Aust Endod J 2019

ORIGINAL RESEARCH

Antimicrobial effectiveness of grape seed extract against

Enterococcus faecalis biofilm: A Confocal Laser Scanning
Microscopy analysis
Nicole de Mello Fiallos, DDS, MSc1; Doglas Cecchin, DDS, PhD2; Carolina Oliveira de Lima, DDS, MSc1 ;
Raphael Hirata, Jr., DDS, PhD3; Emmanuel Joa~o Nogueira Leal Silva, DDS, PhD1 ; and
1
Luciana Moura Sassone, DDS, PhD
1 School of Dentistry, Rio de Janeiro State University (UERJ), Rio de Janeiro, Brazil
2 Department of Endodontics, Passo Fundo University (UPF), Passo Fundo, Brazil
3 Department of Microbiology, Rio de Janeiro State University (UERJ), Rio de Janeiro, Brazil

Keywords
biofilm, confocal laser scanning microscopy,
Enterococcus faecalis, grape seed extract, root
canal irrigants.
Correspondence
Dr Emmanuel Jo~ao Nogueira Leal Silva, Rua
i, RJ,
Herotides de Oliveira, 61/902, Icaraı, Nitero
Brazil. Email: nogueiraemmanuel@hotmail.com
doi: 10.1111/aej.12390
(Accepted for publication 22 October 2019.)
Raphael Hirata Jr died on 18th November, 2018.
Abstract
This study evaluated the antimicrobial effectiveness of 6.5% Vitis vinifera grape
seed extract (GSE) against Enterococcus faecalis biofilm using confocal laser scan-
ning microscopy (CLSM). Saline solution (SS), 5.25% sodium hypochlorite
(NaOCl) and 2% chlorhexidine (CHX) were used for comparison. Dentin discs
were inoculated with E. faecalis strain establishing a 3-week-old biofilm. Discs
(n = 10) were exposed to 5.25% NaOCl, 2% CHX, 6.5% GSE and SS (negative
control) for 10 min. Discs were stained with the fluorescent LIVE/DEAD-
BacLightTM dye and analysed using CLSM. The proportion of dead cells in bio-
film was analysed using one-way ANOVA and Tukey tests (P < 0.05). A higher
proportion of dead cells was found in GSE group compared with CHX and SS
(P < 0.05). NaOCl group was associated with the highest proportion of dead
cells (P < 0.05). GSE presented antimicrobial activity against E. faecalis; how-
ever, NaOCl was the most effective irrigant solution. GSE was more effective
than CHX and SS.

Generally, root canal treatments are performed using

Introduction
The objective of primary endodontic infection’s treat-
ment is to promote an effective root canal disinfection,
reducing bacterial load to levels compatible with peri-
radicular tissue healing and preventing microbial
recolonisation of the treated canal (1). Current tech-
niques include the mechanical preparation of the root
canal system using different nickel–titanium rotary or
reciprocating systems associated to irrigants with antimi-
crobial/tissue dissolving properties (2). However, an
anterior study has reported that more than half of denti-
nal walls have remained unprepared (3,4), a critical chal-
lenge for any available shaping protocol. In fact,
mechanical preparation systems are able to act only on
the most central portion of root canal (canal lumen), thus
leaving irregularities merely untouched harbouring resi-
dent biofilm (5). Accordingly, irrigation is fundamental
during endodontic treatment as it assists the disinfection
of unprepared root canal walls (6).
sodium hypochlorite (NaOCl) or chlorhexidine (CHX) as
irrigating agents (6). NaOCl has potent antibacterial and
antibiofilm activity and promotes tissue dissolution; how-
ever, NaOCl is highly toxic and can cause allergic reac-
tions (6). Furthermore, NaOCl reduces the mechanical
properties of dentine (7) and promotes structural dam-
ages in organic and inorganic dentin components, partic-
ularly the collagen (8). CHX has been suggested to be
used as an alternative to NaOCl based on its antibacterial
action, substantivity and lower tissue toxicity, when
compared to NaOCl; however, CHX is unable to dissolve
pulp tissue or eradicate biofilm (6).
The limitation of these irrigants has encouraged
research in order to develop alternative substances for
root canal preparation. Advances in materials science
have focused the use of biologically compatible materials
for biomedical applications to minimise the adverse
impact of harmful materials on biological tissue (9). The
vegetal specie Vitis vinifera has the potential to be used as

