Antibacterial Evaluation of Plectranthus amboinicus-infused Glass Ionomer Cement Against Streptococcus mutans: An In-vitro Study

1. Postgraduate Student, Department of Paediatric and Preventive Dentistry, Saveetha Dental College, Chennai, Tamil Nadu, India. 2. Associate Professor, Department of Paediatric and Preventive Dentistry, Saveetha Dental College, Chennai, Tamil Nadu, India.

Abstract

Introduction: Dental caries remains one of the most prevalent oral diseases worldwide and is strongly associated with Streptococcus mutans, while conventional Glass Ionomer Cement (GIC) has limited intrinsic antibacterial activity. Therefore, there is a need to develop modified restorative materials with enhanced antimicrobial properties to reduce bacterial colonisation and improve clinical outcomes.

Aim: The present preliminary in-vitro study aimed to evaluate and compare the antibacterial efficacy of Plectranthus amboinicus aqueous extract-modified GIC with that of conventional GIC against Streptococcus mutans using Colony-Forming Unit (CFU) enumeration and crystal violet assay.

Materials and Methods: This preliminary in-vitro study was conducted in the Department of Microbiology, Saveetha Dental College and Hospitals, Chennai, Tamil Nadu, India, from December 2025 to January 2026. An aqueous extract of Plectranthus amboinicus was prepared and incorporated into Type II GIC (GC Corporation, Tokyo, Japan) to form the test group. At the same time, conventional GIC served as the control group. A total of six cylindrical specimens (5 mm × 2 mm) were fabricated, with three specimens in each group (n=3). Each specimen was incubated with a standardised Streptococcus mutans suspension. Antibacterial activity was evaluated using serial dilution and CFU enumeration. Biofilm biomass was assessed using a crystal violet assay with absorbance measured at 592 nm. All experiments were performed in triplicate for each group. Statistical analysis was carried out using an Independent samples t-test (Welch’s correction), with a p-value <0.05 considered statistically significant.

Results: The P. Amboinicus modified GIC demonstrated consistently lower colony counts and optical density values than the control at all tested dilutions, indicating enhanced antibacterial performance. Mean CFU values in the modified group were reduced from 222.33 to 198.00 CFU/mL at 10^-6 dilution, from 205.33 to 147.67 CFU/mL at 10^-5, and from 143.67 to 95.33 CFU/mL at 10^-4, with all differences statistically significant (p<0.001). Similarly, mean OD values decreased from 2.137 in the control to approximately 1.48 in the modified GIC group (p<0.001), reflecting reduced biofilm biomass.

Conclusion: Incorporation of Plectranthus amboinicus aqueous extract into glass ionomer cement significantly enhances its antibacterial efficacy against Streptococcus mutans in-vitro. This modification may represent a promising approach to improving the antimicrobial profile of restorative materials and potentially reducing the risk of secondary caries. However, further studies are required to evaluate its physicochemical properties, biocompatibility, and long-term clinical performance.

Antibacterial activity, Bioactive restorative material, Dental caries prevention, Herbal dental materials

Dental caries remains a prevalent global oral health challenge, affecting individuals of all age groups and socioeconomic backgrounds. The disease is primarily initiated by the colonisation of cariogenic microorganisms, particularly Streptococcus mutans, which adheres to the tooth surface, metabolises fermentable carbohydrates, and produces acids that demineralise enamel (1),(2). The biofilm-forming capacity and acidogenic nature of S. Mutans make it one of the most virulent pathogens responsible for caries initiation and progression (3),(4). Conventional preventive strategies such as fluoride application and mechanical plaque removal have significantly reduced caries prevalence, yet recurrent caries around dental restorations termed “secondary caries” remains a major clinical concern (5).

