Xanthophyll

Methanol (MeOH), hydrochloric acid (HCl), sodium carbonate, sodium sulphate, acetone, phosphate buffered saline (PBS) were purchased from Mallinckrodt Chemicals (Phillipsburg, NJ). Folin-Ciocalteu reagent, quercetin, ascorbic acid, ferulic acid, chlorogenic acid, caffeic acid, p-coumaric acid and syringic acid, xanthophyll, zeaxanthin, β-cryptoxanthin, dichlorofluorescein- diacetate (DCFH-DA), were purchased from Sigma (St. Louis, MO). 2, 2-Azobis-amidinopropane (ABAP) was purchased from Wako Chemicals (Richmond, VA). Gallic acid was purchased from ICN Biomedical Inc. (Costa Mesa, CA). Ethyl acetate, triflouroacetic acid, and ethanol were purchased from Mallinckrodt (Paris, KS). Sodium hydroxide, hexane, acetonitrile, magnesium carbonate, tetrahydrofuran were obtained from Fisher Scientific (Pittsburgh, PA). MDA human breast cancer cell lines and HepG2 liver cancer cell lines are provided by the American Type Culture Collection (ATCC, Rockville, MD). Williams’ medium E (WME), α-MEM, Hanks’ Blanced Salt Solution (HBSS) were purchased from Gibco Life Technologies, and Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Lawrenceville, GA).

The P. yezoensis was a kind gift from Professor Taejun Han (Ghent University Global Campus, Incheon, Korea). Astaxanthin and xanthophyll were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). For extract preparation, the P. yezoensis was washed with tap water to remove the salts and a dried sample (100 g) was pulverized, followed by extraction with 80% methanol (MeOH, 1:10, w/v) for 30 min by sonication. Then, it was filtered by Whatman filter paper and the solvents of filtrates were evaporated by the vacuum rotary evaporator. In this study, the water fraction of the P. yezoensis extract (PYE) was isolated as the supernatant and then lyophilized using a freeze-dryer (TD5508, Ilshin Lab, Co., Ltd., Yangju, Korea). The PYE, Astaxanthin, and xanthophyll were dissolved in dimethyl sulfoxide (DMSO) before use in the experiments.

Chromatographic analyses were performed on an Agilent HPLC system, 1200 series (Agilent Technologies, Inc. Waldbronn, Germany) equipped with double-beam photometer (Variable Wavelength Dector). UV absorbance was monitored at 470 nm. Quantification was analyzed by integration of the peak areas at 470 nm. The separation was performed using a Kromasil 100-5C₁₈ column (particle size, 5 μm; 250 * 4.6 mm; Teknokroma, Barcelona, Spain) with the temperature maintained at 30 °C, and the injection volume was 10 μL. The mobile phase consisted of A: 0.05% Trifluoracetic Acid H₂O and B: acetonitrile, and the flow rate was 1.0 mL/min. A gradient of 0–20 min from 10–0% and 20–30 min at 100% B was used. The PYE and astaxanthin (Sigma-Aldrich, St. Louis, MO, USA) were used as the sample and standard at a concentration of 10 mg/mL and 0.1 μg/mL of each of astaxanthin and xanthophyll (Sigma-Aldrich, St. Louis, MO, USA) was used for HPLC analysis to identify the peaks.