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Antifungal activity of propolis against Candidaspecies isolated from cases of chronic periodontitis

Abstract

This research evaluated the fungistatic and fungicidal activities of red propolis alcoholic extract (RPAE) against different Candida species isolated from chronic periodontitis cases, and compared with chlorhexidine (CHX). Nineteen samples of Candida species (C. albicans [n = 12], C. tropicalis [n = 5] andC. glabrata [n = 2]) isolated from chronic periodontitis cases were analyzed. The fungistatic and fungicidal activity of both RPAE and CHX were evaluated using fluconazole and C. parapsilosis (ATCC 6258) as a control. Fungistatic activity was analyzed based on the Clinical and Laboratory Standards Institute (CLSI) reference procedure to determine the minimum inhibitory concentrations. Fungicidal activity was established according to the absence of fungal growth on Sabouraud Dextrose Agar medium. The fungistatic and fungicidal activities of RPAE were observed, respectively, at 32-64 μg/mL and 64-512 μg/mL for C.albicans, 64 μg/mL and 64-256 μg/mL for C. glabrata, and 32-64 μg/mL and 64 µg/mL for C. tropicalis. CHX fungistatic activity was observed at concentrations of 0.003-1.92 µg/mL for C. albicans, 1.92 µg/mL for C. glabrata, and 0.03-1.92 µg/mL for C. tropicalis. Fluconazole fungistatic activity ranged between 1-64 μg/mL, and fungicidal activity occurred at 8-64 μg/mL, for the threeCandida species analyzed. All the Candidaspecies were susceptible to RPAE antifungal activity, but five samples ofC.albicans, one ofC.tropicalis and one ofC.glabrata were resistant to fluconazole antifungal activity. CHX showed fungistatic activity against all the Candida species analyzed. The antifungal potential of these substances suggests that they can be applied as an alternative treatment for diseases affected by these species.

Candida ; Propolis; Chlorhexidine; Periodontitis


Introduction

The presence of fungi, bacteria and viruses in dental biofilm can contribute to the progression of periodontal disease. Some authors have reported the prevalence of bacteria associated with periodontitis,1Ferreira FB, Torres SA, Rosa OP, Ferreira CM, Garcia RB, Marcucci MC, et al. Antimicrobial effect of propolis and other substances against selected endodontic pathogens. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007 Nov;104(5):709-16. but other studies have related the presence of fungi such as Candida. These fungi form part of an individual’s microbiota, especially in areas of the mucosa, such as the oral cavity, and play an important role both in health and in the development of oral diseases.2Tamai R, Sugamata M, Kiyoura Y. Candida albicans enhances invasion of human gingival epithelial cells and gingival fibroblasts by Porphyromonas gingivalis. Microb Pathog. 2011 Oct;51(4):250-4.

Subgingival colonization by C. albicans, C. dubliniensis, C. glabrata and C. tropicalis has been described by other authors.3Pizzo G, Barchiesi F, Falconi Di Francesco L, Giuliana G, Arzeni D, Milici ME, et al. Genotyping and antifungal susceptibility of human subgingival Candida albicans isolates. Arch Oral Biol. 2002 Mar;47(3):189-96. Tamai et al.2Tamai R, Sugamata M, Kiyoura Y. Candida albicans enhances invasion of human gingival epithelial cells and gingival fibroblasts by Porphyromonas gingivalis. Microb Pathog. 2011 Oct;51(4):250-4. found thatC.albicans may boost the infectious effect of periodontitis by acting in conjunction with anaerobic bacteria. Individuals susceptible to primary infection have been reported to use an antifungal prophylaxis to prevent cases of recurrence.4Azevedo RVP, Komesu MC, Candido RC, Salvetti C, Rezende FHC. Candida sp in the oral cavity with and without lesions: maximal inhibitory dilution of propolis and Periogard. Rev Microbiol. 1999 Oct-Dec;30(4):335-41. Among the different chemical agents used, chlorhexidine has broad-spectrum antiseptic action. It is used in maintaining periodontal therapy, and as a treatment adjuvant in scaling and root planing procedures.5Guarnelli ME, Franceschetti G, Manfrini R, Trombelli L. Adjunctive effect of chlorhexidine in ultrasonic instrumentation of aggressive periodontitis patients: a pilot study. J Clin Periodontol. 2008 Apr;35(4):333-41. The fungistatic activity of 0.12% chlorhexidine has also been reported as preventing the growth of C. albicans on denture acrylic resin.6Pusateri CR, Monaco EA, Edgerton M. Sensitivity of Candida albicans biofilm cells grown on denture acrylic to antifungal proteins and chlorhexidine. Arch Oral Biol. 2009 Jun;54(6):588-94.

