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  • Natural products have been always the most important and pro

    2021-12-08

    Natural products have been always the most important and productive sources of lead compounds for the development of drugs [[15], [16], [17]]. A variety of potent α-glucosidase inhibitors have been identified from natural products, such as quercetin, resveratrol, isorhamnetin, calein C and curcumin [[18], [19], [20]]. However, the insufficient understanding of the interaction between such bioactive molecules and α-glucosidase has made it becoming a difficult task to efficiently discover excellent drug leads. Therefore, it is indispensable for drug-protein system and therapeutic applications to explore the inhibitory behaviors of small molecules binding to α-glucosidase, which can provide useful information to elucidate interaction mechanism between ligands and the protein at molecule level. So far, the development of a variety of spectroscopic methods, such as ultraviolet visible Methyllycaconitine citrate (UV–vis), fourier transform infrared spectroscopy (FT-IR), fluorescence spectroscopy and circular dichroism (CD), has greatly accelerated the discovery of drug leads due to some advantages, such as high sensitivity and reproducibility, time and cost efficiency, and providing useful information to guide drug design [21]. Currently, most of these techniques are employed as an important means to investigate the protein-drug system and the conformational changes of the protein target, which has been integrated with in vitro and in vivo biological assay to discuss the interaction characteristics of ligands binding to their targets [22,23]. For example, Xu et al. applied fluorescence spectroscopy to study the interaction mode between Salvia miltiorrhiza and the active site of human serum albumin [24]. Additionally, molecular modelling also serves as a widely used method to predict the potential binding mechanism between bioactive molecules and the corresponding protein target [25,26]. In our previous study, the natural product salvianolic acid C (SAC) (shown in Fig. 1) identified from the traditional Chinese medicine Salvia miltiorrhiza, was discovered as a potent α-glucosidase inhibitor based on an integrated method [27]. However, the inhibitory behavior of compound SAC was limited to the enzymatic activity evaluation. In this study, several spectroscopic methods were combined and applied to investigate the interaction mechanism between the inhibitor SAC and α-glucosidase. The enzyme kinetics, fluorescence spectroscopy and molecular modelling studies were employed to determine the inhibitory effect, inactivation behavior, thermodynamic parameters and potential binding mode. Meanwhile, the synchronous fluorescence spectra, FT-IR spectroscopy was performed to elucidate the conformational changes of α-glucosidase mediated by SAC.
    Materials and methods
    Results and discussion
    Conclusions In this study, a combined method including inhibition kinetics, UV–vis, fluorescence spectrum, FT-IR spectrum and molecular modelling technique was performed to explore the inhibitory effect of SAC on α-glucosidase and their interaction mechanism. Consequently, SAC showed a distinctly inhibitory activity in a concentration-dependent manner with the IC50 value of 3.03 ± 0.27 μM. SAC acted as a mixed-competitive inhibitor with the Ki and Ki' values of 1.5 and 8.8 μM, respectively when binding to α-glucosidase, and the inhibition effect presented a reversible mechanism. The activity of α-glucosidase could be significantly inactivated by SAC in a dose-dependent manner through the first-order kinetics process. The fluorescence quenching assay suggested that SAC could interact with the enzyme and effectively quench its intrinsic fluorescence in a static quenching mechanism coupled with the formation of α-glucosidase-SAC complex. The number of binding site (n) revealed that only one single site existed for SAC binding to the protein. The calculated thermodynamic parameters ΔHo and ΔSo were 99.73 ± 0.05 kJ mol−1 and 453.80 ± 0.13 J mol−1 K−1, respectively, indicating that the binding of SAC to α-glucosidase was dominated mainly by the hydrophobic interaction. The obtained data from synchronous fluorescence and FT-IR spectra suggested that SAC could interact with α-glucosidase and induce the rearrangement and secondary structural changes of the protein. Moreover, the results of molecular docking and dynamics simulations further validated that SAC could bind to the active site of α-glucosidase mainly driven by the hydrophobic interaction and the generated hydrogen bonds. The described results illustrated the interaction mechanism between α-glucosidase and its inhibitor SAC, which revealed that SAC could become a promising drug lead for type 2 diabetes mellitus. Also, such findings provide some valuable information to guide us to design novel α-glucosidase inhibitors derived from SAC.