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Tesi Phd Casacchia.Pdf Università della Calabria Dipartimento di Farmacia e Scienze della salute e della nutrizione Dottorato di ricerca “Medicina traslazionale” XXX ciclo Estratti naturali ad azione polivalente sui pathway di alterazioni metaboliche Settore scientifico disciplinare Bio15 – Biologia farmaceutica Coordinatore Ch.mo Prof. Sebastiano Andò Supervisore Dottoranda Ch.mo Prof. Giancarlo Statti dr. Teresa Casacchia 2014 -2017 ad Alessia e Francesco Indice Lista tabelle i Lista figure iii Introduzione 1 Capitolo 1 Sistematica, tassonomia e proprietà biologiche 3 1.1 Storia tassonomica M. comosum 3 1.1.1 Le specie del genere Muscari 6 1.1.2 Muscari comosum (L.) Mill. dalla botanica all’etnobotanica 7 1.1.3 Componente fitochimica nel M. comosum 10 1.1.4 Proprietà medicinali ed utilizzi terapeutici 16 1.1.4.1 Attività antiossidante e antiobesità in vitro 17 1.1.4.2 Attività antinfiammatoria 18 1.1.4.3 Attività antimutagena, anticancerogena e citotossica 19 1.1.4.4 Attività antibatterica e antivirale 19 1.1.4.5 Attività neuroprotettiva 20 1.2 Storia tassonomica Citrus L. 21 1.2.1 Caratteristiche botaniche del genere Citrus L. 22 1.2.2 Componente fitochimica in Citrus L. 24 1.2.3 Proprietà medicinali ed utilizzi terapeutici 26 1.2.3.1 Attività antiossidante e antiobesità 27 1.2.3.2 Attività antiperlipidemica 28 1.2.3.3 Attività antipertensiva 29 1.2.3.4 Attività antiperglicemica 30 1.2.3.5 Proprietà antitumorali 31 1.2.3.6 Attività antimicrobica 32 1.3 Storia tassonomica Citrullus lanatus (Thunb.) Matsum. & Nakai 33 1.3.1 Caratteristiche botaniche Citrullus lanatus 33 1.3.2 Caratteristiche fitochimiche Citrullus lanatus 34 1.3.3 Proprietà medicinali ed utilizzi terapeutici 38 1.3.3.1 Attività antiperglicemica 39 1.3.3.2 Attività antiperlipidemica 40 1.3.3.3 Attività antipertensiva 40 1.3.3.4 Attività antibatterica 41 1.3.3.5 Attività anticarcinogenica 42 1.3.3.6 Attività controllo disfunzione erettile 42 1.3.3.7 Attività lassativa 43 Capitolo 2 Sindrome metabolica e fattori di rischio 44 2.1 Criteri di definizione della Sindrome metabolica 46 2.2 Fattori di rischio 46 2.2.1 Obesità 46 2.2.2 Insulino-resistenza 47 2.2.3 Dislipidemia 48 2.2.4 Ipertensione 49 Capitolo 3 Materiali e metodi 53 3.1 Materiali 54 3.1.1 Reagenti per saggi chimici 57 3.2 Metodi 58 3.2.1 Estrazione e preparazione dei campioni 58 3.2.2 Analisi chimica 62 3.2.2.1 Determinazione dei polifenoli totali metodo Folin-Ciocalteau 62 3.2.2.2 Determinazione dei flavonoidi totali 63 3.2.2.3 Dosaggio licopene 64 3.2.2.4 Determinazione della L- citrullina 65 3.2.2.5 Determinazione della Vit. C 66 3.2.2.6 HPLC – UV- DAD 66 3.2.2.7 GC-MS 66 3.2.2.8 HPLC 66 3.2.2.9 Attività antiossidante metodo DPPH 68 3.2.2.10 Attività antiossidante metodo βcarotene bleaching test (BCB) 68 3.2.3 Saggi enzimatici in vitro 69 3.2.3.1 Determinazione dell’inibizione della lipasi pancreatica 69 3.2.3.2 Determinazione dell’inibizione dell’attività dell’enzima α-amilasi 70 3.2.3.3 Coltura cellulare saggio MTT (MCF-7) 70 3.2.4 Disegno di studio in vivo 71 3.2.4.1 Variabili antropometriche 72 3.2.4.2 Istologia