Inhibitory effects of cranberry polyphenols on formation and acidogenicity of Streptococcus mutans biofilms

Inhibitorye¡ects of cranberrypolyphenolson formationandacidogenicityofStreptococcusmutansbio¢lmsSimone Duarte1, Stacy Gregoire1, Ajay P. Singh2, Nicholi Vorsa2,3, Karen Schaich4, William H. Bowen1 &Hyun Koo1

1Eastman Department of Dentistry Center for Oral Biology, University of Rochester Medical Center, Rochester, NY, USA; 2Department of Plant Biology

and Plant Pathology, Rutgers University, New Brunswick, NJ, USA; 3Philip E. Marucci Center for Blueberry and Cranberry Research and Extension,

Rutgers University, Chatsworth, NJ, USA; and 4Department of Food Science, Rutgers University, New Brunswick, NJ, USA

Correspondence: Hyun Koo, University of

Rochester Medical Center, Eastman

Department of Dentistry and Center for Oral

Biology, 625 Elmwood Ave., Box 683,

Rochester, NY 14620, USA. Tel.:11 585 273

4216; fax:11 585 276 0190; e-mail:

[email protected]

Received 29 November 2005, revised 4 January

2006, accepted 5 January 2006.

First published online 15 February 2006.

doi:10.1111/j.1574-6968.2006.00147.x

Editor: Stefan Schwarz

Keywords

Streptococcus mutans; biofilms; glucans;

cranberry; proanthocyanidins; glycolysis.

Abstract

Cranberry fruit is a rich source of polyphenols, and has shown biological activities

against Streptococcus mutans. In the present study, we examined the influence of

extracts of flavonols (FLAV), anthocyanins (A) and proanthocyanidins (PAC)

from cranberry on virulence factors involved in Streptococcus mutans biofilm

development and acidogenicity. PAC and FLAV, alone or in combination, inhibited

the surface-adsorbed glucosyltransferases and F-ATPases activities, and the acid

production by S. mutans cells. Furthermore, biofilm development and acidogeni-

city were significantly affected by topical applications of PAC and FLAV

(Po 0.05). Anthocyanins were devoid of any significant biological effects. The

flavonols are comprised of mostly quercetin glycosides, and the PAC are largely

A-type oligomers of epicatechin. Our data show that proanthocyanidins and

flavonols are the active constituents of cranberry against S. mutans.

Introduction

Streptococcus mutans is generally regarded as a primary

microbial agent in the pathogenesis of dental caries although

additional acidogenic microorganisms may be involved

(Loesche, 1986; Bowen, 2002; Beighton, 2005). This bacter-

ium synthesizes extracellular glucans from sucrose using

glucosyltransferases (GTFs) (Loesche, 1986; Bowen, 2002).

Glucans promote the accumulation of cariogenic strepto-

cocci (and other oral microorganisms) on the tooth surface,

and are critical for the formation and structural integrity of

biofilms (Dibdin & Shellis, 1988; Schilling & Bowen, 1992;

Cury et al., 2000; Marsh, 2004). Furthermore, S. mutans

carries out glycolysis of multiple carbohydrates efficiently.

However, within the biofilms effective neutralization cannot

occur because of limited access by saliva; the low pH values

in the plaque matrix contribute to demineralization of tooth

enamel and selection of aciduric (acid-tolerant) organisms,

such as mutans streptococci. Streptococcus mutans has

developed mechanisms to alleviate the influences of acid-

ification by increasing proton-translocating F-ATPase activ-

ity in response to low pH (Sturr & Marquis, 1992; Quivey

et al., 2000). F-ATPase transports protons (H1) out of cells

in association with ATP hydrolysis to maintain intracellular

pH more alkaline than the extracellular environment pH

(Sturr & Marquis, 1992). Therefore, there are several ave-

nues for chemotherapeutic intervention other than attempt-

ing to eliminate mutans streptococci selectively, which

include inhibition of glucan production by GTFs and F-

ATPase activity.

