INTRODUCTION:

Enterococcus faecalis is a gram-positive bacteria often found in persistent root canal infections, even after endodontic treatment¹. These bacteria can survive difficult conditions and form biofilms that protect colonies from antimicrobial agents, leading to chronic inflammation of the periapical tissue and often treatment failure ². E. faecalis can also infiltrate dentin tubules and persist in a dormant state, contributing to root canal damage such as resorption and chronic periapical inflammation ³. Understanding of roles E. faecalis It is essential to improve the success of endodontic therapy and prevent recurrent infections.

One of the main problems in endodontic care is the presence and spread of bacteria such as Enterococcus faecalis within the root canal, which often leads to treatment failure ⁴. These bacteria can survive in extreme conditions and form biofilms that protect them from irrigation and antimicrobial agents. Bacteria that are not wholly eliminated can spread to the dentin tubules and periapical tissue, causing reinfection, chronic inflammation, and resorption of root tissue ⁵. This phenomenon shows the need for more effective irrigation techniques and materials to target resistant bacteria and clean biofilms thoroughly⁶.

This study is essential in endodontics, especially in exploring natural irrigation solutions based on Moringa oleifera against Enterococcus faecalis infections that often cause root canal treatment failures. Moringa oleifera, which is known for its antibacterial and antioxidant properties, has the potential to be a safe and effective irrigation alternative compared to more aggressive chemicals ⁷. This study will evaluate how Moringa oleifera affects changes in the surface of the root canal, such as biofilm reduction and bacterial infiltration, as well as its impact on the survival of E. faecalis. The findings from this study could provide a more environmentally friendly and low-risk solution for patients while improving the success of endodontic therapy by better controlling persistent bacterial infections ⁸. The significance of this research also lies in developing irrigation methods that can be widely applied in dental practice to improve long-term clinical outcomes.

Sodium hypochlorite (NaOCl), EDTA, and chlorhexidine gluconate (CHX) are commonly used irrigation materials to kill Enterococcus faecalis⁹. However, CHX has drawbacks, such as tooth staining and high cost. Therefore, irrigation materials that are safer, less toxic, and still effective in antibacterial terms are needed ¹⁰. Moringa oleifera has been known to have antibacterial properties, especially against Gram-positive bacteria, and has the potential to be used as an irrigation ingredient ⁷. Previous research has shown that moringa leaves can help remineralize enamel and dentin and prevent erosion ¹¹. Still, there have been no specific studies on their effects on cell morphology, roughness, and the release of calcium ions in exposed root canals E. faecalis. This study aims to analyze the effect of moringa leaf irrigation solution on these factors on the surface of the dental root canal.

MATERIALS AND METHODS:

This study used 14 specimens of mandibular premolar teeth that were intact, caries-free, and had straight root canals. Enterococcus faecalis ATCC 29212, moringa leaf irrigation solution (Moringa oleifera), and CHX 2% were used in the study. The specimens were divided into 7 groups, namely 5 treatment groups with different concentrations of moringa leaf extract 1 positive control group (CHX 2%), and 1 negative control group. Each group required 1 sample, and the incubation time for all groups was set for 24 and 48 hours to evaluate the results.

Extraction and Irrigation Solutions Preparation :

The method of making irrigation solutions from moringa leaves (Moringa oleifera) begins with cleaning and drying the leaves, which are then crushed into fine powder. Moringa leaf powder is soaked in 96% ethanol for 24-48 hours and then filtered to separate the extract from the solid residue. The extract is evaporated using a rotary evaporator to remove ethanol solvents, resulting in a viscous extract. This extract is then dissolved in a sterile according to the desired concentration and stored in a sterile container until ready for use ¹².

Culture and toxicity cell assay:

Enterococcus faecalis cultures were performed on Mueller Hinton Agar (MHA) using the T scratch method, with zig-zag inoculation in a petri dish divided into three parts and incubated for 24 hours at 37°C under anaerobic conditions. Bacterial identification is carried out through Gram staining, where the bacterial smear is fixated and stained using crystal violet, iodine, alcohol, and safranin. Gram-positive E. faecalis will be violet, while Gram-negative bacteria will be pink after staining. The suspension of E. faecalis was then prepared by mixing a bacterial colony into a NaCl solution and equalizing it with a 0.5 McFarland solution (1.5 x 10⁸ CFU/mL). Gram staining is also used to assess the toxicity of the tested material against bacterial cells by observing morphological changes under a microscope after the staining process is complete ¹³.

