3.3. Molecular Docking for Anti-Skin Aging Activity EvaluationThe test ligands used in this study were compounds found in Indonesian ginger extracts. The control ligand used was ascorbic acid. Ascorbic acid was chosen as a control ligand because it can prevent aging by inhibiting the formation of reactive oxygen species and stratum corneum formation so that dead skin cells can be replaced quickly [31]. In addition, ligand preparation produces ligands whose geometric structure is optimized. These conditions are expected to increase the accuracy and validity of the molecular docking results. The results of the ligand preparation are saved in *pdbqt format. The results of the preparation of the test and control ligands used are listed in Table S2. The receptors used were collagenase (2TCL), hyaluronidase (2PE4), elastase (3F19), and tyrosinase (5M8R). Information about the coordinates of each receptor’s active site was generated in the grid box from Autodock Vina. The coordinates of each active site can be seen in Table 1.Most of the test ligands’ affinity energies in each complex had higher negative values than the crystal and control ligands (Table S3). This indicated that the test ligands that were used had the potential to be inhibitors of the selected receptor. For example, the d-Corlin ligand appeared in four receptor complexes with the highest negative affinity energies for the collagenase, hyaluronidase, elastase, and tyrosinase complexes, namely −14.6; −13.9; −16.0; and −13.4 kcal/mol (Table 2). The 20-α-dhydrodydrogesterone also appeared in four receptor complexes with an average affinity energy of −14.6 kcal/mol (Table 2), but was most likely not chosen because the compound is listed as a potent drug causing miscarriage and bleeding in pregnant women [32]. The catechin ligand appeared in four complexes, namely the collagenase complex with an affinity energy of −8.8 kcal/mol, the hyaluronidase complex with an affinity energy of −7.9 kcal/mol, the elastase complex with an affinity energy of −9.7 kcal/mol, and the elastase complex with an affinity energy of −8.4 kcal/mol (Table 2). Unfortunately, despite having a low affinity energy, the abundance of d-Corlin and catechin in Indonesian ginger extracts was low. Therefore, this study selected ligands showing a reasonable negative affinity energy and a high enough abundance in Indonesian ginger rhizome extracts.The 6-gingerol, 8-shogaol, 4-shogaol, 10-shogaol, and 6-paradol had an average affinity energy of −6.9; −6.8; −6.7; −6.7; and −6.5 kcal/mol, respectively (Table 2). The decrease in affinity energy of gingerol, shogaol, and paradol compounds possibly occurred due to the decreasing the -OH group in gingerol and the double-bond change in shogaol. In another study, the different constituents of gingerol, shogaol, and paradol caused differences in their antioxidant activity. According to Dugasani et al., 6-gingerol has higher antioxidant activity than shogaol and paradol compounds [33]. However, this study found that the abundance of 6-gingerol, 8-shogaol, 4-shogaol, 10-shogaol, and 6-paradol was very low. The compounds octinoxate, 6-gingerdione, p-cymene, and ethyl cinnamate had an average affinity energy of −6.9; −6,7; −6.2; and −6.2 kcal/mol (Table 2). Based on the results of the molecular docking analysis, octinoxate, 6-gingerdione, p-cymene, and ethyl cinnamate were most likely to be potential anti-skin aging compounds in the Indonesian ginger rhizome extracts. These four compounds had negative affinity energies and showed a high abundance in the three Indonesian ginger rhizome extracts. However, octinoxate, 6-gingerdione, p-cymene, and ethyl cinnamate still had to pass the physicochemical tests. 3.4. Visualization and Determination of Physicochemical Properties of Ligands Based on admetSAR ParametersA comparison of the test and control ligand complex’s interaction is one of the appropriate parameters to determine which test ligands have the greatest potential as anti-aging compounds. The comparison resulted in the percentage of binding site similarity (%BSS), which was observed from the similarity of amino acid residues that interacted between the test and control ligands. The similarity of binding sites is helpful in drug development, the analysis of protein–ligand complexes, and drug function prediction in chemistry [34]. Ligand–receptor interactions can occur in hydrophobic or hydrogen bonds. Hydrophobic interactions occur because of the contact between the alkyl chains in both the ligands and the receptors. Hydrogen bonds are formed