American cranberry (Vaccinium macrocarpon Ait., Erica-

ceae) is a widely consumed fruit in North America, and has

been recognized to have several biological properties which

may provide human health benefits (Sobota, 1984; Ofek

et al., 1996; Ahuja et al., 1998), including effects on virulence

factors of S. mutans involved in the pathogenesis of dental

caries. Cranberry juice and a high-molecular weight non-

dialyzable material (NDM) extracted from cranberry inhibit

the formation of biofilms and coaggregates of oral bacteria,

the activity of GTFs, and the bacterial adherence on apatitic

surfaces (Weiss et al., 2002; Steinberg et al., 2004; Yamanaka

et al., 2004; Duarte et al., 2005). Recently, we have shown

that cranberry juice disrupts the accumulation and acido-

genicity of S. mutans biofilms in vitro without killing the

organisms (Duarte et al., 2005).

FEMS Microbiol Lett 257 (2006) 50–56c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Cranberry fruit is a unique and rich source of various

classes of potentially bioactive flavonoids (polyphenols).

Flavonoids are a large group of polyphenolic natural com-

pounds that are universally distributed in higher plants; they

are built upon a C6–C3–C6 flavone skeleton in which the

three-carbon bridge between the phenyl groups is com-

monly cyclized with oxygen. Several classes are differentiated

according to the degree of unsaturation and degree of

oxidation of the three-carbon segment. Four phenolic

classes identified in cranberry include phenolic acids, an-

thocyanins, flavonols and flavan-3-ols, which consist of

monomers and the polymer classes of proanthocyanidins.

Anthocyanins, flavonols and proanthocyanidins are among

the most abundant flavonoid classes (Vvedenskaya & Vorsa,

2004), and have been associated with the health promoting

benefits of cranberry and its products (Cunningham et al.,

2004).

Therefore, the aim of the present study was to examine

the influence of anthocyanins, flavonols, and proanthocya-

nidins extracts from cranberry on (i) glucans production by

purified glucosyltransferases (GTFs) adsorbed to sHA, (ii)

membrane-associated F-ATPase and glycolytic activities,

and (iii) viability, development, polysaccharide composition

and acidogenicity of S. mutans biofilms.

Materials and methods

Cranberry constituents

The flavonol (FLAV)-, anthocyanin (A)-, and proanthocya-

nidin (PAC)-rich fractions were prepared from fruit of the

cranberry variety ‘Stevens’ harvested at Rutgers University,

PE Marucci Center during September/October 2003 and

kept frozen at � 20 1C. Fractions were eluted from a

Sephadex LH-20 column in following sequence: anthocya-

nins with water, flavonols with 60% methanol, and

proanthocyanidins with 80% acetone/water. Fractions were

characterized using HPLC-Photodiode/electrochemical de-

tection, LC-MS, and MALDI-TOF.

A cranberry extract was provided by NIH/NCCAM

approved contractor, which was diluted to a final concentra-

tion of 20 mg of dry-weight mL�1 (CE solution); this is the

usual concentration of cranberry extract in most of the

products available commercially, such as cranberry cocktail

juice. The content of various cranberry polyphenols in CE

solution is subjected to variation depending on several

factors, such as seasonal and varietal effects; concentrations

of the cranberry polyphenols in the CE solution in these

experiments were estimated to be 300–500 mg mL�1 (for

PAC), 50–200 mg mL�1 (for A), and 30–125 mg mL�1 (for

FLAV). We tested each of the extracts alone or in combina-

tion as follows: PAC1A, PAC1FLAV, A1FLAV, and

A1PAC1FLAV. A 10% ethanol [volume in volume (v/v)]

solution was used as a vehicle control; the pH of all solutions

was adjusted to 5.5 using 5 mM potassium phosphate buffer.

The bioassay data reported here utilized the highest con-

centration of each of the extracts (125 mg mL�1 of FLAV,

500 mg mL�1 of PAC, and 200 mg mL�1 of A).

