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The aim of the work was to study a dose-dependent effect of inhaled carnosine (10, 50 or 100 mg/kg/day) in mice exposed to cigarette smoke as a model of chronic obstructive pulmonary disease (COPD). A dose-dependent loading of the dipeptide in lung tissue and bronchoalveolar lavage (BAL) was firstly demonstrated by LC-ESI-MS analysis. Cigarette smoke exposure induced a significant lung inflammation and oxidative stress in mice which was dose-dependently reduced by carnosine. Inflammation was firstly evaluated by measuring the cytokines content in the BAL. All the measured cytokines were found significantly higher in the smoke group in respect to control, although the data are affected by a significant variability. Carnosine was found effective only at the highest dose tested and significantly only for keratinocyte-derived cytokine (KC). Due to the high variability of cytokines, a quantitative proteomic approach to better understand the functional effect of carnosine and its molecular mechanisms was used. Proteomic data clearly indicate that smoke exposure had a great impact on lung tissue with 692 proteins differentially expressed above a threshold of 1.5-fold. Protein network analysis identified the activation of some pathways characteristic of COPD, including inflammatory response, fibrosis, induction of immune system by infiltration and migration of leukocyte pathways, altered pathway of calcium metabolism and oxidative stress. Carnosine at the tested dose of 100 mg/kg was found effective in reverting all the pathways evoked by smoke. Only a partial reverse of the dysregulated proteins was evident at low- and mid-tested doses, although, for some specific proteins, indicating an overall dose-dependent effect. Regarding the molecular mechanisms involved, we found that carnosine upregulated some key enzymes related to Nrf2 activation and in particular glutathione peroxidase, reductase, transferase, SOD, thioredoxins, and carbonyl reductase. Such mechanism would explain the antioxidant and anti-inflammatory effects of the dipeptide.

This systematic review investigates the potential of bergamot, a polyphenol-rich citrus fruit, in improving osteosarcopenic obesity, a condition characterized by the simultaneous presence of osteoporosis, obesity, and sarcopenia. Bergamot extracts have been suggested to possess several pharmacological properties, including anti-inflammatory and antioxidant effects, which could be useful in the management of age-related diseases and neuromuscular health. The review highlights the promising effects of bergamot extracts on skeletal muscle mass and function, particularly in the context of obesity, metabolic syndrome, osteosarcopenic obesity, and osteoporosis. Furthermore, some studies have shown that bergamot extracts can improve the metabolic balance, endothelial function, and maximal oxygen uptake in athletes, highlighting their potential benefits for skeletal muscle health. Taken together, these results suggest that bergamot extracts, especially those rich in polyphenols, may be a valuable adjunct in the management of osteosarcopenic obesity and other associated clinical conditions involving pro-inflammatory effects on organs and tissues.

Policosanols (PCs) are bioactive compounds extracted from different natural waxes. In this work, the purification, characterization and assessment of the antioxidant and anti-inflammatory activity was carried out on PCs from an innovative source, i.e. a waxy material from supercritical-fluid extraction (SFE) of non-psychoactive Cannabis sativa L. (hemp) inflorescences. Starting from this material, PCs were obtained by microwave-assisted trans-esterification and hydrolysis, followed by preparative liquid chromatography under normal phase conditions. The purified product was characterized using high-performance liquid chromatography (HPLC) with an evaporative light scattering detector (ELSD). In vitro cell-free and cell-based antioxidant and anti-inflammatory assays were then performed to assess their bioactivity.HPLC-ELSED analysis of the purified mixture from hemp wax revealed C26OH and C28OH as the main compounds. In vitro assays indicated an inhibition of intracellular reactive oxygen species (ROS) production, a reduction of nuclear factor kappa B (NF-κB) activation and of the activity of the neutrophil elastase. Immunoblotting assays allowed us to hypothesize the mechanism of action of the compounds of interest, given the higher levels of MAPK-activated protein kinase 2 (MK2) and heme oxygenase-1 (HO-1) protein expression in the PC pretreated HaCaT cells. In conclusion, even if more research is needed to unveil other molecular mechanisms involved in hemp PC activity, the results of this work suggest that these compounds may have potential for use in oxinflammation processes.

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.

