Razvoj metode za določitev permeabilnosti sestavin propolisa skozi kožo prašičjega uhlja in vitro

Nabernik, Urša (author), Žakelj, Simon (mentor)

Propolis je naravna, lepljiva snov, ki jo najdemo v čebeljih panjih. Poznan je predvsem zaradi svoje učinkovitosti - ima protivirusne lastnosti, pomaga pri celjenju ran, ščiti razpokano kožo idr. Zaradi svoje kompleksne in variabilne sestave je preučevanje njegovih sestavin oteženo, vendar pa vedno bolj zaželeno. Poleg identificiranja posameznih komponent propolisa se večkrat izvajajo tudi testi permeabilnosti spojin, ki jih nanesemo na kožo. V ta namen se lahko uporabljajo in vivo ali in vitro testi, najpogosteje pa se v primeru propolisa uporabljajo in vitro testi s Franzovimi difuzijskimi celicami, kjer opazujemo prehod sestavin/e iz donorske na akceptorsko stran skozi izbrano membrano. Namen diplomske naloge je bil razviti HPLC analizno metodo, s katero bi določili vsebnost posameznih sestavin propolisa po prehajanju skozi kožo. Pomemben vidik pri razvoju metode je izbira pogojev, ki ustrezajo številčnosti in raznolikosti sestavin propolisa in omogočajo čim boljšo ločitev na eni ter čim hitrejšo analizo na drugi strani. Metodo smo razvijali postopno - preizkusili smo različne mobilne faze, koncentracije spojin, kolone, vse dokler nismo našli najboljše kombinacije. Nato smo izvedli poskus, ki je vključeval tri oblike propolisa: tinkturo propolisa, mazilo s propolisom ter suh propolis. Poskus smo izvajali s Franzovimi difuzijskimi celicami, za membrano pa smo izbrali kožo prašičjega uhlja, ker je ta najbolj podobna človeški koži. Zanimala nas je primerjava permeabilnosti sestavin iz različnih donorskih medijev, kakor tudi v kolikšni meri in če sploh sestavine prehajajo skozi kožo in katere so te sestavine. Po končani analizi je bilo v kromatogramih vidnih preveč vrhov, da bi se lahko osredotočili na vse, zato smo izbrali pet večjih dobro ločenih vrhov – pet neznanih sestavin, ki smo jih na akceptorski strani zaznali pri večini donorskih medijev in smo jih želeli opazovati ter določiti. Rezultati eksperimentalnega dela so pokazali, da sestavine propolisa najbolj prehajajo kožo iz tinkture in mazila, medtem ko so bile koncentracije na akceptorski strani v primeru suhega propolisa komaj zaznavne. S pomočjo spektrov smo uspeli identificirati derivat krisina, pri primerjavi ostalih sestavin z retencijskim časi in UV spektri referenc pa nismo bili uspešni. V okviru diplomske naloge smo dokazali, da propolis prehaja kožo v pomembnih koncentracijah, za bolj podrobne rezultate (identifikacija večjega števila sestavin) pa bi namesto HPLC morali uporabiti LC-MS/MS sistem.

• Language: Slovenian
• Keywords: Propolis, permeabilnost, Franzova difuzijska celica, HPLC
• Work type: Bachelor thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-107981
• Publication date in RUL: 11.06.2019

Secondary language

• Language: English
• Title: Development of method for determination of permeability of propolis constituents through porcine ear skin in vitro

Abstract

Propolis is a natural, sticky substance, found in beehives. It is primarily known for its effectiveness – it has antiviral properties, it helps healing wounds and protects cracked skin. Due to its complexity and diverse structure, examination of propolis is difficult, but highly needed. Tests of permeability through skin are common. In vivo or in vitro tests may be used for this purpose. In vitro tests with Franz diffusion cells are more likely to be used in a case like ours. The purpose of this study was to develop an HPLC analytical method, which would help us determine the content of propolis components passing through the skin. The chromatographic conditions needed to be optimised for the diversity of propolis components and to provide a reasonably good separation in a reasonably fast analysis. For this reason we tested multiple mobile phases, concentrations of compounds and different columns, until we found a satisfactory combination. Then we carried out an experiment, which included three donor solutions: tincture of propolis, propolis ointment and dry propolis. The experiment was carried out with Franz's diffusion cells, and because of the similarity to human skin, we chose porcine ear skin for the membrane. What we most wanted to see was whether the components of propolis permeate through skin at all and if they do, how much and what components are those. We also wanted to examine the difference in permeability from different donor solutions. After the analysis there were too many visible peaks in the chromatograms to focus on all of them, so we selected five largest and well-separated peaks – five unknown ingredients that we were able to detect on most acceptor sides in case of most donor solutions. The results of the experimental work showed best permeability through skin in the case of tincture of propolis and propolis ointment. Permeability from dry propolis was barely detectable. Using UV-spectra, we were able to identify the derivative of chrysin. This study allowed us to see that propolis permeates in significant concentrations, but for more detailed results (identification of all components) it would be necessary to use LC-MS/MS system.

• Keywords: Propolis, permeability, Franz diffusion cell, HPLC