IOP was extracted as previously described (10). Briefly, Inonotus obliquus (IO) was crushed, and fat was removed using petroleum ether, followed by traditional hot water extraction (4, 30). The above process was repeated three times before adding 1% trichloroacetic acid for deproteinization. The extract was then collected and concentrated, and three volumes of absolute ethanol were added, followed by overnight incubation at 0°C. The precipitate was centrifuged to obtain the crude extract, which was washed 2–3 times with absolute ethanol. To purify the crude polysaccharide, first, DEAE-52 cellulose was soaked in 0.5 mol/L hydrochloric acid for 1 h. Then, impurities in the resin were washed away bay 0.5 mol/L hydrochloric acid, and the resin was eluted with distilled water, 4–5 times its volume, until it reached a neutral pH. Finally, the resin was buffer with distilled water at a flow rate of 5 mL/min for 2 h. Afterwards, the sample was prepared for loading. The crude polysaccharide was dissolved in distilled water, heated, and shaken. The mixture was centrifuged at 12,000 rpm for 2 min, and the supernatant was collected for loading. Subsequently, 2% sodium nitrite solution was added to the polysaccharides at a ratio of 1:2 (polysaccharides: 2% sodium nitrite solution), and the mixture was heated in a water bath at 80°C. Activated carbon was added, and the mixture was stirred, heated for 1 h, and incubated at 4°C overnight. Finally, the supernatant was centrifuged, and the solution was freeze-dried to obtain IOP.

First, a standard curve was plotted. Here, 1 mg of standard dextran (Yuanye Bio-Technology Co., Ltd., Shanghai, China) was weighed into a 1 mL volumetric flask. The flask was then topped up to 1 litre. Standard dextran solutions of 8 mg/mL, 4 mg/mL, 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, and 0.125 mg/mL, each 1 mL, were then prepared. Thereafter, 200 μL of 6% phenol (Komio Chemical Reagents Co., Ltd., Tianjin, China) and 1 mL of concentrated sulfuric acid (Hushi Laboratorial Equipment Co., Ltd., Shanghai, China) were added. The mixture was left to stand for 10 min, shaken well, and allowed to stand further at room temperature for 20 min. The absorbance was measured at 490 nm. Distilled water used as a blank and the same colorimetric procedure was followed. A standard curve of polysaccharide concentration (x-axis) and absorbance (y-axis) was then plotted.

The sample content was then determined. Distilled water (200 μL) was added into 1 mg of IOP obtained in section 2.1. The absorbance was measured in triplicate as described in the preceding section. The polysaccharide content was calculated using the standard curve.

The molecular weight of IOP was determined using the high-performance gel permeation chromatography (HPGPC) method. First, the mobile phase solution was prepared by dissolving 11.688 g of NaCl in purified water and transferring it to a 1 L volumetric flask. After sonication for 10 min, the mixture was filtered through a 0.22 μm pore membrane. The standard solution was then prepared. Eight dextran standard samples (2 mg each) were weighed and dissolved in 1 mL of the mobile phase solution to prepare a 2 mg/mL solution. The solution was transferred to 1.8 mL sample vials. The sample (2 mg) was dissolved in 1 mL of the mobile phase solution, sonicated for 10 min, and centrifuged at 12000 rpm for 10 min. The supernatant was filtered through a 0.22 μm membrane filter and transferred to a 1.8 mL sample vial. Finally, the samples were then analyzed. In summary, the standard samples were placed in the sample tray for chromatographic analysis under the following experimental conditions; mobile phase: 0.2 M NaCl solution; chromatographic column: BRT105-103-101 gel permeation column (8× 300 mm); flow rate: 0.8 mL/min; column temperature: 40°C; injection volume: 25 μL; detector: refractive index detector (RID-10A); analysis time: 60 min. The retention time was obtained and the standard curves of lgMp-RT (Mp peak molecular weight), lgMw-RT (Mw average molecular weight), and lgMn-RT (Mn number-average molecular weight) were plotted. The retention time of the sample was used to calculate the molecular weight (Mp, Mw, and Mn).

