Cyanidin chloride

In the present study, different chemicals for the MAE as well as for the quantification of anthocyanins, total phenolic compounds, and antioxidant activity have been employed as follows: methanol of HPLC purity (Fischer Chemical, Loughborough, UK), Milli-Q water from a Milli-Q water purification system (Millipore, Bedford, MA, USA), hydrochloric acid (Panreac, Barcelona, Spain), sodium hydroxide (Panreac, Barcelona, Spain), formic acid (Scharlau, Barcelona, Spain), anhydrous sodium carbonate (Panreac Química, Castellar del Valles, Barcelona, Spain), Folin–Ciocalteu reagent (Merck KGaA, EMD Millipore Corporation, Darmstadt, Germany), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sigma-Aldrich, San Luis, MO, USA). Furthermore, for the quantification of anthocyanins, total phenolic compounds, and antioxidant activity, three standards were employed respectively: cyanidin chloride, gallic acid, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), all of them supplied by Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

Methanol (HPLC grade) was purchased from Fischer Chemical (Loughborough, United Kingdom). Water was obtained from a Milli-Q water purification system from Millipore (Bedford, MA, USA). Hydrochloric acid and sodium hydroxide (both analytical grade) employed for the adjustment of pH were obtained from Panreac (Barcelona, Spain). The reagents necessary for the determination of total phenolic compounds were anhydrous sodium carbonate (Panreac, Barcelona, Spain), and Folin–Ciocalteu reagent (Merck Millipore, Darmstadt, Germany). The phenolic standard (gallic acid) and the anthocyanin standard (cyanidin chloride) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

The following chemicals were purchased from Sigma-Aldrich (Steinheim, Germany): 1,1-diphenyl-2-picrylhydrazyl (DPPH), Trolox (TE), Folin-Ciocalteu’s phenol reagent, sodium carbonate, sodium nitrate, ammonium nitrate, hydrochloric acid, aluminum chloride, sodium hydroxide, glucose, acetic acid, acetonitrile, methanol, gallic acid, quercetin, chlorogenic acid, rutin, and cyanidin chloride.

Anthocyanin and stilbene content analysis was performed by the method HPLC-MS as described [45 (link),47 (link),48 (link)]. Briefly, for anthocyanins 100 mg fresh cells tissue were subsequently homogenized using a mortar and a pestle in 1 mL of 1% (v/v) hydrochloric acid in methanol. Then, shredded tissue was extracted for 1 d at 4 °C. For stilbenes 100 mg of the dried shredded cells tissue were extracted for 2 h at 60 °C in 3 mL of methanol. Then, anthocyanin and stilbene extracts were filtered through a 0.25-um nylon membrane for further analysis. Next, samples were separated on Shim-pack GIST C18 column (150 mm, 2.1-nm i.d., 3-_m part size; Shimadzu, Japan) the on HPLC LC-20AD XR analytical system (Shimadzu, Japan), equipped with an SPD-M20A photodiode array detector. Liquid chromatography-high-resolution mass spectrometry for qualification of all components was performed using a 1260 Infinity analytical system (Agilent Technologies, Santa Clara, CA, USA) as described [24 (link),44 (link)].
The commercial standard cyanidin chloride, petunidin chloride, delphinidin chloride, malvidin chloride, t-resveratrol, t-piceid, t-piceatannol, ε-viniferin were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used as the control.

Chemical standards of (+)-catechin, (-)-epicatechin, cyanidin chloride and cyanidin 3-O-glucocide were purchased from Sigma-Aldrich. The standard of (+)-epicatechin was purchased from Nacalai USA. Standards of 4β-(S-cysteinyl)-catechin and 4β-(S-cysteinyl)-epicatechin were synthesized using procyanidin dimers B3 or B2 and L-cysteine as described previously^{29 (link)}. FBLL (PI 546481), Black Mexican (PI162573) and Arequipa (PI 571427) seeds were ordered from USDA. Suntava and Osage seeds were ordered from Burpee Gardens and Baker Creek Heirloom Seeds, respectively. Maize seeds rich in phlobaphene (P1-rr) in the A619 background were provided by Drs. Nan Jiang and Erich Grotewold at Michigan State University. Tobacco (Nicotiana tabacum, cv Xanthi) seeds constitutively expressing AtPAP1 were obtained from Dr. De-Yu Xie at North Carolina State University. BZ1 and bz1 maize seeds both in the W23 (C1, R-r) genetic background were provided by Dr. Virginia Walbot at Stanford University.

LC-MS was optimized with standards before running actual samples. Anthocyanin standards were purchased from Sigma Aldrich Company, St. Louis, MO, USA and consisted of Cyanidin Chloride (MW-322.70 g/mol), Keracyanin Chloride (Cyanidin-3-O-rutinoside chloride, MW-630.98 g/mol), Kuromanin Chloride (Cyanidin 3-O-glucoside chloride, MW-484.84 g/mol). (St. Louis, MO, USA). All the anthocyanin standards were dissolved in 80% methanol. Then, standards were further diluted to 100 µM using 0.2% Glacial Acetic Acid, 50% Acetonitrile and water.
Frozen TC were ground using a mortar and pestle. Then, TC juice was filtered using 0.22 µm filter unit. Tart cherry juice was freeze-dried using a vacuum desiccator. To extract anthocyanin, 1 g of freeze-dried tart cherry was dissolved with 10 mL methanol/water/acetic acid (85:15:0.5, v/v/v) and the tube was placed on a rocker at 4 °C overnight in the dark due to the sensitivity of anthocyanins to light. The sample was then vortexed, sonicated, and filtered through a 0.22 µm filter unit. Next, 1 mL of sample was dried for 3 h in the vacuum drier and 20 µL of anthocyanins were dissolved in 100 µL of 0.05% acetic acid and filtered using 3 K filter units (Millipore). Filtrate was re-diluted 10 times with 0.05% acetic acid for LC-MS analysis. The anthocyanin analyzed graph is included in Figure S1.

Solvents of HPLC-gradient grade, Folin–Ciocalteu reagent, bovine serum albumin, and standards of gallic acid, cyanidin chloride, (−)-epicatechin, and (+)-catechin were supplied by Sigma-Aldrich (St. Louis, MO, USA). Malvidin-3-glucoside chloride standard was purchased from Extrasynthese (Genay, France). The solutions were prepared in deionized water produced by a Milli-Q system (Merck Millipore, Darmstadt, Germany).