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With the advent of new analytical technologies and the urgent environmental problem, reopening investigations into polluting waste matrices becomes a priority. Olive mill wastewater is a pollutant and phytotoxic by-product of olive oil production. An untargeted UHPLC-QTOF analysis of three olive mill wastewaters from three different olive cultivars was performed, and modern informatic platforms were involved to characterize the chemical components in-depth. Data elaboration and statistical analysis confirmed the differences between samples and revealed a total of 364 annotated compounds, including iridoids, phenolic compounds, flavonoids, lignans, cinnamic acid derivatives, and pyrrolidine derivatives. Many of these metabolites, including compounds with known antioxidant and bioactive potential, are scarcely reported in olive products and by-products. The outcomes of this work could be useful for rethinking olive mill wastewater as a source of bioactive compounds to develop and optimize new detoxification strategies.

The aim of this research was to develop innovative cheeses fortified with vitamin D3 (VD3). Formulation studies and analyses of textural properties and chemicals were carried out for these developments. Two traditional Italian varieties of cheese (giuncata and burrata) were studied. For giuncata, the fortification of milk for cheese production provided a VD3 retention level of 43.9 ± 0.6% in the food matrix. For burrata, the VD3 ingredient was incorporated into the creamy inner part after mixing, maintaining the textural quality of the product (adhesiveness 4.3 ± 0.4 J × 10−3; firmness 0.7 ± 0.0 N; and cohesiveness 0.8 ± 0.2). The optimized enrichment designs allowed to obtain homogenous contents of VD3 during the production of giuncata (0.48 ± 0.01 µg/g) and burrata cheeses (0.32 ± 0.02 µg/g). Moreover, analyses revealed the high stability of VD3 during the storage of the two fortified cheese types (2 weeks, 4 °C). These fortification designs could be implemented at an industrial scale to obtain new cheese types enriched in VD3 and thus contribute to the reduction in VD deficiency prevalence.

Currently, the number of patients with neurodegenerative pathologies is estimated at over one million, with consequences also on the economic level. Several factors contribute to their development, including overexpression of A2A adenosine receptors (A2AAR) in microglial cells and up-regulation and post-translational alterations of some casein kinases (CK), among them, CK-1δ. The aim of the work was to study the activity of A2AAR and CK1δ in neurodegeneration using in-house synthesized A2A/CK1δ dual anta-inhibitors and to evaluate their intestinal absorption. Experiments were performed on N13 microglial cells, which were treated with a proinflammatory CK cocktail to simulate an inflammatory state typical of neurodegenerative diseases. Results showed that the dual anta-inhibitors have the ability to counteract the inflammatory state, even if compound 2 is more active than compound 1. In addition, compound 2 displayed an important antioxidant effect similar to the reference compound ZM241385. Since many known kinase inhibitors are very often unable to cross lipid bilayer membranes, the ability of A2A/CK1δ double anta-inhibitors to cross the intestinal barrier was investigated by an everted gut sac assay. HPLC analysis revealed that both compounds are able to cross the intestinal barrier, making them promising candidates for oral therapy.

This study assessed the impact of full and late flowering stages on the polyphenols and enzyme inhibitory properties of Hypericum perforatum from Poland. Recognizing the significance of phenolic compounds in disease prevention and melatonin’s emerging protective role, we employed an UHPLC-MS/MS system to quantify 38 phenolic compounds, not typical of St. John’s wort, and to develop a new method for melatonin quantification. Afterward, the extracts were tested for their antioxidant capabilities (using phosphomolybdenum, DPPH, ABTS, FRAP, CUPRAC and ferrous chelating assays). Moreover, we investigated enzymes (acetylcholinesterase, butyrylcholinesterase and tyrosinase) involved in neurodegenerative disorders and (α-amylase and α-glucosidase) in diabetes. This study recognized the importance of phenolic compounds in disease prevention and explored the emerging protective role of melatonin, taking into account the floral ontogeny of the plant. Indeed, the full-flowering plant contained the greatest concentration of phenolic compounds (a total of 65,276.5 µg/g): hyperoside (18,726.59 µg/g), isoquercitrin (11,895.02 µg/g) and delphindin-3.5-diglucoside (10,619.51 µg/g), and showed the highest inhibitory enzyme activity. Moreover, only full-flowering St. John’s wort contained melatonin (40 ng/g). Our results offer additional perspectives on the chemical-biological characteristics of H. perforatum and scientific knowledge that testifies to the importance of considering plant growth conditions for the development of nutraceuticals.

