You are on page 1of 9

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
Arunajatesan Subbiya, Krishnan Mahalakshmi,1 Sivan Pushpangadan, Kesavaram
Padmavathy,1 Paramasivam Vivekanandan, and Vridhachalam Ganapathy Sukumaran

Author information ► Article notes ► Copyright and License information ►

This article has been cited by other articles in PMC.

Go to:

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
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
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.
Go to:


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.,
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

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
Go to:

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.
Go to:

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]
Go to:

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.
Go to:

Source of Support: Nil
Conflict of Interest: None declared

Go to:

1. Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis: Its role in root canal
treatment failure and current concepts in retreatment. J Endod. 2006;32:93–8. [PubMed]

2. Sundqvist G, Figdor D, Persson S, Sjögren U. Microbiologic analysis of teeth with failed

endodontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med
Oral Pathol Oral Radiol Endod. 1998;85:86–93. [PubMed]

3. Peciuliene V, Balciuniene I, Eriksen HM, Haapasalo M. Isolation of Enterococcus faecalis in

previously root-filled canals in a Lithuanian population. J Endod. 2000;26:593–5. [PubMed]

4. Peciuliene V, Reynaud AH, Balciuniene I, Haapasalo M. Isolation of yeasts and enteric

bacteria in root-filled teeth with chronic apical periodontitis. Int Endod J. 2001;34:429–
34. [PubMed]

5. Hancock HH, 3rd, Sigurdsson A, Trope M, Moiseiwitsch J. Bacteria isolated after

unsuccessful endodontic treatment in a North American population. Oral Surg Oral Med
Oral Pathol Oral Radiol Endod. 2001;91:579–86. [PubMed]

6. Pinheiro ET, Gomes BP, Ferraz CC, Sousa EL, Teixeira FB, Souza-filho FJ. Microorganisms
from canals of root-filled teeth with periapical lesions. Int Endod J. 2003;36:1–11. [PubMed]

7. Kishen A, George S, Kumar R. Enterococcus faecalis-mediated biomineralized biofilm

formation on root canal dentine in vitro. J Biomed Mater Res A. 2006;77:406–15. [PubMed]

8. Young GR, Parashos P, Messer HH. The principles of techniques for cleaning root
canals. Aust Dent J. 2007;52(1 Suppl):S52–63. [PubMed]
9. Pataky L, Iványi I, Grigár A, Fazekas A. Antimicrobial efficacy of various root canal
preparation techniques: An in vitro comparative study. J Endod. 2002;28:603–5. [PubMed]

10. Paiva SS, Siqueira JF, Rôças IN, Carmo FL, Leite DC, Ferreira DC, et al. Molecular
microbiological evaluation of passive ultrasonic activation as a supplementary disinfecting
step: A clinical study. J Endod. 2013;39:190–4. [PubMed]

11. Dunavant TR, Regan JD, Glickman GN, Solomon ES, Honeyman AL. Comparative
evaluation of endodontic irrigants against Enterococcus faecalis biofilms. J
Endod. 2006;32:527–31. [PubMed]

12. Spangberg L, Engström B, Langeland K. Biologic effects of dental materials. 3 Toxicity

and antimicrobial effect of endodontic antiseptics in vitro. Oral Surg Oral Med Oral
Pathol. 1973;36:856–71.[PubMed]

13. McComb D, Smith DC. A preliminary scanning electron microscopic study of root canals
after endodontic procedures. J Endod. 1975;1:238–42. [PubMed]

14. Torabinejad M, Khademi AA, Babagoli J, Cho Y, Jhonson WB, Bozhilov K, et al. A new
solution for the removal of the smear layer. J Endod. 2003;29:170–5. [PubMed]

15. Oyedemi SO, Pirochenva G, Mabinya LV, Bradly G, Afolayan AJ. Compositions and
comparisons of antimicrobial potencies of some essential oils and antibiotics against
selected bacteria. Afr J Biotechnol. 2008;7:4140–6.

