Preparation and Characterization of Quercetin-Loaded Zein Nanoparticles by Electrospraying and Study of In Vitro Bioavailability.

Quercetin is a hydrophobic flavonoid with high antioxidant activity. However, for biological applications, the bioavailability of quercetin is low due to physiological barriers. For this reason, an alternative is the protection of quercetin in matrices of biopolymers as zein. The objective of this work was to prepare and characterize quercetin-loaded zein nanoparticles by electrospraying and its study of in vitro bioavailability. The physicochemical parameters such as viscosity, density, and electrical conductivity of zein solutions showed a dependence of the ethanol concentration. In addition, rheological parameters demonstrated that solutions of zein in aqueous ethanol present Newtonian behavior, rebounding in the formation of nanoparticles by electrospraying, providing spherical, homogeneous, and compact morphologies, mainly at a concentration of 80% (v/v) of ethanol and of 5% (w/v) of zein. The size and shape of quercetin-loaded zein nanoparticles were studied by transmission electron microscopy (TEM), observing that it was entrapped, distributed throughout the nanoparticle of zein. Analysis by Fourier transform-infrared (FT-IR) of zein nanoparticles loaded with quercetin revealed interactions via hydrogen bonds. The efficacy of zein nanoparticles to entrap quercetin was particularly high for all quercetin concentration evaluated in this work (87.9 ± 1.5% to 93.0 ± 2.6%). The in vitro gastrointestinal release of trapped quercetin after 240 min was 79.1%, while that for free quercetin was 99.2%. The in vitro bioavailability was higher for trapped quercetin (5.9%) compared to free quercetin (1.9%), than of gastrointestinal digestion. It is concluded, that the electrospraying technique made possible the obtention of quercitin-loaded zein nanoparticles increasing their bioavailability. PRACTICAL APPLICATION: This type of nanosystems can be used in the food and pharmaceutical industry. Quercetin-loaded zein nanoparticles for its improvement compared to free quercetin can be used to decrease the prevalence of chronic degenerative diseases by increasing of the bioavailability of quercetin in the bloodstream. Other application can be as an antioxidant system in functional foods or oils to increase shelf life.

Effect of Temperature and pH on the Secondary Structure and Denaturation Process of Jumbo Squid Hepatopancreas Cathepsin D.

BACKGROUND: Cathepsin D is a lysosomal enzyme that is found in all organisms acting in protein turnover, in humans it is present in some types of carcinomas, and it has a high activity in Parkinson's disease and a low activity in Alzheimer disease. In marine organisms, most of the research has been limited to corroborate the presence of this enzyme. It is known that cathepsin D of some marine organisms has a low thermostability and that it has the ability to have activity at very acidic pH. Cathepsin D of the Jumbo squid (Dosidicus gigas) hepatopancreas was purified and partially characterized. The secondary structure of these enzymes is highly conserved so the role of temperature and pH in the secondary structure and in protein denaturation is of great importance in the study of enzymes. The secondary structure of cathepsin D from jumbo squid hepatopancreas was determined by means of circular dichroism spectroscopy. OBJECTIVE: In this article, our purpose was to determine the secondary structure of the enzyme and how it is affected by subjecting it to different temperature and pH conditions. METHODS: Circular dichroism technique was used to measure the modifications of the secondary structure of cathepsin D when subjected to different treatments. The methodology consisted in dissecting the hepatopancreas of squid and freeze drying it. Then a crude extract was prepared by mixing 1: 1 hepatopancreas with assay buffer, the purification was in two steps; the first step consisted of using an ultrafiltration membrane with a molecular cut of 50 kDa, and the second step, a pepstatin agarose resin was used to purification the enzyme. Once the enzyme was purified, the purity was corroborated with SDS PAGE electrophoresis, isoelectric point and zymogram. Circular dichroism is carried out by placing the sample with a concentration of 0.125 mg / mL in a 3 mL quartz cell. The results were obtained in mdeg (millidegrees) and transformed to mean ellipticity per residue, using 111 g/mol molecular weight/residue as average. Secondary-structure estimation from the far-UV CD spectra was calculated using K2D Dichroweb software. RESULTS: It was found that α helix decreases at temperatures above 50 °C and above pH 4. Heating the enzyme above 70°C maintains a low percentage of α helix and increases ß sheet. Far-UV CD measurements of cathepsin D showed irreversible thermal denaturation. The process was strongly dependent on the heating rate, accompanied by a process of oligomerization of the protein that appears when the sample is heated, and maintained a certain time at this temperature. An amount typically between 3 and 4% α helix of their secondary structure remains unchanged. It is consistent with an unfolding process kinetically controlled due to the presence of an irreversible reaction. The secondary structure depends on pH, and a pH above 4 causes α helix structures to be modified. CONCLUSION: In conclusion, cathepsin D from jumbo squid hepatopancreas showed retaining up to 4% α helix at 80°C. The thermal denaturation of cathepsin D at pH 3.5 is under kinetic control and follows an irreversible model.