Vol.

20 No.

2001

Effect

of propolis

and

formation

propolis-containing
of dental

toothpaste

plaque

MASARU SAT01, SHUU FUJIWARA2,

on the

in vitro

MOTOHIKO NAGAYAMA1,

RYOZO YAMAGUCHI3, CHIKAKO TOKUDA1, HIROSHI TAKEUCHI1,

HIDEO YAMADA4, HIROYUKI SUGIM0T04 and

KIYOSHI OKIHARA4

Abstract : The effect of propolis collected in Brazil on the adsorption

of Streptococcus
sanguis to saliva-coated
hydroxyapatite
and on the coaggregation
reaction between S.
sanguis and Fusobacterium
nucleatum
was studied using radio-labeled
bacterial
cells.
The antibacterial
activity of a propolis-containing
toothpaste
against
cariogenic
and
plaque-forming
bacteria was also investigated.
An ethanolic solution of propolis (10 and
20 mg/mL)
significantly
reduced bacterial
adherence
to saliva-coated
hydroxyapatite
(25.3% and 29.6%, p<0.05).
Coaggregation
reaction
was also significantly
inhibited
by 42.9% (10 mg/mL, p<0.01) and 56.2% (20 mg/mL, p<0.01).
The toothpaste
completely
inhibited the growth of 20 bacterial strains,
including Streptococcus,
Actinomyces
and
Lactobacillus
at a concentration
range of 3-7 mg/mL.
In addition to previously
reported
effects of propolis
on mutans streptococci,
propolis
used in the present study was
shown to possess remarkable
potency to inhibit plaque formation
and development.
Propolis
and propolis-containing
toothpaste
might represent
potent inhibitory
agents
against dental plaque formation.
Key words

: propolis, plaque
rial activity

formation,

bacterial

adherence,

coaggregation,

their coaggregation
Introduction
Dental plaque is the most significant
causative factor in oral infectious
diseases,
such as
dental caries and periodontitisl1).
In the initial
stages of plaque formation,
saliva-derived
components
cover tooth surfaces,
with this thin
layer refered to as the pellicle.
The pioneer
bacterial
species adsorb onto the pellicle with
specific ligands and/or
receptors
on their cell
surfaces to form micro-colonies2,3).
The change
of environment
caused by their metabolites
and
1Department

of

Oral

Pathology

, Asahi

University

antibacte-

ability with other bacteria

promote the establishment of secondary colonizers in dental plaque4). The secondary colonizers also actively coaggregate
with different
bacteria, resulting in the formation of mature
dental plaque with complex bacterial flora4,5)
In recent years, much
attention has been
focused on exploration and utilization of plantderived components
and natural products which
possess anti-caries and/or anti-plaque properties because they are expected to be safer and to
show a reliable pattern of activity6-11).Propolis
is a resinous hive product collected by bees that
School

of

Dentistry

(Chief:

Prof.

HIROSHI

TAKEUCHI)
(
:

2Department
of Prosthetic
Dentistry
, Asahi
FUJII)

)
University

(
:
)
31nstitute
of Radioisotope
, Asahi
University
School
(
:
4Research
and
Development
Department
, Yamada
YAMADA)
(
20001211

School

of Dentistry

of Dentistry

(Chief:

Apiculture

Center

(Chief:

Prof.

ATsusHI

Prof.

Incorporated

TERUHISA

FuJITA)
(Chief:

HIDEO

6
contains

various

secreted

plant-derived

beeswax12).

propolis
vary
depending
samples12),
propolis
has
various
tensive
Ikeno
activity
anti-caries
dental
broad
In

mutans

the

present

in Brazil

bacterial

for
study,
was

and
In

may

vivo

addition

to

different
due

of dental

of

propolis-containing

plaque.

to

37.

to

for
the

both
of
formation
The

sample
its

pellicle

There

were

for

activity

as

against

tions

bacteria

room

liquid

subjected

and

radioactivity

system

scintillation

Control
for

30

min

described
were

disks
and

for

Three
the

were
suspenFollow-

washed
to

an

was

Aloka,
measured
(LSC-900,

immersed
was
to

times

automatic

(ASC-113,

counter
were

After

they
cell

radioactivity

above.
run

min.

