Phosphate buffer

ARS and CAT from bovine liver were purchased from Beijing Solarbio Ltd. Na₂HPO₄·12H₂O and NaH₂PO₄·2H₂O were purchased from Tianjin Damao Chemical Reagent Factory. Phosphate buffered solution (PBS, pH 7.2–7.4) for intracellular oxidative stress analysis was from Beijing Macgene Ltd., Beijing, China. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, and penicillin/streptomycin were all purchased from Thermo Fisher Scientific, Waltham, MA, USA. Dimethyl sulfoxide (DMSO) was provided by from Biofroxx (Germany). Hydrogen peroxide was bought from Sinopharm Chemical Reagent Co., Ltd. Phosphate buffer (a mixture of Na₂HPO₄·12H₂O and NaH₂PO₄·2H₂O solution, pH 7.4) was chosen to control the solution pH. All the water solutions involved in this work were prepared by using ultrapure water, and stock solutions were preserved at 0–4 °C.

Instant dark tea was provided by Hunan Tea Industry Group Co. Ltd. (Changsha, China) with the fermentation of Eurotium Cristatum. Carboxylic acid (Trolox) and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonicacid) diammonium salt (ABTS) were purchased from Shanghai Maclin Biochemical Reagent Co., Ltd. (Shanghai, China). Rutin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and phosphate buffer were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Fluorescein and 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH) were purchased from Aladdin Reagent Co. Ltd. (Shanghai, China). Folin-Ciocalteu reagent was purchased from Beijing Solarbio Science and Technology Co. Ltd. (Beijing, China). (-)-Epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), (-)-gallocatechin gallate (GCG), (-)-epicatechin gallate (ECG), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-catechin gallate (CG), (+)-catechin (C), theobromine (TB), theacrine, and gallic acid were purchased from Aladdin Industrial Co. Ltd. (Shanghai, China) with the HPLC purity ≥98%. Simvastatin was purchased from Tianjin Huairen Pharmaceutical Co. Ltd. (Tianjin, China).

The freeze-dried samples from the optimal combination in the orthogonal experiment and the negative control were ground to a powder using an M20 universal mill (IKA, Staufen, Germany). Then, 10 g of the freeze-dried SSF powder were extracted with 200 mL of 70% (by volume) ethanol for 2 h in an ultrasonic extractor (KH-600TDV; Hechuang, Kunshan, PR China). Afterwards, the samples were centrifuged (Heraeus^TM, Multifuge^TM X1R; Thermo Fisher Scientific) at 25 155×g and 4 °C for 10 min, and the supernatants were collected. The residue was then suspended in 100 mL of 70% (by volume) ethanol, ultrasonicated and centrifuged under the same conditions. The supernatants were combined, filtered through Whatman no. 1 paper (GE Healthcare, Chicago, IL, USA) and freeze-dried. An aliquot comprising 5 mg of the freeze-dried sample was stored at –20 °C and dissolved in 1 mL of phosphate buffer (0.1 M, pH=6.8; Sinopharm Chemical Reagent Co., Ltd, Shanghai, PR China) immediately before analysis.