Hypocrella bambusae homogeneous polysaccharide HBPs as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine preparation, and discloses a hypocrella bambusae homogeneous polysaccharide HBPs, which is prepared from the following components in percentage by mass: 52.501% of D-glucose, 34.367% of D-mannose, 12.677% of D-galactose and 0.455% of N-acetyl-D-glucosamine, and the weight-average molecular weight of the hypocrella bambusae homogeneous polysaccharide HBPs is 23.545 KDa. The components of the hypocrella bambusae homogeneous polysaccharide are fixed, the yield is relatively high, the hypocrella bambusae homogeneous polysaccharide has the effect of improving immunity, the safety is good, and a basis is provided for developing a potential new drug for enhancing the immune function of intestinal mucosa.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparation technology, and relates to a homogeneous polysaccharide HBPs of *Russula ovata*, its preparation method and application. Background Technology

[0002] Immunodeficiency is characterized by an abnormal state in which the immune system exhibits impaired or absent function in response to antigenic stimulation. Consequently, individuals become susceptible to bacterial, viral, and fungal infections, which can lead to severe clinical manifestations such as respiratory infections, urinary tract infections, sepsis, and meningitis. Immunosuppression commonly occurs after bacterial, viral, or fungal infections, after organ transplantation, during severe illness, or during immunosuppressive therapy. Patients undergoing treatment for malignant tumors or organ transplantation often receive immunosuppressive therapy. Commonly used antitumor drugs, such as cyclophosphamide (CY), can significantly impair immune responses, disrupt IMB function, and cause dysbiosis within the GM. Immunomodulators like levamisole hydrochloride and thymosin can also cause adverse reactions, including fever, vascularization, allergic reactions, and neutropenia. In severe cases, these reactions can be fatal. Therefore, the development of safe, effective, and natural immunomodulators is crucial for maintaining immune homeostasis and IMB integrity.

[0003] Hypocrella banbusae (Berk. et Br.) Sacc., a fungus belonging to the subgenus Mycelium, is distributed in northwestern Yunnan and southeastern Tibet, growing in subalpine and alpine fir forests at altitudes of 2600 to 3500 meters. Traditionally, it has been used in Traditional Chinese Medicine to treat rheumatoid arthritis, gastrointestinal diseases, and fungal skin diseases. Although Hypocrella banbusae is rich in chemical components and several extracts (A, B, and C) have been identified, its main active polysaccharide components remain poorly understood. Studies have shown that polysaccharides extracted from Rhododendron simsii possess immunomodulatory activity. Preliminary studies indicate that polysaccharides from Rhododendron amurense can modulate intestinal immunity, suggesting potential probiotic activity. However, it remains unclear whether the polysaccharides of Hypocrella banbusae can alleviate cyclophosphamide (Cy)-induced immunosuppression and intestinal mucosal damage by modulating the gut microbiota and metabolism. Furthermore, the composition and activity of bamboo red fungus polysaccharides obtained by different extraction methods vary considerably, which limits their application in actual production. Summary of the Invention

[0004] This invention addresses the technical problem of the varying polysaccharide composition of *Russula baicalensis* affecting its application by providing a homogeneous polysaccharide HBPs from *Russula baicalensis*, its preparation method, and its application. The prepared homogeneous polysaccharide has a fixed composition and a high yield, enhances immunity, and has good safety, providing a basis for developing potential new drugs to enhance intestinal mucosal immune function.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a homogeneous polysaccharide (HBP) from *Russula ovata*, wherein the homogeneous polysaccharide is composed of the following components in mass percentage: D-glucose 52.501%, D-mannose 34.367%, D-galactose 12.677%, and N-acetyl-D-glucosamine 0.455%, and its weight-average molecular weight is 23.545 kDa. Its structural unit has the following structural formula: .

[0006] Secondly, the present invention provides a method for preparing the above-mentioned homogeneous polysaccharide HBPs from *Russula ovata*, comprising the following steps: 1) Boil the crushed bamboo red fungus in water, cool, filter, and obtain bamboo red fungus polysaccharide extract; 2) The polysaccharide extract of *Russula ovata* was subjected to alcohol precipitation to obtain crude polysaccharide precipitated with alcohol; 3) The crude polysaccharide precipitated with alcohol was deproteinized using the Sevag method to obtain total polysaccharide of *Russula basilica*. 4) The total polysaccharides of *Rhododendron simsii* were separated and purified by ion exchange chromatography and gel column chromatography, then concentrated and freeze-dried to obtain homogeneous polysaccharides (HBPs) of *Rhododendron simsii*.