Specimen Preparation:

A total of 14 intact, caries-free mandibular premolar teeth with straight root canals were used as specimens. Access to the pulp chamber is made with a diamond round bur, and the teeth are washed under running water to clean the remaining tissue²⁴. The apical part of the tooth is covered with red night and planted in plasticine, then soaked in a 500 mg amoxicillin solution for 24 hours. After immersion, the teeth are washed and inoculated with bacteria in BHI medium for 24-48 hours at 37°C²⁴. Root canal preparation is carried out with the crown down technique using Protaper SX, S1, S2, S3, F1, F2, and F3²² files⁵. Irrigation was carried out using moringa leaf irrigation solution at various concentrations (75%, 50%, 25%, 12.5%, 6.25%) for the treatment group, CHX 2% for positive control, and aqueous for negative control. The specimens were incubated at 37°C for 24 and 48 hours, and the teeth were divided into seven groups for examination in the two durations ¹⁴.

Calcium Ion Evaluation:

Assessment of calcium ion release from the root canal wall of the tooth was carried out by dividing the teeth vertically using a carborundum disk, resulting in two halves of the tooth. After cutting, the specimen is immersed in a saline solution to clean any tissue or debris that may be attached, then carefully dried so that no liquid affects the analysis. Specimens are prepared for analysis using Scanning Electron Microscopy (SEM) to scan the surface. Energy Dispersive X-ray Spectroscopy (EDX) is used to detect and measure the release of calcium ions. This examination identifies the distribution and release rate of calcium ions from the surface of the root canal wall after interaction with the test material ¹⁵.

Roughness Assessment:

The root canal's surface roughness was analyzed by vertically dividing the teeth using a carborundum disk, which was soaked in a saline solution. The surface roughness of the root canal was studied using Atomic Force Microscopy (AFM), which examined the changes in the surface of the dentin during demineralization. AFM works by touching a microscope on the surface of the dentin using a pyramidal tip made of Si3N4 (DNP-20; Veeco), and the device is controlled with the help of easy scan software ¹⁶.

Data Analysis

The results of the test data on the effect of moringa leaf irrigation solution (Moringa oleifera) on the release of calcium ions, the morphology of Enterococcus faecalis cells, and the surface roughness of the dental root canal were analyzed and processed using t-test and Kruskal-Wallis test with Statistical Product and Service Solution (SPSS) software.

RESULT:

This study observed the release of calcium ions from dental tissue using moringa leaf irrigation solution (Moringa oleifera) on 14 extracted premolar tooth specimens, with five treatment groups, positive control (CHX 2%), and negative control. After being tested with 24 and 48 hours of incubation time, the results showed that a 50% concentration of moringa leaf extract with 24 hours of incubation provided the best calcium ion release of 11.12%, while the concentration of 12.5% had the highest release of 20.78%. At 48-hour incubation, a concentration of 12.5% showed the lowest calcium ion release of 0.24%, and a concentration of 25% resulted in the highest release of 50.63%.

Table 1. Calcium ion release values are distributed from the root canal surface

Concentration	Calcium Value
Concentration	24 Hours	48 Hours
75%	16.89	28,95
50%	11,12	25,21
25%	14,31	50,63
12,5%	20,78	0,24
6,25%	18,01	36,06
CHX 2%	29,05	10,83

The results of SEM/EDX analysis showed that moringa leaf irrigation solution affected the release of calcium ions on the surface of the tooth root canal, with a concentration of 75% resulting in the highest calcium release after 24 hours (Figure 1), while a concentration of 50% showed an even release. After 48 hours (Figure 2), 75% and 25% concentrations still showed high calcium release, while concentrations of 50% and 12.5% showed lower erosion. CHX 2% results in the lowest calcium release at both times. Statistical analysis using the Kruskal-Wallis and t-test showed no significant difference in calcium release based on concentration or incubation time. The morphology of E. faecalis cells that experienced toxicity was also presented to facilitate comparison, but the statistical results showed no significant differences.

