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Angela CONTE

Professore Ordinario
Dipartimento di Scienze della Vita sede ex-Agraria

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Pubblicazioni

2023 - Effect of Ripening and In Vitro Digestion on Bioactive Peptides Profile in Ras Cheese and Their Biological Activities [Articolo su rivista]
Helal, A.; Cattivelli, A.; Conte, A.; Tagliazucchi, D.
abstract

The effect of ripening and in vitro digestion on the biological activities, peptide profiles and release of bioactive peptides in Ras cheese has been investigated. Ras cheese ripening largely influenced the extent of protein hydrolysis. The advancement in ripening resulted in an increase in total peptides (from 0.97 to 2.46 mmol leucine/g in samples at 30 and 180 days of ripening, respectively) and bioactive peptides concentration, especially angiotensin-converting enzyme (ACE)-inhibitory, dipeptidyl-peptidase-IV-(DPP-IV)-inhibitory and antioxidant peptides. In vitro gastro-intestinal digestion further promoted protein hydrolysis and the release of bioactive peptides. Digested Ras cheese at 90 and 180 days of ripening displayed the highest bioactive peptides intensity. The variations in bioactive peptides amount during ripening and in vitro digestion were correlated with the changes in ACE-inhibitory, DPP-IV-inhibitory and antioxidant activities. The highest amounts of VPP and IPP were detected in digested Ras cheese at 90 days of ripening (17.44 and 36.50 mg/kg of cheese, respectively), whereas the highest concentrations of APFPE were found in undigested and digested 180-day ripened Ras cheese (82.09 and 52.01 mg/kg of cheese, respectively). The present investigation underlined potential differences in the biological effect after the ingestion of Ras cheese at different ripening times.

2023 - Quercetins, Chlorogenic Acids and Their Colon Metabolites Inhibit Colon Cancer Cell Proliferation at Physiologically Relevant Concentrations [Articolo su rivista]
Cattivelli, A.; Conte, A.; Tagliazucchi, D.
abstract

Several studies have suggested that a phenolic-rich diet may be protective against colon cancer. Most phenolic compounds are not absorbed in the small intestine and reach the colon where they are metabolized by gut microbiota in simple phenolic acids. In this study, the anti-proliferative activity of quercetins, chlorogenic acids, their colon metabolites and mixtures of parent compounds/metabolites was assessed by using two colon cancer cell lines (Caco-2 and SW480) at physiologically relevant concentrations. Chlorogenic acids, quercetin and the metabolite 3-(3 & PRIME;,4 & PRIME;-dihydroxyphenyl)acetic acid exerted remarkable anti-proliferative activity against Caco-2, whereas quercetin derivatives and metabolites were the most active against SW480. Tested compounds arrested the cell cycle at the S phase in both the cell lines. The mixtures of parent compounds/metabolites, which mimic the colon human metabotypes that slowly or rapidly metabolize the parent compounds, similarly inhibited cell growth. SW480 cells metabolized parent phenolic compounds more rapidly and extensively than Caco-2, whereas colon metabolites were more stable. These results suggest that dietary phenolic compounds exert an anti-proliferative effect against human colon cancer cells that can be further sustained by the colon metabolites. Therefore, gut microbiota metabolism of phenolic compounds may be of paramount importance in explaining the protective effect of phenolic-rich foods against colon cancer.

2023 - Red-skinned onion phenolic compounds stability and bioaccessibility: A comparative study between deep-frying and air-frying [Articolo su rivista]
Cattivelli, A.; Di Lorenzo, A.; Conte, A.; Martini, S.; Tagliazucchi, D.
abstract

In this study, the effect of deep-frying and air-frying on the stability and gastro-intestinal release of red-skinned onion phenolic compounds have been assessed by high-resolution mass spectrometry. Forty-four phenolic compounds were identified and quantified in raw onion. Both the frying treatments caused an increase in total phenolic compounds, which was more evident after air-frying (47.4% vs 18.6% increase). This increase was mainly a consequence of the water loss observed during frying. Quercetin was the most important phenolic compound detected both in raw and air-fried samples (99.5 ± 1.7 and 133 ± 2 μmol/100 g, respectively) while deep-fried onion was richer in quercetin-4’-O-glucoside and quercetin-3-O-glucoside-4'-O-glucoside (48.7 ± 0.9 and 41.8 ± 0.3 μmol/100 g, respectively). After digestion, 38.6%, 60.5% and 89.6% of total phenolic compounds were bioaccessible in raw, deep-fried and air-fried onion. Air-fried onion exhibited the highest concentration of bioaccessible phenolic compounds (206 ± 1 μmol/100 g). Oxidative degradation and hydrolysis reactions of quercetin-hexosides occurred during in vitro digestion. Hence, air-frying can be considered a healthy cooking method able to preserve and release after digestion most of the health-promoting compounds found in raw onion and with a lower amount of fats and polar toxic compounds compared to deep-frying.

2022 - Application of a Combined Peptidomics and In Silico Approach for the Identification of Novel Dipeptidyl Peptidase-IV-Inhibitory Peptides in In Vitro Digested Pinto Bean Protein Extract [Articolo su rivista]
Martini, S.; Cattivelli, A.; Conte, A.; Tagliazucchi, D.
abstract

The conventional approach in bioactive peptides discovery, which includes extensive bioassay-guided fractionation and purification processes, is tedious, time-consuming and not always successful. The recently developed bioinformatics-driven in silico approach is rapid and cost-effective; however, it lacks an actual physiological significance. In this study a new integrated peptidomics and in silico method, which combines the advantages of the conventional and in silico approaches by using the pool of peptides identified in a food hydrolysate as the starting point for subsequent application of selected bioinformatics tools, has been developed. Pinto bean protein extract was in vitro digested and peptides were identified by peptidomics. The pool of obtained peptides was screened by in silico analysis and structure–activity relationship modelling. Three peptides (SIPR, SAPI and FVPH) were selected as potential inhibitors of the dipeptidyl-peptidase-IV (DPP-IV) enzyme by this integrated approach. In vitro bioactivity assay showed that all three peptides were able to inhibit DPP-IV with the tetra-peptide SAPI showing the highest activity (IC50 = 57.7 µmol/L). Indeed, a new possible characteristic of peptides (i.e., the presence of an S residue at the N-terminus) able to inhibit DPP-IV was proposed.

2022 - Cooking and In Vitro Digestion Modulate the Anti-Diabetic Properties of Red-Skinned Onion and Dark Purple Eggplant Phenolic Compounds [Articolo su rivista]
Cattivelli, A.; Conte, A.; Martini, S.; Tagliazucchi, D.
abstract

The intake of phenolic-rich foods is an emerging preventive approach for the management of type 2 diabetes, thanks to the ability of these compounds to inhibit some key metabolic enzymes. In this study, the influence of cooking and in vitro digestion on the α-glucosidase, α-amylase, and dipeptidyl-peptidase IV (DPP-IV) inhibitory activity of red-skinned onion (RSO) and dark purple eggplant (DPE) phenolic fractions was assessed. The applied cooking procedures had different influences on the total and individual phenolic compounds gastrointestinal bioaccessibility. DPE in vitro digested phenolic fractions displayed no inhibitory activity versus α-amylase and DPP-IV, whereas the fried DPE sample exhibited moderate inhibitory activity against α-glucosidase. This sample mainly contained hydroxycinnamic acid amides that can be responsible for the observed effect. Contrariwise, raw and cooked in vitro digested RSO phenolic fractions inhibited all three enzymes but with different effectiveness. Fried and raw RSO samples were the most active against them. Statistical analysis pointed out that quercetin mono-hexosides (mainly quercetin-4′-O-hexoside) were responsible for the inhibition of α-glucosidase, whereas quercetin dihexosides (mainly quercetin-3-O-hexoside-4′-O-hexoside) were responsible for the DPP-IV-inhibitory activity of RSO samples. An accurate design of the cooking methods could be essential to maximize the release of individual phenolic compounds and the related bioactivities.

2022 - In Vitro Bioaccessibility and Antioxidant Activity of Phenolic Compounds in Coffee-Fortified Yogurt [Articolo su rivista]
Helal, A.; Cattivelli, A.; Conte, A.; Tagliazucchi, D.
abstract

Yogurt is considered one of the most popular and healthy dairy products, and has been exploited as a delivery matrix for phenolic compounds. In this study, coffee powder was added to yogurt as a functional ingredient to produce coffee-fortified yogurt. Total phenolic compounds, antioxidant activity and individual hydroxycinnamic acids have been identified and quantified through mass spectrometry. The results from coffee-fortified yogurt were compared with fermented coffee and plain yogurt. Coffee-fortified yogurt had higher total phenolic content and antioxidant activity compared to plain yogurt. However, the total phenolic compounds found in coffee-fortified yogurt represented only 38.9% of the original content in coffee. Caffeoylquinic acids were the most abundant phenolic compounds in coffee. Fermented coffee and coffee-fortified yogurt displayed lower amounts of individual phenolic compounds with respect to coffee (69.8% and 52.4% of recovery, respectively). A protective effect of the yogurt matrix on total and individual coffee phenolic compounds has been observed after in vitro digestion, resulting in a higher bioaccessibility in comparison with digested fermented coffee. Moreover, coffee-fortified yogurt showed the highest antioxidant values after digestion. These findings clearly demonstrate that coffee-fortified yogurt can be considered a significant source of bioaccessible hydroxycinnamic acids, besides its health benefits as a fermented dairy product.

2021 - Black, green, and pink pepper affect differently lipid oxidation during cooking and in vitro digestion of meat [Articolo su rivista]
Martini, S.; Cattivelli, A.; Conte, A.; Tagliazucchi, D.
abstract

Lipid oxidation products generated during meat digestion may contribute to the apparent epidemiological link between red meat intake and the risk of cardiovascular diseases and colorectal cancer. The aim of this work was to assess the lipid oxidation inhibitory activity of black, green, and pink pepper during cooking and in vitro digestion of meat. Peppers were characterized for their phenolic profiles by LC-ESI-MS and the antioxidant properties. Pink pepper showed the highest phenolic content and antioxidant activities. Then, the peppers were added to meat either before or after cooking, and the meat was subjected to in vitro digestion. Pink pepper added before cooking was the most effective, with an inhibition of 80% and 72% in lipid hydroperoxides and TBA-RS formation after digestion, respectively. These findings suggest that peppers, particularly pink pepper, can be used to minimize lipid oxidation in the gastro-intestinal tract and for the design of healthy dietary patterns.

2021 - Domestic cooking methods affect the stability and bioaccessibility of dark purple eggplant (Solanum melongena) phenolic compounds [Articolo su rivista]
Martini, S.; Conte, A.; Cattivelli, A.; Tagliazucchi, D.
abstract

Eggplant is an important component of the Mediterranean Diet, which becomes edible after cooking. This study determined the fate of dark purple eggplant phenolic compounds after baking, boiling, frying, grilling and digestion. Thirty-seven phenolic compounds were identified and quantified in raw eggplant. Frying determined a 74% increase in total hydroxycinnamic acids whereas a decrease was observed after boiling (27%), grilling (51%), and baking (60%). After digestion, 45%, 33% and 22% of total phenolic compounds resulted bioaccessible in baked, grilled and fried dark purple eggplant. Fried eggplant displayed the highest amount of phenolic compounds (751.46 mg/100 g) after digestion. The cooking methods differently affected the release of individual phenolic compounds. Baking and grilling resulted in higher amount of bioaccessible caffeoylquinic acids whereas frying in di-caffeoylquinic acids and hydroxycinnamic acid-amides. A careful design of the cooking method may be pivotal to modulate the release of specific phenolic compounds.

2021 - Influence of cooking methods on onion phenolic compounds bioaccessibility [Articolo su rivista]
Cattivelli, A.; Conte, A.; Martini, S.; Tagliazucchi, D.
abstract

The impact of domestic cooking (baking, boiling, frying and grilling) and in vitro digestion on the stability and release of phenolic compounds from yellow-skinned (YSO) and red-skinned onions (RSO) have been evaluated. The mass spectrometry identification pointed out flavonols as the most representative phenolic class, led by quercetin-derivatives. RSO contained almost the double amount of phenolic compounds respect to YSO (50.12 and 27.42 mg/100 g, respectively). Baking, grilling and primarily frying resulted in an increased amount of total phenolic compounds, especially quercetin-derivatives, in both the onion varieties. Some treatments promoted the degradation of quercetin-3-O-hexoside-4′-O-hexoside, the main compound present in both the onion varieties, leading to the occurrence of quercetin-4′-O-hexoside and protocatechuic acid-4-O-hexoside. After in vitro digestion, the bioaccessibility index for total phenolic compounds ranged between 42.6% and 65.5% in grilled and baked YSO, respectively, and between 39.8% and 80.2% in boiled and baked RSO, respectively. Baking contributed to the highest amount of bioaccessible phenolic compounds for both the onion varieties after in vitro digestion. An in-depth design of the cooking process may be of paramount importance in modulating the gastro-intestinal release of onion phenolic compounds.

2020 - Effect of ripening and in vitro digestion on the evolution and fate of bioactive peptides in Parmigiano-Reggiano cheese [Articolo su rivista]
Martini, Serena; Conte, Angela; Tagliazucchi, Davide
abstract

The influence of ripening and in vitro digestion on the peptidomic profile of Parmigiano-Reggiano (PR) cheeses was investigated. Ripening and in vitro digestion thoroughly modified the peptidomic profile of the three cheeses. Twenty-six bioactive peptides were identified in undigested PR. Some peptides were degraded and others released during ripening. After digestion, 52 bioactive peptides were identified. Semi-quantitative data suggested that bioactive peptides released after digestion can be clustered in 5 groups according to the ripening time. VPP and IPP peptide levels in undigested samples were in the range of 4.52–11.34 and 0.66–4.24 mg kg−1, with the highest amounts found in 18-month ripened PR. YPFPGPI peptide was absent in undigested PRs but was released after digestion, especially in the 12-month-old sample (20.18 mg kg−1). The present study suggests possible differences in bioactive peptide levels after digestion as a function of the duration of ripening of PR cheese.

2020 - Mediterranean diet vegetable foods protect meat lipids from oxidation during in vitro gastro-intestinal digestion [Articolo su rivista]
Martini, S.; Conte, A.; Bottazzi, S.; Tagliazucchi, D.
abstract

Meat lipids oxidation during digestion gives rise to a post-prandial oxidative stress condition, which negatively affects human health. Mediterranean Diet vegetables contain high amount of phenolic compounds, which potentially may reduce the oxidative phenomena during digestion. In vitro co-digestion of turkey meat with a typical Mediterranean Diet salad containing tomato, onion, black olives, extra-virgin olive oil (EVOO) and basil, dose-dependently reduced lipid peroxidation. Onion and EVOO were more effective in limiting oxidation than the other foods, resulting in negligible concentrations of lipid hydroperoxides after digestion. Specific phenolic classes dominated the phenolic profile of the different foods, such as flavonols and anthocyanins in onion, phenolic acids in tomato and basil, and tyrosol-derivatives in black olives and EVOO. The correlation between lipid peroxidation inhibition, phenolic constituents and antioxidant properties was evaluated by principal component analysis (PCA). Flavonols and anthocyanin were the major contributors to the bioactive response of vegetable foods.

2019 - Antiproliferative Activity and Cell Metabolism of Hydroxycinnamic Acids in Human Colon Adenocarcinoma Cell Lines [Articolo su rivista]
Martini, Serena; Conte, Angela; Tagliazucchi, Davide
abstract

In this study, we investigated the antiproliferative activity and the stability and metabolic fate of the main dietary hydroxycinnamates, using two colonic adenocarcinoma cell models (Caco-2 and SW480). Dihydrocaffeic and dihydroferulic acids were the most effective against cell proliferation in both cell lines with IC50 values of 71.7 ± 1.1 and 83.1 ± 1.1 μmol/L, respectively (P < 0.05) in Caco-2. At 200 μmol/L, caffeic and ferulic acids inhibited SW480 proliferation by 40.8 ± 1.6 and 59.9 ± 1.3%, respectively. Hydroxycinnamic acids with a catechol-type structure were degraded in Caco-2 cell medium, resulting in the production of H2O2. Intracellular Caco-2 UDP-glucuronosyltransferases and catechol-O-methyltransferases were able to form glucuronide and methyl conjugates. However, only the sulfate conjugates were detected after incubation with SW480. In addition, simple hydroxycinnamates were released from quinic and aspartic conjugates. The remarkable effect of dihydrocaffeic and dihydroferulic acids against cell proliferation is of paramount importance, since these compounds are the main metabolites detectable at the colonic level.

2019 - Bioactivity and cell metabolism of in vitro digested sweet cherry (Prunus avium) phenolic compounds [Articolo su rivista]
Martini, Serena; Conte, Angela; Tagliazucchi, Davide
abstract

In this study, the bioaccessibility of phenolic compounds after in vitro gastrointestinal digestion of two cherry cultivars was assessed. The phenolic profile was modified during in vitro digestion, with a considerable decrease of total and individual phenolic compounds. Hydroxycinnamic acids and especially coumaroylquinic acids showed the highest bioaccessibility. Isomerisation of caffeoylquinic and coumaroylquinic acids was observed after in vitro digestion. Modification of the phenolic profile after digestion resulted in an increased or decreased scavenging activity depending on the assay. In vitro digested phenolic-rich fractions also showed antiproliferative activity against SW480 but no effect against Caco-2 cell lines. Both Caco-2 and SW480 cell lines were able to metabolise cherry phenolic compounds with remarkable differences. An accumulation of glycosylated flavonols was observed in SW480 medium. In conclusion, phenolic compounds from cherries and especially hydroxycinnamic acids were efficiently released and remained bioaccessible after in vitro digestion, resulting in antioxidant and antiproliferative activities.

2019 - Comparative peptidomic profile and bioactivities of cooked beef, pork, chicken and turkey meat after in vitro gastro-intestinal digestion [Articolo su rivista]
Martini, Serena; Conte, Angela; Tagliazucchi, Davide
abstract

This study was designed to investigate the potential contribution of bioactive peptides to the biological activities related to the consumption of pork, beef, chicken and turkey meat following in vitro gastro-intestinal digestion. After extraction of the peptidic fractions from digested samples, the bioactivities were evaluated by in vitro antioxidant activity as well as angiotensin-converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) inhibition assays. Pork and turkey meat appeared to be the best sources of antioxidant peptides. Pork was found to be the best source of DPP-IV-inhibitory peptides whereas chicken meat supplied peptides with the highest ACE-inhibitory activity. The comprehensive analysis of the peptidomic profile of digested samples was performed by nano-LC-ESI-QTOF MS/MS analysis. A total of 217, 214, 257 and 248 peptides were identified in digested pork, beef, chicken and turkey meat, respectively. Chicken and turkey meat showed the highest similarity in peptide sequences with 202 common peptides. Sixty-two peptides matched with sequences with previously demonstrated biological activity. In particular, 35 peptides showed ACE-inhibitory activity and 23 DPP-IV inhibitory activity. Twenty-two bioactive peptides were commonly released from the different types of meat. The relative amount of identified bioactive peptides were positively correlated to the biological activities of the different digested meats. Biological significance: The present study describes for the first time a comprehensive peptide profile of four types of meat after in vitro gastro-intestinal digestion. The peptide inventory was used to identify 62 bioactive peptides with ACE- and DPPIV-inhibitory and antioxidant activities. The bioactivity analysis revealed interesting and significant differences between the studied meats. The originality of this work lay in the description of intrinsic differences in physiological functions after the ingestion of meat proteins from different species. In a context in which the current research scene relates meat consumption to the onset of chronic pathologies, this peptide profiling and bioactivity analysis shed light on the possible health benefits of peptides released from meat proteins. In fact, this paper represents a sort of detailed peptide list that may help to predict which peptides could be generated after meat intake and detectable at gastro-intestinal level. It also provides a thorough investigation of novel biological activities associated to meat protein hydrolysates, giving a new positive aspect to meat consumption.

