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ORIGINAL ARTICLE
Year : 2020  |  Volume : 38  |  Issue : 4  |  Page : 343-349
 

Comparison of cytotoxicity and smear layer removal efficacy of triphala (an Indian ayurvedic herbal formulation) and 5.25% sodium hypochlorite as root canal irrigants: An in vitro study


Department of Pediatric and Preventive Dentistry, Amrita School of Dentistry, Amrita Institute of Medical Sciences, Amrita University, Kochi, Kerala, India

Date of Submission01-Jul-2020
Date of Decision11-Aug-2020
Date of Acceptance01-Dec-2020
Date of Web Publication5-Jan-2021

Correspondence Address:
Dr. R Balagopal Varma
Department of Pediatric and Preventive Dentistry, Amrita School of Dentistry, Amrita Institute of Medical Sciences, Amrita University, Ponnekara, Kochi - 682 041, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISPPD.JISPPD_297_20

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   Abstract 


Introductio: Healing potential of plants is an age-old idea that has recently attained renewed interest. Considering the ineffectiveness, potentially harmful effects, and safety concerns of commonly used synthetic irrigants, the herbal alternatives for endodontic usage might prove to be advantageous. Aim: The aim of this study was to evaluate the adequacy of smear layer removal and cytotoxicity potential of triphala in comparison to sodium hypochlorite. Materials and Methods: The study was conducted in two parts: the first part of the study was cytotoxicity assessment studied using Alamar blue assay. L929 mouse fibroblasts were seeded in 96-well plates at a density of 5000 cells/well and treated with different concentrations of triphala and NaOCl for a period of 24 and 48 h. The percentage of cell viability was then quantified using an Alamar blue assay. The optical density was measured at 570 nm and compared with 620 nm, which was considered as a reference wavelength. The second part of the study was smear layer assessment at the coronal, middle, and apical third of twenty human premolar teeth using scanning electron microscope. Results: The Alamar blue reagent cytotoxicity study suggested that triphala showed no cytotoxic properties against the normal mouse fibroblast cells whereas sodium hypochlorite showed a significant cytotoxic effect against the L929 cell lines with the IC50 concentration at 1.8%, respectively, after the treatment of 24 h of incubation at 37°C temperature. Triphala was as effective as sodium hypochlorite in smear layer removal in the coronal and middle third of the root, but sodium hypochlorite showed better smear layer removal in the apical third. Conclusion: Triphala can be considered as a superior irrigant with good antibacterial efficacy and least cytotoxicity potential compared to conventional hypochlorite irrigating agent and provide adequate clearing of smear layer in the coronal and middle third, and further studies are warranted to alter the properties of liquid to make it more cleansable in the apical third of the root.


Keywords: Cytotoxicity, herbal irrigant, L929 mouse fibroblasts, primary research, scanning electron microscopy, smear layer removal, sodium hypochlorite, triphala


How to cite this article:
Reshma Raj V P, Varma R B, Sureshkumar J, Kumaran P, Xavier AM, Madhavan M. Comparison of cytotoxicity and smear layer removal efficacy of triphala (an Indian ayurvedic herbal formulation) and 5.25% sodium hypochlorite as root canal irrigants: An in vitro study. J Indian Soc Pedod Prev Dent 2020;38:343-9

How to cite this URL:
Reshma Raj V P, Varma R B, Sureshkumar J, Kumaran P, Xavier AM, Madhavan M. Comparison of cytotoxicity and smear layer removal efficacy of triphala (an Indian ayurvedic herbal formulation) and 5.25% sodium hypochlorite as root canal irrigants: An in vitro study. J Indian Soc Pedod Prev Dent [serial online] 2020 [cited 2021 Apr 23];38:343-9. Available from: https://www.jisppd.com/text.asp?2020/38/4/343/306217





   Introduction Top


Endodontic treatment aims in the complete debridement of the root canal space to achieve three-dimensional cleaning, shaping, and proper obturation with an adequate seal.[1] The smear layer is an aggregation of both organic and inorganic components with a mixture of bacteria and their byproducts.[2] It prevents the proper cleansing and close adherence of sealer cement onto the canal walls due to the penetration of irrigants and intracanal medicaments leading to the blockage of dentinal tubules, thus advocating its removal.

