ORIGINAL RESEARCH ARTICLE
|Year : 2018 | Volume
| Issue : 4 | Page : 443-449
|Exploring the role of Morinda citrifolia and Triphala juice in root canal irrigation: An ex vivo study
Ekta Choudhary1, KR Indushekar1, Bhavna Gupta Saraf1, Neha Sheoran1, Divesh Sardana2, Ankur Shekhar3
1 Department of Paediatric Dentistry, Sudha Rustagi College of Dental Sciences and Research, Faridabad, Haryana, India
2 Department of Paediatric Dentistry, Sudha Rustagi College of Dental Sciences and Research, Faridabad, Haryana, China
3 Private Practitioner, Kapoor Dental Care, Model Town, New Delhi, India
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|Date of Submission||02-Mar-2018|
|Date of Decision||13-Apr-2018|
|Date of Acceptance||18-Apr-2018|
|Date of Web Publication||27-Jul-2018|
| Abstract|| |
Background: The present ex vivo study explores the role of Indian medicaments in endodontic irrigation in an attempt to search for a safe alternative to sodium hypochlorite (NaOCL).
Aim: To evaluate the efficacy of commercial preparations of Morinda citrifolia juice (MCJ) and Triphala juice against Enterococcus faecalis and Candida albicans.
Materials and Methods: The study was conducted on 84 permanent extracted human teeth. After decoronation and biomechanical preparation, inoculated (with E. faecalis and C. albicans) root sections were divided randomly into four experimental (MCJ, Triphala juice, 1% NaOCl, and 2% chlorhexidine [CHX]) and two control groups (preservative control and distilled water). Colony-forming units (CFUs) obtained for each group were counted at baseline (S0) and after irrigation at 1 and 3 days (S1and S2, respectively). Mean of Log CFU at S0, S1, and S2was compared for each irrigant using Friedman's two-way ANOVA.
Results: There was a significant decrease in microbial counts of both microbes in all groups at S1, but only CHX could demonstrate further decrease in the microbial counts of both microorganisms at S2.
Conclusion: The overall antimicrobial effects of different irrigants were maximum for CHX, whereas MCJ and Triphala juice also showed significant reductions. The herbal irrigants hold the promise of becoming efficient irrigants and warrant further research.
Keywords: Chlorhexidine; endodontic irrigant; root canal treatment; sodium hypochlorite
|How to cite this article:|
Choudhary E, Indushekar K R, Saraf BG, Sheoran N, Sardana D, Shekhar A. Exploring the role of Morinda citrifolia and Triphala juice in root canal irrigation: An ex vivo study. J Conserv Dent 2018;21:443-9
|How to cite this URL:|
Choudhary E, Indushekar K R, Saraf BG, Sheoran N, Sardana D, Shekhar A. Exploring the role of Morinda citrifolia and Triphala juice in root canal irrigation: An ex vivo study. J Conserv Dent [serial online] 2018 [cited 2019 Feb 21];21:443-9. Available from: http://www.jcd.org.in/text.asp?2018/21/4/443/237744
| Introduction|| |
The etiology of root canal infections is polymicrobial consisting of a mixed consortium of more than 600 species of microorganisms organized in biofilms.,Enterococcus faecalis has been reported to be the one of the most prevalent microorganisms in infected root canals and in retreatment cases of apical periodontitis, with a reported prevalence from 24% to 77%. In addition, the incidence of Candida albicans also increases with secondary infections and is mostly found in association with E. faecalis. Both these microorganisms have notorious reputation of being most persistent and resistant, especially in secondary infections, owing to multiple virulent factors.