Table 2 reports the toxicity results of moringa leaf irrigation solution (Moringa oleifera) and CHX 2% against the cell morphology of Enterococcus faecalis bacteria after 24 and 48 h incubation. At 24-hour incubation, 25% and 50% concentrations showed high toxicity with a smaller cell area, while concentrations of 6.25% showed the lowest toxicity with a cell area of 230,435 μm². After 48 hours, the 75% concentration showed a significant increase in toxicity, with the cell area decreasing drastically to 54,643 μm², while the 50% concentration experienced a decrease in toxicity with a cell area of 533,207 μm². CHX 2% as a positive control showed consistently high toxicity at both incubation times. These results showed that moringa leaf extract was effective as an antibacterial, especially at a concentration of 50% after 48 hours of incubation.

Table 2. Toxicity evaluation of cell area of E. faecalis

Concentration	Cell Morphology (mm)
Concentration	24 h	48 h
75%	109.262	54.643
50%	96.731	533.207
25%	54.805	64.475
12,5%	134.596	102.773
6,25%	230.435	63.972
CHX 2%	141.462	333.397

Figure 3 shows the cell toxicity profile of Enterococcus faecalis bacteria after exposure to moringa leaf irrigation solution with a concentration of 50% at two different incubation times, namely 48 hours (figure a) and 24 hours (figure b). In Figure a (48 hours), the number of bacterial cells is less spread out in a stretched manner, indicating higher toxicity and more substantial antibacterial effectiveness after 48 hours of incubation. On the contrary, in Figure b (24 hours), the bacterial cells still look dense and numerous, indicating that at the time of 24-hour incubation, the toxicity of the moringa leaf irrigation solution has not reached its maximum effectiveness. These results showed that moringa leaf irrigation at a concentration of 50% was more effective in killing E. faecalis after 48 hours compared to 24 hours.

Figure 3. Bacterial Cell Toxicity Profile E. faecalis under the influence of moringa leaf irrigation solution 50%). A (48 hours) and B (24 hours)

Table 3 reports the results of the study showing changes in the surface roughness of the root canal after being given moringa leaf irrigation solution at various concentrations, measured based on the Roughness average (Ra) value. Surface roughness is presented as a percentage and depicted in a diagram for easy comparison. At 24-hour incubation, a concentration of 50% resulted in a change in roughness of 0.1095 μm. Meanwhile, after 48 hours of incubation, a concentration of 12.5% resulted in a significant decrease in roughness of 0.0905 μm. These results showed that the most effective antibacterial effect in lowering the surface roughness of the root canal occurred at a concentration of 12.5% after 48 hours of incubation.

Table 3. Roughness value of root canal extraction of moringa leaves based on incubation time

Concentration	Roughness Average (mm)
Concentration	24 h	48 h
75%	0,1425	0,24
50%	0,1095	0,22
25%	0,38	0,17
12,5%	0,255	0,0905
6,25%	0,187	0,0985
CHX 2%	0,185	0,18

Figure 4. AFM Profile of Root Canal Surface Roughness, concentration 50% a. 24 hours; b.48 hours. 30x Magnification

Figure 4 shows the surface roughness profile of the dental root canal using Atomic The results showed that the irrigation solution of moringa leaf (Moringa oleifera) with a concentration of 50% caused a change in the surface roughness of the dental root canal after 24 hours and 48 hours of incubation, with a lower roughness at 24 hours compared to 48 hours. AFM images show smoother surfaces at 24 hours and rougher surfaces at 48 hours, signaling increased surface degradation over time. Statistical analysis using the T-test showed that although there was a difference in the decrease in surface roughness, the difference was not statistically significant (p>0.05 = 0.380). Overall, the antibacterial activity of moringa leaf extract was more effective in reducing roughness and increasing toxicity to E. faecalis after 48 hours of incubation.

