Effects of Compounds Found in Propolis on Streptococcus mutans Growth and on Glucosyltransferase Activity

Propolis, a resinous bee product, has been shown to inhibitthe growth of oral microorganisms and the activity of bacterium-derivedglucosyltransferases (GTFs). Several compounds, mainly polyphenolics,have been identified in this natural product. The present studyevaluated the effects of distinct chemical groups found in propolison the activity of GTF enzymes in solution and on the surfaceof saliva-coated hydroxyapatite (sHA) beads. Thirty compounds,including flavonoids, cinnamic acid derivatives, and terpenoids,were tested for the ability to inhibit GTFs B, C, and D fromStreptococcus mutans and GTF from S. sanguinis (GTF Ss). Flavonesand flavonols were potent inhibitors of GTF activity in solution;lesser effects were noted on insolubilized enzymes. Apigenin,a 4',5,7-trihydroxyflavone, was the most effective inhibitorof GTFs, both in solution (90.5 to 95% inhibition at a concentrationof 135 µg/ml) and on the surface of sHA beads (30 to 60%at 135 µg/ml). Antibacterial activity was determined byusing MICs, minimum bactericidal concentrations (MBCs), andtime-kill studies. Flavanones and some dihydroflavonols, aswell as the sesquiterpene tt-farnesol, inhibited the growthof S. mutans and S. sobrinus; tt-farnesol was the most effectiveantibacterial compound (MICs of 14 to 28 µg/ml and MBCsof 56 to 112 µg/ml). tt-Farnesol (56 to 112 µg/ml)produced a 3-log-fold reduction in the bacterial populationafter 4 h of incubation. Cinnamic acid derivatives had negligiblebiological activities. Several of the compounds identified inpropolis inhibit GTF activities and bacterial growth. Apigeninis a novel and potent inhibitor of GTF activity, and tt-farnesolwas found to be an effective antibacterial agent.

INTRODUCTION

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Introduction

Glucosyltransferase (GTF; EC 2.4.1.5) enzymes produced by Streptococcusmutans have been recognized as virulence factors in the pathogenesisof dental caries (13, 45, 54). GTF enzymes catalyze the formationof soluble and insoluble ${alpha}$ -linked glucans from sucrose and contributesignificantly to the dental plaque matrix polysaccharide composition(41). Dental plaque is essentially a biofilm. Glucans promotethe adherence and accumulation of cariogenic streptococci onthe tooth surface and play an essential role in the developmentof pathogenic dental plaque related to caries-forming activity(19, 44, 54). S. mutans produces at least three GTFs: GTF B,which synthesizes a polymer of mostly insoluble ${alpha}$ 1,3-linked glucan;GTF C, which synthesizes a mixture of insoluble ${alpha}$ 1,3-linked glucanand soluble ${alpha}$ 1,6-linked glucan; and GTF D, which synthesizes ${alpha}$ 1,6-linked soluble glucan (2, 20, 21). An additional GTF enzymefrom S. sanguinis (GTF Ss) may also be involved in the developmentof dental plaque (39, 50). S. sanguinis colonizes the toothsurface early in plaque formation, and its GTF catalyzes predominantly ${alpha}$ 1,6-linked soluble glucan (9). Enzymatically active GTFs arepresent in the soluble fraction of whole human saliva and arealso incorporated into the salivary pellicle that is formedon the tooth surface (41, 42). Furthermore, the GTFs incorporatedinto an experimental pellicle demonstrate distinct physicaland kinetic properties compared to the same enzymes in solution;GTFs C and D express enhanced enzymatic activity (43, 47, 51).A large proportion of the glucans synthesized by these surface-adsorbedGTFs is retained on the pellicle and may provide binding sitesfor S. mutans, contributing to the in situ formation of dentalplaque (43, 44, 49). Therefore, inhibition of GTFs both in solutionand adsorbed to the pellicle of tooth surface is one of thestrategies by which to prevent dental caries and other plaque-relateddiseases.