To achieve successful endodontic therapy, chemomechanical preparation has a major role to play. Owing to the limitations of mechanical instruments to reach far and beyond in narrow isthmuses, accessory canals, and dentinal tubules, it becomes essential that an irrigant with good penetrability and bactericidal activity is used to inhibit microorganisms present in biofilms, dissolve necrotic pulp remnants, inactivate endotoxins, and remove smear layer. Till now, many irrigants have been used such as 0.5%–6% sodium hypochlorite (NaOCl), 0.2%–2% chlorhexidine (CHX), 3%–30% hydrogen peroxide (H2O2), 2%–5% iodine potassium iodide, 0.5% dequalinium acetate, 10%–17% ethylenediaminetetraacetic acid (EDTA), 10%–50% citric acid, proteolytic enzymes, sodium hydroxide, urea, potassium hydroxide, local anesthetic solutions, and normal saline. Various other newer irrigants such as mixture of tetracycline acid and detergents (MTAD), tetraclean, electrochemically activated solutions, ozonated water, and photon-activated disinfection have also been tried, all with some advantages and disadvantages. NaOCl has remained the gold standard against which efficacy of other irrigants is adjudged, however; if used injudiciously, NaOCl is very toxic and destructive to intraoral soft tissues, periradicular vasculature, and cancellous bone, where it can elicit severe inflammatory responses and degradation of the organic components of these tissues.
Morinda citrifolia juice (MCJ) and “Triphala” juice are well-known preparations in the Indian system of medicine. MCJ has been relatively new to endodontics and has been suggested as an alternative to NaOCl because of its chelating ability to remove smear layer and antimicrobial activities, especially against anaerobic bacteria such as E. faecalis and C. albicans., Similarly, Triphala has also been suggested as an alternative to NaOCl as a root canal irrigant in addition to its other uses in dentistry, namely, anticaries agent and mouth rinse., Although herbal extracts of MCJ and Triphala juice have been used in various studies, the preparation of extract is a technique-sensitive and cumbersome process and fresh extracts need to be prepared every time before use. On the other hand, commercial herbal preparations are readily available, easy to use and have comparatively longer shelf life. Thus, the present ex vivo study was done to investigate the antimicrobial activity of commercial preparations of MCJ and Triphala juice, used as endodontic irrigants, compared to 1% NaOCl and 2% CHX against mix culture of E. faecalis and C. albicans.
| Materials and Methods|| |
The present ex vivo study was carried out in the Department of Paedodontics and Preventive Dentistry and the Department of Microbiology of the institute. The present study was done on 84 permanent single-rooted extracted human teeth. Gross debris and blood were removed from the extracted teeth, and surfaces were disinfected with 5% NaOCl and 6% H2O2 followed by rinsing and storing in normal saline till further use.
Sample size calculation
Sample size calculation was done using G*Power 3.1 software (Heinrich-Heine-Universität, Düsseldorf, Germany). There were four experimental irrigant groups and two control groups, all of which were evaluated at three time intervals, i.e., at baseline (S0), immediately after chemomechanical preparation (S1), and after 72 h of incubation (S2). For six groups, ANOVA (for repeated measures, within interactions) was used with the following parameters:
- Effect size f = 0.25
- α err probability = 0.05
- Power (1-β err probability) = 0.90.
The resultant total sample size was 60, i.e., minimum 10 for each group. In case of four experimental groups only, if power was increased to 0.95, then the resultant total sample size was 60, i.e., minimum 15 for each group. Keeping both these values in mind, the sample size for four experimental groups was kept 16 teeth each and for two control groups at 10 each, thus making a total of 84.
- All permanent single-rooted teeth indicated for extractions.
Exclusion criteria (as determined radiographically)
- Root canals with fillings or posts
- Teeth with root resorption or perforations
- Root caries or fracture
- Calcified root canals
- Multiple canals or canal bifurcations.
Root canal preparation
All the teeth were decoronated with a diamond disc using a low-speed straight hand-piece to obtain root segments of 11 mm length, in which the patency was determined by inserting No. 15 K-file till its tip was just visible at the apex. Working length in all root sections was taken as 10 mm and root canals were prepared up to size 25 with K-files with simultaneous irrigation of normal saline. The root canals were filled with 17% EDTA for 3 min to remove smear layer followed by 2 mL irrigation with normal saline. Root section surfaces were then coated with two coats of acrylic varnish.