DISCUSSION:

This study showed that the irrigation solution of moringa leaf (Moringa oleifera) affected the release of calcium ions from dental tissue, with a concentration of 50% at 24-hour incubation, resulting in the best release of calcium ions of 11.12%. In comparison, the concentration of 12.5% showed the highest release of 20.78%. At 48-hour incubation, the concentration of 12.5% had the lowest calcium release of 0.24%, while the concentration of 25% experienced a significant increase in release to 50.63%. These results align with previous studies that reported that natural ingredients with antibacterial properties can affect the demineralization of complex tissues, including teeth ¹⁷. In addition, a longer incubation time also increases the effectiveness of irrigation, but it can lead to greater surface degradation ¹⁸.

The results showed that the moringa leaf irrigation solution affected the release of calcium ions on the surface of the tooth root canal, with a concentration of 75% resulting in the highest release after 24 and 48 hours, while the concentrations of 50% and 12.5% showed lower erosion. CHX 2% as a positive control resulted in the lowest calcium release at both times. Statistical analysis showed no significant difference in calcium release based on concentration or incubation time, although changes in the morphology of E. faecalis cells undergoing toxicity were observed. These findings are consistent with previous studies that show that natural ingredients often affect the release of ions from dental tissue, but the differences are not always statistically significant ¹⁹.

This study showed that Moringa oleifera and CHX 2% irrigation solution significantly affected the morphological toxicity of E. faecalis cells. Concentrations of 25% and 50% indicated higher toxicity at 24 hours, while after 48 hours, concentrations of 75% showed the most significant increase in toxicity. Figure 3 supports these results, with toxicity more potent at 48 hours than at 24 hours. These findings are consistent with previous studies that suggest that moringa leaf extract has effective antibacterial potential, especially after a longer incubation time.

Studies have demonstrated that Moringa oleifera possesses antimicrobial solid properties due to its bioactive compounds, including flavonoids, tannins, and phenolic acids, which are known to disrupt bacterial cell walls and inhibit bacterial growth ²⁰. In the context of dental research, the antibacterial effect of moringa is particularly effective against Gram-positive bacteria such as Enterococcus faecalis, a common pathogen in endodontic infections, often linked to its biofilm-forming ability and resistance to standard treatments ²¹.

Furthermore, the enhanced antibacterial activity observed after longer incubation periods can be attributed to the increased contact time between the active compounds in moringa and the bacterial cells, leading to more significant damage to the bacterial membrane and cellular components ²². This prolonged interaction may also allow for better penetration into bacterial biofilms, which are notoriously difficult to eradicate ²³. Previous research has similarly noted that prolonged exposure to natural plant extracts increases their efficacy against resistant bacterial strains, further supporting the effectiveness of moringa as an alternative or adjunctive treatment in combating resistant bacteria in dental infections ²⁴.

The results showed that moringa leaf irrigation solution (Moringa oleifera) affected the change in the surface roughness of the tooth root canal, with a concentration of 50% resulting in a change in roughness of 0.1095 μm after 24 hours of incubation. A concentration of 12.5% showed a significant decrease in roughness of 0.0905 μm after 48 hours. AFM images confirm that the surface of the root canal is smoother at 24 h and rougher at 48 h, which suggests surface degradation increases with incubation time. These findings align with previous research that indicates that plant extracts such as moringa leaves can serve as effective antibacterials ²⁵. Still, longer incubation times often increase the degradation of tooth tissue surfaces associated with ion release and morphological changes ²⁶.

CONCLUSION:

The conclusion of this study shows that moringa leaf irrigation solution (Moringa oleifera) effectively affects the release of calcium ions, the toxicity of Enterococcus faecalis cells, and changes in the surface roughness of the root canal. A concentration of 50% at 24 hours provides the best calcium release, while a concentration of 12.5% shows a significant decrease in roughness after 48 hours. In addition, a concentration of 75% showed the highest toxicity to E. faecalis at 48 hours. These results align with previous studies that showed the effectiveness of natural ingredients in influencing demineralization and cell toxicity, especially with longer incubation times.

ACKNOWLEDGMENTS:

This research is funded by the Directorate of Research, Technology, and Community Service, Directorate General of Higher Education, Research, and Technology, Ministry of Education, Culture, Research, and Technology, following the Fundamental Research Program Implementation Contract-Regular Number: 094/E5/PG.02.00.PL/2024

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interest.

