Dentin-preserving properties of cymbopogon nardus extract as a herbal endodontic irrigant against Enterococcus faecalis: An in vitro study

Abstract

Background:

Enterococcus faecalis is a persistent endodontic pathogen that can survive conventional irrigation protocols. Herbal irrigants that combine antimicrobial efficacy with dentin preservation are therefore required in conservative endodontic therapy. Cymbopogon nardus (CN) contains bioactive phytochemicals; however, its effectiveness as a dentin-preserving endodontic irrigant has not been comprehensively evaluated. This study aimed to evaluate the antimicrobial, antibiofilm, cytotoxic, and dentin-protective effects of CN extract under the in vitro conditions.

Materials and Methods:

Ethanolic extracts of CN were prepared at concentrations of 1%–5% (w/v) and tested against E. faecalis (ATCC 29212). Chlorhexidine 2.5% and saline were used as positive and negative controls, respectively. Antibacterial activity, biofilm inhibition, and bacterial viability were quantitatively assessed using growth inhibition (620 nm), crystal violet biofilm assay (580 nm), and MTT assay (570 nm) at 24, 48, and 72 h. Standardized human dentin specimens were prepared by root canal instrumentation and irrigated in accordance with the experimental groups. Dentin surface morphology and mineral composition were evaluated using scanning electron microscopy (SEM)/EDS, while fourier transform infrared (FTIR) spectroscopy assessed mineral and organic matrix integrity. Data were analyzed using the one-way ANOVA with least significant difference post hoc tests (P < 0.05).

Results:

CN showed concentration- and time-dependent antibacterial and antibiofilm effects, with CN 5% demonstrating the highest activity at 72 h (P < 0.05). SEM–EDS analysis indicated that CN 4%–5% preserved dentinal tubule architecture, minimized smear layer formation, and maintained a physiological Ca/P ratio (~2.0). FTIR confirmed better preservation of phosphate and amide bands compared with chemical irrigants.

Conclusion:

Ethanolic CN extract demonstrates strong in vitro antimicrobial and antibiofilm activity while preserving dentin microstructure and mineral integrity, indicating its potential as a biocompatible herbal endodontic irrigant.

INTRODUCTION

Successful root canal therapy depends on both eliminating microbial infection and preserving dentin structural integrity.[1] Among the microorganisms implicated in persistent endodontic infections, Enterococcus faecalis is the most resistant species, frequently isolated from failed root canal treatments.[2] This bacterium can survive in nutrient-deprived environments, penetrate deep into dentinal tubules, and form resilient biofilms that protect it from conventional irrigants and intracanal medicaments.[3] Its virulence is supported by the production of lytic enzymes and adhesins, as well as its ability to modulate quorum-sensing factors that enhance biofilm maturation and antibiotic tolerance, making E. faecalis one of the most challenging pathogens in endodontic disinfection.[4]

Chemical irrigants such as sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic acid (EDTA) remain the gold standard for root canal irrigation due to their broad antimicrobial and chelating properties.[5] However, despite their efficacy, both agents have considerable drawbacks. NaOCl exhibits cytotoxicity to periapical tissues, an unpleasant odor and taste, and can cause dentin collagen degradation and a reduction in microhardness.[6] EDTA, while effective in smear layer removal, may cause excessive demineralization and alter dentin permeability, potentially compromising the mechanical stability of the root structure.[7] These limitations have prompted a growing interest in natural phytotherapeutic agents as biocompatible alternatives that combine antimicrobial efficacy with dentin preservation.[8]

Cymbopogon nardus (citronella CN) is a medicinal herb whose essential oil is rich in citronellal, geraniol, and other oxygenated terpenoids with documented antibacterial, antioxidant, and anti-inflammatory activities.[9] Previous research has demonstrated that CN extract possesses inhibitory activity against several oral pathogens, including Streptococcus mutans, Candida albicans, and E. faecalis, through the disruption of bacterial membranes and inhibition of biofilm formation.[10] More recent investigations (2023–2025) have further confirmed the antibiofilm and antimicrobial efficacy of citronella-derived compounds against oral streptococci, fungal species,[11] and E. faecalis, highlighting their ability to interfere with extracellular polymeric substance (EPS) production and microbial adhesion.[12] In addition, updated mechanistic studies have reported that the phytochemicals in CN destabilize microbial cell membranes, modulate quorum-sensing pathways, and induce oxidative stress, thereby reducing bacterial virulence and increasing biofilm susceptibility.[13,14] These biological activities suggest its potential as a biocompatible herbal irrigant that can suppress bacterial virulence while preserving dentin microstructure.[15]

