Antibacterial Efficacy of Herbal Solutions in Disinfecting Gutta Percha Cones Against Enterococcus Faecalis

Abstract

Objective:

Even when endodontic procedures are performed with the utmost care, reinfection can occur. One possible explanation is that contaminated gutta-percha cones were used in the root canals. The study's goal is to assess the antimicrobial activity and efficacy of Neem, Turmeric, and Calotropis Procera in disinfecting gutta percha cones prior to obturation.

Materials and Methods:

80 Gutta percha (GP) cones were contaminated with 0.5ml Ferlands standard Enterococcus Faecalis for 10 minutes.20 cones each were treated up to 5 minutes in their prepared neem, turmeric, calotropis and sodium hypochlorite solutions. Then, they placed in BHI broth and vertex is done for 1 minute. One loopful of broth was stroked on 20 blood agar plates of 5 each neem, turmeric, calotropis and sodium hypochlorite. The plates were then incubated for 24 hours aerobically at 37°C and the colony forming units were counted with a digital colony counter.

Results:

Mean colony forming units were compared in all the groups and there was a statistically significant difference present among the groups (p<0.01). Sodium Hypochlorite (NaOCL)as control group showed average of 5 colony forming units(CFU). Turmeric had the least average of 55 CFU and was the most effective of the experimental groups. Neem and Calotropis had the higher average of 130 CFU and 200 CFU respectively, making it the least effective of the solutions tested.

Conclusion :

All of the herbal solutions tested positive for disinfection of GP points. When compared to Neem and Calotropis, Turmeric was the most effective and has superior antibacterial activity.

INTRODUCTION

The outcome of endodontic treatment is determined by maintaining a sterile sequence from preparation of the access cavity to permanent restoration of the tooth. Both foreign and endogenous oral microbial flora must be considered by the dentist.[1] Every equipment and material inserted in the root canals should be sterile for the best infection control.[2]

The primary goal of root canal filling is to prevent reinfection of the root canal system and limit the growth of bacteria that may persist following chemicomechanical treatment.[3] The most commonly employed core material for the obturation of root canal systems is Gutta Percha (GP) cones.[4]

GP cones are created under aseptic circumstances and have antibacterial qualities, particularly due to the zinc oxide component,[5] but they can be contaminated by aerosols, inappropriate storage, and physical handling.[2]

Microorganisms have been found in freshly opened boxes, according to studies. Contamination can develop as a result of incorrect storage, aerosol exposure, or handling during and/or after the manufacturing process.[6] As a result, before using GP points as a filling material, a fast chairside disinfection protocol (CDP) of GP points is required.

Staphylococcus genus is determined to be the most prevalent bacteria infecting gutta percha cones in their packaging and after handling with gloves, according to numerous investigations.[7] Another organism that has been proven to be the most resistant intracanal pathogen in unsuccessful root canals and used as a gold standard bacterium in endodontic research is Enterococcus faecalis.[8]

Because GP cones are heat labile, both moist and dry heat sterilization are ineffective as they modify the GP structure.[2] As a result, chemical disinfectants such as ethyl alcohol, paraformaldehyde, sodium hypochlorite, and formocresol are commonly employed for cold sterilization, which takes 1–25 min.[9]

Immersion of GP points in a 5.25% sodium hypochlorite (NaOCl) solution for 1 min has been found to be the most effective method for disinfecting them without causing topographic alterations. However, crystal deposition on the surface of GP cones has been documented at all concentrations, obstructing the sealers' bond with the GP cones and resulting in microleakage.[9]

Herbal treatments have grown in popularity as people look for less-expensive, more convenient, and vital alternatives. However, there is a paucity of data on the use of disinfection agents in endodontic treatment. As a result, herbal medicaments such as neem, turmeric, and Calotropis were used in the current study. The goal of the study was to examine the efficacy of neem, turmeric, and Calotropis procera in disinfecting gutta percha cones before obturation.

MATERIALS AND METHODS

Preparation of Herbal Disinfectants

Preparation of neem solutions

Neem solutions were made by combining 25 g of 99% fresh neem leaf powder (The Indian Neem Tree Company, Mumbai, India) with 50 ml of pure ethanol. After macerating the mixture for 1–2 min, the extract was filtered through a muslin cloth for obtaining a coarse residue and through a filter paper for obtaining a finer residue.

Preparation of turmeric solutions

In 500 ml ethanol, 40 g turmeric powder (RYM Exports, Mumbai, India) was dissolved. For a coarse residue, muslin cloth was used and for a finer residue, filter paper was used.