Glass Ionomer Cement (GIC) has long been favoured as a restorative material owing to its excellent adhesion to tooth structure, biocompatibility, and fluoride-releasing ability (6). Despite these advantages, traditional GICs exhibit limited antibacterial efficacy, which may compromise the longevity of restorations, particularly in patients with high caries risk. The incorporation of antimicrobial agents into GIC has been proposed as an effective strategy to enhance its antibacterial potential without impairing mechanical integrity. Various agents such as chlorhexidine, silver nanoparticles, and herbal extracts have been studied, with varying degrees of success (7). In recent years, the search for natural and biocompatible alternatives has driven interest toward phytochemicals derived from medicinal plants, which offer broad-spectrum antimicrobial activity with minimal cytotoxicity (8),(9).

Among these plants, Plectranthus amboinicus (Lour.) Spreng., commonly known as Indian borage or Cuban oregano, has garnered significant attention for its potent antimicrobial, anti-inflammatory, and antioxidant properties. This aromatic perennial herb, belonging to the Lamiaceae family, is rich in secondary metabolites such as carvacrol, thymol, eugenol, and rosmarinic acid compounds well documented for their inhibitory effects on pathogenic bacteria (10),(11). Several in-vitro studies have demonstrated that P. Amboinicus extracts exhibit notable antibacterial activity against oral pathogens including S. Mutans, S. Sobrinus, and Lactobacillus acidophilus (12),(13),(14). The mechanism of action is believed to involve disruption of bacterial cell membranes, interference with enzymatic systems, and inhibition of glucosyltransferase activity, thereby reducing biofilm formation and acid production (14).

Herbal-modified dental materials represent an emerging area within restorative dentistry, aligning with the growing interest in biologically derived and sustainable therapeutic approaches. When incorporated into restorative matrices such as GIC, plant-derived bioactive compounds may enhance antibacterial properties and influence interactions at the material-tissue interface (9). The incorporation of Plectranthus amboinicus extract into GIC is hypothesised to provide antimicrobial effects that could potentially reduce bacterial colonisation at restoration margins. Additionally, the polyphenolic constituents of P. amboinicus possess antioxidant properties, which may contribute to the biological behaviour of the modified material. However, these effects require further investigation through detailed in-vitro and in vivo studies.

Furthermore, the global rise in antibiotic resistance underscores the necessity of identifying novel antimicrobial agents from natural sources (World Health Organisation, 2023). Phytochemicals with broad-spectrum activity and low propensity for inducing resistance could play a pivotal role in preventive dentistry and restorative biomaterials. Studies have reported synergistic effects between plant essential oils and conventional restorative materials, enhancing both antibacterial and physicochemical properties (14),(15). For instance, integration of Ocimum sanctum and Azadirachta indica extracts into GIC formulations demonstrated promising inhibitory effects on S. mutans growth while maintaining acceptable compressive strength (8),(16). However, there remains a paucity of data specifically examining the impact of P. amboinicus-infused GIC against S. mutans.

The potential of P. amboinicus in dental applications extends beyond antibacterial efficacy. Its bioactive terpenoids and phenolic acids have been associated with anti-inflammatory and wound healing properties, which could facilitate pulp and dentin repair when used in restorative procedures (10). These pharmacological attributes, coupled with the plant’s abundance and low toxicity, make it a compelling candidate for incorporation into dental cements.

The present preliminary in-vitro study aimed to evaluate the antibacterial efficacy of Plectranthus amboinicus aqueous extract-modified GIC against Streptococcus mutans. The objective was to compare the antibacterial activity of the modified GIC with that of conventional GIC using CFU enumeration and crystal violet assay.

The null hypothesis (H0) stated that there would be no significant difference in antibacterial efficacy between Plectranthus amboinicus-modified GIC and conventional GIC.

The alternative hypothesis (H1) proposed that the modified GIC would demonstrate significantly greater antibacterial efficacy against Streptococcus mutans.

Material and Methods

This preliminary in-vitro experimental study was conducted in the Department of Microbiology, Saveetha Dental College and Hospitals, Chennai, Tamil Nadu, India, from December 2025 to January 2026. The study was designed to evaluate and compare the antibacterial efficacy of Plectranthus amboinicus-modified GIC with that of conventional GIC against Streptococcus mutans. The study protocol was reviewed and approved by the Institutional Scientific Review Board (SRB), Saveetha Dental College and Hospitals, Chennai, India (Approval No.: SRB/SDC/PEDO-2403/25/451). As the present study was conducted in-vitro and did not involve human participants or animal subjects, formal ethical clearance was not mandatory; however, Institutional approval was obtained before the commencement of the study.

