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Year : 2019  |  Volume : 37  |  Issue : 3  |  Page : 275-281

Indigenous irrigants as potent antimicrobials in endodontic treatment: An in vitro study

1 Department of Pedodontics and Preventive Dentistry, Sibar Institute of Dental Sciences, Guntur, Andhra Pradesh, India
2 Department of Pedodontics and Preventive Dentistry, Drs. Sudha and Nageshwar Rao Siddhartha Institute of Dental Sciences, Krishna, Andhra Pradesh, India
3 Department of Pharmacy, Birla Institute of Technology and Science Pilani, Hyderabad, Telangana, India

Date of Web Publication30-Sep-2019

Correspondence Address:
Dr. Parvathi Satti
Department of Pedodontics and Preventive Dentistry, Sibar Institute of Dental Sciences, Guntur - 522 509, Andhra Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JISPPD.JISPPD_112_19

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Background: The success of an endodontic treatment depends on effective disinfection and complete sealing of root canal. The irrigants that are currently used in the field of endodontics have certain limitations, so the quest for an ideal root canal irrigant continues. Nowadays, the use of herbal extracts such as Triphala and liquorice are used for their potent antimicrobial activity and less side effects. Aim: This study aims (1) to evaluate the antimicrobial efficacy of Triphala and liquorice against Enterococcus faecalis.(2) To determine any cytotoxic effect on isolated human periodontal ligament (PDL) fibroblasts. Materials and Methods: The antimicrobial efficacy of Triphala and liquorice extracts was analyzed at different concentrations (12.5, 25, 50, 75, and 100 mg/ml) using the well-diffusion method. Three percent sodium hypochlorite (NaOCl) and distilled water were taken as positive and negative controls. Minimum inhibitory concentration of the active extract was determined by the broth dilution assay. Human PDL fibroblast tissue culture was used to assess the cytotoxicity of the preparations. The data thus obtained were subjected to statistical analysis. Results: The results showed that the mean antimicrobial efficacy of Triphala and liquorice at 50 mg/ml is 20.33 and 9.33, respectively, which are statistically significant (P < 0.0001) as compared with a concentration 12.5 and 25 mg/ml. 50 mg/ml showed significant results (P < 0.001) on comparing with hypochlorite. Triphala and liquorice showed no cytotoxic effect as compared to NaOCl on human PDL fibroblasts. Conclusion: Among the three tested materials Triphala showed the highest antimicrobial efficacy followed by NaOCl and liquorice.

Keywords: Enterococcus faecalis, liquorice extract, MTT reagent, periodontal ligament fibroblasts, sodium hypochlorite, Triphala extract

How to cite this article:
Satti P, Kakarla P, Jogendra Avula SS, Muppa R, Kiran Rompicharla SV, Biswas S. Indigenous irrigants as potent antimicrobials in endodontic treatment: An in vitro study. J Indian Soc Pedod Prev Dent 2019;37:275-81

How to cite this URL:
Satti P, Kakarla P, Jogendra Avula SS, Muppa R, Kiran Rompicharla SV, Biswas S. Indigenous irrigants as potent antimicrobials in endodontic treatment: An in vitro study. J Indian Soc Pedod Prev Dent [serial online] 2019 [cited 2022 Oct 1];37:275-81. Available from: http://www.jisppd.com/text.asp?2019/37/3/275/268173

   Introduction Top

Endodontic infections are polymicrobial in nature dominated by obligate anaerobic bacteria. Enterococcus faecalis is such a persistent organism that plays a major role in the etiology of periradicular lesions after root canal treatment. It can even survive in restricted conditions due to biofilm formation and physicochemical properties of the organism that helps it to modify the prevailing environmental and nutritional conditions. Therefore, the choice of instrumentation and irrigating solutions that permit bacterial neutralization and toxin inactivation without negative interference with the healing process is fundamental for the success of endodontic treatment.[1]

Thus, irrigation is considered imperative as it assists in cleaning the root canal system areas that are inaccessible by instruments, which in turn reduces the number of bacteria and control the periapical disease.[2],[3]

