Olive Oil Waste as a Source of Functional Food Ingredients: Assessing Polyphenolic Content and Antioxidant Activity in Olive Leaves.

Around two million tons of olive oil are produced in Europe annually, with Portugal being among the top five European olive oil-producing countries. Olive oil production results in a substantial amount of waste in the form of olive leaves. These discarded olive leaves contain valuable phenolic compounds with antioxidant, anti-inflammatory, hypoglycaemic, neuroprotective, and antiproliferative properties. Due to their richness in polyphenols with health-promoting properties, olive leaves can be considered a potential functional food ingredient. Thus, sustainable practices for reusing olive leaf waste are in demand. In this study, the polyphenolic content in olive leaves from different Portuguese locations was determined using HPLC-UV-Vis after defining the best fit-for-purpose liquid extraction strategy. The differences in the in vitro antioxidant activity in these samples were determined by several methodologies based on radical scavenging (against 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), 2,2-diphenyl-2-picrylhydrazyl (DPPH), and peroxyl radical (ORAC)) and on reducing properties (cupric-reducing antioxidant capacity (CUPRAC), and Folin-Ciocalteu assay (FC)), to unveil the relationship between the profile and quantity of polyphenols with antioxidant mechanisms and their capacity. At last, the stability of extracted compounds upon lyophilization and exposition to surrogate biological fluids was assessed, envisioning the future incorporation of olive leaves extracted compounds in food products.

Microcarrier-based fluorescent yeast estrogen screen assay for fast determination of endocrine disrupting compounds.

The presence of endocrine-disrupting compounds (EDCs) in water poses a significant threat to human and animal health, as recognized by regulatory agencies throughout the world. The Yeast Estrogen Screen (YES) assay is an excellent method to evaluate the presence of these compounds in water due to its simplicity and capacity to assess the bioaccessible forms/fractions of these compounds. In the presence of a compound with estrogenic activity, Saccharomyces cerevisiae cells, containing a lacZ reporter gene encoding the enzyme ß-galactosidase, are induced, the enzyme is synthesised, and released to the extracellular medium. In this work, a YES-based approach encompassing the use of a lacZ reporter gene modified strain of S. cerevisiae, microcarriers as solid support, and a fluorescent substrate, fluorescein di-ß-d-galactopyranoside, is proposed, allowing for the assessment of EDCs' presence after only 2 h of incubation. The proposed method provided an EC50 of 0.17 ± 0.03 nM and an LLOQ of 0.03 nM, expressed as 17ß-estradiol. The assessment of different EDCs provided EC50 values between 0.16 and 1.2 × 103 nM. After application to wastewaters, similar results were obtained for EDCs screening, much faster, compared to the conventional 45 h spectrophotometric procedure using a commercial kit, showing potential for onsite high-throughput screening of environmental contamination.

Cryo-XPS for Surface Characterization of Nanomedicines.

Nanoparticles used for medical applications commonly possess coatings or surface functionalities intended to provide specific behavior in vivo, for example, the use of PEG to provide stealth properties. Direct, quantitative measurement of the surface chemistry and composition of such systems in a hydrated environment has thus far not been demonstrated, yet such measurements are of great importance for the development of nanomedicine systems. Here we demonstrate the first use of cryo-XPS for the measurement of two PEG-functionalized nanomedicines: a polymeric drug delivery system and a lipid nanoparticle mRNA carrier. The observed differences between cryo-XPS and standard XPS measurements indicate the potential of cryo-XPS for providing quantitative measurements of such nanoparticle systems in hydrated conditions.

Combining orthogonal measurements to unveil diclofenac encapsulation into polymeric and lipid nanocarriers.

The quantification of the drug associated to nanoparticle carriers, often expressed in terms of encapsulation efficiency, is a regulatory requirement. The establishment of independent methods to evaluate this parameter provides a means for measurement validation, which is critical in providing confidence in the methods and enabling the robust characterization of nanomedicines. Chromatography is traditionally used to measure drug encapsulation into nanoparticles. Here, we describe an additional independent strategy based on analytical centrifugation. The encapsulation of diclofenac into nanocarriers was quantified based on the mass difference between placebo (i.e. unloaded) and loaded nanoparticles. This difference was estimated using particle densities measured by differential centrifugal sedimentation (DCS) and size and concentration values measured by particle tracking analysis (PTA). The proposed strategy was applied to two types of formulations, namely poly(lactic-co-glycolic acid) (PLGA) nanoparticles and nanostructured lipid carriers, which were analysed by DCS operated in sedimentation and flotation modes, respectively. The results were compared to those from high performance liquid chromatography (HPLC) measurements. Additionally, X-ray photoelectron spectroscopy analysis was used to elucidate the surface chemical composition of the placebo and loaded nanoparticles. The proposed approach enables the monitoring of batch-to-batch consistency and the quantification of diclofenac association to PLGA nanoparticles from 0.7 ng to 5 ng of drug per 1 µg of PLGA, with good linear correlation between DCS and HPLC results (R2 = 0.975). Using the same approach, similar quantification in lipid nanocarriers was possible for a loading of diclofenac ≥1.1 ng per 1 µg of lipids, with results in agreement with the HPLC method (R2 = 0.971). Hence, the strategy proposed here expands the analytical tools available for evaluating nanoparticles encapsulation efficiency, being thus significant for increasing the robustness of drug-delivery nanocarriers characterization.

