A boletus xylan palmitate, a preparation method and application thereof

Abstract

The application discloses a brown ring lactarius xylolipalm and a preparation method and application thereof. The brown ring lactarius xylolipalm is xylolipalm formed by taking alpha-(1→4)-xylose as a main chain, alpha-(1→)-glucose as a branch chain and a palmitic acid molecule through an ester bond. The application further discloses a preparation method of the brown ring lactarius xylolipalm and an application thereof in immune regulation, and the brown ring lactarius xylolipalm can be used as a dietary supplement or a food additive for immunodeficiency patients or tumor patients.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biology, and particularly relates to a Suillus luteus xylan palmitate and a preparation method and application thereof. BACKGROUND

[0002] As a natural resource with both nutritional and medicinal values, edible fungi have their cell walls mainly containing polysaccharides with immunomodulatory, anti-tumor, anti-inflammatory and other biological activities, and the development and application of polysaccharides in the fields of functional foods and biological medicines have become a research hotspot. As an excellent mycorrhizal edible fungus, Suillus luteus is rich in polysaccharides, proteins, flavonoids, ergosterol and other active ingredients, and can be used for soil remediation by regulating soil microbial community diversity, enriching nutrients and reducing heavy metal content, and has a high comprehensive utilization value, providing a high-quality raw material basis for the development of active polysaccharides.

[0003] However, the current research and application of Suillus luteus polysaccharides still face many technical bottlenecks: first, the structural properties and biological functions of polysaccharides are highly dependent on the extraction conditions, and the existing common water-soluble alcohol extraction process often focuses on high molecular weight polysaccharide components, but ignores the mining of low molecular weight polysaccharides in alcohol-soluble components. Related studies have confirmed that low molecular weight polysaccharides have better water solubility and stronger cell permeability, and usually exhibit more excellent biological activity, and their development potential has not been fully released; second, polysaccharides are difficult to directly enter the body blood stream as macromolecular compounds after oral administration, and their biological functions need to rely on the regulation of intestinal flora and their metabolites, but the interaction mechanism of Suillus luteus polysaccharides and intestinal flora, especially the specific action path of immune function mediated by key regulatory pathways of intestinal flora, has not formed a clear and systematic research conclusion, resulting in a lack of clear mechanism support in the development of immune regulation and anti-tumor related products, which restricts the process of technology transformation and industrialization application.

[0004] Therefore, it is a key requirement to develop a preparation process for Suillus luteus alcohol-soluble polysaccharides, to clarify their structural characteristics, to reveal their specific mechanism of enhancing immune function through intestinal flora metabolism, to obtain Suillus luteus polysaccharide raw materials with high biological activity, clear action target and industrial application potential, which is a key requirement to solve the current development bottleneck of Suillus luteus polysaccharides, to promote the industrialization application of Suillus luteus polysaccharides in immune regulation and anti-tumor related products, and to promote the efficient utilization of active ingredients of edible fungi and technological innovation in the field of biological medicine. SUMMARY

[0005] The present application provides a brown ring milk cow liver xylan palmitate (SLEP) and a preparation method and application thereof, the xylan palmitate prepared by the preparation method solves the technical bottlenecks of "unclear structure, low utilization rate of alcohol-soluble low molecular weight components and unclear mechanism" in the development of brown ring milk cow liver polysaccharide, provides a brown ring milk cow liver xylan palmitate with clear structural characteristics and high biological activity, and simultaneously discloses a preparation method, an immune improvement mechanism based on intestinal flora microecological regulation and an application direction, thereby providing high-quality active raw materials for the field of biological medicines.

[0006] To achieve the above object, the present application provides the following technical scheme: a brown ring milk cow liver xylan palmitate, the brown ring milk cow liver xylan palmitate takes alpha-(1→4)-xylose as a main chain, alpha-(1→)-glucose as a branch chain, and forms a repeating unit through the main chain and the branch chain, and forms a xylan palmitate through an ester bond with a palmitic acid molecule.

[0007] Preferably, the main chain repeating unit of the brown ring milk cow liver xylan palmitate comprises nine xyloses, and the branch chain repeating unit of the brown ring milk cow liver xylan palmitate comprises two glucoses.

[0008] Preferably, the two glucoses of the branch chain repeating unit of the brown ring milk cow liver xylan palmitate are both connected to the third carbon of the xylose on the main chain.