Bacterial strains

The bacterial strains used for the production of GTFs were:

Streptococcus anginosus KSB8 (Fukushima et al., 1992),

which harbors the gtfB gene (for GTF B production) and S.

mutans WHB 410 (Wunder & Bowen, 1999), in which the

genes for GTF B, D and fructosyltransferase were deleted

(for GTF C). The S. mutans UA159, a proven virulent

cariogenic pathogen and the strain selected for genomic

sequencing (Ajdic et al., 2002), was used for F-ATPase,

glycolytic pH drop, and biofilm studies. The cultures were

stored at –801 C in low molecular weight medium (Koo

et al., 2003) containing 20% glycerol.

GTF B and C assays

The GTF B and C enzymes (EC 2.4.1.5) were prepared from

culture supernatants and purified to near homogeneity by

hydroxyapatite column chromatography as described by

Venkitaraman et al. (1995) and Wunder & Bowen (1999).

GTF activity was measured by the incorporation of

[14C]glucose from labeled sucrose (NEN Research Products,

Boston, MA) into glucans (Venkitaraman et al., 1995; Koo

et al., 2000). The GTF enzyme added to each sample for all

assays was equivalent to the amount required to incorporate

1 to 1.5 mmol of glucose over the 4 h reaction (1.0–1.5 U).

The activities of GTFs were determined with the enzymes

adsorbed to hydroxyapatite beads (Macro-Prep Ceramic

Hydroxyapatite Type I, 80 mm, Bio-Rads, Hercules, CA)

coated with clarified whole saliva (sHA) (free of GTF

activity; Koo et al., 2000), in the presence of test agents

(CE, PAC, A, FLAV, PAC1A, PAC1FLAV, A1FLAV, PA-

C1A1FLAV) or to the vehicle control (10% ethanol, v/v) as

described elsewhere (Koo et al., 2000). The final pH of the

test agents was 5.5. The activities of GTFs in solution were

not examined in this study because their enzymatic activities

are affected by the acidic pH of the test agents (pH 5.5),

which is below the optimum pH for the enzymes in solution

(DpH 6.5). In contrast, surface-adsorbed GTFs are active

over a much broader pH range (pH 4.7–7.5) (Schilling &

Bowen, 1988).

F-ATPase and glycolytic pH-drop assays

ATPase assay was performed using permeabilized cells of S.

mutans UA159 prepared as described by Belli et al. (1995). F-

ATPase activity was assayed in terms of the release of

inorganic phosphate in the following reaction mixture:

FEMS Microbiol Lett 257 (2006) 50–56 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

51Inhibitory effects of cranberry polyphenols

100 mmol of Tris-maleate buffer (pH 7.0) containing 5 mM

ATP, 10 mmol MgCl2, permeabilized cells, and the test

agents (CE, PAC, A, FLAV, PAC1A, PAC1FLAV, A1FLAV,

PAC1A1FLAV or the vehicle control). The released phos-

phate was then determined by the method of Bencini et al.

(1983).

The effects of cranberry compounds on glycolysis were

measured by standard pH drop with dense cell suspensions

as previously described by Belli et al. (1995). Cells of S.

mutans UA159 from suspension cultures were harvested,

washed once with salt solution (50 mM KCl plus 1 mM

MgCl2), and resuspended in salt solution containing the test

agents, alone or in combinations. The pH was adjusted to

7.2 with 0.1 M KOH solution, sufficient glucose was added

to give a concentration of 1% [weight in volume (w/v)], and

the decrease in pH was assessed by means of a glass electrode

over a period of 2 h (Futura Micro Combination pH

electrode, 5 mm diameter, Beckman Coulter, Inc., CA) (Belli

et al., 1995).

