In vitro evaluation of anti-adherence activity of the ...

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the& Ocimum& basilicum& L.& (basil)& extract& & and& of& the& Coriandrum& sativum& L.& (coriander)& extract& in& biofilm& of& Streptococcus& mutans&.
& & &

& IN'VITRO'EVALUATION'OF'ANTI> ADHERENCE'ACTIVITY'OF'THE' OCIMUM&BASILICUM&L.'(BASIL)' EXTRACT'AND'OF'THE'CORIANDRUM& SATIVUM&L.'(CORIANDER)'IN'ACRYLIC' SURFACE'OF'REMOVABLE' ORTHODONTIC'APPLIANCES' & & &

ABSTRACT' & AIM:& The& aim& of& this& study& is& evaluate& the& antimicrobial& activity& of& the& Ocimum& basilicum& L.& (basil)& extract& & and& of& the& Coriandrum& sativum& L.& (coriander)& extract& in& biofilm& of& Streptococcus& mutans& colonized& in& specimens& confectioned& in& acrylic& used& to& confection& removable& orthodontic& appliances.& MATERIAL' AND' METHODS:& To& perform&this&work,&specimens&were&confectioned&(spherical&discs&in& sterile& acrylic)& which& were& immersed& in& extracts& pure& and& in& series& dilutions& (1:2& a& 1:128)& by& 24,& 48& and& 72& hours.& For& each& time& of& exposition,& the& disintegration& of& bacterial& films& was& performed& by& sonication& in& saline& solution.& RESULTS:& The& results& obtained& were& satisfactory& for& the& extracts& studied& on& the& inhibition& of& biofilm& formation.& CONCLUSION:& We& conclude& that& polyphenols& present& in& the& extracts& interfered& on& the& glucan& synthesis& engine,& possibly& inhibiting& the& enzymes& (glucosyl]& and& fructosyl& transferase)& which& synthesize&the&extracellular&polymers,&also&acting&as&antioxidant,&and& therewith&they&presented&antimicrobial&activity.& & ' ' ' ' ' &' FERREIRA,&Fabíola&Iahn*& ' BERNARDI,&Adilson&Cesar&Abreu**& KEYWORDS' LUNARDI,&Nadia***& & BOECK'NETO,&Rodolfo&Jorge****& BOECK,&Eloisa&Marcantonio***&

Biofilm.&Vegetal&extracts.&Orthodontic&appliances.&

& & & & & & School&of&Dentistry,&Araraquara&University&Center,&Araraquara,&SP,&Brazil*& Department&of&Microbiology,&School&of&Dentistry,&Araraquara&University&Center,&Araraquara,&SP,&Brazil**& Department&of&Orthodontics,&School&of&Dentistry,&Araraquara&University&Center,&Araraquara,&SP,&Brazil***& Department&of&Surgery,&School&of&Dentistry,&Araraquara&University&Center,&Araraquara,&SP,&Brazil****& Correspondence:[email protected]&(BOECK&EM)&|&Received&02&Feb&2015&Received'in'revised'form&04&Feb&2015&Accepted&08&Feb&2015&

&

637

INTRODUCTION

solid# surfaces# represent# more# preferably# for#

In# the# last# years# there# was# scienti/ic#

colonization# by# some# microorganisms.#

advance# involving# studies# on# the# medicinal#

Therefore,# the# use# of# orthodontic# appliances#

plants#in#order#to# obtain#new# composites# with#

and# prosthetic# devices# contribute# to# the#

therapeutic# properties1.# The# appearance# and#

retention# of# the# bio/ilm# and# feed8.# Bacterial#

spread# of# microorganisms# resistant# to#

cells#adhered#to# the# surface#of#materials#are#a#

antimicrobials# available# in# the# Market# have#

signal# or# a# target# for# speci/ic# bacterial#

been# reported# through# decades,# what#

receptors#to#be#liked5,6.

encourages# the# search# for# new# sources# of#

#

substances#for#this#purpose,# like#plants#used#in#

several# dentistry# specialties# is# very# important#

the#traditional#medicine2,3.

