Protective Effect ApplePolyphenolson Rampant Caries

The Japanese Society for Dental Health

NII-Electronic Library Service

TheJapaneseSociety for Dental Health

MfiigastskE'la･ JDentHtth58/113-124,2008

Originaltii

Protective Effect of ApplePolyphenolson Rampant Caries in Hamsters

Fumio MATSUDAIRA, Jose Geraldo de Oliveira CORDEIRO,

Hidenori YAMADA and Akio YANAGIDA*

Abstraet: The effect of apple polyphenols {APP) on rampant caries was investigated in hamsters, Sixty male hamsters

were divided into 3 groups and all orally infected with Streptococcus mutans. For a 12-week period, all animals received the

cariogenic diet 2eOO (5696 of sucrose), At the age of 42 days, two groups received a supplement of powdered APP obtained

fronn immature applcs at low (O.05% APP) and high (O,296 APP) concentrations, and one group served as a control {SUgroup}. Plaque extent and caries destructiveness were assessed using a O-5 scoring reference. In all moLar surfaces, an in-

creasing earies protection was found in the APP groups. The score distribution fer plaque and caries per hamster was

most significantly reduced in the O,2% APP group. Categorically, significant differences in plaque scores and in caries

scores existed between the O.0596 APP and SU groups (p=O.O03), and the O.29t) APP and SU groups (p<O.OOI), but net be-

tween the two APP groups, During the animal experiment, the iiumbers of mutans streptococci in colony forming units

(CFU)fml and their relationships with the numbers of caries score 5 per hamster were determined in three bacterial re-

coveries: MS Recovery-1 (prior to treatment with APP), MS Recovery-2 (in the middle of lhe experiment with APP) and}vlS Recovery-3 {prior to sacrifices). At }L{S Recovery-2, positive assocititions were found in the SU group (r= O.67: p< O.OOI),

in the O.05% APP group <r =: 0.59; p<O.Ol> and a negative association existed in thc O,2% APP group (r = -

O.18; ns). At MS

Recovery-3, decreasing pesitive associations were found in the SU (r =: O.76: p<0,OOI), O.05% APP (r =O.30; ns} and 02%

APP (r=O.17: ns) groups. The results suggest that APP affected the numbers of mutans streptococci in CFUfml, con-

trelled plaque forination and prevenLed extensive caries destruction.

Key words: Apple polyphenols, Mutans streptococcL Dental plaque. Rampant earies, Prevention

J Dent Hlth 58: II3-I24, 2e08

(Received: September 27, 20061Accepted: January 22, 2008)

Introduction

The cariogenic combination sucrose-mutans strepto-

cocci leads to a rapid and progressive destruction of the

tooth surfacesi). A very destructive co"dition whieh af-

fects both the primary and permanent dentitions hasbeen defined as rampant caries2L For adolescents and

adults, rampant caries is manifest in population minori-ties3). Since long ago, this characteristic pattern of caries

seems to affect humans and many factors have been as-

sociated with its development`). At a higher risk of de-

veloping rampant caries are those individuals living in

areas hampered by bad socioeconomic indieators and

whose families cannot afford dental care clue to low fam-

ilyincome5),

Possible caries-protective compounds other than fiuo-

ride are present in p]antsS'7). Phyto-metabolites also

kiiown as polyphenols are naturatly occurring antioxi-

dailt substances most cummonly found in the skins of

many fruits, vegetables and herbs. Over the past two

decades, an increasing number of studies have shownthat the polyphenols present in leaves of tea have antim-icrobiat propertiesS), Tea polyphenols have shown caries-

preventive effects both in vitro and in vivo studies-iU.

DepartmenlofPreventiveDentistry,TsurumiUniversitySchoelofDental)Iedicine.Yoknhama,Japan'DivisionefStructuraLBiologyandAnalytiealScicncc.SchootofPharmaeyTekyoUmivcrsityoH)harmacyandLifeSciencc,Tokyo,Japan

II3




MvezaEkki'S JDentIIIth58(2),2008

Like tea, some fruitjuices andbeverages such as cocoa

and coffee have also exhibited anticaries properties'2).

Recently, it has been found that the phenolic content ef

immature apples is about 20 times higher than that of

edible mature onesB). Polyphenols from immature apples

have inhibited the cellular adherence of mutans strepto-

cocci in vitroi`) and human plaque formation in vivoi5}.

With exception of the direct effect of iruits on caries

produced in animals, information on the relationship be-

tween fruit polyphenols and caries prevention is un-

known.

The aim of this study was to investigate the effect of

apple polyphenols (APP) extracted from immature ap-

ples on the development of rampant caries in hamsters

subjected to an extreme cariogenic challenge with mu-

tans streptococci and a sucrose-rich diet.

Materials and Methods

Applepolyphenols

The APP compounds were provided by the Institute

for Production Research and Development of Nikka

Whisky DistilLing Co., Ltd. All procedures for the extrac-

tion and sample preparation of APP as well as informa-

tion on the chemical structures of these compounds are

describedelsewherei:･i'LiS).

Bacteriapreparation

Bacterial strains of Streptococctts mutans NCTCI0449

suspended in Brain Heart Infusion (BHI) (Wako PureChemical Industries, Lrd,, Tokyo) broth were incubated

at 37℃ for 18 hours. Aftcr incubation, the broth was

centrifuged at 6000 r.p.m. for 15 minutes, The superna-

tant fluids were discarded, and the sediment solutiens

were repeatedly mixed gently and washed with steril-

ized O,85% saline by means of a pipette, and then centri-

fuged. Final solutions with the bacterial strains (approxi-mately 10S colony forming units (CFU)fml) suspended in

saline were obtained and stored at L

20℃ . These proce-

dures started 3 days preceding the animal experiment

and were repeated for a few days afterwards.

