ISSN: 0972-0707

Journal of
Conservative
Dentistry
Volume 18 Issue 5 Sep - Oct 2015

Official Publication of
Indian Association of
Conservative Dentistry and
Endodontics
Online full text at
www.jcd.org.in
Original Article

Smear layer removal efficacy of combination of

herbal extracts in two different ratios either alone
or supplemented with sonic agitation: An in vitro
scanning electron microscope study
Naveen Chhabra, Hitesh Gyanani, Laxmikant Kamatagi1
Department of Conservative Dentistry and Endodontics, K. M. Shah Dental College and Hospital, Sumandeep Vidyapeeth,
Vadodara, Gujarat, 1Department of Conservative Dentistry and Endodontics, P. M. N. M. Dental College and Hospital,
Bagalkot, Karnataka, India

Abstract
Objectives: The study aimed to evaluate the effectiveness of the combination of two natural extracts in varying ratios for
removal of smear layer either alone or supplemented with sonic agitation.
Materials and Methods: Fifty extracted single-rooted teeth were collected, disinfected and decoronated below the cementoenamel
junction to obtain standardized root length of 10 mm. Root canals were instrumented using rotary files at working length 1
mm short of the apex. Specimens were divided into six groups according to the irrigation protocol as follows: Group A –
Distilled water, Group B – 17% ethylenediaminetetraacetic acid, Group C – Herbal extracts in 1:1 ratio, Group D – Herbal
extracts in 1:1 ratio supplemented with sonic agitation, Group E – Herbal extracts in 2:1 ratio, Group F – Herbal extracts in
2:1 ratio supplemented with sonic agitation. Specimens were longitudinally sectioned and evaluated under scanning electron
microscope for smear layer removal efficacy. Obtained scores were statistically analyzed using one-way analysis of variance
and post-hoc test.
Results: Among all, Group B showed the best results followed by Group F. Remaining other groups showed inferior outcome
(P < 0.05).
Conclusion: The combination of two extracts in 2:1 ratio was slightly better than 1:1 ratio and the smear layer removal efficacy
was further improved when accompanied with sonic agitation.
Keywords: Agitation; Citrus aurantifolia; endodontic irrigation; root canal therapy; Sapindus mukorossi; smear layer

INTRODUCTION system is the most accepted method to achieve complete

disinfection.[1]
Root canal therapy is aimed to completely eradicate
microbes and three dimensionally seal the endodontic Mechanical preparation inadvertently forms an
system. Chemo-mechanical debridement of the root canal amorphous layer termed as “smear layer” over prepared
dentinal walls that may hold the microorganisms as well.
Address for correspondence: Thus, elimination of smear layer from the root canal
Dr. Naveen Chhabra, Department of Conservative
Dentistry and Endodontics, K. M. Shah Dental College walls is highly desirable to obtain long-term endodontic
and Hospital, Sumandeep Vidyapeeth, Piparia, Waghodia, success.[2]
Vadodara - 391 760, Gujarat, India.
E-mail: drnaveenchhabra@yahoo.com This is an open access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-ShareAlike 3.0 License, which
Date of submission : 17.04.2015
Review completed : 28.06.2015 allows others to remix, tweak, and build upon the work non-commercially,
Date of acceptance : 05.07.2015 as long as the author is credited and the new creations are licensed under
the identical terms.
Access this article online For reprints contact: reprints@medknow.com
Quick Response Code:
Website:
www.jcd.org.in
How to cite this article: Chhabra N, Gyanani H, Kamatagi L.
Smear layer removal efficacy of combination of herbal extracts
in two different ratios either alone or supplemented with sonic
DOI: agitation: An in vitro scanning electron microscope study. J
10.4103/0972-0707.164035
Conserv Dent 2015;18:374-8.

