A Comparative Evaluation of Antimicrobial Efficacy of Triphala and Calcium Hydroxide as Intracanal Medicament: An In Vitro Study.

Background: The goal for placement of intracanal medicament during pulpal therapy procedures is to eradicate the bacteria and the exotoxins from the infected root canal, thereby preventing and controlling pulpal and periradicular infections. Chemo-mechanical preparation is considered an essential step in root canal disinfection; but to accomplish the total elimination of bacteria in highly complex root canal anatomy is difficult. Intracanal medicaments help eliminate bacteria by remaining in the root canal, and calcium hydroxide has been recommended as one of the most effective antimicrobial dressings during endodontic therapy. The herbal agents offer an alternative to synthetic compounds. They have been considered either non-toxic or less toxic. The rapid increase in antibiotic-resistant strains and side effects caused by synthetic drugs have prompted researchers to look for herbal alternatives. Aim and Objective: The in vitro study aims to evaluate and compare the antibacterial activity of triphala and calcium hydroxide against Enterococcus faecalis as an intracanal medicament. Materials and
Methods: The antimicrobial efficacy of triphala and calcium hydroxide was evaluated against E. faecalis cultured and incubated for various time frames in 42 intact, caries-free, human, single-rooted teeth with straight roots and mature apices. The comparison of the number of counted colonies after each incubation time and based on the type of drug used inside the canal is evaluated using non-parametric Kruskal-Wallis test and Mann-Whitney U test, respectively. Statistical analysis was done using SPSS version 14.0.
Results: Efficacy of calcium hydroxide against Enterococcus faecalis was best in the first 72-hr time duration compared with triphala. Triphala showed significant antimicrobial activity against Enterococcus faecalis that was closely parallel to the calcium hydroxide group in one week. Calcium hydroxide and triphala had complete eradication of Enterococcus faecalis in a given one-month time duration. Copyright:

INTRODUCTION

The goal for placement of intracanal medicament in pulpal treatment procedures is to eliminate the bacteria and the exotoxins produced from the infected root canal, which is directed towards controlling pulpal and periradicular infections. The endodontic failure is a common problem in dentistry. The success of endodontic and re-endodontic treatments depends on many factors. These include periodontal disease, root fractures, residual necrotic pulp tissue, presence of periradicular infection, broken instruments, mechanical perforations, root canal underfillings, root canal overfillings, missed canals or unfilled canals.[1] The major factors responsible for endodontic treatment failure are the persistent microbial infection in the root canal system and peri-radicular tissue.[23] Some specific endodontic pathogens were sentenced to be the primary cause for endodontic failure. These are polymicrobial in nature and dominated by gram-negative anaerobic rods. Enterococcus faecalis (E. faecalis), a facultative anaerobic gram-positive coccus, is the most common species cultured from non-healing endodontic cases and is found in 4%–40% of primary care endodontic infections.[4] This microorganism can even survive in an environment with scantily available nutrients and minimal commensality with other bacteria.[5] E. faecalis invades the dentinal tubules and may survive chemo-mechanical instrumentation and intracanal medication; it can colonize the tubules and reinfect the obturated root canal.[1] Total cleaning of the root canal system using mechanical instrumentation is ineffective due to highly complex root canal anatomy.[5] Complete chemomechanical preparation is an inevitable step in root canal disinfection. However, total elimination of microorganism is difficult to accomplish. By remaining in the root canal, intracanal medicaments may help to eliminate surviving bacteria.[6]

Since the introduction of calcium hydroxide (Hermann 1920), it has been used in endodontic treatment procedures. It is strongly alkaline and has a pH of approximately 12.5. It has numerous properties such as antimicrobial activity, tissue-dissolving ability, tooth resorption inhibition, and repair induction by hard tissue formation. Because of such effects, calcium hydroxide has been recommended as one of the most effective antimicrobial dressings during endodontic therapy.[7] It has been used mainly as a root canal medicament, with safe and satisfactory clinical outcomes,[8] and has been reported as the best interappointment medication available against residual microbial flora.[9]