Animal experiment

The schedule of the animal experiment is presented

in Fig. 1. Sixty newly weaned 21-day-old male golden

hamsters were obtained from Saitama Experimental

Animals Supply Co., Ltd, weighed. and distributed into

cages made of solid plastic walLs with a stainless steel

114

bottom and a mesh lid. On day 1, all animals received

sierilized distilled water containing a solution of O.02%

streptomycin and were fed a solid stock diet CE-2 (CleaJapan, Inc.) for about 24 hours, On day 2, all the animal$

were skin-marked with picric acid and divided into 3

groups (2'3 animals per cage} having similar body

weights (mean weight =

50.2 ± 3.1 g}.

Then, all animals were given a powdered caries-

promoting diet 2000i7', provided by Clea Japan, Inc. Af-

terwards, all animals were each infected orally with

streptomycin-resistant strains of S, mutans NCTCI0449.

Disposable mini-syringes were used to instill approxi-

mately 02 ml of the fluids containing S, mutans into the

mouth and cheek pouches of the animals. A micro-

applicator with a cotton pellet on the tip was also used

to spread the fluids containing the bacterial strains over

alt of the animals' molars. These procedures were re-

peated daily for 6 consecutive days. In addition, O.4 rnl of

the strains was added to 200 ml of drinking water and

given to all animals for about 48 hours, After the bacte-

rial challenge was ovcr, sterilized distilled water was

made available ad libitum to all animals. Cages were

changed once a week, the animals' weight was meas-

ured weekly, and diet and water intakes were moni-

tored every day. All animals were fed the diet 2000 until

they were 105 days oicl {Fig. 1).

Bacterial recoveries

A period of 12 days was allowed for the implantation

and recovery of the cariogenic baeteria. On day l4,

plaque samples were taken by swabbing the molar

teeth of the animals with a cotton pellet soaked in 1 ml

of 1% peptone sotution {pH 7.0). The presence of mutans

streptococci was then confirmed by the selective me-

dium mitis salivarus agar {DIFCO Laboratories, Detroit,

Michigan, USA) containing 5% sucrose and bacitracin

(O.2 u/mt). Additionally, oral swabs were taken again 3

times respectively when the animals were 41, 74 and 104

days old. Later, the numbers of CFU/ml of S, mutans in

the groups were assessed for each occasion,

Diet preparation and experimental groups

Extracts of APP were obtained from the juice of im-

mature apples and transiormed into powder by a spray-

drier, APP at concentrations of O.2% and O,05% were

then added to the diet 2000 by Clea Japan, Inc. At the

age of 42 days, all animals' body weight were measured




Hamsters'ageindaysProcedure

212223242526272829ttt34ttt4142tttttt74ttttttttt104105106107ant

I-Iousing+CE-2diet+O.02C/)streptomyeininthedrinkingwater

wdits#lvaw}wWl'

Skin-marking+divisioninto3groupswithts.ffpt lsimilarbedyweight tevegew

InocutationofS,mutans gemde

･"e-vademasas

(O.2mlfhamsterwithmini-syringes)$.-.-.rgvaeWmsuge eutde /

edi"

InoculationofS,'mutans :ge:: l(O.4mlf200mlofthedrinkingwater) vabffe.tstevwnypt-"rt

1Feedingwithdiet2000(allanimals) JL1veem!ere'1/wavet.vat.uat1-./:･"tte$.x'eeg:mdi-"tidiefiut･ldi..:enttajdileffm-emesxstebem::di-wde-t;le'taj.t::di-vaesva-scAv:vedieevee#･:za･e::edi1:/"vaww･ee-¢evermvawpt-eveevth1 leenpt

Checkingofbacterialimplantation I/ .e-efeermt

Takingofplaquesamples(MSrecovety1)1111 s';･Balancingofbodyweightinthe3groups -.tevee.tevaWvenn･t･#tt.tt

TreatmentwithAPP(addedtothediet2000of2groups)

//:1..;tMnfiw::i$:;;::::1vain.nyt..didiee:1･/e//vt-sutw-dete･estWVI.t･-ab#aleaeeevttt

Takingofplaquesamples(MSrecovery2) 1,;ee

Takingofplaquesamples(MSrecovery3) /:k'..t'e4-tti1t.t.t

Lastmeasurementofbodyweight 1 f,lf,l-ttt･t.ort

Sacrificeswithch!oroforminhalationandre- esut"t-sltee･stgedi

movalofjaws il:;'''ttse･tttt

Note: Diet and water were

was measured weeklv. "given

ad libitum and monitored every day; cages were changed

Fig.1 Schedule of lhe animal experiment

once a week; body weight

and 3 new experimental groups (20 animals each) with

balanced body weight (mean initial weight=96,8 ± 8.6 g)

were formed as followsi

I. SU group (control group with diet 2000 only, 56%

sucrose)

II. O.05% APP group (low polyphenolic group. 56%

sucrose + O.05% APP)

III. O.2% APP group (high polyphenolic group, 56%

sucrose +O.2% APP)

Smooth surface plaque extent

Animals were killed by chloroform inhalation at 12

weeks after the bacterial challenge, at the age of 106-

107 days.

Immediately after each animal was killed, their jawswere dissected out, slightly rinsed in tap water to re-

move blood, and stored moistened in sealed plastic jars.Afterwards, each section containing the 3 molar teeth

(hereinafter: quadrant> of the upper and lower jaws

were stained with a 1% solution of Neutral Redi8) (WakoI'ure Chemical Industries, Ltd. Osaka), Then, each

stained quadrant was immersed in water and shaken for10 seconds, dried with absorbent paper, and puffed with

compressedair.