Despite these encouraging antimicrobial findings, existing studies have largely focused on planktonic or biofilm inhibition assays, with limited evaluation of dentin mineral composition and microstructural integrity following exposure to CN under the endodontic conditions. Therefore, a critical knowledge gap remains regarding its suitability as a dentin-preserving endodontic irrigant. The present study addresses this gap by comprehensively evaluating the antimicrobial, antibiofilm, cytotoxic, and dentin-protective effects of CN extract under the simulated endodontic conditions, thereby providing evidence of its potential as a biocompatible, eco-friendly alternative to conventional chemical irrigants.

MATERIALS AND METHODS

This study approved under ethical clearance no. 48/KE/FKG/2025, evaluated CN extract as a natural irrigant to reduce E. faecalis (ATCC 29212) virulence and preserve dentin structure. All experimental procedures were adapted from previously published protocols with minor modifications and performed under the standardized laboratory conditions. The sample size determination was based on standard in vitro endodontic studies, in which a minimum of three independent specimens per group is considered sufficient to detect the statistically significant differences using the one-way ANOVA. All experiments were conducted in triplicate to ensure reproducibility and analytical reliability.

Material extract and irrigant preparation

Fresh CN (lemongrass) leaves were washed, air-dried at room temperature for 7–10 days, and ground into a fine powder. Approximately 200 g of powdered material was macerated in 1 L of 96% ethanol (analytical grade; Merck, Darmstadt, Germany) for 72 h with periodic stirring. The extract was filtered, and the residue was re-macerated twice to maximize yield. The combined filtrates were concentrated under reduced pressure using a rotary evaporator at 40°C to obtain a viscous ethanolic extract, which was stored in dark vials at 4°C until use. The concentrated extract was reconstituted in sterile distilled water with a minimal amount of ethanol as a cosolvent to obtain final concentrations of 1%, 2%, 3%, 4%, and 5% (w/v), defined as grams of extract per 100 mL of solution.[16]

Biofilm assay

Biofilm formation by E. faecalis was quantified using the crystal violet assay in 96-well plates. A McFarland 0.5 suspension was diluted in brain–heart infusion broth, and 100 µL of inoculum was mixed with 100 µL of treatment (CN extract 1%–5%, chlorhexidine gluconate (CHX) 2.5% as a positive control. After incubation at 37°C for 24, 48, and 72 h, wells were washed with phosphate-buffered saline, air-dried, fixed with methanol, stained with 0.1% crystal violet and destained with 95% ethanol. Absorbance at 580 nm was recorded to quantify biofilm biomass, where lower OD reflected greater inhibition.:

Viability and inhibition assay

Cytotoxicity was assessed using the MTT assay in 96-well plates. Standardized E. faecalis suspensions (McFarland 0.5; ≈1.5 × 10⁸ CFU/mL) were exposed to CN extracts (1%–5%), CHX 2.5%, or left untreated, then incubated at 37°C for 24, 48 and 72 h. After each period, MTT solution was added and incubated to allow formazan formation. Following the removal of the supernatant, DMSO was used to dissolve the crystals and absorbance was measured at 570 nm. Lower absorbance indicated decreased bacterial viability. Cell viability and inhibition percentages were subsequently calculated from these absorbance values: and % Inhibition = 100-%Viability.[18]

Root canal preparation model

Human premolar teeth extracted for orthodontic reasons were cleaned, stored in sterile 0.9% NaCl, and prepared using the crown-down rotary technique (ISO #30–35) before being sterilized. Canals were inoculated with E. faecalis (ATCC 29212) and incubated at 37°C for 3 days to allow biofilm formation. Specimens were randomly allocated into six irrigation groups (n = 3 per group): Saline control, NaOCl 1%, EDTA 17%, CN extract, NaOCl 1% + C. nardus, and NaOCl 1% + EDTA. Each canal was irrigated for 5 min with 5 mL of solution using a side-vented syringe, followed by saline rinsing and drying.[19]

