Journal of Ethnopharmacology 102 (2005) 371376

The effect of bee propolis on oral pathogens and human gingival broblasts
Sule Sonmez a, , Levent Kirilmaz b , Mine Yucesoy c , Banu Y ucel d , Berna Yilmaz e
c a Ege University, School of Dentistry, Department of Periodontology, Bornova-35100, Izmir, Turkey Ege University, School of Pharmacy, Department of Biopharmaceutics Pharmatokinetics, Bornova-35100, Izmir, Turkey Dokuz Eyl ul University, School of Medicine, Department of Microbiology and Clinical Microbiology, Inciralti-35340, Izmir, Turkey d Ege University, School of Agriculture, Department of Zootechnics, Bornova-35100, Izmir, Turkey e Ege University, School of Medicine, Department of Medical Biology, Bornova-35100, Izmir, Turkey b

Received 5 November 2004; received in revised form 1 June 2005; accepted 20 June 2005 Available online 3 August 2005

Abstract Propolis is one of the few natural remedies that have maintained its popularity over a long period of time. The aim of this study is to investigate the antimicrobial properties of six propolis solutions and evaluate their cytotoxicity on gingival broblasts at different dilutions. Two different solutions of powder propolis (Sigma) and Turkish propolis were prepared and propylene glycol (PG) and alcohol were used as solvents for each propolis sample. In addition to the four propolis solutions, two other propolis samples of far geographic regions (USA and Australia) were included in the study. The antibacterial effects of six solutions on oral pathogen microorganisms were tested and their cytotoxic effects on human gingival broblasts were evaluated by MTT assay. The effective dilutions of the six propolis samples on periodontopathogen microorganisms were found to be cytotoxic to gingival broblasts. All solutions had strong antifungal activity and the effective dilutions were safe for gingival broblasts. Propolis could have a promising role in the future medicine, if appropriate solutions can be prepared being strongly antibacterial and non-cytotoxic as well. 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Propolis; Antibacterial activity; Fibroblast culture; Cytotoxicity; MTT

1. Introduction Natural products have been used for thousands of years in folk medicine for several purposes. Among them, propolis has attracted increased interest due to its antimicrobial activity against a wide range of pathogenic microorganisms. Propolis, sometimes also referred to bee glue, is the generic name for the resinous substance collected by honeybees (Apis mellifera) from various plant sources (Burdock, 1998; Kujumgiev et al., 1999; Almas et al., 2001). The word propolis is derived from the Greek pro-, for or in defense, and -polis, the city, referring to the defense of the city or the hive (Santos et al., 2002). It is a strongly adhesive, resinous substance col

Corresponding author. Tel.: +90 232 3881105; fax: +90 232 3880325. E-mail address: sule@bornova.ege.edu.tr (S. Sonmez).

lected, transformed and used by bees to seal holes in their honeycombs, smooth out the internal walls and protect the entrance against intruders (Molan, 2001). Honeybees collect the resin from cracks in the bark of trees and leaf buds. This resin is masticated, salivary enzymes are added and the partially digested material is mixed with bee wax and used in the hive. The precise composition of raw propolis varies with the source. In general, it is composed of resin (50%), wax and essential-aromatic oils (30%) mixed with the salivary secretion of bees (10%), pollen (5%) and various other substances (5%) including amino acids, minerals, ethanol, Vitamins A, B complex, E and the highly active mixture of compounds known as bioavonoids (Marucci, 1995). Advances in understanding the etiology and pathogenesis of periodontitis have led to increasingly effective pharmacological interventions. In this regard, various medications

0378-8741/$ see front matter 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.06.035

