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Can herbal products be alternative root canal irrigation solutions in primary

teeth? An in vitro study

Article in Pediatric Dental Journal · October 2022

DOI: 10.1016/j.pdj.2022.09.001

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Research Paper

Can herbal products be alternative root canal

irrigation solutions in primary teeth? An in vitro
study

Semih Ercan Akgun a, Ipek Arslan a,*, Sema Aydinoglu a,

Dilara Nil Gunacar b, Sengul Alpay Karaoglu c, Emine Yurteri d,
Seyma Suyabatmaz c
a
Recep Tayyip Erdogan University, Faculty of Dentistry, Department of Pediatric Dentistry, Rize, Turkey
b
Recep Tayyip Erdogan University, Faculty of Dentistry, Department of Oral and Maxillofacial Radiology, Rize,
Turkey
c
Recep Tayyip Erdogan University, Faculty of Art and Science, Department of Biology, Rize, Turkey
d
Recep Tayyip Erdogan University, Faculty of Agriculture, Department of Agriculture and Natural Sciences, Rize,
Turkey

article info abstract

Article history: Objectives: To evaluate antimicrobial properties and smear layer removal efficiency of
Received 24 January 2022 various plant extracts and apple cider vinegar (AV).
Received in revised form Methods: A total of 112 primary molars roots were randomly divided into two groups
31 August 2022 (n ¼ 56) for antimicrobial test and infected with E faecalis and Candida albicans then divided
Accepted 5 September 2022 again into 7 groups (n ¼ 8) as physiological saline (PS), NaOCI, white tea (WT), Helichrysum
Available online xxx arenarium (HA), AV, Citrus lemon (CL), and Anzer thyme (AT). Disinfecting abilities were
measured with ImageJ. In the smear layer removal efficiency test, prepared 70 roots were
Keywords: divided into 7 groups (n ¼ 10) as in the antimicrobial test also 2 subgroups were designed as
Herbal with or without EDTA usage (n ¼ 5). All samples were examined under SEM. Open dentin
Vinegar tubule percentage was evaluated with ImageJ.
Primary teeth Results: HA and CL showed higher antibacterial activity against E. faecalis than PS (p ¼ 0.038,
Root canal treatment p ¼ 0.009) Antifungal activity of AV against C. albicans was higher than saline (p < 0.001). AV
Smear layer in the coronal third and CL in the middle third removed more smear layer than saline and
NaOCI (p < 0.05). In the coronal region, HA and AV showed similar smear layer removal
efficacy to NaOCl þ EDTA (p > 0.05). CL, HA, and AV presented statistically similar results to
NaOCl þ EDTA in smear layer removal efficacy in the middle region (p > 0.05).
Conclusions: CL and AV have antimicrobial efficacy against E. faecalis and C. albicans
respectively and smear layer removal activity.
© 2022 Japanese Society of Pediatric Dentistry. Published by Elsevier Ltd. All rights reserved.

* Corresponding author.
E-mail address: ipek.arslan@erdogan.edu.tr (I. Arslan).
https://doi.org/10.1016/j.pdj.2022.09.001
0917-2394/© 2022 Japanese Society of Pediatric Dentistry. Published by Elsevier Ltd. All rights reserved.