Routinely used endodontic irrigants include sodium hypochlorite (NaOCl), ethylenediaminetetraacetic acid (EDTA), and chlorhexidine (CHX). Even though NaOCl is more commonly used, it has certain detrimental effects such as unpleasant taste, toxicity, and its inability to remove the smear layer by itself, as it dissolves only the organic material. EDTA also effectively demineralizes the smear layer but has to be used along with NaOCl to remove the organic part and does not have disinfective ability. CHX is also frequently used to irrigate the canals due to its wide spectrum antimicrobial activity, biocompatibility, and ability to disinfect the infected root canals, but it does not have a tissue dissolving capability.[2] Due to the constant increase in antibiotic-resistant strains and the side effects caused by irrigants, it has promoted the researchers to explore for herbal alternatives which gained greater attention owing to their beneficial effects.[1]

Ayurveda is a traditional, natural system of medicine which uses herbal remedies. It is gaining popularity as these herbs used are free of side effects and also have antimicrobial activity and medicinal value.[4] One such herbs which have numerous medicinal values are Triphala and liquorice.[5]

Triphala is comprised of fruits of three medicinal trees, Amalaki-Emblica officinalis, Vibhitaki-Terminalia bellirica, and Haritaki-Terminalia chebula. T. belerica was the most active antioxidant followed by E. officinalis and T. chebula. The major ingredients of T. belerica are ellagic and gallicacid and in T. chebula, gallic acid is the major ingredient. Tannic acid represents the major constituent of the ripe fruit of T. chebula, T. bellirica, and E. officinalis. The phenolic nature of these active ingredients may be responsible to scavenge the free radicals generated by the bacteria.[1] It possesses analgesic, antiarthritic, antiseptic, hypoglycemic, anti-aging, antiviral, anti-inflammatory, and antibacterial effects. Antibacterial activity of Triphala is complex yet it inhibits the cell division or damage to the cell walls of the bacterium. When used as a mouth rinse, Triphala showed no evidence of staining of teeth. All these properties along with biological activities make it a prospective Ayurvedic drug for the treatment of dental diseases.[6]

Glycyrrhiza glabra commonly known as liquorice (mulethi) is generally regarded as safe by the Food and Drug Administration when used as food flavoring and sweetening agent. Recent research suggests that the anti-adherence, antimicrobial, anti-inflammatory properties of liquorice and its various bioactive ingredients provided the potential benefits to treat oral diseases, including dental caries.[7],[8]

Thus, considering the beneficial effects of Triphala and liquorice, the present in vitro study was undertaken to evaluate the antimicrobial efficacy of Triphala and liquorice at various concentrations against E. faecalis in comparison to NaOCl and also assess the cytotoxicity against human periodontal ligament (PDL) fibroblasts.

   Materials and Methods Top

The in vitro microbiological study was conducted where Triphala and liquorice were selected as experimental groups at different concentrations 12.5, 25, 50, 75, and 100 mg/ml, NaOCl and distilled water as positive and negative controls, respectively, and their antimicrobial activity against E. faecalis was carried out using agar well-diffusion method.

Extract preparation

Triphala and liquorice extracts available in powder form were procured and mixed at different concentrations of 12.5, 25, 50, 75, and 100 mg with 10 ml of distilled water in Jiffy's centrifuge tubes. These five concentrations of extracts were later immersed in a thermostatic water bath at a temperature of 60°C for 20 min, then allowed to cool and centrifuged at 2500 rpm for 10 min. Thus, the obtained supernatants were used to analyze the antimicrobial efficacy.[9]

Agar diffusion test

A pure culture of E. faecalis was grown on Mueller-Hinton agar, which was inoculated into Mueller-Hinton broth (HiMedia, Mumbai, India), incubated at 37°C overnight and then streaked onto the agar plates. These extracts were then loaded into the wells (6-mm diameter) along with 3% NaOCl and distilled water at different concentrations. All the plates were then incubated in anaerobic jar at 37°C for 24 h, and the diameter of the growth inhibition zone was measured using inhibition zone reader template [Figure 1]a, [Figure 1]b,[Figure 1]c.[10]
Figure 1: (ac) The zone of inhibition with Triphala, liquorice, sodium hypochlorite, and distilled water after 24 h against E. faecalis

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Determination of minimal inhibitory concentration

