*2.4. Molecular Docking Studies for Potential Natural Product Metabolites as Inhibitors of Glycation*

Some of the important phenolic compounds present in garlic extracts were analyzed for their inhibitory role in the glycation reaction. Sudlow Site I is the primary binding site for glucose in pyranose form. The interaction of the glucose ring form and other ligands (considered in this study) with the SAL subsite of the Sudlow Site I are summarized in Table 1. Figures 9 and 10 provide insight into the most stable 2D and 3D docked conformations of the ligands at the SAL subsite obtained via exhaustive molecular docking application. The orientation of the glucopyranose at the SAL subsite was obtained from the crystal of glucose-bound has submitted by Wang et. al. [20] to the protein data bank (PDB ID: 4iw2). The binding efficiency reported in Table 3 is a result of multiple interactions of the ligands with the target active site.

The blue dotted interaction of Arg257 with glucopyranose (Figure 9a) represents an altered confirmation state of glucopyranose. The 2D interaction images in Figures 9 and 10 represent the type and count of interactions specific ligand undergoes. The 3D images of docked ligands in Figures 9 and 10 represent the polarity distribution at the docking site. The pink environment in the close vicinity of the ligands represents hydrogen donors or an electronegative environment; however, the green cloud near the ligands is due to amino acid with a side chain rich in hydrogen acceptors and results in an electropositive environment. The white cloudy areas around the ligands constitute the neutral space due to the presence of hydrophobic amino acids.

*cation* 

the ligands with the target active site.

are denoted by "–" and "+" signs. Different GE concentrations were used in µg/mL. The 5 mm of AG was used as negative control. The *t* test was adopted for the comparison between the groups.

*2.4. Molecular Docking Studies for Potential Natural Product Metabolites as Inhibitors of Gly-*

Some of the important phenolic compounds present in garlic extracts were analyzed for their inhibitory role in the glycation reaction. Sudlow Site I is the primary binding site for glucose in pyranose form. The interaction of the glucose ring form and other ligands (considered in this study) with the SAL subsite of the Sudlow Site I are summarized in Table 1. Figures 9 and 10 provide insight into the most stable 2D and 3D docked conformations of the ligands at the SAL subsite obtained via exhaustive molecular docking application. The orientation of the glucopyranose at the SAL subsite was obtained from the crystal of glucose-bound has submitted by Wang et. al. [20] to the protein data bank (PDB ID: 4iw2). The binding efficiency reported in Table 3 is a result of multiple interactions of

**Figure 9.** 2D and 3D interaction models of ligands with HSA. Ligands used are (**a**) glucose, (**b**) catechin, (**c**) quercetin, and (**d**) caffeic acid. **Figure 9.** 2D and 3D interaction models of ligands with HSA. Ligands used are (**a**) glucose, (**b**) catechin, (**c**) quercetin, and (**d**) caffeic acid.

The blue dotted interaction of Arg257 with glucopyranose (Figure 9a) represents an

10 represent the type and count of interactions specific ligand undergoes. The 3D images