del tessuto grasso 72 3.2.4.3 Misura biochimica del sangue 72 3.2.4.4 Homa Index 73 3.2.4.5 Western Blot: enzimi gluconeogenici/gluconeogenesi 73 3.2.4.6 Perfusione secondo metodica Langendorff 73 3.2.4.7 Statistica 76 Capitolo 4 Risultati e discussioni 77 4.1 Premessa 77 4.2 Muscari comosum (L.) Mill. 78 4.2.1 Analisi chimica 78 4.2.1.1 Polifenoli, flavonoidi totali, attività antiossidante 78 4.2.1.2 HPLC e GC-MS 83 4.2.2 Saggi enzimatici in vitro 86 4.2.2.1 Inibizione di lipasi e amilasi pancreatica di bulbi M. comsum 87 4.2.2.2 Attività antiproliferativa su cellule MCF-7 (MTT test) M. comosum 87 4.2.3 Saggi in vivo 88 4.2.3.1 Animali, parametri antropometrici e metabolici 88 4.2.3.2 Istologia dei tessuti adiposi 93 4.2.3.3 Cuore isolato e perfuso secondo Langendorff 96 4.3 Citrus L. 98 4.3.1 Analisi chimica 99 4.3.1.1 Polifenoli, flavonoidi totali, attività antiossidante 100 4.3.1.2 HPLC determinazione naringina, naringenina ed esperedina 109 4.3.2 Saggi enzimatici 110 4.3.2.1 Inibizione di lipasi e amilasi pancreatica 110 4.3.3 Animali, parametri antropometrici e metabolici 115 4.4 Citrullus lanatus (Thunb.) Matsum. & Nakai 120 4.4.1 Analisi chimica 121 4.4.1.1 Polifenoli, flavonoidi e attività antiossidante 121 4.4.1.2 Saggi enzimatici 125 Conclusioni 126 Bibliografia 131 Lista tabelle tab. 1 Classificazione Cronquist e APGIII tab. 2 Criteri diagnostici per sindrome metabolica tab. 3 Origine, periodo di raccolta e sigla di M. comosum (2014) tab. 4 Origine, periodo di raccolta e sigla Citrus 2014-2015) tab. 5 Origine, periodo di raccolta e sigla Citrus (2015- 2016) tab. 6 Origine, periodo di raccolta, sigla Citrullus lanatus (2015 – 2016) tab. 7 Resa di estrazione di bulbi M. comosum (2014) tab. 8 Resa di estrazione di bulbi, gambi e fiori M. comosum (Sila, 2015) tab. 9 Resa di estrazione di bulbi M. comosum crudi e cotti (Sila, 2015) tab. 10 Resa di estrazione di polpa Citrus (2014-2015) tab. 11 Resa di estrazione di buccia Citrus (2014-2015) tab. 12 Resa di estrazione di foglie Citrus (2014-2015) tab. 13 Resa di estrazione di bucce Clementina, Tarocco, Tacle® (2015 -2016) tab. 14 Resa di estrazione di polpa Clementina, Tarocco, Tacle® (2015 – 2016) tab. 15 Resa di estrazione di foglie Clementina, Tarocco, Tacle® (2015 – 2016) tab. 16 Resa di estrazione di buccia Citrullus lanatus tab. 17 Resa di estrazione di polpa Citrullus lanatus tab. 18 Gradiente di solventi tab. 19 Dosi estratto M. comosum tab. 20 Contenuto in polifenoli, flavonoidi totali e attività antiossidante (DPPH test) di bulbi M. comosum di diversa provenienza (anno 2014) tab. 21 Contenuto in polifenoli, flavonoidi totali e attività antiossidante (DPPH test) di bulbi, fiori e gambi M. comosum di origine calabrese (Sila, 2015) tab. 22 Contenuto in polifenoli e attività antiossidante (β-carotene bleaching test) di bulbi, fiori e gambi M. comosum di origine calabrese (Sila, 2015) tab.23 Contenuto in polifenoli e flavonoidi totali e attività antiossidante (DPPH test) in bulbi M. comosum crudi, bolliti e cotti a vapore tab. 24 Contenuto in polifenoli e flavonoidi totali e attività antiossidante (β-carotene bleaching test) in bulbi