Broth dilution method was adopted to determine minimum inhibitory concentration (MIC) of the active extract. The inoculum of the test bacterium was prepared by diluting the overnight culture of bacterium in Mueller-Hinton broth to a level of 1.5 × 107 CFU/ml. 2 ml of extracts diluted in two-fold concentration were added to a sterile glass tube containing 0.5 ml Mueller-Hinton broth. The tubes were then incubated at 37°C for 18–24 h. Due to the turbidity and dark color of the extracts, 0.1 ml of mixture in the tubes were spread onto the surface of Mueller-Hinton agar plates and then they were incubated at 37°C for 18–24 h.[10] Bacterial colonies were not detected for Triphala and liquorice extracts at 50, 75, 100 mg/ml. Thus, the lowest concentration, i.e., 50 mg/ml was taken as MIC, which was further used to test the cytotoxicity of the extracts on human PDL fibroblasts.

Culturing of periodontal ligament fibroblasts

The cytotoxic effect of the four groups against human PDL cells was analyzed using MTT ASSAY- 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. After obtaining the institutional ethical committee approval and informed patient consent, sound teeth extracted for orthodontic purpose were selected for the study with the exclusion of carious and periodontally compromised teeth. Human PDL cells were cultured using an explant technique.[11] The extracted teeth were placed in Hanks' balanced salt solution to maintain the viability of PDL cells. Teeth were rinsed in sterile phosphate-buffered saline (PBS), and PDL tissue was scraped from the middle-third of the roots. The explants were cultured on a 60 mm culture dishes and were grown in α Methyl Modified Eagles Medium (α MEM) supplemented with 10% fetal calf serum and antibiotics (streptomycin and amphotericin). Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air. After confluence (1 week), the cells were passaged using 0.125% trypsin ethylenediaminetetraacetic. Passages numbers 3–6 were used in this study.[12]

Cell culture and cytotoxicity testing (MTT assay)

The fibroblasts were plated separately using 96 well plates with the concentration of 1 × 104 cells/well in an α MEM with 10% fetal bovine serum, 50 μg/ml streptomycin and 3 μg/ml amphotericin in carbon dioxide incubator at 37°C with 5% CO2. The cells were later washed with 200 μL of PBS and then treated with various test concentrations in serum-free media and incubated for 24 h. The medium was aspirated from cells at the end of the treatment period. 0.5 mg/ml MTT prepared in PBS was added and incubated at 37°C for 4 h using CO2 incubator in a dark environment. After incubation period, the medium containing MTT was discarded from the cells and washed using 200 μl of PBS. Thus, the formed crystals were dissolved with 100 μl of dimethyl sulfoxide and mixed thoroughly. The viable cells with active metabolism convert MTT into a purple-colored formazan product with an absorbance maximum near 570 nm, and this color formation serves as a useful and convenient marker of the viable cells. The absorbance was measured at 570 nm using microplate reader.[13] Thus, the data obtained were subjected to statistical analysis using Kruskal–Wallis test, post hoc Mann–Whitney U-test, and independent samples t-test.

   Results Top

In the present study, the antimicrobial efficacy of herbal extracts against E. faecalis and their cytotoxic effect against human isolated PDL cells were evaluated. Triplicates were performed for each test solutions, and the averages of the three values were taken. P < 0.05 was considered as statistically significant.

The mean difference in the zones of inhibition on comparing Triphala and liquorice at different concentrations with 50 mg/ml was mentioned in [Table 1], respectively. This represents that Triphala and liquorice at 50 mg/ml concentration have higher antimicrobial efficacy when compared to 12.5 and 25 mg/ml concentrations of each extracts and have slightly lower antimicrobial efficacy when compared to 75 and 100 mg/ml concentration.
Table 1: Comparison of zone of inhibition at different concentrations

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Intergroup comparison values of Triphala with liquorice, hypochlorite, and distilled water were 11, 5.66, and 20.33, respectively, and the mean difference of zone of inhibition on comparing liquorice with hypochlorite and distilled water was 5.34 and 9.33, respectively [Table 2]. This illustrates that Triphala has superior antimicrobial efficacy than other experimental groups and liquorice have less antimicrobial efficacy when compared to hypochlorite and more when compared with distilled water.
Table 2: Comparison of antimicrobial efficacy between Triphala (50 mg/ml), liquorice (50 mg/ml), sodium hypochlorite, distilled water

