β carotene

High-performance liquid chromatography (HPLC)-grade authentic standard reagents, including α-carotene, β-carotene, lutein, zeaxanthin, and β-cryptoxanthin, were purchased from Extrasynthese (Genay, France). HPLC-grade methanol, acetone, and methyl tert-butyl ether were purchased from Beijing Solarbio Science & Technology Co. Ltd. (Beijing, China). All aqueous solutions were prepared using ultra-high-purity water (18.2 MΩ. cm) obtained from a Milli-Q water purification system (Millipore Corporation, Bedford, MA, USA), filtered through a 0.22 μm filter.

The cold-pressed oil, a kind gift from a local producer from Bihor County (in the Northern region of Romania), was obtained by processing sea buckthorn berries, more precisely Mara variety. The oil was stored at room temperature, away from humidity and light, until use (typically within 15 days).
Tween-20 (Art. No. P1379), pepsin from porcine gastric mucosa (Art. No. P6887), pancreatin from porcine pancreas (Art. No. P7545), cholesterol esterase from porcine pancreas (Art. No. 26745) and bovine bile extract (Art. No. B8631) were purchased from Sigma-Aldrich (Steinheim, Germany).
β-Carotene, β-cryptoxanthin, and zeaxanthin standards were purchased from Extrasynthese (Lyon, France), while zeaxanthin monopalmitate, zeaxanthin dipalmitate, and β-cryptoxanthin palmitate were obtained by semi-synthesis and purified by HPLC-PDA, as previously reported [22 ,23 ].
All used chemicals and reagents were of analytical or HPLC grade. The water used for all experiments was treated in a Milli-Q water purification system.

The ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) diammonium salt, potassium peroxodisulfate, 2,4,6-tris(2-pyridyl)-s-triazine, Trolox^®, aminoguanidine hydrochloride, methylglyoxal solution, L-arginine, hexane, 5-hydroxymethyl-L-furfural, BHT (2,6-Di-tert-butyl-4-methylphenol), (+)-catechin, rutin, quercetin, D-glucose, citric, L-ascorbic, oxalic, gallic and hydroxybenzoic acid were purchased from Sigma-Aldrich (Switzerland). Acetone and trifluoroacetic acid were bought from Acros Organics (France). Methanol and acetonitrile were obtained from Fine Chemicals (Netherlands), ethyl acetate from Alfa Aesar (Germany). Acetic and sulfuric acids were from Thermo Scientific (Germany). Lycopene, β-carotene, lutein and zeaxanthin were bought from Extrasynthèse (France) and absolute ethanol from Alcosuisse (Switzerland). Fructose and saccharose were obtained from Merck (Germany).
Folin and Ciocaulteu reagent, sodium dihydrogen phosphate mono-hydrate, di-sodium hydrogen phosphate anhydrous and sodium azide were purchased from Chempur (Poland), BSA (Bovine Serum Albumin) from Pol-Aura (Poland). The pectinase Pectinex Ultra SP-L was obtained from Novozymes (Denmark).
Deionized water (Milli-Q purification system, BlancLabo, Switzerland) was used for chromatography.
All reagents used were of analytical grade or higher. All solvents were of HPLC grade.

Reagents and solvents used in extractions and sample preparation were of analytical, HPLC or LC/MS grade and were purchased from Merck Life Science (Merck KGaA, Darmstadt, Germany). Carotenoid standards, β-carotene, lycopene, β-cryptoxanthin, lutein, and zeaxanthin were purchased from Extrasynthese (Genay, France). The fatty acid methyl ester (FAME) standard (37 component FAME Mix, SUPELCO, catalog No: 47885-U) was purchased from Supelco Inc. (Bellefonte, PA, USA).

Chemicals and reagents were obtained from VWR International (Milan, Italy) and Sigma-Aldrich Chemical Co., Ltd. (Milan, Italy). Lycopene and β-carotene were purchased from Extrasynthese (Genay-France). Acarbose was purchased from Serva (Heidelberg, Germany).

HPLC-DAD separation was performed using a Shimadzu LC20 AT HPLC system (Shimadzu Corporation, Kyoto, Japan) with an SPDM20A diode array detector and a YMC C30 reversed-phase column (250 mm length, 4.6 mm inner diameter and 5 μm particle size). The experimental conditions for the separation and identification by HPLC-DAD were the same as described in a previous study [62 (link)]. Quantification of carotenoids was performed using external calibration with standards of β-carotene, lutein, β-cryptoxanthin, and zeaxanthin purchased from Extrasynthese (Lyon, France) in the range of 1–100 μg/mL. The HPLC-DAD analysis was performed three times for the tested sample and data are expressed in mg/100 g F.W (fresh weight) and presented as the mean ± SD of these three measurements.

Plasma samples of 200 μL were mixed with 10 μL 8-apocarotenal in ethanol [0.0001% (w/v), (Sigma-Aldrich, St. Luis, MO, USA)] as internal standard, 1 mL ethanol containing 0.01% (w/v) butylhydroxytoluene, and 4 mL hexane:dichloromethane [4:1 (v/v)]. The extraction process was repeated if the plasma samples contained precipitated material. After stirring and centrifugation, the supernatants rich in carotenoids were collected and kept in liquid nitrogen. Subsequently, the samples were evaporated to dryness and the residues were re-dissolved in 200 μL hexane:acetone:ethanol:toluene [10:7:6:7 (v/v/v/v)] solution and filtered through a 0.22-μm filter for high-performance liquid chromatography (HPLC) analysis. A C30 carotenoid column [S5 μm, 250 × 2.0 mm I.D. (YMC, Wilmington, NC, USA)] was used at 30 °C, and the SPD-M10 vp diode array detector (Shimadzu, Kyoto, Japan) was set at 460 nm. Carotenoids {[lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene, and lycopene (cis- and trans-isomers)], Extrasynthese, Genay, France} were quantified by determining peak areas in the HPLC chromatograms, calibrated against known standards.