## 3 Results and Discussion
### Molecular Docking  
As one of the important tools to investigate the active site of a protein and to elucidate the interactions between ligands and the biological molecule, molecular docking was conducted. The most abundant compounds of willow bark and leaves extracts were selected to study their binding affinity and interactions to AChE enzyme. While docking results have shown a number of possible ligand conformations, only the lowest binding energy was chosen. Results of docking analysis are shown in Table 5. The more negative the binding energy value, the better affinity the ligand has for the receptor. The inhibition constant (Ki) was also predicted for the selected ligands, which brings about additional information along with energy values. A very good linear correlation between pKi, where pKi = -logKi, and docked binding energies was observed (R\({}^{2}\) = 1). The standard, donepezil, showed the strongest binding energy toward AChE and the largest number of residues (Table 5). The nature of the interactions is presented in Figure 3. \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Compound**} & \multicolumn{2}{c}{**Binding Energy**} & \multicolumn{2}{c}{**Inhibition**} & \multirow{2}{*}{**Interaction Site**} \\  & **(Kcal/mol)** & & & \\ \hline \multirow{3}{*}{Epicatechin} & \multirow{3}{*}{\(-9.33\)} & \multirow{3}{*}{145.64} & Tyr 72, Trp 86, Tyr 124, Trp 286, \\  & & & & 295, Phe 297, Tyr 337, Phe 338, \\  & & & & Tyr 341 \\ \multirow{3}{*}{Salicin} & \multirow{3}{*}{\(-7.38\)} & \multirow{3}{*}{3890} & Tyr 72, Asp 74, Tyr 124, Trp 286, \\  & & & Phe 295, Phe 297, Phe 338, Tyr 341 \\ \multirow{3}{*}{Chlorogenic acid} & \multirow{3}{*}{\(-7.87\)} & \multirow{3}{*}{1700} & Tyr 72, Trp 86, Gly 121, Tyr 124, \\  & & & & Ser 203, Trp 286, Phe 295, Phe 297, \\ \multirow{3}{*}{Rutin} & \multirow{3}{*}{\(-0.87\)} & \multirow{3}{*}{232030000} & Tyr 337, Phe 338, Tyr 341, His 447 \\  & & & & Gly 345, Ser 347 \\ \multirow{3}{*}{Donepezil} & \multirow{3}{*}{\(-11.43\)} & \multirow{3}{*}{4.2} & Trp 86, Gly 121, Tyr 124, Ser 203, \\  & & & Trp 286, Phe 295, Phe 297, Tyr 337, \\ \multirow{3}{*}{Chlorogenic acid} & \multirow{3}{*}{\(-7.87\)} & \multirow{3}{*}{1700} & Tyr 338, Tyr 341, His 447 \\  & & & & Tyr 341, His 447 \\ \multirow{3}{*}{Rutin} & \multirow{3}{*}{\(-0.87\)} & \multirow{3}{*}{232030000} & \multirow{3}{*}{Gly 345, Ser 347} \\  & & & Trp 86, Gly 121, Tyr 124, Ser 203, \\ \multirow{3}{*}{Donepezil} & \multirow{3}{*}{\(-11.43\)} & \multirow{3}{*}{4.2} & Trp 286, Phe 295, Phe 297, Tyr 337, \\  & & & Phe 338, Tyr 341, His 447 \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Summary of molecular docking studies of selected compounds against acetylcholinesterase (AChE). Rutin demonstrated the weakest binding affinity toward AChE among the studied ligands. Previous reports also indicate that rutin was not inhibiting AChE and are consistent with our data [40; 41]. Epicatechin, salicin and chlorogenic acid showed good affinity toward AChE in comparison with the standard inhibitor donepezil. Indeed, epicatechin showed the lowest binding energy among the ligands. In addition, these three ligands interact with important amino acid residues at the active site of AChE [42]. In order to get insight into the interactions of these ligands with the active site of AChE, 3D and 2D structures of AChE-ligand complexes are shown in Figures 4-6. Figure 4: Active site of the AChE enzyme with epicatechin: (**a**) 3D view; (**b**) 2D view. Figure 3: Active site of the AChE enzyme with donepezil: (**a**) 3D view; (**b**) 2D view. AChE has a narrow 20 A gorge with two binding sites: the catalytic active site at the bottom of the structure and the peripheral anionic site (PAS) near the entrance of the gorge. Consequently, ligands that bind to either one or two of the sites represent promising inhibitors of AChE in treatment of AD [43].