Hyssopus harzianum polysaccharide as well as preparation method and application thereof

Abstract

The invention provides hyssopus harzianum polysaccharide as well as a preparation method and application thereof. The hyssop officinalis polysaccharide is prepared from galactose, arabinose and rhamnose. The molecular weight of the hyssop officinalis polysaccharide is 149 kDa, and the molar ratio of the galactose to the arabinose to the rhamnose is 59.59: 34.53: 5.88. The research clarifies that HCBP-0.1-W is arabinogalactan targeting TLR4 for the first time, and provides a theoretical basis for clinical application of HCBP-0.1-W as an immunologic adjuvant by activating dendritic cells and improving CTX-induced immunosuppression. Besides, by deeply researching the immunological enhancement effect of the hyssopus harzianum polysaccharide, the material basis of the aqueous extract of the hyssopus harzianum polysaccharide for treating related diseases can be clarified, and a new thought can be provided for development and utilization of the hyssopus harzianum.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide extraction technology, and more specifically, to a hard-tipped sage polysaccharide, its preparation method, and its application. Background Technology

[0002] Polysaccharides are biological macromolecules composed of multiple monosaccharides linked by glycosidic bonds. They are also known as polysaccharides and are widely found in animals, plants, and microorganisms. Different polysaccharides have a variety of important biological functions, such as antioxidant, antitumor, antiviral, and pharmacological effects that regulate dyslipidemia and dysglucose.

[0003] *Hyssopus cuspidatus* Boriss. is a perennial herb containing various bioactive components, such as volatile oils, flavonoids, and phenolic acids. Current research mainly focuses on the effects of *Hyssopus cuspidatus* in treating asthma, anti-inflammation, and immunosuppression, while research on its immune-enhancing effects is relatively scarce. Polysaccharides, as active substances with immune-enhancing effects in water extracts, have yet to be studied in relation to *Hyssopus cuspidatus* polysaccharides. Researching the effects of *Hyssopus cuspidatus* polysaccharides is of great significance for the development and utilization of *Hyssopus cuspidatus*. Summary of the Invention

[0004] The main objective of this invention is to provide a polysaccharide of vanilla scabra, its preparation method and application, in order to solve the problem that the existing technology has limited understanding of the structure and function of vanilla scabra polysaccharide and its application is relatively limited.

[0005] To achieve the above objectives, according to a first aspect of the present invention, a hard-tipped vanilla polysaccharide is provided, which is composed of galactose, arabinose, and rhamnose; the hard-tipped vanilla polysaccharide has the following properties: Figure 6 The structure shown; the molecular weight of the above-mentioned hard-tipped vanilla polysaccharide is 149 kDa; the molar ratio of the above-mentioned galactose, the above-mentioned arabinose and the above-mentioned rhamnose is 59.59:34.53:5.88.

[0006] To achieve the above objective, according to a second aspect of the present invention, a method for preparing the above-mentioned hard-tip vanilla polysaccharide is provided, the method comprising:

[0007] Degreasing and impurity removal were performed on the hard-tip angelica to obtain pretreated hard-tip angelica;

[0008] The pretreated *Hedyotis diffusa* was subjected to water extraction and alcohol precipitation to obtain crude polysaccharide;

[0009] The crude polysaccharide was decolorized and deproteinized to obtain a polysaccharide mixture.

[0010] The above polysaccharide mixture was purified to obtain the above-mentioned hard-tipped vanilla polysaccharide.

[0011] Furthermore, the above-mentioned degreasing and impurity removal steps for the above-mentioned hard-tipped angelica include:

[0012] The above-mentioned hard-tipped angelica was soaked in the above-mentioned organic reagent, filtered and dried to obtain the above-mentioned pretreated hard-tipped angelica;

[0013] Preferably, the above-mentioned organic reagent is selected from any one or more of the following: petroleum ether or 80-95% ethanol;

[0014] Preferably, the mass-to-volume ratio of the above-mentioned *Hedyotis diffusa* and the above-mentioned organic reagent is 1:3 to 6;

[0015] Preferably, the soaking time is 1 to 2 hours;

[0016] Preferably, the soaking temperature is 50–60°C.

[0017] Furthermore, the above-mentioned water extraction and alcohol precipitation steps include:

[0018] Water was mixed with the above-pretreated hard-tip angelica to extract polysaccharides, resulting in a solution containing polysaccharides;

[0019] The above-mentioned polysaccharide-containing solution was mixed with ethanol to precipitate the polysaccharide, thus obtaining the above-mentioned crude polysaccharide;

[0020] Preferably, the mass-to-volume ratio of the pretreated hard-tip angelica to the water is 1:20-45;

[0021] Preferably, the extraction temperature of the polysaccharide is 75–100°C; the extraction time is 0.5–2.5 h.

[0022] Preferably, the final concentration of the ethanol is 60-80%;

[0023] Preferably, the precipitation time of the polysaccharide is 20-26 hours.

[0024] Further, choose any one of the following decolorizing agents to perform the above decolorization: polyamide, activated carbon, or hydrogen peroxide;

[0025] Preferably, the mass ratio of the above-mentioned decolorizing agent to the above-mentioned crude polysaccharide is 2-3:1;

[0026] Preferably, the decolorization time is 2 to 6 hours;

[0027] Preferably, the sevage method is used for the above-mentioned protein removal treatment.