Bergamot (Citrus bergamia) is a fruit with anti-inflammatory and antioxidant properties that contains similar bioactive composition to its leaf, evidencing its therapeutic potential in the treatment of chronic diseases. The aim was to evaluate the treatment effect of bergamot leaf extract (BLE) on the redox/inflammatory scenario in different organs of obese rats with metabolic syndrome. After detection of metabolic syndrome, male Wistar rats were allocated (n = 10/group) for the treatment with BLE by gavage (50 mg/kg): Control, Control+BLE, High Sugar fat (HSF), and HSF+BLE. Evaluations included: metabolic-nutritional profile; tissues function and redox/inflammatory parameters. The HSF group presented metabolic syndrome, cardiac, hepatic and renal dysfunction; inflammation; and oxidative stress. BLE decreased oxidative stress and inflammation levels in adipose tissue, heart, liver and kidneys, as well as decreased levels of triglycerides, insulin and insulin resistance. BLE act in all target organs of obesity, improving the redox/inflammatory scenario in a diet-induced metabolic syndrome.

The excessive consumption of diets rich in sugar and fat is associated with metabolic manifestations involving adipose tissue and the liver. Bergamot, due to its antioxidant and anti-inflammatory properties, has been used to treat metabolic disorders. This work aimed to verify the effect of Bergamot leaves extract (BLE) on the crosstalk in the adipose tissue–liver axis of obese rats. For 20 weeks, Wistar rats were distributed into two groups: control (Control) and high sugar–fat (HSF) diet groups. Afterwards, the animals were redistributed into three groups for 10 weeks: control diet + vehicle (Control, n = 08), HSF + vehicle (HSF, n = 08), and HSF + BLE (HSF + BLE, n = 08). The BLE was carried out daily by gavage (50 mg/kg). The HSF group presented obesity, hyperglycemia, hypertriglyceridemia, insulin resistance, hepatic microvesicular steatosis, higher inflammation and oxidative stress in the liver and adipose tissue. In comparison to the HSF group, HSF + BLE animals showed protection by reducing the triglyceride levels, insulin resistance, inflammation and oxidative stress in hepatic and adipose tissues. BLE acted on the inflammation and oxidative stress in the adipose tissue–liver axis in obese rats when compared to the HSF group, which may have reflected on the improvement of insulin resistance and dyslipidemia.

Abstract Chronic thromboembolic pulmonary hypertension (CTEPH) is a vascular disease characterized by the presence of organized thromboembolic material in pulmonary arteries leading to increased vascular resistance, heart failure and death. Dysfunction of endothelial cells is involved in CTEPH. The present study describes for the first time the molecular processes underlying endothelial dysfunction in the development of the CTEPH. The advanced analytical approach and the protein network analyses of patient derived CTEPH endothelial cells allowed the quantitation of 3258 proteins. The 673 differentially regulated proteins were associated with functional and disease protein network modules. The protein network analyses resulted in the characterization of dysregulated pathways associated with endothelial dysfunction, such as mitochondrial dysfunction, oxidative phosphorylation, sirtuin signaling, inflammatory response, oxidative stress and fatty acid metabolism related pathways. In addition, the quantification of advanced oxidation protein products, total protein carbonyl content, and intracellular reactive oxygen species resulted increased attesting the dysregulation of oxidative stress response. In conclusion this is the first quantitative study to highlight the involvement of endothelial dysfunction in CTEPH using patient samples and by network medicine approach.

The present paper reports a sustainable raw material obtained from the by-products derived from the industrial production of bergamot (Citrus × Bergamia Risso & Poiteau) essential oils. The procedure to obtain the raw material is designed to maintain as much of the bioactive components as possible and to avoid expensive chemical purification. It consists of spray-drying the fruit juice obtained by squeezing the fruits, which is mixed with the aqueous extract of the pulp, i.e., the solid residue remained after fruit pressing. The resulting powder bergamot juice (PBJ) contains multiple bioactive components, in particular, among others, soluble fibers, polyphenols and amino-acid betaines, such as stachydrine and betonicine. LC-MS analysis identified 86 compounds, with hesperetin, naringenin, apigenin and eridictyol glucosides being the main components. In the second part of the paper, dose-dependent anti-inflammatory activity of PBJ and of stachydrine was found, but neither of the compounds were effective in activating Nrf2. PBJ was then found to be effective in an in vivo model of a metabolic syndrome induced by a high-sugar, high-fat (HSF) diet and evidenced by a significant increase of the values related to a set of parameters: blood glucose, triglycerides, insulin resistance, systolic blood pressure, visceral adipose tissue and adiposity index. PBJ, when given to control rats, did not significantly change these values; in contrast, they were found to be greatly affected in rats receiving an HSF diet. The in vivo effect of PBJ can be ascribed not only to bergamot polyphenols with well-known anti-inflammatory, antioxidant and lipid-regulating effects, but also to the dietary fibers and to the non-phenolic constituents, such as stachydrine. Moreover, since PBJ was found to affect energy homeostasis and to regulate food intake, a mechanism on the regulation of energy homeostasis through leptin networking should also be considered and deserves further investigation.

