Phloridzin

HPLC-grade methanol and acetonitrile were obtained from Fisher Scientific (Pittsburgh, PA, USA). The chemical standards for quercetin-3-O-galactoside (≥90%), catechin (≥98%), and phloridzin (≥99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Procyanidin B1 (>95%) and procyanidin B2 (>95%) were purchased from Solarbio (Beijing, China). Procyanidin C1 (>95%) and quercetin-3-O-glucoside (>98%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). Folin-Ciocalteu reagent (2 mol/L), 4-dimethylaminocinnamaldehyde (DMAC), dimethyl sulfoxide (DMSO), alpha-glucosidase (EC 3.2.1.20), and 4-nitrophenyl-α-D-glucopyranoside (PNPG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The mass spectrometry LockSpray calibration solution was leucine-encephalin and it was commercially available from Waters Corporation (ESI⁻: 554.2615, ESI⁺: 556.2771, Waters, UK). Double-distilled water (ddH₂O) was used in all experiments and samples for UPLC-Q-TOF/MS were filtered through a 0.22 μm membrane before injection. All the other reagents were of analytical grade bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Trilobatin, phloridzin, canagliflozin, empagliflozin were purchased from Sigma (Sigma, St. Louis, MO, USA). Cell-light EDU Appollo567 in vitro kit was purchased from RIBOBIO (Guangzhou, China). Rat SGLT2 primary antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). HNF4α and HBXIP primary antibody was from Proteintech (Rosemont, IL, USA). Specific anti-mouse and anti-rabbit HRP-conjugated second antibodies were obtained from Santa Cruz Biotechnology (Texas, CA, USA). ECL Chemiluminescence Detection Reagent was obtained from Millipore Corporation (Billerica, MA, USA). BCA protein assay kit and other chemicals were purchased from Biyotime (Shanghai, China).

Standards of (+)-catechin, (−)-epicatechin, p-coumaric acid, ferulic acid, ellagic acid, quercetin 3-rutinoside,
quercetin, cyanidin 3-glucoside, phloridzin, and phloroglucinol with
purity >99% were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Castalagin was kindly provided by Dr. S. Quideau (Bordeaux, France).
Methanol and acetonitrile were from J.T. Baker (Deventer, The Netherlands),
formic acid from Honeywell (Barcelona, Spain), and hydrochloric acid
and sodium acetate from Panreac (Barcelona, Spain). Ascorbic acid
was from Acros Organic (Geel, Belgium). Water was deionized using
a Milli-Q-system (Millipore, Bedford, MA, USA).
Strawberries
(Fragaria x ananassa) cv. Primoris were from Huelva
(Spain) and harvested ripe (80% red) in June 2018. Apples (Malus domestica) cv. Golden Delicious were from Zaragoza
(Spain) and harvested in November 2019.

The phenolic compounds were analyzed by HPLC (Waters 2695; C18 column (250 × 4.6 mm × 5 μm size particle) and the absorbance was measured at 280 nm by a Waters 2478 Dual λ Absorbance Detector. The mobile phase of acidified water containing 1% formic acid (A) and acetonitrile (B) was used. The setting procedure of the gradient was as follows: 0 min, 20% B; 17 min, 21.5% B; 17.5 min, 68% B; 40 min, 68.3% B; 41 min, 100% B; 51 min, 20% B, and held for 2 min. The volume of injection was 20 μL and the flow rate was 1.0 mL/min. All HPLC-grade standards (ferulic acid, epicatechin, catechins, phloridzin, phloretin, caffeic acid, rutin, and chlorogenic acid) were purchased from Sigma Chemicals. Identification of each peak was performed on the basis of comparing their retention times with their known standards. The concentration of phenolic contents was expressed as milligrams of each compound per liter of juice.

The standard of polyphenols (purity ≥ 98%), including chlorogenic acid, caffeic acid, rutin, catechin, epicatechin, phloridzin, kaempferol 3-O-glucoside, apigenin glucoside, phloretin, procyanidin b1, and procyanidin b2, were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). Reagents used for the antioxidant tests such as gallic acid, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,3,5-triphenyltetrazolium chloride (TPTZ), anhydrous ferric chloride, hydrochloric acid, and sodium acetate were purchased from Sigma–Aldrich (Milan, Italy). Methanol (MeOH) and water (LC-MS grade) were purchased from Carlo Erba reagents (Milan, Italy), whereas formic acid (98–100%) was purchased from Fluka (Milan, Italy).

Crabapple leaf samples (approximately 0.8–1.0 g fresh weight) were subjected to extraction with 10 mL extraction solution (methanol: water: formic acid: trifluoroacetic acid= 70: 27: 2: 1)58 (link) at 4 °C in the dark for 72 h, shaking every 6 h. The supernatant was isolated by filtration through filter paper and a further filtration through a 0.22 μm Millipore^TM filter (Billerica, MA, USA). For the HPLC analysis, trifluoroacetic acid: formic acid: water (0.1: 2: 97.9) was used as mobile phase A and trifluoroacetic acid: formic acid: acetonitrile: water (0.1: 2: 48: 49.9) was used as mobile phase B. The gradients used were as follows: 0 min, 30% B; 10 min, 40% B; 50 min, 55% B; 70 min, 60% B; 30 min, 80% B. Detection was performed at 520 nm for anthocyanin and 350 nm for flavonol58 (link), respectively. All samples were analyzed in three biological triplicates (extracted from three different batches of leaves).
In this study, we employed HPLC-ESI (±)-MS2 analysis to identify the kinds of compounds by standards and comparing their spectroscopic data to literature59 60 (link). Cyanidin-3-O-glucoside, quercetin-3-O-glucoside, avicularin, phloridzin, quercetin (Sigma-Aldrich, Germany), Procyanidin B2 (Sigma-Aldrich, UK) was used as standards.