Serum Metabolomics and Proteomics to Study the Antihypertensive Effect of Protein Extracts from Tenebrio molitor

Hypertension is the leading risk factor for premature death worldwide and significantly contributes to the development of all major cardiovascular disease events. The management of high blood pressure includes lifestyle changes and treatment with antihypertensive drugs. Recently, it was demonstrated that a diet supplemented with Tenebrio molitor (TM) extracts is useful in the management of numerous pathologies, including hypertension. This study is aimed at unveiling the underlying mechanism and the molecular targets of intervention of TM dietary supplementation in hypertension treatment by means of proteomics and metabolomics techniques based on liquid chromatography coupled with high-resolution mass spectrometry. We demonstrate that serum proteome and metabolome of spontaneously hypertensive rats are severely altered with respect to their normotensive counterparts. Additionally, our results reveal that a diet enriched with TM extracts restores the expression of 15 metabolites and 17 proteins mainly involved in biological pathways associated with blood pressure maintenance, such as the renin-angiotensin and kallikrein-kinin systems, serin protease inhibitors, reactive oxygen scavenging, and lipid peroxidation. This study provides novel insights into the molecular pathways that may underlie the beneficial effects of TM, thus corroborating that TM could be proposed as a helpful functional food supplement in the treatment of hypertension

Caratterizzazione chimica di zucca e radicchio a tutela di prodotti tipici del Delta del Po

