Magnolol and Luteolin Inhibition of α-Glucosidase Activity: Kinetics and Type of Interaction Detected by In Vitro and In Silico Studies

Abstract

Magnolol and luteolin are two natural compounds recognized in several medicinal plants widely used in traditional medicine, including type 2 diabetes mellitus. This research aimed to determine the inhibitory activity of magnolol and luteolin on α-glucosidase activity. Their biological profile was studied by multispectroscopic methods along with inhibitory kinetic analysis and computational experiments. Magnolol and luteolin decreased the enzymatic activity in a concentration-dependent manner. With 0.075 µM α-glucosidase, the IC₅₀ values were similar for both compounds (~ 32 µM) and significantly lower than for acarbose (815 μM). Magnolol showed a mixed-type antagonism, while luteolin showed a non-competitive inhibition mechanism. Thermodynamic parameters suggested that the binding of magnolol was predominantly sustained by hydrophobic interactions, while luteolin mainly exploited van der Waals contacts and hydrogen bonds. Synchronous fluorescence revealed that magnolol interacted with the target, influencing the microenvironment around tyrosine residues, and circular dichroism explained a rearrangement of the secondary structure of α-glucosidase from the initial α-helix to the final conformation enriched with β-sheet and random coil. Docking studies provided support for the experimental results. Altogether, the data propose magnolol, for the first time, as a potential α-glucosidase inhibitor and add further evidence to the inhibitory role of luteolin.

Keywords: circular dichroism; diabetes mellitus; enzymatic kinetics; hyperglycaemia; luteolin; magnolol; molecular docking; natural polyphenols; α-glucosidase inhibitors.

Figure 1

Chemical structures of magnolol and luteolin and the reference compound acarbose.

Figure 2

Glucosidase activity expressed as a percentage of maximal activity in the presence of magnolol, luteolin, and acarbose.

Figure 3

Enzyme kinetics observed with different concentrations of α-glucosidase and 2 mM pNPG (A). Insert: graph “v versus [α-glucosidase]” (B).

Figure 4

Enzyme kinetics of 0.075 µM α-glucosidase in the presence of increasing concentrations of magnolol (A), luteolin, (B) and acarbose (C).

Figure 5

α-Glucosidase activity in the presence of magnolol, luteolin, and acarbose (linear scale) (A). The plots “v versus [α-glucosidase]” of magnolol (B), luteolin (C), and acarbose (D) are reported. α-Glucosidase concentration: 0.075 µM, pNPG concentration: 2 mM.

Figure 6

Michaelis–Menten and Lineaweaver–Burk graphs of magnolol (A,B), luteolin (D,E), and acarbose (G,H) of α-glucosidase activity with different substrate concentrations (0.25–2.5 mM). Graphs (C,F,I) are the secondary plots of “slope versus magnolol, luteolin, and acarbose concentrations”.

Figure 7

Kinetic time courses for the relative activity of 0.75 µM α-glucosidase with 2 mM pNPG in the presence of magnolol (A), luteolin, (B) and acarbose (C). Semilogarithmic plot analysis for magnolol (D,G), luteolin (E,H), and acarbose (F,I) considering the lowest and highest concentrations.

Figure 8

Fluorescence spectra of α-glucosidase in the presence of magnolol (A), luteolin (B), and acarbose (C), pH = 6.8 at 298 K. Concentrations of: α-glucosidase 0.35 µM, magnolol 0, 0.045, 0.15, 0.25, 0.35, 0.45, 0.50, 0.60, 0.725 µM, luteolin 0, 0.125, 0.25, 0.5, 1, 5, 7.5, 10 µM, and acarbose 0, 0.42, 0.503, 0.586, 0.669, 0.752, 0.918, 1.08, for curves a → i, a → j, a → h. Insets: Stern–Volmer plots for fluorescence quenching of α-glucosidase with magnolol (D), luteolin (E) and acarbose (F) at 298, 304 and 310 K.

Figure 9

Synchronous fluorescence spectra of α-glucosidase with inhibitors (pH 6.8, T = 298 K) at ∆λ = 15 nm, magnolol (A), luteolin (B), and acarbose (C), and at ∆λ = 60 nm, magnolol (D), luteolin (E), and acarbose (F). Concentrations of: magnolol 0.0, 0.045, 0.15, 0.25, 0.35, 0.45, 0.50, 0.60, 0.725, 0.85, 0.95 µM; luteolin 0.0, 0.125, 0.25, 1, 5, 7.5, 10, 12,5, 15 µM, and acarbose 0.0, 0.42, 0.503, 0.586, 0.669, 0.752,0.918,1.08, 1.23, 6.4, 11.56, 22 µM, for curves a → m, a → j, a → k, respectively.

Figure 10

Circular dichroism spectra of α-glucosidase (1 µM) in the presence of increasing concentrations of magnolol (A), luteolin (B), and acarbose (C). Inserts: secondary structure contents (D–F; pH = 6.8).

Figure 11

Chemical structures and inhibitory activities of chromones without sugar kaempferol (1) and quercetin (2) and of compounds 3–5 based on sugar (O-galloyl) as α-glucosidase inhibitors.

Figure 12

Docking positioning of the α-glucosidase inhibitors magnolol and luteolin at the ABS2 binding site (A). Only the most relevant residues are depicted. The amino acids included in ABS2 are listed. Ligplot (B) reports the most relevant contacts between α-glucosidase and magnolol in ABS2. Ligplot (C) reports the most relevant contacts between the α-glucosidase and luteolin in ABS2. Hydrophobic and polar residues are shown in green and pink, respectively, while the negative- and positive-charged amino acids are detailed by red and blue circles.