ENDODONTOLOGY Volume: 27 Issue 1 June 2015

ORIGINAL RESEARCH

Comparison of Antibacterial Efficacy of Calcium Hydroxide

with and without Addition of Herbal Medicaments
against Enterococcus Faecalis
Bhuvan Shome Venigalla1, Karthik Prasad L2, Veerandar singh T3,
Jyotsna SV4, Kiran Ghatole5
_____________________________________________________________________________________________________________________________________________________________

1Professor

and HOD, 2PG student, 3,4,5Senior lecturer, Sri Sai college of dental surgery, Vikarabad, Telangana.

Corresponding Author:
E-mail: bhudent@yahoo.co.in

Abstract
Aim: The aim of this study is to compare the antibacterial efficacy of Calcium hydroxide with and without addition of herbal
medicaments like turmeric and neem extracts against Enterococcus faecalis.
Materials and Methodology: Calcium hydroxide and herbal extracts were prepared and the samples were divided into 6 groups.
Group A Calcium hydroxide + saline, Group B Calcium hydroxide + Turmeric extract, Group C Calcium hydroxide +
Neem extract, GROUP A1 Normal saline, GROUP B1 Turmeric extract, GROUP C1 - Neem extract. Enterococcus faecalis are
grown in Brain heart infusion broth (BHI). 3 wells, 4mm in diameter are punched in each media plate and are filled with
respective groups of equal volume. After incubation for 24-48hrs, at 37C, inhibition zones are measured with a sliding caliper.
Results: All the statistical analysis was done using SPSS version 18. A p-value of <0.05 was considered statistically significant.
Comparison of mean zone of inhibition was done using ANOVA with post-hoc Games Howell test.
Conclusion: Within the limitations of the study, all materials showed antibacterial activity against Enterococcus faecalis but
Calcium hydroxide and saline combination showed best efficacy against E.faecalis while Turmeric showed least efficacy.
Keywords: Calcium hydroxide, Turmeric extract, Neem extract, Enterococcus.faecalis, Zone of inhibition.

Hence, the aim of the present study was to

compare the antibacterial efficacy of calcium
hydroxide with and without addition of herbal
medicaments like turmeric and neem extracts against
Enterococcus. faecalis

Introduction
The main purpose of root canal therapy is to
eliminate micro-organisms and their products from
root canal system to prevent reinfection. Complete
elimination of bacteria from root canal system by
instrumentation alone is not achieved1.
Results of several studies have clearly
shown that a variety of micro-organisms reside in
root canal. Enterococccus faecalis has been isolated
from almost 2977% teeth undergoing endodontic
procedures2. Additional methods like chemical
solutions are required to disinfect and kill microorganisms. Chemical treatment of root canal system
includes
Rinses
Irrigants
Interappointment medicaments.
Calcium hydroxide is the most commonly
used endodontic medicament and it eliminates most
microorganisms due to high pH (12.8) when used as
7 day dressing3. But, antimicrobial activity of
calcium hydroxide changes with the type of vehicle
and the manner in which it is dispensed. Furthermore,
E.feacalis has found to be resistant to calcium
hydroxide preparation4.
In order to overcome the shortcomings of
present medicaments, herbal extracts like turmeric
and neem have been explored in the present study in
combination with calcium hydroxide powder. The
micro-organism used in the study is Enterococcus.
faecalis.

Materials and Methods

Preparation of Calcium Hydroxide Mix: Calcium
hydroxide powder mixed with sterile normal saline in
the ratio of 1:1 (wt/vol) to obtain paste5.
Preparation of Aqueous Solution of Turmeric: The
rhizomes were washed with distilled water, and then
cut into irregular large pieces. They were then dried
in an oven by tray drying process at a temperature of
455 C for a period of about 9-10 days till they were
completely moisture-free. These irregular pieces were
ground to obtain a coarse powder.
Coarse powder of Rhizomes was than
extracted by maceration process.250 gms of coarse
powder of the Curcuma longa rhizome was placed in
a large glass chamber.
1000 ml of sterile distilled water was added
to a glass chamber prepare the aqueous extract. The
glass chamber was closed with a glass lid to prevent
evaporation of the menstruum and this system was
allowed to stand for 7 days with occasional stirring.
The liquid obtained was stored in a refrigerator at
4C in a beaker6.
Preparation of Neem Extract: Mature fresh
Azadirachta indica leaves were collected and
weighed in a sterile disposable cup. 25gms of fresh
38

Comparison of antibacterial efficacy of calcium hydroxide

with / without addition of herbal medicaments against Enterococcus.faecalis
neem leaves were added to 50ml of distilled water.
Mixture was macerated for 1-2 mins and care was
taken to prevent temperature rise beyond 45-50 C.
Muslin cloth was used to filter coarse
residue from extract. The process was repeated using
coarse residue and 25ml ethanol. The extract was
placed in a hot water bath to facilitate evaporation of
ethanol until the volume reaches to 25ml. The
prepared extract was then stored in airtight amber
coloured container6.
Test For Antibacterial Assay: A loopful of bacterial
colonies from stock blood agar plate of Enterococcus
faecalis was mixed with 1 ml dialyzed Brain Heart
Infusion Broth (equivalent to 0.5 McFarland Unit) ,
and was poured and spread with sterile glass beads on
the prepared agar plates.

