Browsing by Author "Andò, Sebastiano"

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Action of the E2/ERβ/PTEN signaling in the metabolic reprogramming of TCam 2, human melanoma cell line
(2019-03-21) De Rose, Daniela; Andò, Sebastiano; Aquila, Saveria
I tumori maligni più abbondanti nella popolazione maschile di età compresa tra i 17 ed i 45 anni, sono i tumori delle cellule germinali (GCTs). Essi comprendono un gruppo eterogeneo di neoplasie in termini istologici, di marker d’espressione ed età di manifestazione. I tumori delle cellule germinali testicolari negli adolescenti e negli adulti (TGCTs) possono essere classificati in tumori seminomatosi (GCT di tipo II) e non seminomatosi. Nel nostro studio prenderemo in considerazione il GCT di tipo II, utilizzando come modello sperimentale la linea cellulare TCam2, ad oggi unica al mondo ampiamente caratterizzata e comprendente tutte le caratteristiche del seminoma umano, originata dalla lesione primaria di un seminoma testicolare sinistro di un paziente di 35 anni. La difficoltà di avere un modello cellulare valido per i tumori seminomatosi è il motivo principale che rende il tumore testicolare uno dei tumori meno studiati. La ricerca sul cancro testicolare continua ad investigare e studiare terapie volte ad indurre la morte nelle cellule tumorali. Recentemente, il metabolismo energetico è considerato un obiettivo innovativo nelle terapie antitumorali, in quanto le alterazioni metaboliche sono una caratteristica comune dei tessuti cancerosi. Il fenotipo metabolico maggiormente caratterizzante e per prima osservato nelle cellule cancerose è quello conosciuto come Effetto Warburg, che prevede la produzione di ATP attraverso la glicolisi invece che attraverso la fosforilazione ossidativa, anche in presenza di normali concentrazioni di ossigeno (Barger JF et al. 2010). Tuttavia, la riprogrammazione metabolica nei tumori si estende oltre l'Effetto Warburg. In effetti, la teoria classica sul metabolismo delle cellule tumorali (aumento dell'attività glicolitica e down-regolazione della fosforilazione ossidativa) è ancora oggetto di indagini in quanto numerosi studi hanno dimostrato che le cellule tumorali possono vivere in un ampio spettro di stati bioenergetici che variano dalla predominanza del fenotipo glicolitico, glicolitico parzialmente ossidativo, fino a quello prevalentemente fosforilativo (Smolková K et al. 2011). Gli estrogeni ed i loro recettori, sono in grado di modulare diversi aspetti del metabolismo cellulare come quello glucidico o lipidico, un’alterazione dei loro pathways trasduzionali è stata correlata infatti allo sviluppo di malattie metaboliche (Faulds Malin Hedengran, 2012). Nel nostro precedente studio abbiamo evidenziato un link tra ERβ/PTEN che attivato dall’estradiolo, induce la morte di tali cellule mediante autofagia e necroptosi (Guido C. et al. 2012). Poiché, morte cellulare e metabolismo energetico sono strettamente correlati, abbiamo ipotizzato che il link E2/ERβ/PTEN possa indurre una alterazione anche nella riprogrammazione metabolica nelle cellule di SE. Il ruolo di PTEN nella sopravvivenza e proliferazione cellulare è stato già riportato, inoltre PTEN è in grado di influenzare alcuni pathways metabolici come il metabolismo del glucosio (Madeline B, 2002), ed il metabolismo lipidico (Qiu W. 2008; Juan Liu, 2012; Ana Ortega-Molina and Manuel Serrano, 2013). Lo scopo di questo studio è quello di investigare un potenziale cross-talk funzionale tra E2, ERβ e PTEN nell’interferire sulla riprogrammazione metabolica delle cellule TCam2 di seminoma umano, così da ampliare le nostre conoscenze sul ruolo e sulla regolazione del gene PTEN oltre che sulla biologia di questo tipo di tumore. I nostri dati evidenziano un nuovo ruolo dell’ERβ come tumor suppressor, indicando che il meccanismo attraverso cui l’E2 induce la morte delle cellule TCam2 avviene anche attraverso l’alterazione della riprogrammazione metabolica in cooperazione con il gene PTEN. Ad oggi, il metabolismo di questa linea cellulare non è stato ancora investigato e pertanto il nostro lavoro contribuirà a migliorare le conoscenze su questo aspetto della biologia del seminoma umano. Concludendo, i nostri risultati supportano l’idea di una dipendenza estrogenica del tumore testicolare come già riportato in letteratura, indicando l’ERβ come possibile target terapeutico per il trattamento di questa condizione patologica.
Activated FXR inhibits leptin signaling and counteracts tumor-promoting activities of cancer-associated fibroblasts in breast malignancy
(2017-06-12) Vircillo, Valentina; Andò, Sebastiano; Catalano, Stefania
Cancer-associated fibroblasts (CAFs), the principal components of the tumor stroma, play a central role in cancer development and progression. As an important regulator of the crosstalk between breast cancer cells and CAFs, the cytokine leptin has been associated to breast carcinogenesis. The nuclear Farnesoid X Receptor-(FXR) seems to exert an oncosuppressive role in different tumors, including breast cancer. In this study, we demonstrated, for the first time, that the synthetic FXR agonist GW4064, inhibiting leptin signaling, affects the tumor-promoting activities of CAFs in breast malignancy. GW4064 inhibited growth, motility and invasiveness induced by leptin as well as by CAF-conditioned media in different breast cancer cell lines. These effects rely on the ability of activated FXR to increase the expression of the suppressor of the cytokine signaling 3 (SOCS3) leading to inhibition of leptin-activated signaling and downregulation of leptin-target genes. We further extend our data investigating whether FXR agonist may directly influence CAF phenotype. We demonstrated that FXR is expressed in different CAFs and treatment with GW 4064 is able to induce the transcription of key FXR target genes, including SHP (Small Heterodimer Partner) and BSEP (Bile Salt Export Pump). Interestingly, FXR activation is able to significantly reduce CAF motility, without influencing their proliferation capabilities. Accordingly, IPA (Ingenuity Pathway Analysis) on FXR-modulated genes highlighted cellular movement as the most significantly represented biologic process and evidenced a marked reduction in the activity of Rho signaling and Integrin proteins, with activation z-score of -1, -0,5 respectively. Moreover, our data showed a reduction in stress fibers formation in GW 4064 -treated CAFs. Activated FXR is able to reduce tumor promoting effects of CAFs on breast cancer cells, due to the ability of GW 4064 to reduce CAF secreted soluble factors, including IGF-1 (Insulin Growth Factor-1), FGF-9 (Fibroblast Growth Factor 9), TGF-3 (Transforming Growth Factor Beta 3) and others key mediators involved in the crosstalk tumor-stroma. Indeed, our data demonstrate how ER-breast cancer cell lines, MCF-7 and T47D, cocoltured with conditioned media derived from GW4064-treated CAFs, exhibit a significantly reduced anchorage-independent growth and migration. In vivo xenograft studies, using MCF-7 cells alone or co-injected with CAFs, showed that GW4064 administration markedly reduced tumor growth. Thus, FXR ligands might represent an emerging potential anti-cancer therapy able to block the tumor supportive role of activated fibroblasts within the breast microenvironment
Advanced and smart nanocarriers for pharmaceutical applications
(Università della Calabria, 2020-04-17) Mazzotta, Elisabetta; Andò, Sebastiano; Muzzalupo, Rita
The application of nanotechnology in drug formulation has made significant headway over the last decade. Nanoscale controlled release systems allow to revalue and reformulate old drugs through targeted delivery to the desired pathological site, improving therapeutic efficiency and reducing side effects. This innovative modality of drug delivery aims to create personalized, safer and effective treatments. A great interest of the academic and industrial research is focused on the design and development of advanced nanocarriers since is a profitable way in terms of costs, times and risks than the discovery of new drugs. Herein, an extensive variety of nanocarriers systems composed of different materials including lipids, polymers and non-ionic surfactants have been proposed for different pharmaceutical applications. Specifically, the carriers have been tailor-made designed to improve drug photostability, enhance skin permeation and realize smart tools for tumor target therapy.