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are delivered into periodontal pockets to suppress or eradicate the pathogenic microbiota or modulate the inammatory response, thereby limiting tissue destruction (Bollen and Quirynen, 1996; Greenstein and Polson, 1998). Current researches reveal that propolis, a natural and old remedy, seems to be promising in suppressing the oral pathogenic microorganisms. Antibacterial activity (Kujumgiev et al., 1999; Koo et al., 2000a,b; Tichy and Novak, 2000; Sorkun et al., 2001; Borelli et al., 2002; Castaldo and Francesco, 2002; Kartal et al., 2003; Stepanovic et al., 2003), antiviral activity (Huleihel and Isanu, 2002; Yildirim et al., 2004), antifungal activity (Ota et al., 2001; Sawaya et al., 2002) of propolis and its effects on the microorganisms responsible of caries (Koo et al., 2000a,b, 2002) have been reported in various studies. In a comprehensive study on propolis of different geographic origin, Kujumgiev et al. (1999) have investigated the antibacterial activity on Escherichia coli, antifungal activity on Candida albicans and antiviral activity on Avian inuenza and found out that all samples were active against the fungal and gram positive bacterial test strains, and most showed antiviral activity. Santos et al. (2002) investigated the inhibitory activity of Brazilian propolis on Aa, Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia and found out that all of the assayed bacterium species were susceptible to propolis extract. Koo et al. (2000a,b) compared the antimicrobial effects of Arnica montana, a perennial herbaceous plant, to propolis extract on 15 oral pathogen microorganism and stated that propolis extracts showed in vitro antibacterial activity and inhibition of cell adherence while Arnica extract was only slight active. Taking into consideration of the results of the aforementioned studies, the present study was designed to compare the antimicrobial effects of Turkish propolis, powder propolis from Sigma and two other propolis samples of far geographic regions (USA and Australia) on oral pathogen microorganisms and their cytotoxic effects on human gingival broblasts.

to 10 ml with 70% ethyl alcohol or 10 ml PG. Four propolis solutions prepared at different concentrations can be summarized as: (1) 10% alcoholic propolis (Sigma), (2) 10% propylene glycol propolis (Sigma), (3) 10% alcoholic Turkish propolis and (4) 10% propylene glycol Turkish propolis. Besides the prepared propolis tinctures, two other propolis samples from far geographical regions were also included in this study: (5) Australian propolis: non-alcoholic 30% liquid propolis containing 300 mg/ml bee propolis extract in water soluble base (propolis 30% liquid, non-alcoholic, Natures Goodness, Narellean, Australia) and (6) USA propolis: nonalcoholic 20% propolis containing 150 mg/ml bee propolis extract in water soluble base (20% propolis extract, GloryBee Foods, Inc., OR, USA). 2.2. Bacterial strains and susceptibility testing Porphyromonas gingivalis of the National Collection of Type Cultures (London, UK) (NCTC) 11834-5A, Prevotella intermedia NCTC 13070-02, Campylobacter rectus NCTC 11489-05 and Fusobacterium nucleatum NCTC 11326-04 were included in the present study as anaerobic bacteria. The effect of the solutions in 1/1281/2048 dilutions against anaerobic bacteria were determined by agar dilution method using Brucella agar (Oxoid) supplemented with Vitamin K, haemin and lysed blood, which has been accepted as a reference method for investigating susceptibility of anaerobic bacteria (National Committee For Clinical Laboratory Standards (NCCLS), 2001). Lyophilized bacteria were cultured on anaerobic blood agar, thioglycolate broth and cooked meat broth. Pure cultures were obtained after microscopic and macroscopic investigation of the colonies of the bacteria. After subculturing to thioglycolate broth, the isolates were incubated under anaerobic conditions (using anaerobic jar and gas pack system (Oxoid)) for 48 h in order to investigate susceptibilities. After 48 h, the turbidity of bacterial suspensions were adjusted to 0.5 McFarland turbidity standard and were diluted by 1/10 ratio. Ten microlitres from each bacterial suspension were inoculated onto Brucella agar plates containing 1/1281/2048 dilutions of solutions, which were prepared on the same day. Agar plates without solutions were also prepared and used as growth controls. All isolates and solution combinations were studied twice. The results were read after the incubation of plates under anaerobic conditions at 37 C for 48 h. The concentration which causes a marked decrease (prominent inhibition of growth or no growth) compared to growth control was determined as minimal inhibitory concentration (MIC) value by following the endpoint denition of National Committee for Clinical Laboratory Standards (NCCLS, 2001). 2.3. Fungal strains and susceptibility testing In this study, Candida albicans ATCC 90028, Candida parapsilosis ATCC 90018 and Candida krusei ATCC 6258 isolates were used (National Committee For Clinical Lab-

