Efficacy of herbal irrigants versus conventional irrigants in smear layer removal: A Scanning electron microscopy study

Abstract

Aim:

The aim of this study was to compare the efficacy of herbal irrigants (Panchvalkal extract and grape seed extract) with conventional irrigants (5.25% sodium hypochlorite [NaOCl] and 17% ethylenediaminetetraacetic acid [EDTA]) in smear layer removal using scanning electron microscopy.

Materials and Methods:

This in vitro study was conducted on 48 extracted human maxillary central incisors randomly divided into three groups (n = 16): Group I – Panchvalkal extract (5%), Group II – grape seed extract (5%), and Group III – 5.25% NaOCl with 17% EDTA. Following standard biomechanical preparation and irrigation using side-vented needles, samples were sectioned longitudinally and examined under scanning electron microscopy at ×2000. Smear layer removal was assessed at coronal, middle, and apical levels. Data were analyzed using one-way ANOVA and Tukey’s post hoc test.

Results:

Statistically significant differences were observed among the groups (P < 0.05). Group III demonstrated the highest smear layer removal efficacy at coronal, middle, and apical levels, followed by Group I, whereas Group II showed the least effectiveness, particularly at the apical level. Smear layer removal was greatest at the coronal level and least at the apical level across all groups.

Conclusion:

Within the limitations of the present study, conventional irrigants (5.25% NaOCl with 17% EDTA) demonstrated superior smear layer removal efficacy compared to herbal irrigants. Panchvalkal extract showed moderate efficacy and may serve as a potential biocompatible alternative endodontic irrigant.

INTRODUCTION

The success of root canal therapy is determined by thorough debridement, effective microbial elimination, and the establishment of a fluid-tight seal. During root canal instrumentation, a smear layer is inevitably formed, consisting of dentin debris, pulp tissue remnants, microorganisms, and endotoxins that adhere to the canal walls and occlude dentinal tubules.[1,2] Morphologically, this amorphous layer is approximately 1–2 μm thick and consists of two distinct phases: a superficial film on the canal wall and deeper smear plugs that penetrate the dentinal tubules up to 40 μm. The presence of these components fundamentally alters the structural characteristics of the root canal dentin. This layer has been shown to adversely affect dentin permeability and compromise the penetration of irrigants, medicaments, and sealers into dentinal tubules.[3] The role of the smear layer in endodontic outcomes remains controversial. While some argue that it may temporarily block bacterial penetration into dentinal tubules, the majority of evidence suggests that its presence supports bacterial survival, reduces disinfection efficacy, and interferes with the bonding of root canal sealers.[4,5] Therefore, its removal is now widely recommended as an essential step in enhancing root canal disinfection and improving the sealing ability of obturation materials.[6,7]

Mechanistically, NaOCl exerts its tissue-dissolving effect through saponification and the formation of hypochlorous acid, which rapidly breaks down organic cellular components. Conversely, ethylenediaminetetraacetic acid (EDTA) induces calcium extraction from the dentinal hydroxyapatite matrix, effectively decalcifying the inorganic debris. Among the herbal alternatives, Panchvalkal extract possesses a high tannin and phenolic content, which is theorized to denature microbial proteins and exert a strong astringent effect on organic tissues. Similarly, grape seed extract is highly concentrated with oligomeric proanthocyanidins (OPCs), which actively cross-link dentinal collagen and disrupt bacterial cell membranes, potentially aiding in superficial debris clearance.

Standard chemical irrigants continue to be the primary approach for addressing the smear layer. Because of its strong tissue-dissolving capabilities and excellent antimicrobial properties, sodium hypochlorite (NaOCl) is the most frequently utilized irrigating solution in clinical practice.[8] While NaOCl is highly effective, it lacks the capacity to eliminate the inorganic constituents of the smear layer. Consequently, it must be supplemented with a chelator such as EDTA.[9] EDTA can clean mineralized debris by binding the calcium ions in the dentin, thus opening dentinal tubules.[10] NaOCl and EDTA are used in succession, which means they are used together to remove the smear layer completely.[11] Regardless of their effectiveness, these irrigants have significant drawbacks. Despite its widespread clinical acceptance, NaOCl presents notable disadvantages, including high cellular toxicity, an unpleasant odor, and an acrid taste. Furthermore, prolonged exposure or the utilization of high concentrations can induce detrimental alterations in the physical properties of dentin, ultimately diminishing its flexural strength.[12] Likewise, EDTA can result in dentin erosion when overused, and it is also not antimicrobial.[13,14] These drawbacks have promoted the need to identify other safeguarded, biocompatible, and environmentally friendly irrigants.

