The phenol–sulphuric acid method was used to measure the total sugar content in GP and GP-1, using glucose as the standard reference (24). The Bradford method was employed to measure protein content, using bovine serum albumin as the standard (19). The m-hydroxydiphenyl method, with galacturonic acid as the standard, was used to measure the glucuronide content of GP-1 (25).

High-performance gel permeation chromatography (HPGPC) was used to determine the homogeneity and molecular weight (Mw) of GP-1 (10, 26). The chromatographic setup included a sequence of BRT105-104-102 gel columns, each measuring 8 mm by 300 mm. The mobile phase used was a 0.05 M NaCl solution, with a flow rate of 0.6 mL per minute and a column temperature maintained at 40 °C. A 20 μL sample injection volume was used, and detection was carried out with a differential detection technique. The relative molecular weights of GP-1 (5,000, 11,600, 23,800, 48,600, 80,900, 148,000, 273,000, 409,800, and 667,800) were calculated using the standard dextran curve. 2.3.3. Analysis of monosaccharide composition.

To verify the monosaccharide composition of GP-1, high-performance liquid chromatography (HPLC) was utilized, with precolumn derivatization using 1-phenyl-3-methyl-5-pyrazolone (PMP) as outlined in an earlier study Sun et al. (27). Around 2 mg of polysaccharide was fully broken down with 1.0 mL of 2 M trifluoroacetic acid (TFA) at 100 °C over a period of 10 h. Following the removal of TFA using vacuum rotary evaporation, the dried residue was dissolved in 300 μL of distilled water and underwent PMP derivatisation. The derivatised samples underwent three extractions with 600 μL of chloroform and were subsequently analyzed using an HPLC system featuring a Hedra C18 column (5 μm, 4.6 mm × 250 mm) linked to an Agilent 1,100 system (Agilent, Santa Clara, CA, USA). Detailed experimental procedures are provided in Supplementary material S2.

To identify the glycosidic linkages in GP-1, we used a methylation analysis method that was slightly modified from a reported procedure (28, 29). We transferred 3 mg of GP-1 to a glass reaction vial and added 1 mL of anhydrous dimethylsulfoxide (DMSO). Methylating reagent A (anhydrous alkaline solution) was rapidly added, and the vial was sealed. The mixture was then subjected to ultrasonic dissolution, followed by the addition of methylating reagent B, an iodomethane solution. The reaction was carried out at 30 °C in a magnetically stirred water bath for a duration of 60 min. To terminate the methylation reaction, 2 mL of ultrapure water was added. The methylated polysaccharide was subjected to hydrolysis using 1 mL of 2 M trifluoroacetic acid (TFA) and incubated for 90 min, after which it was dried. The resulting residue was dissolved in 2 mL of ultrapure water and subsequently reduced with 60 mg of sodium borohydride for a duration of 8 h. The reaction mixture was neutralized with ice-cold acetic acid, and the product was concentrated through rotary evaporation prior to being dried at 101 °C. Subsequently, 1 mL of acetic anhydride was added, and the reaction was carried out at 100 °C for 1 h, followed by a cooling phase. Following the addition of 3 mL of toluene, the mixture underwent concentration under reduced pressure, a procedure that was repeated four to five times to eliminate excess acetic anhydride. Subsequently, the acetylated product was dissolved in 3 mL of dichloromethane (CH2Cl2), transferred to a separatory funnel, and subjected to vigorous agitation with a minimal volume of distilled water. The upper aqueous phase was discarded, and this extraction process was repeated four times. The dichloromethane layer was then dried using an appropriate amount of anhydrous sodium sulfate, and its volume was adjusted to 10 mL. The acetate esters of the methylated sugar alcohols were analysed using a GC–MS system (GC–MS-QP2010).

GC–MS conditions: RXI-5 SIL MS column (30 mm × 0.25 mm, 0.25 μm); temperature program began at 120 °C, ramped at 3 °C/min up to 250 °C, where it was held for 5 min; injection port and detector temperatures were set to 250 °C and helium was used as the carrier gas at a flow rate of 1 mL/min.

GP-1 (1 mg) was finely ground with dried KBr and pressed into a pellet. The IR spectrum was recorded using a Nicolet FT-IR spectrometer (Nicolet IR100, Thermo Scientific, USA) in the wavelength range of 4,000–400 cm⁻¹ (30).

