Določevanje stabilnosti izbranih sekundarnih metabolitov gliv Cordyceps spp. s tekočinsko kromatografijo

Čeh, Kaja (avtor), Marolt, Gregor (mentor), Markelj, Jernej (komentor)

V diplomskem delu sem želela določiti temperaturno in UV stabilnost izbranih sekundarnih metabolitov, in sicer adenozina, kordicepina in pentostatina, v posušeni in zmleti glivi Cordyceps spp. Za določanje vsebnosti metabolitov sem uporabila tekočinsko kromatografijo visoke ločljivosti. Najprej sem optimizirala ločbo analitov z uporabo reverzno-fazne (nepolarne) stacionarne faze C18 in polarne mobilne faze, sestavljene iz fosfatnega pufra in metanola. Določila sem mejo zaznave in mejo določitve za posamezne učinkovine, pri čemer so meje zaznave za pentostatin, adenozin in kordicepin znašale 0,026, 0,016 in 0,022 mg/L, meje določitve pa 0,086, 0,053 in 0,072 mg/L. Določila sem, da sta pentostatin in adenozin v vzorcu pod mejo zaznave in določitve. Nato sem preverila ponovljivost HPLC analiz ter ponovljivost priprave vzorcev in ugotovila, da sta ustrezni. Nadaljevala sem z raziskavo temperaturne in UV stabilnosti vzorcev. Umeritvene premice za temperaturno in UV stabilnost sem pripravljala v koncentracijskem območju med 0,2 in 1 mg/L. Vzorce sem shranjevala v različnih pogojih: v zmrzovalniku (–21 °C), hladilniku (3,3 °C), pri sobni temperaturi (23 °C), pri 40 °C in pri 80 °C. Po 18, 62, 159, 328 in 495 urah sem izvedla ekstrakcijo in pripravila ekstrakte za HPLC analizo. Ugotovila sem, da je imel vzorec pri času 0 dni najvišji masni delež kordicepina, in sicer 18,303 mg/g vzorca, medtem ko se je njegova koncentracija ob shranjevanju v hladilniku po 495 urah znižala za 4,7 %. Pri raziskavi UV stabilnosti sem testirala dva različna tipa embalaže, in sicer rjavo plastiko in prozorno steklo. Posodice z vzorcem, pokrite z izbranima materialoma, sem izpostavila UV svetlobi za 1, 2, 3 in 5 ur. Ugotovila sem, da se je masni delež kordicepina skozi celotno UV študijo vidno zmanjševal. V vzorcu, ki je bil pokrit z rjavo plastiko, se je po 5 urah bolj znižal masni delež, in sicer za 9,6 %, medtem ko se je pri vzorcu, pokritem s steklom, masni delež znižal za 6,5 %. Z dvostranskim t-testom parov sem ugotovila, da se masni delež kordicepina, glede na vrsto embalaže, signifikantno ne razlikuje. Zato je rezultat povprečje znižanih masnih deležev in znaša 8,0 %. Primerjala sem UV obsevanje v komori z obsevanjem sonca in ugotovila, da bi izpostavitev vzorcev najvišjemu sončnemu obsevanju v osrednji Sloveniji povzročila 1,9- krat hitrejše znižanje masnega deleža kordicepina, kot v UV komori, če predpostavimo, da vsebnost kordicepina linearno pada z naraščanjem UV obsevanja.

• Jezik: Slovenski jezik
• Ključne besede: Cordyceps spp., stabilnost, kordicepin, adenozin, pentostatin, tekočinska kromatografija visoke ločljivosti.
• Vrsta gradiva: Diplomsko delo/naloga
• Tipologija: 2.11 - Diplomsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-168612
• COBISS.SI-ID: 234410755
• Datum objave v RUL: 18.04.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Determining stability of selected secondary metabolites from Cordyceps spp. by liquid chromatography

Izvleček

In my thesis, I aimed to determine the temperature and UV stability of selected secondary metabolites, namely adenosine, cordycepin, and pentostatin, in a dried and ground Cordyceps spp. For the determination of metabolite content, I used a high performance liquid chromatography. First, I optimized the separation of analytes using a reverse-phase (non-polar) C18 stationary phase and a polar mobile phase consisting of phosphate buffer and methanol. I determined the limit of detection and limit of quantification for each compound, with detection limits for pentostatin, adenosine, and cordycepin being 0,026, 0,016, and 0,022 mg/L, and quantification limits being 0,086, 0,053, and 0,072 mg/L. I found that pentostatin and adenosine in the sample were below the detection and quantification limits. I then verified the repeatability of the HPLC analyses and the repeatability of sample preparation, finding them suitable. I proceeded with the study of the temperature and UV stability of the samples. Calibration curves for temperature and UV stability were prepared in a concentration range between 0,2 and 1 mg/L. Samples were stored under different conditions: in a freezer (–21 °C), refrigerator (3,3 °C), at room temperature (23 °C), at 40 °C, and at 80 °C. After 18, 62, 159, 328, and 495 hours, I performed extraction and prepared extracts for HPLC analysis. The initial mass fraction of cordycepin concentration in the sample was 18,303 mg/g, while the concentration decreased by 4,7 % when stored in the refrigerator for 495 hours. In the UV stability study, I tested two different types of packaging, brown plastic and clear glass. Sample containers covered with these materials were exposed to UV light for 1, 2, 3, and 5 hours. I found that the mass fraction of cordycepin decreased with time throughout the UV study. In the sample covered with brown plastic, the mass fraction decreased more after 5 hours, by 9,6 %, while in the sample covered with glass, the mass fraction decreased by 6,5 %. Using a paired two-tailed t-test, I determined that the mass fraction of cordycepin does not differ significantly depending on the type of packaging. Therefore, the result is the average of the reduced mass fractions and amounts to 8,0 %. I compared UV exposure in a chamber with sunlight exposure and found that exposure of the samples to the highest solar radiation in central Slovenia would cause a 1,9-times faster decrease in the mass fraction of cordycepin compared to the UV chamber exposure, assuming that the cordycepin content decreases linearly with increasing UV exposure.

• Ključne besede: Cordyceps spp., stability, cordycepin, adenosine, pentostatin, high performance liquid chromatography.