[0028] Further, the purification process includes: subjecting the polysaccharide mixture to anion exchange chromatography and gel filtration chromatography to obtain the above-mentioned hard-tip senna polysaccharide;

[0029] Furthermore, the packing material for the above anion exchange chromatography is selected from any one of the following: DEAE SFF;

[0030] Furthermore, the packing material for the above gel filtration chromatography is selected from any one of the following: Sephadex G-75.

[0031] To achieve the above objectives, according to a third aspect of the present invention, a hard-tipped vanilla polysaccharide prepared by the above-described preparation method is provided.

[0032] To achieve the above objectives, according to a fourth aspect of the present invention, the above-described vanilla polysaccharide, or the above-described preparation method, or the above-described vanilla polysaccharide is provided in the preparation of a medicament for immunomodulation.

[0033] Using the technical solution of this invention, polysaccharides from *Hedyotis diffusa* were extracted and purified to obtain a homogeneous polysaccharide (designated HCBP-0.1-W in this application). This polysaccharide is composed of galactose, arabinose, and rhamnose; it has the following properties... Figure 6 The structure shown is as follows; the molecular weight of the polysaccharide is 149 kDa; the molar ratio of the above galactose, the above arabinose and the above rhamnose is 59.59:34.53:5.88.

[0034] This polysaccharide possesses immunomodulatory effects, significantly promoting the maturation of bone marrow-derived dendritic cells (BM-DCs), upregulating the expression of co-stimulatory molecules (CD40, CD86, MHC II), and enhancing the secretion of pro-inflammatory cytokines (IL-6, TNF-α, IL-12p40, IL-1β). Its effects depend on the TLR4 / NF-κB signaling pathway. Furthermore, this polysaccharide can reverse weight loss, immune organ atrophy, and leukopenia, and restore the balance of immune cell subsets. Metabolomics analysis further reveals that HCBP-0.1-W enhances immune function by regulating amino acid metabolism pathways. This research not only elucidates the material basis for the therapeutic effects of its aqueous extract on related diseases and provides a theoretical basis for its clinical application as an immunoadjuvant, but also offers new insights for the development and utilization of *Hedyotis diffusa*. Attached Figure Description

[0035] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0036] Figure 1The isolation and purification of polysaccharides according to embodiments of the present invention, as well as the HPGPC chromatogram, Fourier transform infrared spectrum, and monosaccharide composition of the purified polysaccharides, are shown. Figure 1 In this context, A represents the fractionation elution curve of HCBP-0.10 dextran gel chromatography column; Figure 1 In the figure, B represents the HPGPC chromatogram of HCBP-0.1 and HCBP-0.1-W; Figure 1 In this context, C represents the Fourier transform infrared spectrum of HCBP-0.1-W; Figure 1 In the figure, D indicates the determination of monosaccharide composition (correspondence of peak shape: 1: GlcN 2: Man 3: Rha 4: GlcA 5: GalA 6: NAG 7: Glc 8: Gal 9: Ara 10: Fuc).

[0037] Figure 2 SEM images of the polysaccharide HCBP-0.1-W at different magnifications (180, 500 and 3K) and the total ion chromatogram of HCBP-0.1-W after methylation are shown according to embodiments of the present invention. Figure 2 Table A in the table contains SEM images. Figure 2 B in the figure represents the total ion chromatogram.

[0038] Figure 3 The proton NMR spectrum of polysaccharide HCBP-0.1-W according to an embodiment of the present invention is shown. 1 H-NMR)( Figure 3 A) and carbon spectrum ( 13 C-NMR)( Figure 3 (B in the middle).

[0039] Figure 4 The HSQC spectrum of polysaccharide HCBP-0.1-W according to an embodiment of the present invention is shown. Figure 4 A) and 1H- 1H COSY spectrum ( Figure 4 (B in the middle).

[0040] Figure 5 The HMBC spectrum of polysaccharide HCBP-0.1-W according to an embodiment of the present invention is shown. Figure 5 A) and NOSY spectrum ( Figure 5 (B in the middle).

[0041] Figure 6 A structural model of the polysaccharide HBCP-0.1-W according to an embodiment of the present invention is shown.

[0042] Figure 7The effect of HCBP-0.1-W on DC maturation according to an embodiment of the present invention is shown - the average fluorescence intensity of the expression of surface molecules CD40, CD86, and MHC II in DCs; Figure 7 A in the equation is CD40; Figure 7 B in the code is CD86; Figure 7 C in the text represents MHC II.

[0043] Figure 8 The effect of HCBP-0.1-W on DC maturation according to an embodiment of the present invention is shown - ELISA detection of the secretion of cytokines IL-6, TNF-α, IL-12p40 and IL-1β (*p<0.05, **p<0.01, ***p<0.001); Figure 8 The A in the text represents the cytokine IL-6; Figure 8 In this context, B represents the cytokine TNF-α; Figure 8 The C in the text represents the cytokine IL-12p40; Figure 8 The D in the text represents the cytokine IL-1β.