Rice is one of the major staple foods consumed worldwide and due to the presence of γ-oryzanol (γ-OZ) it is well-recognized as functional food. For this reason, the most appropriate varieties' identification in term of content of γ-OZ has become essential in order to assess their potential nutraceutical exploitation. This study reports a suitable and versatile procedure to obtain the isotopologues of sitosteryl ferulate, one of the major γ-OZ components, namely 3-O-(3-OC2H3-feruloyl)-β-sitosterol (14-d3) and 3-O-(3-OC2H3-8-2H-feruloyl)-β-sitosterol (14-d4) for use as analytical tool.

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.

A multidisciplinary investigation on Achillea moschata Wulfen (Asteraceae) is outlined herein. This work, part of the European Interreg Italy–Switzerland B-ICE 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.

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.

The qualitative profile of thinned apple polyphenols (TAP) fraction (≈24% of polyphenols) obtained by purification through absorbent resin was fully investigated by LC-HRMS in positive and negative ion mode and using ESI source. A total of 68 polyphenols were identified belonging to six different classes: flavanols, flavonols, dihydrochalchones, flavanones, flavones and organic and phenolic acids. The antioxidant and anti-inflammatory activities were then investigated in cell models with gene reporter for NRF2 and NF-κB and by quantitative proteomic (label-free and SILAC) approaches. TAP dose-dependently activated NRF2 and in the same concentration range (10–250 µg/mL) inhibited NF-κB nuclear translocation induced by TNF-α and IL-1α as pro-inflammatory promoters. Proteomic studies elucidated the molecular pathways evoked by TAP treatment: activation of the NRF2 signaling pathway, which in turn up-regulates protective oxidoreductases and their nucleophilic substrates such as GSH and NADPH, the latter resulting from the up-regulation of the pentose phosphate pathway. The increase in the enzymatic antioxidant cellular activity together with the up-regulation of the heme-oxygenase would explain the anti-inflammatory effect of TAP. The results suggest that thinned apples can be considered as a valuable source of apple polyphenols to be used in health care products to prevent/treat oxidative and inflammatory chronic conditions.

Enocianina is an anthocyanin-rich extract obtained from grape pomace. It is widely used as a colorant in the food industry and, in addition to anthocyanins, it also contains a variety of polyphenols. To understand whether enocianina, besides its coloring effect, may offer potential health benefit applications, we aimed to fully characterize the profile of four commercial enocianinas and assess their radical scavenging, enzymatic, antioxidant, and anti-inflammatory activities. LC-ESI-MS/MS analysis identified 90 phytochemicals. The relative content of each anthocyanin was assessed by a semi-quantitative analysis, with malvidin derivatives being the most abundant. UV-VIS spectroscopy detected total amounts of polyphenols and anthocyanins of 23% and 3.24%, respectively, indicating that anthocyanins represent a minor fraction of total polyphenols. Multiple linear regression analysis indicated that the radical scavenging activity is related to the total polyphenol content and not to anthocyanins. All four enocianinas dose-dependently activate Nrf2, and such activity was correlated with catechol-containing polyphenol content. Finally, all enocianinas showed dose-dependent anti-inflammatory activity, which at the highest concentrations tested was closely related to the total polyphenol content and was explained by radical scavenging, Nrf2 activation, and other mechanisms related to the polyphenolic components.

Over the years secondary metabolites have been considered as lead molecules both in their natural form and as templates for medicinal chemistry. Some secondary metabolites such as polyphenols and flavan-3-ols exert beneficial effects after a modification by the microbiota. Synthetic precursors of some of these modified compounds, in turn, carried a γ-alkylidenebutenolide moiety which characterizes a large class of bioactive natural products endowed with a wide range of biological activities. For these reasons stereoselective preparation of γ-alkylidenebutenolide continues to be an important issue for organic chemists. Our objective is to synthetize the novel compound (Z)-5-(3′,4′-bis(benzyloxy)benzylidene)furan-2(5H)-one in a stereocontrolled-one-pot reaction. The product was obtained in good yield. Furthermore, the theoretical investigation of the transition states suggests a new procedure to achieve Z-isomer of β-unsubstituted γ-alkylidenebutenolide.

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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.

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.

