ORIGINAL ARTICLE
Comparison of the Antimicrobial Effect of Egyptian Propolis vs New Zealand Propolis on Streptococcus mutans and Lactobacilli in Saliva Ghada A. Elbaza/Iman I. Elsayadb Purpose: To evaluate the antimicrobial effect of Egyptian propolis vs New Zealand propolis on Streptococcus mutans and lactobacilli in saliva. Materials and Methods: The strains used for the experiment were isolated from 12 patients having a high caries index. The ethanolic extract (EEP) of pure Egyptian propolis was obtained by dissolving 20 g of propolis in 70% aqueous ethanol to a final volume of 100 ml. The commercial New Zealand propolis, combined with antibacterial agents, was an ethanolic extract of propolis in lozenge form; this was dissolved in distilled water to obtain an EEP. The EEP was further fractioned using a liquid-liquid extraction technique with hexane and chloroform solvents. The antimicrobial properties of the two propolis types and their fractions on Streptococcus mutans and lactobacilli were examined separately by determining minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Twelve clinical isolates were obtained from the collected saliva of all patients, one (Streptococcus mutans and Lactobacilli) from each patient, for susceptibility testing. Results: The MIC values of the New Zealand propolis were lower than the MIC values of the Egyptian propolis, indicating that the New Zealand propolis and hexane fractions (H-fr) in general had stronger antimicrobial effects. In addition, its antimicrobial action was greater on S. mutans than on lactobacilli, except with H-fr they were the same. Conclusion: The commercial New Zealand propolis hexane fraction had the strongest antimicrobial action. The EEP had a more potent antimicrobial effect on S. mutans than on lactobacilli. Key words: bactericidal effect, lactobacilli, propolis, Streptococcus mutans Oral Health Prev Dent 2012; 10: 155-160
D
isease prevention is the ultimate goal in restorative dentistry (Featherstone, 2000; Pitts, 2003). Dental diseases are among the most prevalent and costly diseases affecting industrialised societies, yet they are highly preventable. Dental caries is the most prevalent disease affecting humans, and its incidence is particularly high during childhood (Araujo and Figueiredo, 1997). One of the primary causes of dental caries is Streptococcus mutans. There are several virulence factors that make these bacteria cariogenic, including adhesion, acidogenicity and
a
Lecturer, Department of Pedodontics, Faculty of Dentistry, Suez Canal University, Egypt.
b
Associate Professor, Department of Operative Dentistry, Faculty of Oral and Dental Medicine, Cairo University, Egypt.
Correspondence: Ghada AbdelHameed Elbaz, 16 Sybawaye Elmasry Street, Nasr City, Cairo, Egypt. Tel: +2-022-261-6747, Fax: +2-064322-4471. Email:
[email protected]
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Submitted for publication: 16.01.11; accepted for publication: 30.09.11
acid tolerance. These bacteria produce glucosyltransferases and synthesise glucans from sucrose (in particular, water-insoluble glucans), which mediate the adherence of S. mutans and other oral bacteria on tooth surfaces and contribute to the formation of dental plaque. When a drop in the pH occurs after the fermentation of carbohydrates, the probability of enamel demineralisation increases, leading to cariogenesis (Banas, 2004). Success in inhibiting any of these previous steps would lead to caries prevention (James and Tagg, 1991; Matsumoto et al, 1999; Yanagida et al, 2000; Radcliffe et al, 2002; Matsumoto et al, 2003). Both Streptococcus mutans and lactobacilli play an essential role in the pathogenesis of caries and consequently are a prime target for the prevention of this disease by antibiotics and vaccines (Loesche et al, 1989). More recently, several natural products have been introduced on the market with
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claims of being anticariogenic. Some natural products have been suggested for use in developing countries as an economic, safe and probably effective alternative for prevention and treatment of caries. Propolis, a natural resinous substance collected by honeybees from buds and exudates of plants to be used as a protective barrier in the beehive, is currently incorporated in food and beverages to improve health and to prevent several diseases. Besides its use in folk medicines, it possesses various corroborative pharmacological activities, such as antibacterial, antiviral, antifungal, anaesthetic, anti-inflammatory, hypotensive, immunostimulatory and cytostatic properties (Bankova et al, 2000). Moreover, propolis may also prevent dental caries (Ikeno et al, 1991; Park et al, 1998; Koo et al, 1999; Koo et al, 2000a; Koo et al, 2000b; Koo et al, 2000c; Koo et al, 2002a; Koo et al, 2002b; Koo et al, 2002c; Duarte et al, 2003), as previous in vitro and in vivo studies have shown that propolis inhibits the growth of mutans streptococci in vitro (Ikeno et al, 1991; Park et al, 1998) and reduces the incidence of caries and dental plaque accumulation in vivo (Koo et al, 1999; Koo et al, 2002a; Koo et al, 2002b). Two mechanisms of action have been associated with the anti-caries/anti-plaque