[0044] Figure 9 The effect of HCBP-0.1-W on DC maturation according to an embodiment of the present invention is shown. Statistical analysis results of FITC-Dextran phagocytosis detected by flow cytometry are presented (*p<0.05, **p<0.01, ***p<0.001).

[0045] Figure 10 The effect of HCBP-0.1-W on DC maturation according to an embodiment of the present invention is shown - flow cytometry detection of CCR-7 expression and the statistical results of cell counting in Transwell migration assay (*p<0.05, **p<0.01, ***p<0.001). Figure 10 In this context, A represents the expression level of CCR-7; Figure 10 In this context, B represents the cell count statistics from the Transwell migration experiment.

[0046] Figure 11 The effects of HCBP-0.1-W on the proliferation and responsiveness of DCs according to embodiments of the present invention are shown (*p<0.05, **p<0.01, ***p<0.001); Figure 11 In this context, A represents the effect of HCBP-0.1-W treatment on the proliferation capacity of CD4+ T cells; Figure 11 In the middle B, the effect of HCBP-0.1-W treatment on the proliferation capacity of CD8+ T cells is indicated. Figure 11 In this context, C indicates the effect of HCBP-0.1-W treatment on the immune response capacity of CD4+ T cells; Figure 11In this context, D represents the effect of HCBP-0.1-W-treated DCs on the immune response capacity of CD8+ T cells.

[0047] Figure 12 It is shown that TAK-24 and BAY 11-7082, according to embodiments of the present invention, can inhibit the effect of HCBP-0.1-W on CD86 and CD40 expression (*p<0.05, **p<0.01, ***p<0.001).

[0048] Figure 13 An embodiment of the present invention illustrates how HCBP-0.1-W enhances DC maturation by relying on the TLR4-NF-κB signaling pathway.

[0049] Figure 14 A possible mechanism for activating DCs using HCBP-0.1-W according to an embodiment of the present invention is shown.

[0050] Figure 15 The effects of HCBP-0.1-W treatment according to embodiments of the present invention on the expression levels of proteins MYD88, IKKα, IKKβ, P-IKKα / β, P65, P-P65, P38 and P-P38 are shown (*p<0.05, **p<0.01, ***p<0.001).

[0051] Figure 16 The photographic images of the spleen and thymus of mice treated with HCBP-0.1-W according to an embodiment of the present invention and the statistical results of their organ indices are shown. Figure 16 Image A shows a mouse spleen. Figure 16 In the figure, B represents the statistical results of the mouse spleen index. Figure 16 C in the figure represents the mouse thymus. Figure 16 In the figure, D represents the statistical results of the mouse thymus index (*p<0.05, **p<0.01, ***p<0.001).

[0052] Figure 17 The body weight curves of mice treated with HCBP-0.1-W according to embodiments of the present invention are shown (*p<0.05, **p<0.01, ***p<0.001).

[0053] Figure 18 The immunohistochemical statistical results of CD4+ T cells, CD8+ T cells and DCs in mouse spleen after treatment with HCBP-0.1-W according to an embodiment of the present invention are shown (*p<0.05, **p<0.01, ***p<0.001).

[0054] Figure 19Shows the immunohistochemical photographing results of CD4+ T cells, CD8+ T cells and DCs in the spleen of mice after HCBP-0.1-W treatment according to an embodiment of the present invention.

[0055] Figure 20 Shows the enrichment diagram of the metabolic pathways of CTX and HCBP-0.1-W differential metabolites according to an embodiment of the present invention.

[0056] Figure 21 Shows the heat map of the correlation analysis of differential metabolites with cytokines and immune cells according to an embodiment of the present invention.

[0057] Figure 22 Shows the fractional elution curve diagram of DEAE SFF anion exchange chromatography column of HCBP-C according to an embodiment of the present invention. Detailed implementation manners

[0058] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0059] Term explanation:

[0060] GlcN: Full name is Glucosamine, Chinese name is glucosamine, an amino derivative of glucose, commonly found in chitin and cartilage, an important component of joints and soft tissues, and commonly used in joint health supplements.

[0061] Man: Full name is Mannose, Chinese name is mannose, a hexose aldehyde sugar, widely present in plants, especially in mannan, and participates in various biosynthetic processes.

[0062] Rha: Full name is Rhamnose, Chinese name is rhamnose, a naturally occurring methyl pentose, often found in the secondary metabolites of plants, and participates in the composition of cell walls.

[0063] GlcA: Full name is Glucuronic acid, Chinese name is glucuronic acid, an oxidation derivative of glucose, with a carboxylic acid group, participates in the body's detoxification process, and is excreted out of the body by binding with toxins to form soluble glucuronic acid conjugates.

[0064] GalA: Full name is Galacturonic acid, Chinese name is galacturonic acid, an oxidation derivative of galactose, an important component of pectin, and plays a role in plant cell walls.

[0065] NAG: full name N-Acetylglucosamine; an acetylated derivative of glucosamine, it is a component of chitin and bacterial cell walls, and has multiple functions in organisms.

[0066] Glc: short for glucose; a major hexose aldose, it is a core substance of energy metabolism in organisms and is widely found in nature.

