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Lietuvos Sveikatos Mokslų Universitetas Medicinos LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS MEDICINOS AKADEMIJA FARMACIJOS FAKULTETAS FARMAKOGNOZIJOS KATEDRA RUGILĖ OKULEVIČIŪTĖ LIETUVOJE AUGINAMŲ NAMINIŲ SLYVŲ (PRUNUS DOMESTICA L.) VAISIŲ FENOLINIŲ JUNGINIŲ KOKYBINĖS IR KIEKINĖS SUDĖTIES ĮVAIRAVIMO TYRIMAS Magistro baigiamasis darbas Darbo vadovas Dr. Mindaugas Liaudanskas KAUNAS, 2019 2 LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS MEDICINOS AKADEMIJA FARMACIJOS FAKULTETAS FARMAKOGNOZIJOS KATEDRA TVIRTINU: Farmacijos fakulteto dekanė: prof. dr. Ramunė Morkūnienė Data LIETUVOJE AUGINAMŲ NAMINIŲ SLYVŲ (PRUNUS DOMESTICA L.) VAISIŲ FENOLINIŲ JUNGINIŲ KOKYBINĖS IR KIEKINĖS SUDĖTIES ĮVAIRAVIMO TYRIMAS Magistro baigiamasis darbas Darbo vadovas Dr. Mindaugas Liaudanskas Data Recenzentas Darbą atliko Magistrantė Rugilė Okulevičiūtė Data Data KAUNAS, 2019 3 TURINYS SANTRAUKA....................................................................................................................... 7 SUMMARY .......................................................................................................................... 8 SANTRUMPOS .................................................................................................................... 9 ĮVADAS ................................................................................................................................ 8 DARBO TIKSLAS IR UŽDAVINIAI ................................................................................... 9 1. LITERATŪROS APŽVALGA ..................................................................................... 10 1.1 Slyvų (Prunus L.) genties augalų apibūdinimas, kilmė, paplitimas ir morfologiniai požymiai .............................................................................................................................. 10 1.2 Naminių slyvų (Prunus domestica L.) vaisių ir lapų cheminės sudėties tyrimai ..... 11 1.3 Slyvų vaisių fenolinių junginių ir antocianinų ekstrakcijos, kokybinės ir kiekinės analizės metodai ............................................................................................................................ 14 1.4 Slyvų vaisiuose kaupiamų junginių biologinio poveikio tyrimai ............................ 17 1.5 Literatūros apžvalgos apibendrinimas .................................................................... 19 2. TYRIMO METODIKA ................................................................................................ 20 2.1. Tyrimo objektas .................................................................................................... 20 2.2. Naudoti reagentai .................................................................................................. 20 2.3. Naudota aparatūra ................................................................................................. 21 2.4. Slyvų vaisių ėminių ištraukų paruošimas ............................................................... 21 2.5. Tyrimo metodai ..................................................................................................... 22 2.5.1. Bendro antocianinų kiekio nustatymas ............................................................. 22 2.5.2. Antocianinų nustatymas taikant efektyviąją skysčių chromatografiją................ 23 2.5.3. Bendro proantocianidinų kiekio nustatymas UV–regimosios šviesos spektrofotometrijos metodu........................................................................................................ 23 2.5.4. Fenolinių junginių nustatymas taikant ultra–efektyviąją skysčių chromatografiją . ........................................................................................................................ 24 2.5.5. Antioksidacinio aktyvumo in vitro nustatymas ................................................. 24 2.5.6. Tyrimo duomenų analizė .................................................................................. 25 3. REZULTATAI IR JŲ APTARIMAS............................................................................ 27 3.1. Slyvų vaisių ėminių antocianinų ekstrakcijos sąlygų nustatymas ........................... 27 3.2. Antocianinų kiekinės sudėties slyvų vaisių ėminiuose įvairavimas ........................ 28 3.2.1. Slyvų vaisių bendro antocianinų kiekio įvairavimo tyrimas .............................. 29 3.2.2. Slyvų vaisių antocianinų kokybinės ir kiekinės sudėties įvairavimo tyrimas ..... 30 3.3. Slyvų vaisių proantocianidinų kokybinės ir kiekinės sudėties įvairavimas ............. 33 3.4. Slyvų vaisių ėminių fenolinių junginių kiekinės ir kokybinės sudėties įvairavimas 36 4 3.5. Slyvų vaisių ėminių ištraukų antioksidacinio aktyvumo in vitro nustatymas UV– regimosios šviesos spektrofotometriniu metodu ............................................................................. 