Sample size: A total of six specimens were included in the study, with three specimens in each group (n=3 per group). As this was a preliminary in-vitro study, a sample size of three specimens per group was considered adequate to obtain preliminary data, consistent with similar in-vitro studies evaluating antibacterial properties of modified GICs (5).

Study Procedure

1. Preparation of Plectranthus amboinicus extract: Fresh leaves of Plectranthus amboinicus were collected from healthy, pesticide-free plants and thoroughly washed under running tap water, followed by rinsing with sterile distilled water. The leaves were blotted dry and weighed using a digital balance, and approximately 10 g of fresh leaves were used for extraction. The weighed leaves were added to 500 mL of distilled water in a sterile borosilicate vessel and subjected to decoction by boiling, followed by gentle simmering for 10-15 minutes to facilitate the release of water-soluble phytoconstituents, which is a commonly accepted method for aqueous herbal extraction.

After heating, the mixture was allowed to cool to room temperature and filtered through multiple layers of sterile muslin cloth, followed by Whatman No. 1 filter paper to obtain a clear aqueous extract. The filtrate was collected in sterile, airtight glass containers, and the final volume obtained after filtration was approximately 470-480 mL, accounting for minor evaporation losses during the decoction process, ensuring consistency. The extract was stored at 4°C and used within 48 hours of preparation to minimise degradation of active constituents. No preservatives were added.

The final volume: The prepared aqueous extract corresponded to an approximate concentration of 2% w/v (fresh weight basis) and was used as the modifying agent for incorporation into GIC. The extract concentration was standardised based on the ratio of plant material to solvent during preparation (15) (Table/Fig 1).

No phytochemical characterisation (such as quantification of phenols, flavonoids, or specific bioactive compounds), physicochemical evaluation of the modified material, or sterility testing of the extract was performed in the present preliminary study, which is acknowledged as a limitation. Further studies are required to evaluate these parameters.

2. Preparation of GIC specimens: Type II GIC (GC Corporation, Tokyo, Japan) was used in the study. The control group specimens were prepared according to the manufacturer’s instructions using the standard powder-to-liquid ratio of 1 scoop powder to 1 drop liquid.

For the test group, the aqueous extract of Plectranthus amboinicus was incorporated by replacing 50% of the liquid component of the GIC with the prepared extract in a 1:1 ratio (volume-to-volume basis). The modified liquid was then mixed with the GIC powder using the manufacturer-recommended powder-to-liquid ratio to maintain acceptable consistency and handling characteristics. The powder and modified liquid were mixed using an agate spatula for approximately 30-45 seconds to obtain a homogeneous paste.

The replacement of 50% of the GIC liquid with the aqueous extract may have influenced the acid-base setting reaction, pH, and physicochemical behaviour of the material. These parameters were not evaluated in the present preliminary study; therefore, the antibacterial findings should be interpreted cautiously.

The mixed material was immediately transferred into pre-sterilised cylindrical stainless steel moulds measuring 5 mm in diameter and 2 mm in thickness. A minimal amount of sterile petroleum jelly was applied to the inner surfaces of the moulds as a separating medium to facilitate easy removal of the set specimens. Care was taken to ensure that only a thin layer was used to avoid any potential interference with the experimental outcomes (Table/Fig 2),(Table/Fig 3).

The specimens were allowed to set under aseptic conditions at room temperature. Following complete setting, the specimens were carefully removed from the moulds and stored in a desiccator until further testing to prevent moisture contamination.

