Valorization of the olive by-products phenolic compounds and their evaluation as therapeutic agents for ocular surface inflammatory diseases

Resumen

This thesis aimed to valorize an environmentally hazardous agro-industrial by-product, the olive pomace, as source of phenolic compounds as potential therapeutic agents for dry eye disease. The effect of crude olive pomace extracts and their major pure phenolic compounds (oleuropein and hydroxytyrosol), alone or in combination, was studied in the inflammatory and oxidative processes of the ocular surface and evaluating them as future topical ophthalmic products. For this purpose, a sequential extraction process was applied to olive pomace to obtain extracts rich in the phenolic compounds of interest (expressed as mg per g dry extract), as well as high chemical antioxidant activity. Supercritical carbon CO2 extraction (to remove the residual olive oil) was followed by pressurized liquid extraction (to recover the phenolic compounds). This last step was optimized through design of experiments (circumscribed central composite design). The factors were temperature, ethanol percentage in water, and solid/liquid ratio. Subsequently, selected olive pomace extracts based on the optimization study, together with pure oleuropein, hydroxytyrosol, and low doses of their mixture were tested in vitro on two human ocular surface (corneal and conjunctival) epithelial cell lines for their anti-inflammatory and antioxidant effect, using as stimuli TNF-α to induce inflammation and UV-B to induce oxidative stress, respectively. In addition, the immunosuppressive effect of the most potent extract (namely, OPT3) and pure compound (hydroxytyrosol) was tested on lymphocytes CD4+ T cells activated with phytohemagglutinin-M. Their anti-inflammatory effect was also studied through topical application in a desiccating stress-induced dry eye disease mouse model. Finally, the stability and accessibility of the olive phenolic compounds were assessed under different pretreatment and storage conditions of the raw material. Two extracts were prepared by conventional solid-liquid extraction and used as references: CONV using freeze-dried material (reference for the in vitro study) and CONV-2 using freeze-dried material defatted with supercritical carbon CO2 (reference for the extraction optimization study). Also, for the aqueous phenolic solutions, long-term storage stability at 4 different conditions and ocular surface safety through genotoxicity assay (comet) were performed based on industrial guidelines followed by EMA. In 3 times shorter extraction time and 1.6 times less solvent consumption compared to CONV-2, an increase of 2 to 5-fold was observed in the phenolic extract richness, depending on the response. In addition, different optimal extraction conditions were observed for each key compound and three extracts were selected: OPT1, which showed the highest chemical antioxidant activity, OPT2, which had the highest oleuropein content in dry extract, and OPT3, with the highest content in hydroxytyrosol. Among the different extracts screened in vitro, CONV and OPT3 demonstrated improved antioxidant and anti-inflammatory activity. On corneal epithelial cells, both extracts and hydroxytyrosol reduced the levels of most measured interleukins/chemokines (IL-1β, IL-6, IL-8, IP-10, and IL-17A) dose-dependently, whereas on conjunctival epithelial cells all treatments decreased IP-10 production. In addition, on both cell lines, all treatments reduced reactive oxygen species production. Compared to CONV, the OPT3 extract demonstrated antioxidant and anti-inflammatory activity at 10 to 40 times and 2 to 40 times lower concentrations, respectively. Thus, it was selected to be further tested in vivo and in vitro on CD4+ T cells, together with hydroxytyrosol. Both treatments inhibited the proliferation of stimulated CD4+ T cells and decreased corneal fluorescein staining, IP-10 and TNF-α gene expression in lacrimal functional unit tissues, and lymphocyte count in cervical lymph nodes of mice exposed to desiccating stress. Among the pretreatment/storage methods of the raw material, lyophilization and supercritical carbon CO2 increased the extract richness in the phenolic compounds measured. Hydroxytyrosol and oleuropein demonstrated different stability profiles as pure aqueous solutions or as part of an extract. Additionally, oleuropein also had a distinct degradation profile. No genotoxic effect was detected for any of the treatments. In conclusion, an efficient, selective, scalable, and sustainable sequential extraction process was proposed, able to maximize the phenolic recovery from an agro-industrial by-product, reducing its environmental impact and using exclusively green solvents. In addition, olive pomace extracts and pure compounds (principally hydroxytyrosol) can modulate the inflammatory, oxidative, and immune responses on a cellular level at the ocular surface. Also, their topical application in a mouse dry eye model enhanced clinical signs and reduced inflammation in the ocular surface. Thus, a high-value application was demonstrated for the olive pomace compounds as potential ocular surface therapy. All treatments were proved to be safe for ophthalmic application at the concentrations selected and their industrial stability evaluation was established, necessary for their future approval as pharmaceutical products.