The main molecular mechanisms explaining the well-established antioxidant and reducing activity of N-acetylcysteine (NAC), the N-acetyl derivative of the natural amino acid l-cysteine, are summarised ...and critically reviewed. The antioxidant effect is due to the ability of NAC to act as a reduced glutathione (GSH) precursor; GSH is a well-known direct antioxidant and a substrate of several antioxidant enzymes. Moreover, in some conditions where a significant depletion of endogenous Cys and GSH occurs, NAC can act as a direct antioxidant for some oxidant species such as NO
2
and HOX. The antioxidant activity of NAC could also be due to its effect in breaking thiolated proteins, thus releasing free thiols as well as reduced proteins, which in some cases, such as for mercaptoalbumin, have important direct antioxidant activity. As well as being involved in the antioxidant mechanism, the disulphide breaking activity of NAC also explains its mucolytic activity which is due to its effect in reducing heavily cross-linked mucus glycoproteins. Chemical features explaining the efficient disulphide breaking activity of NAC are also explained.
A fully-detailed LC-MS qualitative profiling of red grape skin, extracted with a mixture of ethanol and water (70:30 v:v) has permitted the identification of 65 compounds which can be classified into ...the following chemical classes: organic and phenolic acids (14 compounds), stilbenoids (1 compound), flavanols (21 compounds), flavonols (15 compounds) and anthocyanins (14 compounds). The extraction yield obtained with water at different temperatures (100 °C, 70 °C, room temperature) was then evaluated and the overall polyphenol content indicates that EtOH:H2O solvent is the most efficient and selective for polyphenol extraction. However, by analyzing the recovery yield of each single polyphenol, we found that water extraction under heating conditions is effective (extraction yield similar or even better in respect to the binary solvent) for some polyphenolic classes, such as hydrophilic procyanidins, phenolic acids, flavonol glucosides and stilbenoids. However, according to their lipophilic character, a poor yield was found for the most lipophilic components, such as flavonol aglycones, and in general for anthocyanins. The radical scavenging activity was in accordance with the polyphenol content, and hence, much higher for the extract obtained with the binary solvent in respect to water extraction. All the tested extracts were found to have an anti-inflammatory activity in the R3/1 cell line with NF-kb reporter challenged with 0.01 µg/mL of IL-1α, in a 1 to 250 µg/mL concentration range. An intriguing result was that the EtOH:H2O extract was found to be superimposable with that obtained using water at 100 °C despite the lower polyphenol content. Taken together, the results show the bioactive potentialities of grape skin extracts and the possibility to exploit this rich industrial waste. Water extraction carried out by heating is an easy, low-cost and environmentally friendly extraction method for some polyphenol classes and may have great potential for extracts with anti-inflammatory activities.
Reactive carbonyl species (RCS) formed by lipidperoxidation as free forms or as enzymatic and non-enzymatic conjugates are widely used as an index of oxidative stress. Besides general measurements ...based on derivatizing reactions, more selective and sensitive MS based analyses have been proposed in the last decade. Untargeted and targeted methods for the measurement of free RCS and adducts have been described and their applications to in vitro and ex vivo samples have permitted the identification of many biological targets, reaction mechanisms and adducted moieties with a particular relevance to RCS protein adducts. The growing interest in protein carbonylation can be explained by considering that protein adducts are now recognized as being involved in the damaging action of oxidative stress so that their measurement is performed not only to obtain an index of lipid peroxidation but also to gain a deeper insight into the molecular mechanisms of oxidative stress. The aim of the review is to discuss the most novel analytical approaches and their application for profiling reactive carbonyl species and their enzymatic and non-enzymatic metabolites as an index of lipid-oxidation and oxidative stress. Limits and perspectives will be discussed.
Display omitted
Plant secondary metabolites, known as phytochemicals, have recently gained much attention in light of the "circular economy", to reutilize waste products deriving from agriculture and food industry. ...Phytochemicals are known for their onco-preventive and chemoprotective effects, among several other beneficial properties. Apple phytochemicals have been extensively studied for their effectiveness in a wide range of diseases, cancer included. This review aims to provide a thorough overview of the main studies reported in the literature concerning apple phytochemicals, mostly polyphenols, in cancer prevention. Although there are many different mechanisms targeted by phytochemicals, the Nrf2 and NF-κB signaling pathways are the ones this review will be focused on, highlighting also the existing crosstalk between these two systems.