• Group I - Conventional GIC (Control group)

• Group II - Plectranthus amboinicus-modified GIC (Test group)

3. Bacterial strain and inoculum preparation: The antibacterial activity was evaluated against Streptococcus mutans (ATCC 25175), a standard reference strain widely recognised for its cariogenic potential. The bacterial strain was obtained from the institutional microbiology laboratory. The organism was cultured on Brain Heart Infusion (BHI) agar and incubated at 37°C for 24 hours to obtain fresh, isolated colonies. A standardised bacterial suspension was prepared by transferring selected colonies into sterile saline solution and adjusting the turbidity to match the 0.5 McFarland standard, corresponding to approximately 1.5×10⁸ CFU/mL. This standardised inoculum ensured reproducibility and uniform bacterial concentration across all experimental procedures (Table/Fig 4).

4. Antibacterial testing procedures:

a. Serial dilution and CFU enumeration: The antibacterial effect of the GIC specimens was evaluated using three independent specimens per group (n=3). Each specimen was incubated with 1 mL of standardised Streptococcus mutans suspension (0.5 McFarland standard Ëœ1.5×10⁸ CFU/mL) in 9 mL of sterile saline, resulting in a total volume of 10 mL. The tubes were incubated at 37°C for 24 hours.

Following incubation, serial tenfold dilutions (10^-1 to 10^-6) were prepared to obtain countable bacterial colonies. Aliquots from the selected dilutions were plated on Brain Heart Infusion agar and incubated under standard conditions (at 37°C for 48 hours). Colony counts from the plated dilutions were recorded using a digital colony counter and compared between the study groups. The CFU/mL was calculated using the formula:

CFU/mL=(Number of colonies × dilution factor)/volume plated (mL)

However, CFU/mL calculations were not used as the primary outcome measure in the present preliminary study; only raw mean colony counts obtained from plated aliquots were used for statistical analysis.

All measurements were performed for each specimen, and mean CFU values were calculated for each group (17) (Table/Fig 5).

b. Crystal violet assay for biofilm mass: Following incubation with the GIC specimens, the bacterial suspensions were centrifuged at 3000 rpm for 10 minutes. The resulting pellets were washed twice with sterile Phosphate-Buffered Saline (PBS; pH 7.4) to remove residual medium. The pellets were then fixed with methanol for 10 minutes and stained with 0.1% crystal violet for 15 minutes.

Excess stain was removed by rinsing with PBS, and the bound dye was solubilised using 33% glacial acetic acid. Absorbance was measured at 592 nm using a UV-Visible spectrophotometer. The crystal violet assay was used to assess the biomass of adherent biofilm-forming bacterial cells attached to the surface. Lower absorbance values indicated reduced adherent biofilm biomass in the presence of the modified GIC. The assay was not used to directly distinguish viable from non viable bacterial cells. In this assay, cells attached to the surface of the culture plate retain the crystal violet dye, whereas dead or non adherent cells are removed during the washing steps and therefore do not contribute to the final staining signal. Consequently, the assay evaluates total adherent biofilm biomass based on the amount of crystal violet dye retained by adherent cells.

The serial dilution procedure was performed specifically for colony count enumeration to obtain countable bacterial colonies. In contrast, the crystal violet assay was used as an overall assessment of biofilm biomass following incubation with the respective GIC specimens and was not intended to represent dilution-dependent measurements.

STATISTICAL ANALYSIS

All quantitative data (colony counts and optical density values) were expressed as Mean±Standard Deviation (SD). Intergroup comparisons between conventional GIC (control) and modified GIC (test) were performed using an Independent samples t-test with Welch’s correction. Serial dilutions were performed as part of the standard microbiological procedure to obtain countable CFU and were not considered independent experimental groups. A p-value <0.05 was considered statistically significant. Statistical analysis was carried out using Statistical Package for Social Sciences (SPSS) Statistics for Windows, version 26.0 (IBM Corp., Armonk, NY, USA). Given the exploratory nature of this preliminary study, adjustments for multiple comparisons were not applied, and findings should be interpreted cautiously.

