Gallocatechin gallate

Epigallocatechin gallate (EGCG, ≥98%), gallocatechin gallate (GCG, ≥98%), epicatechin (EC, ≥98%), catechin (C≥98%), catechin gallate (CG, ≥98%), epicatechin gallate (ECG, ≥98%), and caffeine (≥98%) were purchased from Sigma, USA. Theasinensin A (≥98%) monomer was prepared at the Tea Research Institute of the Chinese Academy of Agricultural Sciences. Acetonitrile (chromatographically pure) and methanol (chromatographically pure) were purchased from Merck, Germany. Analytical reagents such as copper chloride, ascorbic acid, methanol, and phosphoric acid were purchased from Zhejiang Nade Scientific Instrument Co., Ltd., China. Black tea, oolong tea, and green tea samples were purchased from different areas in China. The Milli-Q ultrapure water was used.

TPHK is an typical and high economic efficiency green tea in China. We collected green tea samples (TPHK, Anhui) from 2015 to 2020 with the same tea variety and grade tea fresh leaves. Tea samples were stored in a 4 °C refrigerator.
( + )-Catechin (C), (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-gallocatechin (GC), (−)-epicatechin gallate (ECG), (−)-gallocatechin gallate (GCG), (−)-epigallocatechin gallate (EGCG), GA, caffeine, quercetin, kaempferol and myricetin (M) were purchased from Sigma‒Aldrich (St. Louis, MO, USA). HPLC-grade methanol and acetonitrile were purchased from Tedia Co., Ltd. (Fairfield, OH, USA).

The following reagents were used during the process: dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS), caffeine, 4-phenylbutyrate, glycine betaine, (±) catechin, (−) epicatechin, (−) gallocatechin gallate, epigallocatechin gallate, luteolin, kaempferol, resveratrol, piceid, caffeic acid phenethyl ester (CAPE), curcumin and α-tocopherol were supplied by Sigma-Aldrich (St. Louis, MI, USA). OptiCHO free serum media and l-glutamine were supplied by Thermo Fisher Scientific (Ulm, Germany). Further well known chaperon chemicals were also assessed as reference (Appendix B).
The cell line used was a stable recombinant DG44 CHO cell line that expressed an IgG monoclonal antibody, provided by Fujifilm Diosynth. This cell line was then left to grow in suspension in an OptiCHO™ free serum media in a 125 mL Erlenmeyer Flask (Corning, NY, USA) containing 33 mL of culture media, supplemented with 8 mM l-glutamine and 100 mg/L hygromycin B. Culture was maintained in a Infors HT Multitron cell shaking incubator (Bottmingen, Switzerland) set at 130 rpm with 5% CO₂ modified atmosphere at 37 °C. Cells were seeded into new media at 0.2 × 10⁶ viable cells/mL every three to four days for a maximum of 16 passages prior to use.

Green tea (dry leaves) was purchased from the local tea market (Hubei Province, China), the chemical composition of which was shown in Table 4. Standard compounds: (−)-gallocatechin (≥98%, GC), (−)-epigallocatechin (≥98%, EGC), (+)-catechin (≥98%, C), EC (≥97%), EGCG (≥98%), (−)-gallocatechin gallate (≥98%, GCG), ECG (≥98%), (−)-catechin gallate (≥98%, CG), theobromine (≥95%), theophylline (≥95%), caffeine (≥95%) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Myricetin-3-O-glucoside (≥99%, M-glu), quercetin-3-O-glucoside (≥99%, Q-glu), kaempferol-3-O-glucoside (≥98%, K-glu) were purchased from Chengdu DeSiTe Biological Technology Co., Ltd. (Chengdu, China). HPLC grade methanol and acetonitrile were purchased from Sigma-Aldrich (Shanghai, China). Polyamide (particle size 74–174 µm) and formic acid (≥99%, HPLC grade) were bought from Aladdin (Shanghai, China). The Ultrapure water was prepared by an EASYPure II UV Ultra Pure Water System (Barnstead International, Dubuque, IA, USA).

Catechins were extracted as described by Wang et al. (2014 (link)). Tea sample (0.2 g, dry weight) was extracted with 5 ml 70 % methanol in a water bath at 70 °C for 10 min with intermittent shaking. The supernatant was placed in a 10 ml volumetric flask, and the extraction step was repeated to reach the final volume of 10 ml. The extracts were filtered through a 0.45-μm Millipore filter before the injection was made. Each experiment was performed in triplicate.
The extracts were analyzed using an Agilent 1100 HPLC system. Separations were carried out using a Phenomenex RP-MAX 4 μm 250 mm × 4.6 mm i.d. C12 reverse phase column maintained at 40 °C, eluted at 1 ml min⁻¹ with a 60 min gradient of 4–25 % gradient of acetonitrile in water containing 1 % formic acid and monitored at 280 nm (Del Rio et al. 2004 (link)).
The standard chemicals, including catechin (C), epicatchin (EC), gallocatechin (GC), epigallocatechin (EGC), Epicatechin gallate (ECG), gallocatechin gallate (GCG), epigallocatechin gallate (EGCG) were purchased from Sigma Chemical Company (St. Louis, MO, USA).

Catechin, epiCatechin gallate (ECG), EC epiCatechin (EC), epigalloCatechin (EGC), galloCatechin (GC), Catechin gallate (CG), EGCG, and galloCatechin gallate (GCG) were acquired from Sigma-Aldrich Co. (St. Louis, MO, United States). High-Performance Liquid Chromatography (HPLC)-grade methanol was obtained from Merck (Darmstadt, Germany). All other chemicals and reagents were of analytical grade. FBT is mainly consumed in Shaanxi province, Hunan province, Guizhou province, Zhejiang province, and Guangxi Zhuang Autonomous Region in China. From these main producing areas, five FBT samples were collected from large factories and named as SX (Shaanxi Xianxi Lamu Tea Co., Ltd.), HN (Anhua Yuntiange Tea Industry Co., Ltd.), GZ (Guizhou Fanjin Tea Industry Co., Ltd.), ZJ (Zhejiang Wuyi Luotuo Jiulong Brick Tea Co., Ltd.), and GX (Guangxi Jinhua Tea Co., Ltd.). All tea samples used in this study were produced in 2018.

Acetonitrile, formic acid, and methanol were all HPLC grade and from Merck (Darmstadt, Germany). Authentic standards were purchased from Sigma–Aldrich (Shanghai, China), including caffeine, gallic acid, (+)-Catechin (C), (−)-epicatechin (EC), (−)-gallocatechin (GC), (−)-epigallocatechin (EGC), (−)-catechin gallate (CG), (−)-epicatechin gallate (ECG), (−)-gallocatechin gallate (GCG), (−)-epigallocatechin gallate (EGCG). Acetic acid was purchased from Aladdin Co., Ltd (Shanghai, China). Ethyl caprate (decanoate; 99 %) and n-alkanes (C7-C40) were purchased from Beijing Zhongsheng Ruitai Technology Co., Ltd. (Beijing, China).