The Open Dentistry Journal




ISSN: 1874-2106 ― Volume 13, 2019

Dentinal Tubule Disinfection with Propolis & Two Extracts of Azadirachta indica Against Candida albicans Biofilm Formed on Tooth Substrate



Dakshita Joy Sinha1, Paridhi Garg1, *, Anurag Verma2, Vibha Malik3, Edgar Richard Maccune3, Agrima Vasudeva1
1 Conservative Dentistry & Endodontics, Kothiwal Dental College & Research Centre, Moradabad (U.P.)- 244001, India
2 School of Pharmaceutical Sciences, IFTM University, Moradabad, India;
3 Dr. Vibha Pathlabs, Moradabad, India

Abstract

Aim:

This study evaluates the disinfection of dentinal tubules using Propolis, Azadirachta indica (alcoholic and aqueous extracts), 2% chlorhexidine gel and calcium hydroxide against Candida albicans biofilm formed on tooth substrate.

Materials & Method:

One hundred and five human teeth were infected with Candida albicans for 2 days. Samples were divided into 7 groups. Group I- Propolis, Group II- Alcoholic extract of Azadirachta indica, Group III- Aqueous extract of Azadirachta indica, Group IV- 2% Chlorhexidine, Group V- Calcium hydroxide, Group VI- Ethanol and Group VII- Saline (negative control). At the end of 1,3 and 5 days, the antimicrobial efficacy of medicaments against Candida albicans was assessed at the depths of 200 µm and 400 µm.

Results:

The overall percentage inhibition of fungal growth (at 200 µm and 400 µm depth) was 99.2% with 2% chlorhexidine gel. There was no statistical difference between propolis, alcoholic extract of Azadirachta indica (neem) and 2% chlorhexidine.

Conclusion:

Propolis and alcoholic extract of Azadirachta indica performed equally well as that of 2% Chlorhexidine.

Keywords:: Azadirachta indica, calcium hydroxide, Candida albicans, chlorhexidine, dentinal tubule disinfection, propolis.


Article Information


Identifiers and Pagination:

Year: 2015
Volume: 9
First Page: 369
Last Page: 374
Publisher Id: TODENTJ-9-369
DOI: 10.2174/1874210601509010369

Article History:

Received Date: 05/12/2014
Revision Received Date: 26/08/2015
Acceptance Date: 13/09/2015
Electronic publication date: 12/11/2015
Collection year: 2015

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© Sinha et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.


* Address correspondence to this author at the Department of Conservative Dentistry & Endodontics, Kothiwal Dental College & Research Centre, Moradabad (U.P.)- 244001, India; Tel: 9997052495; E-mail: paridhigarg03@gmail.com




INTRODUCTION

Dentinal tubules are exposed to microbial invasion when a breach in the integrity of the overlying enamel or cementum occurs, following a cariogenic or a periodontal insult. If this invasion cannot be overcome by the host defences, it results in pulpal necrosis, infection of the root canal system and periapical disease [1Love RM, Jenkinson HF. Invasion of dentinal tubules by oral bacteria Crit Rev Oral Biol Med 2002; 13(2): 171-83.
[http://dx.doi.org/10.1177/154411130201300207] [PMID: 12097359]
]. One of the important objectives of root canal treatment is the elimination of the microorganisms from the root canal system [2Sirén EK, Haapasalo MP, Ranta K, Salmi P, Kerosuo EN. Microbiological findings and clinical treatment procedures in endodontic cases selected for microbiological investigation Int Endod J 1997; 30(2): 91-5.
[http://dx.doi.org/10.1111/j.1365-2591.1997.tb00680.x] [PMID: 10332242]
]. Several hundred microbial species are known to inhabit the oral cavity but relatively small and selected group of microbes is involved in the invasion of dentinal tubules and subsequent infection of the root canal space. Gram-positive organisms dominate the tubule microflora in both carious and non-carious dentin. It is difficult to deliver intracanal medicaments in the depth of dentinal tubules, leading to survival of the microbes. If opportunistic conditions occur, they may become a focal source of reinfection [3Wu MK, Dummer PM, Wesselink PR. Consequences of and strategies to deal with residual post-treatment root canal infection Int Endod J 2006; 39(5): 343-56.
[http://dx.doi.org/10.1111/j.1365-2591.2006.01092.x] [PMID: 16640632]
].

