Tea tree (Camellia sinensis) leaves contain catechins, among which the most important one is (-)-epigallocatechin gallate (EGCG). In pharmacy, catechins are of interest due to their strong antioxidant activity, which results from scavenging and neutralizing radicals, chelating metal ions, oxidizing phenolic groups, and inducing the body's own antioxidant mechanisms.
Due to its structural properties, EGCG has very low bioavailability. It has an ester bond and 8 free phenolic groups in its structure, which makes the molecule highly hydrophilic (calculated logP = 2.25), hindering its passage through the lipid bilayer. It is also prone to rapid conversion through hydrolysis and oxidation, as well as undergoing metabolism by cell enzymes under biological conditions.
In the literature, there are several examples of improving the bioavailability of EGCG, including by incorporating it into nanoparticles, with co-administration with other substances, and chemically modifying the molecule, primarily by attaching alkyl chains to the free phenolic groups through ether and ester bonds.
As part of this thesis, we investigated the possibility of improving the bioavailability of epigallocatechin gallate (EGCG). Our hypothesis was that we could enhance the pharmacokinetic properties of EGCG by increasing its lipophilicity through the esterification of some phenolic groups in the molecule,
Using acyl chlorides, we synthesized acetyl and butyryl derivatives of EGCG. Due to the structural and chemical characteristics of EGCG, we were unable to obtain pure products, as it would require lengthy proceadures and costly equipment and reagents. Instead, we prepared fractions that differed in the degree of esterification.
We then evaluated the stability of the starting compounds and prepared mixtures in methanol. Methanolic solutions with a molar concentration of approximately 200 μM were prepared. Protected from sunlight, the solutions were kept at room temperature, and samples were taken at time points: t = 0, 30 min, 2 h, and 4 h. We analyzed the samples using Ultra High Performance Liquid Chromatography (UHPLC).
We also assessed the stability with the Caco-2 cell model (epithelial cells derived from human colon adenocarcinoma). Their advantage is that in culture in vitro they can form a monolayer with properties similar to intestinal epithelium, making them a model for drug absorption and other compounds. Ethanol solutions of the starting compound and prepared mixtures were added to the cells grown on six-well cell culture plates. Samples were taken after 30 minutes, 2 hours, and 4 hours and analized with UHPLC.
The permeability of the prepared mixtures through the cell membrane was evaluated by adding the prepared mixtures to Caco-2 cells grown on six-well plates and incubating them in a incubator with CO2 for 5 hours. The cells were then lysed, and the presence of products was measured with UHPLC.
All the prepared compounds slowed down the conversion of EGCG, but within four hours, a decrease in concentrations of all products was observed, indicating that we were unable to prepare stable derivatives of EGCG through acylation. In the presence of Caco-2 cells the decrease in concentrations of all measured compounds was even faster than in methanol, due to the presence of cellular esterases.
In the analysis after the compound permeability test, we detected EGCG in only one sample, which could be a sign of improved permeability.
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