*2.8. Molecular Docking*

The phenolic compounds identified through analysis in the n-butanol fraction of *A. bilimbi* crude leaves methanolic extract were considered as possible molecules that contribute to the antioxidant properties. Hence, they were docked into the active site of the crystal structure of xanthine oxidase. The 3D structure for bovine xanthine oxidase, co-crystallized with standard inhibitor, quercetin (PDB ID: 3NVY), was obtained from a protein data bank (PDB) (http://www.rcsb.org/) [22]. Software including Molecular Graphics Library MGLTools 1.5.6 (The Scripps Research Institute, San Diego, CA, USA), AutoDock Tools 4.2 (www.scripps.edu), (The Scripps Research Institute, San Diego, CA, USA) [23], and Discovery Studio Visualizer 4.0 (www.accelrys.com) Bovia, San Diego, CA, USA) [24] were used for the molecular docking experiments. Crystallographic waters and co-crystallized ligand were removed, and polar hydrogens were added to a macromolecule using AutoDock 4.2, after which the structure was saved in the PDBQT file format that contains a protein structure with hydrogen in all polar residues. The 2D structures of ligands were drawn through ChemDraw® (PerkinElmer, Waltham, MA, USA) and converted to 3D format using ChemBio3D Ultra® 14 Suit (PerkinElmer, Waltham, MA, USA), which were further subjected to energy minimization using molecular mechanics 2 (MM2) force field and saved in PDB file format. The ligands were then prepared for docking by computing the charges, and the structures were saved in PDBQT file format via AutoDock 4.2. Molecular docking experiment of ligands was performed following the protocol described by Zhang et al. [25]. The size and center of the coordinates of the grid box was first validated by re-docking the co-crystallized ligand into the active sites of the receptor. The rotational bonds of the ligands were considered to be flexible, while those of the receptor were kept rigid. A grid box was centered on binding site of the co-crystallized ligand. The box size was set to 50, 50 and 50 Å3 (x, y and z, respectively) and the grid spacing to 0.375 Å. The grid maps for atoms were calculated and genetic algorithm (GA) was used for searching; the population size was set to 150, 100 runs, and 5 million energy evaluations. The ligands (identified compounds) were then docked into the active site of the enzyme using the grid box parameter obtained from the re-docking of co-crystallized ligand as reference.