The antimicrobial activities of natural derivatives, such as propolis, have been researched over recent years as alternatives for new therapeutic strategies. The presence of flavonoids, as well as phenolic, aromatic and diterpene acids, in the composition of propolis, has been associated with various biological attributes, including its antifungal properties.7Oliveira AC, Shinobu CS, Longhini R, Franco SL, Svidzinski TI. Antifungal activity of propolis extract against yeasts isolated from onychomycosis lesions. Mem Inst Oswaldo Cruz. 2006 Aug;101(5):493-7.,8Sawaya AC, Palma AM, Caetano FM, Marcucci MC, Cunha IBS, Araujo CE, et al. Comparative study of in vitro methods used to analyse the activity of propolis extracts with different compositions against species of Candida. Lett Appl Microbiol. 2002 Sep;35(3):203-7.

The antifungal activity of Brazilian red propolis against differentCandida species has already been reported in the literature.7Oliveira AC, Shinobu CS, Longhini R, Franco SL, Svidzinski TI. Antifungal activity of propolis extract against yeasts isolated from onychomycosis lesions. Mem Inst Oswaldo Cruz. 2006 Aug;101(5):493-7.,8Sawaya AC, Palma AM, Caetano FM, Marcucci MC, Cunha IBS, Araujo CE, et al. Comparative study of in vitro methods used to analyse the activity of propolis extracts with different compositions against species of Candida. Lett Appl Microbiol. 2002 Sep;35(3):203-7.,9Trusheva B, Popova M, Bankova V, Simova S, Marcucci MC, Miorin PL, et al. Bioactive constituents of brazilian red propolis. Evid Based Complement Alternat Med. 2006 Jun;3(2):249-54. This propolis variety is obtained from exudates collected by bees (Apis mellifera) from the surface ofDalbergia ecastophyllum in the Northeast of Brazil, and contains a high concentration of flavonoids.1010 Daugsch A, Moraes CS, Fort P, Park YK. Brazilian red propolis--chemical composition and botanical origin. Evid Based Complement Alternat Med. 2008 Dec;5(4):435-41. The antifungal activity of this red propolis (from Paraiba State, Brazil) proved effective against Trichophyton tonsurans, T. rubrum and T. mentagrophytes;1111 Siqueira AB, Gomes BS, Cambuim I, Maia R, Abreu S, Souza-Motta CM, et al. Trichophyton species susceptibility to green and red propolis from Brazil. Lett Appl Microbiol. 2009 Jan;48(1):90-6.however, the present study is the first to investigate the antifungal activity of red propolis against Candida species isolated from periodontal pockets.

The chemical composition of red propolis alcoholic extract indicates a predominance of flavonoids, such as rutin, liquiritigenin, daidzein, pinobanksin, quercetin, luteolin, dalbergin, isoliquiritigenin, pinocembrin, pinobanksin-3-acetate, biochanin A and formononetin, the last being the predominant component.1010 Daugsch A, Moraes CS, Fort P, Park YK. Brazilian red propolis--chemical composition and botanical origin. Evid Based Complement Alternat Med. 2008 Dec;5(4):435-41.

Thus, the aim of this study was to evaluate the in vitro fungistatic and fungicidal activities of Brazilian red propolis alcoholic extract against theCandida species isolated from cases of periodontitis. Its effects were compared with those of chlorhexidine, an agent considered the gold standard for antifungal periodontal treatment.