Here, 600ul of D₂O reagent was added to 100 mg IOP in a Nuclear Magnetic Resonance (NMR) tube. After dissolving the IOP, ID (¹H and ¹³C) and 2D (HSQC, HMBC, COSY) spectra were measured using a 600 MHz Bruker (Germany). A zg30 pulse sequence was used for ¹H, and the sample was scanned 32 times. A zgpg30 pulse sequence was used at ¹³C, and the test scans were performed 1,024 times. The Cosygpppqf, noesygpphpp and Hsqcedetgp pulse sequences were used for the two-dimensional spectra, and four test scans were performed.

LS411N cells were cultured to prepare a single cells’ suspension. After culturing, the culture medium was poured out, and the residual culture medium was washed using PBS. The cell monolayer was separated into single cells using 0.25% trypsin for 1 min. The digestion was terminated by adding a culture medium containing 10% FBS. The cell suspension was poured into a 15 mL centrifuge tube and centrifuged at 300–400 × g for 5 min at 4°C. The supernatant was discarded, and the cell pellet was washed two times with PBS through centrifugation at 300–400 × g for 5 min at room temperature. Then the cells were resuspended in flow loading buffer for counting and viability determination. The cell density was adjusted to 1 × 10⁷ cells /mL.

Flow cytometry experiment was then performed. First, 1–3 × 10⁶ cells were collected, washed twice with precooled PBS, centrifuged at 300 g for 5 min (model DM0412S, SCILOGEX, United States), and the supernatant was discarded. A few cells were picked from the sample tube and one blank tube to set up two single-staining tubes. Cells were washed once with precooled PBS, the supernatant was discarded after centrifugation, and the cells were resuspended in 500 μL of precooled 1× Binding Buffer working solution. Next, 5 μL of Annexin V-FITC (United Biology, Hangzhou, China) and 10 μL of PI staining solution were added to each sample tube, and 5 μL Annexin V-FITC or 10 μL PI was added to the two single-staining tubes, vortexed gently, mixed, and incubated at room temperature in the dark for 5 min. Flow cytometric assessment was then performed (model CytoFLEX, Beckman coulter, United States), and the voltage of FSC, SSC, FITC and PI channels was adjusted using a blank tube. Under this voltage, the compensation between FITC and PI was adjusted with a single staining tube before loading the sample. IOP of 100 μg/mL and 200 μg/mL were used to detect the apoptosis rate experiment.

Forty-eight Sprague Dawley (SD) rats (female: 150 ± 10 g, male: 180 ± 10 g) were purchased from the Chengdu Dashuo Experimental Animal Co., Ltd. (Chengdu, China). The rats were housed under constant conditions (temperature 25 ± 3°C, humidity 75 ± 5%), were provided with enough food and water, and were reared at 12 h light and 12 h darkness cycle. All laboratory procedures, including those related to animal handling, welfare and euthanasia, were carried out in accordance with the guidelines and regulations of ARRIVE, and the protocol for the animal studies study was approved by the Animal Care Office of Chengdu Normal University (No: CDNU-2021092614 M). After 7 days of acclimation, the mice were randomly divided into the following four groups (n = 12): control-female group, control-male group, IOP-female group and IOP-male group. The IOP group received 100 mg/kg by gavage (31), while the control group received the same proportion of normal saline. After 4 weeks of treatment, the rats were anaesthetized with ether and sacrificed by neck dislocation. Cecal contents were collected in a sterile environment and stored in a refrigerator at −80°C after quick freezing in liquid nitrogen.

Whole blood was centrifuged at 3000 g for 10 min to obtain serum. Serum testosterone (T) and estradiol (E2) levels were measured using an ELISA kit (Hepeng Biotechnology Co., Ltd., Shanghai, China), according to the manufacturer’s instructions.

Numerical results of normally distributed variables were expressed as mean ± standard deviation. Data were analyzed using IBM SPSS Statistics v26.0 and GraphPad Prism 8 (GraphPad InStat Software, United States) software. α diversity analysis was analyzed using t-test, the linear discriminant analysis effect size (LEfSe) analysis initially employed the Kruskal–Wallis rank sum test to identify species exhibiting significant differences in abundance across different groups. Subsequently, the Wilcoxon rank sum test was utilized to assess intergroup disparities. Finally, linear discriminant analysis (LDA) was applied for data reduction and evaluation of the impact of significantly distinct species (LDA score). The remaining analyses were conducted using one-way analysis of variance (ANOVA). A significance level of p < 0.05 was considered statistically significant.