Before microbial inoculation, the root sections were individually placed in glass bottles, with numbering on each section, and autoclaved twice with 24-h interval in between, at 121°C at 15 psi for 20 min to sterilize the root canals, which was confirmed by culture methods. To verify the sterilization process, size 25 sterile paper point was inserted in the individual canal of randomly chosen one out of every five root specimens. These paper points were transported in sterile saline (1.5 mL) in individual sterile glass testtubes, vortexed, cultured, and confirmed for zero colony-forming units (CFU). After confirming sterility of root canals, microbial inoculation in each root section was done.
The following procedural steps were carried out using sterile materials and instruments, in Laminar Air Flow station, the working surface of which was swabbed with 2% lysol with UV lamp then switched on for 30 min before procedure.
Microbial strains used in the study, viz., E. faecalis (Microbial Type Culture Collection and Gene Bank, Chandigarh, India; MTCC 2729) and C. albicans (Microbial Type Culture Collection and Gene Bank, Chandigarh, India; MTCC 1637) were cultured on brain–heart infusion (BHI) agar (HiMedia Laboratories, Mumbai, India) and Sabouraud Dextrose Agar (SDA) (HiMedia Laboratories, Mumbai, India), respectively [Figure 1]a.
|Figure 1: (a) Colonies of Candida albicans and Enterococcus faecalis on Sabouraud Dextrose Agar and brain–heart infusion culture plates, respectively. (b) Discoloration produced in root specimens irrigated with Morinda citrifolia juice. (c) Discoloration produced in root specimens irrigated with Triphala|
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One milliliter each, of 24-h-old broth suspension, of both E. faecalis and C. albicans was collected in a testtube. Root apices were sealed with a temporary cement (Cavitemp, AMMDENT Mohali, India), and 10 μL of mixed culture suspension was inoculated in each root canal through micropipette sterile tips, by placing them 1 mm short of working length, and simultaneously pulling upward to deliver the culture up to the coronal end of all root sections. A 5 μL of each broth suspension was separately inoculated on respective culture media plates after doing serial dilutions in normal saline to obtain 1:1010 dilution. The CFU/mL thus obtained was recorded thrice, and their mean was taken as the baseline count for that respective microorganism.
Coronal access was also sealed with temporary cement. Then, each root section was placed in a single-sterile glass testtube filled with 2 mL normal saline, in upright position with root apex downward, and incubated at 37°C for 48 h. Root sections were removed and the surface was rinsed with sterile normal saline.
The inoculated root sections were divided randomly into six irrigant groups (four experimental and six controls) which are as follows:
- Group 1 (n = 16): MCJ (Basic Ayurveda, Ghaziabad, India)
- Group 2 (n = 16): Triphala juice (Basic Ayurveda, Ghaziabad, India)
- Group 3 (n = 16): 1% NaOCl (HypoSol™ Prevost Denpro Limited)
- Group 4 (n = 16): 2% CHX
- Group 5 (n = 10): Preservative control group (0.1% sodium benzoate, 0.1% potassium sorbate and citric acid [to establish pH = 7, as determined using pH indicator papers, at the same level as in commercial herbal extracts])
- Group 6 (n = 10): Distilled water – negative control group.
The cervical and apical seals were removed before instrumentation. Further enlargement of each root canal was done in a fixed period of 15 min, using K-files in step-back technique, to obtain apical enlargement of size 40 at the established working length, with simultaneous irrigation of total 3 mL of respective irrigant, done by inserting the tip of irrigating needle (30-G) almost 1 mm short of working length. Care was taken that tip of needle does not bind to the walls of the canal and the irrigation was passive, i.e., space was there for the irrigant to flow out coronally. On completion of the mechanical enlargement, 2 mL of irrigant solution was used to rinse off any debris in the canals, for another 5 min. Thus, the total volume of each irrigant was fixed, i.e., 5 mL with a fixed contact time of 20 min (15 min bio-mechanical preparation (BMP) + 5 min after rinse) in the root canal, in all the cases. Sterile normal saline (2 mL) was used as final rinse.