Herbal irrigants, owing to their antimicrobial, antioxidant, and biocompatible properties, are emerging as potential substitutes.[15] Panchvalkal extract is a traditional Ayurvedic formulation composed of the bark of five medicinal plants and exhibits astringent, antiseptic, antimicrobial, wound-healing, and anti-inflammatory properties.[16] Grape seed extract, derived from Vitis vinifera, is rich in polyphenolic compounds such as proanthocyanidins, which demonstrate strong antioxidant, antibacterial, and antifungal effects.[17] Furthermore, grape seed extract has shown the ability to disrupt microbial biofilms and improve dentin surface cleanliness. Even though chemical irrigants are unequivocally established as the gold standard, the pursuit of biocompatible alternatives holds substantial clinical significance. Exploring safer substitutes is particularly critical when managing patients with chemical sensitivities or when treating teeth with wide open apices, as the inadvertent periapical extrusion of highly cytotoxic solutions in these scenarios could trigger severe tissue necrosis. The smear layer removal efficacy of these herbal irrigants was evaluated and compared with a conventional irrigation regimen consisting of 5.25% NaOCl in combination with 17% EDTA, which is widely regarded as the gold standard for canal debridement.[18,19] The assessment of dentin surface cleanliness was carried out using scanning electron microscopy.[20]

MATERIALS AND METHODS

Study design and setting

The current in vitro scanning electron microscopy investigation was conducted within the Department of Conservative Dentistry and Endodontics at (NIMS Dental College and Hospital).

Sample size and selection criteria

A total of 48 freshly extracted human maxillary central incisor teeth with intact crown structures were collected (from the Department of Oral and Maxillofacial Surgery, NIMS Dental College and Hospital). Teeth were analyzed by radiovisiography in the buccal and proximal direction to confirm noncomplicated root canal anatomy, presence of single root canal, and complete root formation. All the teeth were cleaned thoroughly to remove hard and soft debris and blood on the external root surface using an ultrasonic cleaner. The specimens were autoclaved at 121°C under 15 lbs of pressure for 30 min, followed by storage in normal saline prior to the experimental procedures.

Inclusion criteria

1. Single rooted teeth with single canal

2. Root with closed apex

3. Noncarious teeth

4. Sound root portion with no fractures or cracks

5. No root resorption.

Exclusion criteria

1. Presence of calcification in root canal

2. Teeth with root length < 16 mm

3. Endodontically treated teeth

4. Complicated root canal anatomy.

Randomization and grouping

The prepared teeth were randomly divided into three equal categories (n = 16 per group) according to the respective irrigating solutions:

• Group I: Irrigated using 5% Panchvalkal extract

• Group II: Irrigated using 5% grape seed extract

• Group III (control): Irrigated with a standard regimen of 5.25% NaOCl and 17% EDTA.

To formulate the herbal solutions, Panchvalkal extract was prepared utilizing the bark from five distinct medicinal trees (Ficus bengalensis, Ficus religiosa, Ficus infectoria, Ficus racemosa, and Thespesia populnea), yielding a solution heavily concentrated with hydrolyzable tannins, flavonoids, and phytosterols. The 5% grape seed extract (V. vinifera) predominantly comprised OPCs, linoleic acid, and a diverse range of polyphenolic antioxidants.

Preparation of samples

The working length was standardized for all samples. Root canal preparation was carried out using standard endodontic procedures with hand files and rotary instruments. Following access cavity preparation, canal patency was confirmed using a size 10 K-file. The coronal third was flared, and biomechanical preparation was performed using a crown-down technique with ProTaper Gold rotary nickel–titanium files up to a master apical file size of 30/0.06 [Figures 1a and 1b]. Between each instrument change, the canals were copiously irrigated to prevent debris accumulation. Irrigation was done using side-vented endodontic irrigating needles during instrumentation.