[0067] Gal: full name Galactose; a six-carbon aldose, usually combined with glucose to form lactose, which is a common sugar in dairy products.

[0068] Ara: full name Arabicinose; a pentose aldose commonly found in plant polysaccharides, such as gum arabic, which participates in the construction of cell walls.

[0069] Fuc: Full name Fucose; a six-carbon deoxyglucose, it is an important component of alginate and certain glycoproteins, and participates in cell recognition and signal transduction.

[0070] Bone marrow-derived dendritic cells (DCs) are a type of dendritic cell derived from the bone marrow. They play a crucial role in antigen presentation in the immune system. Dendritic cells are specialized antigen-presenting cells, capable of capturing, processing, and presenting antigens to T cells, thereby initiating and regulating adaptive immune responses. Bone marrow-derived dendritic cells typically differentiate from hematopoietic stem cells, undergoing a series of developmental processes to eventually mature and participate in immune surveillance and regulation. They are distributed in various tissues, including the skin, lymph nodes, and spleen.

[0071] CD40 is a transmembrane protein belonging to the tumor necrosis factor receptor (TNF-R) family. It is primarily expressed on the surface of antigen-presenting cells, such as dendritic cells, B cells, and macrophages. By binding to its ligand CD40L, CD40 activates downstream signaling pathways, promoting the proliferation, differentiation, and antibody production of immune cells. Upregulation of CD40 is generally associated with enhanced immune responses, while downregulation may lead to immunosuppression.

[0072] CD86: A co-stimulatory molecule in the B7 family, primarily expressed on the surface of antigen-presenting cells. CD86 binds to CD28 on T cells, providing a second signal essential for T cell activation and proliferation. Upregulation of CD86 typically reflects enhanced T cell activation and immune responses, which is beneficial in anti-infection and anti-tumor processes. Conversely, downregulation of CD86 may lead to insufficient T cell activation, affecting the effectiveness of immune surveillance and immune responses.

[0073] IL-6 (interleukin-6): A multifunctional cytokine involved in regulating immune responses, inflammation, and hematopoiesis. It plays an important role in both acute-phase responses and chronic inflammation. Increased IL-6 levels are commonly associated with both acute and chronic inflammatory diseases.

[0074] TNF-α (tumor necrosis factor-α) is a pro-inflammatory cytokine primarily secreted by macrophages, playing a crucial role in immune responses, apoptosis, and inflammation. Studying changes in its expression can help identify the mechanisms underlying inflammation-related diseases and develop anti-TNF therapeutic strategies.

[0075] IL-12p40 (interleukin-12p40): A subunit of the IL-12 and IL-23 cytokines, it plays an important role in inducing and maintaining Th1 cell responses. Changes in IL-12p40 are associated with a variety of autoimmune diseases and infections. Studying its expression can reveal the mechanisms of immune system dysregulation and help in the development of targeted therapies.

[0076] IL-1β (interleukin-1β): A potent pro-inflammatory cytokine involved in regulating immune and inflammatory responses, primarily secreted by activated macrophages. IL-1β is elevated in various inflammatory and autoimmune diseases; studying its expression changes helps to understand the inflammatory mechanisms of these diseases and may provide targets for anti-inflammatory therapies.

[0077] MHC II (major histocompatibility complex II molecules) are a class of protein complexes that play a crucial role in the immune system, primarily found on the surface of antigen-presenting cells (such as dendritic cells, macrophages, and B cells). The main function of MHC II molecules is to present exogenous antigens to CD4+ T cells, thereby initiating an immune response. Upregulation of MHC II expression is usually associated with an enhanced immune response, helping to more effectively recognize and eliminate pathogens. Downregulation, on the other hand, may lead to a weakened immune response, making the body more susceptible to infection.

[0078] CCR7, short for CC Chemokine Receptor 7, is a G protein-coupled receptor primarily expressed on the surface of certain leukocytes, such as dendritic cells and T cells. Its main function is to mediate the migration of these immune cells to lymph nodes. Through binding to its ligands CCL19 and CCL21, CCR7 plays a crucial role in immune surveillance and adaptive immune responses. Upregulation of CCR7 is typically associated with the activation and migration of immune cells, enhancing the body's immune surveillance capabilities and antigen presentation efficiency.

[0079] CD8+ T cells, also known as cytotoxic T cells or killer T cells, are T lymphocytes with CD8 molecules on their surface. They are primarily responsible for recognizing and directly killing infected cells and cancer cells. When viruses or other pathogens infect cells, these cells present abnormal antigens. CD8+ T cells recognize these antigens and bind to MHC class I molecules, triggering the killing process. They induce apoptosis in target cells by releasing perforin and granzymes.

[0080] CD4+ T cells, also known as helper T cells, are T lymphocytes with the CD4 molecule on their surface. They play a "helper" role in the immune system, primarily by secreting cytokines to regulate and promote the activation and function of other immune cells, such as B cells, CD8+ T cells, and macrophages. CD4+ T cells function by recognizing antigens presented on MHC class II molecules, which are usually derived from extracellular pathogens. These two types of cells work synergistically in the immune system; CD8+ T cells are responsible for directly eliminating infected or mutated cells, while CD4+ T cells support the overall immune response by coordinating and enhancing other immune responses.