First microbial sample (S1) was taken immediately by inserting sterile size 40 paper point to full working length and left for 30 s. Paper points were then transported in 1.5 mL sterile normal saline in a sterile test tube. The tubes with paper points were vortexed for 1 min and were inoculated on the surface of both BHI agar plates and SDA, by spreading 100 μL of vortexed saline with L-glass spreader on each and incubating at 37°C for 48 h in case of E. faecalis and at 37°C for 72 h in case of C. albicans. After completion of respective incubation periods, the culture plates were subjected to colony counts and expressed as CFU/mL.
The root specimens were sealed and incubated for 72 h at 37°C in the same manner as before. After 72 h, temporary filling material was removed at both ends and the root canals were filled with sterile normal saline. The second microbial sample (S2) was taken in the same manner as for first sample (S1). All the steps for microbial culture were also same as done for sample S1.
The CFUs obtained for each group were counted thrice and then noted in a proforma. The mean of all the three readings was taken as the final count for analysis. These data were subjected to statistical analysis and CFUs at baseline, S1, and S2 were tabulated and compared for each irrigant, against both the microorganisms.
The collected data were analyzed using SPSS (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp.,). The CFU/mL of test microorganisms was converted into Log10 values.
For computing the percentage change, following formulae were used between different time intervals
- % reduction from Log10S0 to Log10S1= ([Log10S0− Log10S1]/Log10S0) × 100
- % change from Log10S1 to Log10S2= ([Log10S2− Log10S1]/Log10S1) × 100
- % reduction from Log10S0 to Log10S2= ([Log10S0− Log10S2]/Log10S0) × 100.
For intragroup comparison of variables at different time intervals, Friedman's two-way analysis of variance by ranks was used with P = 0.05. The significance was further tested with post hoc Wilcoxon signed-rank test. For intergroup comparison of different irrigants, one-factor Kruskal–Wallis test was done.
| Results|| |
[Table 1] summarizes the mean CFU/mL of E. faecalis and C. albicans at different time intervals (S0, S1, and S2) after use of different endodontic root canal irrigants. There was a decrease in microbial counts of both E. faecalis and C. albicans in all six groups at S1 time. After initial decrease in microbial counts at S1, only CHX could demonstrate further decrease in microbial counts of both microorganisms at S2 whereas other irrigants showed increased counts compared to S1, thus demonstrating the substantivity property of CHX. The decrease in microbial counts was found to be statistically significant (P< 0.001) on intergroup and intragroup comparisons. [Figure 2] compares the effect of different root canal irrigants on mean microbial counts (CFUs/mL) of E. faecalis and C. albicans, respectively, at different time intervals.
|Table 1: Comparison of overall effect of different endodontic irrigants against Enterococcus faecalis and Candida albicans expressed as mean percentage change between S0, S1, and S2|
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|Figure 2: (a) Overall effect of different irrigants against Enterococcus faecalis at different time intervals. (b) Overall effect of different irrigants against Candida albicans at different time intervals|
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| Discussion|| |
In the present study, 1% NaOCl was not effective in completely removing either of the two tested microorganisms, even after 20 min contact period. The effect against C. albicans was more pronounced immediately after contact, compared to E. faecalis, but the overall microbial count reduction was similar for both. It brought a decrease of 87.05% in E. faecalis counts as compared to 94.48% against C. albicans after chemomechanical preparation. This is in agreement with the results of Siqueira et al., 1997 where even the 4% NaOCl failed to eradicate E. faecalis completely in all canals, probably because of lack of contact in dentinal tubules which may be attributed to its high surface tension., Sen et al., 1999 evaluated the antifungal properties of 1% NaOCl and 5% NaOCl against C. albicans using cylindrical dentin tubes and found that C. albicans was more resistant in the presence of the smear layer than in the absence of the smear layer. When the smear layer was absent, NaOCl started to display antifungal activity after 30 min.
