Dynamic flow-through approach to evaluate readily bioaccessible antioxidants in solid food samples.

Release of bioactive compounds from food matrices is regarded as the first step towards their human bioavailability. The objective of this work was the implementation of an affordable and robust flow-through device for expedient dynamic leaching experiments aiming at the assessment of readily bioaccessible antioxidant compounds in solid food commodities. A simple configuration is proposed using commercially available devices containing regenerated cellulose filters placed in polypropylene holders to entrap the solid sample, featuring a disposable, single use extraction chamber. The kinetic extraction profile of fast leachable antioxidants from different food matrices was evaluated using the ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) assay, fitting a first-order reaction model for readily bioaccessible compounds (R>0.9). The leaching rate constant values associated to the fast leachable antioxidant compounds were 0.060-0.446min-1 and 0.105-0.210min-1 for water and ethanol/water (1:1, v/v) applied as extractants, respectively. Furthermore, no statistically significant differences were found between the estimated values of bioaccessible antioxidant compounds by the kinetic model and the values attained using conventional batch-wise extraction methodology, ranging from 3.37 to 60.3 µmol of Trolox ((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) per g of sample. Extension of the method using U. S. Pharmacopeia surrogate biological media (stomach (pH 1.2) and intestinal (pH 7.5) fluids without enzymes) to NIST-1570a spinach leaves provided gastrointestinal compartment-dependent kinetic leaching rates (0.120 and 0.198min-1, respectively) and total antioxidant content (45.5 and 52.5µmol of Trolox per g of sample, respectively).

Myoglobin microplate assay to evaluate prevention of protein peroxidation.

The current therapeutic strategies are based on the design of multifunctional drug candidates able to interact with various disease related targets. Drugs that have the ability to scavenge reactive oxygen species (ROS), beyond their main therapeutic action, may prevent the oxidative damage of biomolecules. Therefore, analytical approaches that monitor in a continuous mode the ability of drugs to counteract peroxidation of physiologically relevant biotargets are required. In the present work, a microplate spectrophotometric assay is proposed to evaluate the ability of selected cardiovascular drugs, including angiotensin-converting enzyme (ACE) inhibitors, ß -blockers and statins to prevent protein peroxidation. Myoglobin, which is a heme protein, and peroxyl radicals generated from thermolysis of 2,2'-azo-bis(2-amidinopropane) dihydrochloride at 37 °C, pH 7.4 were selected as protein model and oxidative species, respectively. Myoglobin peroxidation was continuously monitored by the absorbance decrease at 409 nm and the ability of drugs to counteract protein oxidation was determined by the calculation of the area under the curve upon the myoglobin oxidation. Fluvastatin (AUC50=12.5 ± 1.2 µM) and enalapril (AUC50=15.2 ± 1.8 µM) showed high ability to prevent myoglobin peroxidation, providing even better efficiency than endogenous antioxidants such as reduced glutathione. Moreover, labetalol, enalapril and fluvastatin prevent the autoxidation of myoglobin, while glutathione showed a pro-oxidant effect.