[0081] TLR4 (Toll-like receptor 4): A member of the Toll-like receptor family, it primarily recognizes bacterial lipopolysaccharide (LPS), a molecule found in the cell walls of Gram-negative bacteria. Activation of TLR4 is a crucial step in the innate immune response, initiating signaling pathways and triggering inflammatory and immune responses.

[0082] NF-κB (nuclear factor κB): A family of transcription factors involved in regulating the expression of a variety of genes, especially those related to immune and inflammatory responses. NF-κB is activated when cells are stimulated (such as by bacterial infection, cytokines, stress, etc.), thereby entering the cell nucleus and promoting the transcription of specific genes.

[0083] MyD88, short for Myeloid differentiation primary response 88, is an important adapter protein that plays a crucial role in innate immune responses, particularly in Toll-like receptor (TLR) and IL-1 receptor signaling. Upregulation of MyD88 is often associated with enhanced immune responses and may lead to stronger inflammatory responses.

[0084] The IKK (IκB kinase) complex comprises IKKα and IKKβ subunits, which play a crucial role in the NF-κB signaling pathway. Activation and phosphorylation of IKKα / β lead to the degradation of IκB protein, releasing NF-κB into the nucleus and initiating gene transcription. IKK phosphorylation is commonly associated with inflammation, immune responses, and certain cancers.

[0085] p65 protein: a subunit of the NF-κB transcription factor. Phosphorylation and activation of p65 are markers of NF-κB signaling pathway activation. Its upregulation is often associated with enhanced expression of inflammatory genes involved in various immune and inflammatory responses.

[0086] p38 protein: p38 is a member of the mitogen-activated protein kinase (MAPK) family and is involved in cellular stress responses, inflammation, and apoptosis. Phosphorylation and activation of p38 are generally associated with cellular responses to inflammatory factors and regulate cellular survival and apoptosis under stress conditions.

[0087] As mentioned in the background section, the structure and function of *Hedyotis diffusa* polysaccharide in the prior art are unclear, thus limiting its application. In this invention, the inventors attempted to purify *Hedyotis diffusa* polysaccharide and analyze its structure, further exploring its application in immunomodulation, and thus proposed a series of protection schemes for this invention.

[0088] In a first typical embodiment of the present invention, a hard-tipped vanilla polysaccharide is provided, which is composed of galactose, arabinose, and rhamnose; the hard-tipped vanilla polysaccharide has the following properties: Figure 6 The structure shown; the molecular weight of the above-mentioned hard-tipped vanilla polysaccharide is 149 kDa; the molar ratio of the above-mentioned galactose, the above-mentioned arabinose and the above-mentioned rhamnose is 59.59:34.53:5.88.

[0089] The *Hedyotis diffusa* polysaccharide with the above-described structure and composition significantly promotes the maturation of bone marrow-derived dendritic cells (BM-DCs), upregulates the expression of co-stimulatory molecules (CD40, CD86, MHC II), and enhances the secretion of pro-inflammatory cytokines (IL-6, TNF-α, IL-12p40, IL-1β), with its effects dependent on the TLR4 / NF-κB signaling pathway. In vivo experiments showed that in a cyclophosphamide (CTX)-induced immunosuppressed mouse model, the above-described *Hedyotis diffusa* polysaccharide (i.e., HCBP-0.1-W of this application) could reverse weight loss, immune organ atrophy, and leukopenia, and restore the balance of immune cell subsets. Metabolomics analysis further revealed that the above-described *Hedyotis diffusa* polysaccharide (i.e., HCBP-0.1-W of this application) enhances immune function by regulating amino acid metabolism pathways.

[0090] In a second typical embodiment of the present invention, a method for preparing the above-mentioned hard-tip sage polysaccharide is provided, the method comprising:

[0091] Degreasing and impurity removal were performed on the hard-tip angelica to obtain pretreated hard-tip angelica;

[0092] The pretreated *Hedyotis diffusa* was subjected to water extraction and alcohol precipitation to obtain crude polysaccharide;

[0093] The crude polysaccharide was decolorized and deproteinized to obtain a polysaccharide mixture.

[0094] The above polysaccharide mixture was purified to obtain the above-mentioned hard-tipped vanilla polysaccharide.

[0095] This preparation method ensures precise control of the polysaccharide components and molecular weight uniformity of *Hedyotis diffusa*. The *Hedyotis diffusa* polysaccharide HCBP-0.1-W extracted using this method exhibits immunomodulatory effects, activating specific immune signaling pathways in vivo and enhancing the function of immune cells.

[0096] In a preferred embodiment of the present invention, the steps of defatting and removing impurities from the above-mentioned hard-tipped angelica include: soaking the above-mentioned hard-tipped angelica in the above-mentioned organic reagent, filtering and drying to obtain the above-mentioned pretreated hard-tipped angelica.