Please cite this article as: Akgun SE et al., Can herbal products be alternative root canal irrigation solutions in primary teeth? An in vitro
study, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2022.09.001
2 pediatric dental journal xxx (xxxx) xxx
1. Introduction
Dental caries and pulpitis are still among the major problems
in childhood. In cases where pulpitis cannot be controlled,
tooth loss may occur at an early age, which may result in
psychological and physiological problems. Root canal therapy
(RCT) is a frequently preferred therapeutic method to treat
pulpitis. Elimination of microorganisms and their products
from the root canal system is one of the key factors for the
success of RCT [1]. Although many microorganisms have been
reported to be associated with pulpitis, in most cases of RCT
failure, Enterococcus faecalis (E. faecalis) and Candida albicans (C.
albicans), which can withstand restricted nutritional condi-
tions can commonly be isolated [2].
Mechanical preparation is conducted to reduce the count
of microorganisms in root canals. It has long been known that
mechanical preparation alone is not sufficient for proper
elimination due to the presence of microorganisms found in
lateral canals, dentinal tubules, or anastomoses where files
cannot reach [3]. Also, a smear layer is formed that can pro-
vide an environment to maintain the vitality of microorgan-
isms in the root canal following the mechanical preparation
[4]. Additionally, the smear layer creates a physical barrier to
reduce dentin permeability and the sealing ability of root
canal filling materials. To overcome these, combined therapy
with mechanical preparation and irrigation with antibacterial
solutions are recommended for the success of endodontic
therapy [5].
Various irrigation solutions such as physiological saline
(PS), ethylenediaminetetraacetic acid (EDTA), chlorhexidine
(CHX), and sodium hypochlorite (NaOCl) are used for RCT in
primary teeth [6]. NaOCl has been preferred the most for many
years due to its high antibacterial effect and organic tissue-
dissolving ability. Despite its advantages, it is not recom-
mended to be used alone due to its insufficiency in dissolving
inorganic tissues. Accordingly, a combination with chelating
agents, which can solve inorganic components, especially
EDTA, is advised. However, many adverse effects of these
chemical irrigation solutions have been reported [6]. There-
fore, the need for safer alternative irrigation solutions has
arisen [7].
There has been increased interest in herbal products
recently in dentistry due to their high therapeutic efficacy and
fewer adverse effects [8]. Camellia sinensis (White Tea-WT), CL,
and Thymus species have been investigated, albeit limitedly,
in various areas of dentistry to date because of their thera-
peutic effects [9e11]. To the best of our knowledge there are no
studies about Helichrysum arenarium (HA) in dentistry. Apple
vinegar (AV), known to have many therapeutic effects, has
been investigated in a few studies as an irrigation solution
[12,13]; nevertheless, all of these studies were conducted on
permanent teeth and no studies were found on deciduous
teeth.
Therefore, the aims of this in vitro study were to evaluate
the antibacterial activity of WT, HA, AV, CL, and Thymus
praecox Opiz subsp. Caucasicus [Wild. Ex Ronniger] Jalas var.
Caucasicus (Anzer Thyme-AT), and to investigate their smear
layer removal ability.
Please cite this article as: Akgun SE et al., Can herbal products be alternative root canal irrigation solutions in primary teeth? An in vitro
study, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2022.09.001
2. Materials and methods
2.1. Sample size calculation
The minimum sample size for microbiologic evaluation was
defined from the study of Almeida et al. It was determined
that a minimum of 8 samples should be taken in each group
with 95% confidence (1-a) and 99.9% test power (1-b) [14]. To
determine smear layer removal efficacy, it was seen that a
minimum of five samples should be included in each group
with 95% confidence and 97.9% test power [15].
2.2. Preparation of herbal products
The dried samples of WT, HA, CT, and AT were weighed as 5 g
each and 100 mL of methanol was added. Extraction was
performed in a soxhlet extractor (E-816, BUCHI, Flawil,
Switzerland) for 1 h. After the solvent was removed using the
evaporator, the extract was kept at 20  C until required for
analysis. AV was obtained commercially (Kemal Kükrer,
Eskis‚ehir, Turkey).
2.3. Minimum inhibitory concentrations (MIC) and
minimum bactericidal/fungicidal concentrations (MBC/MFC)
For the determination of MIC values, 96-well enzyme-linked
immunosorbent assay (ELISA) plates were used. One hun-
dred microliters of Brain Heart Infusion (BHI) broth was added
to all wells of the plates prepared for E. faecalis, and 100 mL of
DeMan Rogosa Sharp (MRS) broth was added to those pre-
pared for C. albicans. After placing 100 mL of test solutions into
the first wells of the columns, a serial dilution was performed
from the first column to the last column. From the suspen-
sions prepared in 1 McFarland scale using a spectrophotom-
eter (1100RS, Cole-Parmer, IL, USA), 10 mL of suspension in E.
faecalis and C. albicans groups were placed in each well. Plates
prepared for E. faecalis were incubated for 24 h and those
prepared for C. albicans for 48 h at 37  C. The MIC values of
tested solutions were determined as the lowest concentration
inhibiting the visible growth of bacteria/fungus. To ascertain
the MBC and MFC values, upper concentrations of MIC values
were subcultured on the Mueller-Hinton Agar (MHA) plates for
E. faecalis and Potato Dextrose Agar (PDA) plates for C. albicans.
