Effectiveness of hop extract in the protection of yeast Saccharomyces cerevisiae against oxidative stress

Namen diplomske naloge je bil proučiti, ali je ekstrakt hmelja učinkovit pri zaščiti kvasovk Saccharomyces cerevisiae pred oksidativnim stresom. Priprava vodne raztopine etanolnega ekstrakta hmelja, ki smo jo uporabljali za analize, sestoji iz mešanja vzoreca hmelja z etanolom, filtriranja, evaporacije etanola, homogenizacije dobljene suhe snovi etanolnega ekstrakta z vodo in centrifugiranja. Dobljeno vodno raztopino etanolnega ekstrakta hmelja smo nato v različnih koncentracijah dodali kvasnim kulturam ter vzorce inkubirali. Nato smo celicam dodali 2\u27,7\u27-diklorodihidrofluorescein diacetat (H2DCFDA), ki se je ob prehodu skozi celično membrano v celici hidroliziral v DCFH. Le ta pa se je ob prisotnosti reaktivnih kisikovih zvrsti oksidiral do fluorescentne oblike (DCF). Z merjenjem ravni oksidacije v kvasnih celicah smo nato ugotavljali kateri vzorec določene koncentracije raztopine etanolnega ekstrakta hmelja ima največji antioksidativni potencial, pri čemer pomeni večja intenziteta fluorescence večjo znotrajcelično oksidacijo. Prišli smo do zaljučka, da vzorec z najvišjo koncentracijo vodne raztopine etanolnega ekstrakta hmelja rezultira v največji antioksidativni aktivnosti. Torej je naša domneva, da ekstrakt hmelja pozitivno vpliva na kvasovke ter jih ščiti pred povišanjem ravni znotrajcelične oksidacije, pravilna. V raziskavi pa se nismo poglobili v to, ali je za zaščito kvasnih celic pred oksidativnim stresom odgovoren le ksantohumol. V vzorcu vodne raztopine etanolnega ekstrakta hmelja, ki smo ga dali na analizo HPLC, so namreč poleg ksantohumola prisotne tudi druge spojine, katerim se v okviru svoje diplomske naloge nismo posvetili.The purpose of this work was to study whether hop extract is effective in protecting the yeast Saccharomyces cerevisiae from oxidative stress. Preparation of aqueous solution of the ethanol hop extract used for the analyses consists of mixing the sample hops with ethanol, solution filtration, ethanol evaporation, homogenization the resulting dry matter of the ethanol extract with water and centrifugation. Aqueous solution of ethanol hop extract was then added to yeast cultures in various concentrations and samples were incubated. 2\u27,7\u27-dichlorodihydrofluorescein diacetate (H2DCFDA) was then added to the cultures, which hydrolyzed to DCFH in the cell upon passage through the cell membrane. In the presence of reactive oxygen species, it oxidized to the fluorescent form dichlorofluorescein (DCF). By measuring the oxidation level in the yeast cells, we determined which concentration of ethanol hop extract solution, had the highest antioxidant potential, where a higher fluorescence intensity means higher intracellular oxidation level. We found out that the highest concentration of aqueous solution of ethanol hop extract results in the highest antioxidant activity. Therefore, our assumption that hop extract has a positive effect on yeast cells and protects them from increasing levels of intracellular oxidation, is correct. In research, however, we did not focus, whether, xanthohumol alone is responsible for protecting yeast cells from oxidative stress. In a sample of an aqueous solution of ethanol hop extract, which was given to HPLC analysis, there are other compounds beside xanthohumol, which were not addressed in the graduation thesis