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162]

Original Article

Carnosic Acid as an intracanal medicament performs

better than triple antibiotic paste and calcium
hydroxide to eradicate Enterococcus faecalis from
root canal: An in vitro confocal laser scanning
microscopic study
Ashwini Dessai, Neeta Shetty, Vishwas Saralaya1, Srikant Natarajan2, Kundabala Mala
Departments of Conservative Dentistry and Endodontics and 2Oral Pathology and Microbiology, Manipal College of Dental Sciences,
Mangalore, Manipal Academy of Higher Education, Manipal, 1Department of Microbiology, Kasturba Medical College, Manipal Academy
of Higher Education, Manipal, Karnataka, India

Abstract
Background: Carnosic acid is an herbal derivative with potent antioxidant, anti‑inflammatory, antimicrobial, and anticancer properties.
Aim: Comparative evaluation of the antimicrobial potential of carnosic acid, calcium hydroxide, and triple antibiotic paste as
intracanal medicaments against Enterococcus faecalis.
Settings and Design: Department of Conservative Dentistry and Microbiology, an in vitro study.
Materials and Methods: Fifty‑two extracted single‑rooted human teeth were decoronated and chemomechanical preparation
was performed. The specimens were secured in the center of screw‑capped vials and autoclaved. A strain of E. faecalis was
inoculated into the canals and grown for 72 h. The teeth were divided into: Group I‑Ca(OH)2, Group II‑ triple antibiotic
paste (TAP), Group III‑Carnosic acid, and Group IV‑Negative control. The medicaments were applied in the canal and left for
14 days. The specimens were sectioned transversely at three levels to create dentinal discs and observed under the confocal
laser scanning microscopic (CLSM). Images were analyzed, and quantification of bacteria was done using the Image J software.
Statistical Analysis: Mean percentage of live/dead bacteria was analyzed using One‑way ANOVA and Post hoc Tukey test.
Results: Mean percentages of live and dead bacteria were seen under CLSM in Group I, Group II, and Group III
were (4.44 ± 2.87, 4.56 ± 2.93, 1.61 ± 1.90), and (4.59 ± 3.04, 4.25 ± 2.98, 1.70 ± 1.99), respectively, with least
mean percentages for live and dead bacteria in carnosic acid (Group III).
Conclusion: Carnosic acid showed better antimicrobial efficacy against E. faecalis than TAP and Ca(OH)2 by showing a low
percentage of both live and dead bacteria.
Keywords: Calcium hydroxide; carnosic acid; confocal laser scanning microscopy; Enterococcus faecalis; intracanal
medicaments; microorganisms

Address for correspondence:

Dr. Neeta Shetty, INTRODUCTION
Department of Conservative Dentistry and Endodontics,
Manipal College of Dental Sciences, Mangalore, Manipal
Academy of Higher Education, Manipal, Light House Hill Road, The microorganisms that enter the root canal system
Mangalore ‑ 575 001, Karnataka, India. are responsible for the initiation and sustenance
E‑mail: neeta.shetty@manipal.edu
This is an open access journal, and articles are distributed under the terms
Date of submission : 16.06.2021 of the Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0
Review completed : 18.12.2021 License, which allows others to remix, tweak, and build upon the work
Date of acceptance : 21.12.2021 non‑commercially, as long as appropriate credit is given and the new
Published : 02.05.2022 creations are licensed under the identical terms.
Access this article online For reprints contact: WKHLRPMedknow_reprints@wolterskluwer.com
Quick Response Code:
Website:
How to cite this article: Dessai A, Shetty N, Saralaya V,
www.jcd.org.in
Natarajan S, Mala K. Carnosic Acid as an intracanal medicament
performs better than triple antibiotic paste and calcium hydroxide to
DOI: eradicate Enterococcus faecalis from root canal: An in vitro confocal
10.4103/jcd.jcd_317_21
laser scanning microscopic study. J Conserv Dent 2022;25:20-5.

20 © 2022 Journal of Conservative Dentistry | Published by Wolters Kluwer - Medknow