The constant increase in antibiotic-resistant strains of microorganisms and the deleterious effects caused by synthetic drugs has made researchers look for herbal alternatives that have been considered non-toxic or less toxic. Triphala is an Indian ayurvedic herbal formulation consisting of dried and powdered fruits.[4] Triphala is a combination of powders of three dried fruits, Terminalia chebula (haritaki/chebulic myrobalan), Terminalia bellirica (vibhitaki/bibhitaki/beleric myrobalan), and Emblica officinalis (amla/gooseberry) in equal proportions. Being primarily used in ayurveda, triphala has been proven to be a potent antimicrobial agent with much-desired antibacterial, antiviral, and antifungal actions. It also possesses antihistamine, anti-inflammatory, antioxidant, antitumor, blood pressure-lowering, cholesterol-lowering, digestive, diuretic, and laxative properties.[4] To overcome the harmful effects of drug resistance and side effects associated with the commonly used antibiotics, an alternative treatment method to cure such infections by the use of traditional medicinal herbs like triphala, which is a potent antibacterial agent and is clinically safer, was developed.[45]

This in vitro study aimed to evaluate and compare the antibacterial activity of Triphala and calcium hydroxide against Enterococcus faecalis as an intracanal medicament. The purpose of this in vitro study was to identify the following:

(i) The antibacterial activity of calcium hydroxide against Enterococcus faecalis as an intracanal medicament.

(ii) The antibacterial activity of triphala against Enterococcus faecalis as an intracanal medicament.

(iii) Compare the antibacterial activity of calcium hydroxide and triphala against Enterococcus faecalis.

MATERIALS AND METHODS

The present in vitro study was carried out in the Department of Pedodontics and Preventive Dentistry, in association with the Department of Microbiology after obtaining ethical clearance from the institutional research committee to evaluate and compare the antibacterial activity of triphala and calcium hydroxide against Enterococcus faecalis as an intracanal medicament. In this experimental in vitro study, 42 intact, caries-free, human, single-rooted teeth with straight roots and mature apices that had been recently extracted in the process of orthodontic treatment or because of periodontal problems were selected.

The 42 samples were then divided into four experimental groups [Figure 1].

Figure 1

Distribution of samples based on four experimental groups

The teeth samples were debrided and cleaned. All hard and soft tissues attached to the teeth were carefully removed using scaling curettes. The specimens were sectioned below the cementoenamel junction to obtain a standard 8–12 mm tooth length. Endodontic file sizes 10 and 15 were used to ensure that the roots had only one canal and the canal was patent. Each tooth was radiographed to confirm the presence of a single patent canal. For topical disinfection, specimens were immersed in 5.25% sodium hypochlorite for 30 minutes. The teeth were then stored in sterile 0.9% physiological saline (0.9% sodium chloride solution). All canals were then primarily prepared using similar mechanical and chemical procedures. To do so, first, a coronal pre-flaring was done using Gates Glidden drills numbers 2 and 3 in order with no lateral pressure and passive up and down motion. The entire working length of the canals was then prepared up to size 35 with hand instrumentation. Recapitulation was performed with a hand file size of 15 between every two files. Irrigation was done thoroughly with 5.25% sodium hypochlorite. After canal preparation, all the specimens' apical foramina were sealed with cyanoacrylate glue to prevent bacterial microleakage. To eliminate the smear layer, samples were placed in an ultrasonic bath. Teeth were first placed in 17% EDTA (ethylenediaminetetraacetic acid) at a pH of 7.8 for four minutes and then another four minutes in 5.25% sodium hypochlorite (NaOCl) and were later irrigated with sterile distilled water for 10 minutes. The teeth were separately placed in 2-ml microtubes containing 0.5 ml brain heart infusion broth (BHI) and then were sterilized in an autoclave for 20 minutes at 121°C and 15 psi pressure. Samples in aseptic condition were separately incubated in microtubes containing BHI for 24 hours in aerobic conditions at 37°C to reassure sterilization. After that, three samples were randomly selected, and a microbial culture was prepared from their BHI. No bacterial growth was considered as the proof of correct sterilization (negative control- 3 specimens). After confirmation of sterilization, all experiment phases were performed in aseptic conditions using sterile gloves and instruments.

To induce a controlled and standard infection in all samples, a resistant bacterium named Enterococcus faecalis that is mainly responsible for the failure of endodontic treatments was employed. This gram-positive facultative anaerobic microorganism was procured from Microbiology with the ID number American Type Culture Collection [ATCC] 29212.

Enterococcus faecalis was cultured in BHI medium in a CO2 incubator at 37°C for 48 hours. A pure suspension of E. faecalis bacteria with a concentration of 1.5 × 108 CFU/ml was prepared using spectrophotometry with turbidity adjusted to be equivalent to a 0.5 McFarland BaSO4 standard close to the flame. The standardized bacterial suspension was injected into the canals in equal portions (0.5 CC). Then, all microtubes were stored in an incubator at 37°C in aerobic conditions for 48 hours.