Methodology

Fungi samples

Candida samples (twelve isolates ofC.albicans [63%], five of C.tropicalis [26%] and two of C.glabrata [11%]) were obtained from periodontal pockets of patients with chronic periodontitis seen in the Faculdade de Odontologia of the Centro Universitário Cesmac [Clinical Dental School of the Cesmac University Center]. All isolates were preserved in mineral oil,1212 Sherf A. A method for maintaining Phytomonas sepedomica for long periods without transfer. Phytopathology. 1943;31:30-2. taxonomically confirmed, and then seeded on CHROMagar Candida® (BD, Sao Paulo, Brazil) in a Petri dish kept at 37°C for 48 h.1313 Yucesoy M, Marol S. Performance of CHROMAGAR candida and BIGGY agar for identification of yeast species. Ann Clin Microbiol Antimicrob. 2003 Oct;2:8. doi: 10.1186/1476-0711-2-8.A sample of C.parapsilosis(ATCC 6258) was used as the control.

This study was approved by the Research Ethics Committee of Centro Universitário Cesmac, Alagoas, Brazil (protocol number: 723/09).

Evaluation of antifungal activity

The antifungal agents evaluated were fluconazole (Pfizer®, Sao Paulo, Brazil), chlorhexidine and red propolis alcoholic extract (lot code PRDE0906 – Pharmanéctar®, Belo Horizonte, Brazil). According to the certificate of analysis provided by the manufacturer of the red propolis alcoholic extract, the chemical composition of the product is consistent with that found by Daugsch et al.1010 Daugsch A, Moraes CS, Fort P, Park YK. Brazilian red propolis--chemical composition and botanical origin. Evid Based Complement Alternat Med. 2008 Dec;5(4):435-41. The procedure was performed according to standards published in document M27-A3 by the Clinical and Laboratory Standards Institute (CLSI).1414 Clinical and Laboratory Standards Institute (CLSI). Reference method for broth dilution testing of yeasts. Approved standard document M27-A3. Wayne (PA): CLSI; 2008. The culture medium used was bicarbonate-free RPMI 1640 (Sigma-Aldrich, St. Louis, USA) with L-glutamine, buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) (Sigma-Aldrich, St. Louis, USA). The medium was sterilized by filtration through a 0.22 µm Millipore membrane.

The commercial antifungal agent was dissolved in sterile distilled water and prepared in concentrations ranging from 0.125 to 64 μg/mL. The lyophilized red propolis alcoholic extract, originally from Paraiba (Brazil’s Northeast region), was solubilized in 70% ethyl alcohol (v/v), according to Sawayaetal.,8Sawaya AC, Palma AM, Caetano FM, Marcucci MC, Cunha IBS, Araujo CE, et al. Comparative study of in vitro methods used to analyse the activity of propolis extracts with different compositions against species of Candida. Lett Appl Microbiol. 2002 Sep;35(3):203-7. to obtain concentrations from of 4 to 2048 μg/mL. Chlorhexidine was prepared at concentrations from 0.003 to 1.92 μg/mL, corresponding to 0.000003 and 0.000192%, respectively.

All Candida isolates were plated on Sabouraud Dextrose Agar medium contained in test tubes and maintained at 28°C (± 1ºC), for 48 h, to standardize the inoculum. Thereafter, suspensions were prepared in 5 ml of 0.145 mol/L sterile saline solution, then vortexed for 15 seconds. The cell density was adjusted to an equivalent of 0.5 on the McFarland scale and standardized in a spectrophotometer at 530 nm to obtain 90% transmittance. The suspensions were then diluted sequentially in a RPMI 1640 liquid medium to obtain concentrations of 1:100 to 1:20, resulting in a concentration of 2-5 x 103cells/mL.

Sterile microtiter plates (TPP, Trasandingen, Switzerland) containing 96 U-wells were used to perform the test procedure. 100 µL of fluconazole, red propolis and chlorhexidine were deposited on separate plates, in rows from 1 to 10, and each concentration was deposited in a row. 100 µL of RPMI 1640 medium was deposited in well rows 11 and 12, in which the controls and the sterilization medium were grown, respectively. These plates were stored at -20°C until use.