[0009] Preferably, the ester bond is formed between the palmitic acid molecule and the terminal xylose on the main chain.

[0010] Preferably, the molecular weight of the brown ring milk cow liver xylan palmitate is (6.2±2)×10³ Da.

[0011] Preferably, the molar ratio of xylose:palmitic acid:glucose in the brown ring milk cow liver xylan palmitate is 1.00:0.03:0.28.

[0012] The present application also provides a preparation method of the brown ring milk cow liver xylan palmitate, and the preparation method comprises the following specific steps:

[0013] S1, dry brown ring milk cow liver fruiting bodies are added to hot water at 70 DEG C, soaked, concentrated by a rotary evaporator, and a brown ring milk cow liver concentrated solution is obtained.

[0014] S2, 4 times the volume of anhydrous ethanol is added to the brown ring milk cow liver concentrated solution obtained in step S1, uniformly mixed, and then left overnight, centrifuged, and the supernatant is collected, ethanol is removed, an appropriate amount of water is added for redissolution, and then frozen and dried to obtain the brown ring milk cow liver xylan palmitate.

[0015] Preferably, the step S1 is that the boletus edulis basidioma is added into hot water for soaking, the ratio of material liquid is 1:15-1:20 g / mL, the soaking time is 3-4 hours, the concentration temperature of the rotary evaporator is 55-60 DEG C, and the vacuum degree is 0:08-0:09 MPa; the step S2 is that the centrifugal speed is 4000 r / min, the centrifugal time is 15 min, and the ethanol in the supernatant is removed by rotary evaporation under the conditions of 45-50 DEG C and 0:08-0:09 MPa.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] 1. Structural innovation: the present application first determines the structure of xylan palmitate from boletus edulis, the main branch connection mode (alpha-(1→4)-xylose main chain, alpha-(1→)-glucose branch chain) and the esterification modification characteristics, filling the gap in the structural research of this kind of polysaccharide conjugate, and providing a clear basis for the correlation between quality control and activity;

[0018] 2. Process superiority: the present application provides an optimized and efficient preparation process, which can efficiently extract alcohol-soluble polysaccharide components, has stable product yield and high purity, does not require complex equipment, is suitable for industrial mass production, and solves the problem of waste of alcohol-soluble components in the traditional process;

[0019] 3. Mechanism clarity: the present application first discloses the action chain of 'polysaccharide conjugate-intestinal flora quorum sensing-T cell immunity', determines that lactobacillus is the key flora and homoserine lactone is the core signal molecule, provides clear scientific support for its application in the field of immune regulation, and avoids the disadvantages of 'ambiguous action and uncontrollable effect';

[0020] 4. Wide application: the xylan palmitate of the present application has alcohol and water solubility and low toxicity, can be applied in multiple fields of 'immune aid-biomedicine', is suitable for immunocompromised people, tumor patients and other people, has a broad market prospect, and promotes the transformation of boletus edulis from 'edible resource' to 'high-value medicinal resource';

[0021] Other features and advantages of the present application will become apparent from the following detailed description of exemplary embodiments thereof, which should be considered in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the related art, the following will briefly introduce the drawings needed to be used in the embodiment or related art description. Obviously, the drawings in the following description are only embodiments of the present application, and those skilled in the art can obtain other drawings according to the provided drawings without any creative labor.

[0023] Figure 1 This is a high-performance gel permeation chromatogram of xylan palmitate from Boletus chapensis according to the present invention;

[0024] Figure 2 The infrared spectrum of xylan palmitate from *Boletus chalcogenide* according to this invention;

[0025] Figure 3 This is a gas chromatography-mass spectrometry (GC-MS) chromatogram of xylan palmitate from Boletus chalcogenide of the present invention;

[0026] Figure 4 This is a 200× scanning electron microscope image of xylan palmitate from Boletus chalcogenide of the present invention.

[0027] Figure 5 Nuclear magnetic resonance imaging of xylan palmitate from *Boletus chalcogenide* in this invention. 1 H spectrum;

[0028] Figure 6 Nuclear magnetic resonance imaging of xylan palmitate from *Boletus chalcogenide* in this invention. 13 C spectrum;

[0029] Figure 7 This is the COSY NMR spectrum of xylan palmitate from *Boletus thaliana* according to the present invention.