because# it# pints# both# for# prevention# and#

#

Several# microorganisms# of# dental#

treatment# of# diseases.# In# order# to# help# the#

bio/ilm# are# associated# to# the# caries# and#

conventional# methods# of# oral# hygiene# (tooth#

periodontal# diseases.# S.# mutans# is# one# of# the#

brushing,#dental#/loss#or#tape)#several#chemical#

most# related# oral# microorganisms.# It# is#

agents# have# been# studied,# among# them# the#

believed# that# it# is# involved# to# the# initial#

chlorhexidine# gluconate.# However,# the#

development# of# these# problems# because# it# is#

frequent# use# and# for# long# time# of# this#

able#to#colonize#teeth4.

substance# presents# some# undesirable# effects.#

#

The# bio/ilm# formation# is# seen# as# a#

Therewith,# the# curative# effect# of# some#

process# of# growth# well# regulated,# which#

medicinal# plants# was# noticed# out;# they# are#

presents#as#a#result#the#formation#of#a#complex#

used# for# thousand# years# and# are# base# for#

community# of#microorganisms.# This# formation#

studies#for#production#of#new#drugs9.

involves# some# physical,# chemical# and#

#

biological# interactions,# which# results# in#

therapeutic#resources#for#treatment#of#diseases#

adherence,# colonization# and# growth5.# The#

and# serve# both# as# the# known# home# medicine#

bio/ilm# might# be# formed# during# hours,# and#

a n d# a s# r a w# m a t e r i a l# t o# e l a b o r a t e#

because# they# are# tolerant# to# the# microbial#

phytotherapy10.

agents,#they#are#dif/icult#to#remove.

#

#

Microorganisms,# when# associated# to#

is# evaluated# through#determination# of#a#small#

bio/ilms,# become# more# resistant6.# The# safest#

quantity#of#substances# necessary#to# inhibit#the#

way# to# remove# this# microorganism# is# the#

growth# of# microorganismGtests;# this# value# is#

mechanical# one;#however,# the#chemical#agents#

known# as# Minimum# Inhibitory# Concentration#

are#useful7.#The#presence#of#retentive#areas#on#

(MIC).##A#very#relevant#aspect#to#determine#the#

Bacterial# micro/ilm# control# within# the#

Medicinal# plants# constitute# important#

Microbial# activity# of# vegetable# extracts#

JRD010Journal0of0Research0in0Dentistry,0Tubarão,0v.03,0n.01,0jan/feb.02015

638

MIC# of# vegetable# extracts# is# the# concern#

#

regarding# to# the# toxicological,# microbiological#

Coriandrum# sativum# L.# (coriander)# extracts#

and# legal# features# related# to# the# natural#

were# used,# and# they# were# obtained# from# the#

composites#or#their#combination11,12.

enterprise#Biotae/SP.

#

#

Owing# to# the# use#of#plants,# many# times#

The# Ocimum# basilicum# L.# (basil)# and#

In# this# study,# the# S.# mutans# (ATCC#

in# an# empirical# way,# this# work# evaluated# the#

25175)#standard#strains#were#used.#They#were#

action#in# vitro# of#antiGadherence# action#of#two#

assigned#by# Professor# Alessandra#N.S.# Rastelli,#

v e g e t a b l e# e x t r a c t s ,# t h e# b a s i l# e x t r a c t#

of#the# Dentistry# School# of#Araraquara# G#Unesp.#

(Ocimumbasilicum# L.)# and# the# coriander#

The# strain# was# refrigerated# and# conserved# in#

extract# (CoriandrumSativum# L.)# which#

cryoval#with# glycerin#nutrient# broth#40%,# and#

presented# great# potential# of# application# as#

reactivated#at#the#moment#of#use.

antimicrobial# agent,# as# medicinal# agent,# as#

#

/lavor# in# food# and# also# fragrance# in#

agar# (TSA)# and# Tryptic# soy# broth# (TSB)# were#

pharmaceutical#products13.

prepared# according# to# the# provider’s#

The# culture# medium# used,# Tryptic# soy#

directions,#with#0.25#%#glucoses#added. MATERIAL-AND-METHODS !

The# study# was# performed# um# the#

Microbiology# Laboratory# of# the# University# Center#of#Araraquara#G#Uniara.# #To#evaluate#the# effect#of#the#antimicrobial#agent#on#the#cells#of# the#bio/ilm. #

It# was# confected# 100# specimens# in#

acrylic,# equal# to# the# removable# orthodontic# appliances# in# the# measures# of# 0.5mm# by# 0.3mm.# They# were# washed# in# water# and# neutral# soap,# rinsed# in# sterile# distilled# water# and,# next#they#stay# in#chlorhexidine#10%#for#2# hours.#After#this#period,#they#were#rinsed#again# in# sterile# distilled# and# sterilized# under# ultraviolet# light# in# biological# cabin# by# a# half# hour.#The#specimens#were#stored#aseptically#in# sterile#test#tubes.

