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Pergularia tomentosa L. (P. tomentosa) has been largely used in Tunisian folk medicine as remedies against skin diseases, asthma, and bronchitis. The main objectives of this study were to identify phytochemical compounds that have antioxidant and antimicrobial properties from the stem, leaves, and fruit crude methanolic extracts of P. tomentosa, and to search for tyrosyl-tRNA synthetase (TyrRS), topoisomerase type IIA, and Candidapepsin-1 (SAP1) enzyme inhibitors through molecular docking study. Phytochemical quantification revealed that fruit and leaves extracts displayed the highest total flavonoids (582 mg QE/g Ex; 219 mg QE/g Ex) and tannins content (375 mg TAE/g Ex; 216 mg TAE/g Ex), also exhibiting significant scavenging activity to decrease free radicals for ABTS, DPPH, β-carotene, and FRAP assay with IC50 values (> 1 mg/mL). Additionally, promising antimicrobial activities towards different organs have been observed against several bacteria and Candida strains. From the liquid chromatography-mass spectrometry (LC-MS) analysis, five polyphenolic compounds, namely digitoxigenin, digitonin glycoside and calactina in the leaves, kaempferol in the fruit, and calotropagenin in the stems, were identified. They were also analyzed for their drug likeliness, based on computational methods. Molecular docking study affirmed that the binding affinity of calactin and actodigin to the active site of TyrRS, topoisomerase type IIA, and SAP1 target virulence proteins was the highest among the examined dominant compounds. Therefore, this study indicated that P. tomentosa methanolic extracts displayed great potential to become a potent antimicrobial agent and might be a promising source for therapeutic and nutritional functions. These phytocompounds could be further promoted as a candidate for drug discovery and development.

Moradyn is an Italian purple corn variety whose cobs represent a rich source of polyphenols. At the industrial level, they are used to produce a dried extract (MCE) by the addition of 20% Arabic gum. In order to evaluate the extract solid-state stability, an innovative accelerated stress protocol was developed following the isoconversion approach. The degradation kinetics of cyanidin-3-O-glucoside (C3G), the most suitable marker to monitor the overall MCE degradation status, was monitored under five temperature–humidity (RH) combinations. These data were used to build a mathematical model, able to estimate the C3G stability at 25 °C and 30% RH, whose predictiveness was further assessed by comparing the predicted vs. experimental C3G isoconversion time. Finally, by applying this model, the expiry date of the extract was calculated to be within 26–33 days, confirming that the addition of 20% Arabic gum is insufficient to stabilize MCE and highlighting the need of a new formula in order to prolong MCE shelf-life.

A liquid chromatographic MS-compatible method was applied to the structural elucidation of Teicoplanin for identification CRS components. The method, previously developed by our group, involves the use of LiChrospher 100 RP-18 column with a mobile phase composed of ammonium formate 25 mM at pH 6.00 and acetonitrile (ACN). All the peaks with a 0.10% UV area, largely above the disregard limit of 0.15% as fixed by EMA, were considered and submitted to MS/MS fragmentation experiments. The study of MS/MS spectrum collected for Teicoplanin complex major component (namely A2-2) allowed to elucidate the fragmentation pathway and enabled the successful identity assignment of all the 42 detected species. Elution order was also rationalized. An in house batch sample of Teicoplanin was analyzed and, while the 86% of the detected species were structurally identical to those in Teicoplanin for identification CRS, five new derivatives were revealed and structurally characterized. In both the Teicoplanin samples, all the considered species were found to have a Teicoplanin-like structure that allows their classification as closely related impurities, with a significant implication in their qualification threshold.

Avocado industry produces enormous by‐products and represents an important source of raw material in food and non‐food applications. This review aims to focus the attention on the valorisation of avocado wastes recycling to produce different materials with important industrial and environmental impacts. In fact, peels and seeds can be reused in food and cosmetic fields due to the presence of bioactive ingredients such as polyphenols; thanks to their antioxidant and antinflammatory activities. In addition, both peels and seeds can be reused in the production of carbonaceous materials with important consequences for environmental pollution in the removal of contaminants from water. Interesting applications of avocado wastes in photocatalysis and biofuel production are also discussed. All the utilisations attest the great potential of recycling avocado by‐products, a little known resource.

The reuse of byproducts from agricultural and food industries represents the key factor in a circular economy, whose interest has grown in the last two decades. Thus, the extraction of bioactives from agro-industrial byproducts is a potential source of valuable molecules. The aim of this work was to investigate the in vitro capacity of byproducts from a new Italian corn variety, named Moradyn, to inhibit the accumulation of advanced glycation end products (AGEs) involved in several chronic age-related disorders. In addition, the hypoglycemic effect of Moradyn was tested by in vitro enzymatic systems. A Moradyn phytocomplex and its purified anthocyanin fraction were able to inhibit fructosamine formation and exhibited antiglycative properties when tested using BSA-sugars and BSA-methylglyoxal assays. These properties could be attributed to the polyphenols, mainly anthocyanins and flavonols, detected by RP-HPLC-DAD-ESI-MSn. Finally, a Moradyn phytocomplex was submitted to a simulated in vitro digestion process to study its bioaccessibility. Moradyn could be considered as a promising food ingredient in the context of typical type 2 diabetes risk factors and the study will continue in the optimization of the ideal formulation to preserve its bioactivities from digestion.