Preparation of C. procera solutions

Powdered leaves of C. procera were separated into three parts, each of which was dissolved in acetone, ethanol, and water. The extracts of ethanol and acetone were evaporated, while the aqueous extract was freeze dried. Weighed dried extracts were diluted in 10% dimethylsulfoxide (DMSO) to make a stock solution, from which smaller quantities could be made.

Artificial Contamination of Gutta Percha Cones

In this investigation, Enterococcus faecalis strain ATCC 29212 was cultured overnight in nutritional broth. Using a sterile tweezer, 80 gutta percha cones of various sizes (Size 80, Neoendo) were removed from freshly opened boxes and immersed for 10 min in 0.5 ml of Ferlands standard E. faecalis microbiological suspensions. After that, the cones were placed on sterile absorbent paper and left to air dry for 10 min. Following drying, 80 contaminated gutta percha cones were divided into four groups, each with 20 gutta percha cones as given below:

-control group: 20 contaminated GP cones submerged in 5.25% NaOCl for 5 min;

-study group A: 20 contaminated GP cones submerged in neem preparations for 5 min;

-study group B: 20 contaminated GP cones submerged in turmeric preparations for 5 min; and

-study group C: 20 contaminated GP cones submerged in Calotropis preparations for 5 min.

All GP cones were taken from the solution and placed in Brain Heart Infusion (BHI) broth, where they were vortexed for 1 min. Before immersing the treated cones in BHI broth, they were placed on absorbent paper to remove any excess medication. The gutta percha cones were taken from the disinfecting solutions after immersion and placed on absorbent paper to dry. Twenty blood agar plates with five each of neem, turmeric, Calotropis, and NaOCl were stroked with one loopful of broth. After that, the plates were incubated aerobically at 37°C for 24 h and the colony-forming units were counted using a digital colony counter (Labmate).

RESULTS

The results obtained in this study are shown in Tables 1 and 2. The antibacterial activities of herbal extracts were summarized using descriptive statistics such as mean, standard deviation (SD), and range. Kruskal–Wallis and Mann–Whitney U-tests were used to examine the data.

Table 1.

Mean comparison of colonies among neem, Calotropis, turmeric, and hypochlorite

Variables	Sample size	Min.	Max.	Mean	SD	K-W test value	P
Neem	20	127.00	133.00	130.00	1.69	74.234	<0.001 Significant
Calotropis	20	197.00	204.00	199.95	2.16
Turmeric	20	53.00	59.00	55.95	1.50
Hypochlorite	20	2.00	7.00	4.20	1.47

SD=Standard deviation, K-W=Kruskal-Wallis

Table 2.

Mean comparison of colonies between variables

Variables	Mean±SD difference	P
Neem and calotropis	69.95±0.47	<0.001 Significant
Neem and turmeric	74.05±0.19	<0.001 Significant
Neem and hypochlorite	125.80±0.22	<0.001 Significant
Calotropis and turmeric	144.00±0.66	<0.001 Significant
Calotropis and hypochlorite	195.75±0.69	<0.001 Significant
Turmeric and hypochlorite	51.75±0.03	<0.001 Significant

DISCUSSION

The presence of facultative and resistant bacteria species such as E. faecalis, Candida albicans, and Staphylococcus aureus is the leading cause of root canal treatment failure. E. faecalis is a gram-positive, facultative anaerobe that survives in the root canal system undetected. It causes endodontically treated teeth to fail (5% in untreated teeth and 29%–77% in root-filled teeth).[10]

Surface imperfections in the planes of gutta percha cones are filled by root canal sealers. These openings and massive abnormalities form a broad contact between the root canal wall and gutta percha, allowing the molecules that serve as nutrition for the bacteria in the root canal system to flow out.[11] The number of organisms increases throughout ordinary clinical practice, despite the fact that the quantity of organisms is fairly modest at the time of packaging.[12]

Contamination of gutta percha cones can occur if they are handled incorrectly during retreatment or if the master cone is not checked for fit before the final rinse. Microorganisms have been found in 5%–19% of freshly opened gutta percha sachets, according to studies. Gomes et al.[13] were able to confirm this. E. faecalis is a gold standard to represent other potential microorganisms because it is the most resistant intracanal pathogen in failed root canals. As a result, E. faecalis was chosen for this study.[14]

Despite the fact that a variety of chemicals and disinfecting agents are available, they are known to take a long time to disinfect, ranging from 3 to 15 min.

The control in this investigation was NaOCl, which was completely efficient in disinfecting gutta percha cones. The average colony-forming unit in NaOCl was 5. This was comparable to Bloomfield et al.'s[15] discovery that 5.25% NaOCl was effective after 10 min of contact with Bacillus subtilis vegetative forms. With the liberation of active chlorine, NaOCl has antibacterial and sporicidal properties. The germination and outgrowth of bacterial spores may be inhibited by chlorine.