Fungi are chemoorganotroph eukaryotic microorganisms that may be found in 2 basic forms: molds and yeasts. Candida species have surface molecules that mediate adherence to host tissues. Candida species are also able to bind to collagen types I and IV. C.albicans produces hydrolytic enzymes that may be involved in the damage to the periradicular tissues [4Siqueira JF Jr, Sen BH. Fungi in endodontic infections Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004; 97(5): 632-41.
[http://dx.doi.org/10.1016/j.tripleo.2003.12.022] [PMID: 15153878]
]. Enzymes include secreted aspartyl proteinase, collagenase, aminopeptidases, glucosaminidases, acid and alkaline phosphatases, hyaluronidase, and chondroitin sulfatase, all of which have some effect in the degradation of extracellular matrix proteins [5Calderone RA, Fonzi WA. Virulence factors of Candida albicans Trends Microbiol 2001; 9(7): 327-35.
[http://dx.doi.org/10.1016/S0966-842X(01)02094-7] [PMID: 11435107]
]. It has been shown that a collagenolytic enzyme produced by this fungal species may degrade the human dentin collagen [6Hagihara Y, Kaminishi H, Cho T, Tanaka M, Kaita H. Degradation of human dentine collagen by an enzyme produced by the yeast Candida albicans Arch Oral Biol 1988; 33(8): 617-9.
[http://dx.doi.org/10.1016/0003-9969(88)90138-0] [PMID: 3075886]
]. C. albicans is often described as a dimorphic fungus that exists in blastospore and hyphal forms. It has been consistently suggested that the transition from the blastospore to the hyphal form represents a change from a commensal to a pathogenic state [7San-Blas G, Travassos LR, Fries BC, et al. Fungal morphogenesis and virulence Med Mycol 2000; 38(Suppl. 1): 79-86.
[http://dx.doi.org/10.1080/mmy.38.1.79.86] [PMID: 11204167]
, 8Phan QT, Belanger PH, Filler SG. Role of hyphal formation in interactions of Candida albicans with endothelial cells Infect Immun 2000; 68(6): 3485-90.
[http://dx.doi.org/10.1128/IAI.68.6.3485-3490.2000] [PMID: 10816502]
]. but it has not been found to be true [9Cotter G, Kavanagh K. Adherence mechanisms of Candida albicans Br J Biomed Sci 2000; 57(3): 241-9.
[PMID: 11050778]
]. The properties of a growing hypha may confer the ability to invade host tissues and escape phagocytosis by macrophages [7San-Blas G, Travassos LR, Fries BC, et al. Fungal morphogenesis and virulence Med Mycol 2000; 38(Suppl. 1): 79-86.
[http://dx.doi.org/10.1080/mmy.38.1.79.86] [PMID: 11204167]
]. However, although transformation from yeast to hyphal form may be important, it is not always a prerequisite for infection to occur [10Marsh P, Martin MV. Oral microbiology. 4th ed. Oxford, England: Wright 1999.]. Indeed, most infections caused by C.albicans are populated by both yeast and filamentous morphologic forms, suggesting that both have a role in the development and progression of the disease [11Calderone R, Suzuki S, Cannon R, et al. Candida albicans: adherence, signaling and virulence Med Mycol 2000; 38(Suppl. 1): 125-37.
[http://dx.doi.org/10.1080/mmy.38.s1.125.137] [PMID: 11204138]
].

Two percent chlorhexidine is one of the most versatile irrigants and an intracanal medicaments in vital and non-vital teeth. Its efficacy is based on the interaction between the positive charge of the molecule and the negatively charged phosphate groups on the microbial cell wall, which allows the CHX molecule to penetrate into the microbes with toxic effects [12Lindskog S, Pierce AM, Blomlöf L. Chlorhexidine as a root canal medicament for treating inflammatory lesions in the periodontal space Endod Dent Traumatol 1998; 14(4): 186-90.
[http://dx.doi.org/10.1111/j.1600-9657.1998.tb00835.x] [PMID: 9796483]
]. It also retains its antimicrobial activity in the presence of blood and other organic matter [13Gélinas P, Goulet J. Neutralization of the activity of eight disinfectants by organic matter J Appl Bacteriol 1983; 54(2): 243-7.
[http://dx.doi.org/10.1111/j.1365-2672.1983.tb02613.x] [PMID: 6687886]
].

Propolis is a brownish resinous substance collected mainly from plants, by the bees. It is a potent antibacterial, antiviral, antifungal and antiprotozoan activities [14Ferreira FB, Torres SA, Rosa OP, et al. Antimicrobial effect of propolis and other substances against selected endodontic pathogens Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007; 104(5): 709-16.
[http://dx.doi.org/10.1016/j.tripleo.2007.05.019] [PMID: 17964476]
]. The main chemical elements present in propolis are flavonoids, phenolics, and various aromatic compounds [15Kandaswamy D, Venkateshbabu N, Gogulnath D, Kindo AJ. Dentinal tubule disinfection with 2% chlorhexidine gel, propolis, morinda citrifolia juice, 2% povidone iodine, and calcium hydroxide Int Endod J 2010; 43(5): 419-23.
[http://dx.doi.org/10.1111/j.1365-2591.2010.01696.x] [PMID: 20518935]
].

Azadirachta indica (Neem) is a tree with medicinal value. Each part of the tree has been explored in phytotherapy. The leaves contain alkaloids, glycosides, saponins, flavonoids, steroids, anthraquinone and tannic acid, which are of medicinal value [16Kausik B, Chattopadhyey I, Benerjee RK, Bandyopdyey U. Biological activities and medicinal properties of neem Curr Sci 2002; 82: 1336-44.].