[0030] Figure 8 The HSQC nuclear magnetic resonance spectrum of xylan palmitate from *Boletus thaliana* is shown below.

[0031] Figure 9 The nuclear magnetic resonance (HMBC) spectrum of xylan palmitate from *Boletus thaliana* is shown below.

[0032] Figure 10 This is a structural formula of xylan palmitate from Boletus chalcogenide of the present invention;

[0033] Figure 11 This is another structural formula of the xylan palmitate of *Boletus chapensis* according to the present invention;

[0034] Figure 12 This is the third structural formula of the xylan palmitate of *Boletus chapensis* in this invention;

[0035] Figure 13 The figure shows the effect of xylan palmitate from *Boletus thaliana* on tumor weight and tumor inhibition rate in tumor-bearing mice.

[0036] Figure 14 The effect of *Boletus chalcogenide* xylan palmitate on the diversity of gut microbiota in tumor-bearing mice is shown in the Venn diagram.

[0037] Figure 15Ternary diagram of influence of brown ring milk cow liver xylan palmitate on intestinal flora diversity of tumor-bearing mice of the present application;

[0038] Figure 16 Significance analysis diagram of influence of brown ring milk cow liver xylan palmitate on intestinal lactobacillus of tumor-bearing mice of the present application;

[0039] Figure 17 Heat map of influence of brown ring milk cow liver xylan palmitate on intestinal flora metabolite content of tumor-bearing mice of the present application;

[0040] Figure 18 Volcano plot of influence of brown ring milk cow liver xylan palmitate on intestinal flora metabolite content of tumor-bearing mice of the present application;

[0041] Figure 19 KEGG enrichment bubble chart of influence of brown ring milk cow liver xylan palmitate on intestinal flora metabolism of tumor-bearing mice of the present application;

[0042] Figure 20 Influence diagram of brown ring milk cow liver xylan palmitate on peripheral blood T cell subpopulation distribution of tumor-bearing mice of the present application; DETAILED DESCRIPTION

[0043] Please refer to Figures 1-20 The present application provides a technical solution:

[0044] I. Structural characteristics of brown ring milk cow liver xylan palmitate:

[0045] Brown ring milk cow liver xylan palmitate is a polysaccharide conjugate with alcohol and water solubility. Its core structural characteristics are analyzed by various detection methods, and the specific parameters are as follows:

[0046] Chemical composition and proportion: by gas chromatography-mass spectrometry analysis, brown ring milk cow liver xylan palmitate is composed of xylose (Xylose, Xyl), palmitic acid (Palmitic acid, PA) and glucose (Glucose, Glc), and the molar ratio of the three is 1.00:0.03:0.28.

[0047] Molecular weight and physicochemical properties: by liquid exclusion chromatography detection, the average molecular weight of brown ring milk cow liver xylan palmitate is (6.2±2)×10³ Da, which belongs to low molecular weight polysaccharide conjugate; scanning electron microscope observation shows that its microstructure is dense and easy to absorb water due to changes in environmental humidity.

[0048] Main branched chain structure and connection mode: the nuclear magnetic resonance analysis result shows that the brown ring lactarius liver mushroom xylan palmitate takes alpha-(1→4)-xylose as the main skeleton (main chain), connects glucose as the branch chain through alpha-(1→) glycosidic bond, and forms covalent combination with palmitic acid molecule through ester bond to form " polysaccharide-lipid" conjugate structure, and the structure is the key basis for its alcohol-soluble and water-soluble.

[0049] II. The preparation method of the brown ring lactarius liver mushroom xylan palmitate is as follows:

[0050] The present application optimizes the design of the preparation process according to the extraction demand of the brown ring lactarius alcohol-soluble polysaccharide conjugate, the steps are clear and the repeatability is strong, which is suitable for industrialized production, and the specific process is as follows:

[0051] Raw material pretreatment: select dry brown ring lactarius fruiting body without mildew and impurities.

[0052] Hot water extraction and concentration: according to the ratio of 1:15-1:20 g / mL, the brown ring lactarius liver mushroom is added into 70 DEG C constant temperature water bath, soaked and stirred for 3-4 h; after the extraction is completed, the clear extraction liquid is collected; the extraction liquid is transferred into a rotary evaporator, and is concentrated under reduced pressure at 55-60 DEG C and vacuum degree of 0.08-0.09 MPa, to obtain brown ring lactarius liver mushroom concentrated liquid.