Propolis, a resinous substance collected by Apis mellifera beesfrom various plant sources and mixed with secreted beeswax,is a multifunctional material used by bees in the construction,maintenance, and protection of their hives (8, 17). Propolisis a nontoxic natural product with multiple pharmacologicaleffects and a complex chemical composition (8, 17). Severalcompounds have been identified in propolis, and three distinctchemical groups have been reported to be present: (i) flavonoidaglycones, (ii) cinnamic acid derivatives, and (iii) terpenoids(3, 4, 40, 46). Flavonoids have been considered the main biologicallyactive compounds in propolis (1, 6, 17). Propolis exhibits awide range of biological activities, including antimicrobial,antiinflammatory, anesthetic, and cytostatic properties (8,17). Previously, we have demonstrated that two chemically distincttypes of propolis from Brazil inhibited the activity of GTFsand the growth of S. mutans in vitro (28, 29, 30). Furthermore,topical application of propolis twice daily (27) or its inclusionin drinking water available ad libitum (25) reduced the incidenceof dental caries in rats. Nevertheless, information on the biologicalproperties of specific compounds, which could be useful forprevention of oral diseases, is sparse. Because propolis hasbeen shown to reduce the incidence of dental caries in rats,the purpose of the present study was to explore the effectsof several compounds identified in propolis on the activityof individual, purified GTF enzymes and on the growth of S.mutans.

MATERIALS AND METHODS

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Materials and Methods

Test compounds. The compounds used in this study are classified into three groups:(i) flavonoids (flavonols, flavones, flavanones, and dihydroflavonols),(ii) cinnamic acid derivatives, and (iii) terpenoids. The flavonols(kaempferide, kaempferol, galangin, isorhamnetin, rhamnetin,myricetin, fisetin, and rutin), flavones (apigenin, acacetin,baicalein, chrysin, luteolin, and tectochrysin), flavanones(pinocembrin, sakuranetin, and isosakuranetin), cinnamic acidderivatives (ferulic acid, p-coumaric acid, and caffeic acid),and terpenoids (tt-farnesol, ß-caryophyllene, terpineol,and syringaldehyde) were all obtained from Extrasynthese Co.,Genay, France. Protocatechuic acid, vanillin, and benzoic acidwere obtained from Sigma-Aldrich Co. The dihydroflavonols, pinobanksin,pinobanksin-7-methyl ether, and pinobanksin-3-acetate, werekindly provided by E. Wollenweber (Darmstadt, Germany). Allof the chemical compounds were dissolved in dimethyl sulfoxide(DMSO)-ethanol (1:4, vol/vol) or ethanol (99.9%; high-performanceliquid chromatography grade) just prior to performance of theassays. Appropriate solvent controls were always included. Table1 summarizes all of the test compounds used in this study andtheir chemical structures.

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TABLE 1. Chemical structures of compounds used in this study

Bacterial strains. The bacterial strains used for the production of GTFs were (i)Streptococcus milleri KSB8, which harbors the gtfB gene fromS. mutans GS-5 (for GTF B production); (ii) S. milleri NH5,which contains the gtfD gene S. mutans GS-5 (for GTF D); (iii)S. mutans WHB410 (53), in which the genes for GTFs B and D andfructosyltransferase were deleted (for GTF C); and (iv) S. sanguinis10904. For susceptibility and time-kill assays, the bacterialstrains used were S. mutans GS-5, S. mutans UA159, and S. sobrinus6715. The cultures were stored at -80°C in brain heart infusionor tryptic soy broth (TSB) containing 20% glycerol. The S. millericonstructs were a kind gift from Howard K. Kuramitsu (StateUniversity of New York, Buffalo), and S. mutans UA159 was obtainedfrom Robert E. Marquis (University of Rochester, Rochester,N.Y.).