CHX used in the present study was found to be a more efficient antifungal agent as opposed to antibacterial. The minimum inhibitory concentration (MIC) of CHX to inhibit C. albicans has been determined to be <0.63 μg/mL. The contact time of CHX for negative cultures of C. albicans has been determined to be in the range of 30–1800 s.
It is known that mechanical instrumentation alone can reduce the bacterial counts by approximately 50% and the subsequent flushing action of irrigant is effective in bringing up to 98% bacterial reduction. However, this is directly related to the volume and frequency of irrigation as well as to the depth of the irrigating needle. These findings are replicated in the present study where distilled water was taken as negative control. It brought a reduction of 76.94% and 77.83% in the microbial counts of E. faecalis and C. albicans, respectively, immediately after biomechanical preparation. There is no residual anti-microbial action, and so, an increase of 24.88% and 36.98% was seen in the microbial counts of E. faecalis and C. albicans, respectively. The overall efficacy of distilled water against E. faecalis and C. albicans, respectively, was 71.35% and 71.11% which is almost equal. Since distilled water lacks any inherent antimicrobial properties, the reduction in microbial count could be attributed to effect of biomechanical preparation; hence, equal effects were observed for both the tested microorganisms. Since the commercial preparations of these herbal juices contain added preservatives, a positive control group containing same preservatives as indicated by the manufacturer was taken to assess their antimicrobial action, if any. The preservatives included salts of weak acids, i.e., 0.1% sodium benzoate and 0.1% potassium sorbate and citric acid, at pH of 7. Inhibition of microbial growth by weak acid preservatives has been proposed to be due to a number of actions including, membrane disruption,, inhibition of essential metabolic reactions, stress on intracellular pH homeostasis, and accumulation of toxic anions. Citric acid inhibits microbial growth due to its Ca 2+-chelating activity. In the present study, preservatives also showed bacterial and fungal reductions. Better results with preservatives alone compared to herbal extracts may also be due to neutralization of some preservative action by herbal components.
MCJ (Noni, Indian Mulberry, Ba Ji Tian, Nono or Nonu, Cheese Fruit, Nhau) is a traditional folk medicinal plant used by Polynesians for over 2000 years. It is believed to express its antimicrobial activity due to the presence of acubin, L-asperuloside, alizarin, and some anthraquinones. Murray et al., 2008 investigated the antimicrobial effectiveness of MCJ to E. faecalis and found its MIC to be 6%. They showed that MCJ was effective in the removal of smear layer, especially in combination with EDTA but not to the same extent as NaOCl and EDTA. However, the combination was not 100% effective in smear removal and MCJ actions were neutralized with addition of CHX. The authors however recommended MCJ as an irrigant because it is biocompatible oxidant, not likely to cause serious injuries if extruded out. Jainkittivong et al., 2009 demonstrated the antifungal effect of MCJ against C. albicans which varied with different concentrations and time intervals. The results of the present investigation showed that MCJ brought a reduction of 73.78% in the microbial counts of E. faecalis immediately after chemomechanical preparation. However, it lacked any residual antimicrobial activity which allowed a growth of approximately 30% in the microbial counts, thereby averaging the overall effect of MCJ against E. faecalis at 66.69% microbial reduction. Similar results were seen by Kandaswamy et al., 2010, where MCJ brought a reduction of only 69% in the microbial counts of E. faecalis from day 1 to day 5, at different depths. They have used it in gel form, with prolonged contact period. In the present study, MCJ in the solution form was used in limited contact of 20 min. This may be the reason for comparatively lesser percentage reduction in microbial counts in pre- and post-application of medicament.
