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Table of Contents   
ORIGINAL RESEARCH  
Year : 2020  |  Volume : 31  |  Issue : 3  |  Page : 376-381
Comparative evaluation of various herbal and synthetic solutions on disinfection of guttapercha: An in vitro study


Department of Conservative Dentistry and Endodontics, Meghna Institute of Dental Sciences, Nizamabad, Telangana, India

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Date of Submission03-Oct-2017
Date of Decision19-Nov-2018
Date of Acceptance23-May-2020
Date of Web Publication06-Aug-2020
 

   Abstract 


Background: Although gutta-percha cones are usually supplied in aseptic packages, once opened and used, they may be contaminated. Chair side disinfection of these cones is important and critical for success of endodontic therapy. Gutta-percha being heat labile, chemical disinfection is the only viable alternative for the sterilization of these cones. Aim: The aim of the study was to evaluate an effective method of disinfection of gutta-percha cones using various herbal and chemical solutions after intentional contamination with Staphylococcus aureus and Enterococcus faecalis. Materials and Methods: 150 gutta-percha cones were selected for the study. Based on the contaminants used, 140 gutta-percha cones were divided into 2 groups: Group A and Group B with 70 cones in each group and the remaining 10 cones (Group VIII) which were uncontaminated served as negative control. The cones from Group A were contaminated with Staphylococcus aureus (S. aureus) and Group B were contaminated with Enterococcus faecalis (E. faecalis). The contaminated cones from both groups were subdivided into 7 groups with 10 cones in each group. Group I cones were disinfected with Q Mix 2 in 1, Group II with 3% sodium hypochlorite, Group III with chitosan, Group IV with aloe vera juice, Group V with amla juice, and Group VI with pancha tulsi. Group VII consisted of gutta-percha cones without any disinfection in both Group A and Group B which served as positive control. The disinfection time was 1 min. All the cones were then incubated in thioglycollate media for 7 days. The thioglycollate media was subcultured and colony forming units were counted. The data were analyzed by one-way ANOVA and post-hoc Tukey tests using SPSS 17.0 software. Results: There was a statistically significant difference present in the mean colony forming units among all the groups (P < 0.05). Conclusion: Pancha Tulsi and Q Mix 2 in 1 possessed superior antibacterial activity when compared to other experimental solutions.

Keywords: Aloe Vera Juice, Amla Juice, Chitosan, Digital colony counter, Guttapercha, PanchaTulsi, QMix™ 2 in 1, Sodium Hypochlorite

How to cite this article:
Sailaja PM, Ahmed S, Devi KS, Shiva S. Comparative evaluation of various herbal and synthetic solutions on disinfection of guttapercha: An in vitro study. Indian J Dent Res 2020;31:376-81

How to cite this URL:
Sailaja PM, Ahmed S, Devi KS, Shiva S. Comparative evaluation of various herbal and synthetic solutions on disinfection of guttapercha: An in vitro study. Indian J Dent Res [serial online] 2020 [cited 2020 Sep 23];31:376-81. Available from: http://www.ijdr.in/text.asp?2020/31/3/376/291492



   Introduction Top


Microorganisms are the main cause of pulpal and periapical diseases. A successful endodontic therapy aims to eliminate the microorganisms in the root canal and prevents its return. Therefore, maintaining the chain of asepsis is extremely important to prevent bacterial contamination of the root canal system. Based on modern-day infection control concepts, the instruments and materials used during endodontic treatment, need to be free of contaminating microorganisms.[1] Obturation is the final stage of endodontic treatment which promotes healing and prevents percolation or ingress of microorganisms into the periapical area.[2] Gutta-percha cones are considered to be the best core material for the obturation of root canal system, regardless of the method used.[3] Although GP cones are manufactured under aseptic conditions and present potential antimicrobial properties especially owing to zinc oxide component,[4] the risk of GP contamination either by handling or aerosols and from physical sources during the storage process does not guarantee sterility.[5]

According to Pang et al., GP cones exposed in clinics can be contaminated (19.4%), and the contaminating bacteria were all vegetative bacterial cells (Staphylococcus species) rather than resistant bacterial spores; this result justifies the need of gutta-percha disinfection.[6] Various studies have reported that Enterococcus faecalis is found to be the most resistant intracanal pathogen in failed root canal cases that serves as a gold standard bacterium in the endodontic research.[7] Due to the superior virulence of Enterococcus faecalis, it was selected in this study to represent the other possible organisms that may get contaminated with gutta-percha cones.