REFERENCES:

1. Asmah N, Suniarti DF, Bachtiar EW, Margono DA, Gani BA. Chemical compounds Antibacterial of Citrus aurantifolia Ethanol Extract to Inhibit the Early Biofilm Formation and Growth of Enterococcus faecalis Root Canal Isolate. Research Journal of Pharmacy and Technology. 2022; 15(6): 2667-74. http://dx.doi.org/10.52711/0974-360X.2022.00446

2. Nugroho JJ, Kayla TA, Rachmuddin A. Antibacterial effectiveness of garlic (Allium sativum Linn.) extract nanoparticles against E. faecalis. Makassar Dental Journal 2024; 13(2): 184-87. http://dx.doi.org/10.4103/tdj.tdj_3_19

3. Oli AK, Javaregowda PK, Jain A, Kelmani CR. Mechanism Involved in Biofilm Formation of Enterococcus faecalis. Focus on Bacterial Biofilms: Intech Open; 2022. http://dx.doi.org/10.5772/intechopen.103949

4. Cancio V, Carvalho Ferreira Dd, Cavalcante FS, et al. Can the Enterococcus faecalis identified in the root canals of primary teeth be a cause of failure of endodontic treatment? Acta Odontologica Scandinavica. 2017; 75(6): 423-28. http://dx.doi.org/10.1080/00016357.2017.1328742

5. Kanarek P, Breza-Boruta B, Rolbiecki R. Microbial composition and formation of biofilms in agricultural irrigation systems-a review. Ecohydrology and Hydrobiology. 2023. http://dx.doi.org/10.1016/j.ecohyd.2023.10.004

6. Yao Y, Habimana O. Biofilm research within irrigation water distribution systems: Trends, knowledge gaps, and future perspectives. Science of the total environment. 2019; 673: 254-65. http://dx.doi.org/10.1016/j.scitotenv.2019.03.464

7. Dzuvor CK, Pan S, Amanze C, et al. Bioactive components from Moringa oleifera seeds: production, functionalities and applications–a critical review. Critical Reviews in Biotechnology. 2022; 42(2): 271-93. http://dx.doi.org/10.1080/07388551.2021.1931804

8. Nunes LP, Nunes GP, Ferrisse TM, et al. Antimicrobial photodynamic therapy in endodontic reintervention: A systematic review and meta-analysis. Photodiagnosis and Photodynamic Therapy. 2022; 39: 103014. http://dx.doi.org/10.1016/j.pdpdt.2022.103014

9. Ozkan HB, Cobankara FK, Sayin Z, Ozer F. Evaluation of the antibacterial effects of single and combined use of different irrigation solutions against intracanal enterococcus faecalis. Acta Stomatologica Croatica. 2020; 54(3): 250. http://dx.doi.org/10.15644/asc54/3/3

10. Bukhari S, Babaeer A. Irrigation in endodontics: a review. Current Oral Health Reports 2019; 6: 367-76. https://link.springer.com/article/10.1007/s40496-019-00241-6

11. Younis SH, Obeid RF, Ammar MM. Subsurface enamel remineralization by Lyophilized Moringa leaf extract loaded varnish. Heliyon. 2020; 6(9). http://dx.doi.org/10.1016/j.heliyon.2020.e05054

12. Soraya C, Batubara FY, Nasroen SL, Jakfar S, Gani BA. Role of Moringa oleifera irrigation solution on the cell metabolism change of Streptococcus mutans. Journal of Advanced Pharmaceutical Technology and Research. 2024; 15(3): 200-07. http://dx.doi.org/10.4103/JAPTR.JAPTR_442_23

13. Soraya C, Syafriza D, Gani BA. Antibacterial effect of Moringa oleifera gel to prevent the growth, biofilm formation, and cytotoxicity of Streptococcus mutans. Journal of International Dental and Medical Research. 2022; 15(3): 1053-61.

