Keracyanin chloride

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.

In this experiment, the chemicals purchased from Sigma-Aldrich (St. Louis, MO, USA) were as follows: caffeic acid, keracyanin chloride, kuromanin chloride, Folin-Ciocalteau reagent, quercetin, pepsin, and pancreatin. Aluminium chloride hexahydrate and potassium acetate were obtained from Junsei Chemical (Tokyo, Japan). Citric acid, sodium alginate, sodium acetate, potassium chloride, and sodium carbonate were purchased from Daejung Chemicals & Metals (Seoul, Korea).

Phenolic standards (gallic acid (≥98%), (+)-catechin (≥99%), (-)-epicatechin (≥98%), caffeic acid (≥98%), coutaric acid (≥90%), quercetin (≥95%), and myricetin glycosides (≥99%)) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Keracyanin chloride (≥98%) were obtained from Sigma Chemical Co. as the anthocyanin standard. For HPLC analysis, phosphoric acid, 85 wt.% in H₂O were obtained from Sigma Chemical Co. TPTZ (2,4,6-tripyridyl-s-triazine), and FeCl₃·6H₂O used for the FRAP assay were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). All solvents used in this study were HPLC grade.