#

From# the# cryovals# (micro# tubes# for#

conservation)# the# TSA# plates# were# prepared# with# handle# 10µl.# The# plates# TSA# were# incubated# during# 24# hours# at# 35G37°C# in# microaerophilia.# After# the# incubation# period,# with#a#bacteriological#needle,#thereabout#3#or#4# colonies# grown# in# the# TSA# plates# were# transferred#to#Falcon#tubes#(15ml)#with#5#–#10# ml#of#TSB. #

The# tubes# were# incubated# by# 16/18#

hours#(one#night)#at#35G37#°C. #

After# the# incubation# period,# the# tubes#

were# submitted# to# centrifugation# at# by# 5# minutes# at# 2500# rpm.# The# supernatant# was# discarded# and# the# biomass# obtained# was# resuspended#in#10#ml#of#TSB#in#order#to#obtain# a#turbidity#correspondent#to# 0.5#in#the#scale#of#

JRD010Journal0of0Research0in0Dentistry,0Tubarão,0v.03,0n.01,0jan/feb.02015

639

Mac# Farland,# that# corresponds# to# 1.5x10⁸#

expected# of#the# pharmacological# agent.# At# the#

(Figure#1).

end# of# dilution# of# 100µl# of# a# bacterial# suspension# prepared# 1.5x10⁸# UFC/ml,# a#

Figure01.0Representation0of0Mac0Farland0scale.

specimen#was#added#in#all# the#wells# of#culture# (Figure#3). Figure0 2.0 Microtiter0 plate0 with0 960 wells.0 Lines0 A,0 B0 and0 C,0 column0 10 correspond0 to0 the0 basil0extract;0 lines0 D,0 E0 and0F,0column0 10 correspond0 to0the0 coriander0extracts;0both0 extracts0in0 their0 concentrated0 form0and0 other0dilutions0until0the0column08.

#

A# concentrated# stock# solution# was#

prepared# for# each# pharmacological# agent# evaluated,# for#posterior# dilution,# as# illustrated# in# the# Figure# 2.# On# the# /irst# line# of# wells# (correspondent# to# the# position# vertical# 1)# in# plates#of#microtiter#plates#of#96#wells#(Sarstedt,# Newton,# NC,# USA),# 200µl# of# stock# solution#

Figure0 3.0 Plates0 containing0 specimens0 inoculated0 in0 TSB0 broth0 +0 extract.

p r e p a r e d# w e r e# d i s t r i b u t e d# f o r# e a c h# pharmacological# agent:# lines# A,# B# and# C# the# basin#extract#and#lines#D,#E#and#F#the#coriander# agent# and# the# positive# and# negative# controls# (the#/irst#one#the#S.#mutans#suspension#and#the# second# the# TSB# pure#broth).# From# the# second# line# of# wells# in# vertical# (2),# 100µl# of#TSB# with# 0.25%# glucose# were# distributed# for# each# well# of#culture.# #

Serial# dilutions# were# carried# out,#

transferring# 100# µl# of# the# line# of# wells# 2,# and# consecutively# until# obtain# the# concentration#

#

The# plates# were# incubated# at# 35G37°C#

by#24,#48#and#72#hours#with#no#agitation.#

JRD010Journal0of0Research0in0Dentistry,0Tubarão,0v.03,0n.01,0jan/feb.02015

640

#

The#positive#control#was#that#which#was#

no# exposed# to# any# pharmacological# agent;# in#

Figure05.0Serial0dilution0of0the0sample0in0NaCl0and0plating.

other# words,# only# the# culture# medium# plus# S.# mutans#suspension,# while#the#negative#control# was#the#specimen#with#the#culture#medium.#All# the#experiments# were# carried# out# in#triplicate# and# for# each# time# of# contact# (24,# 48# and# 72# hours). #

After# the# incubation# period,# with# a#

sterilized# tweezers,# the# specimens# were# removed#from#their#wells#and#washed#in#saline# solution# (0.09%)# to# remove# non# adhered# bacteria.# After# the# process# of# washing,# the# specimens# referent# to# each# incubation# time# and#each#dilution#were#placed#in#conical#plastic# tubes# 15# ml# (Falcon),# containing# 2.0# ml# of# saline# solution# (0.09%)# and# agitated# by# 60#

!