Owing to their physical and chemical nature, gutta-percha cones are not amenable to physical methods of sterilization like hot air oven and autoclaving, necessitating a rapid chair side disinfection of the cones by using chemical solutions.[8] Hence, cold sterilization using chemical disinfectants such as ethyl alcohol, paraformaldehyde, sodium hypochlorite, and formocresol are routinely used, which usually take 1–25 min for disinfection.[9] The disinfectant solutions used in this study were QMix™ 2 in 1 (Dentsply, Tulsa Dental), 3% sodium hypochlorite (Prevest DenPro; India), and chitosan (Everest Bio; India), aloe vera juice (The Unati Co-op. Marketing-Cum Processing Ltd, India.), amla juice (The Unati Co-op. Marketing-Cum Processing Ltd, India), pancha tulsi (Deltas Pharma, India).

QMix™ 2 in 1 is an irrigating solution, contains a mixture of a bisbiguanide antimicrobial agent, polyamine carboxylic acid calcium chelating agent, saline, and a surfactant.[10] Sodium hypochlorite (NaOCl) solution is a broad spectrum antimicrobial agent and has a sterilizing action on artificially contaminated gutta-percha cones, mainly when used at a concentration of 5.25%.[11]

Chitosan, a versatile hydrophilic polysaccharide derived from chitin, has a broad antimicrobial spectrum to which gram-negative, gram-positive bacteria and fungi are highly susceptible.[12]

Herbal solutions which are commonly available in Indian market have shown dramatic results as antimicrobials, anti-cancer, anti-diabetic, immune modulatory, respiratory diseases, liver disorders, and cosmetics agents.[13],[14],[15] The popularity of herbal medications has increased due to the search for cheaper, more accessible and natural form of alternatives. However, data regarding their use as disinfection agents in endodontic practice is still lacking.[10] Thus, herbal solutions like aloevera juice, amla juice, pancha tulsi were used in the current study.

Therefore, the purpose of the present study was to compare the efficacy of the herbal solutions and chemical solutions in the disinfection of GP cones contaminated with Enterococcus faecalis and Staphylococcus aureus.


   Materials and Methods Top


150 GP cones (2% taper, Size 80, Dentsply) were taken from freshly opened boxes under sterile conditions.

Test organisms

Microbial suspension of E. faecalis (ATCC2912) and S. aureus (ATCC6538) of approximately 108 CFU/ml in Trypticase Soy broth (HiMedia Laboratories) were used for this study. Both the strains were grown in 8 mm culture tubes and incubated at 37°C for approximately 23 h.

Artificial contamination of GP cones

140 GP cones were divided into 2 groups: Group A and Group B with 70 cones in each group. Group A with 70 cones were contaminated with 20 ml of microbial suspension of S. aureus for 30 min. Group B with 70 cones were contaminated with 20 ml of microbial suspension of E. faecalis for 30 min. They were subsequently transferred for air drying to sterile dishes containing sterile 4 × 4 gauze pads.

Disinfection of GP cones

After artificial contamination GP cones were immersed in the respective disinfectant solutions for 1 min. Based on the disinfectant used, GP cones from both the groups were subdivided into 7 groups with 10 cones in each group.

Group I A- 10 contaminated cones (S. aureus) immersed in Q Mix 2 in 1.

Group II A- 10 contaminated cones (S. aureus) immersed in 3% sodium hypochlorite.

Group III A-10 contaminated cones (S. aureus) immersed in chitosan.

Group IV A- 10 contaminated cones (S. aureus) immersed in aloe vera juice.

Group V A- 10 contaminated cones (S. aureus) immersed in amla juice.

Group VI A- 10 contaminated cones (S. aureus) immersed in pancha tulsi.

Group VII A-10 contaminated cones (S. aureus) without any disinfectant and served as positive control.

Group I B – 10 contaminated cones (E. faecalis) immersed in Q Mix 2 in 1.

Group II B-10 contaminated cones (E. faecalis) immersed in 3% sodium hypochlorite.

Group III B-10 contaminated cones (E. faecalis) immersed in chitosan.

Group IV B- 10 contaminated cones (E. faecalis) immersed in aloe vera juice.