374 © 2015 Journal of Conservative Dentistry | Published by Wolters Kluwer - Medknow


Chelating agents such as 17% ethylenediaminetetraacetic
acid (EDTA),[3] 10% citric acid,[4] tannin, and maleic acid[5] are
used for smear layer removal. Literature also supports the
use of 17% EDTA solution followed by sodium hypochlorite
(NaOCl) as final flush.[6] Citric acid is a weak organic acid,
belonging to the chelating agent’s category has been
claimed to be as efficacious as EDTA in the removal of
smear layer.[7,8]
Efficacy of irrigating solutions can be effectively
increased by incorporation of surfactant as it reduces
the surface tension, thus, increasing the wettability of
the prepared surface. High wettability also enables the
irrigating solution for the deeper penetration into the
dentinal tubules. Furthermore, studies have shown that
irrigating solutions with detergents eradicate bacteria
in direct contact faster than agents without detergent.
It may be related to weakening of cohesive forces
in extracellular polymeric substances and bacterial
membranes.[3]
Synthetic chemical endodontic irrigating solutions with
their huge list of adverse effects have led the researchers
to explore various natural substitutes for the same.[9]
Extracts of Azadirachta indica, Ocimum sanctum, Syzygium
aromaticum, Cinnamomum zeylanicum and Citrus aurantifolia
have been explored as a possible endodontic irrigating
solution.[10-12] C. aurantifolia contains 88% water, 6-8% citric
acid, 2% potassium citrate, and other substances. Owing
to the citric acid as a basic constituent in C. aurantifolia,
researchers have evaluated it for its possible role in the
endodontic smear layer removal; though, the results were
disagreeable.[12,13] Furthermore, the role of natural fruit
juices including C. aurantifolia on the exposed root dentin
surfaces in the elicitation of dentinal hypersensitivity has
been confirmed and is linked with the removal of existing
smear layer on the exposed dentin.[14]
Sapindus mukorossi (Sapindaceae), commonly known as
“ritha,” “aritha” or “soapnut” is found throughout India.
The major constituents of its fruit’s pericarp are saponins
(10-11.5%), sugars (10%), and mucilage. Higher content
of saponins results in greater surfactant action. Hence,
the extract of soapnut is utilized to clean the skin of oily
secretion and is even used as a cleanser for washing hair
and as a hair tonic. The powdered seeds have also been
employed in the treatment of dental caries, arthritis,
common cold, constipation, and nausea.[15]
Agitation techniques improve the cleaning efficacy of
irrigating solutions by enhancing the irrigation dynamics.
This results in the maximum elimination of dentin debris,
remnant pulp tissues and biofilms from the root canal wall.
Studies have concluded that there is a minimal extrusion
of the irrigating solution beyond apex when using sonic
agitation as compared to other agitation techniques.[16]
Journal of Conservative Dentistry | Sep-Oct 2015 | Vol 18 | Issue 5
Chhabra, et al.: Smear layer removal efficacy of herbal extracts
Furthermore, the efficacy of smear layer removal was better
as compared to manual agitation.[17]
In the light of above context, we hypothesized that the
combination of C. aurantifolia and Sapindus mukorrossi
extracts accompanied with sonic agitation during final wash
should result in better smear layer removal. Hence, the
aim of this study is to test the unique combination of two
herbal extracts in two different ratios for the elimination of
endodontic smear layer either alone or supplemented with
sonic agitation.
MATERIALS AND METHODS
Fifty intact single rooted human permanent teeth having
a single canal and fully developed apices, indicated for
extraction due to orthodontic/periodontal reasons were
selected for the study. The teeth were cleaned using
ultrasonic scaler followed by sterilization under autoclave
as suggested in previous studies.[18,19] Thereafter, teeth
were stored in distilled water until use. The teeth were
decoronated below the cementoenamel junction using the
diamond disc to obtain standardized root length of 10 mm.
Working length was measured 1 mm short of the apical
foramen and glide path was established using size 15 K file
(Mani Inc., Japan). All the samples were randomly divided
into six groups out of which Group A (negative control)
and Group B (positive control) consisted of 5 samples each
and remaining 4 experimental groups had 10 samples,
respectively [Table 1].
Root canals were prepared till apical size 35, 0.06 taper using
K3XF™ nickel titanium files (SybronEndo Corp., Orange,
USA) in Crown down manner. During instrumentation,
each root canal was irrigated using 2 ml of test solution
corresponding to its group. The apical foramen of each root
was coated with cyanoacrylate glue before embedding the
roots into a polyvinyl-siloxane impression material filled
transparent tube to obtain the closed system. The extracts
of C. aurantifolia and S. mukorrossi were procured directly
from the manufacturer (Navchetana Kendra, New Delhi,
India) and the aqueous solution of the combination was
prepared in different ratios in the laboratory. The 1:1 ratio
solution of the combination was prepared in a dilution
of 0.1 g/ml of C. aurantifolia and 0.1 g/ml of S. mukorrossi
extract dissolved in distilled water. Similarly the 2:1 ratio of
the combination was prepared in a dilution of 0.12 g/ml of
C. aurantifolia and 0.06 g/ml of S. Mukorossi extract dissolved
in distilled water. The prepared solutions were filtered
using Whatman filter paper and the final irrigating solution
obtained.
For all the groups, the final rinse irrigation was done
according to group protocol [Table 1]. Each specimen was
irrigated using 3 ml of corresponding irrigating solution
375
Chhabra, et al.: Smear layer removal efficacy of herbal extracts