[0007] Preferably, the boiling time in step 1) is 1-2 hours.

[0008] Preferably, the alcohol precipitation in step 2) uses ethanol with a concentration of 80-95%, the precipitation temperature is 4-25 °C, and the time is 2-12 h.

[0009] Preferably, in step 3), when removing protein using the Sevag method, a mixture of chloroform and n-butanol is used for extraction; the volume ratio of chloroform to n-butanol is 3~5:1.

[0010] Preferably, the ion exchange chromatography column in step 4) is DEAE-52 cellulose, the flow rate of the ion exchange column chromatography is 50 mL / min, and the gradient elution program is: 0-40 min water, 0.1 mol / L NaCl 20-40 min, 0.3 mol / L NaCl 15-25 min; the packing material of the gel column is Sephadex-100.

[0011] Thirdly, the present invention provides the application of the above-mentioned homogeneous polysaccharide HBPs from *Russula ovata* in the preparation of drugs that enhance intestinal mucosal immune function.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: The homogeneous polysaccharide HBPs of *Russula ovata* of this invention are novel homogeneous polysaccharide compounds discovered for the first time. They have a fixed composition and a high yield, providing a basis for quality control and standardized production of *Russula ovata* homogeneous polysaccharides. Furthermore, they have an immune-enhancing effect and show no significant toxicity at a concentration of 400 μg / ml, providing a basis for developing potential new drugs to improve intestinal immunity. Attached Figure Description

[0013] Figure 1 The hydrogen spectrum of homogeneous polysaccharide HBPs from *Rhodotorula buergerianum*. Figure 2 The carbon spectrum of homogeneous polysaccharide HBPs from *Rhododendron simsii*. Figure 3 HH-COSY spectrum of homogeneous polysaccharide HBPs from *Rhodotorula buergerianum*; Figure 4 HMBC pattern of homogeneous polysaccharide HBPs from *Rhodotorula buergerianum*; Figure 5 HSQC spectrum of homogeneous polysaccharide HBPs from *Russula ovata*; Figure 6 NOESY spectrum of homogeneous polysaccharide HBPs from *Rhodotorula buergerianum*.

[0014] Figure 7 The effects of *Rhododendron molle* polysaccharide in cy-induced RAW264.7 cells were investigated, including: (A) cell viability; (B) phagocytic activity; (C) scratch assay for cell migration; (D) nitric oxide (NO) production; (E) levels of pro-inflammatory cytokines; and (F) Western blot (WB) analysis showing phosphorylation levels of ERK, p38, JNK, p65, and IκBα.

[0015] Figure 8 The study aimed to improve the effect of *Rhododendron molle* polysaccharide in cytotoxic mice, including: (A) experimental protocol; (B) changes in body weight; (C) spleen index; (D) thymus index; (E) CD4⁺ / CD8⁺ T cell ratio and CD19⁺ B cell percentage as determined by flow cytometry and immunofluorescence; (F–I) serum IgA, IgG, IgM and intestinal SIgA levels; and (J–K) hematoxylin-eosin (H&E) staining results of spleen and thymus tissues.

[0016] Figure 9To investigate the effect of *Rhododendron molle* polysaccharide on the intestinal improvement of cy-induced mice, the following were observed: (A) representative images of colon tissue, disease activity index (DAI) score, and colon length; (B) intestinal H&E staining; (C) histopathological score; (D) intestinal AB-PAS staining; (E) goblet cell quantification; (F) intestinal TNF-α, IL-2, IL-6, and IL-10 levels measured by ELISA; (G) Western blot analysis and quantification results of Occludin and ZO-1 in colon tissue; and (H) immunofluorescence analysis of Occludin, ZO-1, and MUC2 in colon tissue. Detailed Implementation

[0017] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the test methods in the following embodiments are conventional methods.