*Molecules* **2022**, *27*, 1868

**Table 3.** *Cont.*

#### **Ligand/Inhibitor Name (Common Name) IUPAC Name, Mol Formula, and PubChem ID Binding Affinity Kcal/Mol Number of Hydrogen Bonds Other Interactions \*** Caffeic Acid (E)-3-(3,4 dihydroxyphenyl) prop-2-enoic acid Mol formula: C9H8O<sup>4</sup> PubChem ID:689043 −6.6 3 (1 × Arg 222, 2 × Ser 192) 12 (1 × Arg 218, 1 × Po 4603, 1 × Glc 602, 1 × Leu 238, 1 × Tyr 150, 1 × Lys 195, 1 × Glu 153, 1 × Ala 291, 1 × His 288, 1 × Phe 157,1 × Gln 196, 1 × Lys 199) Gallic Acid 3,4,5 trihydroxybenzoic acid Mol formula: C7H6O<sup>5</sup> PubChem ID: 370 −6.4 4 (2 × Lys 199, 1 × Gln 196, 1 × Glu 153) 10 (1 × His 242, 1 × Gln 196, 1 × Ser 192, 1 × Lys 195, 1 × Tyr 150, 1 × Ala 291, 1 × Phe 157, 1 × His 288, 1 × Glu 292, 1 × Arg 257) m-Coumaric Acid E)-3-(3 hydroxyphenyl)prop-2 enoic acid Mol formula: C9H8O<sup>3</sup> PubChem ID: 637541 −6.3 4 (1 × Arg 222, 1 × PO 4603, 1 × Glc 602, 1 × Ser 192) 6 (1 × Arg 222, 1 × PO 4603, 1 × Glc 602, 1 × Ser 192, 1 × Gln 196 Unfavorable donor–donor, 1 × Gln 196 Unfavorable donor–donor) Quercetin 2-(3,4 dihydroxyphenyl)- 3,5,7 trihydroxychromen-4 one Mol formula: C15H10O<sup>7</sup> PubChem ID: 5280343 −8.1 4 (1 × Ser 192, 1 × Lys 199, 1 × Arg 257, 1 × Arg 222) 17 (1 × Lys 195, 1 × Lys 199, 1 × Gln 196, 1 × His 242, 1 × Tyr 150, 3 × Ala 291, 2 × Leu 238, 1 × Leu 260, 1 × Ala 261, 1 × Ser 287, 1 × Ile 290, 1 × Leu 219,1 × Arg 222, 1 × Glu 153) Pyrogallol benzene-1,2,3-triol Mol formula: C6H6O<sup>3</sup> PubChem ID: 1057 −5.4 3 (2 × Ser 192, 1 × Gln 196) 8 (1 × Tyr 150, 1 × Glu 153, 1 × Phe 157, 1 × Lys 199, 1 × Ala 291, 1 × Lys 195, 1 × His 242, 1 × Arg 257) Dihydroxybenzoic acid 2,3-dihydroxybenzoic acid Mol formula: C7H6O<sup>4</sup> PubChem ID: 19 −6.2 3 (1 × Arg 222, 1 × Ser 287, 1 × Arg 257) 9 (1 × Leu 260, 1 × Ile 290, 1 × Leu 238, 1 × Ala 291, 1 × Arg 257, 1 × Leu 219, 1 × Ile 264, 1 × Ala 261, 1 ×

\* Van der Waals, polar, pi–pi interactions, carbon–hydrogen bonds, pi–sigma, pi–alky, etc.

Tyr 150)

**Figure 10.** 2D and 3D interaction models of ligands with HSA. Ligands used are (**a**) gallic acid, (**b**) dihydroxy benzoic acid, (**c**) pyrogallol, and (**d**) m-coumaric acid. **Figure 10.** 2D and 3D interaction models of ligands with HSA. Ligands used are (**a**) gallic acid, (**b**) dihydroxy benzoic acid, (**c**) pyrogallol, and (**d**) m-coumaric acid.

environment. The white cloudy areas around the ligands constitute the neutral space due

### **Table 3.** Ligands. Common name refers to the compound name used in the study. **3. Discussion**

**Ligand/Inhibitor Name (Common Name)** 

> Glucose (Cyclic form)

> > Catechin

**IUPAC Name, Mol Formula, and PubChem ID Binding Affinity Kcal/Mol Number of Hydrogen Bonds Other Interactions \*** The core cause of many lethal diseases is thought to be connected to metabolic disturbances and inflammatory changes. Diabetes mellitus, characterized by hyperglycemia, is a severe health concern that affects people all over the world [21]. Long-term hyperglycemia has been linked to diabetes complications and biomolecule glycation [22–24].

(3R,4S,5S,6R)-6-(hydroxymethyl)oxane-2,3,4,5-tetrol Mol formula: C6H12O6 PubChem ID: 5793 −6.2 6 (3 × Arg257, 1 × Arg221, 1 × Tyr150, 1 × His242) 1 (1 × Lys199) Leu238 and Ala291 (Hydrophobic interactions) (2S,3R)-2-(3,4-dihy-13 Nutrition looks at how people might use their food choices to reduce their disease risk and manage their illnesses. If a person's diet lacks the right nutritional balance, they are more prone to develop a variety of health problems. When a person consumes excess or very little amounts of a nutrient, it might cause sickness. A balanced diet, according to growing data, may help you avoid problems including heart disease, cancer, osteoporosis, and type 2 diabetes [25].

droxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol Mol formula: C15H14O6 PubChem ID: 73160 −6.7 1 (1 × His 288) (1 × Arg 257, 1 × His 288, 1 × Glc 602, 1 × Ala 291, 1 × Tyr 150, 1 × Glu 153, 1 × Glu 292, 1 × Lys 195, 1 × Ala 191, 1 × Phe 157, 1 × Ser 192, 1 × Lys 436, 1 × Glu 188 Carbon H Bond) Garlic has been reported to have potential therapeutic properties due to its biochemical constituents. Garlic extract can be used against glycation and AGE-related health complications linked with chronic diseases in diabetic patients due to its broad therapeutic potential [26,27]. Compounds such as quercetin, pyrogallol, caffeic acid, gallic acid, m-coumaric acid, and their derivatives are among the beneficial chemicals found in garlics. A high quantity of quercetin has been found in garlic, which is a potent antioxidant compound [28]. The antioxidant capabilities of garlic were well studied in the present investigation through various in vitro methods.

Biochemical analysis of this study showed the presence of phenolic compounds and flavonoids in a respectable amount in garlic extract [28]. According to the current findings, methanolic extract of garlic has a considerable potential to scavenge free radicals, as estimated using DPPH. Garlic also showed high reducing activity that increased with

higher concentrations of the extract. It is generally known that the breakdown of H2O<sup>2</sup> produces hydroxyl free radicals in the blood. The strong antioxidant properties are attributed to the polyphenolic compounds and polysaccharides. The polysaccharides and polyphenolic compounds in garlic might prevent free radical formation, making it an effective antioxidant.

Lowering chronic inflammation may help to postpone, prevent, and possibly treat a variety of chronic illnesses, including cancer [29]. Besides, many ROS, free radicals including NO, superoxide (O<sup>2</sup> – ), and their reaction product peroxynitrite (ONOO– ) are produced in excessive quantities during the host's response to infections and inflammatory circumstances [16]. Furthermore, oxidative stress and inflammation are connected in glycation pathways. Identifying alternatives to non-steroidal anti-inflammatory drugs and developing innovative, effective, and safe anti-inflammatory medicines has long been a key priority. External stress as well as several compounds may cause protein denaturation that leads to the loss of structural integrity of proteins and thus loss of their functions [30,31]. Our results suggest that garlic extract can inhibit the heat-induced denaturation of HSA.

Several diseases such as familial amyloidosis, Alzheimer's, pancreatic islet amyloidosis, etc. are caused by protein aggregation in the circulation and in organs [32]. These aggregates undergo further reactions and form amyloid fibrils that contain cross beta structures. In glycation reactions, reducing carbohydrates non-enzymatically and covalently binds to lysine and arginine groups of proteins as well as with the N terminus of polypeptides [33]. Increased levels of these aggregates may cause neurological degeneration. It has been evident that protein glycation can induce aggregation. Intense browning was observed in the polyacrylamide gel electrophoresis glycated samples of albumin [1,33].

Glycation-induced microenvironment structural alterations were observed through spectral studies that included UV spectra and AGE-specific fluorescence. These alterations were inhibited when garlic extract was present in the reaction mixture during the incubation of the glycation reaction. However, a significant inhibition in microenvironment alterations was observed at higher concentrations of the extract. Consequently, extracts lent protection from structural alterations to the protein.

Further in-depth analysis of structural alterations in glycated proteins, including the inhibitory effect of garlic extract on these changes, was investigated using CD. CD analysis of glycated HSA showed protein destabilization and reduction in α-helix structure [34]. It has been reported that changes in CD spectra of glycated HSA are based on glucose concentration. It has also been observed that upon HSA glycation there was a partial denaturation with alterations in structural integrity at different glucose concentrations (1 mg/mL and 5 mg/mL) [35]. Some authors showed that glycated albumin was more favored in a β-sheet conformation structure [36]. These previous studies showed coherence with our CD results of native and glycated HSA. Moreover, CD experimental analysis revealed that garlic extract provided protection of protein secondary structure alterations and inhibited the conversion of α-helix to β-pleated sheet structure. There are possibilities of the interaction of the metabolites or compounds present in garlic extract (Table 3) with the glycation sites on the HSA molecule, causing inhibition in secondary structure alterations (conversion of a-helix to β-sheet). These inhibitions in structural alteration of glycated HSA are highly important for their functional integrity of the protein.