Lab-on-Valve Automated and Miniaturized Assessment of Nanoparticle Concentration Based on Light-Scattering.

Nanoparticles (NPs) concentration directly impacts the dose delivered to target tissues by nanocarriers. The evaluation of this parameter is required during NPs developmental and quality control stages, for setting dose-response correlations and for evaluating the reproducibility of the manufacturing process. Still, faster and simpler procedures, dismissing skilled operators and post-analysis conversions are needed to quantify NPs for research and quality control operations, and to support result validation. Herein, a miniaturized automated ensemble method to measure NPs concentration was established under the lab-on-valve (LOV) mesofluidic platform. Automatic NPs sampling and delivery to the LOV detection unit were set by flow programming. NPs concentration measurements were based on the decrease in the light transmitted to the detector due to the light scattered by NPs when passing through the optical path. Each analysis was accomplished in 2 min, rendering a determination throughput of 30 h-1 (6 samples h-1 for n = 5) and only requiring 30 µL (≈0.03 g) of NPs suspension. Measurements were performed on polymeric NPs, as these represent one of the major classes of NPs under development for drug-delivery aims. Determinations for polystyrene NPs (of 100, 200, and 500 nm) and for NPs made of PEGylated poly-d,l-lactide-co-glycolide (PEG-PLGA, a biocompatible FDA-approved polymer) were accomplished within 108-1012 particles mL-1 range, depending on the NPs size and composition. NPs size and concentration were maintained during analysis, as verified for NPs eluted from the LOV by particle tracking analysis (PTA). Moreover, concentration measurements for PEG-PLGA NPs loaded with an anti-inflammatory drug, methotrexate (MTX), after their incubation in simulated gastric and intestinal fluids were successfully achieved (recovery values of 102-115%, as confirmed by PTA), showing the suitability of the proposed method to support the development of polymeric NPs targeting intestinal delivery.

Exploiting Kinetic Features of ORAC Assay for Evaluation of Radical Scavenging Capacity.

The analysis and interpretation of data retrieved from Oxygen Radical Absorbance Capacity (ORAC) assays represent a challenging task. ORAC indexes originate from different mathematical approaches often lacking correct elucidation of kinetic features concerning radical scavenging reactions by antioxidant compounds. In this work, the expression of ORAC values as area under fluorescein (FL) decay curves (AUC) and lag time are critically compared. This multi-parametric analysis showed the extension of radical scavenging reactions beyond the lag time period for caffeic acid, gallic acid, reduced glutathione and quercetin, extending their antioxidant protection of FL. Ethanol delayed the reaction of both FL and antioxidant compounds with free radical species generated from 2,2'-azobis(2-amidinopropane) dihydrochloride thermolysis. Trolox equivalent values, commonly used to express ORAC values, were more affected by the differences in radical scavenging kinetics between the reference and the tested antioxidant compounds when calculated from AUC than from lag time. These findings stressed the importance of choosing calibrator compounds presenting ORAC kinetics similar to samples to prevent biased estimation of the antioxidant capacity. Additionally, the framework proposed here provides a sustainable analytical method for the evaluation of antioxidant capacity, with an AGREE score of 0.73.

Sample preparation and chromatographic methods for the determination of protein-bound uremic retention solutes in human biological samples: An overview.

Protein-bound uremic retention solutes, such as indole-3-acetic acid, indoxyl sulfate, p-cresol and p-cresol sulfate, are associated with the development of several pathologies, namely renal, cardiovascular, and bone toxicities, due to their potential accumulation in the human body, thus requiring analytical methods for monitoring and evaluation. The present review addresses conventional and advanced sample treatment procedures for sample handling and the chromatographic analytical methods developed for quantification of these compounds in different biological fluids, with particular focus on plasma, serum, and urine. The sample preparation and chromatographic methods coupled to different detection systems are critically discussed, focusing on the different steps involved for sample treatment, namely elimination of interfering compounds present in the sample matrix, and the evaluation of their environmental impact through the AGREEprep tool. There is a clear trend for the application of liquid-chromatography coupled to tandem mass spectrometry, which requires protein precipitation, solid-phase extraction and/or dilution prior to analysis of biological samples. Furthermore, from a sustainability point of view, miniaturized methods resorting to microplate devices are highly recommended.