The specificity of peptide activity depends on the sequence of amino acids and structure, so it is easy to understand the great degree of biodiversity that peptides can cover. Accordingly, the literature shows that peptides carry out various activities, including antiproliferative, antihypertensive, antimicrobial, antioxidant, anticholesterolemic, opioid and antidiabetic activities. However, the potential activity of peptides is strongly quenched by their low bioavailability. The aim of this work is to provide some insights on the possibility of increasing the bioavailability of peptides. Two key points have been investigated. The results demonstrate that N-terminus acetylation and C-terminus amidation can strongly protect peptides from proteolytic degradation. Furthermore, the activity of the peptides can be reduced by the formation of complexes, for example through hydrophobic interactions. In particular peptides containing cysteine can dimerize with the formation of a disulfide bridge. The possibility of decreasing complexes formation by solubilizing peptides in a dissociation mixture (containing glycine, urea, dithiothreitol and beta-mercaptoethanol), is discussed.

Leonurus cardiaca L. (Lamiaceae) is a perennial herb distributed in Asia and Southeastern Europe and has been used in traditional medicine since antiquity for its role against cardiac and gynecological disorders. The polar extracts obtained from L. cardiaca aerial parts contain several compounds among which alkaloids, iridoids, labdane diterpenes, and phenylethanoid glycosides play a major role in conferring protection against the aforementioned diseases. On the other hand, the antioxidant activities and the enzyme inhibitory properties of these extracts have not yet been deeply studied. On the above, in the present study, crude and purified extracts were prepared from the aerial parts of L. cardiaca and have been chemically characterized by spectrophotometric assays and HPLC-DAD-MS analyses. Notably, the content of twelve secondary metabolites, namely phenolic acids (chlorogenic, caffeic, caffeoylmalic and trans-ferulic acids), flavonoids (rutin and quercetin), phenylethanoid glycosides (verbascoside and lavandulifolioside), guanidine pseudoalkaloids (leonurine), iridoids (harpagide), diterpenes (forskolin), and triterpenes (ursolic acid), has been determined. Furthermore, the extracts were tested for their antioxidant capabilities (phosphomolybdenum, DPPH, ABTS, FRAP, CUPRAC, and ferrous chelating assays) and enzyme inhibitory properties against cholinesterase, tyrosinase, amylase, and glucosidase. The purified extracts contained higher phytochemical content than the crude ones, with caffeoylmalic acid and verbascoside as the most abundant compounds. A linear correlation between total phenolics, radical scavenging activity, and reducing power of extracts has been found. Notably, quercetin, caffeic acid, lavandulifolioside, verbascoside, chlorogenic acid, rutin, and ursolic acid influenced the main variations in the bioactivities found in L. cardiaca extracts. Our findings provide further insights into the chemico-biological traits of L. cardiaca and a scientific basis for the development of nutraceuticals and food supplements.

