NatPROB’s researchers presented their investigation at IADR 2010, July 14-17, Barcelona – Spain

Antifungal activity of the Brazilian red propolis against Candida spp

P.L. ROSALEN(1,5), V.C.P.P. QUEIROZ(1), V.F. FURLETTI-GÓES(1), S.M.D. ALENCAR(2), M. IKEGAKI(3), H. KOO(4,5), and M.C.T. DUARTE(1),

(1)University of Campinas, Piracicaba, SP, Brazil, (2)University of São Paulo, Piracicaba, SP, Brazil, (3)Federal University of Alfenas, Alfenas, MG, Brazil, (4)University of Rochester School of Medicine and Dentistry, Rochester, NY, (5)NatPROB-Natural Product Research Group in Oral Biology

Natural products have been a valuable source for development of new drugs and propolis from Apis mellifera emerges as a promising oral antifungal agent. Objective: This study evaluated the antimicrobial potential of Brazilian red propolis against Candida spp. Methods: The ethanolic extract of red propolis (EEP) and its respective fractions: hexane, dichloromethane and ethyl acetate were produced and chemically characterized by HPLC and GC/MS as type 13. The EEP and its fractions were tested against Candida collection (C. albicans CBS562, C. dubliniensis CBS7987, C. krusei CBS573, C. glabrata CBS07, C. parapsilosis CBS604, C. tropicalis CBS94), by Minimum Inhibitory Concentration (MIC) and Minimum Fungicide Concentration (MFC) grown in planktonic and biofilm forms. The biofilm morphology was evaluated by scanning electron microscope (SEM). Nystatin and vehicle were used as controls and the experiments were performed in triplicate of three independent assays. Results: The dichloromethane fraction showed the best fungicide result against all Candida tested on planktonic cells (minimum/maximum MIC values: 0.2 µg/mL for CBS573 / 7.8 µg/mL for CBS562 and CBS604; and minimum/maximum MFC values: 125 µg/mL for CBS562 and CBS7987 / 500 µg/mL for CBS604; respectively) and fungistatic action against Candida tested on forming biofilms (minimum/maximum MIC values: 1.0 µg/mL CBS07 and 31.3 µg/mL CBS604, respectively; excepting for CBS 573). The EEP had higher MIC/MFC values than dichloromethane did and the other fractions were negligible. The SEM assays showed that the dichloromethane fraction and EEP were the only ones capable of altering the forming biofilm, however they poorly affected the mature biofilm. Conclusion: The Brazilian red propolis has clearly demonstrated its antifungal activity against Candida spp. and although the dichloromethane fraction has shown greater effect it may be concluded that there is still bioactive compounds in EEP that can justify a synergistic effect against Candida spp. (FAPESP 08/51048-5; CNPq 302222/2008-1, Brazil).

Influences of tt-farnesol on Streptococcus mutans metabolism and multispecies-biofilm development

H. KOO(1), J.-G. JEON(2), J. XIAO(3), and S. GREGOIRE(3),

(1)University of Rochester School of Medicine and Dentistry and NatPROB-Natural Product Research Group in Oral Biology, Rochester, NY, (2)Chonbuk National University, School of Dentistry and NatPROB-Natural Product Research Group in Oral Biology, Jeon-ju, South Korea, (3)University of Rochester School of Medicine and Dentistry, Rochester, NY

Trans-trans farnesol (tt-farnesol) is a naturally occurring sesquiterpene alcohol commonly found in propolis and citrus fruits, which exhibits bioactivity against several pathogenic microorganisms, including Streptococcus mutans. Objective: We investigated whether tt-farnesol (i) affects cell membrane-permeability and acid production by S. mutans UA159 cells, and (ii) influences the formation of biofilms by this bacterium in the presence of other oral bacteria. Methods: The effects of tt-farnesol on the bacterial physiology were determined by proton-permeability and glycolytic pH-drop assays. Mixed-species biofilms comprised of S. mutans  UA159, Actinomyces naeslundii 12104 and S. oralis 35037 were initially established on saliva-coated hydroxyapatite (sHA) discs for 43h; from this point, biofilms (43h-old) were treated twice daily (60s exposure) with 1 mM tt-farnesol or vehicle control until the end of experimental period (91h). Results: In general, tt-farnesol (concentrations ranging from 0.25 to 1 mM) was highly effective in disrupting the proton permeability and the glycolytic pH-drop of S. mutans cells (P < 0.05). Furthermore, topical applications of 1 mM tt-farnesol significantly reduced the biomass and altered the microbial composition by significantly reducing the proportion of S. mutans   in the biofilms (vs. vehicle-treated biofilms; P < 0.05). Conclusion: Our data indicate that tt-farnesol may affect the establishment and competitiveness of S. mutans  in a mixed-species environment by disrupting the acidurance and glycolytic activity of this bacterium; this naturally occurring compound could be a potentially useful adjunctive agent to the current anti-caries chemotherapeutic strategies (NIH/NIDCR 1R01 DE018023).

