## 3 Results and Discussion
As shown in Figure 3a and Table 1, compounds **1** and **2** exhibited significant dose-dependent neuraminidase inhibition, with IC\({}_{50}\) values of 56.0 uM and 203.3 uM, respectively. The IC\({}_{50}\) values suggested that compound **1** was four times more active than compound **2**. The IC\({}_{50}\) of the positive control (quercetin) was 21.0 uM under the same conditions. The inhibition of neuraminidase by compound **1** is represented in Figure 3b. When the initial velocity was plotted against the concentration of enzyme in the presence of varying concentrations of compound **1**, a family of straight lines was obtained, all of which had their axes in the origin. The line gradients were reduced as the inhibitor concentration was increased, demonstrating that the chemical was a reversible inhibitor. \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Compounds**} & \multicolumn{3}{c}{**BNA**} \\ \cline{2-4}  & **IC\({}_{50}\)\({}^{\text{a}}\) (\(\mu\)g/mL)** & **Kinetic Mode** & **K\({}_{\text{1}}\) (\(\mu\)M)** & **K\({}_{\text{1S}}\) (\(\mu\)M)** \\ \hline Compound **1** & 56.0 \(\pm\) 0.7 & Mixed type I (28.4) & 27.7 & 92.0 \\ Compound **2** & 203.3 \(\pm\) 2.8 & NT\({}^{\text{c}}\) & NT & NT \\ Quercetin \({}^{\text{d}}\) & 21.5 \(\pm\) 0.8 & NT & NT & NT \\ \hline \hline \end{tabular}  
* All compounds were examined by repeating the experiments thrice. \({}^{\text{a}}\) IC\({}_{50}\) values of the compounds represent the concentration responsible for 50% loss in enzymatic activity. \({}^{\text{b}}\) Value of the inhibition constant; \({}^{\text{c}}\) NT denotes not tested; \({}^{\text{d}}\) Quercetin was a positive control.  
\end{table} 
Table 1: Inhibitory effects of isolated compounds on bacterial neuraminidase (BNA) activity. Figure 3: Inhibitory activity and mechanism of action of compounds against bacterial neuraminidase (BNA). (**a**) Dose-dependent inhibition of BNA by compound **1**, compound **2**, and quercetin (positive control). (**b**) Reversibility of the inhibitory effect of compound **1**. (**c**) Lineweaver–Burk plots for BNA inhibition by compound **1**. (**d**) Dixon plots of BNA inhibition by compound **1**. (Inset) Tendencies of the intercept and slope from each straight line of the Lineweaver–Burk plot. Our data reveal interesting facets in the structure-activity relationship of the compounds. Better inhibition was observed when a hydroxy group was present at C-3\({}^{\prime}\). This could be seen by comparing C-3 hydroxy-substituted compound **1** (IC\({}_{50}\) = 56.0 \(\upmu\)M) with the glucosyl analog of compound **2** (IC\({}_{50}\) = 203.3 \(\upmu\)M). As shown in Figure 3c,d, the inhibition kinetics analyzed using Dixon and Lineweaver-Burk plots showed that compound **1** (\(K_{\rm i}\) = 28.4 \(\upmu\)M) was a mixed type-I inhibitor, because increasing the concentration of the substrate yielded a family of lines that intersected at a non-zero point on both the x- and y-axes (Figure 3d). As a result of the mixed nature of the inhibition, the inhibitor had different degrees of affinity for the substrate-bound and free enzymes. Since the inhibitor could bind to either the free enzyme or the enzyme-substrate complex, we sought to separate the inhibition constants for these two states. Type-I inhibition occurred when the inhibitor was bound to the free enzyme, while type-II inhibition occurred when the inhibitor was bound to the enzyme-substrate complex. Both the free enzyme and the enzyme-substrate complex states are referred to as the enzymatic state. In order to conduct this analysis, the concentrations of the inhibitor and substrate were changed based on equations (2) through (4). The equilibrium constants for the two distinct binding events, inhibitor binding to the free enzyme (\(K_{\rm I}\)), and inhibitor binding to the enzyme-substrate complex (\(K_{\rm IS}\)), were determined using secondary plots of \(K_{\rm m}/V_{\rm max}\) and \(1/V_{\rm max}\) versus compound **1**'s concentration, respectively.