1. INTRODUCTION:

The endodontic procedure encompasses a meticulous three-dimensional filling of the root canals following comprehensive disinfection achieved through a combined chemical and mechanical approach. Its objective is to enhance the recovery of periradicular tissues and eradicate causative agents implicated in pulpal and periradicular pathologies¹. Gram-negative bacteria like Porphyromonas gingivalis, Fusobacterium nucleatum, Porphyromonas endodontalis, Tannerella forsythia, various species of Prevotella, and Treponema, as well as Gram-positive bacteria such as Olsenella uli, Pseudoramibacter alactolyticus, different Streptococcus species, Actinomyces species, various Propionibacterium species, and Cutibacterium acnes, are frequently identified within the endodontic biofilm^2. Treating biofilm infections has become challenging due to the rise of drug-resistant bacteria, rendering conventional antibiotic therapy ineffective for common infectious conditions. Factors such as the exopolysaccharide matrix limiting antibiotic penetration, as well as the reduced oxygen levels and resulting nutritional gradient within biofilms, contribute to their heightened resistance³. Various approaches have been employed to minimize the bacterial population within the root canal system. Present knowledge underscores the necessity for thorough root canal debridement due to the uncertainty surrounding medicament efficacy in the presence of debris⁴. Advances in instrumentation techniques and irrigation protocols have enhanced the management of most cases in a single visit, yet unfavorable outcomes may stem from factors such as persistent infections or inaccessible areas. In such instances, employing intracanal medicaments post-debridement provides a clinical advantage, confirming symptom improvement before completing treatment and enhancing overall effectiveness⁵. Persistent infection is a major factor leading to endodontic failure in teeth that have undergone root canal treatment⁶. Another notable challenge experienced by dentists is the inter-appointment flare-up, which presents as post-treatment discomfort with pain and/or swelling shortly after root canal procedures. This condition requires prompt attention, often resulting in unscheduled visits for urgent treatment. In endodontic treatment, the utilization of intracanal medicament is pivotal, hinging on the efficacy of antimicrobial agents to eradicate microorganisms and alleviate post-operative discomfort⁷.

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The intracanal medicaments commonly used in endodontics are calcium hydroxide (Ca(OH)₂), triple antibiotic paste, chlorhexidine gluconate, ledermix paste, antibiotics, and halogen salts with each having its limitations⁸. Despite being introduced in the 1990s, Ca(OH)₂ continues to be considered the gold standard in endodontic treatment. When applied in the root canal following chemomechanical preparation, Ca(OH)₂ effectively eliminates apical exudates owing to its antibacterial properties. Ca(OH)₂ exhibits broad-spectrum bactericidal effects, which are primarily linked to its alkaline properties, as only a few microorganisms can thrive in an environment with a pH exceeding 1Top of Form1⁹. However, despite these antimicrobial qualities, Ca(OH)₂ demonstrates limited efficacy towards polymicrobial infections and bacteria within biofilms. Microorganisms like Enterococcus faecalis and Candida albicans frequently persist in cases where endodontic treatment has failed¹⁰. Gram-positive cocci, including E. faecalis, have the ability to infiltrate dentinal tubules, establish biofilms, adhere to serum collagen, and impede lymphocyte function as a defense mechanism against eradication. Additionally, they inhibit the host proton pumps, which confers resistance across a broad pH spectrum, extending up to approximately pH 11.5¹¹. This adaptability allows E. faecalis to endure the alkalinity associated with Ca(OH)2, potentially persisting after completion of root canal treatment and frequently contributing to persistent and recurring infections¹². Another microorganism implicated in persistent post-treatment apical periodontitis is C. albicans. The capacity of C. albicans to transition between blastospore and hyphal forms (known as thigmotropism), allowing for easier penetration of host tissue and avoiding macrophage phagocytosis, is a key component of its pathogenicity. C. albicans is capable of establishing persistent infections due to its rapid biofilm formation within 48 hours and its ability to deeply infiltrate dentinal tubules. It is also resilient in a variety of pH ranges, highly alkaline environments, and ecologically challenging situations^13,14. The probable interaction between organisms like E. faecalis and fungi within the root canal biofilm could exacerbate the existing infection, highlighting the necessity for innovative treatment approaches¹⁵.

