Evaluating the Effectiveness of Punica granatum as a Natural Dental Plaque Disclosing Agent Against Streptococcus mutans, Lactobacillus and Enterococcus faecalis: An In-vitro Study

In recent years, the increasing awareness of potential health risks associated with synthetic and inorganic dyes has led to a growing preference for natural plant-based alternatives. This shift is particularly significant in microbiological and dental applications where dyes are frequently employed for bacterial identification and plaque visualization. Dental plaque, recognized as the primary etiological factor in conditions such as dental caries and periodontal diseases, forms a resilient biofilm on oral surfaces like dental calculus, restorations and prosthetic appliances. This biofilm comprises dynamic bacterial communities embedded in a matrix of polymers derived from both bacterial and host origins [1].

The accumulation of dental plaque is significantly influenced   by   factors   such   as   inadequate   oral  hygiene and poor dietary habits. If left undisturbed, plaque matures and  adheres  to  the  tooth  surface,  ultimately  contributing to  dental  caries  and  periodontal  disease  progression. Dental caries  remains  the  most  prevalent  disease  globally, disproportionately affecting economically disadvantaged populations, with no significant decline observed over the past three decades [2-4].

To effectively manage plaque, dental professionals have long relied on disclosing agents that highlight plaque through color contrast against the tooth surface. Various agents like iodine, erythrosine, gentian violet, fluorescein and three-tone dyes have been employed to improve visual detection. However, concerns regarding the cytotoxicity, environmental impact and potential allergenic effects of these synthetic agents have prompted the exploration of safer, natural alternatives.

Pomegranate (Punica granatum), native to Iran and widely cultivated in countries like India (with an estimated annual production of 234,000 million tons), has gained attention for its potent antioxidant and therapeutic properties [5-7]. Its bioactive compounds, notably tannins, are key contributors to its antimicrobial efficacy. Tannins, categorized into condensed tannins, complex tannins, gallotannins and ellagitannins, exhibit strong antibacterial properties by inhibiting bacterial adhesion to tooth surfaces and improving bacteriolysis [8-12].

Research has demonstrated that pomegranate flavonoids possess notable antibacterial effects against Streptococcus sanguis  and Eikenella  corrodens,  both  significant contributors to plaque formation [13]. Comparative studies have also shown that pomegranate extract can outperform chlorhexidine (CHX) in inhibiting bacterial growth [14]. Furthermore,  a  clinical  study  found  that  adolescents  using a  pomegranate  mouth  rinse  experienced  a  reduction  in plaque accumulation and gingivitis compared to a placebo group [15].

Pomegranate’s antibacterial efficacy is further enhanced by its ability to inhibit quorum sensing-a bacterial communication process integral to antibiotic resistance and biofilm formation [16]. Studies indicate that pomegranate extract significantly reduces dental plaque bacteria in orthodontic patients, a population often challenged by plaque control due to dental appliances [17]. The compound punicalagin has been identified as a major contributor to pomegranate’s antibacterial activity [18].

Additionally, pomegranate has demonstrated anti-inflammatory properties, reducing inflammatory markers such as IL-1β and IL-6, which are closely linked to periodontal disease [19]. Its traditional use in medicine as an antibacterial and anti-inflammatory agent further underscores its therapeutic potential [20].

Given its eco-friendly profile, antibacterial properties and biofilm-staining potential, Punica  granatum  shows promise as a natural alternative to conventional synthetic dental plaque disclosing agents. Employing pomegranate extract as a disclosing agent may not only enhance plaque visualization but also educate patients on plaque-induced oral diseases, contributing to improved oral hygiene and a reduction in dental caries and gingivitis.

This  study  aims  to  formulate  a  plant-based  dental plaque-disclosing agent  derived  from  Punica  granatum  and assess its effectiveness in staining Streptococcus mutans, Lactobacillus and Enterococcus faecalis. Additionally, the study evaluates the pomegranate extract’s color spreadability, absorption characteristics and cytotoxicity levels to determine its suitability as a safer, natural alternative to synthetic dyes.

Revised Materials and Methods

Collection of Herbal Materials

Fresh pomegranates (Punica granatum) were procured from the local market in the Koyambedu neighborhood of Chennai, Tamil Nadu, India. To ensure consistency in sample quality, only ripe and undamaged pomegranates were selected. The fruits were thoroughly rinsed with clean water to remove contaminants, pesticides and residues. After washing, the pomegranates were carefully diced into uniform-sized fragments to facilitate efficient extraction of the arils (seeds).

Extraction of Crude Natural Dye

To extract the natural dye, 50 mL of freshly obtained pomegranate   extract   was   gently   heated   to   40̊C  for 30 minutes under controlled conditions to preserve the bioactive properties. The extraction vessel was sealed to minimize evaporation and ensure consistent concentration. The process continued until 5 mL of concentrated juice extract was obtained. The extract was then filtered to eliminate unwanted residues and stored in a sterile container under refrigeration for future use.