Biofilm assays

Biofilms of S. mutans UA159 were formed on saliva-coated

hydroxyapatite discs placed in a vertical position (HAP

ceramic – calcium hydroxyapatite, 0.500 diameter ceramic –

Clarkson Calcium Phosphates, Williamsport, PA) in batch

cultures at 37 1C and 5% CO2 (Koo et al., 2003; Chatfield

et al., 2005). Biofilms of S. mutans were formed in ultra-

filtered (Amicon 10 kDa molecular weight cut-off mem-

brane; Millipore Co., MA, USA) tryptone-yeast extract

broth with addition of 30 mM sucrose containing [14C-

glucose]-sucrose (0.74 kBq mL�1) (Koo et al., 2003). The

biofilms were grown undisturbed for 24 h to allow initial

biofilms formation. At this point (24 h-old), the biofilms

were treated twice daily (10 a.m. and 4 p.m.) until the fifth

day of the experimental period (126 h-old biofilm) with one

of the test agents (CE, PAC, A, FLAV, PAC1A, PAC1FLAV,

A1FLAV, PAC1A1FLAV) or vehicle control. The biofilms

were exposed to the treatments for 1 min, double-dip rinsed

in sterile saline solution and transferred to fresh culture

medium as detailed elsewhere (Koo et al., 2003). Each

biofilm was exposed to the respective treatment a total of

eight times. The treated biofilms were analyzed for biomass

(dry weight) and bacterial viability (colony forming units –

CFU mg�1 of biofilm dry weight). The polysaccharide com-

position (extracellular water-soluble and insoluble glucans,

and intracellular iodophilic polysaccharides) was deter-

mined by colorimetric assays and scintillation counting as

detailed by Koo et al. (2003). The acid production by the

biofilms that were treated with the test agents (or control)

was determined by glycolytic pH drop after addition of

glucose solution (final concentration of 1%, w/v) by means

of a glass electrode (Futura Micro Combination pH elec-

trode, 5 mm diameter, Beckman Coulter, Inc, CA) (Belli

et al., 1995).

Statistical analyses

The data were analyzed using ANOVA, and the F-test was used to

test any difference among the groups. When significant

differences were detected, pairwise comparisons were made

between all the groups using Tukey’s method to adjust for

multiple comparisons. Triplicates from at least two separate

experiments were conducted in each of the assays. Statistical

software JMP version 3.1 (SAS Institute, Cary, NC) was used

to perform the analyses. The level of significance was set at 5%.

Results and discussion

The basic chemical structures of three of the most abundant

flavonoid classes found in cranberry are shown in Fig. 1. The

PAC extract consisted of monomers and polymers with the

degree of polymerization ranging from dimers to octamers.

MALDI-TOF mass spectra analysis confirmed A-type oligo-

mers of epicatechin from trimers to octamers which is

similar to those from cranberry reported by Foo et al.

(2000), except with the addition of octamers. The flavonols

were mostly quercetin glycosides, including the most abun-

dant flavonol, quercetin-3-b-galactoside consistent with a

previous study by Vvedenskaya et al. (2004). Cranberry

offers to be a source of some unique PAC and FLAV,

e.g. epicatechin-(4b ! 8, 2b ! O ! 7)-epicatechin-(4b! 8)-epicatehin and quercetin-3-a-arabinopyranoside.

The anthocyanin fraction which gives the characteristic red

color of cranberry is composed largely of galactoside and

arabinoside conjugates of cyanidin and peonidin (Hong &

Wrolstad, 1990). We focused on each of the components at

concentrations normally found in cranberries to identify the

most effective compound(s).