Crude polysaccharides were purified and a gradient elution curve was obtained according to the method described in section 2.1 (Figure 1A). Four distinct elution peaks which correspond to elution with distilled water, 0.2 M NaCl, 0.5 M NaCl, and 1.0 M NaCl solutions were obtained. Elution with distilled water generated the highest polysaccharide content. Therefore, the polysaccharide fraction obtained from the first elution with distilled water was collected, freeze-dried, and used in subsequent analyses.

The total sugar content of IOP is shown in Figure 1B. The regression equation for the relationship between absorbance and polysaccharide concentration was y = 0.09021 + 0.48593x (where x represents the concentration of dextran in mg/mL and y represents the absorbance value at 490 nm for various concentrations of glucose). The coefficient of determination (R²) was 0.99572. By substituting the absorbance values of IOP measured at 490 nm into the dextran standard equation, the total sugar content of IOP was determined to be 96.2%, implying that a highly pure IOP was obtained.

As shown in Table 1 and Figure 2 the peak molecular weight (Mp) at the peak apex was 4,489 Da. The average molecular weight (Mn), calculated by considering the number of molecules weighted by their abundance, was 4,549 Da. The average molecular weight (Mw), which was calculated by considering the mass of the molecules weighted by their abundance, was 4,828 Da. The polydispersity coefficient (PD), which represents the dispersion of the substance, and is the ratio of Mw to Mn (PD = Mw/Mn), was 1.061. Generally, a PD value below 1.2 indicates a narrow dispersion. Thus, the polymer had a relatively uniform dispersion.

In the present study, the IOP structure was analyzed based on 1D (¹H and ¹³C) NMR and 2D (HSQC, HMBC, COSY) NMR spectra. The ¹³C NMR and HSQC results (Figures 3A,C) revealed no obvious chemical shift signal of uronic acid within δ160–190 ppm, which demonstrated that IOP was a neutral substance. From the hydrogen spectrum of NMR (Figure 3B), it was found that the ¹H NMR spectrum of IOP had a significant hydrogen signal in the range of 4.3–6.0 abnormal hydrogen, which is at δ5.29 ppm. Based on the NMR spectrum analysis of the polysaccharide material, the 2D HSQC (Figure 3C) revealed residual sugar in α configuration, which was confirmed by correlation signal with anechoic carbon at 5.29/99.37. From the overall analysis of the hydrogen and carbon spectra, the main IOP structure is composed of a six-carbon pyopyrum residual sugar of α configuration. Considering the extremely weak signal at other positions, the nuclear magnetic situation of the residual sugar of 5.29 ppm hetero-hydrogen, which is the majority, is discussed here. Preliminary findings showed that the signal is generated by the Glc glucose residue.

The chemical shifts at each position of the above major monosaccharide residues were identified by analyzing the 2D NMR spectra of HSQC (Figure 3C), HMBC (Figure 3D), and ¹H-¹H COSY (Figure 3E) in combination with the ¹H and ¹³C NMR spectra. It is worth noting that the chemical shift of the anomer hydrogen and anomer carbon signals of the monosaccharide glucose residues is δ5.29/99.37 ppm. In the ¹H-¹H COSY spectrum, a correlation signal was found at δ5.29/3.51 ppm, validated by the H1/H2 signal in the residual sugar. The relevant information of δ3.53/76.57 ppm can be found (Figure 3E). The C2 chemical shift of this monosaccharide residue was 76.57 ppm. Using the same method, the chemical shift of H3 related to H2 was determined by ¹H-¹H COSY spectrum to be 3.51/3.72 ppm (Figure 3C), and the chemical shift of C3 corresponding to H3 in HSQC was found to be 71.30 ppm. The chemical shifts of H4/C4, H5/C5 and H6/C6 of the main monosaccharide residues were 3.29/69.31, 3.56/72.97 and 3.75/60.30, respectively. According to the HMBC spectrum (Figure 3D), there was a strong interference signal (3.75/99.80) at the position 99.37 of abnormal carbon and H-6 (3.75 ppm), which confirmed that the connection between the abnormal position of glucose and the C-6 position of IOP residue was the main component of IOP. The above results confirmed that the backbone of the IOP molecule is a linear structure composed of α-D-type glucose connected by the 1 → 6 position. The structure of the IOP backbone is shown in Figure 3F.