© 2019 Australian Society of Endodontology Inc 1

Antimicrobial Effectiveness of GSE
an alternative for conventional root canal irrigants. It is
noticed the potential of the Vitis vinifera grape seed
extract (GSE) as an antimicrobial and antifungal alterna-
tive as well as in therapeutic activities, such as antioxi-
dants, immunomodulators and antimutagens (10).
Besides these properties, GSE has great applicability in
the dental field for enhancement of dentin biomechani-
cal properties and biostability (7). Thus, the use of GSE as
an endodontic irrigant is promising, and studies are
needed to better elucidate its effect against bacteria bio-
film involved in endodontic disease.
For the mentioned reasons, this study evaluated the
antibacterial effectiveness of GSE against Enterococcus fae-
calis biofilm through the confocal laser scanning micro-
scopy (CLSM) analysis. NaOCl, CHX and saline solution
(SS) were used as reference for comparison. The null
hypothesis tested was that there are no differences in the
antibacterial effectiveness amongst the tested irrigants.
Materials and methods
Specimen and irrigant preparation
This study was approved by the Local Ethics Committee.
The previously autoclaved teeth were donated by the
Human Teeth Bank – Biobank of the School of Dentistry
of the State University of Rio de Janeiro. Extracted
human single-rooted teeth were collected and analysed
clinically and radiographically. Roots with anatomic
irregularities, fracture lines, with curvature or showing
previous endodontic treatment were excluded. Speci-
mens were prepared according a previously published
study (11) with some modifications. After the initial
decontamination, the teeth were sectioned to obtain 44
dentin discs approximately 0.5 mm wide, 6 mm in diam-
eter and 8 mm in length using a low-speed sectioning
saw (ISOMET 1000; Buehler, Illinois, USA), under con-
tinuous water cooling. The crowns and apical portions of
the tooth were sectioned off and discarded. The dentin
discs were then decontaminated with 2.5% NaOCl for
5 min, followed by 6% citric acid for 5 min for smear-
layer removal and then bidistilled water. Finally, the
specimens were subjected to UV rays from a laminar flow
chamber for 15 min. Two discs, randomly selected, were
incubated in brain heart infusion (BHI) broth for 24 h at
37°C to confirm that there was no bacterial contamina-
tion.
Bacterial strain, biofilm generation and treatment of
infected specimens
Enterococcus faecalis (ATCC 29212) was plated in BHI broth
and incubated in anaerobic conditions (37°C/24 h).
2 © 2019 Australian Society of Endodontology Inc
N. M. Fiallos et al.
Then, bacterial colonies were diluted in fresh BHI broth
and incubated to match the optical density of 1.18 absor-
bance to 405 nm on the spectrophotometer, equivalent
to 3 9 108 CFU mL 1. The sterilised discs were settled in
sterilised 24-well culture plates with further inoculation
using 3.0 mL of E. faecalis suspension anaerobically for
21 days at 37°C for biofilm formation. During this period,
every 48 h or 72 h, old BHI was discarded and renewed
to remove dead cells and allow cell viability. After these
procedures, specimens were removed from the wells and
gently rinsed with sterile phosphate-buffered saline
(PBS) for 1 min in order to remove loosely attached
planktonic bacteria and gently settled in other 24-well
sterile culture plates. Infected specimens were randomly
divided into three experimental and one control groups