Results

Colony Count Results

Descriptive analysis of colony count data demonstrated consistently lower Streptococcus mutans counts in the modified GIC group compared to the control group. Serial dilutions were performed to obtain countable colonies, and the values presented represent mean colony counts obtained from plated aliquots at the respective dilution levels (Table/Fig 6).

Independent samples t-tests demonstrated statistically significant differences in colony counts between group I and group II under the present experimental conditions. At 10^-6, 10^-5, and 10^-4 dilutions, t-values were 23.085, 61.165, and 51.265, respectively, with two-sided p-values <0.001 for each comparison (Table/Fig 7). These findings indicate lower recoverable colony counts in group II compared to group I; however, they should be interpreted cautiously due to the preliminary study design and small sample size.

Optical Density (OD) Values - Crystal Violet Assay

Optical density measurements obtained from the crystal violet assay demonstrated lower overall biofilm biomass measurements in the modified GIC group compared to the control group under the present experimental conditions (Table/Fig 8).

Intergroup differences in optical density measurement demonstrated statistically significant differences under the present experimental conditions. Mean OD differences (approximately 0.66 units) had very narrow 95% confidence intervals, indicating reduced biofilm biomass in group II compared to group I (Table/Fig 9).

However, the unusually high t-values observed in the present study should be interpreted cautiously, as the low within-group variability likely reflects the uniformity of triplicate measurements obtained under controlled in-vitro experimental conditions rather than true biological variability.

Discussion

The present preliminary in-vitro study evaluated the antibacterial performance of Plectranthus amboinicus-modified GIC against Streptococcus mutans, a key pathogen associated with dental caries. The modified GIC demonstrated consistently lower colony counts and reduced optical density values compared to the conventional material, indicating a measurable reduction in bacterial growth and biofilm formation under experimental conditions. These findings suggest that incorporation of plant-derived extracts may enhance the antibacterial properties of restorative materials. However, the extent of reduction observed in this study should be interpreted with caution, as the clinical significance of these findings cannot be directly inferred from in-vitro data alone (9),(16).

Although previous studies have attributed the antimicrobial activity of Plectranthus amboinicus to various bioactive constituents, the present study did not include phytochemical analysis or evaluation of the stability of these compounds within the GIC matrix. Therefore, the exact mechanism underlying the observed antibacterial effect warrants further investigation (9),(13),(14),(18).

The findings of the present study are in agreement with previous reports demonstrating the antimicrobial potential of Plectranthus amboinicus against oral microorganisms (14). Earlier studies have reported that both aqueous and essential oil extracts of this plant exhibit antibacterial activity, which has been attributed to the presence of various bioactive compounds (14),(16),(19). These compounds have been suggested to exert antimicrobial effects through mechanisms such as disruption of bacterial cell membranes, alteration of membrane permeability, and interference with enzymatic systems involved in biofilm formation. However, such mechanisms were not directly evaluated in the present study. The observed reduction in colony counts and biofilm biomass in this study indicates an overall antibacterial effect of the modified GIC under the present experimental conditions; however, the exact mechanism underlying this effect requires further investigation. In addition, the crystal violet assay used in the present study reflects total biofilm biomass and does not differentiate between viable and non viable bacterial cells.

Similar findings have been reported in studies evaluating plant-based modifications of restorative materials, where incorporation of herbal extracts such as Ocimum sanctum, Azadirachta indica, and Salvadora persica into GIC has demonstrated enhanced antibacterial properties against cariogenic microorganisms (15),(16),(17),(18). Previous investigations have suggested that plant-derived additives may improve the antimicrobial profile of GIC while maintaining acceptable handling characteristics, particularly when used in controlled concentrations. Systematic reviews have also highlighted the potential of phytochemical incorporation as an emerging strategy for developing bioactive restorative materials; however, variations in extract type, concentration, and methodology across studies limit direct comparison of outcomes (20). The performance of the P. amboinicus modified GIC in this study is consistent with those observations, demonstrating a pattern of bacterial inhibition comparable to that reported for other herbal and bioactive restorative systems. Therefore, while the present findings are consistent with existing literature, further standardised studies are required to evaluate the effects of such modifications on both antimicrobial efficacy and material properties.