[0053] Alcohol-soluble separation and purification: slowly add 4 times the volume of anhydrous ethanol to the concentrated liquid (so that the volume fraction of ethanol in the system reaches 80%), and stir until the ethanol is completely mixed, and then stand overnight (12-16 h) at room temperature, so that the non-alcohol-soluble impurities are fully precipitated; the next day, centrifuge at a speed of 4000 r / min for 15 min, discard the bottom precipitate, and collect the upper alcohol-soluble supernatant; the supernatant is transferred into a rotary evaporator again, and the ethanol is evaporated at 45-50 DEG C and vacuum degree of 0.08-0.09 MPa to obtain an alcohol-free concentrate; add an appropriate amount of deionized water to the alcohol-free concentrate, and centrifuge to collect the supernatant to obtain a xylan palmitate aqueous solution.

[0054] Freeze-dried product: the pre-cooled sample is placed in a freeze dryer, and freeze-dried for 24-48 h to obtain the brown ring lactarius liver mushroom xylan palmitate finished product.

[0055] III. The mechanism of the brown ring lactarius liver mushroom xylan palmitate in improving the intestinal flora microecology of immune damage:

[0056] Through the tumor-bearing mouse (immune damage model) animal experiment, the present application systematically reveals the action path of the brown ring lactarius liver mushroom xylan palmitate in regulating the intestinal flora microecology and mediating the quorum sensing system, and finally enhancing the immune function, and the specific experimental results are as follows:

[0057] Experimental design and grouping: SPF Kunming mice (body weight 25 ± 2g) were selected, and after adaptive feeding for 1 week, they were randomly divided into 5 groups (10 in each group): blank control group (normal mice without immune damage), model control group (tumor-bearing modeling without intervention), positive control (CTX) group (tumor-bearing modeling + 30 mg / kg cyclophosphamide intraperitoneal injection), SLEP low-dose group (tumor-bearing modeling + 100 mg / kg gavage), SLEP high-dose group (tumor-bearing modeling + 200 mg / kg gavage).

[0058] Intervention process: On days 1-7, the low / high dose groups were gavaged with the corresponding concentration of SLEP solution every day, and the blank group and the model group were gavaged with the same volume of normal saline; on day 8, except for the blank group, the rest of the groups were subcutaneously injected with 2x10 6 S180 tumor cells in the right front limb axillary to construct a tumor-bearing model; on days 9-21, the blank group and the model group continued to be gavaged with normal saline, the CTX group was intraperitoneally injected with cyclophosphamide, and the low / high dose groups maintained the original dose of gavage; after the experiment, the tumor size, intestinal flora, and immune indicators were detected.

[0059] Regulation of intestinal flora diversity and structure: Through high-throughput sequencing analysis of the V3V4 region of the 16S rRNA gene, the results show that SLEP can restore the intestinal flora diversity of immune-damaged mice and significantly increase the abundance of Lactobacillus, confirming that the xylan palmitate can improve the imbalance of intestinal flora structure by enriching beneficial bacteria (Lactobacillus).

[0060] Mediation of the quorum sensing system of intestinal flora: Liquid chromatography-mass spectrometry (LC-MS) was used to detect the intestinal flora metabolites in the feces of mice, and the results showed that SLEP can regulate cell communication between bacteria, inhibit the proliferation of harmful bacteria, and promote the function of beneficial bacteria.

[0061] Repair of immune damage and anti-tumor effect: Flow cytometry detection shows that SLEP gavage intervention can significantly increase the proportion of CD4 + T cells in the peripheral blood of tumor-bearing mice, improve the immune damage state, and the tumor inhibition rate reaches 56.02%.

[0062] Four, application direction of xylan palmitate of Xerula badius:

[0063] Based on the above structural characteristics, preparation process and mechanism of action, the xylan palmitate of Xerula badius of the present application can be applied in the following fields, and the specific scenarios are as follows:

[0064] Immune regulation field: It can be used as an immune adjuvant raw material for the auxiliary intervention of immunodeficient patients and tumor patients. For example, it can be used in combination with tumor radiotherapy and chemotherapy adjuvant drugs to reduce intestinal mucosa damage caused by radiotherapy and chemotherapy, while enhancing T cell immune activity and improving treatment tolerance; or it can be made into capsules alone for intestinal microecological repair of patients with chronic immunodeficiency diseases to reduce the recurrence rate of infection.