Sodium hypochlorite has been the most common irrigant used because of its antimicrobial potential and its ability to dissolve organic material. However, it is a potent irritant to the periapical tissues and inherently possesses many disadvantages such as burning of surrounding tissues, unpleasant taste, staining of instruments, high toxicity, poor smear layer removal, corrosiveness to instruments, and reduction in elastic modulus and flexural strength of dentin.[3] These disadvantages have prompted researchers to look for other alternatives.

The healing potential of plants is an age-old idea that has recently attained renewed interest. Triphala is validated to be a safe herbal cocktail of active constituents with beneficial physiological effects and curative properties such as antioxidant activity, anti-inflammatory, and radical scavenging as an advantage over synthetic drugs. The major advantages include its low toxicity, lack of microbial resistance, increased shelf life, easy availability, and cost-effectiveness.[4] Hence, this study aimed to explore newer irrigation solutions that provide adequate smear layer removal and less cytotoxicity, which would probably be as effective as sodium hypochlorite.


   Materials and Methods Top


This study was conducted in a tertiary care hospital in Kochi, Kerala, in collaboration with the Nanotechnology and Microbiology Department. Triphala powder was made by procuring high standard raw products such as fruits of three medicinal plants: Terminalia bellirica, Terminalia chebula, and Emblica officinalis (Ayurveda Depot, Trivandrum), followed by drying and powdering into a uniform mix [Figure 1].
Figure 1: Fruits of three medicinal plants Terminalia bellirica, Terminalia chebula, and Emblica officinalis

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Triphala powder was made into a solution by dissolving in 10% dimethyl sulfoxide (DMSO), which was added to improve the efficiency of herbal products. It did not interfere with the antibacterial property which was confirmed with the disc diffusion method. This solution was stirred for 2 min and then passed through fast filter paper. The strained liquid was collected and samples were prepared.

Part 1: Assessment of efficacy of smear layer removal

Twenty single-rooted human mandibular premolars having a single canal and fully developed apices freshly extracted for orthodontic purposes were selected for this study. The teeth were cleaned using an ultrasonic scaler, followed by sterilization under autoclave, and thereafter stored in distilled water until use. The teeth were decoronated below the cement-enamel junction using a diamond disc to obtain a standardized root length of 10 mm. Working length was measured 1 mm short of apical foramen, and the glide path was established using a 15K file. All the samples were then randomly divided into two groups: Group A (control) and Group B (intervention).

Root canals were prepared until apical size 35, 0.06 taper using nickel–titanium files in a crown-down manner. During instrumentation, each root canal was irrigated using 2 ml of the solution corresponding to its group. The apical foramen of each root was coated with cyanoacrylate glue before embedding the roots into a polyvinyl siloxane impression material-filled transparent tube to obtain a closed system. For each specimen, final rinse irrigation was done using a 3-ml corresponding irrigating solution using a 30G side-vented needle, followed by rinsing with sterile water, and dried using sterile absorbent paper points.

Two longitudinal grooves were prepared on the buccal and lingual surfaces of each root specimen using a diamond disc, avoiding the penetration into the canal. The roots were split into two halves with a chisel and coded as the test and intervention. The coded specimens were mounted on metallic stubs with carbon strip backing and were examined independently by two observers under scanning electron microscopy (SEM) [Figure 2]. To avoid any observational bias, the readings were independently evaluated by a third examiner.
Figure 2: Scanning electron microscope and its imaging

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After a general evaluation of the canal wall, three SEM photomicrographs were taken at a magnification of ×2000 for evaluation of the smear layer at the center of the coronal, middle, and apical third of each specimen. The scores were attributed according to the rating system developed by Torabinejad et al., 2002 [Table 1].[5]
Table 1: Smear layer evaluation criteria

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The final scores of apical, middle, and coronal third of each specimen were tabulated. The mean and standard deviation of these values were calculated. The intergroup and intragroup comparisons of the mean and standard deviation of the three areas were calculated separately. The collected data were subjected to one-way analysis of variance to compare the mean of the groups. The post hoc test was performed to find the interrelationship between the two groups of significant differences.

