Journal of Ethnopharmacology 80 (2002) 1 /7 www.elsevier.

com/locate/jethpharm

Review

Antibacterial activity of Brazilian propolis and fractions against oral anaerobic bacteria
F.A. Santos a,b, E.M.A. Bastos c, M. Uzeda b, M.A.R. Carvalho a, L.M. Farias a, E.S.A. Moreira a,*, F.C. Braga d
a

Departamento de Microbiologia, Laboratorio de Biologia de Microrganismos, Instituto de Ciencias Biologicas da Universidade Federal de Minas Gerais, Caixa Postal 486, Avenida Antonio Carlos 6627, CEP 31270 901, Belo Horizonte, MG, Brazil b Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil c Fundacao Ezequiel Dias-Belo Horizonte, Belo Horizonte, Minas Gerais, Brazil d Faculdade de Farmacia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Received 1 May 2001; received in revised form 24 December 2001; accepted 29 December 2001

Abstract Propolis collected from a cerrado area in Minas Gerais State, Brazil, was subjected to chromatography on silica gel column and to partition between immiscible solvents. Propolis aqueous-ethanolic extract and fractions obtained were tested for inhibitory activity against periodontitis-causing bacteria. All of the assayed bacterium species were susceptible to propolis extract. The two fractionation methodologies yielded fractions which were active against bacteria, with minimum inhibitory concentrations (MIC) ranging from 64 to 1024 mg/ml. TLC and HPLC analyses of the extract and of active fractions showed the presence of phenolic compounds of varied polarity. None of the assayed fractions was more active than the extract, suggesting that the antibacterial activity is probably due to the synergistic effect of several compounds. # 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Propolis; Antibacterial activity; Anaerobes; Phenolic compounds

1. Introduction Propolis is a resinous material collected by bees from plant buds and exudates, which is employed for construction and repair of the honeycomb (Ghisalberti, 1979; Asis, 1991). Propolis has been used in folk medicine for curing infections for hundreds, perhaps thousands of years (Cheng and Wong, 1996). Several biological activities have been described for propolis, including antibacterial (Grange and Darvey, 1990; Ikeno et al., 1991; Kujumgiev et al., 1993; Menezes et al., 1997), antifungal (Valdes et al., 1987), antiprotozoan (Scheller et al., 1977), antiviral (Amoros et al., 1992), antitumor (Grunberger et al., 1988), immunomodulation (Dimov et al., 1992) and antiinflammatory (Dobrowolski et al., 1991) activities, among others. Propolis is a complex mixture of chemical constituents and its composition is dictated by the constituents of the plant
* Corresponding author. Fax: '55-31-3499-2730. E-mail address: spangler@mono.icb.ufmg.br (E.S.A. Moreira).

material making up the native vegetation and by the season of collection (Ghisalberti, 1979; Vanhaelen and Vanhaelen-Fastre, 1979; Bankova et al., 1992; Mar cucci, 1995; Cheng and Wong, 1996; Oliveira and Bastos, 1998; Bankova et al., 2000). The development of new therapies for the treatment of the diseases of the oral cavity is of great relevance, since the systemic administration of antimicrobials has been reported to cause the development of multiresistant microorganisms, interbacterial transfer of resistance determinants, and side effects (Walker, 1996). Actinobacillus actinomycetemcomitans is a Gram-negative bacterium associated with a variety of infectious diseases such as brain abscess, urinary tract infections, periodontal diseases and special cases of localized juvenile periodontitis (Slots, 1982; Kaplan et al., 1989). Species of Fusobacterium belong to the human microbiota, which is normally found in the oral cavity, colon, genital tract and upper respiratory tract. Fusobacterium species increase in number in the gingival pockets as periodontal disease progresses (Tanner et al., 1979;