40 3.6. Slyvų vaisių ėminių fenolinių junginių kiekinės sudėties ir ištraukų antioksidantinio aktyvumo in vitro koreliacinių ryšių įvertinimas ............................................................................ 43 3.7. Slyvų vaisių ėminių sudėties palyginimas naudojant klasterinę analizę .................. 46 3.8. Poskiepio įtakos skirtingų veislių slyvų vaisių ėminiuose kaupiamų fenolinių junginių kiekiui tyrimas ................................................................................................................. 49 3.9. Rezultatų apibendrinimas ...................................................................................... 51 4. IŠVADOS .................................................................................................................... 52 5. PRAKTINĖS REKOMENDACIJOS ............................................................................ 54 6. LITERATŪRA............................................................................................................. 55 7. PRIEDAI........................................................................................................................ 8 SANTRAUKA R. Okulevičiūtės magistro baigiamasis darbas/ mokslinis vadovas dr. Mindaugas Liaudanskas; Lietuvos sveikatos mokslų universiteto Medicinos akademijos Farmacijos fakulteto Farmakognozijos katedra. – Kaunas. Pavadinimas - Lietuvoje auginamų naminių slyvų (Prunus domestica L.) vaisių fenolinių junginių kokybinės ir kiekinės sudėties įvairavimo tyrimas. Darbo tikslas – ištirti Lietuvoje auginamų naminių slyvų (Prunus domestica L.) vaisių fenolinių junginių kokybinės ir kiekinės sudėties įvairavimą ir nustatyti jų ištraukų antioksidacinį aktyvumą in vitro. Darbo uždaviniai: Nustatyti tinkamiausias antocianinų ekstrakcijos sąlygas. UV–regimosios šviesos spektrofotometriniu ir ESC metodais nustatyti slyvų vaisių ėminių antocianinų kiekinę ir kokybinę sudėtį. UESC–MS metodu nustatyti slyvų vaisių ėminių fenolinių junginių kiekinę ir kokybinę sudėtį. Nustatyti slyvų vaisių ištraukų antioksidacinį aktyvumą in vitro. Įvertinti koreliacinius ryšius tarp slyvų vaisių fenolinių junginių kiekio ir jų ištraukų antioksidacinio aktyvumo in vitro. Įvertinti poskiepio įtaką slyvų vaisiuose kaupiamų fenolinių junginių kiekinei sudėčiai. Tyrimo objektas ir metodai: tirti naminių slyvų (Prunus domestica L.) šių veislių vaisių ėminiai: 'Čačanska najbolje' (Prunus cerasifera Ehrh. ir 'Vangenheimo vengrinė' poskiepių), 'Čačanska Rana', 'Dambrovicų vengrinė', 'Edinburginė' (skirtingų poskiepių), 'Favorita del Sultano', 'Herman', 'Jubileum' (skirtingų poskiepių), 'Kijevo vėlyvoji', 'Kometa' ,'Opal', 'Rausvė', 'Stanley', 'Uleno renklodė', 'Valor' (skirtingų poskiepių), 'Viktorija', 'Violeta', 'Zarechnaya Rannyaya'. Bendras antocianinų ir proantocianidinų kiekis, antioksidacinis aktyvumas buvo vertintas UV–regimosios šviesos spektrofotometriniu metodu, individualių fenolinių junginių kiekiai nustatyti UESC–MS metodu. Tyrimo rezultatai ir išvados: Didžiausias bendras antocianinų kiekis buvo išgautas, taikant atitinkamai šias sąlygas: naudojant 50 proc. (v/v) etanolį, 678 W ultragarso galią 73 minutes ir naudojant 83,64 proc. (v/v) etanolį, 678 W ultragarso galią 40 min. UESC–MS metodu slyvų vaisiuose identifikuoti ir kiekybiškai įvertinti šie fenoliniai junginiai: cianidin-3-O-gliukozidas, cianidin-3-O- rutinozidas, peonidin-3-O-gliukozidas, peonidin-3-O-rutinozidas, procianidinai A2 ir C1, katechinas, avikuliarinas, hiperozidas, izokvercitrinas, izoramnetin-3-O-rutinozidas, rutinas, kvercetinas, chlorogeno rūgštis. Stipriausiu antioksidaciniu aktyvumu, įvertinus ABTS, FRAP ir CuPRAC metodais, pasižymėjo 'Čačanska najbolje' (abiejų poskiepių), 'Kometa', 'Opal' ir 'Zarechnaya Rannyaya' veislių slyvų vaisių ištraukos. Nustatytas stiprus koreliacinis ryšys tarp bendro antocianinų kiekio ir antioksidacinio aktyvumo, nustatyto ABTS ir FRAP metodais. Poskiepis turi įtakos kai kurių veislių slyvų vaisiuose kaupiamų tam tikrų fenolinių junginių kiekiui. SUMMARY The title of the master thesis. Qualitative and quantitative composition variability of phenolic compounds in European plum (Prunus domestica L.) fruits. The aim of the research. To determine qualitative and quantitative composition variability of phenolic compounds in fruits of European plum, which grow in Lithuanian climatic conditions and to evaluate their extracts antioxidant activity in vitro. The objectives of the research. To ascertain the most appropriate extraction conditions of anthocyanins from samples of European plum fruits. To determine qualitative and quantitative composition of anthocyanins in European plum fruit samples suing UV–Vis spectrophotometric and HPLC methods of analysis. To determine qualitative and quantitative composition of
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