GTF enzymes. All of the purification procedures were carried out with bufferscontaining the protease inhibitor phenylmethylsulfonyl fluoride(PMSF; final concentration, 1 mM) and NaN₃ (final concentration,0.02%), which was added as a preservative. Neither of the reagentshad any adverse effects on enzyme activity or stability.

GTFs B, D, and Ss were obtained from culture supernatants andpurified to near homogeneity by hydroxyapatite column chromatographyas described by Venkitaraman et al. (51) and Vacca-Smith etal. (50). For GTF C isolation from S. mutans WHB410 (53), cellpellets were harvested from low-molecular-weight broth (2.5%Tryptone, 1.5% yeast extract, 0.3% glucose, 0.1% fructose, and0.1% sorbitol, which had been ultrafiltered through a membranewith a 10-kDa molecular size cutoff) cultures of S. mutans WHB410(ftf gtfD gtfB mutant derivative of S. mutans UA130) grown indialysis tubing (43). The cells were washed twice in 20 mM potassiumphosphate buffer, pH 7.5, containing 1 mM PMSF and 0.02% sodiumazide. The cells were then resuspended in 30 ml of 50 mM potassiumphosphate buffer, pH 7.5, containing 0.1% Triton X-100, 2 Murea, 500 mM NaCl, 0.02% sodium azide, and 1 mM PMSF and incubatedat 25°C for 2 h with gentle agitation. The cell suspensionwas centrifuged at 10,000 x g for 15 min at 4^oC. The supernatant,which was used as the source of GTF C, was carefully collectedand dialyzed against 50 mM potassium buffer, pH 7.5, containing1 mM PMSF and 0.02% sodium azide. The dialyzed preparation waspurified by hydroxyapatite column chromatography as detailedby Venkitaraman et al. (51).

The purity of the enzyme preparations was analyzed by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis in a HoeferMighty Small SE245 system (Hoefer Scientific Instruments, SanFrancisco, Calif.) and silver staining (36). Prestained standardswere purchased from Bio-Rad Laboratories. Protein concentrationwas determined by the method of Lowry et al. (33), and bovineserum albumin (Sigma Chemical Co., St. Louis, Mo.) was usedto construct standard curves.

GTF activity was measured by the incorporation of [¹⁴C]glucosefrom labeled sucrose (NEN Research Products, Boston, Mass.)into glucans (16, 51). The GTF enzyme added to each sample forall assays was equivalent to the amount required to incorporate1.0 to 1.5 µmol of glucose over the 4-h reaction (1.0to 1.5 U).

Assays of activity of GTF in solution and adsorbed onto a saliva-coated hydroxyapatite (sHA) surface. For solution assays, purified GTFs B, C, D, and Ss were mixedwith a twofold dilution series of the test compounds (concentrationsranging from 125 to 500 µM) and incubated with a [¹⁴C]glucose-sucrosesubstrate (0.2 µCi/ml; 200.0 mmol of sucrose per liter,40 µmol of dextran 9000 per liter, and 0.02% sodium azidein adsorption buffer consisting of 50 mM KCl, 1.0 mM KPO₄, 1.0mM CaCl₂, and 0.1 mM MgCl₂ [pH 6.5]) to a final concentrationof 100 mmol of sucrose per liter (200-ml final volume). Forthe control, the same reaction was carried out with ethanol-DMSO(final concentrations of 7.5 and 1.25%, vol/vol) or ethanol(final concentration of 5%, vol/vol) replacing the test agentsolutions. The samples were incubated at 37°C with rockingfor 4 h. After incubation, ice-cold ethanol (1.0 ml) was addedand the samples were stored for 18 h at 4°C for precipitationof glucans. Radiolabeled glucan was determined by scintillationcounting (16, 51).