Rapid and sustainable HPLC method for the determination of uremic toxins in human plasma samples.

Protein-bound uremic toxins, mainly indoxyl sulfate (3-INDS), p-cresol sulfate (pCS), and indole-3-acetic acid (3-IAA) but also phenol (Pol) and p-cresol (pC), are progressively accumulated during chronic kidney disease (CKD). Their accurate measurement in biomatrices is demanded for timely diagnosis and adoption of appropriate therapeutic measures. Multianalyte methods allowing the establishment of a uremic metabolite profile are still missing. Hence, the aim of this work was to develop a rapid and sensitive method based on high-performance liquid chromatography with fluorescence detection for the simultaneous quantification of Pol, 3-IAA, pC, 3-INDS, and pCS in human plasma. Separation was attained in 12 min, using a monolithic C18 column and isocratic elution with acetonitrile and phosphate buffer containing an ion-pairing reagent, at a flow rate of 2 mL min-1. Standards were prepared in plasma and quantification was performed using the background subtraction approach. LOQ values were ≤ 0.2 µg mL-1 for all analytes except for pCS (LOQ of 2 µg mL-1). The method proved to be accurate (93.5-112%) and precise (CV ≤ 14.3%). The multianalyte application of the method, associated to a reduced sample volume (50 µL), a less toxic internal standard (eugenol) in comparison to the previously applied 2,6-dimethylphenol and 4-ethylphenol, and a green extraction solvent (ethanol), resulted in the AGREE score of 0.62 which is in line with the recent trend of green and sustainable analytical chemistry. The validated method was successfully applied to the analysis of plasma samples from control subjects exhibiting normal levels of uremic toxins and CKD patients presenting significantly higher levels of 3-IAA, pC, 3-INDS, and pCS that can be further investigated as biomarkers of disease progression.

Insights on Ultrafiltration-Based Separation for the Purification and Quantification of Methotrexate in Nanocarriers.

The evaluation of encapsulation efficiency is a regulatory requirement for the characterization of drug delivery systems. However, the difficulties in efficiently separating nanomedicines from the free drug may compromise the achievement of accurate determinations. Herein, ultrafiltration was exploited as a separative strategy towards the evaluation of methotrexate (MTX) encapsulation efficiency in nanostructured lipid carriers and polymeric nanoparticles. The effect of experimental conditions such as pH and the amount of surfactant present in the ultrafiltration media was addressed aiming at the selection of suitable conditions for the effective purification of nanocarriers. MTX-loaded nanoparticles were then submitted to ultrafiltration and the portions remaining in the upper compartment of the filtering device and in the ultrafiltrate were collected and analyzed by HPLC-UV using a reversed-phase (C18) monolithic column. A short centrifugation time (5 min) was suitable for establishing the amount of encapsulated MTX in nanostructured lipid carriers, based on the assumption that the free MTX concentration was the same in the upper compartment and in the ultrafiltrate. The defined conditions allowed the efficient separation of nanocarriers from the free drug, with recoveries of >85% even when nanoparticles were present in cell culture media and in pig skin surrogate from permeation assays.

Assessment of immunoglobulin capture in immobilized protein A through automatic bead injection.

The repeatable immobilization of molecular recognition elements onto particle surfaces has a strong impact on the outcomes of affinity-based assays. In this work, an automatic method for the immobilization of immunoglobulin G (IgG) onto protein A-Sepharose microbeads was established through the flow programming features of the portable lab-on-valve platform using micro-bead injection spectroscopy. The reproducible packing of protein A-microbeads between two optic fibers was feasible, allowing on-column probing of IgG retention. The automation of solutions handling and the precise control of time of IgG interaction with the beads rendered repeatable immobilization cycles, within a short timeframe (<2 min). The proposed method featured the preparation of disposable immunosorbents for downstream analytical applications, such as immunosensing or microenrichment of target analytes. In-situ quantification of IgG@protein A-microbeads was carried out using a horseradish peroxidase-labeled detection IgG. The colorimetric oxidation of 3,3',5,5'-tetramethylbenzidine was monitored on-column. Quantitation of mouse and human IgG immobilized@protein A-microbeads was achieved for loading masses between 0.1 and 0.4 µg per ca. 5.5 mg of sorbent. The implemented detection strategy allowed the quantification of human IgG in certified human serum (ERM®- DA470k/IFCC) and spiked saliva, yielding recoveries of 102-108% and requiring minimal volume (1-15 µL) from serum and saliva.

New Insights into the Anti-Inflammatory and Antioxidant Properties of Nitrated Phospholipids.