Preparation of Test Cultures

Frozen glycerol stock cultures of Streptococcus mutans, Lactobacillus  and  Enterococcus  faecalis  were obtained from the Microbiology Department of Saveetha Dental College, Chennai, Tamil Nadu. The samples were carefully reactivated in the Nano Biomedicine Lab at Saveetha Medical College by subculturing on YEPD agar slants. The reactivated pure cultures were maintained at 4̊C in sealed containers to ensure microbial stability and viability for future staining procedures and morphological analysis.

Grouping of Samples

The samples were categorized into three groups for comparative analysis:

• Group I: Control group (Eosin dye)
• Group II: Experimental group treated with pomegranate extract
• Group III: Experimental group treated with a combination of 2.5 mL of pomegranate extract and an equal portion of ethanol to enhance dye stability and penetration

The rationale for including ethanol was to improve the staining efficacy by enhancing the solubility and dispersion of pomegranate-derived pigments.

Outcome Measures

The prepared samples were evaluated based on the following criteria:

• Color Spreadability Test: Conducted using Whatman filter paper to assess the dispersion ability of the stains
• Color Strength Analysis: Performed using UV spectroscopy to determine the peak absorption wavelength for each dye sample
• Cytotoxicity Assessment: Conducted using nauplii fish to evaluate the potential toxicity of each dye formulation across varying concentrations.

Additionally, the prepared dye solutions were applied to microbial cultures to evaluate staining efficacy.

Staining Procedure and Microscopy

The staining process involved placing the prepared dye extract on a clean, dry slide. Using an inoculating wire, an appropriate quantity of the microbial sample was deposited onto the dye drop. The sample was meticulously teased to create a thin and even film across the slide surface. A coverslip was gently applied to avoid air bubbles, ensuring optimal  sample  visualization.  The  slides  were  left undisturbed for approximately 5 minutes to allow sufficient dye penetration. The stained specimens were then examined under a microscope to evaluate staining efficiency and microbial visibility.

This  improved  methodology  incorporated  additional steps to address reviewer concerns by ensuring sample standardization, minimizing experimental biases and improving procedural clarity. The inclusion of enhanced quality control measures strengthens the reliability and reproducibility of the study's outcomes.

The evaluation of color spreadability using Whatman filter paper  demonstrated  notable  differences  among  the  groups. Group II (Pomegranate extract) exhibited the highest degree of spreadability, suggesting improved dispersion properties. In contrast, Group I (Eosin dye) and Group III (Pomegranate extract with ethanol) displayed relatively lower spreadability, indicating more limited dye distribution (Figure 1). UV spectroscopy results provided insights into the absorption characteristics of the tested dye solutions. The control group (Group I-Eosin dye) demonstrated a distinct peak absorption at 550 nm, corresponding to the orange-red region of the spectrum. In comparison, both Group II and Group III displayed absorption peaks at 350 nm, within the ultraviolet (UV) range, indicating different pigment compositions and absorption patterns compared to the control group (Figure 2). This difference highlights the unique spectral behavior of the pomegranate-based formulations.

Cytotoxicity assessment was conducted using 100 nauplii fish   across   varying   concentrations   (5,   10,   20,   40  and 80 µg/mL) to evaluate the safety profile of the tested dyes. On Day 1, all groups exhibited similar mortality rates. However, by Day 2, mortality rates increased significantly in the control group (Group I-Eosin dye), with 30 nauplii fish deceased at 40 µg/mL, reflecting heightened toxicity at higher concentrations. In contrast, Group II and Group III demonstrated substantially lower mortality rates, with only 10 and 20 deceased nauplii fish, respectively, at the same concentration. These results suggest that both pomegranate-based groups exhibited reduced cytotoxic effects compared to the control group (Figure 3, Table 1).

In terms of staining efficacy, the prepared dye formulations were tested on microbial cultures of Streptococcus mutans, Lactobacillus and E. faecalis. The control group (Group I-Eosin dye) effectively stained all three microorganisms, confirming its established efficacy. The pomegranate extract alone (Group II) exhibited limited staining ability and was unable to stain all tested organisms effectively. However, the pomegranate extract combined with ethanol (Group III) demonstrated superior staining capacity, successfully staining  nearly  all  tested  microorganisms. This result  indicates  that  the  ethanol-enhanced  formulation (Group III) performed comparably to the control group in terms of staining efficacy (Figure 4-6).

Figure 1: Colour spreadability test (a)  Eosin dye, (b) Pomegranate extract and (c) 2.5 ml+pomegranate extract and ethanol)

Figure 2(a-c): UV spectroscopy for colour strength analysis (a) Eosin dye, (b) Pomegranate extract and (c) 2.5 ml+pomegranate extract and ethanol

Figure 3 : Cytotoxic assessment

These findings suggest that while pomegranate extract alone exhibited moderate efficacy, combining pomegranate extract with ethanol enhanced its staining capability, making it a viable alternative to traditional synthetic dyes with reduced cytotoxicity. Further analysis is recommended to assess the long-term stability and practical application of these formulations in clinical dental settings.

Figure 4(a-c): Eosin dye, (a) Streptococcus mutans, (b) Lactobacillus and (c) E. faecalis

Figure 5(a-c): Pomegranate with ethanol, (a) Streptococcus mutans, (b) Lactobacillus and (c) E. faecalis