br Results Four HT esters butyrate caprylate laurate

Results Four HT esters (butyrate, caprylate, laurate and stearate esters) were chosen as representative compounds of increasing lipophilicity (Fig. 1). We will refer to them as C4HT, C8HT, C12HT and C18HT, respectively. They have been synthesized, purified and characterized according to our previous report [12]. Conventional (sub-micrometer) POPC liposomes, as well as giant ones, have been used as simplified membrane models for investigating the interaction of the HT esters with lipids, and for assessing the function of these antioxidants in a reconstructed cell-like system. POPC was chosen because of its structure (saturated and unsaturated chains, i.e., a good mimics of egg yolk lecithin that is used as animal membrane model), convenient-to-work phase transition temperature (ca. −2.5°C), and easy formulation of stable liposomes by different methods.
Discussion Previous results on the antioxidant properties of four sets of hydroxytyrosyl esters [12] or analogues [11] when tested in Diperodon HCl synthesis in culture, showed a marked dependence of their activity on their structure, in particular on the length of hydrophobic chain. A major factor can simply be the limited aqueous solubility [10], [38]. Consequently, cut-off effect could derive from the decrease of available compound concentration. Although this fact can help the rationalization of data in the case of very long tails, as also revealed in this study, the activity variations in the case of intermediate-length chain can be more difficult to explain. The details of structure-activity relationship of lipophilic derivatives of natural products – in this specific case, antioxidants – are still elusive. Here we have studied HT esters of different lipophilicity by means of specific designed liposome-based assays. We aimed to mimic the administration of HT antioxidants to cells in culture by the use of a simplified artificial system made of liposomes, allowing the study of HT esters features in more accurate way. In addition to their traditional use as membrane models, we have introduced novel experimental design using liposomes as cellular model. We show how the fine interplay between their self-associative behaviour, their solubility and their ability to bind or cross the lipid membrane determines the overall observed pattern. The first important observation was that when a very hydrophobic HT ester, like the C18HT, is administered to an aqueous phase from a concentrated ethanol solution, it might display an anomalous lack of activity (or an erratic behaviour) not due to chemical reasons, but to physical ones. In particular, we have observed the irreversible formation of kinetically trapped macroscopic C18HT aggregates. In addition to its low monomer solubility (ca. 7.8μM, Fig. 2), it appears from our evidences that the desorption of monomeric C18HT from the aggregate is kinetically hindered. One of the reasons for this behaviour can be the C18 chain physical state (expected to be solid-like at room temperature). More extensive and temperature-dependent investigations are required to further explore and clarify the dynamics of C18HT. So far the absent (or very low) antioxidant behaviour of C18HT in bulk solution (see Table 1) seems to be strongly connected with its inability to disaggregate during the time of analysis. In particular we argue that various factors such as the lipid/antioxidant ratio, the vesicle size, the mixing procedure, and other apparently minor experimental details might contribute to determine the complex and sometimes erratic C18HT pattern we revealed (e.g., compare the lack of C18HT insertion in Laurdan experiments, Section 2.2.2, with the not negligible C18HT activity in the DPH-based assay, Section 2.3.1). Note that also the C18HT-like vitamin E results (presented in this work) might be affected by similar factors. This work therefore represents a warning for future studies on these very hydrophobic compounds – which requires special caution for a proper handling and measurements.