Ca(OH)2 is believed to have many of the properties of an ideal root canal dressing, mainly due to its alkaline pH [17Tronstad L, Andreasen JO, Hasselgren G, Kristerson L, Riis I. pH changes in dental tissues after root canal filling with calcium hydroxide J Endod 1981; 7(1): 17-21.
[http://dx.doi.org/10.1016/S0099-2399(81)80262-2] [PMID: 6938618]
, 18Torneck CD, Moe H, Howley TP. The effect of calcium hydroxide on porcine pulp fibroblasts in vitro J Endod 1983; 9(4): 131-6.
[http://dx.doi.org/10.1016/S0099-2399(83)80031-4] [PMID: 6574198]
]. It is microbicidal [19Safavi KE, Dowden WE, Introcaso JH, Langeland K. A comparison of antimicrobial effects of calcium hydroxide and iodine-potassium iodide J Endod 1985; 11(10): 454-6.
[http://dx.doi.org/10.1016/S0099-2399(85)80086-8] [PMID: 3865992]
] and neutralizes the remaining tissue debris in the root canal system. [20Hasselgren G, Olsson B, Cvek M. Effects of calcium hydroxide and sodium hypochlorite on the dissolution of necrotic porcine muscle tissue J Endod 1988; 14(3): 125-7.
[http://dx.doi.org/10.1016/S0099-2399(88)80212-7] [PMID: 3268627]
] Ca(OH)2 also promotes an alkalinizing osteogenic environment on the surrounding tissues through the continuous release of OH- ions. [17Tronstad L, Andreasen JO, Hasselgren G, Kristerson L, Riis I. pH changes in dental tissues after root canal filling with calcium hydroxide J Endod 1981; 7(1): 17-21.
[http://dx.doi.org/10.1016/S0099-2399(81)80262-2] [PMID: 6938618]
, 21Ringel AM, Patterson SS, Newton CW, Miller CH, Mulhern JM. In vivo evaluation of chlorhexidine gluconate solution and sodium hypochlorite solution as root canal irrigants J Endod 1982; 8(5): 200-4.
[http://dx.doi.org/10.1016/S0099-2399(82)80354-3] [PMID: 6955420]
] Furthermore, Ca(OH)2 mediates the neutralization of lipopolysaccharides [22Safavi KE, Nichols FC. Alteration of biological properties of bacterial lipopolysaccharide by calcium hydroxide treatment J Endod 1994; 20(3): 127-9.
[http://dx.doi.org/10.1016/S0099-2399(06)80057-9] [PMID: 7996084]
] and thus helps in cleansing the root canal [20Hasselgren G, Olsson B, Cvek M. Effects of calcium hydroxide and sodium hypochlorite on the dissolution of necrotic porcine muscle tissue J Endod 1988; 14(3): 125-7.
[http://dx.doi.org/10.1016/S0099-2399(88)80212-7] [PMID: 3268627]
].

The aim of this study is to evaluate the dentinal tubule disinfection with Propolis, Alcoholic & aqueous extract of Azadirachta indica, 2% Chlorhexidine and calcium hydroxide against Candida albicans biofim formed on tooth substrate. There has been no study evaluating the dentinal tubule disinfection with Azadirachta indica till date.

MATERIALS & METHOD

Preparation of Dentine Specimens

The model proposed by Haapasalo & Ørstavik (1987) [23Haapasalo M, Ørstavik D. In vitro infection and disinfection of dentinal tubules J Dent Res 1987; 66(8): 1375-9.
[http://dx.doi.org/10.1177/00220345870660081801] [PMID: 3114347]
] was modified. One hundred and five single-rooted human mandibular premolar teeth freshly extracted for orthodontic reasons were selected for the study.

A rotary diamond disc was used to decoronate the teeth below the cementoenamel junction and the apical part of the root to obtain 6 mm of the middle third of the root (Fig. 1). Cementum was removed from the root surface. Gates Glidden drills no. 3 in a slow-speed handpiece was used to standardize the internal diameter of the root canals. The specimens were placed in an ultrasonic bath of 17% ethylenediaminetetraacetic acid for 5 min followed by 3% NaOCl for 5 min to remove organic and inorganic debris. The traces of chemicals used were removed by immersing the dentine specimens in an ultrasonic bath containing distilled water for 5 min. All the specimens were then sterilized in an autoclave for two cycles. The first cycle at 121oC and the second with the specimens immersed in 1 mL of tryptone soya (TS) broth in individual microcentrifuge tubes [24Kandaswamy D, Venkateshbabu N, Gogulnath D, Kindo AJ. Dentinal tubule disinfection with 2% chlorhexidine gel, propolis, morinda citrifolia juice, 2% povidone iodine, and calcium hydroxide Int Endod J 2010; 43(5): 419-23.
[http://dx.doi.org/10.1111/j.1365-2591.2010.01696.x] [PMID: 20518935]
].

Fig. (1)

Prepared specimens.