(n = 10). The three experimental groups were exposed to
one of the following solutions: 5.25% NaOCl (Asfer,
LDTA Chemical Industry, Santa Maria, SP, Brazil), 2%
CHX (Biodynamic, Ibipor~ a, Paran
a, Brazil) and GSE V
ıtis
vinifera (BIOVEVA, Dallas, TX, USA). Each specimen was
immersed in 2 mL of the tested solution for 10 min and
then washed for 1 min using 5 mL PBS in order to
remove the residual irrigating solution. To ensure the
correct concentration of NaOCl, the solution was tested
before its use by iodometric titration method (12). The
GSE has its pharmaceutical form in 100 mg capsules,
with 85% polyphenols. These were dissolved in DMSO
(dimethyl sulphoxide) and 70% ethyl alcohol to obtain
the desired concentration. The control group was treated
with SS.
Confocal Laser scanning microscopic analysis
After irrigation, samples were washed with 2 mL sterile
PBS solution for 1 min to remove loosely attached plank-
tonic bacteria. The specimens were then stained with
LIVE/DEAD® BacLightTM fluorescence dye (Invitrogen
Molecular Probes, Eugene, OR, USA) for 30 min. This kit
consists in two nucleic acid-binding dyes used to differen-
tiate live and dead cells. Bacteria with intact cell mem-
brane stain green, whereas the damaged membranes
stain red. A fresh mix at 1:1 ratio was prepared and
diluted in PBS at 1:50 ratio. Once stained, the discs were
washed with PBS solution to remove excess of dye. The
samples were mounted on 25-mm slides and coverslips
and positioned for confocal analysis. Samples were anal-
ysed using the LEICA TCS-SPE confocal microscope
(Leica, Wetzlar, Germany) using 259 oil lens. Four con-
focal images were obtained from each sample, corre-
sponding to each of its quadrants scanned with 2 lm step
size at a resolution of 512 9 512 pixels. A total of 160
confocal laser scanning microscopic operative field stacks
were evaluated.
N. M. Fiallos et al.
Simultaneous dual-channel imaging was used to dis-
play green and red fluorescence (live and dead, respec-
tively). Emission wavelength for green fluorescence
ranged in 510–550 and 580–690 nm for the red fluores-
cence. The excitation and emission wavelengths of the
laser power were, respectively, 488 nm-30% for SYTO9
(green fluorescent) and 532 nm-18% for propidium
iodide (red fluorescent). The pinhole aperture was
488 nm-82,77 lmol L 1 and 532-90,24 lmol L 1,
always using airy = 1. The objective numerical aperture
was 0.6. The gain used was 663 (green) and 766 (red).
The software ‘LAX X’ (Leica, Wetzlar, Germany) was
used for image acquisition and biofilm quantification.
The ratio of red to green and red fluorescence indicates
the rate of dead cells in each sample, by which percent-
age of dead cells could be obtained.
Statistical analysis
A bell-shaped distribution of the data was confirmed
using the Shapiro–Wilk test (P > 0.05); therefore, statisti-
cal analysis was performed using one-way ANOVA. Post
hoc pair-wise comparisons were performed using Tukey’s
test for multiple comparisons (P < 0.05). BioEstat 5.0
(Mamirua Institute, Solim~ oes, Brazil) was used as the
analytical tool.
Results
Data regarding the percentage of dead cells are shown in
Table 1. A significant greater proportion of dead cells was
found in the GSE group compared with CHX and SS
(P < 0.05). The highest proportion of dead cells was
found in NaOCl group (P < 0.05). No statistical signifi-
cant difference was observed between CHX and SS