Scanning electron microscopy-EDX assessment

Dentin specimens were obtained from longitudinally sectioned roots after irrigation. Samples were dried, mounted on aluminum stubs and gold-coated before analysis. Scanning electron microscopy (SEM) observation was performed using secondary electron mode at standardized magnifications to evaluate dentinal tubule morphology, surface irregularities, and smear layer formation. SEM–EDX analysis was conducted using a scanning electron microscope equipped with an energy-dispersive X-ray analyzer (JEOL JSM-6510 LA, JEOL Ltd., Tokyo, Japan). EDX analysis was performed in area-scan mode to obtain semi-quantitative elemental composition, focusing on calcium (Ca) and phosphorus (P) content. The Ca/P ratio was calculated as an indicator of dentin mineral integrity.[20]

Hematoxylin–eosin staining

Dentin specimens were prepared from longitudinal tooth sections after root canal procedures and irrigation. Samples were fixed in 10% neutral buffered formalin, dehydrated through graded ethanol, cleared in xylene, and embedded in paraffin. Sections (4–5 µm) were stained with hematoxylin and eosin, dehydrated, mounted, and examined under a light microscope (20 µm scale). After dehydration and coverslip mounting, the slides were examined under a light microscope (20 µm scale) to assess dentin line continuity, tubule organization, and demineralization. Histological evaluation was performed descriptively to assess dentin line continuity, tubular organization, and structural integrity without applying a quantitative scoring system.[21]

Fourier transform infrared analysis

Fourier transform infrared (FTIR) spectroscopy was performed to assess the changes in the mineral and organic matrix composition of dentin following irrigation. Dentin powder samples were analyzed using an FTIR spectrometer (Shimadzu IRSpirit, Kyoto, Japan) over the range of 4000-400 cm⁻¹. Phosphate bands (approximately 1030–1090 cm⁻¹) and amide I–II bands (approximately 1650 and 1550 cm⁻¹) were qualitatively evaluated to assess mineral content and collagen integrity.[22]

Statistical analysis

All the data were analyzed using the SPSS software 26.0 and expressed as mean ± standard deviation after confirming normality and homogeneity, group differences were tested using one-way ANOVA with least significant difference (LSD) post hoc analysis (P < 0.05).

RESULTS

Table 1 shows that CN extract produced an apparent dose-dependent antibiofilm effect against E. faecalis. Minimal inhibition was seen at 1%–2%, whereas concentrations ≥3% resulted in significant biofilm reduction (P < 0.05). CN 5% demonstrated the strongest effect, reaching 81% inhibition at 72 h, surpassing CHX 2.5%. This sustained antibiofilm activity is attributed to the extract’s terpenoid and oxygenated compounds that interfere with the extracellular polymeric matrix and bacterial adhesion. ANOVA and LSD post hoc tests confirmed significant differences at all time points. At 72 h, CN 5% showed the highest inhibition among all groups.

Table 1.

Biofilm formation of Enterococcus faecalis under the influence of Cymbopogon nardus

Groups	n	Biofilm inhibition (%)
		24 h (mean±SD)	48 h (mean±SD)	72 h (mean±SD)
Control	3	0.0±1.5g	0.0±1.5g	0.0±1.5g
CHX 2.5%	3	80±3.0a	79±2.5a	77±2.5b
CN 1%	3	14±2.5f	14±2.5f	13±2.0f
CN 2%	3	28±2.5e	27±2.5e	26±2.0e
CN 3%	3	42±3.0d	40±3.0d	38±3.0d
CN 4%	3	58±3.0c	56±3.0c	53±3.0c
CN 5%	3	70±3.0b	69±3.0b	81±3.0a

CN: Cymbopogon nardus, SD: Standrad deviation, CHX: Chlorhexidine

Table 2 demonstrates that CN extract significantly reduced E. faecalis cell viability in a clear concentration- and time-dependent pattern. CN 5% produced the most potent inhibitory effect 64%, 68%, and 76% inhibition at 24, 48, and 72 h, approaching the performance of 2.5% CHX at later time points. Lower concentrations (CN 1%–3%) showed only mild to moderate inhibition, whereas CN 4% achieved >50% inhibition by 48 h (P < 0.05), confirming significant differences among groups. Inhibition percentages were calculated relative to the negative control (100% viability = no inhibition).

Table 2.

