## 3 Results and Discussion
### Biosynthesis of AgNP  
In the current study, the biosynthesis of AgNP using a green tomato extract obtained by subcritical water treatment as a reducing agent is reported. A solution of silver nitrate salt (AgNO\({}_{3}\)) in deionized water was applied as the silver source (Ag). The Surface Plasmon Resonance (SPR) peak of AgNP ranges from 300 to 600 nm and depends on the environmental conditions, such as pH, size, shape, and the concentration of the extract or silver nitrate [32]. It has already been demonstrated that by varying key conditions/parameters in the biosynthesis of AgNP, it is possible to dramatically change the characteristics of the particles obtained [33]. In pre-formulation assays, it was possible to establish an optimal value for TE, and with this fixed concentration, different experimental conditions were tested, namely, the effects of the temperature, pH, reaction time, and concentration of AgNO\({}_{3}\) (Figure 1). This procedure allowed the selection of the optimal conditions for the biosynthesis of AgNP using this TE. The effect of the temperature on the SPR peak using 1.70% (\(v/v\)) TE and 1.67 mM AgNO\({}_{3}\) is presented in Figure 3A. The reaction was carried out for 24 h in the dark at pH 11. As indicated in Table 1, the AgNP produced at 37 \({}^{\circ}\)C were smaller in size and had a lower PdI than those produced at room temperature. The zeta potential of AgNP produced at both temperatures was negative (\(<\)-20 mV), indicating that they can be considered stable. The intensity of the SPR peak was higher at 37 \({}^{\circ}\)C, which is in accordance with what has been found in other studies that correlated higher temperatures with an increased production rate of monodisperse and smaller nanoparticles [34]. \begin{table}
\begin{tabular}{c c c c c} \hline \multicolumn{1}{c}{**Manipulated Parameter**} & \multicolumn{1}{c}{**Z-Average (nm)**} & \multicolumn{1}{c}{**PdI**} & \multicolumn{1}{c}{**Zeta Potential (mV)**} \\ \hline \multirow{6}{*}{Time} & 15 min & 163 \(\pm\) 22 & 0.32 \(\pm\) 0.08 & \(-\)22 \(\pm\) 1 \\  & 30 min & 235 \(\pm\) 47 & 0.29 \(\pm\) 0.04 & \(-\)28 \(\pm\) 1 \\  & 60 min & 222 \(\pm\) 25 & 0.30 \(\pm\) 0.03 & \(-\)28 \(\pm\) 1 \\  & 90 min & 191 \(\pm\) 27 & 0.29 \(\pm\) 0.02 & \(-\)29 \(\pm\) 1 \\  & 120 min & 290 \(\pm\) 35 & 0.36 \(\pm\) 0.07 & \(-\)29 \(\pm\) 3 \\  & 150 min & 246 \(\pm\) 34 & 0.34 \(\pm\) 0.04 & \(-\)32 \(\pm\) 0 \\ \hline \multirow{2}{*}{Temperature} & Room & 51 \(\pm\) 1 & 0.34 \(\pm\) 0.05 & \(-\)29 \(\pm\) 1 \\  & 37 \({}^{\circ}\)C & 35 \(\pm\) 0 & 0.31 \(\pm\) 0.03 & \(-\)21 \(\pm\) 3 \\ \hline \multirow{3}{*}{AgNO\({}_{3}\)} & 1 mM & 23 \(\pm\) 1 & 0.61 \(\pm\) 0.01 & n.d. \\  & 5 mM & 29 \(\pm\) 6 & 0.50 \(\pm\) 0.19 & n.d. \\  & 10 mM & \textgreater{}1000 & - & n.d. \\ \hline \multirow{3}{*}{pH} & pH 8 & 245 \(\pm\) 5 & 0.43 \(\pm\) 0.17 & \(-\)22 \(\pm\) 2 \\  & pH 9 & 226 \(\pm\) 28 & 0.51 \(\pm\) 0.02 & \(-\)22 \(\pm\) 1 \\ \cline{1-1}  & pH 11 & 160 \(\pm\) 3 & 0.32 \(\pm\) 0.03 & \(-\)31 \(\pm\) 1 \\ \cline{1-1}  & pH 12 & \textgreater{}1000 & - & n.d. \\ \hline \end{tabular}
\end{table} 
Table 1: The reaction parameters, mean size, PdI, and zeta potential of AgNP obtained from the TE. Data represent the mean \(\pm\) standard deviation of three measurements. n.d.: no data. Figure 3B depicts the SPR peaks resulting from different concentrations of silver nitrate tested in reaction mixtures with 0.5% (\(\
u/v\)) TE at room temperature. The reaction time was 2.5 h and carried out in the dark at pH 11. The SPR peak observed for the 1 mM silver nitrate concentration is narrower. The size difference in AgNP produced with 1 mM and 5 mM silver nitrate is minimal (Table 1). When the AgNO\({}_{3}\) concentration is higher (10 mM), a reduction in the SPR peak may indicate the depletion of biomolecules in the TE, which cannot maintain AgNP formation [35]. The effect of the reaction time was evaluated using 1.67% (\(\
u/v\)) TE and 1.67 mM AgNO\({}_{3}\) at 65 \({}^{\circ}\)C in the absence of light. The pH of the solution was 11 (Figure 3C).