0378-8741/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 0 0 3 - X

F.A. Santos et al. / Journal of Ethnopharmacology 80 (2002) 1 /7

Bolstad et al., 1996). Several black-pigmented Gramnegative anaerobes such as Porphyromonas gingivalis and Prevotella intermedia , are usually isolated from the gingival crevice of patients with periodontitis (Dahlen, 1993; Rodrigues et al., 1999). Despite increasing use of propolis worldwide (Marcucci, 1995; Cheng and Wong, 1996) only few studies have been carried out to determine the inhibitory effect of propolis against anaerobic bacteria of odontological relevance (Santos et al., 1999). Within this context, the aim of the present work was to evaluate the activity of propolis against nine periodontal disease-causing bacteria. Some propolis fractions were also evaluated, in order to verify whether they possess antibacterial activity.

70 8C, for 30 min protected from light. The resulting aqueous-ethanol extract was filtered through a Whatman 1 paper and concentrated at 50 8C, and the obtained resin was dissolved in 95% ethanol to a final concentration of 100 mg/ml (Menezes et al., 1997). This final solution was employed for the antimicrobial assays. 2.2. Column chromatography A portion of crude propolis (7 g) was dissolved in 80% ethanol (3 ) 70 ml) at 70 8C for 30 min. This extract was concentrated in a rotary evaporator under reduced pressure at 70 8C to approximately 50 ml and partitioned with CH2Cl2 (3 ) 50 ml). The organic layer was concentrated in a rotary evaporator at 40 8C and the resulting residue was dissolved in CH2Cl2 (2 ml) and was submitted to chromatography on a silica gel column (Silica gel 60 Merck 0.2 /0.5 mm; 50) 600 mm I.D.). Elution was performed with solvents of increasing polarity and solvents were removed using a rotary evaporator, at a temperature 50 8C: n -hexane (21; 60 mg), n -hexane/CH2Cl2 (1:1, 4 l, 120 mg), CH2Cl2 (3 l, 220 mg), CH2Cl2/EtOAc (1:1, 3 l, 1700 mg), EtOAc (2 l, 190 mg), EtOAc/MeOH (1:1, 4.5 l, 560 mg) and MeOH (7 l, 170 mg).

2. Material and methods 2.1. Propolis samples Crude samples of bee (Apis mellifera ) propolis were collected from an experimental apiary located in a cerrado area, in the city of Cachoeira da Prata, Minas Gerais State, Brazil. An aliquot of crude propolis (7 g) was dissolved in 80% ethanol (3 ) 70 ml) by shaking at

Fig. 1. Fractionation of propolis aqueous-ethanolic extract by partition between immiscible solvents.

F.A. Santos et al. / Journal of Ethnopharmacology 80 (2002) 1 /7

Table 1 Susceptibility of nine oral anaerobic strains to propolis fractions obtained from propolis aqueous-ethanolic extract (PAE) partition between immiscible solvents Bacterial strainsa MIC (mg/ml) PAE Aqueous fraction Aa FDC Y4 Aa ATCC 29 523 Fn ATCC 10 953 Fne ATCC 25 386 Pg ATCC 33 277 Pi ATCC 25 611 Pn ATCC 33 563 El ATCC 25 559 Pa ATCC 27 337
a

NaHCO3 fraction  1024  1024  1024  1024 256 256 256  1024 256

Na2CO3 fraction 1024 512 512 512 256 256 512 512 256

NaOH fraction  1024  1024  1024  1024  1024  1024  1024  1024 1024

Final residue Meropenem Penicillin G  1024  1024  1024  1024 1024 512 512  1024 512 0.5 0.5 0.06 0.03 1 2 2 8 8 4 4 4 1 0.03 0.06 0.03 4 4

1024 1024 1024 256 256 256 512 1024 128

 1024  1024  1024  1024  1024  1024  1024  1024  1024

Aa, A. actinomycetemcomitans ; Fn, F. nucleatum ; Fne, F. necrophorum ; Pg, P. gingivalis ; Pi, P. intermedia ; Pn, P. nigrescens ; El, E. lentum ; Pa, P. anaerobius .