due to the electrostatic attraction between hydrogen atoms attached to more electronegative atoms or groups and other electronegative atoms with lone pairs of electrons. In this case, a hydrogen bond occurred between the amino acid residue from the receptor and the ligand.The amino acid residues of collagenase that interacted hydrophobically with ascorbic acid were Leu-81, Val-115, Tyr-137, Ser-139, and Tyr-140, as well as Ala-82, Arg-114, His-118, Glu-119, and Pro-138 which hydrogen-bonded (Figure 4a). The Leu-81, Ala-82, His-118, Pro-138, and Tyr-140 residues are directly involved in the ligand binding process on collagenase. Molecular docking to collagenase yielded various %BSS. The octinoxate ligand had a 90%BSS because it interacted with nine amino acid residues (Leu-81, Ala-82, Arg-114, Val-115, His-118, Glu-119, Pro-138, Ser-139, and Tyr-140) that interacted with ascorbic acid (Figure 4b). Only the N-atom at Leu-81 in collagenase hydrogen-bonded with the O-atom in octinoxate, and the distance between them was 2.97 Å (Figure 4b).The amino acid residues Pro-62, Met-71, Ile-73, Val-127, Tyr-202, and Trp-321 of hyaluronidase interacted hydrophobically while Asn-37, Tyr-75, Asp-129, Glu-131, Tyr-247, and Tyr-286 hydrogen-bonded with ascorbic acid (Figure 4c). According to Chao et al., Tyr-75, Asp-129, Glu-131, Tyr-202, Tyr-247, Tyr-286, and Trp-321 are residues that play a critical role in hyaluronidase’s active site [35]. The octinoxate ligand had hydrophobic interactions with amino acid residues Asn-37, Pro-62, Ile-73, Tyr-75, Asp-129, Glu-131, Tyr-202, Tyr-247, and Tyr-286; it hydrogen-bonded with Asn-39 at a distance of 3.04 Å and with Trp-321 at a distance of 2.99 Å (Figure 4d). The %BSS for the octinoxane ligand was 83%.Ascorbic acid interacted hydrophobically and hydrogen-bonded with amino acid residues in the elastase enzyme. The amino acid residues His-218, His-222, His-228, Phe-237, and Tyr-240 were hydrogen-bonded, while the residues Thr-215, Pro-238, and Thr-239 interacted hydrophobically (Figure 4e). The octinoxate ligand had only hydrogen-bonded with Leu-181 at a distance of 2.52 Å (Figure 4f). Octinoxate had a 50%BSS because it only had four of the eight interactions that were the same as the ascorbic acid–elastase interaction.In tyrosinase, ascorbic acid hydrogen-bonded with His-192, His-215, His-224, Glu-360, His-377, Asn-378, His-381, Gly-389, and Ser-394; it interacted hydrophobically with Phe-362, Glu-390, and Val-391 (Figure 4g). The octinoxate had a 46%BSS because it only interacted with six of the thirteen ascorbic acid–tyrosinase interactions (Figure 4h). This was due to ascorbic acid ligands hydrogen-bonding with amino acid residues of tyrosinase, whereas octinoxate only interacted hydrophobically.The structure and shape similarity between ligand and substrate [36], as well as the interaction of the ligand and amino acid residues [37], are the things that determine the inhibitory ability of the ligand–receptor activity. Most of the interactions formed between the test ligands and the receptors were hydrophobic interactions. In contrast to the test ligands, most of the interactions between the control ligand and the amino acid residues of the receptors were hydrogen bonds. All hydrogen bonds formed between ligands and amino acid residues are >1.85 Å long.In addition to the parameters previously described, several parameters are related to the physicochemical properties of compounds in the body. These parameters are contained in Lipinski’s rules and the admetSAR test. Lipinski’s rules and the admetSAR test have several parameters, including relative atomic mass, partition coefficient value (log p), number of hydrogen-bond acceptors and donors, number of rotatable bonds, mutagenesis, carcinogenicity, and eye irritation. All ligands with the potential for anti-skin aging passed Lipinski’s rules (Table 3). However, all ligands, except for octinoxate and 20-α-dhydrodydrogesterone, could cause eye irritation, while catechin, 8-shogaol, 6-gingerdione, and 10-shogaol had the potential to cause mutation. Therefore, the use of these ligands must be considered in order to avoid adverse effects on the body. Based on the values of compound abundance, affinity energy, Lipinski’s rules, and the admetSAR test, octinoxate in the Indonesian ginger rhizome extracts was the only compound with anti-skin aging activity potential and was abundant in the EE ginger rhizome extract. Therefore, the EE ginger rhizome extract was the ginger rhizome extract with the greatest potential for anti-skin aging.