*2.5. Dynamic Molecular Docking of Active Components of DGS with Key Targets*

The binding ability of compounds from DGS to VEGFR2 was validated in the present study using the same molecular docking to provide evidence for the proangiogenic effect of DGS with a virtual approach. All 37 components of DGS were docked to the binding pocket of VEGFR2, and an agonist of VEGFR2 was used as the positive control. All compounds were docked into the active pocket of VEGFR2, and all exhibited binding energies were greater than −5 kcal/mol. When the binding energy of agnuside was used as a control [22], nine compounds had binding energies greater than that of agnuside, as shown in Table 2. The binding modes of agnuside and salvianolic acid C with VEGFR2 were visualized separately using Pymol, which showed that the binding of agnuside to VEGFR2 was mainly via hydrogen bonding and pi-sigma interactions (Figure 6A). On the contrary, salvianolic acid C displayed pi–pi stacked interactions in addition to hydrogen bonding and pi–sigma interactions (Figure 6B).

Based on the docking results, salvianolic acid C and agnuside with VEGFR2 were further selected for MD simulation to examine their stability in the binding pocket. Root mean square deviation (RMSD) serves as an important basis for measuring system stability. In this study, the mean RMSD of the salvianolic acid C–VEGFR2 system after balance was 2.63 ± 0.323 Å (Figure 7A). Meanwhile, the mean RMSD of the agnuside–VEGFR2 system after balance was 2.36 ± 0.44 Å (Figure 7A). The mean RMSD of the two systems is <3 Å, which is completely acceptable in the protein system. Subsequently, the flexibility changes and root mean square fluctuation (RMSF) values of amino acid residues in VEGFR2 were evaluated. Figure 7B,F, the amino acid residues that interact with the ligand for more than 30% in the RMSF diagram. The RMSF values of most of the amino acid residues involved in the interaction were small, thereby indicating that the stability of the entire system was increased after salvianolic acid C and agnuside are combined with VEGFR2. The 2D visualization analysis of the salvianolic acid C–VEGFR2 system showed that salvianolic acid C formed hydrogen bonds with VEGFR2 via LYS-920, ASN-923, ARG-842, CYS-919, GLU-917, and GLU-885; furthermore, it forms Pi–Pi stacking interaction with PHE-1047 (Figure 7C,D). The 2-D visual analysis of the agnuside–VEGFR2 system showed that agnuside formed hydrogen bonds with VEGFR2 through VAL-914, THR-916, GLU-885, VAL-899, ASP-1046, and ILE-1025; moreover, it formed π–cation stacking with LYS-868 (Figure 7G,H). All the interaction durations exceed 30% of the whole simulation time.


**Table 2.** Docking information of VEGFR2 with the corresponding compounds.

**Figure 6.** Molecular docking of Agnuside and Salvianolic Acid C to VEGFR2 protein: (**A**) Agnuside binding model with VEGFR2, yellow dashed lines represent hydrogen bonding interactions, green dashed lines represent π–Sigma interactions. (**B**) Salvianolic Acid C binding model with VEGFR2, yellow dashed lines represent hydrogen bonding interactions, green dashed lines represent π–Sigma interactions and red dashed lines represent π–π stacked interactions.

**Figure 7.** Dynamics molecular docking of two protein–ligand complexes, Salvianolic Acid C–VEGFR2 (**A**–**D**) and Agnuside–VEGFR2 (**E**–**H**): (**A**) RMSD of Salvianolic Acid C–VEGFR2. (**B**) RMSF of Salvianolic Acid C–VEGFR2. (**C**) Protein–Ligand Contacts Diagram of Salvianolic Acid C–VEGFR2; Y axis suggests that percentage of the simulation time the specific interaction is maintained; Values over 1.0 are possible as some protein residue may make multiple contacts of the same subtype with the ligand. (**D**) A schematic of detailed Salvianolic Acid C atom interactions with the VEGFR2 residues. (**E**) RMSD of Agnuside–VEGFR2. (**F**) RMSF of Agnuside–VEGFR2. (**G**) Protein–Ligand Contacts Diagram of Agnuside–VEGFR2. (**H**) A schematic of detailed Agnuside atom interactions with the VEGFR2 residues.
