α tocopherol

Cannabidiol was purchased from Medcolcanna Organics Inc. (Calgary, AB, Canada), refined hemp oil from Molpharma (Ustroń, Poland), Glycerol from PPH Micropharm (Zabierzów, Poland), SLS, and α-Tocopherol from Sigma-Aldrich (St. Louis, MO, USA). The soybean phospholipids (Lipoid P 75) and the egg yolk phospholipids (Lipoid E 80) were very kindly gifted by Lipoid GmbH (Ludwigshafen, Germany). Kolliphor HS15 and ELP; Tween 20, and 80; sodium deoxycholate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Water for injection was purchased from B. Braun Melsungen AG (Melsungen, Germany). All organic solvents used in the studies were of analytical or high-performance liquid chromatographic grade and were purchased from Avantor Performance Materials Poland (Gliwice, Poland).

α-Tocopherol was prepared by dissolving 4 mg of α-Tocopherol ( 96%, Sigma–Aldrich, Steinheim, Germany) in 10 µl of dimethyl sulfoxide (DMSO; Calbiochem, CA, USA), followed by 990 µl of brain–heart infusion (BHI) broth to achieve a final concentration of 4 mg ml⁻¹ and subjected to 45 s of sonication.

NEs were prepared according to the spontaneous emulsification technique [30 (link),31 (link)]. The effects of varying amounts of medium-chain triglyceride (MCT, Lipoid GmbH, Germany), α-tocopherol and oleic acid (Lipoid GmbH, Ludwigshafen, Germany) on the accelerated stability and characterization of the formulations were investigated. The formulation codes and their contents are presented in Table 2. After determining the optimum amounts for these components for formulation development, NEs with the extracts were prepared. The required amounts of MCT, L-α-phosphatidylcholine (soy, PC, 95%, Avanti Polar Lipid Inc., Alabaster, AL, USA), α-tocopherol (Sigma-Aldrich, St. Louis, MO, USA) and oleic acid were dissolved in ethanol (30 mL, Isolab, Wertheim, Germany) to form the oil phase. The amount of PC (600 mg) was kept constant across all formulations. For extract-loaded NEs, the extracts (200 mg) were added to the oil phase. The aqueous phase (80 mL) was prepared by dissolving the required amount of polysorbate 80 (2% w/v, Isolab, Wertheim, Germany) and sodium benzoate (0.2% w/v) in purified water. Emulsification was carried out by gradually adding the oil phase to the aqueous phase while stirring with a magnetic stirrer at 500 rpm at room temperature. The mixture was evaporated in a round-bottom flask with a rotary evaporator at 43 °C until the final volume reached 20–30 mL.

Garlic is harvested from Taşköprü District of Kastamonu Province. Glutathione and acetonitrile were purchased from Sigma Aldrich and Merck, respectively. NIH/3T3 mouse embryo fibroblast cell line (ATCC CRL-1658), Caco-2 human colorectal cancer cell line (ATCC HTB- 37), and MCF-7 human breast adenocarcinoma (ATCC HTB-22) were purchased from American Type Culture Collection (ATCC). Dulbecco’s modified Eagle’s medium/Nutrient Mixture Ham’s F-12 (DMEM/F-12), fetal bovine serum (FBS), penicillin-streptomycin solution, phenazine methosulfate (PMS) and trypsin/EDTA (0.25%) were obtained from Sigma-Aldrich (St Louis, MO, USA). 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) was purchased from Biomatik (USA). Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), trolox, and α-tocopherol, N,N-dimethyl-p-phenylenediamine (DMPD), CuCl₂, 2,2′-Bipyridyl were obtained from Sigma-Aldrich GmbH (Sternheim, Germany).

Analytical-grade solvents and reagents, including anhydrous sodium sulfate, chloroform, n-hexane, methanol, sulfuric acid, diethyl ether, ethanol (99.9% purity), potassium hydroxide, and zinc powder, were purchased from Chempur (Piekary Śląskie, Poland). Chromatographic-grade solvents and reagents, including silylating agents, such as pyridine (anhydrous, 99.8% purity, and N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane), n-hexane (≥95% purity), heptane (≥99% purity), and isopropanol (99.5% purity), and standards, such as α-tocopherol (≥96% purity), γ-tocopherol (≥96% purity), δ-tocopherol (≥96% purity), (≥98% purity), and 5-α-cholestane (≥97% purity), were purchased from Sigma-Aldrich (Poznań, Poland). Helium for gas chromatography (99.999% purity) was purchased from Eurogaz-Bombi (Olsztyn, Poland). Deionized water was prepared using an HLP 5 deionizer (Hydrolab, Gdańsk, Poland).

1,1-Diphenyl-2-picryl-hydrazyl (DPPH) radicals, neocuproine (2,9-dimethyl-1,10-phenanthroline), ascorbic acid, BHT, BHA, α-tocopherol, Trolox, Folin Ciocalteu reagent and Ellman's reagent (5,5-dithio-bis-(2-nitrobenzoic acid) were purchased from Sigma-Aldrich GmbH, Steinheim, Germany. Standard phenolic compounds for LC-MS/MS were purchased from Sigma.