The data presented in Fig. 2 indicate that FLAV

(125 mg mL�1) and PAC (500mg mL�1), alone or in combi-

nation, significantly inhibited the activities of surface ad-

sorbed GTF B and C (30–60% inhibition). Combination of

fractions PAC1FLAV and PAC1A1FLAV approached the

effectiveness of CE solution, suggesting a synergistic action

between the fractions. The inhibition of GTF B and C has

many implications for biofilms development because large

proportion of the insoluble glucans synthesized by these

surface-adsorbed enzymes is retained on the pellicle pro-

moting accumulation of Streptococcus mutans and other

cariogenic bacteria on the tooth surface, and contributing

to the formation of the matrix of the biofilms (Schilling &

Bowen, 1992). These enzymes are critical in the expression

of virulence by S. mutans in the pathogenesis of dental caries

(Yamashita et al., 1993). We have shown that flavonols

aglycones, such as quercetin, myricetin and kaempferol, are

effective GTF inhibitors (Koo et al., 2002); the inhibitory


52 S. Duarte et al.

effects could be associated with the presence of an unsatu-

rated double bond between C-2 and C-3 (Fig. 1), which may

provide a site for nucleophilic addition by side chains of

aminoacids in GTFs. It is noteworthy that anthocyanins,

which lack a double bond between C-2 and C-3 (Fig. 1),

exhibited only modest inhibitory activities (Fig. 2). PAC are

known to bind proteins forming protein–polyphenol com-

plexes (Haslam, 1996; Bravo, 1998), which could inhibit the

activity of GTFs. The protein binding capacity of PACs

depends on their degree of polymerization and access to

the proteins (Bravo, 1998). Further research with individual

compounds is needed to elucidate the mechanistic details of

GTF inhibition by these groups of flavonoids.

The activity of S. mutans membrane associated F-ATPase

was also inhibited by PAC, alone or in combinations

(4 85% of inhibition, Fig. 3). Flavonols also significantly

inhibited the activity of F-ATPase (Po 0.05), although the

inhibitory effect was modest compared to that of PAC. F-

ATPase comprises two major complexes, the F1 water-

soluble catalytic site and the proton-conducting hydropho-

bic F0 complex (Sturr & Marquis, 1992). Flavonoids have

been shown to inhibit various forms of ATPases (Havsteen,

1983). Quercetin is a known non-competitive inhibitor of

proton-translocating F-ATPases by binding to F1 catalytic

complex (Zheng & Ramirez, 2000); whether its glycosides

display similar mechanisms of enzyme inhibition remain to

be elucidated. In contrast, little is known on the effects of

PAC on the ATPases; this is the first study showing that PAC

effectively inhibit the activity of F-ATPase of bacterial

membranes.

Proton translocating F-ATPases (H1-ATPase) play major

roles in protecting S. mutans against environmental stress

caused by acidification of the biofilms. By taking up or

releasing H1 as they synthesize or hydrolyze ATP, F-ATPases

help maintain DpH across the membrane, which is critical

for the optimum function of glycolysis. Enolase and other

enzymes of the glycolytic pathway and the sugar transport

system are sensitive to cytoplasmic acidification (Belli et al.,

A

O

OH

R

HO

OH

O glycoside

Cyanidin:R=OHPeonidin:R=OCH3Glycoside=galactose,glucose,arabinose

epicatechin−(4β 8, 2β O 7)−epicatechin−(4β 8)−epicatehin

PAC

OH

OH

HO O

OH

OH

OH

OH

HOO

OH

OH

O

OH

HO

O

OH

HO

A-Type interflavan bond

OOH

HO O

R

OH

O glycoside

R'

Kaempferol:R=R'=HQuercetin:R=OH,R'=HMyricetin:R=R'=OH

glycoside=galactose,glucose,arabinose,rhamnose

FLAV

. .

Fig. 1. Structure of main classes of flavonoids

found in cranberry. FLAV, flavonols; A, anthocya-

nins; PAC, proanthocyanidins.