Hyaluronic acid (HA) is a glycosaminoglycan very common in commercial products from pharmaceuticals to cosmetics due to its widespread distribution in humans and its diversified physico-chemical ...proprieties. Despite its extended use and preliminary evidence showing even also opposite activities to the native form, the precise cellular effects of HA at low-molecular-weight (LWM-HA) are currently unclear. The ‘omics sciences currently in development offer a new and combined perspective on the cellular and organismal environment. This work aims to integrate lipidomics analyses to our previous quantitative proteomics one for a multi-omics vision of intra- and extra-cellular impact of different concentrations (0.125, 0.25, and 0.50%) of LMW-HA (20–50 kDa) on normal human dermal fibroblasts by LC-high resolution mass spectrometry (LC-HRMS). Untargeted lipidomics allowed us to identify 903 unique lipids mostly represented by triacylglycerols, ceramides, and phosphatidylcholines. According to proteomics analyses, LMW-HA 0.50% was the most effective concentration also in the lipidome rearrangement especially stimulating the synthesis of ceramides involved in skin hydration and reparation, cell signaling, and energy balance. Finally, integrative analyses showed 25 nodes covering several intra- and extra-cellular functions. The more complete comprehension of intra- and extra-cellular effects of LMW-HA here pointed out will be useful to further exploit its features and improve current formulations even though further studies on lipids biosynthesis and degradation are necessary.
Carnosine is an endogenous β-alanyl-L-histidine dipeptide endowed with antioxidant and carbonyl scavenger properties, which is able to significantly prevent the visible signs of aging and photoaging. ...To investigate the mechanism of action of carnosine on human skin proteome, a 3D scaffold-free spheroid model of primary dermal fibroblasts from a 50-year-old donor was adopted in combination with quantitative proteomics for the first time. The label free proteomics approach based on high-resolution mass spectrometry, integrated with network analyses, provided a highly sensitive and selective method to describe the human dermis spheroid model during long-term culture and upon carnosine treatment. Overall, 2171 quantified proteins allowed the in-depth characterization of the 3D dermis phenotype during growth and differentiation, at 14 versus 7 days of culture. A total of 485 proteins were differentially regulated by carnosine at 7 days, an intermediate time of culture. Of the several modulated pathways, most are involved in mitochondrial functionality, such as oxidative phosphorylation, TCA cycle, extracellular matrix reorganization and apoptosis. In long-term culture, functional modules related to oxidative stress were upregulated, inducing the aging process of dermis spheroids, while carnosine treatment prevented this by the downregulation of the same functional modules. The application of quantitative proteomics, coupled to advanced and relevant in vitro scaffold free spheroids, represents a new concrete application for personalized therapies and a novel care approach.
Background: The viral main protease (Mpro) of SARS-CoV-2 has been recently proposed as a key target to inhibit virus replication in the host. Therefore, molecules that can bind the catalytic site of ...Mpro could be considered as potential drug candidates in the treatment of SARS-CoV-2 infections. Here we proposed the application of a state-of-the-art analytical platform which combines metabolomics and protein structure analysis to fish-out potential active compounds deriving from a natural matrix, i.e., a blueberry extract. Methods: The experiments focus on finding MS covalent inhibitors of Mpro that contain in their structure a catechol/pyrogallol moiety capable of binding to the nucleophilic amino acids of the enzyme’s catalytic site. Results: Among the potential candidates identified, the delphinidin-3-glucoside showed the most promising results. Its antiviral activity has been confirmed in vitro on Vero E6 cells infected with SARS-CoV-2, showing a dose-dependent inhibitory effect almost comparable to the known Mpro inhibitor baicalin. The interaction of delphinidin-3-glucoside with the Mpro pocket observed was also evaluated by computational studies. Conclusions: The HRMS analytical platform described proved to be effective in identifying compounds that covalently bind Mpro and are active in the inhibition of SARS-CoV-2 replication, such as delphinidin-3-glucoside.