Candida spp. are pathobionts, as they can switch from commensals to pathogens, responsible for a variety of pathological processes. Adhesion to surfaces, morphological switch and biofilm-forming ability are the recognized virulence factors promoting yeast virulence. Sessile lifestyle also favors fungal persistence and antifungal tolerance. In this study, we investigated, in vitro, the efficacy of two urinary cranberry metabolites, 5-(3′,4′-dihydroxy phenyl)-γ-valerolactone (VAL) and 4-hydroxybenzoic acid (4-HBA), in inhibiting C. albicans adhesion and biofilm formation. Both the reference strain SC5314 and clinical isolates were used. We evaluated biomass reduction, by confocal microscopy and crystal violet assay, and the possible mechanisms mediating their inhibitory effects. Both VAL and 4-HBA were able to interfere with the yeast adhesion, by modulating the expression of key genes, HWP1 and ALS3. A significant dose-dependent reduction in biofilm biomass and metabolic activity was also recorded. Our data showed that the two cranberry metabolites VAL and 4-HBA could pave the way for drug development, for targeting the very early phases of biofilm formation and for preventing genitourinary Candida infections.

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. Keywords: Advanced lipoxidation end products (ALEs), Human serum albumin (HSA), RAGE, Pull-down assay, VC1 domain, Reactive Carbonyl Species (RCS), 4-hydroxy-trans− 2-nonenal (HNE), Acrolein (ACR) and malondialdehyde (MDA)

Advanced glycation end-products (AGEs) and advanced lipoxidation end-products (ALEs), particularly carboxymethyl-lysine (CML), have been largely proposed as factors involved in the establishment and progression of heart failure (HF). Despite this evidence, the current literature lacks the comprehensive identification and characterization of the plasma AGEs/ALEs involved in HF (untargeted approach). This work provides the first ex vivo high-resolution mass spectrometry (HR-MS) profiling of AGEs/ALEs occurring in human serum albumin (HSA), the most abundant protein in plasma, characterized by several nucleophilic sites and thus representing the main protein substrate for AGE/ALE formation. A set of AGE/ALE adducts in pooled HF-HSA samples was defined, and a semi-quantitative analysis was carried out in order to finally select those presenting in increased amounts in the HF samples with respect to the control condition. These adducts were statistically confirmed by monitoring their content in individual HF samples by applying a targeted approach. Selected AGEs/ALEs proved to be mostly CML derivatives on Lys residues (i.e., CML-Lys12, CML-Lys378, CML-Lys402), and one deoxy-fructosyl derivative on the Lys 389 (DFK-Lys 389). The nature of CML adducts was finally confirmed using immunological methods and in vitro production of such adducts further confirmed by mass spectrometry.

The aim of the study is to compare the qualitative and semi-quantitative profile of the polyphenol fraction purified from the leaf (BLPF) and fruit (BFPF) of bergamot (Citrus bergamia), and to evaluate their antioxidant and anti-inflammatory activity. The analytical qualitative profile was carried out by LC-ESI/MS using three different approaches: targeted (searching analytes already reported in bergamot extract), semi-targeted (a selective search of 3-hydroxy-3-methylglutarate [HMG] derivatives involved in the cholesterol reducing activity of BPF) and untargeted. A total number of 108 compounds were identified by using the three approaches, 100 of which are present in both the extracts thus demonstrating a good qualitative overlapping of polyphenols between the two extracts. The antioxidant activity was higher for BLPF in respect to BFPF but when normalized in respect to the polyphenol content they were almost overlapping. Both the extracts were found to dose dependently inhibit cell inflammation stimulated with IL-1α. In conclusion, the comparison of the qualitative and quantitative profile of polyphenols as well as of the antioxidant and anti-inflammatory activity of bergamot leaf and fruit well indicates that leaf is a valid source of bergamot polyphenol extraction and an even richer source of polyphenol in respect to the fruit.

In the present paper, the extracellular antioxidant activity of N-acetyl-cysteine (NAC) is explained by considering its ability to regenerate the free form of albumin Cys34 by breaking the disulfide bond of the cysteinylated form (HSA-Cys). NAC’s capability to regenerate albumin Cys34 (HSA-SH) was studied by MS intact protein analysis in human plasma and in a concentration range of NAC easily achievable after oral and i.v. administration (5–50 µg/mL). NAC dose-dependently broke the HSA-Cys bond to form the dimer NAC-Cys thus regenerating Cys34, whose reduced state was maintained for at least 120 min. Cys was faster in restoring Cys34, according to the reaction constant determined with the glutathione disulfide (GSSG) reaction, but after 60 min the mixed disulfide HSA-Cys turned back due to the reaction of the dimer Cys-Cys with Cys34. The explanation for the different rate exchanges between Cys-Cys and Cys-NAC with Cys34 was given by molecular modeling studies. Finally, the Cys34 regenerating effect of NAC was related to its ability to improve the total antioxidant capacity of plasma (TRAP assay). The results well indicate that NAC greatly increases the plasma antioxidant activity and this effect is not reached by a direct effect but through the regenerating effect of Cys34.