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.

Kolaflavanone, a biflavonoid derived from medicinal plant Garcinia, is an inhibitor of mitotic kinesin Eg5.

Mitotic kinesin Eg5 remains a validated target in antimitotic therapy because of its essential role in the formation and maintenance of bipolar mitotic spindles. Although numerous Eg5 inhibitors of synthetic origin are known, only a few inhibitors derived from natural products have been reported. In our study, we focused on identifying novel Eg5 inhibitors from medicinal plants, particularly Garcinia species. Herein, we report the inhibitory effect of kolaflavanone (KLF), a Garcinia biflavonoid, on the ATPase and microtubule-gliding activities of mitotic kinesin Eg5. Additionally, we showed the interaction mechanism between Eg5 and KLF via in vitro and in silico analyses. The results revealed that KLF inhibited both the basal and microtubule-activated ATPase activities of Eg5. The inhibitory mechanism is allosteric, without a direct competition with adenosine-5'-diphosphate for the nucleotide-binding site. KLF also suppressed the microtubule gliding of Eg5 in vitro. The Eg5-KLF model obtained from molecular docking showed that the biflavonoid exists within the α2/α3/L5 (α2: Lys111-Glu116 and Ile135-Asp149, α3: Asn206-Thr226; L5: Gly117-Gly134) pocket, with a binding pose comparable to known Eg5 inhibitors. Overall, our data suggest that KLF is a novel allosteric inhibitor of mitotic kinesin Eg5.

Bioremediation treatment improves water quality for Nile tilapia (Oreochromis niloticus) under crude oil pollution.

Despite favorable publicity of bioremediation as an affordable technology for cleanup of crude oil, public concerns on ecological safety in the presence of residual oil remain a global challenge. In this study, effects of crude oil exposure at sublethal concentration (0.25% v/v) and bioremediation treatment were investigated on histology and biochemical parameters of organs (gills, liver, kidney, and brain) of juvenile Nile tilapia (Oreochromis niloticus). Ten percent (10%) of mixed bacterial culture was used for bioaugmentation treatment. Ninety juvenile fish were used for study, and experiments were carried out in triplicates for three different groups. Malondialdehyde (MDA), an index of lipid peroxidation, was assayed as biomarker for oxidative stress. Activities of antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)), level of non-enzymatic antioxidant (reduced glutathione (GSH)), and activity of brain acetylcholinesterase (AChE) were assayed in selected fish organs as markers for environmental stressor. Histological examination of fish organs was done for all groups. Results of treated groups were compared with those of the control. Levels of MDA significantly increased with significant inhibition of antioxidant enzyme activities in the polluted group. Activities of SOD, CAT, and AChE and levels of GSH in fish organs in the bioaugmentation group were similar to results obtained in the control. Remarkably, the bioaugmentation group showed minimal or no lesions which suggested the efficacy of bioremediation technique in alleviating crude oil toxicity and preserving normal physiology of fish. This study provides deeper insights into the ecosafety of bioremediation treatment and can be extrapolated to other species of fish.

Physicochemical properties of malted finger millet (Eleusine coracana) and pearl millet (Pennisetum glaucum).

Germinated and raw finger millet (Eleusine coracana) and pearl millet (Pennisetum glaucum) were investigated for their physicochemical (pH, total titratable acidity (TTA), proximate, mineral analysis), phytochemical, and antioxidant properties. The results showed that there were decreases in pH (8.50-7.60) with a corresponding increase in TTA (0.0038-0.18 g/L) during germination of the millets. Proximate composition of the millets revealed slight increases in protein (7.61%-7.81%; 10.57%-11.87%) and crude fiber (5.54%-8.81%; 1.07%-2.55%) with reductions in fat (3.84%-2.73%; 7.69%-2.30%) after germination for finger and pearl millet, respectively. The millets were found to be rich sources of minerals, which include magnesium (1,028.42-1,763.50 ppm), calcium (36.42-4,158.40 ppm), sodium (150.00-510.00 ppm), potassium (470.00-4,500 ppm), zinc (20.00-40.00 ppm), and iron (66.00-121.00 ppm) which either decreased or increased with germination. The results of the phytochemical composition revealed that during germination, alkaloid (36.03-74.53 mg/g) and saponin (4.46-31.91 mg/g) contents were found to increase while there were reductions in tannin (0.88-1.64 mg/g) and phytate (7.00-17.72 mg/g) content of the flour. For finger millet, DPPH ranged from 70.00% to 72.14% and pearl millet (49.95%-64.01%), while for FRAP, pearl millet (53.69-53.76 mg/g) demonstrated better activity compared to finger millet (46.91-53.54 mg/g). Findings from this work may suggest that further studies should be carried out on germinated finger and pearl millets to examine their abilities to serve as functional foods.

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.

Interaction of Aldehyde dehydrogenase with acetaminophen as examined by spectroscopies and molecular docking.