The lowest concentration without bacterial/fungal growth
was determined as the MBC/MFC value.
2.4. Preparation of teeth
A total of 186 deciduous human molar teeth without any ab-
normalities such as calcification, obliteration, physiologic or
pathologic resorption in the root were used. The teeth were
stored temporarily at room temperature in 0.1% thymol so-
lution until required for use. All debris and tissue remnants
were cleaned and removed under water. To standardize the
roots, the crown part of the teeth was decoronated using a
diamond disk (SH 393190, Sunshine Diamonds, Langenhagen,
Germany) to achieve a 12 ± 0.5 mm standard root length. The
root canal system was checked using 10 K-file (Dentsply
 pediatric dental journal xxx (xxxx) xxx
Maillefer, Ballaigues, Switzerland) until the apical end, and the
working length was adjusted to 1 mm short of the apical fo-
ramen. Root canal instrumentation was performed using
ProTaper Universal files (Dentsply Maillefer, Ballaigues,
Switzerland) up to F2 by the same operator. Root canals were
irrigated with 2 mL of distilled water after each file during
instrumentation. Following these procedures, the roots were
randomly divided into two groups as antimicrobial activity
(n ¼ 116) and smear layer removal ability (n ¼ 70). In the
antimicrobial activity group, 3 mL 17% EDTA (Imicryl Ltd.,
Konya, Turkey) was used to remove the smear layer. The
apical foramens were sealed using fluid composite resin
(Estelite Flow Quick, Tokuyama Dental, Japan), and the root
surfaces were varnished with two layers of nail polish (L'Oreal
Jet-Set Diamond, Paris, France) to obtain a closed canal sys-
tem. All specimens were autoclaved at 121  C under 15 psi
pressure for 30 min. All procedures after this stage were per-
formed under aseptic conditions in a laminar flow hood (MN
120, Nüve, Ankara, Turkey).
In the smear layer removal ability group, longitudinal
grooves were formed on the buccal and lingual/palatal sur-
faces of the roots using a diamond disk without extending to
the root canal.
2.5. Determination of the antibacterial effect
2.5.1. Preparation of bacterial and fungicidal culture
A suspension of E. faecalis (American Type Culture Collection
[ATCC] 29212) in BHI broth (Merck, Darmstadt, Germany) and
C. albicans (ATCC 60193) in MRS broth (Merck, Darmstadt,
Germany) were prepared for 24e48 h at 37  C in a bacteriologic
incubator (Dabi Atlante, SP, Brazil). The degree of turbidity of
the inoculums was adjusted to 1.0 McFarland scale, which
corresponds to 3  108 cfu/mL.
2.5.2. Bacterial inoculation of the root canals
The total of 116 sterilized roots at 121  C in an autoclave were
randomly placed into the wells of 24-well culture plates pre-
pared for E. faecalis (n ¼ 56) and C. albicans (n ¼ 56). Two extra
samples for each group were added to the wells to determine
biofilm formation. The suspensions containing the relevant
microorganisms (50 mL) were inoculated into the root canals of
each sample. BHI broth for E. faecalis and MRS broth for C.
albicans were added, 1 mL for each, into the wells and incubated
at 37  C for 10 days. The media in the wells was renewed every
Fig. 1 e Software colony analysis with the ImageJ program. a. The image of the petri b. CFU percentages of the total image.
Please cite this article as: Akgun SE et al., Can herbal products be alternative root canal irrigation solutions in primary teeth? An in vitro
study, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2022.09.001
3
48 h. At the end of the tenth day, two randomly selected
samples from both groups were examined using scanning
electron microscopy (SEM) (JSM-6610, JEOL, Peabody, Massa-
chusetts, USA) at 5000 and 10,000 magnifications to confirm
biofilm formation.
2.5.3. Irrigation of teeth
After 10 days incubation period, the roots were divided into
seven subgroups (n ¼ 8) for the E. faecalis (n ¼ 56) and C. albi-
cans (n ¼ 56) groups. NaOCI, WT, HA, AV, CT, and AT solutions
were prepared freshly according to the MBC/MFC values. For
each root, a total of 6 mL irrigation solution was applied to the
root canal via a 27-gauge needle.
2.5.4. Root canal sampling
At the end of this period, an #F2 paper point was placed in the
root canals for 30 s to completely absorb the root canal con-
tent. The sample was transferred to an Eppendorf tube and
1 mL of sterile saline was added and vortexed for 1 min. The
material was diluted to 103. Aliquots of 100 mL solution were
cultivated on MHA medium for E. faecalis and PDA medium for
C. albicans and incubated at 37  C for 24e48 h.
2.5.5. Software colony forming unit (cfu) counting
In the calculation of cfus, the ImajeJ 1.53 (Wayne Rasband,
National Institutes of Health, USA) image analysis program
was used. An image of the Petri plate, whose cfu count was to
be made, was opened using the ImageJ program. A circle was
drawn around the edge of the Petri plate and a region of in-
terest (ROI) was determined. Under the “Edit” tab, “Clear
Outside” was used to remove the outer boundaries of the Petri
plate. Under the “Image” tab, “Type” was chosen to convert
the image to 8-bit. To make colonies appear as black spots
with a white background, the threshold located “Image” tab
under “Adjust” was set. For all of these steps, “Analyze Parti-
cles” was selected under the “Analyze” tab. The cfu count was
calculated as the ratio of the area of microorganisms grown in
the Petri plates to the total surface area of the Petri plates
(Fig. 1). For each samples, three replicates were assayed.
2.6. Determination of smear layer removal
A total of 70 teeth were divided into seven groups (n ¼ 10),
similar to the groups in the antimicrobial activity test. In each
group, five samples were irrigated with only the tested
4 pediatric dental journal xxx (xxxx) xxx