[0065] Biological medicine field: It can be used as a precursor of active pharmaceutical ingredients to further develop intestinal microecological regulation drugs. For example, for "intestinal flora imbalance related immune diseases" (such as inflammatory bowel disease combined with immunosuppression), it is made into enteric-coated tablets (to avoid damage by gastric acid) to achieve the dual treatment effect of "intestinal microecological repair-immune function enhancement" by targeted regulation of intestinal lactobacilli and quorum sensing pathway.

[0066] Embodiment:

[0067] The application will be further described in detail below in combination with the accompanying drawings and through specific embodiments. The following embodiments are only descriptive and not limiting, and cannot limit the protection scope of the application.

[0068] A preparation method of Suillus bovinus xylan palmitate, dry Suillus bovinus fruiting bodies free of mold and impurities are selected. The Suillus bovinus is added into a 70°C constant temperature water bath at a solid-liquid ratio of 1:15 g / mL, soaked and stirred for 3h; after the extraction is completed, the clear extraction liquid is collected; the extraction liquid is transferred into a rotary evaporator for reduced pressure concentration, 4 times the volume of anhydrous ethanol is slowly added into the concentrated liquid, and the mixture is left to stand at room temperature overnight; the next day, the upper alcohol-soluble supernatant is collected, and after the ethanol is removed, the mixture is freeze-dried to obtain the final Suillus bovinus xylan palmitate product (SLEP).

[0069] The polysaccharide content of SLEP is detected by the phenol-sulfuric acid method, and the result is 86.35±4.36%.

[0070] The average molecular weight of SLEP is determined, and the result is shown in Figure 1 On the high-performance gel permeation chromatogram detected by liquid exclusion chromatography, a single, narrow and symmetrical peak appears, proving that the molecular weight distribution of SLEP after purification is uniform, and the purity is high. The retention time 11.693 min is substituted into the standard curve (y = -0.4601x + 9.1698, R2=0.9984, y represents the logarithmic value of the molecular weight, and x represents the corresponding retention time) to obtain the average molecular weight of SLEP, which is about 6.2×10 3 Da.

[0071] The main characteristic functional groups of SLEP are determined, and the result is shown in Figure 2 . The characteristic absorption peaks of polysaccharide are 3404.72 cm-1 2930.09 cm -1 and 1404.49 cm -1 , representing the stretching vibration of OH, the stretching vibration of CH, and the deformation vibration of CH, respectively. 1632.82 cm -1 The strong absorption peak at 1000-1200 cm⁻¹ is attributed to the bending vibration absorption of OH groups, while the strong absorption peak at 1000-1200 cm⁻¹ is attributed to the bending vibration absorption of OH groups. -1 The absorption peak at 876.67 cm⁻¹ is due to the presence of COC glycosidic bonds in the polysaccharide. Additionally, the absorption peak at 876.67 cm⁻¹... -1 The absorption peaks indicate that the SLEP mainly consists of α-glycosidic bonds.

[0072] The monosaccharide composition of SLEP was determined, and the results are shown in [Figure number missing]. Figure 3 The peak with a retention time of 9.0 min identified xylose, palmitic acid at 9.6 min, and glucose at 9.7 min. Based on their relative contents, the molar ratio of xylose, palmitic acid, and glucose in SLEP is 1.00:0.03:0.28.

[0073] Figure 4 The image shows the microstructure of SLEP magnified 200 times. SLEP exhibits a dense, irregular aggregate structure with wrinkled surfaces, making it prone to absorbing water and moisture, which may be closely related to its structural composition and physicochemical properties.

[0074] Figure 5 For SLEP 1 The 1H spectrum shows a chemical shift at 4.79 ppm, which can be attributed to the presence of a proton signal from D₂O. A signal range of 5.20 ppm indicates that the SLEP primarily contains anomeric hydrogen signals from α-glycosidic bonds, a signal value of 6.53 ppm indicates the presence of ester bonds formed between fatty acid compounds and hydroxyl groups, and signal peaks from 3.0 ppm to 4.5 ppm indicate the presence of H₂–H₆ in these sugar residues.