PBS,

were

then

108
disks

contain30

temperature.

disks

combustion

for

at

per
HAP

with

at

PBS

dpm

radio-labeled

the

cells/mL.

solutions

times
a

at

and

Aloka).

antibacterial

min

Japan)

using

nol

in

reaction,
PBS

Tokyo,

30,000

of
24

in

saliva-coated

propolis

2001

centrifugation

109

ethanolic

disks

30

by

resuspended

The

mg/mL

for

approximately

in
20

the

the

a concentration

approximately

immersed

ing

and

of

and

again

at

condition

harvested

times

immersed
10

20 No.

aerobic

cells.

washing

toothpaste

plaque-forming

were

sample

which
play
and develop-

were

radio-labeled

on
and

cells

washed

col-

effect

an

concentration

with

a propolis

under
The

and

Vol.

England)

Ci/mL

sion

its

five

in

etha-

measured
determina-

experiment.

was

investigated.

Materials

Buckinghamshire,
3

ing

in

show
health

examined

adherence

and

effects

properties.

ment

also

inhibitory

rat.

dental

coaggregation
reaction,
important
roles
in the

cariogenic

the

propolis

activity
biological

lected

in

12-14)

glucosyltransferase

streptococci

potency
caries,

beneficial

the
and

hypo-

properties

reported

on the growth
of

including

anti-inflammatory,

immuno-stimulatory

et a1.15) first

of propolis

of

region
of
to exhibit

activities,

antiviral,

and

and

the components

on collection
been shown

pharmacological

antibacterial,

components

Although

and Methods

1. Propolis and propolis-containing

toothpaste
Propolis
and a propolis-containing
toothpaste
were supplied
by Yamada
Apiculture
Center,
Inc. (Okayama,
Japan).
The propolis
was collected in Brazil and pulverized
and extracted
with ethanol.
The extract was filtered and then
adjusted to 10% (w/v) with ethanol.
The toothpaste
(Yamada
Yohojo
Propolis
Hamigaki
;
Yamada
Apiculture
Center, Inc) contains
the
same 10% ethanolic solution of propolis sample
at a concentration
of 2% (w/w).
2. Effect of propolis on adherence
of Streptococcus San guis to saliva-coated
hydroxyapatite
disks
Hydroxyapatite
(HAP) disks (diameter:
7 mm,
thickness 3.5 mm) were purchased
from Pentax
Co. Ltd., (Tokyo, Japan).
The disks were held
with small surgical
forceps and hung from a
silicon cap, and autoclaved.
Parotid saliva was
collected
from a healthy
male volunteer
and
sterilized
by filtration.
The disks were immersed in saliva for 30 min and then washed 3
times with phosphate buffered saline (PBS ; 0.01
M, pH 7.3).
S. sanguis
ATCC10556 was grown in Tryptic
Soy Broth (Difco, Detroit, MI) containing
[methyl
-3H] thymidine
(Amersham
Pharmacia
Bioteh,

3. Effect of propolis
on the coaggregation
reaction between S. San guis and Fusobacterium
nucleatum
The influence of propolis on the coaggregation
reaction
was investigated
as previously
described16). Briefly, saliva-coated
HAP disks were
incubated in Tryptic Soy Broth inoculated
with
S. sanguis for 5 days.
The medium was exchanged every 24 h. Following
incubation,
the
disk surfaces were confirmed
to be completely
covered with bacterial
cells by scanning electron
microscopy.
F. nucleatum ATCC25586 was grown
in Tryptic Soy Broth enriched with 0.5% (w /v )
Yeast Extract (Difco) containing
[5,6-3H] uracil
(Amersham
Pharmacia
Biotech) at a concentration of 3,2 Ci/mL.
The cells were collected and
washed 4 times and resuspended
in PBS (approximately
109 cells/mL).
The streptococcal
cell-covered
disks were washed
3 times with
PBS and then immersed
in ethanolic
solutions
containing
propolis
(10 and 20 mg/mL)
for 30
min.
One group of disks were immersed
in a
radio-labeled
F. nucleatum
cell suspension
for
30 min without washing,
and another group was
washed 3 times with PBS and then immersed
in
a cell suspension.
Following
the reaction,
the
disks were washed 3 times with PBS and radioactivity
was measured
as described
above.
Control disks were immersed
in ethanol for 30
min instead of propolis solution.

Vol.

20 No.

4.

Antibacterial

total

listed

20

4,

and

Broth

then

resuspended
of

mL.