using 30-G side vent needles (Max-I-Probe™, Dentsply, New

Delhi, India) with the flow rate of approximately 5 ml/min.
Irrigation was supplemented with sonic agitation using
EndoActivator® (Dentsply Tulsa Dental Specialities, Tulsa,
USA) in Group D and Group F. For all the groups, root canal
of specimens was finally rinsed with sterile distilled water
and dried using sterile absorbent paper points.

Two longitudinal grooves were prepared on the buccal and

lingual surfaces of each root using a diamond disc, avoiding
penetration into the canal. The roots were split into two
halves with a chisel and coded according to groups. The coded
specimens were then mounted on metallic stubs with carbon
strip backing and subsequently examined independently
by two observers under scanning electron microscope
(SEM). After general evaluation of the canal wall, three SEM
photomicrographs were taken at magnification of ×1000 for
evaluation of smear layer at the center of the coronal, middle,
and apical thirds of each specimen [Figure 1]. Cleanliness was
evaluated using 5-point scoring system by Hülsmann et al.[7]
[Table 2] and the results were tabulated.

The collected data were subjected to one-way analysis of

variance to compare the means of the groups. The post-
hoc test (Tukey) was performed to find the interrelationship
between different groups of significant difference
(P < 0.05; confidence interval 95%).

RESULTS

Group B showed the best outcome and showed statistically
significant difference from other groups except Group F at
all three levels that were comparable to Group B (P < 0.05).
There was statistically insignificant difference in the smear
layer removal efficacy among the Group C and Group E.
Figure 1: Figure shows representative scanning electron
microscope images (×1000) of different groups (A-F). The
alphabet and numeric value in the figure label depict the
group, and area of the root canal imaged, respectively as
follows: 1 – Coronal third, 2 – Middle third, 3 – Apical third

Table 1: Group distribution

Group Description
A Distilled water will be used as root canal irrigant during instrumentation, as well as final rinse irrigant for 5 min (negative control)
B 17% EDTA will be used as root canal irrigant during instrumentation as well as final rinse for 5 min (positive control)
C Combination of Citrus aurantifolia (Navchetana Kendra, New Delhi, India) and Sapindus mukorossi (Navchetana Kendra, New Delhi, India)
extracts will be used as root canal irrigant during instrumentation as well as final rinse irrigant in 1:1 ratio for 5 min
D Combination of Citrus aurantifolia (Navchetana Kendra, New Delhi, India) and Sapindus mukorossi (Navchetana Kendra, New Delhi, India) extracts
will be used as root canal irrigant during instrumentation, as well as final rinse irrigant in 1:1 ratio for 5 min supplemented with sonic agitation
E Combination of Citrus aurantifolia (Navchetana Kendra, New Delhi, India) and Sapindus mukorossi (Navchetana Kendra, New Delhi, India)
extracts will be used as root canal irrigant during instrumentation as well as final rinse irrigant in 2:1 ratio for 5 min
F Combination of Citrus aurantifolia (Navchetana Kendra, New Delhi, India) and Sapindus mukorossi (Navchetana Kendra, New Delhi, India) extracts
will be used as root canal irrigant during instrumentation as well as final rinse irrigant in 2:1 ratio for 5 min supplemented with sonic agitation
EDTA: Ethylenediaminetetraacetic acid

Table 2: Smear layer evaluation criteria

Score Description
1 No smear layer and all dentinal tubules open
2 A small amount of smear layer and some dentinal tubules open
3 Homogenous smear layer covering the root canal wall and only a few dentinal tubules open
4 Complete root canal wall covered by a homogeneous smear layer and no open dentinal tubules
5 Heavy homogeneous smear layer covering the complete root canal wall

376 Journal of Conservative Dentistry | Sep-Oct 2015 | Vol 18 | Issue 5

 Chhabra, et al.: Smear layer removal efficacy of herbal extracts

However, Group F differed significantly from Group D at

middle and apical third sections of the root (P < 0.001)
[Figures 2-4].