properties of propolis: the first is the antimicrobial activity against cariogenic bacteria and the second is the inhibition of glucosyltransferase enzyme activity (Koo et al, 2000a). The chemical composition of propolis is complex, typically consisting of waxes, resins, water, inorganics, phenolics and essential oils; the exact composition of propolis is dependent upon the source plant(s) (Bankova et al, 1992; Marcucci, 1995; Markham et al, 1996; Burdock, 1998). Flavonoids and (hydroxyl) cinnamic acid derivatives have been considered the primary biologically active compounds of propolis (Burdock, 1998). Nevertheless, its composition is highly variable, depending on its geographical origin (Greenaway et al, 1990; Bankova et al, 1992; Park et al, 1997). Analyses of the chemical composition of propolis have identified at least 300 compounds (Castro, 2001). Abd El Hady and Hegazi (2002) found 104 different chemicals in three samples of propolis collected from different areas in Egypt, which were grouped into seven categories: aliphatic acids, aromatic acids, esters, di- and triterpenes, flavonoids, sugars and miscellaneous. Walker and Crane (1987) listed 149 compounds isolated from samples collected from around the world, and again, acids and flavonoids were well represented.
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This study was conducted to evaluate the antimicrobial effect of Egyptian propolis vs New Zealand propolis on Streptococcus mutans and lactobacilli in saliva. The null hypothesis is that there is no difference between the antimicrobial effect of Egyptian and New Zealand propolis.
MATERIALS AND METHODS Propolis samples and fractionation The propolis samples were obtained from two different sources, an ethanolic extract of pure crude Egyptian bee propolis and commercial New Zealand bee propolis lozenges (International Medical Guide, Egypt). Egyptian bee propolis
The Egyptian crude samples were taken from hybrid Carniolan honeybee (Apis mellifera) colonies to obtain honeybee propolis. Honeybee propolis was collected from camphor (Eucalyptus spp.) trees. Three beehives were located at the Beekeeping Department, Plant Protection Research Institute, Dokki, Giza, Egypt. Propolis samples were dehydrated, ground and extracted in absence of bright light at room temperature, (20 g of propolis and 70% ethanol to a final volume of 100 ml). After a week, extracts were filtered to obtain an ethanolic extract of propolis EEP (Sforcin et al, 2000). The EEP was serially fractioned with hexane and chloroform solvents (Duarte et al, 2003). The ethanolic extracts of the isolated fractions were obtained by dissolving 1 g of these fractions in 10 ml of 80% (v/v) ethanol. New Zealand bee propolis
Each propolis lozenge contains 93 mg ethanolic extract of New Zealand bee propolis in an all-natural base of raw sugar, glucose, water, honey, oil of lemon, menthol and peppermint oil with no artificial flavours or preservatives. The lozenges were dissolved in distilled water to form an aqueous ethanolic extract solution (90% v/v). To obtain the hexane and chloroform fractions, the same technique was used as described above.
Isolation of bacterial strains The strains used for the experiment were isolated from 12 patients having a high caries index
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(DMFT ≥ 7) after they had given informed consent. The patients, all with permanent dentition, were selected from the outpatient clinic of the Operative Dentistry Department, Faculty of Oral and Dental Medicine, Cairo University and the Pedodontic Department, Faculty of Dentistry, Suez Canal University. The protocol of the research project follows the guidelines of the human ethics committee at Suez Canal University, Faculty of Dentistry, which approved this project. Each patient’s medical history was recorded to make sure that no antibiotics had been used recently prior to sampling. Before examination, the teeth were cleaned using cotton pellets and air from the triple syringe. Patients were examined while sitting in an upright position on the dental chair, and a plane dental mirror and explorer No. 23 (HU-Freidy; Chicago, IL, USA) were used to detect dental caries. The criteria used for the dental caries examination were published by the WHO (1971). In permanent teeth, the DMF index was used (D = decayed tooth, filling indicated; M = missing tooth due to caries; F = filled tooth). From each patient, fresh whole saliva was collected in a graduated plastic tube under masticatory stimulation by having the subject chew sugarfree gum. The freshly collected saliva samples were cultured on plates of selective media: mitis salivarius bactiracin agar and Rogosa agar for the primary isolation of Streptococcus mutans and lactobacilli, respectively. The cultured mitis salivarius agar plates were incubated aerobically in a candle jar (5% to 10% CO2), whereas the cultured Rogosa agar plates were incubated anaerobically. All plates were kept at 37ºC for 48 h. The suspected bacterial growth was identified according to Ruoff et al (2003). Twelve clinical isolates were obtained from the saliva collected from all patients, one (Streptococcus mutans and lactobacilli) per patient for the susceptibility testing.