2019 - Protocatechuic and 3,4-Dihydroxyphenylacetic Acids Inhibit Protein Glycation by Binding Lysine through a Metal-Catalyzed Oxidative Mechanism [Articolo su rivista]
Tagliazucchi, Davide; Martini, Serena; Conte, Angela
abstract

The mechanism of inhibition of advanced glycation end product (AGE) formation by protocatechuic acid and 3,4-dihydroxyphenylacetic acid (DHPA) has been studied using a widespread applied in vitro model system composed of bovine serum albumin (BSA) and supraphysiological glucose concentrations. Protocatechuic acid and DHPA inhibited the formation of Amadori compounds, fluorescent AGEs (IC50 = 62.1 ± 1.4 and 155.4 ± 1.1 μmol/L, respectively), and Nε-(carboxymethyl)lysine (IC50 = 535.3 ± 1.1 and 751.2 ± 1.0 μmol/L, respectively). BSA was pretreated with the two phenolic acids, and the formation of BSA–phenolic acid adducts was estimated by nanoflow liquid chromatography–electrospray ionization–quadrupole time-of-flight mass spectrometry. Results showed that the tested phenolic acids bound key sites of glycation in BSA through a metal-catalyzed oxidative mechanism. The antiglycative activity mechanism involved the formation of BSA–phenolic acid adducts, and it is unlikely that this occurs in vivo. These results raise the problem to design in vitro models closer to physiological conditions to reach biologically sound conclusions.

2019 - Shared polygenic risk and causal inferences in amyotrophic lateral sclerosis [Articolo su rivista]
Bandres-Ciga, S.; Noyce, A. J.; Hemani, G.; Nicolas, A.; Calvo, A.; Mora, G.; Arosio, A.; Barberis, M.; Bartolomei, I.; Battistini, S.; Benigni, M.; Borghero, G.; Brunetti, M.; Cammarosano, S.; Cannas, A.; Canosa, A.; Capasso, M.; Caponnetto, C.; Caredda, C.; Carrera, P.; Casale, F.; Cavallaro, S.; Chio, A.; Colletti, T.; Conforti, F. L.; Conte, A.; Corrado, L.; Costantino, E.; D'Alfonso, S.; Fasano, A.; Femiano, C.; Ferrarese, C.; Fini, N.; Floris, G.; Fuda, G.; Giannini, F.; Grassano, M.; Ilardi, A.; La Bella, V.; Lattante, S.; Logroscino, G.; Logullo, F. O.; Loi, D.; Lunetta, C.; Mancardi, G.; Mandich, P.; Mandrioli, J.; Manera, U.; Marangi, G.; Marinou, K.; Marrali, G.; Marrosu, M. G.; Mazzini, L.; Melis, M.; Messina, S.; Moglia, C.; Monsurro, M. R.; Mosca, L.; Occhineri, P.; Origone, P.; Pani, C.; Penco, S.; Petrucci, A.; Piccirillo, G.; Pirisi, A.; Pisano, F.; Pugliatti, M.; Restagno, G.; Ricci, C.; Rita Murru, M.; Riva, N.; Sabatelli, M.; Salvi, F.; Santarelli, M.; Sideri, R.; Simone, I.; Spataro, R.; Tanel, R.; Tedeschi, G.; Tranquilli, S.; Tremolizzo, L.; Trojsi, F.; Volanti, P.; Zollino, M.; Abramzon, Y.; Arepalli, S.; Baloh, R. H.; Bowser, R.; Brady, C. B.; Brice, A.; Broach, J.; Campbell, R. H.; Camu, W.; Chia, R.; Cooper-Knock, J.; Cusi, D.; Ding, J.; Drepper, C.; Drory, V. E.; Dunckley, T. L.; Eicher, J. D.; Faghri, F.; Feldman, E.; Kay Floeter, M.; Fratta, P.; Geiger, J. T.; Gerhard, G.; Gibbs, J. R.; Gibson, S. B.; Glass, J. D.; Hardy, J.; Harms, M. B.; Heiman-Patterson, T. D.; Hernandez, D. G.; Jansson, L.; Kamel, F.; Kirby, J.; Kowall, N. W.; Laaksovirta, H.; Le Ber, I.; Lumbroso, S.; Macgowan, D. J. L.; Maragakis, N. J.; Mouzat, K.; Murphy, N. A.; Myllykangas, L.; Nalls, M. A.; Orrell, R. W.; Ostrow, L. W.; Pamphlett, R.; Pickering-Brown, S.; Pioro, E.; Pliner, H. A.; Pulst, S. M.; Ravits, J. M.; Renton, A. E.; Rivera, A.; Robbrecht, W.; Rogaeva, E.; Rollinson, S.; Rothstein, J. D.; Salvi, E.; Scholz, S. W.; Sendtner, M.; Shaw, P. J.; Sidle, K. C.; Simmons, Z.; Singleton, A. B.; Stone, D. C.; Sulkava, R.; Tienari, P. J.; Traynor, B. J.; Trojanowski, J. Q.; Troncoso, J. C.; Van Damme, P.; Van Deerlin, V. M.; Van Den Bosch, L.; Zinman, L.; Stone, D. J.
abstract

Objective: To identify shared polygenic risk and causal associations in amyotrophic lateral sclerosis (ALS). Methods: Linkage disequilibrium score regression and Mendelian randomization were applied in a large-scale, data-driven manner to explore genetic correlations and causal relationships between >700 phenotypic traits and ALS. Exposures consisted of publicly available genome-wide association studies (GWASes) summary statistics from MR Base and LD-hub. The outcome data came from the recently published ALS GWAS involving 20,806 cases and 59,804 controls. Multivariate analyses, genetic risk profiling, and Bayesian colocalization analyses were also performed. Results: We have shown, by linkage disequilibrium score regression, that ALS shares polygenic risk genetic factors with a number of traits and conditions, including positive correlations with smoking status and moderate levels of physical activity, and negative correlations with higher cognitive performance, higher educational attainment, and light levels of physical activity. Using Mendelian randomization, we found evidence that hyperlipidemia is a causal risk factor for ALS and localized putative functional signals within loci of interest. Interpretation: Here, we have developed a public resource (https://lng-nia.shinyapps.io/mrshiny) which we hope will become a valuable tool for the ALS community, and that will be expanded and updated as new data become available. Shared polygenic risk exists between ALS and educational attainment, physical activity, smoking, and tenseness/restlessness. We also found evidence that elevated low-desnity lipoprotein cholesterol is a causal risk factor for ALS. Future randomized controlled trials should be considered as a proof of causality. Ann Neurol 2019;85:470–481.

2018 - Bioaccessibility, bioactivity and cell metabolism of dark chocolate phenolic compounds after in vitro gastro-intestinal digestion [Articolo su rivista]
Martini, Serena; Conte, Angela; Tagliazucchi, Davide
abstract

The bioaccessibility of phenolic compounds after in vitro gastro-intestinal digestion of dark chocolate, dark chocolate enriched with Sakura green tea and dark chocolate enriched with turmeric powder was studied. The phenolic profile, assessed by accurate mass spectrometry analysis, was modified during in vitro gastro-intestinal digestion, with a considerable decrease of total and individual phenolic compounds. Phenolic acids showed the highest bioaccessibility with hydroxycinnamic acids displaying higher bioaccessibility (from 41.2% to 45.1%) respect to hydroxybenzoic acids (from 28.1% to 43.5%). Isomerisation of caffeoyl-quinic acids and galloyl-quinic acids as well as dimerization of (epi)gallocatechin were also observed after in vitro gastro-intestinal digestion. Antioxidant activity increased after the gastric step and rose further at the end of the digestion. Furthermore, in vitro digested phenolic-rich fractions showed anti-proliferative activity against two models of human colon adenocarcinoma cell lines. Cell metabolism of digested phenolic compounds resulted in the accumulation of coumaric and ferulic acids in the cell media.

2018 - Biological activities and peptidomic profile of in vitro-digested cow, camel, goat and sheep milk [Articolo su rivista]
Tagliazucchi, Davide; Martini, Serena; Shamsia, Sherif; MOHAMED IBRAHIM HELAL, Ahmed; Conte, Angela
abstract

In vitro digestibility, selected biological activities and digested products of proteins from skimmed cow, camel, goat and sheep milk were compared. The harmonised in vitro INFOGEST digestion model and mass spectrometry were combined to identify peptides. Goat milk had the highest digestibility, sheep milk the highest angiotensin-converting enzyme (ACE)-inhibitory activity after digestion and cow milk the highest dipeptidyl peptidase-IV (DPP-IV)-inhibitory activity. After in vitro digestion of the milk samples, 522 peptides were identified. Goat and sheep milk showed the highest peptide sequence similarity with 151 common peptides. Thirteen, forty-three and twenty peptides with previously demonstrated antioxidant, ACE-inhibitory and DPP-IV-inhibitory activities, respectively, were found. Nineteen bioactive peptides in common were released from the different milk types. Despite limitations related to analysis of one milk sample for each species, possible differences in physiological functions after ingestion of milk from different species are suggested; this requires confirmation by in vivo testing.

2018 - Comprehensive evaluation of phenolic profile in dark chocolate and dark chocolate enriched with Sakura green tea leaves or turmeric powder [Articolo su rivista]
Martini, Serena; Conte, Angela; Tagliazucchi, Davide
abstract

Recently, a huge number of studies have confirmed the important role of chocolate polyphenols in human health, underlining its beneficial effects especially in the treatment of cardiovascular diseases. However, a thorough evaluation of chocolate phenolic profile is still lacking. This study aimed at a comprehensive characterization of dark chocolate phenolic profile, using non-targeted mass spectrometry identification. This approach allowed a tentative identification of 158 individual phenolic compounds: 67 were newly detected in dark chocolate, among these 38 were observed for the first time in chocolate as well as in cocoa beans or products. Ellagitannins, which have never been reported in cocoa or chocolate, represented about the 10% of the phenolic profile of dark chocolate. The enrichment of dark chocolate with Sakura green tea leaves or turmeric powder influenced and modified the phenolic profile, resulting in a phenolic concentration increase. In this way, this functional chocolate might maximize the beneficial effect of chocolate consumption, combining the positive health effects of chocolate, turmeric and green tea and, at the same time, reducing the amount of sugars and calories introduced with chocolate.

2018 - The paradoxical effect of extra-virgin olive oil on oxidative phenomena during in vitro co-digestion with meat [Articolo su rivista]
Martini, Serena; Cavalchi, Martina; Conte, Angela; Tagliazucchi, Davide
abstract

Extra-virgin olive oil is an integral part of the Mediterranean diet and its consumption has been associated with a reduction risk of chronic diseases. Here we tested the potential of extra-virgin olive oil to limit the oxidative phenomena during in vitro gastro-intestinal co-digestion with turkey breast meat. The extra-virgin olive oil was particularly rich in oleuropein aglycone isomers, which represented the 66.8% of total phenolic determined with MS/MS experiments. Meals supplemented with extra-virgin olive oil equivocally affected lipid peroxidation. At low concentration (2.5% respect to meat), a significant inhibition of lipid oxidation was observed, whereas lipid peroxidation was greatly enhanced when the amount of extra-virgin olive oil was increased in the gastro-intestinal system. The inhibitory effect observed at 2.5% extra-virgin olive oil was due to the antioxidant properties of extra-virgin olive oil phenolic compounds. At high concentration, extra-virgin olive oil phenolic compounds (especially hydroxytyrosol-derivative) behaved as pro-oxidants increasing the generation of lipid hydroperoxides from meat. At the same time, the presence in the digestive system of catalysers from meat induced the peroxidation of extra-virgin olive oil fatty acids, which was further intensified by the pro-oxidant activity of extra-virgin olive oil phenolic compounds. Our study underlined the importance of the timing and amount of consumption of extra-virgin olive oil as well as its phenolic composition in limiting the peroxidative phenomena on meat lipids during digestion.

2017 - Angiotensin-converting enzyme inhibitory peptides from goats' milk released by in vitro gastro-intestinal digestion [Articolo su rivista]
Tagliazucchi, Davide; Shamsia, Sherif; Helal, Ahmed; Conte, Angela
abstract

The aim of this study was to identify the angiotensin-converting enzyme (ACE) inhibitory peptides released after in vitro gastro-intestinal digestion of skimmed goats' milk. The experimental approach involved a recently developed harmonised static in vitro digestion model, with mass spectrometry (MS) to identify bioactive peptides. Peptides in the post-pancreatic digest were extracted by ultrafiltration and isolated by reversed-phase high-performance liquid chromatography followed by MS. Among the identified sequences, eighteen were identical to known bioactive peptides with ACE-inhibitory activity. Peptides with dipeptidyl peptidase IV-inhibitory and antioxidant activities were also identified. The antihypertensive tripeptides valine-proline-proline and isoleucine-proline-proline were released from goats’ milk protein during in vitro gastro-intestinal digestion at concentrations of 1829.8 ± 216.4 and 141.4 ± 15.1 ug L-1, respectively. This research underlines the suitability of the harmonised digestive model system to study the release of short bioactive peptides during gastro-intestinal transit.

2017 - Chocolate melanoidins as a carrier for the delivery of phenolic compounds in the gut [Poster]
Martini, S.; Conte, A.; Tagliazucchi, D.
abstract

Emerging evidence from in vitro and in vivo studies suggested that the gastro-intestinal tract may be the key site for the biological action of food melanoidins [1]. In this work, we tested high molecular weight melanoidins (HMWM) extracted from dark chocolate for their ability to release low molecular weight polyphenols following in vitro digestion. Water soluble HMWM were extracted by ultrafiltration (10 kDa) from three different types of dark chocolate (dark 70% cocoa (DC), dark 70% cocoa and 8% turmeric (TDC), dark 62% cocoa and 2% Sakura green tea (GTDC)). HMWM have been chemically characterized for their content in total polyphenols, protein, and polysaccharides [2]. HMWM were in vitro digested and separated from low molecular weight fractions (LMWF) by ultrafiltration. Digested HMWM were chemically characterized and LMWF were characterized for their total phenolic content. Individual phenolic compounds in the LMWF were identified and quantified by HPLC-ESI-QTOF-MS/MS. Results showed that melanoidins are carbohydrate-based structures with the phenolic content ranging from 8.62 ± 1.0 in TDC to 12.79 ± 0.4 g/100 g HMWM in GTDC. The in vitro digestion caused a general decrease in the polysaccharide, protein and phenolic content. Digestion resulted in the release of phenolic compounds from HMWM to LMWF. The highest amount was recovered in the LMWF of GTDC (436.2 ± 87.6 mg/L), whereas the lowest amount in the LMWF of TDC (242.1 ± 17.1 mg/L). The most representative classes of polyphenols released from HMWM were stilbenes (resveratrol and derivatives) and phenolic acids (hydroxybenzoic, benzoic, quinic, cinnamic, coumaric and ferulic acids). As expected, food matrices influenced the chemical composition of HMWM. Specific phenolic compounds were detected in the LMWF from GTDC (quercetin-3-rutinoside) and TDC (curcumin, cathecol and protocatechuic acid).

2017 - Identification and in vitro bioaccessibility of dark chocolate phenolic compounds [Poster]
Martini, S.; Conte, A.; Tagliazucchi, D.
abstract

Polyphenols have become an intense focus of research interest because of their perceived health-beneficial effects such as anti-atherogenic, anti-inflammatory and anti-tumoral effects. Cocoa (Theobroma cacao) is a major economically important international crop and has been associated with several nutritional benefits [1]. The aim of the present study was to identify and quantify phenolic compounds from three different types of dark chocolate using HPLC-ESI-QTOF-MS/MS. Phenolic compounds were also identified and quantified after in vitro gastro-intestinal digestion. Three different types of dark chocolate (dark 70% cocoa (DC), dark 70% cocoa and 8% turmeric (TDC), dark 62% cocoa and 2% Sakura green tea (GTDC)) were in vitro digested with a simulated gastro-intestinal procedure [2]. Polyphenols were extracted from un-digested samples with methanol-water-formic acid solution (70:28:2 v/v) followed by a second extraction step with acetone. Total polyphenols were determined by Folin-Ciocalteau assay whereas antioxidant activity by FRAP methods. Individual phenolic compounds were identified and quantified using mass spectrometry. Total polyphenol concentration in the un-digested samples varied from 2.6 ± 0.1 in DC to 3.4 ± 0.2 g/100g of chocolate in GTDC. The antioxidant activity ranged between 6.3 ± 0.1 to 15.4 ± 0.7 mmol trolox/100g in DC and GTDC, respectively. As expected, monomeric and oligomeric flavan-3-ols were the most representative compounds in all the three samples, followed by resveratrol and derivative. In addition, some phenolic acids (caffeoylquinic, protocatechuic, vanillic acids) and ellagic acid derivative (ellagic acid, ellagic acid hexoside) were also identified. The addition of green tea and turmeric resulted in the presence of gallate flavan-3-ols (such as epigallocatechin-3-gallate, epigallocatechin and epicatechin gallate) and curcuminoids, respectively. In vitro digestion resulted in a decrease in total polyphenols and in the individual phenolic profile modification.

2017 - PEPTIDOMIC PROFILE OF IN VITRO DIGESTED BOVINE, CAMEL, GOAT AND SHEEP MILK [Abstract in Atti di Convegno]
Tagliazucchi, Davide; Martini, Serena; Sherif, Shamsia; Ahmed, Helal; Conte, Angela
abstract

Milk and dairy products have long traditions in human nutrition since they are a rich source of nutrients such lipids, proteins, amino acids, vitamins and minerals. Nowadays, milk may also be considered as a source of health-promoting compounds. Bioactive peptides deriving from milk proteins may play an important role in the prevention and treatment of metabolic syndrome and its complications [1]. This study aimed to characterize the peptidomic profile of in vitro digested skimmed bovine, camel, goat and sheep milk. Milk samples were in vitro digested following the harmonized INFOGEST protocol [2]. The peptide fractions were extracted from fully digested milk by ultrafiltration (cut-off 3 kDa) and the permeate were characterized by nanoflow-LC-ESI-QTOF MS/MS analysis. Digestibility and biological activities of the peptide fractions were also determined at the end of the digestion. The results showed a faster and more efficient gastric and duodenal degradation of goat milk proteins than camel, bovine and sheep milk. More than 100 peptides were identified in the permeate of digested milk, most of them arising from beta-casein. Goat, sheep and bovine milk showed the highest similarity in peptide sequences than camel milk. Peptides with previously demonstrated biological activities were found in the permeate of all the milk samples after in vitro digestion. Biological activities analysis showed that bovine milk peptides were the most effective in scavenging hydroxyl radical and in the inhibition lipid peroxidation. Bovine milk was also the best source of DPPIV-inhibitory peptides (IC50=6.87 mg of peptides/mL) whereas sheep milk was the best source of ACE-inhibitory peptides (IC50=625.4 μg of peptides/mL). The quantitative diversity in the identified bioactive peptides explained the observed differences in the biological activities.