Figure 1.

(a) Standardization of working length, (b) Preparation of root canal

Irrigation protocol

Each group received its respective irrigant during canal preparation:

• Group I: Panchvalkal extract (5%)

• Group II: Grape seed extract (5%)

• Group III: 5.25% NaOCl followed by 17% EDTA.

Irrigation was performed throughout the procedure using side-vented needles to ensure effective delivery of irrigants into the canal.

Sample processing and scanning electron microscopy analysis

Following root canal instrumentation, each tooth was cleaved longitudinally. To render the nonconductive dentin surfaces electrically conductive for precise electron imaging, the specimens were placed in a vacuum chamber and subjected to gold–palladium sputter coating using an ion sputtering device. This process deposits an ultra-thin metallic layer, minimizing thermal damage and preventing electron charge accumulation on the samples during microscopy. The prepared samples were then examined under the scanning electron microscopy at a standardized ×2000 magnification to visually assess and score the clearance of the smear layer within the apical, middle, and coronal root segments.

Statistical analysis

Intergroup comparison was performed using one-way analysis of variance (ANOVA) to evaluate whether there was a statistically significant difference in smear layer removal efficacy. To identify specific group differences, post hoc multiple comparison test (Tukey’s honestly significant difference [HSD] test) was performed.

RESULTS

A total of 48 recently extracted, single-rooted human maxillary central incisors with similar anatomical features were randomly allocated into three equal groups (n = 16) [Table 1 and Graph 1] to evaluate the cleaning efficacy of different endodontic solutions:

Table 1.

Distribution of samples and allocation of group name

Final irrigating solution used	Group name	Number of samples, n (%)
Panchvalkal extract	Group I	16 (33.33)
Grape seed extract	Group II	16 (33.33)
Sodium hypochlorite (5.25%)+17% EDTA	Group III	16 (33.33)
Total		48 (100)

EDTA: Ethylenediaminetetraacetic acid

Graph 1.

Sample distribution among study groups (n = 48)

• Group I: Irrigated with Panchvalkal extract

• Group II: Irrigated with grape seed extract

• Group III: Irrigated with 5.25% NaOCl and 17% EDTA.

To quantify the extent of smear layer eradication, the dentinal walls at the apical, middle, and coronal thirds were observed using scanning electron microscopy [Figure 2]. The resulting data were subjected to a one-way ANOVA and a Tukey’s HSD post hoc test to identify statistically significant variations among the experimental cohorts.

Figure 2.

Scanning electron microscopy photomicrograph (×2000) showing smear layer removal efficacy for all the groups at all the levels of root canal dentin: (a) Coronal, (b) Middle, and (c) Apical thirds

Descriptive statistics [Table 2 and Graph 2] for the coronal region indicated means of 0.75 ± 0.45 (Group I), 1.00 ± 0.00 (Group II), and 0.31 ± 0.48 (Group III). For the middle root canal space, the respective mean values were 0.69 ± 0.48, 1.69 ± 0.48, and 0.50 ± 0.52. Similarly, the apical third demonstrated means of 1.69 ± 0.48, 2.00 ± 0.00, and 1.06 ± 0.93 for Groups I, II, and III, respectively. Regardless of the anatomical level evaluated, Group II consistently registered the highest mean smear layer scores.

Table 2.

Intergroup comparison of mean distribution of coronal, middle, and apical sections of all the groups