Fig. 2 e Examination of open dentin tubules with ImageJ program, a. SEM image of the root canal dentine, b. Applying the
“Threshold” adjustment, c. Calculation of open dentinal tubules ratio to the total surface.

irrigation solution, and five samples were irrigated with EDTA used for post hoc group comparisons to compare the inter-
as final irrigation. The study by Teixeira et al. [16] was taken as group values of antimicrobial effects. The smear layer efficacy
a reference for irrigation with tested solutions. Accordingly, of the coronal and middle thirds was analyzed using one-way
the teeth were irrigated with 3 mL of test solution for 3 min, analysis of variance (ANOVA) followed by the Tukey test
then 3 mL of distilled water for 1 min. Afterwards, five because the data were normally distributed. The
randomly selected samples were irrigated additionally with KruskaleWallis test and Bonferroni-adjusted ManneWhitney
3 mL of 17% EDTA for 3 min, then with 3 mL of distilled water U test were used to analyze apical thirds. The differences be-
for 1 min. The concentrations of the irrigation solutions were tween the thirds were assessed using the Friedman test and
selected as the highest MBC and MFC values determined for E. repeated measures ANOVA for non-normally and normally
faecalis and C. albicans. distributed data, respectively. Student t-test was used to
compare groups with or without EDTA.
2.6.1. SEM
After irrigation with the test solutions, the root canals were
dehydrated with ascending grades of ethanol (25%, 50%, 75%, 3. Results
and 100%) for 15 min and were dried using sterile paper points.
All samples were kept in liquid nitrogen and divided into two 3.1. MIC and MBC/MFC values
using a chisel. Then, samples were sputter-coated with gold
(SC7620 “Mini”, Polaron Sputter Coater, Quorum Technologies, 3.1.1. E. faecalis
Newhaven, England) and examined using SEM (JSM-6610; The MIC and MBC/MFC values of the tested solutions are
JEOL, Peabody, Massachusetts, USA). Images were obtained at shown in Table 1. The MIC values of WT, HA, and AT
1000 magnification from the coronal, middle, and apical (0.125 mg/mL) were lower than AV and CL (0.250 mg/mL). HA
third regions. and AT had lower MBC values (0.125 mg/mL) than WT, AV, and
AT. NaOCl had the lowest values of both MIC and MBC
2.6.2. Software image analysis (0.025 mg/mL).
The ImageJ image analysis program was used to analyze SEM
images. Before the analysis, SEM information was removed 3.1.2. C. albicans
from each image. For better calculations, the digital photos The MIC values of WT, CL, and AT (0.125 mg/mL) were lower
were converted to an 8-bit format. Under the “Edit” tab, than AV and CL (0.250 mg/mL). AT had a lower MFC value
“Invert” was used to highlight the dentinal tubules, which (0.125 mg/mL) than herbal products. NaOCl had the lowest
enhanced the contrast. Following the tabs “Image,” “Adjust,” values of both MIC and MFC (0.025 mg/mL).
and “Threshold,” the image was converted the background to
white and dentinal tubules to black. All of these steps, under
the “Analyze” tab, “Analyze Particles” was selected and the
ratio of the total surface area of open dentinal tubules to the
Table 1 e MIC and MBC/MFC values of test solutions.
surface area of the image was determined (Fig. 2). For each
samples, three replicates were assayed. Test E. faecalis C. albicans
solution
MIC MBC MIC MFC
2.7. Statistical analysis NaOCI 0.025 mg/mL 0.025 mg/mL 0.025 mg/mL 0.025 mg/mL
WT 0.125 mg/mL 0.250 mg/mL 0.125 mg/mL 0.250 mg/mL
The data were analyzed using the Statistical Package for the HA 0.125 mg/mL 0.125 mg/mL 0.250 mg/mL 0.250 mg/mL
Social Sciences (SPSS), version 20 software package (IBM Corp, AV 0.250 mg/mL 0.500 mg/mL 0.250 mg/mL 0.500 mg/mL
CL 0.250 mg/mL 0.250 mg/mL 0.125 mg/mL 0.250 mg/mL
Armonk, NY, USA). The normality of the data distribution was
AT 0.125 mg/mL 0.125 mg/mL 0.125 mg/mL 0.125 mg/mL
determined using the Shapiro-Wilk test. The level of signifi-
cance was set to p < 0.05. The data obtained from antimicrobial *PS: physiological saline, NaOCI: sodium hypochlorite, WT: white
tea, HA: Helichrysum arenarium, AV: apple vinegar, CL: citrus lemon,
activity were not normally distributed. The KruskaleWallis
AT: Anzer thyme.
test and Bonferroni-adjusted ManneWhitney U test were

Please cite this article as: Akgun SE et al., Can herbal products be alternative root canal irrigation solutions in primary teeth? An in vitro
study, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2022.09.001
 pediatric dental journal xxx (xxxx) xxx 5

Table 2 e Antimicrobial activities of tested solutions against E. faecalis and C. albicans.

Test Solution E. faecalis C. albicans
Mean ± SD Med (MineMax) Mean ± SD Med (MineMax)
a a
PS 11.14 ± 0.91 10.8 (8.32e15.23) 12.57 ± 1.88 10.53 (5.38e21.11)
NaOCI 0±0b 0 (0e0.01) 0±0b 0 (0e0.01)
WT 0.68 ± 0.12a,c 0.69 (0.11e1.27) 1.14 ± 0.15a,c 1.23 (0.54e1.79)
HA 0.50 ± 0.05b,c 0.51 (0.30e0.72) 1.09 ± 0.19a,c 1.00 (0.29e1.83)
AV 0.73 ± 0.08a,c 0.77 (0.39e1.05) 0.17 ± 0.04b,c 0.16 (0.02e0.44)
CL 0.44 ± 0.05b,c 0.46 (0.18e0.69) 0.80 ± 0.13a,c 0.89 (0.15e1.22)
AT 0.92 ± 0.13a,c 0.84 (0.49e1.82) 0.79 ± 0.13a,c 0.72 (0.38e1.31)

p value p<0.001 p<0.001

*The cfu count was calculated as the ratio of the area of microorganisms grown in the Petri plates to the total surface area of the Petri plates.
**KruskaleWallis test was performed (p < 0.05).
***PS: physiological saline, NaOCI: sodium hypochlorite, WT: white tea, HA: helichrysum arenarium, AV: apple vinegar, CL: citrus lemon, AT:
Anzer thyme., Mean ± SD: mean ± standard deviation, Med (MineMax): median (minimumemaximum).
****There was no statistical difference between irrigation solutions shown with similar superscript in the same column (p > 0.05).

