Effect of Azadirachta indica Paste on Lactobacillus rhamnosus in Dentinal Tubules and Its Cytotoxicity for Dental Pulp Stem Cells
Abstract
Objectives: Azadirachta indica var. siamensis Valeton, also known as neem, possesses various beneficial biological properties, including antibacterial activity. Neem paste (NZ-paste) was developed to enhance its application. This study aimed to investigate the antibacterial activity of NZ-paste against Lactobacillus rhamnosus (L. rhamnosus), its penetration ability, and its cytotoxicity for human dental pulp stem cells (hDPSCs).
Materials and Methods: The active compounds of the neem extract were identified using high-performance liquid chromatography (HPLC). The rheological and physicochemical characteristics of 20mg/g NZ-paste were evaluated by pH measurement, penetration testing, and rheometer analysis. The antibacterial activity of NZ-paste against L. rhamnosus was assessed using agar disc diffusion and live/dead BacLight® assays in dentin, and confirmed by confocal laser stereomicroscopy (CLSM). The methyl thiazolyl tetrazolium assay was used to determine its cytotoxicity for hDPSCs.
Results: HPLC analysis identified azadirachtin, quercetin, nimbin, and nimbidin as the main bioactive compounds. NZ-paste had a smooth, fluid-like texture, was water-soluble, and exhibited a pH of 7.09. Although its inhibition zone was comparable to that of Ca(OH)₂, NZ-paste demonstrated superior antibacterial efficacy in the dentinal tubule invasion model, as indicated by the lowest live/dead ratio confirmed by CLSM. NZ-paste at concentrations up to 10mg/mL did not affect hDPSC viability.
Conclusion: The novel NZ-paste exhibits antibacterial activity against L. rhamnosus, with suitable penetration ability and favorable biocompatibility with hDPSCs at low concentrations.
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11. Mahapatra S, Young CY, Kohli M, Karnes RJ, Klee EW, Holmes MW, et al. Antiangiogenic effects and therapeutic targets of Azadirachta indica leaf extract in endothelial cells. Evid Based Complement Alternat Med. 2012;2012:303019.
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13. Gupta Y, Sharma D, Sharma Y. Bactericidal activity of different parts of Azadirachta indica on probiotic microbes. Asian J Pharm Clin Res. 2016;9(1):50-3.
14. Tasanarong P, Patntirapong S, Aupaphong V. The inhibitory effect of a novel neem paste against cariogenic bacteria. J Clin Exp Dent. 2021 Nov;13(11):e1083-8.
15. Arici M, Ersoz NB, Toker ÖS, Yilmaz MT, Cankurt H, Durak MZ, et al. Microbiological, steady, and dynamic rheological characterization of boza samples: temperature sweep tests and applicability of the Cox-Merz rule. Turk J Agric For. 2014;38(3):377-87.
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18. Shah S, Venkataraghavan K, Choudhary P, Mohammad S, Trivedi K, Shah SG. Evaluation of antimicrobial effect of azadirachtin plant extract (Soluneem (™)) on commonly found root canal pathogenic microorganisms (viz. Enterococcus faecalis) in primary teeth: A microbiological study. J Indian Soc Pedod Prev Dent. 2016 Jul;34(3):210-6.
19. Usman A, Fitzsimmons-Thoss V, Tawfike A. Anti-bacterial, anti-oxidant and cytotoxic activities of nimbin isolated from African Azadirachta indica seed oil. Adv J Chem. 2020;2(2):81-90.
20. Jaisinghani RN. Antibacterial properties of quercetin. Microbiol. Res. 2017 May;8(1):6877.
21. Valek Zulj L, Martinez S. Copper corrosion inhibition by Azadirachta indica leaves extract in 0.5 M sulphuric acid. Mater Lett. 2007 Jan;61(1):148-51.
22. Nguyen TLA, Bhattacharya D. Antimicrobial activity of quercetin: an approach to its mechanistic principle. Molecules. 2022 Apr;27(8):2494.
23. Mackenzie CG, Mackenzie JB, Beck P. The effect of pH on growth, protein synthesis, and lipid-rich particles of cultured mammalian cells. J Biophys Biochem Cytol. 1961 Jan;9(1):141-56.
24. Kok FS, Muhamad II, Lee CT, Razali F, Pa'e N, Shaharuddin S. Effects of pH and temperature on the growth and β -glucosidase activity of Lactobacillus rhamnosus NRRL 442 in anaerobic fermentation. Int Rev of Chem Eng. 2012 May;4(3):293-9.