Cell# viability# was# determined# by# serial#

dilution#of#the#sample#in#saline#solution,#and#10# µl# of# each# dilution# were# spread# by# the# Drop# method#in#plates# Petri#(90x15#mm)#containing# tryptic# soy# agar# (TSA).# The# plates# were# incubated# at# 35G37ºC# in# bacteriological# incubator#by#until#48#hours#(Figure#5).#

seconds# in# a# tube# shaker# (vortex),# and#

RESULTS

submitted#to# sonication#in#the#frequency#40±2# kHz#by#5#minutes#(Figure#4). Figure0 4.0 Ultrasound0 tank.0 Tubes0 with0 specimens0 are0 sonicated0 in0 order0to0provoke0disintegration0of0bacterial0bioYilm.

Antimicrobial# activity# of# Ocimum# basilicum# L.# (basil)# and#Coriandrum#sativum#L.# (coriander)#extracts#in#bio/ilm#of#S.#mutans#was# conducted#evaluating#the#determination#of#the# Inhibitory# Minimum# Concentration# (IMC)# of# the#pure# extract# and#diluted# on# the# formation# of# the# bio/ilm# in# acrylic# specimens# as# used# in# appliances#in#orthodontics.# #

The# determination# od# the# Inhibitory#

Minimum# Concentration# (IMC)# by# the# microdilution# broth# method# showed# that# the# /luid#extract# of#Ocimun# basilicum#L.# and#of#the# Coriandrum# sativum# L.# presented# inhibitory# activity# on# the# adherence# face# the# S.# mutans# strain.# The# microorganism# did# not# adhere# to#

JRD010Journal0of0Research0in0Dentistry,0Tubarão,0v.03,0n.01,0jan/feb.02015

641

the#surface# of#specimen#and# consequently# did#

and#Figure#6).

not#form#the#bio/ilm#on#its#surface#(Frames#1,#2# Frame01.0Evaluation0of0inhibition0of0the0formation0of0bioYilm0of0Ocimum#basilicum#L.0extracts0(basil).

Extract0concentration/incubation0time 240hours 480hours 720hours

Pure Neg. Neg. Neg.

1:2 Neg. Neg. Neg.

1:4 Neg. Neg. Neg.

1:8 Neg. Neg. Neg.

1:16 Neg. Neg. Neg.

1:32 Neg. Neg. Neg.

1:64 Neg. Neg. Neg.

1:128 Neg. Neg. Neg.

1:16 Neg. Neg. Neg.

1:32 Neg. Neg. Neg.

1:64 Neg. Neg. Neg.

1:128 Neg. Neg. Neg.

Frame02.0Evaluation0of0inhibition0of0the0formation0of0bioYilm0of0Coriandrum#sativum#L.0extracts0(coriander).

Extract0concentration/incubation0time 240hours 480hours 720hours

Pure Neg. Neg. Neg.

1:2 Neg. Neg. Neg.

1:4 Neg. Neg. Neg.

1:8 Neg. Neg. Neg.

Figure06.0No0development0of0colonies0in0TSA0representative0of0each0dilution0and0extract.0Only0observing0the0inoculum0mark0in0each0triplicate.0

