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J Conserv Dent. 2013 Sep-Oct; 16(5): 454–457.

doi: 10.4103/0972-0707.117507

PMCID: PMC3778630

Antibacterial efficacy of Mangifera indica L. kernel and Ocimum


sanctum L. leaves against Enterococcus faecalis dentinal
biofilm
Arunajatesan Subbiya, Krishnan Mahalakshmi,1 Sivan Pushpangadan, Kesavaram
Padmavathy,1 Paramasivam Vivekanandan, and Vridhachalam Ganapathy Sukumaran

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Abstract
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INTRODUCTION
Enterococcus faecalis plays a major role in the etiology of persistent periradicular lesions
after root canal treatment.[1] It is frequently found in high percentage of root canal failures
and is able to survive in the root canal as single organism or as a major component of the
mixed flora.[2,3,4,5,6] E. faecalis’ mode of growth is by formation of biofilm, an adaptive
process that enables microorganisms to survive in severely harsh conditions.[2,7] To mimic
the clinical scenario the present study was aimed to assay the antibacterial activity on mature
biofilm.
Various investigations have demonstrated that thorough and complete debridement of root
canal system with all its ramifications and anatomical irregularities is impossible with
mechanical driven or hand instrumentation to eliminate the microorganisms and their
byproducts.[8] Hence, endodontic preparation should be supported by irrigants for enhanced
disinfection.[9,10] The use of conventional irrigants especially sodium hypochlorite (NaOCl)
is highly efficient in eliminating E. faecalis biofilm.[11] But, the major disadvantage of
NaOCl is its tissue toxicity and in addition it does not remove all of the smear
layer,[12,13,14] for which reason plant products that are consumed orally for varied
medicinal purpose are assayed for their antibacterial properties.
Ocimum sanctum L., known as ‘tulsi’ in Hindi and ‘holy basil’ in English, is traditionally
used as a medicinal plant in day-to-day practice in Indian homes for various ailments. The
essential oil extracted from the tulsi leaves contains eugenol, a phenolic compound which
may be attributed to its antimicrobial, antidiabetic, and anticancer properties.[15,16,17]
Quantitative variations have been seen in the composition of essential oils of O. sanctum L.
growing in different parts of India. Extracts from the leaves of O. sanctum L. have been
found to inhibit in vitrogrowth of Mycobacterium tuberculosis, Staphylococcus aureus,
Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi, Salmonella typhimurium,
Bacillus cereus, Bacillus subtilis, and Streptococcus pyogenes.[18,19,20]
Mangifera indica L. is commonly called mango in English. Mangiferin, a major C-
glucosylxanthone is found to occur in the M. indica stem bark, leaves, heartwood, roots, and
fruits. The antibacterial activity of mango kernel may be attributed to the tannins present in
them.[21]
The present study therefore was aimed to assess and compare the antibacterial efficacy of M.
indica L. kernel (mango kernel) and O. sanctum L. leaves (tulsi) extracts with the
conventional irrigants (5% NaOCl and 2% chlorhexidine) against E. faecalis biofilm.
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MATERIALS AND METHODS


Antibacterial assay was carried out against E. faecalis culture isolated from retreated root
canal. E. faecalisATCC 29212 was used as control.

E. faecalis isolation from retreated root canal


E. faecalis was isolated from clinical samples of root canal retreatment. Species
identification of E. faecaliswas carried out using standard microbiological methods including
growth in 6.5% NaCl, heat tolerance at 42°C, magenta pink colored colonies on MacConkey
agar, bile esculin hydrolysis, arginine hydrolysis, and mannitol fermentation.[22]
Methanol extracts of dried and finely pulverized mango kernel (Group A: Impcops Ltd,
Chennai, India) and two different tulsi leaves (Group B: Impcops Ltd, Chennai, India and
Group C: Khadi Gramodyog Chennai, India) powder were obtained by continuous hot
percolation method in a soxhlet apparatus. The extracts were then concentrated and dried
under reduced pressure. The stock solutions of the methanol free extracts were prepared in
10% dimethyl sulfoxide (DMSO) (S.D Fine Chem Private Ltd.).[23] Agar well diffusion
assay was done as per Clinical and Laboratory Standards Institute (CLSI) guidelines to
evaluate the efficacy of the herbal extracts.[24] Ten microliter of 2% chlorhexidine (Asep-
RC) and 5% NaOCl (Prime Dental, India) were placed on sterile discs (Himedia, India) for
the assay. DMSO (10%) was also assayed to check if they showed any significant zone of
inhibition. Vancomycin (30 μg) (Himedia) was used as control. The whole assay was
performed in triplicate. As the herbal extracts demonstrated promising results on planktonic
counterparts of E. faecalis, the assay was further extended to 3 week in vitro biofilm formed
on tooth samples.
Broth microdilution assay was performed to determine the minimum inhibitory concentration
(MIC) and minimum bactericidal concentration (MBC) of the different herbal extracts.

Biofilm formation on the root canal


The tooth samples for biofilm formation was prepared and sterilized as per Prabhakar et al.,
2010.[23]
E. faecalis culture was prepared in Mueller-Hinton Broth (MHB) and the turbidity was
adjusted to 0.5 McFarland standard to obtain a cell density of 1.5×108 cells/mL. The broth
culture was dispensed into the tissue culture plates containing gamma sterilized tooth
samples at a volume of 2 mL/well. The culture plates were incubated at 37°C for 3 weeks. To
avoid nutrition depletion and accumulation of toxic end products, every alternate day the
media was replaced with sterile MHB.
At the end of third week, the purity of the culture was checked by gram staining and the
entire group was exposed for 10 min to different concentrations of herbal extracts as shown
in Table 1. Group F served as control with absence of the herbal extracts and conventional
irrigants. Each group comprised 10 tooth samples.