Part 2: Cytotoxicity study

About 200-μl cell suspension was seeded in a 96-well plate at required cell density (20,000 cells per well) without the test agent, and the cells were allowed to grow for about 24 h [Figure 3]. Appropriate concentrations of the test agent were added to it, following which the plate was incubated for 24 h at 37°C in a 5% CO2 atmosphere. After the incubation period, the plates were removed from the incubator and 10 μL of Alamar blue reagent was added to a final concentration of 0.5 mg/mL of total volume and wrapped with aluminum foil to avoid exposure to light and was incubated for 2–3 h. The absorbance on a spectrophotometer value or an ELISA reader at 570 and 600 nm was used as the reference wavelength [Figure 4]. The IC50 value was determined using a linear regression equation, i.e., Y = Mx + C. (Here, Y = 50 and M and C values were derived from the viability graph.)
Figure 3: 20,000 cells were seeded in a 96-well ELISA plate

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Figure 4: Direct microscopic observations of the drug-treated images using inverted biological microscope after the drug treatment period of 24 h

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Concentrations used for the study

In this study, two test compounds, namely T and S, were used to check the cytotoxicity. The study was done on the L929 mouse fibroblast cell line (ATCC # CCL-1 CCL-1). The used concentrations of the compounds to treat the cells are shown in [Table 2].
Table 2: Details of drug concentrations used for the study

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Analysis of potential cytotoxicity of the irrigation solutions under investigation was conducted as follows. First, the optical densitometric readings of each of the four wells were determined and the arithmetic mean ± standard errors were calculated. Second, an arbitrary unit of 100 was assigned to the optical densitometric readings obtained from the control wells and converted into percentages. The control, ½, ¼, 1/8, and 1/16 dilutions were plotted as the abscissa of a linear regression graph with corresponding percentages of optical densitometric values as the ordinates. Using the InStat statistical software, a linear regression line was fitted onto the converted values and dilution on the abscissa that produced 50% cytotoxicity (cytotoxic dilution 50% [CD50]) as determined by interpolation on the regression line. These CD50 values were used as an expression of cytotoxicity potentials of the substance and were used for comparison accompanied with the corresponding correlation coefficient values (r2). The effect of irrigation solution was evaluated by comparing the mean optical density value of treated cells to the mean OD of untreated control wells. To statistically analyze the cytotoxicity of triphala and NaOCl, the Mann–Whitney test was applied.


   Results Top


An ideal root canal agent should efficiently remove the smear layer adhered to the radicular dentinal structure to guarantee a better bond of root canal sealer and the obturating material. Hence, to understand the cleansing efficacy, the smear layer removal at each segment of the root was carefully evaluated and compared with other segments.

Cytotoxicity study using L929 mouse fibroblast cells when exposed to NaOCl at all concentrations (5, 10, 20, 40, and 80) showed a significant decrease in cell viability to 8.46, 8.24, 7.89, 7.37, and 6.70 [Graph 3] [Table 6] as compared to triphala (94.90, 83.61, 78.24, 73.21, and 62.82). This indicates that sodium hypochlorite showed a significant cytotoxic effect against the L929 cell lines with the IC50 concentration at 1.8% after the treatment of 24 h of incubation at 37°C. The results suggest that triphala does not possess toxic potential properties against the normal healthy fibroblast cells.
Table 3: Comparison of smear layer removal at apical third using triphala and sodium hypochlorite

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Table 4: Comparison of smear layer removal at middle third using triphala and sodium hypochlorite

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Table 5: Comparison of smear layer removal at coronal third using triphala and sodium hypochlorite

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Table 6: Viability percentage of L929 mouse fibroblasts after 24 h treatment of test (triphala) and control (sodium hypochlorite) reagents

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Triphala was as effective as sodium hypochlorite in smear layer removal in the coronal and middle third of the root [Graph 2] [Table 4] and [Table 5], but sodium hypochlorite showed better smear layer removal in the apical third (P < 0.05) [Graph 1] [Table 3].