Sudlow Site I is made up of three subsites: the SAL subsite, deep-seated at the bottom of the Sudlow Site I and comprised of hydrophobic residues Leu-238 and Ala-291 and the indomethacin (IMD) and 3'-azido-3'-deoxythymidine (ADT) subsites situated near the opening of the Sudlow Site I are rich in positively charged residues, Arg-218, Lys-195, and Glu-292 [20]. In blood plasma, the D-glucose found in blood plasma is a mixture of two anomers—i.e., α-D-glucopyranose and β-D glucopyranose [37]. The mutarotation between the open aldehyde chain form and ring form is quick and dependent upon medium conditions. Thus, glucose can potentially react, as an open aldehyde form or closed ring form, within a short time frame [37]. Wang Yu et al. reported the mechanism of glucose interaction with HSA [20]. Two molecules of glucopyranose were found to interact

subsequently at the Sudlow Site I [20]. The first molecule gets bottom deep-seated at the SAL subsite, and this configuration is stabilized by the hydrophobic interactions involving Leu238 and Ala291 (Figure 9a). The second glucopyranose molecule is held at the entrance of the Sudlow Site I marked by IMD and AZT subsites. This region is rich in positively charged residues Lys-195, Arg-218, and Glu-292. The oxygen atom bound to C5 (carbon 5 of the second glucopyranose molecule) is attacked (protonated) by the Lys199. This releases C1 atom, which undergoes a covalent interaction with the Lys195, providing strong stability to the second glucopyranose molecule bound to HSA. The presence of a stable ligand at the SAL subsite of the Sudlow Site I reduces the propensity of protonation of the second glucopyranose molecule by the Lys199. It can be observed that quercetin and gallic acid undergoes a conventional hydrogen bonding interaction with Lys199. In both cases the Lys199 serves as a hydrogen donor; hence, this hydrogen is not available for protonation of C5 of the second glucopyranose molecule. Therefore, no covalent interaction could be established between the second glucopyranose and Lys195. This makes the second glucose molecule, at the entrance of Sudlow Site I, more vulnerable for replacement by a more stable competitor. Since quercetin, gallic acid, catechin, and caffeic acid offer more stable interaction as compared with a non-covalently bound glucose molecule (Table 3), they can offer strong competition.

Quercetin appears to be the most promising inhibitor of glucose interaction since it efficiently prevents the formation of a covalent bond by directly interacting with the Lys199. Interaction of quercetin with the SAL subsite is stabilized by the hydrophobic interaction with Ala291 and Leu238—the same amino acids involved in the stabilization of the first glucopyranose molecule at the SAL subsite. The 3-4 dihydroxyphenyl ring bound to C2 of the deep seated 3,5,7-trihydroxychromen-4-one of quercetin at the SAL subsite (Figure 9c) extends well beyond the Lys199 wall, as described by Wang et al. [20]. This sterically hinders the settlement of the second glucopyranose molecule at the opening of the Sudlow Site I, hence inhibiting the glucose interaction. Gallic acid also prevents covalent bond formation (even more efficiently than quercetin due to two possible hydrogen bond interaction); however, due to the small size of trihydroxy benzoic acid, it remains deepseated at the SAL subsite and well within the wall defined by the Lys199, thereby incapable of preventing the entry of the second glucopyranose molecule at Sudlow Site I.

Uncontrolled levels of blood glucose together with oxidative stress create conditions with high possibilities for the formation of intermediary metabolites of glycation and AGEs. These AGEs and AGE-related metabolites exert structural and functional alterations of blood proteins, contributing to further complications in patients with hyperglycemia as well patients with other inflammatory diseases. In our study, we proved that the natural products that are present in garlic extract have antioxidant and anti-glycation properties and lend protection against the structural destabilization of proteins such as HSA. As a result, this research will help researchers better grasp the link between glycation and natural products that could be beneficial for disease prevention mechanisms in humans.
