Stability study and validation of a liquid chromatography tandem mass spectrometry method for the quantitative analysis of polyphenols in fish feed ingredients.

The perception of polyphenols as a safe, healthy, and sustainable solution for replacing synthetic antioxidants has been an important factor for their rapid growing in the global food market. Therefore, it is essential to use reliable methods for their quantification in commercial products intended for animal or human consumption. The purpose of this study is to evaluate the performance of some solvents used for the extraction of selected polyphenols, explore their stability under different experimental conditions, and validate a liquid chromatography tandem mass-spectrometry method for their quantification in commercial fish feed ingredients by using the standard addition method. The regression models for gallic acid, hydroxytyrosol, catechin, oleuropein, carnosol and carnosic acid were linear in the range 0-30 µg/mL, limit of detection and quantification around 0.03 and 0.1 µg/mL, respectively, and accuracy within ± 15 % of the nominal concentrations. The method was successfully applied to the determination of specific polyphenols in commercial fish feed ingredients supplemented with polyphenols from olive and rosemary extracts.

Effect of cryotherapy duration on experimentally induced connective tissue inflammation in vivo.

Objectives: This study tested the hypothesis that cryotherapy duration influences lipopolysaccharide (LPS)-induced inflammation in a rat model. Materials and Methods: Six Wistar rats (Rattus norvegicus albinus) were used. Five sites were selected per animal and divided into 5 groups: a negative control group (NC), 2 positive control groups (PC1 and PC2), and 2 experimental groups (E1 and E2). Cryotherapy was applied for 1 minute (E1) or 5 minutes (E2). An acute inflammatory response was induced in the PC and E groups via subcutaneous administration of 0.5 mL/kg. In the PC2 group, a catheter was inserted without additional treatment. For the E1 and E2 groups, 2.5°C saline solution was administered through the implanted catheters for 1 and 5 minutes, respectively. The rats were sacrificed, and samples were obtained and processed for histological analysis, specifically examining the presence of polymorphonuclear neutrophils and hemorrhage. The χ2 test was used to compare the presence of acute inflammation across groups. Dependent variables were compared using the linear-by-linear association test. Results: Inflammation and hemorrhage varied significantly among the groups (p = 0.001). A significantly higher degree of acute inflammation was detected (p = 0.0002) in the PC and E1 samples than in the E2 group, in which cryotherapy was administered for 5 minutes. The PC and E1 groups also exhibited significantly greater numbers of neutrophils (p = 0.007), which were essentially absent in both the NC and E2 groups. Conclusions: Cryotherapy administration for 5 minutes reduced the acute inflammation associated with LPS and catheter implantation.

Effective Removal of Metal ion and Organic Compounds by Non-Functionalized rGO.

Effective removal of heavy metals from water is critical for environmental safety and public health. This work presents a reduced graphene oxide (rGO) obtained simply by using gallic acid and sodium ascorbate, without any high thermal process or complex functionalization, for effective removal of heavy metals. FTIR and Raman analysis show the effective conversion of graphene oxide (GO) into rGO and a large presence of defects in rGO. Nitrogen adsorption isotherms show a specific surface area of 83.5 m2/g. We also measure the zeta-potential of the material showing a value of -52 mV, which is lower compared to the -32 mV of GO. We use our rGO to test adsorption of several ion metals (Ag (I), Cu (II), Fe (II), Mn (II), and Pb(II)), and two organic contaminants, methylene blue and hydroquinone. In general, our rGO shows strong adsorption capacity of metals and methylene blue, with adsorption capacity of qmax = 243.9 mg/g for Pb(II), which is higher than several previous reports on non-functionalized rGO. Our adsorption capacity is still lower compared to functionalized graphene oxide compounds, such as chitosan, but at the expense of more complex synthesis. To prove the effectiveness of our rGO, we show cleaning of waste water from a paper photography processing operation that contains large residual amounts of hydroquinone, sulfites, and AgBr. We achieve 100% contaminants removal for 20% contaminant concentration and 63% removal for 60% contaminant concentration. Our work shows that our simple synthesis of rGO can be a simple and low-cost route to clean residual waters, especially in disadvantaged communities with low economical resources and limited manufacturing infrastructure.