Susceptibility testing The antimicrobial activity of the two types of propolis extracts on the isolated caries-associated strains (Streptococcus mutans and lactobacilli) was examined separately by determining the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) in accordance with the Clinical and Laboratory Standard Institute guidelines (CLSI, 2005) and Koo et al (2000b). To determine MIC, the starting inoculum of the test strains was 3 x 108 CFU/ml in Mueller Hinton broth, and
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the test extract concentrations ranged from 3200 mg/ml (first concentration) to 12.5 mg/ml for EEP and from 1600 mg/ml (first concentration) to 6.25 mg/ml for the other fractions of propolis (in a series of 2-fold dilutions); the control was ethanol 0.6% v/v. MIC was determined as the lowest concentration causing inhibition of bacterial growth. To determine MBC, an aliquot (50 μl) of all incubated tubes with concentrations higher than MIC was subcultured on BHI agar. The agar was supplemented with 5% of defibrinated sheep blood. MBC was determined as the lowest concentration that causes the killing of bacteria and prevents visible growth on the agar (Koo et al, 2000b). Three separate experiments were conducted for each concentration of the extracts tested.
RESULTS The MIC and MBC values at which S. mutans growth was inhibited and killed, respectively, are shown in Table 1. The MIC and MBC values at which lactobacilli growth was inhibited and killed, respectively, are shown in Table 2. The MIC values of EEPs and their hexane (H-fr) and chloroform (Chlo-fr) fractions for the New Zealand propolis lozenges were similar for S. mutans and lactobacilli were similar, with the exception of H-fr for lactobacilli, which was less. The MIC values of the Egyptian propolis were the lowest with H-fr for both S. mutans and lactobacilli. The MIC values of the New Zealand propolis were lower than the MIC values of the Egyptian propolis, indicating that the New Zealand propolis had a more potent antimicrobial effect. For the New Zealand propolis, the MBC values were from 2 to 12 times greater than the MIC values. In addition, its antimicrobial action was greater on S. mutans than on lactobacilli, except with H-fr, they were the same. The EEP of the pure Egyptian propolis had a stronger antimicrobial effect with a lower MIC on S. mutans than on lactobacilli. Most of the pure Egyptian propolis extracts did not show any antimicrobial action except for the H-fr.
DISCUSSION The results allow partial confirmation of the null hypothesis, because although for Egyptian propolis, there is no MBC except for H-fr, some of the MIC values between Egyptian and New Zealand propolis are similar. Thus, both Egyptian and New Zealand
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Elbaz and Elsayad Table 1 MIC and MBC values in μg/ml of propolis and their fractions for Streptococcus mutans Pure Egyptian propolis
Commercial New Zealand propolis
Fractions
Fractions
EEP
H-fr
Chlo-fr
EEP
H-fr
Chlo-fr
MIC
100–200
25–50
200–400
25–50
25–50
25–50
MBC
*
200–400
*
100–200
50–100
100–200
*No bactericidal action was observed at the starting inoculum.
Table 2 MIC and MBC values in μg/ml of propolis and their fractions for lactobacilli Pure Egyptian propolis
Commercial New Zealand propolis
Fractions
Fractions
EEP
H-fr
Chlo-fr
EEP
H-fr
Chlo-fr
MIC
200–400
25–50
200–400
25–50
12.5–25
25–50
MBC
*
200–400
*
200–400
50–100
200–400
*No bactericidal action was observed at the starting inoculum.
