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Peron, Gregorio (2018) Metabolomics in natural products research: application to in vivo
bioactivity studies involving nutraceuticals.
[Ph.D. thesis]

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Abstract (english)

The analysis of urinary metabolite changes can provide information on the effects of food supplements or health-promoting products on healthy subjects or animal models. Specimen collection is non-invasive, long-term experiments can be easily conducted and urinary biomarkers of oxidative stress can also be measured, offering the opportunity to study the effects of a nutritional intervention and evaluate the redox status of the considered organism. In this thesis, three natural extracts were selected, namely Polygonum cuspidatum Sieb. et Zucc. (rich in resveratrol), Vaccinium macrocarpon Aiton (cranberry, rich in type A procyanidins) and green Coffea canephora Pierre ex Froehn. beans (GCBE, rich in chlorogenic acids), on the basis of their distribution on the market and on the basis of the information regarding their in vivo activity actually available in literature. In the first part of the work, the effects of P. cuspidatum dry extract were studied in healthy adult rats during a 49-days supplementation, using a combined 1H NMR and UPLC-HRMS metabolomics approach. Because of the reported antioxidant activity of resveratrol, the urinary amounts of two oxidative stress biomarkers were measured by targeted HPLC–MS/MS analyses and, due to the supposed “anti-aging” effects of resveratrol, multivariate models were designed in order to compare the aging effects between control and treated animals. Specific biomarkers were then selected and identified, and their amounts in urine were monitored throughout the experimental period.
UPLC-MS metabolomics approaches were used to evaluate the mode of action of cranberry against uropathogenic Escherichia coli in two independent experiments, using an animal model and enrolling healthy adult volunteers, respectively. The experimental design was similar for the two trials, and the aim was to observe if the results obtained from the first animal experiment were reproducible in humans, being cranberry supplements claimed for treatment of UTIs in human consumers. In the first experiment, healthy Sprague−Dawley rats were orally supplemented with a standardized cranberry extract for 35 days, to mimic a prolonged consumption of cranberry by healthy subjects. 24-h urinary outputs were collected weekly during the experiment, and samples were subjected to UPLC−MS analysis using an untargeted approach. In a second experiment on the same animal model, a single dose of cranberry was administered to animals and the changes of urinary composition at 2, 4, 8, and 24 h after extract administration were monitored. Anti-adhesive properties of all the urine samples were studied. Furthermore, the markers related to cranberry intake were discovered using a multivariate data analysis approach. Finally, a specific chromatographic method was developed for the measurement of unmodified PAC-A in urine. In the experiment involving human volunteers, these consumed an oral sachet containing 360 mg of dry cranberry extract and 100 mg of quercetin. Urine samples were collected at 2, 4, 6, 8 ad 24 hours after product administration and the anti-adhesive properties of urine samples were tested using an in vitro assay on E. coli. In order to correlate possible observed bioactivity with modification of urinary composition, LC-MS-based targeted and untargeted metabolomics approaches were used.
Finally, a clinical trial on a small number of healthy adult volunteers was performed to study the effects of a prolonged (30 days) supplementation with 400 mg of green coffee bean extract. The 24-h urinary samples were collected weekly, and analyzed by LC-MS. Multivariate data analysis approaches were applied and also targeted analysis were performed to measure urinary oxidative stress biomarkers, namely allantoin and 8-hydroxydeoxyguanosine (8-OHdG), in order to assess the potential antioxidant activity of GCBE in vivo.

