Antimicrobial Efficacy of a Novel Root Canal Irrigant Made from Coconut and Bromelain Extract against Biofilm Forming Pathogens–An In vitro Evaluation

Jerry Jose¹, Ajitha Palanivelu²

¹Postgraduate Student, Department of Conservative Dentistry and Endodontics, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, India.

²Professor, Department of Conservative Dentistry and Endodontics, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, India.

*Corresponding Author E-mail: ajitharijesh@gmail.com

ABSTRACT:

Objective: Sodium hypochlorite is shown to be a gold standard intracanal irrigant which has shown to have adequate antimicrobial properties and tissue dissolving properties. The toxicity of these irrigants are seen to be high towards various cells. Natural irrigants provide an alternative to show adequate antimicrobial activity and lesser cytotoxicity potential. The current study proposes a novel intracanal irrigant made from coconut and bromelain extract. Material and Methods: 10grams of sodium hydroxide was dissolved in deionized water followed by which 60ml of coconut oil was added to the warm water bath (4⁰C). The final product was left to dry and crushed manually to powder. This powder was then taken in measured quantity to make a final product containing 20% of coconut and 4% of bromelain. The obtained irrigant was then evaluated against Enterococcus faecalis using Agar well diffusion method. Results: For the natural irrigant, the maximum zone of inhibition was seen highest at 22mm against Enterococcus faecalis and 25mm against Candida Albicans. For 3% sodium hypochlorite when compared against Enterococcus faecalis and Candida Albicans showed a maximum zone of inhibition at 33mm against Enterococcus faecalis and 35mm against Candida Albicans. Conclusion: The study shows that the novel irrigant derived from coconut and bromelain extract has a good antimicrobial efficacy against Enterococcus faecalis and Candida albicans. It can be concluded that it has a potential application as a root canal irrigant for root canal disinfection.

KEYWORDS: Root canal irrigants, Cocos, Bromelains, Enterococcus faecalis, Candida Albicans.

INTRODUCTION:

Success to an endodontic treatment depends on various factors with irrigation being of uttermost significance. Endodontic infections are one of the major infections seen in the oral cavity which is seen to be associated with a wide variety of microorganisms, mostly anaerobic and some facultative bacteria.¹ The inflammation of the dental pulp forms the initial pathogenesis by the involvement of microorganisms which then progress to infect the periapical region and surrounding structures.

The success of endodontic treatment depends on multiple factors in which root canal irrigants play a significant role. The goal of endodontic treatment is to properly disinfect the canal space by both physical and chemical means so that it creates a favourable aseptic sterile environment for the filling of the root canal space.² The ideal property of a root canal irrigant is that it should be nontoxic in nature, should not harm the periapical tissue region if extruded as well having the ability to inactivate endotoxins released by microbes as well as preventing the formation of smear layer or dissolving it completely.³

Currently used root canal irrigants in endodontics such as Sodium hypochlorite, Chlorhexidine and MTAD has many disadvantages such as unpleasant taste, discolouration, cytotoxic to  stem cells.⁴ Many studies have been carried out extensively in order to negate this side effects of conventional irrigants and paving them to find alternatives in the form of natural products which is proven more biocompatible as well as exhibiting a similar efficacy to the current gold standard sodium hypochlorite. Natural products such as propolis, Miswak has shown some amount of success to a certain extend.^5,6 They showed to exhibit similar antimicrobial activity as well as negating the adverse effects of conventional irrigants. Though the addition of these irrigants there is no currently suitable alternative in order to replace this. In ordinance to this the search for alternative natural root canal irrigants seem to be crucial. The current aim of the present study was to develop a novel natural root canal irrigant derived using coconut and bromelain extract and compare its antimicrobial efficacy against 3% Sodium hypochlorite.

MATERIALS AND METHODS:

The invitro study was conducted in the research laboratory of the university and the protocol was approved by the scientific review board of the university before the study began.

1) Preparation of the Novel Irrigant:

For the preparation of the novel root canal irrigant, the irrigant was made by using 10 grams of sodium hydroxide which was then dissolved in 25 ml of deionized water followed by which 60ml of virgin coconut oil (VCO) on warming bath (40ºC) with continuous mixing by glass rod until hardening. The final product was left to dry on filter paper and then crushed to powder manually. 20 grams of the formulated powder was then added to 100ml of deionized water with continuous mixing until it dissolves completely, followed by which 4gm of bromelain powder were added to the mixture to obtain the final solution which consist of 20% coconut and 4% bromelain (MCB).⁷ For sterility check the prepared solution was again sterilized using syringe driven filters (Pore size – 0.22µm and Diameter 30mm). The conventional irrigant used for comparison was 3% Sodium hypochlorite (Parcan, Septodont, USA). These irrigants were then subjugated to check the antimicrobial activity against Enterococcus faecalis and Candida albicans.