Table 1

Concentration of test solution used to assay on Enterococcus faecalisbiofilm

Quantitative assay
After 10 min, the biofilm formed on the root canal surface were removed with sterile scalpel
and inoculated into 1 mL of MHB and gently vortexed. Spread plate method of inoculation
was performed with 10 μL of the MHB onto Mueller-Hinton agar (MHA) plates, incubated
at 37°C for 24 h. Colony forming units (CFU)/mL was calculated by viable plate count
method.

Statistical analysis
Statistical analysis was performed by using one-way analysis of variance (ANOVA) with
Tamhane's post hoc testing to evaluate the overall significance of CFU/mL between and
within the different test groups. P< 0.05 (95% confidence level) was considered statistically
significant.
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RESULTS
Table 2 shows the zone of inhibition, MIC, and MBC of different test groups for E.
faecalis clinical isolate and E. faecalis ATCC 29212. Among the three herbal groups tested,
mango kernel (group A) showed maximum inhibition in proximity with the conventional
irrigant 5% NaOCl. While, high zone of inhibition was observed for mango kernel compared
to vancomycin. High zone of inhibition was observed for 2% chlorhexidine.

Table 2

Susceptibility of Enterococcus faecalis planktonic cells to different test solutions: Agar


diffusion assay/broth microdilution assay

In the qualitative assay on E. faecalis 3 weeks old biofilm; group A, B, and C showed few
colonies when compared to the control (group F). Table 3 shows the results of quantitative
assay. In the quantitative assay, all the three herbal groups (A, B, and C) have shown
significantly higher reduction (P = 0.0001) of CFU/mL. The mean CFU/mL for the three
herbal groups was not significantly high compared to 5% NaOCl (P < 0.05). The
antibacterial activity observed for E. faecalis clinical isolate was at par with E.
faecalis ATCC 29212. Also, 5% NaOCl and 2% chlorhexidine showed 100% reduction.

Table 3

Quantitative assay on Enterococcus faecalis 3 week old biofilm


Statistical significance was observed for groups A (mango kernel), B (Impcops tulsi), and C
(Khadi tulsi) when compared to the conventional irrigants with regard to antibacterial
activity against E. faecalisplanktonic and biofilm.
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DISCUSSION
Endodontic infection by E. faecalis represents a biofilm mode of growth where progression
of the infection and treatment failure are due to the high adaptability of this bacterial biofilm
towards reactive compounds. The capacity of E. faecalis to form calcified biofilm on root
canal dentine may attribute to their persistence after endodontic treatment.[7] Three weeks
old E. faecalis biofilm on dentinal canal was preferred in the present study to mimic their
usual endodontic niche.
Several studies have reported antibacterial efficacy for mango kernel on different bacterial
species at a very high concentration (50 mg/ml).[25,26,27,28,29] However, the present study
demonstrates antibacterial activity of mango kernel extract against E. faecalis at a very low
concentration (5 mg/mL) and this activity at very low concentration may be attributed to the
method of extraction.
In the agar well diffusion assay, group B (Impcops tulsi) demonstrated inhibition at 5 mg/mL
concentration, but group C did not reveal any zone at 5 mg/mL and the antibacterial activity
was demonstrated at a slightly high concentration when microbroth dilution was performed
and this could be attributed to the shelf life of the tulsi powder that was marketed by Khadi.
The present study reports the efficacy of the herbal extracts against E. faecalis isolated from
the teeth with failed root canal therapy. E. faecalis ATCC 29212 served as control. Bacteria
sequestered in biofilm are shielded and are often difficult to kill than their planktonic
counterparts.[30] The concentration of the herbal alternatives used for 3 weeks old biofilm
was six times the MIC valve, so as to achieve the biocide gradient of the extracts throughout
the biofilm.[31] The three plant extracts possessed excellent antibacterial efficacy both on
planktonic cells and biofilm. The eugenol, a phenolic compound present in the tulsi leaves
and tannins present in mango kernel may be attributed to their antibacterial properties.[17,21]
Previous study has reported 5% NaOCl to be superior over triphala and MTAD.[22]
Conversely, the present study demonstrates the antibacterial efficacy of mango kernel almost
equivalent to 5% NaOCl, which may well be replaced by this potential herbal extract as
endodontic irrigant to overcome the deleterious effects of the conventional irrigants (NaOCl
and chlorhexidine) on dentine.[32,33]
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CONCLUSION
The activity of mango kernel was highly significant, comparable to the conventionally used
5% NaOCl. In spite of showing antibacterial effect in low concentrations, conventional
irrigants can be toxic to tissues. On the contrary, these herbal formulations may be used even
at higher concentrations as there are no deleterious side effects reported. Within the scope of
the study, present investigation reveals that M. indicakernel and leaves of Ocimum
sanctum may be used as natural antibacterial agent in destroying the E. faecalis biofilm since
promising results were obtained at a very low concentration.
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Footnotes
Source of Support: Nil
Conflict of Interest: None declared

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