At test time, 100 µL of standardized inoculum was deposited in each well, and the microtiter plates were incubated at room temperature (28 ± 1°C) for 48 h until the interpretation of the results. The determination of the minimum inhibitory concentrations (MIC) of fluconazole, red propolis and chlorhexidine was performed by visual observation of each well to identify the reduction in fungal growth. Considering the total growth (100%) in the control well, the percentage growth reduction was attributed to the remaining wells.

The MIC for fluconazole was regarded as the lowest concentration, inhibiting growth by ≥ 50%. The interpretive CLSI breakpoints for fluconazole were: susceptible [S]: ≤ 8 µg/mL; susceptible dose dependent [SDD]: from 16 to 32 µg/mL; and resistant [R]: ≥ 64 µg/mL. The MICs for chlorhexidine and red propolis were regarded as the lowest concentrations, inhibiting fungal growth by 100%.

The minimum fungicidal concentration (MFC) was determined as follows: the contents of the wells showing 100% inhibition of fungal growth were transferred from the microtiter plate to Petri dishes containing Sabouraud Dextrose Agar medium. The dishes were kept at room temperature (28 ± 1°C) for 72 h to determine the development of Candida colonies. The MFC corresponded to the lowest fungistatic concentration preventing fungal growth.1515 Favre B, Hofbauer B, Hildering KS, Ryder NS. Comparison of in vitro activities of 17 antifungal drugs against a panel of 20 Dermatophytes by using a microdilution assay. J Clin Microbiol. 2003 Oct;41(10):4817-9. The MIC and MFC of fluconazole, red propolis and chlorhexidine were determined, as well as the concentration capable of inhibiting either half or all of the fungal growth (MIC50 and MIC100, respectively).

Results

All the samples of C. albicans, C. tropicalis andC. glabrata were confirmed in regard to aspects of purification and taxonomy. No fungistatic activity of fluconazole was observed for any isolate of any species analyzed; for this reason, these results are not shown in Table 1. Sensitivity to fluconazole was observed in nine isolates of C.albicans, four ofC. tropicalis and one of C. glabrata (MIC ≤ 8 μg/mL). Two isolates ofC.albicans were SDD, demonstrating MICs of 16 and 32 μg/mL. In addition, five samples ofC.albicans, one ofC.tropicalis and one ofC.glabrata were resistant to the fungicidal activity of fluconazole.

Table 1
Mean results of fungistatic activity of fluconazole, red propolis alcoholic extract and chlorhexidine against Candidaspecies isolated from cases of chronic periodontitis.

The fungistatic activity of chlorhexidine was observed in concentrations of 0.003-1.92 μg/mL, 1.92 μg/mL and 0.03-1.92 μg/mL forC.albicans,C.glabrata and C. tropicalis, respectively.

The fungistatic activity of red propolis alcoholic extract was 32-64 µg/mL forC. albicans, 64 µg/mL for C. glabrata and 32-64 µg/mL for C. tropicalis. The fungicidal activity was 64-512 µg/mL for C. albicans, 64-256 µg/mL for C. tropicalis, and 64 µg/mL for C. glabrata.

The fungicidal activity of red propolis alcoholic extract and of chlorhexidine was observed for all Candida species. tested. The MFC was 0.003 µg/mL for chlorhexidine. The results are shown in Tables 1 and 2.

Table 2
Mean results of fungicidal activity of fluconazole and red propolis alcoholic extract against Candida species isolated from cases of chronic periodontitis.

Discussion

The frequency rates of C. albicans, C. tropicalis and C. glabrata in oral infections have been previously reported by other authors, who confirmed the susceptibility of the Candida species associated with chronic periodontitis to fluconazole.1616 Furlletti VF, Mardegan RC, Obando-Pereda GA, Aníbal PC, Duarte MCT, Gonçalves RB, et al. Susceptibility of Candida spp. oral isolates for azolic antifungals and amphotericin B. Braz J Oral Sci. 2008 Apr-Jun;7(25):1543–9.Similar results were observed in the present study.