DISCUSSION

C. aurantifolia has been previously tested for smear layer

removal capabilities and found unsatisfactory.[12] The
negative outcome may be related to the poor wettability
of the solution. The present study employed C. aurantifolia
and S. mukorrossi in two different ratios to determine the
effect of surfactant in the removal of smear layer. The Figure 2: Smear layer score in coronal third (P < 0.05)
results portrayed 2:1 concentration to be more effective.
Reduced effect of 1:1 solution may be related to the
reduced effective concentration of the citric acid in the
prepared irrigant. Other different proportions of the tested
extract combinations with further reduced concentration
of S. mukorossi may be evaluated to determine the optimal
proportion.

Emulsification activity of the bio-surfactants like S.

mukorrossi is one of the important properties that dissolve
the water-insoluble substances/hydrocarbons.[20] This
property may be primarily responsible for the better smear
Figure 3: Smear layer score in middle third (P < 0.05)
layer removal effect when used in combination with lime
extract.

The present experiment utilized an aqueous solution of

the extract combination; however, the effect of ethanolic
extract solutions may be further evaluated to confirm the
smear layer removal capabilities of the tested solution.

The root canal system behaves as a closed end channel,

being apically surrounded by bone socket. Therefore, our
study also utilized the closed apical model to mimic the
clinical situation. Sonic agitation was opted over ultrasonic
method in this study as EndoActivator consist of polymer
based tips that do not damage the prepared dentinal
walls also, the higher frequency generated by ultrasound
can result in the greater extrusion of debris. In this trial,
the sonic agitation performed better in Group F (2:1
concentration); however, 1:1 concentration accompanied
with sonic agitation, that is, Group D failed to do so. Hence,
the results partially coincide with the previous research,[19]
but the primary factor responsible for the better outcome
remains the concentration of the combination extract
solution.
The optimal contact time of the irrigant is essential to
obtain adequate canal cleaning effect. Although, the closed
apical model provided good contact time for the irrigating
solution, the results of the study were not as good as
for 17% EDTA group except the Group F, which showed
comparable results. This may be related to the presence of
reduced available concentration of citric acid in the tested
Figure 4: Smear layer score in apical third (P < 0.05)
solutions. Five minutes contact time for all irrigants was
decided in this trial considering the clinical scenario.
SEM images of all the groups showed the presence of debris
over the prepared dentin surfaces that may probably be
linked to the residues of other organic components of the
solution and the nonuse of any debris dissolving solution
like NaOCl.
Complete apical penetration and constant replenishment
of irrigating solution are essential to achieve thorough
cleaning and may be achieved using apical negative pressure
irrigation systems. Further researches may be designed to
explore the effect of such devices in improving the efficacy
of the tested solution. Also the effect of other adjuncts
such as canal brush[21] and self-adjusting file system[22] may
be evaluated.