[0097] In a preferred embodiment of the present invention, the organic reagent is selected from any one or more of the following: petroleum ether or 80-95% ethanol. The above organic solvents can remove impurities from *Hedyotis diffusa*. Petroleum ether can remove non-polar components such as fats, oils, waxes, chlorophyll, and fat-soluble pigments. 80-95% ethanol can remove small-molecule water-soluble impurities, such as small-molecule sugars (including monosaccharides, oligosaccharides, and oligosaccharides), phenols, tannins, organic acids (interfering components in some plants), and some alcohol-soluble pigments. Soaking *Hedyotis diffusa* in the above organic reagents can more thoroughly remove impurities and fats.

[0098] In a preferred embodiment of the present invention, the mass-to-volume ratio of the above-mentioned *Hedyotis diffusa* and the above-mentioned organic reagent is 1 g:(3-6) mL. In a preferred embodiment of the present invention, the soaking time is 1-2 h; in a preferred embodiment of the present invention, the soaking temperature is 50-60 °C. Soaking *Hedyotis diffusa* according to the above parameters has the beneficial effect of effectively removing impurities and fats from *Hedyotis diffusa*, significantly improving the polysaccharide extraction rate, and reducing the generation of by-products.

[0099] It should be noted that when multiple organic reagents are used for soaking, after the first soaking with the first organic reagent (this process is called the first round of soaking) is completed, the first organic reagent is replaced with the second organic reagent, and the soaking step of the first organic reagent is repeated (this process is called the second round of soaking). Before using the second organic reagent for the second round of soaking, the solvent from the first round of soaking needs to be removed to obtain filter residue. In a preferred embodiment of the present invention, the mass-to-volume ratio of the filter residue to the above-mentioned organic reagent is 1 g:(3-6) mL.

[0100] In a preferred embodiment of the present invention, each soaking process needs to be repeated 3 times; this makes the removal of impurities more thorough and ensures the purity of the subsequent polysaccharide extraction.

[0101] In a preferred embodiment of the present invention, the above-mentioned water extraction and alcohol precipitation steps include:

[0102] Water was mixed with the above-pretreated hard-tip angelica to extract polysaccharides, resulting in a solution containing polysaccharides;

[0103] The above-mentioned polysaccharide-containing solution was mixed with ethanol to precipitate the polysaccharide, thus obtaining the above-mentioned crude polysaccharide.

[0104] A polysaccharide of *Hedyotis diffusa* with a specific chemical composition was obtained through water extraction and alcohol precipitation. This polysaccharide consists of galactose, arabinose, and rhamnose in a molar ratio of 59.59:34.53:5.88, with a molecular weight of 149 kDa. This specific structure of the polysaccharide is more conducive to subsequent functional research and product development, ensuring the consistency and reliability of its biological activity.

[0105] In a preferred embodiment of the present invention, the mass-to-volume ratio (g / mL) of the pretreated *Hedyotis diffusa* and the water is 1:20-45; in a preferred embodiment of the present invention, the extraction temperature of the polysaccharide is 75-100°C; and the extraction time of the polysaccharide is 0.5-2.5 h.

[0106] In a preferred embodiment of the present invention, the final concentration of the ethanol is 60% to 80%; in a preferred embodiment of the present invention, the precipitation time of the polysaccharide is 20 to 26 hours.

[0107] In a more preferred embodiment of the present invention, the water extraction step is repeated three times before the alcohol precipitation step is performed.

[0108] In a preferred embodiment of the present invention, any one of the following decolorizing agents is selected for the above decolorization: polyamide, activated carbon, or hydrogen peroxide. In a preferred embodiment of the present invention, the mass ratio (g / g) of the above decolorizing agent to the above crude polysaccharide is 2-3:1; in a preferred embodiment of the present invention, the decolorization time is 2-6 hours. The above decolorization method has the beneficial effects of high selectivity, mild conditions, and low polysaccharide loss rate.

[0109] In a preferred embodiment of the present invention, the deproteinization is performed using the sevage method. This method offers the advantages of being simple to operate, operating under mild conditions, and achieving a high polysaccharide retention rate.

[0110] In a preferred embodiment of the present invention, the purification includes: performing anion exchange chromatography and gel filtration chromatography on the polysaccharide mixture to obtain the above-mentioned hard-tip senna polysaccharide; in a preferred embodiment of the present invention, the packing material for the anion exchange chromatography is selected from any one of the following: DEAE SFF; in a preferred embodiment of the present invention, the packing material for the gel filtration chromatography is selected from any one of the following: Sephadex G-75.

[0111] Anion exchange chromatography and gel filtration chromatography were used to effectively remove pigments and proteins from the polysaccharide extract, thus improving the purity of the polysaccharide. This purification method is not only simple to operate, but also maintains the integrity of the polysaccharide structure, avoiding potential damage to the polysaccharide caused by high temperatures and extreme pH values.

[0112] In a third typical embodiment of the present invention, a polysaccharide of *Hedyotis diffusa* prepared by the above-described preparation method is provided. This polysaccharide exhibits excellent immunomodulatory functions, such as promoting the maturation of dendritic cells (DCs), enhancing their antigen-presenting capacity, and activating immune activity. Furthermore, it can activate immune cells through the TLR4 / NF-κB signaling pathway, restore immune organ function, regulate immune cell subsets, thereby reversing immunosuppression and demonstrating its great potential as an immunomodulator.