Contamination of the Specimens

C. albicansATCC 10231 (Himedia, Mumbai L. No. 443-207) was grown in saboraud dextrose agar (SDA) (Himedia, Mumbai) for 24h (Fig. 2). The culture was suspended in 5 mL of SDA broth and incubated for 4 h at 37oC and its turbidity adjusted to 0.5 McFarland standard with sterile SDA broth [25Tyagi SP, Sinha DJ, Garg P, Singh UP, Mishra CC, Nagpal R. Comparison of antimicrobial efficacy of propolis, Morinda citrifolia, Azadirachta indica (Neem) and 5% sodium hypochlorite on Candida albicans biofilm formed on tooth substrate: An in-vitro study J Conserv Dent 2013; 16(6): 532-5.
[http://dx.doi.org/10.4103/0972-0707.120973] [PMID: 24347888]
]. Each dentine block was placed in pre-sterilized microcentrifuge tubes containing 1 mL of the TS broth. 50 µL of the inoculums containing the C. albicans was transferred into each of the microcentrifuge tubes. At the end of 24 h, the dentine specimens were transferred into fresh broth containing C. albicans. All procedures were carried out under laminar flow. Purity of the culture was checked by subculturing 5 µL of the broth from the incubated dentine specimens in SDA broth on SDA plates [26Vaghela DJ, Kandaswamy D, Venkateshbabu N, Jamini N, Ganesh A. Disinfection of dentinal tubules with two different formulations of calcium hydroxide as compared to 2% chlorhexidine: As intracanal medicaments against Enterococcus faecalis and Candida albicans: An in vitro study J Conserv Dent 2011; 14(2): 182-6.
[http://dx.doi.org/10.4103/0972-0707.82625] [PMID: 21814363]
]. Contamination of the dentine specimens were carried out for a period of 2 days.

Fig. (2)

Candida albicans cultured on Sabouraud dextrose Agar.



Antimicrobial Assessment

At the end of 2 days, the specimens were irrigated with 5 mL of sterile saline to remove the incubation broth. Then, the specimens were assigned into 7 groups (n = 15 dentine blocks each).

Group 1- Propolis (Herbal Biosolutions, Delhi) was prepared by diluting a 33% commercially available alcoholic extract using warm saline in a ratio 2:1, to form an 11% alcoholic extract [27Shingare P, Chaugule V. Comparative evaluation of antimicrobial activity of miswak, propolis, sodium hypochlorite and saline as root canal irrigants by microbial culturing and quantification in chronically exposed primary teeth Germs 2011; 1(1): 12-21.
[http://dx.doi.org/10.11599/germs.2012.1004] [PMID: 24432254]
]. Then, it was made into gel using Hydroxypropyl methylcellulose (Himedia).

Group 2- Alcoholic extract of Azadirachta indica was prepared using 25 g of fresh neem leaves powder of 99% purity (The Indian Neem Tree Company, Mumbai) was added to 50 ml of absolute ethanol (Sterling Chemicals and Alcohols Pvt. Ltd., Mumbai). Mixture was macerated for 1-2 min, then extract was filtered through muslin cloth for coarse residue and then through filter paper for finer residue [28Dubey S, Chaodary M, Gupta P. Comparative study of the antimicrobial efficiency of neem leaf extract, sodium hypochlorite and biopure MTAD: An in vitro study Ind J Dent Adv 2012; 4: 740-3.]. It was made into a gel by using HPMC.

Group 3- Aqueous extract of Azadirachta indica (Neem) was prepared by mixing 25 g of dry powder of neem leaves (The Indian Neem Tree Company, Mumbai) with 100 ml of sterile distilled water in a round bottom flask with occasional shaking. The extracts were then filtered through a muslin cloth for coarse residue and then through a Whatman no. 1 filter paper and kept in an airtight amber‑colored container [29Nayak A, Nayak RN, Somya GB, Kishore B, Mithun K. Evaluation of antibacterial and anticandidial efficacy of aqueous and alcoholic extract of Neem (Azadirachta indica) an in vitro study IJRAP 2011; 2: 230-5.] It was made into a gel by using HPMC.

Group 4- 2% Chlorhexidine diacetate powder (2% solution was made and then gel was prepared by mixing it with HPMC as a thickening agent and distilled water) [30Krithikadatta J, Indira R, Dorothykalyani AL. Disinfection of dentinal tubules with 2% chlorhexidine, 2% metronidazole, bioactive glass when compared with calcium hydroxide as intracanal medicaments J Endod 2007; 33(12): 1473-6.
[http://dx.doi.org/10.1016/j.joen.2007.08.016] [PMID: 18037062]
].

Group 5- Calcium Hydroxide + HPMC (150 mg of calcium hydroxide powder was mixed with 0.15 ml of the vehicle, i.e., distilled water) [31Simon ST, Bhat KS, Francis R. Effect of four vehicles on the pH of calcium hydroxide and the release of calcium ion Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995; 80(4): 459-64.
[http://dx.doi.org/10.1016/S1079-2104(05)80371-3] [PMID: 8521110]
].

Group 6- Absolute Ethanol

Group 7- Sterile Saline (Negative Control)

Hydoxypropyl methyl cellulose (HPMC) was used as a thickening agent for Groups 1,2,3,4 and 5 to standardize all the test solutions, which is an inert polymer [32Fernandez-Vigo J, Refojo MF, Verstraeten T. Evaluation of a viscoelastic solution of hydroxypropyl methylcellulose as a potential vitreous substitute Retina 1990; 10(2): 148-52.
[http://dx.doi.org/10.1097/00006982-199004000-00012] [PMID: 2402557]
].