Nitro-fatty acids (NO2 -FA) have been widely studied with regard to their identification, structural characterization, and biological actions. NO2 -FA could also be present endogenously esterified to phospholipids (PL), and NO2 -PL were already detected in cardiac mitochondria from diabetic rats and cardiomyoblasts subjected to starvation. However, the biological actions of NO2 -PL have been overlooked. In this study, we evaluate the antioxidant and anti-inflammatory potential of the nitrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) formed in vitro by incubation with NO2 BF4 , in a well-recognized mimetic model of nitroxidative stress. Nitrated POPC showed anti-radical ability to reduce both 2,2-diphenyl-1-picrylhydrazyl radical (DPPH• ) (IC20 = 225 ± 4 µg/mL; Trolox equivalent (TE) = 86 ± 6 µmol Trolox/g lipid) and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS•+ ) (IC50 = 124 ± 2 µg/mL; TE = 152 ± 9 µmol Trolox/g lipid). Also, higher lag times were achieved in oxygen radical absorbance capacity (ORAC) assay for nitrated POPC, indicating a faster reaction with oxygen-derived radicals (TE = 1.03 ± 0.22 and TE = 1.30 ± 0.16 mmol Trolox/g lipid for nonmodified and nitrated POPC, respectively). Nitrated POPC showed the ability to inhibit lipid oxidation induced by the hydroxyl radical generated under Fenton reaction conditions, monitored by electrospray ionization (ESI) mass spectrometry (MS) using phosphatidylcholine (PtdCho) liposomes as a model of cell membrane. Nitrated POPC showed anti-inflammatory potential, as assessed by the inhibition of inducible nitric oxide synthase (iNOS) expression in RAW 264.7 macrophages activated by the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) in a well-described in vitro model of inflammation. Altogether, this study provides new clues regarding the antioxidant and anti-inflammatory potential of nitrated POPC, which should be explored in depth.

Fully automatic flow-based device for monitoring of drug permeation across a cell monolayer.

A novel flow-programming setup based on the sequential injection principle is herein proposed for on-line monitoring of temporal events in cell permeation studies. The permeation unit consists of a Franz cell with its basolateral compartment mixed under mechanical agitation and thermostated at 37 °C. The apical compartment is replaced by commercially available Transwell inserts with a precultivated cell monolayer. The transport of drug substances across epithelial cells genetically modified with the P-glycoprotein membrane transporter (MDCKII-MDR1) is monitored on-line using rhodamine 123 as a fluorescent marker. The permeation kinetics of the marker is obtained in a fully automated mode by sampling minute volumes of solution from the basolateral compartment in short intervals (10 min) up to 4 h. The effect of a P-glycoprotein transporter inhibitor, verapamil as a model drug, on the efficiency of the marker transport across the cell monolayer is thoroughly investigated. The analytical features of the proposed flow method for cell permeation studies in real time are critically compared against conventional batch-wise procedures and microfluidic devices.

Sexuality Education Websites for Adolescents: A Framework-Based Content Analysis.

The web has unique potential for adolescents seeking comprehensive sexual health information. As such, it is important to understand the nature, scope, and readability of the content and messaging provided by sexuality educational websites. We conducted a content analysis of 14 sexuality education websites for adolescents, based on the 7 essential components (sexual and reproductive health and HIV, relationships, sexual rights and sexual citizenship, pleasure, violence, diversity, and gender) of the International Planned Parenthood Framework for Comprehensive Sexuality Education. A majority of content across all sites focused on sexual and reproductive health and HIV, particularly pregnancy and STI prevention, and other information about STIs and HIV. No other topic comprised more than 10% of content coverage across a majority of sites. The authors found little discussion of gender issues, sexual rights, sexual diversity, or sexual violence. Most sites provided brief references to sexual pleasure, generally moderated with cautionary words. Language used implied a heterosexual female audience. Reading levels for most sites were above the 9th-grade level, with several at the college level. These findings have implications for enhancing online sexuality education and broadening the coverage of essential topics.

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.

Insights on antioxidant assays for biological samples based on the reduction of copper complexes-the importance of analytical conditions.

Total antioxidant capacity assays are recognized as instrumental to establish antioxidant status of biological samples, however the varying experimental conditions result in conclusions that may not be transposable to other settings. After selection of the complexing agent, reagent addition order, buffer type and concentration, copper reducing assays were adapted to a high-throughput scheme and validated using model biological antioxidant compounds of ascorbic acid, Trolox (a soluble analogue of vitamin E), uric acid and glutathione. A critical comparison was made based on real samples including NIST-909c human serum certified sample, and five study samples. The validated method provided linear range up to 100 µM Trolox, (limit of detection 2.3 µM; limit of quantification 7.7 µM) with recovery results above 85% and precision <5%. The validated developed method with an increased sensitivity is a sound choice for assessment of TAC in serum samples.