Table 3 e Pairwise comparison results (p-values) of antimicrobial activity of irrigation solutions against E. faecalis and C.
albicans.
E. faecalis C. albicans
NaOCl WT HA AV CL AT NaOCl WT HA AV CL AT
PS <0.001 0.110 0.006 0.330 0.001 1.000 <0.001 1.000 0.594 <0.001 0.100 0.071
NaOCl 0.041 0.518 0.011 1.000 0.001 0.002 0.005 1.000 0.045 0.065
WT 1.000 1.000 1.000 1.000 1.000 0.100 1.000 1.000
HA 1.000 1.000 1.000 0.192 1.000 1.000
AV 1.000 1.000 1.000 1.000
CL 0.550 1.000

*p-values obtained after Bonferroni correction.

**Significantly different p values are shown in bold.
***PS: physiological saline, NaOCI: sodium hypochlorite, WT: white tea, HA: Helichrysum arenarium, AV: apple vinegar, CL: citrus lemon, AT:
Anzer thyme.

3.1.3. Antimicrobial efficacy against E. faecalis and C.
albicans
Antimicrobial activities against E. faecalis and C. albicans are
illustrated in Table 2. It was found that the antimicrobial ef-
ficacy of NaOCl was significantly higher than for PS against
both E. faecalis and C. albicans (p < 0.001). HA and CL showed
significantly more antibacterial efficacy than PS (p ¼ 0.006 and
p ¼ 0.001, respectively) and similar efficacy to NaOCl (p > 0.05)
against E. faecalis. AV was observed to have greater antifungal
efficacy than PS (p < 0.001). There were no statistical differ-
ences between the other groups (p > 0.05) (Table 3).
3.2. Smear layer removal efficacy
(p > 0.05). In the apical region, AV and AT had a significantly
higher percentage of open dentin tubules than NaOCl
(p ¼ 0.018 and p ¼ 0.022, respectively) and had similar results
to NaOCl þ EDTA (p > 0.05). There were no statistically sig-
nificant differences between the groups with EDTA in all re-
gions (p > 0.05). Decreased open dentin tubule percentages
from coronal to the apical thirds were found but not all dif-
ferences were statistically significant. Further, the compari-
son of open dentine tubules between with or without EDTA
groups were shown. PS, NaOCl, HA and AT in the coronal re-
gion, PS, NaOCl and AV in the middle region and NaOCl in the
apical region had significantly less open dentin tubules than
same irrigants combined with EDTA (p < 0.05).

Representative SEM images (1000) of root canal walls of all

groups without and with EDTA are presented in Figs. 3 and 4,
respectively. The percentages of open dentin tubules are
illustrated in Table 4. In the coronal region, AV irrigation
revealed a highly significant difference compared with PS and
NaOCl (p ¼ 0.023 and p ¼ 0.019, respectively), also HA and AV
showed similar smear layer removal efficacy to NaOCl þ EDTA
(p > 0.05). In the middle region, CL irrigation presented more
open dentin tubule percentages than PS and NaOCl (p ¼ 0.049
and p ¼ 0.036, respectively). Furthermore, CL, HA, and AV
presented statistically similar results to NaOCl þ EDTA
4. Discussion
Elimination of organic and inorganic remnants and microbial
contents in root canals is one of the important points for
successful endodontic treatment [1]. Irrigation has great
importance because of the remaining area without instru-
mentation, especially the more complex root canal system of
primary teeth with its wide number of branches and micro-
canals [1]. NaOCI, CHX, and EDTA are the most frequently
used agents as auxiliary irrigation solutions in the

Please cite this article as: Akgun SE et al., Can herbal products be alternative root canal irrigation solutions in primary teeth? An in vitro
study, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2022.09.001
6 pediatric dental journal xxx (xxxx) xxx

Fig. 3 e Representative SEM images from groups of the coronal, middle, and apical thirds at £1000 magnification. CL, HA,
and AV presented statistically similar results to NaOCl þ EDTA (p > 0.05).

Please cite this article as: Akgun SE et al., Can herbal products be alternative root canal irrigation solutions in primary teeth? An in vitro
study, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2022.09.001
 pediatric dental journal xxx (xxxx) xxx 7

Fig. 4 e Representative SEM images from groups with EDTA of the coronal, middle, and apical thirds at £1000 magnification.
There were no statistically significant differences between the groups with EDTA in all regions (p > 0.05).

Please cite this article as: Akgun SE et al., Can herbal products be alternative root canal irrigation solutions in primary teeth? An in vitro
study, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2022.09.001
8 pediatric dental journal xxx (xxxx) xxx

Table 4 e The percentages of open dentin tubules by groups and regions.