propolis have an antimicrobial action, albeit of different potencies. In terms of health promotion, propolis extract may represent a new option, as this substance is easily obtained and inexpensive, in addition to showing long-term beneficial effects (Oliveira, 2004). The chemical composition of propolis is complex; it depends on the flora in the areas where it is collected (Bankova et al, 2000). It is generally recognised that Gram-positive bacteria are more susceptible to the antibacterial action of propolis than Gram-negative bacteria (Mirzoeva et al, 1997; Drago et al, 2000; Sforcin et al, 2000). Although the antimicrobial properties of propolis have been evaluated in several investigations, it is difficult to compare the results of different studies due to the different methods used (Drago et al, 2000). However, the results of this study reveal that the New Zealand propolis lozenges had a potent antimicrobial activity. This may also be attributed to the action of other ingredients, such as honey, menthol and other essential oils present in the New Zealand propolis lozenges. It is well established that the hydrogen peroxide activity in honey correlates with floral sources, and the antibacterial activity of honey is mainly due to the enzymatic formation of hydrogen peroxide (Molan, 1992; Baltrusaityte et al, 2007). Menthol, which is a compound obtained from peppermint oil, also has a proven antibacterial action (Saeed et al, 2006). While essential oils derive their antibacterial
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effect from their unique chemical makeup, each single, pure essential oil consists of several, sometimes hundreds of distinct natural chemicals. Many of these have antimicrobial activity and show synergistic effects (Ncube et al, 2008). Several compounds have been identified in propolis, falling into three distinct chemical groups: 1. flavonoid aglycones, 2. cinnamic acid derivatives and 3. terpenoids (Bankova et al, 1995; Park et al, 1998). The antibacterial activity of propolis has been attributed to the phenolic compounds, especially flavonoids, phenolic acids and their esters (Loesche et al, 1989). Some prenylated p-coumaric acids isolated from Brazilian propolis have been shown to possess antibacterial activity (Aga et al, 1994). Bankova et al (1995; 1996; 2000) reported the antibacterial activity of volatile compounds and diterpenic acids from Brazilian propolis. Park et al (1998) showed the presence of different flavonoids and the inhibitory effects of propolis on cariogenic bacteria. Hegazi and Abd El Hady (2002) investigated the chemical composition of two types of Egyptian propolis collected from two different areas; they identified 75 compounds, with 22 being new for propolis. The new esters belonged to 4-methoxyhydrocinnamic acid, hydroferulic acid, ferulic acid and 2,6-bis-(pentanyloxy)-4-pentanylphenethanol. They also found that one type had higher antioxidant, antibacterial and antifungal activity than the other type.
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The findings of this study confirm and extend previous observations on the anti-caries properties of propolis (Hayacibara et al, 2005). Here, it was shown that H-fr, followed by Chlo-fr were the most effective extracts, indicating that the putative biologically active compounds are mostly apolar. In general, H-fr from both propolis samples showed the most potent antibacterial effect in vitro. Although the Egyptian propolis did not show any antimicrobial action with EEP and Chlo-fr at the starting concentration of inoculum, it might have shown such an effect at a higher concentration of the starting inoculum. However, using propolis from two different geographic origins was a major limitation in this study. This was unavoidable due to difficulties in importing fresh crude New Zealand propolis; thus, commercial New Zealand propolis lozenges were used. Furthermore, some of the antimicrobial action of the New Zealand propolis may be due to the added antibacterial substances in the lozenges. Because well-characterised reference bacterial strains were not available, many clinical isolates were tested to overcome the variation in the susceptibility pattern among different bacterial strains.
CONCLUSIONS Under the conditions of this study, it can be concluded that both propolis types can inhibit S. mutans and lactobacilli growth. The hexane fractions contain most of the active compounds of propolis and should be the fractions of choice for further chemical characterisation and isolation. The EEP had a more potent antimicrobial effect on S. mutans than on lactobacilli.
ACKNOWLEDGEMENTS The authors would like to thank Dr Mohammad Sherief, Professor of Microbiology at Al-Azhar University, and Dr Maha Fathy Professor of Microbiology at Ain Shams University for their great help during this study.