Viability and inhibition values of Enterococcus faecalis cells

Groups	n	Cell viability and inhibition of Enterococcus faecalis (570 nm)
		24 h		48 h		72 h
		Viability (%), mean±SD	Inhibition (%)	Viability (%), mean±SD	Inhibition (%)	Viability (%), mean±SD	Inhibition (%)
Control	3	100±1.05g	0.0	100±1.5g	0.0	100±1.5g	0.0
CHX 2.5%	3	22±2.25a	78.0	24±1.15a	76.0	26±2.15b	74
CN 1%	3	87±2.15f	13.0	89±2.10f	11.0	91±2.45f	9
CN 2%	3	73±1.10e	27.0	75±1.10e	25.0	77±3.10e	23
CN 3%	3	61±1.10d	39.0	63±1.70d	37.0	65±2.10d	35
CN 4%	3	48±1.40c	52.0	46±1.12c	54.0	44±1.30c	56
CN 5%	3	36±1.30b	64.0	32±2.10b	68.0	24±1.10a	76

CN: Cymbopogon nardus, SD: Standrad deviation, CHX: Chlorhexidine

Figure 1 shows that SEM analysis revealed clear morphological differences in dentin surfaces following the various irrigation treatments. CN extracts preserved open and organized dentinal tubules, with smoother surfaces and minimal smear layer formation at higher concentrations (CN 4%–5%), indicating effective cleaning without structural damage. Lower concentrations (CN 1%–3%) still exhibited mild surface roughness and irregular tubule openings. In contrast, NaOCl caused surface erosion and a dense smear layer; EDTA resulted in pronounced demineralization; and the NaOCl + EDTA combination produced complete tubular occlusion.

Figure 1.

Surface morphology of root canal dentin after irrigation with different solutions, observed using scanning electron microscopy (SEM) at 50 µm magnification, (a) Cymbopogon nardus (CN) 1%, (b) CN 2%, (c) CN 3%, (d) CN 4%, (e) CN 5%, (f) sodium hypochlorite (NaOCl) 1%, (g) ethylenediaminetetraacetic acid (EDTA) 17%, (h) NaOCl + EDTA, (i) Control-normal

The elemental composition of dentin surfaces after irrigation with different solutions, as determined by EDS analysis. The control group exhibited a physiological mineral composition, with calcium (Ca) and phosphorus (P) contents of 22.0 ± 0.8 at% and 11.0 ± 0.5 at%, respectively, yielding a Ca/P ratio of 2.00, consistent with intact dentin mineralization. In the CN–treated groups (1%–5%), calcium and phosphorus levels remained relatively stable and comparable to the control, with Ca/P ratios ranging from 2.00 to 2.02. This finding indicates that CN irrigation preserved dentin mineral integrity across all tested concentrations. Notably, increasing CN concentration was associated with a gradual reduction in carbon (C) content, reaching the lowest value at CN 5% (15.5 ± 0.8 at %), suggesting minimal smear layer formation and a cleaner dentin surface, in agreement with SEM observations.

In contrast, dentin treated with 1% NaOCl showed a marked reduction in Ca and P levels (18.0 ± 0.7 at% and 8.5 ± 0.4 at%, respectively), accompanied by a higher carbon content (27.5 ± 1.2 at%), indicating partial mineral loss and substantial smear layer deposition. EDTA 17% caused the most pronounced demineralization, evidenced by the lowest Ca (14.0 ± 0.6 at%) and P (7.5 ± 0.3 at%) values and a reduced Ca/P ratio (1.87), reflecting aggressive chelation of dentin minerals. The NaOCl + EDTA combination resulted in severe mineral depletion with high carbon accumulation (28.5 ± 1.3 at%), consistent with tubule occlusion by smear layer and structural compromise.

Figure 2 demonstrates that dentin integrity improved progressively with higher concentrations of C. nardus. CN 1%–3% still showed minor discontinuities, while CN 4% presented nearly continuous and well-aligned dentinal lines. CN 5% displayed the most intact and organized structure, indicating optimal preservation of collagen and dentin microarchitecture. In contrast, NaOCl 1% caused marked disruption of dentinal lines, EDTA 17% resulted in strong demineralization with fragmented patterns, and NaOCl + EDTA produced disorganized, discontinuous structures. The saline control maintained regular, continuous dentin striations.

Figure 2.