Groups Coronal Middle Apical
PS 0.12 ± 0.01/0.11 (0.07e0.17)a,y 0.09 ± 0.01/0.09 (0.09e0.10)a,y 0.07 ± 0.01/0.07 (0.07e0.10)
NaOCI 0.07 ± 0.01/0.07 (0.07e0.09)a,A,y 0.04 ± 0.01/0.04 (0.04e0.06)a,B,y 0.01 ± 0.01/0.01 (0.01e0.02)a,C,y
WT 0.64 ± 0.15/0.56 (0.20e1.03)a,b, 0.41 ± 0.07/0.43 (0.25e0.63)a,b 0.33 ± 0.12/0.26 (0.14e0.81)
HA 1.23 ± 0.61/0.33 (0.19e3.05)a,c,A,y 0.71 ± 0.31/0.28 (0.17e1.67) 0.64 ± 0.30/0.17 (0.12e1.49)B
AV 2.10 ± 0.60/2.18 (0.34e3.72)b,c,A 1.02 ± 0.41/0.50 (0.22e2.30)y 0.78 ± 0.36/0.26 (0.21e2.04)b
CL 1.39 ± 0.12/1.56 (1 .02e1.61)A 1.22 ± 0.26/0.85 (0.70e1.98)b,c,A 0.30 ± 0.06/0.26 (0.15e0.49)B
AT 0.40 ± 0.09/0.30 (0.23e0.70)a,b,y 0.36 ± 0.03/0.36 (0.28e0.48)a,b 0.33 ± 0.03/0.29 (0.25e0.46)b
NaOCI þ EDTA 2.84 ± 0.66/2.16 (1.23e4.65)c 1.55 ± 0.36/1.59 (0.60e2.52)c 0.27 ± 0.08/0.31 (0.01e0.52)
p value p < 0.001 p ¼ 0.001 p ¼ 0.003
PS þ EDTA 1.48 ± 1.30/1.04 (0.34e3.69)A,z 1.16 ± 0.80/1.47 (0.26e1.91)z 0.31 ± 0.44/0.12 (0.07e1.11)B,
NaOCI þ EDTA 2.84 ± 1.49/2.16 (1.23e4.65)A,z 1.55 ± 0.82/1.59 (0.60e2.52)z 0.27 ± 0.19/0.31 (0.01e0.52)B,z
WT þ EDTA 1.45 ± 0.75/1.59 (0.39e2.16) 0.73 ± 0.45/0.65 (0.40e1.50) 0.30 ± 0.16/0.28 (0.09e0.50)
HA þ EDTA 3.23 ± 1.30/3.67 (1.52e4.81)A,z 1.35 ± 0.40/1.37 (0.85e1.76)A 0.38 ± 0.18/0.30 (0.20e0.59)B
AV þ EDTA 2.66 ± 0.83/2.87 (1.22e3.31) 1.32 ± 0.61/1.22 (0.50e2.05)z 1.05 ± 0.58/0.73 (0.50e1.77)
CL þ EDTA 2.02 ± 1.20/1.85 (0.53e3.36) 1.28 ± 0.75/1.69 (0.44e1.99) 0.34 ± 0.09/0.37 (0.23e0.45)
AT þ EDTA 2.02 ± 1.26/2.16 (0.69e3.38)z 0.80 ± 0.44/0.69 (0.47e1.56) 0.32 ± 0.17/0.28 (0.08e0.55)
p value p ¼ 0.448 p ¼ 0.081 p ¼ 0.061

*PS: physiological saline, NaOCI: sodium hypochlorite, WT: white tea, HA: Helichrysum arenarium, AV: apple vinegar, CL: citrus lemon, AT: Anzer
thyme.
Mean ± standard deviation/median (minimumemaximum).
**ANOVA and Tukey test were used in the evaluation of the coronal and middle thirds, KruskaleWallis and Bonferroni corrections were per-
formed in the apical third. Student t-test was used to compare groups with and without EDTA.
***There was a statistically significant difference between the groups shown with different lowercase superscripts in the same column and
between the regions in the same row with uppercase superscripts (p < 0.05). Different superscript symbols show the statistically differences
between the groups with and without EDTA.