2.3. Partition between immiscible solvents A portion of crude propolis (7 g) was dissolved in 80% EtOH (3 )70 ml) at 70 8C for 30 min. This extract was concentrated in a rotary evaporator under reduced pressure, at 70 8C, to approximately 50 ml and partitioned with CH2Cl2 (3 ) 50 ml). The organic layer was successively partitioned with 5% NaHCO3 aqueous solution (3 ) 30 ml), 5% Na2CO3 aqueous solution (3 ) 30 ml) and 10% NaOH aqueous solution (3 ) 30 ml), according to the sequence depicted in Fig. 1. Solvents were removed in a rotary evaporator, at a maximum temperature of 70 8C. 2.4. TLC analysis Thin-layer chromatography (TLC) analysis of propolis aqueous-ethanolic extract and its fractions were performed on silica gel plates (Merck TLC plates).

Samples were dissolved in MeOH (100 mg/ml) and 5 ml aliquots were applied to the TLC plates. Elution was performed with CHCl3/EtOAc (60:40, v/v) and the plates were visualized under visible and ultraviolet light (254 and 360 nm), after spraying with 5% AlCl3 in methanol solution (Wagner et al., 1984). 2.5. HPLC analysis Propolis aqueous-ethanolic extract and its fractions were analyzed by high-pressure liquid chromatography (HPLC). Analyses were carried out in a Merck-Hitachi apparatus (Germany). An ODS column (250 ) 4.0 mm I.D., 5 mm) was employed (Merck, Germany) at temperature of 30 8C, flow rate of 1.0 ml/min and wavelength of 254 nm. A linear gradient of H2O'0.1% H3PO4 (A) and CH3CN'0.1% H3PO4 (B) was employed: 0 min, 90% A 10% B; 60 min, 10% A 90% B, followed by 5 min of isocratic elution. Solvents used

Table 2 Susceptibility of nine oral anaerobic strains to propolis fractions obtained from propolis aqueous-ethanolic extract (PAE) silica gel column chromatography Bacterial strainsa MIC (mg/ml) PAE Aa FDC Y4 Aa ATCC 29 523 Fn ATCC 10 953 Fne ATCC 25 386 Pg ATCC 33 277 Pi ATCC 25 611 Pn ATCC 33 563 El ATCC 25 559 Pa ATCC 27 337
a

HEXb  1024  1024  1024  1024  1024  1024  1024  1024  1024

HEX/DCM  1024  1024  1024  1024  1024  1024  1024  1024  1024

DCM  1024  1024  1024 256 512 128 256  1024 256

DCM/EtOAc 1024 1024 1024 64 512 64 128  1024 64

EtOAc 1024 1024 1024 64 1024 256 512  1024 128

EtOAc/ MeOH  1024  1024  1024 64 1024 128 1024  1024 64

MeOH  1024  1024  1024 64 1024 128 1024  1024 256

1024 1024 1024 256 256 256 512 1024 128

Aa, A. actinomycetemcomitans ; Fn, F. nucleatum; Fne, F. necrophorum ; Pg, P. gingivalis ; Pi, P. intermedia ; Pn, P. nigrescens ; El, Eubacterium lentum ; Pa, P. anaerobius. b HEX, Hexane; HEX/DCM, Hexane/Dichloromethane; DCM, Dichloromethane; DCM/EtOAc, Dichloromethane/Ethyl acetate; EtOAc/MeOH, Ethyl acetate/Methanol; MeOH, Methanol.

