, 2003). It has been reported that ASTA has a high antioxidant

activity: 10 times higher than other carotenoids such as lutein, canthaxantin, and β-carotene and 100 times higher than α-tocopherol (Goto et al., 2001 and Naguib, 2000). This potent antioxidant activity has been observed to modulate biological functions ranging from lipid peroxidation to tissue protection against light damage (McNulty et al., 2007 and Santocono et al., 2006). At the same time, ASTA displays interesting anti-inflammatory effects TGF-beta inhibitor by preserving redox-sensitive (and essential) structures of human lymphocytes, although the applied dose apparently hinders lymphocyte proliferation (Bolin et al., 2010). As fatty acids are potent inducers of oxidative stress and as reported by many authors that ASTA has an important and prominent antioxidant activity, we propose Antidiabetic Compound Library to evaluate the oxidative

stress caused by a mixture of fatty acids previously used by our group, and the possible ASTA protective role of oxidative stress induced by the FA mixture. Astaxanthin (ASTA) and most of other chemicals were purchased from Sigma–Aldrich Chemical Company (St. Louis, MO, USA), excepting the RPMI-1640 culture medium, pluronic acid, Vybrant MTT Cell Proliferation kit and acetoxymethylester (Fura-2 AM) which were from Life Technologies (California, USA). Common reagents for buffers (e.g. PBS) and regular laboratory solutions were obtained from Labsynth (Diadema, SP, Brazil). The Ethical Committee of the Universidade Cruzeiro do Sul (protocol number 030/07) approved the experimental procedure of this study. Around 30 healthy adult women and men (mean age 27.0 ± 9.0) were included in the present study. All subjects did not present systemic or topical therapeutic regimen at least for the last 2 months. Subjects with a smoking history, alcohol habits, obesity or any other

systemic diseases were excluded of the study (based on an anamnesis protocol). Lymphocytes were obtained through the collection of human peripheral blood by venipuncture procedure in vacuum/siliconized tubes containing 0.1 mM EDTA. Peripheral blood Bcl-w lymphocytes were isolated under sterile conditions by using a density gradient present in the reagent Histopaque 1077 (Sigma–Aldrich) according to the manufacturer’s instructions. After centrifugation, lymphocytes were counted in a neubauer chamber using Trypan blue (1%). Lymphocytes (1 × 106/mL) were cultured in 5 mL of RPMI 1640 supplemented as described above. The cells were treated with 0.3 mM of the fatty acid mixture added or not of 2 μM of ASTA solubilized in DMSO and cultured at 5% CO2 for up to 24 h at 37 °C. After this period, the cells were collected, centrifuged and stored at −80 °C. To perform the assays of enzymes activities and oxidative damages in biomolecules, cells were defrosted and immediately used.