Phosphoric acid

Chromatographic 2-octanol was obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China), and 4,7-diphenyl-1,10-phenanthroline was obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Folin phenol and a starch content detection kit were obtained from Beijing Solebar Technology Co., Ltd. (Beijing, China). Carbazole was obtained from Shanghai Maclean Biochemical Technology Co., Ltd. (Shanghai, China). Potassium hydrogen phthalate, trichloroacetic acid, phosphoric acid, ethanol, ferric chloride, ascorbic acid, sodium carbonate, gallic acid, phenol, concentrated sulfuric acid, sucrose, potassium sulfate, copper sulfate pentahydrate, boric acid, hydrochloric acid and galacturonic acid were obtained from Chengdu Kelong Chemical Co., Ltd. (Chengdu, China). All reagents were analytical reagents.

Hydroxyethyl cellulose (3400–5000 mPa.s, 25°C), azone, glycerol, triethanolamine, and ethyl p-hydroxybenzoate were purchased from Aladdin-Reagent Co. Ltd. (Shanghai, China). Centella asiatica total glycosides (47.95%), asiaticoside standard (≥98% pure), and madecassoside standard were obtained from Pusi Biotechnology Co., Ltd. (Chengdu, China). β-Cyclodextrin, propylene glycol, sodium hydroxide, polyethylene glycol, and absolute ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Acetonitrile was purchased from Tedia (Tedia, Fairfield, OH, USA). 2,4-Dinitrochlorobenzene was purchased from Huamai Technology Co., Ltd. (Wuhan, China). Recombinant human epidermal growth factor solution was purchased from Shenzhen Watsin Genetech Ltd. Dibasic sodium phosphate was purchased from Chengdu Jinshan Chemical Reagent Co., Ltd. Potassium dihydrogen phosphate was purchased from Sangon Biotech (Shanghai) Co., Ltd. Nutrient agar and eosin methylene blue were purchased from Beijing Solarbio Science and Technology Co., Ltd. Phosphoric acid, methanol, n-butanol, acetic anhydride, sulfuric acid, formaldehyde solution, sodium chloride, sodium citrate, and citric acid were purchased from Chengdu Kelong Chemical Reagent Factory (Chengdu, China). The diabetes-inducing agent streptozotocin (STZ) (S0130) was purchased from Sigma-Aldrich (Sigma-Aldrich, Shanghai, China).

Fresh pork hind legs and pork backfat were purchased from Yibin Daily Shopping Supermarket (Sichuan, China), divided into pieces of the same size, and stored in the refrigerator at −18 °C. κ-CAR, EWP, WG, and SPI used in this research were food-grade and purchased from Henan Wanbang Industrial Co., Ltd., Shangqiu, China. Sodium chloride (NaCl), urea, β-mercaptoethanol, trichloroacetic acid, sodium hydroxide (NaOH), and phosphoric acid were purchased from Chron Chemicals (Chengdu, China). All reagents were of analytical grade.

Cu plate with a thickness of ~0.2 mm was first polished by using 3000 mesh sandpaper and then cleaned in acetone, ethanol, and deionized water in sequence. Further, it was electropolished in 85% phosphoric acid (Chron Chemicals) solution at 3.0 V versus counter-electrode of another Cu plate for 5 min. After that, the polished Cu plate was ultrasonically cleaned in Ar-saturated ultrapure water (Milli-Q, 18.2 MΩ) to remove the surface residual particles.