M. comosum crudi, bolliti e cotti a vapore tab. 25 Contenuto in polifenoli e flavonoidi totali e attività antiossidante (DPPH test) in bulbi M. comosum silani (2015, popolazione). tab. 26 Contenuto in polifenoli e flavonoidi totali e attività antiossidante (β-carotene bleaching test) in bulbi M. comosum silani (2015, popolazione). tab. 27 Inibizione di lipasi e amilasi pancreatica di estratti di bulbi crudi e cotti M. comosum tab. 28 Inibizione di lipasi e amilasi pancreatica di estratti di bulbi crudi M. comosum (Sila, 2015, popolazione). tab. 29 Variabili antropometriche nei ratti alimentati con SD (dieta standard) o HFD (dieta ad alta percentuale di grassi) o HFD + Lc (20 mg) o HFD + Lc (60 mg) per 12 settimane tab. 30 Variabili metaboliche in ratti alimentati con SD (dieta standard) o HFD (dieta ad alta percentuale di grassi) o HFD + Lc (20 mg) o HFD + Lc (60 mg) per 12 settimane tab. 31 Campioni e sigle Citrus tab. 32 Contenuto in polifenoli e flavonoidi totali in estratto di bucce Citrus tab. 33 Contenuto in polifenoli e flavonoidi totali in estratto di polpa Citrus tab. 34 Contenuto in polifenoli e flavonoidi totali in estratto di foglie Citrus tab. 35 Contenuto in polifenoli, flavonoidi totali ed attività antiossidante (DPPH) in estratto di bucce Citrus tab 36 Contenuto in flavonoidi totali ed attività antiossidante (β-carotene bleaching test) in estratto di bucce Citrus 1 tab. 37 Contenuto in polifenoli, flavonoidi totali ed attività antiossidante (DPPH test) in estratto di polpa Citrus tab. 38 Contenuto in flavonoidi totali ed attività antiossidante (β-carotene bleaching test) in estratto di polpa Citrus tab.39 Contenuto in polifenoli, flavonoidi totali ed attività antiossidante (DPPH test) in estratto di foglie Citrus tab.40 Contenuto in flavonoidi totali ed attività antiossidante (β-carotene bleaching test) in estratto di foglie Citrus tab. 41 Contenuto in polifenoli, flavonoidi totali ed attività antiossidante (DPPH test) in estratto di buccia Clementina, Tarocco, Tacle® tab.42 Contenuto in polifenoli, flavonoidi totali ed attività antiossidante (DPPH test) in estratto di polpa Clementina, Tarocco, Tacle® (2015) tab. 43 Contenuto in polifenoli, flavonoidi totali ed attività antiossidante (DPPH test) in estratto di foglie Clementina, Tarocco, Tacle® tab. 44 Contenuto in polifenoli, flavonoidi totali ed attività antiossidante (DPPH test) in buccia Tacle® (storage) tab. 45 Contenuto in polifenoli, flavonoidi totali ed attività antiossidante (DPPH test) in polpa Tacle® (storage) tab. 46 Contenuto in esperedina, naringenina, naringina in buccia Tacle® (1 °C e 90 - 95% RH) tab. 47 Contenuto in esperedina, naringenina, naringina in polpa Tacle® (1 °C e 90 - 95% RH) tab. 48 Inibizione di amilasi e lipasi pancreatica in bucce Citrus tab. 49 Inibizione di amilasi e lipasi pancreatica in polpa Citrus. tab. 50 Inibizione di lipasi e amilasi pancreatica in foglie Citrus tab. 51 Inibizione di lipasi e amilasi pancreatica in buccia Clementina, Tarocco, Tacle® tab. 52 Inibizione di lipasi e amilasi pancreatica in polpa Clementina, Tarocco, Tacle® tab.
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