## 2 Materials and Methods
### Evaluation of the Antimicrobial Properties of the Developed Toothpastes  
The development of a natural toothpaste has to be acceptable in terms of its organoleptic characteristics (appearance, color flavor), meaning it must meet the physicochemical and microbiological specifications set for this type of product (pH, viscosity, dispersion of silica particles, total aerobic microbial count, molds, yeast, and absence of pathogens). For this reason, the newly developed natural products successfully passed both accelerated and long term stability tests according to protocol by ICH Topic Q1 A(R2): 40 \(\pm\) 2 \({}^{\circ}\)C/75% RH for 3 months, 50 \(\pm\) 2 \({}^{\circ}\)C/75% RH for 1 month, 25 \(\pm\) 2 \({}^{\circ}\)C/60% RH for 12 months, 5 \(\pm\) 3 \({}^{\circ}\)C for 6 months, and Freeze/Thaw Cycle Tests (4) to be considered stable over time, before conducting the antimicrobial property test. Three equally stable versions of natural toothpaste were achieved, each with a different natural antimicrobial agent. The natural toothpastes, SAMPLE A (0.1% BOOC), SAMPLE B (0.1% Microsilver BG), and SAMPLE C (0.5% Cranberry LG), met all the required specifications for such a formulation. Therefore, a comparative in vitro efficacy study of the three natural antimicrobial agents compared to a negative control (SAMPLE D) as well as a commercial synthetic toothpaste as a positive control (SAMPLE E) was conducted. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Commercial Name of the Natural** & & **Content** & **Physical Characteristics of the Natural Antimicrobial Agent** \\
**Antimicrobial Agent** & **INCI Name—Composition** & **(\% _w/w_)** & **Natural Antimicrobial Agent** \\ \hline Biosecure Organic & Glycerin, Citrus Reticulata Fruit & & Brown, slightly viscous liquid \\ Oral Care & Extract, Citrus Aurantium Amara & & consisting of a mixture of citrus \\ (Sharon) & Fruit Extract, Citrus Aurantium & 0.1\% & extracts in glycerin (extracts from: \\ (Sharon) & Sinensis Peel Extract, Ascorbic Acid, & & mandarin fruit, bitter orange fruit, \\  & Citric Acid, Lactic Acid, Water & & sweet orange peel) \\ \hline Microsilver BG & Silver & 0.1\% & Grey powder \\ (BioGate) & & & \\ \hline Cranberry LG & Glycerin, Vaccinium Macrocarbon & & \\ (Givaudan) & Extract, & 0.5\% & Colorless solution of cranberry extract \\  & Water & & in glycerin \\ \hline \hline \end{tabular}
\end{table} 
Table 2: The composition, content (\(\%\)\(w/w\)) and physical characteristics of the tested antimicrobial agents. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Antimicrobial Natural** & & & & **Viscosity** \\
**Toothpaste** & & & & **(Brookfirld DVI+, Spindle:** \\  & & & & **F–96, rpm: 1.5, T: 20 \({}^{\circ}\)C)** \\ \hline Biosecure Organic & & & & \\ Oral Care (0.1\% \(w/w\)) & Yellowish gel & & & \\  & & & flavor (mint) & \\ \hline Microsilver BG & & & & \\ (0.1\% \(w/w\)) & Grey gel & & & \\  & & & flavor (mint) & \\ \hline Cranberry LG & & & & \\ (0.5\% \(w/w\)) & Transparent gel & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: The physical–chemical characteristics of the developed natural antimicrobial toothpastes with antimicrobial agents. The method that was used in this experiment was the disc diffusion test based on Kirby-Bauer Test [32]. The tested toothpaste was placed in Petri dishes in whichspecific microorganisms selected for the study had been selectively cultivated. After incubation, the circular area formed around the discs (diameter), known as the inhibition zone, was measured, demonstrating the inhibitory effect of the product on the microorganisms used in the efficacy test. The larger the inhibition zone, the greater the antimicrobial action of the product, always compared with the reference systems and control samples [33; 34; 35]. More specifically, a sample of the product diluted with water at a 1:1 ratio (to simulate the clinical conditions) was added to sterile filter paper discs and placed on top of a pre-inoculated agar plate surface with bacteria or fungi.