Discovery of a septin-4 covalent binder with antimetastatic activity in a mouse model of melanoma.

Cancer spreading through metastatic processes is one of the major causes of tumour-related mortality. Metastasis is a complex phenomenon which involves multiple pathways ranging from cell metabolic alterations to changes in the biophysical phenotype of cells and tissues. In the search for new effective anti-metastatic agents, we modulated the chemical structure of the lead compound AA6, in order to find the structural determinants of activity, and to identify the cellular target responsible of the downstream anti-metastatic effects observed. New compounds synthesized were able to inhibit in vitro B16-F10 melanoma cell invasiveness, and one selected compound, CM365, showed in vivo anti-metastatic effects in a lung metastasis mouse model of melanoma. Septin-4 was identified as the most likely molecular target responsible for these effects. This study showed that CM365 is a promising molecule for metastasis prevention, remarkably effective alone or co-administered with drugs normally used in cancer therapy, such as paclitaxel.

Treatment with bergamot (Citrus bergamia) leaves extract attenuates leptin resistance in obese rats.

Low-grade chronic inflammation in obesity is associated with leptin resistance. In order to alleviate this pathological condition, bioactive compounds capable of attenuating oxidative stress and inflammation have been researched, and bergamot (Citrus bergamia) presents these properties. The aim was to evaluate the effect of bergamot leaves extract on leptin resistance in obese rats. Animals were divided into 2 groups: control diet (C, n = 10) and high sugar-fat diet (HSF, n = 20) for 20 weeks. After detecting hyperleptinemia, animals were divided to begin the treatment with bergamot leaves extract (BLE) for 10 weeks: C + placebo (n = 7), HSF + placebo (n = 7), and HSF + BLE (n = 7) by gavage (50 mg/kg). Evaluations included nutritional, hormonal and metabolic parameters; adipose tissue dysfunction; inflammatory, oxidative markers and hypothalamic leptin pathway. HSF group presented obesity, metabolic syndrome, adipose tissue dysfunction, hyperleptinemia and leptin resistance compared to control group. However, the treated group showed a decrease in caloric consumption and attenuation of insulin resistance. Moreover, dyslipidemia, adipose tissue function, and leptin levels showed an improvement. At the level of the hypothalamus, the treated group showed a reduction of oxidative stress, inflammation and modulation of leptin signaling. In conclusion, BLE properties were able to improve leptin resistance through recovery of the hypothalamic pathway.

Targeting Nrf2 and NF-κB Signaling Pathways in Cancer Prevention: The Role of Apple Phytochemicals.

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.

Bergamot (Citrus bergamia) leaf extract improves metabolic, antioxidant and anti-inflammatory activity in skeletal muscles in a metabolic syndrome experimental model.

Metabolic Syndrome (MetS), inflammation and oxidative stress contribute to impairment of skeletal muscle function. Bergamot (Citrus bergamia) leaf extract (BLE) has shown protective effects against comorbidities associated with MetS through its anti-inflammatory and antioxidant effects. The aim of this work was to elucidate the antioxidant and anti-inflammatory activity of BLE in skeletal muscles in an experimental model of MetS. Once metabolic syndrome was diagnosed, animals were divided into groups receiving different treatments for 10 weeks, including control diet (n = 10), control + BLE (n = 10), High Sugar-fat diet (HSF) (n = 10), HSF + BLE (n = 10). Evaluation included nutritional, metabolic and hormonal analyses, along with measurements of inflammatory status and oxidative stress in soleus and extensor digitorum longus (EDL) muscles. BLE showed positive metabolic effects, with a reduction of plasma triglycerides and insulin resistance and an increase in high-density lipoprotein cholesterol, and protective activity against oxidative stress and inflammation in Soleus and EDL muscles in animals with MetS.

An integrated metabolomic and proteomic approach for the identification of covalent inhibitors of the main protease (Mpro) of SARS-COV-2 from crude natural extracts.

