Antibacterial efficacy of herbal extracts as photosensitizers in photodynamic root canal disinfection

Abstract

Context:

Photodynamic root canal disinfection is a valuable adjunct in endodontics. However, the standard photosensitizer often causes tooth discoloration. Natural photosensitizers, such as curcumin (CUR) and Beta vulgaris (BV), can be explored for their antimicrobial efficacy against Enterococcus faecalis biofilms.

Aims:

This study evaluates the antibacterial effectiveness of BV extract as a photosensitizer in antimicrobial photodynamic therapy (aPDT) against E. faecalis biofilms, comparing it to CUR and toluidine blue (TB).

Settings and Design:

A comprehensive study protocol outlining the objectives and methodologies was submitted to the Institutional Ethics Committee and received ethical approval (Approval No. 2024-25/1114).

Subjects and Methods:

Fifty-six single-rooted extracted teeth were prepared using Protaper Gold to #F3, irrigated with 3% sodium hypochlorite (NaOCl), and finalized with 17% ethylenediaminetetraacetic acid. The teeth were contaminated with E. faecalis suspension and divided into seven groups: NaOCl alone (positive control), NaOCl with TB, CUR, or BV; B. Vulgaris alone; CUR solvent alone; and Saline (negative control). Photosensitizers (0.5 mg/mL) were activated with a 640 nm diode laser for 120 s. Bacterial load was assessed through colony-forming unit counts.

Statistical Analysis Used:

Data were subjected to statistical analysis using SPSS v22, with analysis of variance and post hoc Tukey’s tests (P < 0.05).

Results:

NaOCl combined with CUR-PDT showed the highest CFU reduction, followed by NaOCl with TB and BV. Nonphotoactivated photosensitizers were less effective in comparison.

Conclusions:

Photoactivated CUR and BV extracts demonstrated superior antimicrobial activity compared to their nonphotoactivated forms, suggesting their potential as alternative photosensitizers in aPDT.

INTRODUCTION

The primary objective of endodontic treatment is to eliminate microorganisms responsible for pulpal and periapical diseases.[1] To achieve this, various disinfection protocols are employed, including mechanical instrumentation followed by root canal irrigation and the use of intracanal medicaments.[2] Sodium hypochlorite (NaOCl) is the most commonly recommended and widely used irrigant due to its antibacterial and tissue-dissolving properties.[3] However, its penetration depth into dentinal tubules is limited, ranging from 40 to 309 µm.[4] In contrast, Enterococcus faecalis can colonize dentinal tubules at depths exceeding 1000 µm.[5]

To enhance the effectiveness of conventional chemo-mechanical root canal preparation, advanced methods such as high-power lasers and photodynamic therapy (PDT) have been introduced.[6,7] Antimicrobial photodynamic therapy (aPDT) combines a nontoxic photosensitizer, a light source, and molecular oxygen to selectively target and eliminate microorganisms.[8] Lasers enhance access to difficult-to-reach areas due to their superior penetration into dentin tissue.[9] In particular, diode lasers have been employed for complete disinfection, achieving 99% bacterial elimination through temperature-induced protein denaturation.[10]

A photosensitizer is a light-sensitive chemical with low toxicity in the absence of light. It selectively targets bacterial cells and, upon light activation, destroys both infected tissues and bacteria.[11] Toluidine blue (TB) is a blue-colored photosensitizer, it possesses amphiphilic properties, a positive charge, a low molecular weight, and a peak absorption range of 620–660 nm.[12] Phenothiazine-based dyes, such as TB, are among the most commonly used photosensitizers in PDT and have demonstrated favorable results. However, as dyes, they may alter tooth color, potentially affecting dental esthetics.[13]

Various natural photosensitizers are used for photoactivated root canal disinfection, including curcumin (CUR), which exhibits excellent antimicrobial properties.[14] A study by Pourhajibagher et al. reported that CUR-aPDT reduced E. faecalis colony-forming unit (CFU)/mL by 99.6%. A major advantage of CUR-mediated aPDT is its ability to provide strong antibacterial effects without staining the teeth.[15]

Beta vulgaris (BV) (beetroot) is recognized as a functional food due to its rich nutritional profile, which includes micronutrients, carbohydrates, and bioactive compounds such as betalains, the key pigments.[16] BV has been widely studied for its bioactive properties, including antioxidant, anti-inflammatory, antitumor, hepatoprotective, and cognitive-enhancing effects.[17] A study by El-Beltagi et al. found that beetroot extract exhibits strong antibacterial properties, demonstrating greater effectiveness against Gram-positive bacteria than Gram-negative bacteria.[18]

To date, no reports in the literature have explored the use of BV extract as a photosensitizer in root canal disinfection. Therefore, the present study aims to evaluate the antibacterial effectiveness of BV extract as a photosensitizer in aPDT against E. faecalis biofilm and compare its efficacy with TB and CUR.