2017 - Phenolic compounds profile and antioxidant properties of six sweet cherry (Prunus avium) cultivars [Articolo su rivista]
Martini, Serena; Conte, Angela; Tagliazucchi, Davide
abstract

Sweet cherry (Prunus avium) fruits are a nutritionally important food rich in dietary phenolic compounds. The aim of this study was to investigate the phenolic pro file and chemometric discrimination of fruits from six cherry cultivars using a quantitative metabolomics approach, which combine non-targeted mass spectrometry and chemometric analysis. The assessment of the phenolic fingerprint of cherries allowed the tentative identi fication of 86 compounds. A total of 40 chlorogenic acids were identi fied in cherry fruit, which pointed out hydroxycinnamic acid derivatives as the main class of phenolics by number of compounds. Among the compounds detected, 40 have been reported for the first time in sweet cherry fruit. Hydroxycinnamic acids are also the quantitatively most represented class of phenolic compounds in the cherry cultivars with the exception of Lapins and Durone della Marca where the most representative class of phenolic compounds were anthocyanins and flavan-3-ols, respectively. This non-targeted approach allowed the tentative identi fication of the cultivar-compound relationships of these six cherry cultivars. Both anthocyanins and colorless phenolic compounds profile appeared to be cultivar-dependent. In detail, anthocyanins and flavonols patterns have the potential to be used for the determination of a varietal assignment of cherries

2016 - Bioaccesssibility and bioactivity of polyphenols extracted from six cherry cultivars [Poster]
Martini, S.; Tagliazucchi, D; Conte, A
abstract

Introduction There is a possible association between consumption of fruit and vegetables and reduced risk of cancer, particularly cancer of the digestive tract. This anti-cancer activity has been attributed in part to polyphenols present in foods [1]. Cherries in particular are a rich source of polyphenols, especially anthocyanins and hydroxycinnamic acids [2]. Method Cherries from six different cultivars (Durone della Marca, Celeste, Durone Nero I, Bigarreau, Lapins and Moretta) were in vitro digested with a procedure that mimicked the physiochemical conditions of the gastro-intestinal tract [3]. After digestion, polyphenol-rich extracts were obtained by preparative chromatography on C18 column. Polyphenols were also extracted from un-digested cherries with methanol-water-formic acid solution (70:28:2 v/v) and chromatographed on C18 column. Total polyphenols and anthocyanins concentration were determined by Folin-Ciocalteau assay and pH differential method, respectively [3]. Polyphenols were identified using liquid chromatography mass spectrometry. The digested and un-digested preparations were assessed for their cytotoxic and anti-proliferative activities using two human colon adenocarcinoma cell lines (Caco-2 and SW480) and IC50 values of anti-proliferative activity were determined. Results / Discussion / Conclusion The total polyphenol concentration in the un-digested samples varied from 70.2mg/100g of cherry in the cultivar Durone della Marca to 280.6mg/100g of cherries in the cultivar Durone Nero I. The total anthocyanin content varied from 0.12mg/100g of cherry in the cultivar Durone della Marca to about 17mg/100g of cherry in the two darker cultivars (Lapins and Moretta). In vitro digestion resulted in a decrease in both total polyphenols and anthocyanins. Total polyphenols decrease ranged from 56 to 80%, whereas total anthocyanin decrease was more than 90% in all the tested cultivars. Mass spectrometry showed that the gastro-intestinal digestion decreased the content of hydroxycinnamic acids and anthocyanins. Other classes of polyphenols are more stable to digestion. Breakdown products of polyphenols are also present. Digested and un-digested polyphenol-rich extracts showed no cytotoxic activity. Un-digested polyphenol-rich extract from the cultivar Bigarreau showed the highest anti-proliferative effect (IC50 of 7.1 ± 1.1 µg polyphenols/mL) on the SW480 cells. The in vitro digestion increased the anti-proliferative activity of cherry extracts. Polyphenol-rich extracts from digested Durone della Marca and Celeste were the most active with IC50 values of 1.7 ± 0.8 and 3.5 ± 1.2 µg polyphenols/mL, respectively. All of the tested extracts showed no anti-proliferative activity on Caco-2 cells. Our results showed that cherry polyphenols were effective anti-proliferative agents on the more undifferentiated colon adenocarcinoma cells SW480 and that their digestion increased the effect. The increase may be due to the formation of polyphenol metabolites. A great difference was observed between the cultivars used. Further research is required to identify the compounds responsible for the observed anti-proliferative effect.

2016 - Bovine milk antioxidant properties: effect of in vitro digestion and identification of antioxidant compounds [Articolo su rivista]
Tagliazucchi, Davide; Helal, Ahmed; Verzelloni, Elena; Conte, Angela
abstract

Milk proteins contained encrypted in their sequence biologically active components that can be released by enzymatic hydrolysis. Among the biological activities recognized in milk components, the antioxidant activity is of great interest. The objective of the present study was to analyse the antioxidant properties of whole, semi-skimmed and skimmed milk during simulated gastrointestinal digestion and to identify the compounds responsible for the antioxidant activity. Simulated digestion increased the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS)+ radical scavenging activity of milk. In digested whole milk, the main contribution to ABTS+ radical scavenging activity was due to high molecular weight fraction (>3 kg.mol−1). For semi-skimmed and skimmed milk, the main contribution was due to low molecular weight fraction (<3 kg.mol−1). Twelve major peaks were collected from low molecular weight fraction of digested skimmed milk by reversed-phase high-performance liquid chromatography and evaluated for their ABTS+ radical scavenging activity. Among the different fractions, three (F2, F3 and F5) showed high ABTS+ and hydroxyl radical scavenging activity and lipid peroxidation inhibitory capacity. The compounds (free amino acids and peptides) present in these fractions were identified with nano-LCQTOF MS/MS analysis. The amino acids tryptophan and tyrosine seemed fundamental in the ABTS+ and hydroxyl radical scavenging capacities whereas the amino acids phenylalanine and histidine played an important role in the lipid peroxidation inhibitory activity of the peptides. The results reported in this study suggested that milk proteins could act as a carrier for the delivery of antioxidant compounds in the gastrointestinal tract possibly protecting the gastrointestinal tract itself from the oxidative damage.

2016 - Colon-available dietary melanoidins exhibit cytotoxic activity on human colon adenocarcinoma cell lines [Poster]
Tagliazucchi, D.; Martini, S.; Conte, A
abstract

Introduction Emerging evidence from in vitro and in vivo studies suggested that the gastro-intestinal tract might be the key site for the biological action of food melanoidins [1]. Recently, Vitaglione et al. [2] reviewed the possible mechanisms by which coffee melanoidins may influence the risk of colorectal cancer development. In this work, we tested the water-soluble high molecular weight melanoidins (HMWM) obtained from different food sources for their cytotoxic activity against colon cancer cell lines. Method HMWM were obtained by ultrafiltration (cut-off 10 kDa) from instant coffee, cocoa brew, dark beer and instant barley coffee beverages [2]. HMWM have been chemically characterized for their content in total polyphenols, protein, and polysaccharides as well as their spectroscopic properties. The value of Kmix 420nm (defined as the absorption at 420nm of a solution of HMWM at a concentration of 1 g/L) provides information on the relative amount of melanoidins (Kmix 420nm) respect to other compounds found in the HMWM of beverages. Cytotoxic activity against two colon cancer cell lines (Caco-2 and SW480) was assayed by MTT assay. HMWM obtained from the above beverages submitted to in vitro gastro-intestinal digestion [3], were subjected to the same chemical analysis and assayed for their cytotoxic activity. Results / Discussion / Conclusion Barley coffee had the highest HMWM content (83.3g/100g of dry matter) followed by coffee (34.4g/100g of dry matter), cocoa (28.1g/100g of dry matter) and dark beer (20.0g/100g of dry matter). The HMWM of coffee had the highest Kmix 420nm value, suggesting that this fraction is relatively rich in melanoidins. On the contrary, despite the highest content in HMWM, barley coffee was the sample with the lowest melanoidins content. Protein content of HMWM ranged from 6.1% (dark beer) to 10.6% (coffee) whereas polysaccharides content ranged from 42% (dark beer) to 61% (cocoa). Cocoa and coffee HMWM had the highest phenolic content (6 and 12%, respectively). The in vitro digestion caused a general decrease in the polysaccharide and protein content of all the analysed HMWM because of the action of amylolytic and proteolytic enzymes. The analysis of the content of polyphenols showed a decrease in these components as a result of the digestion. On the contrary, the in vitro digestion caused an increase in the Kmix 420nm of all the HMWM investigated suggesting that this process resulted in an enrichment in melanoidins. The effect of HMWM on the human colon adenocarcinoma Caco-2 and SW480 cell cultures was investigated. Un-digested HMWM had no significant cytotoxic effect against both the cell lines. However, the in vitro digestion greatly increased the cytotoxic activity of HMWM against both the cell lines. Coffee HMWM were the most active against Caco-2 cells whereas cocoa HMWM were found to be most active against SW480 cells. Considering that the colon accumulates its content over at least 24 h in a maximum volume of 2 l, it is possible to estimate the concentrations of HMWM reaching daily the colon with diet according to the different food matrices [1]. Data showed that, the consumption of coffee, cocoa and barley coffee beverages may result in intestinal concentrations of HMWM, which are cytotoxic against Caco-2 and SW480 cell lines.

2016 - Composition and properties of peptides that survive standardised in vitro gastro-pancreatic digestion of bovine milk [Articolo su rivista]
Tagliazucchi, Davide; Helal, Ahmed; Verzelloni, Elena; Bellesia, Andrea; Conte, Angela
abstract

There are conflicting results on the release and conservation of bioactive protein sequences during in vitro digestion. Only the peptides that are released and survive may exert their action. Peptides &lt;3 kDa were determined by mass spectrometry after standardised in vitro gastro-pancreatic digestion of bovine milk. At a degree of digestion of 30.7%, 384 peptides were obtained from 2 to 25 residues in length. Five other peptides were recovered after reduction of disulphide bonds. Due to the presence of proline residues at the last or second last position at the C-terminus, a high number of peptides with ACE-inhibitory activity were obtained. Peptides with opioid, antimicrobial, immunomodulatory and antithrombotic activities were recovered, with many precursors and degradative products. Peptides with opioid and antimicrobial activities may be the result of evolutionary processes. The peptides obtained were similar to those released in the human jejunum.

2016 - Release of angiotensin converting enzyme-inhibitory peptides during in vitro gastro-intestinal digestion of camel milk [Articolo su rivista]
Tagliazucchi, Davide; Shamsia, Sherif; Conte, Angela
abstract

Angiotensin-converting enzyme (ACE)-inhibitory peptides released from camel milk after simulated gastro-intestinal digestion were identified. The hydrolysis degree increased during digestion. The highest ACE-inhibitory activity was found in the post-pancreatic &lt;3 kDa fraction. Peptides responsible for the biological activity were isolated by reversed-phase high-performance liquid chromatography and identified by mass spectrometry. Among the identified sequences, 17 were identical to known bioactive peptides with ACE-inhibitory activity. Based on previous structureeactivity relationship studies, the sequence of some peptides allowed us to anticipate the presence of biological activities. The antihypertensive tripeptide isoleucine-proline-proline (IPP) was identified and quantified in digested camel milk. The amount of released IPP was 2.56 ± 0.15 mg L1 of milk. For the first time, we showed that IPP is released during the gastro-intestinal digestion of camel milk k-casein. This research provides the basis to increase the exploitation of the health benefits of camel milk.

2015 - Characterization of dietary melanoidins and evaluation of the cytotoxic activity against colon cancer cells [Poster]
Tagliazucchi, Davide; Bellesia, Andrea; Martini, Serena; Conte, Angela
abstract

Emerging evidence from in vitro and in vivo studies suggested that the gastro-intestinal tract may be the key site for the biological action of food melanoidins. Recently, Vitaglione et al. reviewed the possible mechanisms by which coffee melanoidins may influence the risk of colorectal cancer development. In this work, we tested high molecular weight melanoidins (HMWM) extracted from different food sources for their cytotoxic activity against colon cancer cell lines. Water soluble HMWM were extracted by ultrafiltration (cut-off 10 kDa) from instant coffee, cocoa brew, dark beer and instant barley coffee. HMWM have been chemically characterized for their content in total polyphenols, protein, and polysaccharides, antioxidant activity as well as their spectroscopic properties. These extracts were assessed using two colon cancer cell lines: Caco-2 and SW480. Cytotoxic activity was assayed by MTT and data are presented as the dose that inhibited 50% control growth (IC50). All of the assessed HMWM were cytotoxic against Caco-2 cell line. Coffee HMWM were the most effective (IC50 of 1042 and 857 µg/mL after 24 and 48h of treatment, respectively) followed by dark beer, barley coffee and cocoa HMWM. Meanwhile, they are less effective against SW480 with coffee HMWM that were the most effective (IC50 of 2038 µg/mL after 48h) followed by cocoa, dark beer and barley coffee. Considering that the colon accumulates its content over at least 24h in a maximum volume of 2 litres, and that the daily intake of coffee melanoidins range between 0.5 and 2.0 g , it is possible to calculate a hypothetical concentration of coffee melanoidins in the colon between 0.25 and 1 mg/mL, which are values comparable to the IC50 for the cytotoxic activity against colon cancer cells.

2015 - Gastro-pancreatic release of phenolic compounds incorporated in a polyphenols-enriched cheese-curd [Articolo su rivista]
Helal, A.; Tagliazucchi, Davide; Verzelloni, Elena; Conte, Angela
abstract

As functional food, enriched cheese has recently been developed. The main objectives of this study were to investigate the role of casein in the retention of polyphenol during curd formation and the release of polyphenols during in vitro gastro-pancreatic digestion of polyphenols-enriched cheese and their contribution to the antioxidant activity of digested curd. Polyphenols showed high retention coefficient in curd. The retention coefficient of polyphenol was related to the binding affinity to casein and to their hydrophilicity. The polyphenols should be added before milk coagulation since the binding decreases as casein molecules aggregate. During in vitro gastro-pancreatic digestion steps, polyphenols were released from curd due to the dilution in gastric fluid and to casein proteolysis. The addition of polyphenols to curd determined a relevant increase of antioxidant activity respect to the curd control even a part of polyphenols is degraded by alkaline pH of pancreatic fluid. Our results suggested the possibility of producing highly nutritive value cheese with high release of the polyphenols during digestion. In addition, the whey, which contains polyphenols, can be involved in different products to maximize its utilization.

2015 - Identification of ACE-inhibitory peptides from Phaseolus vulgaris after in vitro gastrointestinal digestion [Articolo su rivista]
Tagliazucchi, Davide; Martini, Serena; Bellesia, Andrea; Conte, Angela
abstract

The objective of this study was to identify the angiotensin I-converting enzyme (ACE)-inhibitory peptides released from thermally treated Phaseolus vulgaris (pinto) whole beans after in vitro gastrointestinal digestion. The degree of hydrolysis increased during digestion reaching a value of 50% at the end of the pancreatic digestion. The&lt;3 kDa fraction of the postpancreatic sample showed high ACE-inhibitory activity (IC50=105.6±2.1 mug of peptides/mL). Peptides responsible for the ACE-inhibitory activity were isolated by reverse-phase high-performance liquid chromatography (HPLC). Three fractions, showing the highest inhibitory activity, were selected for tandem mass spectrometry (MS/MS) experiments. Eleven of the identified sequences have previously been described as ACE-inhibitors. Most of the identified bioactive peptides have a hydrophobic amino acid, (iso)leucine or phenylalanine, or proline at the C-terminal position, which is crucial for their ACE-inhibitory activity. The sequence of some peptides allowed us to anticipate the presence of ACE-inhibitory activity.

2014 - Bioaccessibility of polyphenols and cinnamaldehyde in cinnamon beverages subjected to in vitro gastro-pancreatic digestion [Articolo su rivista]
A., Helal; Tagliazucchi, Davide; Verzelloni, Elena; Conte, Angela
abstract

The present study was undertaken to investigate the in vitro bioaccessibility of polyphenols and cinnamaldehyde in cinnamon beverage and formulations during simulated gastro-pancreatic digestion. Cinnamon polyphenols post-pancreatic bioaccessibility was 79.8%. The decline in total polyphenols was caused by the precipitation of proanthocyanidins by pepsin. The addition of sweeteners increased the polyphenols bioaccessibility decreasing the interaction between pepsin and tannins. The addition of bovine milk had no effect on polyphenols post-pancreatic bioaccessibility. Quercetin-3-rhamnoside, syringic and coumaric acids were found to be poor bioaccessible whereas kaempferol and cinnamaldehyde were stable during digestion. The addition of sweeteners did not affect the bioaccessibility of these compounds. The final bioaccessibility was affected by milk only for cinnamaldehyde. The present study demonstrates that cinnamon beverage provides a significant source of dietary bioaccessible polyphenols and that the modality of consuming cinnamon could represent a crucial factor since the addition of sweeteners improve their bioaccessibility whereas milk has no effect on their bioaccessibility.

2013 - Cocoa extract inhibits in vitro α-glucosidase activity: the role of polyphenols and melanoidins [Abstract in Atti di Convegno]
Tagliazucchi, Davide; Bellesia, Andrea; Conte, Angela
abstract

Studies on the health benefits of cocoa have recently focused on the anti-diabetic potential of cocoa extract. The intake of cocoa has been related to a reduction in the post-prandial levels of blood glucose in diabetic rats. A possible explanation for these observations is that cocoa extract possesses the ability to inhibit enzymes involved in the digestion of carbohydrate, attenuating the post-prandial increase in glycemia. Using an in vitro model, the activity of rat intestinal α-glucosidase has been determined in the absence and presence of cocoa extract and fractions. The addition of cocoa extract inhibited α-glucosidase activity with an IC50 of 7.9 mg/mL. The cocoa extract was further fractionated by ultrafiltration in a polyphenolic–rich fraction (<10 KDa) and a melanoidins-rich fraction (>10 KDa). Both the fractions were able to inhibit α-glucosidase with an IC50 of 2.3 and 3.9 mg/mL, respectively. To identify the bioactive compounds, the polyphenolic–rich fraction was further fractionated with Sephadex LH-20 columns to separate tannins from low molecular weight flavanols. Furthermore, the melanoidins-rich fraction was separated in two fractions using ultrafiltration at 30 KDa. All the obtained fractions were characterized for their ability to inhibit α-glucosidase and for their content in polyphenolic compounds.

2013 - Effect of cinnamon beverage formulations on the bioaccessibility of polyphenols and cinnamaldehyde during in vitro gastro-pancreatic digestion [Abstract in Atti di Convegno]
A., Helal; Tagliazucchi, Davide; Verzelloni, Elena; Conte, Angela
abstract

The present study was undertaken to investigate the in vitro bioaccessibility of phenolic compounds and cinnamaldehyde in cinnamon beverage and formulations during simulated gastro-pancreatic digestion. Cinnamon polyphenol bioaccessibility was 79.8% at the end of the digestion. This decline in total polyphenols was found during the gastric step of the digestion and was caused by the precipitation of cinnamon proanthocyanidins by pepsin. The addition of sweeteners increased the polyphenol bioaccessibility to value near 90% decreasing the interaction between pepsin and tannins. The addition of bovine milk gave rise to an immediate decrease in cinnamon polyphenol concentration and negatively affect their post-pancreatic bioaccessibility. Quercetin-3-rhamnoside, syringic and coumaric acids were find to be poor bioaccessible whereas kaempferol and cinnamaldehyde were stable during digestion. The addition of sweeteners did not affect the bioaccessibility of these compounds. Milk addition caused an immediate decrease in the concentration of monomeric phenolic compounds partially due to the interaction with milk protein as probed by fluorescence spectroscopy. The final bioaccessibility was affected by milk only for cinnamaldehyde. The present study demonstrates that cinnamon beverage provide a significant source of dietary bioaccessible polyphenols and that the modality of consuming cinnamon could represent a crucial factor since the addition of sweeteners improve their bioaccessibility whereas milk negatively affect it.

2013 - Effect of grape variety on the evolution of sugars, hydroxymethylfurfural, polyphenols and antioxidant activity during grape must cooking [Articolo su rivista]
Tagliazucchi, Davide; Verzelloni, Elena; A., Helal; Conte, Angela
abstract

Grape must cooking is a traditional practice used for the production of foodstuff worldwide such as traditional balsamic vinegar. The aim of the present work was to reveal the effect of grape variety on the evolution of the main chemical components in grape must during cooking. On this purpose, two grape must varieties (red Lambrusco and white Trebbiano grapes) were cooked and analyzed. The monosaccharides concentration decreased because cooking resulting in the formation of 5-hydroxymethylfurfural. At the end of cooking, the antioxidant activity and polyphenols concentration was higher and the 5-hydroxymethylfurfural was lower in Lambrusco than in Trebbiano must. Additional changes involved degradation of monomeric anthocyanins resulting in the formation of the corresponding phenolic acids. From a health point of view, the high antioxidant activity and polyphenols concentration and the low 5-hydroxymethylfurfural concentration make cooked red Lambrusco must a safer raw starting material for making traditional balsamic vinegar.

2013 - Mechanism of inhibition of protein glycation by polyphenols [Abstract in Atti di Convegno]
Bellesia, Andrea; Tagliazucchi, Davide; Conte, Angela
abstract

We have observed that protocatechuic (PC) and 3,4-dihydroxyphenylacetic (DHPA) acids, found in human plasma after the consumption of foods rich in anthocyanins and flavanols, inhibit the formation of advanced glycation endproducts (AGEs). We have investigated the mechanisms of inhibition incubating for 7 days bovine serum albumin (BSA) (50 mg/mL) and glucose (0.8 mol/L) in the presence and absence of these polyphenols at various concentrations. Fluorescence emission at 405 nm was used to evaluate AGEs formation. Carboxymethyl-lysine (CML), Amadori products and quinoproteins were determined by ELISA, NBT assay and electrophoresis, respectively. Both PC and DHPA were able to decrease the formation of Amadori products, fluorescent AGEs and CML. These decreases were concentration dependent. Quinoproteins formation increases with the increase of polyphenol concentration. BSA was also incubated with 0.5 and 1.0 mmol/L of phenols in the absence of glucose. After 7 days of incubation the unbound polyphenols were removed by ultrafiltration. The phenols-treated BSA was incubated with glucose for 7 days. No significant differences are observed in the inhibition of AGEs formation between phenol pre-incubated and non pre-incubated BSA. These results and the formation of quinone modified BSA suggest that polyphenols are pre-Amadori inhibitors of AGEs formation. Phenols bind BSA and decrease glucose binding. They are oxidized (pH 7.5, 37°C) in the reactive quinones intermediates and react with BSA amino groups forming quinone modified BSA and decreasing the AGEs formation.