[0075] Figure 6 For SLEP 13 C-spectrum. Based on the above results, fatty acid anodic carbon signals were observed at around 135.33 ppm, while α-glycosidic bond anodic carbon signals were found at 93.20 ppm. The signals of C2 to C6 in the conjugate residues were mainly distributed between 53.85 ppm and 72.49 ppm.

[0076] Figure 7 The correlation of adjacent hydrogen atoms in SLEP is shown, and the relevant cross-absorption signals are labeled. Here, A represents α-Xylp, and B represents α-Glcp. The letter subscripts and numbers represent the hydrogen atom indices of the sugar residues, with the first digit representing the x-axis and the second the y-axis.

[0077] Figure 8 The chemical shifts of the C1-C6 of the xylose and glucose residues in SLEP were analyzed.

[0078] Figure 9 The existence of A1,4, A3B1 cross signals was identified, and the results showed that the SLEP of X. badius mainly consisted of α-(1→4)-Xylp and α-(1→)-Glcp, and α-(1→)-Glcp linked the third carbon atom of the main chain xylose as a branch.

[0079] As shown in Figure 10 , Figure 11 and Figure 12 , the structure of SLEP was deduced according to the above results. SLEP has α-(1→4)-Xylp as the main chain, and α-(1→)-Glcp links the third carbon atom of the main chain xylose as a branch, and covalently links a palmitic acid molecule. Among them, the connection of the two glucose molecules in the repeating unit with xylose has multiple possibilities, such as Figure 10 two glucoses are connected to the 3rd and 6th xyloses, as shown in Figure 11 two glucoses are connected to the 2nd and 8th xyloses, and Figure 12 two glucoses are connected to the 4th and 5th xyloses. In addition to the above three possibilities, there are also various arbitrary combinations of arrangements.

[0080] Figure 13 The tumor weights and inhibition rates of tumor-bearing mice after different treatments are shown. The results show that the tumor weights of the cyclophosphamide group and the SLEP group are significantly reduced compared with the model group, and the tumor inhibition rates are 57.37%, 38.83% and 56.35%, respectively. Cyclophosphamide is a common chemotherapeutic drug that can indiscriminately eliminate immune cells and tumor cells, and has a certain positive effect on the inhibition of transplanted tumor growth, but the immune capacity of the body is also damaged. SLEP can effectively improve the body's anti-tumor immune capacity.

[0081] As shown in Figure 14 , each circle represents a group, and the numbers in the overlapping (non-overlapping) part of the ellipse represent the number of shared (unique) OTUs. As mentioned above, the four experimental groups have a total of 507 OTUs, while the unique OTUs of the blank group, model group, CTX group and SLEP high dose group are 103, 135, 98 and 80, respectively, indicating that these experimental groups show extensive intestinal microbial community diversity, which may be the reason for the difference in anti-tumor immune capacity of different groups.

[0082] Figure 15A ternary phase diagram showing the top 10 intestinal microbial genera. Compared with the model group, the abundance of Lactobacillus in the blank group and the SLEP group was significantly increased, indicating that it was closely related to the progression of solid tumors in mice, which may be the main reason for the enhanced anti-tumor immunity of tumor-bearing mice after SLEP intervention.

[0083] Figure 16 The results showed that the abundance of intestinal Lactobacillus in the SLEP group was significantly higher than that in the model group (p<0.05), which was consistent with the results of previous studies, indicating that Lactobacillus was a key intestinal flora for SLEP to regulate the immune capacity of tumor-bearing mice.

[0084] Non-target metabolomics was used to detect metabolites with significant differences between the model group and the SLEP high-dose group, as shown in Figure 17 . The color from blue to red represents the increase in the relative content of metabolites, and the horizontal direction represents the sample information (3 parallel samples). There were 43 metabolites with significant differences (p<0.05) in the SLEP high-dose group compared with the model group, of which 15 were down-regulated and 28 were up-regulated. However, the function of these metabolites in tumor-bearing mice needs further analysis.

[0085] In the volcano plot Figure 18 , each point represents a metabolite. Metabolites with significant up-regulation (down-regulation) compared with the model group are represented by red dots (blue dots), while metabolites with no significant difference are represented by gray. The results are consistent with Figure 17 .