SUBJECTS AND METHODS

The study was approved by the Institute’s research ethics committee (ethical approval number: 2024-25/1114).

Sample size determination

The sample size was determined using G*power v 3.0.1 software (Franz Faul universitat, Kiel, Germany). A total sample size of 56 (n = 56) is obtained with an effect size of 0.60, power of the study 0.80, and type-I (α) error 0.05.

Sample preparation

Fifty-six extracted single-rooted teeth with a single canal, intact and fully developed roots, and a root curvature of <30° were selected. The occurrence of a single canal was verified using radiographs taken in both buccolingual and mesiodistal directions. The specimens were decoronated with a disc to standardize the root length at 12 mm. The working length was determined with a #10 K-file (Mani, Tochigi, Japan) positioned 0.5 mm short of the apices, ensuring apical patency. Rotary instrumentation was performed using ProTaper Gold rotary files #F3 (Dentsply Maillefer, India) and the canals were irrigated with 1 mL of 3% NaOCl (Prime Dental Products pvt ltd, India) and normal saline (0.9% v/w, Lifusion, India) after each instrumentation. To remove the smear layer, the canals were finally irrigated with 1 mL of 17% ethylenediaminetetraacetic acid (Ammdent Dental Products, India) for 1 min, followed by a rinse with 5 mL of saline. The apical foramen was sealed with glass ionomer cement (GC Fuji II, Tokyo, Japan).

The prepared samples were dried using paper points and autoclaved at 121°C for 30 min to ensure sterility. Following sterilization, the samples were transferred to test tubes filled with sterile brain heart infusion (BHI) broth (HiMedia Laboratories, Mumbai, India) and were incubated at 37°C for 48 h. To verify sterility, a random selection of four samples was incubated separately in sterile BHI broth, and no bacterial growth was detected within 24 h.

A preserved stock culture of E. faecalis (ATCC 29212) was revived by transferring it into BHI broth and incubating it at 37°C for 24 h. A bacterial suspension equivalent to 0.5 McFarland standard (1.5 × 10⁸ CFU/mL) was made by adjusting the optical density (OD₆₀₀) to 0.08–0.1 using a spectrophotometer. A 1 mL aliquot of the bacterial suspension was carefully introduced into each tube using a sterile syringe. The specimens were incubated at 37°C under anaerobic conditions for 3 weeks, with the BHI broth replaced every 48 h. Biofilm formation on the root canal surfaces was verified by sampling and performing CFU counts.

Experimental groups

After a 3-week incubation period, the media was discarded. To eliminate planktonic bacteria, the roots were carefully washed with sterile saline. The specimens were subsequently assigned to seven experimental groups (n = 8) using simple randomization:

• Group 1 (positive control): Root canal irrigation with NaOCl

• Group 2: Root canal irrigation with NaOCl followed by PDT using TB (NaOCl + TB)

• Group 3: Root canal irrigation with NaOCl followed by PDT using CUR (NaOCl + CUR)

• Group 4: Root canal irrigation with NaOCl followed by PDT using BV (NaOCl + BV)

• Group 5: Root canal irrigation with BV solvent (dimethyl sulfoxide [DMSO]) for 120 s (BV solvent)

• Group 6: Root canal irrigation with CUR solvent (DMSO) for 120 s (CUR solvent)

• Group 7 (Negative control): Irrigation of root canal with 5 mL of sterile normal saline for 30 s.

Determination of preliminary antimicrobial activity of photosensitizer

MIC and disc diffusion assay were carried out for BV extract to determine the antimicrobial activity. According to the disc diffusion assay, BV showed a zone of inhibition measuring 18 mm (75 μg/mL) [Figure 1]. The MIC for both the aqueous extract of CUR (0.8 μg/mL) and the ethanolic extract of BV (0.8 μg/mL) were determined.

Figure 1.

Disc diffusion showing inhibition zone of beta vulgaris extract

Preparation of photosensitizers

A solution of TB (HiMedia Laboratories, Mumbai, India) at 0.5 mg/mL concentration was prepared with normal saline. CUR and BV extracts (AGHP Enterprises, Chennai, India) were dissolved in DMSO to achieve a concentration of 0.5 mg/mL.

Primary irrigation with sodium hypochlorite

In Groups 1–4, the root canals were irrigated with 5 mL of 3% NaOCl using a 30-gauge, 25 mm side vented needle (Neoendo, Orikam, India) positioned 1 mm from the apex for 30 s, followed by a final rinse with sterile saline for 30 s.