Senia et al.[16] found that after 1 min of exposure to 5.25% NaOCl, gutta percha cones contaminated with B. subtilis spores are disinfected. To destroy all microorganisms, 5.25% NaOCl took from 15 s to 1 min, whereas 0.5% took half an hour.

In this study, we evaluated the effectiveness of NaOCl in disinfecting GP cones. In addition to the current finding, Short et al.[17] did a scanning electron microscopy (SEM) research in which they discovered that chloride crystal formation by GP cones immersed in 5.25% and 2.5% NaOCl interferes with bonding.

Herbal treatments have grown in popularity as people look for less-expensive, more accessible, and natural options. However, data on their application in endodontics and disinfection of GP cones is still insufficient.

The traditional medicinal plant Azadirachta indica (neem) may be the most effective. Neem leaves are well known for their anti-inflammatory, antipyretic, analgesic, antibacterial, antifungal, antiviral, and antioxidant effects. Neem had the greatest effect on adhesion of E. faecalis to dentin. The infected gutta percha cones immersed in neem preparations yielded an average of 130 colony-forming units in this investigation.

Curcuma longa, also known as turmeric, is a herbal remedy that is used because of its low side effects.[18] Turmeric is a natural antibiotic with anti-inflammatory, antioxidant, anticarcinogenic, antimutagenic, anticoagulant, antidiabetic, antifertility, antibacterial, and antifungal properties.[19] Curcuminoids are the constituents of turmeric. These substances are polyphenols, which have a strong antioxidant effect.[20] The infected gutta percha cones immersed in turmeric preparations showed an average of 55 colony-forming units in this study.

C. procera is a resinous golden brown to dark brown material that is used as a natural antibiotic. It also has antioxidant and anti-inflammatory properties. C. procera leaves are traditionally used to treat jaundice. The infected gutta percha cones immersed in Calotropis preparations yielded an average of 200 colony-forming units in this investigation.

Turmeric had the least amount of colonies among the experimental groups, when compared to other disinfecting solutions. Within the mean colony-forming units, there was a statistically significant difference (P < 0.01) between the study groups. Turmeric has substantial antibacterial action against E. faecalis, according to this study. This is similar to the result of Neelakantan et al., who reported that curcumin's antibacterial activity was comparable to NaOCl, and that herbal medicine might be employed in endodontics to treat root canal failure.[18]

Nadia et al.[21] conducted a similar investigation and discovered that the ionic, resin, and ethanolic fractions of turmeric are 100% effective against all gram-positive bacteria tested, including those resistant to most of the broad-range antibiotics utilized.

Neem was determined to be the second most effective disinfecting solution among the experimental groups, when compared to other disinfecting solutions. Shenoi et al.[9] tested neem, aloe vera, and turmeric gels for their antibacterial properties against Escherichia coli, E. faecalis, and S. aureus. Neem gel showed decontamination equivalent to NaOCl across all herbal extracts used in GP cone decontamination for 3 min.

Calotropis was the least effective disinfecting solution among the experimental groups in our current investigation, when compared to other disinfecting solutions. Similarly, Oncag et al.[22] and Awawdeh et al.[23] conducted studies to demonstrate the efficacy of propolis and aloe vera as intracanal medicaments; however, sterilization of gutta percha and its effect on gutta percha topography have received less attention.

Athiban et al.[24] demonstrated aloe vera gel's antibacterial activity against three microorganisms by producing effective inhibition zones that were nearly equal to 5.25% NaOCl. As a result, herbal products can be used as an alternative for chemical disinfectants.

Limitations

Polymicrobial infections are common in root canals. This study, on the other hand, looks at the efficiency of herbal treatments against E. faecalis in disinfecting gutta percha cones before obturation. More research is needed to assess the disinfectant's effectiveness against other bacteria and organisms such as fungus, viruses, and yeasts. In addition, more research into the specific mechanism of disinfection action of herbal treatments is required in the future.

CONCLUSION

Within the study's parameters, all the herbal solutions tested showed disinfectant activity against E. faecalis. In the mean colony-forming units, there was a statistically significant difference (P < 0.01) between the study groups. Turmeric was the most effective disinfectant against E. faecalis, followed by neem. C. procera had the least disinfectant activity in the experimental groups against both bacteria.

Financial support and sponsorship

Department of Conservative dentistry and Endodontics, Government Dental College and Hospital, Hyderabad

Conflicts of interest

There are no conflicts of interest.