The use of natural products in dentistry has been on the rise of late owing to their non-cytotoxic, antimicrobial, and anti-inflammatory properties¹⁶. Utilization of plant extracts that exhibit broad biological activities can be beneficial in polymicrobial infections like endodontic infections. Phytochemicals serve as the most abundant reservoir of novel therapeutics, even though active constituents within plants may exist in low concentrations¹⁷. Alstonia scholaris (AS), often called the devil tree, blackboard tree, or saptaparna, belongs to the Apocynaceae family. It is an indigenous tropical evergreen tree found in India. It is recognized for its antimicrobial, anti-inflammatory, antioxidant, pain-relieving, and anti-ulcer properties. Extracts from this plant showed antibacterial activity against E. faecalis^18-24. Ficus racemosa (FR), belonging to the Moraceae family, is widely known by names such as cluster fig, gular, or audumbar. This plant, native to India, exhibits antidiabetic, analgesic, antipyretic, anti-inflammatory, and antimicrobial properties. FR extracts showed antibacterial efficacy directed against Candida albicans and Staphylococcus aureus^25-27. Naturally occurring phytochemicals are less cytotoxic in addition to being anti-bacterial, anti-inflammatory, and antioxidant, making them a good alternative to conventional intracanal medicaments. This study aimed to evaluate the penetration depth of AS and FR in comparison to calcium hydroxide when used as intracanal medicaments, utilizing a Confocal Laser Scanning Microscope (CLSM) for analysis.

2. MATERIALS AND METHODS:

2.1 Plant Material Collection and Extraction Process:

Bark samples of A. scholaris and F. racemosa were collected from Shobhavana Garden in Tenkamijar, Karnataka, then air-dried and ground into a fine powder. To prepare the aqueous extracts, 0.3g of the powder was mixed with 30ml of sterile distilled water and left to steep for 60 minutes.

2.2 Preparation of samples:

For this investigation, thirty-six human mandibular first and premolars having closed apices and single roots were chosen. This was confirmed with a radiograph. The University's Institutional Review Board and Ethics Committee gave its approval to the study procedure. Patients were informed about the procedure, and written consent was obtained prior to the extraction of teeth for orthodontic purposes. Following extraction and debris removal, the teeth were soaked in a 0.5% sodium hypochlorite (NaOCl) solution, adhering to OSHA guidelines²⁸Top of Form. A uniform root segment measuring 13 mm was prepared by decoronating the tooth using a dental double-sided diamond disc (NMD) attached to a micromotor (NSK) with water cooling. Apical patency was achieved by inserting a size 10K hand file (Mani Inc, Japan) into the canal, extending 1mm past the apical foramen. The working length was determined by positioning the instrument tip 0.5mm short of the apical foramen. Circumferential filing was done up to 15K file to obtain glide path. Biomechanical preparation was performed using Protaper universal rotary files (Dentsply Maillefer, Ballaigues, Switzerland) to F3 size, with alternating irrigation of NaOCl and saline solutions. The samples were subsequently divided randomly into three groups, each containing 12 specimens, for the application of intracanal medicaments: Group I – calcium hydroxide (CH); Group II - Alstonia scholaris (AS); Group III - Ficus racemosa (FR). The medicaments were mixed with a 0.1 wt% fluorescent Rhodamine B dye (Sigma Aldrich) in a 1:1 ratio and delivered into the canal using a lentulospiral (Mani Inc, Japan).

2.3 Sectioning of samples:

Apical transverse sections were obtained for each specimen, positioned 3mm from the apex, utilizing a hard tissue microtome (LEICA SP 1600). A slice with a thickness of 0.1mm was carefully generated. Subsequently, the samples were individually mounted on glass slides for observation under the Confocal Laser Scanning Microscope (CLSM) (Carl Zeiss).

2.4 Confocal laser scanning microscope investigation:

Images were captured using a Confocal Laser Scanning Microscope set at a wavelength of 461nm with 10x magnification. To determine the penetration extent, each image was segmented into four similar parts, and the penetration depth was determined in individual sections. The average of these four measurements was taken as the sample's penetration depth. This evaluation was conducted using the digital measuring ruler feature in the Zeiss Microsystems software (Zen Blue, Carl Zeiss AG, Oberkochen, Germany). (Figures 1-3).

Figure 1. Confocal laser scanning microscope image of sample from Group I: Calcium hydroxide (CH)

Figure 2. Confocal laser scanning microscope image of sample from Group II - A. scholaris (AS)

Figure 3. Confocal laser scanning microscope image of sample from Group III - F. racemosa (FR)

2.5 Statistical analysis:

Data analysis was performed using SPSS Statistics for Windows, version 25.0. The analysis utilized an ANOVA test to determine significance. P-value less than 0.05 was regarded as statistically significant.

3. RESULTS:

The experimental conditions of this in vitro study demonstrated that the test groups, AS and FR, exhibited penetration depths similar to the control group (CH). No statistically significant differences were found between the groups (Tables 1 and 2). The greatest penetration depth was observed in Group II, A. scholaris (AS), followed by Group I, calcium hydroxide (CH) (Figure 4).

