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Title: Synthesis and structure/antioxidant activity relationship of lipophilic hydroxytyrosyl esters and their analogues
Authors: Balducci, Valentina
metadata.dc.contributor.advisor: Gambacorta, Augusto
Incerpi, Sandra
Keywords: hydroxytyrosyl
Issue Date: 25-Feb-2013
Publisher: Università degli studi Roma Tre
Abstract: The olive tree (Olea Europea) is widely studied for its alimentary use: the fruits and the oil are essential components in the mediterranean diet of a large part of european’s population, whereas fruits and leaves are important for their secondary metabolites, known for their protective (antioxidant) properties. These properties are attributed to the phenolic compounds of olive tree. Hydroxytyrosol (here named 3,4-DHE) is a small hydrosoluble molecule (log P = 0.08), which is present in the leaves, fruits and, in small amount, in olive oil (113.7 381.2 mg/kg). It acts as an efficient scavenger of peroxyl radicals, preventing oil oxidation and therefore contributing to improve its shelf life. Previous studies on hydroxytyrosol’s biological properties gave information about its bioavailability in humans, its nontoxicity in vivo, its antiatherogenic properties (mainly by protecting LDL from oxidation damage), its antiplatelet aggregation ability and its anti-inflammatory effects. All these properties can be related to the ability of 3,4-DHE to interact with redox sensitive cellular elements, such as by inactivating NF-κB pathways. As for the radical scavenging and biological properties mentioned above, 3,4-DHE seems to be a good antioxidant, safe enough for industry preparation. Nevertheless, easy incorporation of 3,4-DHE as an additive in food and cosmetics, as oil matrices, is still a challenge. The hydrophilic character stands as a serious disadvantage, first of all for the difficulties in extracting it from aqueous solutions, then for the reduction of its effectiveness in stabilizing fats and oils. Therefore, more lipophilic 3,4-DHE derivatives could promote hydroxytyrosol’s properties with respect to the dispersion medium. Derivatization of 3,4-DHE demonstrated to be a promising tool to increase its solubility in lipophilic preparations. Several studies have highlighted that 3,4-DHE derivatives possess antioxidant properties. However a systematic study of the structure-activity relationships, required for a fine tuning of the lipophilicity that maximizes its antioxidant properties, is still lacking. An accurate analysis of the influence of structural rearrangements on the antioxidant activity of 3,4-DHE may help to better understand the basic aspects of the behavior of lipophilic phenolic antioxidants. Furthermore, it may bring worthy information in order to build up efficient antioxidants for industrial employment. The aim of the present PhD project has been to explore the structure - activity relat ionship of four complete series of lipophifilic hydroxytyrosyl derivatives, obtained by: esterification of the alcoholic moiety with fatty acids of increasing acyl chain length small structural modifications of hydroxytyrosol main scaffold. The project was divided into three parts:  Preparation of (Figure 1): 1. hydroxytyrosyl esters of fatty acids by chemical synthesis; 2. a new hydroxytyrosol superior homologue (named 3,4-DHP) having a propyl alcoholic chain and the complete series its fatty acid esters (C2- C18); 3. a new hydroxytyrosol isomer (named 2,3-DHE) having the catecholic hydroxyls in 2,3 position with respect to the alcoholic chain and the complete series of its fatty acid esters (C2-C18); 4. a new catechol obtained by pooling together the two previous rearrangements (named 2,3-DHP) and the complete series of its fatty acid esters (C2-C18).  Evaluation of antioxidant activity of all the compounds obtained, by in vitro ABTS assay and by H2DCF assay in cultured L6 myoblasts.  Study of the interaction between a representative series of hydroxytyrosyl esters derivatives and lipid bilayer by using lipid vesicles (liposomes), representing the simplest membrane model. Figure1. Molecules under study in this thesis The results obtained indicate that the derivatization of hydrophilic catechols, by regio-selective esterification with linear fatty acid on the alkylic alcohol, can effectively maintain or even enhance their antioxidant properties. Lipophilicity can be modulated, by the choice of an adequate acyl chain length, to cooperate with antioxidant activity. It emerged that the dependence between hydrophobicity and the antioxidant capacity is nonlinear, but characterized by a clear cut-off effect for longer chain esters. We investigated the reasons of the decreased activity for longer alkyl chains. In ethanol (used for ABTS assay), it was found that esters having chains longer than C12-C14 can in part assume folded structures, thus interfering with the catechol moiety’s radical scavenging ability. Comparative analysis of the four sets of molecules under study suggested that shifting the hydroxyl to ortho-meta position with respect to the alcoholic chain can successfully affect the stability and the antioxidant activity of lipophilic phenols, by creating an intramolecular hydrogen bond that in compounds with meta-para catechols is not allowed. The formation of this new bond is particularly facilitated by elongating the alcoholic side chain from C2 to C3. Viability assessment of 24h-preincubated myoblasts with all the compounds confirmed the nontoxicity of these substances, as already reported in literature for hydroxytyrosol. ROS production assay in cells described a similar activity pattern for the four sets of compounds (higher antioxidant activities for short-medium size esters, and a drop for longer chains), demonstrating that in a biological environment rearrangements of hydroxytyrosol structure have minor influence in the activity, instead, the only involved parameter is lipophilicity (that is related to the length of the ester chain). The determination of the size-dependence on the critical aggregation concentrations reveled that these amphiphilic catechols self-associate in aqueous solutions, like classical surfactants, and - interestingly – that the 50% of 10 μM stearate ester (the concentration used for our experiments) forms supramolecular structures in aqueous solution. However, this could only partially explain the drop of activity for long chain esters in cells. Activity assays performed with liposomes clarified that medium-long esters are unable to protect the intravesicular environment from induced oxidation giving low values of DCF assay. Notwithstanding, medium-long esters strongly interact with the membrane where they are soluble, and they can efficiently protect it from oxidative damage as shown by measures of antioxidant activity with DPH probe. In conclusion, the performed studies demonstrate that it is possible to modulate the length of the acyl chain in order to maximizes the defense from oxidative injure either in the intravesicular environment or in the bilayer, supposing for analogy, that the results obtained with liposomes could fit for cells. Moreover, rational design of hydroxytyrosol structure brought new information about improving its efficiency, which may be useful in the market of food preservants.
Access Rights: info:eu-repo/semantics/openAccess
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T - Tesi di dottorato

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