## 2 Results
### Molecular Dynamics Simulation of \(\beta\)-Glucuronidase Inhibition  
To examine the structural stability and its variations of the **1**-6 LEL complex, MD was performed after docking computations with a period of 100 ns, using the Desmond package (Schrodinger 2020-1, New York, NY, USA). The general conditions of the simulation and its stabilization are obtained by root-mean-square deviation (RMSD) analysis [28]. The higher stability of the protein-ligand complex was demonstrated by a lower RMSD value, whereas decreased stability was indicated by an increased RMSD value [28]. The RMSD value of the **1**-6 LEL complex increased rapidly during the initial equilibration fluctuation for 5 ns, slowly increasing from 5 to 38 ns, after which the RMSD was maintained between 2.2 and 2.4 A, until the simulation was completed (Figure 6A). A maximum RMSD of 2.6 A was observed for the target protein of \(\beta\)-glucuronidase, indicating that the **1**-6 LEL complex maintained stability throughout the MD period. The flexibility and fluctuation of each residue in \(\beta\)-glucuronidase over the 100 ns simulation were represented by the root-mean-square fluctuation (RMSF) value used to predict the ligand binding-induced structural alterations in the protein structure [29]. Higher RMSF values represent greater flexibility in MD simulations [30]. The RMSF plot of the complex between compound **1** and \(\beta\)-glucuronidase is displayed in Figure 6B, where the peaks represent the \(\beta\)-glucuronidase regions that fluctuated the most during the 100 ns simulation. The RMSF values of amino acid residues in the allosteric site of \(\beta\)-glucuronidase were less than 1.3 A. In contrast, the RMSF values of residues in the active site of \(\beta\)-glucuronidase fluctuate slightly more, between 2.0 and 3.5 A, suggesting that the protein structure in ligand-bound conformations is stable during MD simulation. Hence, compound **1** may function as a non-competitive inhibitor of \(\beta\)-glucuronidase, which is consistent with the results of the kinetic study. Figure 5: **The 3D docking poses (A) and 2D interaction diagrams (B) of \(\beta\)-glucuronidase inhibition mediated by compound 1. Green: hydrogen bonds, violet and pink: hydrophobic, orange: electrostatic, and light green: van der Waals interactions. **Figure 7 presents the protein-ligand contact diagram between compound **1** and \(\beta\)-glucuronidase. The hydroxy groups of **1** established hydrophobic contact with Leu435, polar interactions with Thr191 and His192, a positive charge with Lys400, and a negative charge with Asp436; these residues of the \(\beta\)-glucuronidase allosteric site contributed to the binding interactions of 24, 17, 13, 28, and 98%, respectively. In addition, the lactone ring of **1** was linked with Thr438 via polar interactions; Arg272 and Arg439 through \(\pi-\)cation interactions, accounting for 12 and 41% of the contribution, respectively. The MD results revealed the crucial role of the lactone ring in the \(\beta\)-glucuronidase inhibitory activity mediated by active compound **1**. Figure 6: Molecular dynamics simulation of active compound **1** and \(\beta\)-glucuronidase (PDB ID: 6 LEL) complex: RMSD ((**A**), protein RMSD: green line and RMSD of **1**: red line) and RMSF (**B**). Figure 7: Protein–ligand contact analysis between compound **1** and \(\beta\)-glucuronidase complex ((**A**), timeline and (**B**), bar chart presentations) and 2D interaction diagram (**C**). The ligand torsion plot that characterizes the conformational evolution of each rotatable bond (RB) in compound 1 during the 100 ns simulation trajectory is displayed in Figure 8A, where the 2D chemical structure of compound **1** with colored RB is followed by a same-colored dial and bar plots. A total of four RBs were observed in compound **1**, with the potential values of 3.36 kcal/mol and 7.16 kcal/mol for hydroxy groups and the linkage between the phenol ring and lactone ring, respectively.