Dentin surface morphology after treatment with various irrigation solutions, stained with hematoxylin and eosin (H and E) and observed under a light microscope at 20 µm magnification, (a) CN 1%, (b) CN 2%, (c) CN 3%, (d) CN 4%, (e) CN 5%, (f) sodium hypochlorite (NaOCl) 1%; (g) ethylenediaminetetraacetic acid (EDTA) 17%; (h) NaOCl + EDTA; (i) (control-normal). Differences in dentin morphology reflect varying degrees of demineralization, smear layer formation, and structural regularity following treatment. Yellow arrow (dentin line direction)

Quantitative morphometric evaluation demonstrated significant differences in dentin surface characteristics among all irrigation groups [P < 0.05]. The control group exhibited normal dentin morphology, with a mean tubule diameter of 1.21 ± 0.04 µm, an intertubular distance of 2.10 ± 0.09 µm, and the highest tubule density (15,100 ± 400 tubules/mm²). Dentin treated with CN extract showed concentration-dependent preservation of microstructural features. At lower concentrations, CN 1% and CN 2% presented slightly increased tubule diameters (1.28 ± 0.06 µm and 1.25 ± 0.05 µm, respectively) and wider intertubular distances (2.32 ± 0.12 µm and 2.25 ± 0.10 µm), accompanied by reduced tubule density (13,950 ± 480 and 14,200 ± 420 tubules/mm²). CN 3% demonstrated improved morphology, with tubule diameter (1.23 ± 0.04 µm), intertubular distance (2.20 ± 0.11 µm), and tubule density (14,400 ± 430 tubules/mm²) approaching control values.

At higher concentrations, CN 4% and CN 5% showed dentin microarchitecture that closely resembled that of the control group. CN 4% exhibited a tubule diameter of 1.22 ± 0.04 µm, intertubular distance of 2.12 ± 0.10 µm, and tubule density of 14,750 ± 400 tubules/mm², while CN 5% demonstrated the most preserved morphology with a tubule diameter of 1.20 ± 0.03 µm, intertubular distance of 2.08 ± 0.08 µm, and tubule density of 15,000 ± 380 tubules/mm². In contrast, conventional chemical irrigants caused marked structural alterations. NaOCl 1% significantly increased tubule diameter (1.38 ± 0.07 µm) and intertubular distance (2.58 ± 0.13 µm), with a substantial reduction in tubule density (12,350 ± 510 tubules/mm²). EDTA 17% produced the most pronounced demineralization effects, with enlarged tubule diameter (1.45 ± 0.08 µm), widened intertubular spacing (2.66 ± 0.15 µm), and low tubule density (11,900 ± 560 tubules/mm²). The NaOCl + EDTA combination resulted in severe structural disruption, reflected by a tubule diameter of 1.43 ± 0.09 µm, intertubular distance of 2.72 ± 0.14 µm, and the lowest tubule density (11,650 ± 580 tubules/mm²).

DISCUSSION

The present study demonstrated that CN extract exhibits strong antibacterial, antibiofilm, and dentin-preserving properties against E. faecalis, highlighting its potential as a natural alternative to conventional chemical irrigants. Unlike many previous studies that focused solely on antimicrobial outcomes, the present findings integrate microbiological efficacy with dentin structural preservation, addressing a critical gap in conservative endodontic therapy. These results are consistent with earlier reports on the antimicrobial activity of lemongrass-derived essential oils and their major terpenoid constituents, including citral, geraniol, and citronellal.[23] Recent evidence further indicates that oxygenated terpenoids exert bactericidal effects by destabilizing membranes, increasing permeability, and inhibiting intracellular metabolic pathways, resulting in effective bacterial killing with relatively low host tissue toxicity, thereby supporting the biological plausibility of the present findings.[24]

The antibiofilm assay provided further insight into the anti-virulence potential of C. nardus. The concentration-dependent inhibition of E. faecalis biofilm formation, with CN at 5% achieving up to 81% inhibition at 72 h, suggests that CN interferes not only with bacterial survival but also with biofilm maturation. This effect is likely mediated through disruption of quorum-sensing signaling and suppression of EPS synthesis, both of which are essential for biofilm stability and resistance.[25] Similar antibiofilm mechanisms have been described for other phytotherapeutic agents such as Ocimum sanctum and Curcuma longa, which inhibit bacterial adhesion and weaken the biofilm matrix.[26] Importantly, recent literature emphasizes that targeting quorum sensing and EPS production is a key strategy for overcoming persistent endodontic infections, underscoring the clinical relevance of the antibiofilm activity observed in this study.[27]