Advanced functionalized materials for multipurpose applications
(Università della Calabria, 2021-03-21) Pantuso, Elvira; Andò, Sebastiano; Nicoletta, Fiore
Advanced mass spectrometric approaches for the determination of food quality and development of new methodologies for enrichment of nutraceuticals in functional foods
(2018-02-27) Bartella, Lucia; Andò, Sebastiano; Di Donna, Leonardo
The research works presented in this Ph.D. thesis are based on the nutraceuticals and functional foods research. The first part of the thesis deals with three main topics: 1. Structural characterization of new HMG-flavonoids in bergamot juice Two unknown 3-hydroxy-3-methylglutaryl flavanone glycosides were identified and characterized by the combination of liquid chromatography and tandem mass spectrometry experiments. 2. Preparation of extracts enriched in HMG-flavonoids A novel food-grade procedure to obtain HMG-flavonoids enriched extracts with an high purity grade was developed. The purification procedure was tested on different citrus fruits, i.e. bergamot, sour orange and chinotto. 3. Development of a new procedure for the enrichment of foodstuffs in hydroxytyrosol New functional foods are presented. They were obtained by the enrichment of foodstuffs generally used in daily diet, i.e. flour, whole wheat flour and sugar, with the nutraceutical compound hydroxytyrosol. The second part concerns the development of advanced mass spectrometric analytical procedure for quality assessment of foods, through the quantification of nutraceutical compounds and the determination of target quality molecules. The works presented in this field are the following: 1. Determination of Hydroxytyrosol and Tyrosol derivatives compounds in Extra Virgin olive oil by Microwave Hydrolysis and LC-MS/MS analysis A novel method to determine the absolute amount of hydroxytyrosol and tyrosol derivatives in EVOO samples was developed. The procedure consists in a simple and fast microwave assisted acidic hydrolysis reaction, combined with the use of liquid chromatography- tandem mass spectrometry analysis. 2. Absolute Evaluation of Hydroxytyrosol in Extra Virgin Olive Oil by Paper Sray Tandem Mass Spectrometry A novel and fast methodology for the evaluation of the absolute amount of Hydroxytyrosol in extra virgin olive oil was set up. The method is based on the exploitation of paper spray ionization (PS) tandem mass spectrometry (MS/MS). 3. Discriminant Analysis of Vegetables Oils by Paper Spray Mass Spectrometry and Statistical Approach An innovative approach for the characterization of triacylglycerols profile of different vegetable oils was developed. The method employs the paper spray ionization technique that in combination with chemometric analysis has allowed to discriminate the extra virgin olive oil from the others vegetable oils commonly used as adulterants. 4. Quantitative Evaluation of Caffeine in beverages and drugs by Paper spray tandem Mass Spectrometry A simple and fast method for the quantification of caffeine in beverages and drugs is presented. Also this analytical procedure is based on the use of paper spray tandem mass spectrometry.
Advanced materials for pharmaceutical and biomedical purposes
(2018-02-27) Mellace, Silvia; Andò, Sebastiano; Cassano, Roberta
L'innovazione nella chimica dei materiali e nella nanotecnologia hanno sinergicamente contribuito all'avanzamento e allo sviluppo dei Drug Delivery Systems (DDS) creando devices in grado di proteggere farmaci e sostanze biologicamente attive, aumentarne l'assorbimento, modificarne e migliorarne la penetrazione intracellulare e la distribuzione. Il presente lavoro ha avuto come obiettivo la progettazione, la preparazione e la caratterizzazione di materiali che potrebbero contribuire a trasformare e ottimizzare le performances di vari agenti terapeutici. In sintesi, sono stati preparati diversi dispositivi medici e DDS, utilizzando perlopiù polimeri, poiché esiste un grande potenziale nella combinazione di molecole bioattive con i polimeri, per affrontare sfide in campo farmaceutico e biomedico. Nella progettazione di questi nuovi materiali che sono biocompatibili e biologicamente attivi l’attenzione è stata anche focalizzata sull'applicazione di metodi in vitro e in silico per una previsione efficace ed efficiente dell’attività in vivo. In conclusione, la sintesi di nuovi materiali, l'adozione di polimeri naturali come carriers e una migliore comprensione del rapporto struttura-funzione hanno permesso lo sviluppo di DDS e devices con incoraggianti risultati sperimentali che suggeriscono un possibile utilizzo per una futura sperimentazione in ambito clinico
Anticancer drugs: a detailed computational analysis of "non classical" compounds mechanism of action
(Università della Calabria, 2020-02-05) Ponte, Fortuna; Andò, Sebastiano; Sicilia, Emilia
Metal containing drugs have attracted an enormous deal of interest for their use in cancer therapy. Transition metal compounds’ richness offers extraordinary opportunities for the design of anticancer compounds, possessing pharmacokinetic properties inaccessible to purely organic compounds. The most successful and evident proof of their pivotal role is represented by cisplatin that, together with its carboplatin and oxaliplatin derivatives, continues to be routinely used worldwide in clinical practice. However, it is well known that the use of such drugs for fighting cancer is accompanied by severe side effects and intrinsic or acquired resistance that drastically limit their successful action. Therefore, decades of research efforts have been devoted to the search and the synthesis of safer and more effective and selective agents, either containing platinum or alternative metals, acting with similar or different mechanisms. In order to accomplish this aim is of decisive importance the elucidation of the mechanism of action of the drugs. Molecular simulations, or in silico experiments, are able to provide detailed information at atomistic resolution, rarely accessible to experiments, that can complement laboratory experiments. The increasing accuracy of computational approaches and the growing performance of computer performance, allow to properly describe reaction paths and involved molecular orbitals, calculate electronic properties, simulate spectra without any limitation except those connected with the adopted level of theory and compuatational protocol. The main aim of the present work was the detailed investigation, in the framework of the Density Functional Theory, of the mechanism of action of “non classical” platinum and transition metal non-platinum compounds, for some of them in collaboration with experimentalists, and the rationalization of their behaviors. In the next paragraphs all the studied systems will be shortly described together with the motivations that have prompted us to study such systems. Both “non classical” platinum(IV) prodrugs, non-platinum drugs and photoactivatable Pt(II) and Pt(IV) complexes have been examined. In the development of new platinum-based anticancer drugs, is of great interest the emerging class of "dual action" Pt(IV) prodrugs that, undergoing a reductive elimination process, which is the key step for their activation, are able to release the active Pt(II) complexes and bioactive axial ligands that together lead to cell death. Indeed, the two axial ligands, in turn, can be chosen to possess physico-chemical and biopharmaceutical properties or even facilitate the incorporation into a drug delivery system. According to the research lines mentioned above, the use of drug delivery systems has also grown, and many different strategies have been examined to encapsulate platinum drugs within macromolecules, including macrocyclic species, which are responsible for creating supramolecular host-guest structures. The encapsulation slows down and prevents the drug degradation by proteins and peptides. One of the most widely studied class of synthetic supramolecular macrocycles are Calix[n]arenes (CX), whose property, as molecular hosts and delivery systems, are of increasing interest. Photodynamic Therapy (PDT) is a non-toxic therapeutic technique, clinically approved and minimally invasive, used for the treatment of several types of cancers based on the generation of reactive oxygen species (ROS), that acts as cytotoxic agents. In PDT applications three components are required: a photosensitizer (PS), a light of a specific wavelength and tissue oxygen. A promising approach to increase the effectiveness of anticancer therapy is the combination of multimodal treatment methods into a single system. Recently, a strategy has been proposed providing the possibility to combine the classical Pt-based chemotherapy with photodynamic therapy (PDT) treatment. This approach involves the functionalization of a photosensitizer (PS) with a therapeutic drug such as cisplatin-like compounds.
