Antimicrobial effect of propolis and other substances against
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Antimicrobial effect of propolis and other substances against selected endodontic pathogens Flaviana Bombarda de Andrade Ferreira (UNOPAR – Londrina), Sergio Aparecido Torres (Bauru Dental School – USP), Odila Pereira da Silva Rosa (Bauru Dental School – USP), Cláudio Maniglia Ferreira (UNIFOR - Fortaleza), Roberto Brandão Garcia (Bauru Dental School – USP), Maria Cristina Marcucci (UNIBAN – São Paulo), Brenda Paula Figueiredo de A. Gomes (Piracicaba Dental School – UNICAMP) BRAZIL Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontics, v. 104, p. 709-16, 2007
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Literature Review Bacteria and the inflammatory reactions induced by their metabolic products, enzymes, and toxins are the main cause of endodontics diseases. Kakehashi; Stanley; Fitzgerald, 1965 Miller, 1894 – different bacteria observed inside root canal Crawford; Shankle, 1961 – prevalence of streptococci Möller, 1966 –prevalence of anaerobic microorganisms
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Bacteroides - SUNDQVIST et al., 1979, WINKELHOFF; CARLEE; GRAAFF, 1985, GOMES; LILLEY; DRUCKER, 1996 SIQUEIRA JR. et al., 1998 Fusobacterium nucleatum - BYSTRÖM; CLAESSON; SUNDQVIST, 1985, SJÖGREN et al., 1991, SIQUEIRA JR.; UZEDA, 1996 GOMES; LILLEY; DRUCKER, 1996 SJÖGREN et al., 1997 Actinomyces israelii - HAPPONEN et al., 1985, NAIR et al., 1990 FIGDOR et al. 1992 BARNARD; DAVIES; FIGDOR, 1996 SJÖGREN et al., 1997 Clostridium perfringens - SUTTER et al., 1979 THORNSBERRY, 1990 CARLSSON; FRÖLANDER; SUNDQVIST, 1977 KONTAKIOTIS; NAKOU; GEORGOPOULOU, 1995 GOMES; LILLEY; DRUCKER, 1996 Literature Review – root canal microbiota
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Bacterial persistence after instrumentation BYSTRÖM; SUNDQVIST, 1981, BYSTRÖM; CLAESSON; SUNDQVIST, 1985, SIDNEY, 1996, GOMES; LILLEY; DRUCKER, 1996 Intra-canal dressings NAIR et al., 1990, SUNDQVIST, 1992 SJÖGREN et al., 1997 Literature Review - Endodontics
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The need for medication increases in those cases where an infection resists to regular treatments and the therapy cannot be successfully completed owing to the presence of pain or constant exudation. Ferreira et al., 2004 Literature Review - Endodontics
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Strong antiseptics Formocresol, CPMC (camphorated paramonochlorophenol) Dangerous to living tissues VANDER WALL; DOWSON; SHIPMAN, 1972, SPANGBERG; RUTBERG; RYDINGE, 1979 LEWIS; CHESTNER, 1981 Formocresol - VANDER WALL; DOWSON; SHIPMAN, 1972 OHARA; TORABINEJAD; KETTERING, 1993 CPMC - STEVENS; GROSSMAN, 1983 HAAPASALO; ØRSTAVIK, 1987 Antimicrobial results
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Calcium hydroxide HEITERSAY, 1975 BYSTRÖM; CLAESSON; SUNDQVIST, 1985, GEORGOPOULOU; KONTAKIOTIS; NAKOU, 1993 SUSUKI et al., 1999 HAAPASALO; ØRSTAVIK, 1987, SIQUEIRA JR.; UZEDA, 1996 BYSTRÖM; CLAESSON; SUNDQVIST, 1985, SJÖGREN et al., 1991 Excellent biologic and antimicrobial properties
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EEP – ethanolic extract of propolis A new substance as intracanal dressing STOJKO et al., 1978 SCHELLER et al., 1978 GAFAR et al., em 1986 SILVEIRA et al., 1988 IKENO; IKENO; MIYAZAWA, 1991 MARCUCCI, 1995 BRETZ et al., 1998 KEDZIA, 1990, FOCHT et al., 1993, AGA et al., 1994, BANKOVA et al., 1995 GOMES et al., 1998, GEBARA; MAYER, 1998 Beehive resinous compound Excellent antimicrobial properties Antibacterial Antiviral Antifungal Antiprotozoan
