Biotechnological Synthesis and Molecular Encapsulation of Natural Stilbenes and Lipophilic Derivatives for Biomedical and Food Applications

Resum

Stilbenes are phenolic compounds whose many bioactivities make them of great interest to human health, such as antimicrobial, antioxidant, anti-inflammatory, cardioprotective, anticarcinogenic and anti-obesity activity. They are secondary metabolites produced by certain plants to protect them against biotic or abiotic stress, with the enzyme stilbene synthase being key in this biosynthesis process. Within this group of bioactive compounds, resveratrol is the most researched, but there are other lesser-known stilbenes with greater activity. However, the reduced production, low water solubility, ease of degradation and low bioavailability of this family of bioactive compounds hinder their successful application in the food, cosmetic and pharmaceutical industries. In this Doctoral Thesis, several strategies have been investigated with the aim to overcome these limitations through the use of different methodologies. In order to address the problems in obtaining natural stilbenes, a method to increase the enzymatic synthesis of piceatannol was developed and the expression of a novel recombinant stilbene synthase found by phylogenetic analyses was optimised, which also led to the discovery of the origin and evolution of these relevant proteins in different kingdoms. In addition, some of the most promising resveratrol analogues were characterised and encapsulated, increasing their solubility and stability in aqueous solutions by means of inclusion complexes with cyclodextrins. The stoichiometry and encapsulation constants were determined, as well as the type of cyclodextrin that formed the most stable complexes. Furthermore, to address the bioavailability problems of stilbenes, hydrophobic stilbene derivatives were synthesised that could act as precursors to better carry stilbenes to target tissues. The optimised synthesis processes achieved high yields with high specificity. These derivatives were physicochemically characterised, encapsulated in cyclodextrins, and their biological properties were analysed in vitro and in vivo. It could be observed that hydrophobic derivatives of stilbenes could be susceptible to hydrolysis during the digestive process. However, the use of inhibitors effectively counteracted this hydrolysis, improving their preservation. The effect of the addition of encapsulating agents in this process was also evaluated, and inclusion complexes characterised. In general, the hydrophobic stilbene derivatives were more stable, less metabolizable and more permeable than their original stilbenes, supporting the structural modification of stilbenes. Cyclodextrins were also found to interfere with in vitro antioxidant activity determination methods, potentially leading to misinterpretations of activity in their presence. This interference seems to be caused by the encapsulation of the reagents involved in the reactions. Moreover, structure-activity relationships were established between the cytotoxic activity of several resveratrol analogues, enabling the selection stilbenes with the greatest therapeutic potential. In vivo studies with free and/or encapsulated hydrophobic stilbene derivatives showed beneficial effects on obesity and cancer, with no harmful effects observed in individuals after administration. Finally, models of functional foods enriched with one of these derivatives, in its free and encapsulated form, were developed, monitoring the quality parameters and stability of the bioactive ingredient under different storage conditions, in order to check the industrial application feasibility. Overall, the findings of this Doctoral Thesis present different innovative approaches to address the current problem that restricts the use of stilbenes, increasing their production, characterisation, solubility, stability and bioavailability, so that they can be used effectively in biomedicine and food.