Glabridin, an isoflavan from licorice root, upregulates paraoxonase 2 expression under hyperglycemia and protects it from oxidation.

SCOPE: Hyperglycemia is associated with oxidative stress, which accelerates cardiovascular complications. This study investigates the potential of glabridin to regulate paraoxonase 2 (PON2) levels, in vivo, and explores the glabridin protective effect on PON2 through tryptophan-fluorescence quenching and mass spectrometry. METHODS AND RESULTS: Adult mouse offspring of saturated fatty acids fed mothers, which developed hyperglycemia after exposure to a high fat diet in their adult life, had lower levels of heart PON2 mRNA and protein expression than did the control mice (64 and 26%, respectively). Glabridin supplementation significantly upregulated PON2 mRNA and protein expression in the liver (2.1-fold and 2.6-fold, respectively) and heart (2.5-fold and 1.6-fold, respectively) in these mice. In vitro studies demonstrated that the fluorescence quenching of PON2 by glabridin was a result of the formation of a glabridin-PON2 interaction. The binding constant (7.61 × 10(5) M(-1) ) and the ΔG (-33.55kJ/mol) indicated that this interaction was driven by a hydrophobic force, which confers protection against CuSO4 -induced PON2 oxidation. CONCLUSION: Such results indicate that glabridin preserves the anti-atherogenic abilities of PON2 by maintaining its levels, in vivo. The glabridin-PON2 interaction may be the mechanism by which glabridin protects PON2 from oxidation, thus contributing to the protection of PON2 activity in hyperglycemia.

The synthesis and analysis of S-nitorsylated paraoxonase 1.

Post-translational modification (PTM) of proteins plays a crucial role in health and disease by affecting numerous aspects of protein structure, function, stability and subcellular localization. Protein S-nitrosylation is one type of PTM that involves the covalent modification of cysteine sulfhydryl groups with nitric oxide (NO) and has a regulatory impact similar to phosphorylation. The enzyme paraoxonase 1 (PON1) is associated with high-density lipoprotein (HDL), and is responsible for many of HDL's antiatherogenic properties. The enzyme contains a free thiol group at Cys-284 which can also be modified covalently. As part of our effort to study the effect of PTMs on PON1 activities and properties and its implication for cardiovascular disease, we examined PON1's ability to undergo S-nitrosylation on its free Cys-284. Recombinant (re) PON1 was trans-S-nitrosylated by several NO donors, glutathione-NO (GSNO) was found to be the most effective. The S-nitrosylated rePON1 was analyzed by Q-TOF LC/MS and by Saville-Griess assay: the two analytical methods revealed closely similar results. rePON1 was also nitrosylated by nitrosylated human serum albumin (HSA-NO) via protein-protein trans-nitrosylation. HSA-NO transferred an NO group to rePON1 much more efficiently than GSNO with the formation of a higher than 70% rePON-NO when incubated with a 40-fold excess of a HSA-NO/HSA mixture. RePON1-NO was relatively stable: storage for 3days at 37°C resulted in only 25% decomposition. This is the first report of PON1's S-nitrosylation via GSNO and HSA-NO.

Maillard-conjugate based core-shell co-assemblies for nanoencapsulation of hydrophobic nutraceuticals in clear beverages.

Maillard reaction based protein-polysaccharide conjugates (PPC) are typically better emulsifiers than the proteins used to form them. Such conjugates have been proposed as encapsulation agents, but their potential as nanoencapsulation agents in clear beverages has not yet been proposed. In this research we formed casein-maltodextrin (CN-MD) conjugates and co-assembled them with hydrophobic nutraceuticals to create nanovehicles for the enrichment of clear beverages. Conjugation was validated using gel electrophoresis and by ortho-phthaldialdehyde (OPA) assay. Vitamin D (VD) was nano-entrapped by the conjugates, and compared to controls based on an unconjugated CN-MD mixture and unencapsulated VD. The following advantages of the conjugates were shown: the diameters of conjugate-VD nanoparticles was smaller and their solution was more transparent than that of the vitamin dispersion or the protein-polysaccharide mixture with VD. Conjugates conferred better protection against oxidation to both VD and epigallocatechin gallate (EGCG) than the CN-MD mixture. Conjugates were more colloidally stable at the CN original pI, allowing the formation of nanoparticles with mean diameter below 30 nm when mixed with VD, even at the original pI of CN. Conjugates provided protection against degradation at low pH and during shelf life. Entrapment efficiency was measured using Nile red (NR), a fluorescent model for a hydrophobic nutraceutical. Protein affinity to the hydrophobic compound was not diminished due to conjugation, and 90% entrapment efficiency was obtained under the conditions tested. During simulated gastric digestion, Nile red was not released from the conjugates, suggesting potential application in enteric delivery.

Beta-casein-based nanovehicles for oral delivery of chemotherapeutic drugs: drug-protein interactions and mitoxantrone loading capacity.

Beta-casein (beta-CN), a major milk protein, is amphiphilic and self-associates into micelles in aqueous solutions. We have recently introduced a novel oral drug delivery system based on beta-CN nanoparticles. The current research builds on and complements this work by studying the interactions of mitoxantrone (MX) and beta-CN as they co-assemble into nanoparticles, using absorption and emission spectra, static and dynamic light scattering, and fluorescent emission of both MX and tryptophan 143 (Trp143) of beta-CN. The optimal loading molar ratio was 3.3 MX/beta-CN at 1 mg/mL beta-CN, and the association constant was (2.45 +/- 1.76) x 10(5) M(-1) based on beta-CN Trp143 fluorescence; independent MX fluorescence results provided supporting values. In these conditions a bimodal particle distribution was obtained (174.4 nm, 45.9%; 485.1 nm, 54.1%). The gastric digestibility of beta-CN suggests possible targeting to stomach tumors. Hence, beta-CN nanoparticles have potential to serve as effective vehicles of hydrophobic drugs for oral delivery preparations. From the clinical editor: Beta-casein (b-CN) is an amphiphilic milk protein that self-associates into micelles in aqueous solutions and can be utilized as a novel oral drug delivery system. This study investigates the basic properties of a mitoxantrone delivery system based on the above principles.