[0086] The differential metabolites obtained by statistical analysis of these positive and negative ion modes were enriched by KEGG database for pathway analysis, as shown in Figure 19 . Each bubble represents a metabolic pathway, and the difference in metabolites may be related to changes in cellular pathways. Compared with the model group, the most significant signal pathway affected by the SLEP high-dose group in the intestinal metabolism was quorum sensing. Bacterial quorum sensing (QS) refers to a sensing phenomenon that occurs only when the number of bacteria reaches a certain density. As a mode of intercellular communication that changes microbial interactions, it can regulate the formation of biofilms, the secretion of public goods, and the synthesis of antibacterial substances, directly or indirectly affecting the adaptation of microbial communities. This indicates that SLEP exerts an immune-enhancing effect by affecting the quorum sensing system of intestinal flora, ultimately inhibiting the growth of solid tumors in the body.

[0087] Figure 20 The proportion of peripheral blood CD4 + and CD8 + T cells in the blank group, model group, CTX group, and SLEP high-dose group mice is shown. The results show that compared with the blank group, the CD4 +The proportion of cells was significantly reduced (p<0.05), while the proportion of CD4 + T cells in the SLEP group was significantly higher than that in the model group (p<0.05), indicating that SLEP may have a stronger immune regulatory effect on CD4 cells. The proportion of CD8 + T cells was relatively stable, but compared with the model group, the proportion of CD8 + T cells in the CTX group and the SLEP group was significantly reduced (p<0.05). In combination with the previous results, it can be speculated that CTX showed an active inhibitory effect on both T cell subpopulations, while SLEP enhanced the activity of CD4 + T cells, activated and recruited CD8 + T cells to exert a direct killing effect on tumor cells.

[0088] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A xylan palmitate from *Boletus chapensis*, characterized in that: The *Boletus chalcogenide* xylan palmitate has α-(1→4)-xylose as the main chain and α-(1→)-glucose as the side chain, with the main chain and side chain forming repeating units, and is a xylan palmitate formed by ester bonds with palmitic acid molecules; the molecular weight of the *Boletus chalcogenide* xylan palmitate is (6.2±2)×10³Da; the molar ratio of xylose:palmitic acid:glucose in the *Boletus chalcogenide* xylan palmitate is 1.00:0.03:0.

28.

2. The xylan palmitate of *Boletus chalcogenide* according to claim 1, characterized in that: The main repeating unit of the *Boletus chalcogenide* xylan palmitate comprises nine xyloses, and the branch repeating unit of the *Boletus chalcogenide* xylan palmitate comprises two glucose molecules.

3. The *Boletus chalcogenide* xylan palmitate according to claim 2, characterized in that: Both glucose molecules in the repeating unit of the xylan palmitate branched chain of *Boletus chapensis* are attached to the third carbon of the xylose in the main chain.

4. The xylan palmitate of *Boletus chalcogenide* according to claim 1, characterized in that: The ester bond is formed between the palmitic acid molecule and the terminal xylose in the main chain.

5. The method for preparing xylan palmitate from *Boletus chalcogenide* as described in any one of claims 1-4, characterized in that: The preparation method includes the following specific steps: S1. The dried Boletus chalcogenide fruiting bodies were added to hot water at 70°C and soaked. The solution was then concentrated using a rotary evaporator to obtain a concentrated Boletus chalcogenide liquid. S2, add 4 times the volume of anhydrous ethanol to the concentrated liquid of *Boletus circinus* obtained in step S1, mix well, let stand overnight, centrifuge, collect the supernatant to remove ethanol, add an appropriate amount of water to reconstitute, and freeze dry to obtain *Boletus circinus* xylan palmitate.

6. The method for preparing xylan palmitate from *Boletus chalcogenide* according to claim 5, characterized in that: In step S1, the ratio of the fruiting bodies of *Boletus globosum* to hot water for soaking is 1:15-1:20 g / mL, and the soaking time is 3-4 hours. The concentration temperature in the rotary evaporator is 55-60℃ and the vacuum degree is 0.08-0.09 MPa. In step S2, the centrifugation is performed at a speed of 4000 r / min for 15 min, and the ethanol is removed from the supernatant by rotary evaporation at 45-50℃ and a vacuum degree of 0.08-0.09 MPa.

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