[Downloaded free from http://www.jcd.org.in on Saturday, December 24, 2022, IP: 163.53.207.162]
of periapical disease.[1] The reinfected root canal is
dominated by anaerobic bacteria microflora, especially
facultative anaerobes like Enterococcus faecalis.[2] Apart
from its resistance to antibiotics, other specific virulence
factors: quorum‑sensing, adhesins, secretion factors,
capsular polysaccharide, and collagen‑binding protein are
attributed to the persistence of E. faecalis in the root canal
microenvironment system.[3,4]
Calcium hydroxide (Ca(OH)2 is the intracanal medicament
of choice for many years, owing to its antimicrobial
properties. When mixed into a paste, it has a solubility
of <0.2% and a pH of 12.5. Ca(OH)2 exhibits its antimicrobial
activity by releasing hydroxyl ions (OH‑), thereby creating a
nonconducive alkaline environment for the microorganisms
to survive. However, the rate of hydroxyl ion diffusion is
prolonged due to the buffering capacity of the dentin.[5]
The polymicrobial nature of microorganisms is responsible
for root canal infections. Therefore, triple antibiotic
paste (TAP), a combination of antibiotics (Metronidazole,
ciprofloxacin, and minocycline), is routinely used to
eliminate the microorganisms.[6] The major concern with
the usage of this drug is its potential to discolor the tooth,
bacterial resistance, and altered root dentin structure.[7,8]
Plant extracts and their components possessing
antimicrobial properties are used in medical treatments.
Rosmarinus officinalis L.(Lamiaceae) is an edible evergreen
shrub that possesses antioxidant, anti‑inflammatory,
anticancer, and antimicrobial properties for medicinal
and culinary purposes. The main extracts are: Rosmarinic
acid, carnosic acid, carnosol, ursolic acid, oleanolic
acid, genkwanin, apigenin, and luteolin.[9] An in vitro
study evaluated commercially available rosemary
extract formulations against a few bacterial species.
The Gram‑positive bacteria were more sensitive than
Gram‑negative bacteria, especially to oil‑soluble extracts
that contained carnosic acid.[10] There is a paucity of
research regarding its use as an intracanal medicament.
Hence, we planned to conduct this preliminary study to
evaluate the antimicrobial efficacy of Carnosic acid and
compare it with that of TAP and Ca(OH)2 when used as
intracanal medicaments against E. faecalis using confocal
laser scanning microscopy (CSLM). The null hypothesis
stated no difference in the antimicrobial efficacy of the
various experimental intracanal medicaments.
MATERIALS AND METHODS
The present study was conducted in the Department of
Conservative Dentistry and Endodontics, Department of
Microbiology and School of life sciences after obtaining
the Institutional Ethics Committee clearance (protocol ref
no: 16091).
Journal of Conservative Dentistry | Volume 25 | Issue 1 | January-February 2022
Dessai, et al.: Carnosic acid as intracanal medicament
Selection and preparation of samples
Fifty‑two extracted human single‑rooted teeth with Type I
root canal anatomy were chosen for the study. Teeth with
restorations, stains, cracks, noncarious lesions, attrition,
white spot lesions, and hypoplasia were excluded from the
study. The collected teeth were stored in 0.01% sodium
hypochlorite (NaOCl) solution (Vishal Dentocare, Gujarat,
India) until use. Debris, soft tissue, and calculus were
mechanically removed from the root surface, following
which teeth were decoronated using a highspeed
diamond bur (TF‑21, Mani Inc, Japan) under water
cooling. The root length of all the teeth was maintained
at 16 mm. After confirming apical patency with #15
K‑file (Dentsply Maillefer, Ballaigues, Switzerland), root
canals were enlarged to #20 K‑file (Dentsply Maillefer,
Ballaigues, Switzerland). Chemomechanical preparation
of the samples was performed using 2.5% NaOCl to
flush out debris and to shape the canals. Protaper rotary
nickel‑titanium system (Dentsply Maillefer, Ballaigues,
Switzerland) up to the size F3 finishing file was used. On
completion of instrumentation, the canals were irrigated
with 17% ethylene diamine tetraacetic acid to ensure smear
layer removal, followed by 2.5% NaOCl and a final rinse
with sterile saline for a minute. The apical foramina were
sealed with cyanoacrylate, and specimens were secured in
the center of screw‑capped vials and autoclaved twice at
121°C for 20 min.
Bacterial introduction and biofilm generation
E. faecalis strain ATCC 29212 was the microorganism
used in this study. It was cultivated in the microbiology
laboratory. A single colony of E. faecalis was collected from
the agar plate and suspended in the sterile brain heart
infusion broth supplemented with 1.5%(wt/vol) agar and
incubated anaerobically at 37°C for 24 h. The plastic
vials containing the autoclaved teeth were opened in
a Biohazard Cabinet (ESCO Airstream) to maintain the
aseptic environment. The experimental root canals
were inoculated with 10 µl of E. faecalis suspension using
sterile 1 ml tuberculin syringes. Specimens were then
placed in stainless steel boxes and incubated at 37°C for
72 h within Orbital Incubator (Sanyo). The specimens were
then divided into four experimental groups according to
the intracanal medicaments to be used.
Preparation of intracanal medicaments