Separation of anti-acidogenic substances form Polygonum cuspidatum

J.-G. JEON(1,2), K.-W. CHANG(1), J.-E. KIM(1), and S. PANDIT(1),

(1)Chonbuk National University, School of Dentistry, Jeon-ju, South Korea, (2) NatPROB-Natural Product Research Group in Oral Biology

OBJECTIVES: The aim of the present study was to separate anti-acidogenic substances from Polygonum cuspidatum, based on high-performance liquid chromatography and glycolytic pH-drop assay, and then prove the inhibitory effects of the separated substances on S. mutans   UA 159. MATERIAL AND METHODS: The anti-acidogenic substances from P. cuspidatum were separated by a series of liquid-liquid fractionations and then normal-phase liquid chromatography. The effectiveness of the separated substances regarding acidogenicity of Streptococcus mutans UA 159 was investigated by glycolytic pH-drop assay. The chemical composition and amounts of the components consisted of the substances was also studied by qualitative–quantitative chromatographic analysis. RESULTS: Among the substances separated from P. cuspidatum, F3 showed the strongest inhibitory effect on the acidogenicity by S. mutans  UA 159 without displaying any bactericidal activity. F3 dose-dependently decreased the acidogenicity by S. mutans cell, even at 12.5 ug/ml (p < 0.05). Furthermore, the anti-acidogenic effect of F3 was similar to that of sodium fluoride at 25 ug/ml after 45 min incubation (p > 0.05). F3 was mainly consisted of resveratrol and emodin, C14H12O3 and C14H4O2(OH)3CH3, respectively, which constituted approximately 60 % of F3 weight. CONCLUSION: Based on the results, F3 can be considered a promising agent for the control of acidogenicity by S. mutans and subsequent dental caries formation.

Effects of Brazilian propolis on morning halitosis and oral microbiota

M.F. HAYACIBARA(1), M. FAVERI(2), G. CANCINE(1), H. KOO(3,6), M. FERES(2), L.C. FIGUEIREDO(2), M. IKEGAKI(4), P.L. ROSALEN(5,6), and R.M. HAYACIBARA(1),

(1)Universidade Estadual de Maringa, Maringa, Brazil, (2)Universidade de Guarulhos, Guarulhos, Brazil, (3)Eastman Dental Center, Rochester, NY, (4)EFOA, Alfenas, Brazil, (5)University of Campinas, Piracicaba, SP, Brazil, (6) NatPROB-Natural Product Research Group in Oral Biology

Propolis is a resinous substance collected from various plants sources by honeybees displaying several pharmacological properties. Objective: The aim of this study was to evaluate the effects of a propolis-based mouthrinse on the concentration of volatile sulphur compounds (VSCs) and on the microbial profile of the tongue dorsum. Methods: A three-step double-blind, crossover, and randomized study was conducted in 10 dental students with healthy periodontium, who refrained from mechanical plaque control and rinsed twice daily the following mouthrinses: I- Placebo (P); II: 3% Ethanolic Extract of Propolis type-3 (EEP); III: 0.12% Chlorhexidine (CHX) for a 5-day period. Each experimental period was followed by a 21-day washout interval. Morning halitosis was assessed by VSC concentrations. Microbiological samples were obtained from the dorsum of the tongue at baseline and at the end of the period and analyzed using the checkerboard DNA-DNA hybridization technique for 39 bacterial species. Results: CHX and EEP groups showed significantly lower levels of VSCs when compared with P group (p<0.05, ANOVA and Tukey tests), even in the absence of mechanical plaque control. Moreover, there were no statistical differences for VSC measurements between CHX and EEP groups (p>0.05). Subjects rinsing with EEP solutions showed significant reduction on the counts of 10 species, including VSCs producers such as Prevotella nigrescens, Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola. Total counts of organisms present on the tongue surface decreased significantly after rinsing with EEP and CHX compared to placebo (p<0.05, Wilcoxon test). There was a positive correlation between decrease of bacterial and reduction of VCSs concentration for the EEP and CHX groups (r= 0.88, p=0.04 and r=0.71, p=0.049, respectively). Conclusion: The use of propolis-based mouthrinse effectively reduced morning malodor,

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