The medicaments were placed inside the canals and sealed at both ends with paraffin wax. They were then incubated in an aerobic environment for 37oC. At the end of 1, 3, and 5 days (n=5 each), harvesting of dentine was carried out at two depths (200 and 400 µm) with Gates Glidden drills no 4 and 5, respectively [24Kandaswamy D, Venkateshbabu N, Gogulnath D, Kindo AJ. Dentinal tubule disinfection with 2% chlorhexidine gel, propolis, morinda citrifolia juice, 2% povidone iodine, and calcium hydroxide Int Endod J 2010; 43(5): 419-23.
[http://dx.doi.org/10.1111/j.1365-2591.2010.01696.x] [PMID: 20518935]
]. The collected dentine shavings were transferred into 1 mL of sterile SDA broth and incubated in an aerobic environment at 37oC for 24 h. After 24 h, the contents of each tube were serially diluted, 100 µL of the broth in 100 µL of sterile saline five times. 50 ìL of the dilution was then plated on SDA plates and incubated for 24 h. Colonies were counted and readings were tabulated. The results were statistically analysed by ANOVA and post hoc tukey test.

RESULTS

The current study showed that 5 medicaments studied exerted antifungal activity. Contamination of the dentin blocks was confirmed when debris samples harvested from the saline group (negative control) yielded positive growth. Table 1 shows the antifungal activity, measured at 2 depths (200 and 400 μm) and at 3 time intervals (1, 3, and 5 days).

Table 1

Mean & Standard deviation of colony counts for different intracanal medicaments at 200 and 400 µm depths at different time intervals.




There was no significant difference between Propolis and Neem (Alc. extract); Propolis and 2% Chlorhexidine; Neem (Alc.) and 2% Chlorhexidine and Ethanol and Saline, whereas all the other groups had significant differences. On the basis of above analysis, the following order of antimicrobial efficacy of different medicaments was observed (Fig. 3):

Fig. (3)

Graph indicating the antimicrobial efficacy of different groups.



DISCUSSION

The in vitro model developed by Haapasalo & Ørstavik (1987) [23Haapasalo M, Ørstavik D. In vitro infection and disinfection of dentinal tubules J Dent Res 1987; 66(8): 1375-9.
[http://dx.doi.org/10.1177/00220345870660081801] [PMID: 3114347]
] was modified and used to assess the efficacy of endodontic medicaments in the disinfection of dentinal tubules. Human permanent teeth were used instead of the bovine teeth as suggested by Basrani et al. [33Basrani B, Tjäderhane L, Santos JM, et al. Efficacy of chlorhexidine- and calcium hydroxide-containing medicaments against Enterococcus faecalis in vitro Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003; 96(5): 618-24.
[http://dx.doi.org/10.1016/S1079-2104(03)00166-5] [PMID: 14600699]
], as human dentin blocks would simulate the clinical scenario better. Lynne et al. [34Lynne RE, Liewehr FR, West LA, Patton WR, Buxton TB, McPherson JC. In vitro antimicrobial activity of various medication preparations on E. faecalis in root canal dentin J Endod 2003; 29(3): 187-90.
[http://dx.doi.org/10.1097/00004770-200303000-00006] [PMID: 12669878]
] further modified this model to include quantitative analysis of microbes in the dentinal tubules to define a percentage of reduction in CFU in infected dentine before and after the application of intracanal medicaments. Since apical dentine is mostly sclerotic and physically impedes microbial invasion, mid-root sections were taken. Invasion of coronal and mid-root dentin occurs more readily whereas the extent and depth of invasion are significantly less in apical dentin, because of less patent dentinal tubules [35Love RM. Regional variation in root dentinal tubule infection by Streptococcus gordonii J Endod 1996; 22(6): 290-3.
[http://dx.doi.org/10.1016/S0099-2399(96)80261-5] [PMID: 8934987]
] The samples were tested at two depths, 200µm and 400µm, because calcium hydroxide is known to penetrate only upto 200 to 300 µm [36Lin S, Tsesis I, Zukerman O, Weiss EI, Fuss Z. Effect of electrophoretically activated calcium hydroxide on bacterial viability in dentinal tubules--in vitro Dent Traumatol 2005; 21(1): 42-5.
[http://dx.doi.org/10.1111/j.1600-9657.2004.00293.x] [PMID: 15660756]
]. Thus, disinfection can be assessed better at these two depths. Three time periods have been taken to assess the disinfection because the effective antimicrobial action of the tested intracanal medicaments decreased after 48 hours [37Neelakantan P, Sanjeev K, Subbarao CV. Duration-dependent susceptibility of endodontic pathogens to calcium hydroxide and chlorhexidene gel used as intracanal medicament: an in vitro evaluation Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007; 104(4): e138-41.
[http://dx.doi.org/10.1016/j.tripleo.2007.04.035] [PMID: 17689115]
], therefore, time periods chosen to evaluate the antimicrobial assessment were first and third day. Panagiotis et al. [38Beltes PG, Pissiotis E, Koulaouzidou E, Kortsaris AH. In vitro release of hydroxyl ions from six types of calcium hydroxide nonsetting pastes J Endod 1997; 23(7): 413-5.
[http://dx.doi.org/10.1016/S0099-2399(97)80292-0] [PMID: 9587291]
] have shown that a 5-day period is adequate for Ca(OH)2 pastes to release the OH- ions, so an additional time period of 5 days was also taken.