Group I‑Commercially available CA(OH)2 paste (Calcicur,
VOCO America. Inc) was used as an intracanal medicament.
Preparation of triple antibiotic paste
Group II‑The ingredients of TAP: Ciprofloxacin (Ciplox
500 mg tablet, Cipla India), Metronidazole (Metrogyl
400 mg tablet, JB Chemicals and Pharmaceuticals) and
minocycline (Minoz, 100 mg tablet, Ranbaxy India) Each
of these powders were prepared separately by removing
21
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Dessai, et al.: Carnosic acid as intracanal medicament
the enteric coatings of the tablet and crushing it to a fine
powder using sterile mortar and pestle. The three antibiotic
powders were weighed individually using a digital weighing
machine to obtain a desired 1:1:1 proportion. Then the
powders were mixed to obtain Triple antibiotic powder.[6]
One mg powder was mixed with one ml of sterile water to
obtain a paste consistency.
Preparation of carnosic acid paste
Group III‑≥91% Carnosic acid (Sigma Aldrich) is available
in a powder form, 10 mg of powder was dispensed
on the paper pad and mixed with two drops (0.1 ml) of
propylene glycol to a paste consistency.
Group IV‑No intracanal medicament was placed (Control).
Treatment of biofilms
Thirty‑nine samples were irrigated with sterile saline
for 2 min and dried with sterile paper points, and
randomly divided into three groups (n = 13). Control
specimens (n = 13) were placed in the incubator for the
entire duration of the experiment [Table 1]. The root canals
were filled with the allocated experimental intracanal
medicament using a Lentulospiral (Dentsply Maillefer,
Ballaigues, Switzerland). Zinc oxide eugenol was used to
seal the coronal access cavity.
Preparation of specimens for microbiological
analysis
The root canals were washed with 20 ml sterile saline and
dried with absorbent paper points on the 14th day.
Confocal laser scanning microscopic
examination
The specimens (n = 13 in each group) were sectioned
transversely to obtain dentin discs of approximately 1 mm
thickness at three levels (coronal, middle, and apical),
using a rotating diamond disk under water cooling. After
the specimens were washed using phosphate‑buffered
saline, they were stained with fluorescent LIVE/DEAD
BacLight Bacterial Viability stain [Molecular Probes,
Eugene, OR, USA]. The SYTOTM 9 (ThermoFisher Scientific,
Table 1: Comparison of coronal, middle, and apical live and dead bacterial counts (expressed as a percentage) using the
one‑way ANOVA test (P=0.05)
Intra‑canal level
Calcium hydroxide
Coronal 5.04±3.01∞
Middle 5.02±2.6∞,ⴊ
Apical 3.28±2.84∞,ⴊ
Coronal 5.44±3.41∞
Middle 4.92±2.68∞,ⴂ
Apical 3.4±2.84ⴂ
ⴆ,ⴃ,∞,ⴂ,ⴊ
: Similar symbols represent significant difference (P<0.05) between the subgroups using Posthoc Tukey test. SD: Standard deviation
22 Journal of Conservative Dentistry | Volume 25 | Issue 1 | January-February 2022
USA) (green‑fluorescent stain) that labels live bacteria and
Propidium Iodide (PI) (Himedia, India) (red‑fluorescent stain)
that labels dead bacteria were used. These fluorescent
stains were diluted in saline to give final concentrations
of 10 and 60 µmol L−1, respectively. The specimens were
placed in the tubes containing the stain and stored in the
dark for 30 min. The specimens were rinsed thoroughly
using PBS solution and blot dried before confocal laser
scanning microscopic (CLSM) analysis.
A confocal laser scanning microscope [Leica DMi] was
used to view the specimen cross‑sections. The coronal,
middle, and apical thirds of the root specimens were
scanned at ×40 magnification. The excitation/emission
wavelengths were 480/500 nm for SYTO9 and 490/635 nm
for PI. CLSM images were acquired and analyzed using
Leica Application Suite (Leica Microsystems, Germany) at a
resolution of 1024 × 1024 pixels. Images were processed
for background noise reduction (Leica Application Suite
software). Quantification of the CLSM images was done
using the Image J software. The area percentage of live
cells (green fluorescence) and dead cells (red fluorescence)
was calculated at coronal, middle, and apical third levels
for all samples.
Statistical analysis
The mean percentage of live and dead bacteria was
statistically analyzed using One‑way ANOVA and Post
hoc Tukey test. Paired t‑test was applied for intragroup
comparison of the colonization variation between the
coronal, middle, and apical thirds. Analysis of data was
performed using SPSS version 20.0 software (IBM Corp,
Somers, NY) and the P value was set at P ≤ 0.05.
RESULTS
From the data collected using open J software analyses
of CLSM images, it revealed that among the intracanal
medicament groups, Carnosic acid group showed very
low live bacterial percentage (1.61 ± 1.90) followed by
Ca(OH)2 group (4.44 ± 2.87) and TAP group (4.56 ± 2.93).
The Control group had the highest live bacterial
Medicament group (mean±SD) P
Triple antibiotic paste Carnosic acid Saline
Live bacterial percentage
5.09±3.22ⴃ 3.07±2.53ⴆ 12.43±1.43ⴆ,ⴃ,∞ <0.001
4.62±2.94ⴃ,ⴂ 1.28±0.99ⴆ,ⴂ,ⴊ 10.08±2.48ⴆ,ⴃ,∞ <0.001
3.98±2.74ⴃ,ⴂ 0.49±0.47ⴆ,ⴂ,ⴊ 11.62±1.2ⴆ,ⴃ,∞ <0.001
Dead bacterial percentage
5.4±3.23ⴃ 3.18±2.7 1.68±0.55∞,ⴃ <0.001
5.03±2.94ⴃ,ⴊ 1.45±1ⴂ,ⴊ 1.23±0.6∞,ⴃ <0.001
2.33±1.7ⴊ 0.46±0.41ⴂ,ⴊ 0.7±0.44 <0.001
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Dessai, et al.: Carnosic acid as intracanal medicament