The interaction of acetaminophen, a non-substrate anionic ligand, with Aldehyde Dehydrogenase was studied by fluorescence, UV-Vis absorption, and circular dichroism spectroscopies under simulated physiological conditions. The fluorescence spectra and data generated showed that acetaminophen binding to ALDH is purely dynamic quenching mechanism. The acetaminophen-ALDH is kinetically rapid reversible interaction with a binding constant, Ka, of 4.91×103 L mol-1. There was an existence of second binding site of ALDH for acetaminophen at saturating acetaminophen concentration. The binding sites were non-cooperative. The thermodynamic parameters obtained suggest that Van der Waal force and hydrogen bonding played a major role in the binding of acetaminophen to ALDH. The interaction caused perturbation of the ALDH structures with an obvious reduction in the α-helix. The binding distance of 4.43 nm was obtained between Acetaminophen and ALDH. Using Ficoll 400 as macro-viscosogen and glycerol as micro-viscosogen, Stoke-Einstein empirical plot demonstrated that acetaminophen-ALDH binding was diffusion controlled. Molecular docking showed the participation of some amino acids in the complex formation with -5.3 kcal binding energy. With these, ALDH might not an excipient detoxifier of acetaminophen but could be involved in its pegylation/encapsulation.

Spectroscopic characterisation of interaction of ferulic acid with aldehyde dehydrogenase (ALDH).

Interaction of a pharmacological important phenolic, ferulic acid, with Aldehyde dehydrogenase (ALDH) at the simulative pH condition, was studied using spectroscopic approach. Ferulic acid caused a decrease in the fluorescence intensity formed from ALDH-ferulic acid complex resulting in mixed inhibition of ALDH activity (IC50=30.65µM). The intrinsic quenching was dynamic and induced altered conformation of ALDH and made the protein less compact but might not unfold it. ALDH has two binding sites for ferulic acid at saturating concentrations having association constant of 1.35×103Lmol-1 and a dissociation constant of 9.7×107Lmol-1at 25°C indicating ALDH-ferulic acid complex formation is more favourable than its dissociation. The interaction was not spontaneous and endothermic and suggests the involvement of hydrophobic interactions with a FRET binding distance of 4.49nm. Change in pH near and far from isoelectric points of ferulic acid did not affect the bonding interaction. Using trehalose as viscosogen, the result from Stoke-Einstein hypothesis showed that ferulic acid-ALDH binding and dissociation equilibrium was diffusion controlled. These results clearly suggest the unique binding properties and lipophilicity influence of ferulic acid.

Catalysis of Silver catfish Major Hepatic Glutathione Transferase proceeds via rapid equilibrium sequential random Mechanism.

Fish hepatic glutathione transferases are connected with the elimination of intracellular pollutants and detoxification of organic micro-pollutants in their aquatic ecosystem. The two-substrate steady state kinetic mechanism of Silver catfish (Synodontis eupterus) major hepatic glutathione transferases purified to apparent homogeneity was explored. The enzyme was dimeric enzyme with a monomeric size of 25.6 kDa. Initial-velocity studies and Product inhibition patterns by methyl glutathione and chloride with respect to GSH-CDNB; GSH-ρ-nitrophenylacetate; and GSH-Ethacrynic acid all conforms to a rapid equilibrium sequential random Bi Bi kinetic mechanism rather than steady state sequential random Bi Bi kinetic. α was 2.96 ± 0.35 for the model. The pH profile of Vmax/KM (with saturating 1-chloro-2,4-dinitrobenzene and variable GSH concentrations) showed apparent pKa value of 6.88 and 9.86. Inhibition studies as a function of inhibitor concentration show that the enzyme is a homodimer and near neutral GST. The enzyme poorly conjugates 4-hydroxylnonenal and cumene hydroperoxide and may not be involved in oxidative stress protection. The seGST is unique and overwhelmingly shows characteristics similar to those of homodimeric class Pi GSTs, as was indicated by its kinetic mechanism, substrate specificity and inhibition studies. The rate- limiting step, probably the product release, of the reaction is viscosity-dependent and is consequential if macro-viscosogen or micro-viscosogen.

Catalytic activity of human indoleamine 2,3-dioxygenase (hIDO1) at low oxygen.

A cytokine-inducible extrahepatic human indoleamine 2,3-dioxygenase (hIDO1) catalyzes the first step of the kynurenine pathway. Immunosuppressive activity of hIDO1 in tumor cells weakens host T-cell immunity, contributing to the progression of cancer. Here we report on enzyme kinetics and catalytic mechanism of hIDO1, studied at varied levels of dioxygen (O2) and L-tryptophan (L-Trp). Using a cytochrome b5-based activating system, we measured the initial rates of O2 decay with a Clark-type oxygen electrode at physiologically-relevant levels of both substrates. Kinetics was also studied in the presence of two substrate analogs: 1-methyl-L-tryptophan and norharmane. Quantitative analysis supports a steady-state rather than a rapid equilibrium kinetic mechanism, where the rates of individual pathways, leading to a ternary complex, are significantly different, and the overall rate of catalysis depends on contributions of both routes. One path, where O2 binds to ferrous hIDO1 first, is faster than the second route, which starts with the binding of L-Trp. However, L-Trp complexation with free ferrous hIDO1 is more rapid than that of O2. As the level of L-Trp increases, the slower route becomes a significant contributor to the overall rate, resulting in observed substrate inhibition.