Section	Group	n	Mean	SD	SE	95% CI (Lower)	95% CI (Upper)	Minimum	Maximum
Coronal	Group I	16	0.75	0.45	0.11	0.51	0.99	0.00	1.00
	Group II	16	1.00	0.00	0.00	1.00	1.00	1.00	1.00
	Group III	16	0.31	0.48	0.12	0.06	0.57	0.00	1.00
	Total	48	0.69	0.47	0.07	0.55	0.82	0.00	1.00
Middle	Group I	16	0.69	0.48	0.12	0.43	0.94	0.00	1.00
	Group II	16	1.69	0.48	0.12	1.43	1.94	1.00	2.00
	Group III	16	0.50	0.52	0.13	0.22	0.78	0.00	1.00
	Total	48	0.96	0.71	0.10	0.75	1.17	0.00	2.00
Apical	Group I	16	1.69	0.48	0.12	1.43	1.94	1.00	2.00
	Group II	16	2.00	0.00	0.00	2.00	2.00	2.00	2.00
	Group III	16	1.06	0.93	0.23	0.57	1.56	0.00	2.00
	Total	48	1.58	0.71	0.10	1.38	1.79	0.00	2.00

SD: Standard deviation, SE: Standard error, CI: Confidence interval

Graph 2.

Intergroup comparison of mean distribution of coronal, middle, and apical sections of all the groups

Statistical analysis [Table 3] revealed that the debridement efficacy of Group III was vastly superior overall. In both the coronal and apical thirds, Group III significantly outperformed Group I (P = 0.006 and P = 0.014, respectively) as well as Group II (P < 0.001 for both anatomical regions). Interestingly, within the middle third, Group III and Group I produced comparable cleaning results (P = 0.532), whereas Group II differed significantly from the other two solutions (P < 0.001). Additionally, the coronal comparison between Groups I and II lacked statistical significance (P = 0.159). Collectively, the evidence firmly establishes Group III as the most capable protocol for comprehensive smear layer eradication.

Table 3.

Intergroup comparison of mean difference of coronal, middle, and apical sections of all the groups

Section	Comparison	Mean difference	SE	P	95% CI (Lower)	95% CI (Upper)
Coronal	Group I versus Group II	−0.25	0.13	0.159	−0.57	0.07
	Group I versus Group III	0.44*	0.13	0.006*	0.11	0.76
	Group II versus Group III	0.69*	0.13	<0.001*	0.36	1.01
Middle	Group I versus Group II	−1.00*	0.17	<0.001*	−1.42	−0.58
	Group I versus Group III	0.19	0.17	0.532	−0.23	0.61
	Group II versus Group III	1.19*	0.17	<0.001*	0.77	1.61
Apical	Group I versus Group II	−0.31	0.21	0.317	−0.83	0.20
	Group I versus Group III	0.63*	0.21	0.014*	0.11	1.14
	Group II versus Group III	0.94*	0.21	<0.001*	0.42	1.45

*Significance level established at P<0.05. CI: Confidence interval, SE: Standard error

DISCUSSION

The primary objective of the present investigation was to evaluate and contrast the smear layer removal capabilities of two herbal alternatives against a standard irrigation protocol (5.25% NaOCl and 17% EDTA) via scanning electron microscopy.[21] To achieve this, a prospective, experimental in vitro model was utilized to systematically assess the cleaning efficacy of these various endodontic solutions.[22] The study followed a comparative longitudinal design, where 48 extracted human teeth were standardized and randomly assigned to three experimental groups based on the final irrigating solution used: Group I (Panchvalkal extract), Group II (grape seed extract), and Group III (17% EDTA + 5.25% NaOCl).[23]

Mechanical instrumentation serves two purposes: removal of infected tissue and shaping the canal to facilitate obturation.[24] Even with modern technological progress, mechanical instrumentation is inherently limited by the intricate anatomy of the root canal network. Anatomical complexities such as isthmuses, fins, dentinal tubules, and accessory canals serve as inaccessible reservoirs for microbes, shielding them from endodontic files.[25,26] Consequently, no current instrumentation technique can fully prepare all surfaces of the canal, and studies have shown that 30%–40% of canal walls remain untouched after preparation.[27] Mechanical preparation alone is insufficient, as it cannot dissolve necrotic pulp tissue remnants or adequately remove the smear layer, a tenacious film composed of organic and inorganic debris generated during instrumentation.[28] The smear layer occludes dentinal tubules and protects microorganisms from the action of irrigants and intracanal medicaments, thereby necessitating the use of chemical irrigation for effective disinfection and smear layer removal.[29,30]