The microorganism used in the study was Candida albicans (ATCC 10231) since it is the most commonly encountered fungus in the root canals, 21% in primary infections [39Baumgartner JC, Watts CM, Xia T. Occurrence of Candida albicans in infections of endodontic origin J Endod 2000; 26(12): 695-8.
[http://dx.doi.org/10.1097/00004770-200012000-00003] [PMID: 11471635]
] and 18% in cases of retreatments [40Siqueira JF Jr, Rôças IN. Diversity of endodontic microbiota revisited J Dent Res 2009; 88(11): 969-81.
[http://dx.doi.org/10.1177/0022034509346549] [PMID: 19828883]
]. Dimorphic switching is observed in this species, which is a pivotal factor for biofilm formation and the pathogenic potential of C. albicans. There is an initial period of adherence (0-2 h) followed by microcolony formation (2-4 h). Dimorphic switching occurs thereafter with a transition from budding-yeast forms to filamentous pseudo- and true hyphal forms (4-6 h). The complexity of the biofilm increases with time, taking on three dimensional architecture with spatial heterogeneity as it matures (8-48 h) [25Tyagi SP, Sinha DJ, Garg P, Singh UP, Mishra CC, Nagpal R. Comparison of antimicrobial efficacy of propolis, Morinda citrifolia, Azadirachta indica (Neem) and 5% sodium hypochlorite on Candida albicans biofilm formed on tooth substrate: An in-vitro study J Conserv Dent 2013; 16(6): 532-5.
[http://dx.doi.org/10.4103/0972-0707.120973] [PMID: 24347888]
], which is why the 48 h biofilm model was used. Sen et al. [41Sen BH, Safavi KE, Spångberg LS. Growth patterns of Candida albicans in relation to radicular dentin Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997; 84(1): 68-73.
[http://dx.doi.org/10.1016/S1079-2104(97)90298-5] [PMID: 9247954]
] investigated the growth patterns of C.albicans in relation to human radicular dentin and observed blastospores and hyphal structures on the root canal walls of all specimens. Dentin colonization may be an important step during infection of the root canal system. The invasion of dentinal tubules can protect microbial cells from the effects of intracanal procedures and may play an important role in the establishment of persistent root canal infections. Yeasts are on average 1 to 6 µm in diameter, whereas hyphae are usually 1.9 to 2.6 µm in diameter [42Waltimo TM, Sirén EK, Torkko HL, Olsen I, Haapasalo MP. Fungi in therapy-resistant apical periodontitis Int Endod J 1997; 30(2): 96-101.
[http://dx.doi.org/10.1111/j.1365-2591.1997.tb00681.x] [PMID: 10332243]
]. It has also been proposed that the contact-sensing (thigmotropism) ability of hyphal structures of Candida makes dentinal invasion inevitable. Therefore, on the basis of this invasive affinity to dentin, C.albicans is considered a dentinophilic microorganism [43Ørstavik D, Haapasalo M. Disinfection by endodontic irrigants and dressings of experimentally infected dentinal tubules Endod Dent Traumatol 1990; 6(4): 142-9.
[http://dx.doi.org/10.1111/j.1600-9657.1990.tb00409.x] [PMID: 2133305]
]. Thus, the means to eradicate C.albicans from root canals becomes important for complete disinfection and to avoid reinfection.