Application of computational methodologies toward the identification of novel therapeutics and photodegradation studies for the design of light-stable formulations
(Università della Calabria, 2021-06-10) Occhiuzzi, Maria Antonietta; Andò, Sebastiano; Grande, Fedora; Iole, Giuseppina
Benefits of advanced techniques and data processing for the analysis of complex biological and food matrices
(2019-03-03) Spatari, Claudia; Andò, Sebastiano; Ragno, Gaetano
Durante i tre anni di dottorato in Medicina Traslazionale, il mio lavoro di ricerca si è concentrato sull'applicazione di tecniche analitiche avanzate e sull'elaborazione dei dati per lo studio di matrici complesse biologiche e alimentari. Recenti studi hanno dimostrato che in circa il 10% dei campioni di latte materno acquistato online è presente DNA bovino che dimostra adulterazione intenzionale. I rischi associati al consumo da parte del neonato di latte umano adulterato con contaminanti animali o altro sono molti e gravi, tra cui carenza di ferro, disidratazione, aumento del potenziale di carico di soluto renale (PRSL) e reazioni allergiche, motivo per il quale risulta fondamentale la ricerca di eventuali adulteranti nel latte materno. L'applicazione di tecniche chemiometriche sullo spettro IR del latte materno si è dimostrata molto efficace nel tracciare anche minime variazioni nella composizione e nelle caratteristiche del latte umano, essendo in grado di sfruttare le informazioni non specifiche memorizzate nello spettro IR. In questo caso, l'elaborazione delle impronte digitali spettrali ATR-FTIR mediante regressione PCA e PLS, è stata in grado di rilevare l'aggiunta fraudolenta di acqua o latte di mucca. In particolare, la tecnica PLS-DA è risultata essenziale per riconoscere il latte materno puro dal latte adulterato. Un'ulteriore definizione di quattro modelli PLS1 ha consentito inoltre la determinazione della quantità di adulteranti specifici aggiunti. La ricerca sulle matrici alimentari è stata estesa a una serie di oli commestibili al fine di verificare la loro fotostabilità. Tecniche di irradiazione forzata, in combinazione con analisi spettroscopica UV-vis, FTIR-ATR e cromatografica HPLC-DAD, hanno mostrato cambiamenti quantitativi e qualitativi dei principali acidi grassi. La ricerca si è concentrata in seguito sull'olio di lino, data la sua grande importanza come alimento funzionale. Anche in questo caso, la spettroscopia UV e l'HPLC hanno rivelato cambiamenti significativi nella concentrazione di acidi grassi, ridotti al 35% dopo 48 ore di esposizione alla luce. La quantità di lignani, altri importanti componenti nutraceutici, ha invece mostrato una significativa stabilità. La fotoprotezione dell’olio di lino è stata in seguito dimostrata dall’uso di contenitori di vetro ambrato associato all’aggiunta di ascorbil palmitato quale agente antiossidante. I risultati ottenuti sono importanti per aumentare la durata di conservazione dell'olio di semi di lino o di altri oli commestibili, mediante l'adozione di formulazioni appropriate e accurata protezione fisica. Il monitoraggio dei farmaci nei fluidi biologici ha rappresentato un altro argomento importante del mio lavoro di tesi. Particolare attenzione è stata rivolta allo sviluppo di un metodo analitico per il monitoraggio della quantità dell’anestetico bupivacaina nel sangue del cordone ombelicale. La determinazione del farmaco è stata definita mediante un metodo di estrazione SPE seguito da analisi HPLC e GC-MS. Questo studio sta procedendo con l'analisi di un gran numero di campioni reali, al fine di valutare la sicurezza degli anestetici somministrati durante il parto naturale e nel contempo ottimizzare il protocollo terapeutico attualmente utilizzato in partoanalgesia. Un'esperienza fondamentale durante il dottorato è rappresentata dal periodo trascorso presso un laboratorio di ricerca dell'Università di Utrecht in Olanda. Qui ho avuto l'opportunità di studiare per un semestre (dal 26 settembre 2016 al 27 Marzo2017) presso il laboratorio di ricerca guidato dal prof. Rolf Sparidans. In tale occasione ho approfondito le mie conoscenze sull'uso della cromatografia LC-MS/MS applicata allo studio della farmacocinetica di nuovi farmaci antitumorali. In particolare ho partecipato allo studio di un saggio bioanalitico del farmaco lorlatinib, un inibitore ALK di terza generazione. La preparazione del campione e l'ottimizzazione delle condizioni cromatografiche sono state le fasi più impegnative del lavoro essendo stato il primo test sviluppato e validato per questo prodotto. Una procedura LC-MS / MS è stata ottimizzata e validata sul sangue di topi al fine di stabilirne le proprietà farmacocinetiche Ancora presso il gruppo di ricerca dell’Unical, parte dell’attività di ricerca è stata svolta su matrici farmaceutiche. In particolare si è studiata la stabilità in soluzione acquosa e alla luce di una 1,4-diidropiridina (siglata M3) di nuova sintesi, data la nota scarsa solubilità e fotolabilità di questa classe di farmaci. Una serie di tensioattivi, tra questi i tween, è stata testata per promuovere la solubilità in acqua, ottenendo risultati incoraggianti con l'uso del tensioattivo non ionico Tween20. Successivamente, la fotostabilità del complesso M3-Tween20 è stata studiata vagliando la capacità fotoprotettiva del materiale di diversi contenitori: quarzo, PET blu, PET ambrato, vetro scuro rivestito. La migliore fotoprotezione è stata garantita dal PET ambrato e dal contenitore in vetro scuro. I risultati ottenuti dimostrano che l'uso combinato di tensioattivi e contenitori specifici rappresenta una strategia interessante da applicare ai farmaci fotolabili e a carattere prevalentemente lipofilo
Breast tumor microenvironment and endocrine resistance: dissecting the molecular link
(Università della Calabria, 2023-07-04) Caruso, Amanda; Catalano, Stefania; Andò, Sebastiano
Charatterization of store-operated calcium entry in the neuroimmune response evoked by ischemic preconditioning in mice subjected to middle cerebral artery occlusion
(Università della Calabria, 2021-06-10) Frisina, Marialaura; Andò, Sebastiano; Amantea, Diana
