A154 1 1

The pH of the aspirated stomach contents was determined using a calibrated portable pH meter (FiveEasy22-Meter, Mettler-Toledo International Trading (Shanghai) Co., China) having an accuracy of 0.01. The stomach content was homogenized and filtered through four layers of gauze. Then, the amount of glucose in the stomach content was determined using a glucose kit (glucose oxidase method, A154–1-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Serum glucose level was determined by using the commercially available kits (A154-1-1) from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to manufacturers’ instruction.

The effects of 3,3′,4,5′-TMS on glucose consumption potency were determined by a glucose assay kit, which was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China, A154-1-1). HepG2 cells (7 × 10⁵ cells/well) were seeded on 6-well culture plates and cultured overnight. The cells were treated with DXMS plus D-glucose for 48 h and different concentrations of 3,3′,4,5′-TMS for 16 h. According to the manufacturer’s instructions, the supernatants were collected, and their glucose concentrations were measured by the kit. The contents of cell glucose were determined by the glucose oxidase method, which causes an amaranth tint in the sample. The absorbance at 505 nm was detected with a microplate spectrophotometer. The result was calculated as below: glucose consumption = glucose concentration of blank–glucose concentration of each treatment group.

Intracellular TG and TCH concentrations were measured according to the instructions of the TG (F001-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and TCH assay kits (F002-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and the values were corrected using the protein concentration of the samples. The glucose concentration in the medium was measured according to the instructions of the glucose assay kits (A154-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and the values were corrected using the cell numbers of the samples. The cell numbers of the samples were tested by an automated cell counting chamber (SD100-002, Nexcelom Bioscience, Shanghai).

The level of fasting blood glucose was measured by a glucose oxidase method-based assay kit (A154-1-1, Jiancheng Bioengineering Institute, Nanjing, China). In brief, serum samples (2.5 µL) were incubated with 250 µL of the working solution containing 4-aminoantipyrine, glucose oxidase, and sodium 3,5-dichloro-2-hydroxybenzenesulfonate at 37°C for 10 min protected from light, followed by the detection of the absorbance at 505 nm with a microplate reader (EMax Plus, Molecular Devices, Sunnyvale, CA, USA). Fasting insulin concentration was determined in rat serum via radioimmunoassay (outsourced by Chemclin Biotechnology Corporation Limited, Beijing, China). Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated based on the formula: HOMA-IR  =  fasting insulin (μU/mL) × fasting blood glucose (mmol/L)/22.5 [23 (link)].

Based on previous results, wheat straw was selected as the substrate for the following analyses: In order to examine the combined impacts of RuXyn and cellulase on the breakdown of wheat straw, a mixture containing 0.78 U of RuXyn and 5% wheat straw was subjected to preincubation in a 0.1 M citric acid buffer (5 mL, pH 6.0) at 40 °C for 90 min. Subsequently, the liquid component was discarded, and the remaining solid residues were subjected to two washes with distilled water. These residues were then utilized as the substrate for cellulase. In a parallel manner, a control group containing inactive RuXyn was subjected to a comparable incubation process.
The leftover substances were placed in a 0.1 M citric acid solution (5.0 mL, pH 5.0) with the addition of 1 mg of cellulase mixture derived from Trichoderma sp. (C8272, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China; 10 U/mg). Afterwards, the mixture was placed in an incubator at a temperature of 37 °C for a duration of 90 min. Glucose quantification was carried out by utilizing a glucose oxidase-based commercial glucose analysis kit (A154-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).