   Discussion Top


The integral part of irrigation entails the proper reagents and instrumentation in facilitating the entire removal of pulp tissue and/or microorganisms. The accessory features in the root canal, such as fins, cul-de-sacs, and intercanal communications, are easily colonized by microorganisms following tooth infection.[6] These bacterial clusters were termed “biofilm” by Costerton et al. in 1978.[7] In reviewing the microbial efficacy of sodium hypochlorite, Estrela et al. 2002[8] suggested that it was similar to calcium hydroxide because of its high pH, which interferes in the integrity of the bacterial cytoplasmic membrane, the cellular metabolism, and the degradation of phospholipids present in the root canal. A comprehensive literature review by Moriam et al.(2012)[9] observed that 5.25% sodium hypochlorite was more effective in dissolving organic tissue in a short time, had a more alkaline pH, and had a greater antibacterial action. Hence, we opted for the use of 5.25% sodium hypochlorite in our study.

The usage of herbal therapies has expanded significantly in many disciplines including dentistry. Triphala is a novel drug gifted by Ayurveda with an array of therapeutic activities and the potential to treat a variety of human ills with minimal or no side effects. This may be ascribed to its formulation, which contains three different medicinal plants in equal proportions: Terminalia bellirica, T. chebula, and E. officinalis. The strong antioxidant activity of triphala may be partly responsible for many of the biological properties. T. bellirica was the most active antioxidant, followed by E. officinalis and T. Chebula.[10] Variability in the herbal source, processing, bioavailability, digestion, and absorption of herbal components are the main factors that cause variability in their efficacy, besides the inherent variation in the gut microbiota that acts on the ingestion and transformation of components of herbal remedies into compounds with increased bioabsorption and bioactivity.[11] This variation has further raised questions, and hence, commercially available high-quality raw fruits were used for the preparation of Triphala.

Triphala can be prepared in two proportions:

  1. Equal proportions of T. bellirica, T. chebula, and E. officinalis (1:1:1 ratio)
  2. One proportion of T. bellirica, two proportions of T. chebula, and four proportions of E. officinalis 1:2:4 ratio).[12]


Raw fruits were dried and powdered separately and prepared into a solution by dissolving 60 mg/ml in 10% DMSO (HiMedia, Mumbai) similar to the study by Prabhakar et al., 2010.[13] Straight single-rooted mandibular premolar teeth were selected with root length of approximately 20–22 mm and curvature <5° according to Schneider to maintain standardization and avoid anatomic variation which was confirmed using radiograph.[14] A principal factor affecting the prognosis of root canal therapy is the failure to obtain a hermetic three-dimensional seal of the root canal space. Shahravan et al. in 2007 in their systematic review and meta-analysis observed that smear layer removal significantly improves the apical and coronal seal of the obturated root canal space and is independent of the type of obturation, site of leakage, the sealer type, the type of dye used for testing, and the duration of the test.[15] Although conflicting studies exist in deciding the inclusion or elimination of smear layer, Alamoudi, 2019,[16] demonstrated the importance of removing the smear layer to enhance the adaptation of filling materials and proper disinfection of the canal system.

According to Khademi et al.,[17] the minimum instrumentation size needed for penetration of irrigants to the apical third of the root canal is a #30 file. Therefore, a 0.30-mm apical size was chosen for the study. We used 27G needle tips which have a small bore size and a side venting, which can penetrate deeply into the apical third, increase adherence of irrigant to the canal walls, prevent the forceful passage of irrigants through the apical foramen, and increase the efficiency of the irrigant in smear layer removal. In this study, the SEM determined the effectiveness of various irrigants to remove the smear layer and examined the morphologic details of prepared root canals.

The cytotoxicity analysis with Alamar blue assay showed no cytotoxic properties against the L929 mouse fibroblast cells with triphala, whereas sodium hypochlorite showed a significant cytotoxic effect against the L929 cell lines after 24 h of incubation at 37°C.

Numerous studies have shown a higher cytotoxicity and caustic effects of 5.25% sodium hypochlorite compared to its 0.5 and 1% concentrations on healthy tissues.[18] This is in agreement with the earlier studies on the toxicity of sodium hypochlorite.[18],[19],[20] The sodium hypochlorite toxicity was attributed to its high pH, which interferes with the cytoplasmic membrane integrity.[8] According to Alamar blue assay findings in the present study, the cell viability decreased significantly when the cells were exposed to a higher concentration of sodium hypochlorite at 24 h. In recent years, there has been an increased tendency to use plant-derived alternative irrigation solutions with pharmaceutical properties. However, a lacuna exists in the literature studying the toxic nature of triphala, and ours is the pioneer study in this field.

