**2. Materials and Methods**

#### *2.1. Materials and Reagents*

Fresh yam (*Dioscoreae Rhizoma*), which was plucked in November and purchased from Jiujiang, Jiangxi, China, was powdered with a high-speed disintegrator (XL-20B, Zhengzhou Fengli Crushing Equipment Co., Ltd., Zhengzhou, China). Fetal bovine serum (FBS) and Dulbecco's modified eagle medium (DMEM) were purchased from Solarbio Science and Technology Co., Ltd. (Beijing, China). The cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kyushu Island, Japan). The reactive oxygen species kit, superoxidase dismutase kit, and malondialdehyde kit were purchased from Beyotime Biotechnology (Shanghai, China). Hydrogen peroxide (H2O2) was purchased from Sigma (Sigma, Burlington, MA, USA). The IEC-6 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Antibodies used in this study were purchased from Cell Signalingas (Bever, MA, USA). All the other chemicals used in the present study were analytical reagent grade.

#### *2.2. Extraction and Purification of Polysaccharides*

The yam was cleaned and dried for 12 h and then peeled and sliced the next day. The sliced yam (thickness = 3 mm) was placed in a tray, dried in an oven (VYJG-9420, Hangzhou Yijie Technology Co., Hangzhou, China) at 55 ◦C for 48 h, taken out and ground into powder (60 mesh) with a high-speed disintegrator, after soaking in ethanol (95%) overnight to remove fat, color, and small molecules, the precipitate was collected the next day and heated in a water bath in a ventilated area to remove the ethanol and dry completely. The obtained powder was placed in a self-sealing bag and prepared for later experiments. A total of 300 g of yam powder was weighed, and 6 L of ultrapure water was added at a ratio of 1:20. After extraction at 80 ◦C for 3 h, the above process was repeated twice, and the extracts were combined, centrifuged, and concentrated to 1/10 of the original volume. After adding 95% ethanol (concentrate:ethanol = 1:5.33) overnight precipitate at 4 ◦C. The next day, the precipitate was taken and re-solubilized, then the solution pH was adjusted to 4.5 with hydrochloric acid, and glycosylase was added at 55 ◦C for 1 h, followed by pH adjustment to 6.5 with sodium hydroxide, amylase was added at 95 ◦C for 2 h, finally papain was added at 65 ◦C for 1.5 h, high temperature (100 ◦C) inactivated for 30 min, and proteins were removed 3 times by Sevage method [27], (polysaccharide liquid:chloroform:n-Butyl alcohol = 25:4:1). Then the solution was centrifuged, and the upper layer was gathered to obtain the polysaccharide solution with proteins removed, and then the solution was subjected to dialysis (Mw:8000, tap water for 24 h, distilled water for 24 h, ultrapure water for 24 h) to remove minor molecules and the inorganic ions. The yam polysaccharide solution was concentrated under reduced pressure with a vacuum pump, and four times the volume of anhydrous alcohol was added for 24 h. After centrifugation, the precipitate was placed in a lyophilizer to obtain refined yam polysaccharide.

#### *2.3. Structure Characterization*

2.3.1. Determination of the Content of Total Carbohydrate, Uronic Acid and Protein

Carbohydrate content was determined by phenol sulfate method according to the method of Huang et al. [28]. In short, prepare 0.1 mg/mL standard glucose solution was prepared, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mL was pipetted in a test tube, and then add distilled water to 1 mL, after that, 1 mL of 3% phenol was added, gradually add 4 mL of concentrated sulfuric acid, shake while adding, measuring the absorbance at 490 nm after reaction for 30 min at room temperature.

Content of the uronic acid was determined by Carbazole-sulfate method [29]. In brief, galacturonic acid standard solution was configured in the same way, then 6 mL of superior pure sulfuric acid was added in an ice bath, shaking while adding, water bath at 85 ◦C for 25 min, taken out and cooled to room temperature, 0.2 mL of 0.1% carbazole-ethanol (25 mg carbazole dissolved in 25 mL ethanol) was added, and the reaction was carried out for 2 h. The absorbance was measured at 530 nm.

Content of protein was measured with Coomassie brilliant blue method [30]. Configure 0.1 mg/mL of BSA standard solution, and Komas brilliant blue solution (100 mg of Komas brilliant blue dissolved in 50 mL of 90% ethanol, add 100 mL of 85% phosphoric acid, distilled water volume to 1000 mL), 5 mL of Komas brilliant blue was added to the standard solution and CYP solution, plug the cap and invert 3–5 times, measure the absorbance at 595 nm.

#### 2.3.2. Determination of Molecular Weight (Mw)

Molecular weight (Mw) was evaluated on a high-performance liquid chromatography system with an ultraviolet absorption and refractive index detector.

The chromatographic column of UltrahydrogelTM-1000 was eluted with ultrapure water at a flow rate of 0.6 mL/min. The column and RI detector were maintained at 35 ◦C. Glucose and dextrans (T-10, T-25, T-40, T-50, T-70, and T-500) were used as standards. We prepared 1 mg/mL CYP solution and added the polysaccharide solution to the injection vial by using a syringe combined with a 0.22 μm aqueous-phase needle filter for determination.

#### 2.3.3. Monosaccharide Composition

The monosaccharide composition of CYP was determined based on the method of Xu et al. [31] with few changes. CYP (5 ± 0.05 mg) was put into a tube, and 0.5 mL of 12 M H2SO4 was added in an ice bath with magnetic stirring for 0.5 h to dissolve it completely. After 2 mL of ultrapure water was added, tubes were placed in an oil bath at 105 ◦C for 4 h. After heating, polysaccharide solution was fixed the volume with a 250 mL volume volumetric flask, 1 mL of sample was taken out and was filtered through 0.22 μm membrane for HPAEC analysis. Water and sodium hydroxide were used as the eluent and at a flow rate of 0.25 mL/min.

#### 2.3.4. Scanning Electron Microscope (SEM) Analysis

CYP micromorphology was observed by SEM. In a typical procedure, polysaccharide samples were placed on a conductive plate and held in place. After CYP was sprayed with gold powder, SEM was used to record images at different magnifications (500–2000 times) at an acceleration voltage of 5 kV [32].