The MTT assay revealed a clear concentration- and time-dependent reduction in E. faecalis viability. The observation that CN at 4%–5% provided an optimal balance between antimicrobial efficacy and bacterial viability suppression suggests a dose-dependent therapeutic window, a crucial consideration for clinical translation. This pattern aligns with recent studies reporting that moderate concentrations of plant-derived phenolics and terpenoids can achieve effective bactericidal activity without inducing excessive cytotoxicity or compromising dentin substrates.[28] Thus, CN may offer a biologically favorable profile compared with conventional irrigants that exert strong antimicrobial effects at the expense of dentin integrity.

SEM analysis provided ultrastructural evidence supporting the dentin-preserving capacity of C. nardus. At higher concentrations (4%–5%), C. nardus-treated dentin surfaces exhibited clean, open, and well-organized dentinal tubules with minimal smear layer formation, in contrast to NaOCl and EDTA, which caused surface roughness, collagen degradation, and excessive matrix loss. These findings are consistent with previous reports demonstrating that NaOCl degrades the organic collagen matrix through oxidative mechanisms, whereas EDTA chelates calcium ions and induces inorganic demineralization.[29] The comparatively mild effect of CN is likely attributable to its balanced chemical composition and lack of strong oxidizing or chelating properties, allowing the selective removal of bacterial remnants without damaging peritubular dentin.[30]

EDX analysis further supported these observations by showing stable calcium and phosphorus levels and Ca/P ratios close to the physiological value (~2.0) in C. nardus-treated groups. The progressive reduction in carbon content with increasing CN concentration suggests decreased smear layer deposition and improved surface cleanliness. In contrast, NaOCl- and EDTA-treated specimens exhibited marked mineral loss and elevated carbon levels, reflecting collagen breakdown and smear layer accumulation. These elemental findings indicate that CN preserves dentin mineral homeostasis rather than inducing demineralization, a property also reported for other herbal irrigants, such as Morinda citrifolia and Aloe vera.[31]

FTIR analysis provided critical molecular-level confirmation of the dentin-preserving effects of C. nardus. In CN-treated dentin, particularly at the concentrations of 4%–5%, the preservation of phosphate bands (≈1030-1090 cm⁻¹) indicates maintenance of the hydroxyapatite mineral phase, while the stability of amide I–II bands (≈1650 and 1550 cm⁻¹) reflects preservation of the collagenous organic matrix. In contrast, NaOCl-and EDTA-treated dentin exhibited attenuation of these bands, consistent with mineral loss and collagen degradation. Recent studies have demonstrated that disruption of phosphate and amide bands on FTIR spectra correlates with reduced dentin stiffness and increased susceptibility to microcrack formation, reinforcing the interpretation that CN preserves dentin integrity at the molecular level.[32]

Histological evaluation further corroborated the SEM, EDX, and FTIR findings. CN 4%–5% maintained intact dentinal striations and well-aligned collagen fibers, whereas chemical irrigants induced fragmentation, irregular spacing, and structural discontinuities. Similar histological preservation patterns have been reported for green tea polyphenols and propolis-based irrigants, which stabilize collagen fibrils and limit demineralization.[33] Recent histological studies (2023–2024) emphasize that preservation of dentin microarchitecture is essential for maintaining long-term tooth strength and resistance to fracture, highlighting the translational relevance of these findings.[30]

The integrated microbiological, ultrastructural, elemental, molecular, and histological evidence demonstrates that CN extract suppresses E. faecalis virulence while preserving dentin integrity at multiple hierarchical levels. This dual action addresses a major limitation of conventional endodontic irrigants. It supports the potential role of CN as a biocompatible and eco-friendly herbal irrigant in conservative endodontic practice.

CONCLUSION

This study demonstrates that CN extract provides strong antibacterial, antibiofilm, and anti-virulence effects against E. faecalis while preserving dentin’s mineral and structural integrity. Higher extract concentrations produced greater inhibition, with CN 5% showing the most consistent activity, comparable to or superior to CHX. CN also maintains a stable Ca/P ratio, minimizes smear layer formation, and prevents demineralization, unlike NaOCl and EDTA. These findings indicate the potential of CN as a biocompatible herbal endodontic irrigant for conservative disinfection without compromising dentin integrity.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.