Table 1: Mean Zone of Inhibition (in mm) of

different experimental groups.

The samples were divided into 6 groups.

Group A calcium hydroxide + saline
Group B calcium hydroxide + turmeric extract
Group C calcium hydroxide + neem extract
Group A1 Normal saline
Group B1 Turmeric extract
Group C1 Neem extract
Agar Plate Diffusion Method
The antibacterial effect was evaluated on
blood agar for Enterococcus faecalis ATCC29212.
Three wells, 4 mm in diameter were punched in each
selected media plate containing 20 ml agar and filled
with its respective medicament of equal volume. (Fig
1) All the groups are mixed until a paste consistency
was obtained.
After incubation for 24-48hrs, at 37C,
inhibition zones were measured with a sliding caliper
and the mean number of three perpendicular
diameters was obtained to calculate the inhibition
area.

Table 2: Intergroup comparison of zone of

inhibition between different groups.

Results
Statistical analysis:
Comparison of mean zone of inhibition was
done using ANOVA with post-hoc Games Howell
test. A summary of results is given in Fig 2, Table-1,
Table -2 and chart-1. The zone of inhibition of
Group A (16.43mm) against Enterococcus. Faecal is
was significantly higher than other groups. There was
no statistical significance in the difference in zone of
inhibition between Group B (15.37mm) and Group C
(14.25mm). Amongst all experimental groups, Group
B1 (1.5mm) showed least zone of inhibition followed
by C1 (10.55mm) where the difference was
statistically significant when compared between other
groups.

39

Comparison of antibacterial efficacy of calcium hydroxide

with / without addition of herbal medicaments against Enterococcus.faecalis
Antibacterial activity of Ca(OH)2 can be
attributed due to its high pH (12.5-12.8) and its
ability to dissociate into hydroxyl ions which causes
damage to bacterial cytoplasmic membrane, protein
denaturation and damage to DNA7. But, Ca(OH)2
showed decreased antibacterial activity over a period
of time which may be attributed to several factors8:
1) Buffering action on the alkalinity of Ca(OH)2 by
dentin and its components.
2) Colonization of E. faecalis within dentinal
tubules.
3) Decreased diffusibility of hydroxyl ions in
dentinal tubules.
Studies with agar diffusion test have
revealed that the calcium hydroxide had pronounced
antibacterial activity against facultative and anaerobic
bacteria 9. Curcumin (Turmeric) a polyphenolic
compound is known to strongly inhibit bacterial cell
proliferation by inhibiting the assembly dynamics of
Z-ring, needed for bacterial cell division and thus,
having a potent antibacterial activity against a
number of pathogenic bacteria. Curcumin is stable at
a pH of 6.5 but highly unstable at neutral-basic pH
conditions10.
Turmeric
(yellow
colour
due
to
xanthophylls) when comes in contact with fatty acids
or base liquids like (Ca(OH)2), carotene pigment gets
more activated and turmeric turns red11.
Neem is a biocompatible antioxidant. It acts
on oral microflora because of its anti-adherence
activity by altering bacterial adhesion. Also,
Nimbidin and Nimbolide in neem have been found to
possess antibacterial and antifungal properties which
cause lysis of bacterial cell wall12.
Turmeric and Neem are stable at pH 6.1-6.5,
so they have neutralizing action on Calcium
hydroxide which has high alkaline pH ranging 12.512.8.
In the present study, the
antibacterial
activity of Calcium hydroxide
and saline
combination was more than the other agents for
E.faecalis, the difference being
statistically
significant (p>0.001) Whereas, turmeric extract
alone showed weak antibacterial activity.

Chart 1: Chart showing zone of inhibition of

different groups

Fig. 1: Agar plates filled with experimental

groups.

In the comparative analysis between the groups,

Group A showed highest zone of inhibition
than Group B, C, B1, C1 which was
statistically significant.
Group B showed no statistical significant
difference with Group C and higher zone of
inhibition than Group B1, C1 which was
statistically significant.
Group C showed higher zone of inhibition than
Group B1, C1 which was statistically
significant.

Fig. 2: Agar plates showing Zone of Inhibition of

different groups
Discussion
Investigators have noted that bacteria in
instrumented, unobturated canals can multiply and
reach their pre-treatment numbers in 2 days1. E.
faecalis being a resistant microorganism plays an
important role in persistent periapical lesions. E.
faecalis will grow in a wide range of pH (4.69.9),
with the optimum being 6.5- 7.5.
40

Comparison of antibacterial efficacy of calcium hydroxide

with / without addition of herbal medicaments against Enterococcus.faecalis
A study by Vibha hegde et al. Showed that
neem leaf extract showed the highest zone of
inhibition against E.faecalis and C.Albicans13.
Another study by Mithra N. Hegde et al. showed that
the aqueous extract of turmeric showed mild activity
against Enterococcus faecalis14. In contrast a study
by Anurag singhal et al. Showed that calcium
hydroxide and neem combination is highly effective
against E.faecalis15.

10.

Conclusion
Under the limitations of the study, the following
can be concluded:
All the materials showed antibacterial activity
against Enterococcus faecalis.
But, calcium hydroxide and saline combination showed best efficacy against E. faecalis (P
> 0.001) and turmeric showed least efficacy.

13.

However, in vivo studies will also be required for

recommending ideal clinical protocols using these
materials.

15.

11.

12.

14.

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