Methylation and Acetylation Enhanced the Antidiabetic Activity of Some Selected Flavonoids: In Vitro, Molecular Modelling and Structure Activity Relationship-Based Study

Abstract

Flavonoids have been reported to exert antihyperglycemic effects and have potential to enhance the current therapy options against type 2 diabetes mellitus. However, the structure activity relationships (SAR) studies of flavonoids against this disease have not been thoroughly comprehended. Hence, in the present study, 14 structurally related flavonoids viz. wogonin, techtochrysin, norwogonin, isoscutellarein, hypolaetin, kaempferol, quercetin, methyl ether of wogonin, acetate of wogonin, acetate of norwogonin, 8-hydroxy-7-methoxyflavone, chrysin, (+)-catechin and (-)-epicatechin were taken into account for in vitro antidiabetic evaluation. Cell viability of RIN-5F pancreatic cells and 3T3-L1 pre-adipocyte cells was initially tested, then an insulin secretion assay of RIN-5F as well as adipogenesis and glucose uptake measurements of adipocyte were investigated. Subsequently, protein expressions study through adipokines measurement (leptin, adiponectin, TNF-α, RBP-4) via enzyme-linked immunosorbent assay (ELISA) kit, Western blotting analysis against GLUT4 and C/EBP-α as well as molecular docking against GLUT1 were analyzed. The results from cell culture antidiabetic assays (insulin secretion, adipogenesis, and glucose uptake), protein expressions and molecular docking pointed that the methoxy group at position C-8 is responsible for antidiabetic property of selected flavonoids via glucose uptake mechanism indicated by up regulation of GLUT4 and C/EBP-α expressions. The mechanism could be enhanced by the addition of an acetate group at C-5 and C-7 of the flavone skeleton.

Keywords: 3T3-L1 pre-adipocytes; RIN-5F pancreatic cells; SAR; adipokines; flavonoids; molecular docking; type 2 diabetes mellitus.

Figure 1

Pathophysiology of type 2 diabetes mellitus (T2DM).

Figure 2

Structures of the tested flavonoids; (a) MN1-MN12, (b) (+)-Catechin (MN13), (c) (-)-Epicatechin (MN14). Wogonin (MN1) → R₅ = OH, R₇ = OH, R₈ = OMe. Methyl ether of wogonin (MN2) → R₅ = OMe, R₇ = OMe, R₈ = OMe. Acetate of wogonin (MN3) → R₅ = OAc, R₇ = OAc, R₈ = OMe. Techtochrysin (MN4) → R₅ = OH, R₇ = OMe. 8-Hydroxy-7-methoxyflavone (MN5) → R₇ = OMe, R₈ = OH. Chrysin (MN6) → R₅ = OH, R₇ = OH. Norwogonin (MN7) → R₅ = OH, R₇ =OH, R₈ = OH. Acetate of norwogonin (MN8) → R₅ = OAc, R₇ = OAc, R₈ = OAc. Isoscutellarein (MN9) → R₅ = OH, R₇ = OH, R₈ = OH, R_4′ = OH. Hypolaetin (MN10) → R₅ = OH, R₇ = OH, R₈ = OH, R_3′ = OH, R_4′ = OH. Kaempferol (MN11) → R₃ = OH, R₅ = OH, R₇ = OH, R_4′ = OH. Quercetin (MN12) → R₃ = OH, R₅ = OH, R₇ = OH, R_3′ = OH, R_4′ = OH.

Figure 3

The percentage of cell viability of RIN-5F ranging from 0.39–25 μg/well in 1 h. Mean ± standard deviation (S.D.), n = 3, small letters represent Dunnet’s test. Mean values that do not share a letter are significantly different (p < 0.05).

Figure 4

The effect of the flavonoid on insulin secretion activity of RIN-5F pancreatic cells. Mean ± S.D., n = 3, means that do not share a letter are significantly different (p < 0.05).

Figure 5

The percentage of cell viability of 3T3-L1 preadipocyte cells measured by MTT assay, ranging from 0.78 to 100 μg/well for 48 h. Small letters represent Dunnet’s test. Mean ± S.D., n = 3, means values that do not share a letter are significantly different (p < 0.05).

Figure 6

Quantification of lipid droplet after treatment. Small letters represent Tukey’s test. Mean ± S.D., n = 3, means values that do not share a letter are significantly different (p < 0.05).

Figure 7

Lipid droplets formations of 3T3-L1 adipocyte cells stained with Oil red O. Magnification 40×. (NT: non-treated, MN1: wogonin, MN2: methyl ether of wogonin, MN3: acetate of wogonin, MN4: techtochrysin, MN5: 8-hydroxy-7-methoxyflavone, MN6: chrysin, MN7: norwogonin, MN8: acetate of norwogonin, MN9: isoscutellarein, MN10: hypolaetin, MN11: kaempferol, MN12: quercetin, MN13: (+)-catechin and MN14: (-)-epicatechin).

Figure 8

The effect of flavonoids on glucose uptake measurement of adipocyte cells. Small letters represent Tukey’s test. Mean ± S.D., n = 3, means values that do not share a letter are significantly different (p < 0.05).

Figure 9

Adipokines measurement of adipocyte after flavonoids treatment at 12.5 μg/mL; (a) leptin, (b) adiponectin, (c) TNF-α, (d) RBP-IV. Mean ± S.D., n = 3, mean values that do not share a letter are significantly different (p < 0.05).

Figure 10

The density of protein bands of GLUT4 and C/EBP-α per β-actin. Mean ± S.D., n = 3, mean values that do not share a letter are significantly different (p < 0.05).

Figure 11

Image of protein bands of GLUT4, C/EBP-α and β-actin (internal control) on polyvinylidene difluoride (PVDF) membrane.

Figure 12

Binding interactions of glucose (a) with active site residues of GLUT1 (PDB ID: 5EQI). The compounds are coloured black while amino acids are green. Green and purple dashes depict hydrogen bond and hydrophobic bond, respectively. (b) Surface structure of glucose in active site of GLUT1. Blue and brown colours indicate polar and hydrophobic regions, respectively.

Figure 13

Binding interactions of wogonin (MN1) (a), methyl ether of wogonin (MN2) (b), acetate of wogonin (MN3) (c), and techtochrysin (MN4) (d) with active site residues of GLUT1 (PDB ID: 5EQI). The compounds are coloured black while amino acids are green. Green and purple dashes depict hydrogen bond and hydrophobic bond, respectively.

Figure 14

Binding interactions of norwogonin (MN7) (a) and acetate of norwogonin (b) with active site residues of Glut1 (PDB ID: 5EQI). The compounds are coloured black while amino acids are green. Green and purple dashes depict hydrogen bond and hydrophobic bond, respectively.

Figure 15

Binding interactions of kaempferol (MN11) (a), (-)-epicatechin (MN14) (b) with active site residues of Glut1 (PDB ID: 5EQI). The compounds are coloured black while amino acids are green. Green and purple dashes depict hydrogen bond and hydrophobic bond, respectively.

Figure 16

Methyl and acetate groups act as the key role in the antidiabetic activity for the glucose uptake mechanism.