H class

All chromatographic evaluations were performed using ACQUITY UPLC Classic, H‐Class, or I‐Class instruments equipped with ACQUITY photodiode array detectors (Waters, Milford, MA). ACQUITY UPLC BEH C₁₈, CSH C₁₈, HSS T3, and Atlantis PREMIER BEH C₁₈ AX columns (1.7 or 1.8, 2.5 and 5 μm, 2.1 × 50 mm) were obtained from Waters (Milford, MA).

Quantitative analyses of the α-CBT-diols and β-CBT-diols isolated from WTFs were performed using ultra-HPLC (UPLC; H-CLASS, Waters, MA, USA). The chromatographic column was an Acquity UPLC BEH C18 column (1.7 μm, 50 mm × 2.1 mm). The mobile phase A and B were acetonitrile and pure water, respectively. The isogradient elution procedure was as follows: 0–8 min, 50% A and 50% B. The column temperature, injection volume, flow rate, and detection wavelength were 40 °C, 5 μL, 0.3 mL/min, and 200 nm, respectively. The chromatograms of α-CBT-diols and β-CBT-diols are shown in Figure 6. The calibration curve equation used for α-CBT-diols and β-CBT-diols determination were y = 20910x − 21414 (r² = 0.9997, n = 8, with a linear range of 10–1000 μg/mL), and y = 15297x − 14940 (r² = 0.9996, n = 8, with a linear range of 10–1000 μg/mL), respectively.

Protein liquid chromatography-mass spectrometry (LC–MS) was carried out by Analytical Services in the School of Chemistry at Cardiff University. The analysis was performed on a Waters Synapt G2-Si quadrupole time-of-flight mass spectrometer coupled to a Waters Acquity H-Class ultraperformance liquid chromatography (UPLC) system. The column used was a Waters Acquity UPLC Protein C4 BEH column (300 Å, 1.7 µm, 2.1 by 100 mm), held at 60 °C. A gradient of H₂O containing 0.1% CHO₂H and acetonitrile containing 0.1% CHO₂H was employed. Data was collected in positive electron spray ionization mode and analyzed using Waters MassLynx software version 4.1. Maximum entropy 1 software was used to generate deconvoluted mass spectra (Figs. S17–19).

The previously extracted C3G (Cui et al., 2021 (link)) was used in this study. We harvested black chokeberry “Fu Kangyuan No.1” from Liaoning FuKangyuan Black Chokeberry Technology Co., Ltd. and transported it at 4°C to Shenyang Agriculture University. Black chokeberry (500 g) was homogenized and extracted for 90 min in an ultrasonic bath (45°C, 500 W) with 0.1% HCl‐acidified ethanol at 1:5 (w/v) ratio. The extraction solution underwent vacuum filtration to remove the dross and rotary evaporation to remove the alcohol. It was then purified through D101 absorbent resins (Beijing Solarbio Science and Technology Co., Ltd.). To further isolate and purify the C3G, an LC‐3000 semi‐preparative HPLC (Beijing Tong Heng Innovation Technology Co., Ltd.) equipped with a Dikma Platisil C18 (300 × 10 mm, 10 μm) column and a G4212B diode‐array detector (Agilent Technologies) was used. Finally, the extract was freeze‐dried into powder and the purity of the powder was calculated (98.28%) by comparing its chromatogram with the chromatogram of a C3G standard using UPLC (H‐Class, Waters Corp.), as described in our previous paper (Cui et al., 2021 (link)), and then the powder was stored at −20°C until further use.

A Fusion trihybrid LC-MS/MS system with electron transfer dissociation (ETD) and high energy collision-induced dissociation (HCD) (Thermo, San Jose, CA) with a binary ultrahigh performance high pressure LC pump (H class, Waters), a 70 min reverse-phase 0.1% triflouroaceticacid/acetonitrile gradient at 0.25 mL/min, and a Zorbax C8 column (300-SB, 2.1 × 100 mm, 1.8 μm, Agilent, San Jose, CA) at 60 °C was employed to collect 3–6 technical replicates per sample, where only the last 3 replicates, collected in randomized fashion several months later than the initial technical replicates, are the focus of this work. Data-dependent acquisition was performed where the top 5 most abundant ions were selected with a unique MS/MS scan function with 120k mass resolution in the MS dimension.

The FPMB lyophilized powder (5 mg) was accurately weighed. A sample solution of 1.0 mg/mL was prepared from the lyophilized powder of FPMB in a 10 mL volumetric flask. The sample solution and standard solution were diluted with 30% methanol 5 times. The diluted solution was filtered with an organic membrane of 0.5 µm. The filtered solution was then analyzed by HPLC (H-CLASS, Waters Technologies, Inc., Shanghai, China). The type of chromatographic column was ALPHA1-2 LD plus (Bio-labs Instruments Co., Ltd., Guangzhou, China). The chromatographic conditions included the mobile phase: methanol (A), ultra-pure water (B), and 1% acetic acid solution (C): flow rate: 1.0 mL/min; injection volume: 10 μL; elution conditions: 0–30 min, 30–80% A, 1% acetic acid solution (C) always maintained at 10%; detection wavelength: 350 nm; and column temperature: 30 °C.