DISCUSSION

cost16# opened# up# ways# to# use# base# plants#

In#the# ast#years#it#has# become# common#

products# as# a# supporting# to# the# mechanical#

place# the# use# of# phytotherapy# as# alternative#

therapy.# Phytoterapics# are# free# of# side# effects#

method# to# treat# buccal# diseases14,15.# The#

and# they# can# be# used# for# a# long# period17.#

inef/icacy# of# traditional# methods# and# the# low#

However,#the#use#of#medicinal# plants#might#be#

#

aggressive,# and# there# would# be# caution#

JRD010Journal0of0Research0in0Dentistry,0Tubarão,0v.03,0n.01,0jan/feb.02015

642

Bezerra# et# al.16# (2013)# described# that#

regarding# to# the# toxicological,# microbiological#

#

and# legal# features# relative# to# the# natural#

microorganisms#of#tooth#bio/ilm#are#associated#

composites#and#their#combination.11#Rasekh#et#

to# caries.# However,# some# bacteria# are# more#

al.10# in# 2011,# evaluated# the# acute# and# sub#

active# than# others,# and# the# Streptococcus#

chronic# toxicity# of# the# hydro# alcoholic# extract#

mutans#is#one#of#the#oral#microorganisms#more#

of# O.# basilicum# L.# in# mice# and# they# suggested#

related#to#the#initial#caries#because#it#is#able#to#

that# the# hematologic# system# could# be# as# a#

colonize#teeth,# produce#intra#and#extra#cellular#

target#to#the#oral#toxicity#of#this#plant.

polysaccharides,# is# highly# acidogenic# further#

#

metabolize#several##salivary#glycoproteins#.#

In# Brazil,# there# are# more# than# 260#

medicinal#plants#cataloged#and#distributed#into#

#

19#different#indications#for#use#in#dentistry18,19.#

precursor# step# of# all# the# process# of#

Regarding#to# the#world#market,#80%#of#people#

development# of# the# bio/ilm# in# surfaces# in#

use#plants#to#heal#illnesses.#Authors#like#Cragg#

contact# with# water;# this# pellicle# is# formed# by#

&# Newma12# (1999)# believe# that# the# use# of#

adsorption# of# organic# molecules# dissolved# in#

substances# derivated# from# herbs# represent#

the# aqueous# medium# on# the# surface.# Next,# a#

25%# of# medical# prescription# in# industrialized#

displacement#of# microorganisms# and#particles#

countries.#

occurs#to#the#surface25.#Therefore,#the#adhesion#

#

In# the# literature,# most# of# publications#

depends# basically#on#the#factors#which#involve#

with# phytoterapics# is# related# to# the#

characteristics# of# the# microorganism,# of# the#

antimicrobial# and# antifungal# activities# by# the#

surface#and#of#the#medium#conditions12.

signi/icant# advance# of#development# of# natural#

#

d e r i v a t i v e s# w i t h# b a c t e r i a l# o r# f u n g a l#

i t# a l w a y s# s h o u l d# b e# c o n s i d e r e d# t h e#

activity20123.# The# ideal# characteristics# of# an#

experimental# conditions# and# the# in/luence# of#

antimicrobial:# low# toxicity,# and# knowing# the#

factors# like# temperature# and# surface#

inter# action# of# the# product# with# oral#

composition# of# the# material# in# the# process# of#

epithelium;# low# permeability,# no# provoking#

bacterial# adhesion.# For# some# researchers,#

unbalances#which#could#lead#to#other#diseases#

bacterial# adhesion# and# bio/ilm# production#

r e c u r r e n t ;# a n d# g o o d# r e t e n t i v e n e s s#

result#from#a#multi#factorial#process12.

(substantivity)#to#could#be#slowly#released.24

#

#

microbial#agents,# the#bio/ilm#cells# (sessile)#are#

The# action# of# phytotherapy# against# S.#

The# formation#of# conditioning# /ilm# is# a#

In#any# study#on#the# bio/ilm# production,#

Because# they# are#very#resistant#to# antiG

mutans# is# reported# successfully# in# the#

very#dif/icult#to#remove26.

literature20124.

#

High# concentrations# of# antimicrobial#

are#not#also# too# much#effective,# because#these#

JRD010Journal0of0Research0in0Dentistry,0Tubarão,0v.03,0n.01,0jan/feb.02015

643

agents#cannot#kill#the#cells#due#to#the#limitation#

#

of# the# drug# diffusion;# not# all# the# good#

the# concentrated# extracts# seem# to# have# antiG

antibiotics#are#effective#against#the#bio/ilm.#

caries# properties# that# can# be#effective,# even#in#

#

the# presence# of# saccharose# and# in# very# low#

In# the# case# of# the# oral# bio/ilm,# several#

Phenolic# composites# (polyphenolics)#in#

agents# are# available# in# the# Market# with#

doses.

different#ways# of# action,# and#they#were#tested#

#

to# intervene# on# the# bio/ilm# formation# and#

antimicrobial# activity# of# phenolic# composites#

metabolism.# However,# due# to# the# various#

present# in# extract# of# plants# is# not# completely#

undesirable# side# effects# associated# to# these#

understood.# However,# several# studies# have#

agents,#the# research#for# alternative#agents# is# a#

suggested# that# these# composites# might#

need27.