Mpro represents one of the most promising drug targets for SARS-Cov-2, as it plays a crucial role in the maturation of viral polyproteins into functional proteins. HTS methods are currently used to screen Mpro inhibitors, and rely on searching chemical databases and compound libraries, meaning that they only consider previously structurally clarified and isolated molecules. A great advancement in the hit identification strategy would be to set-up an approach aimed at exploring un-deconvoluted mixtures of compounds such as plant extracts. Hence, the aim of the present study is to set-up an analytical platform able to fish-out bioactive molecules from complex natural matrices even where there is no knowledge on the constituents. The proposed approach begins with a metabolomic step aimed at annotating the MW of the matrix constituents. A further metabolomic step is based on identifying those natural electrophilic compounds able to form a Michael adduct with thiols, a peculiar chemical feature of many Mpro inhibitors that covalently bind the catalytic Cys145 in the active site, thus stabilizing the complex. A final step consists of incubating recombinant Mpro with natural extracts and identifying compounds adducted to the residues within the Mpro active site by bottom-up proteomic analysis (nano-LC-HRMS). Data analysis is based on two complementary strategies: (i) a targeted search applied by setting the adducted moieties identified as Michael acceptors of Cys as variable modifications; (ii) an untargeted approach aimed at identifying the whole range of adducted peptides containing Cys145 on the basis of the characteristic b and y fragment ions independent of the adduct. The method was set-up and then successfully tested to fish-out bioactive compounds from the crude extract of Scutellaria baicalensis, a Chinese plant containing the catechol-like flavonoid baicalin and its corresponding aglycone baicalein which are well-established inhibitors of Mpro. Molecular dynamics (MD) simulations were carried out in order to explore the binding mode of baicalin and baicalein, within the SARS-CoV-2 Mpro active site, allowing a better understanding of the role of the nucleophilic residues (i.e. His41, Cys145, His163 and His164) in the protein-ligand recognition process.

Identification and quantification of ionising radiation-induced oxysterol formation in membranes of lens fibre cells.

Ionising radiation (IR) is a cause of lipid peroxidation, and epidemiological data have revealed a correlation between exposure to IR and the development of eye lens cataracts. Cataracts remain the leading cause of blindness around the world. The plasma membranes of lens fibre cells are one of the most cholesterolrich membranes in the human body, forming lipid rafts and contributing to the biophysical properties of lens fibre plasma membrane. Liquid chromatography followed by mass spectrometry was used to analyse bovine eye lens lipid membrane fractions after exposure to 5 and 50 Gy and eye lenses taken from wholebody 2 Gy-irradiated mice. Although cholesterol levels do not change significantly, IR dose-dependant formation of the oxysterols 7ß-hydroxycholesterol, 7-ketocholesterol and 5, 6-epoxycholesterol in bovine lens nucleus membrane extracts was observed. Whole-body X-ray exposure (2 Gy) of 12-week old mice resulted in an increase in 7ß-hydroxycholesterol and 7-ketocholesterol in their eye lenses. Their increase regressed over 24 h in the living lens cortex after IR exposure. This study also demonstrated that the IR-induced fold increase in oxysterols was greater in the mouse lens cortex than the nucleus. Further work is required to elucidate the mechanistic link(s) between oxysterols and IR-induced cataract, but these data evidence for the first time that IR exposure of mice results in oxysterol formation in their eye lenses.

N-Acetylcysteine Regenerates In Vivo Mercaptoalbumin.

