensitivity of bacterial species found in the oral cavity: Streptococcus mutans, Streptococcus salivarius, Streptococcus mitis and Lactobacillus casei front of propolis extracts and aqueous alcohol, diluted respectively in alcohol and distilled water and diluted pollen in alcohol, at concentrations of 5% and 50%.In vitro study -by determining the Maximum Inhibitory Dilution 8 [12] In vitro and ex vivo study of the action of different concentrations of extracts of propolis against the microorganisms present in human saliva Portuguese/Rev.Bras.Farmacogn In phase 1, were evaluated in vitro, the action of three different concentrations of propolis extract compared to industrialized products Periogard®, Listerine®, Malvatricin® and Parodontax®, while in phase 2 was evaluated the action, ex vivo, propolis extract in the same concentrations used in phase 1, front of microorganisms that occur in saliva.In vitro study (lab) e ex vivo (experimental) 8 [13] Effect of a propolis extract on Streptococcus mutans counts in vivo English/J Appl Oral Sci Evaluating in vivo antimicrobial activity of the extract prepared with propolis produced by bees Melipona compressipes fasciculata (Ministry of agriculture, registration number 0005/731) and used as mouthwash on S. Mutans concentration present in the oral cavity of young individuals.

Table 2 .
Action of propolis on oral cavity microorganisms, as revised publications.