Tocopherol

HPLC-grade acetonitrile, ethyl acetate, and n-hexane were purchased from Fisher Scientific (Lisbon, Portugal). L-ascorbic acid, tocopherol, sugar, and organic acid standards were purchased from Sigma (St. Louis, MO, USA). Racemic tocol (50 mg/mL) was purchased from Matreya (Pleasant Gap, PA, USA). Water was treated in Milli-Q water purification system (TGI Pure Water Systems, Greenville, SC, USA).

To evaluate the antioxidant activity, the method of reduction of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was used [28 ]. The methanolic extracts were evaluated at concentrations of 3.125 to 1000 ppm. The DPPH (Sigma-Aldrich) was prepared to 125 μM in methanol, 100 μL of each sample was taken, and 100 μL of DPPH was added; the samples were allowed to stand for 30 min protected from light. The absorbance at 517 nm was measured using a spectrophotometer (Jenway 320d). As a positive control, a solution of tocopherol (Sigma-Aldrich) was used and as negative control EtOH; the reduction percentage was calculated using
$\begin{array}{c}\%\text{Reduction}=\frac{\text{Absorbancy\hspace{0.17em}negative\hspace{0.17em}control}–\text{Absorbancy\hspace{0.17em}sample}}{\text{Absorbancy\hspace{0.17em}negative\hspace{0.17em}control}}\times 100.\end{array}$

The BMSCs were plated in 96-well plates (5 × 10⁴ cells/ml, 100 μl per well) and grown overnight. The cells were treated with 200 µM H₂O₂ and different concentrations of tocopherol (Sigma, United States) (from 1 to 200 µM) for 4 h. Then, each well was cultured with a normal medium for 24 h. The cell viability was stained with Cell Counting Kit-8 assays (CCK-8, Dojindo, Japan), and the OD value was determined at 450 nm by using a microplate reader (Bio-Rad, United States).

Antarctic krill (moisture content, 78.90 ± 0.75%) was supplied by Liaoyu Group Co., Ltd. (Dalian, China) and frozen at −23 °C. A part of them were used to prepare krill powder by a vacuum freeze dryer (Scientz-12N, Ningbo Science Biotechnology Co. Ltd, Ningbo, China) for 48 h and stored at −23 °C. The other part of them were used for extraction krill oil by SDE after thawed.
The DE (purity > 99.9%) was purchased from Hangzhou Airco Refrigerant Technology Co., Ltd. (Hangzhou, china). The standards of 37 fatty acid methyl esters (FAMEs), astaxanthin (≥97% HPLC), l-α-phosphatidylcholine (PC), l-α-phosphatidylethanolamine (PE), tocopherol and retinol were purchased from Sigma-Aldrich (Shanghai, China). All other reagents and solvents were provided by Fisher Scientific (Shanghai, China).

Nelfinavir (chemical name: 3-Isoquinolinecarboxamide, N-(1,1-dimethylethyl)decahydro-2-[(2R,3R)-2-hydroxy-3-[(3-hydroxy-2-methylbenzoyl)amino]-4-(phenylthio)butyl]-(3S,4aS,8aS), was dissolved in DMSO to a final concentration of 50 mM and stored at -20°C. Its structure is well defined [54 (link)]. It was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. It was used at the indicated concentration, and added to the culture medium every 48 hours. Cycloheximide, MG-132, tocopherol, diphenyleneiodonium (DPI), (4-2-amino-ethyl)-benzolsulphonyl-fluoride hydrochloride (AEBSF), NG-nitro-l-arginine methyl ester (L-NAME) and allopurinol were all purchased by Sigma (Saint Louis, MO, USA) and used at indicated concentrations.

Analytical-grade reagents were purchased for this study: p-nitrophenyl-α-d-glucopyranoside (PNPG), tocopherol, hydroxybiphenyl, α-glucosidase (14.5 U/mg), acarbose, PBS (Sigma, Shanghai, China), ethanol, petroleum ether, methanol, DMSO, methanol (HPLC grade), 98% sulfuric acid, anthrone, sodium chloride, phosphate buffer, sodium carbonate, and sodium hydroxide (Nanjing Chemical Reagent Co., Ltd., China). AB-8 macroporous adsorption resin was purchased from Bengbu Liaoyuan New Materials Co., Ltd. Anhydrous glucose, DEAE cellulose, and dialysis bags were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Pectinase was purchased from Kandin Chemical Reagent Co., Ltd. (Guangzhou, China). DMEM was purchased from Thermo Fisher Scientific Instrument Co., Ltd. (Waltham, MA, USA).