The phytochemical constituents in the aqueous methanolic leaf extract of Triclisia gilletii responsible for its nephroprotective potentials against ethane-1,2-diol induced nephrolithiasis as previously investigated in our laboratory were elucidated. The extract was prepared using 80% aqueous methanol in 72 h, Phytochemical contents of aqueous methanolic extract of Triclisia gilletii (TGME) was identified using both a Thermo Scientific DSQII single quadrupole gas chromatography (GC) and a Thermo Scientific liquid chromatography (LCQ Fleet system) tandem mass spectroscopy. The chromatogram acquisition, detection of mass spectral peaks and their waveform processing were performed using Xcalibur MS Software (Thermo Scientific Inc.). GC-MS analysis revealed the presence of phenols, fatty acids, vitamins and steroids. Likewise, for LC-MS analysis kaempferol and dihydrovomifoliol-O-glucoside were detected. The identified constituents have possible contributively effect on the acclaimed pharmacological potential of Triclisia gilletii against ethane-1,2-diol induced nephrolithiasis.

The interest towards in-flow biocatalytic processes using immobilised enzyme reactors (IMERs) has grown significantly over the past ten years. In an IMER, the enzyme is immobilized on a suitable chromatographic support, where the catalytic reaction occurs. Interfacing IMERs to a second dimension in integrated analytical systems represents a valuable way to monitor reactions with the advantage of automation, reproducibility, and robustness. This review explores the recent achievements on the development and applications of different analytical set-up for the in-line and on-line monitoring of in-flow bioreactions. The preparation of IMERs is briefly described together with the analytical platforms used to study enzymatic substrate specificity, reaction optimization and kinetic parameters. The review is divided in two parts, one devoted to spectroscopic and spectrometric methods and the other to separative methods. The selected examples illustrate that integration of IMERs in analytical platforms may widespread the industrial applications of biocatalyzed reactions.

The agri-food sector produces a huge amount of agri-food wastes and by-products, with a consequent great impact on environmental, economic, social, and health aspects. The reuse and recycling of by-products represents a very important issue: for this reason, the development of innovative recovery and extraction methodologies must be mandatory. In this context of a circular economy, the study of green extraction techniques also becomes a priority in substitution of traditional extraction approaches. This review is focused on the recovery of chlorogenic acids from agri-food wastes, as these compounds have an important impact on human health, exhibiting several different and important healthy properties. Novel extraction methodologies, namely microwave and ultrasound-assisted extractions, supercritical fluid extraction, and pressurized-liquid extraction, are discussed here, in comparison with conventional techniques. The great potentialities of these new innovative green and sustainable approaches are pointed out. Further investigations and optimization are mandatory before their application in industrial processes.

Biopharmaceutical drugs are large, complex protein molecules derived from living cells. The characterization of the protein-based products is a complicated and challenging task necessary to guarantee their efficacy and safety. A major concern in manufacturing protein biopharmaceuticals is their propensity to form aggregates. The separation of proteins by size-exclusion chromatography (SEC) is a well-established method for the characterization of macromolecules and their aggregates. The demand for increased resolution and faster analysis in SEC has led to the development of small, more rigid fully porous particles and superficially porous particles with different pore sizes. The coupling of SEC with three or four detectors has enhanced protein characterization by generating accurate molecular weights (absolute determination) and by giving insight into the degradation pathways and products associated with aggregation. The intent of this review is to report the continuous developments of SEC stationary phases and their applications on the characterization of intact proteins, their variants or aggregates and protein complexes. Thus, the most recent applications of SEC with the focus on proteins and protein aggregates are reported and discussed.

In this work we explored the feasibility of different CE-ESI-MS set-ups for the analysis of conformational states of an intact protein. By using the same background electrolyte at quasi physiological conditions (50 mM ammonium bicarbonate, pH 7.4) a sequential optimization was carried out, initially by evaluating a sheath-liquid interface with both a single quadrupole (SQ) and a time-of-flight (TOF) mass spectrometer; then a sheathless interface coupled with high-resolution QTOF MS was considered. Beta2-microglobulin has been taken as a model, as it is an amyloidogenic protein and its conformational changes are strictly connected to the onset of a disease. The separation of two conformers at dynamic equilibrium is achieved all the way down to the MS detection. Notably, the equilibrium ratio of the protein conformers is maintained in the electrospray source after CE separation. Strengths and weaknesses of each optimized set-up are emphasized and their feasibility in unfolding studies is evaluated. In particular, ESI-TOF MS can assign protein forms that differ by 1 Da only and sheathless interfacing is best suited to preserve protein structure integrity. This demonstrates the CE-ESI-MS performance in terms of separation, detection and characterization of conformational species that co-populate a protein solution.