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The mean difference of cell viability on comparing Triphala, liquorice and distilled water with hypochlorite was 89.67 and 90.53, respectively [Table 3] and [Table 4]. This shows that hypochlorite is highly toxic, and Triphala and liquorice are nontoxic to the PDL fibroblasts.
Table 3: Cytotoxicity at different concentrations on periodontal ligament fibroblasts

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Table 4: Comparing the cytotoxicity between Triphala (50 mg/ml), liquorice (50 mg/ml), sodium hypochlorite, distilled water

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   Discussion Top

NaOCl is used as the popular irrigant till date, due to its strong proteolytic potential as it results in sufficient tissue lysis. The use of higher concentrations increase its ability to dissolve the necrotic tissue and shorten the time needed for the inhibition of bacterial growth, but it causes damage to periapical tissues. The main disadvantages of NaOCl are high toxicity, corrosive to instruments, inability to remove smear layer and reduction in elastic modulus, and flexural strength of dentin.[14],[15],[16] As irrigation with NaOCl is still not predictably effective, researchers have focused on searching for an alternative solution.[12]

In endodontics, because of the cytotoxic reactions of the most of the commercial available irrigants used and their inability to eliminate bacteria from dentinal tubules, the trend of recent medicine attends to use biologic medication extracted from natural plants.[17] Medicinal plants such as Triphala and liquorice are such herbal extracts with more pronounced therapeutic values.[18]

E. faecalis, a saprophytic component of enteric flora is selected for the present study as it is the most common bacterium isolated from endodontic retreatment of apical periodontitis, either as a single organism or as a major component of the flora.[1],[19]E. faecalis has the capacity to produce biofilms, where they aggregate and coaggregate with other microorganisms and are embedded in the extracellular matrix.[19] Distel et al.[20] have revealed that E. faecalis can colonize medicated root canals with biofilm formation. That is the reason for selection of E. faecalis in studies concerning to the evaluation of endodontic irrigation solutions for cleaning the root canal system.

The antimicrobial efficacy of any natural or synthetic agent can be evaluated using broth dilution method, agar dilution method, disc diffusion method, cup plate method or agar well-diffusion method, and ditch-plate method. However, agar well-diffusion method was used in the present study as it depends on the diffusion of the tested material to such an extent that growth of the added microorganism is prevented entirely in a zone around the hole containing the test material. Similarly, Kriplani et al.[21] also employed agar well-diffusion method to evaluate the antimicrobial efficacy of different groups as it is more reliable, acceptable, and easy to perform.

In the present study, the mean zone of inhibition diameter using Triphala at a concentration of 50 mg/ml was 20.33 mm. Similarly, in the study conducted by Bag et al.,[22] hot aqueous extract of Triphala was found to be more potent against E. coli strains with an inhibitory zone diameter ranged from 20 to 21 mm. Thus, a hot aqueous extract of Triphala was used in the study as water is a high polarity solvent, and almost all compounds in the plant would be soluble in water. On the contrary, the mean zone of inhibition diameter using liquorice at 50 mg/ml concentration was 9.33 mm. In a study conducted by Badr et al.[18] Liquorice extract showed the largest zone of inhibition (3.97 ± 0.24) when compared to mixture of liquorice and calcium hydroxide and CaOH alone. Similarly, Chittrarasu et al.[23] determined that liquorice extract has higher activity than calcium hydroxide against enterococci and better activity on biofilms. The antimicrobial effect of liquorice extract against E. faecalis may be related to the glycyrrhizin.[24] The mode of action of antibacterial effects of saponins seems to involve membranolytic properties, rather than simply altering the surface tension of the extracellular medium, thus being influenced by microbial population density.[25],[26]

Minimal inhibitory concentration (MIC) of Triphala was found to be 50 mg/ml. However, this finding was found to be disagreement to a study conducted by Prabhakar et al.[27] where MIC of Triphala is 3.125 mg/ml. On the other hand, the MIC of liquorice was found to be 50 mg/ml in the present study. This finding was also found in disparity to a previous study conducted by Ajagannanavar et al.,[5] comparing the effect of aqueous and alcoholic liquorice root extract with CHX against Streptococcus mutans and Lactobacillus acidophilus where MIC of aqueous liquorice root extract for S. mutans was established at 25 mg/ml and L. acidophilus at 50 mg/ml and the MIC of alcoholic liquorice root extract for S. mutans was established at 12.5 mg/ml and L. acidophilus at 6.25 mg/ml. This difference in the results may be attributed to the method of aqueous extract preparation.