The procedures to examine atl the teeth undera rni-

croscope were as follows,

The maxitlary and mandibular quadrants were

mounted on blocks of utility wax fixed to glass Petridishes and examined under a stereo-rnicroscope at mag-

nifications of 10 × to 20 × ,

Then, each quadrant was positioned about 1 cm paral-

lel to the stage, and photographs of their buccal, lingual

and occlusal aspects were taken at magnifications oi

l5x to 20 × .

At the same time, to facilitate further checking, a

video camera with a zoom function was also used. All

the molar surfaces were filmed and the video images

1.l5




Nnl th tilk'tifi J Dent Hlth 58 (2}. 2008

PIaqueScoresReference(O-5)

ScoreOScore1Score2Score3 Score4SeereS

o o o e. ee)deNoplaque Smallareawithplaque(E])Smallareascovering<V3ofthesuhaceeLargeareascovering>V2ofthe$uhace(2ormore

Almostcompletelyceveredwithplaque

Cenlyone$malr,cleanportion)

Comp[etelycoveredwithplaque(necleanportion)

cleanportions)

CariesScoresReference(O-5}

ScoreOScore1Score2Score3 Seore4Score5

o o (e)e.e eNocarles Smalicatity(!E)Smail(!DLange

A[mostcompletelydestroyedCompletelyde$troyed{nointact

eatitjes cavities bycariesport]on)

oracavity>li2ofthe(enlyonesmall,

<1/3efthesuface intact

surface(2ormereportion)

intactpertions)

Fig.2 Scoring references from O-5 for recerding

and caries sevcrity on the molar surfacesplaque

accumulation extent

were transferred to a computer using a video-eapture

software.

Later, the images were color-printed and the scores

were checked and calibrated.

Dental caries

After the plaque accumulation had been scored, the

upper and lower jaws were autoclaved and gent]y

cleaned to remove soft tissues and debris with a small

soft toothbrush under tap water.

PIaque on the molar surfaces was removed by scrap-

ing carefully the molar surfaces with a sharpened dental

explorer. Then, the cleaned jaws were placed into small

bottles containing a O.0696 murexide solutioni") (Wako

Pure Chemical Industries, Ltd., Osaka) and stored in a

dark room for 24 hours.

Later, the murexide-stained samples were genily

washed once in tap water and dried at 50℃ in an oven,

The procedures to examine the teeth under a micro-

scope and to facilitate further checking were similar to

those described above, Furthermore, only the stained

red areas which could be detected with an explorer

116

were recorded as caries.

Plaque and caries scores

For scoring the smooth surface pLaque extent and car-

ies severity. the 0-5 scoring reference shown in Fig. 2

was used. In each hamster, 12 molars were scored,

which corresponded to 36 suriaces (buccal, lingual and

occlusal}, Thus, the maximum score per animal would be

180. Based on past experience20}, an effort was made to

use scores in a O-5 scale according to the extent of

plaque accumulation and destructiveness by caries, As

illustrated in Fig. 2, for seoring plaque and caries, a

Score O was given to surfaces with no plaque at all or

caries-free, respectively. Likewise, Score 1 was given to

surfaces with only a very smatl area (up to 1 '5

of the

surface) covcred with plaque and incipient caries. Score

2 was given to plaque covering less than 113 of a surface

and mild caries; Score 3 for grades ranging from moder-

ate to severe -'ith at least 2/5 of a surface being cLean or

intact; Score 4 was given to areas showing about V4 of

the surface being clean or intact: Score 5 was given

when all the surface was covered with piaque and com-




Table 1 Animals'growth conditions in each group

Experimental Group

Number of

animalsInitial

weighr (g)Weight gain (g)

MeanSD pt,Iean

suO.05(,,6APP

O.2%APP

zo2017 96.896.996.89,19,27.985.389.386.5

SD17.415.013,6

pletely destroyed by caries (Fig. 2},

Statistical analysis

To compare the numbers of surfaces affected by

plaque and caries in the groups, both parametric and

non-parametric methods were used. For the evaluation

of the tooth surfaees affected according to their localiza-

tion (e.g., buccal, lingual, or oeclusal surfaces) the scores

for plaque extent and caries severity were anatyzed sta-

tistica]ly using a complete randomized Analysis of V' ari-

ance (ANOV'A). Thc Kruskal-Wallis test was used to

compare differences in categorical data. Posz-hoc com-

parisons (Scheffe test; Tukey HSD test} were also used

when necessary. The same procedures were used for

comparing the recoveries of mutans streptococei duringthe experiment.

Additionally, linear regression was used to determinethe relationships between caries score 5 and the recov-

eriesofmutansstreptococci.

All tests were periormcd using a version 14 of SPSSfor Windows (SPSS Inc., Japan).

Results

Animals' growth eonditions

All animals remained healthy throughout the experi-

ment Accidentally, however, 3 animals from the O.2%APP group were lost one week prior to sacrifices. Thus,the number of hamsters in the O.296 APP group was re-

duced to 17.

The experimental groups, number ef animals per

group and the animals' growth conditions in each groupare given in Table 1. 0verall, the mean body weight

gain was 86,3± 15.1 g. Furthermore. body weight gain

was almost similar within the three groups and no sta-

tistically signifieant difference was found among the

three groups (Table 1).