The variation in the MICs obtained for fluconazole for samples of C. albicans (≥ 64 μg/mL, and a resistance profile) have been previously reported by other authors.1717 Koga-Ito CY, Komiyama EY, Martins CAP, Santos SSF, Balducci I, Jorge AOC. Phenotypic characterization of Candida spp. isolates from chronic periodontitis patients Brazilian. Braz J Oral Sci. 2008 Apr-Jun;7(25):1531-4. The decrease in the susceptibility profiles of non-albicans samples to fluconazole may be attributed to this antifungal agent, used both prophylactically and as a treatment. A national survey in Taiwan showed increasing rates of fluconazole resistance, from 1.9% in 2002 to 17.1% in 2006, mainly among non-blood or non-albicans isolates.1818 Yang YL, Wang AH, Wang CW, Cheng WT, Li SY, Lo HJ, et al. Susceptibilities to amphotericin B and fluconazole of Candida species in Taiwan Surveillance of Antimicrobial Resistance of Yeasts 2006. Diagn Microbiol Infect Dis. 2008 Jun;61(2):175-80.

A similar susceptibility of C. albicans to Brazilian red propolis alcoholic extract in different concentrations (32-512 µg/mL) was observed in this study for C.albicans to Mexican propolis (at higher concentrations of 0.06 to 32 mg/mL),1919 Quintero-Mora ML, Londono-Orozco A, Hernandez-Hernandez F, Manzano-Gayosso P, Lopez-Martinez R, Soto-Zarate CI, et al. Effect of Mexican propolis extracts from Apis mellifera on Candida albicans in vitro growth. Rev Iberoam Micol. 2008 Mar;25(1):22-6. Spanish. unlike the susceptibility of C. glabrata to Iranian propolis (approximately 5000 µg/mL)2020 Shokri H, Khosravi AR, Yalfani R. Antifungal efficacy of propolis against fluconazole-resistant Candida glabrata isolates obtained from women with recurrent vulvovaginal candidiasis. Int J Gynaecol Obstet. 2011 Aug;114(2):158-9. and of C. tropicalis to Chilean propolis (197-476 µg/mL).2121 Herrera CL, Alvear M, Barrientos L, Montenegro G, Salazar LA. The antifungal effect of six commercial extracts of Chilean propolis on Candida spp. Cien Inv Agr. 2010 Abr;37(1):75-84.

Sforcin et al.2222 Sforcin JM, Fernandes-Júnior A, Lopes CAM, Funari SRC, Bankova V. Seasonal effect of brazilian propolis on Candida albicans and Candida tropicalis. J Venom Anim Toxins. 2001;7:139-44.reported that C.albicans was more susceptible to propolis from Sao Paulo (Brazil) thanC.tropicalis. Our results were similar for MIC50; however, in regard to fungicidal activity, the susceptibility of C.tropicaliswas higher. The antifungal effect of Brazilian propolis from the northeastern region, as observed in this study, is consistent with the findings of other studies that used Brazilian propolis from the southeastern region; however, the antifungal activity was observed at higher concentrations for the propolis from the Southeast of Brazil (between 3 and 12 µg/mL).2323 Ota C, Unterkircher C, Fantinato V, Shimizu MT. Antifungal activity of propolis on different species of Candida. Mycoses. 2001 Nov;44(9-10):375-8.

Most biological activities of propolis are related to the alcoholic extract, because when the extract is prepared in ethyl alcohol, a greater amount of active compounds are extracted and the inhibitory effect is greater.2424 Agüero MB, Svetaz L, Baroni V, Lima B, Luna L, Zacchino S, et al. Urban propolis from San Juan province (Argentina): Ethnopharmacological uses and antifungal activity against Candida and dermatophytes. Ind Crops Prod. 2014 Jun;57:166-73. doi: 10.1016/j.indcrop.2014.03.009. When water is used as the solvent, the antifungal and antibacterial activities are weaker, compared with the alcoholic extract.8Sawaya AC, Palma AM, Caetano FM, Marcucci MC, Cunha IBS, Araujo CE, et al. Comparative study of in vitro methods used to analyse the activity of propolis extracts with different compositions against species of Candida. Lett Appl Microbiol. 2002 Sep;35(3):203-7.

