Propolis is a sticky, wax-like natural substance found in beehives. It possesses antioxidant, anti-bacterial, anti-viral and anti-inflammatory properties, some new studies are investigating its immunomodulatory activity. This wide range of therapeutic indications is supposed to be attributed to flavonoids in propolis, such as galangin, chrysin, naringenin, pinocembrin and others. Due to the complexity and variability of the composition of propolis, its analysis is difficult. So far, there has been no development of sufficiently good analytical methods capable of determining it and no permeability studies were performed to determine the absorption of propolis or its constituents yet. Good absorption of the active substances in the small intestine is crucial for achieving potential therapeutic effects.
For this thesis, we focused primarily on the development of the HPLC analytical method, which would detect as many propolis constituents as possible, separate them well, but would also be time-efficient. Various mobile phases, columns, and sample concentrations were tested. The developed HPLC analytical method was then used to try to determine the qualitative composition of propolis and for analysing propolis components that passed through the tissue in permeability studies.
The permeability studies were performed in Sweetana-Grass type two-cell diffusion cells. The rat small intestine was used, as it represents a simple and good model for predicting intestinal absorption in humans. Due to the complex composition and poor solubility of propolis, the method had to be adjusted accordingly.
By comparing the chromatograms of propolis tincture and some of the reference compounds (based on retention times and UV spectra), we identified chrysin, galangin, pinocembrin and naringin in our sample. HRMS analysis of the sample additionally confirmed the presence of naringenin, sacuranetin, gingerol, acacetin, abrectorin, salvigenin, CAPE (Caffeic acid phenethyl ester) and icaridin. Studies of permeability have shown that a very small percentage of propolis constituents are absorbed through the small intestinal mucosa. Among them, we detected chrysin, a naringenin derivative, a pinocembrin derivative and sakuranetin. Of the seven substances selected for analysis, three exhibited good permeability (including an identified naringenin derivative) and four were identified as poorly permeable. Besides low permeability, low solubility, or even precipitation of some substances in the intestinal lumen can cause poor intestinal absorption. For this reason the kinetic solubility of propolis constituents was also tested in biorelevant medium FaSSIF.
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