2. Methodology 2.1. Propolis samples Six different concentrations of propolis solutions from four different origins were used in this study: (1) Turkish propolis: pine originated (Pinus spp.) propolis made by honey bees (Apis mellifera) was collected from Kemalpasa district of Izmir province (west of Anatolia) in November, 2003 and were kept desiccated pending processing. (2) Powder propolis (Sigma, Germany) (P-8904). Four tinctures were prepared with the rst two propolis groups at 10% concentration and 70% ethyl alcohol and propylene glycol (PG) was used as a solvent, respectively. One gram of propolis was weighed and added to 9 ml of 70% ethyl alcohol or 9 ml of (PG) and kept at a cool and dark place for 1 week by shaking occasionally. The solution was ltered using a quantitative lter paper (Schleicher & Sch ull, Dassel, Germany) and then adjusted

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oratory Standards, 2002). The activities of the solutions in 1/641/8000 dilutions against Candida isolates were investigated with reference microdilution method following the NCCLS M27-A2 standards by using RPMI 1640 medium (Sigma) buffered with 0.165 M morpholinepropanesulfonic acid (MOPS) with l-glutamine and phenol red and without sodium bicarbonate (2002). Candida suspensions were prepared from the colonies of the strains which had grown on Saboraud-dextrose agar after 24 h of incubation. Yeast suspensions were inoculated into microplate wells which contained 1/641/8000 dilutions of solutions. Microplates were evaluated after incubation at 37 C for 48 h. The concentration which totally inhibited the growth of yeasts was taken as MIC value. 2.4. Gingival broblast cell culture Human gingival tissue samples were obtained during maxillary premolar extraction for orthodontic reasons from 10 healthy donors (1218 years old), with their consent or their parents, in accordance with the declaration of Helsinki. Tissue sample was washed in PBS, minced and incubated overnight at 37 C in 5 ml of 253 U/ml Collagenase type CLSII (Biochrom, Germany). Cells were spun at 225 G for 5 min, washed with PBS and cultured. Gingival broblasts were maintained using DMEM (Sigma, UK) with 10% FCS (Biochrom), supplemented with antibiotics (100 U/ml penicillin and 100 g/ml streptomycin) (Biochrom) and 200 mM l-glutamine (Biochrom). The cells were grown as monolayer cultures in 25 TC-asks (Greiner, Germany) and incubated at 37 C in a humidied atmosphere of 5% CO2 and 95% air. The media were replaced every 2 or 3 day until conuence was reached. For subcultivation, cells were detached from the culture asks with 0.25% Trypsin/EDTA Solution (Sigma, UK) for 35 min. Cell viability assays were performed using third to fth-passage of gingival broblasts. Cell morphology was visualized with phase contrast microscopy (Olympus, Japan). 2.5. Cellular viability assay (MTT assay) Conuent gingival broblast cell monolayers in 96-well plastic plates were incubated with various concentrations (1/641/8000) of six different propolis samples in growth medium and were observed microscopically for morpholologic changes. Cell survival was analyzed by using a nonradioactive cell proliferation assay system (MTT assay) consisting of 3-(4,5-dimethylthiaziazol-2-yl) 2,5-diphenyl tetrazolium bromide. The cells were plated at 2 105 cell/ml per well onto 96-well plates in concentrations of six different propolis samples [1/641/8000] were added with serum-free DMEM in a total volume of 100 l/well. After further culture for 24 h at 37 C in a humidied atmosphere of 5% CO2 in air, broblast growth-induction activity was determined by the MTT solution (5 mg/ml) was added to each well of the plate and plates were incubated for 4 h at 37 C in a humidied