In our study, the coronal third presented the least amount of smear with 100% removal in the sodium hypochlorite group and 90% in the triphala group, followed by the middle third with 100% removal in the sodium hypochlorite group and 60% in the triphala group and the most amount of smear in the apical third of the canals with a 60% removal in the sodium hypochlorite group and 10% removal in the triphala group. This is in agreement with the study done by Susan et al. in 2019[21] who compared smear layer removal with different solutions of triphala when used in specific irrigant protocols in curved canals. In another study, Sowjanyaa et al., 2017,[22] showed maximum sealer penetration depth in the coronal and middle thirds than in the apical thirds of root canals studied by confocal laser microscopy, and better removal of the smear layer was seen in the coronal thirds than in the apical thirds of root canals, as found in our study. There are more dentinal tubules with large diameters in the coronal area that allows a better flow of a higher volume of irrigants, further improving the efficiency of smear layer removal.

In contradiction, the study by Prabhakar et al., 2013,[23] and Rathakrishnan, 2015,[24] found moderate to heavy smear layer in specimens irrigated with 5% sodium hypochlorite, especially in the apical third. They also found moderate to thin smear layer in the triphala group in the middle and coronal sections, and more smear layer in the apical third, which is in agreement with our study.

The higher penetration depth of sodium hypochlorite may be the reason for better smear layer removal in the apical, middle, and coronal third of the root compared to the triphala group. Triphala solution was found to be more viscous, which in turn might have resulted in the collection of the solution along with debris in the apical third, as our specimen was the closed-end system. In a similar study by Bhargava et al., 2015,[25] herbal irrigants, amla, and triphala showed promising results in smear layer cleansing, but maximum efficacy was shown by EDTA gel. Other important properties for better smear layer removal and more sealer penetration depth into the dentinal tubules are the surface tension and the pH of the final irrigating solution. The lower the surface tension values of the irrigant, the more the sealer penetration depth into the narrow canals as it improves dentin wetting ability. This low surface tension value is the probable reason for the better cleansing and smear layer removal in triphala (the surface tension value of citric acid is 68.34 dyne/cm).


   Conclusion Top


Within the limitations and the experimental conditions of this study, the following conclusions can be made:

  • Triphala does not have toxic potential properties against the normal healthy fibroblast cells, whereas NaOCl exhibited cytotoxic properties and should be used with caution
  • Triphala was as effective as sodium hypochlorite in smear layer removal properties in the coronal and middle third of the root, but there was better smear layer removal property in the apical third by sodium hypochlorite.


Based on our result, triphala can be advocated as an efficient irrigating agent and a viable alternative to sodium hypochlorite. Synthetic chemical substances used as irrigants in endodontics help in disinfection of the canal system but possess undesirable properties such as toxicity, allergic potential, unacceptable taste, and cost factor. There is a major drift in the trend toward the use of native herbal medicines with the right pharmacological properties due to less toxicity and cost-effectiveness.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Kuruvilla A, Jaganath BM, Krishnegowda SC. A comparative evaluation of smear layer removal by using EDTA, etidronic acid, and maleic acid as root canal irrigants-¯: An in vitro scanning electron microscopic study. J Conserv Dent 2015;18:247-51.  Back to cited text no. 1
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Prabhakar J, Balagopal S, Priya MS, Selvi S, Senthilkumar M. Evaluation of antimicrobial efficacy of Triphala (an Indian Ayurvedic herbal formulation ) and 0.2% chlorhexidine against Streptococcus mutans biofilm formed on tooth substrate: An in vitro study. Indian J Dent Res 2014;25:0-4.  Back to cited text no. 4
    
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Marion JJ, Manhaes F. Efficiency of different concentrations of sodium hypochlorite during endodontic treatment. Literature review. Dent Press Endod 2012;2:32-7.  Back to cited text no. 9
    
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Prakash S, Shelke AU. Role of Triphala in dentistry. J Indian Soc Periodontol 2014;18(2):2–5.  Back to cited text no. 10
    
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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.  Back to cited text no. 13
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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