25. Zand V, Mokhtari H, Hasani A, Jabbari G. Comparison of the penetration depth of conventional and nano-particle calcium hydroxide into dentinal tubules. Iran Endod J. 2017;12(3):366
26. Manca ML, Manconi M, Meloni MC, Marongiu F, Allaw M, Usach I, et al. Nanotechnology for natural medicine: formulation of neem oil loaded phospholipid vesicles modified with argan oil as a strategy to protect the skin from oxidative stress and promote wound healing. Antioxidants (Basel). 2021 Apr;10(5):670.
27. Lin S, Tsesis I, Zukerman O, Weiss EI, Fuss Z. Effect of electrophoretically activated calcium hydroxide on bacterial viability in dentinal tubules–in vitro. Dent Traumatol. 2005 Feb;21(1):42-5.
28. Lin S, Zuckerman O, Weiss EI, Mazor Y, Fuss Z. Antibacterial efficacy of a new chlorhexidine low-release device to disinfect dentinal tubules. J Endod. 2003 Jun;29(6):416-8.
29. Bhardwaj A, Ballal S, Velmurugan N. Comparative evaluation of the antimicrobial activity of natural extracts of Morinda citrifolia, papain and aloe vera (all in gel formulation), 2% chlorhexidine gel and calcium hydroxide, against Enterococcus faecalis: An in vitro study. J Conserv Dent. 2012 Jul;15(3):293-7.
30. Phan TN, Buckner T, Sheng J, Baldeck JD, Marquis RE. Physiologic actions of zinc related to inhibition of acid and alkali production by oral streptococci in suspensions and biofilms. Oral Microbiol Immunol. 2004 Feb;19(1):31-8.
31. Zarei M, Kazemi Z, Javidi M, Shayani Rad M, Karimpour S, Forghani M. Comparison of tubular penetration of nano ZnO, AH26 and kerr pulp canal sealer EWT using scanning electron microscope. J Dent Mater Tech 2020;9(3):147-51.
32. Anila B, Murali H, Cheranjeevi J, Kapil RS. Lesion sterilization and tissue repair (LSTR): A review. J Sci Dent. 2020 Aug;4(2):49-55.
33. Tabari K, Hosseinpour S, Parashos P, Khozestani PK, Rahimi HM. Cytotoxicity of selected nanoparticles on human dental pulp stem cells. Iran Endod J. 2017;12(2):137-42.
34. Pati R, Das I, Mehta RK, Sahu R, Sonawane A. Zinc oxide nanoparticles exhibit genotoxic, clastogenic, cytotoxic and actin depolymerization effects by inducing oxidative stress responses in macrophages and adult mice. Toxicol Sci. 2016 Apr;150(2):454-72.
35. Raghavendra G, Mahija D, Sreevani T, Yamala A, Bindu PH. Cell viability studies of green synthesised ZnO nanoparticles for antibacterial properties. Am J Mater Synth Process. 2017;2(5):56-60.
36. Kushwaha P, Ahmad N, Dhar YV, Verma A, Haldar S, Mulani FA, et al. Estrogen receptor activation in response to azadirachtin a stimulates osteoblast differentiation and bone formation in mice. J Cell Physiol. 2019 Dec;234(12):23719-35.
37. Kushwaha P, Khedgikar V, Haldar S, Gautam J, Mulani FA, Thulasiram HV, et al. Azadirachta indica triterpenoids promote osteoblast differentiation and mineralization in vitro and in vivo. Bioorg Med Chem Lett. 2016 Aug;26(15):3719-24.
38. Robertson J, McGoverin C, Vanholsbeeck F, Swift S. Optimisation of the protocol for the LIVE/DEAD® BacLightTM bacterial viability kit for rapid determination of bacterial load. Front Microbiol. 2019 Apr;10:801.
2. World Health Organization. Global oral health status report: towards universal health coverage for oral health by 2030. Available at: www.who.int/publications/i/item/9789240061484/Accessed November 18, 2022.
3. Centers for Disease Control and Prevention. Oral Health Surveillance Report: Trends in Dental Caries and Sealants, Tooth Retention, and Edentulism, United States, 1999–2004 to 2011–2016. Atlanta: Centers for Disease Control and Prevention, 2019.
4. Mohammadi Z, Dummer PM. Properties and applications of calcium hydroxide in endodontics and dental traumatology. Int Endod J. 2011 Aug;44(8):697-730.
5. Cwikla SJ, Bélanger M, Giguère S, Progulske-Fox A, Vertucci FJ. Dentinal tubule disinfection using three calcium hydroxide formulations. J Endod. 2005 Jan;31(1):50-2.