The present results can also be viewed within the broader context of bioactive and antibacterial restorative materials, which include calcium silicate based cements, bioactive glass containing composites, and nanoparticle functionalised resins (21),(22). These materials typically act through ion release, pH elevation, or surface modifications that hinder bacterial colonisation, but some pose concerns related to cytotoxicity, cost, or aesthetic alteration. In contrast, the use of a plant-derived aqueous extract such as P. amboinicus offers a more natural and potentially safer alternative, with the present data indicating substantial antibacterial benefit and no observable adverse effect on the basic handling of the GIC in-vitro (11).

Within the limitations of this preliminary in-vitro study, the observed reduction in colony counts and biofilm biomass suggests that incorporation of Plectranthus amboinicus extract may contribute to the antibacterial activity of the modified GIC. GIC is known for its hydrophilic nature and ion exchange properties, which may influence the distribution of incorporated agents within the matrix. However, the impact of extract incorporation on the material’s physicochemical and mechanical properties was not assessed in this study and requires further investigation (23).

This modification may offer a potential approach to reducing bacterial colonisation at restoration margins and lowering the risk of secondary caries under in-vitro conditions; however, these findings should be interpreted with caution. Further research is required to standardise extract preparation through phytochemical characterisation and to evaluate the release kinetics of active constituents from the modified GIC. In addition, studies involving multispecies biofilm models, long-term antibacterial assessment, and in-vivo or ex-vivo conditions are necessary to better establish clinical applicability. Comprehensive evaluation of physicochemical properties, fluoride release, mechanical strength, and cytocompatibility with oral tissues is also essential prior to clinical translation. Based on the findings of the present study, the null hypothesis was rejected, as the modified GIC demonstrated significantly greater antibacterial activity compared to conventional GIC.

Limitation(s)

The present pilot in-vitro study has several limitations that must be considered while interpreting the findings. The study was conducted using a single bacterial species under controlled laboratory conditions, which does not replicate the complexity of multispecies oral biofilms, salivary interactions, pH fluctuations, and mechanical stresses present in the oral environment. The extract preparation was not subjected to phytochemical characterisation, and the concentration of active constituents was not quantified, which may affect reproducibility. Additionally, sterility testing of the extract and evaluation of its stability during incorporation into the GIC matrix were not performed. The sample size was limited, and although triplicate specimens were used, the low variability observed may reflect controlled experimental conditions rather than true biological variation. Furthermore, adjustments for multiple comparisons were not applied, which may increase the risk of Type I error. Therefore, the statistical findings should be interpreted cautiously and regarded as preliminary and hypothesis-generating rather than confirmatory. The study also did not assess the physicochemical properties, fluoride release, pH changes, setting characteristics, or mechanical performance of the modified material.

Despite these limitations, the present findings contribute to the growing body of research exploring plant-derived modifications of restorative materials. The observed reduction in Streptococcus mutans counts and biofilm biomass suggests a potential antibacterial effect of Plectranthus amboinicus modified GIC under in-vitro conditions.

However, the present study findings should be interpreted with caution, and further well-designed studies are required to establish the clinical relevance and material performance of such modifications.

Conclusion

Plectranthus amboinicus-modified GIC demonstrated lower Streptococcus mutans colony counts and reduced adherent biofilm biomass measurements compared with conventional GIC under the conditions of this preliminary in-vitro study. These findings suggest a potential antibacterial effect of the plant extract when incorporated into GIC. Further studies with larger sample sizes, comprehensive physicochemical characterisation, and biological validation are required to confirm these findings and evaluate the clinical applicability of the modified material.

DOI and Others

DOI: 10.7860/JCDR/2026/87813.24403

Date of Submission: Jan 29, 2026
Date of Peer Review: Mar 24, 2026
Date of Acceptance: Jul 15, 2026
Date of Publishing: Sep 01, 2026

Author declaration:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? NA
• For any images presented appropriate consent has been obtained from the subjects. NA

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