For surface assays, the GTFs were adsorbed to hydroxyapatitebeads coated with clarified whole saliva (free of GTF activity)as detailed elsewhere (30, 43, 51). The sHA beads were exposedto sufficient enzyme to saturate the surface as determined experimentally.Following adsorption of the enzyme, the beads were washed threetimes with buffer to remove the loosely bound material and exposedto 300 µl of the twofold dilution series of test (or control)compounds for 30 min at the concentrations described above.The beads were washed and exposed to 300 µl of [¹⁴C]glucose-sucrosesubstrate (final concentration, 100.0 mmol of sucrose per liter).The radiolabeled glucan formed was collected and quantifiedby scintillation counting (16, 51). All of the solution andsurface assays were done in quadruplicate in at least threedifferent experiments.

For the compound with the highest activity, inhibition curves(concentration versus activity; concentrations ranged from 62.5µM to 1 mM) for all of the GTFs were plotted and 50% inhibitoryconcentrations (IC₅₀s; the concentrations of test compoundsrequired to inhibit enzymatic activity by 50%) were calculatedfrom regression lines (10). For this assay, a one-way layoutexperimental design was used in a four by two by seven (enzymeby state by dose) factorial scheme. An analysis of variancewas carried out, and qualitative treatments were compared byusing Tukey's test at a 5% level of significance (P < 0.05).A nonlinear regression was applied in order to evaluate theeffects of different concentrations.

Susceptibility testing. The MIC and minimum bactericidal concentration (MBC) of eachtest compound were determined in accordance with the NCCLS guidelines(37, 38) and Koo et al. (29). The broth microdilution and macrodilutionmethods (in TSB) were used for the antibacterial tests. Thestarting inoculum was 5 x 10⁵ CFU/ml, and the concentrationsof test compounds ranged from 15.6 to 500 µM (twofolddilutions). The MICs and MBCs were determined in quadruplicatein at least three different experiments.

Time-kill assays. For the compound with the highest level of antibacterial activity,time-kill studies were performed by the broth macrodilutionmethod (37). The starting inoculum of S. mutans GS-5 and UA159and S. sobrinus 6715 was 1 x 10⁶ to 5 x 10⁶ CFU/ml. The finalconcentration of the antibacterial agent was four times theMIC (or MBC). Tubes containing the microorganisms and the testcompound in TSB were incubated in 5% CO₂ at 37°C; sampleswere removed for determination of viable counts at 0 and 30min and 1, 2, 4, 8, and 24 h. Serial dilutions (10^-1 to 10^-4)were prepared in sterile 0.9% sodium chloride solution. Thediluted sample (50 µl) was plated onto tryptic soy agarwith a spiral plater (Autoplate model 3000; Spiral Biotech,Inc., Bethesda, Md.). The plates were incubated in 5% CO₂ at37°C for 48 h, when the number of colonies was determined.Killing curves were constructed by plotting the log₁₀ CFU permilliliter versus time over 24 h. All of the assays were donein quadruplicate on at least three occasions. A bactericidaleffect was defined as a $>=$ 3-log₁₀ decrease in the number of CFUper milliliter from the original inoculum.

The potential for drug carryover to produce falsely low viabilitycounts was minimized by dilution of inocula and plating of smallvolumes of diluted samples (50 µl). In addition, no evidenceof drug carryover was detected at the lowest dilution used forplating (10^-1).