The myrrh-like furanosesquiterpene isofuranodiene (IFD) is the main constituent of wild celery (Smyrnium olusatrum L., Apiaceae), an overlooked vegetable that was cultivated during the Roman Empire. In the present study, we investigated the protective effects of IFD pre-treatment against oxidative stress and inflammatory response in an animal model of ischemic stroke. IFD was isolated by the crystallization of Smyrnium olusatrum essential oil, and its structure and purity were confirmed by NMR and HPLC analyses. Acute pre-treatment of IFD (10 mg/kg i.p.) significantly reduced the levels of the inflammatory cytokines IL-1β and TNF-α, the expression of pNF-κB/NF-κB, and the lipid peroxidation indicator MDA. Finally, IFD boosted a faster recovery and better scores in grid-walking and modified neurological severity scores (mNSS) tests. Taken together, these findings indicate IFD as a promising lead compound for the discovery of new treatments of brain ischemia.

Fecal proteomics allows for the identification of proteins and peptides present in stools and is useful in finding possible new biomarkers for diagnosing and/or monitoring gastrointestinal (GI) disorders. In the present study, we investigated the fecal proteome in healthy and diseased cheetahs (Acinonyx jubatus). Captive individuals of this species frequently show gastrointestinal disorders characterized by recurrent episodes of diarrhea, rare episodes of vomiting and weight loss, associated with Helicobacter spp. infection. Fecal proteomic evaluation has been performed by two-dimensional electrophoresis followed by liquid chromatography-tandem mass spectrometry. In healthy cheetahs, the results showed the presence of the following proteins: collagen alpha-1 (II) chain, transthyretin, IgG Fc-binding protein, titin, dystonin, isopentenyl-diphosphate Delta-isomerase 1, sodium/potassium-transporting ATPase subunit alpha-1 and protein disulfide-isomerase A6. The presence of albumin isoforms was found only in diseased cheetahs. The present paper reports the study of the fecal proteome in the cheetah, evidences some differences between healthy and diseased patients and confirms, once again, the potential of fecal proteomics for the study of the GI environment, with promising developments regarding the identification of new diagnostic/monitoring markers.

The yeast Wickerhamomyces anomalus has several applications in the food industry due to its antimicrobial potential and wide range of biotechnological properties. In particular, a specific strain of Wickerhamomyces anomalus isolated from the malaria mosquito Anopheles stephensi, namely WaF17.12, was reported to secrete a killer toxin with strong anti-plasmodial effect on different developmental stages of Plasmodium berghei; therefore, we propose its use in the symbiotic control of malaria. In this study, we focused on the identification/characterization of the protein toxin responsible for the observed antimicrobial activity of the yeast. For this purpose, the culture medium of the killer yeast strain WaF17.12 was processed by means of lateral flow filtration, anion exchange and gel filtration chromatography, immunometric methods, and eventually analyzed by liquid chromatography-tandem mass spectrometry (LC–MS/MS). Based on this concerted approach, we identified a protein with a molecular weight of approximately 140 kDa and limited electrophoretic mobility, corresponding to a high molecular weight β-glucosidase, as confirmed by activity tests in the presence of specific inhibitors.

Canine intestinal lymphangiectasia (IL) is a condition characterized by variably severe gastrointestinal signs, frequently associated with laboratory abnormalities; the research for markers allowing a better understanding of the severity degree and/or obtaining an early diagnosis and/or monitoring is continuously progressing. In the present study, we investigated possible new diagnostic/follow-up markers in IL dogs, namely, serum C-reactive protein, serum bacterial lipopolysaccharide, serum cleaved cytokeratin 18, serum citrulline, and zonulin (in both serum and feces). A fecal proteomic study looking for possible confirmation and/or new marker candidates was also performed. All markers in both substrates, with the exception of serum citrulline, significantly differed between diseased and control dogs. Fecal proteomics allowed the retrieval of three proteins in IL dogs (Fc fragment of IgG-binding protein; transthyretin; proproteinase E) that were not previously found in clinically healthy subjects. Although further studies are needed, C-reactive protein, bacterial lipopolysaccharide, cleaved cytokeratin 18, and zonulin (in both serum and feces) resulted as promising markers for canine IL; similarly, fecal proteomics represents a road worthy of being pursued in the search for candidate biomarkers.