[0113] In a fourth typical embodiment of the present invention, an application is provided for the use of the above-described vanilla polysaccharide or the vanilla polysaccharide prepared by the above-described preparation method in the preparation of a drug for immunomodulation. The above-described polysaccharide has a high molecular density and a regular branched structure, which not only increases its solubility in water but also enhances its stability in vivo circulation, thus contributing to improved bioavailability and making it more suitable for drug development and application.

[0114] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0115] Example

[0116] 1.1 Materials and Animals

[0117] The medicinal material used in this study, *Hyssopus cuspidatus* Boriss., was collected from farmers in Altay, Xinjiang (batch number: 20190820) and Xinjiang Weicaoji Medicinal Materials Co., Ltd. (batch number: 20240227). It is the dried aerial part of *Hyssopus cuspidatus*, a plant of the genus *Hyssopus* in the family Lamiaceae.

[0118] Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and CCL19 were purchased from PeproTech. PE / CY7 Anti-Mouse CD40, APC Anti-Mouse CD86, and FITC Anti-Mouse MHC-II were purchased from Elabscience Biotechnology Co., Ltd. Hematoxylin-eosin staining kit, DAB color development kit, and HRP-labeled goat anti-rabbit secondary antibody were purchased from Sevier Biotechnology Co., Ltd. ELISA kit was purchased from Elabscience Biotechnology Co., Ltd.

[0119] Animals: 48 SPF-grade BALB / c mice, 6 - 8 weeks old, weighing 18 - 22 g, were provided by Nanjing Mobowen Biotechnology Co., Ltd. Production License Number: SCXK(Yu)2020 - 0005, Experimental Animal Use License: SYXK(Xin)2023 - 0001.

[0120] 1.2 Isolation and purification study of Hyssopus cuspidatus polysaccharide

[0121] The crude polysaccharide of Hyssopus cuspidatus was extracted by the following method:

[0122] 1) Weigh the powdered medicinal materials and place them in a beaker. Add petroleum ether at 60°C at a material-liquid ratio of 1:5 and soak for 2 h. Repeat 3 times. For the residue after removing the solvent, add 95% ethanol at a material-liquid ratio of 1:5 and ultrasonicate for 1 h. Repeat 3 times. After cooling, filter by suction, and dry the filtered powder to obtain the pretreated Hyssopus cuspidatus powder sample.

[0123] 2) Take the pretreated Hyssopus cuspidatus powder sample, use water as the solvent, extraction temperature of 100°C, material-liquid ratio of 1:25 (g / mL), extract 3 times, each extraction for 1 h, final ethanol concentration of 75%, precipitate with alcohol overnight, and take the precipitate and dry it to obtain.

[0124] 3) Take the crude polysaccharide, dissolve it in water, add 2 g of polyamide per 50 ml, oscillate for decolorization for 4 h, use the sevage method to remove protein, after complete dialysis with a 3000 Da dialysis bag, perform freeze-drying to obtain a light yellow Hyssopus cuspidatus crude polysaccharide powder, named HCBP-C.

[0125] Through optimizing the technological conditions, a light yellow crude polysaccharide was extracted from Hyssopus cuspidatus, named HCBP-C. Subsequently, DEAE SFF ion exchange column was used for separation and purification. After treatment with 0.5 mol / L sodium hydroxide and hydrochloric acid, a polysaccharide solution of 100 mg / ml was prepared and then gradient elution was carried out.

[0126] Four components were obtained by gradient elution (see Figure 22The samples were HCBP-W, HCBP-0.05, HCBP-0.1, and HCBP-0.2, with yields of 2.29%, 5.15%, 11.08%, and 6.48%, respectively. After dialysis and freeze-drying, HCBP-0.1, with the highest yield, was selected as the primary sample. To further improve its purity, HCBP-0.1 was subjected to secondary chromatography on a Sephadex G-75 gel column, ultimately yielding purified sample HCBP-0.1-W with a yield of 3.18%. Figure 1 (A) in the middle.

[0127] 1.3 Results of molecular weight and homogeneity determination of polysaccharide HCBP-0.1-W

[0128] The molecular weight and homogeneity of HCBP-0.1-W samples were determined using an HPGPC system. This analytical system employed three Waters Ultrahydrogel columns (sizes 250, 1000, and 000, 30 cm × 7.8 mm) and RID was used for detection. 3 mM sodium acetate (0.05% sodium azide) was used as the mobile phase, with a flow rate of 0.5 mL / min, a column temperature of 40 °C, and a sample loading volume of 20 μL. A series of dextran standards (5.2 kDa, 4.86 kDa, 14.8 kDa, 23.8 kDa, and 66.8 kDa) were used to construct a molecular weight standard curve. The molecular weight distribution of HCBPs was accurately determined and the sample homogeneity was evaluated by analyzing the peak time and peak shape characteristics.

[0129] A standard equation was established based on molecular weight and elution time, yielding Log(Mw) = -8.0801T + 91.6393. The relative molecular mass was calculated based on the chromatographic retention time. Figure 1 As shown in B, the HPGPC spectrum of HCBP-0.1-W shows a single symmetrical peak with an elution time of 49.9 min. After calculation by software, it is determined that HCBP-0.1-W is a homogenized pure polysaccharide with a molecular weight of 149 kDa.