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Toxic to tumoral cells Promotes cartilaginous and bone tissue regeneration Prevent stomach ulcer Prevents tooth decay Immunomodulatory Anesthetic EEP – ethanolic extract of propolis Excellent biologic properties STOJKO et al., 1978 SCHELLER et al., 1978 GAFAR et al., em 1986 SILVEIRA et al., 1988 IKENO; IKENO; MIYAZAWA, 1991 MARCUCCI, 1995 BRETZ et al., 1998
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EEP – ethanolic extract of propolis When used as pulp-capping agent, EEP induced hard tissue bridge similar to calcium hydroxide Bretz et al., 1998 EEP exert 10 times less toxicity than calcium hydroxide on pulpal and periodontal fibroblast cell culture Al-Shaher et al., 2004
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EEP – ethanolic extract of propolis Propolis solution promoted a new storage media for avulsed teeth, leaving more viable periodontal periodontal ligament cells than Hank’s solution, milk or saline. Martin; Pileggi, 2004 Propolis on dentinal surfaces promoted dentinal tubules occlusion suggesting its uses for dentinal hypersensitivity. Almas et al., 2001 EEP induced antiinflamatory and analgesic effects in mouse models, inhibiting nitric oxide production, better than diclofenac, and decreasing the chemotaxis of PMFN leucocytes. Paulino et al., 2006, Ta-No et al., 2006, Naito et al., 2007
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EEP – ethanolic extract of propolis Propolis decreased the recurrences and improved the quality of life in patients who suffered from recurrent aphthous stomatitis. Samet et al., 2007 EEP inhibited oral candidiasis in denture-bearing patients. Santos et al., 2005 Two weeks of subgingival irrigation with propolis as an adjuvant to periodontal treatment was more effective than conventional treatment, by clinical and microbiological parameters. Gebara et al., 2003
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Propolis – oral antimicrobial effect Periodontopathogens isolates Prevotella intermedia / P. nigrescens were susceptible to propolis as to penicillin, erythromycin, meropenemand metronidazol. Santos et al., 2002 Against Staphylococcus aureus, EEP showed inhibition of lipase and coagulase activity, and a negative interaction with adhesion and consequent biofilm formation. Scazzocchio et al., 2006 Two weeks of subgingival irrigation with propolis as an adjuvant to periodontal treatment decreased the sites with Porphyromonas gingivalis and yeasts. Gebara et al., 2003
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Broth macro and micro dilution methods FINEGOLD; BARON 1986, SUTTER, 1987 BRUMFITT; HAMILTON-MILLER; FRANKLIN, 1990, IKENO; IKENO; MIYAZAWA, 1991, OHARA; TORABINEJAD; KETTERING, 1993, SIQUEIRA; LOPES; UZEDA, 1996, GOMES et al., 1998 Literature Review – microbiological methods Microdilution ROSENBLATT et al., 1979, THORNSBERRY, 1990, GOLDSTEIN; CITRON; GOLDMAN, 1992, FOCHT et al., 1993, SHAPIRO; MEIER; GUGGENHEIM, 1994. The susceptibility of anaerobic root canal infection is ideally determined by dilution tests, which is relatively scarce. ROSA et al., 2002
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CULTURE MEDIA Brucella Wadsworth Anaerobic Bacteriology Manual - Sutter et al., 1985 Murray, 1978 Finegold; Baron, 1986 Goldstein; Citron; Goldman, 1992 Van Horn; Warren; Baccaglini, 1997 RCM INCQS - Oswaldo Cruz Fondation (Rio de Janeiro, Brazil) D’Antonio, 1984 Literature Review – microbiological methods