Antimicrobial photodynamic therapy

In groups 2-4, the specific photosensitizers were introduced into the root canals and left for 120 s before laser exposure. PDT was performed for 120 s using an endodontic diode laser with a wavelength of 640 nm (Novolase Dual Wave Endo Diode, Novolase Technologies, India) at an output intensity of 240 mW/cm². The laser irradiation was carried out using an endodontic tip with a 200 µm diameter, inserted into the canal 1 mm short of the working length.

Sample collection and colony counting

The roots were irrigated with sterile saline, followed by scraping the dentinal shavings using # 2 GG drills for 30 s. Each GG drill was transferred to a test tube containing 1 mL of sterile saline and agitated using a vortex mixer for 1 min to dislodge any bacteria. The resulting bacterial suspensions were serially diluted and plated onto BHI agar using the spread plate technique. Following a 24-h incubation period at 37°C, the CFUs were enumerated using a digital colony counter (Singhla Scientific Laboratories, India) [Figure 2].

Figure 2.

Original images showing colony forming units (CFUs) of all experimental groups, (a) Sodium hypochlorite (NaOCl) + curcumin (CUR), (b) NaOCl + toluidine blue (TB), (c) NaOCl + beta vulgaris (BV), (d) NaOCl, (e) CUR alone, (f) BV alone, (g) Saline

The CFUs were determined using the following formula:

CFU/mL = (Number of bacterial colonies counted on plate × dilution factor)/volume of culture plate.

Statistical analysis

Data analysis was conducted using Statistical Package for the Social Sciences (SPSS, Version 22, IBM, Chicago, Illinois). A one-way analysis of variance (ANOVA) was used to compare the means between groups, followed by a post hoc Tukey’s test to assess differences between the experimental groups. The confidence intervals were set at 95%, and a significance level of P < 0.05 was used.

RESULTS

This study was conducted with seven groups, each consisting of eight samples. As per Graph 1, the mean value of the negative control group (344 CFUs/mL) against E. faecalis biofilm was significantly higher than that of all the experimental groups. Among the experimental groups, the NaOCl + CUR PDT group (63.25 CFUs/mL) had the lowest mean value, followed by the NaOCl + TB PDT group (67.00 CFUs/mL), NaOCl + BV PDT group (114.75 CFUs/mL), NaOCl group (117.75 CFUs/mL), CUR solvent group (127.75 CFUs/mL), and BV solvent group (192.12 CFUs/mL), respectively.

Graph 1.

Mean values of each experimental group

Table 1 shows the results of intergroup comparison using one-way ANOVA test. A highly significant statistical difference (P = 0.000) was noted when all the 7 experimental groups were compared with each other. Table 2 demonstrates the comparison of two groups at a time using Tukey’s post hoc test. Accordingly, a highly significant statistical difference was noted between all the groups, except, NaOCl (positive control) and NaOCl + BV PDT as well as CUR solvent. Similarly, no significant difference was seen between NaOCl + CUR PDT and NaOCl + TB blue PDT groups and NaOCl + BV PDT and CUR solvent groups.

Table 1.

Analysis of variance test

	Sum of squares	df	Mean square	F	Significant
Between groups	451015	6	75169.1	183.68	0.000*
Within groups	20052.8	49	409.24
Total	471067	55

*P value is significant

Table 2.

Tukey’s post hoc test

Groups	Mean difference	Significant	95% CI
			Lower bound	Upper bound
NaOCl (positive control)
NaOCl + TB PDT	50.75	0.000*	19.66	81.84
NaOCl + CUR PDT	54.5	0.000*	23.41	85.59
NaOCl + BV PDT	3	1	−28.09	34.09
BV solvent	−74.37	0.000*	−105.47	−43.28
CUR solvent	−10	0.95	−41.09	21.09
Negative control group (saline)	−225.87	0.000*	−256.97	−194.78
NaOCl + TB PDT
NaOCl + CUR PDT	3.75	1	−27.34	34.84
NaOCl + BV PDT	−47.75	0.000*	−78.84	−16.66
BV solvent	−125.12	0.000*	−156.22	−94.03
CUR solvent	−60.75	0.000*	−91.84	−29.66
Negative control group (saline)	−276.62	0.000*	−307.72	−245.53
NaOCl + CUR PDT
NaOCl + BV PDT	−51.5	0.000*	−82.59	−20.41
BV solvent	−128.87	0.000*	−159.97	−97.78
CUR solvent	−64.5	0.000*	−95.59	−33.41
Negative control group (saline)	−280.37	0.000*	−311.47	−249.28
NaOCl + BV PDT
BV solvent	−77.37	0.000*	−108.47	−46.28
CUR solvent	−13	0.85	−44.09	18.09
Negative control group (saline)	−228.87	0.000*	−259.97	−197.78
BV solvent
CUR solvent	64.37	0.000*	33.28	95.47
Negative control group (saline)	−151.5	0.000*	−182.59	−120.41
CUR solvent
Negative control group (saline)	−215.87	0.000*	−246.97	−184.78