Figure 1: Preparation of the novel irrigant and assessment of antimicrobial activity using agar well diffusion method.

1) Determination of Antimicrobial Action by Agar Well Diffusion Method:

A 50µL of microbial inoculum was taken using a micropipette and loaded on the agar plates evenly. The inoculation was conducted by evenly streaking the swab over the entire surface of the plate three times rotating the petri plates after each application. Finally, the periphery of the agar surface was swabbed evenly. Five wells of 7mm size and 4mm depth were made at equal distance.

2) Antimicrobial efficacy against Enterococcus faecalis:

The test sample was carried out in triplicates for each concentration. Purity checks were performed at the time of inoculation of test samples. Clinical bacterial strain of Enterococcus faecalis (ATCC 29212), was chosen based on their clinical and pharmacological importance. The bacterial microorganisms were cultured on nutrient agar by using spread plate technique and were incubated for 24 hours at 37°C aerobically. The bacterial strains were allowed to grow in Mueller-Hinton agar (MHA) plates at 37°C (the bacteria were grown in the nutrient broth at 37°C and maintained on nutrient agar slants at 4°C. The stock cultures were maintained at 4°C. Sterile spreader was used for inoculation of this organisms across respective media. Bacterial broth culture was prepared to a density of 10⁸cells/ml and the aliquot was spread evenly by L rod onto Muller Hinton agar followed by which the plated medium was allowed to dry at room temperature for 30 min.

3) Antimicrobial efficacy against Candida albicans:

The test sample was carried out in triplicates for each concentration. Clinical grade fungal strain extracted from extracted tooth (Candida albicans) (ATCC 10231) were chosen based on their clinical and pharmacological importance. The fungal stock cultures were incubated for 24hours at 37°C on potato dextrose agar (PDA) medium, following low temperature storage at 4°C. A suspension of Sabouraud dextrose broth was made to achieve a final density of 10⁴ CFU/mL. The fungal strains were grown in         Sabouraud dextrose       agar and        PDA       media, respectively,               at                            28°C.      The         stock         cultures            were maintained at 4°C followed by which formation of clear inhibition on the agar plates were measured.

Statistical Analysis:

The statistical analysis was carried out using SPSS v21.0 (IBM Corp., Armonk, USA) The analysis of data was done using independent t-test for each concentration. When p value was less than 0.05 it was considered statistically significant.

RESULTS:

The test was conducted in triplicates with each concentration recorded specifically for Enterococcus faecalis and Candida albicans. The zone of inhibition of natural irrigant against Enterococcus faecalis (Figure 2, Table 1) and Candida albicans (Figure 3, Table 1). The data was then converted to mean and standard deviation (Table 2, Table 3) the effect of the irrigants against different microbes was seen to be statistically significant

(p<0.05)

Figure 2: Zone of inhibition seen with natural irrigant derived from coconut and bromelain extract against Enterococcus faecalis

Figure 3: Zone of inhibition seen with natural irrigant derived from coconut and bromelain extract against Candida albicans

Table 1: Zone of inhibition of Natural root canal irrigant and 3% Sodium hypochlorite against Enterococcus faecalis and Candida albicans

Zone of Inhibition against Enterococcus faecalis and Candida albicans
	Against E. faecalis			Against C. albicans
	25µ	50µ	100µ	25µ	50µ	100µ
Natural root canal irrigant against E. faecalis	12mm	14mm	22mm	13mm	14mm	27mm
3% Sodium hypochlorite	20mm	25mm	30mm	25mm	30mm	35mm

Table 2: Mean and Standard Deviation on the comparison of different concentrations of the groups against Enterococcus faecalis.

Groups	Zone of Inhibition Mean±S.D.	Mean Difference	t value	p value
25µl coconut and bromelain extract	11.00±1.00	-10.33	-9.803	0.001*
25µl 3% Sodium hypochlorite	21.33±1.52
50µl coconut and bromelain extract	14.00±1.00	-12.33	-6.345	0.003*
50µl 3% Sodium hypochlorite	26.33±3.21
100µl coconut and bromelain extract	22.33±1.52	-5.00	-1.819	0.143
100µl 3% Sodium hypochlorite	27.33±4.50

Table 3: Mean and Standard Deviation on the comparison of the different concentrations of the groups against Candida albicans.