Il progetto seguito durante questo dottorato di ricerca, in collaborazione con il CUM (Consorzio Uomini di Massenzatica, provincia di Ferrara), si è prefisso lo scopo di affermare la qualità, il valore nutritivo e la composizione di alcuni prodotti identitari del territorio di Massenzatica. In primo luogo sono stati individuati come oggetto di studio due produzioni provenienti dai terreni di tale Consorzio: la zucca, nelle varietà Delica (Cucurbita maxima) e Violina (Cucurbita moschata), ed il radicchio rosso (Cichorium intybus) nelle tipologie tondo e lungo. Il progetto, della durata di tre anni, ha portato come primo risultato ad una completa caratterizzazione in termini chimico-bromatologici di zucca e radicchio prodotti e coltivati a Massenzatica, ottenendo tutti i parametri utili alla compilazione della loro etichetta nutrizionale. E' stato effettuato un accurato confronto con competitors presenti sul mercato, per individuare se e quali differenze esistano in relazione al territorio di produzione e coltivazione. La rielaborazione statistica, mediante l’analisi delle componenti principali, dei dati relativi ai tre anni, ha evidenziato una buona differenziazione in relazione al territorio di produzione e coltivazione. Per il radicchio, durante l’ultimo anno di progetto, è stato avviato uno studio biomolecolare mirato alla differenziazione dei campioni di Massenzatica dai competitors a marchio IGP. I risultati ottenuti confermano quanto emerso dalle analisi chimico-bromatologiche ed incoraggiano quindi un approccio multidisciplinare per studi di questo tipo. L’Organizzazione Mondiale della Sanità promuove una dieta sana ed equilibrata poiché esistono testimonianze di come gli alimenti possano influenzare la risposta immunitaria dell’organismo, prevenire l’insorgere di malattie cardiovascolari, degenerative, diabete e diversi tipi di cancro. In quest’ottica, per entrambe le matrici considerate, è stato effettuato uno studio mirato alla determinazione del profilo qualitativo dei metaboliti secondari di interesse nutraceutico. L’utilizzo della tecnica HPLC-UV-Vis-APCI-MS/MS ha permesso l’ottenimento dei profili cromatografici di zucca Delica e Violina di Massenzatica, evidenziando la presenza di 11 carotenoidi. Nello specifico, sono stati selezionati due carotenoidi (luteina e beta-carotene) ai fini della quantificazione. I risultati ottenuti evidenziano come entrambi i composti target siano presenti in maggiori quantità nella zucca Delica rispetto alla Violina. I carotenoidi sono suscettibili alla degradazione durante la lavorazione e, proprio per questo motivo, luteina e beta-carotene sono stati monitorati in campioni di zucca cotti al forno e a vapore. Un completo profilo in termini di apporto di carotenoidi in seguito all’introduzione della zucca nella dieta, è stato ottenuto grazie agli studi di bioaccessibilità in vitro. Il completo profilo polifenolico per entrambe le tipologie di radicchio rosso prodotto e coltivato a Massenzatica `e stato effettuato mediante analisi HPLC-MS/MS operando sia in modalità ESI positiva che negativa e permettendo l’identificazione di un totale di 36 composti. Sono stati selezionati sette composti target (cianidina-3-O-glucoside, acido dicaffeiltartarico, acido 5-caffeilchinico, apigenina-7-O-glucoside, kaempferolo-7-O-glucoside, quercetina-3-O-(6”-malonil)-glucoside, acido 3-caffeilchinico) rispetto ai quali `e stata effettuata la quantificazione, utile per i successivi studi di bioaccessibilità. Tutte le molecole selezionate sono infatti risultate bioaccessibili durante le fasi salivare, gastrica e duodenale e sono stati individuati tre diversi comportamenti, relazionabili alla posizione della glicosilazione.The objective the project has set during this PhD, in collaboration with the CUM (Consorzio Uomini di Massenzatica, province of Ferrara), is to affirm the quality, nutritional value and composition of some of the identity products of the territory of Massenzatica. First of all, two products were identified as the subject of the study from the territory of the Consortium: pumpkin, in the Delica Cucurbita maxima) and in the Violina (Cucurbita moschata) varieties, and red chicory (Cichorium intybus) in the round and long ecotypes.The project, which lasted three years, led at first to a complete characterization of the pumpkin and the red chicory produced and cultivated in Massenzatica in chemical and bromatological terms, obtaining all the parameters useful for the compilation of their nutritional label. A careful comparison was made between competitors in the market, to identify whether and what differences exist in relation to the territory of production and cultivation.The statistical re-elaboration, through the analysis of the main components, of the data achieved during these three years, has shown a good differentiation in relation to the territory of production and cultivation. For the red chicory, during the last year of the project, a biomolecular study was launched aimed at differentiating Massenzatica samples from PGI competitors. The World Health Organization promotes a healthy and balanced diet because it’s evident how food can affect the body’s immune response, prevent the onset of cardiovascular disease, degenerative diseases, diabetes and different types of cancer. In this perspective, for both the considered matrices, a study was carried out aimed at determining the quality profile of secondary metabolites of nutraceutical interest. The use of the HPLC-UV-Vis-APCI-MS/MS technique allowed to obtain the chromatographic profiles of Delica and Violina pumpkin of Massenzatica, highlighting the presence of 11 carotenoids.Specifically, two carotenoids (lutein and beta-carotene) were selected for the quantification. The obtained results show that both target compounds are present in bigger quantities in the Delica pumpkin than in the Violina one. Carotenoids are susceptible to degradation during food processing and, for this reason the concentration of lutein and beta-carotene were monitored in ovencooked and steam-cooked pumpkins. A complete profile was obtained in terms of carotenoid count after the introduction of pumpkin into the diet through in vitro bioaccessibility studies. The complete polyphenolic profile for both types of the red chicory produced and cultivated in Massenzatica was carried out thanks to the HPLC-MS/MS analysis, operating in both positive and negative ESI modes and allowing the identification of a total amount of 36 compounds.Seven target compounds (cyanidin-3-O-glucoside, dicaffeiltartaric acid, 5-caffeilquinic acid, apigenina-7-O-glucoside, kaempferolo-7-O glucoside, quercetin-3-O-(6”-malonyl)-glucoside, 3-caffeilquinic acid) were selected for the quantification, useful for subsequent bioaccessibility studies. All the molecules selected were bioaccessible during the salivary, gastric and duodenal phases and three different behaviors were identified, related to the position of glycosylation

HPLC-UV/Vis-APCI-MS/MS Determination of Major Carotenoids and Their Bioaccessibility from "Delica" (Cucurbita maxina) and "Violina" (Cucurbita moschata) Pumpkins as Food Traceability Markers