We dissolved 20 mg of GP-1 in 0.5 mL of deuterium oxide and subjected it to freeze-drying. This procedure was repeated three times to guarantee adequate exchange of labile hydrogen. The GP-1 sample was then dissolved in 0.5 mL of heavy water and analysed using 1D and 2D NMR spectroscopy using a 600 MHz NMR (AAVANCE NEO 600 M, Bruker, Germany).

The Congo red staining technique was employed to characterize the triple-helix structure of GP-1 (31). The polysaccharides were dissolved in distilled water at a concentration of 1 mg/mL, together with a 100 μM Congo red solution. The polysaccharide solution was combined in equal parts with Congo red reagent, and then the NaOH concentration of the mixture was adjusted using a 1 M NaOH solution within the range of 0–0.5 M. Spectral scanning in the 400–900 nm range was used to find the maximum absorption wavelength of the samples at various NaOH concentrations (32).

SEM was used to examine the morphology of GP-1. The sample was placed on a copper stub and covered with a thin gold layer through sputter coating before analysis at 2.0 kV (33).

Male Wistar rats, weighing between 180 and 220 grams and aged eight weeks, were obtained from Shanghai Shilaike Laboratory Animal Co., Ltd. in Shanghai, China, and kept at the Nanjing University of Chinese Medicine’s animal facility. The animals were kept at room temperature (20 °C–25 °C) with constant relative humidity under a 12-h light/dark cycle. Prior to the experiment, the rats were allowed to adjust to the lab environment for a week, with unlimited access to food and water. All experimental protocols and procedures were approved by the Experimental Animal Ethics Committee of the Nanjing University of Chinese Medicine (ethics approval number: 202207A019). Furthermore, the animal studies followed the guidelines set by the National Research Council’s Guide for the Care and Use of Laboratory Animals.

The rats were randomly divided into control (CON), model (MOD), low-dose GP (0.84 g/kg) and high-dose GP (3.36 g/kg) groups. All rats except those in the CON group were placed in a glass cylinder (diameter = 31 cm and depth = 48 cm) maintained at 37 °C ± 1 °C (the tank diameter and water depth were adjusted visually according to the size of the rat) and forced to swim until exhaustion. They were given 75 g/kg body weight of feed every other day to induce an immunocompromised state (23). After two weeks, the rats in the CON group were given sterile physiological saline through intragastric feeding once a day for two weeks. Meanwhile, the rats in the other groups were administered oral GP daily for the same duration.

The rats were anesthetized with sodium pentobarbital through an intraperitoneal injection 24 h after the last dose. Blood was drawn from the abdominal aorta, and the rats were euthanized. Intestinal tissues and feces were gathered.

The ileum tissue was preserved in 10% neutral formalin, set in paraffin, and then stained using haematoxylin and eosin (H&E). For histopathological analysis, microscopic images were taken with a 100 × objective lens to examine the length and spacing of intestinal villi.

Blood samples were centrifuged at 4 °C for 10 min at 3,500 × g to isolate the sera. The levels of cytokines (IL-2, IL-4, TNF-α and IFN-γ) and immunoglobulins (IgA and IgG) in the sera were quantified using an ELISA kit provided by Nanjing Lapuda Biotechnology Co., Ltd. (China).

The GPC chromatogram exhibited a relatively uniform and symmetric peak, indicating that GP-1 is a highly pure and homogenous polysaccharide (Figure 1C). The GPC analysis revealed that the Mw of GP-1 was 7.6 kDa. Its relatively low molecular weight endows it with superior water solubility and dispersion performance. Comparison with the monosaccharide standard confirmed that GP-1 is composed of glucose (Figures 2A,B). Neutral polysaccharides from G. uralensis are primarily composed of glucans, as described in a previous study (12). However, significant differences in Mw were observed, which can be attributed to variations in the raw materials and processing methods used.