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OBJECTIVES Evaluate the minimal inhibitory concentration (CIM) and minimal bactericidal concentration (CBM) of antimicrobial medicaments: formocresol, Camphorated paramonochlorophenol (CPMC), 10% calcium hydroxide, 10% ethanolic extract of propolis
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against bacterial reference strains: Fusobacterium nucleatum, Prevotella intermedia, Actinomyces israelii, Clostridium perfringens, Enterococcus faecalis, by means of macrodilution method, in Brucella, Reinforced Clostridial Medium (RCM) and Brain Heart infusion (BHI) broths. OBJECTIVES
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MATERIAL AND METHODS
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Antimicrobials Formocresol (Probem, Brazil) Camphorated paramonochlorophenol (CPMC) (S.S.White, USA) 10% calcium hydroxide (Biodinâmica, Brazil) 10% ethanolic extract of propolis (EEP) Pure ethanol 95% (Merck, Germany) - control
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Propolis extraction and analysis The solid propolis, obtained from the state of Rio Grande do Sul (Brazil), was dissolved in an extractor in boiling ethanol. This extract was cooled and the wax removed by filtration. The concentration of this filtrate was done in a rotary evaporator at room temperature until a thick paste was formed. By adding ethanol to this extract in a volumetric balloon, a 10% concentration of EEP was obtained. The EEP was analyzed using high-performance liquid chromatography (HPLC) to detect its components. The selected sample for the test showed high levels of the coumarates (3,5-diprenyl-4- hydroxycinnamic acid), an H component not yet identified, caffeic acid, and benzoic acid.
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Table I. Chemical composition of the ethanolic extract of propolis (EEP) Component tR (min)* mg/mL 2,4-Dihydroxybenzoic acid — 0.00 4-Hydroxybenzoic acid — 0.00 Caffeic acid 2.80 0.15 Benzoic acid 6.43 2.82 Ferulic acid — 0.00 p-Coumaric acid — 0.00 “H” compound 11.01 2.63 3-Prenyl-4-hydroxycinnamic acid 19.31 0.19 2,2-Dimethyl-6-carboxyethenyl- 2H-1-Benzopiran 22.40 1.81 3,5-Diprenyl-4- hydroxycinnamic acid 28.59 5.39 “E” compound 31.71 0.03 6-Propenoic-2,2-dimethyl-8-prenyl- 2H-1-benzopiran acid 33.01 0.13 “G1” compound — 0.00 3-Methoxy-4- hydroxycinnamaldehyde — 0.00 2-[1-Hydroxymethyl]vinyl-6-acetyl-5- hydroxy-coumaran — 0.00 “K”compound — 0.00 “L1” compound — 0.00 “L2” compound — 0.00 *Retention time of each compound in the chromatogram, in minutes.
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EEP sample chromatography (HPLC)
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Culture media For anaerobes: Brucella broth Reinforced Clostridial Medium (RCM) broth RCM blood agar For Enterococcus faecalis – facultative anaerobe: Brain Heart infusion (BHI) broth BHI agar
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Microorganisms Fusobacterium nucleatum - ATCC 25586 Prevotella intermedia - ATCC 33563 Actinomyces israelii - ATCC 12102 Clostridium perfringens - ATCC 13124 Enterococcus faecalis – ATCC 29212 Lyophilized strains were obtained from INCQS - Oswaldo Cruz Fondation (Rio de Janeiro, Brazil), reconstituted in RCM broth and checked for bacterial purity.