Edited Ethnopharmacology 80 F.A. Santos et al. / Journal of by Foxit Reader (2002) 1 /7

were of HPLC grade (Merck, Germany) and were degassed by sonication before use. Samples were dissolved in methanol to concentrations of 10 and 5 mg/ml, for propolis aqueous-ethanolic extract and fractions, respectively. The solutions were centrifuged at 10 000 rpm before injection to HPLC. 2.6. Bacterial strains A total of nine microbial strains were tested: A. actinomycetemcomitans ATCC 29 523 and FDC Y4, Fusobacterium nucleatum ATCC 10 953, Fusobacterium necrophorum ATCC 25 386, P. gingivalis ATCC 33 277, P. intermedia ATCC 25 611 and Prevotella nigrescens ATCC 33 563, Eubacterium lentum ATCC 25 559 and Peptostreptococcus anaerobius ATCC 27 337. All strains were kept in our laboratory and cryopreserved at (86 8C. For experiments bacteria were inoculated in Brucella agar supplemented with 0.5% yeast extract, hemine (5 mg/ml), menadione (1 mg/ml) and 5% horse blood. Incubation was performed at 37 8C, in anaerobic conditions, for 5 days in Brewer-like anaerobic jars (90% N2, 5% CO2 and 5% H2). 2.7. Determination of the minimum inhibitory concentration (MIC)

Copyright(C) by Foxit Software Company,2005-2008 For Evaluation Only.

Tests were performed by the agar dilution method, according to the NCCLS guidelines (NCCLS, 1997). For the antibacterial assays, propolis aqueous-ethanolic extract and fractions were dissolved in 95% EtOH to a final concentration ranging from 50 to 100 mg/ml. The antimicrobials meropenem and penicillin G were used as positive controls (concentrations from 0.03 to 32 mg/ml). To increase concentrations of antimicrobials, propolis aqueous-ethanolic extract and fractions were added to the Brucella agar medium. The inoculum was standardized using the Steer replicator (ca. 105 CFU/ml). Plates were incubated in anaerobic environment for 48 h. Control plates contained no drug. For testing propolis aqueous-ethanolic extract, control plates containing the culture medium plus 2% of 95% EtOH were also included. The results were expressed as MIC, i.e. the minimum concentration which completely inhibited bacterial growth. All experiments were performed in duplicates. The data were submitted to analysis of variance using ANOVA test.

Fig. 2. TLC chromatogram developed in chloroform/ethyl acetate 60:40 visualized under ultraviolet light (365 nm) and sprayed with 5% aluminum chloride. (A) Fractions obtained from propolis aqueousethanolic extract partition between immiscible solvents: (1) propolis aqueous-ethanolic extract, (2) aqueous solution, (3) acid-rich fraction, (4) strong phenol-rich fraction, (5) weak phenol-rich fraction, (6) nal residue, (7) quercetin, (8) kaempferol; (B) fractions obtained from propolis aqueous-ethanolic extract silica gel column chromatography: (1) propolis aqueous-ethanolic extract, (2) hexane fraction, (3) hexane/ dichloromethane (1:1) fraction, (4) dichloromethane fraction, (5) dichloromethane/ethyl acetate (1:1) fraction, (6) ethyl acetate fraction, (7) ethyl acetate/methanol (1:1) fraction, (8) methanol fraction, (9) quercetin, (10) kaempferol; (C) fractions that exhibited antibacterial activity: (1) propolis aqueous-ethanolic extract, (2) acid-rich fraction, (3) strong phenol-rich fraction, (4) dichloromethane/ethyl acetate (1:1) fraction, (5) ethyl acetate fraction, (6) ethyl acetate/methanol (1:1) fraction, (7) quercetin, 8-kaempferol.