Flavonoids are largely responsible for antifungal activity.9Trusheva B, Popova M, Bankova V, Simova S, Marcucci MC, Miorin PL, et al. Bioactive constituents of brazilian red propolis. Evid Based Complement Alternat Med. 2006 Jun;3(2):249-54.,1010 Daugsch A, Moraes CS, Fort P, Park YK. Brazilian red propolis--chemical composition and botanical origin. Evid Based Complement Alternat Med. 2008 Dec;5(4):435-41. The susceptibility of differentCandida species is dependent on the chemical composition of the propolis; however, Sforcin et al.2222 Sforcin JM, Fernandes-Júnior A, Lopes CAM, Funari SRC, Bankova V. Seasonal effect of brazilian propolis on Candida albicans and Candida tropicalis. J Venom Anim Toxins. 2001;7:139-44. found no change in the susceptibility ofC.albicans and C. tropicalis. Thus, the fungicidal activity of red propolis alcoholic extract found in this study could be attributed to its chemical composition.2525 Mello BCBS, Petrus JCC, Hubinger MD. Concentration of flavonoids and phenolic compounds in aqueous and ethanolic propolis extracts through nanofiltration. J Food Eng. 2010 Feb;96(4):533-9.

The use of propolis in dentistry has been proposed as an aid in the treatment of oral infections. Koo et al.2626 Koo H, Gomes BP, Rosalen PL, Ambrosano GM, Park YK, Cury JA. In vitro antimicrobial activity of propolis and Arnica montana against oral pathogens. Arch Oral Biol. 2000 Feb;45(2):141-8. evaluated the antimicrobial activity of red propolis alcoholic extract from Minas Gerais, southeastern Brazil (10% w/v) and concluded that propolis may have an effect on periodontal disease, because of its antifungal activity against C.albicans and other microorganisms, and because it inhibits the formation of biofilm in vitro.

Chlorhexidine is known to have excellent antifungal activity in vitro against all isolates of C. albicans, C. glabrata and C. tropicalis. Furthermore, fungistatic and fungicidal activities were found at a concentration of 1.92 μg/mL. Dodwad and Kukreja2727 Dodwad V, Kukreja BJ. Propolis mouthwash: a new beginning. J Indian Soc Periodontol. 2011 Apr;15(2):121-5. found that chlorhexidine (0.2%) can inhibit dental biofilm formation better than propolis contained in mouthwash solutions. The fungistatic activity of chlorhexidine has also been reported recently in the literature. Whereas the MIC90 for differentCandida species, includingC.albicans,C.glabrata andC.parapsilosis, was 6.25 mg/L,2828 Salim N, Moore C, Silikas N, Satterthwaite J, Rautemaa R. Chlorhexidine is a highly effective topical broad-spectrum agent against Candida spp. Int J Antimicrob Agents. 2013 Jan;41(1):65-9. our results showed an activity of 1.92 µg/mL. Ellepola et al. 2929 Ellepola AN, Joseph BJ, Khan ZU. Effects of subtherapeutic concentrations of chlorhexidine gluconate on germ tube formation of oral Candida. Med Princ Pract. 2012;21(2):120-4. found that chlorhexidine interferes with the formation of the germ tube ofC.albicans, in subtherapeutic concentrations (0.00125, 0.0025 and 0.005%). According to Pizzoet al.,3030 Pizzo G, Giuliana G, Milici ME, D’Angelo M. Effect of antimicrobial mouthrinses on the in vitro adhesion of Candida albicans to human buccal epithelial cells. Clin Oral Investig. 2001 Sep;5(3):172-6.chlorhexidine causes changes in the epithelial cell surface of the oral mucosa, thus interfering in the colonization of different Candida species, and indicating a reduction in the adhesion of these fungi.