Plaque and caries scores by surface localization

The mean scores for plaque extent and carics sever-

ity by moLtth site in each group were calculated for thewhole mouth by quadrant (MLM2M", by molar (M], M:, M3),and by rnolar surface (BuecaL Occlusal, LinguaD. Overalldata showed a tendency toward lower p]aque scores

and lower :aries scores for all molar surfaces in the APP

groups, particular]y in the 0.2% APP group,

For the whole niouth, out of a possible maximum

score of 180 per animal, the plaque scores {mean± SD)were 128.5± 29.3, l20.7± 23.3 and 114.8± 20,4, respee-

tively for the SU, O,0596 APP and O.2V6 APP groups.

Likewise, respectively, the caries scores {mean± SD)

were 124.1± 302, 115.6±･ 25.2, and 109,1± 21.4, for the

SU, O.05% APP and O.296 APP groups. Analysis of V'ari-

ance (ANOVA) found no significant differences when

the ptaque scores and caries scores were compared be-tween the groups.

Vlaque and caries scores regardless of surface local-

ization

Tables 2 and 3 .cr. ive the distribution of the number of

affected surfaces according to the plaque and earies

scores frem 5-0 for each group, respectively. and show

the statistical relationships obtained from the Kruskal-

Wallis and Tukey HSD tests for comparisons among the

.oroups.

Plaguc cxtent

The pcrcentages of plaque score 5 per group were

35.7%, 29.6% and 27.6% for the SU O.05% APP, and

O.2% APP groups. respectively. Likewise, the percent-

ages of plaque score 4 for each group were also highestin the SU group, and lowest in the O.296 APP

.vroup. In

contrast the pcrcentages of plaque score 2 per groupwere 15.4e/6, 1829,o' and 24.3% for the SU, 0.05% APP

and O.2% APP groups. respectively. The percentages of

animals with a plaque score 4 were highest in the SU

group (17.8%> and lowest in the O,2% iXPP group

(13.9%). The hi.ahest percentages of plaque score 3 were found

li7




[[n2th'th ft aS J Deiit Hlth 58{Z),20e8

Table 2 Distribution of affeeted surface nurnber according to the plaque scores in each group

Experimenlal Group ,lNumbers

of Plaque Scores {%)

5257

(35.7)

213 (29.6)169 (27.6)

3 2 1O Totalp-value

suO.05%ilPP

O.296APP

128([7,8) 224(3Ll) 111(15,1)

104(14.4) 269(37.4) 131(18.2)

85 (1:l.9) 209 (34.2) 149 (24,3)32).4)

?/ ;'i: =P=OO03 ]p<oooJ p<oooi o o 612 L･ p= e･16,1 1 J ,

Notes: p= p-values obtained by TLtkey HSD test comparisons; P =p-value

obtained by the Kruskal-"' allis test

Table 3 Disrribution of affected surface number according to lhe caries scores ineach greup

Experimental

Group

Numbcrs of C]aries scores (9.t)

5 4 3 2 l oTotalp-value

suO.059,6APP

O.2%APP

2:ll(32.1) I:i2(l8.3) 231(321) I07(14.9)

193(26,8) I08(i5,O) 261(36.3} 125(17.4)

148(24.2) 9104,9) 200(32.7) M5(23.7)

13(1,8)22

(3,1)16 (2.6)12

[?I:.l i:: ! p - OO03 1 p < o.ool l p < o.ool12 (2.e) 612 lp=O･2461 1

Notes: p = p-values obtainedby TLikey HSD test comparisons: P

in the O,05% APP group. No group had ptaque score O

and plaque score 1 were praetically inexistent in the

three groups (Tabte 2).

The Kruskal-Waltis test found a high[y significant dif-

ference (p<O.OOI) am{}ng the groups. Pesr-hoc compari-

sons with the Tukcy HSD test found significant differ-

ences between the StJ and O.059G APP groups {p == O.O03)

and the SU and 02% AI'P groups (p<O.OOI), but not be-

tween the O.05% APP and O.2% APP groups {p-O.164)(Table 2).

Cariesseverity

The distribution of caries scores per group was simi-

lar te that described above for plaquc extent. Of the

three groups, the O.2% APP group had the towest num-

ber of caries score 5 (most severe caries) and the highest

number of caries score 2 (mild caries). The percentages

of earies score 5 per group were 32,1%, 26.8% and

24.2 96 for the SU, O.05% ,ALI'P and 0296 APP groups, re-

spectively. The percentages of caries score 4 were also

highest in rhe SU group (18.3%) and lowest in tha O.2%

APP group (14.9%). Conversely, the percentages of car-

ies score 2 per group were 14,99'6, 17.4?6 and 23.7% for

the SU, O.05% APP and O.2% APP groups, respectjvely

(Table 3).

Among the groups, the KruskaLWallis tcst found a

highly signiiicant difference (p<O.OOI). Tukey HSD test

comparisons found significant differences betwcen the

118

= p-value obtained by the Kruskal-Wallis rest

Fig. 3 Exarnples of caries scores (Csc) on a O-5 scale of se-

veritv found in some lower molars of the SU and

high polyphenolic (O,2% APP) groups

Frem the first lower molars on the left to the third

molars en the right, total scores for the three mo-

lars were 12 for the SU group and 7 for the O,2%

APP group

SU and O.0596 APP groups (p==O.O03) and the SU and

e.296 AI'I' groups (p<O.OOI), but not between the O.05%

APP and O.2% APP groups (p = O.246) (Table 3}.

Fig. 3 shows some examples of caries scores for the

molar surfaces of animals in the SU and O.2% APP

groups. In tbe 02% APP group, dental caries was less

dcstructive than in Lhe other two groups (Fig. 3}.