*P value is significant. PDT: Photodynamic therapy, TB: Toluidine blue, CUR: Curcumin, BV: Beta vulgaris, CI: Confidence interval

DISCUSSION

The goal of endodontic therapy is to minimize the microbial load in the root canal system through chemo-mechanical preparation. However, even after this procedure, 40%–60% of root canals still harbor culturable microbial species.[19] Endodontic diseases are biofilm-mediated infections, most commonly associated with E. faecalis, which is frequently found in persistent infections and root canal failures.[14] Hence, our study utilized E. faecalis biofilm contamination to evaluate disinfection efficacy.

Adjunctive procedures like aPDT have been used to enhance root canal disinfection, as they can overcome bacterial resistance, penetrate biofilms effectively, and aid in the elimination of E. faecalis from the root canal system.[20] Combining photosensitizers with traditional irrigation solutions has shown superior antimicrobial effects compared to conventional methods like NaOCl alone.[21] Therefore, NaOCl was used as the primary irrigant, followed by photoactivation with photosensitizers. Additionally, diode lasers improve dye penetration into dentinal tubules, particularly in the apical third, enabling deeper antimicrobial diffusion and enhanced root canal disinfection.[22]

This study evaluated the antimicrobial activity of B. vulgaris and CUR extracts in comparison to TB, both with and without PDT. Photoactivation was performed using a diode laser at 640 nm for 120 s. To ensure consistency, all photosensitizers were used at a standardized concentration of 0.5 mg/mL. The selected photoactivation time and photosensitizer concentration were based on the recommendations of Mozayeni et al.[23]

The results showed that aPDT with all three photosensitizers significantly reduced E. faecalis CFUs. CUR, with and without photoactivation, exhibited the highest antimicrobial activity compared to the other two photosensitizers. TB with photoactivation demonstrated antibacterial activity nearly equivalent to CUR PDT, while B. Vulgaris with photoactivation showed better antibacterial activity than its nonphotoactivated counterpart.

TB is among the most extensively studied photosensitizers in aPDT. Research has shown that TB-mediated aPDT when combined with NaOCl, enhances antibacterial effects against E. faecalis. Similarly, our study consistently yielded the same results with TB.[23] Studies have also demonstrated that CUR exhibits potent antibacterial activity against E. faecalis biofilm at 460 and 660 nm.[24,25] Likewise, in our study, CUR exhibited superior antibacterial efficacy against E. faecalis biofilm.

Studies have highlighted the versatility of B. vulgaris extracts and pigments as photosensitizers, with applications in antibacterial treatments and cancer therapy.[18,26] The efficacy of B. vulgaris as a photosensitizer in PDT is primarily attributed to its high content of betalain pigments, particularly betanin. Upon photoactivation, these pigments generate reactive oxygen species, leading to cytotoxic effects on targeted cells.[26] Yasmin et al. reported that beetroot extract exhibited superior antibacterial activity against E. faecalis in its planktonic form.[27] Additionally, a study by Nouairi et al. found that the absorption maximum of beetroot extract ranges from 480 to 540 nm.[28] Notably, a wavelength of 640 nm was used in this study. In contrast, our study found that photoactivated B. vulgaris exhibited lower antibacterial activity compared to the other two photosensitizers. Therefore, further research should explore activating B. vulgaris extract at its peak absorption wavelength to maximize its effectiveness.

Variations in our study results compared to other studies may be attributed to factors such as the concentration of the photosensitizer, wavelength of light, and bacterial growth conditions. Future research should focus on conducting similar studies with larger sample sizes, varying concentrations, pre-irradiation and irradiation durations, light sources, and energy levels. Furthermore, the effectiveness of these natural extracts should be evaluated in in vivo clinical settings, as CUR and B. vulgaris are low-cost phytochemicals that cause minimal tooth discoloration.

CONCLUSIONS

Within the limitations of this study, it can be concluded that CUR, TB, and BV extract with photoactivation demonstrated superior antimicrobial activity compared to their non-photoactivated counterparts. Therefore, CUR and BV phytochemical extracts can be recommended as alternative photosensitizers in aPDT against E. faecalis biofilm.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.