RESULTS

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Results

The effects of the most active compounds on the activity ofGTFs are shown in Tables 2 and 3. In general, flavonols andflavones reduced the activity of all of the enzymes tested insolution (40 to 95% inhibition) and on a surface (15 to 60%inhibition) at a concentration of 500 µM. Apigenin (a4',5,7-trihydroxyflavone) displayed the most potent inhibitionof GTF activity. Apigenin inhibited 90.5 to 95% of the activityof all of the GTFs tested in solution at a concentration aslow as 500 µM (135 µg/ml). The inhibitory effectof apigenin on surface-adsorbed enzymes was not as potent asthat observed when the same enzymes were in solution. Nevertheless,it was an effective inhibitor (30 to 60% inhibition at a concentrationof 500 µM). The inhibitory effects of apigenin on GTFsare illustrated in Fig. 1. Apigenin reduced the activity ofall of the enzymes in solution in direct proportion to the amountadded in the reaction test (r² values ranging from 0.92 to 0.99).The IC₅₀s of apigenin for the GTFs in solution were 58 µM(16 µg/ml) to 98 µM (26 µg/ml). The IC₅₀sfor surface-adsorbed enzymes were noticeably higher; the IC₅₀sfor GTFs B and C were 478 µM (128 µg/ml) and 458µM (122 µg/ml); those for GTFs D and Ss were >1mM. It is conceivable that an IC₅₀ of this agent would not beachieved for surface-adsorbed GTFs D and Ss. Surface-adsorbedGTFs B and C were inhibited significantly more at all of theconcentrations tested than were GTFs D and Ss (P < 0.05).The flavone baicalein and the flavonols myricetin and rhamnetinalso proved to be effective inhibitors of GTFs in solution (70to 90% at 500 µM) and adsorbed on an sHA surface (19 to40% at 500 µM).

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TABLE 2. Effects of a selected flavone and a selected flavonol on the activities of streptococcal GTFs in solution and adsorbed onto an sHA surface^a

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FIG. 1. Effects of apigenin on the activities of streptococcal GTFs in solution (A) and adsorbed onto an sHA surface (B). Symbols: ${diamondsuit}$ , GTF B; ${blacksquare}$ , GTF C; ${triangleup}$ , GTF D; ${circ}$ , GTF Ss. The data shown are mean values ± standard deviations. Percent inhibition was calculated by considering the control (DMSO-ethanol; final concentrations of 7.5 and 1.25% [vol/vol], respectively) to contain maximum GTF activity. At each concentration of apigenin, means labeled with symbols (_* and ${delta}$ ) are not significantly different from each other at P < 0.05.

The flavanones, the dihydroflavonols, and some of the terpenoids(tt-farnesol and ß-caryophyllene) tested in this studyshowed moderate inhibitory effects (8 to 45% for GTFs in solutionand 7 to 24% for GTFs on a surface at a concentration of 500µM); the cinnamic acid derivatives showed negligible effectson GTF enzymes. In some cases, the activity of GTFs was enhanced,e.g., by cinnamic acid derivatives and some terpenoids (e.g.,protocatechuic acid and terpineol).

The MICs and MBCs of the test compounds for S. mutans GS-5 andUA159 and S. sobrinus 6715 are shown in Table 4. Some of theflavanones and dihydroflavonols, as well as tt-farnesol (a terpenoid),displayed antibacterial activity. All of the flavanones inhibitedbacterial growth; pinocembrin was the most effective of them,with an MIC of 250 µM (64 µg/ml) for all of thestrains tested. Pinocembrin showed a bactericidal effect againstS. sobrinus 6715 at 500 µM (128 µg/ml). The dihydroflavonolpinobanksin-3-acetate also inhibited the growth of S. sobrinus6715 and S. mutans strains (MIC of 500 µM or 157 µg/ml).Of all of the test compounds, tt-farnesol was the most effectiveantibacterial agent. The MICs were 125 µM (28 µg/ml)for S. mutans strains and 62.5 µM (14 µg/ml) forS. sobrinus 6715. The MBCs were 500 µM (112 µg/ml)for S. mutans strains and 250 µM (56 µg/ml) forS. sobrinus 6715. Chlorhexidine (a positive control) yieldedMICs of 1.1 to 2.2 µM (1 to 2 µg/ml) and an MBCof 8.9 µM (8 µg/ml). Flavonols, flavones, and cinnamicacid derivatives did not show any antibacterial activity atthe concentrations used in this assay, with the exception ofbaicalein (MIC of 500 µM).

