Epigallocatechin gallate (egcg)

Pharmacological agents were EGCG (98%, Yuanye, Shanghai, China), GCG (98%, Yuanye, Shanghai, China), TFDG (99.12%), and TSA (91.4%) as test samples (generous gifts from the Tea Research Institute of Chinese Academy of Agricultural Sciences, Hangzhou, China) and kojic acid (99%, Macklin, Shanghai, China) as a positive control.
Unless specified otherwise, all chemicals and laboratory ware used for cell culture were purchased from GIBCO (Grand Island, NY, USA), Corning (Corning, NY, USA), GENOM (Jiaxing, China), and Beyotime (Shanghai, China). α-MSH (A1025) was purchased from Apexbio (Houston, TX, USA), and L-DOPA was purchased from Macklin (Shanghai, China). EASY spin Plus cell RNA rapid extraction kit was purchased from Aidlab (Beijing, China). The reverse transcription kit (MR05201M) and fluorescence quantitative kit (MQ00401S) were purchased from Monad (Suzhou, China). Primary antibodies against TYR (TU343218), TRP1 (TD13325), TRP2 (TA5303), MITF (TU326886), p-MITF (Ser180, TA3027), and MC1R (TD4992) were purchased from Abmart (Shanghai, China). Primary antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AG019), secondary antibodies of goat anti-mouse IgG labeled with horseradish peroxidase (HRP), goat anti-rabbit IgG labeled with HRP, and other relevant reagents used for protein expression detection were purchased from Beyotime (Shanghai, China).

Gelatin (type-A, pI = 8.0) and EGCG (purity ≥ 98%, HPLC) were purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Poly γ-glutamic acid (γ-PGA; MW = 600 kDa) was obtained from Nanjing Shineking Biotech Co., Ltd. (Nanjing, China). EGCG (purity ≥ 90%) was kindly supplied by Anhui Agricultural University (Anhui, China). Simulated gastric fluid (SGF; pH 2.1 with pepsin) and simulated intestinal fluid (SIF; pH 7.2, with trypsin) were purchased from Yuanye Microbes (Shanghai, China). 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and DPPH free radical scavenging capacity assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (A153-1-1, Nanjing, China). The total antioxidant capacity assay kit using the ABTS method was purchased from the Beyotime Institute of Biotechnology (S0119, Nantong, China). Caco-2 (human colorectal cancer cells) lines were purchased from ProCell Life Science and Technology Co., Ltd. (Wuhan, China), while the Caco-2 cell-specific medium was purchased from Yifeixue Biotechnology (Nanjing, China).

EGCG (purity ≥98%, high-performance liquid chromatography) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China) and stored at 4°C. Prior to the experiments, EGCG was dissolved in dimethylsulfoxide (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) and kept at −20°C until ready for use.

Chitosan (CS) and carboxymethyl cellulose (CMC) were obtained from Shanghai Macklin Biochemical Technology. Montmorillonite (MMT, K10) and the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Paraffin, span 80, glutaraldehyde and isopropanol were obtained from Aladdin Biochemical Technology, Shanghai, China. EGCG (purity ≥ 98%, high-performance liquid chromatography) was obtained from yuanye Bio-Technology, Shanghai, China. A Cell Counting Kit-8 (CCK8) was purchased from Dojindo China Co., Ltd. (Shanghai, China). BCIP/NBT alkaline phosphatase color development kit and enhanced BCA protein assay kit were obtained from Beyotime Biochemical Technology, Shanghai, China. The calcium colorimetric assay kit and alkaline phosphatase (ALP) kit were obtained from Jiancheng Biochemical Technology, Nanjing, China. All other cell culture products and reagents were purchased from GIBCO unless otherwise specified. All chemicals were of reagent grade and used without any further purification. Ultrapure water (18.2 MΩ, Millipore Co., USA) was used in all solutions and reagents throughout the experiment.

The clinical isolate A.hydrophila AH08 was kindly provided by Prof. Defang Chen (Sichuan Agriculture University) and was used throughout the in vitro and in vivo studies. The test strain was routinely grown at 30 °C in trypticase soy broth (TSB) with rotary shaking at 150 rpm. All bacterial culture mediums were purchased from Haibo Bio (Qingdao, China). EGCG was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO) (Biosharp) to prepare stock solutions at concentrations of 80 mg/mL.