[0018] Example 1: Extraction of homogeneous polysaccharide HBPs from *Russula ovata* (1) The specific method for extracting total polysaccharides from *Phyllostachys edulis* is as follows: The dried whole plant of *Russula ovata* from Zhaotong, Yunnan Province was collected. 10 kg of *Russula ovata* was chopped, added to 10 times its volume of distilled water, and simmered at 100℃ for 1 hour. After cooling, the mixture was filtered, and the filtrate was collected to obtain a polysaccharide extract. The polysaccharide extract was concentrated using a rotary evaporator to obtain a fluid extract. The fluid extract was dispersed and dissolved in 1 L of distilled water, and 90% ethanol was added. After standing for 24 hours, the mixture was centrifuged to obtain a precipitate. The ethanol was evaporated to obtain crude polysaccharide precipitate.

[0019] The crude polysaccharide was precipitated with alcohol, and then extracted with 1 / 5 volume of distilled water using the Sevag method. The mixture was then added to a chloroform-n-butanol mixture (4:1 volume ratio) and shaken for 20 min. The extract was transferred to a separatory funnel, allowed to stand, and the supernatant was collected and centrifuged for 1 min to remove residual protein precipitate. This process was repeated 3-5 times to obtain a crude polysaccharide solution of *Russula ovata* after protein removal. This solution was concentrated and dried under reduced pressure to obtain 0.4864 kg of total polysaccharide from *Russula ovata*.

[0020] (2) Isolation and purification of homogeneous polysaccharides: A DEAE-52 cellulose column was used, connected to a fraction collector and a peristaltic pump. The total polysaccharides of *Russula baicalensis* were eluted sequentially with distilled water, 0.1 mol / L NaCl, and 0.3 mol / L NaCl at a flow rate of 50 mL / min for 35 min, 25 min, and 20 min, respectively. The eluent was collected in 10 mL test tubes and labeled. The absorbance was measured at 490 nm using the phenol-sulfuric acid method, and a scatter plot was plotted. The eluent (0-80 Tubes of 0-0.3 mol / L NaCl) was collected, concentrated, dialyzed through a 3500 Da dialysis bag, and freeze-dried to obtain the eluted fractions HBP-1 (27.0 g), HBP-2 (16.0 g), and HBP-3 (4.0 g). Take 10 g of HBP-1 polysaccharide component, dissolve it in 3 mL of distilled water, centrifuge (12000 rpm) for 10 min, and further separate and purify the supernatant by passing it through a dextran gel Superdex-100 column. Combine the solutions and concentrate them by rotary evaporation and freeze-dry to obtain the component named HBPs with a mass of 35 mg.

[0021] Example 2: Structural Identification of Homocystis jirovecii Polysaccharide (HBPs) (1) Homogeneity, molecular weight, and chain conformation analysis were performed using high-performance gel permeation chromatography (HPGPC). This was conducted using a Shimadzu LC-20AT chromatography system (Tokyo, Japan) equipped with a refractive index detector (RID). The experimental setup was a sample concentration of 2 mg / mL, a flow rate of 0.5 mL / min, and a column temperature maintained at 25 °C. Chromatographic separation was performed using a Shodex SB-804 HQ analytical column (7.8 × 300 mm) and an Ohpak SB-G guard column, following the previously described procedure (Wu et al., 2024). In addition, the molecular weight of HBPs was determined using gel permeation chromatography (GPC) at a concentration of 1 mg / mL. The instrument used was a Wyatt Technology U3000 liquid chromatography system, equipped with an Optilab T-rEXRID detector manufactured by Thermo Fisher Scientific, USA. A 300 × 8 mm Ohpak SB-803HQ gel filter column was used, and the samples were separated in tandem. The column temperature was maintained at 45 °C, and the injection volume was set to 100 μL. Analysis was performed for 75 minutes using a constant-phase mobile phase (containing 0.02% NaN3 and 0.1 M NaNO3) at a flow rate of 0.6 mL / min.

[0022] (2) Analysis of the monosaccharide composition of HBPs by ion chromatography: The monosaccharide content of HBPs was investigated using PMP derivatization. The hydrolysis process involved heating 1.0 mg of accurately weighed HBPs sample in an oil bath with 2 mL of 4 M trifluoroacetic acid (TFA) at 110 °C for 4 hours. After hydrolysis, the reaction mixture was dried by vacuum evaporation. The dried material was reconstituted with deionized water, followed by the addition of 200 µL of 0.6 M NaOH and 400 µL of 0.5 M PMP-dimethyl ether. The mixture was incubated at 70 °C for 1 hour. The acid was then neutralized by adding 400 µL of 0.3 M hydrochloric acid. The aqueous phase was collected after chloroform extraction, centrifuged, and filtered through a 0.22 µm filter before HPLC analysis. Analysis was performed using a Hadesil C18-Bio column at 25 °C, a flow rate of 1.0 mL / min, and a detection wavelength of 250 nm. Gradient elution was performed for 60 minutes using acetonitrile concentrations ranging from 17% to 20%.