percentage (11.38 ± 2.00). The highest mean dead bacterial
percentage values were for the Ca(OH)2 group (4.59 ± 3.04)
followed by the TAP paste group (4.25 ± 2.98), Carnosic
acid group (1.70 ± 1.99), and the least in the control
group (1.20 ± 0.66) [Figure 1]. Interestingly, the Carnosic
acid group exhibited a low percentage of both live and
dead bacteria in the present study.
present deep within the tubules.[13] This could explain why
a more significant number of dead cells were observed at
the periphery/at the circumpulpal dentin than the deeper
parts of the dentin, as seen in the CLSM images.
According to Mohammadi and Abbott, tetracyclines present
in the TAP (minocycline) can create a strong and reversible

The intragroup comparison showed that the live mean

bacterial percentage was highest in the coronal third of
the root canal and least in the apical third among all the
experimental groups. Similarly, the mean dead bacterial
percentage was highest in the coronal third section and
least in the apical one‑third [Table 1].

The intergroup comparison showed that the live and dead

bacteria percentage was comparable in all three groups in
the coronal regions [Figure 2]. The live bacterial percentage
was significantly more in the middle and apical regions in
Ca(OH)2 and TAP groups than in the Carnosic acid group,
with no significant difference in Ca(OH)2 and TAP groups.
A similar observation was noted with the dead bacteria
percentage (P = 0.001) [Figure 2 and Table 1]. Carnosic
acid showed the lowest live and dead bacteria in all three
sections of root.

DISCUSSION

Chemomechanical preparation of the canal system will

eradicate most organisms and their biofilms, but some
chronic organisms in the root canal are challenging to
eradicate, especially E. faecalis.[11] The study results imply
that the antibacterial activity of Ca(OH)2 and TAP was
similar in all three regions of the roots [Table 1]. The
antibacterial effect produced by calcium hydroxide is
primarily dependent on the diffusion of the hydroxyl ions
into the dentinal tubules and their high pH.[5] Therefore,
it is evident that to achieve any antibacterial effect within
the tubules, the ionic diffusion capacity of Ca(OH)2 must
exceed the dentin’s inherent buffering ability.[12] According
to Siqueira et al., the bacterial cells at the periphery of
the colonies can have a protective effect on the colonies

Figure 2: CLSM images showing live (L)/dead (D)/

Figure 1: Live and dead bacterial percentage values
Consolidated(C) bacteria for different groups at coronal
third level:1A- Ca(OH)2 (L),1B- Ca(OH)2 (D), 1C- Ca(OH)2
(C),2A- TAP (L),2B- TAP(D), 2C- TAP(C),3A-Carnosic
acid(L),3B- Carnosic acid(D),3C- Carnosic acid(C). CLSM
images showing live (L)/dead (D)/Consolidated(C) bacteria
for different groups at middle third level.: 1D- Ca(OH)2
(L),1E- Ca(OH)2 (D), 1F- Ca(OH)2 (C),2D- TAP (L),2E-
TAP(D), 2F- TAP(C),3D-Carnosic acid(L),3E- Carnosic
acid(D),3F- Carnosic acid(C). CLSM images showing live
(L)/dead (D)/Consolidated(C) bacteria for different groups
at apical third level. 1G- Ca(OH)2 (L),1H- Ca(OH)2 (D),
1I- Ca(OH)2 (C),2G- TAP (L),2H- TAP(D), 2I- TAP(C),3G-
Carnosic acid(L),3H- Carnosic acid(D),3I- Carnosic acid(C)