3. Results and discussion The in vitro antibacterial activity of Brazilian propolis aqueous-ethanolic extract was evaluated against nine strains of periodontitis-causing bacteria (Table 1). More than 180 substances have already been identified as propolis constituents and phenolic compounds are regarded as responsible for propolis biological activities (Marcucci, 1995; Bankova et al., 2000). Propolis aqueous-ethanolic extract was fractionated employing two different processes (chromatography on silica gel column and partition between immiscible solvents) and the

F.A. Santos et al. / Journal of Ethnopharmacology 80 (2002) 1 /7

Fig. 3. HPLC chromatograms of propolis fractions that exhibited antibacterial activity. (A) Propolis aqueous-ethanolic extract; (B) Acid-rich fraction; (C) Strong phenol-rich fraction; (D) Dichloromethane/ethyl acetate (1:1) fraction and (E) Ethyl acetate fraction. HPLC conditions: see experimental.

activity of the resulting fractions were determined against the same strains (Tables 1 and 2). All the assayed bacterial species were susceptible to propolis aqueous-ethanolic extract and fractions derived from both fractionation processes (Tables 1 and 2). P. anaerobius , P. gingivalis and P. intermedia were the bacteria most sensitive to propolis as well as acid- and

strong phenol-rich fractions, with MIC values ranging from 128 to 256 mg/ml. Medium polarity fractions (CH2Cl2/EtOAc 1:1; EtOAc), obtained from the silica gel column and acid- and strong phenol-rich fractions, from the partition between immiscible solvents showed the lowest MIC values (Tables 1 and 2). ANOVA indicated no significant difference in the susceptibility

F.A. Santos et al. / Journal of Ethnopharmacology 80 (2002) 1 /7

profile of the most active fractions (Tables 1 and 2) in comparison to propolis aqueous-ethanolic extract (P 5 0.05). None of the assayed fractions was more active than propolis aqueous-ethanolic extract, suggesting that the antibacterial activity is probably caused by the synergistic effects of different compounds, corroborating previously reported results (Marcucci, 1995; Kujungiev et al., 1999). Propolis mechanism of action appears to be complex and a simple analogy cannot be made to the mode of action of classic antibiotics (TakisiKikuni and Schilcher, 1994). Among the fractions from the partition between immiscible solvents (Table 1) the one obtained by partition with sodium carbonate solution, corresponding to the strong phenolic constituents, was the most active and exhibited a MIC value similar to that of propolis aqueous-ethanolic extract. The aqueous fractions from this procedure exhibited no inhibitory activity on the tested bacteria (Table 1). Several fractions from the silica gel column showed antibacterial activity and those eluted with CH2Cl2/ EtOAc (1:1) and EtOAc were the most active (Table 2). The less polar fractions from this column (hexane and hexane/CH2Cl2) had no effect on the assayed strains. A. actinomycetemcomitans FDC Y4 and ATCC 29 523, F. nucleatum and E. lentum were the species least susceptible to the fractions from silica gel column (MIC values range from 1024 to 1024 mg/ml). Meropenem and penicillin G were employed as positive controls and showed MIC values ranging from 0.03 to 8 and 0.03 to 4 mg/ml, respectively. In comparison to these values, the minimum inhibitory concentrations of propolis aqueous-ethanolic extract and its fractions appear to be not significant. It has to be stressed, however, that the propolis antibacterial activity is very significant, since many of the assayed bacteria present resistance against antibiotics in clinical use (Walker, 1996). Propolis antibacterial activity has been attributed to phenolic compounds, especially flavonoids, phenolic acids and their esters (Ghisalberti, 1979). 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. In order to characterize the chemical profile, the bioactive fractions of propolis were analyzed by TLC and HPLC. The presence of phenolic compounds in the active fractions was evidenced by the intense fluorescence produced under UV360 light after spraying the TLC plates with aluminum chloride solution. All the active fractions showed strong fluorescent spots of a

wide range of polarity (Fig. 2). Quercetin and kaempferol were used as reference compounds. The HPLC profile of propolis aqueous-ethanolic extract indicates its complex composition with several peaks of varied retention times (Fig. 3). The chromatograms obtained for the active fractions did not show similar patterns of composition. All of them, however, contained a great number of substances (Fig. 3). In the present work we demonstrated the antibacterial activity of propolis and its fractions against several oral anaerobes, including A. actinomycetemcomitans , F. nucleatum , P. gingivalis and P. intermedia , species frequently associated with destructive periodontitis (Slots, 1982; Kaplan et al., 1989; Dahlen, 1993; Rodri gues et al., 1999). Since these bacteria may be resistant to several antibiotics, the antimicrobial activity reported here is of relevance and propolis may constitute, in the future, an alternative for treating these pathogens.