[0023] The results showed that the homogeneous polysaccharide HBPs of *Russula ovata* consisted of D-glucose (Glc, 52.501%), D-mannose (Man, 34.367%), D-galactose (Gal, 12.677%), and N-acetyl-D-glucosamine (GlcNAc, 0.455%), with a weight-average molecular weight of 23.545 kDa.

[0024] (3) Gas chromatography-mass spectrometry (GC-MS) analysis of samples after methylation, hydrolysis, and acetylation, as well as infrared spectroscopy and 1H NMR spectroscopy. Figure 1 ), 13C NMR spectrum ( Figure 2 The DEPT135 one-dimensional and two-dimensional spectra were used to assign glycosidic bond signals to the polysaccharide. The main chain linkage of the polysaccharide was determined to be: →3)-α-D-Glcp-(1→(A), α-D-Glcp-(1→(B), α-D-Glcp-(1→(C), →6)-α-D-Glcp-(1→(D), →2)-α-D-Manp-(1→(E), →2,6)-α-D-Manp-(1→(F), →6)-α-D-Manp-(1→(G), →2)-β-D-Galf-(1→(H), →2,6)-β-D-Galf-(1→(I), →6)-β-D-Galf-(1→(J), α-D-Galp-(1→(K), and α-D-Manp-(1→(L).

[0025] The analysis results are shown in Table 1-2.

[0026] Table 1. Analysis of methylated sugar alcohol acetyl ester (PMAA) results for HBPs Table 2. Hydrogen and carbon signal attribution of HBPs The present invention also performed a series of structural characterizations, including: methylation assays to analyze its sugar residues and NMR spectra analysis to determine the glycosidic bond linkage mode. Figure 3 HH-COSY spectrum of homogeneous polysaccharide HBPs from *Rhododendron molle*. Figure 4 The HSQC spectrum of homogeneous polysaccharide HBPs from *Rhododendron molle*. Figure 5 HMBC map of homogeneous polysaccharide HBPs from *Rhododendron molle*. Figure 6 The NOESY spectrum of homogeneous polysaccharide HBPs from *Russula ovata* is shown. The structural formulas of the structural units of *Russula ovata* homogeneous polysaccharide HBPs obtained through various analytical methods are as follows: .

[0027] Example 3: Inhibitory effect of homogeneous polysaccharide from *Rhododendron molle* on RAW264.7 cells. 3.1 Reagents DMEM medium (Procell), McCoy's 5A medium (BI), FBS fetal bovine serum (BI), antibiotics (Solepro), CCK-8 (Solepro), PBS, and homogeneous polysaccharide of *Rhododendron simsii* dissolved and diluted in complete cell culture medium, prepared fresh for use.

[0028] 3.2 Consumables and Instruments Haier Medical Low-Temperature Storage Box (Qingdao Haier Special Electric Appliances Co., Ltd.). Analytical Balance (Restaurant Scientific Instruments Co., Ltd.), Thermo Clean Bench, Water-jacketed CO2 Cell Culture Incubator (Thermo), Microplate Reader. Pipettes, Pipettes (5 ml and 10 ml sizes), 6-well plates, 96-well plates, Transwell 24-well plates.

[0029] 3.3 Experimental Methods 3.3.1 Cell proliferation experiment study: RAW 264.7 mouse macrophages were cultured in DMEM containing 10% fetal bovine serum (FBS) under standard culture conditions (37°C, 5% CO2). Cells were aliquoted into 96-well plates and incubated with HBP at doses ranging from 25 to 800 µg / mL. Positive controls (1 µg / mL LPS) and blank controls (culture medium only) were also included. After 24 hours of incubation, MTT solution (Promega, Madison, Wisconsin, USA) was added, followed by overnight incubation. Absorbance at 490 nm was recorded using a Thermo Fisher Scientific microplate reader located in Waltham, Massachusetts, USA. Each treatment was performed in triplicate.