Human serum albumin (HSA) represents the most abundant plasma protein, with relevant antioxidant activity due to the presence of the sulfhydryl group on cysteine at position 34 (Cys34), the latter being one of the major target sites for redox-dependent modifications leading to the formation of mixed disulfide linkages with low molecular weight thiols. Thiolated forms of HSA (Thio-HSA) may be useful as markers of an unbalanced redox state and as a potential therapeutic target. Indeed, we have previously reported that albumin Cys34 can be regenerated in vitro by N-Acetylcysteine (NAC) through a thiol-disulfide breaking mechanism, with a full recovery of the HSA antioxidant and antiplatelet activities. With this case study, we aimed to assess the ability of NAC to regenerate native mercaptoalbumin (HSA-SH) and the plasma antioxidant capacity in subjects with redox unbalance, after oral and intravenous administration. A placebo-controlled crossover study, single-blinded, was performed on six hypertensive subjects, randomized into two groups, on a one-to-one basis with NAC (600 mg/die) or a placebo, orally and intravenously administered. Albumin isoforms, HSA-SH, Thio-HSA, and glutathione levels were evaluated by means of mass spectrometry. The plasma antioxidant activity was assessed by a fluorimetric assay. NAC, orally administered, significantly decreased the Thio-HSA levels in comparison with the pre-treatment conditions (T0), reaching the maximal effect after 60 min (-24.7 ± 8%). The Thio-HSA reduction was accompanied by a concomitant increase in the native HSA-SH levels (+6.4 ± 2%). After intravenous administration of NAC, a significant decrease of the Thio-HSA with respect to the pre-treatment conditions (T0) was observed, with a maximal effect after 30 min (-68.9 ± 10.6%) and remaining significant even after 6 h. Conversely, no effect on the albumin isoforms was detected with either the orally or the intravenously administered placebo treatments. Furthermore, the total antioxidant activity of the plasma significantly increased after NAC infusion with respect to the placebo (p = 0.0089). Interestingly, we did not observe any difference in terms of total glutathione corrected for hemoglobin, ruling out any effect of NAC on the intracellular glutathione and supporting its role as a disulfide-breaking agent. This case study confirms the in vitro experiments and demonstrates for the first time that NAC is able to regenerate mercaptoalbumin in vivo, allowing us to hypothesize that the recovery of Cys34 content can modulate in vivo oxidative stress and, hopefully, have an effect in oxidative-based diseases.

The Burden of Impaired Serum Albumin Antioxidant Properties and Glyco-Oxidation in Coronary Heart Disease Patients with and without Type 2 Diabetes Mellitus.

Human serum albumin (HSA) has an important antioxidant activity due to the presence of the reduced cysteine at position 34, which represents the most abundant free thiol in the plasma. In oxidative-based diseases, HSA undergoes S-thiolation (THIO-HSA) with changes in the antioxidant function of albumin that could contribute to the progression of the disease. The aim of this study was to verify, for the first time, the different burdens of THIO-HSA, glycated HSA (GLY-HSA), and advanced glycation end products (AGE) accumulation both in type 2 diabetes mellitus (T2DM) patients and in non-diabetic patients, with or without coronary heart disease (CHD). In this study, we assessed the presence of modified forms of HSA, THIO-HSA, and GLY-HSA by means of mass spectrometry in 33 patients with both T2DM and CHD, in 31 patients with T2DM and without CHD, in 30 patients without diabetes with a history of CHD, and 27 subjects without diabetes and CHD. All the patients' anthropometric and clinical data were recorded including age, sex, duration of diabetes, body mass index (BMI), blood pressure, and history of CHD defined with anamnestic data. Metabolic parameters, such as fasting plasma glucose (FPG), glycated hemoglobin (HbA1c), lipids, pentosidine, AGE, receptor for advanced glycation end-products (RAGE) and its soluble form (sRAGE), were measured. AGE and pentosidine are significantly higher in T2DM patients with and without CHD with respect to non-diabetic patients with CHD and control subjects. RAGE levels are significantly higher in T2DM patients with respect to non-diabetic patients, and among T2DM patients, the group with CHD showed significantly higher RAGE levels than those without CHD (217 ± 171 pg/mL and 140 ± 61 pg/mL, respectively). Albumin isoforms discriminate between non-diabetic patients with CHD and T2DM patients with and without CHD and control subjects, with GLY-HSA levels higher in T2DM with and without CHD, and THIO-HSA higher in CHD patients without T2DM. Finally, we demonstrated that the oxidized forms of HSA can increase the expression of the inflammatory cytokine Tumor Necrosis Factor-alpha (TNFα) in monocytic cells. In patients with CHD, GLY-HSA and THIO-HSA have a different prevalent distribution, the first one prevailing in patients with T2DM and the second one in patients without T2DM. These findings suggest that albumin quality and homeostasis balance between glyco-oxidation and thiolation might have an impact on the antioxidant defense system in cardiovascular diseases.

Polyphenols from Thinned Young Apples: HPLC-HRMS Profile and Evaluation of Their Anti-Oxidant and Anti-Inflammatory Activities by Proteomic Studies.