Triphala powder (IMPCOPS Ltd, Chennai, India) was made into a solution by dissolving 60 mg of powder in 1 ml of 10% dimethyl sulfoxide (DMSO). The calcium hydroxide paste used in the study was RC Cal (Prime dental products PVT. LTD). After the incubation period, the teeth specimens were divided into six groups of six teeth each and one positive control group of three teeth. Samples were extracted from the microtubes using sterile gloves, and instruments and were fixed using a hemostat. Understudy materials (triphala and calcium hydroxide) were then inserted into the canals according to the following grouping:

Group 1: Addition of triphala and storing the specimens in the CO2 incubator for 72 hours

Group 2: Addition of calcium hydroxide and storing the specimens in the CO2 incubator for 72 hours

Group 3: Addition of triphala and storing the specimens in the CO2 incubator for one week

Group 4: Addition of calcium hydroxide and storing the specimens in the CO2 incubator for one week

Group 5: Addition of triphala and storing the specimens in the CO2 incubator for one month

Group 6: Addition of calcium hydroxide and storing the specimens in the CO2 incubator for one month.

Three specimens numbered 1, 2, 3 in the positive control group were stored in the CO2 incubator without any additives for 72 hours, one week, and one month, respectively for evaluating the duration of action of the medicament to eliminate the microorganisms from the root canal.

After completing the required incubation period, a 40 size Hedstrom (H) file was used to collect the debris from the root canal to observe and evaluate the microorganisms that had penetrated the dentin. Debris on the H file was quickly transferred into the microtubes containing sterile brain heart infusion (BHI) broth. Microtubes were stored for 48 hours in a CO2 incubator, after which samples were removed and cultured in a bile esculin agar medium. Colours were counted after 72 hours, Enterococcus faecalis was observed under the light microscope, and the type of bacteria was confirmed. The presence of other bacteria (positive control three specimens) was also evaluated to detect any possible contamination during the process. The comparison of a number of counted colonies after each incubation time and based on the type of drug used inside the canal was evaluated using nonparametric Kruskal–Wallis test, and Mann–Whitney U test, respectively, and statistical analysis was done using SPSS version 14.0.

RESULT

Statistically significant difference in the total number of CFUs/ml was observed using triphala as an intracanal medicament in different storage periods in the incubator (P < 0.001). In comparison, triphala treated group incubated for 72 hours showed a mean value of 41,136.17 CFUs and a standard deviation of 2,604.65. One-week incubated triphala group showed a mean value of 687.2 CFUs and a standard deviation of 114.5, while one-month incubated triphala group showed 0 value [Table 1]. In a comparison of a total number of CFUs/ml in calcium hydroxide–treated groups in the different incubation periods, 72-hour calcium hydroxide group showed a mean value of 29,784.6 and a standard deviation of 4,548.63, while one-week calcium hydroxide group showed a mean value of 517.5 and a standard deviation of 123.7, and the calcium hydroxide group incubated for one month showed a 0 value. The result infers a statistically significant difference in the total number of CFUs/ml when using calcium hydroxide in different periods of storage in the incubator with a P value of <0.001 which is highly significant [Table 2].

Table 1

Comparison of total number of CFUs/ml in triphala-treated groups in different time period of storage in the incubator

TRIPHALA GROUP	MEAN	S.D.	MINI	MAX	P
72 Hours (Group 1)	41,136.17	2,604.65	0	100,000	0.001 (S)
1 Week (Group 3)	687.2	114.5	0	1,000
1 Month (Group 5)	0	0	0

Table 2

Comparison of total number of CFUs/ml in calcium hydroxide group in different time period of storage in the incubator using non-parametric Kruskal–Wallis test

CALCIUM HYDROXIDE GROUP	MEAN	S.D.	MINI	MAX	P
72 Hours (Group 2)	29,784.60	4,548.63	0	100,000	0.001 (S)
1 Week (Group 4)	517.5	123.7	0	1,000
1 Month (Group 6)	0	0	0

Comparing the CFU/ml for triphala (group 1) and calcium hydroxide (group 2) in 72 hours of storage in the incubator, group 1 showed a mean value of 41,136.17 and a standard deviation of 2,604.65 while group 2 showed a mean value of 29,784.5 and a standard deviation of 4,548.63. The result showed a statistically significant difference (P < 0.001). [Table 3]

