Dl α tocopherol acetate

Extraction procedure and saponification were performed from 10 mg of ground, freeze-dried leaf tissue according to the recently described protocol (Demurtas et al. 2023 (link)). LC separation was performed using an LC-PhotoDiode Array (PDA) system (Accela, Thermo Fisher Scientific, Thermo Fisher Scientific, Waltham, MA, USWaltham, MA, US) and a carotenoid C30 column (100 × 3 mm², 3-µm particle size) (YMC, Dinslaken, Germany), the column temperature was 25 °C. Elution system was A, MeOH; B, MeOH/water (4:1 v/v) with 0.2% ammonium acetate; and C, tert-Butyl methyl ether (C), and gradient was 0 to 1.2 min 95% A, 5% B; 3.5 min 80% A, 5% B; 6.8 min 30% A, 5% B, 65% C; 16 min 95%, 5%. Injection volume was 10 µL, chromatographic flux after equilibration was 0.8 ml/min and the total run time 18 min. PDA data were recorded in the 200–700 nm range. Carotenoids were measured based on their absorption spectra as detected by the PDA, integrated at their individual λmax and normalized to DL-α-tocopherol acetate (Sigma–Aldrich, Cat. No. T3376-5G) at 285 nm, as recently described (Demurtas et al. 2023 (link)).

Two different colors (red and yellow) of fresh and healthy bell pepper used in this study were purchased from local farmers in Chuncheon, Republic of Korea, and transported to the laboratory. The bacterial strains used were L. monocytogenes (ATCC 19118), S. Typhimurium (ATCC14028), and L. rhamnosus GG (ATCC 53103) from the American type of the culture collection located in University Blvd, Manassas, VA, United States. The culture media used were tryptic soy broth (TSB) and tryptic soy agar (TSA); xylose–lysine–desoxycholate agar (XLD); Polymyxin acriflavine lithium chloride ceftazidime aesculin mannitol (PALCAM) agar; and de Man, Rogosa, and Sharpe agar and broth (MRSA and MRSB, respectively) from Oxoid (Basingstoke, Hampshire, UK). Sterile peptone water was purchased from BactoTM, Difco, MD, United States. Phosphate-buffered saline (PBS; Corning Mediatech, Inc., Corning, NY, USA), sodium benzoate, ascorbic acid, and DL-α-tocopherol acetate were obtained from Sigma Aldrich, the Republic of Korea.

After expansion in their optimum media, the different cell types were trypsinized and transferred to organoid media as described by Unno et al.¹⁶ . Briefly; 5000 cells were resuspended in organoid media containing low percentage matrigel (5%) then plated in to 96-well ultralow attachment plates (Corning no. 3474). A hunderd microliters of fresh media was added to the cultures every four days or every two days once the treatments commenced. Treatments were added to the following final concentrations; 40 μM DL-αTocopherol-Acetate (Sigma no. T3376), 40 μM RRR-γ-Tocopherol (Sigma no. T1782) and/or 1.3 μM Seleno-L-methionine (Sigma no. S3132) representing the mean concetrations attained in the blood plasma of the SELECT subjects^{21 (link)}. N-acetyl cysteine (NAC; Sigma no. A9165) and Etomoxir sodium salt hydrate (Eto; Sigma no. E1905) were used at various concentrations as indicated in the figures. Organoid growth was captured by brightfield microscopy using Zeiss Axioskop/Nuance microscope (Carl Zeiss Inc. Oberkochen, Germany).

Isopropyl β-D-1-thiogalactopyranoside (IPTG), quercetin, ursolic acid, capsaicin, limonin, ellagic acid, simvastatin, DL-α tocopherol acetate, and dimethyl sulfoxide (DMSO) were procured from Sigma Aldrich (St. Louis, MO). Ni-NTA resin was bought from Thermo Scientific (USA). BIOMOL® Green reagent was bought from Enzo (New York, USA). All other reagents used were of analytical grade.

To determine the capacity of EPA inclusion in enrichments to raise the n-3 LC-HUFA content in Artemia sp., five experimental enrichments (EPA 0–4) with increasing EPA content (0%–60%) were formulated (Table 1). The enrichments were prepared by mixing increasing amounts of a commercial oil (Incromega EPA 500TG; Croda, Barcelona, Spain) containing 63% EPA and 8% DHA in total fatty acids (TFA) with a second commercial oil (Oleic Oil; Sigma–Aldrich, Madrid, Spain) containing 77% oleic acid (OA, 18 : 1n-9) in TFA. Additionally, every enrichment preparation was mixed with constant amounts of soya lecithin (Korot SL, Alcoy, Spain) containing 54% linoleic acid (18 : 2n-6) and trace amounts of EPA and DHA as emulsifier. Also, enrichment preparations were fortified with 3 g kg⁻¹ vitamin E (DL-α-tocopherol acetate; Sigma–Aldrich, Madrid, Spain) and 2.5 g kg⁻¹ vitamin C (L-ascorbic acid, Asc; Sigma–Aldrich, Madrid, Spain) to prevent the oxidation according to Betancor et al. [24 (link)], Hamre et al. [13 (link)], and Roo et al. [10 (link)]. Once prepared, experimental enrichments were stored in a fridge at 4°C until used. During the feeding trial, three subsamples of each experimental enrichment (ca. 100 g), newly hatched Artemia sp. (ca. 100 g), and enriched Artemia sp. (ca. 100 g) were collected and analyzed for proximate and fatty acid composition (Table 2).

HFL-1 (CCL-153; ATCC, Manassas, VA, USA), IMR-90 (CCL-186; ATCC), and WI-38 (CCL-75; ATCC) human lung fibroblasts and A549 human lung adenocarcinoma cells (CCL-185; ATCC) were used in this study. HFL-1 was cultured in F12–K medium (21127022; Thermo Fisher Scientific, Waltham, MA, USA). IMR90 and WI-38 cells were maintained in Eagle's minimum essential medium (10-009-CV; Corning Cellgro, Manassas, VA, USA). A549 cells were cultured in the Roswell Park Memorial Institute 1640 medium (10-040-CVRC; Corning Cellgro). All media were supplemented with 10% fetal bovine serum (16000044; Thermo Fisher Scientific) and 1% antibiotic-antimycotic (15240062; Thermo Fisher Scientific). Cells were grown in 5% CO₂ at 37 °C. VitC (l-ascorbic acid; A92902; Sigma-Aldrich, St. Louis, MO, USA) and NAC (A9165; Sigma-Aldrich) were dissolved in distilled water, vitamin D (VitD) (Cholecalciferol; C9756; Sigma-Aldrich) was dissolved in ethanol, and vitamin E (VitE) (DL-α-Tocopherol acetate; T3376; Sigma-Aldrich) was dissolved in chloroform, according to the manufacturer's instructions.