Activity of the Antioxidant Defense System in a Typical Bioinsecticide-and Synthetic Insecticide-treated Cowpea Storage Beetle Callosobrochus maculatus F. (Coleoptera: Chrysomelidae).

The non-enzymatic and enzymatic antioxidant defense systems play a major role in detoxification of pro-oxidant endobiotics and xenobiotics. The possible involvement of beetle non-enzymatic [α-tocopherol, glutathione (GSH), and ascorbic acid] and enzymatic [catalase (CAT), superoxide dismutase (SOD), peroxidase (POX), and polyphenol oxidase (PPO)] antioxidant defense system on the insecticidal activity of synthetic insecticides (cypermethrin, 2,2-dicholorovinyl dimethyl phosphate, and λ-cyhalothrin) and ethanolic plant extracts of Tithonia diversifolia, Cyperus rotundus, Hyptis suaveolens leaves, and Jatropha Curcas seeds was investigated. 2,2-Dicholorovinyl dimethyl phosphate (DDVP; 200 ppm, LC50 = 13.24 ppm) and T. diversifolia (20,000 ppm) resulted in 100% beetle mortality at 96-hour post-treatment. The post-treatments significantly increased the beetle α-tocopherol and GSH contents. Activities of CAT, SOD, POX, and PPO were modulated by the synthetic insecticides and bioinsecticides to diminish the adverse effect of the chemical stresses. Quantitative and qualitative allelochemical compositions of bioinsecticides and chemical structure of synthetic insecticides possibly account and for modulation of their respective enzyme activities. Altogether, oxidative stress was enormous enough to cause maladaptation in insects. This study established that oxidative imbalance created could be the molecular basis of the efficacy of both insecticides and bio-insecticides. Two, there was development of functional but inadequate antioxidant defense mechanism in the beetle.

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.

Some physicochemical properties of two major soluble hepatic glutathione transferases of tilapia (Tilapia zilli).

Two distinct glutathione transferases from the liver of adult Tilapia zilli were identified and purified to apparent homogeneity by ion-exchange chromatography on DEAE-cellulose and by gel filtration on Sephadex G-150. These major GST forms labeled tzGST1 and tzGST2 accounted for approximately 42% of the activity detectable with 1-chloro-2,4-dinitrobenzene (CDNB) as a typical electrophilic substrate. Apparent subunit and molecular mass values, substrate specificities and sensitivity to inhibitors as well as kinetic studies were used to differentiate the GST forms. SDS/PAGE indicated subunit molecular masses of 22.0 kDa (tzGST1) and 26.1 kDa (tzGST2) while native molecular weight by gel-filtration on sephadex G-100 indicated native molecular masses of 46.8 kDa and 48.0 kDa for tzGST1 and tzGST2 respectively. They appeared to be homodimers. Inhibition studies showed that tzGST1 was more sensitive to ethacrynic acid (EA), hematin, tributyltinacetate (TBTA), triethyltinbromide (TETB), and triphenyltinchloride (TPTC) than tzGST2 with TPTC being the most potent inhibitor. T. zilli GSTs could conjugate CDNB, DCNB, ρ-NBC, and EA with GSH but displayed no observable conjugating activity with NBDCl. The K(m) and V(max) for tzGST1 and tzGST2 with CDNB were 0.56 ± 0.05 mM; 0.24 ± 0.03 µmol/min/ml and 0.91 ± 0.07 mM; 0.14 ± 0.05 µmol/min/ml respectively while K(m) and V(max) with GSH were 0.46 ± 0.02 mM; 0.19 ± 0.20 µmol/min/ml and 1.32 ± 0.15 mM; 0.21 ± 0.07 µmol/min/ml respectively. Denaturation and renaturation studies with guanidine hydrochloride (Gdn-HCl) revealed that concentration of 4.0 M Gdn-HCl completely denatured tzGST1 and the possible isoenzyme was able to renature to 92% of the original activity. The renaturation process was dependent on temperature. The outcome of this study indicated that tzGSTs are possible GST isoenzymes actively present and involve in the detoxification process in the liver of tilapia when the subject is exposed to chemical toxins. The wide range of chemical toxins encountered in the polluted environment may have directed the selection of multiple tilapia GST isoforms with broad substrate specificity via gene duplication. Consequently, tzGST1 has a better chemical toxin bio-transforming capacity than tzGST2 due to its higher affinity for its substrates--a form of adaption to the polluted environment.