2012 - Antioxidant activity of in vitro digested bovine milk [Abstract in Atti di Convegno]
Verzelloni, Elena; A., Helal; Tagliazucchi, Davide; Conte, Angela
abstract

The objective of the present study was to analyze the antioxidant properties of different types of bovine milk subjected to simulated gastro-pancreatic digestion. Whole, semi-skimmed, and skimmed milk were digested in sequence with pepsin (300 U/ml; pH 2.5; 2h) then with pancreatin (0.8 g/l; pH 7.5; 2h) and bile salts (5 mg/ml). At the end of digestion, samples were subjected to ultrafiltration (3 KDa) and the high (HMW) and low (LMW) molecular weight fractions analyzed with ABTS assay. All milk types contain antioxidants with whole milk having the highest amount (622.3 ± 44.5 mg vitamin C/L). About 90% of antioxidant activity was in the HMW fraction. The degree of protein hydrolysis (DH) after digestion was different considering the different milk types (20.8 ± 0.4, 24.3 ± 0.3 and 30.7 ± 0.8 in whole, semi-skimmed and skimmed milk, respectively). Pancreatic digestion caused an increase in the antioxidant activity for all the analyzed milk (post-pancreatic ABTS value of 3374.3 ± 104.6, 2657.1 ± 39.6 and 2751.2 ± 46.9, in whole, semi-skimmed and skimmed milk, respectively). The distribution of the antioxidant activity between the LMW and HMW fractions was different considering the diverse milk types. The percentage of antioxidant activity in the LMW fraction increased to 38%, 79% and 90% in whole, semi-skimmed and skimmed milk, respectively, according to the increase in the DH. HPLC analysis of LMW fractions revealed that all the digested milk had the same peptides pattern but the peptides concentrations in skimmed milk was higher than in semi-skimmed and whole milk. On-line HPLC-ABTS analysis of LMW fractions showed that two peptides are the major contributor to the antioxidant activity of this fraction. The two peptides were identified by MS/MS. Our data suggest that digestion of milk increased the antioxidant activity, potentially reducing the oxidative stress in the gastro-intestinal tract.

2012 - Antioxidant properties of polyphenols incorporated in casein/sodiumcaseinate films [Articolo su rivista]
A., Helal; Tagliazucchi, Davide; Conte, Angela; S., Desobry
abstract

Radical-scavenging activity (RSA) of sodium caseinate (NaCAS) films with 0e30% added casein and antioxidants was measured. Tannic acid and catechin were added to the films as model antioxidants. RSA was measured using 2,2-diphenyl-1-picrylhydrazyl and 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); the two methods gave similar results for RSA measurement. Film casein content most influenced initial RSA, while increasing casein level resulted in an apparent decrease of RSA due to quenching. During storage, a good stability of RSA was observed. The surface antioxidant activity is of primary interest for food contact materials; a decrease in film surface RSA occurred during the first 20 days of storage, followed by an increase in surface RSA during the remainder of the 90-day storage period, especially at high relative humidity. This phenomenon was due to plasticizing of NaCAS, with possible network alteration over long storage times

2012 - Casein-antioxidants interaction, nutritional effects and technological applications [Abstract in Atti di Convegno]
A., Helal; Tagliazucchi, Davide; Verzelloni, Elena; S., Desobry; Conte, Angela
abstract

Casein-antioxidants interaction, nutritional effects and technological applications

2012 - Manufacturing of antioxidant packaging Na-caseinate edible films [Abstract in Atti di Convegno]
A., Helal; Tagliazucchi, Davide; Conte, Angela; S., Desobry
abstract

Manufacturing of antioxidant packaging Na-caseinate edible films

2012 - Pomegranate ellagitannins inhibit in vitro carbohydrate digestion [Abstract in Atti di Convegno]
Tagliazucchi, Davide; Verzelloni, Elena; Bellesia, Andrea; Conte, Angela
abstract

Pomegranate is an ancient fruit studied for its anti-diabetic properties. The aim of the present work was to identify the α-glucosidase inhibitors in a pomegranate juice polyphenol-rich extract (PPRE). The PPRE, obtained using C18 solid-phase extraction, contained 5.87 ± 0.26 mmol of ellagic acid equivalent (EAE)/L and showed an inhibitor activity on rat intestinal α-glucosidase with an IC50 value of 922.2 ± 7.1 μmol of EAE/L. The PPRE was fractioned in three different fractions using C18 columns and glucosidase inhibitory activity was recovered in an ellagitannins-enriched fraction. After HPLC separations punicalagin, punicalin and ellagic acid were identified as α-glucosidase inhibitors with IC50 values of 140.2 ± 1.1, 191.4 ± 1.3 and 380.9 ± 3.5 μmol/L, respectively. Kinetic analysis showed that the inhibition is of mixed type with a Ki of 903.0 ± 74.7 μmol/L. Pomegranate ellagitannins interact directly with the α-glucosidase and the non-specific binding of ellagitannins may alters the protein structure reducing the velocity of the catalysis and altering the accessibility to the active site of the substrate. The inhibitory effect on carbohydrate hydrolysis of PPRE was tested against a real food system (potatoes) using an in vitro digestion protocol. The presence of PPRE produced a decrease in the amount of released glucose. During digestion punicalin and punicalagin concentration decreased as a consequence of their binding to salivary or potatoes proteins and of their hydrolysis. Despite this loss, the PPRE retained high inhibitory activity. In conclusion, our data are consistent with the hypothesis that pomegranate juice can be considered a very useful food supplement to ameliorate postprandial hyperglycemia linked to type 2 diabetes and hyperglycemia-induced vascular complications.

2012 - The Type and Concentration of Milk Increase the in Vitro Bioaccessibility of Coffee Chlorogenic Acids [Articolo su rivista]
Tagliazucchi, Davide; A., Helal; Verzelloni, Elena; Conte, Angela
abstract

Coffee with different types and concentrations of milk was digested with pepsin (2 h) and pancreatin (2 h) to simulate gastropancreatic digestion. Chlorogenic acids (CGAs) were determined by high-performance liquid chromatography− electrospray ionization−tandem mass spectrometry in ultrafiltrate (cutoff 3 kDa) to evaluate their bioaccessibility. After digestion, bioaccessible CGAs decreased from 80.2 to 53.0 and 69.5 μmol/200 mL in coffee without milk and coffee-whole milk, respectively. When whole, semiskimmed, skimmed, or diluted milk were present, the increase in bioaccessibility was dependent on fat content (r = 0.99, p < 0.001). No relationship was observed between bioaccessibility and proteins, carbohydrates, and calcium content. The addition of milk to coffee caused an immediate decrease in the bioaccessibility due to CGAs binding to proteins. After digestion, 86−94% of bound CGAs remained in the high molecular weight fraction. Casein bound 5-caffeoylquinic acid with high affinity (KD of 37.9 ± 2.3 μmol/L; n = 0.88 ± 0.06).

2012 - The first tract of alimentary canal as an extractor. Release of phytochemicals from solid food matrices during simulated digestion [Articolo su rivista]
Tagliazucchi, Davide; Verzelloni, Elena; Conte, Angela
abstract

The release of some phytochemicals (polyphenols, flavonoids, anthocyanins, carotenoids) from peach, plums, prunes, walnuts and tomatoes using an in vitro model simulating oral-gastro-hepato-pancreatic digestion has been investigated. Most of phytochemicals are extracted during the oral phase, and the amount of bioaccessible polyphenols, flavonoids and anthocyanins increased during gastric digestion in all the analyzed fruits. The transition in the hepatopancreatic environment caused a decrease in total polyphenols, whereas flavonoids (except anthocyanins) were not degraded. In most of the tested fruits, the hepatopancreatic digestion did not affectthe extraction of polyphenols and flavonoids except in walnuts, for which this phase caused an important release of both of them. Our results suggest that the first tract of alimentary canal may act as an extractor of phytochemicals where the antioxidantcompounds are progressively released from food matrix and are made available for the absorption or to exert their biological effects in this area.

2011 - Antiglycative and antioxidative properties of coffee fractions [Articolo su rivista]
Verzelloni, Elena; Tagliazucchi, Davide; D., Del Rio; L., Calani; Conte, Angela
abstract

In this work the inhibitory activity of coffee low molecular weight compound (LMWC) and high molecular weight compound (HMWC) fractions against in vitro advanced glycation end products (AGEs) formation was investigated. The HMWC fraction was characterized for its content in total phenolic groups, proteins and carbohydrates. The chlorogenic acids of LMWC fraction were identified by liquid chromatography coupled with tandem mass spectrometry. HMWC inhibited bovine serum albumin glycation by acting as radical scavenger and Fe-chelator in the post-Amadori phase of the reaction and by inhibiting dicarbonyl reactive compounds production during glucose autoxidation. LMWC fraction was able to inhibit protein glycation and dicarbonyl reactive compounds formation more than HMWC fraction. Chlorogenic acids are the main responsible for the antiglycative activity of LMWC fraction. This study clearly shows that coffee contains molecules with in vitro antiglycative activity and in particular chlorogenic acids are of particular interest for their known bioavailability in vivo.

2011 - Antiglycative and neuroprotective activity of colon-derived polyphenol catabolites [Articolo su rivista]
Verzelloni, Elena; C., Pellacani; Tagliazucchi, Davide; S., Tagliaferri; L., Calani; L. G., Costa; F., Brighenti; G., Borges; A., Crozier; Conte, Angela; D., Del Rio
abstract

Dietary flavonoids and allied phenolic compounds are thought to be beneficial in the control of diabetes and its complications, because of their ability to inhibit oxidative stress, protein glycation and to act as neuroprotectants. Following ingestion by humans, polyphenolic compounds entering the large intestine undergo extensive metabolism by interaction with colonic microbiota and it is metabolites and catabolites of the parent compounds that enter the circulatory system. The aim of this study was to investigate the inhibitory activity of some colonic microbiota-derived polyphenol catabolites against advanced glycation endproducts (AGEs) formation in vitro and to determine their ability, at physiological concentrations, to counteract mild oxidative stress of cultured human neuron cells. This study demonstrated that ellagitannin-derived catabolites (urolithins and pyrogallol) are the most effective antiglycative agents, whereas chlorogenic acid-derived catabolites (dihydrocaffeic acid, dihydroferulic acid and feruloylglycine) were most effective in combination in protecting neuronal cells in a conservative in vitro experimental model. In conclusion, some polyphenolic catabolites, generated in vivo in the colon, were able in vitro to counteract two key features of diabetic complications, i.e. protein glycation and neurodegeneration. These observations could lead to a better control of these events, which are usually correlated with hyperglycemia.

2011 - Effect of colon-derived polyphenol metabolites on in vitro protein glycation [Abstract in Rivista]
Conte, Angela; Verzelloni, Elena; D., Del Rio; Tagliazucchi, Davide
abstract

Polyphenols are thought to be beneficial in the control of diabetic complications because of their ability to inhibit oxidative stress and protein glycation. Polyphenols undergo extensive metabolism during their absorption though the first-intestinal tract. Polyphenol catabolites produced by microbia of colon enter the circulatory system. The aim was to investigate the inhibitory activity of colonic-derived catabolites against protein glycation. Protein glycation was measured, after a two weeks incubation under physiological condition (pH 7.4; 37°C) of bovine serum albumin (40 mg/mL) and glucose (30 mmol/L) in presence or absence of polyphenol catabolites, with fluorescence at the excitation and emission maxima of 355 and 405 nm, respectively. A dose dependent inhibition of glycation was observed with urolithin A and B (catabolites of pomegranates and raspberries) and dihydroferulic acid (from coffee.) The IC50 of urolithin A is 12mM. Urolithin B and dihydroferulic acid are less effective. The catabolites were also tested in association, based on the dietary sources. The pomegranate-derived catabolites urolithin A, B and pyrogallol, inhibit glycation by 37 and 44% at 1 and 2µmol/L respectively, suggesting a synergic action between catabolites. The red wine-derived catabolites in association show a lower antiglycative activity (15% inhibition at 2µmol/L) while coffee catabolites are inactive. Polyphenol colon-catabolites inhibit protein glycation at concentrations observed in blood after consumption of a portion of pomegranate or raspberry. These observations suggest that some polyphenols may contribute to reduce the hyperglycemia-correlated pathologies.

2011 - Effect of cow milk on the in vitro bioaccessibility of coffee chlorogenic acids [Relazione in Atti di Convegno]
Tagliazucchi, Davide; Verzelloni, Elena; A. M. I., Helal; Conte, Angela
abstract

To study the effect of cow milk on coffee chlorogenic acids (CQAs) bioaccessibility during simulated gastro-intestinal digestion, 50% coffee with 50% water (C), or with 50% whole milk (CM50), or with 25% whole milk-25% water (CM25), or with 10% whole milk- 40% water (CM10), or with 50% semi-skimmed milk (CSSM50), or with 50% skimmed milk (CSM50) were digested in sequence with pepsin (300 U/ml; pH 3; 2h) then with pancreatin (0.8 g/l; pH 7.5; 2h) and bile salts (5 mg/ml). At the end of the digestion periods, samples were subjected to ultrafiltration and the low molecular weight fractions were analyzed with LC-MS. The bioaccessible CQAs decreased from 91.4 ± 1.3 to 85.2 ± 5.4 and to 61.2 ± 2.0 micromol/100ml at the end of gastric and intestinal digestions respectively in sample C, suggesting CQAs degradation. After the two digestion periods higher recovery of CQAs were obtained with CM50, CM25, CM10 and CSSM50 samples respect to C. For example at the end of the digestion periods, the bioaccessible CQAs in CM50 and CSSM50 were 80.0 ± 4.6 and 67.7 ± 1.0 micromol/100 ml respectively. Similar results were obtained with 5-caffeoylquinic acid. These data suggest that the milk fats have a protective effect on CQAs degradation during digestion.

2011 - Interaction of colon-derived polyphenol metabolites with bovine serum albumin [Abstract in Atti di Convegno]
Tagliazucchi, Davide; D., Del Rio; Verzelloni, Elena; L., Calani; A., Crozier; Conte, Angela
abstract

Following ingestion, polyphenols entering the large intestine undergo extensive metabolism by colonic microbiota and metabolites of the parent compounds enter the circulatory system. It is of limited relevance attempting to unravel the mechanism of action of intact parent polyphenols. Their colonic metabolites are more plausible candidates for investigation of putative biological activity. The aim of this work was investigating the interaction between BSA and polyphenol metabolites using tryptophan fluorescence quenching. Fluorescence spectra of BSA were recorded before and after the addition of selected metabolites, under physiological conditions (37°C and pH 7.4), in the range of 300-550 nm at an excitation wavelength of 294 nm. Calculation of KSV demonstrated that, for all the tested metabolites (with the exception of 3-hydroxyphenylacetic and 3,4-dihydroxyphenylacetic acids), the type of quenching was static, suggesting the formation of a complex between the molecules and the fluorophore (tryptophan). Urolithins had the lowest KD value and bound BSA with higher affinity with respect to the other tested compounds. The binding stoichiometry was 1:1 for all the tested metabolites, suggesting the existence of a single binding site in the protein. Competitive binding experiments with quercetin showed a displacement of urolithins from the complex, demonstrating that site I (the hydrophobic binding pocket located in the subdomain IIA of BSA, which is known to bind a variety of ligands) is likely to be the preferred binding site for these molecules. The affinity of BSA for urolithins lets hypothesise that these phenolic metabolites could circulate in vivo as protected by plasma proteins. In the presence of solid tumors there is a reduction in tumor venous blood pH below 7, as a consequence of lactate accumulation. This acidic environment may cause the release of urolithins due to pH-induced decreased affinity, and these phenolic metabolites can exert local anticancer effects.

2010 - Changes in major antioxidant compounds during aging of traditional balsamic vinegar [Articolo su rivista]
Verzelloni, Elena; Tagliazucchi, Davide; Conte, Angela
abstract

Traditional balsamic vinegar (TBV) shows antioxidant capacity that increases passing from cask 5, containing the youngest vinegar, to cask 1 containing the oldest vinegar. This increase in antioxidant capacity is a consequence of both the concentration of compounds already present and of new antioxidants formation and is positively related to the increase in the polyphenolic content and in the Maillard reaction/caramellisation products. During TBV ageing, some reactions involving flavonoids and tannins take place. Tannins can undergo acid-catalyzed cleavage of their interflavonoid bonds with subsequent condensation of other flavonoid molecules. In addition, the low pH, the decrease of the water content and the presence of aldehydes, may promote flavonoids polycondensation. These reactions explain the observed increase in polymeric phenolic compounds and the decrease in monomeric flavonoids. During TBV ageing an increase in the browning index is observed as a consequence of the polycondensation reactions of flavonoids and of brown melanoidins accumulation.

2010 - Contribution of melanoidins to the antioxidant activity of traditional balsamic vinegar during ageing [Articolo su rivista]
Tagliazucchi, Davide; Verzelloni, Elena; Conte, Angela
abstract

The contribution of high molecular weight melanoidins to the overall antioxidant activity of traditional balsamic vinegar has been determined. High molecular weight melanoidins are gradually synthesized and accumulated during traditional balsamic vinegar ageing contributing significantly to the browning of the vinegar. The increase in antioxidant activity of traditional balsamic vinegar during ageing is largely due to the formation of high molecular weight melanoidins and only in lower lesser part to the concentration of polyphenols deriving from the grape or to the formation of low molecular weight Maillard reaction or caramelisation products during must cooking and vinegar ageing. During ageing, low molecular weight compounds are progressively incorporated into the melanoidins skeleton and may contribute significantly to the antioxidant activity of high molecular weight melanoidins. Among these compounds, it has been shown that antioxidant phenolic compounds are progressively incorporated into the melanoidins skeleton during traditional balsamic vinegar ageing.

2010 - Effect of dietary melanoidins on lipid peroxidation during simulated gastric digestion: their possible role in the prevention of oxidative damage [Articolo su rivista]
Tagliazucchi, Davide; Verzelloni, Elena; Conte, Angela
abstract

The ability of high molecular weight melanoidins extracted from coffee, barley coffee and dark beer to inhibit lipid peroxidation during simulated gastric digestion of turkey meat has been investigated. Results showed that melanoidins decrease the synthesis of lipid hydroperoxides and secondary lipoxidation products. Coffee melanoidins at 3 mg/ml reversed the reaction and broke down hydroperoxides to concentration lower than the initial value. Barley coffee and dark beer melanoidins were less effective and even at 12 mg/ml did not reverse the reaction. The proposed mechanism of action involved Fe2+ chelating capacity, heme binding ability and radical scavenging activity. Melanoidins were characterized for their content in total proteins, carbohydrates and phenolics and the relationship between the chemical composition and the antioxidant activity of dietary melanoidins was investigated. Coffee melanoidins that contain more phenolics and proteins respect to the other melanoidins, showed greater antioxidant activity respect to the other melanoidins tested.

2010 - Effect of prolonged phenytoin administration on rat brain geneexpression assessed by DNA microarrays [Articolo su rivista]
Mariotti, V.; Melissari, E.; Amar, S.; Conte, Angela; Belmaker, R. H.; Pellegrini, S.; Agam, G.
abstract

Preliminary clinical trials have recently shown that phenytoin, an antiepileptic drug, may also be beneficial for treatment of bipolar disorder. To examine molecular mechanisms of action of phenytoin as a potential mood stabilizer, DNA microarrays were used to study the effect of phenytoin on gene expression in the hippocampus and frontal cortex of Sprague–Dawley rats. While our particular interest is in bipolar disorder, this is the first DNA microarray study on the effect of phenytoin in brain tissue, in general. As compared with control rats, treated rats had 508 differentially expressed genes in the hippocampus and 62 in the frontal cortex. Phenytoin modulated the expression of genes which may affect neurotransmission, e.g. glutamate decarboxylase 1 (Gad1) and {gamma}-aminobutyric acid A receptor, alpha 5 (Gabra5). Phenytoin also exerted an effect on neuroprotection-related genes, namely the survival-promoting and antioxidant genes v-akt murine thymoma viral oncogene homolog 1 (Akt1), FK506 binding protein 12-rapamycin associated protein 1 (Frap1), glutathione reductase (Gsr) and glutamate cysteine ligase catalytic subunit (Gclc). The expression of genes potentially associated with mechanisms of mood regulation such as adenylate cyclase-associated protein 1 (Cap1), Glial Fibrillary Acidic Protein (Gfap) and prodynorphin (Pdyn) was also altered. Some of the above genes are regarded as targets of classical mood stabilizers and their modulation supports the clinical observation that phenytoin may have mood-stabilizing effects. The results may provide new insights regarding the mechanism of action of phenytoin and genes found differentially expressed following phenytoin administration may play a role in the pathophysiology of either bipolar disorder or epilepsy.