atmosphere of 5% CO2 in air. The medium in each well was changed to 1000 l dimethylsulfoxide (DMSO) and mixed thoroughly to dissolve the dark blue crystals. After 10 min at room temperature to ensure that all crystals were dissolved. The plates were read with ELISA reader (EL X 808), using test wavelength of 570 nm and a reference wavelength of 630 nm. In the present study, MTT assay, which presents the metabolic and mitochondrial activity of treated cells, was chosen to measure the cell viability of gingival broblast culture treated with propolis solutions. The reduction of tetrazolium salts by active cells is widely accepted as a reliable way to examine cell viability. In the present study, however, the dark brown color of propolis solutions interfered with the intracellular purple formazan crystals and impeded the spectrophotometric quantication of viable cells. For this reason morphological screening was preferred instead.

3. Results The aim of the present study was to nd out the safe concentration of propolis which is effective against oral pathogen microorganisms and non-cytotoxic to gingival broblasts as well. Fig. 1a shows the intercellular purple formazan of viable gingival broblasts before the application of propolis solutions. MTT assay had demonstrated that the 1/21/128 dilutions of all propolis samples were cytotoxic for gingival broblasts. 1/256 dilutions of alcoholic propolis (Sigma) (Fig. 1b), PG-propolis (Sigma) (Fig. 1c), alcoholic Turkish propolis (Fig. 1d) and PG-Turkish propolis (Fig. 1e) were safe for gingival broblasts, where 30% non-alcoholic-AU propolis (propolis 30% liquid, non-alcoholic, Natures Goodness) and 20% non-alcoholic-USA propolis (20% propolis extract, GloryBee Foods, Inc.) had to be diluted to 1/1024 in order to see viable cells in the broblast tissue culture (Fig. 1f and g, respectively). Dilutions of solutions (MIC values) that inhibited the growth of anaerobic bacteria are presented in Table 1. 1/128 dilutions of 10% alcoholic propolis (Sigma), 10% PG-propolis (Sigma), 10% alcoholic Turkish propolis, 10% PG-Turkish propolis and 1/128 to 1/256 dilutions of 30% nonalcoholic-AU propolis and 20% non-alcoholic-USA propolis were found to be effective in inhibiting periodontopathogens, but the mentioned concentrations of these propolis solutions were cytotoxic to gingival broblasts. MIC values of six different solutions for Candida species can be observed in Table 2. 1/256 to 1/512 dilutions of 10% alcoholic propolis (Sigma), 10% PG-propolis (Sigma), 10% alcoholic Turkish propolis, 10% PG-Turkish propolis and 1/1024 to 1/2048 dilutions of 30% non-alcoholic-AU propolis and 20% non-alcoholic-USA propolis inhibited all the Candida species and the mentioned concentrations of the propolis solutions were found to be safe for gingival broblasts.

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Fig. 1. Crystal formazan formation in viable gingival broblasts (V), dead gingival broblasts (D) (scale bar=50 m) (200 magnication). (a) Viable gingival broblasts before the application of propolis solutions. (b) Viable gingival broblasts after 10% alcoholic propolis (Sigma) application (1/256 dilution). (c) Viable gingival broblasts after 10% PG-propolis (Sigma) application (1/256 dilution). (d) Viable gingival broblasts after 10% alcoholic Turkish propolis application (1/256 dilution). (e) Viable gingival broblasts after 10% PG-Turkish propolis application (1/256 dilution). (f) Viable gingival broblasts after 30% non-alcoholic-AU propolis application (1/1024 dilution). (g) Viable gingival broblasts after 20% non-alcoholic-USA propolis application (1/1024 dilution).