[0030] 3.3.2 Experimental study on phagocytosis: Phagocytosis of RAW 264.7 cells was quantified using a neutral red uptake method and a previously established protocol. Cells were cultured at a density of 1.5 × 10⁶ cells per well. 5 Cells were exposed to 50–200 µg / mL HBPs at a density of [number missing] cells and treated at 37 °C for 24 hours. A negative control and a positive control (containing 1 µg / mL LPS) were also included. After culturing at 37 °C for another 1.5 hours, neutral red solution was added. Cells were then washed three times with PBS, discarding any residual liquid at the bottom. Next, cells were lysed by adding 150 µL of a mixture of ethanol, AA, and water (1:1:2 ratio) to each well and incubated at 37 °C for 2 hours. The ability of cells to phagocytose harmful microorganisms was assessed by measuring absorbance at 570 nm.

[0031] 3.3.3 Detection of nitric oxide production and cytokine secretion: The levels of NO and cytokines were determined according to the relevant kit instructions. The incubation conditions for RAW 264.7 cells were as follows: HBPs (50-200 µg / mL), LPS (1 µg / mL), or culture medium alone as a blank control. Incubation time was 24 hours at 37 °C. After collecting the cell supernatant, the NO concentration was measured at 540 nm using Griess reagent, and the cytokine levels were quantified at 450 nm using enzyme-linked immunosorbent assay (ELISA). Each experiment was repeated three times.

[0032] 3.3.4 Receptor Blockade Assay Following the approach of Li et al. (2019), HBPs and LPS36-mediated macrophage receptor activation were investigated. Briefly, 1.5 × 10⁻⁶ samples were placed in each well. 5 RAW 264.7 cells were cultured for 24 hours. In the experiment, the cells were divided into four groups: one group served as a blank control (culture medium only), one group was activated with LPS (1 µg / mL), one group was treated with HBPs (200 µg / mL), and the last group underwent receptor blockade. To inhibit the receptor, cells were pretreated for 1 hour with a TLR4 inhibitor (Resatorvid, 1 µM), a TLR2 inhibitor (C29, 10 µM), or a combination of both. Afterward, cells were treated with HBPs (200 µg / mL) or LPS (1 µg / mL) and cultured for another day at 37°C and 5% CO2. After collecting the supernatant, NO production was measured at 540 nm using the Griess method.

[0033] 3.3.5 WB Analysis RAW264.7 cells were treated with HBPs at 37°C for 24 hours at doses ranging from 50 to 200 µg / mL. Cells were then lysed on ice in RIPA buffer containing protease and phosphatase inhibitors to extract total protein. The BCA assay was used to determine protein levels. 20 µg of protein was transferred to a PVDF membrane and electrophoresed on a 10% SDS-PAGE gel. The membrane was then incubated in 5% bovine serum albumin for 1 hour. Afterward, primary antibodies against IκBα, p-IκBα, p65, p-p65, p38, p-p38, ERK, p-ERK, JNK, and p-JNK were incubated overnight at 4°C. After three washes each time, the membrane was immersed in TBST for 10 minutes, followed by incubation with secondary antibodies for 1 hour. The final step was a ten-wash series of TBST washes. Protein bands detected by enhanced chemiluminescence reagents were captured using an Alliance Mini HD 9 imaging system (UVITEC Limited, UK). GAPDH was used as an internal control to normalize protein expression.

[0034] 3.3.6 Animal Experiments Male BALB / c mice were purchased from Beijing Vital River Biotechnology Co., Ltd., at an age of 8 weeks and weighing approximately 24 grams. They were housed in a controlled environment of a designated pathogen-free (SPF) facility, with the temperature maintained between 22 and 23 °C, humidity between 50% and 60%, and a light cycle of 12 hours. The use of animals in this invention has been reviewed and approved by the Animal Ethics Committee of Henan University of Traditional Chinese Medicine (KIB202406007). After one week of acclimatization, mice were divided into six groups: (1) control group (orally administered saline); (2) model group (orally administered CTX 80 mg / kg / day for five days, followed by saline); (3) low-dose HBPs group (treated with CTX as in the model group, followed by HBPs 100 mg / kg by gavage); (4) medium-dose HBPs group (treated with CTX as in the model group, followed by HBPs 200 mg / kg by gavage); (5) high-dose HBPs group (treated with CTX as in the model group, followed by HBPs 300 mg / kg by gavage); and (6) positive control group (treated with CTX as in the model group, followed by levamisole 40 mg / kg by gavage). Mouse weight was recorded every two days.