Table 3

Comparison of total number of CFUs/ml when using triphala (group 1) and calcium hydroxide (group 2) in 72 hours of storage in the incubator

TIME DURATION	DRUG	MEAN	S.D.	MIN	MAX	P
72 hrs	Triphala (Group 1)	41,136.17	2,604.65	0	100,000	0.001 Significant
72 hrs	Calcium hydroxide (Group 2)	29,784.50	4,548.63	0	100,000

The number of CFU/ml when using triphala (Group 3) in one week of storage in the incubator showed a mean value of 687.2 and a standard deviation of 114.5, and the calcium hydroxide group showed a mean value of 517.5 and a standard deviation of 123.7. The result shows no statistical significance in the total number of CFUs/ml when using calcium hydroxide and triphala in one week of storage in the incubator [Table 4]. The number of CFU/ml when using triphala and calcium hydroxide in one one month of storage in the incubator showed 0 value.

Table 4

Number of CFU/ml when using triphala (group 3) and calcium hydroxide (group 4) and in one week of storage in the incubator

TIME DURATION	DRUG	MEAN	S.D.	MINIMUM	MAXIMUM	P
1 Week	Triphala	687.2	114.5	0	1000	Not significant 0.12
1 Week	Calcium hydroxide	517.5	281.6	0	1000

DISCUSSION

An endodontic procedure usually focuses on removing the diseased tissue, eliminating bacteria present in the canals and in dentinal tubules to prevent post-endodontic recontamination. Clinically, biomechanical preparation assist with antimicrobial irrigant alone is inadequate for the complete elimination of bacteria and their byproducts, as it is present in great variety and in high numbers, and also due to anatomical variations of the root canal. In addition, these microorganisms can organize themselves in a biofilm, and mostly because of this, conventional endodontic therapy tends to fail in a higher percentage.[10] Enterococcus faecalis (E. faecalis) was the most common species that was recovered from root filled teeth with chronic apical periodontitis.[9] This shows increased resistance by formation of biofilm. E. faecalis can adhere to the root canal walls, accumulate, and form communities organized in biofilm, which helps it to resist destruction by enabling the bacteria to become a thousand times more resistant to phagocytosis, antibodies, and antimicrobials than non-biofilm producing organisms.[11] Thus in the present study, E. faecalis (ATCC 29212) was chosen as a test organism because it was a facultative organism that was non-fastidious, easy-to-grow, and efficiently and rapidly colonizes tubules[12]

According to a 2013 study by Podbielski et al.,[13] intracanal medication between appointments has been unequivocally shown to contribute to a favorable outcome in treating endodontic infections. In the present study, the efficacy of two different medications against E. faecalis was evaluated.

Nobuyuki Kawashima et al.[14] and Ramille Arujo Lima et al.[15] stated that calcium hydroxide has been used worldwide as intracanal medication as it has great value in endodontics; also it is used for several clinical conditions. The bactericidal effects are caused by its high pH (12.5–12.8), which prevents the growth and survival of bacteria and also denatures and detoxifies bacterial products, such as lipopolysaccharide. Calcium hydroxide is reported to be effective in controlling the inflammatory exudates from the periapical region and the mechanism behind this action may be related to its antimicrobial properties, contraction of capillaries, and formation of an apical plug.[12] Thus, in the present study, the standard drug calcium hydroxide was used as one of the intracanal medicaments for comparing its efficacy against E. faecalis.

Triphala is highly valued in ayurveda and other traditional systems of Indian medicine and is proven to be safe for use, and does not have any side effects. The dried and powdered mixture of triphala is constituted by amla, vibhitakai and haritakai in a 1:1:1 ratio. It has known beneficial physiologic effects apart from anti-diabetic, anti-inflammatory, antioxidant, and anti-bacterial actions. Triphala contains many potent antimicrobial chemical compounds like phenol, quinoline and tannins and has added advantage over the traditional root canal medicaments.[716] Triphala is an excellent chelating agent, and it contains fruits that are rich in citric acid that may aid in the removal of the smear layer. The significant advantages of using herbal alternatives were easy availability, cost-effectiveness, increased shelf life, low toxicity, and lack of microbial resistance reported so far.[9] Thus in the present study, the efficacy of calcium hydroxide (the standard intracanal medicament) and triphala (a herbal alternative) against E. faecalis was evaluated and compared.