S. Sonmez et al. / Journal of Ethnopharmacology 102 (2005) 371376 Table 1 Dilutions of solutions which inhibited the growth of anaerobic bacteria Propolis solutions 10% Alcoholic (Sigma) 10% PG (Sigma) 10% Alcoholic (Turkish) 10% PG-Turkish 30% Non-alcoholic (Australian)a 20% Non-alcoholic (USA-P)b
a b

375

Porphyromonas gingivalis >1/128 >1/128 >1/128 >1/128 1/128 1/256

Prevotella intermedia >1/128 >1/128 >1/128 >1/128 1/128 1/256

Campylobacter rectus >1/128 >1/128 1/128 1/128 1/256 1/256

Fusobacterium nucleatum >1/128 >1/128 >1/128 >1/128 1/128 1/128

Propolis 30% liquid, non-alcoholic, Natures Goodness, Narellean, Australia. 20% propolis extract, GloryBee Foods, Inc., OR, USA.

Table 2 MIC values of solutions for Candida species Propolis Solutions 10% Alcoholic (Sigma) 10% PG (Sigma) 10% Alcoholic (Turkish) 10% PG-Turkish 30% Non-alcoholic (Australian)a 20% Non-alcoholic (USA-P)b
a

Candida albicans 1/512 1/256 1/512 1/256 1/1024 1/2048

Candida parapsilosis 1/512 1/512 1/512 1/512 1/1024 1/2048

Candida krusei 1/256 1/256 1/256 1/256 1/1024 1/1024

Propolis 30% liquid, non-alcoholic, Natures Goodness, Narellean, Australia. b 20% propolis extract, GloryBee Foods, Inc., OR, USA.

4. Discussion and conclusion In spite of the chemical composition of propolis from different geographical locations, all samples exhibit signicant antibacterial and antifungal activity. Kujumgiev et al. (1999) tested the antibacterial activity of 11 different propolis samples from different geographical regions and found out that all samples were active against gram-positive bacterial test strains. In the present study, 30% non-alcoholic Australian propolis and 20% non-alcoholic USA propolis were effective in inhibiting the periodontopathogen microorganisms but the same concentrations were cytotoxic to gingival broblasts. Ten percent concentrations of raw Turkish propolis and Sigma propolis dissolved in 70% ethyl alcohol and propylene glycol were not as effective as Australian and USA propolis extracts but were safe for gingival broblasts. In preparing the Turkish propolis and Sigma propolis solutions, it is preferred to keep the concentration at minimum limits to nd out the safe and effective lowest concentrations, which will not disturb gingival broblasts. However, higher concentrations of these solutions might be effective against these microorganisms. The results of the study of Sforcin et al. (2000), who investigated the seasonal effect on Brazilian antibacterial activity, showed that there was a slight difference between the antibacterial activities of the propolis extracts which is not statistically signicant. The authors stated that the slight difference may be due to the chemical composition varying between the samples collected in close but different regions, mainly due to the local ora of the bees. If the slight seasonal effect on the antibacterial activity of propolis solutions is not taken into consideration, as stated

by Sforcin et al., the regional composition but especially the low concentrations might be the reason why the prepared propolis solutions of the present study did not exhibit the same antibacterial effect as the Australian and USA propolis solutions. On the other hand, Bankova et al. (2000), Bosio et al. (2000) and Hegazi et al. (2000) declare that the chemical composition of propolis is very complex and depends on the ora in the area where it was collected. Therefore, variations seen in the antimicrobial activity of the propolis solutions used in this study, which are from different origins, are not surprising. All of the propolis solutions used in the present study were effective at low concentrations in inhibiting Candida albicans, Candida parapsilosis and Candida krusei and the effective dilutions are not cytotoxic to gingival broblasts. These results are in parallel with the studies testing the antifungal activity of various propolis solutions (Kujumgiev et al., 1999; Ota et al., 2001; Sawaya et al., 2002). It can be concluded that propolis is a natural and a strong antifungal substance. The results of the present study showed that 30% nonalcoholic-AU propolis and 20% non-alcoholic-USA propolis presented the best antibacterial activity on periodontal pathogens at 1/128 dilutions, but were found to be cytotoxic on gingival broblasts. 10% alcoholic propolis (Sigma), 10% PG-propolis (Sigma), 10% alcoholic Turkish propolis and 10% PG-Turkish propolis, which were prepared to use in this study, did not presented the same antibacterial activity on periodontal pathogens at 1/256 dilutions as the two foreign propolis solutions, but let the gingival broblasts stay alive. It is suggested that more trials are needed to reach the appropriate propolis solutions, which are less cytotoxic but present strong antibacterial effects. All the researches mentioned have shown clearly the potential antibacterial and antifungal activities of propolis on oral pathogen microorganisms. Current opinion is that the use of standardized preparations of propolis is safe and less toxic than many other synthetic medicines. Since the anaerobic microora associated with aggressive periodontitis may be resistant to several antibiotics, the reported antimicrobial activity is of relevance. In the future, propolis may constitute an alternative for treating these pathogens if less cytotoxic but strongly antibacterial concentrations can be found.

