Spectroscopic and in silico evaluation of hesperetin, aglycone flavanone, as a prospective regulatory ligand for human salivary α-amylase.

The insight into the binding mechanism of hesperetin, an aglycone flavanone, with human salivary α-amylase (HSAA), simulated under physiological salivary condition, was explored using various spectroscopic approaches and in silico method. Hesperetin effectively quenched the intrinsic fluorescence of HSAA and the quenching was mixed quenching mechanism. The interaction perturbed the HSAA intrinsic fluorophore microenvironment and the enzyme global surface hydrophobicity. The negative values of ΔG for thermodynamic parameters and in silico study revealed the spontaneity of HSAA-hesperetin complex while the positive values of enthalpy change (ΔH) and entropy change (ΔS) showed noticeable involvement of hydrophobic bonding in the stabilization of the complex. Hesperetin was a mixed inhibitor for HSAA with a KI of 44.60 ± 1.63 µM and having apparent inhibition coefficient (α) of 0.26. Macromolecular crowding, given rise to microviscosity and anomalous diffusion, regulated the interaction. Sodium ion (Na+) created high ionic strength, also, modulated the interaction. The in silico study proposed the preferential binding of hesperetin at the active cleft domain of HSAA with the least energy of -8.0 kcal/mol. This work gives a novel insight on the potentials of hesperetin as a future prospective medicinal candidate in the management of postprandial hyperglycemic condition.Communicated by Ramaswamy H. Sarma.

Human serum albumin subdomain IB is physiologically adapted for payloading homopterocarpin to human aldehyde dehydrogenase: Combinatorial in vitro and in silico approaches.

The in vitro interactions of homopterocarpin, a potent antioxidant and anti-ulcerative isoflavonoid, with human serum albumin (HSA) and human aldehyde dehydrogenase (hALDH) were explored using various spectroscopic methods, in silico and molecular dynamic (MD) studies. The result showed that homopterocarpin quenched the intrinsic fluorescences of HSA and hALDH. The interactions were entropically favorable, driven primarily by hydrophobic interactions. The proteins have one binding site for the isoflavonoid. This interaction  increased the proteins hydrodynamic radii by over 5% and caused a slight change in HSA surface hydrophobicity Homopterocarpin preferentially binds to HSA subdomain IB with a binding affinity of -10.1 kcal/mol before interaction stoke with hALDH (-8.4 kcal/mol). HSA-homopterocarpin complex attained pharmacokinetic-pharmacodynamics reversible equilibration time faster than ALDH-homopterocarpin. However, the probable and eventual therapeutic effect of homopterocarpin is the mixed inhibition ALDH activity having a Ki value of 20.74 µM. The MD results revealed the stabilization of the complex in HSA-homopterocarpin and ALDH-homopterocarpin from their respective spatial structures of the complex. The findings of this research will provide significant benefits in understanding the pharmacokinetics characteristics of homopterocarpin at the clinical level.

Experimental and computational modeling of interaction of kolaviron-kolaflavanone with aldehyde dehydrogenase.

Aldehyde dehydrogenases (ALDHs) are a diverse family of enzymes that catalyze the NAD(P)+-dependent detoxification of toxic aldehyde compounds. ALDHs are also involved in non-enzymatic ligand binding to endobiotics and xenobiotics. Here, the enzyme crucial non-canonical and non-catalytic interaction with kolaflavanone, a component of kolaviron, and a major bioflavonoid isolated from Garcinia kola (Bitter kola) was characterized by various spectroscopic and in silico approaches under simulated physiological condition. Kolaflavanone quenched the intrinsic fluorescence of ALDH in a concentration dependent manner with an effective quenching constant (Ksv) of 1.14 × 103 L.mol-1 at 25 °C. The enzyme has one binding site for kolaflavanone with a binding constant (Ka) of 2.57 × 104 L.mol-1 and effective Forster resonance energy transfer (FRET) of 4.87 nm. The bonding process was enthalpically driven. The reaction was not spontaneous and was predominantly characterized by Van der Waals forces and hydrogen bond. The flavonoid bonding slightly perturbed the secondary and tertiary structures of ALDH that was 'tryptophan-gated'. The interaction was regulated by both diffusion and ionic strength. Molecular docking showed the binding of kolaflavanone was at the active site of ALDH and the participation of some amino acid residues in the complex formation with -9.6 kcal mol-1 binding energy. The profiles of atomic fluctuations indicated the rigidity of the ligand-binding site during the simulation. With these, ALDH as a subtle nano-particle determinant of kolaviron bioavailability and efficacy is hereby proposed.

Insecticides and Bio-insecticides Modulate the Glutathione-related Antioxidant Defense System of Cowpea Storage Bruchid (Callosobruchus maculatus).

The possible cellular involvements of cowpea storage bruchid (Callosobruchus maculatus (Fab.) [Coleoptera: Chrysomelidae]) glutathione and its related enzymes system in the cellular defense against insecticides (Cypermethrin and λ-cyhalothrin) and bio-insecticides (ethanolic extract of Tithonia diversifolia, Cyperus rotundus, Hyptis suavolens leaves, and Jatropha curcas seed) were investigated. The results showed that the effect of insecticides and bio-insecticides on the C. maculatus is a function of oxidative and nitrosative stresses generated in vivo. A significant (p < 0.05) increase in carbonyl protein (CP) and lipid peroxidation (LPO) contents in bio-insecticides and insecticides exposed groups compared to the control indicates the extent of vital organs damage. These stresses caused similar and significant increase of glutathione peroxidase and glutathione synthetase in response to insecticides and bio-insecticide exposure in a dose-dependent manner. There was no post-translational modification of glutathione transferases expression induced. The alterations of the insect glutathione-dependent antioxidant enzyme activities reflect the presence of a functional defense mechanism against the oxidative and nitrosative stress and are related firmly to the glutathione demands and metabolism but appear inadequate by the significant reduction in glutathione reductase (GR) activity to prevent the damages. Exogenous application of reduced glutathione (GSH), to complement the in vivo demand, could not protect against the onslaught.