1995). By inhibiting the activity of these enzymes, CE

solution and its bioactive components (PAC and to a lesser

extent FLAV) can affect the activity of acid sensitive glyco-

lytic enzymes. Acid sensitization can be readily seen in

glycolytic pH-drop experiments in which cells are given

excess glucose. Streptococcus mutans cells are able to rapidly

degrade glucose and lower the pH value of the suspension to

some minimum value at which they can no longer maintain

a cytoplasmatic pH compatible with glycolytic enzymes. As

shown in Fig. 4, the presence of CE solution and, especially,

PAC (alone or in combinations) sensitized cells of S. mutans

to acidification so that the final pH value was about 4.7–4.9,

compared with about 3.7 for cells not exposed to the test

agents (vehicle control). Whether these extracts can actually

prevent enamel demineralization awaits further evaluation

since the final pH values (4.7–4.9) are still slightly below the

critical pH for enamel dissolution (pH 5.0–5.5).

Viability of the biofilms as assessed by determination of

CFU mg�1 of biofilm dry weight was not impacted by

cranberry extract and its components (data not shown).

Nevertheless, short-term topical application of PAC and

FLAV (one-minute exposure, twice daily) significantly dis-

rupted the accumulation and polysaccharide composition of

S. mutans biofilms compared with the control (Table 1,

Po 0.05), reducing both the biomass (dry weight) and total

amount of insoluble glucans. In contrast, the influence of

10

0

20

30

40

50

60

70

80

90

100

Vehicl

e contro

l AFLAV

A+FLAV

PAC

A+PAC

FLAV+PAC

A+FLAV+P

AC

Crude e

xtra

ct

Test Agents

% o

f E

nzy

mat

ic A

ctiv

ity

Fig. 3. Influence of cranberry components on F-ATPase of permeabi-

lized cells of Streptococcus mutans UA159. The concentrations of each

of the extracts were 125mg mL�1 (FLAV, flavonols), 500mg mL�1 (PAC,

proanthocyanidins), and 200 mg mL�1 (A, anthocyanins). The percentage

of inhibition was calculated considering the vehicle control as 100% F-

ATPase activity. Values (SD, N = 9) connected by lines are not significantly

different from each other (P4 0.05, ANOVA, comparison for all pairs using

Tukey test).

3

3.5

4

4.5

5

5.5

6

6.5

7

7.5

0 20 40 60 80 100 120 140Time (min)

pH

AFLAVPACA+FLAVA+PACFLAV+PACA+FLAV+PACcrude extractVehicle control

Fig. 4. Influence of cranberry components on glycolytic pH drop of

Streptococcus mutans UA159 in suspensions. The concentrations of

each of the extracts were 125 mg mL�1 (FLAV, flavonols), 500 mg mL�1

(PAC, proanthocyanidins), and 200 mg mL�1 (A, anthocyanins). Values

(N = 9, SD not shown) from PAC, A1PAC, FLAV1PAC, A1FLAV1PAC

and crude extract solution are not significantly different from each other

at each time point (P4 0.05, ANOVA, comparison for all pairs using Tukey

test). Values (N = 9, SD not shown) from A, FLAV, A1FLAV and vehicle

control are not significantly different from each other at time points

t10 min, t20 min, t30 min, t40 min, t50 min (P4 0.05, ANOVA, comparison for all

pairs using Tukey test).

0

10

20

30

40

50

60

70

80

90

100

Vehicl

e contro

l AFLAV

A+FLAV

PAC

A+PAC

FLAV+PAC

A+FLAV+P

AC

Crude e

xtra

ct

Test Agents

% o

f E

nzy

me

Act

ivit

y

Fig. 2. Influence of cranberry components on the activities of glucosyl-

transferase B adsorbed onto a saliva-coated hydroxyapatite surface. The

concentrations of each of the extracts were 125 mg mL�1 (FLAV, flavo-

nols), 500mg mL�1 (PAC, proanthocyanidins), and 200 mg mL�1 (A, an-

thocyanins). The percentage of inhibition was calculated considering the

vehicle control as 100% glucosyltransferases (GTF) activity. A similar

inhibitory profile was obtained for GTF C. Values (SD, N = 9) connected

by lines are not significantly different from each other (P4 0.05, ANOVA,

comparison for all pairs using Tukey test).