groups (P > 0.05). Fig. 1 shows representative images of
each tested group.
Discussion
One of the most important characteristic of an endodon-
tic irrigant is its antimicrobial capacity. In the present
Table 1 Mean and standard deviation of dead cells (%) after 10 min of
exposure to irrigant solutions
Solution Mean  SD (%)
Saline solution 40  20C
CHX 47  7.5C
GSE 72  8.8B
NaOCl 87  1.4A
Superscript different letters represent statistical significant differences
(P < 0.05).
© 2019 Australian Society of Endodontology Inc
Antimicrobial Effectiveness of GSE
study, the antimicrobial activity of different solutions was
evaluated by CLSM images. The use of CLSM to deter-
mine bacterial viability in dentistry has been increasing
over the years (11,13–15). CLSM has some advantages,
as the low probability to generate artefacts and the possi-
bility to quantify the biofilm’s thickness and determinate
the bacteria viability (11,13,14). However, this method
has also some disadvantages, as the impossibility to reuse
the specimens, since the energy generated by the laser
makes this process irreversible by the gradual loss of fluo-
rescence, difficulties in the preparation of specimen and,
also, for does not allow greatly increased fields as in scan-
ning electronic microscopy (14).
Enterococcus faecalis was used in the present study due
to its ability to survive isolated in root canals without
support of other bacteria (16) and because it is a well-
documented model in literature (17,18). Also, E. faecalis
is able to penetrate deeply into the dentine tubules and
form biofilm that influence its resistance to being killed
by antibacterial agents (19). In endodontic infections,
microorganisms are organised as sessile biofilms, that rep-
resents a significant increase in the resistance against
antimicrobial agents (20). Anterior studies showed that
the mature biofilm of single species of E. faecalis is estab-
lishment with 3 weeks (21 days) (21,22). Therefore, in
the present study the antimicrobial effectiveness of the
tested irrigants was performed in 21-day E. faecalis bio-
film-infected dentine similar with other studies
(11,23,24).
The 6.5% GSE group presented significantly higher
proportion of dead bacteria than CHX and control groups.
The antimicrobial properties of GSE against Gram-nega-
tive and Gram-positive bacteria have already been
demonstrated in the literature (7,10,25). The antimicro-
bial activity of GSE has been specifically attributed to the
presence of phenolic compounds, which are capable of
disrupt the cell wall and bacterial plasma membrane
(10,25). Moreover, the high concentrations of flavonoids
and their derivates, and stilbenes, were thought to be
responsible for this activity (25). The findings of the pre-
sent study are in accordance with a previous study that
showed lower antimicrobial activity of GSE when com-
pared to NaOCl (26). In contrast, Cecchin et al. (7)
showed a higher antimicrobial activity of GSE when
compared to 2.5% NaOCl in straight root canal prepara-
tion. The variability of experimental designs (e.g. antimi-
crobial evaluation methodologies and/or irrigant
concentrations) could explain the discrepancies amongst
the studies. However, this study is the first one to evalu-
ate GSE antimicrobial effectiveness using CLSM and a
biofilm model.
The GSE was dissolved in DMSO and 70% ethyl alco-
hol. One may argue that DMSO or ethyl alcohol could
3
Antimicrobial Effectiveness of GSE N. M. Fiallos et al.