Table 1. Results for depth of penetration of three groups

	N	Min.	Max.	Mean	Std. Deviation
Group I - CH	12	454.88	912.65	570.5619	121.62674
Group II - AS	12	491.32	766.21	588.7506	77.10990
Group III - FR	12	326.68	805.84	561.1913	129.30288

Min: Minimum; Max: Maximum

Table 2. Comparison between the groups

ANOVA
Penetration depth
	Sum of Squares	df	Mean Square	F	Sig.
Between Groups	4712.627	2	2356.314	0.189	0.829
Within Groups	412040.609	33	12486.079
Total	416753.236	35

*There is no statistically significant difference in Penetration depth between the three groups

Figure 4. Graphical representation of the depth of penetration of 3 groups

4. DISCUSSION:

The role of microorganisms in endodontic conditions, particularly apical periodontitis, is well recognized. Apical periodontitis results from an infection within the root canal system and involves a complex interaction between the host’s immune response and the invading microbes. This interaction ultimately leads to inflammation, which causes bone resorption surrounding the root of the affected tooth²⁹. Despite efforts to eliminate bacteria through instrumentation, complete bacterial elimination is often not achievable. Moreover, systemic factors like uncontrolled type II diabetes, compromised immune function, and the presence of chronic infections such as periapical cysts or sinus tracts can hinder healing in apical periodontitis and raise the risk of treatment failure^4,5.

To address the challenges posed by root canal complexities and bacterial resistance, a clinical strategy involves the use of intracanal medicaments, which may necessitate additional appointments. One such medicament is calcium hydroxide (Ca(OH)₂), which exerts its antimicrobial effects by releasing hydroxyl ions in aqueous solution³⁰. Free radicals generated by hydroxyl ions impair bacterial cell membranes, impede DNA replication, and induce mutations. Additionally, the alkaline pH of calcium hydroxide disrupts enzyme function, affecting both structural proteins and cellular metabolism. Furthermore, hydroxyl ions can diffuse through dentin, raising the pH to around 9.0, a process referred to as trans-dentinal medication. This multi-faceted approach helps combat endodontic infections and promotes successful treatment outcomes³¹. Although Ca(OH)₂ has antimicrobial effects, its efficacy as an intracanal medicament can be compromised by factors like the buffering capacity of dentin, which lowers its high pH and may permit bacterial growth inside the dentinal tubules³². Research indicates that the application of Ca(OH)₂ may not lead to a notable decrease in post-treatment pain after endodontic procedures. Furthermore, the extrusion of Ca(OH)₂ beyond the apex of the tooth can cause harmful effects³³. In addition, Ca(OH)₂ demonstrates limited effectiveness against specific pathogens such as E. faecalis and C. albicans, which are frequently associated with endodontic infections. Recent investigations employing both culture-based techniques and polymerase chain reaction (PCR) have consistently detected E. faecalis and C. albicans as predominant species in cases of failed root canal treatments, emphasizing the need to address their resistance to Ca(OH)₂ in endodontic therapy^34–37. The ability of E. faecalis to invade dentinal tubules to depths ranging from 821.91 µm to 1061.79 µm further illustrates the difficulty in completely eliminating these microorganisms from infected root canal systems. Additionally, both fungi and bacteria have been observed to co-aggregate, forming complex biofilm communities that provide mutual protection and tolerance against antimicrobial medications^{38, 39}. Given these complexities, an intracanal medicament with potent antimicrobial activity and maximum penetration into the dentinal tubules would be ideal for effectively combating these microbial communities and preventing their invasion and dissemination inside the intricate anatomy of the tooth’s root canal system.