Groups	Zone of Inhibition Mean±S.D.	Mean Difference	t value	p value
25µl of Natural irrigant	12.66±1.52	-12.33	-8.488	0.001*
25µl 3% Sodium hypochlorite	25.00±2.00
50µl of Natural Irrigant	15.33±1.52	-14.66	-7.546	0.002*
50µl 3% Sodium hypochlorite	30.00±3.00
100µl coconut and bromelain extract	26.66±1.52	-6.33	-4.359	0.01*
100µl 3% Sodium hypochlorite	33.00±2.00

DISCUSSION:

The root canal system is a complex morphology which is shown to habitat a wide array of microorganisms.⁸ Enterococcus faecalis is one of the normal habitants of the root canal system shown to be the primary cause of 4 to 40% of primary endodontic infections. Polymerisation chain analysis has exhibited the presence of Enterococcus faecalis to be up to 66-77% in the root canal system.⁹ Similar to this another one such organism is Candida albicans which is a fungal organism found as normal commensals in the oral cavity. It was found to have significant involvement in dental caries and periodontal diseases. A recent study by Mergoni et al. had found that the prevalence of Candida albicans was the most prevalent organism which was seen up to 8.2% of the total microflora.^10,11

Sodium hypochlorite is one of the main compounds used for root canal irrigation which is known to be a halogenated compound known for its antibacterial action, proteolytic and dissolution capacity, and debridement properties.¹² It has many adverse effects with studies showing it has a deproteinizing action on the collagen fibres.¹³ Another common feature seen clinically with sodium hypochlorite is accident by extrusion of the irrigant periapically. In a recent review by Guivarc’h et al.,¹⁴ it was seen that sodium hypochlorite had multiple adverse effects on an event of extrusion periapically such as tissue swelling, facial hematomas as well as tissue necrosis regardless of the concentration of the irrigant. It is well known that one of the advantages of sodium hypochlorite is its tissue dissolving property but this has been seen to be dependent largely on the concentration of the irrigant.¹⁵

Another commonly used root canal irrigant used alternatively to sodium hypochlorite is chlorhexidine gluconate which is a synthetic cationic biguanide consists of two symmetric 4-cholorophenyl rings and two biguanide groups connected by a central hexamethylene chain.¹⁶ CHX is bactericidal which causes precipitation of cytoplasmic contents resulting in cell death.¹⁷ It has antibacterial as well as antifungal activity with various studies proving this to great extent. One of its major properties of CHX being its substantivity.^18,19 Siqueira et al.²⁰ compared the effectiveness of 2.5% NaOCl and 0.12% CHX as irrigants and found the levels of microbial reduction to be almost similar to each other. Another important step was the combination of NaOCl and CHX for root canal irrigation which was shown to enhance their antimicrobial properties by synergistic effect but in turn exhibiting adverse effects for instance when irrigating with sodium hypochlorite and chlorhexidine it there is an orange-brown precipitate formation called parachloroanaline (PCA) which can occlude the dentinal

tubules and compromise the seal of the obturation materials. This precipitate is of concern has shown to have cytotoxicity against rats and humans as well.²¹

Various natural products are available in dentistry which has shown good antimicrobial activity. Herbs such as Morinda citrafolia, Propolis, Orange Oil, Green Tea, Tea Tree oil, Aloe vera gel are seen to exhibit various medicinal properties with the potential of application in various medicinal purposes.²² One of the natural products which less reported is the coconut extract. Literature had shown its significant effect as oil pulling for mouth washes.²³ Ogbulo et al. in their experimental study using coconut extract against Candida isolates and found that that they have 100% zone of inhibition was seen in comparison to fluconazole. Peedikayil et al. who had done an in vivo study against chlorhexidine on Streptococcus mutans found that coconut oil was equally effective as chlorhexidine for reduction against Streptococcus mutans.²⁴ Virgin coconut oil is shown to exhibit antimicrobial action mainly due to fatty acids such as 12C lauric acid, 8C caprylic acid, 10C capric acid.^25,26 The presence of free fatty acids specifically 12C lauric acid has one of the most active antimicrobial FFA which is shown to be present in the as a minor sebum component,²⁷ as well as being one of components of human breast milk protecting the new born from infections.^28,29