[0035] 3.3.7 Determination of immune organ indices and immunoglobulins At the end of the 21-day experiment, the spleen and thymus of euthanized mice were aseptically removed and immediately weighed. Serum immunoglobulin concentrations were determined using an ELISA assay. Prior to analysis, blood samples collected from the orbital vein were centrifuged at 3000 rpm for 10 minutes. Serum samples were then stored at -80 °C. Similar to the method used to assess sIgA levels in blood using a dedicated ELISA kit, sIgA levels in the rectal mucosa were also measured. Immediately after euthanasia, the rectum was carefully excised, and the mucosal tissue was gently scraped and homogenized in ice-cold PBS. The homogenate was centrifuged at 3000 rpm for 10 minutes; the supernatant was stored at -80 °C for subsequent sIgA level determination.

[0036] 3.3.8 Flow cytometry analysis Aseptic spleens were rapidly extracted and immediately placed in cold PBS, followed by euthanasia. Cells were mechanically separated using a cell filter and then transferred to culture dishes containing PBS to prepare single-cell suspensions. Red blood cells were first lysed with ammonium chloride buffer at room temperature for three to five minutes, then neutralized with an equal volume of PBS. The mixture was then centrifuged at 300 g for five minutes. Cells were then labeled with CD4-specific markers in a dark room at 4 °C. + CD8 + T cells and CD19 + B cells were treated with fluorescently labeled antibodies for 30 minutes. The antibody-to-label ratio was 0.25 µg / 100 µL PBS (containing 1% BSA). Cells were resuspended in 200 µL PBS and washed twice with PBS before flow cytometry analysis. Dead cells and cell debris were excluded based on the characteristics of forward and side scattering. Data were analyzed using FlowJo software developed by BD Biosciences. Further immunophenotyping studies were performed when necessary.

[0037] 3.3.9 Histological examination Immediately after euthanasia, the jejunum was removed and fixed in 4% paraformaldehyde for 24 hours, then dehydrated in gradually increasing concentrations of ethanol (75%, 85%, 95%, 100%). Samples were washed with xylene and then embedded in paraffin. 5-micrometer thick sections were cut from the tissue blocks using a Leica Biosystems RM2235 rotary microtome. Paraffin sections were dewaxed with xylene before rinsing with distilled water, and then rehydrated through a gradient of 100% to 75% ethanol. After hematoxylin staining for three to five minutes, the sections were differentiated, treated with iron blue, and counterstained with eosin for ten seconds. Following staining, the sections were dehydrated with 100% ethanol, cleared with xylene, and mounted with synthetic resin. Morphological characteristics were observed and recorded using a conventional optical microscope.

[0038] 3.3.11 Data Analysis Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism (version 9). One-way ANOVA was followed by Tukey's multiple comparison test to analyze differences among multiple groups. For datasets that did not conform to a normal distribution, the Kruskal-Wallis test was performed followed by Dunn post-hoc comparisons. P < 0.05 was considered statistically significant, expressed using significance indicators: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ##p < 0.01, ###p < 0.001.

[0039] 3.4 Experimental Results Homocystis jirovecii polysaccharide showed good biocompatibility with RAW264.7 cells at HBP concentrations ≤300 μg / mL. HBP treatment (50, 100, and 200 μg / mL) dose-dependently enhanced the phagocytic capacity of macrophages. Figure 7 -B). Simultaneously promotes cell migration in a dose-related manner ( Figure 7 -C). Pro-inflammatory cytokines also increase with increasing HBP concentration ( Figure 7 -E). At the highest HBP concentration (200 μg / mL), the phosphorylation level of the kinase was comparable to that observed under LPS stimulation, indicating that these inflammatory pathways were significantly activated. Figure 7 -F).

