Chlorophyll a

Acetone, acetonitrile, isopropanol, ethanol, methanol and n-hexane (all HPLC grade) were purchased from Sigma–Aldrich (Milano, Italia). Water was purified with a Milli-Q system (Millipore, Bedford, MA, USA). All other reagents were of analytical grade. Tocopherol (α, γ and δ-tocopherols) and phenolic acids (tyrosol and hydroxytyrosol) standards were purchased from Sigma–Aldrich (Milano, Italia). Chlorophyll A was purchased from Sigma–Aldrich (Milano, Italia).

Spirulina (Arthrospira) platensis was purchased from Martin Bauer GmbH (Vestenbergsgreuth, Germany). Hemin was obtained from Frontier Scientific (Logan, UT, USA), and chlorophyllin, chlorophyll a, chlorophyll b, crystal violet, thiazolyl blue tetrazolium bromide (MTT), dimethylsulfoxide (DMSO), cell culture media, and supplements were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetone, methanol, dioxane, acetonitrile, and glutaraldehyde (25% solution) used in all experiments were obtained from Penta (Prague, Czech Republic). methanol and acetonitrile were obtained from Merck (Darmstadt, Germany).

The main chemicals used in this study included ninhydrin, anthrone, concentrated sulfuric acid, and acetone (AR, China national pharmaceutical group, Shanghai chemical reagent Co., Ltd.); Folinphenol (AR, Sigma, USA); methanol and ether (AR, Fisher Scientific, Ireland); chlorophyll a, and chlorophyll b (standard products, Sigma-Aldrich, USA); lutein and β-carotene (standard products, Shanghai Yuanye Biotechnology Co., Ltd.).

Pigment standards including chlorophyll a, violaxanthin, vaucheriaxanthin, and β-carotene were purchased from Sigma-Aldrich Chemical Co. (Shanghai, China; http://www.sigmaaldrich.com). Silica gel (200–300 mesh) was obtained from Qing Dao Marine Chemical Co. (Qingdao, China). HPLC-grade solvents used for HPLC analysis were purchased from Guangzhou Runhao Biotech Co. (Guangzhou, China), such as methanol, acetonitrile, acetic ether, and dichloromethane. Other analytical solvents (n-hexane, methanol, and acetone) used in extraction and isolation of violaxanthin were purchased from Guangzhou Runhao Biotech Co. (Guangzhou, China). Deionized water was prepared by a Milli-Q water purification system (Millipore Corp., Bedford, MA, USA).
Chemicals used for their antioxidant activity including 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), potassium persulfate, ferrous chloride, potassium ferricyanide, trichloroacetic acid, hydrogen peroxide, ascorbic acid, sodium dihydrogen phosphate, and disodium hydrogen phosphate were obtained from Sangon Biotech Co. (Shanghai, China).

Pigments were extracted using 98% methanol buffered with 0.5 M ammonium acetate and analysed by HPLC-DAD according to a protocol modified from Ensminger et al.14 . An Agilent high performance liquid chromatography (HPLC) system (Böblingen, Germany) with a quaternary pump (model 1260), autosampler (model 1260, set to 4 °C), column oven (model 1260, set to 25 °C), and photodiode array detector (model 1290, recording absorption at 290 nm, 450 nm and 656 nm wavelength) was used for reverse-phase chromatography using a C₃₀-column (5 μm, 250*4.6 mm; YMC Inc., Wilmington, NC, USA). Three solvents (A: 100% methanol, B: 60% methanol buffered with 20 mM ammonium acetate, C: 100% methyl-tert-butyl-ether) were used to run a gradient starting with 40% A and 60% B. Solvent B was gradually replaced by solvent A to a minimum of 5% B; afterwards solvent A was gradually replaced by solvent C until the solvent mixture consisted of 45% A, 5% B and 50% C. Peaks were quantified using standards for chlorophyll a, chlorophyll b and β-carotene from Sigma Aldrich (Oakville, ON, Canada). Standards for violaxanthin, antheraxanthin and zeaxanthin were obtained from DHI Lab products (Hørsholm, Denmark). ChemStation B.04.03 software (Agilent Technologies, Böblingen, Germany) was used for peak integration.

Chlorophyll a (Sigma) was dissolved in methanol (ChemPur, Karlsruhe, Germany) to the concentration of 1 × 10⁻⁵ M and one of 3 concentrations of NaF, matching those in previously tested soils and MS media. Samples were incubated at room temperature in darkness for 10 days. Each day, spectrophotometric assay was performed using Cary 5000 UV-Vis NIR spectrophotometer for absorbance measurements and Cary Eclipse spectrophotometer for fluorescence measurements. Similarly, pheophytin (from ChromaDex, Longmont, CO, USA) was dissolved in methanol at concentrations of 1 × 10⁻⁵ M and its absorption was measured. All measurements were performed at temperatures of 25 ± 1 °C. Absorbance was tested in the wavelength range from 310 to 750 nm, emission was tested from 600 to 800 nm, and excitation was tested from 300 to 750 nm.