6. Tasanarong P, Dechatiwongse NAT, Techanitiswad T, Koontongkaew S. Reduction of viable bacteria in dentinal tubules treated with a novel medicament (Z-Mix). J Dent Sci. 2016 Dec;11(4):419-26.
7. Kneist S, Schmidt F, Callaway A, Willershausen B, Rupf S, Wicht M, et al. Diversity of Lactobacillus species in deep carious lesions of primary molars. Eur Arch Paediatr Dent. 2010 Aug;11(4):181-6.
8. Lima KC, Coelho LT, Pinheiro IVA, Rocas IN, Siqueira JF. Microbiota of dentinal caries as assessed by reverse-capture checkerboard analysis. Caries Res. 2011 Dec;45(1):21-30.
9. American Academy of Pediatric Dentistry. Pulp therapy for primary and immature permanent teeth. The reference manual of pediatric dentistry. Chicago, III: American Academy of Pediatric Dentistry. 2022; 415-23.
10. Santos PSD, Pedrotti D, Braga MM, Rocha RO, Lenzi TL. Materials used for indirect pulp treatment in primary teeth: a mixed treatment comparisons meta-analysis. Braz Oral Res. 2017 Dec;18(31): e101.
11. Mahapatra S, Young CY, Kohli M, Karnes RJ, Klee EW, Holmes MW, et al. Antiangiogenic effects and therapeutic targets of Azadirachta indica leaf extract in endothelial cells. Evid Based Complement Alternat Med. 2012;2012:303019.
12. Alqahtani SM. Antimicrobial efficacy of neem and liquorice with chlorhexidine on Streptococcus sanguis, Streptococcus mutans, Lactobacillus and Actinomyces naeslundii–An in vitro study. bioRxiv. 2020 Sep;2020-9.
13. Gupta Y, Sharma D, Sharma Y. Bactericidal activity of different parts of Azadirachta indica on probiotic microbes. Asian J Pharm Clin Res. 2016;9(1):50-3.
14. Tasanarong P, Patntirapong S, Aupaphong V. The inhibitory effect of a novel neem paste against cariogenic bacteria. J Clin Exp Dent. 2021 Nov;13(11):e1083-8.
15. Arici M, Ersoz NB, Toker ÖS, Yilmaz MT, Cankurt H, Durak MZ, et al. Microbiological, steady, and dynamic rheological characterization of boza samples: temperature sweep tests and applicability of the Cox-Merz rule. Turk J Agric For. 2014;38(3):377-87.
16. International Organization for Standardization. ISO 10993-5: 2009-Biological evaluation of medical devices- Part 5: Tests for in vitro cytotoxicity. http://www.iso.org/standard/36406.html
17. Rex KGR, Pavithra V, Bhavya R. Isolation and purification of nimbidin from Azadirachta indica of western ghats and evaluate antioxident, anti-bacterial and anti-diabetic properties. Int J Pharm Sci Res. 2021;12(7):3975-84.
18. Shah S, Venkataraghavan K, Choudhary P, Mohammad S, Trivedi K, Shah SG. Evaluation of antimicrobial effect of azadirachtin plant extract (Soluneem (™)) on commonly found root canal pathogenic microorganisms (viz. Enterococcus faecalis) in primary teeth: A microbiological study. J Indian Soc Pedod Prev Dent. 2016 Jul;34(3):210-6.
19. Usman A, Fitzsimmons-Thoss V, Tawfike A. Anti-bacterial, anti-oxidant and cytotoxic activities of nimbin isolated from African Azadirachta indica seed oil. Adv J Chem. 2020;2(2):81-90.
20. Jaisinghani RN. Antibacterial properties of quercetin. Microbiol. Res. 2017 May;8(1):6877.
21. Valek Zulj L, Martinez S. Copper corrosion inhibition by Azadirachta indica leaves extract in 0.5 M sulphuric acid. Mater Lett. 2007 Jan;61(1):148-51.
22. Nguyen TLA, Bhattacharya D. Antimicrobial activity of quercetin: an approach to its mechanistic principle. Molecules. 2022 Apr;27(8):2494.
23. Mackenzie CG, Mackenzie JB, Beck P. The effect of pH on growth, protein synthesis, and lipid-rich particles of cultured mammalian cells. J Biophys Biochem Cytol. 1961 Jan;9(1):141-56.