Cerebral ischemia is one of the leading causes of death and long-term disability worldwide. Currently approved therapies for ischemic stroke are limited to reperfusion through mechanical recanalization and/or pharmacological thrombolysis; however, only a small percentage of eligible patients may benefit from this treatment due to its contraindications and, furthermore, it does not provide neuroprotective effects. In this context, several studies have highlighted the potential of inducing ischemic tolerance by stimulating endogenous neuroprotection. To this aim, brain ischemic preconditioning (PC), namely a sublethal ischemic event able to increase the resistance of the brain against a subsequent, more intense ischemic insult, has been considered as a useful experimental paradigm to investigate the mechanisms implicated in brain tolerance. A deep comprehension of endogenous neuroprotection elicited by ischemic PC represents a promising approach to identify novel targets that can be translated into stroke therapy. One of the main factors involved in the progression of neuronal damage during cerebral ischemia is the alteration of cellular Ca2+ homeostasis. Indeed, cytosolic Ca2+ overload due to increased membrane permeability or to its leak from intracellular organelles could result in neuronal demise. Detrimental effects involve the activation of a series of Ca2+-dependent enzymes that degrade cellular components or activate death pathways, and the formation of cytotoxic products that cause irreversible mitochondrial damage and cellular demise. The main objective of the present research work was to investigate the involvement of store-operated calcium entry (SOCE) in brain ischemia and ischemic preconditioning in mice subjected to focal cerebral ischemia. Following an ischemic insult and depletion of Ca2+ stores, the endoplasmic reticulum Ca2+ sensor stromal interaction molecule (STIM)1 interacts with the Ca2+ selective plasmamembrane channel Orai1, to promote SOCE, that may protect neurons by re-establishing Ca2+ homeostasis or could also be the source of excessive Ca2+ influx, thus causing nonexcitotoxic neuronal death. This Ca2+ influx is regulated by SOCE-associated regulatory factor (SARAF) that associates with STIM1 and promotes a slow Ca2+- dependent inactivation of SOCE, or directly interacts with Orai1 to promote SOCE activation in the absence of STIM1. Furthermore, SOCE represents the main source of Ca2+ in immune cells, regulating several of their critical functions. Besides the pivotal role played by immune mediators in the evolution of cerebral ischemic damage, it has been demonstrated that the innate immune system is also an essential component of the delayed ischemic tolerance elicited in the brain by ischemic PC. Therefore, we investigated whether central and peripheral innate immune responses contribute to PC-induced ischemic tolerance and if modulation of different SOCE components occurs in ischemic damage (1h middle cerebral artery occlusion, MCAo, followed by 24h of reperfusion) and/or in neuroprotection conferred by ischemic PC (15 min MCAo, 72h before) in C57BL/6J adult male mice. Ischemic PC significantly reduced histological damage and neurological deficits produced in mice by a more severe ischemia of 1h. Western blot analysis revealed that Orai1 expression is not affected by the ischemic insult preceded or not by the PC stimulus in the frontoparietal ischemic cortex. However, Orai1 expression was detected in neurons, but also in Ly6B.2+ myeloid cells infiltrating the ischemic hemisphere. By contrast, STIM1 and SARAF expression, mainly found in NeuN+ neurons, was significantly reduced in the ischemic cortex. Interestingly, ischemic PC prevented SARAF downregulation in the ischemic cortex, thus suggesting that this regulatory factor may play a crucial role in SOCE-mediated tolerance. To assess the immunomodulatory effects of ischemic PC, we performed ELISA assay to demonstrate that cerebral damage was associated with increased protein levels of the proinflammatory cytokine IL-1β in the ischemic cortex, while this effect was prevented by the PC stimulus. Regarding alternatively-activated phenotypes, western blot analysis revealed a significant elevation of the expression of Ym1, marker of M2-polarized microglia/macrophages, in the ischemic cortex as compared to contralateral tissue. Interestingly, ischemic PC further increased Ym1 expression in the ipsilateral cortex as compared to MCAo group. Immunohistochemical analysis revealed that the majority of Ym1+ cells are mainly amoeboid CD11b+ myeloid cells, very likely monocytes/macrophages infiltrating from blood vessels. Thus, elevated brain infiltration of these phenotypes is very likely involved in the protective effects of ischemic PC. The involvement of the peripheral immune response was confirmed by the evidence that the 70% increase in spleen weight observed after 1h MCAo was abolished in mice pre-exposed to PC. Accordingly, flow cytometry analysis revealed that PC significantly attenuates elevation of neutrophil counts (Ly-6G+ events) induced by 1h MCAo in blood. Since the Ca2+-selective plasmamembrane channel Orai1 is crucial in the recruitment of immune cells during inflammation, we have analysed its expression in the whole population of circulating leukocytes and in neutrophils, demonstrating that the number of Orai1+ cells, mainly corresponding to Ly-6G+ neutrophils, was significantly enhanced in the blood after the ischemic insult, as compared to sham, regardless of whether mice received or not ischemic PC. In conclusion, this research project reaffirms that cerebral ischemic tolerance induced by PC involves both central and peripheral modulation of the innate immune system, further underscoring the relevance of exploiting immunomodulatory approaches for the development of effective stroke therapies and originally demonstrates that preventing SARAF downregulation could represent an important neuroprotective mechanism aimed at preserving SOCE functions, making SARAF a valuable target to protect neurons from the ischemic damage.