Journal of Conservative Dentistry | Sep-Oct 2015 | Vol 18 | Issue 5 377

Chhabra, et al.: Smear layer removal efficacy of herbal extracts
Although, the therapeutic use of both the extracts owing
to its various medicinal values is well-established, the
chemical analysis of the combination of the extracts needs
to be established to confirm the biocompatibility issues.
CONCLUSION
Within the limitations of the present in vitro research, it
can be concluded that the experimental solution showed
mixed results. The 2:1 ratio was slightly superior to 1:1 and
sonic activation improved the effectiveness of the solution.
Further researches with the different combination ratios,
contact time and extract type may be designed to validate
the research outcome.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
REFERENCES
1. Haapasalo M, Endal U, Zandi H, Coil JM. Eradication of endodontic
infection by instrumentation and irrigation solutions. Endod Topics
2005;10:77-102.
2. Cergneux M, Ciucchi B, Dietschi JM, Holz J. The influence of the smear
layer on the sealing ability of canal obturation. Int Endod J 1987;
20:228-32.
3. Lui JN, Kuah HG, Chen NN. Effect of EDTA with and without surfactants
or ultrasonics on removal of smear layer. J Endod 2007;33:472-5.
4. Götze Gda R, Cunha CB, Primo LS, Maia LC. Effect of the sodium
hypochlorite and citric acid association on smear layer removal of
primary molars. Braz Oral Res 2005;19:261-6.
5. Ballal NV, Kandian S, Mala K, Bhat KS, Acharya S. Comparison of the
efficacy of maleic acid and ethylenediaminetetraacetic acid in smear
layer removal from instrumented human root canal: A scanning electron
microscopic study. J Endod 2009;35:1573-6.
6. Goldman M, Goldman LB, Cavaleri R, Bogis J, Lin PS. The efficacy of
several endodontic irrigating solutions: A scanning electron microscopic
study: Part 2. J Endod 1982;8:487-92.
378 Journal of Conservative Dentistry | Sep-Oct 2015 | Vol 18 | Issue 5
7. Hülsmann M, Heckendorff M, Lennon A. Chelating agents in root canal
treatment: Mode of action and indications for their use. Int Endod J
2003;36:810-30.
8. Di Lenarda R, Cadenaro M, Sbaizero O. Effectiveness of 1 mol L-1 citric
acid and 15% EDTA irrigation on smear layer removal. Int Endod J
2000;33:46-52.
9. Pashley EL, Birdsong NL, Bowman K, Pashley DH. Cytotoxic effects of
NaOCl on vital tissue. J Endod 1985;11:525-8.
10. Dutta A, Kundabala M. Antimicrobial efficacy of endodontic irrigants
from Azadirachta indica: An in vitro study. Acta Odontol Scand 2013;
71:1594-8.
11. Gupta A, Duhan J, Tewari S, Sangwan P, Yadav A, Singh G, et al.
Comparative evaluation of antimicrobial efficacy of Syzygium aromaticum,
Ocimum sanctum and Cinnamomum zeylanicum plant extracts against
Enterococcus faecalis: A preliminary study. Int Endod J 2013;46:775-83.
12. Bolhari B, Sharifian MR, Aminsobhani M, Monsef Esfehani HR, Tavakolian P.
Assessing the efficacy of Citrus aurantifolia extract on smear layer removal
with scanning electron microscope. Iran Endod J 2012;7:88-97.
13. Sharifian MR, Shokouhinejad N, Monsef-Esfahani HR, Aligholi M,
Amjadi M. Antimicrobial effect of Citrus aurantifolia extract on
Enterococcus faecalis within the dentinal tubules in the presence of
smear layer. J Dent Med 2011;24:148-55.
14. Corrêa FO, Sampaio JE, Rossa Júnior C, Orrico SR. Influence of natural
fruit juices in removing the smear layer from root surfaces — An in vitro
study. J Can Dent Assoc 2004;70:697-702.
15. Aneja KR, Joshi R, Sharma C. In vitro antimicrobial activity of Sapindus
mukorossi and Emblica officinalis against dental caries pathogens.
Ethnobotanical Lealf 2010;14:402-12.
16. Desai P, Himel V. Comparative safety of various intracanal irrigation
systems. J Endod 2009;35:545-9.
17. Mancini M, Cerroni L, Iorio L, Armellin E, Conte G, Cianconi L. Smear
layer removal and canal cleanliness using different irrigation systems
(EndoActivator, EndoVac, and passive ultrasonic irrigation): Field
emission scanning electron microscopic evaluation in an in vitro study.
J Endod 2013;39:1456-60.
18. Nakamura VC, Kataoka SH, Gavini G, Ferrari PH, Cai S. FE-SEM
evaluation of dental specimens prepared by different methods for in vitro
contamination. Int J Dent 2012;2012:748471.
19. Kuruvilla A, Jaganath BM, Krishnegowda SC, Ramachandra PK, Johns DA,
Abraham A. A comparative evaluation of smear layer removal by using
EDTA, etidronic acid, and maleic acid as root canal irrigants: An in vitro
scanning electron microscopic study. J Conserv Dent 2015;18:247-51.
20. Balakrishnan S, Varughese S, Deshpande AP. Micellar characterization
of saponin from Sapindus Mukorossi. Tenside Surf Det 2006;43:262-8.
21. Kamel WH, Kataia EM. Comparison of the efficacy of smear clear with
and without a canal brush in smear layer and debris removal from
instrumented root canal using WaveOne versus ProTaper: A scanning
electron microscopic study. J Endod 2014;40:446-50.
22. Çapar ID, Aydinbelge HA. Effectiveness of various irrigation activation
protocols and the self-adjusting file system on smear layer and debris
removal. Scanning 2014;36:640-7.