Acknowledgements The authors would like to thank Gilvania Ferreira S. Santos for technical help. This study was supported by grants from CNPq and FAPEMIG.

References
Aga, H., Shibuya, T., Sugimoto, T., Kurimoto, M., Nakajima, S.H. 1994. Isolation and identication of antimicrobial compounds in Brazilian propolis. Bioscience, Biotechnology and Biochemistry 58, 945 /946. Amoros, M., Sauvager, F., Girre, L., Cormier, M. 1992. In vitro antiviral activity of propolis. Apidologie 23, 231 /240. Asis, M., 1991. El Oro purpura de las Abejas. Centro de Informacion y Documentacion Agropecuario de Cuba. Bankova, V., Dyulgerov, A., Popov, S., Evstatieva, L., Kuleva, L., Pureb, O., Zamjansan, Z. 1992. Propolis produced in Bulgaria and Mongolia: phenolic compounds and plant origin. Apidologie 23, 79 /85. Bankova, V., Christov, R., Kujumgiev, A., Marcucci, M.C., Popov, S. 1995. Chemical composition and antibacterial activity of Brazilian propolis. Zeischrift fur Naturforschung 50c, 167 /172. Bankova, V., Marcucci, M.C., Simonova, S., Nikolova, N., Kujumgiev, A. 1996. Antibacterial Diterpenic acids from Brazilian propolis. Zeischrift fur Naturforschung 51c, 277 /280. Bankova, V., Castro, S.L., Marcucci, M.C. 2000. Propolis: recent advances in chemistry and plant origin. Apidologie 31, 3 /15. Bolstad, A.I., Jensen, H.B., Bakken, V. 1996. Taxonomy, biology, and periodontal aspects of Fusobacterium nucleatum . Clinical Microbiology Reviews 9, 55 /71. Cheng, P.C., Wong, G. 1996. Honey bee propolis: prospects in medicine. Bee World 77, 8 /15. Dahlen, G.G. 1993. Black-pigmented Gram-negative anaerobes in periodontitis. FEMS Immunology and Medical Microbiology 6, 181 /192. Dimov, V., Ivanovska, N., Bankova, V., Nikolov, N., Popov, S. 1992. Immunomodulatory action of propolis: IV. Prophylatic activity against Gram-negative infections and adjuvant effect of water soluble derivative. Vaccine 10, 817 /823.