The present study demonstrated that AS and FR have a depth of penetration comparable to the conventional intracanal medicament used in endodontics indicating that they can be applied in endodontic practice. The depth of penetration of AS was seen to be higher than that of FR and Ca(OH)₂. Studies on AS have identified a variety of compound classes in different plant parts through phytochemical investigations^40-42. Iridoids, coumarins, and flavonoids have been detected in the leaves, whilst terpenoids are isolated from the root and bark portions. Alkaloids, phlobatannins, simple phenolic compounds, steroids, saponins, and tannins have been identified across all parts of the plant. Alkaloids are particularly abundant and significant in AS, with over 70 different types reported in various plant parts like the root, stem bark, leaves, fruit, and flowers. Among these, the leaves contain the highest concentration of alkaloids. This diverse array of phytochemicals underscores the potential pharmacological importance of AS in traditional medicine and pharmaceutical research⁴³. The alkaloid components of AS, particularly vallesamine, picrinine, and scholaricine, have shown notable anti-inflammatory and pain-relieving effects. These compounds are believed to alleviate peripheral pain by either suppressing or modulating the release of endogenous pro-inflammatory agents such as prostaglandins, serotonin, and histamine. The effects observed may be attributed to the inhibition of key inflammatory mediators, including 5-lipoxygenase (5-LOX), cyclooxygenase-1 (COX-1), and cyclooxygenase-2 (COX-2), as supported by findings from both in vitro studies and animal models. Compounds that act as dual inhibitors of COX and 5-LOX have been reported to reduce the production of both leukotrienes and prostaglandins, potentially enhancing anti-inflammatory outcomes. Furthermore, alkaloids have been shown to boost the activity of antioxidant enzymes, helping to prevent lipid peroxidation and neutralize free radicals⁴⁴. Methanol, butanol, and petroleum extracts obtained from the leaves, stem, and root bark demonstrated antibacterial properties against both gram-positive bacteria, such as Streptococcus species including Streptococcus mutans, and gram-negative bacteria like Enterobacter, Klebsiella, Neisseria, Proteus, and Pseudomonas, among others. Additionally, Monoterpenoid Indole Alkaloids and Pentacyclic Triterpenoids isolated from A. scholaris were effective against E. faecalis^18-20.

FR demonstrates antipyretic, anti-inflammatory, and antimicrobial properties^45,46.The wide range of pharmacological activities is attributed to various bioactive compounds, including glauanol, beta-sitosterol, tiglic acid, glauanol acetate, glucose, hentriacontane, taraxasterol esters, friedelin, lupeol acetate, and phytosterols. The anti-inflammatory characteristics of FR leaf petroleum ether extract were investigated, revealing its potential anti-serotonin action, which may contribute to its anti-inflammatory effects. It operates by exerting a similar effect to glucocorticoids, albeit on a time-delayed mechanism. This suggests that the extract's active components—steroids and triterpenoids—possess some affinity for glucocorticoid receptors⁴⁷. Additionally, FR bark extract had a stronger COX-1 inhibitory impact, indicating its potential anti-inflammatory properties⁴⁸. The ethanol extract of FR stem bark demonstrated notable free radical scavenging effects that increased with the dosage, highlighting its antioxidant capabilities. Furthermore, studies on cytotoxicity revealed that several other components of FR, particularly the bark, displayed low toxicity levels and were potentially safe for human consumption. Additionally, the stem bark demonstrated high efficacy against bacterial strains such as Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, Alcaligenes faecalis, Bacillus cereus and Streptococcus faecalis⁴⁹. Antimicrobial activity was seen against C. albicans as well^25-27. Quorum sensing (QS) represents a mode of communication among bacteria, whereby gene expression is controlled by the density of cells and signaling molecules. In the formation of an oral biofilm, QS plays a crucial role by orchestrating bacterial actions to form intricate and functional communities. Ethanolic extract of FR mediated repression of Pseudomonas aeruginosa Las R gene thereby inhibiting QS. Further studies should be undertaken to evaluate if FR has a similar effect against endodontic biofilms⁵⁰.

The experimental plant-derived phytochemicals used as intracanal medicaments in the present study showed adequate penetration into the dentinal tubules. Bacteria such as E. faecalis are the primary contributors to post-treatment apical periodontitis and are often responsible for the failure of endodontic therapies. These could be potentially eliminated by the use of these phytochemicals as intracanal medicaments between appointments. In addition to the good depth of penetration, these also have anti-inflammatory, analgesic, and antioxidant properties which can prove beneficial to the patient in reducing post-treatment pain and flare-ups. Further studies must be undertaken to assess these properties along with studies on cytotoxicity, antibacterial and antibiofilm activity of these phytochemicals. Further comprehensive phytochemical investigations are essential to identify the active principles and precisely elucidate the mechanisms of action.

5. CONCLUSION:

Within the constraints of this study, it can be concluded that Alstonia scholaris and Ficus racemosa demonstrate penetration depths comparable to that of calcium hydroxide as an intracanal medicament. Further investigations are warranted to evaluate their efficacy in this role. The use of these plant products as intracanal medicaments represents a promising frontier in endodontic treatment, owing to their demonstrated depth of penetration, antimicrobial, anti-inflammatory, analgesic, and antioxidant properties.

6. CONFLICT OF INTEREST:

The authors declare no conflicts of interest regarding this research.

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Received on 06.06.2025 Revised on 24.10.2025

Accepted on 30.12.2025 Published on 05.06.2026

Available online from June 06, 2026

Research J. Pharmacy and Technology. 2026;19(6):2744-2750.

DOI: 10.52711/0974-360X.2026.00392

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