2010 - From balsamic to healthy: traditional balsamic vinegar melanoidins inhibit lipid peroxidation during simulated gastric digestion of meat [Articolo su rivista]
Verzelloni, Elena; Tagliazucchi, Davide; Conte, Angela
abstract

In this work traditional balsamic vinegar (TBV) melanoidins were characterized for chemical composition and antioxidant activity and their antiperoxidative effect during an in vitro gastric digestion of turkey meat was studied. The most important constituents of TBV melanoidins were carbohydrates (51% w/w) of which glucose (35% w/w) and fructose (10% w/w) are the main representatives, hydroxymethylfurfural (HMF) (7.2% w/w), phenolic groups (4.6% w/w) and proteins (1.2% w/w). The antioxidant capacity of melanoidins was studied, measuring lipid hydroperoxides and secondary lipoxidation products formed during in vitro gastric digestion of turkey meat. The most important mechanisms in their antioxidant activity resulted radical scavenging and Fe2+-chelating activities. Pepsin inhibiting ability has been excluded.. TBV melanoidins were also able to bind heme under gastric conditions potentially preventing its absorption and prooxidant and cytotoxic effects. Our results support the idea that TBV melanoidins may have a role in oxidative damage prevention. Fe2+-chelating and heme-binding activities as well as mechanisms of antioxidant activity of TBV melanoidins were also compared with coffee, barley coffee and dark beer melanoidins.

2010 - In vitro bio-accessibility and antioxidant activity of grape polyphenols [Articolo su rivista]
Tagliazucchi, Davide; Verzelloni, Elena; D., Bertolini; Conte, Angela
abstract

The bio-accessibility (the release of compounds from solid food matrices) of grape polyphenols using an in vitro model simulating gastro-intestinal conditions has been investigated. In vitro studies are needed to unravel the factors affecting the release of antioxidants during digestion. The amount of bio-accessible polyphenols, flavonoids and anthocyanins increases during gastric digestion. The transition in the intestinal environment causes a decrease in all the analyzed classes of polyphenols followed by a renewal in the extraction of polyphenols and flavonoids but not of anthocyanins. The stability under gastro-intestinal conditions of pure phenolic acids, flavonoids and resveratrol has been analyzed and discussed. Changes in antioxidant activity during digestion were correlated to the changes in polyphenols concentration as well as to the pH. Our results suggest that the gastro-intestinal tract may act as an extractor where polyphenols are progressively released from solid food matrix and made available for the absorption or to exert their biological effects in the gastro-intestinal tract.

2008 - Antioxidant properties of traditional balsamic vinegar and boiled must model systems [Articolo su rivista]
Tagliazucchi, Davide; Verzelloni, Elena; Conte, Angela
abstract

Traditional balsamic vinegar (TBV) is a natural product prepared with cooked and concentrated locally grown grape must. It has been demonstrated that TBV contains phenols and shows antioxidant activity. In this study we investigated the antioxidant properties of TBV in relation to its content in phenolic compounds, polymeric tannins and Maillard reaction products (MRPs). Results show that TBV has a high antioxidant activity measured with both FRAP and ABTS assay that is higher or equal to those obtained in some red wine. About 45% of the antioxidant activity of TBV is due to the total polyphenolic fraction. Among polyphenols, tannins contribute to about 50% of the antioxidant activity of the total polyphenolic fraction. The residual antioxidant activity of TBV is due to the melanoidins (about 45%) synthesized during the boiling of the must and the ageing of TBV and to other compounds such as low molecular weight MRPs. When we investigated the effect of heating on the browning and on the formation of antioxidant MRPs in must model systems we observed a major formation of antioxidant MRPs for the model system containing both amino acids and sugars with respect to the model system containing only sugars. We also tested the effect of some representative phenolic compounds present in must. Only polyphenols were stable in the model solution, however, in our experimental conditions they did not influence the browning and the formation of MRPs. Indipendently of their bioavailability, dietary antioxidants play an important role in protecting the gastrointestinal tract itself from oxidative damage and could protect against a built up of peroxides and their assimilitation in the digestive tract.

2008 - SNPs in neurotrophin system genes and Alzheimer's disease in an Italian population [Articolo su rivista]
Cozza, A; Melissari, E; Iacopetti, P; Mariotti, V; Tedde, A; Nacmias, B; Conte, Angela; Sorbi, S; Pellegrini, S.
abstract

Increasing evidence suggests a role for nerve growth factor (NGFB), brain-derived neurotrophic factor (BDNF), and their receptors, nerve growth factor receptor (NGFR), and neurotrophin tyrosine kinase receptors 1 and 2 (NTRK1 and NTRK2), in Alzheimer's disease (AD). However, genetic association between the neurotrophin system genes and AD has been poorly investigated. We genotyped 21 single nucleotide polymorphisms (SNPs) within these genes in a population of Italian AD patients and healthy controls. We found an allele-wise association of rs2072446 on NGFR with familial AD (fAD, p = 0.047), and a genotype-wise association of rs2289656 on NTRK2 with sporadic AD (sAD, p = 0.0036). rs6336 on NTRK1 resulted associated to early-onset sAD in both allele-wise (p = 0.028) and genotype-wise (p = 0.014) analysis, while rs1048218 on BDNF showed allele-wise association with late-onset sAD (p = 0.047). A trend to association with sAD and/or fAD was observed for other SNPs. Our results suggest that genetic variants of neurotrophin system genes might confer susceptibility to AD.

2007 - Occurence of two NOS isoforms in the developing gut of sea bass Dicentrarchus labrax (L.) [Articolo su rivista]
Pederzoli, A.; Conte, A.; Tagliazucchi, D.; Gambarelli, A.; Mola, L.
abstract

In this work we have examined the appearance and distribution of nitric oxide synthase (NOS), with histochemical, immunohistochemical and biochemical methods, during development of the sea bass (Dicentrarchus labrax) gut. The data showed that both the calcium-calmodulin dependent neuronal isoform (nNOS) and calcium-independent inducible isoform (iNOS) are present in the larval gut of sea bass. The nNOS-immunoreactivity was present in the epithelial cells and enteric nerve cells of gut both in the 8-day-old specimens and in the 24-day-old-larvae. In the adult nNOS-immunoreactivity disappeared from epithelial cells, remaining in the wall intramural neurons and fibers. The iNOS-immunoreactivity was present in the epithelial cells of 24-day-old-larvae and was not detectable in the adult gut. Western blot analysis and determination of NOS activity also demonstrated the presence of the two NOS isoforms, nNOS and iNOS, in the gut of 24-day-old specimens. The presumably different roles played by the two isoforms of enzyme are discussed. The presence of nNOS isoform in the gut enteric neurons of the same larval stages of D. labrax in which we previously demonstrated the presence of substance P and Vasoactive Intestinal Polypeptide (VIP), may suggest that all these three components of the motility control system are already present in the larval phase. Nitric oxide (NO) may be also involved in the early immune response. The present results on the occurrence of iNOS isoform in epithelial gut cells of the same regions in which the gut-associated lymphoid tissue (GALT) will differentiate, may suggest for NO a role in early defence mechanisms, before the establishment of immune responses in GALT. Finally, the developmental and regional differences in nNOS and iNOS expression also suggest a regulatory role in development and differentiation of the sea bass gut.

2007 - Relationship between the antioxidant properties and the phenolic and flavonoid content in traditional balsamic vinegar [Articolo su rivista]
Verzelloni, Elena; Tagliazucchi, Davide; Conte, Angela
abstract

The antioxidant properties of traditional balsamic vinegar as regards its phenolic and flavonoid content comparing it to selected vinegars and red wines have been investigated. The polyphenols were separated from interfering compounds utilizing C18 columns. The polyphenolic content was determined utilizing both Folin–Ciocalteu and peroxidase assays. The antioxidant capacity was quantified using both ABTS and FRAP assays. The results show that traditional balsamic vinegar has lower antioxidant activity and phenolic and flavonoid content than Nero d’Avola but higher than the other tested products. The antioxidant capacity of wines and vinegars is highly correlated with their phenolic content, measured by peroxidase assay and it is also highly correlated with their flavonoid content while in traditional balsamic vinegar and balsamic vinegar this correlation diminishes. The study describes a simple and fast method of separating from other compounds and of measuring polyphenols in the analysis of red wines and vinegars with complex composition such as traditional balsamic vinegar.

2006 - Confronto fra le proprietà antiossidanti e il contenuto fenolico di vini rossi e aceto balsamico tradizionale [Relazione in Atti di Convegno]
Tagliazucchi, Davide; Verzelloni, Elena; Conte, Angela
abstract

Negli ultimi anni i composti polifenolici sono stati studiati per le loro proprietà antiossidanti e i potenziali effetti benefici sulla salute umana. Nel presente lavoro sono state studiate le proprietà antiossidanti e il contenuto polifenolico totale di alcuni campioni di vino rosso e di aceto balsamico tradizionale (ABT). Per determinare il contenuto polifenolico totale sono stati utilizzati il saggio di Folin-Ciocalteu ed il saggio della perossidasi. Sulla base dei nostri risultati, il saggio della perossidasi sembra essere il metodo più preciso per determinare il contenuto polifenolico in matrici estremamente complesse come l’ABT. Considerando i dati ottenuti con il saggio della perossidasi, l’ABT contiene meno polifenoli del Nero d’Avola ma più polifenoli degli altri vini analizzati. Le proprietà antiossidanti dei campioni sono state analizzate in funzione di quello che era il loro contenuto in polifenoli e analizzando quelle che erano le capacità di ciascun campione di inattivare i radicali liberi generati in fase acquosa e di inibire la perossidazione lipidica durante una digestione simulata di carne suina. I risultati mostrano come la capacità antiossidante totale dei campioni era: Nero d’Avola > ABT > Dolcetto d’Alba > Lambrusco. Inoltre, sia il vino rosso sia l’ABT sono in grado di inibire in maniera significativa la formazione di perossidi lipidici durante una digestione simulata di carne suina. In conclusione, l’ABT rappresenta un ottimo condimento che contribuisce positivamente sia sulla qualità dei pasti che sulla quantità totale di antiossidanti introdotti durante i pasti.

2006 - Effect of chain length and aldehydic function on some biological properties of parropolyenes [Articolo su rivista]
Tagliazucchi, Davide; Verzelloni, Elena; E., Pini; G., Ronca; Conte, Angela
abstract

Parropolyenes, also called parroenes, parrodienes and psittacofulvines, are aldehydes costituted of an unsaturated chain ranging from 8 to 18 carbon atoms. These compounds have been isolated from the plumage of parrots and are also synthesized in the laboratory. Parropolyenes have antiproliferative and antioxidant activities. We investigated the effect of chain length and the role of aldehydic function of some parropolyenes by comparing their biological activities with those of their corresponding alcohols. Trans, trans-Δ2,4-hexadienal at a concentration of 100 μM significantly inhibited the growth of SH-SY5Y cells after 72 h of incubation. The corresponding alcohol was less effective. All-trans-Δ2,4,6-octatrienal and all-trans-Δ2,4,6,8,10-dodecapentaenal were more effective since significant inhibition of growth was observed at a concentration of 10 μM. Again, the corresponding alcohols were less effective. All-trans-Δ2,4,6-octatrienal and all-trans-Δ2,4,6,8,10-dodecapentaenal at 1 μM concentration protected 2-deoxyribose from degradation by ferrous ions. Trans, trans-Δ2,4-hexadienal at the same concentration does not show this protective effect. The protection increased with increasing parropolyene chain length. No significant difference was observed between aldehydes and their corresponding alcohols. Parropolyenes increased the rate of lysis of erythrocyte membrane in a lactoperoxidase-potassium iodide-hydrogen peroxide assay. The cytolytic effect increased with increasing parropolyene chain length. In this assay no difference was observed between aldehydes and alcohols. In all assays the effects were concentration dependent.

2006 - RELATIONSHIP BETWEEN THE ANTIOXIDANT PROPERTIES AND THE PHENOLIC AND FLAVONOID CONTENT IN TRADITIONAL BALSAMIC VINEGAR [Relazione in Atti di Convegno]
Verzelloni, Elena; Tagliazucchi, Davide; E., Panicucci; Conte, Angela
abstract

The antioxidant properties of traditional balsamic vinegar as regards its phenolic and flavonoidcontent comparing it to selected vinegars and red wines have been investigated. Thepolyphenols were separated from interfering compounds utilizing C18 columns. Thepolyphenolic content was determined utilizing both Folin-Ciocalteu and peroxidase assays. Theantioxidant capacity was quantified using both ABTS and FRAP assays. The results show thattraditional balsamic vinegar has lower antioxidant activity and phenolic and flavonoid contentthan Nero d’Avola but higher than the other tested products. The antioxidant capacity of winesand vinegars is highly correlated with their phenolic content, measured by peroxidase assay andit is also highly correlated with their flavonoid content while in traditional balsamic vinegar andbalsamic vinegar this correlation diminishes suggesting that other antioxidant compounds arepresent. The study describes a simple and fast method of separating from other compounds andof measuring polyphenols in the analysis of red wines and vinegars with complex compositionsuch as traditional balsamic vinegar.

2006 - Serum Heat Shock Protein in Knee Joint Osteoarthritis Patients Treated with Grass Thermal Therapy [Articolo su rivista]
E., Verzelloni; F., Russo; G., Agostini; P., Manica; Conte, Angela
abstract

In thermal resort of Garniga in Bondone Alp, Trento (Italy), spa therapy of osteoarthritis is carried out with applications on body of fermenting grass from Bondone prairies, 1600 m above sea-level. During application heat is developed and grass compounds are released. The mechanism of therapy is not well known. Heat shock proteins 70 (Hsp 70) and 60 (Hsp 60) were determined in serum of fifteen patients with knee joint osteoarthritis treated with standard therapy (11 applications in 2 weeks). Clinical assessments were carried out and serum was collected before and after the third and the ninth application and after one, three and six months. Hsps 70 and 60, were determined by immunoassay.Funtional impairment (Lequesne’s index), pain rating, quality of life indexes, analgesic and/or NSAID consumption significantly improve after treatment up to the end of the follow-up period. Improvement of physical activity and of psychic and physical wellbeing is also obtained. Significant increasing of Hsp 70 level is observed after the third application with a maximum after the 9th application then decreases progressively. No variations of Hsp 60 serum level are found. No adverse reactions were reported.Conclusion. Heat produced by grass applications determines a controlled repeated heat shock which increases extra-cellular and probably intracellular Hsp 70 at joint level increasing cellular defences and modulating local and general immunity responses. Possible roles of grass-released compounds remain to be investigated.

2006 - Vinegar polyphenols increase the proteolysis and decrease the oxidative stress during digestion by pepsin [Abstract in Atti di Convegno]
Tagliazucchi, Davide; Verzelloni, Elena; Conte, Angela
abstract

Vinegar polyphenols increase the proteolysis and decrease the oxidative stress during digestion by pepsin

2005 - Calcium/calmodulin dependence of nitric oxide synthase from Viviparus aeter immunocytes [Articolo su rivista]
Tagliazucchi, Davide; Conte, Angela
abstract

The calcium ion dependence of soluble and particulate NOS activity from Viviparus ater hemocytes has been investigated. At 2 nM calcium ion concentration the NOS activity measured by citrulline formation is 27.1 ± 2.2 and 9.3 ± 0.8 pmol.min-1.106cell-1 for soluble and particulate NOS respectively. The increase of free calcium ion concentration up to 300 nM increases the enzyme activity to 57.5 ± 4.1 and 23.5 ± 1.2 respectively. The 50% activation of the calcium dependent activity is 91 and 97 nM Ca2+ for soluble and particulate enzyme. Trifluoperazine, an inhibitor of calmodulin dependent enzyme, partially inhibits both activities. The soluble NOS is five time more sensible than particulate NOS. The behaviour of both activities to three NOS inhibitors (7-nitroindazole, S-methylisothiourea sulfate, diphenyleneiodonium) is very similar with not significant differences in IC50 values. The calcium ion dependence of NOS activities in a range of free calcium ion variations which are transiently observed in receptor stimulated cells suggests that NO in V. ater hemocytes has not only a defensive role but also a signalling relevance in cross-talking between hemocytes and other cells.

2005 - Differente tolleranza all’ossigeno e dipendenza dal Ca2+ della NOS nel cervello di tre diverse specie di pesci [Abstract in Atti di Convegno]
Tagliazucchi, Davide; Verzelloni, Elena; Conte, Angela
abstract

nd

2005 - Effect of some phenolic compounds and beverages on pepsin activity during simulated gastric digestion [Articolo su rivista]
Tagliazucchi, Davide; Verzelloni, Elena; Conte, Angela
abstract

The effect of some polyphenols (resveratrol, catechin, epigallocatechin-3-gallate, and quercetin) and beverages (red wine and green tea) on the enzymatic activity of pepsin during the digestion of three different substrates (pork meat, insoluble azocasein, and denatured hemoglobin) has been investigated. The tested polyphenols and beverages increase the initial velocity of the reaction, and the activating effect is concentration dependent. The order of effectiveness of polyphenols in increasing the initial velocity of the reaction is resveratrol >= quercetin > epigallocatechin-3-gallate > catechin. The kinetic data obtained with soluble denatured hemoglobin show that the Km for the substrate is not changed, whereas the V-max of the reaction is increased. Pepsin activity follows a simple Michaelis-Menten kinetic suggesting that k(3) is increased by polyphenols. To the authors' knowledge, the present study is the first demonstration that some polyphenols and related beverages are able to enhance the enzymatic activity of pepsin.

2005 - Il Pianeta Acqua nel Continente Agricoltura (Congresso Nazionale dell’Associazione Italiana delle Società Scientifiche Agrarie – AISSA) [Altro]
Stanca, Am; Arru, Laura; Bignami, Cristina; Conte, Angela; Endrighi, Emiro; Franchini, ; Lofiego, D; Manicardi, Gian Carlo; Orlandini, Stefano; Pellegrini, ; Ulrici, Alessandro; Bacarella, Borin; Dazzi, Espen; Gallerani, Giupponi; Magnani, ; Pecchioni, Nicola; Poni, Rossi; Zanni,
abstract

Nell’era della specializzazione nel settore della Ricerca Scientifica, il Convegno ha l’ambizione di mettere insieme i singoli componenti del mondo scientifico agrario, di farli interagire tra di loro e di tentare di affrontare il problema Acqua in modo interdisciplinare. L’avanzamento delle conoscenze sul ruolo dell’acqua nel “Continente Agricoltura” garantirà ricadute di notevole interesse a breve, medio e lungo termine, per migliorare ulteriormente l’interazione “Organismi viventi di interesse agrario e forestale - Terreno – Atmosfera”. L’obiettivo finale è infatti quello di assicurare per il futuro uno sviluppo sostenibile, grazie alla razionale gestione di un fattore ambientale ed economico primario, l’Acqua.

2005 - Variazione plasmatica delle HSPs 70 in pazienti gonartrosici sottoposti a terapia termale con applicazione di fanghi caldi delle Terme di Castrocaro [Relazione in Atti di Convegno]
G., Ronca; G., Agostini; F., Russo; M., Conti; Tagliazucchi, Davide; Conte, Angela
abstract

Le proteine dello shock termico o heat shock protein (HSP) sono proteine che aumentano nelle cellule in seguito a stress di varia natura, il primo dei quali ad essere stato identificato è stato quello termico. In questa ricerca abbiamo valutato il livello di HSP70 e HSP60 nel plasma di 20 pazienti maschi gonartrosici trattati in ambiente termale con 9 applicazioni di fanghi caldi (50°C) per 20 minuti sul 50-70% della superficie corporea. Le HSP70 aumentavano già alla terza applicazione, l'aumento diventa significativo alla nona applicazione e permane anche dopo 30-35 giorni. L'aumento si correla al miglioramento degli indici patologici della gonartrosi. Le HSP 60 invece non subiscono modificazione. L'applicazione dello stimolo termico in ambiente termale determina un aumento delle difese intracellulari, modula la risposta infiammatoria sia locale che generale diminuendo il dolore e migliorando la funzione articolare.