The

ized

water

medium
10

(5

mg/mL

spotted

v/v

; 1

mg

with

conditions.

tion

was

at

of

plates

1-

were
and

under

anaerobic

inhibitory
the

colony

agar

suspensions
h

as

steril-

Soy

agar

48

minimum

no

with

range

The

for

yield
units/

concentration

defined

which

to

Tryptic

cell

37

our
in

forming

dissolved

step).

was

medium

to

; final

The

(MIC)

the

bacterial

at

of

conditions

colony

added

incubated

tion

in

and

cultures

of propolis

inhibited the coaggregation reaction between F.

nucleatum and S. sanguis by 42.9% (10 mg/mL )
and 56.2% (20 mg/mL) as shown in Table 3.
Both differences were statistically
significant
(p<0.01). When the disks immersed in a propolis
solution (20 mg/mL) were washed with PBS,
the decrease rate was 34.6% (p<0.05).
Propolis-containing
toothpaste inhibited the
growth of 20 strains of cariogenic and plaqueforming Gram-positive bacteria at a concentration range of 3-7 mg/mL, as shown in Table 4.
Strains of S. mutans and S. sobrinus, the major
pathogens of human dental caries, were inhib-

species,

pre-incubated

anaerobic

1 ~108

toothpaste

12

were

under

activity

toothpaste

stock

strains

concentration

the

representing
were

The
Soy

of

strains

Table

laboratory.
Tryptic

anti-plaque

activity

of

in

2001

concentra-

lowest

was

concentra-

observed

Table

after

Formula
toothpaste

incubation.

of propolis-containing

Results
The

formula

Table

1.

(as

of

The

10%

solution

(abrasive),

sodium

E and

present
of

cells

2193,

while

were

10918}210

25.3%

and

0MZ175

those

not

to

the

HAP

of

of
disks

differences

were
also

at

20

mg/mL

Disks

**

dpm

were
; mean

} S.D.
* * * Statistically

shown).
(10

mg/mL

Table

and

Streptococcal

**

dpm

***Statistically

; mean

20

mg/mL)

cell

-covered

3 -5

significant

immersed
of

3-5

significant

in

ethanol

.
determinations

separate

(p<0

also

Effect of propolis
on the coaggregation
S. sanguis
and F., nucleatum.

of

of the adsorption
saliva-coated
hy-

(20

mutans

38.3%

Effect of propolis
of S. sanguis
to
droxyapatite

were

Propolis

Streptococcus
by

propolis

decrease

(p<0.05).

Table

disks.

10295}1099

rates
both

the

14619}

with

and

and

in

adherence

was

treated

mg/mL)

adherence

cells

used
the

disks

The

29.6%,

the

propolis

saliva-coated

significant

reduced

Propolis

of
(10

water.
synthetic

compounds.
the

control

respectively.

statistically

(data

of

that

and
any

inhibited
to

dpm

mg/mL),

2,

strongly

sanguis
average

acid
(humectant),

contain

anti-plaque

Table

study

S.

The

not

or
in

calcium

(stabilizer),

does

shown

in

propolis

2 %w/w),

glycerin

hinokitiol

anti-mocrobial

shown

are

agent),

toothpaste

is

tetradecensulphonic

(foaming

vitamin

As

toothpaste
constituents

ethanolic

carbonate

The

the

major

disks

separate

were

immersed

determinations}S
(p<0

.01)

reaction

****

in
.D.

(P<0.05)

ethanol.

between

.05)

8
Table

Minimum
inhibitory
of propolis-containing
against
forming

cariogenic
bacteria.

concentrations
toothpaste
and

plaque-

Vol.

20 No.

2001

cariostatic
action of propolis
depends
on its
composition,
consequently the region of propolis
samples collections17,18). Thus, we preliminarily
examined
the antibacterial
and anti-glucosyltransferase
activity of the used propolis against
mutans streptococci,
and found that the growth
of 3 strains
of S. mutans
and 1 strain
of S.
sobrinus
was inhibited
at 5 mg/mL
(by paper
disk method) and glucan synthesis was reduced
by approximately
10% at 10 mg/mL
(data not
shown).
In addition
to an inhibitory
activity
against
mutans
streptococci,
the
propolis
sample
strongly inhibited the adsorption
of S. sanguis
and S. mutans
to saliva-coated
HAP disks.
Inhibition
of the adsorption
of streptococci
to
the pellicle has been shown to be an effective
prophylaxic
Given that

against dental plaque f ormation19).