The effect of IOP on the apoptosis of LS411N cells was determined by flow cytometry (Figure 4). IOP increased the apoptosis rate of LS411N cells (Figure 4D, p < 0.01) in a concentration-dependent manner (Figure 4A for control: 10.96%; Figure 4B for 100 μg/mL IOP: 38.49%; Figure 4C for 200 μg/mL IOP: 46.19%). These results indicate that a low dose of IOP is not toxic to the body. At the same time, IOP can promote the apoptosis of cecum cancer cells (LS411N cells) in a dose-dependent manner.

IOP treatment decreased the secretion of E2 in female rats but significantly increased the secretion of this hormone in male rats (Figure 5A). Meanwhile, IOP treatment decreased the secretion of T in both female and male rats, though not significantly (Figure 5B).

The raw data obtained from high-throughput sequencing ranged from 78,105 to 80,586, and the volume of valid tag data after removing chimeras was distributed between 466 and 671. Each group shared 245 ASVs (Figure 6A). The degree of diversity in each group was analyzed using α diversity, whereas the Shannon and Simpson indices were measured using the t-test. The Shannon and Simpson index results showed that the α diversity of males and females in the control groups was significantly different. The Shannon index of females in control and IOP groups was also significantly different. The results generally indicated that the diversity of caecal flora was higher in male than in female rats. However, IOP intake abrogated the difference (Figures 6C,D). Furthermore, β diversity analysis using PCoA plots revealed significant differences in cecal microbiota composition between male and female rats in the control group. This indicates that a shift was observed in the cecal microbiota after IOP intake. Generally, the caecal flora of male and female rats has a certain similarity (Figure 6B).

IOP intake significantly increased the abundance of members in the Lactobacillus (p < 0.001), Roseburia (p < 0.01), and Clostridia_UCG-014 (p < 0.05) genera in female rat. However, no significant differences were observed at the phylum level in the female rats (Figure 7A). In contrast, at the phylum level, the abundance of Desulfobacterota (p < 0.001) decreased significantly in male rats. The abundance of members in the Prevotella (p < 0.05), Prevotellaceae_NK3B31_group (p < 0.05), Alistipes (p < 0.05) and Clostridia_UCG-014 (p < 0.05) genus increased significantly in male rats. However, the abundance of Alloprevotella (p < 0.05), Colidextribacter (p < 0.001), and Oscillibacter (p < 0.05) decreased significantly (Figure 7B).

At the phylum level, the abundance of Desulfobacterota (p < 0.001), Campilobacterota (p < 0.05), Proteobacteria (p < 0.01), and Actinobacteriota (p < 0.01) was significantly higher in male than female rats in the control group. The abundance of Bacteroidota (p < 0.05) was significantly lower in male rats than in female rats. At the genus level top15, the abundance of members in Helicobacter (p < 0.05), Colidextribacter (p < 0.001), and Oscillibacter (p < 0.05) general was significantly higher in male than in female rats. The abundance of Prevotella (p < 0.001) was significantly lower in male than in female rats (Figure 7C).

In the IOP group, the abundance of Proteobacteria (p < 0.05) was significantly higher in the top 10 male rats at the phylum level than in the female rats. In the top15 genus level, the abundance of Alistipes (p < 0.01) was significantly higher than in the female rats, while the abundance of Lactobacillus (p < 0.001) and Roseburia (p < 0.01) was significantly lower than in the female rats (Figure 7D).

LEfSe analysis showed that the abundance and composition of 72 bacterial species were significantly different among the four groups. The dominant bacteria in the control-female group were Prevotella, Oxalobacter, etc. The dominant bacteria in control-male group were Proteobacteria, Colidextribacter, Oceanisphaera, Lgnatzschineria, Bilophila, Dubosiella, Romboutsia, and Peptococcus, Butyricimonas, Anaerofilum, Jeotgalicoccus, Odoribacter, Corynebacterium, Atopostipes, Sporosarcina, Turicibacter, and Harryflintia among others. The dominant bacteria in the IOP-female group were Roseburia, Lactobacillus and Clostridia_UCG_014. The dominant bacteria in the IOP-male group were Prevotellaceae_NK3B31_group, Ruminococcus, Eubacterium_xylanophilum_group, Alistipes, Desulfovibrio and Escherichia a_Shigella, Lachnoclostridium, Lachnospiraceae_UCG_006, Oligella, and Parasutterella among others (Figure 8).