Journal of Conservative Dentistry | Volume 25 | Issue 1 | January-February 2022 23

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Dessai, et al.: Carnosic acid as intracanal medicament
bond with the hard tissues by forming complexes with
trivalent and bivalent cations, resulting in slower release of
the drug over a period of time.[6] Sato et al. demonstrated
that Metronidazole could penetrate the deeper layers
of the dentin and indicated in the treatment of obligately
anaerobic bacteria.[14] Due to the inherent discoloration
potential and its effect on dentin structure, TAP must be
used cautiously.[15]
In the present study, the carnosic acid group has displayed
good antimicrobial efficacy by showing a low percentage of
the live and dead bacterial count, suggesting that carnosic
acid did not allow the bacteria to multiply [Table 1]. The
exact antimicrobial mechanism of carnosic acid is not
entirely known but could be attributed to the lipophilic
nature of the compounds that allows it to insert into the
bacterial membrane.[16]
Carnosic acid interacts with the ethidium bromide
efflux system and acts as a modulator for membrane
permeability.[17] According to Nakagawa et al., carnosic
acid is a potential quorum sensing inhibitor against
Staphylococcus aureus; it inhibits the activation of genes
involved in virulence and biofilm formation. Quorum
sensing inhibition has been suggested as an alternative
approach to combat infection and the same mechanism of
action of carnosic acid may also have been effective against
E. faecalis.[18] The pathogenesis of Enterococcal infection
at the molecular level is oxidative stress due to the
production of free radicals. E faecalis produces superoxide
and hydrogen peroxide, which is essential for its survival.
The antioxidant activity of carnosic acid could have acted
as scavengers of free radicals and curbed its proliferative
ability.[19]
E. faecalis uses dentin serum to adhere to the dentin and enter
the dentinal tubules and multiply, so it can be postulated that
carnosic acid could have prevented the growth of E faecalis
by modifying the dentinal serum.[20] Hence, we speculated
that carnosic acid reduces adhesion and does not provide an
environment to allow microorganism growth. Moreno et al.,
concluded that the antimicrobial efficacy of R. officinalis L.
was associated with their specific phenolic composition.
According to their findings, 25–200 µg of carnosic acid
inhibits bacterial growth.[21]
An intragroup comparison revealed that all the experimental
intracanal medicaments showed the highest dead bacteria
percentage in the coronal third, followed by the middle
and apical third region [Table 1]. Intracanal medicaments
are conveniently and adequately applied in the coronal
third, enabling better action at this region.
Recently, CSLM has emerged as an excellent method to
study the biofilm structure, primarily because it does not
disturb the bacterial ecosystem and permits noninvasive
24 Journal of Conservative Dentistry | Volume 25 | Issue 1 | January-February 2022
investigation of these ecosystems.[22,23] However, we
performed our experiments on an in vitro biofilm model
that may not accurately represent or mimic in vivo biofilm
behavior. The limitation of in vitro models is that they lack
the external factors that challenge in vivo biofilm, primarily
due to the host immune response.
The standardization of fluorescence between the optical
slices and obtaining a consistent balance between the red
and green fluorescent signals is a limitation with CSLM.
The intensity of fluorescence depends on various factors:
The depth of the optical slice, the thickness of the sections,
and the degree of dentin mineralization.[24] Hence, these
parameters need to be adjusted separately for each image,
resulting in slight variations. These shortcomings have
been highlighted previously by Hope and Wilson.[25]
To our knowledge, very few studies have been conducted to
evaluate the effectiveness of carnosic acid as an intracanal
medicament. Further clinical studies are required to evaluate
the potential effect of carnosic acid on clinical outcomes.
Hence within the study’s limitations, it can be concluded
that all three experimental intracanal medicaments are
capable of producing an antimicrobial effect and are
effective against E. faecalis. Since Carnosic acid is a new
biomaterial used in endodontics, further studies evaluating
its physical, chemical, and biological properties and their
effects on substrate would be beneficial.
CONCLUSION
Based on the results obtained, all three intracanal
medicaments tested in the study showed significantly
reduced E faecalis count in 14 days. Carnosic acid is a
promising intracanal medicament with a good antibacterial
effect, where E. faecalis is a significant pathogen.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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