The present thesis deals with the enzymatic modification of selective antioxidant compounds of plant origin (silybin and rutin) with simple fatty acids, esters of fatty acids and dicarboxylic acids by lipase CALB (Novozym®435) in organic solvents, in order to increase their lipophilic nature and modify their biological action. Several parameters were studied concerning their impact on the performance of the enzymatic process. Acetone was the most suitable medium for the studied enzymatic processes, since its use lead to the highest conversion of silybin. Using simple fatty acids for the enzymatic acylation of silybin, it was found that the conversion increased by increasing the fatty chain length from 8 to 16 carbon atoms. Increasing the number of carbon atoms from 16 to 18, resulted in lower performance. The highest conversion was determined when butyric acid was used as acyl donor, a short chain fatty acid with 4 carbon atoms, in the presence of lipase Novozym®435 in acetone after 96 ...
The present thesis deals with the enzymatic modification of selective antioxidant compounds of plant origin (silybin and rutin) with simple fatty acids, esters of fatty acids and dicarboxylic acids by lipase CALB (Novozym®435) in organic solvents, in order to increase their lipophilic nature and modify their biological action. Several parameters were studied concerning their impact on the performance of the enzymatic process. Acetone was the most suitable medium for the studied enzymatic processes, since its use lead to the highest conversion of silybin. Using simple fatty acids for the enzymatic acylation of silybin, it was found that the conversion increased by increasing the fatty chain length from 8 to 16 carbon atoms. Increasing the number of carbon atoms from 16 to 18, resulted in lower performance. The highest conversion was determined when butyric acid was used as acyl donor, a short chain fatty acid with 4 carbon atoms, in the presence of lipase Novozym®435 in acetone after 96 h at 50°C. In all cases, higher conversion yields were determined when fatty acids esters were used as acyl donors, instead of their corresponding fatty acids. Regardless of the acyl donor nature, increase of its concentration led to higher silybin conversion as well as higher reaction initial velocity, due to thermodynamic equilibrium shift towards synthesis. Using acetone as reaction medium, increase of silybin concentration led to higher process productivity. Of the organic solvents studied, acetone and acetonitrile were those that led to high conversion yields for the synthesis of mono-and di- acylated silybin derivatives, irrespectively of the dicarboxylic acid used. Lipase Novozym®435 exhibited higher selectivity for dicarboxylic acids with long carbon chain length (16 carbon atoms). In all cases, increasing of dicarboxylic acid concentration resulted in increased silybin conversion until the amount of the acyl donor exceeded the maximum solubility capacity of the solvent. Increasing silybin concentration led to increased monoester synthesis and reduced formation of corresponding diester. When rutin was used, only mono acylated derivatives were detected, unlike silybin for which both mono-and di-esters were detected. The aim of this study was also to introduce a lipase or esterase capable of catalyzing adequately the acylation of flavonoids (rutin and narigin) and anthocyanins (cyanin), and to investigate the feasibility of improving the catalytic performance by using "directed evolution" methodology. The reaction between nargin and vinyl butyrate in tertiary alcohol was chosen as model reaction. The reaction leads to the formation of narigin butyrate accompanied by vinyl alcohol release, which is automatically tautomerized into acetaldehyde. Acetaldehyde reacts with NBD-H, which is added to the system, and the complex formed can be fast and with sufficient sensitivity determined photometrically. Finally, the effect of the new compounds on cell proliferation, secretion of vascular endothelial growth factor (VEGF) and induction of apoptosis in human leukemia K562 cells was investigated. Additionally, their ability to induce the proteasome activity, a protein complex with important role in maintaining cellular homeostasis and cellular aging, was estimated in order to assess their anti-aging action. It was found that the antiproliferative effect of the new silybin esters with simple fatty acids on human leukemia K562 cells, was significantly retained compared to the parental compound (silybin). Silybin esters with dicarboxylic acids maintained the antiproliferative and anti-angiogenic effect of the parental compound and in some cases the effect was strengthened (ester of silybin with hexadecanedioic acid). The esters of silybin with dicarboxylic acids, did not induce the apoptosis of human leukemia K562 cells in contrast to silybin. In this study, the effect of silybin and its esters with simple fatty and dicarboxylic acids on the proteolytic activity of the proteasome was evaluated, in order to determine whether these substances inhibit the aging phenomenon in vitro. The ester with hexadecanedioic acid led to proteasome induction for all the studied concentrations and the determined proteasome proteolytic activity was higher than the parental compound.
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