Preučevanje delovanja antioksidanta epigalokatehin galata in oksidanta menadiona v celicah z metodo infrardeče spektroskopije

Svet, Luka (author), Jamnik, Polona (mentor), Grdadolnik, Jože (comentor)

V okviru magistrskega dela smo preverili vpliv in delovanje induktorja oksidativnega stresa menadiona na celice kvasovk Saccharomyces cerevisiae, kot modelnega organizma za raziskave oksidativnega stresa ter vlogo antioksidanta epigalokatein-3-galata (EGCG) pri zaščiti celic pred oksidativnim stresom. Celice kvasovke smo izpostavili preučevanim snovem v različnih koncentracijah, in sicer antioksidantu epigalokatehin galatu v koncentraciji 0,1 g/L ter oksidantu menadionu v koncentraciji 60 mmol/L. Vpliv oksidativnega stresa na celice smo preučevali tako, da smo celice najprej izpostavili antioksidantu in nato oksidantu ali pa le oksidantu brez predhodnega tretiranja. Oksidativni stres in zaščitno delovanje antioksidanta ter vplive na fiziološko stanje celic smo preverjali s pomočjo različnih metod, v osnovi pa so te bile: določanje znotrajcelične oksidacije, oksidativnih poškodb lipidov in celične energijske metabolne aktivnosti, določanje kultivabilnosti celic ter analiza absorpcijskih spektrov s pomočjo metode ATR-FTIR. Izkazalo se je, da so znotrajcelična oksidacija in oksidativne poškodbe lipidov v celicah, ki so bile izpostavljene EGCG in nato menadionu, nižje v primerjavi s celicami, ki so bile izpostavljene le menadionu. Celična metabolna aktivnost je bila v celicah, ki so bile izpostavljene oksidativnemu stresu, zaradi aktivacije popravljalnih mehanizmov višja, hkrati pa je EGCG inducirajoče vplival na metabolno aktivnost. Dodatek antioksidanta ni bistveno vplival na zvišanje kultivabilnosti celic kvasovk, se je pa ta zaradi oksidativnega stresa zmanjšala. Spremembe v fiziološkem stanju celic po tretiranju smo potrdili tudi z metodo ATR-FTIR, in sicer so bile razlike med različno tretranimi vzorci najbolj opazne v vsebnosti lipidov in ogljikovih hidratov. Na podlagi vseh rezultatov ocenjujemo, da je ATR-FTIR primerna metoda za oceno sprememb znotrajceličnega redoks stanja po tretiranju celic z oksidantom/antioksidantom.

• Language: Slovenian
• Keywords: oksidativni stres, antioksidant, oksidant, in vivo antioksidativna učinkovitost, polifenoli, epigalokatehin galat, menadion, kultivabilnost, znotrajcelična oksidacija, oksidativne poškodbe lipidov, metabolna aktivnost, infrardeča spektroskopija, FTIR, kvasovka
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [L. Svet]
• Year: 2018
• PID: 20.500.12556/RUL-101718
• UDC: 602.3:582.282.23:547.56:543.42(043.2)
• COBISS.SI-ID: 9009017
• Publication date in RUL: 30.06.2018

Secondary language

• Language: English
• Title: he study of activity of epigallocatechin gallate as an antioxidant and menadione as an oxidant in cells with infrared spectroscopy

Abstract

In the MSc thesis we studied the effects and activity of menadione as an inductor of oxidative stress at cellular level using yeast Saccharomyces cerevisiae as a model organism and the effects of antioxidant epigallocatechin-3-gallate (EGCG) on cells and its role in protecting cells against oxidative stress. The cells where treated with the selected compounds in different concentrations - EGCG was present in cell suspension in the concentration of 0,1 g/L and menadione in the concentration of 60 mmol/L. The effects of oxidative stress on physiological state of cells was studied using two different approaches. In the first approach, the cells were initially treated with EGCG and after that with menadione. In the second manner, the cells were treated with menadione only. Methods used for detecting such stress are various, however we used the following: determining intracellular oxidation, oxidative damage to lipids and cell metabolic energy; CFU assay and analysis of ATR-FTIR spectra. We determined that intracellular oxidation and oxidative damage to lipids in cells first treated with EGCG and then menadione is noticeably lower than in cells treated only with menadione. Cell metabolic energy in cells undergoing oxidative stress is higher due to activation of repair mechanisms. At the same time, we noticed that EGCG increases the metabolic activity of cells. The addition of EGCG did not significantly improve culturability of cells, which decreased due to oxidative stress. Additionally, changes in physiological state of treated cells were confirmed by ATR-FTIR. The differences in spectra between differently treated samples resulted mainly because of changes in lipid and carbohydrate content. Based on all the results we assess, that ATR-FTIR is a suitable method for estimating changes in intracellular redox state after treating cells with an oxidant/antioxidant.

• Keywords: oxidative stress, antioxidant, oxidant, in vivo antioxidative efficiency, polyphenols, epigallocatechin gallate, menadione, cultivability, intracellular oxidation, oxidative damage to lipids, metabolic activity, infrared spectroscopy, FTIR, yeast