2005 - Variazione plasmatica delle HSPs70 in pazienti gonartrosici sottoposti a terapia termale con applicazione di fanghi caldi delle Terme di Castrocaro(FC) [Articolo su rivista]
G., Ronca; G., Agostini; F., Russo; M., Conti; Tagliazucchi, Davide; Conte, Angela
abstract

Le proteine dello shock termico o heat shock protein (HSP) sono proteine che aumentano nelle cellule in seguito a stress di varia natura, il primo dei quali ad essere stato identificato è stato quello termico. In questa ricerca abbiamo valutato il livello di HSP70 e HSP60 nel plasma di 20 pazienti maschi gonartrosici trattati in ambiente termale con 9 applicazioni di fanghi caldi (50°C) per 20 minuti sul 50-70% della superficie corporea. Le HSP70 aumentavano già alla terza applicazione, l'aumento diventa significativo alla nona applicazione e permane anche dopo 30-35 giorni. L'aumento si correla al miglioramento degli indici patologici della gonartrosi. Le HSP 60 invece non subiscono modificazione. L'applicazione dello stimolo termico in ambiente termale determina un aumento delle difese intracellulari, modula la risposta infiammatoria sia locale che generale diminuendo il dolore e migliorando la funzione articolare.

2004 - NO in the developing gut of Dicentrarchus labrax: an early immune role ? [Abstract in Rivista]
Mola, Lucrezia; Pederzoli, Aurora; Gambarelli, Andrea; Tagliazucchi, Davide; Conte, Angela
abstract

It is known that nitric oxide (NO) plays an important role in the immune-neuroendocrine communications. In this issue, we have examined the appearance and distribution of nitric oxide sinthase (NOS) histochemically, immunohistochemically and biochemically during development of the sea bass gut. In 4 and 24-day-old larvae the NOS activity evaluated by NADPH-diaphorase was strong in all epithelial cells and in cell and fibers of intestinal wall, at all gut levels. In the same larval stages and localizations immunoreactive material to antibodies against nNOS and iNOS was present. In the 5-month-old adult gut both enzimatic activity for NADPH-diaphorase and immunoreactivity (IR) to anti-nNOS disappeared from epithelium remaining in the gastro-enteric nervous system. The IR to anti-iNOS completely disappeared. Western blot analysis showed that neuronal (about 150,000 mol. wt band) and inducible (about 135,000 mol wt band) NOS-immunoreactive proteins are present in 24 day-old larvae gut. In the 5 month-old adult gut nNOS and iNOS IR disappeared in the soluble fraction of crude gut homogenates. A small amount of nNOS IR was present in particulate gut fraction. Our data show that both the calcium-calmodulin dependent nNOS and calcium-independent iNOS are present in the larval gut of sea bass. In this species the maturation of cell-mediated immune responses and humoral immune system takes place respectively around the first and second month post hatching (Scapigliati et al., 2002). The presence of inducible NOS in the same regions of the sea bass gut in which the GALT will differentiate, may suggest for NO a role in early defence mechanisms, before of establishment of immune responses in GALT.

2003 - Effect of resveratrol and catechin on PC12 tyrosine kinase activities and their synergistic protection from beta-amyloid toxicity [Articolo su rivista]
Conte, Angela; S., Pellegrini; Tagliazucchi, Davide
abstract

Beta-Amyloid peptide (beta-AP) is the main component of amyloid deposits around the cerebral vessel and in the brain parenchyma in Alzheimer's disease and Down's syndrome. In vitro studies in neuronal cells or in PC12 and Hela cell lines have shown that the aggregate form of beta-AP is toxic. Many genetic and environmental factors including metal ions, proteoglycans, plasma proteins and antioxidants modify beta-AP toxicity. We investigated the effect of two plant polyphenols-resveratrol and catechin-on soluble and particulate tyrosine kinase activity from PC12 cells and the protective action of these compounds against beta-AP (1-41) toxicity. beta-AP (1-41) decreased PC12 viability with an IC50 value of 1.1 +/- 0.14 x 10(-8) M. Resveratrol and catechin protected PC12 cells from beta-AP (1-41) toxicity. With 25 muM resveratrol the IC50 value increased to 2.2 +/- 0.19 x 10(-7) M. In the presence of beta-AP (1-41) resveratrol showed a concentration-dependent biphasic effect, and at a concentration of up to 40 muM it protected PC12 cells from beta-AP (1-41) toxicity. At concentrations higher than 40 muM, an inhibitory activity on cell proliferation appeared. This antiproliferative effect was also seen in the absence of beta-AP (1-41). With 100 muM catechin the IC50 value increased from 1.1 +/- 0.14 x 10(-8) M to 3.2 +/- 0.25 x 10(-7) M beta-AP (1-41). The protective effect was concentration dependent. Resveratrol and catechin had a synergistic protective action. In the presence of 40 muM catechin and 10 muM resveratrol or 20 muM resveratrol and 10 muM catechin, the toxicity determined by 10(-7) M beta-AP (1-41) was almost completely removed. Resveratrol and catechin had different effects on PC12 tyrosine kinase activity. With peptide 1-17 of gastrin as substrate, resveratrol inhibited particulate tyrosine kinases while it had no effect on soluble activity. With the same substrate, catechin increased the activity of soluble fraction while it inhibited particulate activity When peptide 6-20 of cell division kinase p34(cdc2) was utilized, catechin showed an opposite effect, inhibiting soluble tyrosine kinase activity and increasing particulate activity With peptide 6-20, resveratrol inhibited both soluble and particulate activities. These results demonstrate that resveratrol and catechin have different activities on the signal transduction pathway involving protein phosphorylation. These differences may contribute not only to the different effects of these compounds on PC12 growth but also to the synergistic effect against beta-AP (1-41) toxicity The different activity of resveratrol and catechin on signal transduction pathways, as well as the differences in metal chelation, partition coefficient between water and lipids, hydrogen donation redox potential and enzyme inhibition may be at least in part based on synergistic protection against beta-AP (1-41) toxicity.

2003 - Physiologic pH changes modulate calcium ion dependence of brain nitric oxide synthase in Carassius auratus [Articolo su rivista]
Conte, Angela
abstract

Species of the fish genus Carassius survive prolonged anoxia. Nitric oxide (NO) regulates cerebral blood flow in these fish during normoxic conditions whereas adenosine is the main vasoregulating molecule during anoxia. We investigated the calcium ion dependence of Carassius auratus brain NO synthase (NOS) as a function of pH. The physiological pH decrease from 7.2 to 6.8, which takes place during anoxia, greatly decreases NOS activity. This strong pH dependence is mainly due to variation of the calcium sensitivity of the enzyme. The EC50 is 0.15 muM at pH 7.2 and 2.1 muM at pH 6.8 for the soluble enzyme. The particulate enzyme is also dependent on pH variations. The reduced sensitivity to calcium ions at acidic pH decreases both NO and H2O2 production, saving the cells by suppression of the formation of potentially toxic nitrogen and oxygen species. Modulation of NOS activity by variation of its calcium affinity within the range of physiological pH constitutes an important and rapid mechanism to control the formation of NO and H2O2 during normoxia-anoxia and anoxia-normoxia transitions. (C) 2002 Elsevier Science B.V. All fights reserved.

2003 - Relationship between iron and protein content of dishes and polyphenol content in accompanying wines [Articolo su rivista]
G., Ronca; L., Palmieri; S., Maltinti; Tagliazucchi, Davide; Conte, Angela
abstract

The traditional combination of wines and dishes is highly complex, elaborated and refined. The aim of this study was to investigate the possible relationship between the chemical composition of wines and dishes that determines their combination. We determined the content of total polyphenols in 56 wines. The content of total proteins, total lipids, kilocalories, sodium, potassium, calcium, copper and zinc were determined in 44 raw foods and 44 dishes. Nine gourmets independently chose three wines for each food. We correlated the content of the chemical constituents of foods with the phenol content of wines combined with each food by the gourmets. A significant positive correlation was obtained between the phenol content of wines and iron (r = 0.81, p < 0.0001) and total protein content (r = 0.66, p < 0.0001) of foods. Nine gourmets composing a second panel chose three wines for each dish. A significant positive correlation was also obtained between the phenol content of wines and iron (r = 0. 69, p < 0. 000 1), total protein (r = 0. 50, p < 0. 0006) and potassium (r = 0. 45, p < 0. 002) in dishes combined with wines by the second panel of gourmets. Plant phenols decrease the intestinal absorption of iron and have antioxidant activity in the intestinal tract and elsewhere in the body These positive effects compensate the negative antinutritional activity toward protein digestion. The traditional combination of wines and dishes appears to be very favorable since wines poor in phenols are combined with dishes poor in iron and/or proteins to minimize their possible antinutritional effects, while phenol-rich wines are combined with dishes rich in iron to decrease iron absorption and prandial peroxidative stress.

2003 - Synergistic protection of PC12 cells from β-amyloid toxicity by resveratrol and catechin [Articolo su rivista]
Conte, Angela; S., Pellegrini; Tagliazucchi, Davide
abstract

Beta-Amyloid peptide (beta-AP) elicits a toxic effect on neurons in vitro and in vivo. Many environmental factors including antioxidants, metal ions and proteoglycans modify beta-AP toxicity. We have investigated on PC12 cells, the protective effect from beta-AP (1-41) of two plant polyphenols, resveratrol and catechin. PC12 cells treated with 10(-6) M beta-AP (1-41) for 16 h decrease the cell viability at 24% of the control with an IC50 value of 1.1 +/- 0.14 x 10(-8) M. Twenty-five micromolar resveratrol and 50 muM catechin protect PC12 cells from beta-AP (1-41) toxicity with the IC50 value increased at 2.2 +/- 0.19 x 10(-7) M and at 8.9 +/- 0.7 x 10(-8) M, respectively. While the protective effect is concentration dependent for catechin, resveratrol shows a concentration-dependent biphasic effect. Up to 50 muM concentration, resveratrol protects PC12 cells from beta-AP (1-41) toxicity. At concentration higher than 50 muM, an inhibitory activity on cell proliferation appears. This antiproliferative effect is shown also in the absence of beta-AP (1-41). Resveratrol and catechin have a synergistic protective action. In the presence of 50 muM catechin and 10 muM resveratrol or 25 muM resveratrol and 10 muM catechin, the toxicity determined by 10(-7) M beta-AP (1-41) is almost completely abolished. Catechin is more effective than resveratrol in protecting PC12 cells from the toxicity of hydrogen peroxide. The protection from Oxygen Reactive Species (ROS) toxicity is concentration dependent for both resveratrol and catechin. In this case the protection is merely additive and the synergistic effect is not observed. These results demonstrate that resveratrol and catechin protect PC12 cells from beta-AP (1-41) toxicity and that their protective effect is synergistic. Such a protective effect probably is not due only to their antioxidant activity. The different chemical and biological activity shown by these compounds on several cell types and the complexity of the beta-AP (1-41) toxicity may explain the synergistic protective effect and suggest that the utilization of different compounds with synergistic activity may protect more effectively from complex mechanisms of toxicity.

2002 - 50 Hz magnetic fields of varying flux intensity affect cell shape changes in invertebrate immunocytes: the role of potassium ion channels [Articolo su rivista]
Ottaviani, Enzo; Malagoli, Davide; A., Ferrari; Tagliazucchi, Davide; Conte, Angela; Gobba, Fabriziomaria
abstract

The effect induced by exposure to 50 Hz magnetic fields (MFs) in immunocytes from the mussel Mytilus galloprovincialis is evaluated. The whole animal was exposed for 15 and 30 min to MF intensities ranging from 200 to 1,000 microT. The changes in the cellular shape of immunocytes, expressed as shape factor (SF), were studied at different times after addition of the chemotacting substance N-formyl-Meth-Leu-Phe (fMLP). Results show that MFs provoke differing delays in fMLP-induced cellular shape changes: 200 microT are ineffective, while levels from 300 microT upwards cause a significant increase in immunocyte SF values compared to controls. Reactivation of the cells is possible up to an intensity of 600 microT. The use of PCO 400, an opener of ATP-sensitive K+ channels, shows that potassium channels are involved in the effect of MFs on M. galloprovincialis immunocytes.

2002 - Bone and bone-marrow interactions: Haematological activity of osteoblastic growth peptide (OGP)-derived carboxy-terminal pentapeptide. Mobilizing properties on white blood cells and peripheral blood stem cells in mice [Articolo su rivista]
Fazzi, R.; Testi, R.; Trasciatti, S.; Galimberti, S.; Rosini, S.; Piras, F.; L'Abbate, G.; Conte, A.; Petrini, M.
abstract

Osteogenic growth peptide (OGP) increases blood and bone marrow cellularity in mice, and enhances engraftment of bone marrow transplant. Carboxy-terminal pentapeptide (OGP10-14) holds several properties of full-length polypeptide. We evaluated whether synthetic OGP-derived pentapeptide (sOGP10-14) has some activity on peripheral blood cell recovery after cyclophosphamide-induced aplasia, and on stem cell mobilization. Peripheral blood stem cell (PBSC) mobilization was evaluated by administering granulocyte-colony stimulating factor (G-CSF) or sOGP10-14 after cyclophosphamide (CTX) injection. Haematological parameters and CD34/Sca-1 positive cells were sequentially evaluated. Colony-forming tests were performed in bone marrow cells from CTX-, G-CSF- and sOGP10-14-treated mice. sOGP10-14 was able to enhance band cells and monocyte recovery after cyclophosphamide administration. White blood cell (WBC) counts reached the maximum peak by day + 10 but, on day + 7, a significant recovery was already detected in sOGP10-14 treated mice. On day + 10 the WBC increase in sOGP10-14-treated mice was comparable to that found in G-CSF treated ones. Moreover, CD34/Sca-1 positive early precursors were significantly mobilized by sOGP10-14 compared to the control group. In sOGP10-14-treated mice, the colony-forming unit-granulocyte-macrophage-megakaryocyte (GEMM-CFU) and burst-forming unit-erythroid (BFU-E) were significantly increased in bone marrow cells in comparison to mice treated with CTX only. These results suggest a central role of sOGP10-14 in bone and bone marrow interaction, and a possible role of sOGP10-14 as a mobilizing agent. (C) 2001 Elsevier Science Ltd. All rights reserved.

2002 - Effect of lecithin on epicutaneous absorption of diclofenac epolamine [Articolo su rivista]
Conte, Angela; G., Ronca; M., Petrini; G., Mautone
abstract

The epicutaneous application of nonsteroidal antiinflammatory drugs in localized rheumatic diseases results in a highly targeted antiinflammatory action and is associated with reduced systemic effects. The new diclofenac epolamine (DHEP) salt is much more soluble both In water and In lipid solvent than other diclofenac salts. The pharmaceutical addition of lecithin to DHEP leads to the formation of mixed micelles with high affinity to the cellular component, which guarantees the absorption of the active ingredient. We performed a bioavailability randomized, cross-over study to compare the plasma profiles of diclofenamic acid after repeated epicutaneous administration of the new topical formulation with those of the marketed DHEP formulation without lecithin. Based on a randomization list, 12 healthy volunteers were asked to apply one of the two formulations twice a day for 10 consecutive days. The other formulation was given after a washout period of 1 week. Blood samples were collected before the morning epiculaneous dose on days 1, 3, 5 and 8 of treatment and on day 10 at different sampling times until 24 h after the application. The pharmacokinetic analysis showed a significantly higher plasma concentration of diclofenamic acid after the application of DHEP lecithin, which indicates a better saturation of the subcutaneous tissues underlying the application site. This also indicates increased local availability of the active principle. In conclusion, the new DHEP formulation with lecithin should have a therapeutic advantage compared with the formulation without lecithin, even in cases of short- to medium-term treatments.

2002 - Erratum: Differential activity of glycosaminoglycans on colony-forming cells from cord blood (Leukemia Research (1999) 23 (1015-1019) PII: S014521269900123X) [Articolo su rivista]
Conte, A.; Da Prato, I.; Petrini, M.; Testi, R.; Valentini, P.; Volpi, N.
abstract

2002 - Protein kinases mediate nitric oxide-induced apoptosis in the insect cell line IPLB-LdFB [Articolo su rivista]
Malagoli, Davide; Conte, Angela; Ottaviani, Enzo
abstract

The involvement of protein kinases (PKA, PKC and PKB) in nitric oxide (NO)-induced apoptosis with sodium nitroprusside plus N-acetyl-L-cysteine in the IPLB-LdFB cell line from the insect Lymantria dispar was investigated. The presence of protein kinase-like molecules was demonstrated by Western blot analysis. The role of the kinases in programmed cell death was analysed in cytofluorimetric experiments by incubating the insect cells with H-89 (a specific inhibitor of PKA), calphostin C (an inhibitor of PKC) or wortmannin (an inhibitor of phosphatidylinositol 3-kinase). The results show that PKA is correlated with the induction and PKC and PKB with the prevention of NO-induced insect cell death. Moreover, NO-induced apoptosis involves the release of cytochrome c.

2001 - Role of pH on the calcium ion dependence of the nitric oxide synthase in the carp brain [Articolo su rivista]
Conte, Angela
abstract

The role of pH on the calcium dependence of nitric oxide synthase (NOS) of Cyprinus carpio brain was investigated. This fish is known to survive prolonged periods of hypoxia. Under this condition, cerebral blood flow is no longer regulated by nitric oxide (NO). Nitric oxide synthase activity is pH dependent in the range of pH between 7.4 and 6.2 with a decrease when tissue acidifies. At acidic pH, the dependence of the NOS activity on the free Ca2+ concentrations changes considerably and shows an EC50 of 0.13 muM at pH 7.1 and of 5.1 muM at pH 6.2 for the soluble enzyme. The variation in the Ca2+ dependence with acidification is greater for the soluble than for the particulate enzyme. This may be the main factor protecting sudden NO formation mainly during anoxic-normoxic transitions.

1999 - Differential activity of glycosaminoglycans on colony-forming cells from cord blood [Articolo su rivista]
Conte, A.; Da Prato, I.; Petrini, M.; Testi, R.; Valentini, P.; Volpi, N.
abstract

Heparin, heparan sulfate and chondroitin sulfate were evaluated for their possible role on proliferation and differentiation of hematological precursor cells from cord blood. For these purposes, different concentrations of glycosaminoglycans were added to methyl-cellulose in colony assay performed with human cord blood derived cells. A volume of 10 microg/ml heparin induces a significant increase of both granulocyte-monocyte and granulocyte colonies, and a decrease of erythroid-colonies, more evident in the presence of 100 microg/ml. Heparan sulfate-treatment induces a significant increase of all granulocyte-monocyte colonies derived from CFU-granulocyte-monocyte, CFU-granulocyte and CFU-monocyte precursors. A significant decrease of multipotent cells was also observed. On the other hand, chondroitin sulfate induces an increase of granulocyte-colonies and a decrease of erythroid-colonies. Glycosaminoglycans with different structure may be useful to increase the number of specific colonies. The selective and differential binding of glycosaminoglycans with several growth factors and the regulation of their activities is discussed.

1999 - NMR and pressure correlated analysis of metabolic changes in soft-X-rays irradiated yeast cells [Articolo su rivista]
M., Milani; Conte, Angela; Costato, Michele; Salsi, Francesca; G., Baroni; D., Batani; L., Ferraro; I. C. E., Turcu
abstract

An NMR technique for the identification of the energy related molecules, AMP, ADP and ATP, is suitably taylored to follow up the dynamics of the glycolitic metabolism in a suspension of yeast cells. Such a technique proves to be efficient to investigate soft X-ray radiation damage as complementary to other physical and chemical assessment.