Chemical modifications of polyphenols for the development of new molecules foods for the treatment of metabolic disorders
(Università della Calabria, 2020-07-01) Carullo, Gabriele; Andò, Sebastiano; Aiello, Francesca
Metabolic disorders represent the principal cause of death in the industrialized world, affecting several people, because of their incorrect lifestyle. Among them, Type 2 Diabetes Mellitus and related comorbidities, such diabetic wounds, and hypertension represent the most studied examples of metabolism-associated diseases. In this context, the role of food-derived products and hybrid molecules has been investigated in this thesis. Two different braches have been investigated: one regarding food chemistry and one regarding medicinal chemistry. In food chemistry section, wine-making byproducts were used as starting point for the development of food ingredients able to modulate vascular tone in rat aortic rings. In particular, autochthonous (Calabria, southern Italy) white grape pomaces of Mantonico and Pecorello and red grape pomaces of Magliocco dolce (also known as Arvino) have been used. The pomaces were extracted in order to obtain samples, that were chemically-characterized and assayed in rat aorta rings. The results obtained and published demonstrated how these extracts were able to modulate vascular tone by a combination of antioxidant and nitric oxide-mediated effects; in the case of white pomaces, these effects were also assayed when the extract was embedded in a pectin matrix. In line with this, also a functional kefir was produced in order to evaluate its efficacy as anti-inflammatory agent for gastric epithelium, showing how the addition of the Sangiovese pomace extract was able to improve the activity of kefir. In medicinal chemistry section, the role of polyphenols, present in food products, was evaluated in order to drive their activity versus specific molecular pathways. In this field, we used quercetin, in combination with oleic acid, developing GPR40/FFA1 agonists that demonstrated to be useful insulin secretagogue agents. In particular, one derivative was also assayed in wound healing models, demonstrating the role of the receptor also in this proliferation process. In line with this, the GPR120/FFA4 demonstrated interesting pharmacological activities. Their agonists were able to modulate incretin secretion, with interesting anti-diabetic effect. Nevertheless, little structural determinants are present in literature. In this context, in my Spanish experience at University of Seville, I isolated (S)- pinocembrin from honey, and decided to create molecular hybrids, according to a homology model published. The hybrids obtained demonstrated a useful GPR120 agonist activity, promoting wound healing in HaCaT cell line. Furthermore, quercetin and morin were used as template for the development of new KCa1.1 and Cav1.2 channel modulators, with the aim of obtaining new suitable anti-hypertensive candidates. The results obtained showed how quercetin derivatives maintain their activity versus KCa1.1 channel, while morin derivatives demonstrated to activate KCa1.1 channel, behavior not registered in the case of parent compound. These results are now under review for publication.
A computational mechanistic study of potentially evolving platinum based anticancer drugs
(Università della Calabria, 2021) Dabbish, Eslam; Andò, Sebastiano; Sicilia, Emilia
Metals are known to play a fundamental physiological role inside human body affecting many of the biological functions. Analogously, metal based drugs can also have a similar impact. Cisplatin, a simple platinum complex, is well known to be a cytotoxic agent and the first approved and most widely used metal based drug for fighting cancer. Currently, used platinum containing anticancer agents namely cisplatin, carboplatin and oxaliplatin suffer from serious toxic side effects as well as acquired and inherent drug resistance against many types of cancer. Consequently, new platinum anticancer drug families evolved to overcome the current limitations of traditional platinum drugs. Monofunctional platinum complexes, Pt(IV) complexes, platinum complexes targeting mitochondria, platinum idodio derivatives and photoactivated platinum compounds are examples of some of such newly developed platinum based cytotoxic families. Computational chemistry has strongly grown over the past years with both the increase in computers capabilities and the development of new theories and efficient algorithms that can allow to handle bigger models in a reasonable time. Molecular modelling can give a wealth of information about the studied systems in terms of energies, electronic properties, geometries, conformations, structure/activity relationships, reaction mechanisms and many others. By using quantum mechanical methods like Density Functional Theory (DFT) and its time-dependant formulation TD-DFT and molecular dynamics (MD) computational tools, the mechanism of action of some selected examples of non-traditional platinum anticancer drug families have been studied in this thesis. Phenanthriplatin is the most effective member of a new class of platinum anticancer agents (7-40 times more active than cisplatin) known as monofunctional platinum anticancer drugs. In addition, it has started its clinical trials phase. Our computational mechanistic study of phenanthriplatin highlighted the importance of the role played by its unique chemical structure in the drug activation, interaction with DNA and transcription blockage. Targeting of mitochondrial DNA by means of platinum drugs can lead to mitochondrial dysfunction in cancer cells that causes tumour cells growth inhibition and apoptosis. We have undertaken a comparative study between three different isomers of a recently prepared triphenyl phosphonium modified monofunctional platinum complexes for their mechanism of action. Pt(IV) complexes are prodrugs that are reduced inside the body by means of abundant biological reducing agents like ascorbic acid to release the equivalent cytotoxic Pt(II) complexes. This reduction step is considered to be the limiting step for the activity of such class of drugs. In a series of studies, we have carried out a detailed mechanistic study to understand the relation between the nature of Pt(IV) complexes axial and equatorial ligands and the extent and mechanism of reduction by means of ascorbic acid at physiological pH. We highlighted the particular importance and impact of the nature of axial ligands on the reduction process. Photoactivated chemotherapy (PACT) technique allows the localized activation of drugs by means of specific wavelength light. A recently synthesized complex named platicur is a cis-diammineplatinum(II) complex of curcumin in which the Pt(II) centre is bound to a curcumin molecule as the leaving ligand. Upon light irradiation curcumin molecule is released together with the doubly aquated Pt(II) complex that can exert the required cytotoxic effect. In our study, we have provided a deep insight in the photoactivated excited states and their role in the photocleavage mechanism with the release of curcumin.
Computational study of new drugs for photodynamic therapy
(2017-05-05) Pirillo, Jenny; Andò, Sebastiano; Russo, Nino; Mazzone, Gloria
Photodynamic'therapy'(PDT)'is'a'non:invasive'therapeutic'technique' for' the' treatment' of' different' kind' of' tumours' through' the' production' of' reactive'oxygen'species'(ROS)'that'act'as'cytotoxic'agents.'''' PDT'works' through' the' associated' events' of' three' key' components:' photosensitizer' (PS),' visible' or' near:infrared' region' light,' and' molecular' oxygen'in'tissues.'In'general,'after'the'injection'of'a'PS'agent'and'subsequent' accumulation' in' the' target' tissues,' the'agent' is'activated'by' a' radiation'with' appropriate' wavelength' to' give' a' photosensitization' of' the' endogenous' oxygen'(photoreaction'of'type'II)'or'the'production'of'free'radicals'from'the' surrounding'molecules' (photoreaction' of' type' I).' In' both' cases,' a' cytotoxic' species'is'generated,'and'destruction'of'the'cancer'cells'occurs.' Success'in'PDT'relies'on'the'advantage'that'a'non:invasive'technique can'selectively'annihilate'altered'cells'without'undesirable'side'effects.'Recent' research' in'PDT'has' been' focused' on' the'development' of'more' specific' and' powerful'PSs.' In' this' thesis,' the' photophysical' properties' of' different' kinds' of' PSs,' found' to'be'good'candidates' for'PDT,'have'been' investigated.'The'PSs'under' study'differ'in'the'chemical'nature'and'in'the'photosensitization'mechanism:' i)' PSs' based' on' the' nucleobases' analogue' of' DNA' can' be' used' as' UVA' chemotherapeutic'agents,'due'to'their'absorption'in'the'range'300−400'nm;' ii)'PSs'based'on'the'Ru(II)'polypyridine'complex,'which'are'designed'to'give'a' long:lying'excited'state,'can'act'as'PSs'through'both'type'I'and'II'reactions;'iii)' PSs' based' on' aza:BODIPY' core' should' be' of' interest' for' type' II'mechanism.' Other' BODIPY' derivatives,' linked' to' an' antioxidant' molecule' (e.g.' α: tocopherol)' have' been' suggested' to' obtain' a' chemicontrolled' 1O2' photosensitization.' Density' functional' theory' (DFT)' and' time:dependent' DFT' (TD:DFT)' have' been'employed'in'all'the'computations.''