Conclusion

The results of this study show that, similar to chlorhexidine, red propolis alcoholic extract has good fungistatic and fungicidal activity against most samples ofCandida species. This antifungal activity may hold a promise for future applications as an alternative treatment for infections caused by these fungi. Further investigation into the use of red propolis for the prevention and treatment of periodontal diseases is required, including microbiological, randomized controlled trials and longitudinal studies.

References

  • 1
    Ferreira FB, Torres SA, Rosa OP, Ferreira CM, Garcia RB, Marcucci MC, et al. Antimicrobial effect of propolis and other substances against selected endodontic pathogens. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007 Nov;104(5):709-16.
  • 2
    Tamai R, Sugamata M, Kiyoura Y. Candida albicans enhances invasion of human gingival epithelial cells and gingival fibroblasts by Porphyromonas gingivalis. Microb Pathog. 2011 Oct;51(4):250-4.
  • 3
    Pizzo G, Barchiesi F, Falconi Di Francesco L, Giuliana G, Arzeni D, Milici ME, et al. Genotyping and antifungal susceptibility of human subgingival Candida albicans isolates. Arch Oral Biol. 2002 Mar;47(3):189-96.
  • 4
    Azevedo RVP, Komesu MC, Candido RC, Salvetti C, Rezende FHC. Candida sp in the oral cavity with and without lesions: maximal inhibitory dilution of propolis and Periogard. Rev Microbiol. 1999 Oct-Dec;30(4):335-41.
  • 5
    Guarnelli ME, Franceschetti G, Manfrini R, Trombelli L. Adjunctive effect of chlorhexidine in ultrasonic instrumentation of aggressive periodontitis patients: a pilot study. J Clin Periodontol. 2008 Apr;35(4):333-41.
  • 6
    Pusateri CR, Monaco EA, Edgerton M. Sensitivity of Candida albicans biofilm cells grown on denture acrylic to antifungal proteins and chlorhexidine. Arch Oral Biol. 2009 Jun;54(6):588-94.
  • 7
    Oliveira AC, Shinobu CS, Longhini R, Franco SL, Svidzinski TI. Antifungal activity of propolis extract against yeasts isolated from onychomycosis lesions. Mem Inst Oswaldo Cruz. 2006 Aug;101(5):493-7.
  • 8
    Sawaya AC, Palma AM, Caetano FM, Marcucci MC, Cunha IBS, Araujo CE, et al. Comparative study of in vitro methods used to analyse the activity of propolis extracts with different compositions against species of Candida. Lett Appl Microbiol. 2002 Sep;35(3):203-7.
  • 9
    Trusheva B, Popova M, Bankova V, Simova S, Marcucci MC, Miorin PL, et al. Bioactive constituents of brazilian red propolis. Evid Based Complement Alternat Med. 2006 Jun;3(2):249-54.
  • 10
    Daugsch A, Moraes CS, Fort P, Park YK. Brazilian red propolis--chemical composition and botanical origin. Evid Based Complement Alternat Med. 2008 Dec;5(4):435-41.
  • 11
    Siqueira AB, Gomes BS, Cambuim I, Maia R, Abreu S, Souza-Motta CM, et al. Trichophyton species susceptibility to green and red propolis from Brazil. Lett Appl Microbiol. 2009 Jan;48(1):90-6.
  • 12
    Sherf A. A method for maintaining Phytomonas sepedomica for long periods without transfer. Phytopathology. 1943;31:30-2.
  • 13
    Yucesoy M, Marol S. Performance of CHROMAGAR candida and BIGGY agar for identification of yeast species. Ann Clin Microbiol Antimicrob. 2003 Oct;2:8. doi: 10.1186/1476-0711-2-8.
  • 14
    Clinical and Laboratory Standards Institute (CLSI). Reference method for broth dilution testing of yeasts. Approved standard document M27-A3. Wayne (PA): CLSI; 2008.
