Vol. 20 No. 1 2001 5

Effect of propolis and propolis-containing toothpaste on the

formation of dental plaque in vitro

MASARU SAT01, SHUU FUJIWARA2, 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

6 Vol. 20 No. 1 2001

contains various plant-derived components and

secreted beeswax12). Although the components of

propolis vary depending on collection region of

samples12), propolis has been shown to exhibit

various pharmacological activities, including

antibacterial, antiviral, anti-inflammatory, hypo-

tensive and immuno-stimulatory properties 12-14)

Ikeno et a1.15) first reported the inhibitory effects

of propolis on the growth and glucosyltransferase

activity of mutans streptococci and in vivo

anti-caries potency in the rat. In addition to

dental caries, propolis may show different

beneficial activity for dental health due to its

broad biological properties.

In the present study, a propolis sample col-

lected in Brazil was examined for its effect on

bacterial adherence to the pellicle and

coaggregation reaction, both of which play

important roles in the formation and develop-

ment of dental plaque. The antibacterial activity

of propolis-containing toothpaste against

cariogenic and plaque-forming bacteria was

also investigated.

Materials 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 col-lected in Brazil and pulverized and extracted with ethanol. The extract was filtered and then adjusted to 10% (w/v) with ethanol. The tooth-

paste (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 Strepto-

coccus 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 im-mersed 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,

Buckinghamshire, England) at a concentration of

3 Ci/mL under an aerobic condition for 24 h at

37. The cells were harvested by centrifugation

and washed 4 times and resuspended in PBS at

a concentration of approximately 109 cells/mL.

There were approximately 30,000 dpm per 108

radio-labeled cells. The saliva-coated HAP disks

were immersed in ethanolic solutions contain-

ing 10 and 20 mg/mL propolis for 30 min. After

washing the disks 6 times with PBS, they were

again immersed in a radio-labeled cell suspen-

sion for 30 min at room temperature. Follow-

ing the reaction, the disks were washed 3 times

with PBS and then subjected to an automatic

sample combustion system (ASC-113, Aloka,

Tokyo, Japan) and radioactivity was measured

using a liquid scintillation counter (LSC-900,

Aloka). Control disks were immersed in etha-

nol for 30 min and radioactivity was measured

as described above. Three to five determina-

tions were run for the experiment.

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 de-scribed16). Briefly, saliva-coated HAP disks were incubated in Tryptic Soy Broth inoculated with

S. sanguis for 5 days. The medium was ex-changed 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 concentra-tion of 3,2 Ci/mL. The cells were collected and washed 4 times and resuspended in PBS (ap-

proximately 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 radio-activity was measured as described above. Control disks were immersed in ethanol for 30 min instead of propolis solution.

Vol. 20 No. 1 2001 anti-plaque activity of propolis 7

4. Antibacterial activity of the toothpaste

A total of 20 strains representing 12 species,

listed in Table 4, were stock cultures of our

laboratory. The strains were pre-incubated in

Tryptic Soy Broth under anaerobic conditions

and then resuspended in the medium to yield a

concentration of 1 ~108 colony forming units/

mL. The toothpaste was dissolved with steril-

ized water and added to Tryptic Soy agar

medium (5 % v/v ; final concentration range of 1-

10 mg/mL ; 1 mg step). The agar plates were

spotted with bacterial cell suspensions and

incubated at 37 for 48 h under anaerobic

conditions. The minimum inhibitory concentra-

tion (MIC) was defined as the lowest concentra-

tion at which no colony was observed after

incubation.

Results

The formula of the toothpaste is shown in

Table 1. The major constituents are propolis

(as 10% ethanolic solution ; 2 %w/w), calcium

carbonate (abrasive), tetradecensulphonic acid

sodium (foaming agent), glycerin (humectant),

vitamin E and hinokitiol (stabilizer), and water.

The toothpaste does not contain any synthetic

anti-mocrobial or anti-plaque compounds.

As shown in Table 2, the propolis used in the

present study strongly inhibited the adherence

of S. sanguis cells to saliva-coated HAP disks.

The average dpm of control disks was 14619}

2193, while that of those treated with propolis

were 10918}210 (10 mg/mL) and 10295}1099 (20

mg/mL), respectively. The rates of decrease were

25.3% and 29.6%, and both differences were

statistically significant (p

8 Vol. 20 No. 1 2001

Table 4 Minimum inhibitory concentrations

of propolis-containing toothpaste

against cariogenic and plaque-forming bacteria.

ited 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, respec-tively. Whereas S. sanguis ATCC10556 showed a relatively high MIC value (7 mg/mL), other oral streptococci including representative pio-neer species (S. sanguis, S. oralis and S. mitis) were inhibited at almost the same concentra-tions 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

cariostatic action of propolis depends on its

composition, consequently the region of propolis samples collections17,18). Thus, we preliminarily examined the antibacterial and anti-glucosyl-

transferase 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 against dental plaque f ormation19). Given that the propolis sample showed marked

precipitation in water, disks immersed in etha-nol served as controls. The observed effect of

propolis is ascribable to the interaction between the pellicle and propolis components, an inter-action which competitively masked ligands and/ or receptors on the pellicle against S. sanguis.

S. sanguis and F. nucleatum are representa-tive pioneer and secondary colonizers, respec-

tively5) . 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 coag-

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-hydroxycin-namic acid, 3-prenyl-4-dihydrocinnamic acid and 3-prenyl-4-hydroxycinnamic acid, and found that they strongly inhibited the growth, acid produc-tion and synthesis of insoluble glucan of mutans streptococci. Thus, isolation and strac-tural determination of the substance(s) respon-

Vol. 20 No. 1 2001 anti-plaque activity of propolis 9

sible for the observed inhibitory effects should

be required in the future, and a study investi-

gating such matters is currently underway.

Propolis-containing toothpaste completely

inhibited the growth of cariogenic and plaque-

forming bacteria at a concentration range of 3-7

mg / mL. The components other than propolis

such as hinokitiol never showed inhibitory

effects on bacterial growth at concentrations in

the toothpaste. Thus, the observed antibacterial

activity is mainly attributable to propolis. It

has been reported that ethanol extracted propolis

showed antibacterial activity against mutans

streptococci at 800, g/mL, whereas water ex-

tracted propolis did not21) . The observed MIC

value is much lower than those of previously

reported plant-derived compounds7,9,10,11) How-

ever, the broad biological activity of propolis

on oral bacteria might be useful as an inhibi-

tory agent against dental plaque formation.

References

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10 Vol. 20 No. 1 2001

in vitro

1, 2, 1,

3, 1, 1,

4, 4, 4

,

Streptococcus sanguis S.sanguisFusubacterium nucleatum

.

. (10 20mg/

mL)s. sanguis (p