Contribution of flavonoids and iridoids to health properties of edible plants of Arbutas, Cornas, and Vuccinium genera from Mediterranean area
(Università della Calabria, 2020-06-19) Tenuta, Maria Concetta; Tundis, Rosa; Andò, Sebastiano; Deguin, Brigitte; Lallemand, Marie-Christine
Aim of the study: Natural compounds produced by the “nature laboratory” continue to play a leading role in the process of discovering and developing of new drugs, food supplements and/or functional foods. Today, in fact, we are witnessing a renewed interest in these products for the prevention and treatment of numerous diseases, including those associated with oxidative stress such as diabetes and inflammatory diseases. In this context, the present research project aimed at identifying the best techniques that allow the extraction of two classes of active compounds, flavonoids and iridoids, from the fruits and leaves of Arbutus unedo L., Vaccinium corymbosum L., Cornus mas L., and Cornus sanguinea L. Their edible fruits but also their leaves (considered byproducts) represent rich sources of bioactive compounds. The work continued with the evaluation of in vitro biological activity and the fractionation of the most active extracts in order to envisage their potential use as a source of bioactive compounds for the development of functional foods, nutraceuticals and/or food supplements . Materials and methods: The chemical profile of extracts was evaluated by using LC/ESI/QTOF/MS. Four different in vitro tests (DPPH, ABTS, -carotene bleaching test and FRAP), were performed to investigate the antioxidant activity. The potential hypoglycaemic activity was investigated by the inhibition of -amylase and -glucosidase enzymes. The inhibitory effects of the extracts on the production of nitric oxide have been studied by Griess assay. The most active extracts of C. mas and C. sanguinea were subjected to fractionation in order to obtain a separation of flavonoids and iridoids using XAD-16 resin. Obtained fractions were tested to evaluate their ability to reduce the activation of the NF-kB factor. Results: LC/ESI/QTOF/MS analyses detected for the first time in the A. unedo the presence of ellagic acid 4-O--D-glucopiranoside, kaempferol 3-O-glucoside, myricetin, myricetin 3-O-rhamnopiranoside, naringenin 7-O-glucoside, isovitexin 7-O-glucoside, and norbergenine and V. corymbosum the presence of the iridoids scandoside, vaccinoside, geniposide, and dihydromonotropein. The complete chemical characterization of C. sanguinea fruits and leaves was conducted herein for the first time, reporting the presence of flavonoids and iridoids. Worthy of note are the results obtained from the hypoglycaemic activity. The extracts of both Cornus species showed a high inhibition of -glucosidase and a moderate inhibition of -amylase. Of particular note were the results obtained with the hydroalcoholic maceration of leaves and dried fruits of C. mas (TDB and MDB), the ethanol maceration of fresh leaves (PF1) and the hydroalcoholic maceration of dry leaves (SD2) of C. sanguinea, which for this reason were subjected to fractionation using XAD-16 resin. C. sanguinea PF1 (II) and SD2 (II) fractions showed the best antioxidant and NF-KB inhibition activity. Conclusion: All the investigated extracts showed a promising antioxidant, hypoglycaemic and anti-inflammatory potential, confirming the positive contribution of the two classes of compounds under study, flavonoids and iridoids, suggesting their potential use as a rich source of useful bioactive compounds for the formulation of new products to prevent diseases associated with oxidative stress such as inflammatory diseases and diabetes. In particular, results obtained with the C. sanguinea fractions PF1 (II) and SD2 (II) encourage researchers to continue the study with further in vivo studies. Scopo dello studio: I composti naturali prodotti dal “laboratorio natura” continuano ad avere un ruolo di primo piano nel processo di scoperta e sviluppo di nuovi farmaci, integratori alimentari e/o alimenti funzionali. Oggi, infatti, si assiste a un rinnovato interesse nei riguardi di tali prodotti nella prevenzione e nel trattamento di numerose patologie, tra cui quelle associate allo stress ossidativo come diabete e malattie infiammatorie. In questo ambito si inserisce il presente lavoro di ricerca, volto all’identificazione delle migliori tecniche che consentano l’estrazione di due classi di principi attivi, i flavonoidi e gli iridoidi, dai frutti e dalle foglie di Arbutus unedo L., Vaccinium corymbosum L., Cornus mas L. e Cornus sanguinea L. I loro frutti commestibili ma anche le foglie (considerate prodotti di scarto) rappresentano ricche fonti di composti bioattivi. Il lavoro ha quindi riguardato la valutazione dell’attività biologica in vitro e il frazionamento bio-guidato degli estratti risultati più attivi al fine di prospettare un loro potenziale impiego come fonte di composti bioattivi per lo sviluppo di alimenti funzionali, nutraceutici e/o integratori alimentari. Materiali e metodi: Il profilo fitochimico degli estratti è stato valutato mediante LC/ESI/QTOF/MS. Per la determinazione in vitro della capacità antiossidante sono stati eseguiti quattro diversi test (DPPH, ABTS, FRAP e-carotene bleaching test). La potenziale attività ipoglicemizzante è stata investigata mediante l’inibizione degli enzimi -amilasi e -glucosidasi. Gli effetti inibitori degli estratti sulla produzione di ossido nitrico sono stati studiati mediante saggio di Griess. Gli estratti maggiormente attivi di C. mas e C. sanguinea sono stati sottoposti a frazionamento per la separazione di flavonoidi e iridoidi mediante l’impiego della resina XAD-16. Le frazioni ottenute sono state saggiate per valutare la capacità di ridurre l’attivazione del fattore NF-kB. Risultati: L’analisi LC/ESI/QTOF/MS ha rilevato per la prima volta nell’A. unedo la presenza di acido ellagico 4-O--D-glucopiranoside, kaemferolo 3-O-glucoside, miricetina, miricetina 3-O-ramnopiranoside, naringenina 7-O-glucoside, isovitexina 7-O-glucoside, e norbergenina e nel V. corymbosum la presenza degli iridoidi scandoside, vaccinoside, geniposide e diidromonotropeina. La caratterizzazione chimica completa di frutti e foglie del C. sanguinea è stata condotta per la prima volta, riportando la presenza flavonoidi ed iridoidi. Degni di nota sono i risultati ottenuti dall’attività ipoglicemizzante. Gli estratti di entrambe le specie di Cornus hanno mostrato un’alta inibizione dell’-glucosidasi e una moderata inibizione dell’-amilasi. Di particolare rilievo sono stati i risultati ottenuti con la macerazione idroalcolica di foglie e frutti secchi di C. mas (TDB e MDB), la macerazione in etanolo delle foglie fresche (PF1) e la macerazione idroalcolica delle foglie secche (SD2) di C. sanguinea, che per tale motivo sono stati sottoposti a bio-frazionamento mediante l’impiego della resina XAD-16. Le frazioni PF1 (II) e SD2 (II) del C. sanguinea hanno mostrato la migliore attività antiossidante e di inibizione dell’NF-KB. Conclusioni: Tutti gli estratti investigati hanno mostrato un promettente potenziale antiossidante, ipoglicemizzante ed antinfiammatorio, confermando il positivo contributo delle due classi di composti oggetto di studio, i flavonoidi e iridoidi prospettando il loro potenziale impiego come ricca fonte di composti bioattivi utili per la formulazione di nuovi prodotti per prevenire le malattie associate allo stress ossidativo come malattie infiammatorie e diabete. In particolare, i risultati ottenuti con le frazioni PF1 (II) e SD2 (II) del C. sanguinea incoraggiano i ricercatori nel proseguire lo studio con eventuali studi in vivo. Résumé Objet de l’étude : Les substances naturelles produites par le laboratoire « Nature » continuent de jouer un rôle de premier plan dans le processus de découverte et de développement de nouveaux médicaments, compléments alimentaires et/ou aliments fonctionnels. Aujourd’hui, en effet, nous assistons à un regain d'intérêt pour ces produits pour la prévention et le traitement de nombreuses maladies, notamment celles associées au stress oxydatif, telles que le diabète et les maladies inflammatoires. Le présent travail de recherche vise à identifier les meilleures techniques permettant d’extraire deux classes de principes actifs, les flavonoïdes et les iridoïdes, des fruits et des feuilles d’Arbutus unedo L., Vaccinium corymbosum L., Cornus mas L. et Cornus sanguinea L. Leurs fruits comestibles, comme leurs feuilles pourtant actuellement considérées comme des déchets représentent de riches sources de composés bioactifs. Les travaux ont donc porté sur l'évaluation de l'activité biologique in vitro et le fractionnement bio-guidé des extraits les plus actifs afin d'envisager leur utilisation potentielle comme source de composés bioactifs pour le développement d'aliments fonctionnels, et nutraceutiques. Matériels et méthodes : Le profil phytochimique des extraits a été évalué par LC/ESI/QTOF/MS. Pour la détermination in vitro de la capacité antioxydante, quatre tests différents ont été réalisés (DPPH, ABTS, FRAP et-carotene bleaching test). L’activité hypoglycémique potentielle a été étudiée par inhibition des enzymes -amylase et -glucosidase. Les effets inhibiteurs des extraits sur la production d'oxyde nitrique ont été étudiés par le test de Griess. Les extraits les plus actifs de C. mas et C. sanguinea ont été soumis à un bio-fractionnement pour la séparation des flavonoïdes et des iridoïdes au moyen de la résine XAD-16. Les fractions obtenues ont été testées pour évaluer l'aptitude à réduire l’activation du facteur NF-kB. Résultats : L’analyse par LC/ESI/QTOF/MS détectée pour la première fois dans l’A. unedo la présence d’acide ellagique 4-O--D-glucopiranoside, de kaempférol 3-O-glucoside, de myricétine, de myricétine 3-O-rhamnopyranoside, de naringénine 7-O-glucoside, d'isovitexine 7-O-glucoside et de norbergénine, et V. corymbosum la présence d'iridoïdes scandoside, vaccinoside, géniposide et dihydromonotropeine. La caractérisation chimique complète des fruits et des feuilles de C. sanguinea a été réalisée pour la première fois, en indiquant la présence de flavonoïdes et d'iridoïdes. Il convient de noter les résultats obtenus à partir de l’activité hypoglycémique. Les extraits des deux espèces de Cornus ont montré une forte inhibition de l’-glucosidase et une inhibition modérée de l'-amylase. Résultats intéressants ont été obtenus avec la macération hydro-alcoolique de feuilles et de fruits séchés de C. mas (TDB et MDB), la macération en éthanol des feuilles fraîches (PF1) et la macération hydro-alcoolique de feuilles sèches (SD2) de C. sanguinea qui, pour cette raison, ont été soumis à un fractionnement avec la résine XAD-16. Les fractions PF1 (II) et SD2 (II) de C. sanguinea présentaient la meilleure activité inhibitrice des antioxydants et du NF-kB. Conclusion: Tous les extraits étudiés ont montrés un potentiel antioxydants, hypoglycémiants et anti-inflammatoire, confirmant l’apport positif des deux classes de composés étudiés, les flavonoïdes et les iridoïdes, suggérant leur utilisation potentielle comme source riche de composés bioactifs utiles pour la formulation de nouveaux produits pour prévenir les maladies associées au stress oxydatif, telles que les maladies inflammatoires et le diabète. En particulier, les résultats obtenus avec les fractions PF1 (II) et SD2 (II) de C. sanguinea incitent les chercheurs à poursuivre l’étude en proposant éventuellement des études in vivo.