Abstract (italian)

L’analisi del metaboloma urinario e delle sue variazioni può fornire utili informazioni sull’effetto del consumo di prodotti nutraceutici da parte di volontari sani o in modelli animali. I vantaggi di usare l’urina come matrice di studio sono correlati alla scarsa invasività di campionamento, al semplice trattamento pre-analitico dei campioni, alla possibilità di progettare e condurre esperimenti a lungo termine ed alla possibilità di quantificare alcuni markers di stress ossidativo. Sulla base della loro attuale diffusione sul mercato e dei dati di bioattività disponibili in letteratura, per il lavoro di tesi qui presentato sono stati presi in esame tre prodotti nutraceutici, contenenti rispettivamente estratti secchi di Polygonum cuspidatum Sieb. et Zucc. (contenente resveratrolo), Vaccinium macrocarpon Aiton (cranberry, contenente proantocianidine di tipo A) e semi verdi di Coffea canephora Pierre ex Froehn. (GCBE, contenente derivati dell’acido clorogenico). Nella prima parte di lavoro sono stati studiati gli effetti del consumo di un estratto secco di Polygonum cuspidatum per 49 giorni in ratti adulti sani, impiegando un approccio metabolomico combinato NMR/UPLC-MS. Per studiare l’effetto “anti-aging” attribuito al resveratrolo, sono stati sviluppati dei modelli multivariati in cui sono stati posti a confronto il gruppo “trattato” ed il gruppo di controllo; da questi sono stati selezionati ed identificati specifici biomarkers di invecchiamento, i quali livelli urinari sono stati poi monitorati per tutto il corso dell’esperimento.
Un simile approccio UPLC-MS è stato in seguito impiegato per studiare l’attività ex vivo di un estratto secco di cranberry contro un ceppo di Escherichia coli uropatogenico. In questo caso sono stati disegnati due esperimenti indipendenti, in cui gli effetti del trattamento sono stati studiati rispettivamente in ratti sani ed in volontari umani. Nel primo esperimento è stato somministrato un estratto secco di cranberry standardizzato in procianidine per 35 giorni a ratti Sprague-Dawley, per mimare un consumo prolungato di cranberry da parte di pazienti umani. Settimanalmente è stata effettuata la raccolta delle urine delle 24 ore, ed i campioni raccolti sono stati analizzati mediante un approccio untargeted UPLC-MS. Impiegando lo stesso modello animale, è stata effettuata una singola somministrazione di cranberry e la variazione della composizione urinaria è stata studiata a 2, 4, 8 e 24 ore dopo il trattamento. Gli stessi campioni sono stati utilizzati per testarne le proprietà antiadesive contro Escherichia coli e per determinare i markers urinari correlati all’assunzione di cranberry, grazie all’impiego di tecniche statistiche multivariate ed allo sviluppo di specifici metodi cromatografici atti alla determinazione di PAC-A intatte in matrice urinaria. Nel secondo esperimento, ai volontari umani sani è stato chiesto di consumare un integratore contenente 360 mg di estratto secco di cranberry e 100 mg di quercetina. I campioni urinari sono stati raccolti a 2, 4, 6, 8 e 24 ore a seguito del trattamento e le loro proprietà antiadesive sono state testate contro un ceppo di E. coli uropatogenico. In modo da correlare l’attività biologica osservata in vitro con la modifica composizionale indotta dal trattamento, sono stai impiegati degli approcci LC-MS targeted e untargeted.
Infine, è stato condotto uno studio clinico su un piccolo numero di volontari sani per studiare gli effetti di una integrazione con 400 mg di GCBE per 30 giorni. I campioni urinari delle 24 ore sono stati raccolti settimanalmente ed in seguito analizzati mediante LC-MS ed analisi statistica multivariata. Sono state eseguite analisi targeted per quantificare biomarkers urinari di stress ossidativo, al fine di valutare la potenziale attività antiossidante di GCBE in vivo.

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EPrint type:Ph.D. thesis
Tutor:Dall'Acqua, Stefano
Ph.D. course:Ciclo 30 > Corsi 30 > SCIENZE MOLECOLARI
Data di deposito della tesi:07 January 2018
Anno di Pubblicazione:07 January 2018
Key Words:Metabolomics; nutraceuticals; natural products; in vivo bioactivity; Polygonum cuspidatum; Cranberry; Green Coffee Bean Extract
Settori scientifico-disciplinari MIUR:Area 03 - Scienze chimiche > CHIM/08 Chimica farmaceutica
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Chimiche
Codice ID:10561
Depositato il:09 Nov 2018 14:32
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