16. Awasthi PK, Dixit SC, Dixit N, Sinha AK. Eugenol derivatives as future potential drugs. J
Pharm Res. 2008;1:215–20.

17. Prakash P, Gupta N. Therapeutic uses of Ocimum sanctum Linn (Tulsi) with a note on
eugenol and its pharmacological actions: A short review. Indian J Physiol
Pharmacol. 2005;49:125–31. [PubMed]

18. Joshi B, Lekhak S, Sharma A. Antibacterial property of different medicinal

plants: Ocimum sanctum, Cinnamomum zeylanicum, Xanthoxylum armatum and Origanum
majorana. Kathmandu Univ J Sci Eng Technol. 2009;5:143–50.

19. Mishra P, Mishra S. Study of antibacterial activity of Ocimum sanctum extract against
gram positive and gram negative bacteria. Am J Food Technol. 2011;6:336–41.

20. Goyal P, Kaushik P. In vitro evaluation of antibacterial activity of various crude leaf
extracts of Indian sacred plant, Ocimum sanctum L. British Microbiol Res J. 2011;1:70–8.
21. Rajan S, Thirunalasundari T, Jeeva S. Anti-enteric bacterial activity and phytochemical
analysis of the seed kernel extract of Mangifera indica Linnaeus against Shigella dysenteriae
(Shiga, corrig.) Castellani and Chalmers. Asian Pac J Trop Med. 2011;4:294–300. [PubMed]

22. Facklam RR, Carvalho MG, Teixeira LM. History, taxonomy, biochemical characteristics,
and antibiotic susceptibility testing of Enterococci. In: Gilmore MS, editor. The Enterococci:
Pathogenesis, Molecular Biology, and Antibiotic Resistance. Washington: ASM Press; 2002.
pp. 1–54.

23. Prabhakar J, Senthilkumar M, Priya MS, Mahalakshmi K, Sehgal PK, Sukumaran VG.
Evaluation of antimicrobial efficacy of herbal alternatives (Triphala and green tea
polyphenols), MTAD, and 5% sodium hypochlorite against Enterococcus faecalis biofilm
formed on tooth substrate: An in vitro study. J Endod. 2010;36:83–6. [PubMed]

24. Vol. 1. Wayne, PA, USA: CLSI; 2011. Clinical and Laboratory Standards Institute.
Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational

25. Kabuki T, Nakajima H, Arai M, Ueda S, Kuwabara Y, Dosako S. Characterization of novel

antimicrobial compounds from mango (Mangifera indica L.) kernel seeds. Food
Chem. 2000;71:61–6.

26. Mirghani ME, Yosuf F, Kabbashi NA, Vejayan J, Yosuf ZB. Antibacterial activity of mango
kernel extracts. J Appl Sci. 2009;9:3013–9.

27. Sharma A, Chandraker S, Patel VK, Ramteke P. Antibacterial activity of medicinal plants
against pathogens causing complicated urinary tract infections. Indian J Pharm
Sci. 2009;71:136–9.[PMC free article] [PubMed]

28. Engels C, Gänzle MG, Schieber A. Fractionation of gallotannins from mango (Mangifera
indica L.) kernels by high-speed counter-current chromatography and determination of
their antibacterial activity. J Agric Food Chem. 2010;58:775–80. [PubMed]

29. Kaur J, Rathinam X, Kasi M, Leng KM, Ayyalu R, Kathiresan S, et al. Preliminary
investigation on the antibacterial activity of mango (Mangifera indica L: Anacardiaceae)
seed kernel. Asian Pac J Trop Med. 2010;3:707–10.

30. Chai WL, Hamimah H, Cheng SC, Sallam AA, Abdullah M. Susceptibility of Enterococcus
faecalisbiofilm to antibiotics and calcium hydroxide. J Oral Sci. 2007;49:161–6. [PubMed]
31. McBain AJ, Gilbert P, Allison DG. Biofilms and biocides: Are there implications for
antibiotic resistance? Rev Environ Sci Biotechnol. 2003;2:141–6.

32. Sim TP, Knowles JC, Ng YL, Shelton J, Gulabivala K. Effect of sodium hypochlorite on
mechanical properties of dentine and tooth surface strain. Int Endod J. 2001;34:120–
32. [PubMed]

33. Grigoratos D, Knowles J, Ng YL, Gulabivala K. Effect of exposing dentine to sodium

hypochlorite and calcium hydroxide on its flexural strength and elastic modulus. Int Endod
J. 2001;34:113–9. [PubMed]