the propolis sample showed marked

precipitation
in water,
nol served as controls.

disks immersed
The observed

in ethaeffect of

propolis is ascribable
to the interaction
between
the pellicle and propolis
components,
an interaction which competitively
masked ligands and/
or receptors on the pellicle against
S. sanguis.
S. sanguis and F. nucleatum
are representative pioneer and secondary
colonizers,
respectively5) .
They form a corncob
configuration
which is frequently
observed in dental plaque20).
Therefore,
coaggregation
between these species
is believed to play an important
role in the
early
stages
of dental
plaque
development.
Propolis used in the present study also strongly
interfered with the coaggregation
reaction.
The
inhibitory
effect of propolis
on the coagited at 3-5 mg/mL,
except for S. sobrinus
6715.
Other mutans streptococci
(S. cricetus
and S.
rattus) were inhibited at 4 and 6 mg/mL,
respectively.
Whereas S. sanguis
ATCC10556 showed
a relatively
high MIC value (7 mg/mL),
other
oral streptococci
including
representative
pioneer species (S. sanguis,
S. oralis and S. mitis)
were inhibited
at almost the same concentrations with S. mutans. Actinomyces
viscosus, A.
naeslundii
and
Lactobatillus
casei
showed
relatively
higher
MIC values
than
those
of
streptococci.
Discussion
Following
the first report of Ikeno et a1,15)
concerning the anti-caries
properties
of propolis,
several
researchers
demonstrated
that
the

gregation
reaction is attributable
to its ability
to mask receptors and/or ligands on S. sanguis
cell surfaces responsible
for the reaction
with
F. nucleatum.
Although
the propolis
sample
used showed
strong inhibitory
effects on both adsorption
of
streptococci
to the pellicle and coaggregation
reaction,
other propolis
sample
may exhibit
altered influence on such interactions
due to the
difference of components12,17,18)
Nishino et al.21)
isolated
3 cinnamic
acid
compounds
from
Brazilian
propolis,
3, 5-diprenyl-4-hydroxycinnamic acid, 3-prenyl-4-dihydrocinnamic
acid and
3-prenyl-4-hydroxycinnamic
acid, and found that
they strongly inhibited the growth, acid production and synthesis
of insoluble
glucan
of
mutans streptococci.
Thus, isolation
and stractural determination
of the substance(s)
respon-

Vol.

20 No.

sible
be

for

the

anti-plaque

observed

required

gating

2001

in
such

inhibitory

the

future,

matters

and

is

currently

Propolis-containing

effects

should

study

investi-

the

growth

forming

bacteria

of

8)

and

mutans,

Agric

Biol

S.,

concentration

range

of

/ mL.

such

The

as

effects

never

bacterial

activity

Thus,

is
been

that

activity

at

tracted

800,
did

not21)

much

lower

than

It

is

11)

mutans
water

The

of

56

plant-derived

ever,
on

the
oral

tory

biological

bacteria

agent

might

against

be

dental

of

useful

as

plaque

H.,

MIC

13)

propolis

2)

P.,

98`196

Chapman

Embery,

et

an

inhibi-

14)

formation.

M.,

interaction

and

oral

et

Kolenbrander,

E.:

oral

Godowski,

bacteria

oral

coloniza-

Japanese

17

K.

ad-

18)

ecosys-

V.

for

Microbi-

S.,

Clin

et

al:

green

19)

coaggregCrit

K.,

et

caries

in

rats.

action
1989

20)

extract

of

1 : 96`101.

Antibacterial
tea

antibacterial
bacteria.

review.

Bee

substances
against

Strep-

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flavonoids
: 471`474.

Isopentenyl
and

28

on

1983

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propolis.

Phyto-

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et

of

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on
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: 303`309.
caries

com-

Honeybee

10

Vol.

in

vitro

20 No.

2001

1,

2,

3,

1,

4,

4,

1,

1,
4

,
Streptococcus

sanguis

S.sanguisFusubacterium

nucleatum

.
.
mL)s.

sanguis

. 10mg/mL
56.2%(p<0.01)
Lactobacillus

(10

(p<0.05)

.
20

42.9%(p<0.01),20mg/mL

Streptococcus,

3-7mg/mL

20mg/

Actinomyces

.
,

.
: