Rutin

Soil samples used for the strain screening were collected from Herba Epimedii botanical garden in Huangshi, China. Epimedin C, prunin, and hesperetin-7-O-glucoside were obtained from Chengdu Desite Biotechnology Co., Ltd. (Chengdu, China). Icariin, neohesperidin, hesperidin, naringin, and isoquercitrin were purchased from Aladdin (Shanghai, China). Rutin was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). p-nitrophenyl-α-L-rhamnopyranoside (pNPR) was bought from Nantong Feiyu Biological Technology Co., Ltd. (Nanjing, China). Modified BCA Protein Assay Kit and protein markers for SDS-PAGE were produced from Sangon Biotech Engineering Co., Ltd. (Shanghai, China). HPLC-grade acetonitrile was produced from Tianjin Siyou Fine Chemical Co., Ltd. (Tianjin, China). Epimedium water extract (EWE) was provided from Jing Brand Chizhengtang Pharmaceutical Co., Ltd. (Huangshi, China). Two kinds of epimedium water extract were used in this work, one kind of epimedium water extract contained 50% of epimedin C, and another kind of epimedium water extract contained 20% of epimedin C. To make it easier to distinguish, the former was named EWE-I and the latter was named EWE-II. Other chemicals were of reagent grade.

Hibiscus manihot L. was provided and identified by Yulin Xinbang Pharmaceutical Co., Ltd. (Yulin, China). It was dried in a tray at 65 °C, destemmed and crushed (0.42 mm), and stored at 4 °C for further use.
The fermentation strain was Rhizopus arrhizus JHK31. We obtained it through pressure screening. It is stored in the China Center for Type Culture Collection under the deposit number CCTCC M 2023722.
Reagents including rutin (P98%), vitamin C (P99%), aluminum nitrate nonahydrate (P99%), DPPH (P96%), iron trichloride hexahydrate (P99%), trichloroacetic acid (P99%), dibasic sodium phosphate (P99%), and sodium dihydrogen phosphate (P99%) were from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China) provided other chemicals such as ferrous sulfate (P98%), green vitriol (P99%), sodium hydroxide (P96%), Tris (P99.8%), and salicylic acid (P99%). Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China) provided hyperoside (P98%), isoquercitrin (P98%), myricetin (P98%), and quercetin (P98%). Other chemicals such as sodium nitrite (P99%), potassium ferricyanide (P99.5%), and pyrogallol (P99%) were analytically pure. Acetonitrile and methanol were of chromatographic grades. The resin used was LX-83 macroporous resin from Sunresin New Materials Co., Ltd. (Xi’an, China). We used ultrapure water in all experiments.

For total flavonoids estimation, the method of Marinova, Ribarova & Atanassova (2005) was followed with slight modification. Half-gram samples of fresh leaves were homogenized in liquid nitrogen and total flavonoids were extracted in 10 ml of ethanol–water solvent (60%, v/v) (Xilong Scientific Co., Ltd., Shantou, China) in the dark (3 h) and then centrifuged at 15,000 rpm for 10 min. The supernatant (one ml) was mixed with a 0.73M sodium nitrite solution (0.4 ml) (Xilong Scientific Co., Ltd., Shantou, China), a 0.47M aluminum nitrate solution (0.4 ml) (Xilong Scientific Co., Ltd., Shantou, China), 1M sodium hydroxide (four ml) (Guangdong Guanghua Sci-Tech Scientific Co., Ltd., Guangzhou, China) and ethanol–water solvent (60%, v/v) (0.2 ml). The mixture was shaken for 12 min, then its optical density was measured at 510 nm. The total flavonoid content was calculated as milligrams of rutin (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) equivalent per gram of fresh weight.

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).

Acrolein (95%) was acquired from Energy Chemical Co., Ltd. Rutin (98%), 2,4-dinitrophenylhydrazine (DNPH), and ACR-DNPH standard were from Shanghai Macklin Biochemical Co., Ltd. Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), non-essential amino acids (NEAA), penicillin, streptomycin, trypsin-EDTA, and phosphate buffered saline (PBS, pH = 7.4) were bought from Thermo Fisher Scientific Inc. (MA, USA). Simulated saliva (SSF), gastric (SGF), and intestinal (SIF) fluids were from Phygene Biotechnology Co., Ltd. (Fuzhou, China). Cell counting kit-8 (CCK-8), apoptosis detection kit (including Annexin-V fluorescein isothiocyanate, Annexin-V FITC, and propidium iodide, PI), and JC-1 mitochondrial membrane potential assay kit were from Shanghai BestBio Co., Ltd. All the analytical and high-performance liquid chromatography (HPLC) grade reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA). Sephadex^TM LH-20 column fillers were obtained from GE Healthcare Bio-Sciences AB (Uppsala, Sweden).

Folin and Ciocalteu’s phenol reagent (2 N), 2,2-diphenyl-1-picrylhy-drazyl (DPPH) (≥97%), 2,2′-azino-bis-(3-ethylben zothiazoline-6-sulfonic acid) diammonium salt (ABTS) (≥98%), 2,4,6-tripyridyl-s-triazine (TPTZ) (≥99%) and 6-Hydroxy-2,5,7,8-tetramethylchro-man-2-carboxylic acid (Trolox) (≥98%) were obtained from Aladdin (Shanghai, China). The standards, gallic acid (99%), catechin (≥95%) and rutin (98%), used in the colorimetric determination were purchased from Macklin (Shanghai, China), and the anthocyanin standards for UPLC-MS, malvidin-3-O-glucoside (≥90%) and malvidin-3,5-O-diglucosides (≥90%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade reagents, formic acid and acetonitrile were purchased from Fisher Scientific Co. (Fairlawn, NJ, USA). All other analytical-grade chemicals were purchased from Chinese Reagent Network (http://www.labgogo.com/, accessed on: 1 November 2018).

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).