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TABLE 4. MICs and MBCs of antibacterial compounds for S. mutans and S. sobrinus strains^a

The results of the time-kill kinetic studies are summarizedin Fig. 2. tt-Farnesol at four times the MIC (or MBC) rapidlyreduced the viable counts of S. mutans and S. sobrinus within30 min to 1 h of incubation (reduction of 1 log in the numberof CFU per milliliter). tt-Farnesol exerted bactericidal effects(a $>=$ 3-log decrease in the number of CFU per milliliter) on S.sobrinus 6715 and on S. mutans strains between 4 and 8 h ofincubation. Chlorhexidine at 8.9 µM (MBC) displayed bactericidaleffects on S. sobrinus 6715 and S. mutans strains tested after8 h of incubation.

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FIG. 2. Time-kill curves for S. mutans strains by tt-farnesol at four times the MIC (or MBC). Symbols: ${blacktriangleup}$ and ${triangleup}$ , S. mutans GS-5; • and ${circ}$ , S. mutans UA159; ${blacksquare}$ and ${square}$ , S. sobrinus 6715.

DISCUSSION

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Discussion

Dental caries is one of the most common oral diseases worldwide(7). One approach to reducing the incidence of tooth decay isto develop therapeutic agents aimed at preventing the formationof plaque matrix (48), 30 to 40% (dry weight) of which is polysaccharide(12, 24). Indeed, most of the polysaccharide fraction of plaqueis synthesized chiefly by GTFs from sucrose (31, 41), althoughfructan is also present (12, 24). Dental caries results fromevents that occur at the tooth pellicle-plaque interface; enzymaticallyactive GTFs are present in the pellicle. Clearly, it is desirableto determine the effects of potential inhibitors on surface-adsorbedGTF.

The use of natural products has been one of the most successfulstrategies for the discovery of new medicines (22); 78% of newantibiotics and 61% of new antitumor drugs approved by the Foodand Drug Administration or comparable entities in other countriesfrom 1983 to 1994 were natural products or derived from naturalproducts (11). According to Harvey (22), the access to biodiversityis fundamental to expanding the range of natural products tobe used in the search for new drugs. In this context, propolisfrom A. mellifera, a relatively unexplored natural product,could be a valuable resource for exploration of new bioactivecompounds because of the high chemodiversity of this naturalsubstance (3, 6, 18, 27, 40). Recently, we demonstrated thatpropolis reduced dental caries in desalivated rats (27). Therefore,we hypothesized that there are specific compounds in propolisthat can, at low concentrations, inhibit the growth of cariogenicbacteria and the activity of the GTFs, which are associatedwith the pathogenesis of dental caries. This was the first steptoward identifying novel inhibitors of GTF enzymes and S. mutansgrowth.

The results obtained in the present study identified some ofthe compounds that may have been responsible for the previouslyreported effects of propolis on GTFs and bacterial growth (28,29, 30, 40). In general, flavonoids were the most active compounds,displaying distinct biological properties; flavones and flavonolswere effective GTF inhibitors, whereas flavanones and the dihydroflavonolpinobanksin-3-acetate showed antibacterial activity.

Apigenin, a 4',5,7-trihydroxyflavone, was the most effectiveinhibitor of GTFs, especially GTFs B and C. Most of the knownGTF inhibitors tested so far, including currently commerciallyavailable mouthwashes, failed to inhibit surface-adsorbed GTFactivities effectively (48, 53). In contrast, in the presentstudy, apigenin greatly inhibited GTF, especially GTFs B andC, irrespective of whether the enzyme was exposed before orafter adsorption to a surface at concentration as low as 500µM. This level of inhibition has not been observed previously(48, 53). The effective inhibition of GTFs B and C by apigeninmay affect the pathogenic potential of dental plaque relatedto caries, consistent with a reduction in smooth-surface cariesobserved with mutants of S. mutans defective in the productionof either or both GTFs (54).