Methylation analysis is widely used to determine polysaccharide structure. The method involves converting the individual constituent sugars in polysaccharides into partially methylated alditol acetates, which are then analysed and quantified by GC–MS. The proposed method enables the identification of attachment sites and molar ratios of different sugar residues. The results of the methylation analysis are presented in Table 1 and Figure 2C. Based on the GC–MS spectral database (Agilent), retention times of each peak, main characteristic fragments and published spectral analysis results (30, 34), we identified five different glycosidic bonds in GP-1, T-Glcp-(1→, →4)-Glcp-(1→, →6-Glcp-(1→, →3,4)-Glcp-(1 → and →4,6)-Glcp- (1→, with molar ratio of 0.150:0.733: 0.024:0.051:0.041.

As shown in Figure 3A, the FT-IR spectrum of GP-1 exhibited a broad characteristic absorption peak in the range of 3,500–3,000 cm − 1, which can be attributed to the stretching vibration of the O–H bonds in polysaccharides (35). The absorption peak at 2931.97 cm − 1 corresponded to the stretching vibration of the C–H bonds in polysaccharides (36). The absorption peak at approximately 1,100 cm − 1 can be attributed to the stretching vibration of the C–O bonds, indicating the presence of functional groups in the polysaccharide (37). The peak at 1637.76 cm − 1 indicated the presence of free hydroxyl groups in bound water (31). Additionally, the absorption peaks at 1024.17, 1079.23 and 1152.86 cm − 1 indicated the presence of pyranose sugars in the polysaccharide composition of GP-1 (38).

To determine the linkage sequence of each sugar residue in GP-1, we performed 1D and 2D NMR analyses. The proton signals in the ¹H NMR spectrum were primarily concentrated between δ_H 3.0 and 5.5 ppm, with the signals corresponding to the sugar rings appearing in the range of δ_H 3.4–4.0 ppm (Figure 3B). The signal peaks of the main terminal protons at δ_H 5.43, 5.37, 5.36, 5.00 and 4.53 were predominantly distributed in the range of δ_H 4.3–5.5 ppm. In the ¹³C NMR and heteronuclear single quantum coherence (Figures 3C,D) spectra, the carbon signals at δ_C 99.55, 99.59, 99.71, 98.60 and 96.75 ppm were mainly located in the range of δ_C 95–105 ppm. The signal peaks at δ_C 71.51, 73.32, 76.66, 71.15, 60.45, 71.46, 74.11, 73.99, 72.69, 68.47, 71.71, 73.32, 69.30, 72.44, 62.44, 72.44, 74.52, 70.33, 72.85, 66.36, 76.18, 75.74, 70.16, 75.93 and 60.66 ppm were primarily distributed in the range of δ_C 60–85 ppm.

Based on the ¹³C NMR and HSQC spectra (Figure 3E), GP-1 primarily exhibited five continuous oxygenated carbon signals at δ_C 71.51 (CH), 73.32 (CH), 76.66 (CH), 71.15 (CH) and 60.45 (CH₂) and a terminal carbon signal at 99.55 ppm (CH). The anomeric hydrogen signal of β-glycosidically bonded configurations was mainly distributed in the range of δ 4.4–4.8 ppm, whereas that of α-glycosidically bonded configurations primarily appeared in the range of δ 4.8–5.8 ppm (31). The chemical shift of H-1 at 5.43 indicates the presence of an α-glycosidic bond. The heteronuclear multiple bond correlation (HMBC) spectra revealed correlation signals between H-1 and C-4, as well as between H-4 and C-1, indicating that the glycosidic bond in the neutral polysaccharide was linked at the α-(1,4) position. By correlating the results of methylation with those of relevant literature (12, 13), 1,4-Glc was identified as the GP-1 main chain, and the primary connection of the main chain was a: →4)-α-D-Glcp-(1→. The glycosidic bond signalling of GP-1, considering its spatial structure, was further corroborated by correlating the methylation data and HSQC, HMBC, correlation spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY) spectra, as well as extensive literature reports (12, 30, 31, 34). These findings are summarized in Table 2 and Figures 3E–H.

