Introduction

Cavities are related to the demineralization of the hard structure of the teeth by acids of microbial origin as well as several other factors that affect the tooth structure. The oral cavity contains many types of bacteria, of which Streptococcus mutans and Lactobacilli are believed to cause tooth decay.  Restorative cement which includes glass ionomer cement (GICs) is a class of substances [1]. The International Organization for Standardization (ISO) refers to them as "glass polyalkenoate cement," although the phrase "glass ionomer" is acknowledged as an inconsequential term that is frequently employed in dentistry [2]. In recent years, GICs are considered the most commonly employed water-based cement for the concluding cementation of dental crowns, bridges, Braces, and non-traumatic restorations. Glass ionomer is a popular material due to its biocompatibility, fluoride ion release over an extended period, and ability to bond to enamel and dentin [3]. But due to minimal antibacterial effect and lack of physical and mechanical characteristics, recurrence of caries has been reported after restoration, on the other hand, decreased strength of the restorative material was a major drawback. Hence there is a need for use of direct filling materials, thereby modification of GIC came into existence. The addition of different antimicrobial agents to GICs may have therapeutic advantages [4]. Research has focused on the rapid or decreased release of antimicrobial agents such as antibiotics, zinc ions, silver ions, iodine, and most commonly chlorhexidine, the gold standard antibacterial agent [5]. Numerous in vitro investigations supported the idea that when GIC is combined with CHX, its biological qualities are improved. Unfortunately, adding antibacterial agents to restorative materials usually affects their physical and mechanical characteristics over time. If the dose or release is not well managed, they may also have short-term efficacy and be hazardous to nearby tissues [6]. This is likely the cause of the lack of use of GICs in manufacturing when combined with chlorhexidine and other antimicrobials.

Plants were utilized for prevention and therapy for ailments as years pass by, until the invention of chemistry in the sixteen century. A herbal medication known as phytomedicine uses various plant components like extracts as a medicinal and health-improving agent that are considered the least toxic [7]. Based on the World Health Organization, up to eighty percent of people worldwide have a dependency on traditional medicine (herbal) as a form of fundamental medical needs, including flavonoids, phenols, and saponins [8]. Thyme is a little perennial shrub that tends to grow both horizontally and vertically. Rarely does it grow taller than 40 cm. When they get older, the stems turn woody [9]. Thyme leaves are fairly small, usually 2.5 to 5 mm long, and vary widely by variety in terms of form and hair coverage. Thymus vulgaris (T. vulgaris), a large fragrant plant with over 100 varieties worldwide, is extensively utilized in both culinary and medicinal recipes. Due to the numerous therapeutic benefits of its essential oils, commonly referred to as thyme oil, the genus Thymus contains significant medicinal herbs that come highly recommended. Because of their pharmacological and biological characteristics, thymus species are regarded as therapeutic plants. Its primary constituent, thymol, is what gives it its properties [10]. A high concentration of physiologically active substances, including thymol, carvacrol, p-cymene, and -terpinene, is a distinguishing feature of thyme essential oil. It has been demonstrated that carvacrol and thymol possess strong bacteriostatic and bactericidal effects in addition to having a high antioxidant capacity [11]. There is insufficient evidence in the literature for the addition of thyme extract to GIC for restorative purposes, even though thyme extract is effective against salivary environment-induced caries in human mouthwash or toothpaste. Because of the increased likelihood of recurrent caries following restorative therapy, the use of direct filling materials must be done with extreme caution [12], we need a restorative material that can inhibit a broad spectrum of bacteria. Our team has a wealth of knowledge and research expertise, which has resulted in publications of the highest caliber [13-30]. Therefore, the study's objective was to improve the antibacterial capabilities of GICs by modifying them with Thyme leaf extracts. Hence, keeping this in mind the present study was planned to compare and evaluate the antimicrobial activity and compressive strength of thyme-modified GIC with that of conventional Glass ionomer cement where the null hypothesis stated that there is no difference between conventional GIC and thyme-modified GIC.

Materials and Methods

Preparation of thyme leaves extract

Thyme leaves were dried for 5 days. The glassware was adequately washed using distilled water and dried in a hot air oven prior. In the beaker, 0.5 g of Thyme leaves are added to 100 ml of distilled water. The mixture is mixed and boiled in a water bath by covering the beaker for 10 minutes up to the level of 5ml, hence the concentrated thyme leaves extract was prepared. The solution is filtered using filter paper and the filtrate is collected in a separate conical flask. This extract is preserved for further procedure. 

Test pathogens and inoculum preparation

The antimicrobial activity of the synthesized modified Glass ionomer cement with thyme leaves was tested against pathogenic microorganisms Streptococcus mutans and Lactobacillus acidophilus. The bacterial strains were obtained from the Department Of microbiology. Using a sterile complete loop of each pure culture, the facultative strains of S. mutans and Lactobacillus acidophilus were fully grown on Mueller Hinton Agar. Individually injected in tubes holding 5 mL of sterile Mueller Hinton broth, the bacteria were subcultured in the proper culture conditions and incubated at 37 degrees Celsius for 24 hours. Once that was done, the suspension was changed to 0.5 Mcfarland scale = 1.5 × 108 colony-forming unit (CFU).

