*3.5. Molecular Docking*

The structure of vertebrate xanthine oxidase exists as a homodimer with each of its functional unit having four redox sites, viz. an active-site molybdenum center, a pair of spinach ferredoxin-like [2Fe-2S] clusters, and a redox cofactor as flavin adenine dinucleotide (FAD) [36]. Figure 8 shows the active site of bovine xanthine oxidase (PDB code: 3NVY) with quercetin as the co-crystallized ligand. The amino acid residues in the active site include Arg880, Thr1010, Glu808, Phe1009, Ala1079, Phe914, Val1011, Leu1014, Leu873, and Leu648. The 7-OH of quercetin formed hydrogen-bonding interactions with Arg880 and Thr1010. The benzopyran ring displayed pi-pi stacking interactions with Phe914 and Phe1009, while 5-OH formed hydrogen-bonding interaction with the Mo. These interactions are considered as important requirements for the inhibitory activity of flavonoids against xanthine oxidase [36].

**Figure 8.** Binding interaction of quercetin (co-crystallized ligand) with the active site residues of xanthine oxidase (protein data bank (PDB) code: 3NVY). Green and purple dashes represent hydrogen-bonding and hydrophobic interactions, respectively.

Figure 9 shows the binding pose of the afzelechin 3-*O*-alpha-L-rhamnopyranoside in the active site of xanthine oxidase. This compound displayed a binding pose similar to quercetin. The 7-OH formed hydrogen-bonding interactions with Arg880 and Thr1010, while the benzopyran ring exhibited pi-pi stacking interaction with Phe914. These interactions sugges<sup>t</sup> that this compound may exert inhibitory activity against the enzyme. A pi-hydrophobic interaction was observed between the aromatic ring B of the flavan and Leu648. The glycone moiety displayed extensive hydrogen-bonding interactions with Lys771, Glu802, and Asn768, which may also play a role in the xanthine oxidase inhibitory activity of this molecule.

**Figure 9.** Binding interaction of afzelechin 3-*O*-alpha-L-rhamnoside with the active site residues of xanthine oxidase (PDB code: 3NVY). Green and purple dashes represent hydrogen-bonding and hydrophobic interactions, respectively.

The binding pose of cucumerin A in the active site of xanthine oxidase is shown in Figure 10. Cucumerin A displayed similar interaction with quercetin and afzelechin. The 4-OH of the phenyl ethyl group binds into the catalytic pocket, exhibiting hydrogen bonding with Arg880 and Thr1010, as well as pi-pi stacking with Phe914 and Phe1010. The 7-OH of the flavonoid ring formed hydrogen bonding with Ser879, while the glycone moiety interacted with Asp872 as well as Ser876 through hydrogen bonding. These interactions show that cucumerin A could also contribute to the xanthine oxidase inhibitory effect of the butanol fraction of the *A. bilimbi* crude methanolic leaves extract. Figure 11 shows the binding pose of allopurinol, a known inhibitor of xanthine oxidase. The C=O group displayed hydrogen-bonding interaction with Arg 880, while the aromatic ring was stacked by pi-pi hydrophobic interactions with Phe914 and Phe1009. Additional hydrophobic interactions were observed with Ala 1078 and Ala1089. These interactions were similar to what was observed in the case of quercetin, cucumerin A, and afzelechin 3-*O*-alpha-L-rhamnopyranoside. However, it did not exhibit a hydrogen-bonding interaction with Thr1010, as observed with the aforementioned three flavonoids.

**Figure 10.** Binding interaction of cucumerin A with the active site of xanthine oxidase (PDB code: 3NVY). Green and purple dashes represent hydrogen-bonding and hydrophobic interactions, respectively.

**Figure 11.** Binding interactions of allopurinol with the active site residues of xanthine oxidase (PDB code: 3NVY). Green and purple dashes represent hydrogen-bonding and hydrophobic interactions, respectively.

Table 5 shows the docking score of the two phenolic compounds, the co-crystallized ligand and allopurinol. The lower the free binding energy, the greater the interactions of the compound with the enzyme active site residues, which may consequently lead to better inhibitory effect. Cucumerin A and afzelechin 3-*O*-alpha-L-rhamnopyranoside have lower free binding energies than both quercetin and allopurinol. This further suggests that these two compounds could be responsible for the significant xanthine oxidase inhibitory effect of *A. bilimbi* leaves.

