Cyanidin

The standard compounds, including cyanidin-3-O-galactoside (90% purity), cyanidin-3-O-glucoside (95% purity), cyanidin (95% purity), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (98% purity), 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (98% purity), 2,9-dimethyl-1,10-phenanthroline (Neocuproine) (99% purity), potassium persulfate (99% purity), acetonitrile, formic acid, ethanol, methanol, and dimethyl sulfoxide (DMSO), were obtained from Sigma-Aldrich (Darmstadt, Germany). cyanidin-3-O-galactoside and cyanidin-3-O-arabinoside standards were bought from Polyphenols (Sandnes, Norway). HCl, NaNO₂, H₂O₂, and CuCl₂ were purchased from Merck (Darmstadt, Germany). Fetal bovine serum (FBS), Lonza, Dulbecco's Modified Eagle Medium (DMEM), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Lonza Group Ltd. (Basel, Switzerland). Glutamine, penicillin and streptomycin, and amphotericin were purchased from Sigma Chemical Co. (St. Louis, MO). The water used for experiments was treated in a Milli-Q water purification system.

Leaves of tea variety ‘Mooma 1’ and wild type ‘Longjing 43’ (Camellia Sinensis CV ‘Longjing’) were sampled from healthy pants that are grown in the tea garden of Shitai County, Anhui, China (latitude 30.19 N, longitude 4 E, altitude 20 m above mean sea level). A total of 10 buds and leaves were randomly collected from different branches, frozen immediately in liquid nitrogen, and stored at −80 °C for RNA-seq, qRT-PCR and HPLC analysis.
Catechin (C), gallocatechin (GC), epicatechin (EC), epigallocatechin (EGC), epicatechin 3-O-gallate (ECG), epigallocatechin 3-O-gallate (EGCG) were obtained from Shanghai RongHe Pharmaceutical Co. Myricetin 3-O-glucoside, quercetin 3-O-glucoside, kaempferol, petunidin, cyanidin, cyanidin 3-O-glucoside, and delphindin were purchased from Sigma Chemicals Co.

Carrot root samples used for RNA-seq and qPCR were analyzed for anthocyanin concentration. Anthocyanins were extracted from pigmented OP and IP and non-pigmented IP tissues and quantified by HPLC analysis as described previously [9 (link)]. A commercial standard of cyanidin (Sigma-Aldrich, Atlanta, GA, USA) was used for quantitation purposes. Anthocyanin content was expressed as mg of cyanidin equivalents per gram of fresh weight (mg/g fw).

HPLC analysis was carried out by Scistd Testing Co., LTD. (Qingdao, China). Anthocyanins were extracted using 50 mg of powdered petal (flower development stage 2–3) suspended in 5 ml of 0.5% (v/v) HCl‐methanol and incubated for 2 hr at 4°C in the dark. The extracts were then centrifuged for 15 min at 14,000 rpm, and the supernatants were collected and stored at −20°C. The supernatant was diluted with methanol to 5 ml and filtered through a cellulose acetate membrane (Sartorius, Göttingen, Germany). HPLC was performed with Agilent 1260 Infinity LC (Agilent Technologies, USA). Anthocyanidin profile analysis was quantified at the 530 nm wavelength by using a calibration curve from commercial standards of anthocyanidins (pelargonidin, cyanidin, delphinidin, petunidin, peonidin, and malvidin, Sigma, USA).

Acetonitrile, methanol water and dichloromethane (Merck; Darmstadt, Germany) were used for liquid chromatography diode array detection (LC-DAD) analysis and liquid chromatography tandem mass spectrometry (LC-MS/MS). Ethanol absolute and chloroform were obtained from VWR Chemicals (Radnor, PA). Hexane, butylated hydroxytoluene (BHT), formic acid (99% for mass spectrometry) along with analytical standards (chicoric acid, chlorogenic acid, lutein, β-carotene, violaxanthin, neoxanthin, β-cryptoxanthin, and cyanidin) were purchased from Sigma-Aldrich (St. Louis, MO). Ultrapure water was obtained from a Milli-Q Gradient A10 water purification system.

The chemicals were provided by Merck Chemical Co. (St. Louis, MO, USA); hypochlorite solution, Hydrochloric acid, gallic acid, cyanidin, quercetin, and solvents were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), whereas Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) provided reagents, media, and chemicals for biochemical studies. Rosmarinic acid, ursolic acid, quercetin-3-O-rutinoside (Rutin), isocitric acid, and chicoric acid were obtained from Merck Chemical Co. (St. Louis, MO, USA). Rosmarinyl glucoside was obtained from Aobious Inc., 9 Blackburn Drive, Gloucester, MA 01930 (USA). Nepetoidin B, salvigenin, and rosmarinyl glucoside were obtained from BioCrick Biotech, 88 Keyuan Road, Hi-Tech Zone, Chengdu, Sichuan 610042, PRC.

The anthocyanins were determined using high-performance liquid chromatography (HPLC) as previously described (Qi et al., 2013 (link)). For extraction of other flavonoids, freeze-dried flowers were finely ground and 50mg was extracted in 500 μl of MeOH for 48h at 4 °C in darkness. After samples were centrifuged, the supernatants were transferred to fresh tubes and the pellet was resuspended and incubated in 500 μl of 1% MeOH at 4 °C for 24h, and then the supernatant was combined for further HPLC analysis. HPLC was performed as previously described (Qi et al., 2013 (link)). Cyanidin, Cyanidin-galactoside, dihydroquercetin, dihydrokaempferol, (+)-catechin, (–)-epicatechin, luteolin, naringenin, and quercetin were obtained from Sigma-Aldrich China (Shanghai). Standards of afzelechin, (–)-epiafzelechin, (+)-gallocatechin, and (–)-epigallocatechin were purchased from BioBioPha (Yunnan, China). The delphinidin chloride (ChromaDex, Santa Ana, CA, USA), petunidin chloride (ChromaDex), and other flavonoids such as dihydromyricetin (YiFang S&T, Tianjin, China) equivalents were used as standards for quantification. Mean values and SDs were obtained from three biological replicates.