Carotenoids are a widespread group of fat-soluble pigments, and their major nutritional importance comes from their pro-vitamin A activity and their antioxidant capacity. In this study, two different pumpkin cultivars (Cucurbita maxima, also named ‘Delica’ and Cucurbita moschata, also known as ‘Violina’) from the southern Po Delta area were investigated in terms of carotenoid content and the influence of food processing on compositional changes and carotenoid bioaccessibility. Quali- and quantitative determination of carotenoids in sample extracts were performed on a C30 column by means of an online coupled HPLC-UV/Vis-APCI-MS/MS technique. The identification of separated compounds was tentatively achieved by merging (i) chromatographic data, (ii) UV-Vis spectra, and (iii) MS/MS fragmentation spectra. The chromatographic profiles for the two cultivars showed qualitative differences. Two major carotenoids were considered for quantification purposes and further investigations: lutein and b-carotene. Quantification of target carotenoids was performed with external calibration through analytical standards. The concentration of lutein and b-carotene was higher in C. maxima than in the other variety, C. moschata. Carotenoids are susceptible to degradation (isomerization and oxidation) during food processing (i.e., cooking), and the concentration of lutein and b-carotene were monitored in oven-cooked and steam-cooked pumpkins. The steam-cooking process was superior in terms of limiting carotenoid loss. A complete functional profile of pumpkins as a source of carotenoids was gained with the evaluation of their in vitro bioaccessibility and their bioavailability after intake during human digestion. Bioaccessibility of lutein and b-carotene were estimated by an in vitro static digestion model that involved salivary, gastric, and duodenal phases. Bioaccessibility values progressively increased from the salivary to the duodenal phase for both pumpkin varieties and cooking methods. Bioaccessibility of lutein was always lower than b-carotene for both cultivars and for both cooking methods. Bioaccessibility values for lutein and b-carotene changed from 1.93% to 2.34% vs. 4.94% and 8.83% in the salivary phase, from 2.7% to 4.63% vs. 7.83% and 15.60% in the gastric phase, and from 10.04% to 13.42% vs. 25.81% and 35.32% in the duodenal phase. For both target compounds, bioaccessibility in the duodenal phase was more than twice the gastric values, and it underlined that the type of cooking did not influence release from the initial matri

Insights into Structure-Bioaccessibility Relationships of Polyphenols in Red Chicory by HPLC-MS/MS

Two varieties of red chicory from southern Po Delta area have been characterized in terms of polyphenols content. Attention has been mostly paid to phenolic acids and flavonoids1. Qualitative and quantitative determinations have been performed on sample extracts by HPLC-MS/MS. Both glycosylated and ester forms of phenolic acids as well as glycosylated flavonoids were mainly identified. Seven target bioactive compounds were selected and their bioaccessibility from red chicory vegetable matrix was investigated through simulated in-vitro static digestion process2,3. This gastro-intestinal model uses simulated fluids (i.e., salivary, gastric and duodenal) based on electrolytic solutions of different physiological enzymes miming the stages of digestion process. Fractions of polyphenols released from the vegetable matrix were quantitatively determined. Bioaccessibility results show well defined behaviors associated with both specific structures of aglycone or phenolic acid and position of O-glycosyl moiety. Dicaffeoyltartaric acid together with apigenin-7-O-glucoside and kaempferol-7-O-glucoside have similar bioaccessibility trend that is characterized by a higher value for duodenal phase than for gastric one. Quercetin-3-O-(6''-O-malonyl)-glucoside behave in reverse way and gastric bioaccessibility turns higher than duodenal. Lastly, the difference between gastric and duodenal bioaccessibility is ehnanced in the case of cyanidin-3-O-glucoside and two isomers of caffeoylquinic acid. Advanced investigations involved also living biological systems (i.e., cellular lines) and they were aimed at understanding effects of polyphenols on degenerative diseases. Particularly, investigation of the role of polyphenol metabolites and their positive synergic effects together with other nutraceutics was promising for uncovering hidden aspects of bioactives mode of action and their health effects

Insights into structure-bioaccissibility relationships of polyphenols in red chicory by HPLC-MS/MS

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Serum Metabolomics and Proteomics to Study the Antihypertensive Effect of Protein Extracts from Tenebrio molitor