Recoveries of mutans streptecocci

'I"he

mean numbers of colony forming units (in log



TheJapaneseSociety forDentalHealth

1'able4 Bacterial recoveries of rnutans streptococci in log CFUhn

group throughout the eiperiment

l for each

Experimental Group

MS Recoverv 1MS Reeoverv 2MS Recovery 3

Mean (SE) Mean {SE) Mean (SE)

su0.05%APP

O.29,tAPP

6,40 {O.10)

6.52 (O.11)6.72 (O.08)

6.68 (e.15)

6.14 (O.14)5.94 (O.09)]**7.02

(O.18) 16A2 {O.l 5) 1 '

6.48 (O.10)]*

Note: Scheffe post-hec comparisons: ' p < O,05, "

* p < 0,Ol

gne

s,s:9

s.o2

7,s9 T.o{

S.5i

6.o:

:I: 4.5

MSRec2uCs5xSMceunts･SU

o5riO d5 20 25

CariesSeereS30

35

9.0e

8.5r. s.o-e=

7SL9

7ne

6.5:g

6.0:

fi,5

sn

4,5

MSRec2-Cs5x$Meounts-e.OS%APP

osIC l5 2e 2S 30 35

Caries Score 5

g.oS S.5roA

S.O2 zs9

7.0nE

6.s8 6.o$

5.5

5,O

4.5

MSRec2･Cs5xSMceunts-O.2-6APP

o5le a5 20 25 30 35

CariesScore5

9.og

e.sg

s.oE

zse

7.oi

6.58

6.o:

5.E

5.0

Fig. 4

MSRec3-Cs5xSMceunts-SU

9.0e

s.sT.-

8,O!=

7.SLY 7,OS 6,5i

s.ogE

S.Se s,o

4,s

O 5 10 15 20 25 30 35

Caries Score 5

ReLationships betwcen the numbers of

log CFUfml) o

MSRec3-Cs5xSMceunts-O.059tAPP

05fO 15 20 25 30 35

Caries Scere S

9,OS

S.5'm'

8,Og=

7.5L9

7.ee

6.5S 6.0g 5.5i 5.0

4,5

MSRec3-Cs5xSMceunts･O.2g6APP

05tO tS 20 2S 30 35

Caries Seote fi

caries score 5 per hamster and the mean numbers of colony forming units <inf mutans streptococci at MS Recovery-2 and MS Recovery-3 during the anirnal experiment

CFUIml) o[mutans streptococci are given in Table 4.

Data are shown for the three bacterial recoveries

which were checkecl during the animal experiment: MS

Reeovery-1 (prior to treatment with APP), MS

Recovery-2 (in the middle of the experiment with APP)

and MS Recovery-3 {prior to sacrifices). At MS

Recovery-2, significant clifferences existcd bctwecn theSU and e.2% APP groups (p<O,Ol). At ),IS Recovery-3,

significant differences were found between the SU and

O.05% APP groups {p<O.05}, and SU and O.2% APP

groups (p<O,05} (Table 4).

Additionatly, the relationships between the numbers

of caries score 5 per hamster and thc numbers of rnu-

tans streptococci at MS Recovery-2 and at MS

Recovcry-3 were determined and are illustrated in Fig.

4.

At MS Recovery-2, fairly high positive associations

were found for the SU group (r = O.67; r 2= O.45; p< O.OOI}

and for the O.05% APP group (r = O.59; r2=O.35; p<O.Ol),

A low negative associatien was found for the O.2% APP

group (r = -

O.18: r 2=

O.03; ns).

At MS Recovery-3, a high positive association was

found for the SU group (r =0.76; r Z=

O.58; p<O.OOI). Forthe two APP groups, low positive associations were

119




Utigasthft:ri'S JDentHlzh58(2),2008

found for the O.05% APP group (r=O.30; rL'=70.09; ns)

and the 02% APP group (r = O.17; r 2 --

O.03: ns} (Fig. 4).

Discussion

Caries protection by fruit polyphenols

In this study, we tested the possible caries-protective

effect of APP extracted from immature fruits on the de-

velopment of rampant caries in hamsters subjected to

an extreme cariogenic challenge. APP demonstrated a

caries-protective eifect that may be of interest. Appar-ently, APP either at a lower concentration (O.0596 APP)

or at a relatively higher concentration (O.2% APP) were

effective in retarding caries progression. Thus, APP

might be useful for eontrolling the development of ram-

pantcarles.

Furthermore, in this study, plaque formation and car-

ies destructiveness were significantly reduced in both

APP groups. These findings are in Iine with those from

our previous piloz animal experiment (unpublished data),wherein APP at the same concentrations as used in thisstudy were added to the drinking water of hamsterssubjected to a shorter and Iess cariogenic challenge, as

compared with the one of the present study. Lower car-

ies levels were observed in both APP groups regardless

the APP concentration, as compared with a high su-

crose group.

Scoring rampant caries

To study the development of rampant caries in ro-

dents, the harnster model seems appropriate. This is be-cause, in shape, the hamsters' molars are more similar to

those of humans than those of rats. Further, when ihe

hamster is fed a sucrose-rich diet and is infected with S.

mutans, large amounts of plaque are formed on the mo-

lar surfaces. The same phenomenon rarely happens in

ratsi')).

In caries studies with animals, different scoring meth-

ods have been used. A scoring method which has been

comrnonly described uses grades of caries severity

ranging from O to 4. Little effort has been made, hew-

ever, to differentiate in detail the development of ram-

pant caries in animals. For this purpose, the present

study used a Or5 scoring reference, whieh was atso espe-

cially useful and effective to score plaque accumulation

in the hamster. Moreover, the present study used two

different approaches for scoring the deveropment of

120

rampant caries in the animals. One approach was deter-

mination of the mean plaque scores and mean caries

scores by surface localization. The other approach was

screening of the distribution of all scores regardless of

surfaceIocalization.