All the glycosidic bonds were in the α-conformation except for c: β-D-Glcp-1→, which exhibited an isohead carbon signal at 4.53 and adopted a β-conformation. In the HMBC spectrum, a correlation peak was observed between C-4 of the b: →4,6)-α-D-Glcp-(1 → glycosidic bond and H-1 of the a: →4)-α-D-Glcp-(1 → glycosidic bond, indicating the presence of the →4)-α-D-Glcp-(1 → 4,6)-α-D- Glcp-(1 → glycosidic bond. Similarly, a correlation peak was observed between C-4 of the e: →3,4)-α-D-Glcp-(1 → glycosidic bond and H-1 of the a: →4)-α-D- Glcp-(1 → glycosidic bond, indicating the presence of the →4)-α-D-Glcp-(1 → 3,4)- α-D-Glcp-(1 → glycosidic bond. Furthermore, a correlation peak was detected between C-1 of the c: β-D-Glcp-1 → glycosidic bond and H-6 of the d: →6)-α-D- Glcp-(1 → glycosidic bond, indicating the presence of the β-D-Glcp-(1 → 6)-α-D- Glcp-(1 → glycosidic bond, as shown in Figure 3F.

According to the NOESY spectrum, a correlation peak was observed between H-1 of the b: →4,6)-α-D-Glcp-(1 → glycosidic bond and H-4 of the e: →3,4)-α-D-Glcp-(1 → glycosidic bond, indicating the presence of the →4,6)-α-D-Glcp-(1 → 3,4)-α-D- Glcp-(1 → glycosidic bond. Another correlation peak was detected between H-3 of the e: →3,4)-α-D-Glcp-(1 → glycosidic bond and H-1 of the c: β-D-Glcp-1 → glycosidic bond, indicating the presence of the β-D-Glcp-1 → 3,4)-α-D-Glcp-(1 → glycosidic bond. In addition, the correlation signal between H-6 of the b: →4,6)-α-D-Glcp-(1 → glycosidic bond and H-1 of the d: →6)-α-D-Glcp-(1 → glycosidic bond confirmed the presence of the →6)-α-D-Glcp-(1 → 4,6)-α-D -Glcp-(1 → glycosidic bond, as shown in Figure 3H.

Based on these results, we propose that the primary glycosidic bond structure of GP-1 is characterized by a main chain linkage of →4)-α-D-Glcp-(1→, with β-D-Glcp-(1 → 6)-α-D-Glcp-(1 → substitution at the C-6 position of 1,4,6-Glc and β-D-Glcp-(1 → substitutions at the C-3 position of 1,3,4-Glc, as shown in Figure 3I.

α-(1 → 4) Linkage is a typical connection method for certain degradable energy-storing polysaccharides. This type of linkage is more susceptible to enzymatic hydrolysis, which generates oligosaccharide fragments with β-glycosidic bonds that are more readily metabolized by microorganisms. The presence of β-(1 → 6)-side chains enhances the molecular’s three-dimensional conformational flexibility and high branching, enabling it to form a larger contact area when binding to receptors. This increases the likelihood of multi-site interactions, thereby strengthening its binding affinity with immune-related active centers. Additionally, the C-3 β-Glcp side chain alters the stereochemical environment of the main chain, facilitating the formation of unique spatial conformations that improve specific biological activities. These mechanisms contribute to functions such as inhibiting pathogenic viral infections or enhancing the efficiency of host defense signaling transduction (39, 40).

The results of the Congo red experiment are shown in Figure 4A. At low NaOH concentrations, the maximum absorption wavelength of the Congo red-stained polysaccharide solution exhibited a distinct red shift. However, when the NaOH concentration exceeded 0.1 mol/L, a decreasing trend was observed at the maximum absorption wavelength, with no indication of a stable phase throughout the process. The maximum absorption wavelength of the Congo red-polysaccharide solution may not have decreased with increasing NaOH concentration if light protection was not applied during the experiment. As the NaOH concentration increased, the triple-helix structure of the Congo red-GP-1 complex became disrupted, reducing the maximum absorption wavelength. Literature indicates that at low alkali concentrations, a pronounced red shift is typically observed, which can be attributed to the presence of the triple helix, even in the absence of a stabilization zone in the absorption curve (31, 41). Based on these observations, we hypothesized that GP-1 possesses a triple-helix structure.

SEM is a qualitative technique for observing the surface morphology of polysaccharides. As shown in Figure 4B, GP-1 exhibited a smooth reticulated structure composed of many spherical particles stacked on one another, indicating strong intermolecular interactions and tight binding within the sample (30). This result was consistent with its low molecular weight (15).