Hypertension is the leading risk factor for premature death worldwide and significantly contributes to the development of all major cardiovascular disease events. The management of high blood pressure includes lifestyle changes and treatment with antihypertensive drugs. Recently, it was demonstrated that a diet supplemented with Tenebrio molitor (TM) extracts is useful in the management of numerous pathologies, including hypertension. This study is aimed at unveiling the underlying mechanism and the molecular targets of intervention of TM dietary supplementation in hypertension treatment by means of proteomics and metabolomics techniques based on liquid chromatography coupled with high-resolution mass spectrometry. We demonstrate that serum proteome and metabolome of spontaneously hypertensive rats are severely altered with respect to their normotensive counterparts. Additionally, our results reveal that a diet enriched with TM extracts restores the expression of 15 metabolites and 17 proteins mainly involved in biological pathways associated with blood pressure maintenance, such as the renin-angiotensin and kallikrein-kinin systems, serin protease inhibitors, reactive oxygen scavenging, and lipid peroxidation. This study provides novel insights into the molecular pathways that may underlie the beneficial effects of TM, thus corroborating that TM could be proposed as a helpful functional food supplement in the treatment of hypertension

Bioaccessibility and HPLC-MS/MS chemical characterization of phenolic antioxidants in Red Chicory (Cichorium intybus)

Two varieties of red chicory from southern Po Delta area have been characterized in terms of polyphenols content by HPLC-MS/MS. Seven target compounds were selected for determining their bioaccessibility from vegetable matrix. Simulated digestion process was employed for this purpose by means of an in-vitro static model. Salivary, gastric and duodenal phases were realized by using solutions of different physiological enzymes miming the stages of digestion process. Results show well defined behaviors associated with specific structures of aglycone or phenolic acid and position of O-glycosyl moiety. Dicaffeoyltartaric acid together with apigenin-7-O-glucoside and kaempferol-7-O-glucoside have similar bioaccessibility trend that is characterized by a higher value for duodenal phase than for gastric one. Quercetin-3-O-(6″-O-malonyl)-glucoside behaves in reverse way and gastric bioaccessibility turns higher than duodenal. Lastly, the difference between gastric and duodenal bioaccessibility is enhanced in the case of cyanidin-3-O-glucoside and the isomers of caffeoylquinic acid

Rationale behind the optimum efficiency of columns packed with new 1.9 μm fully porous particles of narrow particle size distribution

Columns packed with new commercially available 1.9 fully porous particles of narrow particle sizedistribution (nPSD) are characterized by extremely high efficiency. Under typical reversed phase con-ditions, these columns are able to generate very high number of theoretical plates (in the order of300,000 plates/m and more). In this paper, we investigate the origin of the high performance of thesenPSD columns by performing a series of measurements that include, in addition to the traditional deter-mination of the van Deemter curve, peak parking, pore blocking and inverse size exclusion experiments.Two nPSD columns (both 100 × 3.0 mm) have been considered in this study: the first one, packed withparticles of 80˚A pore size, is commercially available. The second one is a prototype column packed with1.9 fully porous particles of 120˚A pore size.The main conclusion of our study is that these nPSD columns are characterized by extremely low eddydispersion, while longitudinal diffusion and mass transfer kinetics are substantially equivalent to thoseof other fully porous particles of similar chemistr

Rationale behind the optimum efficiency of columns packed with new 1.9μm fully porous particles of narrow particle size distribution

Columns packed with new commercially available 1.9 fully porous particles of narrow particle size distribution (nPSD) are characterized by extremely high efficiency. Under typical reversed phase conditions, these columns are able to generate very high number of theoretical plates (in the order of 300,000plates/m and more). In this paper, we investigate the origin of the high performance of these nPSD columns by performing a series of measurements that include, in addition to the traditional determination of the van Deemter curve, peak parking, pore blocking and inverse size exclusion experiments. Two nPSD columns (both 100×3.0mm) have been considered in this study: the first one, packed with particles of 80Å pore size, is commercially available. The second one is a prototype column packed with 1.9 fully porous particles of 120Å pore size. The main conclusion of our study is that these nPSD columns are characterized by extremely low eddy dispersion, while longitudinal diffusion and mass transfer kinetics are substantially equivalent to those of other fully porous particles of similar chemistry.publisher: Elsevier articletitle: Rationale behind the optimum efficiency of columns packed with new 1.9μm fully porous particles of narrow particle size distribution journaltitle: Journal of Chromatography A articlelink: http://dx.doi.org/10.1016/j.chroma.2016.05.037 content_type: article copyright: © 2016 Elsevier B.V. All rights reserved.status: publishe