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The flavonoid family has been reported to possess a high potential for inhibition of xanthine oxidase (XO). This study concerned the structural aspects of inhibitory activities and binding affinities of flavonoids as XO inhibitors. The result indicated that the hydrophobic interaction was important in the binding of flavonoids to XO, and the XO inhibitory ability increased generally with increasing affinities within the class of flavones and flavonols. The planar structure and the C2═C3 double bonds of flavonoids were advantageous for binding to XO and for XO inhibition. Both the hydroxylation on ring B and the substitution at C3 were unfavorable for XO inhibition more profoundly than their XO affinity. The methylation greatly reduced the inhibition (0.75-3.07 times) but hardly affected the affinity. The bulky sugar substitutions of flavonoids decreased the inhibition (1.69-1.99 times) and lowered the affinities (4.20-9.22 times) to different degrees depending on the conjunction site.

Xanthine oxidase (XO), a key enzyme in purine catabolism, is widely distributed in human tissues. It can catalyze xanthine to generate uric acid and cause hyperuricemia and gout. Inhibition kinetics assay showed that kaempferol inhibited XO activity reversibly in a competitive manner. Strong fluorescence quenching and conformational changes of XO were found due to the formation of a kaempferol-XO complex, which was driven mainly by hydrophobic forces. The molecular docking further revealed that kaempferol inserted into the hydrophobic cavity of XO to interact with some amino acid residues. The main inhibition mechanism of kaempferol on XO activity may be due to the insertion of kaempferol into the active site of XO occupying the catalytic center of the enzyme to avoid the entrance of the substrate and inducing conformational changes of XO. In addition, luteolin exhibited a stronger synergistic effect with kaempferol than did morin at the lower concentration.

The binding mechanism of molecular interaction between diosmetin and human serum albumin (HSA) in a pH 7.4 phosphate buffer was studied using atomic force microscopy (AFM) and various spectroscopic techniques including fluorescence, resonance light scattering (RLS), UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by diosmetin was a static quenching procedure. The binding constants and number of binding sites were evaluated at different temperatures. The RLS spectra and AFM images showed that the dimension of the individual HSA molecules were larger after interaction with diosmetin. The thermodynamic parameters, ΔH° and ΔS° were calculated to be -24.56 kJ mol(-1) and 14.67 J mol(-1) K(-1), respectively, suggesting that the binding of diosmtin to HSA was driven mainly by hydrophobic interactions and hydrogen bonds. The displacement studies and denaturation experiments in the presence of urea indicated site I as the main binding site for diosmetin on HSA. The binding distance between diosmetin and HSA was determined to be 3.54 nm based on the Förster theory. Analysis of CD and FT-IR spectra demonstrated that HSA conformation was slightly altered in the presence of diosmetin.

Betulinic acid (BA), an important pentacyclic triterpene widely distributed in many foods, possesses high antidiabetic activity. In this study, BA was found to exhibit stronger inhibition of α-glucosidase than acarbose with an IC value of (1.06 ± 0.02) × 10 mol L in a mixed-type manner. BA bound with α-glucosidase to form a BA-α-glucosidase complex, resulting in a more compact structure of the enzyme. The obtained concentrations and spectra profiles of the components resolved by the multivariate-curve resolution-alternating least-squares confirmed the formation of the BA-α-glucosidase complex. Molecular docking showed that BA tightly bound to the active cavity of α-glucosidase, which might hinder the entrance of the substrate leading to a decline in enzyme activity. The chemical modification of α-glucosidase verified the results of the computer simulation that the order of importance of the four amino acid residues in the binding process was His > Tyr > Lys > Arg.

The interaction of indigo carmine (IC) with calf thymus DNA in physiological buffer (pH 7.4), using ethidium bromide (EB) dye as a fluorescence probe, was investigated by ultraviolet-visible absorption, fluorescence, and circular dichroism (CD) spectroscopy, coupled with viscosity measurements and DNA-melting studies. Hypochromicity of the absorption spectra of IC and enhancement in fluorescence polarization of IC were observed with the addition of DNA. Moreover, the binding of IC to DNA was able to decrease iodide and single-stranded DNA (ssDNA) quenching effects, increase the melting temperature and relative viscosity of DNA, and induce the changes in CD spectra of DNA. All of the evidence indicated that IC interacted with DNA in the mode of intercalative binding. Furthermore, the three-way synchronous fluorescence spectra data obtained from the interaction between IC and DNA-EB were resolved by parallel factor analysis (PARAFAC), and the results provided simultaneously the concentration information and the pure spectra for the three reaction components (IC, EB, and DNA-EB) of the system at equilibrium. This PARAFAC demonstrated that the intercalation of IC molecules into DNA proceeded by substituting for EB in the DNA-EB complex. The calculated thermodynamic parameters, ΔH° and ΔS°, suggested that both hydrophobic interactions and hydrogen bonds played a predominant role in the binding of IC to DNA.

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