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.

Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS) Profiling of Commercial Enocianina and Evaluation of Their Antioxidant and Anti-Inflammatory Activity.

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.

Gamma-oryzanol reduces renal inflammation and oxidative stress by modulating AGEs/RAGE axis in animals submitted to high sugar-fat diet / Gama-orizanol reduz a inflamação e o estresse oxidativo nos rins pela modulação do eixo RAGE/AGEs em animais submetidos a uma dieta rica em gordura e açúcar

Abstract Introduction: The receptor for AGEs (RAGE) is a multiligand member of the immunoglobulin superfamily of cell surface receptors expressed in many organs, among them, the kidneys. When activated, RAGE leads to a sequence of signaling that results in inflammation and oxidative stress, both involved in kidney disease pathogenesis. Gamma-oryzanol (γOz) comprises a mixture of ferulic acid (FA) esters and phytosterols (sterols and triterpene alcohols) mainly found in rice, with antioxidant and anti-inflammatory activities. Aim: To evaluate the effect of γOz to reduce renal inflammation and oxidative stress by modulating AGEs/RAGE axis in animals submitted to a high sugar-fat diet. Methods: Male Wistar rats (±187g) were randomly divided into two experimental groups: control (n = 7 animals) and high sugar-fat diet (HSF, n = 14 animals) for 20 weeks. After this period, when the presence of renal disease risk factors was detected in the HSF group (insulin resistance, dyslipidemia, increased systolic blood pressure and obesity), the HSF animals were divided to begin the treatment with γOz or continue receiving only HSF for 10 more weeks. Results: No effect of γOz on obesity and metabolic parameters was observed. However, kidney inflammation and oxidative stress decreased as soon as RAGE levels were reduced in HSF + γOz. Conclusion: It is possible to conclude that the gamma- oryzanol was effective in reducing inflammation and oxidative stress in the kidney by modulating the AGEs/RAGE axis.

Resumo Introdução: O receptor para AGEs (RAGE) é um membro multiligante da superfamília das imunoglobulinas dos receptores de superfície celular expresso em muitos órgãos, entre eles, os rins. Quando ativado, o RAGE leva a uma sequência de sinalização que resulta em inflamação e estresse oxidativo, ambos envolvidos na patogênese de doenças renais. O gama-orizanol (γOz) compreende uma mistura de ésteres de ácido ferúlico (AF) e fitoesteróis (esteróis e álcoois triterpenos) encontrados principalmente no arroz, com atividades antioxidantes e anti-inflamatórias. Objetivo: Avaliar o efeito do γOz para reduzir a inflamação renal e o estresse oxidativo pela modulação do eixo RAGE/AGEs em animais submetidos a uma dieta rica em gordura e açúcar. Métodos: Ratos Wistar machos (±187g) foram divididos aleatoriamente em dois grupos experimentais: controle (n = 7 animais) e dieta rica em gordura e açúcar (HSF, do inglês high sugar-fat diet, n = 14 animais) por 20 semanas. Após este período, quando foi detectada a presença de fatores de risco de doença renal no grupo HSF (resistência à insulina, dislipidemia, aumento da pressão arterial sistólica e obesidade), os animais HSF foram divididos para iniciar o tratamento com γOz ou continuar recebendo apenas HSF por mais 10 semanas. Resultados: Não foi observado nenhum efeito do γOz na obesidade e nos parâmetros metabólicos. No entanto, a inflamação e o estresse oxidativo renais diminuíram assim que os níveis de RAGE foram reduzidos em HSF + γOz. Conclusão: É possível concluir que o gama- orizanol foi eficaz em reduzir a inflamação e o estresse oxidativo no rim pela modulação do eixo RAGE/AGEs.

Analytical Profile and Antioxidant and Anti-Inflammatory Activities of the Enriched Polyphenol Fractions Isolated from Bergamot Fruit and Leave.

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.

S-Thiolation Targets Albumin in Heart Failure.