In this study, by surface localizat.ion (e,g., B,O,L and

BOL), a tendeney for lower mean plaque and caries

scores was noted in both APP groups compared to the

SU group, but no significant differences existed between

the three groups. On the other hand, regardless of sur-

face localization, from the score distribution in all ani-

mals, it became apparent that the numbers of scores per

hamster in both APP groups were significantly lower as

compared with those of the SU group. Thus, the use of

these two approaches seems to be reasonable and useful

for scoring rampant caries in animals, because of the

great variability in earies susceptibility found in ham-

sters subjected to such extreme cariogenic challenges.

Furthermore, evaluation of rampant caries in anima]s

has not been investigated probably because most animal

experiments have been terminated in periods up to 8

weeks after a cariogenic chalLenge. In this study, an at-

tempt was made to extend the animal experiment un-

der a severe cariogenic challenge to 12 weeks, in order

to distinctly score the development of rampant caries inthe animals.

Strains of S, mutans NCTCI0449 were tested to de-

velop root surface caries in hamsters over a 24-week pe-riod20) and moderate caries were observed after 8 weeks.

Likewise, in this study, the challenge period was longer

than 8 weeks for all the animals. Although animals of

two groups at the age of 42 days were supplemented

with APP for a 9-week period during the challenge with

S. mutans and diet 2000, only in the high polyphenolic

group the numbers of score 3 were slightly lower thanthose in the SU group, Apparently. even after APP was

added to the diet 2000 of the two groups, moderate car-

ies could not be slowed ever a 12-week period of severe

cariogenic chalLenge (Fig. 1; Table 3).

Mutang streptococci and caries score 5

In this study, contrasting with the O.2% APP group,

the mean numbers of cotony forming units {in log CFU/

ml) of mutans streptococci slightly increased in the SU

group during the animal experiment (Table 4). More-

over, in the SU group, moderate positive associations ex-




isted between the numbers of caries score 5 per ham-

ster and the numbers of mutans streptococci at MSRecovery-2 (r=O,67; p<O.OOI} and MS Recovery-3 (r=O.76; p< O.OOI}, with the points in the scatter plot roughly

concentrated around a straight tine through the data

<Fig. 4).

In the O.05% APP group, moderate to Iow positix,e as-

sociations existed between the two variables at MSRecovery-2 (r=O,59; p<O.Ol) and MS Recovery-3 {r==O.30; ns) and the relationship turned out to be non-linearthrough the data. The non-linear relationship between

the two variables became weaker and more apparent inthe O.2% APP group (Fig. 4), In the O.2% APP group, at

MS Recovery-2, a negative trend was found (r= -O.18;

ns} and at MS Recovery-3 practically no correlation (r =

O.17: ns) existed between the two variables, This sug-

gests that APP added to the high cariogenic diet 2000

might have somewhat affected the relatienship between

the numbers of caries score 5 per hamster and the num-

bers of CFU ef mutans streptococci. Furthermore, these

findings suggest that APP might have some caries-

protective properties that may help control rampant 'carles.

Effect of APP on dental plaque

In this study, plaque formation was reduced in boththe APP groups, This finding is in tine with data from

our previous clinical trial. Matsudaira et al. i5) tested the

effect of O.05% APP on human plaque formation in two

groups of 19-yr- and 20-yr-old girls. The subjects washed

their mouths with O.05% APP solution or a placebo, The

subjects did not use a toothbrush for a periocl oi 3 days

and refrained from any other oral cleaning measures.

Significantly lower plaque scores were found in the sub-

jects who had rinsed their mouths with O.05% APP solu-

tion as compared with the placebo.

Effect of APP on cellular adlierence of mutans strep-

tococci

In this study, the APP added to the diet 2000 (56% su-crose} did not favor an increase in the numbers of mu-

tans streptococci for developing rampant caries duringthe experiment. Because the concentration of APP was

inversely related to the numbers of mutans streptococci,

lower caries scores were found in the APP groups com-

pared with the SU group.

Sucrose has been considered as the culprit in the de-

velopment of dental caries2L). Thc cariogenicity of su-

crose and the important role played by the bacterial en-

zyme gluocosyltransferase (GTF) to mediate the adhe-

sion of sucrose exposed to S. mutans and plaque in vivo

were well-explained by Rella2i).

The inhibitory mechanisms of APP on cellular adher-

cnce of mutans streptococci were studied in vitroi3} andit was found that APP of high molecular weight inhib-ited the cellular adherence of mutans streptococci by in-

terfering with the binding of GTF from cariogenic bac-teria such as S, sobrinus and S. mutans. Both APP and ap-

ple condensed tannins {ACT) markedly inhibited the cel-

lular adherence of growing cells, and their inhibition in-

tensity was dependent on the sampie concentration in

the APP mixturei4}.

Pelyphenols, apples and caries

Polyphenels present in fruits have been extensively

studled22i, The relationship between the content of these

compounds and caries protection has bcen only recently

investigated by Yanagida et al. iS),

who found that the to-tal polyphenolic content present in immature apples (av-erage weight of each fruit was about 10 g) is approxi-mately 20-fold higher than that of edible mat.ure ones

(average weight of each fruit was about 300 g).

Over the past four decades, the cariogenicity of foodsand fruits has been extensively studiedTL However,there is still a paucity of information on protective sub-

stances present in fruits that render them non-

cariogenic4'7]. Because most studies with apples have

used mature fruits, it is possible that the APP content

was diluted in the juice thus reducing its caries-

protective effect. To test the effect of immature fruits

(as edible as possible) on caries in humans deserves fur-therinvestigation.