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S. Sonmez et al. / Journal of Ethnopharmacology 102 (2005) 371376 Koo, H., Cury, J.A., Rosalen, P.L., Ambrosano, G.M.B., Ikegaki, M., Park, Y.K., 2002. Effect of a mouthrinse containing selected propolis on 3-day dental plaque accumulation and polysaccharide formation. Caries Research 36, 445448. Kujumgiev, A., Tsvetkova, I., Serkedjeva, Y., Bankova, V., Christov, R., Popov, S., 1999. Antibacterial, antifungal and antiviral activity od propolis of different geographic origin. Journal of Ethnopharmacology 64, 235240. Marucci, M.C., 1995. Propolis: chemical composition, biological properties and therapeutic activity. Apidologie 26, 8399. Molan, P., 2001. Why honey is effective as a medicine. Bee World 82, 2240. National Committee For Clinical Laboratory Standards, 2001. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria: Approved Standar, fth ed. M11-A5. National Committee for Clinical Laboratory Standards, PA, USA. National Committee For Clinical Laboratory Standards, 2002. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard, second ed. NCCLS document M27-A2, vol. 22, no. 15. National Committee for Clinical Laboratory Standards, Wayne, PA, USA. Ota, C., Unterkircher, C., Fantinato, V., Shimizu, M.T., 2001. Antifungal activity of propolis on different species of Candida. Mycoses 44, 375378. Santos, F.A., Bastos, E.M.A., Uzeda, M.A.R., Carvalho, M.A., Farias, L.M., Moriera, E.S., Braga, F.C., 2002. Antibacterial activity of Brazilian propolis and fractions against oral anaerobic bacteria. Journal of Etnopharmacology 80, 17. Sawaya, A.C., Palma, A.M., Caetano, F.M., Marcucci, M.C., de Silva Cunha, I.B., Araujo, C.E., Shimizu, M.T., 2002. Comparative study of in vitro methods used to analyze the activity of propolis extracts with different compositions against species of Candida. Letters in Applied Microbiology 35, 203207. Sforcin, J.M., Fernandes, A., Lopes, C.A.M., Bankova, V., Funari, A., 2000. Seasonal effect of Brazilian propolis antibacterial activity. Journal of Ethnopharmacology 73, 243249. Sorkun, K., Suer, B., Salih, B., 2001. Determination of chemical composition of Turkish propolis. Zeitschrift f ur Naturforschung 56, 666668. Stepanovic, S., Antic, N., Dakic, I., Svabic-Vlahovic, M., 2003. In vitro antimicrobial activity of propolis and synergism between propolis and antimicrobial drugs. Microbiological Research 158, 353357. Tichy, J., Novak, J., 2000. Detection of antimicrobials in bee products with activity against viridans streptococci. Journal of Alternative Complementary Medicine 6, 383389. Yildirim, Z., Hacievliyagil, S., Kutlu, N.O., Aydin, N.E., Kurkcuoglu, M., Iraz, M., Durmaz, R., 2004. Effect of water extract of Turkish propolis on tuberculosis infection in guinea-pigs. Pharmacological Research 49, 287292.

Acknowledgement This research is supported by Ege University Research Foundation. References

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