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REFERENCES 1. Abd El Hady FK, Hegazi AG. Egyptian propolis: 2. Chemical composition, antiviral and antimicrobial activities of east Nile delta propolis. Z Naturforsch 2002;57:386–394. 2. Aga H, Shibuya T, Sugimoto T, Kurimoto M, Nakajima SH. Isolation and identification of antimicrobial compounds in Brazilian propolis. Biosci Biotech Biochem 1994;58:945– 946. 3. Araújo FB, Figueiredo M C. Promoção de saúde bucal em Odontopediatria. In: Krieger L (ed). Promoção de saúde bucal. São Paulo: Artes Médicas, 1997:283-348. 4. Baltrusaityte V, Venskutonis PR, Ceksteryte V. Food technol Biotechiol 2007;45:201–208. 5. Banas JA: Virulence properties of Streptococcus mutans. Front Biosci 2004;9:1267–1277. 6. Bankova V, Castro SL, Marcucci MC. Propolis: recent advances in chemistry and plant origin. Apidologie 2000;31:3–15. 7. Bankova V, Christov R, Kujumgiev A, Marcucci MC, Popov S. Chemical composition and antibacterial activity of Brazilian propolis. Z Naturforsch 1995;50c:167–172. 8. Bankova V, Christov R, Stoev G, Popov S. Determination of phenolics from propolis by capillary gas chromatography. J Chromatogr 1992;607:150–153. 9. Bankova VS, De Castro SL, Marcucci MC. Propolis: recent advances in search on chemistry and plant origins. Apidologie 2000;31:3–15. 10. Bankova, V, Marcucci MC, Simonova S, Nikolova N, Kujumgiev A. Antibacterial Diterpenic acids from Brazilian propolis. Z Naturforsch 1996;51c, 277–280. 11. Burdock GA. Review of the biological properties and toxicity of bee propolis (propolis). Food Chem Toxicol 1998;36:347– 363. 12. Castro SL. Propolis: biological and pharmacological activities. Therapeutic uses of this bee-product. Annu Rev Biomed Sci 2001;3:49–83. 13. Clinical and Laboratory Standards Institute (Formerly NCCLS) 2005. M2-A9 Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard-Ninth Edition and M100-S16 Performance Standards for Antimicrobial Susceptibility Testing; Sixteenth Informational Supplement. 14. Drago L, Mombelli B, De Vecchi E, Fassina MC, Tocalli L, Gismondo MR. In vitro antimicrobial activity of propolis dry extract. J Chemotherapy 2000;12:390–395. 15. Duarte S, Koo H, Bowen WH, Hayacibara MF, Cury JA, Ikegaki M et al. Effect of a novel type of propolis and its chemical fractions on glucosyltransferases and on growth and adherence of mutans streptococci. Biol Pharm Bull 2003;26:527–531. 16. Featherstone JD. The science and practice of caries prevention. J Am Dent Assoc 2000;131:887–899. 17. Ghisalberti, EL. Propolis: a review. Bee World 1979;60:59– 84. 18. Greenaway W, Scaysbrook T, Whatley FR. The composition and plant origins of propolis: a report of work at Oxford. Bee World 1990;71:107–118. 19. Hayacibara MF, Koo H, Rosalen PL, Duarte S, Franco EM, Bowen WH et al. In vitro and in vivo effects of isolated fractions of Brazilian propolis on caries development J Ethpharm 2005;101:110–115.
159
Elbaz and Elsayad 20. Hegazi AG, Abd El Hady FK. Egyptian Propolis: 3. Antioxidant, antimicrobial activities and chemical composition of propolis from reclaimed lands. Z Naturforsch 2002;57c:395–402. 21. Ikeno K, Ikeno T, Miyazawa C. Effects of propolis on dental caries in rats. Caries Res 1991;25:347–351. 22. James SM, Tagg JR. The prevention of dental caries by BLIS–mediated inhibition of mutans streptococci. NZ Dent J 1991;87:80–83. 23. Koo H, Cury JA, Rosalen PL, Ambrosano GM, Ikegaki M, Park YK. Effect of a mouthrinse containing selected propolis on 3-day dental plaque accumulation and polysaccharide formation. Caries Res 2002a;36:445–448. 24. 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 2000a;45:141–148. 25. Koo H, Pearson SK, Scott AK, Abranches J, Cury JA, Rosalen PL et al. Effects of apigenin and tt-farnesol on glucosyltransferase activity, biofilm viability and caries development in rats. Oral Microbiol Immunol 2002b;17:337–343. 