Mycotoxins are considered one of the most dangerous agricultural and food contaminants. They are toxic and the development of rapid and sensitive analytical methods to detect and quantify them is a very important issue in the context of food safety and animal/human health. The need to detect mycotoxins at trace levels and to simultaneously analyze many different mycotoxin types became mandatory to protect public health. In fact, European Commission regulations specified both their limits in foodstuffs and official sample preparation protocols in addition to analytical methods to verify their presence. Capillary Electrophoresis (CE) includes different separation modes, allowing many versatile applications in food analysis and safety. In the context of mycotoxins, recent advances to improve CE sensitivity, particularly pre-concentration techniques or miniaturized systems, deserve remarkable attention, as they provide an interesting approach in the analysis of such contaminants in complex food matrices. This review summarizes the applications of CE combined with different pre-concentration approaches, which have been proposed in the literature (mainly) in the last ten years. A section is also dedicated to recent microchip–CE devices since they represent the most promising CE mode for this application.

In the last two decades, plants became an interesting alternative for the production of recombinant proteins for human therapy and several antibodies expressed in plants have reached the clinical development stage. Plants are capable of post-translational modifications (PTMs) necessary for protein activity and pharmacokinetics, such as glycosylation. However, there are important kingdom-specific modifications that have to be considered when expressing recombinant proteins. Therefore, there is a need for efficient analytical methods for deep protein characterization starting from the expression platform design until the product approval to guarantee product authenticity, quality and efficacy. Literature lacks of reviews dealing with plant-derived proteins purification and characterization by chromatographic methods, thus the focus of the present review is on this topic for the most representative biotechnological drugs i.e. monoclonal antibodies (mAbs). In the first part, a comprehensive discussion of the methods applied in dowstream processes (extraction and clarification) and a detailed overview of the chromatographic techniques useful for the purification of plant-made mAbs are reported. Among purification techniques, Protein A affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, hydrophobic charge induction chromatography or mixed mode chromatography are described. In the second part, we will discuss analytical platforms based on chromatographic techniques (reverse phase, size exclusion chromatography, ion-exchange chromatography, hydrophilic interaction liquid chromatography) coupled with different detection systems (UV, Fluorescence, MS) used at protein, peptide and glycan level to characterize plant-made mAbs with their unique features.

In the last years, the European Commission has adopted restrictive directives on food quality and safety in order to protect animal and human health. Veterinary drugs represent an important risk and the need to have sensitive and fast analytical techniques to detect and quantify them has become mandatory. Over the years, the availability of different modes, interfaces, and formats has improved the versatility, sensitivity, and speed of capillary electrophoresis (CE) techniques. Thus, CE represents a powerful tool for the analysis of a large variety of food matrices and food-related molecules with important applications in food quality and safety. This review focuses the attention of CE applications over the last decade on the detection of different classes of drugs (used as additives in animal food or present as contaminants in food products) with a potential risk for animal and human health. In addition, considering that the different sample preparation procedures have strongly contributed to CE sensitivity and versatility, the most advanced sample pre-concentration techniques are discussed here.

Teicoplanin is a glycopeptide antibiotic prepared by fermentation from cultures of Actinoplanes teichomyceticus, used as drug of last resort for the treatment of bacterial infections in humans. This study, which is the first in a series of two parts, describes the development of a LC method for the separation of Teicoplanin drug substance and its related impurities compatible with MS detection. The separation conditions for Teicoplanin were set on a LiChrospher 100 RP-18 column under gradient elution with a mobile phase composed of ammonium formate 25 mM at pH 6.00 and ACN. The new method was shown equivalent in terms of selectivity to the one reported in the European Pharmacopoeia Teicoplanin monograph, and was validated according to ICH Q2 R1 guidelines for the drug substance assay. The new method offers similar performance to the compendial one but has the advantage of being fully compatible with MS and it can be proposed as a useful tool also for controlling the quality of Teicoplanin fermentation batches and the occurrence of potential impurities.

A nanomechanical biosensor based on microcantilevers was implemented to test low molecular weight (small) compounds for their ability to stabilize beta(2)-microglobulin (beta(2)-m) in its native conformation. beta(2)-m was immobilized on the top face of silicon microcantilevers and it was demonstrated that pH induced unfolding of the immobilized beta(2)-m drives a specific microcantilever bending. This beta(2)-m microcantilever assay was then implemented to probe the effect of a pilot set small ligands on beta(2)-m conformational stability. Among the tested ligands, congo red was the only one able to protect beta(2)-m from unfolding, that is known to be the primary trigger of its self-polymerization into fibrils and in turn of the onset of amyloidosis. These findings disclose the high potentiality of nanomechanical sensors in the field of protein conformation related diseases, as they bring the unique advantage of directly screening compounds for their specific pharmacological activity rather than for generic binding preferences, effectively shortcutting the identification of the active ones. (C) 2012 Elsevier B. V. All rights reserved.