Electrochemical functionalization strategy for chemical vapor deposited graphene on silicon substrates: grafting, electronic properties and biosensing.

Herein, we present an electrochemical functionalization strategy for high quality single-layer and multilayer chemical vapor deposited (CVD) graphene directly on a Si/SiO2 chip facilitating electronic interfacing. This method avoids oxidation and tearing of graphene basal planes. We demonstrate effective functionalization by D-(+)-biotin (Bio), 4-(phenyldiazenyl)-aniline (Dz), and gallic acid (Gall) using cyclic voltammetry. Raman spectroscopy and XPS are used to demonstrate effective functionalization. In order to evaluate the effect of the electrochemical functionalization on graphene properties, DC electrical conductivity, XPS, mobility, and carrier density analysis are presented. We show that this functionalization strategy does not degrade graphene mobility (103 cm2 V-1s-1). After functionalization we observe a rise in Fermi level of ∼0.06 eV. In addition, we prove sensing capabilities with a CVD graphene monolayer on the biotin/avidin system by electrical resistance measurements and electrochemical impedance spectroscopy reaching a detection of 2.5 ng ml-1. This paper demonstrates an effective strategy to functionalize high quality CVD graphene on a chip compatible with an electronic interface readout.

Structural and energetic basis for novel epicatechin derivatives acting as GPER agonists through the MMGBSA method.

(-)-Epicatechin (Epi) has been demonstrated to activate pathways involved in GPER-stimulated nitric oxide (NO) production via endothelial NO synthase, known as the eNOS/NO pathway. Previous studies combining synthesis of four Epi derivatives demonstrated that Epi and Epi-prop, Epi-4-prop and Epi-5-prop were able to bind GPER by acting as GPER agonists, whereas docking studies allowed observation of structural details of the binding of these derivatives at the GPER binding site. However, due to the nature of past studies, the theoretical methods employed did not allow observation of structural and energetic details linked to ligand binding at the GPER binding site. In this contribution, we explore the structural and energetic changes coupling the binding of Epi and its four derivatives to GPER. To this end, MD simulations on the microsecond scale (1 µs) with an MMGBSA approach were used for each GPER-ligand complex. Energetic analysis demonstrated that incorporation of several aliphatic chains to Epi contributed to increasing the affinity towards the GPER binding site, thus helping to explain the experimental evidence. Structural analysis demonstrated that Epi, Epi-4-prop and Epi-5-prop share more similar interactions at GPER binding sites with similar conformational behavior than with Epi-prop and Epi-Ms. However, Epi-prop had additional residues that could explain its different but related biological effects.

Synthesis of novel (-)-epicatechin derivatives as potential endothelial GPER agonists: Evaluation of biological effects.

To potentially identify proteins that interact (i.e. bind) and may contribute to mediate (-)-epicatechin (Epi) responses in endothelial cells we implemented the following strategy: 1) synthesis of novel Epi derivatives amenable to affinity column use, 2) in silico molecular docking studies of the novel derivatives on G protein-coupled estrogen receptor (GPER), 3) biological assessment of the derivatives on NO production, 4) implementation of an immobilized Epi derivative affinity column and, 5) affinity column based isolation of Epi interacting proteins from endothelial cell protein extracts. For these purposes, the Epi phenol and C3 hydroxyl groups were chemically modified with propargyl or mesyl groups. Docking studies of the novel Epi derivatives on GPER conformers at 14 ns and 70 ns demostrated favorable thermodynamic interactions reaching the binding site. Cultures of bovine coronary artery endothelial cells (BCAEC) treated with Epi derivatives stimulated NO production via Ser1179 phosphorylation of eNOS, effects that were attenuated by the use of the GPER blocker, G15. Epi derivative affinity columns yielded multiple proteins from BCAEC. Proteins were electrophoretically separated and inmmunoblotting analysis revealed GPER as an Epi derivative binding protein. Altogether, these results validate the proposed strategy to potentially isolate and identify novel Epi receptors that may account for its biological activity.