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Badania Związków Fenolowych W Wybranych Gatunkach Szałwii (Salvia Sp.) Metodami Chromatograficznymi HPLC I TLC

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Badania Związków Fenolowych W Wybranych Gatunkach Szałwii (Salvia Sp.) Metodami Chromatograficznymi HPLC I TLC Title: Badania związków fenolowych w wybranych gatunkach szałwii (Salvia sp.) metodami chromatograficznymi HPLC i TLC Author: Dorota Staszek Citation style: Staszek Dorota. (2012). Badania związków fenolowych w wybranych gatunkach szałwii (Salvia sp.) metodami chromatograficznymi HPLC i TLC. Praca doktorska. Katowice : Uniwersytet Śląski Dorota Staszek Badania związków fenolowych w wybranych gatunkach szałwii (Salvia sp.) metodami chromatograficznymi HPLC i TLC praca doktorska Promotorzy pracy: prof. dr hab. Teresa Kowalska Zakład Chemii Ogólnej i Chromatografii Instytut Chemii, Uniwersytet Śląski w Katowicach prof. dr hab. Monika Waksmundzka-Hajnos Katedra Chemii Nieorganicznej Uniwersytet Medyczny w Lublinie Promotor pomocniczy: dr Mieczysław Sajewicz Zakład Chemii Ogólnej i Chromatografii Instytut Chemii, Uniwersytet Śląski w Katowicach Instytut Chemii Uniwersytet Śląski Katowice, 2012 Składam serdeczne podziękowania Pani prof. dr hab. Teresie Kowalskiej oraz Pani prof. dr hab. Monice Waksmundzkiej-Hajnos za opieką naukową poświęcony czas oraz wszechstronną pomoc w trakcie wykonywania badań i redagowania niniejszej pracy Panu dr Mieczysławowi Sajewiczowi za cenne rady oraz pomoc w trakcie prowadzenia badań Rodzicom i bratu za wyrozumiałość, cierpliwość oraz ogromne wsparcie, które dodawało mi sił Koleżankom i kołegom z „ 55 ” za spędzone razem niezapomniane chwile (pracę dedykuje moim ukochanym (Rodzicom Spis treści I. WSTĘP.....................................................................................................................................................................9 II. CZĘŚĆ TEORETYCZNA........................................................................................................................................ 11 1. Charakterystyka botaniczna badanych gatunków szałwii................................................................. 13 1.1. Systematyka szałwi i ......................................................................................................................... 13 1.2. Krótki opis wybranych gatunków szałwii.......................................................................................14 1.3. Wybrane klasy związków chemicznych zawartych w szałwii i ich właściwości farmakologiczne.............................................................................................................................................14 1.3.1. Krótka charakterystyka chemiczna kwasów fenolowych i ich właściwości farmakologiczne.. 14 1.3.2. Krótka charakterystyka chemiczna flawonoidów i ich właściwości farmakologiczne..............15 2. Metody ekstrakcj i.................................................................................................................................. 19 3. Przykłady literaturowe wybranych metod ekstrakcji zastosowanych do wyodrębniania kwasów fenolowych z materiału roślinnego.................................................................................................................23 4. Chemotaksonomia roślin i jej znaczenie dla nowoczesnej systematyki roślin............................... 27 III. CEL PRACY.............................................................................................................................................................29 IV. CZĘŚĆ DOŚWIADCZALNA.................................................................................................................................33 1. Aparatura i odczynniki......................................................................................................................... 35 2. Metodyka prowadzenia badań..............................................................................................................38 2.1. Materiał roślinny................................................................................................................................38 2.2. Zastosowane metody ekstrakcji związków fenolowych............................................................... 38 2.2.1. Ekstrakcja tradycyjna........................................................................................................................ 38 2.2.2. Ciśnieniowa ekstrakcja cieczowa (ASE) z wykorzystaniem różnych ekstrahentów................. 39 2.2.3. Selektywna ekstrakcja kwasów fenolowych...................................................................................39 2.2.4. Selektywna ekstrakcja flawonoidów...............................................................................................41 2.2.5. Wielostopniowa ekstrakcja związków fenolowych.......................................................................41 2.3. Analiza ekstraktów roślinnych techniką chromatografii cienkowarstwowej..............................44 2.3.1. Układy chromatograficzne zastosowane do optymalizacji rozdziału ekstraktów szałwii lekarskiej (S. officinalis) uzyskanych metodą tradycyjną oraz metodą ciśnieniowej ekstrakcji cieczowej........................................................................................................................................................44 2.3.2. Warunki chromatografowania ekstraktu szałwii lekarskiej (S. officinalis) uzyskanego metodą ciśnieniowej ekstrakcji cieczowej techniką chromatografii preparatywnej (PLC)................................ 45 2.3.3. Układy chromatograficzne zastosowane do rozdziału ekstraktów dwudziestu gatunków szałwii uzyskanych metodą ciśnieniowej ekstrakcji cieczowej............................................................................. 45 2.3.4. Specjalne techniki rozwijania w analizie metanolowych ekstraktów szałwii.............................47 2.3.5. Warunki chromatografowania zastosowane podczas określenia aktywności anty oksydacyjnej ekstraktów szałwii przy użyciu stabilnego rodnika DPPH.......................................................................48 2.3.6. Układy chromatograficzne zastosowane do optymalizacji rozdziału ekstraktów uzyskanych metodą selektywnej ekstrakcji związków fenolowych (TLC-MS vs. TLC-LC-MS).............................49 2.3.7. Układy chromatograficzne zastosowane do optymalizacji rozdziału ekstraktów uzyskanych metodą wielostopniowej ekstrakcji związków fenolowych......................................................................50 2.4. Analiza ekstraktów roślinnych techniką wysokosprawnej chromatografii cieczowej...............52 2.4.1. Warunki rozdziału zastosowane podczas porównania metod ekstrakcji oraz do analizy jakościowej i ilościowej ekstraktów uzyskanych metodą ciśnieniowej ekstrakcji cieczowej...............52 2.4.2. Warunki rozdziału zastosowane podczas eksperymentu TLC-LC-MS dla ekstraktów uzyskanych metodą selektywnej ekstrakcji związków fenolowych......................................................... 53 2.4.3. Warunki rozdziału zastosowane podczas analizy jakościowej ekstraktów uzyskanych metodą selektywnej ekstrakcji flawonoidów............................................................................................................54 2.4.4. Warunki rozdziału zastosowane podczas analizy jakościowej ekstraktów uzyskanych metodą wielostopniowej ekstrakcji związków fenolowych.................................................................................... 54 2.5. Oznaczenia spektrofotometryczne sumy zawartości kwasów fenolowych i flawonoidów...........55 2.5.1. Spektrofotometryczne oznaczanie sumy zawartości kwasów fenolowych...................................55 2.5.2. Spektrofotometryczne oznaczanie sumy zawartości flawonoidów................................................55 2.6. Chemometryczna analiza chromatogramów cieczowych zastosowana do wybranych ekstraktów szałwii..........................................................................................................................................56 2.6.1. Eliminacja tła sygnału analitycznego..............................................................................................56 2.6.2. Usuwanie szumu z sygnału analitycznego...................................................................................... 56 2.6.3. Nakładanie sygnałów analitycznych................................................................................................57 2.6.4. Analiza czynników głównych (PCA)..............................................................................................58 2.6.5. Dyskryminacyjna metoda częściowych najmniejszych kwadratów............................................ 58 3.1. Zastosowane metody ekstrakcji składników chemicznych z materiału roślinnego na przykładzie szałwii lekarskiej (S. officinalis).....................................................................................................................60 3.1.1. Porównanie metod ekstrakcji szałwii lekarskiej (S. officinalis) techniką chromatografii cienkowarstwowej (TLC) z detekcją densytometryczną.......................................................................... 60 3.1.2. Porównanie metod ekstrakcji szałwii lekarskiej (S. officinalis) techniką wysokosprawnej chromatografii cieczowej (HPLC/DAD).................................................................................................... 63 3.2. Analiza metanolowych ekstraktów szałwii techniką chromatografii cienkowarstwowej............. 70 3.2.1. Analiza metanolowych ekstraktów szałwii techniką jednokierunkowej, izokratycznej chromatografii cienkowarstwowej.............................................................................................................. 71 3.2.2. Analiza metanolowych ekstraktów szałwii
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