To characterize the high‐value protein content and to discover new bioactive peptides, present in edible organisms, as silkworm pupae, semiquantitative analytical approach has been applied. The ...combination of appropriate protein extraction methods, semiquantitative high‐resolution mass spectrometry analyses of peptides, in silico bioactivity and gene ontology analyses, allowed protein profiling of silkworm pupae (778 gene products) and the characterization of bioactive peptides. The semiquantitative analysis, based on the measurement of the emPAI, revealed the presence of high‐abundance class of proteins, such as larval storage protein (LSP) class. This class of proteins, beside its nutrient reservoir activity, is of great pharmaceutical interest for their efficacy in cardiovascular diseases. Potential allergens were also characterized and quantified, such as arginine kinase, thiol peroxiredoxin, and Bom m 9. This powerful bioanalytical approach proved the potential industrial applications of Bombyx mori pupae, as source of high‐value proteins in a green and “circular” economy perspective.
Protein content by the semiquantitative analyses of Bombyx mori pupae. Identification of high‐value bioactive peptides by mass spectrometry. Potential industrial applications of B. mori pupae, as nutrient reservoir.
A multidisciplinary investigation on
Wulfen (Asteraceae) is outlined herein. This work, part of the European Interreg Italy-Switzerland
project, originated from an ethnobotanical survey performed in ...Chiesa in Valmalenco (Sondrio, Lombardy, Northern Italy) in 2019-2021 which highlighted this species' relevance of use in folk medicine to treat gastrointestinal diseases. In addition, this contribution included analyses of the: (a) phytochemical profile of the aqueous and methanolic extracts of the dried flower heads using LC-MS/MS; (b) morpho-anatomy and histochemistry of the vegetative and reproductive organs through Light, Fluorescence, and Scanning Electron Microscopy; (c) biological activity of the aqueous extract concerning the antioxidant and anti-inflammatory potential through cell-based in vitro models. A total of 31 compounds (5 phenolic acids, 13 flavonols, and 13 flavones) were detected, 28 of which included in both extracts. Covering and secreting trichomes were observed: the biseriate 10-celled glandular trichomes prevailing on the inflorescences represented the main sites of synthesis of the polyphenols and flavonoids detected in the extracts, along with volatile terpenoids. Finally, significant antioxidant and anti-inflammatory activities of the aqueous extract were documented, even at very low concentrations; for the first time, the in vitro tests allowed us to formulate hypotheses about the mechanism of action. This work brings an element of novelty due to the faithful reproduction of the traditional aqueous preparation and the combination of phytochemical and micromorphological research approaches.
Advanced Lipoxidation End-products (ALEs) are modified proteins that can act as pathogenic factors in several chronic diseases. Several molecular mechanisms have so far been considered to explain the ...damaging action of ALEs and among these a pathway involving the receptor for advanced glycation end products (RAGE) should be considered. The aim of the present work is to understand if ALEs formed from lipid peroxidation derived reactive carbonyl species (RCS) are able to act as RAGE binders and also to gain a deeper insight into the molecular mechanisms involved in the protein-protein engagement. ALEs were produced in vitro, by incubating human serum albumin (HSA) with 4-hydroxy-trans- 2-nonenal (HNE), acrolein (ACR) and malondialdehyde (MDA). The identification of ALEs was performed by MS. ALEs were then subjected to the VC1 Pull-Down assay (VC1 is the ligand binding domain of RAGE) and the enrichment factor (the difference between the relative abundance in the enriched sample minus the amount in the untreated one) as an index of affinity, was determined. Computation studies were then carried out to explain the factors governing the affinity of the adducted moieties and the site of interaction on adducted HSA for VC1-binding. The in silico analyses revealed the key role played by those adducts which strongly reduce the basicity of the modified residues and thus occur at their neutral state at physiological conditions (e.g. the MDA adducts, dihydropyridine-Lysine (DHPK) and N-2-pyrimidyl-ornithine (NPO), and acrolein derivatives, N-(3-formyl-3,4-dehydro-piperidinyl) lysine, FDPK). These neutral adducts become unable to stabilize ion-pairs with the surrounding negative residues which thus can contact the RAGE positive residues. In conclusion, ALEs derived from lipid peroxidation-RCS are binders of RAGE and this affinity depends on the effect of the adduct moiety to reduce the basicity of the target amino acid and on the acid moieties surrounding the aminoacidic target.
PDF