  • 15
    Favre B, Hofbauer B, Hildering KS, Ryder NS. Comparison of in vitro activities of 17 antifungal drugs against a panel of 20 Dermatophytes by using a microdilution assay. J Clin Microbiol. 2003 Oct;41(10):4817-9.
  • 16
    Furlletti VF, Mardegan RC, Obando-Pereda GA, Aníbal PC, Duarte MCT, Gonçalves RB, et al. Susceptibility of Candida spp. oral isolates for azolic antifungals and amphotericin B. Braz J Oral Sci. 2008 Apr-Jun;7(25):1543–9.
  • 17
    Koga-Ito CY, Komiyama EY, Martins CAP, Santos SSF, Balducci I, Jorge AOC. Phenotypic characterization of Candida spp. isolates from chronic periodontitis patients Brazilian. Braz J Oral Sci. 2008 Apr-Jun;7(25):1531-4.
  • 18
    Yang YL, Wang AH, Wang CW, Cheng WT, Li SY, Lo HJ, et al. Susceptibilities to amphotericin B and fluconazole of Candida species in Taiwan Surveillance of Antimicrobial Resistance of Yeasts 2006. Diagn Microbiol Infect Dis. 2008 Jun;61(2):175-80.
  • 19
    Quintero-Mora ML, Londono-Orozco A, Hernandez-Hernandez F, Manzano-Gayosso P, Lopez-Martinez R, Soto-Zarate CI, et al. Effect of Mexican propolis extracts from Apis mellifera on Candida albicans in vitro growth. Rev Iberoam Micol. 2008 Mar;25(1):22-6. Spanish.
  • 20
    Shokri H, Khosravi AR, Yalfani R. Antifungal efficacy of propolis against fluconazole-resistant Candida glabrata isolates obtained from women with recurrent vulvovaginal candidiasis. Int J Gynaecol Obstet. 2011 Aug;114(2):158-9.
  • 21
    Herrera CL, Alvear M, Barrientos L, Montenegro G, Salazar LA. The antifungal effect of six commercial extracts of Chilean propolis on Candida spp. Cien Inv Agr. 2010 Abr;37(1):75-84.
  • 22
    Sforcin JM, Fernandes-Júnior A, Lopes CAM, Funari SRC, Bankova V. Seasonal effect of brazilian propolis on Candida albicans and Candida tropicalis. J Venom Anim Toxins. 2001;7:139-44.
  • 23
    Ota C, Unterkircher C, Fantinato V, Shimizu MT. Antifungal activity of propolis on different species of Candida. Mycoses. 2001 Nov;44(9-10):375-8.
  • 24
    Agüero MB, Svetaz L, Baroni V, Lima B, Luna L, Zacchino S, et al. Urban propolis from San Juan province (Argentina): Ethnopharmacological uses and antifungal activity against Candida and dermatophytes. Ind Crops Prod. 2014 Jun;57:166-73. doi: 10.1016/j.indcrop.2014.03.009.
  • 25
    Mello BCBS, Petrus JCC, Hubinger MD. Concentration of flavonoids and phenolic compounds in aqueous and ethanolic propolis extracts through nanofiltration. J Food Eng. 2010 Feb;96(4):533-9.
  • 26
    Koo H, Gomes BP, Rosalen PL, Ambrosano GM, Park YK, Cury JA. In vitro antimicrobial activity of propolis and Arnica montana against oral pathogens. Arch Oral Biol. 2000 Feb;45(2):141-8.
  • 27
    Dodwad V, Kukreja BJ. Propolis mouthwash: a new beginning. J Indian Soc Periodontol. 2011 Apr;15(2):121-5.
  • 28
    Salim N, Moore C, Silikas N, Satterthwaite J, Rautemaa R. Chlorhexidine is a highly effective topical broad-spectrum agent against Candida spp. Int J Antimicrob Agents. 2013 Jan;41(1):65-9.
  • 29
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Publication Dates

  • Publication in this collection
    2015

History

  • Received
    20 Nov 2014
  • Accepted
    14 Mar 2015
  • Reviewed
    27 May 2015
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