biomechanical preparation of root canals. It has been
observed that these solutions exert cytotoxic and genotoxic
effects on oral and periapical tissues. Hence, herbal products
are gaining popularity because of their minimal adverse ef-
fects. In the present study, the antimicrobial effects of WT,
HA, AV, CL, and AT were investigated against E. faecalis and C.
albicans, which are the most commonly associated bacterium
and fungus with recurrent endodontic infections [2], also their
smear layer removal ability.
Intracanal biofilms of E. faecalis and C. albicans are reported
to develop between 1 day and 4 weeks. Microorganisms in
mature biofilms have more resistance than those in immature
biofilms that develop in short periods [17]. Therefore, in this
study, the antimicrobial effectiveness of the irrigation solu-
tions was evaluated in 10-day-old E. faecalis and C. albicans
biofilms. Studies on the anatomy of root apices have demon-
strated that the distance between the apical major foramen
and the minor foramen varied from 0.5 to 1 mm for teeth of
different ages. In particular, physiological resorption often
occurs in the root apices of primary teeth. It can be seen that
the amorphous structure in the root dentin observed in the
apical region of the primary tooth root caused by physiological
resorption. Therefore, in the present study, the roots were
used 1 mm shortened about the working length in the line
with previous studies [18,19].
Image analysis using the ImageJ software was preferred to
investigate both antimicrobial activity and smear layer
removal ability due to its ability to provide quantitative results
and minimise observer bias. These points strengthened the
method section of the present study.
WT, HA, AV, CL, and AT have been shown to have anti-
microbial activity against a range of microorganisms, but
studies on their effectiveness on E. faecalis and C. albicans are
limited [9,10,20,21]. Tea polyphenols, especially catechins,
were known as potent antimicrobial agents [9]. The high
polyphenol content of WT was presented previously [9]. In
previous studies, the antimicrobial activity of WT against E.
faecalis and C. albicans was reported [9,20]. Conflictingly, in the
present study, WT was found to be more effective than saline,
but the difference was not statistically significant. The content
and composition of tea polyphenols are affected by climate,
tea type or harvest season [9]. These influencing factors could
explain the low antimicrobial effects of WT in the present
study.
The high concentration of phenolic components is thought
to be responsible for HA’s antimicrobial effects via disrupting
glucosyltransferase activity [22]. The antifungal effects of HA’s
methanolic extract against C. albicans with a varying concen-
tration range of 10e100 mg/mL in the agar diffusion method
was reported [23]. In Kutluk et al.’s study [21], the antimicro-
bial efficacy of water and ethanol extracts of HA against E.
faecalis and C. albicans was investigated using the micro-
dilution method, and the effectiveness against both microor-
ganisms was reported. In the present study, greater
antimicrobial effects of HA were detected against both mi-
croorganisms than PS, though a significant difference was
found only against E. faecalis. The difference between the
studies may be related to the different antimicrobial suscep-
tibility tests and the fact that the MIC/MBK values in the
literature are much higher than those determined in the
present study. To the best of our knowledge, the present study
is the first to examine the antimicrobial activity of WT and HA
on a tooth model as an irrigation solution, which is a strength
of this study.

