Chlorophyll b

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.).

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.

The following chemical reagents were used in this study: 2′2-azinebis-3-ethylbenzothiazolin-6-sulfonic acid (Merck, Warsaw, Poland), acetic acid (Chempur, Warsaw, Poland), acetonitrile (Sigma-Aldrich, Warsaw, Poland), deionized water (Sigma-Aldrich, Warsaw, Poland), ethyl acetate (Sigma-Aldrich, Warsaw, Poland), hydrochloric acid (Chempur, Warsaw, Poland), magnesium carbonate (Sigma-Aldrich, Warsaw, Poland), methanol (Merck, Warsaw, Poland), ortho-phosphoric acid (Chempur, Warsaw, Poland), and phenolic compound standards, including gallic acid (CAS 149-91-7), chlorogenic acid (CAS 327-97-9), p-coumaric (CAS 501-98-4), caffeic acid (CAS 331-39-5), quercetin-3-O-glucoside (CAS 482-35-9), quercetin-3-O-rutinoside (CAS 207671-50-9), myrycetin (CAS 529-44-2), quercetin (CAS 849061-97-8), cyanidin-3-O-glucoside (CAS 7084-24-4), and cyjanidin-3-O-rutinoside (CAS 18719-76-1), lutein (CAS 127-40-2), zeaxanthin (CAS 144-68-3), β-carotene (CAS 7235-40-7), chlorophyll a (CAS 479-61-8), chlorophyll b (CAS 519-62-0) (Sigma-Aldrich, Supelco, Poland), phosphate buffered saline, and potassium persulfate (Sigma-Aldrich, Warsaw, Poland).

Acetone (HPLC grade) came from Sigma-Aldrich (Poznań, Poland); Acetonitrile (HPLC grade) came from Chempur (Piekary Śląskie, Poland); Carotenoids, vitamin C, chlorophylls, and phenolics standards (HPLC grade 99.5–99.9% pure) came from Fluka and Sigma-Aldrich (Poznań, Poland): β-carotene, caffeic acid, chlorogenic acid, chlorophyll a, chlorophyll b, dehydroascorbic acid, ferulic acid, gallic acid, kaempferol-3-O-glucoside, lutein, l-ascorbic acid, p-coumaric acid, quercetin, quercetin-3-O-rutinoside; Ethyl acetate came from Sigma Aldrich (Poznań, Poland); Magnesium carbonate (ultrapure) came from Chempur (Piekary Śląskie, Poland); Meta-phosphoric acid came from Sigma-Aldrich (Poznań, Poland); Methanol (HPLC grade) came from Chempur (Piekary Śląskie, Poland); Ortho-phosphoric acid came from Chempur (Piekary Śląskie, Poland).

Ammonium acetate, genistein, kaempferol-3-O-glucoside, methanol, quercetin-3-O-glucoside, and tert-butyl methyl ether were purchased from Roth (Carl Roth GmbH, Karlsruhe, Germany). Acetic acid, 2-propanol, and dichloromethane were obtained from Merck (Merck KGaA, Darmstadt, Germany); acetonitrile from J. T. Baker (Fisher Scientific GmbH, Grießheim, Germany) and tetrahydrofurane (THF) from VWR (VWR International GmbH, Darmstadt, Germany). Standards of β-carotene, lutein, and zeaxanthin were from CaroNature (CaroteNature GmbH, Münsingen, Switzerland). Chlorophyll a and chlorophyll b were purchased from Sigma-Aldrich (Sigma Aldrich Chemie GmbH Taufkirchen, Germany). Chrysoeriol was obtained from Phytolab (Phytolab GmbH & Co. KG, Vestenbergsgreuth, Germany) and pheophytin a from LGC Standards (LGC Standards GmbH Wesel, Germany). All chemicals were of HPLC-MS-grade quality.

HPLC grade acetonitrile was procured from J.T. Baker Chemicals (Deventer, Netherlands). Purified water was obtained by a Milli-Q water purification system (Millipore, Bedford, MA, USA). Ethanol (96%) was purchased from Gram–mol d.o.o. (Zagreb, Croatia) and formic acid (98–100%) from T.T.T. d.o.o. (Sveta Nedjelja, Croatia). Commercial standards of quercetin-3-glucoside, kaempferol-3-rutinoside, myricetin, caffeic acid, gallic acid, ferulic acid, sinapic acid, quinic acid, chlorogenic acid, p-coumaric acid, esculetin, scopoletin, α-carotene, β-carotene, chlorophyll a and chlorophyll b were purchased from Sigma–Aldrich (St. Louis, MO, USA). Epicatechin, catechin, epigallocatechin gallate, Epicatechin gallate, apigenin, luteolin and naringenin were obtained from Extrasynthese (Genay, France), while quercetin-3-rutinoside was procured from Acros Organics (Thermo Fisher Scientific, Geel, Belgium). apigenin was dissolved in ethanol with 0.5% (v/v) dimethyl sulfoxide, standards of carotenoids and chlorophylls in n-hexane. Other standards were prepared as a stock solution in methanol, and working standard solutions were prepared by diluting the stock solutions to yield five concentrations.