Conservative Dentistry

ISSN: 0972-0707

Journal of

Conservative Dentistry Volume 16

Issue 5

Official Publication of

Indian Association of Conservative Dentistry and Endodontics Online full text at

www.jcd.org.in

Sep - Oct 2013

Original Article

Antibacterial efficacy of Mangifera indica L. kernel and Ocimum sanctum L. leaves against Enterococcus faecalis dentinal biofilm Arunajatesan Subbiya, Krishnan Mahalakshmi1, Sivan Pushpangadan, Kesavaram Padmavathy1, Paramasivam Vivekanandan, Vridhachalam Ganapathy Sukumaran Departments of Conservative Dentistry and Endodontics, 1Microbiology, Sree Balaji Dental College and Hospital, Chennai, Tamil Nadu, India

Abstract Introduction The Enterococcus faecalis biofilm in the root canal makes it difficult to be eradicated by the conventional irrigants Introduction: with no toxicity to the tissues. Hence, plant products with least side effects are explored for their use as irrigants in the root canal therapy. Aim: To evaluate and compare the antibacterial efficacy of Mangifera indica L. kernel (mango kernel) and Ocimum sanctum L. Aim leaves (tulsi) extracts with conventional irrigants (5% sodium hypochlorite (NaOCl) and 2% chlorhexidine) against E. faecalis dentinal biofilm. Materials and Methods Methods: Agar diffusion and broth microdilution assay was performed with the herbal extracts and conventional irrigants (2% chlorhexidine and 5% NaOCl) against E. faecalis planktonic cells. The assay was extended onto 3 week E. faecalis dentinal biofilm. Results Significant reduction of colony forming units (CFU)/mL was observed for the herbal groups and the antibacterial Results: activity of the herbal groups was at par with 5% NaOCl . Conclusions The antibacterial activity of these herbal extracts is found to be comparable with that of conventional irrigants Conclusions: both on the biofilm and planktonic counterparts. Keywords: Antibacterial activity; Enterococcus faecalis biofilm; mango kernel; root canal; tulsi leaves

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.[2-6] 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 Address for correspondence:

Dr. Krishnan Mahalakshmi, Department of Microbiology, Sree Balaji Dental College and Hospital, Bharath University, Velachery-Tambaram Road, Chennai - 600 100, Tamil Nadu, India. E-mail: [email protected]

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,[12-14] 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 Access this article online Quick Response Code:

Date of submission : 15.01.2013 Review completed : 25.04.2013 Date of acceptance : 13.07.2013

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Journal of Conservative Dentistry | Sep-Oct 2013 | Vol 16 | Issue 5

Website: www.jcd.org.in

DOI: 10.4103/0972-0707.117507

Subbiya, et al.: Antibacterial effect of plant extracts

eugenol, a phenolic compound which may be attributed to its antimicrobial, antidiabetic, and anticancer properties.[15-17]

the assay was further extended to 3 week in vitro biofilm formed on tooth samples.

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 vitro growth of Mycobacterium tuberculosis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi, Salmonella typhimurium, Bacillus cereus, Bacillus subtilis, and Streptococcus pyogenes.[18-20]

Broth microdilution assay was performed to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the different herbal extracts.

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.

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.

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

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.

E. faecalis isolation from retreated root canal

Quantitative assay

MATERIALS AND METHODS

E. faecalis was isolated from clinical samples of root canal retreatment. Species identification of E. faecalis was 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]

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 L 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.

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 g) (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,

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.

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. In the qualitative assay on E. faecalis 3 weeks old biofilm;


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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. 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. faecalis planktonic and biofilm.

Table 1: Concentration of test solution used to assay on Enterococcus faecalis biofilm

Group A§ Group B¶ Group C* Group D¥ Group E** Group F■

Several studies have reported antibacterial efficacy for mango kernel on different bacterial species at a very high concentration (50 mg/ml).[25-29] 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

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E. faecalis ATCC 29212

E. faecalis clinical isolate

37.5 mg/mL 600 mg/mL 1,200 mg/mL 3% 0.01% 2 mL

75 mg/mL 1,200 mg/mL 600 mg/mL 3% 0.01% 2 mL

§: Mango kernel, ¶: Impcops tulsi, *: Khadi tulsi, ¥: Sodium hypochlorite (NaOCl), **: Chlorhexidine, ■: Saline, ATCC: American Type Culture Collection

Table 2: Susceptibility of Enterococcus faecalis planktonic cells to different test solutions: Agar diffusion assay/broth microdilution assay Test group

E. faecalis ATCC 29212 zone of inhibition/ (MIC/MBC)

E. faecal is clinical isolate zone of inhibition/ (MIC/MBC)

A (mango kernel) B (Impcops tulsi) C (Khadi tulsi) 5% NaOCl Vancomycin Chlorhexidine (2%)

24 mm/(6.25 mg/mL) 13 mm/(100 mg/mL) No zone/(200 mg/mL) 29 mm/(0.5%) 18 mm/(2 μg/mL) 30 mm/(0.002%)

24 mm/(12.5 mg/mL) 14 mm/(200 mg/mL) No zone/(100 mg/mL) 29 mm/(0.5%) 18 mm/(2 μg) 33 mm/(0.002%)

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.

Concentration

Test group (N=10)

MIC: Minimum inhibitory concentration, MBC: Minimum bactericidal concentration, NaOCl: Sodium hypochlorite

Table 3: Quantitative assay on Enterococcus faecalis 3 week old biofilm Group

CFU/mL (mean±SD)

% eradication*

A B C D E F

180±44.7 360±89.4 7780±130.4 No growth No growth 1.9×105±7.70065

99.8 99.6 92.2 100 100 NA

A: Mango kernel, B: Impcops tulsi, C: Khadi tulsi, D: Sodium hypochlorite (NaOCl), E: Chlorhexidine, F: Control with absence of herbal extracts and conventional irrigants, *,%: Eradication in comparison to the control F, NA: not applicable, SD: Standard deviation, CFU: Colony forming unit

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]

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. indica kernel 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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3. 4. 5.

6. 7. 8. 9. 10.

11. 12. 13. 14. 15.

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19. 20. 21.

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How to cite this article: Subbiya A, Mahalakshmi K, Pushpangadan S, Padmavathy K, Vivekanandan P, Sukumaran VG. Antibacterial efficacy of Mangifera indica L. kernel and Ocimum sanctum L. leaves against Enterococcus faecalis dentinal biofilm. J Conserv Dent 2013;16:454-7. Source of Support: Nil, Conflict of Interest: None declared.


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