Triphala is found to have a superior antimicrobial efficacy that might be due to the presence of Tannic acid which is bacteriostatic and bactericidal. These findings are similar to the studies conducted by Shakouie et al.[28] revealing that Triphala exhibited better antibacterial activity than NaOCl as an irrigant solution against E. faecalis and Prabhakar et al.[29] who conducted a study to evaluate the antimicrobial efficacy of herbal alternatives (Triphala and green tea polyphenols), MTAD (Mixture of Tetracycline, Acid and Detergent) and 5% NaOCl against E. faecalis biofilm formed on the tooth substrate in which, Triphala achieved 100% killing of E. faecalis at 6 min. However, liquorice showed less antimicrobial efficacy than NaOCl which could be due to the less solubility of liquorice in distilled water which was similar to the study conducted by Ajagannanavar et al.[5]

In the present study, fibroblast cell primary cultures isolated from human periodontal tissue of the extracted teeth were used. According to Hernández-Sierra et al.,[30] primary cultures offer the advantage of having the same characteristics as the original tissue. The cytotoxicity can be determined using methods such as trypan blue dye exclusion assay and MTT assay. However, Avelar-Freitas et al. reported that trypan blue dye exclusion assay has certain limitations like dye can be incorporated by live cells after a short exposure time and personal reliability, related to the expertise of the analyst can affect the results.[31] Thus, MTT an enzyme-based method which relies on a reductive coloring reagent and dehydrogenase in a viable cell was used to determine the cell viability with a colorimetric method in this study. This method is found superior because of its ease in usage, safety, and high reproducibility.[32] The MTT assay determines the functional state of mitochondria that indicates the cell viability. A mitochondrial dehydrogenase enzyme in living cells reduces the yellow tetrazolium salt MTT to blue MTT formazan, which is precipitated in uninjured cells.[33] Similarly, Zhang et al.[34] used MTT assay method to determine the cytotoxicity of MTAD irrigant.

The mean cell viability of Triphala, liquorice, and NaOCl is 99.047, 99.903, and 22.784, respectively. This represents that Triphala and liquorice at 50 mg/ml concentration have no cytotoxic effect and NaOCl has a high cytotoxic effect on PDL fibroblasts [Figure 2]. In a study conducted by Badr et al.[18] liquorice has also shown greater biocompatibility with fibroblasts cells compared to calcium hydroxide, which was severely toxic to the cells. Both the herbal extracts, Triphala and liquorice showed the biocompatibility with human PDL cells.
Figure 2: (a, b, and d) Representative phase micrograph of human periodontal ligament fibroblasts which are arranged irregularly with different size and shape and they have stellate reticulum like morphological appearance (c) illustrates few fibroblast which are plump in nature, and there is no stellate reticulum like shape

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As the aqueous extracts of Triphala and liquorice were used to determine the antimicrobial efficacy at different concentrations against E. faecalis and this efficacy might change when we use other solvent systems like alcohol, because herbs might have the better dissolving capacity in alcohol. Even these in vitro results cannot be extrapolated completely to a clinical scenario as the efficacy of the herbal extracts might be altered and also other properties such as staining and substantivity of herbal extracts should also be evaluated to recommend these herbal extracts as root canal irrigants. Further clinical trials should be carried out to ascertain the effect of these herbal extracts on periodontal pathogens and other caries causing microorganisms.

   Conclusion Top

Herbal extracts (Triphala and liquorice) exhibited significant inhibitory effects against E. faecalis compared to NaOCl and distilled water. These herbal extracts exerted no cytotoxic effects on human PDL cells as compared to NaOCl which showed maximum cytotoxic effect. Hence, indigenous extracts can be promising endodontic irrigants.

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Conflicts of interest

There are no conflicts of interest.

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

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