inactivate#important# enzymes#and#damage#the#

#

Meng# et# al.24# (2000),# Ho# et# al.25# (2001)#

T h e# m e c h a n i s m# r e s p o n s i b l e# b y#

cytoplasmic#membrane.

and# BarbosaGFilho26# (2006)# highlighted# the#

#

importance# to# /ind# effective# and# low# toxic#

basil# and# coriander# extracts# showed# potential#

substances# in# the# run# against# the# resistance#

for#use#in#oral#health#as#prophylaxis#in#the#care#

a n d# t h e# a p p e a r a n c e# o f# p a t h o g e n i c#

of#prosthesis#confectioned#in#acrylic.#

microorganisms.# The# evaluation# of# the#

#

therapeutic# potential# of# medicinal# plants# and#

antioxidant,# it# was# suggested# that# the#

some#of# their# constituents# such# as# /lavonoids,#

antimicrobial# effect# might# be# caused# by# the#

alkaloids,# triterpenes,# sesquiterpenes,# tannins,#

antioxidant# effect.# The# results# indicated# there#

lignans,# among# others,# have# been# studied# as#

are# relations# between#the# antimicrobial# effect#

medicinal#agents.#

and#the#antioxidant#capacity#of#polyphenols.

#

#

The# phytochemical# characteristic# of#

Thereby,# antimicrobial# properties# of#

As# these# polyphenols# are# known# as#

The# data# obtained# in# this# research#

commercial# extracts# provided# by# the#

showed#absence#of#viable#sessile#cells#after#the#

enterprise# Biotae# revealed# the# presence# of#

contact#with#basil#and#coriander#extracts,#what#

/lavonoids#(polyphenols)#e#tannins.

suggests#a#more#metabolic#action#instead#of#an#

Chipault 27# (1962)# describes# that#

antimicrobial# mechanism.# Meanwhile,# more#

antioxidants# are# generally# composites# which#

investigations# are# necessary# because# to#

can# inhibit# the# oxidation# of# lipids# or# other#

distinguish#the#effects#of#polyphenols#from#the#

molecules# by#removal#of#free# radicals#and#that#

antimicrobial#effects,# a#separated#experimental#

several# composites# found# in# all# the# types# of#

environment# is# required,#and#in#this#study,#the#

plants,# vegetables# and# fruit# were# observes#

activity# of# inhibition# to# adherence# was# tested#

because#they#have#the#same#effect.#

thinking# about# the# antimicrobial# action;# we#

#

JRD010Journal0of0Research0in0Dentistry,0Tubarão,0v.03,0n.01,0jan/feb.02015

644

noticed#out#along#the#study#and#the# analisis# of#

4.0 Almeida0 LS,0 Murata0 RM,0 Yatsuda0 R,0 Dos0 Santos0 MH,0

results#a#biochemical#reaction#occurring.

Nagem0 TJ,0 Alencar0 SM,0 et0 al.0 Antimicrobial0 activity0 of0

#

To# verify# the# hypothesis# that# the#

enzymes# produced# by# S.# mutans# can# be#

Rheedia0 brasiliensis0 and0 71epiclusianome0 against0 Streptococcus0mutans.0Phytomed02008;15:866191.

inhibited# by# polyphenols# will# be# necessary#

5.0 Lawrence0 JR,0 Zhu0 B,0 Swerhone0 GD,0 Topp0 E,0 Roy0 J,0

other#studies,#and#thus#con/irm#our#/indings.#

Wassenaar0LI,0et0al.0Community1Level0Assessment0of0the0 Effects0 of0 the0 Broad1Spectrum0 Antimicrobial0 Chlorhexidine0 on0 the0 Outcome0 of0 River0 Microbial0

CONCLUSION

BioYilm0 Development.0 Applied0 and0 Environmental0

•!!Polyphenolic#compounds#present#in#the#basil#

Microbiology02008;74(11):3541–50.0

and#coriander#extracts# inhibited#the#formation#

6.0 Rodrigues0 ACC,0 Guedes0 MLS.0 Utilização0 de0 plantas0

of# bio/ilm# of# S.# mutans# in# vitro# in# the#

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