Group V B- 10 contaminated cones (E. faecalis) immersed in amla Juice.

Group VI B- 10 contaminated cones (E. faecalis) immersed in pancha tulsi.

Group VII B -10 contaminated cones (E. faecalis) without any disinfectant and served as Positive Control.

Group VIII consisted of 10 uncontaminated cones which served as Negative Control.

All the cones were individually transferred to sterile test tubes containing 10 ml of thioglycollate media (HiMedia Laboratories) and incubated at 37°C for 7 days. After 7 days, a micropipette was used to transfer the thioglycollate media to a petridish containing brain heart infusion (BHI) agar. A sterile cotton tip was used to spread the thioglycollate media in a thin layer over BHI agar. The plates were then incubated for 48 h aerobically at 37°C and the colony forming units (CFU) were counted with digital colony counter.

Statistical analysis

The data was statistically analyzed by one-way analysis of variance (ANOVA) followed by post-hoc Tukey test using SPSS 17.0 software. The level of significance was set at P < 0.05.


   Results Top


The results of this study are shown in [Table 1] and [Table 2]. There was a statistical significant difference (P < 0.05) between the test groups and control groups in the mean CFU. Graphic representation of the comparison of mean CFU of S. aureus and E. faecalis among different groups (CFU X 10-1) is shown in [Graph 1].
Table 1: Comparison of the mean number of colonies (X 10-1) among the disinfecting solutions against Staphylococcus aureus using ANOVA and Post Hoc tests

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Table 2: Comparison of the mean number of colonies (X 10-1) among the disinfecting solutions against Enterococcus faecalis using ANOVA and Post Hoc tests

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Among the experimental groups, pancha tulsi exhibited least number of colonies when compared to other disinfecting solutions. Q Mix 2 in group showed the next best disinfectant action on the GP cones. Amla juice was the third most effective disinfecting solution followed by 3% NaOCl and aloe vera juice. Chitosan was least effective among the solutions tested. The VIIth group which served as a positive control (only contamination but no disinfection) showed highest number of colonies. The VIIIth group (without contamination) which served as negative control exhibited least number of colonies among experimental and control groups.

Turbidity in Group A (S. aureus) and Group B (E. faecalis) are illustrated in [Figure 1] and [Figure 2], respectively.
Figure 1: Turbidity in Group A (S. aureus)

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Figure 2: Turbidity in Group B (E. faecalis)

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   Discussion Top


A major objective of endodontic therapy is the establishment of a root canal system free of contaminants, especially microorganisms. Though proper care is taken during biomechanical preparation of the canals, obturation is often accomplished using gutta-percha cones directly from their storage boxes without regard to their sterility.[16] Studies have revealed the presence of microorganisms in 5–19% of freshly opened gutta-percha packs.[11],[17] As GP cones are heat labile, moist and dry heat sterilization cannot be used as it causes alteration of GP structure. Therefore, a rapid and effective chair side decontamination using a chemical agent should be adopted in routine endodontic practice to render them, free of microorganisms.[8],[18] In this study, disinfection treatment was done for 1 min, as it is the minimum chair side time required for the same.

In this study, the cones were intentionally contaminated with two bacteria. Gomes et al. reported that 5.5% of the GP cones taken from the sealed boxes were contaminated and the microorganism most frequently found was Staphylococcus. This is not surprising because it is a common member of the normal flora of skin and mucous membranes.[11]E. faecalis is a fastidious bacterium. It has the ability to resist the high pH of the most commonly used antimicrobial medication: Calcium hydroxide. Also, it has the ability to penetrate deep into dentinal tubules to protect itself from chemomechanical preparation and intracanal medicaments.[19] Thus, because of its diverse survival mechanisms and research that supports its contribution to secondary infections in root canal treatments; it was selected in this study.

The 3 herbal solutions used in this study are proven to be safe, containing active constituents that have beneficial property such as antimicrobial, antioxidant, and anti-inflammatory activity. Shenoi PR et al. evaluated the efficacy of three herbal gels (lemon, tulsi and Green tea) in disinfecting GP cones. They concluded that all the gels showed inhibition zones nearly equal to 5.25% NaOCl.[9] Thus, it would appear prudent to replace the traditional GP disinfecting solutions with these potential herbal solutions.