Intracanal medicament possessing antimicrobial properties between appointments may reduce or eliminate microbes in the root canal system, thereby increasing the predictability and success of root canal treatment [43Ørstavik D, Haapasalo M. Disinfection by endodontic irrigants and dressings of experimentally infected dentinal tubules Endod Dent Traumatol 1990; 6(4): 142-9.
[http://dx.doi.org/10.1111/j.1600-9657.1990.tb00409.x] [PMID: 2133305]
]. In our study, 2% CHX has shown the 99.2% microbial inhibition at depths of 200 and 400 µm from day 1 to 5, which is not in accordance with the other studies in which 100% inhibition has been shown. The possible reasons for this variance might be dentine, dentine components (hydroxyapatite & collagen), heat-killed microbial cells and inflammatory exudates in the root canal system, which are inhibitors of CHX [44Mohammadi Z, Abbott PV. The properties and applications of chlorhexidine in endodontics Int Endod J 2009; 42(4): 288-302.
[http://dx.doi.org/10.1111/j.1365-2591.2008.01540.x] [PMID: 19220510]
]. Yet, it has given maximum inhibition in our study. The explanation for this might be its high microbicidal dosage of 2% and increased diffusion of the medicament into the dentinal tubules [45Bhardwaj A, Ballal S, Velmurugan N. Comparative evaluation of the antimicrobial activity of natural extracts of Morinda citrifolia, papain and aloe vera (all in gel formulation), 2% chlorhexidine gel and calcium hydroxide, against Enterococcus faecalis: An in vitro study J Conserv Dent 2012; 15(3): 293-7.
[http://dx.doi.org/10.4103/0972-0707.97964] [PMID: 22876022]
]. Since, it is a positively charged hydrophobic and lipophilic molecule, it interacts with negatively charged phospholipids and lipopolysaccharides on the cell membrane of fungus and enters the cell through some type of active or passive transport mechanism, which alters the osmotic equilibrium of the cells. This increases the permeability of the cell wall, allowing the CHX molecule to penetrate into the micro-organism, followed by leakage of intracellular constituents, particularly phosphate entities such as adenosine triphosphate and nucleic acids. As a consequence, the cytoplasm becomes congealed, with resultant reduction in leakage; thus, there is a biphasic effect on membrane permeability [15Kandaswamy D, Venkateshbabu N, Gogulnath D, Kindo AJ. Dentinal tubule disinfection with 2% chlorhexidine gel, propolis, morinda citrifolia juice, 2% povidone iodine, and calcium hydroxide Int Endod J 2010; 43(5): 419-23.
[http://dx.doi.org/10.1111/j.1365-2591.2010.01696.x] [PMID: 20518935]
]. It also binds to hydroxyapatite and soft tissues, changing their electrical field to compete with microbial binding, thus decreasing microbial adherence. The result of the present study was similar to that of Krithikadatta et al. [30Krithikadatta J, Indira R, Dorothykalyani AL. Disinfection of dentinal tubules with 2% chlorhexidine, 2% metronidazole, bioactive glass when compared with calcium hydroxide as intracanal medicaments J Endod 2007; 33(12): 1473-6.
[http://dx.doi.org/10.1016/j.joen.2007.08.016] [PMID: 18037062]
], Gomes et al., [46Gomes BP, Souza SF, Ferraz CC, et al. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro Int Endod J 2003; 36(4): 267-75.
[http://dx.doi.org/10.1046/j.1365-2591.2003.00634.x] [PMID: 12702121]
] and Siqueria and Uzeda, [47Siqueira JF Jr, de Uzeda M. Intracanal medicaments: evaluation of the antibacterial effects of chlorhexidine, metronidazole, and calcium hydroxide associated with three vehicles J Endod 1997; 23(3): 167-9.
[http://dx.doi.org/10.1016/S0099-2399(97)80268-3] [PMID: 9594757]
] and Bhardwaj et al. [45Bhardwaj A, Ballal S, Velmurugan N. Comparative evaluation of the antimicrobial activity of natural extracts of Morinda citrifolia, papain and aloe vera (all in gel formulation), 2% chlorhexidine gel and calcium hydroxide, against Enterococcus faecalis: An in vitro study J Conserv Dent 2012; 15(3): 293-7.
[http://dx.doi.org/10.4103/0972-0707.97964] [PMID: 22876022]
] Basrani et al. [33Basrani B, Tjäderhane L, Santos JM, et al. Efficacy of chlorhexidine- and calcium hydroxide-containing medicaments against Enterococcus faecalis in vitro Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003; 96(5): 618-24.
[http://dx.doi.org/10.1016/S1079-2104(03)00166-5] [PMID: 14600699]
] showed that 2% chlorhexidine gel produced a better antimicrobial action as compared to 0.2% chlorhexidine gel or calcium hydroxide mixed with 0.2% chlorhexidine.

In our study, Propolis showed 98.5% microbial inhibition irrespective of depth and time, with no significant difference in comparison to the chlorhexidine group. Propolis exhibits antimicrobial, anti-inflammatory, healing, anesthetic and cariostatic properties [48Garg P, Tyagi SP, Sinha DJ, Singh UP, Malik V, Maccune ER. Comparison of antimicrobial efficacy of propolis, Morinda citrifolia, Azadirachta indica, triphala, green tea polyphenols and 5.25% sodium hypochlorite against Enterococcus faecalis biofilm Saudi Endod J 2014; 4: 122-7.
[http://dx.doi.org/10.4103/1658-5984.138141]
]. Some components present in propolis extract, like flavonoids (quercetin, galangin, pinocembrin), caffeic acid, benzoic acid and cinnamic acid, probably act on the microbial membrane or cell wall site, causing functional and structural damages [25Tyagi SP, Sinha DJ, Garg P, Singh UP, Mishra CC, Nagpal R. Comparison of antimicrobial efficacy of propolis, Morinda citrifolia, Azadirachta indica (Neem) and 5% sodium hypochlorite on Candida albicans biofilm formed on tooth substrate: An in-vitro study J Conserv Dent 2013; 16(6): 532-5.
[http://dx.doi.org/10.4103/0972-0707.120973] [PMID: 24347888]
]. Takaisi- Kikuni and Schilcher [49Takaisi-Kikuni NB, Schilcher H. Electron microscopic and microcalorimetric investigations of the possible mechanism of the antibacterial action of a defined propolis provenance Planta Med 1994; 60(3): 222-7.
[http://dx.doi.org/10.1055/s-2006-959463] [PMID: 8073087]
] have reported that it prevents fungal cell division and also breaks down fungal cell wall and cytoplasm similar to the action of some antibiotics. Kujumgiev et al. [50Kujumgiev A, Tsvetkova I, Serkedjieva Y, Bankova V, Christov R, Popov S. Antibacterial, antifungal and antiviral activity of propolis of different geographic origin J Ethnopharmacol 1999; 64(3): 235-40.
[http://dx.doi.org/10.1016/S0378-8741(98)00131-7] [PMID: 10363838]
] have reported that the antimicrobial action of propolis can be due to flavonoids and esters of phenolic acids. Koo et al. [51Koo H, Gomes BP, Rosalen PL, Ambrosano GM, Park YK, Cury JA. In vitro antimicrobial activity of propolis and Arnica montana against oral pathogens Arch Oral Biol 2000; 45(2): 141-8.
[http://dx.doi.org/10.1016/S0003-9969(99)00117-X] [PMID: 10716618]
] tested the antimicrobial activity of propolis using the agar diffusion method and observed that the propolis extract significantly inhibited all of the microorganisms tested, including facultative and strict anaerobic species. Oncag et al. [52Oncag O, Cogulu D, Uzel A, Sorkun K. Efficacy of propolis as an intracanal medicament against Enterococcus faecalis Gen Dent 2006; 54(5): 319-22.
[PMID: 17004565]
] observed that propolis had good in vitro antimicrobial activity against E. faecalis in the root canals of extracted teeth, suggesting that it could be used as an alternative intracanal medicament.