Specimen preparation

The type II GIC (GC Corporation) was used in the present study. Thyme extract was added after combining the liquid and powder components of conventional GIC in different concentrations and grouped (Table 1). In less than a minute, the prepared specimens were placed in the cylindrical wells using the sterile cement carrier, and the cement layer's upper surface was leveled using a sterile glass slide. The completed cement was then poured into cylindrical molds with a thickness of 2 mm and a diameter of 6 mm. After the cement ignited, the specimens in disc shape were taken out of the mold. We measured and noted the precise specimen dimensions with calipers. Twelve samples were prepared for each group, six for S.mutans and the other six for Lactobacillus. Strains of S.mutans and Lactobacillus were used, and how well the tested groups counteracted bacteria. For the measurement of compressive strength, the cylindrical molds with dimensions of 4.0 mm in diameter and 6.0 mm in height, and compressive strength were assessed in accordance with ISO 9917-1:2007. For each group, twelve specimens were prepared. To create a smooth surface, materials were then added to the mold and leveled. After being removed from the mold an hour later, the samples were kept in deionized water for 24 hours for the evaluation of compressive strength.

Table 1. Grouping

Minimal inhibitory concentration (MIC) assay

The antibacterial effectiveness of modified and unmodified GIC was evaluated using standard strains of S. mutans and Lactobacillus.MHA broth was prepared, and sterilized and 200 µL was added to all four wells. Bacterial suspensions of about 50 µL (S. mutans and Lactobacillus acidophilus ) were added to all 4 wells in the range of 5×10⁵ CFU/ml. The first three wells contain three different concentrations of GIC (2:1:1),(3:1:2), and (3:2:1) and the fourth well is considered the control (Conventional GIC). The incubation is done under suitable conditions for varied time intervals (1h, 2h, 3h, 4h). Using an ELISA reader, the percentage of dead cells is calculated at a wavelength of 540 nm at regular time intervals.

Compressive strength evaluation

Specimens that were deformed or had voids were discarded. Each specimen's diameter was measured using a digital micrometer gauge. The samples were then positioned vertically using the Universal Testing Machine (Instron, ElectroPuls®, E3000 ). At a crosshead speed of 0.5 mm/min, compression load was applied to the specimen's long axes until fracture, and readings were recorded as per the graph.

Statistical analysis

Data entry into an Excel spreadsheet, followed by statistical analysis using SPSS version 24.0, was done with the collected data ( IBM corporation). Descriptive analysis and repeated measure ANOVA were used to calculate the mean MIC values. For compressive strength, the groups were compared by the use of one-way analysis of variance (ANOVA), and the groups were compared pairwise using Tukey's post hoc test at the significance level P  ≤ 0.05 with 95% confidence intervals.

Results and Discussion

Antimicrobial efficacy against S.mutans

Repeated measure ANOVA is used in this study to test the antibacterial effect of modified and unmodified GIC, against S.mutans, first, three thyme-modified groups performed better and also showed statistically significant results when compared with Group IV (control) (Figure 1). Multiple comparison Tukey HSD It was determined by a pairwise comparison test that there was a significant difference between Group IV and any of the other three groups(p<0.05) (Table 2).

Table 2. Pairwise comparison of antimicrobial efficacy on Streptococcus mutans between four groups

 + Mean difference is significant, P value was significant at 0.05,P value was derived from Multiple comparison Tukey HSD Test.

Antimicrobial efficacy against lactobacillus

Antimicrobial activity against lactobacillus, both modified and control groups, showed similar activity proving there were no statistically significant results between conventional GIC and modified GIC. The repeated measure ANOVA linear chart was shown in Figure 2. The pairwise comparison shows there was no statistically significant difference when comparing Group IV with other groups (p>0.05) (Table 3). This proves there was an almost equal antibacterial activity for thyme-modified and conventional groups against Lactobacillus.

Table 3. Pairwise comparison of antimicrobial efficacy on Lactobacillus between four groups

Compressive strength evaluation

The compression load was applied to the specimens and the linear graph values were recorded (Figure 3). One-way analysis of variance (ANOVA) was used to analyze the compressive strength between the groups, and it was discovered that there was a statistically significant difference between the groups with an F value of 718.17 and a p-value of 0.000 (p0.05) (Table 4). The pairwise comparison was done using Tukey's post hoc test, where there was no statistically significant difference between Group IV when compared with group I & II (p>0.05) proving that 2:1:1(Group I) and 3:1:2 (Group II) and Group IV were equally effective, but when comparing Group III with Group IV there was a significant difference (p<0.05) (Table 5) where Group IV(conventional group )has performed with increased compressive strength.

Table 4. Comparison between groups for compressive strength evaluation

*Significant  at  0.05, P value was derived by one way ANOVA

Table 5. Pairwise comparison for evaluation of compressive strength

*significant difference at p=0.05, ⁺significant difference value p<0.05, P value was derived from Tukey Post hoc test.