In the present study, pancha tulsi, Q Mix 2 in 1, amla juice, and 3% NaOCl were shown to possess highest disinfection action against E. faecalis and S. aureus, while aloe vera juice and chitosan had limited disinfection action.

The highest disinfection action of pancha tulsi could be attributed to the presence of more number of bioactive compounds like glycosides, tannins, alkaloids, anthra-quinones, saponins, resins, polysaccharides, steroidal terpens, cardiac glycosides, steroidal ring and flavonoids.[20] The antibacterials present in Tulsi are approved by FDA as food additive. Recent studies have shown that Tulsi is used in the treatment of precancerous lesions and conditions like oral lichen planus, Leukoplakia, etc. It is also considered as a potential intracanal irrigant in contemporary pediatric endodontics.[21]

The current study showed that QMix™ 2 in 1 was the second best disinfectant among the experimental groups. Surface active agent lowers the surface tension of solution and increases their wettability. The potential benefit of bisbiguanide in this mixture is that it prevents the microbial colonization. A polyaminocarboxylic acid-Calcium chelating agent can cause cell wall damage in gram-negative bacteria by chelating and removing divalent cations (Mg +2 and Ca +2) from bacterial cell membrane and increasing its permeability. This could be the probable reason for the better disinfection activity when compared to other test groups. Wang et al. concluded that QMix and 6% NaOCl had stronger antibacterial effects against E. faecalis than 2% NaOCl and 2% CHX.[22]

Amla juice possessed the next better disinfection action and presence of phytochemical components like tannins, terpenoids, alkaloids, flavonoids, various polyphenols, quercetin, gallic acid, pectin, and vitamin C would have contributed to its better disinfection action.[23] Also, studies have shown that amla extract has similar efficacy as EDTA in removal of smear layer.[24]

Senia et al. contaminated gutta-percha cones with cultures of Staphylococcus epidermalis, Carynebacterium xerosis, E. coli, E. faecalis and spores of B. subtilis, immersed them in 5.25% NaOCl, and found that all microorganisms were killed after 1 min.[8] Short et al. identified the presence of crystallization of NaOCl (Chloride crystals) on GP cones after rapid sterilization using 5.25% NaOCl under the scanning electron microscope.[17] Also, in an atomic force microscopy study, Valois et al. reported that a 1 min treatment with 5.25% NaOCl increased the elasticity of gutta-percha cones in comparison with untreated cones.[25] Hence, a lower concentration of NaOCl (i.e., 3% NaOCl) was used in this study. This lower concentration which would require more disinfection time could be the reason for lesser disinfection action of NaOCl.

The disinfection action of aloe vera juice can be attributed to anthra-quinones, saponins, pyrocatechol, cinnamic acid, ascorbic acid, sterols, p-coumaric acid.[26] Though aloe vera juice showed antimicrobial activity, the lesser disinfection action was due to lesser acidic content and lower amount of total monomeric anthocyanins when compared to the other test groups. Recent studies have shown that aloe vera can also be used as canal lubricant and intracanal medicament.[27]

In this study, chitosan showed least disinfection action against both E. faecalis and S. aureus. Various theories have been proposed to explain the mode of action leading to the antimicrobial activity of chitosan. Though the exact mechanism has yet to be elucidated, the intracellular leakage hypothesis is widely accepted. In this mechanism, positively charged chitosan binds to the negatively charged bacterial surface leading to altered membrane permeability, which results in leakage of intracellular constituents causing cell death. However, it has been reported that antimicrobial activity of chitosan is limited to acidic conditions due to the loss of positive charges on the amino group at neutral pH. This restricts the use of chitosan as an antimicrobial agent at neutral pH.[28]


   Conclusion Top


All the tested solutions demonstrated disinfection action against S. aureus and E. faecalis. There was a significant difference found in the number of CFU between Experimental and Control groups. Pancha Tulsi and QMix™ 2 in 1 exhibited good disinfection action against S. aureus and E. faecalis, followed by, amla juice and 3% NaOCl. Hence, herbal alternatives prove to be advantageous considering the several undesirable characteristics of NaOCl.[17],[25] Also, as the herbal alternatives are cheaper and easily available, they can be preferred over the synthetic solutions.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Dr. Pachalla M Sailaja
H. No.: 2-45 / 5/1; PLOT NO.46, Sri Raghavendra Nagar, Thattiannaram, Hyderabad, Telangana - 500 068
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_556_17

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