Gallic acid

The samples were dried by freeze-dryer (Scientz-100F, Ningbo, China), and then ground (30 Hz, 1.5 min) to powder using a grinder (MM 400, Retsch, German). The polyphenol content was determined by the Folin–Ciocalteu method [37 ]. The 0.2 g sample was extracted in 10 mL 70% (v/v) methanol (Merck, Darmstadt, Germany) solution at 4 °C. The 0.1 mL extract was fixed to 0.5 mL with distilled water, and then Folin–Ciocalteu reagent (0.25 mL) and 20% sodium carbonate solution (1.25 mL) were added. After 40 min, the absorbance of the reaction solution was measured at 725 nm (DU730UV VIS, Beckman Coulter, CA, USA). The calibration curve (Figure S5) was prepared with gallic acid (Macklin, Shanghai, China), and mg gallic acid equivalent (GAE) g⁻¹ FW was used to represent the polyphenol content. Each sample was performed in three biological replicates.

CTAC, TEA, xanthine, and xanthine oxidase (XO) were purchased from Sigma-Aldrich. TEOS and NaOH were obtained from Shanghai Lingfeng Chemical Reagent Co., Ltd. Fe(acac)₃ was provided by J&K Scientific. Urea, RhB, iron chloride hexahydrate (FeCl₃·6H₂O) and 3,3′,5,5′-tetramethyl-benzidine (TMB) were bought from Sinopharm Chemical Reagents Co., Ltd. Methoxy PEG silane was purchased from Shanghai Yare Biotech, Co., Ltd. Gallic acid was acquired from Macklin. Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and PBS were provided by Gibco. FITC, 4′, 6-diamidino-2-phenylindole (DAPI), DCFH-DA, CCK-8 assay, calcein-AM, PI, annexin V-FITC, H&E, paraformaldehyde (PFA) and TUNEL apoptosis assay kit were acquired from Beyotime. Primary antibody against GPX4 and a biotinylated secondary antibody (anti-rabbit IgG, HRP-linked antibody) were purchased from Cell Signaling Technology.

To investigate whether the growth improvement of S. litura fed on the diets containing R. terrigena-fermented P. viviparum root powder was caused by the reduction of flavonoids content, the contents of quercetin and gallic acid in P. viviparum precipitates in the cultures with and without R. terrigena inoculation post 7 days at a 200 rpm shaker at 37°C were analyzed by high performance liquid chromatography (HPLC). The precipitates prepared by centrifugation as described above were freeze-dried by using Savant Modulyo D freeze drier (Thermo Fisher Scientific, Waltham, MA, United States). For each treatment, 0.5 g of dry sample was diluted to 50 ml with 30% methanol, and left for 2 h before ultrasonic extraction for 30 min and then used for HPLC analysis with a photodiode array Detector (1,100 series equipment, Agilent Technologies, Santa Clara, CA, United States). A C18 reversed-phase column (3.0 by 150 mm, 2.7 um) was used with the following parameters: 370 nm, 0.4 ml/min flow rate, and 10 μl injection volume for quercetin analysis, and 272 nm, 0.25 ml/min flow rate, and 1 μl injection volume for gallic acid analysis. The mobile phase consisted of 0.1% acetic acid in water and methanol. The peaks of standard quercetin (Macklin, Shanghai, China) and gallic acid (Macklin, Shanghai, China) under the above conditions were identified (Supplementary Figure S2).