The antimicrobial effect of R-limonene and its nano emulsion on Enterococcus faecalis - In vitro study

Abstract

Purpose: This study aimed to evaluate the effectiveness of R-limonene and its nano emulsion formulation against Enterococcus faecalis (E. faecalis) in infected root canals. Materials and Methods: A nano emulsion containing 20% R-limonene was prepared using the phase inversion method. E. faecalis was cultivated, and MIC/MBC values were determined through the macrodilution method. Standardized E. faecalis suspensions were used to infect the root canals ex vivo. Subsequently, R-limonene, nano emulsion containing 20% R-limonene, calcium hydroxide, and sterile saline were applied to the infected root canals for 7 days at the determined MIC/MBC dose. The root canals were sampled, and droplets were plated onto Mueller-Hinton agar plates, and viable bacteria (Colony Forming Units, CFU/mL) were counted. Statistical analyzes were performed based on the data. Results: Statistically similar efficacy was observed between test materials on the root canal samples. All test materials were significantly more effective than the saline control (p<0.05), however, none of the test materials completely eliminated E. faecalis from root canals. Conclusion: Although the effects of R-limonene on E. faecalis in infected root canals appeared to be like calcium hydroxide, with its broad antibacterial spectrum, gutta-percha softening and cleansing benefits, it was considered that it could considered among antimicrobials that could be used especially in endodontic retreatments alone or in synergistic combinations.

Keywords

• Antimicrobial effect
• calcium hydroxide
• Enterococcus faecalis
• nano emulsion
• R-limonene

References

1. Sunde PT, Olsen I, Debelian GJ, Tronstad L. Microbiota of periapical lesions refractory to endodontic therapy. J Endod 2002;28:304-10. google scholar
2. Byström A, Claesson R, Sundqvist G. The antibacterial effect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide in the treatment of infected root canals. Dent Traumatol 1985;1:170-5. google scholar
3. Sinha DJ, Sinha AA. Natural medicaments in dentistry. Ayu 2014;35:113-8. google scholar
4. Tewari R, Kapoor B, Mishra S, Kumar A. Role of herbs in endodontics. Journal of Oral Research and Review 2016;8:95-9. google scholar
5. Vishnuvardhini S, Sivakumar A, Ravi V, Prasad A, Sivakumar J. Herbendodonticsphytotherapy in endodontics: A review. Biomed Pharmacol J 2018;11:1073-82. google scholar
6. Almadi EM, Almohaimede AA. Natural products in endodontics. Saudi Med J 2018;39:124-30. google scholar
7. Jantrawut P, Boonsermsukcharoen K, Thipnan K, Chaiwarit T, Hwang KM, Park ES. Enhancement of antibacterial activity of orange oil in pectin thin film by microemulsion. Nanomaterials (Basel) 2018;8:545. google scholar
8. Seker ID, Akca G, Alacam T. In vitro evaluation of antimicrobial effects of citrus limonum essential oil on some endodontic pathogens. Ann Med Res 2022;29:427-33. google scholar

9. Zahi MR, El Hattab M, Liang H, Yuan Q. Enhancing the antimicrobial activity of d-limonene nano emulsion with the inclusion of s-polylysine. Food Chem 2017;221:18-23. google scholar
10. Zhang Z, Vriesekoop F, Yuan Q, Liang H. Effects of nisin on the antimicrobial activity of d-limonene and its nano emulsion. Food Chem 2014;150:307-12. google scholar
11. Donsı F, Annunziata M, Sessa M, Ferrari G. Nanoencapsulation of essential oils to enhance their antimicrobial activity in foods. LWT - Food Science and Technology 2011;44:1908-14. google scholar
12. Turner S, Love R, Lyons K. An in-vitro investigation of the antibacterial effect of nisin in root canals and canal wall radicular dentine. Int Endod J 2004;37:664-71. google scholar
13. Feng J, Wang R, Chen Z, Zhang S, Yuan S, Cao H, Jafari SM, Yang W. Formulation optimization of d-limonene-loaded nano emulsions as a natural and efficient biopesticide. Colloids Surf A Physicochem Eng Asp 2020;596:124746. google scholar
14. Maté J, Periago PM, Palop A. Combined effect of a nano emulsion of d-limonene and nisin on listeria monocytogenes growth and viability in culture media and foods. Food Sci Technol Int 2016;22:146-52. google scholar
15. Alaçam T, Yoldaş HO, Gülen O. Dentin penetration of 2 calcium hydroxide combinations. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;86:469-72. google scholar
16. Sun J. D-limonene: Safety and clinical applications. Altern Med Rev 2007;12:259-64. google scholar
17. Chikhoune A, Hazzit M, Kerbouche L, Baaliouamer A, Aissat K. Tetraclinis articulata(vahl) masters essential oils: Chemical composition and biological activities. Journal of Essential Oil Research 2013;25:300-7. google scholar
18. Vuuren Sv, Viljoen AM. Antimicrobial activity of limonene enantiomers and 1, 8-cineole alone and in combination. Flavour Fragr J 2007;22:540-4. google scholar
19. Chemat S, Tomao V, Chemat F. Limonene as green solvent for extraction of natural products. In: Mohammad A, editor. Green solvents i: Properties and applications in chemistry. Dordrecht: Springer Netherlands; 2012. p. 175-86. google scholar
20. Uemura M, Hata G, Toda T, Weine FS. Effectiveness of eucalyptol and d-limonene as guttapercha solvents. J Endod 1997;23:739-41. google scholar
21. Li PH, Chiang BH. Process optimization and stability of d-limonene-in-water nano emulsions prepared by ultrasonic emulsification using response surface methodology. Ultrason Sonochem 2012;19:192-7. google scholar
22. Weiss J, Gaysinsky S, Davidson M, McClements J. Nanostructured encapsulation systems: Food antimicrobials. In: Barbosa-Cánovas G, Mortimer A, Lineback D, Spiess W, Buckle K, Colonna P, editors. Global issues in food science and technology. San Diego: Academic Press; 2009. p. 425-79. google scholar
23. Baker J, Hamouda T, Shih A, Myc A, inventors; University of Michigan, assignee. Nontoxic antimicrobial compositions and methods of use. USA patent US-6559189-B2. 2003. google scholar
24. Zahi MR, Liang H, Yuan Q. Improving the antimicrobial activity of d-limonene using a novel organogel-based nano emulsion. Food Control 2015;50:554-9. google scholar
25. Sonu KS, Mann B, Sharma R, Kumar R, Singh R. Physico-chemical and antimicrobial properties of d-limonene oil nano emulsion stabilized by whey protein-maltodextrin conjugates. J Food Sci Technol 2018;55:2749-57. google scholar
26. Wennberg A, 0rstavik D. Evaluation of alternatives to chloroform in endodontic practice. Dent Traumatol 1989;5:234-7. google scholar
27. 0rstavik D, Haapasalo M. Disinfection by endodontic irrigants and dressings of experimentally infected dentinal tubules. Dent Traumatol 1990;6:142-9. google scholar
28. Bodrumlu E, Er O, Kayaoglu G. Solubility of root canal sealers with different organic solvents. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:e67-9. google scholar
29. Rehman K, Khan FR, Aman N. Comparison of orange oil and chloroform as gutta- percha solvents in endodontic retreatment. J Contemp Dent Pract 2013;14:478-82. google scholar
30. Whitworth JM, Boursin EM. Dissolution of root canal sealer cements in volatile solvents. Int Endod J 2000;33:19-24. google scholar