24. Kok FS, Muhamad II, Lee CT, Razali F, Pa'e N, Shaharuddin S. Effects of pH and temperature on the growth and β -glucosidase activity of Lactobacillus rhamnosus NRRL 442 in anaerobic fermentation. Int Rev of Chem Eng. 2012 May;4(3):293-9.
25. Zand V, Mokhtari H, Hasani A, Jabbari G. Comparison of the penetration depth of conventional and nano-particle calcium hydroxide into dentinal tubules. Iran Endod J. 2017;12(3):366
26. Manca ML, Manconi M, Meloni MC, Marongiu F, Allaw M, Usach I, et al. Nanotechnology for natural medicine: formulation of neem oil loaded phospholipid vesicles modified with argan oil as a strategy to protect the skin from oxidative stress and promote wound healing. Antioxidants (Basel). 2021 Apr;10(5):670.
27. Lin S, Tsesis I, Zukerman O, Weiss EI, Fuss Z. Effect of electrophoretically activated calcium hydroxide on bacterial viability in dentinal tubules–in vitro. Dent Traumatol. 2005 Feb;21(1):42-5.
28. Lin S, Zuckerman O, Weiss EI, Mazor Y, Fuss Z. Antibacterial efficacy of a new chlorhexidine low-release device to disinfect dentinal tubules. J Endod. 2003 Jun;29(6):416-8.
29. Bhardwaj A, Ballal S, Velmurugan N. Comparative evaluation of the antimicrobial activity of natural extracts of Morinda citrifolia, papain and aloe vera (all in gel formulation), 2% chlorhexidine gel and calcium hydroxide, against Enterococcus faecalis: An in vitro study. J Conserv Dent. 2012 Jul;15(3):293-7.
30. Phan TN, Buckner T, Sheng J, Baldeck JD, Marquis RE. Physiologic actions of zinc related to inhibition of acid and alkali production by oral streptococci in suspensions and biofilms. Oral Microbiol Immunol. 2004 Feb;19(1):31-8.
31. Zarei M, Kazemi Z, Javidi M, Shayani Rad M, Karimpour S, Forghani M. Comparison of tubular penetration of nano ZnO, AH26 and kerr pulp canal sealer EWT using scanning electron microscope. J Dent Mater Tech 2020;9(3):147-51.
32. Anila B, Murali H, Cheranjeevi J, Kapil RS. Lesion sterilization and tissue repair (LSTR): A review. J Sci Dent. 2020 Aug;4(2):49-55.
33. Tabari K, Hosseinpour S, Parashos P, Khozestani PK, Rahimi HM. Cytotoxicity of selected nanoparticles on human dental pulp stem cells. Iran Endod J. 2017;12(2):137-42.
34. Pati R, Das I, Mehta RK, Sahu R, Sonawane A. Zinc oxide nanoparticles exhibit genotoxic, clastogenic, cytotoxic and actin depolymerization effects by inducing oxidative stress responses in macrophages and adult mice. Toxicol Sci. 2016 Apr;150(2):454-72.
35. Raghavendra G, Mahija D, Sreevani T, Yamala A, Bindu PH. Cell viability studies of green synthesised ZnO nanoparticles for antibacterial properties. Am J Mater Synth Process. 2017;2(5):56-60.
36. Kushwaha P, Ahmad N, Dhar YV, Verma A, Haldar S, Mulani FA, et al. Estrogen receptor activation in response to azadirachtin a stimulates osteoblast differentiation and bone formation in mice. J Cell Physiol. 2019 Dec;234(12):23719-35.
37. Kushwaha P, Khedgikar V, Haldar S, Gautam J, Mulani FA, Thulasiram HV, et al. Azadirachta indica triterpenoids promote osteoblast differentiation and mineralization in vitro and in vivo. Bioorg Med Chem Lett. 2016 Aug;26(15):3719-24.
38. Robertson J, McGoverin C, Vanholsbeeck F, Swift S. Optimisation of the protocol for the LIVE/DEAD® BacLightTM bacterial viability kit for rapid determination of bacterial load. Front Microbiol. 2019 Apr;10:801.
| Issue | Vol 23 (Continuously Published Article-Based) | |
| Section | Original Article | |
| Keywords | ||
| Azadirachta Dental Caries Lactobacillus rhamnosus Microbial Viability | ||
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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
angsapsathit KT, Aupaphong V, Pechyen C, Tasanarong P. Effect of Azadirachta indica Paste on Lactobacillus rhamnosus in Dentinal Tubules and Its Cytotoxicity for Dental Pulp Stem Cells. Front Dent. 2026;23.