1998 - Characterization of Cyprinus carpio brain nitric oxide synthase [Articolo su rivista]
Conte, Angela; Ottaviani, Enzo
abstract

Nitric oxide synthase (NOS) activity is found both in soluble and in particulate fractions of the carp brain. The K-m values for arginine are 2.8 +/- 0.5 and 3.3 +/- 0.4 mu M for the soluble and particulate fractions, respectively. K-i for N-G-monomethyl-L-arginine inhibitor are 2.6 +/- 0.5 and 2.9 +/- 0.6 mu M, and activation energy for the breakdown of the substrate-enzyme complex 8120 +/- 710 and 4620 +/- 450 cal per mole. Carp enzyme shows higher affinity than rat NOS for Ca2+ and for the competitive inhibitor 7-nitroindazole.

1998 - Differential activity of glycosaminoglycans on colony-forming cells from cord blood [Poster]
Da Prato, I.; Valentini, P.; Testi, R.; Volpi, Nicola; Conte, Angela; Petrini, M.
abstract

Differential activity of glycosaminoglycans on colony-forming cells from cord blood

1998 - Lysis of plasma cell membrane by reactive oxygen species and protection by nitric oxide and chondroitin sulfate [Poster]
Conte, Angela; Volpi, Nicola; Ronca, G.
abstract

Lysis of plasma cell membrane by reactive oxygen species and protection by nitric oxide and chondroitin sulfate.

1997 - Interaction of low power laser (LPL) on isolated and integrated cellular biosystems [Articolo su rivista]
Volpi, Nicola; Conte, Angela
abstract

ND

1996 - Brain death criteria knowledge and attitude toward organ donation m medical and non medical hospital staff in the district of naples (italy) [Articolo su rivista]
De Falco, F. A.; Majello, L.; Santangelo, R.; Mastroroberto, G.; Coppeta, D.; Conte, A.
abstract

In the area of Naples the rate of organ donation has remained significantly low during the last decades. Three crucial factors are involved in such a phenomenon: 1) the italian laws concerning brain death and organ donation, which have been modified and updated in recent years, especially as far as the consensus is concerned; 2) the organization of general and university hospitals, in particular in the area of the intensive care units, and the specific attitude of both medical and paramedical professionals; 3) the 'culture' of organs donation among the citizens of the Naples area, where an accurate and detailed information and education campaign appears mandatory, but only after a specific campaign selectively dedicated to the health professionals working in the hospitals network.

1996 - Effects of an Electromagnetic Pulsed Field on Saccharomyces cerevisiae: Cell Growth, CO2 Production, Adenine Nucleotides and Trehalose Content in Samples Submitted to 5-5000 Hz [Articolo su rivista]
Conte, Angela; L., Erspamer; L., Bolognani
abstract

ND

1995 - Biochemical and pharmacokinetic aspects of oral treatment with chondroitin sulfate [Articolo su rivista]
Conte, Angela; Volpi, Nicola; L., Palmieri; I., Bahous; G., Ronca
abstract

Chondroitin sulfate (Condrosulf(R)) was characterized for structure, physicochemical properties and purity. This glycosaminoglycan has a relative molecular mass of about 14,000, a sulfate-to-carboxyl ratio of 0,95 due to the high percentage of monosulfated disaccharides (38 % 6-monosulfate and 55 % 4-monosulfate) and a low amount of disulfated disaccharides (1.1 %) inside the polysaccharide chains No other glycosaminoglycans were detected in the preparation. Chondroitin sulfate was labelled by reduction with sodium H-3-borohydride and administered by oral route in the rat and dog. More than 70 % of radioactivity,vas absorbed and found in urine and tissues. The plasma radioactivity was fractionated by size-exclusion chromatography in three fractions: radioactivity associated with high, intermediate and low molecular mass compounds. The peak value of the concentration of high molecular mass radioactivity compounds in plasma was reached after 1.6 and 2.1 h for the rat and dog, respectively After 36 h the high molecular mass radioactivity compounds were still present in plasma of dog and rat. After 24 h radioactivity was higher in the intestine, liver, kidneys, synovial fluid and cartilage than in other tissues Condroitin sulfate was orally administered to man (healthy volunteer) in a single daily close of 0.8 g and in two daily doses of 0.4 g. The results showed that both forms of administration determined a significant increase of plasma concentration of chondroitin sulfate as compared with predose value over a full 24 h period. Elimination constant values and t(max) (of the first administration in the case of fractionated dose) were almost the same for the two administrations. Some biochemical parameters (number of leukocytes, proteins, sulfated glycosaminoglycans and hyaluronic acid amounts, and N-acetylglucosaminidase activity) of synovial fluid were evaluated in controls and treated osteoarthritic subjects. No variations were observed in the patient who did not receive chondroitin sulfate. Five days of chondroitin sulfate administration led to a significant increase of concentration and molecular mass of hyaluronan and a decrease of a lysosomal enzyme N-acetyl-glucosaminidase. No significant differences in leukocyte count and protein content were detected

1995 - EFFECT OF L-PROPIONYL CARNITINE ON SOME PROPERTIES OF ERYTHROCYTES AND LEUKOCYTES OF ALCOHOL ABUSERS [Articolo su rivista]
Conte, Angela; Bianchi, I; Guazzelli, M; Taponeco, G; Bertelli, A; Ronca, G.
abstract

The effect of L-propionyl carnitine, the carnitine derivative utilized as a more effective drug for membrane protection, on Na-K ATPase activity of erythrocyte gh os ts of alcohol-dependent patients and blood donors has been investigated The effect of L-propionyl carnitine on leukocyte chemotaxis and cytochrome c reduction, a measure of superoxide ion production, was also studied. It has been in fact observed that alcohol is immunotoxic on both the non-specific and the specific immune response. In alcohol-dependent erythrocytes, a significant higher value of the H-1-NMR spin lattice relaxation time (T1) was observed as compared to blood-donor erythrocytes. The in-vitro addition of ethanol increases the T1 values of blood donor erythrocytes, whereas it is without effect on the T1 value of alcohol-dependent erythrocytes. The Na-K ATPase activity is higher in alcohol-dependent erythrocyte ghosts as compared to blood-donor ghosts. A non-significant increase of the Na-K ATPase activity of blood-donor ghosts was observed with the increasing of L-propionyl carnitine concentrations (from 0.2 to 5.0 mM), whereas the Na-K ATPase activity of alcohol-dependent ghosts decreases. From these combined effects the differences of Na-K ATPase activity progressively decrease with the increasing of L-propionyl carnitine concentration, and no significant differences are observed between the two groups at L-propionyl carnitine concentrations higher than 0.5 mM. The in-vitro addition of ethanol increases the enzyme activity to a greater extent in blood-donor ghosts as compared to alcohol-dependent ghosts. This in-vitro activation by ethanol is decreased by the addition of L-propionyl carnitine. The chemotaxis induced by N-formyl-methionyl-leucylphenylalanine and the superoxide anion production stimulated by zymosan is significantly lower in alcohol-dependent neutrophils. L-Propionyl carnitine increases, in a dose-dependent way, both chemotaxis and superoxide anion production of alcohol-dependent neutrophils, and no significant difference was observed between the two groups at 5 mM L-propionyl carnitine. These experimental results suggest that L-propionyl carnitine administration may be useful for reducing some acute and chronic damages due to alcohol ingestion. The protective and modulatory actions of L-propionyl carnitine may be even more evident in cells and tissues different from those investigated in this study and in which ethanol determines several biochemical damages.

1995 - Nitric oxide synthase activity in molluscan hemocytes [Articolo su rivista]
Conte, Angela; Ottaviani, Enzo
abstract

The hemocytes of the freshwater snail Viviparus ater have nitric oxide synthase (NOS) activity, as demonstrated by [H-3]citrulline and nitrite + nitrate formation. The enzyme is NADPH dependent and is competitively inhibited by the mammalian NOS inhibitor N-G-monomethyl-L-arginine (K-i = 4.7 mu M). The K-m for L-arginine is 2.5 mu M. 70% of the total activity is observed at very low free Ca2+ concentration (3 nM). LPS treatment increased total NOS activity 2.4 fold. The activity is partly present in the non-soluble fraction of hemocytes (24% and 8% in non-stimulated and LPS-stimulated snails, respectively). An antiserum to the C-terminal synthetic pentadecapeptide of the rat cerebellar NOS inhibited the enzyme activity in a concentration-dependent manner. This is the first biochemical demonstration of the existance of NOS activity in molluscan hemocytes, the cells responsible for defence mechanisms.

1995 - Nitric oxide: An ancestral immunocyte effector molecule [Articolo su rivista]
Franchini, Antonella; Conte, Angela; Ottaviani, Enzo
abstract

The presence and the role of nitric oxide synthase (NOS) were investigated in the molluscan hemocytes by immunocytochemical, biochemical and functional ap preaches. Using an anti-NOS polyclonal antibody, immunoreactivity was observed in the hemocytes, and this reactivity increased after stimulation of the animals with Escherichia coli, indicating that this enzyme is inducible. The NOS inducibility was also histochemically demonstrated by detection of NADPH-diaphorase activity. Biochemical studies show that the enzyme is 70% cytoplasmatic and 30% membrane bound and that the inducible form is mainly cytoplasmatic. The nitrite + nitrate and citrulline formation, the inhibition by N-omega-nitro-L-arginine, the Km value for arginine, the calcium and co-enzyme dependence show that the molluscan NOS shares the same properties as the NOS isoenzymes so far studied. However, it cannot be identified with any of these enzymes. It appears to be in some way similar to an inducible form of human hepatocyte NOS. Also cytokines are able to induce NOS. In vitro studies have shown that hemocytes produce nitric oxide (NO), a bactericide substance, and that there is a relationship between the NO system and phagocytosis. The presence of NO in the invertebrate hemocyte demonstrates that critical molecules have been conserved over the course of evolution.

1994 - Effects of glycosaminoglycans on U-937 leukemia cell proliferation and differentiation: Structure-function relationship. [Poster]
Volpi, Nicola; Petrini, M; Conte, Angela; Valentini, P; Venturelli, T; Bolognani, Lorenzo; Ronca, G.
abstract

Effects of glycosaminoglycans on U-937 leukemia cell proliferation and differentiation

1994 - Glutathione S-transferases in chlorambucil resistance: Case report [Relazione in Atti di Convegno]
Di Simone, D.; Conte, A.; Mattii, L.; Valentini, P.; Sabbatini, A.; Petrini, M.; Grassi, B.
abstract

1994 - L-PROPIONYL CARNITINE PROTECTS ERYTHROCYTES AND LOW-DENSITY LIPOPROTEINS AGAINST PEROXIDATION [Articolo su rivista]
Bertelli, A; Conte, Angela; Ronca, G.
abstract

The effects of peroxidation on the erythrocytes of rats orally treated with L-propionyl carnitine for 15 day) (50 mg/kg/day/ were investigated. Peroxidation was produced by incubating the cells in the presence of the cytotoxic system: lactoperoxidase-hydrogen peroxide and iodide ions. Lysis of erythrocytes was evaluated by measuring the turbidity following the decrease in absorbance at 600 nm. The 50% of erythrocyte lysis of untreated animals was observed after 16 min and in about 30 min all the cells were lysed. With L-propionyl carnitine-treated rat erythrocytes the time at which 50% of lysis was observed increased to 23 min. L-propionyl carnitine also exerted its protective effect in vitro when incubated with untreated rat erythrocytes or human erythrocytes in the presence of the cytolytic system. The presence of L-propionyl carnitine in the incubation mixture markedly decreased the malonaldehyde formation. The protection was concentration-dependent. To establish if L-propionyl carnitine protects from oxygen reactive species or is able to stabilize the damaged membranes, a latent damage was produced by incubating the erythrocytes with the cytolytic system for a few minutes. The cells were then removed and suspended in buffered saline in the absence or in the presence of different L-propionyl carnitine concentrations L-propionyl carnitine decreased the velocity of lysis of damaged erythrocytes. These data suggest that L-propionyl carnitine protects erythrocytes from oxygen reactive species and also stabilizes the damaged membrane probably by specific binding with protein and/or phospholipid domains. Low density lipoproteins (LDLs) from human blood were peroxidized by exposure to Cu2+ ions in the presence of various L-propionyl carnitine concentrations. The formation of malonaldehyde decreased in the presence of L-propionyl carnitine, These findings provide evidence that L-propionyl carnitine protects cell membrane and circulating lipoproteins from peroxidation and stabilizes the cell membrane damaged by oxygen reactive species.

1994 - effects of glycosaminoglycans on u-937 leukemia-cell proliferation and differentiation - structure-function relationship [Articolo su rivista]
VOLPI, Nicola; PETRINI, M; CONTE, Angela; VALENTINI, P; VENTURELLI, T; BOLOGNANI, L; RONCA, G.
abstract

Glycosaminoglycans (heparins, dermatan sulfate, chondroitin sulfate) with different structures and physicochemical properties were evaluated for their capacity to influence proliferation and differentiation of U-937 cell line. The contrasting and specific effects of glycosaminoglycans (depending on their structures and properties) on a leukemia cell line could help explain the regulation of proliferative and/or differentiative processes of hematopoietic cells in order to clarify the control of development and differentiation of hematopoietic progenitor cells by bone marrow extracellular matrix. Heparin from beef intestinal mucosa, heparan sulfate from beef spleen, dermatan sulfate from beef intestinal mucosa, and chondroitin sulfate from bovine trachea were extracted and purified, and their purity, structures, and physicochemical properties were evaluated. Past-moving heparin was obtained by its selective precipitation as barium salt, and partially desulfated and re-N-sulfated heparin was produced by chemical modifications. Different glycosaminoglycans were tested to evaluate their effects on proliferation and differentiation processes of a monoblastic leukemia cell line (U-937). Heparin and derivatives (from 0.1 to 100 mu g/ml) inhibit eel proliferation; heparan sulfate does not produce modifications, while chondroitin sulfate and dermatan sulfate (from 0.01 to 100 mu g/ml) significantly stimulate cell growth. Cell differentiation was evaluated by cytoenzymatic determination of alpha-naphthyl butyrate esterase and by fluorescein-labeled anti-HLA-DR, anti-CD11b, and anti-CD14 antibodies. Nitro blue tetrazolium reduction and phagocytosis were also evaluated. Heparin and derivatives significantly increase U-937 differentiation. Heparin sulfate has no effect, while chondroitin sulfate and, to a lesser extent, dermatan sulfate, induce a strong decrease of differentiative markers. The regulation of U-937 cell properties appears to be related to charge density and to the amount of N-sulfate and N-acetyl groups. In particular, glycosaminoglycans with lower sulfate-to-carboxyl ratios and N-sulfate group percentages (chondroitin sulfate and dermatan sulfate) stimulate proliferation and produce a decrease of differentiative markers; on the contrary, polysaccharides with high charge density and N-sulfate group amounts (heparin and derivatives) inhibit U-937 proliferation and induce terminal differentiation. A previous paper (N. Volpi, L. Bolognani, A. Conte, and M. Petrini, (1993) Leukemia Res. 17, 789-798) demonstrates dissimilar effects on U-937 cells by chondroitin sulfates with different structures and physicochemical properties. In this study we confirm the importance of glycosaminoglycan structures and physicochemical properties in regulating cell functions. Possible mechanisms of action are discussed.

1993 - Activity of Different Revertant Agents on Multidrug Resistance: in Vitro Evaluation of their Combination [Articolo su rivista]
M., Petrini; L., Mattii; G., Carulli; A., Sabbatini; Conte, Angela; F., Vaglini; B., Grassi; G., Ronca
abstract

The revertant activity of different compounds has been assayed on a multidrug-resistant human breast-cancer cell line (MCF 7/Dx). The calcium-channel blocker nicardipine showed the higher revertant ability when compared to cefoperazone or cyclosporin A at concentrations close to the pharmacological range. Interestingly, nicardipine was able to increase the revertant activities of both cefoperazone and cyclosporin A, but these latter were not able to enhance each other over a plateau. However, a limit of about 70% of growth inhibition of the line cultured in the presence of 60 microM doxorubicin seems to be insuperable at the concentrations employed. The combination of the three drugs brings the concentrations of drugs to the point at which the maximum possible inhibition is reached in the pharmacological range, but the complete reversion of chemoresistance is not reached when the doxorubicin is added at the concentration capable of reducing the cell proliferation by 50%.

1993 - Metabolic Fate of Partially Depolymerized Shark Chondroitin Sulfate in Man [Articolo su rivista]
G., Ronca; Conte, Angela
abstract

Chondroitin sulfates and other glycosaminoglycans are administered as drugs to man by intravenous, intramuscular or oral routes. There are some studies on the pharmacokinetics of heparin, heparan sulfate and dermatan sulfate, whereas few data are available on the metabolic fate of chondroitin sulfate in man. Partially depolymerized chondroitin sulfate (mean mol. wt: 7.5 kd, range 5-10 kd) with a ratio of 1:3 between chondroitin-4-sulfate and chondroitin-6-sulfate has been administered as single administrations of 0.2 and 1.2 g by intramuscular and oral routes respectively to 10 healthy volunteers (5 males and 5 females), aging 25-53 years. After intramuscular administration the plasma level increased to a concentration peak at 90 min. The peak concentration, the elimination half-life and the apparent distribution volume were respectively 3.8 mcg/ml, 275 min and 0.40 ml/g. About 37% of the administered chondroitin sulfate is excreted in the urine during the first 24 h as high- and low-molecular-weight derivatives. After oral administration the concentration peak was observed at 240 min. The concentration at the peak, the elimination half-life and the apparent distribution volume were respectively 4.6 mcg/ml, 310 min and 0.44 ml/g. A peak of mono-, oligo- and polysaccharides with a molecular weight lower than 5 kd derived from partial digestion of exogenous chondroitin sulfate is also present in plasma. This study shows that about 10% and 20% of the orally administered drug is absorbed as high- and low-molecular-weight derivatives respectively. Comparison with the results obtained in experimental animals indicate that the metabolic fate of partially depolymerized chondroitin sulfate is similar in man and in experimental animals.

1993 - Modification of some markers of cartilage matrix metabolism by exogenous chondroitin sulfate [Poster]
Conte, Angela; Volpi, Nicola; Fioravanti, A.; Marcolongo, R; Ronca, G.
abstract

Modification of some markers of cartilage matrix metabolism by exogenous chondroitin sulfate

1993 - REVERSING OF CHLORAMBUCIL RESISTANCE BY ETHACRYNIC-ACID IN A B-CELL PATIENT [Articolo su rivista]
Petrini, M; Conte, Angela; Caracciolo, F; Sabbatini, Anna Maria Teresa; Grassi, B; Ronca, G.
abstract

We evaluated the reversing activity of ethacrynic acid in a B-CLL patient resistant to chlorambucil. The glutathione S-transferase (GST) activity, measured in peripheral blood lymphocytes, resulted extremely elevated. Ethacrynic acid, at pharmacological concentrations, partially reversed chlorambucil resistance and this result appeared related to the increased GST levels.

1993 - Vitamin D3 Administration and Multidrug Resistance in Acute Nonlymphoblastic Leukemia [Articolo su rivista]
M., Petrini; F., Caracciolo; G., Carulli; Conte, Angela; A., Sabbatini; L., Mattii; B., Grassi
abstract

This article reports preliminary results from a pilot study started in 1986 on patients with acute myeloblastic leukemia treated for several months with low-dose arabinosylcytosine and 1(OH)D3. During treatment or at the time of relapse, a monoblastic component was frequently found. A high percentage of patients were P-170-positive. In 2 patients it was possible to show that blasts, previously P-170-negative, became positive after treatment. In these 2 patients, failure of clinical response to antileukemic therapy was associated with this phenotype. The addition of the revertant drug nicardipine to the previously inactive treatment induced a partial response. Thus, previously reported in vitro observations on the differentiating activity of vitamin D3 metabolites, possible induction of multidrug chemoresistance by differentiating agents and the revertant activity of the Ca++ antagonist nicardipine appear to be confirmed in vivo in the reported patients.