Figure 1 CLSM images of infected dentin discs after exposure to NaOCl (a), GSE (b), CHX (c) and saline solution (d). All images are shown at 259 mag-
nification. The green cells represent cells with intact membranes, whereas the red cells are damaged or dead.

have an effect on the E. faecalis biofilm and jeopardise
the results of GSE. However, it is important to emphasise
that previous studies demonstrated that both DMSO and
ethyl alcohol showed lack of antimicrobial activity to
endodontic pathogens such as E. faecalis, especially when
tested in biofilm models (27).
The CLSM images of CHX showed that only the upper
layers of the biofilms were slightly affected by the irrigant
solution and the deeper layers showed live bacterial bio-
film, demonstrating a limited antibiofilm action of CHX.
The poor performance of CHX against biofilm of E. fae-
calis when compared to other endodontic irrigating solu-
tion was already demonstrated in literature (11,13,19).
This may be justified by the inactivation of cationic bis-
biguanides by the organic biofilm matrix and its limited
penetration into the extracellular matrix of the biofilm
(19).
The antimicrobial activity of CHX was evaluated after
10 min based on previous studies (11,24). One of them
states that after 10 min in contact with biofilm of

4 © 2019 Australian Society of Endodontology Inc

N. M. Fiallos et al.
E. faecalis, the effect of both NaOCl and CHX was poor,
and only small amounts of new bacteria were killed
between 10 and 30 min (24) based on those, CHX was
used for 10 min.
In the present study, CLSM images of E. faecalis bio-
films exposed to NaOCl demonstrated the higher ability
to remove biofilm from dentin surfaces with the highest
proportion of dead cells found in this group when com-
pared to the other experimental groups, rejecting the null
hypothesis. The good performance of NaOCl is well docu-
mented in the literature, and these results are in line with
several previous studies that also demonstrated the anti-
biofilm effectiveness of NaOCl (11,13). The ability of
NaOCl to dissolve organic matter and to attack the extra-
cellular matrix of the biofilm (6) may explain these
results. However, the efficacy of NaOCl could be reduced
by the dentin and organic portion of the biofilm (28),
explaining the residual biofilm adhered to the dentin
structure at the end of irrigant exposure, observed in
some samples.
The attractive antimicrobial and tissue dissolution
capacities of NaOCl appear to be intertwined with the
undesired side effects previously described such as its tox-
icity (6) and the untoward effect of degradation the colla-
gen matrix within mineralised dentin (8). Thereby,
studies highlighted alterations in the structural and
chemical composition of human dentin due to prote-
olytic action of NaOCl (8). Due to the small scale, the
hypochlorite anion is able to infiltrate mineralised colla-
gen and to destroy the collagen fibrils. This results in a
mineral-rich, collagen sparse ghost mineral matrix with
reduced flexural strength (8). In consequence, degrada-
tion of organic and inorganic components of dentin
affects negatively the mechanical properties of the den-
tin (7,8) predisposing teeth to fracture under normal
masticatory loads.
Due to the harmful effects of NaOCl on dentin, it is
necessary to search for a biomaterial that could be used
in debridement of the root canal space. Vitis vinifera
(GSE) can be an alternative for the NaOCl. Although GSE
was not effective as NaOCl, in this study, it showed good
antimicrobial activity. Furthermore, GSE did not reduce
the dentin mechanical properties during its use as irrigant
solutions and has been used to treat demineralised den-
tin, increasing mechanical properties and tissue’s stability
against collagenase degradation (29). Also, GSE showed
capacity of improve the bond strength stability of dentin–
resin interfaces (7); besides, the toxicity to human cells is
low (30).
The use of Vitis vinifera (GSE) instead of NaOCl could be
considered especially in endodontic treatment of teeth
presenting thin root structure that will be restored with
adhesive materials and fibre post because GSE is capable
© 2019 Australian Society of Endodontology Inc
Antimicrobial Effectiveness of GSE
to increasing mechanical properties of dentin (29) and
presents a good antimicrobial activity against biofilm of
E. faecalis in this study.
In conclusion, although GSE presented antimicrobial
activity against E. faecalis biofilms, NaOCl was the most
effective irrigant solution, being GSE more effective than
CHX and SS.
Acknowledgements
The authors deny any conflicts of interest related to this
study. This study was financed in part by the Coordenac~ao
de Aperfeicoamento de Pessoal de N
ıvel Superior – Brasil
(CAPES) – Finance Code 001, FAPERJ (Fundac~ ao Carlos
Chagas Filho de amparo  a pesquisa do Estado do Rio de
Janeiro, Grant numbers: E-26/202.839/2015) and CNPq.
Conflict of interest
We, authors, disclose any commercial associations, cur-
rent and within the past 5 years, that might pose a poten-
tial, perceived or real conflict of interest. These include
grants, patent licensing arrangements, consultancies,
stock or other equity ownership, donations, advisory
board memberships, or payments for conducting or pub-
licising the study. We certify that do not have any com-
mercial or associate interest that represents a conflict of
interest in connection with the submitted manuscript.
Authorship declaration
All authors have contributed significantly, and all
authors are in agreement with the manuscript.
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