Design and development of new functional materials for pharmaceutical and biomedical purposes
(Università della Calabria, 2022-02-15) Curcio, Federica; Andò, Sebastiano; Trombino, Sonia
Design, synthesis and characterization of biologically active heterocyclic organic compounds
(2018-04-13) Algieri, Vincenzo; Andò, Sebastiano; De Nino, Antonio
Development and optimization of analytical protocols based on microextraction techniques for clinical screening and environmental control
(2019-03-21) Elliani, Rosangela; Andò, Sebastiano; Tagarelli, Antonio
The development of analytical protocols for the determination of analytes at trace levels in complex matrices (e.g. biological fluids or contaminated water) is a crucial point for the environmental assessment and monitoring as well as for scientific research in the field of disease biomarkers. An essential part of analytical method development is represented by sample preparation due to its significant impact on most of the subsequent steps and the data quality. In recent years, the application of pro-ecological, automated, solvent-free sample preparation approaches or techniques employing a minimal amount of solvents or safe and non-toxic extractants has become one of the most popular research topics in analytical chemistry. In this context, microextraction techniques represent a suitable choice for the extraction of analytes from complex matrices because these techniques use less organic solvent and allow to perform in a single step extraction and concentration of analytes. Moreover, the use of microextraction techniques for sample preparation reduces the number of errors that commonly result from multi-stage procedures, and limits the negative impact on the environment and the health of analytical chemists performing laboratory work. The goal of this Ph.D project was the development and optimization of analytical methods based on the use of microextraction techniques for the assay of disease biomarkers and environmental contaminants in biological fluids and environmental matrices. The microextraction techniques employed in this thesis were solid phase microextraction (SPME) and microextraction by packed sorbent (MEPS). SPME was used to evaluate the applicability of a new fiber (PDMS/DVB/PDMS) as analytical sampling tool for investigation in raw human urine. The PDMS/DVB/PDMS fiber was exploited to develop a DI-SPME-GC-MS method for the assay of polycyclic aromatic hydrocarbons (PAHs) with 2-6 aromatic rings in untreated human urine samples. Moreover, in the light of the increasing demand of faster and easier protocols allowing the assessment of disease biomarkers, SPME was applied to develop a reliable and rapid GC-MS approach for the determination of polyamines in human urine. Indeed, polyamines are widely recognized as among the most important cancer biomarkers for early diagnosis and treatment. SPME was also applied for the extraction of nine phthalates monoesters in urine samples. These compounds are important metabolites of phthalates and their assay can reliably rank exposures to phthalates over a period. MEPS was used to extract organophosphate ester flame retardant in aqueous matrices and, again, monoesters phthalates in urine. In both methods, in order to improve method sensitivity, programmed temperature vaporization (PTV) was chosen as gas chromatographic injection technique. For polyamines and phthalates monoesters, a prior derivatization step with suitable reagents was carried out before gas chromatographic analysis so as to improve chromatographic elution and resolution by decreasing volatility and polarity of analytes. Derivatization reaction was performed directly in aqueous samples using alkyl chloroformates. The combined use of alkyl chloroformate as derivatizing reagent and SPME for analyte extraction was chosen to develop a simple protocol involving minimal sample handling and no consumption of toxic organic solvents. The variables affecting the different steps of the proposed protocols were optimized by the multivariate approach of experimental design which has allowed for the simultaneous investigation of the different factors in the entire experimental domain and the possible synergic effects between variables. In this thesis, experimental design was used to optimize the parameters influencing SPME extraction, MEPS extraction, PTV process and derivatization reaction. Gas chromatographic analyses were carried out using a GC-QqQ-MS instrument in selected reaction monitoring (SRM) acquisition which has allowed to obtain reconstructed chromatograms with well-defined chromatographic peaks and to achieve high specificity through the selection of appropriate precursor-product ion couples, improving the capability in analyte identification. Finally, during the period as visiting Ph.D student at University Duisburg-Essen, Faculty of Chemistry, Instrumental Analytical Chemistry, the object of research activity, coordinated by Professor Torsten C. Schmidt, concerned the extraction of fatty acid methyl esters (FAMEs) in wastewater by solid phase microextraction arrow (SPME arrow).
Eco-friendly extraction of bioactive compounds from olive (drupes and leaves), Stevia and Schizochytrium sp.
(2017-04-13) Santoro, Ilaria; Andò, Sebastiano; Sindona, Giovanni; Nardi, Monica
The use of nutraceutical compounds, as well as their extraction from natural sources, is now the subject of studies in various sectors. The research groups working in this field focus their attention on the optimization of extraction methods that combine the economic aspect with the ecological one. High-value active principles can be recovered from wastes of agri-food farms or industries. The purpose of this work was the evaluation of green extracting techniques and characterization of different bio-active compounds recovered from different matrices. The main goals were:  The development of an easy analytical approach for the identification and assay of Stevia sweeteners in commercially available soft drink based on liquid chromatography coupled to tandem mass spectrometry;  Extraction and characterization of active compounds from Olive (drupes and leaves) wastewaters, monitored by Liquid Chromatography (LC)-Electro Spray Ionization (ESI)-tandem mass spectrometry (MS/MS)  Extraction and characterization of lipids from alghe Schizochytrium sp. and identification of new adducts of fatty acids, monitored by Gas Chromatography/Liquid Chromatography (LC)-Electro Spray Ionization (ESI)-tandem mass spectrometry (MS/MS)
Effect of bergamot essential oil and its constituent linalool on myogenic and neuronally-mediated contractions of human and rat isolated colon: potential benefits in complementary treatment of intestinal diseases
(Università della Calabria, 2020-07-01) Straface, Marilisa; Andò, Sebastiano; Morrone, Luigi
Introduction Bergamot essential oil (BEO) is used in aromatherapy and as an additive to food and drink to promote a citrus flavour. In animal models, BEO can modulate the synaptic functions within the Central Nervous System. However, it is not known if BEO can affect the functions of the gastrointestinal tract, despite being widely used in the food industry. BEO and its components linalool, limonene and linalyl-acetate were therefore examined for their ability to influence neuromuscular contractions of human and rat isolated colon. Material and Methods Human colon was obtained at surgery for bowel cancer following informed consent; mucosa-free strips were cut parallel to the circular muscle. Rat colon (Sprague-Dawley) strips were also cut as circular muscle preparations. In most experiments, each strip was suspended between platinum wire electrodes in tissue baths containing Krebs solution (5% CO2 in O2; 37°C) under tension (1 or 2g of tension for rat and human muscle strips, respectively) for recording of isometric contractions in response to stimulation of cholinergic nerves using electrical field stimulation (EFS) or to the application of exogenous stimulants of smooth muscle contraction (acetylcholine (ACh), 5-hydroxytryptamine (5-HT), substance P (SP) or KCl). Cumulative concentration-response curves were obtained for BEO (10-6 - 10-3 % v/v) and its major components linalool, limonene and linalyl-acetate (10-9 - 10-4 M). The inhibition of the amplitude of the contractions by each agent was expressed in percentage terms as the mean  s.e.m of the numbers of patients or animals. Results In preliminary experiments, BEO and its components reduced contractions of rat colon caused by ACh, 5-HT or SP. Subsequently concentration-dependent inhibition of both KCl-evoked contractions and neuronally-mediated contractions were demonstrated in response to BEO or its components, with greater potency when tested on the latter. The inhibitory effect of BEO on myogenic and neuronally-mediated contractions was associated largely via the actions of linalool (apparent pIC50 5.6 ± 0.4, n= 4 on KCl-evoked contractions; apparent pIC50 6.7 ± 0.2, n = 4 on neuronally-mediated contractions) in human colon. Similar but less potent activity of linalool was obtained in rat colon (apparent pIC50 5.4 ± 0.3, n = 4 on KCl-evoked contractions; apparent pIC50 5.8 ± 0.1 %, n = 4 on neuronally-mediated contractions). Conclusion The results indicated that BEO, largely via the actions of linalool, inhibited both human and rat enteric neurotransmission. Some species differences were found in the ability of these substances to inhibit neuronally-mediated contractions; the rank order in terms of potency (apparent pIC50) in human was: linalool > limonene >> linalyl acetate = BEO, and in rat was: linalyl acetate > limonene = linalool >> BEO. Both BEO and linalool were more potent in human muscle strips, acting at least partly by directly inhibiting muscle contractility. These data provide a potential mechanism for their use as a complementary treatment of gastrointestinal diseases related to increased intestinal motility.