Antifungal activity of propolis against Candida species isolated from cases of chronic periodontitis

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] and C. 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 three Candida species analyzed. All the Candida species were susceptible to RPAE antifungal activity, but five samples of C. albicans, one of C. tropicalis and one of C. 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.


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, 1 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. 2claration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript.
Subgingival colonization by C. albicans, C. dubliniensis, C. glabrata and C. tropicalis has been described by other authors. 3Tamai et al. 2 found that C. 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. 4 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. 5The fungistatic activity of 0.12% chlorhexidine has also been reported as preventing the growth of C. albicans on denture acrylic resin. 6he 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. 7,8he antifungal activity of Brazilian red propolis against different Candida species has already been reported in the literature. 7,8,9This propolis variety is obtained from exudates collected by bees (Apis mellifera) from the surface of Dalbergia ecastophyllum in the Northeast of Brazil, and contains a high concentration of flavonoids. 10The antifungal activity of this red propolis (from Paraiba State, Brazil) proved effective against Trichophyton tonsurans, T. rubrum and T. mentagrophytes; 11 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. 10hus, the aim of this study was to evaluate the in vitro fungistatic and fungicidal activities of Brazilian red propolis alcoholic extract against the Candida 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 of C. 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, 12 taxonomically confirmed, and then seeded on CHROMagar Candida ® (BD, Sao Paulo, Brazil) in a Petri dish kept at 37°C for 48 h. 13 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. 10 The procedure was performed according to standards published in document M27-A3 by the Clinical and Laboratory Standards Institute (CLSI). 14The 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 Sawaya et al., 8 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 10 3 cells/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. 15he 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 (MIC 50 and MIC 100 , respectively).

Results
All the samples of C. albicans, C. tropicalis and C. 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 Table 1.Mean results of fungistatic activity of fluconazole, red propolis alcoholic extract and chlorhexidine against Candida species isolated from cases of chronic periodontitis.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.

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. 16Similar 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. 17The 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. 18 similar susceptibilit y 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), 19 unlike the susceptibility of C. glabrata to Iranian propolis (approximately 5000 µg/mL) 20 and of C. tropicalis to Chilean propolis (197-476 µg/mL). 21forcin et al. 22 reported that C. albicans was more susceptible to propolis from Sao Paulo (Brazil) than C. tropicalis.Our results were similar for MIC 50 ; however, in regard to fungicidal activity, the susceptibility of C. tropicalis was 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). 23ost 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. 24When water is used as the solvent, the antifungal and antibacterial activities are weaker, compared with the alcoholic extract. 8ble 2. Mean results of fungicidal activity of fluconazole and red propolis alcoholic extract against Candida species isolated from cases of chronic periodontitis.Flavonoids are largely responsible for antifungal activity. 9,10The susceptibility of different Candida species is dependent on the chemical composition of the propolis; however, Sforcin et al. 22 found no change in the susceptibility of C. albicans and C. tropicalis.Thus, the fungicidal activity of red propolis alcoholic extract found in this study could be attributed to its chemical composition. 25he use of propolis in dentistry has been proposed as an aid in the treatment of oral infections.Koo et al. 26 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 Kukreja 27 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 MIC 90 for different Candida species, including C. albicans, C. glabrata and C. parapsilosis, was 6.25 mg/L, 28 our results showed an activity of 1.92 µg/mL.Ellepola et al. 29 found that chlorhexidine interferes with the formation of the germ tube of C. albicans, in subtherapeutic concentrations (0.00125, 0.0025 and 0.005%).According to Pizzo et al., 30 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 of Candida 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.
Not determined in nine isolates of C. albicans, four of C. tropicalis and one of C. glabrata (MIC ≤ 8 μg/mL).Two isolates of C. albicans were SDD, demonstrating MICs of 16 and 32 μg/mL.In addition, five samples of C. albicans, one of C. tropicalis and one of C. glabrata were resistant to the fungicidal activity of fluconazole.The fungistatic activity of chlorhexidine was observed in concentrations of 0.003-1.92μg/mL, 1.92 μg/mL and 0.03-1.92μg/mL for C. albicans, C. glabrata and C. tropicalis, respectively.The fungistatic activity of red propolis alcoholic extract was 32-64 µg/mL for C. 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.
a MIC: Minimum Inhibitory Concentration b MIC 50 : Minimum inhibitory concentration able to inhibit 50% of the C. albicans, C. glabrata and C. tropicalis samples.c MIC 100 : Minimum inhibitory concentration able to inhibit 100% of the C. albicans, C. glabrata and C. tropicalis samples.d ND: