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Gervuogių Vaisių Antocianinų Sudėties Tyrimas Efektyviosios Skysčių Chromatografijos (Esc) Metodu

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Gervuogių Vaisių Antocianinų Sudėties Tyrimas Efektyviosios Skysčių Chromatografijos (Esc) Metodu LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS FARMACIJOS FAKULTETAS FARMAKOGNOZIJOS KATEDRA ALIONA GRIŠKEVIČ GERVUOGIŲ VAISIŲ ANTOCIANINŲ SUDĖTIES TYRIMAS EFEKTYVIOSIOS SKYSČIŲ CHROMATOGRAFIJOS (ESC) METODU Magistro baigiamasis darbas Darbo vadovas: lekt. dr. D. Burdulis KAUNAS, 2016 2 LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS FARMACIJOS FAKULTETAS FARMAKOGNOZIJOS KATEDRA TVIRTINU: Farmacijos fakulteto dekanas Vitalis Briedis Data GERVUOGIŲ VAISIŲ ANTOCIANINŲ SUDĖTIES TYRIMAS EFEKTYVIOSIOS SKYSČIŲ CHROMATOGRAFIJOS (ESC) METODU Magistro baigiamasis darbas Darbo vadovas: lekt. dr. Deividas Burdulis Data Recenzentas: Darbą atliko: Magistrantė Aliona Griškevič Data Data KAUNAS, 2016 3 TURINYS SANTRAUKA .................................................................................................................................... 5 SUMMARY ........................................................................................................................................ 6 SANTRUMPOS .................................................................................................................................. 7 ĮVADAS ............................................................................................................................................. 8 DARBO TIKSLAS IR UŢDAVINIAI ............................................................................................... 10 1. LITERATŪROS APŢVALGA .................................................................................................. 11 1.1 Gervuogių genties (Rubus L.) ir rūšių apibūdinimas bei panaudojimas medicinoje .............. 11 1.1.1 Rubus genties apibūdinimas ......................................................................................... 11 1.1.2 Skirtingų gervuogių rūšių morfologinis apibūdinimas .................................................. 11 1.1.3 Paplitimas Lietuvoje .................................................................................................... 13 1.1.4 Gervuogių vaisių ir lapų cheminė sudėtis ..................................................................... 13 1.1.5 Gervuogių vaistinė augalinė ţaliava ir panaudojimas medicinoje ................................. 14 1.2 Antocianinų apibūdinimas, struktūra, biologinės savybės ir panaudojimas .......................... 15 1.2.1 Apibūdinimas............................................................................................................... 15 1.2.2 Paplitimas augaliniame pasaulyje ................................................................................. 15 1.2.3 Cheminės struktūros ypatumai ..................................................................................... 16 1.2.4 Fizikinės ir cheminės savybės ...................................................................................... 18 1.2.5 Farmakologinis poveikis .............................................................................................. 18 2. TYRIMO METODIKA .............................................................................................................. 20 2.1 Tyrimo objektas .................................................................................................................. 20 2.2 Naudoti reagentai ................................................................................................................ 21 2.3 Naudoti antocianinų standartai ............................................................................................ 21 2.4 Naudota aparatūra ............................................................................................................... 21 2.5 Ekstraktų ruošimo metodika ................................................................................................ 22 2.6 Antocianinų tapatybės ir kiekybės nustatymo metodika ....................................................... 22 2.7 Antocianinų kiekybinis nustatymas spektrofotometriniu metodu ......................................... 23 2.8 Nuodţiūvio nustatymas gervuogių vaisiuose ....................................................................... 23 2.9 Statistinis duomenų įvertinimas ........................................................................................... 24 3. TYRIMO REZULTATAI IR JŲ APTARIMAS ......................................................................... 25 4 3.1 Kokybinis antocianinų nustatymas R. caesius L., R. plicatus W. et N. ir R. nessensis H. vaisiuose ........................................................................................................................................ 