NaOCl is the most commonly used solution due to its potent antimicrobial activity and tissue-dissolving capacity.[11] Because NaOCl lacks the ability to break down the inorganic constituents of the smear layer, it necessitates the use of a supplemental chelator like EDTA.[12,13] EDTA cleans mineralized debris by binding calcium ions in dentin, thus opening dentinal tubules. NaOCl and EDTA are used in succession to achieve complete smear layer removal.[9]

In this investigation, the most effective smear layer clearance across the coronal, middle, and apical thirds was achieved by Group III (5.25% NaOCl combined with 17% EDTA). This outcome corroborates the observations of Morago et al.,[31] who concluded that utilizing NaOCl alongside EDTA produces a synergistic cleaning action that significantly improves the opening of dentinal tubules.[31] Hülsmann et al.[32] stated that smear layer retention supports bacterial survival, biofilm maintenance, and apical leakage and emphasized that its removal is essential for improving canal disinfection and treatment prognosis.[33]

In the current investigation, the application of Panchvalkal extract resulted in a moderate reduction of the smear layer. This traditional formulation is well-regarded for its diverse therapeutic benefits, including anti-inflammatory, wound-healing, antimicrobial, antiseptic, and astringent characteristics.[16] However, its smear layer removal efficacy was inferior to conventional irrigants. This inferiority is primarily due to the distinct biochemical limitations of phytochemicals. While tannins possess robust protein-precipitating capabilities, they lack the strong calcium-binding ionic affinity required to effectively dislodge the calcified inorganic matrix of the smear layer, a mechanism inherently possessed by synthetic chelators like EDTA. This inferiority is primarily due to the distinct biochemical limitations of phytochemicals. While tannins possess robust protein-precipitating capabilities, they lack the strong calcium-binding ionic affinity required to effectively dislodge the calcified inorganic matrix of the smear layer, a mechanism inherently possessed by synthetic chelators like EDTA.

Grape seed extract showed the least efficacy, particularly at the apical third. It is rich in polyphenolic compounds such as proanthocyanidins, which demonstrate antioxidant, antibacterial, and antifungal effects, and has shown the ability to disrupt microbial biofilms. However, its smear layer removal ability was lower compared to Panchvalkal extract and conventional irrigants.[17] Our results align with the observations of da Silva et al., concluding that the limited chelating capacity of herbal alternatives results in only mild smear layer elimination.[34] Furthermore, Chhabra et al. similarly noted that while herbal formulations achieve partial debridement, their efficacy remains inferior to that of EDTA.[35]

Irrespective of the irrigant used, maximum smear layer clearance occurred coronally, while the apical third exhibited the least debridement. This discrepancy is likely attributable to the intricate anatomical variations and the restricted fluid dynamics of irrigants reaching the apical region. Similar findings have been reported in studies showing reduced irrigant penetration in the apical region. Therefore, while herbal irrigants offer advantages such as reduced toxicity and better biocompatibility, their effectiveness remains inferior to conventional irrigants.

Although herbal irrigants possess antimicrobial, antioxidant, and biocompatible properties, their chelating ability is limited compared to EDTA, which explains their lower efficacy in smear layer removal. These findings are supported by Divia et al.[36] and Candeiro et al.,[33] who also observed partial smear removal with herbal alternatives, while da Silva et al.[34] and Chhabra et al.[35] confirmed that conventional regimens remain superior for definitive canal debridement.

Limitations

The primary constraint of the current investigation is its in vitro design, which inherently restricts the direct extrapolation of these findings to in vivo scenarios. Because the experiment utilized extracted teeth in a highly controlled laboratory setting, it cannot fully replicate the dynamic biological variables present in the human oral cavity. Consequently, future clinical trials are necessary to validate these irrigation protocols for practical use.

CONCLUSION

The standard combination of NaOCl and EDTA remains unequivocally superior for eradicating the dentinal smear layer. Among the tested natural alternatives, Panchvalkal extract exhibited a moderate cleaning ability, suggesting its potential as a biocompatible adjunct, whereas grape seed extract yielded inferior results. While the biocompatible nature of herbal solutions makes them an attractive prospect, extensive research into optimizing their formulations and concentrations is imperative prior to routine clinical implementation.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.