Bromelain is a mixture of endopeptidases and other components such as phosphatase, glucosidase, peroxidase, cellulase, escharase, and other substances such as protease inhibitors. They show many therapeutic benefits and are used as the treatment regimen of different conditions such as angina pectoris, bronchitis, sinusitis, surgical trauma and also enhancing the absorption of drugs, particularly antibiotics.^30,31 It is said to have antimicrobial activities towards various microorganisms such as Streptococcus mutans, Enterococcus                     fecalis,                  Aggregatibacter actinomycetemcomitans (Aa), and Porphyromonas gingivalis.³² Studies also have shown bromelain to have an effect on the collagen fibres by the depletion of the collagen fibres in turn showing a potential smear layer removal activity.³³ This could implement a proposed chelation effect to the current irrigant in addition to the microbial action.

One of the limitations of the current study is the preliminary analysis in a laboratory environment. This could potentially would not exhibit the same levels of antimicrobial action in a natural tooth.

CONCLUSION:

The current study proposes a novel root canal irrigant derived from coconut and bromelain extract shown to

have significant antimicrobial efficacy against Enterococcus faecalis and Candida albicans in comparison to 3% Sodium Hypochlorite. Further studies are necessitated in order to the current proposed endodontic irrigant as a potential application for root canal disinfection as well as for clinical efficacy and potential.

CONFLICT OF INTEREST:

None.

REFERENCES:

1. Narayanan LL, Vaishnavi C. Endodontic microbiology. J Conserv Dent. 2010 Jan 10; 13(4): 233.

2. Zehnder M. Root Canal Irrigants. J Endod. 2006 May; 32(5): 389– 98.

3. Grossman LI. Endodontic practice. Lea & Febiger; 1965.

4. Mohammadi Z, Shalavi S, Moeintaghavi A, Jafarzadeh H. A Review Over Benefits and Drawbacks of Combining Sodium Hypochlorite with Other Endodontic Materials. Open Dent J. 2017 Dec 26; 11: 661–9.

5. Alharbi A, Aliuddin S, Alharbi TEA, Alharbi S, Alanzi A, AlHarbi A. Herbal Endodontic Irrigants. In 2018.

6. Gupta R, Rai N, Shetty SS. Herbal Irrigants: Underutilized Option in Dentistry. Res J Pharm Technol. 2019; 12(10): 5098.

7. Ghazi Saadallah M, Talal H, Al-Salman, Amer A, Taqa amer. The effect of a new endodontic irrigant solution on push out shear bond strength of a Root Canal Filling System. 2014 Feb 1; 3: 2319– 7463158.

8. Baskaran K, Raj JD, Yang JNC. Comparative Study of Cleaning Efficacy of Different Concentrations of Sodium Hypochlorite on Nickel-Titanium Endodontic Instruments. Res J Pharm Technol. 2017; 10(1): 75.

9. Stuart C, Schwartz S, Beeson T, Owatz C. Enterococcus faecalis: Its Role in Root Canal Treatment Failure and Current Concepts in Retreatment. J Endod. 2006 Feb; 32(2): 93–8.

10. Mergoni G, Percudani D, Lodi G, Bertani P, Manfredi M. Prevalence of Candida Species in Endodontic Infections: Systematic Review and Meta-analysis. J Endod. 2018 Nov; 44(11): 1616-1625.e9.

11. Yuvaraj S, Gheena S, Gopinath P. Comparison of oral candidal flora of diabetics and non diabetics. Res J Pharm Technol. 2016; 9(10): 1645.

12. Sadiq SM, Kareem A, Rasheed N, Mohammad AS. Pharmaceutical Importance of Anti-Microbials. Asian J Pharm Technol. 2017; 7(1): 7.

13. Abuhaimed TS, Abou Neel EA. Sodium Hypochlorite Irrigation and Its Effect on Bond Strength to Dentin. BioMed Res Int. 2017; 2017: 1–8.

14. Guivarc’h M, Ordioni U, Ahmed HMA, Cohen S, Catherine J-H, Bukiet F. Sodium Hypochlorite Accident: A Systematic Review. J Endod. 2017 Jan; 43(1): 16–24.

15. Taneja S, Mishra N, Malik S. Comparative evaluation of human pulp tissue dissolution by different concentrations of chlorine dioxide, calcium hypochlorite and sodium hypochlorite: An in vitro study. J Conserv Dent. 2014 Jan 11; 17(6): 541.