25 3.2 Antocianinų kiekio nustatymas R. caesius L., R. plicatus W. et N. ir R. nessensis H. vaisiuose ........................................................................................................................................ 29 3.3 Antocianinų kiekio kitimo dinamika vaisių nokimo metu .................................................... 30 3.4 Antocianinų kiekio nustatymas skirtingose Lietuvos vietovėse augančių gervuogių vaisiuose....... ......................................... ……………………………………………………………32 4. IŠVADOS .................................................................................................................................. 35 5. PRAKTINĖS REKOMENDACIJOS ......................................................................................... 36 6. LITERATŪROS SĄRAŠAS ...................................................................................................... 37 5 SANTRAUKA A. Griškevič magistro baigiamasis darbas/ mokslinis vadovas lekt. dr. D. Burdulis; Lietuvos Sveikatos mokslų universiteto, Farmacijos fakulteto, Farmakognozijos katedra. – Kaunas. Tikslas: ištirti natūraliose augimvietėse surinktos gervuogių vaisių vaistinės augalinės ţaliavos antocianinų kokybinę ir kiekybinę sudėtį bei įvertinti sudėties kitimo dinamiką vaisių nokimo metu, sudėties įvairavimą skirtingose augavietėse surinktuose mėginiuose. Uţdaviniai: nustatyti antocianinų kokybinę sudėtį gervuogių vaistinės augalinės ţaliavos ekstraktuose; nustatyti antocianinų kiekybinę sudėtį gervuogių vaistinės augalinės ţaliavos ekstraktuose; įvertinti kokybinės ir kiekybinės sudėties įvairavimą skirtingų gervuogių rūšių vaisių ėminiuose; įvertinti antocianinų kiekinės sudėties kitimo dinamiką vaisių nokimo metu; įvertinti antocianinų kiekinės sudėties įvairavimą skirtingose augavietėse surinktuose mėginiuose. Ekstrakcijai pasirinkta maceracija mechaninėje purtyklėje, kaip ekstrahentą naudojant metanolį. Kokybinė ir kiekybinė antocianinų sudėtis R. caesius L., R. plicatus W. et N. ir R. nessensis H. gervuogių vaisių ėminiuose nustatyta efektyviosios skysčių chromatografijos (ESC) metodo pagalba. Bendras antocianinų kiekis R. caesius L. vaisiuose, surinktuose skirtingose Lietuvos vietovėse, nustatytas spektrofotometrijos metodu. Nustatyta, jog visose tirtose gervuogių rūšyse dominuoja du antocianinai: cianidin-3-O-gliukozidas ir cianidin-3-O-rutinozidas. Didţiausias cianidin-3-O-gliukozido ir cianidin-3-O-rutinozido kiekis nustatytas R. caesius L. vaisiuose (1,953 ± 0,452 proc. ir 0,502 ± 0,102 proc., atitinkamai), o maţiausias – R. nessensis H. vaisiuose (1,214 ± 0,225 proc. ir 0,276 ± 0,063 proc.). Nustatyta, jog didţiausias cianidin-3-O-gliukozido ir cianidi-3-O-rutinozido kiekis R. caesius L. vaisiuose yra susikaupęs ketvirtąją jų nokimo savaitę. Didţiausias cianidin-3-O-gliukozido ir cianidin-3- O-rutinozido kiekis nustatytas Balkasodţio kaime (Alytaus r.) augančių gervuogių vaisiuose (3,234 ± 0,448 proc. ir 0,932 ± 0,203 proc., atitinkamai). Maţiausias cianidin-3-O-gliukozido kiekis nustatytas Nemunaityje augančiose gervuogėse (2,166 ± 0,345 proc.), o cianidin-3-O-rutinozido – Obelijoje augančių gervuogių vaisių ėminiuose (0,558 ± 0,146 proc.). 6 SUMMARY A. Griškevič master's thesis / supervised by lect. dr. D. Burdulis; Lithuanian University of Health Sciences, Faculty of Pharmacy, Department of Pharmacognosy. - Kaunas. Objective: to explore qualitative and quantitative composition of anthocyanins in blackberries collected from natural habitats and evaluate the dynamics of anthocyanins in blackberries collected from different habitats on their ripening time. Objectives: to determine the qualitative composition of anthocyanins in blackberry herbal raw material extracts; to determine the quantitative composition of anthocyanins in blackberry herbal raw material extracts; to assess the variation of qualitative and quantitative composition in different types of blackberry fruit samples; to evaluate the variation of quantitative composition of anthocyanins in blackberries during the fruit ripening; to evaluate the variation of quantitative composition of anthocyanin in different habitats collected samples. The maceration by mechanical shaker using methanol was selected for extraction. Qualitative and quantitative composition of anthocyanins in R. caesius L., R. plicatus W. et N. and R. nessensis H. fruits samples was determined using high performance liquid chromatography (HPLC). Total content of anthocyanins in R. caesius L. fruits collected in different areas of Lithuania was determined using spectroscopy. It was found that all investigated blackberry species dominated by two anthocyanins: cyanidin-3-O-glucoside and cyanidin- 3-O-rutinoside. The highest cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside points set R. caesius L. fruits (1.953 ± 0.452 per cent. and 0.502 ± 0.102 per cent., respectively), while the lowest - R. nessensis H. fruits (1.214 ± 0.225 percent. and 0.276 ± 0.063 per cent.).
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