14. Darmawi I, Abidin T, Agusnar H, Gani BA. In Vitro Study of Irrigation solution of Chitosan Nanoparticles to Inhibit the Adhesion and Biofilm Formation of Enterococcus faecalis in the Root Canal. Research Journal of Pharmacy and Technology. 2022; 15(6): 2691-96. http://dx.doi.org/10.52711/0974-360X.2022.00450

15. Sastika DY, Abidin T, Agusnar H, Gani BA. Application of calcium hydroxide with vehicles relate to the pH change, calcium ion diffusion, roughness, and frequency of chemical compound in root canal. Research Journal of Pharmacy and Technology. 2022; 15(7): 2976-82. http://dx.doi.org/10.52711/0974-360X.2022.00496

16. Abidin T, Susilo D, Gani BA. The effectiveness of nano-chitosan high molecular 0.2% as irrigant agent against Enterococcus faecalis with passive ultrasonic irrigant. Journal of Conservative Dentistry and Endodontics. 2022; 25(1): 37-41. http://dx.doi.org/10.4103/jcd.jcd_437_21

17. El Gezawi M, Wölfle UC, Haridy R, Fliefel R, Kaisarly D. Remineralization, regeneration, and repair of natural tooth structure: influences on the future of restorative dentistry practice. ACS biomaterials science and engineering 2019; 5(10): 4899-919. http://dx.doi.org/10.1021/acsbiomaterials.9b00591

18. Chen N, Li X, Shi H, et al. Effect of biodegradable film mulching on crop yield, soil microbial and enzymatic activities, and optimal levels of irrigation and nitrogen fertilizer for the Zea mays crops in arid region. Science of the Total Environment. 2021; 776: 145970. http://dx.doi.org/10.1016/j.scitotenv.2021.145970

19. Noumbissi S, Scarano A, Gupta S. A literature review study on atomic ions dissolution of titanium and its alloys in implant dentistry. Materials. 2019; 12(3): 368. http://dx.doi.org/10.3390/ma12030368

20. Kashyap P, Kumar S, Riar CS, et al. Recent advances in Drumstick (Moringa oleifera) leaves bioactive compounds: Composition, health benefits, bioaccessibility, and dietary applications. Antioxidants. 2022; 11(2): 402. https://doi.org/10.3390/antiox11020402

21. Sopandani P, Iskandar BO, Ariwibowo T, Djamil MS. Antibacterial effects of Moringa oleifera leaf extract against Enterococcus faecalis in vitro. Scientific Dental Journal. 2020; 4(1): 16-20. http://dx.doi.org/10.4103/SDJ.SDJ_43_19

22. Wang S, Liu S, Hao G, et al. Antimicrobial activity and mechanism of isothiocyanate from Moringa oleifera seeds against Bacillus cereus and Cronobacter sakazakii and its application in goat milk. Food Control. 2022; 139: 109067. http://dx.doi.org/10.1016/j.foodcont.2022.109067

23. Hamzah H, Hertiani T, Pratiwi SUT, Nuryastuti T. Efficacy of quercetin against polymicrobial biofilm on catheters. Research Journal of Pharmacy and Technology. 2020; 13(11): 5277-82. https://doi.org/10.5958/0974-360X.2020.00923.3

24. Jubair N, Rajagopal M, Chinnappan S, Abdullah NB, Fatima A. Review on the antibacterial mechanism of plant‐derived compounds against multidrug‐resistant bacteria (MDR). Evidence‐Based Complementary and Alternative Medicine. 2021; 2021(1): 3663315. https://doi.org/10.1155/2021/3663315

25. Ilanko P, McDonnell PA, van Vuuren S, Cock IE. Interactive antibacterial profile of Moringa oleifera Lam. extracts and conventional antibiotics against bacterial triggers of some autoimmune inflammatory diseases. South African Journal of Botany. 2019; 124: 420-35. http://dx.doi.org/10.1016/j.sajb.2019.04.008

26. Aswin SK, Jothishwar S, Nayagam P, Priya G. Scaffolds for biomolecule delivery and controlled release-A Review. Research Journal of Pharmacy and Technology. 2018; 11(10): 4719-30. https://doi.org/10.52711/0974-360X.2022.00446

Received on 08.10.2024 Revised on 03.02.2025

Accepted on 05.04.2025 Published on 01.10.2025

Available online from October 04, 2025

Research J. Pharmacy and Technology. 2025;18(10):4709-4715.

DOI: 10.52711/0974-360X.2025.00677

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.