[0040] Compared with the control group, mice treated with cyclophosphamide (Cy) had a significantly reduced body weight (BW). Figure 8 -B), spleen and thymus indices were also significantly reduced ( Figure 8 -C). High doses (200 and 300 mg / kg) of HBPs significantly reduced CD4+. + / CD8 + T cell ratio ( Figure 8 -D) and CD19 + B cell percentage ( Figure 8 -E) returned to normal. Furthermore, HBPs significantly increased systemic immunoglobulin levels and intestinal SIgA levels ( Figure 8 -F–I). Histological observation of the spleen tissue in the control group showed that its structure was normal, and the red and white pulp regions were clearly distinguishable. Figure 8 -J). Administration of HBPs (100, 200, and 300 mg / kg) gradually restored normal spleen structure, with a clear boundary between the red and white pulp. Similarly, compared to the control group, Cy induced abnormal thymic epithelial structure and atrophy ( Figure 8-K). Treatment with HBPs (100, 200 and 300 mg / kg) gradually normalized the thymus structure.

[0041] HBPs alleviate intestinal mucosal damage and regulate cytokine secretion in Cys-treated mice. Figure 9 Results from the A-group showed that administration of HBPs at doses of 200 and 300 mg / kg significantly reduced Cy-induced weight loss. Furthermore, colonic shortening induced in the immunosuppressed group was significantly improved by HBP supplementation. Histological examination with H&E staining showed that HBP administration enhanced colonic mucosal integrity and significantly protected the intestinal epithelium from Cy-induced damage. Figure 9 -B and 9-C). Cy-induced mice showed significantly reduced mucus secretion, while HBPs treatment effectively restored mucus levels ( Figure 9 -D and 9-E). Cy treatment significantly decreased the concentrations of IL-10, IL-6, IL-2, and TNF-α in the gut; however, administration of HBPs significantly increased these cytokines (-D and 9-E). Figure 9 -F). Further immunofluorescence staining and Western blot analysis confirmed that Cy exposure inhibited the expression of Occludin, MUC2, and ZO-1 proteins in intestinal tissue, while HBP treatment significantly reversed these effects and increased their levels ( Figure 9 -G, Figure 9 -H).

[0042] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. A homogeneous polysaccharide HBPs from *Russula ovata*, characterized in that, The homogeneous polysaccharide from *Russula ovata* is composed of the following components by mass percentage: D-glucose 52.501%, D-mannose 34.367%, D-galactose 12.677%, and N-acetyl-D-glucosamine 0.455%, with a weight-average molecular weight of 23.545 kDa. Its structural unit has the following structural formula: 。 2. The method for preparing homogeneous polysaccharides (HBPs) from *Russula ovata* according to claim 1, characterized in that, Includes the following steps: 1) Boil the crushed bamboo red fungus in water, cool, filter, and obtain bamboo red fungus polysaccharide extract; 2) The polysaccharide extract of *Russula ovata* was subjected to alcohol precipitation to obtain crude polysaccharide precipitated with alcohol; 3) The crude polysaccharide precipitated with alcohol was deproteinized using the Sevag method to obtain total polysaccharide of *Russula basilica*. 4) The total polysaccharides of *Rhododendron simsii* were separated and purified by ion exchange chromatography and gel column chromatography, then concentrated and freeze-dried to obtain homogeneous polysaccharides (HBPs) of *Rhododendron simsii*.

3. The method for preparing homogeneous polysaccharides (HBPs) from *Russula ovata* according to claim 2, characterized in that, The boiling time mentioned in step 1) is 1~2 hours.

4. The method for preparing homogeneous polysaccharides (HBPs) from *Russula ovata* according to claim 2, characterized in that, The alcohol precipitation in step 2) uses ethanol with a concentration of 80-95%, the precipitation temperature is 4-25 ℃, and the time is 2-12 h.

5. The method for preparing homogeneous polysaccharides (HBPs) from *Russula ovata* according to claim 2, characterized in that, In step 3), when removing protein using the Sevag method, a mixture of chloroform and n-butanol is used for extraction; the volume ratio of chloroform to n-butanol is 3~5:

1.

6. The method for preparing homogeneous polysaccharides (HBPs) from *Russula ovata* according to claim 2, characterized in that, The ion exchange chromatography column mentioned in step 4) is DEAE-52 cellulose, the flow rate of the ion exchange column chromatography is 50 mL / min, and the gradient elution program is: 0-40 min water, 0.1 mol / L NaCl 20-40 min, 0.3 mol / L NaCl 15-25 min; the packing material of the gel column is Sephadex-100.

7. The use of the homogeneous polysaccharide HBPs of *Russula ovata* as described in claim 1 in the preparation of drugs to improve intestinal mucosal immunity.

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