Apigenin is a nonmutagenic flavonoid displaying a variety ofantitumor and antiinflammatory effects (32, 34); our study isthe first to show apigenin as a potent inhibitor of GTF activity.The exact mechanism by which apigenin and related flavonoidsact to inhibit GTF activity is unknown, although the data reportedhere provide some insights into the mode of their inhibitoryaction. It appears that inhibition of GTFs depends on the molecularstructure of flavonoids and the physical state of the enzyme.Flavones and flavonols, which have an unsaturated double bondbetween positions C-2 and C-3 (Table 1), showed remarkable inhibitionof GTF activity; in contrast, flavanones and dihydroflavonols,which lack a double bond between C-2 and C-3, exhibited onlymodest inhibitory activities. Results from previous studieshave shown that flavones and flavonols are the main flavonoidsrelated to inhibition of several mammalian enzymes, which suggeststhat a double bond between C-2 and C-3 is required for inhibitoryeffects (15, 52). The presence of a double bond between C-2and C-3 may provide a site for nucleophilic addition by sidechains of amino acids in GTFs. Several amino acid residues havebeen identified as essential for expression of the catalyticactivity of GTFs, especially aspartic acid (26, 35). It is likelythat the side chain of aspartic acid (CH₂COOH) acts as a nucleophileand reacts with flavones and flavonols, causing GTF inhibition.The resistance displayed by surface-adsorbed GTF enzymes maybe related to conformational changes the GTF undergoes duringthe adsorption process, consistent with differences betweenthe adsorbed and soluble forms of the enzyme in terms of physicalor kinetic properties and the products synthesized (31, 43,51). Further research is needed to elucidate the mechanisticdetails of GTF inhibition by these groups of flavonoids.

Several compounds from propolis inhibited S. mutans growth.However, none of them was as potent as chlorhexidine, whichis a clinically proven antimicrobial. Of the compounds tested,tt-farnesol was the most effective antibacterial agent and produceda rapid decrease in S. mutans viable counts. This observationis in agreement with previous reports on the antimicrobial activityof farnesol and related compounds in a variety of other taxa(5, 14). Terpenes such as farnesol have been reported to disruptmembrane function, ultimately reducing cell viability (5). Itis noteworthy that streptococci treated with high concentrationsof tt-farnesol (>10 mM) produced membrane disruption visiblein a phase-contrast microscope; whether the streptococcal membraneis affected at the molecular level by lower concentrations oftt-farnesol (e.g., 0.5 mM) needs to be further elucidated. Recently,farnesol has been shown to be an antifungal agent, displayingquorum-sensing molecule activity (23). We are currently evaluatingthe antibacterial effect of tt-farnesol on sessile bacterialcells or biofilms.

Our data support the hypothesis that the biological activityof propolis, such as caries prevention (27), is related to theeffects of several compounds, as suggested by Amoros et al.(1) and Bonhevì et al. (6), rather than a single compound.Apigenin and tt-farnesol are active compounds in propolis. Itis conceivable that propolis compounds can be exploited commercially.The concentrations used in our study may be readily achievablein the mouth through topical application. Although the toxicologyof these compounds was not studied here, there is no evidencein the literature that apigenin or tt-farnesol has any potentialcellular toxicity or hemolytic effects.

In summary, we have described two new potential anticaries-antiplaqueagents, apigenin and tt-farnesol, each of which exhibits distinctbiological activities. Since some of the biological activitiesof these compounds have been clearly shown, further studiesinvolving animal models are worth performing to evaluate theireffects in vivo. Propolis appears to be a promising source ofnew agents that may prevent dental caries and other oral diseases.

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TABLE 3. Effects of a selected flavanone and a selected dihydroflavonol on the activities of streptococcal GTFs in solution and adsorbed onto an sHA surface^a

ACKNOWLEDGMENTS

We are grateful to Kathleen Scott-Anne for technical assistance.

This research was supported by U.S. Public Health Service grantDE07907 and the Basil G. Bibby Fellowship.

FOOTNOTES

* Corresponding author. Mailing address: University of Rochester Medical Center, Center for Oral Biology, 601 Elmwood Ave., Box 611, Rochester, NY 14642. Phone: (716) 275-1433. Fax: (716) 473-2679. E-mail: Hyun_Koo{at}urmc.rochester.edu.

REFERENCES

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