Caffeic acid

Four wampee fruits samples, W1–W4, were collected in July 2020 from South China, including Guangdong, Guangxi, and Hainan provinces, and the varieties were identified by professionals. Detailed information about W1–W4 is shown in Table 1. All the standards, including o-coumaric acid, gallic acid, gentisic acid, chlorogentic acid, DL-catechin, isoquercitrin, caffeic acid, myricetin, quercein, rutin, kaempferol 7-O-glucoside, quercitrin, and kaempferol, were HPLC-grade (>97%) and purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). 4-nitrophenyl α-D-glucopyranoside (pNPG), 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS), 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), and all mobile phase reagents used for liquid chromatography, including ethanol, acetonitrile, formic acid, and 2-propanol (HPLC-grade), were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Folin–Ciocalteu phenol reagent was purchased from Sigma-Aldrich Chemical Co., Ltd. (St. Louis, MO, USA). Other reagents applied in the study were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Chitosan (CS) (deacetylated degree: 90%; average molecular weight: 1.5 × 10⁵ Da), p-coumaric acid, caffeic acid, ferulic acid, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Fresh pork was purchased from a local market (Yangzhou, China).

Chlorogenic acid, hyperin, rutin, apigenin, quercetin, scopoletin, chrysoeriol, caffeic acid, protocatechin, isorhamnetin, taxifolin, and gentisic acid were purchased from Macklin (Shanghai, China). Methyl alcohol, formic acid, and n-Hexane were purchased from Merck Millipore. The sucrose and stearic acid were purchased from Sigma (St. Louis, MO, USA). Na₂HPO₄, KH₂PO₄, KPO₄, NaCl, CaCl₂, MgSO₄, and NH₄Cl were purchased from Beijing Lanyi Chemical Products Co. Ltd. (Beijing, China). sucrose, stearic acid, agar, peptone, cholesterol, and DMSO were purchased from Lablead (Beijing, China).

Six natural polyphenols (quercetin, caffeic acid, sinapic acid, catechin, ferulic acid, 3,4-dihydroxybenzoic acid), ascorbic acid, HPLC-grade methanol, as well as potassium persulfate, trichloroacetic acid, ferric chloride, and potassium ferricyanide were obtained from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). While DPPH, ABTS, and SAC were supplied by Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China).

Acrylic acid (AA), 3-propyl methacrylate potassium (MASEP), sodium periodate (NaIO₄), Polyethylenimine (PEI), N, N′-bis(acryloyl) cystamine (BAC) and caffeic acid (CA) were purchased from Macklin (China). Alexa Fluor-568 donkey anti-rabbit IgG (H&L) and Alexa Fluor-488 donkey anti-mouse IgG (H&L) and F-actin dye were from Invitrogen. The Cell Counting Kit-8 (CCK-8) was supplied by Dojindo Molecular Technologies (Japan). The primary antibodies against α-actinin (ab9465), connexin 43 (CX-43, ab11370), cardiac troponin T (CTNT, ab10214), CD68 (ab955), CD86 (ab220188), CD206 (ab64693), iNOS (ab49999), TGF-β (ab31013) and von Willebrand factor (vWF, ab6994) were ordered from Abcam. The primary antibody against alph smooth muscle actin (a-SMA, BM0002) was obtained from Boster Biological Technology (Wuhan, China). Male Sprague-Dawley (SD) rats (male, weight 220 ± 20 g, 7–8 weeks) were provided from the Laboratory Animal Center of the Academy of Southern Medical University (Guangzhou, China) under the ethics committee guidelines, and laboratory animals approved by Southern Medical University Animal Ethics Committee (SYXK(粤)2021-0167).

Standards were dissolved in caffeic acid (Macklin, Shanghai, China) solution. Internal standards (IS) were added to each standard and sample for a final concentration of 10 ng/ml to correct for sample and instrument variability. Tissue homogenizations (50 μl) were diluted 12-fold (w/v) by adding 10 μl IS, 500 μl water and 140 μl acetonitrile. Diluted samples were then filtered through Amicon Ultrafilter (Millipore, Billerica, MA, United States) by centrifugation at 15,000 g for 10 min at 4°C. Quantifications of QUIN were determined by HPLC (Waters, Milford, MA, United States) with tandem mass spectrometry (MS/MS) (Thermo Scientific, Waltham, MA, United States). Samples were run in positive ionization mode optimized for QUIN detection. Resultant acquisitions were directly injected into the Waters, equipped with an C18 (waters T3, 2.1 × 100 mm, 1.7 μm) column. The mobile phase consisted of an aqueous component (A: 10 mM ammonium acetate + 0.1% formic acid in ultrapure water) and an organic component (B: 0.1% formic acid in acetonitrile). The elution gradient was used as follows: 100% A for 30 s and 90% B for 6 min. The flow rate was set at 0.25 ml/min and the run time for each sample was 13 min. The concentration of QUIN in each sample was quantified by comparison to the standard curve.