Honokiol as an α-glucosidase inhibitor

Honokiol, a naturally occurring compound from Magnolia obovata Thunb., has many biological activities, but its anti-α-glucosidase activity is still unclear. Therefore, we determined its inhibitory effects against α-glucosidase. Activity assays showed that honokiol was a reversible mixed-type inhibitor of α-glucosidase, and its IC50 value was 317.11 ± 12.86 μM. Fluorescence results indicated that the binding of honokiol to α-glucosidase caused a reduction in α-glucosidase activity. 3D fluorescence and CD spectra results indicated that the binding of honokiol to α-glucosidase caused conformational change in α-glucosidase. Docking simulated the detailed interactions between honokiol and α-glucosidase, including hydrogen and hydrophobic bonds. All findings showed that honokiol could be used as a natural inhibitor to develop α-glucosidase agents.


Introduction
Type 2 diabetes mellitus (T2DM) is a growing health concern with increased prevalence (Xu et al., 2020;ElSayed et al., 2023;Zhou et al., 2023).Now, T2DM has become a significant global health issue (Hu et al., 2024;Li et al., 2024).Epidemiological trends indicate that the prevalence of diabetes could reach an alarming 643 million individuals worldwide by 2030 (Song et al., 2022;Lin et al., 2023).The hallmark clinical feature of T2DM is elevated blood glucose levels, or hyperglycemia, which can result in a spectrum of debilitating complications (Jiang et al., 2020;Ding et al., 2021;Xing et al., 2021).Therefore, management of hyperglycemia is a critical aspect for T2DM patients (Hu et al., 2020).
As far as we know, the detailed inhibitory effects of honokiol on α-glucosidase are still unclear.Hence, the biological activity of honokiol as an α-glucosidase inhibitor was investigated by spectroscopic methods and molecular docking.

Inhibitory activity of honokiol on αglucosidase
First, we assessed the inhibitory activity of honokiol on αglucosidase, as shown in Figure 2.With an increase in the honokiol concentration, the inhibition rate gradually increased, and its IC 50 value was calculated to be 317.11± 12.86 μM, which was lower than that of acarbose (IC 50 = 584.51± 8.56 μM).The potential inhibitory activity of honokiol on α-glucosidase might make it a natural hypoglycemic agent.

Kinetic study
It is very important to clarify the inhibition mode of inhibitors against enzymes for understanding the performance of inhibitors.Hence, the kinetics of honokiol on α-glucosidase were studied.In the plots of the enzyme reaction rate to the enzyme concentration under honokiol (Figure 3A), all lines passed the origin point.This indicated honokiol as a reversible α-glucosidase inhibitor.In the Lineweaver-Burk plots of the enzyme reaction rate to substrate concentration under honokiol (Figure 3B), all lines intersected at the second quadrant.Their slope and Y-intercept were both changed with honokiol concentration.Therefore, it is evident that honokiol was a mixed-type inhibitor.
As a mixed-type inhibitor, honokiol was determined to have an inhibition constant.The fitting plot of slope and Y-intercept versus honokiol (Figures 4A,B) yielded Ki and Kis values of 16.03 and 285.22 μM, respectively.The lower Ki indicated that honokiol tended to bind to substrates.

Fluorescence assay
Based on the fluorescence characteristics of α-glucosidase, the binding of honokiol to α-glucosidase was studied by fluorescence spectroscopy at 298K.In Figure 5, α-glucosidase presented fluorescence with a characteristic peak at 340 nm, while honokiol had very weak fluorescence at 340 nm.With continuous addition of honokiol, α-glucosidase fluorescence gradually decreased.This phenomenon indicated that there were binding interactions between honokiol and α-glucosidase, which could quench the endogenous fluorescence of α-glucosidase.
Subsequently, the binding of honokiol to α-glucosidase was further described by 3D fluorescence (Figures 6A,B).The 3D fluorescence spectra of α-glucosidase had two characteristic peaks due to intrinsic fluorophores and the backbone, which could be reduced by the addition of honokiol.This result was consistent with that of the fluorescence assay.

CD spectra
CD spectra were investigated to evaluate the specific effects of honokiol on α-glucosidase structure.α-Glucosidase showed its own unique CD spectra at 210-222 nm (Figure 7).Honokiol treatment changed the CD spectra of α-glucosidase (Figure 7), which further indicated the binding of honokiol to α-glucosidase.The conformational changes in α-glucosidase were obtained from CD spectral data and showed that honokiol treatment resulted in changes in the α-glucosidase secondary structure content (Table 1).This might be the reason for the inhibition of honokiol on α-glucosidase.

α-Glucosidase inhibitory activity
α-Glucosidase was dissolved in PBS (pH 6.8), and honokiol was dissolved in DMSO.Honokiol solution and α-glucosidase solution were mixed and incubated for an appropriate time, and then a certain amount of substrate p-nitrophenyl-α-D-galactopyranoside Fluorescence spectra of α-glucosidase binding by honokiol.(pNPG) was added.Then, the absorbance of the solution at 405 nm was determined.Then, the α-glucosidase inhibitory effect of honokiol was obtained (Xu et al., 2020;Ali et al., 2023).

Inhibition kinetics
The test procedure for inhibition kinetics followed the same protocol as the α-glucosidase inhibition assay.For enzyme kinetics, the absorbance of a mixture with different concentrations of honokiol and α-glucosidase was recorded.For substrate kinetics, the absorbance of the mixture with different concentrations of honokiol and substrate was recorded (Kaur et al., 2021).

Fluorescence
Fluorescence measurements of α-glucosidase were conducted at an excitation wavelength of 280 nm (Wu et al., 2024).Then, honokiol was added step by step, and the corresponding fluorescence of the mixture was recorded.

CD spectra
CD spectra of α-glucosidase with/without honokiol were also recorded.The concentration of α-glucosidase was 0.1 mg/mL.Honokiol was added to α-glucosidase to prepare their mixture.The data were analyzed using CDNN software (Li et al., 2024).

Molecular docking
The docking of honokiol with α-glucosidase was conducted using SYBYL (Deng et al., 2022;Patil et al., 2022).After being imported into the software, the honokiol structure was hydrotreated and charge-treated.Then, the homology model of αglucosidase was also prepared by hydro-treating and chargetreating.Due to the absence of ligands in the protein, the active pocket of α-glucosidase was produced.Then, the docking of honokiol with α-glucosidase was performed in the default mode.

Statistical analysis
All data were presented as mean ± SD.One-way ANOVA was performed to evaluate the differences between the groups (Zhao et al., 2017;Zhang et al., 2021;Zheng et al., 2021;Sheng et al., 2023).p < 0.05 was considered significant.

Conclusion
As a naturally occurring compound from Magnolia obovata Thunb., honokiol was ascertained for its anti-α-glucosidase activity and inhibition mechanism.So we designed experiments to clarify these properties.Activity assays showed that honokiol was a reversible mixed-type inhibitor of α-glucosidase, and its IC 50 value was 317.11 ± 12.86 μM.Fluorescence, 3D fluorescence, and CD spectra investigations indicated that the binding of honokiol to α-glucosidase caused a reduction in α-glucosidase activity.Docking simulated the detailed interactions between honokiol and αglucosidase.
FIGURE 3 (A) Plots of the enzyme reaction rate to enzyme concentration (B) Lineweaver-Burk plots of the enzyme reaction rate to the substrate concentration.
FIGURE 4 (A,B) Ki and Kis plots of honokiol.