2.3.1. Molecular Docking Study with α-Amylase

Docking studies were carried out to understand the interaction of active compounds **2**–**6** inside the catalytic site of α-amylase. The crystalline structure of porcine pancreatic α-amylase (PDB ID: 1OSE) and active site amino residues are explained briefly in a previ-

ous publication [38]. The selection of the porcine pancreatic α-amylase crystal structure 1OSE in this study was based on three main reasons. First, we have used the porcine pancreatic α-amylase enzyme for our in vitro α-amylase inhibition study. Secondly, the selected protein forms a complex with the acarbose, which we used as a positive control or reference standard in our study. Finally, molecular models for the α-amylases from the human pancreas and human salivary are incredibly similar to the pig pancreatic molecular model. The homology modeling of the porcine and human pancreatic α-amylase is very similar (87.1%) compared with other amylases [39]. The binding energies of tested compounds with α-amylase ranged from −14.52 to −8.94 kcal/mol. The decreasing order of minimal binding energies in the case of α-amylase molecular docking studies are as follows: momordicoside A < kuguaglycoside C < karaviloside VIII < charantoside XV < karaviloside VI < momordicoside L (Table S1). The docking results of the isolated compounds showed the binding site as the same as the binding sites for acarbose. Indeed, the docking analysis predicted that acarbose, a competitive inhibitor of α-amylase, was surrounded by Glu233, Asp300, and Asp197, which are the part of the catalytic residues of α-amylase. [40]. The molecular docking study for 25ξ-isopropenylchole-5(6)-ene-3-*O*-β-D-glucopyranoside (**1**) was previously reported in our study [18]. The binding modes in the active site of α-amylase of all purified compounds, except **1**, are shown in Figure 5A–F. The binding energy and number of hydrogen bonds of the five triterpenes against porcine pancreatic α-amylase are shown in Table S1.

As shown in Figure 5A–F, all five triterpenes bound to the porcine pancreatic αamylase through forming various hydrogen bonds. It was observed that the binding site for karaviloside VI (**2**) was close to the active site, allowing the interaction with Asp300, Ile235, and Trp59. Four hydrogen bonds were formed between compound **2** and the amino acid residues, including Glu240 (3 H-bonds), His201 (1 H-bond). The inhibition constant and binding energy for compound **2** is 18.79 nM and −10.54 kcal/mol (Table S1).

In case of karaviloside VIII (**3**), the docking results predicted that the compound was enfolded in the catalytic domain adopting the same conformation as the acarbose site of α-amylase, as shown in Figure 5B. It was surrounded by fourteen key amino acid residues. The likeliest docked interactions of karaviloside VIII (**3**) and α-amylase is shown in Figure 5B. The ligand was surrounded by amino acid residues located in domain A, making hydrogen bonds with Lys200, His201, Trp59, and Asp356 (Table S1). The principle interaction of karaviloside VIII (**3**) with α-amylase was surrounded by the key catalytic residue Trp59, and the interaction was crucial to inhibit the activity of α-amylase. Overall, the inhibition constant for compound **2** was 1.76 nM.

The refined docking of momordicoside L (**4**) showed weaker interactions than the other compounds because of the steric hindrance of the functional group at position 25, compared to compound **7**. The compound formed fewer H-bonds reflected in the lower binding energy of -8.94 kcal/mol. The 3D figure shows that momordicoside L interacted with sixteen key amino acid residues, including four conventional hydrogen bonds that were established between momordicoside L and Glu233, Ala307, His305 (Figure 5C). The inhibition constant was found to be 278.19 nM.

Figure 5D displays a 3D schematic interaction of momordicoside A (**5**) with α-amylase. Momordicoside A generated the best docking pose with a minimum binding energy of −14.52 kcal/mol, which indicates that momordicoside A showed the most stronger binding affinity with the α-amylase. Momordicoside A was found to anchor at the catalytic site of α-amylase by making ten conventional hydrogen bonds with Glu240, His101, Glu233, Asp197, Gly63 to residues resulting in potent inhibition of α-amylase. The strong inhibition activity of momordicoside A on α-amylase relies on the formation of multiple hydrogen bonds between hydroxyl groups and key residues of α-amylase. The molecular docking of momordicoside A provided supportive data for enzyme inhibition by predicting the binding site of α-amylase. We observed theoretical inhibition constant of 22.5 pM in the case of momordicoside A.

**Figure 5.** The 3D protein-ligand interactions for (**A**) karaviloside VI (**2**), (**B**) karaviloside VIII (**3**), (**C**) momordicoside L (**4**), (**D**) momordicoside A (**5**), (**E**) Charantoside XV (**6**), and (**F**) kuguaglycoside C (**7**) in the binding sites of α-amylase. Black dotted lines indicate hydrogen bonds between compounds and amino acid residues. Ligands in the active sites are denoted in green color. Active site residues are shown in cyans color.

Analysis of molecular docking for the optimized conformation for charantoside XV (**6**) is shown in Figure 5E. The compound was bound to the active site of α-amylase with binding energy −10.64 kcal/mol. charantoside XV interacted with sixteen crucial amino acid residues in domain A of α-amylase Table S1. The compound was able to interact with key amino acid residues, including Glu240, Gly306, by making three conventional hydrogen bonds. Besides, hydrophobic π-alkyl and alkyl interactions of charantoside XV with several amino acid residues, including Trr151, Lys200, Ala307, His201, Leu162, Val163, and Ile235. The inhibition constant of charantoside XV was 15.84 nM. The refined docking of the kuguaglycoside C (**7**) with α-amylase is shown in Figure 5F. The binding energy for compound **7** in the active site of α-amylase is −11.54 kcal/mol. We observed that the compound **7** was able to interact with Asp242, Ser240, Leu246, Asn247, Ser282, Ala281,

Asn302, Glu332, His280, Asp307, Thr310, Ser311, Lys156, Phe314, Leu313, Pro240 in the catalytic site of α-amylase (Figure 5F and Table S2)**.** Also, the interactions calculated for the complex of kuguaglycoside C- α-amylase were effective because kuguaglycoside C (**7**) was buried entirely in the α-amylase binding pocket by forming ten hydrogen bonds with key amino acid residues, including Asp197, Glu233, His305, His299, and Asp300. The 2D figures for all compounds docked are compiled in supporting information (Figure S22).

Overall, the interactions between glucosides on the triterpenes and protein residues were suspected of playing an important role in determining the binding energy among triterpenes bearing the same backbone. Lower binding energy means that the ligand can more easily bind with the protein. Hence, regarding *α-*amylase molecular docking studies, we observed higher binding energy for momordicoside A and formed ten hydrogen bonds with the active site of *α-*amylase. Therefore, according to theoretical studies, momordicoside A was more likely to bind with α-amylase. However, in our in vitro studies, we did not observe a significant difference in the inhibitory activity, but we noticed momordicoside L showed comparatively higher inhibition among the six triterpenes. Therefore, our results suggested that in the inhibition process of triterpenes with α-amylase, lower binding energy does not necessarily lead to a higher inhibition activity, i.e., the inhibition activity of triterpene is affected by not only binding energy but also the chemical structure and glucoside type attached to a different position of triterpene. Besides, molecular docking studies are usually carried out under a theoretical vacuum condition, which deviates from real experimental conditions due to a low number of replications and prediction data [41].