Human serum albumin (HSA) is associated with several physiological functions, such as maintaining oncotic pressure and microvascular integrity, among others. It also represents the major and predominant antioxidant in plasma due to the presence of the Cys34 sulfhydryl group. In this study, we assessed qualitative and quantitative changes in HSA in patients with heart failure (HF) and their relationship with the severity of the disease. We detected by means of mass spectrometry a global decrease of the HSA content in the plasma of HF patients in respect to control subjects, a significant increase of thio-HSA with a concomitant decrease in the reduced form of albumin. Cysteine and, at a lesser extent, homocysteine represent the most abundant thiol bound to HSA. A strong inverse correlation was also observed between cysteine-HSA and peak VO2/kg, an index of oxygen consumption associated with HF severity. Moreover, in HL-1 cardiomyocytes incubated with H2O2, we showed a significant decrease of cell viability in cells treated with thio-HSA in respect to restored native-HSA. In conclusion, we found for the first time that S-thiolation of albumin is increased in the plasma of HF patients and induced changes in the structure and antioxidant function of HSA, likely contributing to HF progression.

Prothrombin is a binding partner of the human receptor of advanced glycation end products.

The receptor for advanced glycation end products (RAGE) plays a key role in mammal physiology and in the etiology and progression of inflammatory and oxidative stress-based diseases. In adults, RAGE expression is normally high only in the lung where the protein concentrates in the basal membrane of alveolar Type I epithelial cells. In diseases, RAGE levels increase in the affected tissues and sustain chronic inflammation. RAGE exists as a membrane glycoprotein with an ectodomain, a transmembrane helix, and a short carboxyl-terminal tail, or as a soluble ectodomain that acts as a decoy receptor (sRAGE). VC1 domain is responsible for binding to the majority of RAGE ligands including advanced glycation end products (AGEs), S100 proteins, and HMGB1. To ascertain whether other ligands exist, we analyzed by MS the material pulled down by VC1 from human plasma. Twenty of 295 identified proteins were selected and associated to coagulation and complement processes and to extracellular matrix. Four of them contained a γ-carboxyl glutamic acid (Gla) domain, a calcium-binding module, and prothrombin (PT) was the most abundant. Using MicroScale thermophoresis, we quantified the interaction of PT with VC1 and sRAGE in the absence or presence of calcium that acted as a competitor. PT devoid of the Gla domain (PT des-Gla) did not bind to sRAGE, providing further evidence that the Gla domain is critical for the interaction. Finally, the presence of VC1 delayed plasma clotting in a dose-dependent manner. We propose that RAGE is involved in modulating blood coagulation presumably in conditions of lung injury.

N-Acetyl-Cysteine Regenerates Albumin Cys34 by a Thiol-Disulfide Breaking Mechanism: An Explanation of Its Extracellular Antioxidant Activity.

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.

Profiling Vaccinium macrocarpon components and metabolites in human urine and the urine ex-vivo effect on Candida albicans adhesion and biofilm-formation.

The aim of this work was to profile, by using an HPLC-MS/MS method, cranberry compounds and metabolites found in human urine after ingestion of a highly standardized cranberry extract (Anthocran®). Two different strategies were adopted for the data analysis: a targeted and an untargeted approach. These strategies allowed the identification of 42 analytes including cranberry components, known metabolites and metabolites hitherto unreported in the literature, including six valerolactones/valeric acid derivatives whose presence in urine after cranberry consumption has never been described before. Absolute concentrations of 26 over 42 metabolites were obtained by using pure available standards. Urine collected at different time points after the last dosage of Anthocran® were tested on the reference strain C. albicans SC5314, a biofilm-forming strain. Fractions collected after 12 h were found to significantly reduce the adhesion and biofilm formation compared to the control (p < 0.05). A similar effect was then obtained by using Anthocran™ Phytosome™, the lecithin formulation containing 1/3 of standardized cranberry extract and formulated to enhance the absorption of the cranberry components. The urinary profile of cranberry components and metabolites in the urine fractions collected at 1 h, 6 h and 12 h after the last capsule intake were then reproduced by using the pure standards at the concentration ranges found in the urine fraction, and tested on C. albicans. Only the mixture mimicking the urinary fraction collected at 12 h and containing as main components, quercetin and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone was found effective thus confirming the ex-vivo results.