F.A. Santos et al. / Journal of Ethnopharmacology 80 (2002) 1 /7 Dobrowolski, J.W., Vohora, S.B., Sharma, K., Shah, S.A., Naqvi, S.A.H., Dandiya, P.C. 1991. Antibacterial, antifungal, antiamoebic, anti inammatory and antipyretic studies on propoli bee products. Journal of Ethnopharmacology 35, 77 /82. Grange, J.M., Darvey, R.W. 1990. Antibacterial properties of propolis (bee glue). Journal of Royal Society of Medicine 83, 159 /160. Ghisalberti, E.L. 1979. Propolis: a review. Bee World 60, 59 /84. Grunberger, D., Banerjee, R., Eisinger, K., Oltz, E.M., Efros, L., Caldwell, M., Esteves, V., Nakanishi, K. 1988. Preferencial cytotoxicity on tumor cells by caffeic acid phenethyl esther isolated from propolis. Experimentia 44, 230 /232. Ikeno, K., Ikeno, T., Miyazawa, C. 1991. Effects of propolis on dental caries in rats. Caries Research 25, 347 /351. Kaplan, A.H., Weber, D.J., Oddone, E.Z., Refect, J.R. 1989. Infection due to Actnobacillus actinomycetemcomitans : 15 cases and review. Review of Infectious Diseases 11, 46 /63. Kujumgiev, A., Bankova, V., Ignatova, A., Popov, S. 1993. Antibacterial activity of propolis, some of its components and analogs. Pharmazie 48, 785 /786. Kujungiev, A., Tsvetkova, I., Serkedjieva, Y., Bankova, V., Christov, R., Popov, S. 1999. Antibacterial, antifungal and antiviral activity of propolis of different geographic origin. Journal of Etnopharmacology 64, 235 /240. Marcucci, M.C. 1995. Propolis: chemical composition, biological properties and therapeutic activity. Apidologie 26, 83 /99. Menezes, H., Bacci, M., Jr, Oliveira, S.D., Pagnocca, F.C. 1997. Antibacterial properties of propolis and products containing propolis from Brazil. Apidologie 28, 71 /76. National Committee for Clinical Laboratory Standards, 1997. Methods for antimicrobial susceptibility testing of anaerobic bacteria, fourth ed. Approved Standard M11-A4. National Commitee for Clinical Laboratory Standards, Wayn, PA. Oliveira, V.C., Bastos, E.M. 1998. Morphological and anatomical aspects of the leaf of Baccharis dracunculifolia DC. (Asteraceae) as regards to the identication of the botanical origin of propolis. Acta Botanica Brasilica 12, 431 /439 (In Portuguese).

Park, Y.K., Koo, M.H., Abreu, J.A.S., Ikegaki, M., Cury, J.A., Rosalen, P.L. 1998. Antimicrobial activity of propolis on oral microorganisms. Current Microbiology 36, 2428. Rodrigues, P.H., Carvalho, S.A., Costa, J.E., Carvalho, M.A.R., Farias, L.M., Petrillo-Peixoto, M.L. 1999. Black-pigmented Gramnegative anaerobes in Brazilian adults with peridontal disease. Anaerobe 5, 267 /268. Santos, F.A., Bastos, E.M.A.F., Uzeda, M., Carvalho, M.A.R., Farias, L.M., Moreira, E.S.A. 1999. Antibacterial activity of propolis produced in Brazil against Actinobacillus actinomycetemcomitans , Fuscbacterium spp. and bacteria from Bacteroides Fragilis group isolated from human and marmoset hosts. Anaerobe 5, 479 /481. Scheller, S., Szaarski, J., Tustanowski, J., Nolewajka, E., Stojko, A., 1977. Biological properities and clinical application of propolis I. Arzeneimittel-Forschung Drug Research 27, 889 /890. Slots, J., 1982. Selective medium for Actinobacillus actinomycetemcomitans . Journal of Clinical Microbiology 15, 606 /609. Takaisi-Kikuni, N.B., Schilcher, H. 1994. Electron microscopic and microcalorimetric investigations of the possible mechanism of the antibacterial action of a dened propolis provenance. Planta Medica 60, 222 /227. Tanner, A.C.R., Haffer, C., Bratthail, G.T., Visconti, R.A., Socransky, S.S. 1979. A study of bacteria associated with advancing periodonitis in man. Journal of Clinical Microbiology 6, 278 /307. Vanhaelen, M., Vanhaelen-Fastre, R. 1979. Propolis */Origine, Mi crographie, Composition Chimique et Activite therapeutique. Journal of Pharmacie Belgique 34, 253 /259. Valdes, V., Rojas, N.M., Morales, C., 1987. Ensayo preliminar de la accion antifungica de extractos de propoleo sobre Candida albicans . Ciencia e Technologia Agricola Apicultura 3, 41 /49. Wagner, H., Bladt, S., Zgainsky, E.M. 1984. Plant Drug Analysis: A Thin Layer Chromatography Atlas. Springer, Berlin, p. 320. Walker, C.B. 1996. The acquisition of antibiotic resistance in the periodontal microora. Periodontology 2000 10, 79 /88.