Apples, more than other fruits, have been associatedwith possible caries-preventive effectsti} and better gingi-val healthLS'2`}. Generally, caries studies with apples have

demonstrated less caries for both the deciduous andpermanent dentitions, but most data have been consid-ered inconclusive or equivocalG-

').

The APP concentration normally found in edible ma-

ture apptes is O.05% w/v. APP even at much higher con-centrations have not shown any side effects or toxicity

whatsoeverii}. Nevertheless, because some poiyphenolsare responsible for the astringent taste and bitterness of12I




Ntiigthithk-ipm'fi･ JDenLHIth58(2),2008

many plants and fruits, it is reasonable to assume that

higher concentrations will be less palarable when these

compounds are put into the oral cavity. In this study, be-

cause in both APP groups plaque and caries scores were

significantly lower than those of the control SU group,

APP at a O.05% concentration rnight be recommended.

Dental caries is still the main cause of tooth loss in

young and adult poputations25]. Routinely practiced

plaque control by the majority of the population is not

sufficient to control caries26', High caries rates are also

strongLy associated with deprivation and poverty3),

Caries is a universal and multifactorial disease that af-

fects people of different ages, races, and socioeconomic

backgrounds. Fruits are also universal and most are in

contact with the teeth either as the fruit flesh or as juice,Most phenolic compounds found in fruits, vegetables

and herbs function as potent antioxidants and can have

great health benefits. However, too iittle is known on the

impact of fruit polyphenols on caries.

In this study, the APP coneentrations (O.05% APP and

O,296 APP) used were the same Ievel as those of the

juice of a mature apple {O,0596 APP) and 4-fold (O.2%APP) to represent a young apple, An increasing caries

protection was observed in the latter.

New research on the relationship between possible

caries-protective compounds other than fluoride and

caries is thu$ required. Like fiuoride, APP might be of

benefit for controlling dental caries in the caries-

susceptibte person. A new series of experiments using

APP as a mouth-rinsing solution are under way to test

the APP effect on caries and root surface caries.

Acknowledgement: This study was supported by a grant

froin Nikka Whisky Distilling Co. Ltd. We gratefully thank

Dr. Fumiko Ozaki for her invaluable suggestions and opin-

ions, We are aiso grateful to Professor Erneritus Dr. Tyuya

Kitamura for his coilaboration andi suggestions,

This study was only possible thanks to the approval of the

Committee for Animal Experiments of Tsurumi University

School of Dental Medicine.References

I) Tanzer JM/ Microbiology of dentai caries, In: Reinhardt R""

editor,ContemporaryoralmicrobioiogyandimmunelogybyJ,

Slots and M. A. Taubman. Mosby-Year Boek, Inc, St. Louis.

1992, pp. 377J424.

122

2' Boue D, Armau E, Tiraby G/ A bacterielogical study of ram-

pant caries in children. J Dent Res 66/ 23-28, 1987,3' O'MuiLane D/ Can prevention eliminate caries? Adv Dent Res

9, 106-109, 1995.4) Stephan RM/Effects of different types ofhuman foods on den-

tal hea]th in experimental anirnals, J Dent Res 45/ 1551-1561,

1966.5)

Watson MR, Horowiaz AM, Garcia 1 et at./ Caries conditiens

among 2-5-year-old irnmigrant [.atvian children related to

parents oral health knowledge, epinions and practices. Com-

munity Dent Oral Epidemiol 27/ 8-15, 1999.

6} Bibby BG/ Fruits and vegetables and dental caries. Clin Prev

Dent 5i 3'IL 1983,

7} Rugg-Gunn AJ: Dental eari ¢ s-the role oi fresh fruit and fruit

products. Ini Nutrition and Dental Health, Oxforcl University

Press, Oxford, 1993, pp. 223-259.8) Hamilton-Miller JMT/ Minireview-Antimicrobial properties of

tea <Camelia sinensis L.). Antimicrob Agent$ Chemother 39:

2375-2377,1995.

9) Otake S, Makimuru M, Kuroki T et aL/ Anticaries effects of

pelyphenolic compounds from Japanese green tca. Caries Res

25: 438-443, 1991.

10) Oeshima T, Minami T, Matsumoto M et aL: Comparison of the

carTostatic effects between regiiiiens to administer oolong tea

polyphenols in SPF rats. Caries Res 32/ 75-80. 1998.11) Onishi M, Ozaki F, Yoshino F et aL/ Experimental evidence ef

caries prei,entive activity of nonfiuoride compoiient of lea, J Dent Hlth 31, 158-16],1981.

12) Kashket S, I'aulino VJ, Lewis DA et al,/ In-vitro inhibition of

glucosyltransferase frem the dentai plaque bacterium Strep-

tococcus mutans by common beverages and food extracts.

Arch Oral Bio] 30: 821-826, 1985,

13} Yanagida A, Kanda T, Shoji T et al,/ Separation and fractiona-

tion of apple procyanidtns by size exclusion chromatography.

J Chromatogr A Sep 3 855: 181-190, 1999.14) Ynnagida A, Kanda T, Tanabe M et al./ Inhibilory effects of

apple polyphenols and related compounds on cariogenic fac-

tors of Mutans StreptococcL J Agric Food Chem 48: 5666-

5671,2000.

15) Matsudaira F, Kitamura C, Yamada H et al,/ Inhibitory effect

of polyphenol extracted frein immature apples on dental

plaque fermation. J Denz Hlth 48: 230-235, 1998,16) Kanda T, Akiyama H, Yanagida A et aL: Inhibitory effects of

applc polyphenol on induced histamine release from RBL-2 H

3 cells and rat rnast cells. Biosci Biotechnel Biochem 62: 1284-

1289,1998.