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MIC – Minimal inhibitory concentration It is the least antimicrobial concentration able to induce the complete inhibition of visible bacterial growth. (JONES et al., 1987) It is the least antimicrobial concentration able to reduce the bacterial population to 0,1% or less of numbers of present cells at the original inoculum. (SCHOENKNECHT; SABATH; THORNSBERRY, 1987) MBC – Minimal bactericidal concentration Methods – Broth macrodilution
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RCM and Brucella broth Antimicrobials MACRODILUTION 2,5 mL of broth in each tube 15 dilution tubes for each medicament Medicaments insertions into tubes, with volume and concentrations previously calculated (serial two fold dilutions) 2,5 mL of inoculum in each tube Subculture and incubation for 24 h at 37ºC in anaerobic environment spectrophotometer readings ? = 540 nm Diluted inocula 5 x 105 UFC/mL
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MACRODILUTION Incubation in anaerobic environment for 24 h at 37º C Spectrophotometer readings of tubes turbidity provided the MIC From each tube 25?L were transferred to small plates with blood agar Incubation in anaerobic environment for 48 h at 37º C Final reading of the plates, providing the results of minimal bactericidal concentration – MBC, by means of broth macrodilution method.
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MBC - MACRODILUTION Positive growth Growth absence
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Results of macrodilution test of 10% calcium hydroxide solution over Actinomyces israelii in RCM broth. Tubes Concentration 1st reading 2nd reading MBC (µg/mL ) optical density – OD OD 1 12500 3.000 3.000 _ 2 6250 2.843 2.907 _ 3 3125 2.242 2.876 _ 4 1562.5 1.944 2.039 _ 5 781.25 1.744 1.227 _ 6 390.62 .709 .752 + 7 195.31 .223 .633 + 8 98 .069 1.149 + 9 49 .106 1.021 + 10 24.5 .051 .868 +
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Minimal Inhibitory concentration and minimal bactericidal concentration of calcium hydroxide at two culture media for anaerobic bacteria in ?g/mL C.p A.i. P.i. F.n.
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1562.5 6250.0 3489.0 6678.0 328.0 760.0 6425.0 7640.0 Minimal Inhibitory and bactericidal concentrations in ?g/mL of all medicaments using the macrodilution method in brain heart infusion broth over Enterococcus faecalis Ca(OH)2 CPMC Formocresol EEP MIC MBC MIC MBC MIC MBC MIC MBC
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RESULTS – CULTURE MEDIA Numbers of colony forming units by mL of tested anaerobic bacteria at two culture broths, at the moment of inocula standardization
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RESULTS – CULTURE MEDIA Strong yellow – RCM broth Weak yellow – Brucella broth
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CONCLUSIONS 1 – The broth macrodilution behaved as a secure method for proposed aims of MIC and MBC determinations. 2 – Every tested drugs exerted inhibitory and bactericidal effect against all bacterial strains. Formocresol, camphorated paramonochlorophenol and ethanolic extract of propolis showed similar inhibitory and bactericidal concentrations, The calcium hydroxide showed numbers a little bit higher.
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CONCLUSIONS 3 – At macrodilution test, formocresol was equally effective over all strains; calcium hydroxide was the least effective over Clostridium perfringens; camphorated paramonochlorophenol was the most effective over Prevotella intermedia, and ethanolic extract of propolis showed the least inhibitory and bactericidal concentrations over Prevotella intermedia in this study.
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CONCLUSIONS 4 - Ethanol was tested because it is the vehicle of ethanolic extract of propolis. Ethanol did not influenced the antimicrobial effect of propolis, showing alone inhibitory and bactericidal concentrations 10 times higher than EEP. 5 – The RCM broth, compared to Brucella broth, promoted better bacterial growth in a less incubation time. Also, RCM broth showed higher inhibitory and bactericidal concentrations for tested drugs.
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Bactericidal concentrations of the tested substances were lower than those used in infected root canals. However, drugs such formocresol and CPMC must be avoided because of their toxicity. The proven antimicrobial activity of EEP against microorganisms detected in pulp-periapical infections allows us to suggest its inclusion in the arsenal of intracanal substances and subjecting it to further research, such as in vivo biologic compatibility tests and ex vivo (animal) studies. CONCLUSIONS
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