Triphala is an Indian Ayurvedic herbal formulation consisting of dried and powdered fruits of three medicinal plants - Terminalia bellerica (Bihara/belericmyrobalan), Terminalia chebula (Harad/chebulic myrobalan), and Emblica officinalis (Amla/gooseberry). Apart from antibacterial activities, Triphala is also a very good-chelating agent because of the fruits that are rich in citric acid and holds promise in the removal of smear layer. Similar to MCJ, Triphala juice also lacked any residual anti-bacterial activity. However, Prabhakar et al., 2010 in an in vitro tooth model showed that Triphala produced complete inhibition of 3-week E. faecalis biofilm after 10 min immersion but had reduced efficacy against 6-week biofilm, probably because of insufficient “in-use” concentration and formation of E. faecalis-mediated bio-mineralized biofilm. They had used Triphala (60 mg/ml in 10% dimethyl sulfoxide) which had minimum inhibitory concentration (MIC).125 mg/ml and minimum bactericidal concentration (MBC) – 5 mg/ml. Similar concentration and preparation were used in another in vitro agar well experiment against E. faecalis by Pujar et al., 2011, which showed that Triphala has some antibacterial effect but did not completely eliminate bacteria; count after 10 min exposure was 2.3 × 104 CFU/ml. Both these findings are in agreement with the results of the present study even though the actual concentration of Triphala cannot be computed in the present study. It is difficult to ascertain whether the E. faecalis counts reduction observed for herbal irrigants is due to the presence of preservatives or if there is any added benefit of the bioactive components. Better results with preservatives alone compared to herbal extracts may also be due to neutralization of some preservative action by herbal components. The better efficacy of distilled water and preservatives may also be due to better flushing action as these are clear solutions and less viscous then herbal extracts which contain pulp and fibers. There may be the presence of additives and flavors in the herbal juices which may provide substrate for growth of microbes during the 72-h incubation period. This is reflected upon by the increase of 45.59% (Triphala) and 30.31% (MCJ) in microbial counts of E. faecalis as compared to only 20.15% (distilled water) and 24.88% (preservatives). These additives may also be the reason for dentinal discoloration observed in the root specimens irrigated with MCJ and Triphala juice.
Thus, the results of present study show that the overall antibacterial efficacy of Triphala against E. faecalis was more, whereas the antifungal effect against C. albicans was more pronounced in case of MCJ. However, both the herbal irrigants were statistically comparable in overall antimicrobial efficacy against the two tested microorganisms. Another finding observed by the investigators of the present study was the discoloration caused by commercial preparations of both the herbal irrigants [Figure 1]b and [Figure 1]c. This may be due to added coloring agent in the commercial products or due to natural color pigments in the MCJ and Triphala juice and needs to be further investigated before recommending the commercial preparations for direct endodontic use. These herbal irrigants also produced discoloration of gloves, were slightly viscous and sticky. In the present study, canals were infected with mixed culture for only 48 h. This may not be enough for formation of biofilm and most of the microorganisms may still be in planktonic state. In clinical situations, many species of microorganisms are present which modify the characteristics of biofilm and alters its resistance to irriganting agents. In the present study, only a mixed culture of two microorganisms is taken which may be different from a clinical picture present in infected root canals.
| Conclusion|| |
The results of the present investigation shows that 2% CHX is the most effective irrigant due to its substantivity followed by 1% NaOCl against the mix culture of E. faecalis and C. albicans, although none of the two was effective in completely disinfecting the root canals. The overall antibacterial effect of different irrigants against E. faecalis was maximum for 2% CHX followed by 1% NaOCl. The rest of irrigants, viz., Triphala, distilled water, preservatives, and MCJ showed comparable antibacterial activity in the same decreasing order. The overall antifungal effect of different irrigants against C. albicans was least for preservatives and maximum for 2% CHX, again followed by 1% NaOCl, Triphala, MCJ, and distilled water in the decreasing order. The herbal irrigants hold the promise of becoming efficient irrigants, but a standardized, easy-to-use preparation should be made available to extend the benefits of nature to dentistry.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Divesh Sardana
Department of Paediatric Dentistry, Faculty of Dentistry Prince Philip Hospital University of Hong Kong, Hong Kong SAR
Source of Support: None, Conflict of Interest: None
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