Azadirachta indica (Neem) has also been used in the study. There has been no study so far of dentinal tubule disinfection with Azadirachta indica (AI), this probably is the first study of its kind. The presence of active constituents such as nimbidin, nimbin, nimbolide, gedunin, azadirachtin, mahmoodin, margolone, and cyclictrisulphide contributes to the antifungal activity of AI [53Ghosh A. Chakrabarti, Roy P, Bhadury S, Nag T, Sarkar S. Bioremidation of heavy metals from neem leaf extract by chelation with dithizone Asian Pharm Clin Res 2009; 2: 87-92.]. These active constituents uncouple mitochondrial oxidative phosphorylation; thus, inhibiting the respiratory chain [54Biswas K, Chattopadhyay I, Banerjee RK, Bandyopadhyay U. Biological activities and medicinal properties of neem (Azadirachta indica) Curr Sci 2002; 82: 1336-45.]. This resulted in its anti-adherence activity by altering the microbial adhesion and the ability of the microorganism to colonize thereby causing maximum reduction in adherence to dentin. Rajashekharan et al. [55Rajasekaran C, Meignanam E, Vijayakumar V, et al. Investigations on antibacterial activity of leaf extracts of Azadirachta indica A.Juss (Meliaceae): A traditional medicinal plant of India Ethnobotanical Leaflets 2008; 12: 1213-7.] have proposed that the inhibitory activities of the leaf extracts of AI were both organism and solvent dependent. Thus, to evaluate the effect of solvent on Candida albicans, both aqueous and alcoholic extracts were taken. Nayak et al. [29Nayak A, Nayak RN, Somya GB, Kishore B, Mithun K. Evaluation of antibacterial and anticandidial efficacy of aqueous and alcoholic extract of Neem (Azadirachta indica) an in vitro study IJRAP 2011; 2: 230-5.] conducted a study in which the minimum inhibitory concentration of aqueous extract of AI was 7.5% as compared to the alcoholic extract of AI which was 3.75%, thus showing alcoholic extract to be better and more efficacious, which is in support of our study.

Calcium hydroxide, the gold standard of intracanal medicament was also taken as a test group. Literature indicates that Candida species are resistant to calcium hydroxide, [26Vaghela DJ, Kandaswamy D, Venkateshbabu N, Jamini N, Ganesh A. Disinfection of dentinal tubules with two different formulations of calcium hydroxide as compared to 2% chlorhexidine: As intracanal medicaments against Enterococcus faecalis and Candida albicans: An in vitro study J Conserv Dent 2011; 14(2): 182-6.
[http://dx.doi.org/10.4103/0972-0707.82625] [PMID: 21814363]
] which has been again verified in our study. Waltimo et al. [56Waltimo TM, Ørstavik D, Sirén EK, Haapasalo MP. In vitro susceptibility of Candida albicans to four disinfectants and their combinations Int Endod J 1999; 32(6): 421-9.
[http://dx.doi.org/10.1046/j.1365-2591.1999.00237.x] [PMID: 10709489]
] evaluated the susceptibility of 7 strains of C. albicans to 4 disinfectants: iodine potassium iodide, chlorhexidine acetate, sodium hypochlorite, and calcium hydroxide. C. albicans cells were highly resistant to calcium hydroxide. It survives in a wide range of pH values, the alkalinity of saturated calcium hydroxide solution may not have any effect on C. albicans. The alkalinity of calcium hydroxide also reduces due to the buffering capacity of dentin. In addition, calcium hydroxide solution may readily display the Ca++ ions necessary for the growth and morphogenesis of Candida. These mechanisms may explain why calcium hydroxide has been found to be ineffective against C. albicans [57Sen BH, Akdeniz BG, Denizci AA. The effect of ethylenediamine-tetraacetic acid on Candida albicans Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000; 90(5): 651-5.
[http://dx.doi.org/10.1067/moe.2000.109640] [PMID: 11077392]
].

Ethanol was also taken as a test group to check if it influenced the activity of propolis or AI. Ethanol showed slight inhibition of microorganism but there was no significant difference between ethanol and saline, which was taken as a negative control. Previously, a study conducted by Ferreira et al., [14Ferreira FB, Torres SA, Rosa OP, et al. Antimicrobial effect of propolis and other substances against selected endodontic pathogens Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007; 104(5): 709-16.
[http://dx.doi.org/10.1016/j.tripleo.2007.05.019] [PMID: 17964476]
] had concluded that Ethanol did not influence the antimicrobial effect of ethanolic extract of propolis.

CONCLUSION

Under the limitations of the study, it can be concluded that Propolis and alcoholic extract of Azadirachta indica performed equally well as that of 2% Chlorhexidine and thus can be probable alternatives to chlorhexidine, but further in vivo and long term studies are warranted.

CONFLICT OF INTEREST

The authors confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS

Declared none.

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