1993 - effects of chondroitin sulfates with different structures on leukemia-cells - U-937 cell-proliferation and differentiation [Articolo su rivista]
Volpi, Nicola; Bolognani, L; Conte, Angela; Petrini, M.
abstract

Chondroitin sulfates extracted and purified by different manufacturers were tested to evaluate their effects on proliferation and differentiation processes of U-937 cells. The different chondroitin sulfates were evaluated for purity, structure and physicochemical properties. The three chondroitin sulfates utilized did not present other contaminant glycosaminoglycans and proteins and had about the same relative molecular mass but different disaccharide patterns and charge density. Chondroitin sulfates with small amounts of disulfated disaccharides and low charge density, at 5 mug/ml concentration, doubled (about + 133%) cell proliferation in comparison to controls. In contrast, chondroitin sulfates with large amounts of disulfated disaccharides and high sulfate to carboxyl ratio were less effective (about + 15%) in stimulating cell proliferation at low concentration. A decrease of U-937 cell proliferation was observed in proportion to the increased amounts of chondroitin sulfate with low sulfate to carboxyl ratio. On the contrary, chondroitin sulfate with large amounts of disulfated disaccharides produced increased cell proliferation depending on concentration. Small amounts (5-10 mug/ml) of chondroitin sulfates with low charge density reduced the differentiative process of U-937 cells. Chondroitin sulfate with large amounts of disulfated disaccharides and high charge density seemed to be able to produce a significant decrease of differentiative processes only at very high concentrations (1000 mug/ml). These contrasting effects of chondroitin sulfates with different disaccharide patterns (and structure) and charge density on a leukemia cell line could help to explain the regulation of proliferative and/or differentiative processes of hemopoietic cells. This is underlined by the changes of types, physicochemical properties and structure of glycosaminoglycans induced by different extracellular factors and agents.

1992 - CoA Protects VLDL Against Peroxidation and Increases the Plasma Triacylglycerol Metabolism [Articolo su rivista]
Conte, Angela
abstract

CoA (coenzyme A) has an antiperoxidative action and protects erythrocytes against oxygen free radicals. The peroxidation favours the uptake of the modified LDL (low-density lipoprotein) by macrophages and has a role in the development of foam cells. A possible relation between the antiperoxidative action of CoA and its normalizing activity on plasma lipids in type IIb and type IV hyperlipoproteinaemias, was investigated in order to see whether CoA protects VLDL (very -low-density lipoproteins) against peroxidation and produces a quicker removal of VLDL from circulation when administered intravenously to rats. The addition of 5 mM CoA to rat hepatocytes in culture was found to produce a significant decrease in VLDL secretion. The plasma clearance was significantly more rapid and the removal of triacylglycerols was significantly enhanced in CoA-treated rats as compared to untreated ones. Furthermore CoA reduced the formation of material reacting with thiobarbituric acid (TBA) in human VLDL peroxidized by exposure to Cu2+. Our study shows that CoA protects VLDL from peroxidation in a significant and concentration-dependent manner and increases plasma triacylglycerol metabolism.

1992 - Effect of CoA on Triglyceride and VLDL Secretion in Cultured Rat Epatocytes [Articolo su rivista]
Conte, Angela; G., Fraticelli
abstract

ND

1992 - Effect of different glycosaminoglycans on HL-60 and U-937 proliferation and differentiation [Poster]
Conte, Angela; Ronca, G.; Petrini, M.; Volpi, Nicola; Bolognani, Lorenzo
abstract

Effect of different glycosaminoglycans on HL-60 and U-937 proliferation and differentiation

1992 - Effect of heparin and D3 metabolite on proliferation and differentiation of U-937 human cell line [Poster]
Conte, Angela; Ronca, G.; Volpi, Nicola; Bolognani, Lorenzo; Petrini, M.
abstract

Effect of heparin and D3 metabolite on proliferation and differentiation of U-937 human cell line

1992 - Effect of mammalian and fish chondroitin sulfate on proliferation and differentiation of human leukemia cell line U-937 [Poster]
Conte, Angela; Ronca, G.; Volpi, Nicola; Bolognani, Lorenzo; Petrini, M.
abstract

Effect of mammalian and fish chondroitin sulfate on proliferation and differentiation of human leukemia cell line U-937

1992 - Effetto di condroitin solfato sulla proliferazione e il differenziamento di cellule leucemiche HL-60 [Poster]
Volpi, Nicola; Bolognani, Lorenzo; Petrini, M.; Valentini, P.; Conte, Angela; Ronca, G.
abstract

Effetto di condroitin solfato sulla proliferazione e il differenziamento di cellule leucemiche HL-60

1992 - Effetto di glicosaminoglicani sulla proliferazione e il differenziamento delle HL-60 e U-937 [Poster]
Volpi, Nicola; Bolognani, Lorenzo; Petrini, M.; Valentini, P; Conte, Angela
abstract

Effetto di glicosaminoglicani sulla proliferazione e il differenziamento delle HL-60 e U-937

1992 - Opposite effects of chondroitin sulfate and D3 metabolite on U-937 proliferation and differentiation [Poster]
Conte, Angela; Volpi, Nicola; Petrini, M.; Ronca, G; Bolognani, Lorenzo
abstract

Opposite effects of chondroitin sulfate and D3 metabolite on U-937 proliferation and differentiation

1992 - Recent Findings on the Regulatory Function of CoA and on the Normalizing Activity on Plasma Lipids of Exogenous CoA [Articolo su rivista]
Conte, Angela; G., Fraticelli; G., Ronca
abstract

In recent years many studies have shown that coenzyme A (CoA) is not only an acyl carrier coenzyme but it also has an important role in the regulation of metabolic functions and cell activities such as transport from the Golgi cisternae. This regulatory role is carried out by CoA, its precursor, catabolites and acylated derivatives. The acylation (myristylation and palmitylation) process of peptides and proteins dependent on CoA seems to be an important regulatory mechanism of cell activities. Furthermore exogenous CoA has been shown to decrease the triacylglycerols, cholesterol and Apo B of plasma lipoproteins in man. This regulatory mechanism acts either on VLDL synthesis and secretion or on their plasma clearance. CoA also protects cell-membrane and plasma lipoproteins against the peroxidative action of oxygen free-radicals.

1991 - Antidyslipaemic Action and Role of CoA in Lipid Metabolism of Mitochondria and Peroxisomes [Articolo su rivista]
Conte, Angela; L., Palmieri; D., Segnini; G., Ronca
abstract

Our study has evaluated the effect of the parenteral administration of CoA on the pattern of haematic lipids and on palmitate oxidation in liver mitochondria and peroxisomes of rats made hyperlipaemic with a high fat diet with or without CoA. Lipid fractions, total cholesterol, triacylglycerols, total lipids and nonesterified fatty acids (NEFA) were determined in blood. Palmitate oxidation was determined in liver peroxisomes and mitochondria incubated in modified Krebs-Henseleit solution with 100 microM 1-14C palmitate in the presence and absence of some cofactors. Our results show that in rats fed with a high fat diet there was an increase of all lipid fractions. The increase of all lipid components was lower in animals treated with CoA. In liver peroxisomes of rats fed with high fat diet an increase in palmitate oxidation, that is higher when CoA is parenterally administered, was observed. In addition, palmitate oxidation in mitochondria of rats treated with CoA reached values higher than those of control and of rats fed with a high fat diet without CoA.

1991 - Effect of Propionyl Carnitine on Cardiac Energy Metabolism Evaluated by the Release of Purine Catabolites [Articolo su rivista]
A., Bertelli; Conte, Angela; G., Ronca; R., Zucchi
abstract

The assessment of purine release in perfusion fluid is a new method (Zucchi et al.) which allows a continuous evaluation of energy metabolism in isolated perfused rat heart. Purine release in fact is related to the imbalance between ATP formed and utilized in myocytes. With this method we have investigated the effect of propionyl carnitine, carnitine and propionate on the working heart. The presence of millimolar concentrations of propionyl carnitine decreases purine release and improves cardiac performance as measured by cardiac output and double product (product of heart rate and aortic systolic pressure). Propionate has no effect, while carnitine slightly decreases purine release. The property shown by propionyl carnitine in decreasing the imbalance between ATP production and utilization and in improving cardiac performance is due to its ability to improve the energy metabolism of cardiomyocytes. This compound supplies oxidizable substrates and intermediates to the tricarboxylic acid cycle. In the presence of propionyl carnitine the myocardium therefore responds better to the sudden requirements of overwork and shows better functional efficiency for longer periods.

1991 - Effect of Propionyl Carnitine on Energy Charge and Adenine Nucleotide Content of Cardiac Endothelial Cells During Hypoxia [Articolo su rivista]
A., Bertelli; Conte, Angela; L., Palmieri; G., Ronca; D., Segnini; G., Yu
abstract

Adenine mucleotide metabolism is very active in endothelial cells. These cells are very rich in xanthine oxidase which may produce oxygen reactive species during ischaemia and reperfusion when a high amount of adenine nucleotides may be catabolized to hypoxanthine. We investigated the effect of propionyl carnitine on energy charge and nucleotide content in cultured endothelial cells during changes in oxygen partial pressure. During hypoxia the adenine nucleotide pool and the energy charge decreased more slowly in the presence of 0.5 mM propionyl carnitine than in the absence of the compound. Furthermore during reoxygenation a more rapid increase of energy charge and adenine nucleotide concentration was observed with propionyl carnitine. These observations suggest that the presence of propionyl carnitine allows the endothelial cells to maintain their functionality and regulatory role on vessel activity for a longer time and decreases the formation of oxygen reactive species due to xanthine oxidase activity on hypoxanthine formed by adenine nucleotide catabolism

1991 - Effects of pulsed electromagnetic fields on the adenine nucleotide pool and energy charge in cells in culture [Articolo su rivista]
Conte, A.; Petrini, M.; Zaniol, P.; Ronca, G.
abstract

1991 - Metabolic Fate of Exogenous Chondroitin Sulfate in Man [Articolo su rivista]
Conte, Angela; M., DE BERNARDI; L., Palmieri; P., Lualdi; G., Mautone; G., Ronca
abstract

Chondroitin sulfate is administered as a drug to man by intravenous, intramuscular or oral route. However, few data are available on the metabolic fate of exogenous chondroitin sulfate in man. After intravenous administration of 0.5 g of chondroitin sulfate to healthy volunteers, the plasma level decreases according to a two-compartmental open model. The half-lives of distribution and elimination are 25.5 +/- 6.6 and 281 +/- 32 min, respectively. The volumes of central and tissue compartments are 6.0 +/- 1.0 and 22.9 +/- 7.7 l, respectively. More than 50% of the administered chondroitin sulfate is excreted with urine during the first 24 h as high and low molecular weight derivatives. After oral administration of 3 g of chondroitin sulfate to 12 healthy volunteers, a main peak (11.4 +/- 3.7 micrograms/ml) preceded by a lower peak is observed after 190 +/- 21 min. The elimination half-life is 363 +/- 109 min. The absolute bioavailability following oral administration calculated from AUC of plasma concentration is 13.2%. A peak of oligo- and polysaccharides with a molecular weight lower than 5000 Daltons derived from partial digestion of exogenous chondroitin sulfate is also present in plasma. These observations indicate that the metabolic fate of exogenous chondroitin sulfate is similar in man and in experimental animals.

1991 - Metabolic Fate of Partially Depolymerized Chondroitin Sulfate Administered to the Rat [Articolo su rivista]
Conte, Angela; L., Palmieri; D., Segnini; G., Ronca
abstract

Partially depolymerized chondroitin sulfate (dCS) was tritiated and given to rats. With both the intramuscular and oral routes of administration the main route of excretion is urine. More than 40% of the radioactivity is present in tissues 24 h after administration. After intramuscular injection, radioactivity plasma levels rapidly increase with a peak at 0.6 h. The separation of the radioactive material on a Biogel P-4 column shows that the radioactivity in the first hour after injection is mainly constituted of dCS with molecular weight higher than 4000 daltons (dCS greater than 4000). The composition of the radioactive material changes with time; after 24 h the dCS greater than 4000 is a few percent of the total radioactivity. A large amount of tritiated water due to exchange and metabolization of dCS is found. Mono-, oligo- and polysaccharides resulting from the breakdown of dCS are also present. After oral administration, plasma radioactivity rapidly increases, with a shoulder and a small peak after 1 h and a large peak after 11 h. A tropism of the radioactivity towards glycosaminoglycan-rich tissues is observed. The presence of dCS greater than 4000 in plasma, synovia and cartilage after oral and intramuscular administrations of dCS may explain the chondroprotective effect of exogenous dCS. In fact, desulfated and sulfated oligo- and polysaccharides have regulatory effects on the synthesis and breakdown of hyaluronate-proteoglycan complexes of cartilage.

1991 - Protective Effect of Propionyl Carnitine Against Peroxidative Damage to Arterial Endothelium Membranes [Articolo su rivista]
F., Bertelli; Conte, Angela; G., Ronca; D., Segnini; G., Yu
abstract

Endothelial cells may be damaged by oxygen reactive species produced by granulocytes, by transition metal ions or by xanthine oxidase, an enzyme present in great quantity in these cells. Since it has been observed that propionyl carnitine protects the heart from peroxidation, we have investigated the effect of this compound on the formation of thiobarbituric acid reactive oxidation products (TBAR) in endothelial membranes. The peroxidation systems used were a mixture of Fe3+ and Fe2+, hydrogen peroxide and Fe2+, or xanthine oxidase-- xanthine. Propionyl carnitine at millimolar concentrations decreases TBAR formation. The protection is concentration-dependent and is almost absent in the presence of propionate and carnitine. From these results it appears that propionyl carnitine may protect not only myocardium but also vessels from peroxidative damage that occurs during ischaemia and reperfusion

1990 - EFFECTS OF DIFFERENT LOW-FREQUENCY ELECTROMAGNETIC-FIELDS ON LYMPHOCYTE-ACTIVATION - AT WHICH CELLULAR-LEVEL [Articolo su rivista]
Petrini, M; Polidori, R; Ambrogi, F; Vaglini, F; Zaniol, P; Ronca, G; Conte, Angela
abstract

It has been shown that low-frequency electromagnetic fields may inhibit or enhance the lymphocyte response to many mitogens. How this happens is not known, and many reports suggest that alterations of surface receptors may be involved. Results presented here indicate that cellular activation is inhibited by a high-intensity EMF after the early phases of signal transduction, but it may be enhanced by a low-intensity EMF.

1990 - Metabolic Fate of Exogenous Chondroitin Sulfate in the Experimental Animal [Articolo su rivista]
L., Palmieri; Conte, Angela; L., Giovannini; P., Lualdi; G., Ronca
abstract

After the administration of tritiated chondroitin sulfate (CS) by oral and intramuscular route, the distribution of radioactivity was investigated in two opportunist omnivorous animals, namely the rat and the dog. More than 70% of the orally administered radioactivity was absorbed. Independently of the administration route, radioactivity was mainly excreted through the urine. Plasma levels showed a rapid increase after oral administration, followed by a large plateau with a maximum at the 14th and 28th h in the rat and in the dog, respectively. A tropism of the radioactivity was observed towards glycosaminoglycan-rich tissues, such as joint cartilage. The analysis of the molecular weight of the radioactive material showed that compounds with a molecular weight corresponding to those of CS, poly-, oligo- and monosaccharides as well as of tritiated water, were present in the plasma, urine, synovial fluid and cartilage. The level of radioactive low molecular weight material, derived from the metabolism of CS and from the exchange reaction, increased with the time after administration. The high molecular weight fraction represented at least 10% of the orally administered CS.

1990 - Metabolizzazione della 4-Carbometossitiazolidina Cloridrato a Cisteina ad Opera di Espettorati ed Omogenati di Polmone Umani e sua Azione sulle Glicoproteine del Muco [Articolo su rivista]
Conte, Angela; G., Ronca
abstract

ND

1990 - Multidrug resistance and electromagnetic fields [Articolo su rivista]
Petrini, M; Mattii, L; Sabbatini, Anna Maria Teresa; Carulli, G; Grassi, B; Cadossi, R; Ronca, G; Conte, Angela
abstract

The effect of low-frequency electromagnetic fields (EMFs) on the multidrug resistant cell line MCF7 dx was studied. In cultures containing 40 mg/ml doxorubicin, incorporation of H-3-thymidine was inhibited by exposure to EMFs. The effect may have occurred via functional inhibition of P-glycoprotein.

1990 - Protection of Adenylate Pool and Energy Charge by L-Carnitine and Coenzyme Q during Energy Depletion in Rat Heart Slices [Articolo su rivista]
Conte, Angela; L., Palmieri; G., Ronca; L., Giovannini; A., Bertelli
abstract

The effect of coenzyme Q, of L-carnitine, and of their combination, on the adenine nucleotide pool and the energy charge has been investigated in rat heart slices subjected to energy depletion and recovery. The addition of coenzyme Q or of L-carnitine alone results in a higher value of the energy charge and of the adenine nucleotide pool after hypoxia, reperfusion and rotenone inhibition of the respiratory chain, as compared to controls without additions. The protective effect is much stronger when the two compounds are given together.

1990 - Protective and Stabilizing Effect of CoA on Cellular Membrane Peroxidation [Articolo su rivista]
Conte, Angela; L., Palmieri; D., Segnini; G., Ronca
abstract

ND

1990 - Synergic and Complementary Effects of L-Carnitine and Coenzyme Q on Long-Chain Fatty Acid Metabolism and on Protection against Anthracycline Damage [Articolo su rivista]
Conte, Angela; L., Palmieri; G., Ronca; L., Giovannini; A., Bertelli
abstract

Exogenous L-carnitine and coenzyme Q are used to protect the heart against anthracycline damage and to enhance energy metabolism in the heart and in the muscle. Though their metabolic function is well known and their effects on anthracycline damage have been largely studied, their combined action has not been investigated. Therefore we have used partially CoQ-depleted bovine mitochondria to evaluate the synergic action of CoQ and carnitine on palmitoylCoA oxidation, as an experimental model in which either CoQ or L-carnitine may be the limiting factor in the oxidation of activated fatty acids. The protective effect exerted by the combined use of L-carnitine and CoQ against damage by the anthracycline derivative doxorubicin has been compared to the protection exerted by each compound alone. The effect was evaluated by assessing oxygen consumption and 14C-leucine incorporation in rat heart slices. The results obtained suggest that the administration of an association of L-carnitine and CoQ exerts a stronger protection against anthracycline damage and induces a greater utilization of fatty acids as compared to the effects of each compound alone.

1989 - Absorption and Excretion in the Experimental Animal of a l4C-Ethylmaleimide Labelled Peptide Fraction of Bovine Factor VIII with Antihaemorrhagic Activity [Articolo su rivista]
Conte, Angela; L., Palmieri; G., Ronca
abstract

In this study the absorption and excretion of a peptide fraction from bovine Factor VIII (Vueffe) with antihaemorrhagic activity were investigated in rats and rabbits. The results obtained suggest that the peptide fraction may be absorbed from the gastrointestinal tract through an active transport or a process of nonselective pinocytosis. The radioactivity elimination was rapid and almost complete within 24 h. In addition no difference was observed between single or repeated dosing in the pharmacokinetic parameters. The evidence following routes of administration shows the good bioavailability of this peptide fraction of low molecular weight and confirms the efficacy after oral administration at very low doses.

1989 - Farmacocinetica, Biotrasformazione e Distribuzione di un Nuovo Antinfiammatorio non Steroideo [Articolo su rivista]
G., Ronca; M., DE BERNARDI; Conte, Angela
abstract

ND

1989 - Phosphocreatine as a possible modulator of the adenylate pool [Relazione in Atti di Convegno]
Ronca, G.; Pezzini, A.; Conte, A.; Galbani, P.; Zucchi, R.; Ronca-Testoni, S.
abstract

1989 - The metabolic fate on tritiated galactosaminglucuronoglycan sulfate [Articolo su rivista]
Palmieri, L.; Conte, A.; Ronca, G.; Giovannini, L.
abstract

1989 - Uric acid and purine compounds in aortic and coronary sinus blood in man [Relazione in Atti di Convegno]
Ronca, G.; Conte, A.; Ronca-Testoni, S.; Zucchi, R.; Poddighe, R.; Mariotti, R.; Limbruno, U.; Mariani, M.
abstract

1988 - A Possible Role of the Creatine Phosphate-Creatine Pool in the Regulation of the Adenylate Pool [Articolo su rivista]
A., Pezzini; Conte, Angela; P., Galbani; S., RONCA TESTONI
abstract

Human lymphoblastoid Raji cells and mouse hybridoma ascites cells incubated with 20 mM creatine showed significant increases in creatine, creatine phosphate and adenine nucleotide levels and in the energy charge. In human erythrocytes in which no variation of the creatine phosphate-creatine pool was observed because of a very low creatine kinase activity, the adenine nucleotide pool and the energy charge were not modified. These observations suggest not only a relationship among the creatine phosphate-creatine pool, the energy charge and the adenylate pool, but also the possibility to increase the energy charge and the adenylate pool in cells with creatine kinase activity by expanding the creatine phosphate-creatine pool.