Protective Effect of Tomato-Oleoresin Supplementation on Oxidative Injury Recoveries Cardiac Function by Improving ß-Adrenergic Response in a Diet-Obesity Induced Model.

The system redox imbalance is one of the pathways related to obesity-related cardiac dysfunction. Lycopene is considered one of the best antioxidants. The aim of this study was to test if the tomato-oleoresin would be able to recovery cardiac function by improving ß-adrenergic response due its antioxidant effect. A total of 40 animals were randomly divided into two experimental groups to receive either the control diet (Control, n = 20) or a high sugar-fat diet (HSF, n = 20) for 20 weeks. Once cardiac dysfunction was detected by echocardiogram in the HSF group, animals were re- divided to begin the treatment with Tomato-oleoresin or vehicle, performing four groups: Control (n = 6); (Control + Ly, n = 6); HSF (n = 6) and (HSF + Ly, n = 6). Tomato oleoresin (10 mg lycopene/kg body weight (BW) per day) was given orally every morning for a 10-week period. The analysis included nutritional and plasma biochemical parameters, systolic blood pressure, oxidative parameters in plasma, heart, and cardiac analyses in vivo and in vitro. A comparison among the groups was performed by two-way analysis of variance (ANOVA). Results: The HSF diet was able to induce obesity, insulin-resistance, cardiac dysfunction, and oxidative damage. However, the tomato-oleoresin supplementation improved insulin-resistance, cardiac remodeling, and dysfunction by improving the ß-adrenergic response. It is possible to conclude that tomato-oleoresin is able to reduce the oxidative damage by improving the system's ß-adrenergic response, thus recovering cardiac function.

Development of a direct ESI-MS method for measuring the tannin precipitation effect of proline-rich peptides and in silico studies on the proline role in tannin-protein interactions.

Tannins are a heterogeneous class of polyphenols that are present in several plants and foods. Their ability to interact and precipitate proline-rich proteins leads to different effects such as astringency or antidiarrheal activity. Thus, evaluation of the tannin content in plant extracts plays a key role in understanding their potential use as pharmaceuticals and nutraceuticals. Several methods have been proposed to study tannin-protein interactions but few of them are focused on quantification. The purpose of the present work is to set up a suitable and time efficient method able to quantify the extent of tannin protein precipitation. Bradykinin, chosen as a model, was incubated with increasing concentrations of 1,2,3,4,6-penta-O-galloyl-ß-d-glucose and tannic acid selected as reference of tannic compounds. Bradykinin not precipitated was determined by a mass spectrometer TSQ Quantum Ultra Triple Quadrupole (direct infusion analysis). The results were expressed as PC50, which is the concentration able to precipitate 50% of the protein. The type of tannin-protein interaction was evaluated also after precipitate solubilisation. The involvement of proline residues in tannin-protein interactions was confirmed by repeating the experiment using a synthesized peptide (RR-9) characterized by the same bradykinin sequence, but having proline residues replaced by glycine residues: no interaction occurred between the peptide and the tannins. Moreover, modelling studies on PGG-BK and PGG-RR-9 were performed to deeply investigate the involvement of prolines: a balance of hydrophobic and H-bond contacts stabilizes the PGG-BK cluster and the proline residues exert a crucial role thus allowing the PGG molecules to elicit a sticking effect.

N-Acetylcysteine as an antioxidant and disulphide breaking agent: the reasons why.

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 NO2 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.

LC-MS/MS Identification of Species-Specific Muscle Peptides in Processed Animal Proteins.

An innovative analytical strategy has been applied to identify signature peptides able to distinguish among processed animal proteins (PAPs) derived from bovine, pig, fish, and milk products. Proteomics was first used to elucidate the proteome of each source. Starting from the identified proteins and using a funnel based approach, a set of abundant and well characterized peptides with suitable physical-chemical properties (signature peptides) and specific for each source was selected. An on-target LC-ESI-MS/MS method (MRM mode) was set up using standard peptides and was then applied to selectively identify the PAP source and also to distinguish proteins from bovine carcass and milk proteins. We believe that the method described meets the request of the European Commission which has developed a strategy for gradually lifting the "total ban" toward "species to species ban", therefore requiring official methods for species-specific discrimination in feed.