54 S. Duarte et al.

the flavonoids on the production of soluble glucans and

intracellular iodophilic polysaccharides was negligible

(P4 0.05, data not shown). As with other assays, the A

extract had no significant effect against biofilms. Although

our mono-species biofilm model does not mimic the com-

plex microbial community found in dental plaque, it is more

advantageous when examining specific actions of test agents

on S. mutans physiology, especially on the glucan-mediated

processes involved in the biofilm development. Clearly, PAC

and FLAV, alone or in combinations, as well as CE solution

reduced the formation and accumulation of S. mutans

biofilms by mostly diminishing the amounts of insoluble

glucans in the biofilms matrix. This observation is consis-

tent with the effective inhibition of GTF B and C observed in

this study.

Furthermore, PAC1FLAV, PAC1A1FLAV and CE solu-

tion treatments also reduced the acidogenicity of the bio-

films (Table 1), although this effect was less than that

observed in glycolytic pH-drop assays using suspension cells

of S. mutans (Fig. 4). Biofilms are known to be more

resistant to antimicrobial agents than cells in suspension

because of higher biomass densities and decreased metabolic

activities in biofilms, which affect the effectiveness of the

therapeutic agents (Lewis, 2001).

Overall, the data show that the fractions in cranberries

that are biologically active against the virulence traits of S.

mutans involved in acidogenicity and biofilm development

are mainly PAC and to a lesser extent FLAV; the A extract, in

contrast, had no significant effects against S. mutans.

Combinations of PAC1FLAV and PAC1A1FLAV displayed

the highest biological activity in vitro and potency compar-

able to crude cranberry extract (CE) solution. The putative

pathways by which flavonols and proanthocyanidins affect

the virulence of S. mutans may involve several routes. We

propose at least three: (1) inhibition of insoluble glucans

synthesis by surface-adsorbed GTF B and C; (2) inhibition

of the proton-translocating F-ATPase activity; and (3) dis-

rupting acid production. Having shown clearly the potential

of cranberry flavonols and proanthocyanidins to interfere

with virulence traits of S. mutans, the identification of the

individual active compound(s) is worthy of exploration.

Acknowledgements

This research was supported by grants from National

Institute for Dental and Craniofacial Research and National

Center for Complementary and Alternative Medicine

1R01DE016139-01 (H.K.) and 5R01AT002058 (N.V.).

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Table 1. Dry-weight, acidogenicity, and total amount of insoluble glucans in the biofilms after treatments

Treatments� Dry-weight (mg) Insoluble glucans (mg) pH (30 min after glucose pulse)

Vehicle control 9.1�0.6a 2.09� 0.31a 4.91�0.04a,b

Anthocyanins (A) 8.7�1.1a,b 1.90� 0.40a,b 4.84�0.03a

Flavonols (FLAV) 7.3�1.4c 1.48� 0.38b,c 4.95�0.04a,b

Proanthocyanidins (PAC) 7.0�1.4c 1.35� 0.32c 5.02�0.11b

A1FLAV 7.7�0.6b,c 1.61� 0.29b,c 5.00�0.04b

A1PAC 7.3�0.5c 1.35� 0.21c 4.98�0.05a,b

FLAV1PAC 6.8�0.9c 1.20� 0.28c 5.39�0.02c

A1FLAV1PAC 7.1�0.8c 1.34� 0.27c 5.19�0.09c

Crude extract 6.8�0.7c 1.22� 0.32c 5.41�0.05c

The concentrations of each of the extracts were 125 mg mL�1 (FLAV), 500mg mL�1 (PAC), and 200mg mL�1 (A).

Values in the same column followed by the same superscripts are not significantly different from each other (P4 0.05, ANOVA, comparison for all pairs

using Tukey test).�Twice daily with one minute exposure for each treatment.



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Inhibitory effects of cranberry polyphenols on formation and acidogenicity of Streptococcus mutans biofilms