In the present study, all three samples from the positive control group demonstrated bacterial growth. However, none of the samples from the negative control groups manifested bacterial growth. Statistical comparison of the number of counted colonies when using triphala for 72 hours (mean 41,136.17 CFUs), one week (mean 687.2 CFUs) and one month (0) showed significant difference (P < 0.001) [Table 1] Jyotsna Srinagesh et al.[7] found that there was a significant reduction in E. faecalis CFUs/ml in the triphala-treated group supporting the present study. Similarly, in a study done by Madhu Pujar et al.,[5] they found that triphala had showed significantly better antibacterial activity against 2-week biofilm of E. faecalis. Maximum activity of triphala extracts for wound healing were found effective against Salmonella, Streptococci, Eschericia coli and Bacillus, whereas, least active against Pseudomonas and Staphylococcus at 250 mg/ml concentration. Similarly, in a study done by Prabhakar J et al.[4] Triphala showed statistically significant antibacterial activity as an irrigating solution against three-week biofilm and showed complete inhibition of bacterial growth, thus supporting the result of the present study.

In the present study, statistical comparison of number of counted colonies when using calcium hydroxide paste for 72 hours (mean 29,784.6 CFUs) (Group 2), one week (mean 517.5 CFUs) (Group 4), and one month (0) (Group 6) showed significant difference (P < 0.001) [Table 2]. Richard E Lynne et al.[17] found that 10% calcium hydroxide had a significant antimicrobial effect against E. faecalis in dentinal tubules, thus supporting the result of the present study against E. faecalis in one-month incubation period.[17] However, this is in contrast to the study conducted by Brenda Paula Figueiredo de Almeida Gomes et al.[18] who found that E. faecalis can survive in the dentinal tubules despite long periods of calcium hydroxide therapy as it has the ability to survive in root canal as a single organism without support from other bacterial species.

In the present study, when comparing triphala (Group 1) and calcium hydroxide (Group 2) in the 72-hour incubation time period, the number of counted E. faecalis colonies was 44,644 ± 37,514 CFUs and (36,096 ± 26,130 CFUs) respectively with statistically significant difference (P < 0.001). Table 3 shows that antibacterial efficacy of Triphala was lower than that of calcium hydroxide in the first 72 hours of incubation period. The result of the present study was supported by Brandle N et al.[19] who stated that the effective treatment time to kill E. faecalis was within 100 minutes using calcium hydroxide and, they recommended that proper endodontic treatment with calcium hydroxide could effectively detoxify E. faecalis and its lipoteichoic acid. And in the present study when comparing Triphala (Group 3) and Calcium hydroxide (Group 4) in one-week incubation time, the number of counted E. faecalis colonies was 124.5 ± 849.3 CFUs and 582.1 ± 891.2, respectively, with statistically non-significant difference (P > 0.12). Thus, by the present study result it was found that even though calcium hydroxide was found highly effective in eliminating E. faecalis due to its alkalinity in the first 72 hours when compared with triphala (group 1), it takes one week to show its antimicrobial property supported by the study done by Sjogren et al.[20] in 1991, who stated that calcium hydroxide must be applied for seven days in the canal for its antimicrobial effect.

There was no E. faecalis colonies counted in the one-month incubation period for both triphala and calcium hydroxide, thus showing that antibacterial efficacy of triphala and calcium hydroxide was effective in eliminating E. faecalis in one month of storage in the incubator. Hence, triphala can be recommended as an herbal alternative for calcium hydroxide to serve as an intracanal medicament due to its antimicrobial effective against E faecalis.

SUMMARY AND CONCLUSION

Based on the data obtained from the present in vitro study it was found that

Efficacy of calcium hydroxide against Enterococcus faecalis was best in first 72 hrs time duration when compared with triphala.

Triphala exhibited significant antimicrobial activity against Enterococcus faecalis that was closely parallel to calcium hydroxide group in one week.

Calcium hydroxide and Ttriphala had complete eradication of Enterococcus faecalis in given one-month time duration.

However, the selection of time interval for recording the colony forming units, which were counted only at a time interval of 72 hours, one week and one month was a limitation of the present study. The specified time of eradication of E. faecalis should have been recorded in two weeks' and three weeks' time duration to get a conclusive data.

Placement of intracanal medicament is regarded as the fundamental step in endodontic treatment procedure. These medicaments play a fundamental role in eliminating endotoxins produced by microbial pathogens Within the limitations of this present in vitro study, triphala and calcium hydroxide exhibited effective antibacterial sensitivity against E. faecalis, and it was concluded that triphala could be used as a safer and effective intracanal medicament with additive or synergistic positive effect.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.