26. Koo H, Rosalen PL, Cury JA, Ambrosano GM, Murata RM, Yatsuda R et al. Effect of a new variety of Apis mellifera propolis on mutans streptococci. Curr Microbiol 2000b;41:192–196. 27. Koo H, Rosalen PL, Cury JA, Park YK, Bowen WH. Effects of compounds found in propolis on Streptococcus mutans growth and on glucosyltransferase activity. Antimicrob Agents Chemother 2002c;46:1302–1309. 28. Koo H, Rosalen PL, Cury JA, Park YK, Ikegaki M, Sattler A. Effect of Apis mellifera propolis from two Brazilian regions on caries development in desalivated rats. Caries Res 1999;33: 393–400. 29. Koo H, Vacca Smith AM, Bowen WH, Rosalen PL, Cury JA, Park YK. Effects of Apis mellifera propolis on the activities of streptococcal glucosyltransferases in solution and adsorbed onto saliva-coated hydroxyapatite. Caries Res 2000c;34:418–426. 30. Loesche WJ, Eklund SA, Mehlisch DF, Burt B. Possible effect of medically administered antibiotics on the mutans streptococci: implications for reduction in decay. Oral Microbiol Immunol 1989;4:77–81. 31. Marcucci MC. Propolis: Chemical composition, biological properties and therapeutic activity. Apidologie 1995;26:83– 99. 32. Markham KR, Mitchell KA, Wilkins AL, Daldy JA, Lu Y. HPLC and GC-MS identification of the major organic constituents in New Zealand propolis. Phytochem 1996;42:205–211. 33. Matsumoto M, Hamada S, Ooshima T. Molecular análisis of the inhibitory effects of oolong tea polyphenols on glucanbinding domain of recombinant glucosyltransferases from Streptococcus mutans MT8148. FEMS Microbiol Letters 2003;228:73–80.
160
34. Matsumoto M, Minami T, Sasaki H, Sobue S, Hamada S, Ooshima T. Inhibitory effects of oolong tea extract on caries-inducing properties of mutans streptococci. Caries Res 1999;33:441–444. 35. Mirzoeva OK, Grishanin RN, Calder PC. Antimicrobial action of propolis and some of its components: the effects on growth, membrane potential and motility of bacteria. Microbiol Res 1997;152:239–246. 36. Molan PC . The antibacterial activity of honey. The nature of antibacterial activity. Bee World 1992;73:59–79. 37. Ncube NS, Afolayan AJ, Okoh AI. Assessment techniques of antimicrobial properties of natural compounds of plant origin: current methods and future trends. Afr J Biotechnol 2008;7:1797–1806. 38. Oliveira EG. Qualidade microbiolǀgica e fƭsico-quƭmica do mel da abelha tiNJba Melƭpona compressipes fasciculata produzida no Estado do Maranhão. 2004. Dissertação (Mestrado) – Centro de Ciências da SaNJde da UFMA. São Luis, 2004. 39. Park YK, Koo MH, Abreu JAS, Ikegaki M, Cury JA, Rosalen PL. Antimicrobial activity of propolis on oral microorganisms. Curr Microbiol 1998;36:2428. 40. Park YK, Koo MH, Ikegaki M, Contado JL. Comparison of the avonoid aglycone contents of Apis mellifera propolis from various regions of Brazil. Arq Biol Tecnol 1997;40:97–106. 41. Pitts NB. Modern concepts of caries measurement. J Dent Res 2004;83(special issue C):C43–C47. 42. Radcliffe C E, Akram N C, Hurrell F, Drucker DB. Effects of nitrite and nitrate on the growth and acidogenicity of Streptococcus mutans. J Dent 2002;30:325–331. 43. Ruoff KL, Whiley RA, Beighton D. Streptococcus. In Murray PR, Baron EJ, Jorgenson JH, Pfaller MA, Yolke RH (eds). Manual of Clinical Microbiology, ed 8. Washington DC: American Society for Microbiology, 2003:405–433. 44. Saeed S, Naim A, Tariq B. In Vitro antibacterial activity of peppermint. Pak J Bot 2006;38:869–872. 45. Sforcin JM, Fernandes Jr A, Lopes CAM, Bankova V, Funari SRC. Seasonal effect on Brazilian propolis antibacterial activity. J Ethnopharmacol 2000;73:243–249. 46. Walker P, Crane E. Constituents of propolis. Apidologie 1987;18:327–334. 47. World Health Organization. Oral Health Surveys, Basic Methods. Bulletin of the World Health Organization. Geneva: WHO, 1971. 48. Yanagida A, Kanda T, Tanabe M, Matsudaira F, Oliveira Cordeiro JG. Inhibitory effects of apple polyphenols and related compounds on cariogenic factors of mutans streptococci. J Agric Food Chem 2000;48:5666–5671.
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