17) Keyes PH, Jordnn HV/ Periodontal lesions on the Syrian

hamsrer-Ill. FindiiLgs related to an infection and transmissible

component. Arch Ora] Biol 9: 377-400, 1964.18) Takashima T/ Influence of dietary fiber oll experimentaL den-

tal caries in golden hamsters.J Dent Hlth 43: 80-91, 1993.19> Navia JM/ Experimentai dental caries. In Animal modeLs in

dental research. The University of Alabama Press, Alabama,

l977. pp. 257-298.20) de Otiveira Cordeiro JG/ Experimental root surface caries in




21)

22)

23}

24)

25)

hamsters the development of the disease after inoculations of

two types of cariogenic bacteria. Bull Tokye Med Dent Univ42i83-103,1995.Rolla G/ W'hy is sucrose so cariogenie?. The role of glucosyl-transierase and polysacharides, Scand J I)ellt Res 97r 115-119,1989,Macheix

JJ, Fleuriet iX, Billot J/ Fruit Phenolics. CRC PressInc, Florida, pp 1-378, 1990.

Siack CL, Martin WJ/ Apples and dental health, Br DemJ 18:

366-371, 1958.Longhurst P, Berman DS/ Apples and gingival health Br DentJ 134i 475-479, 1973,Fejerskev O/ Stralegies in the design of prex,entive programs.

.)Ldv Dent Res 9: 82-88, I995.26) Sutcliffe P: Oral cleanliness and clental caries in the prevontion

of dental disease. In/ In: )v{urray JJ edizor. The prevention of

dental disease. Oxford Medical Publications, Oxford, l989, 2nd

ed. pp 200-217.

Roprint requests to F. MATSUDAIRA. Department ef Pre-venlive Dentistry, Tsurumi University School of Dental

Medicine, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501,

Japan TELi045.581.1001 FAX:042.344.6888

E-mail:matsudfo0616@rhythm,ocn.ne,jp

123



The 　Japanese 　Sooiety 　for 　Dental 　Health

口腔衛生会誌　JDentH ［th58 （2）、2008

リンゴポリフェノールのう蝕抑制効果

松平　文朗　　Jos6　Geraldo　de　Oliveira　CORDEIRO

　　　　　　　山田　秀則　　柳田　顕郎＊

　鶴見大学歯学部予防歯科学講座

＊

東京薬科大学薬学部第一．．一薬品分析化学

　概要 1 リンゴ未熟果実から抽出したポリフェノール APP がグルカン産生に関与するグルコシルトランスフェラ

ーゼの

作用を阻害することが知られている．このリンゴポ T丿フェノー

ル APP のう蝕押制効果を実験期間 12週 li｝」の動物実験で検

討した．3週齢のゴールデンハムスター雄 60匹に 5．muinns （NTCC10449 株）を経凵的に接種，感染させ，高濃度スクロー

ス含有食餌 Diet　2000 を与えた．

　6 週齢のときにポリフェノール APP を食餌 Diet　2000 に添加し，0、05％ APP 添加群と O．2％ APP 添加群を実験群，　APP

無添加群（SU 群）を対照群と定め，ハムスター

を 3 群に分け飼育した．12週後（15週齢）クロロホルム麻酔．ドで屠殺し．

上下顎臼歯を標本とし検索した，頬側．面

・咬合面

・舌側面について，計 3歯面でのプラ

ーク付着とう蝕による歯面崩壊を

検査し，5段階に区分して評価した，

　このプラー

ク付着スコアとう蝕による歯面崩壊スコアでは，重症う蝕（スコア 5）が O．2％ APP 添加群で著しい有意の減

少を示した．う蝕スコアおよびプラーク付着スコアを群間で比較すると，対照の SU 群（APP 無添加群）に比較して，　O．05％

APP 添加群（p＝0，003），0．2％ APP 添加群（p＜ 0，001）で有意の差を認めた．　 APP を添加した 2 群には有意の差は認めら

れなかった．

　統計的検定には Kruska11−Wallis　test および TukeyHSD −testを用いた．

　実験期間中の前期（MS 　Recovery−1 ；APP 添加前），中期（MS 　Recovery−2 ；APP 　i忝加で飼育中），終期（MS 　Recovery−

3 ；屠殺直前）で，5．mutans の CFUIml 量を測定した．　 CFU ．’ml 量と重症う蝕（スコア 5）との相関を調査すると，　 i

・1，i期で

は SU 群で r＝O．67 （p〈0．001），　 O．05％ APP 添加群で r ＝0．59 （p＜0，01）と有意の相関を認めた．終期では SU 群で r ＝O．76

（p 〈0．001）となり，有意の相関を認めた．したがって，SU 群に比較して，実験期問中に 0．2％ APP 添加群では S．　muta ’ns

の CFU 〆m1 量が減少し，重症う蝕（スコア 5）も減少していることが認められた．同様に 0，05％ APP 添加群でも減少傾向

が認められた．

　この結果からリンゴポリフェノール APP はプラーク ri．rの S．　mutans （CFU ！m1 ）量の増減に影響を与え，プラ

ーク付着を

抑制し，重症う蝕を抑制するといえる．

口腔衛生会誌 58 ；113−124，2008

索引用語：リンゴポリフェノール，スクロース，s．　mzatans ，プラーク，重症う蝕

筆者への連絡先：松平文朗　〒 230−8501　神奈川県横浜市鶴見区鶴見 2−1−3　鶴見大学歯学部予防歯科学講座

　　　　　　　　TEL ：045−581−1001 （内線 8368） i’FAX ：042−344−6888／E−mail ； matsudfoO616 ＠rhythm ．ocn ．ne．jp

124

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