16. Noor SSSE, Pradeep. Chlorhexidine: Its properties and effects. Res J Pharm Technol. 2016; 9(10): 1755.

17. Gomes BPFA, Souza SFC, Ferraz CCR, Teixeira FB, Zaia AA, Valdrighi L, et al. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro. Int Endod J. 2003 Apr; 36(4): 267–75.

18. Carrilho MR, Carvalho RM, Sousa EN, Nicolau J, Breschi L, Mazzoni A, et al. Substantivity of chlorhexidine to human dentin. Dent Mater. 2010 Aug; 26(8): 779–85.

19. Shetty SS, Quays DR, John J, Yee NS, Appu DAP, Tze LM, et al. Effect of Oil pulling on Oral Health-A Microbiological Study. Res J Pharm Technol. 2019; 12(1): 1.

20. Siqueira Jr JF, Rôças IN, Paiva SS, Guimarães-Pinto T, Magalhães KM, Lima KC. Bacteriologic investigation of the effects of sodium hypochlorite and chlorhexidine during the endodontic treatment of teeth with apical periodontitis. Oral Surg Oral Med Oral Pathol Oral Radiol Endodontology. 2007; 104(1): 122–130.

21. Kim J-W. Precipitate from a combination of sodium hypochlorite and chlorhexidine. Restor Dent Endod. 2012 Aug; 37(3): 185–6.

22. Almadi E, Almohaimede A. Natural products in endodontics. Saudi Med J. 2018 Feb 8; 39(2): 124–30.

23. Moorthy MK, Nayak BK, Nanda A. Antioxidant and antitumor activity of lactic bacteria isolated from natural beverage-Coconut toddy. Res J Pharm Technol. 2017; 10(12): 4317.

24. Peedikayil FC, Remy V, John S, Chandru TP, Sreenivasan P, Bijapur GA. Comparison of antibacterial efficacy of coconut oil and chlorhexidine on Streptococcus mutans: An in vivo study. J Int Soc Prev Community Dent. 2016; 6(5): 447–52.

25. Shilling M, Matt L, Rubin E, Visitacion MP, Haller NA, Grey SF, et al. Antimicrobial Effects of Virgin Coconut Oil and Its Medium- Chain Fatty Acids on Clostridium difficile. J Med Food. 2013 Dec; 16(12): 1079–85.

26. Joseph D, George J, Mathews MM, Mathew F, Varghese B, Sunny

B. A Compilation on Anti-Diabetic Profile of Cocos nucifera. Res J Pharm Technol. 2019; 12(8): 3791.

27. Nakatsuji T, Kao MC, Fang J-Y, Zouboulis CC, Zhang L, Gallo RL, et al. Antimicrobial Property of Lauric Acid Against Propionibacterium acnes: Its Therapeutic Potential for Inflammatory Acne Vulgaris. J Invest Dermatol. 2009 Oct; 129(10): 2480–8.

28. Gardner AS, Rahman IA, Lai CT, Hepworth A, Trengove N, Hartmann PE, et al. Changes in Fatty Acid Composition of Human Milk in Response to Cold-Like Symptoms in the Lactating Mother and Infant. Nutrients. 2017 Sep 19

29. Devi K, Jayasankari S, Raju LL, Samson R, Ravichandran. A Study to assess the Level of Knowledge and Attitude Regarding Expression and storage of Breast milk among Lactating mothers at Pondicherry Institute of Medical Sciences, Puducherry. Int J Adv Nurs Manag. 2020; 8(3): 194.

30. Pavan R, Jain S, Shraddha, Kumar A. Properties and Therapeutic Application of Bromelain: A Review. Biotechnol Res Int. 2012

31. Ha YJ, Gu JJ, Yoo SK. Characterization of Melanin Isolated and purified by Multi-Stage Enzymatic Reaction from Korean Whole Black Fowl (Gallus gallus domesticus). Res J Pharm Technol. 2018; 11(11): 4821.

32. Praveen NC, Rajesh A, Madan M, Chaurasia VR, Hiremath NV, Sharma AM. In vitro Evaluation of Antibacterial Efficacy of Pineapple Extract (Bromelain) on Periodontal Pathogens. J Int Oral Health JIOH. 2014; 6(5): 96–8.

33. Chauhan K, Shivanna V, Basavanna R. Effect of bromelain enzyme for dentin deproteinization on bond strength of adhesive system. J Conserv Dent. 2015; 18(5): 360.

Received on 13.09.2020              Modified on 15.11.2020

Accepted on 07.12.2020             © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(10):5222-5226.

DOI: 10.52711/0974-360X.2021.00909