α tocopherol

The determination of α-tocopherol was carried out using the method by Martinek [28 (link)].
Reagents. α-Tocopherol (Sigma 3251), absolute ethanol (Merck 1.00983), 2,4,6-tripyridyl-s-triazine (TPTZ, Sigma T 1253), ferric chloride (Merck 1.03943), xylene (Merck 1.08685), and n-propanol (Merck 1.00997) were used.
0.5 mL of plasma, standard solution (200 mg/mL α-tocopherol), and blank (distilled water) were taken into different glass tubes. An equal volume of 0.5 mL absolute ethanol was added to the sample and blank, while 0.5 mL distilled water was added to the standard. To each tube add 0.5 mL xylene. Shake vigorously for at least 30 sec and subject it to centrifugation for 5 min at 2500 rpm, 4°C. After centrifugation, 250 μL of the upper xylene layer from each tube was transferred to test tubes. To each test tube, add 250 μL of TPTZ solution and mix. The samples were measured at 460 nm against the blank within 4 min. Then 50 μL ferric chloride solution was added to each tube, and then the absorbances were measured at 600 nm against the blank.

Groups of 45–50 COCs were matured in vitro in 500 μL medium covered with mineral oil during 24 h at 38.5 °C and 5% CO₂, 5% O₂ and 90% N₂ with maximum humidity. IVM medium consisted of TCM 199 (Gibco, Grand Island, NY, USA) supplemented with 1 μg/mL Folltropin-V^® (Bioniche, Belleville, ON, Canada) (v/v), 5 UI/mL equine chorionic gonadotropin (Biogón^® Plus, Biogénesis Bagó, Provincia de Buenos Aires, Argentina) (v/v), 10% fetal bovine serum (Gibco) (v/v), 5 μg/mL gentamicin (v/v) and 0.2 mM sodium pyruvate (w/v). For α-tocopherol treatment groups, IVM medium was supplemented with different α-tocopherol (Sigma, 258024) concentrations (50, 100 and 200 μM). Antioxidant was first dissolved in absolute ethanol to obtain the stock solution of 0.1, 0.2 and 0.4 M concentration, stored in the dark at 4 °C, and diluted in maturation media to a final concentration of 50, 100 and 200 µM α-tocopherol, respectively, in 0.05% (v/v) ethanol, and prepared 4 h before culture.

α-Tocopherol, neocuproine, DPPH radical, ABTS and DMPD, Trolox and α-Tocopherol were obtained from Sigma-Aldrich (Germany). The other chemicals were used for analytical grade and purchased from Merck or Sigma-Aldrich.

The α-tocopherol content in rat tissues (liver, heart, muscles, and testes) was assessed using high-performance liquid chromatography with a UV-VIS detector. A LiChroCART®250-4 RP-18 (4 × 250 mm; 5 mm) with a precolumn (Merc, col. No. 841071, Darmstadt, Germany) was used. The weighed tissue samples were homogenized with hexane and ethanol. A 10% solution of vitamin C was also added to protect the α-tocopherol from oxidation. The prepared samples were then centrifuged at 2,500 rpm for 15 minutes at 4°C. After cooling, 100 μL of the sample was taken and applied to the chromatography column on which the assay was made. The eluent was a mixture of acetonitrile, hexane, and isopropanol (65 : 14 : 21; v/v/v) and flow rate was 0.8 mL/min. The measurement was performed at a wavelength of UV 292 nm [37 (link)]. All the obtained results were applied to standard curves plotted for α-tocopherol (Sigma Company, St. Louis, MO, USA). The α-tocopherol concentration was expressed as nM/g of tissue.

In the experimental setup, the OVA + CdCl₂ mouse model group were treated concomitantly with the ERK inhibitor U0126 and the antioxidant radical scavenger α-tocopherol. On days 21, 24, and 27, the mice received an intraperitoneal injection daily of 10 mg/kg U0126 (MedChem Express, Monmouth Junction, NJ, USA) dissolved in sterile enzyme-free water with 2% DMSO. Alternatively, α-tocopherol (15 IU/kg) (Sigma Aldrich, St. Louis, MO, USA), dissolved in 50% ethanol with a total volume of 10 μL, was administered orally twice a day from day 21 to day 27. These treatments were administered one hour prior to each instance of intratracheal instillation of CdCl₂ or before the nebulization of ovalbumin (OVA) [25 (link),26 (link),27 (link),28 (link)], as outlined in the experimental protocol depicted in Figure 1.

For carotenoid extraction, 1 g of powdered fruits was suspended in 10 mL of ethyl lactate, and 100 mg of α-tocopherol (Sigma-Aldrich, St. Louis, MO, USA) was added as an antioxidant [52 (link)]. The samples were placed in a water bath at 45 °C for 60 min, centrifuged (4500 rpm, 15 min), and the supernatants were collected. The absorbance was measured at 450 nm and 503 nm. The concentration of carotenoids was calculated according to the following equation [43 (link)];

The results are expressed as the arithmetic mean from three independent experiments in milligrams of carotenoids per 1 g of goji fruits.