Please cite this article as: Akgun SE et al., Can herbal products be alternative root canal irrigation solutions in primary teeth? An in vitro
study, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2022.09.001
 pediatric dental journal xxx (xxxx) xxx
Due to its antimicrobial actions, AV has been accepted as a
potent root canal irrigant [13]. The antimicrobial action of AV is
primarily because of the acetic acid and maleic acid content,
which cause loss of cell integrity [12]. In the present study, the
reduction of E. faecalis achieved by AV was even higher than PS
but not statistically significant. The results of the present study
are in accordance with the study of Guerreiro-Tanomaru
et al.’s study [24], which reported that although AV provided
E. faecalis reduction, it generally had weak antibacterial activ-
ity. In contrast, Mohanty et al. [13] showed the greater anti-
bacterial activity of AV when compared with 5% NaOCI. Yagnik
et al. [25] demonstrated the antifungal activity of AV against C.
albicans and reported that this activity could be explained by
the absence of key enzymes required for candida immunoge-
nicity and glycolysis after exposure to AV. The results of the
present study are in line with the result of Yagnik et al.
Controversially, lower antifungal activity of AV than NaOCl
was reported in a previous study [13]. The discrepancy between
studies may be based on using different antimicrobial evalu-
ation tests and/or the ratio of AV’s active component.
It has been stated in previous studies that lemon contains
phenols, flavonoids, diterpenes, phytosterols, and cardiac
glycosides. The capacity of flavonoids to modulate enzymatic
activities and inhibit cell proliferation has an important place
in the antimicrobial efficacy of lemon [26]. In this study, CL
showed strong antibacterial activity against E. faecalis, similar
to the gold standard NaOCl. Oliveira et al. [13] reported that
the essential oil of CL eliminated 50.7% of E. Faecalis, but was
not similar to those of NaOCl. The formation of biofilm on
acrylic discs rather than in root canals, and using the agar
diffusion method as an antimicrobial susceptibility test could
explain the difference between the studies. In the same study
[13], a wide antifungal effect against C. albicans was found,
similar to 1% NaOCl, whereas in the present study, the fungal
reductions achieved by CL were similar to those of NaOCl and
even higher than those achieved by PS. It was stated previ-
ously that the antifungal potential of lemon against Candidas
was related to the content of monoterpenoids and the type of
Candida strain [27]. Differences between the monoterpenoids
ratio of CLs used in the studies can be found.
Although thymus has been mostly studied because of its
proven antimicrobial activity, very limited studies have been
conducted about the antimicrobial property of AT. It has been
stated that the antimicrobial activity of AT is due to the con-
tent of thymol, carvacrol, g-terpinene, p-Cymene, and terpinyl
acetate [28]. The only one study in the literature about AT
showed that AT essential oil had significant antifungal activity
[28]. In the present study, AT showed a greater reduction in C.
albicans colonies compared with saline, but the difference was
not statistically significant. The use of essential oil and dif-
ferenced between the test methods used may be factors that
explain the disparity in results.
It has been reported that the smear layer formed during
root canal preparation adversely affects endodontic success
and should be removed [1]. To dissolve the smear layer,
various irrigation solutions have been used to date [6]. A
combination of irrigation solutions is recommended for
effective smear layer removal because these solutions have
different effects on the smear layer [6]. In the present study,
herbal products, which could be potential irrigation solutions,
Please cite this article as: Akgun SE et al., Can herbal products be alternative root canal irrigation solutions in primary teeth? An in vitro
study, Pediatric Dental Journal, https://doi.org/10.1016/j.pdj.2022.09.001
9
were evaluated for smear layer removal both alone and in
combination with EDTA.
To dissolve the organic structure and inorganic structure of
the smear layer, NaOCI and EDTA solution are generally rec-
ommended, respectively [29]. In the present study, NaOCI-
EDTA was selected as a positive control and showed supe-
rior cleaning efficacy in root canals as predicted. Effective
smear layer removal activity was found for AV and CL in the
coronal and middle third, respectively, the same as NaOCI-
EDTA, and also even higher than PS and NaOCl.
The effectiveness of AV in smear layer removal has been
studied several times because of its malic acid composition
[30,31]. The findings of the present study are in line with those
of Candeiro et al. [30] who showed the greater smear layer
removal efficacy of AV. By contrast, Spano
et al. [31] reported
that AV was less effective than 15% EDTA. The difference in
the percentage of maleic acid, which is the active component
of AV, and the variability of the solutions’ application time
could explain the difference between the studies.
To the best of our knowledge, this is the first study to
investigate the effectiveness of CL extract in smear layer
removal. The results of the present study concur with the re-
sults of other authors who reported that lemon juice removed
the smear layer and opened the dentin tubules [32]. The citric
acid content of the solutions may have been effective in the
similarity.
In the study of Candeiro et al. [30], which is the only study in
which AV-EDTA was used in combination, it was reported that
AV was the most effective agent in the apical third of the root
canal. It removes the smear layer with or without EDTA. In line
with previous study, it is known that this effect is caused by the
malic acid ingredient in apple cider vinegar. In the present
study, there were no statistically significant differences in the
coronal, middle, and apical thirds of the root canal between
AV-EDTA and the other groups. The reason for the high effi-
ciency in the apical third may be the proportional difference in
the active components of AV used in the studies and the
morphologic difference (permanent/deciduous) of the roots.
To the best of our knowledge, this is the first study to
analyze the smear layer removal efficiency of WT, HA, and AT.
Although WT, HA, and AT removed more smear layer than the
negative control groups, the difference between the groups
was not statistically significant. Differences in the harvest
time of plants, and drying and extraction methods may have
affected the results; the present study may lead to future
studies investigating affecting factors.
It is known that chemical-containing irrigation solutions
such as NaOCI-EDTA have many side effects on oral and
periapical tissues [6]. Safer alternative irrigation solutions
with similar effects continue to be investigated [7]. In this
sense, based on the results of the present study, CL, AV, and
HA are thought to be alternatives to chemical irrigation solu-
tions [5,22,23]. In clinical applications of these herbal solu-
tions; it is thought that using different concentration values of
the solutions, combinations with different herbs, use of irri-
gation activating agents or isolating the active components of
the plant will increase the effectiveness of herbal solutions.
The limitations of this study are that different concentra-
tions of herbal products were not examined. It was considered
that too many groups would be present for proper discussion.
10 pediatric dental journal xxx (xxxx) xxx
In addition, different harvest times were not investigated for
the same reasons. Further, irrigation time can be considered to
be a little longer, since irrigation was performed with the
lowest active ingredient content in the EDTA applied group. In
clinical applications, the concentration of herbal solutions can
be increased to reduce irrigation time. Therefore, further
studies are needed to investigate the affecting factors of herbal
products.
5. Conclusion
Within the limits of the present study, CL and AV can be
considered as potential alternative endodontic irrigation so-
lutions due to their antimicrobial effects against E. feacalis and
C. albicans, respectively, and their smear layer removal activ-
ity. In addition, the association of HA, which has an antibac-
terial effect against E. faecalis, with EDTA, may be considered
among potential irrigation methods. Further studies are
required on these herbal products, which could be alterna-
tives to chemical irrigation solutions.
Ethical approval
This study was approved by the Ethics Committee of the
Recep Tayyip Erdogan University Faculty of Medicine (Protocol
2020/17).
Author contributions
I.A. had conceived the ideas, carried out data analysis, manu-
script writing and manuscript review. S.E.A. conducted the
experiment, carried out literature search and manuscript
writing. S. A. carried out data analysis and manuscript review.
D. N. G carried out data analysis and manuscript review.S.A.K.
conducted the antimicrobial analysis. E.Y. carried out the
extraction of plants. S.S. conducted the antimicrobial analysis
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
This study was supported by RTEU Scientific Research Projects
(TDH-2020-1099).
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