[0130] 1.4 Chemical Composition Analysis of Polysaccharides

[0131] Chemical composition analysis was performed on the polysaccharide sample HCBP-0.1-W extracted and purified from *Hedyotis diffusa*. First, the content of neutral sugars was determined by the phenol-sulfuric acid method and the uronic acid content by the sulfuric acid-carbazole method. The protein content was measured using the BCA method. Then, monosaccharide standards were prepared and derivatized for HPLC analysis. The sample was acid-hydrolyzed in 2M TFA, followed by PMP derivatization, and finally separated and analyzed using a C18 column to determine the monosaccharide composition. The content of each component was calculated using the standard curve method in all experiments.

[0132] Chemical composition analysis showed that HCBP-0.1-W obtained by dextran gel column chromatography contained 97.6% neutral sugars and 2.4% uronic acid, with no detected protein, indicating it is a neutral polysaccharide with a yield of 3.18%. Monosaccharide composition analysis showed (… Figure 1 The monosaccharide composition of HCBP-0.1-W is Rha (rhamnose), Gal (galactose), and Ara (arabinose), with a molar percentage of 5.45:58.36:36.19, suggesting that HCBP-0.1-W is an arabinogalactan.

[0133] 1.5 Structural Analysis of Polysaccharides

[0134] 1.5.1 Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscopy Analysis

[0135] The dried polysaccharide sample was ground together with dried spectral-grade potassium bromide powder, and test pieces were prepared using a hydraulic method, with the thickness of the pressed pieces controlled at 1 mm. The spectral measurement scanning range was set to 4000-400 cm⁻¹. -1 Record the infrared absorption characteristics of the sample.

[0136] The FTIR spectrum of HCBP-0.1-W is in the range of 4000–400 cm⁻¹. -1 The characteristic absorption band of polysaccharides is shown within the range. Figure 1 (C) 3300cm -1 The absorption peaks appearing nearby are usually caused by the stretching vibrations of the abundant hydroxyl groups (-OH) in polysaccharide molecules. Due to the effects of intermolecular and intramolecular hydrogen bonds, the absorption peak of the stretching vibrations of the hydroxyl groups appears at 3300 cm⁻¹. -1 It is located near the infrared spectrum and exhibits a strong and broad characteristic, which is a typical characteristic peak of polysaccharide compounds in the infrared spectrum.

[0137] 2924cm -1 The weak band at 1600 cm⁻¹ is generally caused by the CH stretching vibrations of the methyl (-CH₃) and methylene (-CH₂-) groups in polysaccharide molecules. -1 The strong absorption peak at 1420 cm⁻¹ is due to the C=O stretching vibration of the carboxyl group (-COOH) in the polysaccharide molecule. -1 The vibration is caused by the CH bending vibrations of the methyl (-CH3) and methylene (-CH2-) groups in the polysaccharide molecule. 1377cm -1 The peak at 1076 cm⁻¹ is usually caused by the symmetric bending vibration of the methyl group (-CH₃). -1 This generally reflects the absorption peak of the stretching vibration of CO in polysaccharide molecules.

[0138] Take a small amount of the sample powder after grinding with HCBP-0.1-W, spread it evenly on a 0.8cm×0.8cm conductive adhesive with a toothpick, blow off the floating powder on the sample surface, and observe the shape and surface features of the sample with SEM.

[0139] Based on the SEM images of HCBP-0.1-W at different magnifications (see...) Figure 2 As shown in A), the polysaccharides are all lamellar in shape. The purified HCBP-0.1-W exhibits a regular striped texture and a porous structure, indicating that the polysaccharide has many branches and a high molecular density. The branched structure may present as a striped or fibrous arrangement, and the regular texture can be observed under magnification. The hydroxyl and carboxyl functional groups on the surface of the polysaccharide may form a network structure through hydrogen bonding or electrostatic interactions. This tightly conformed lamellar structure may enhance the mechanical stability of the polysaccharide, increase its specific surface area, and improve its adsorption or sustained-release properties, making it suitable for the preparation of biomolecular pharmaceutical materials.

[0140] 1.5.2 Methylation Analysis

[0141] 20 mg of pre-dried polysaccharide sample was placed in anhydrous DMSO under nitrogen protection, activated with NaH, and then iodomethane was added dropwise. The reaction was carried out in an ice bath at 4 °C. Afterwards, impurities were removed by dialyzing and the product was lyophilized to obtain the methylated product. The methylated polysaccharide was acid-hydrolyzed with 2 mol / L trifluoroacetic acid (TFA) at 120 °C for 4 hours. Residual TFA was removed by rotary evaporation and methanol elution. The hydrolysis product was reduced with sodium borohydride at room temperature for 2 hours. The reaction was terminated by adjusting the pH to neutral with acetic acid, and impurities were removed by rotary evaporation.

[0142] The reduced product was then acetylated by reacting acetic anhydride in pyridine at 90 °C for 2 hours. Extraction with dichloromethane and concentration under reduced pressure were performed. The final acetylated product was analyzed by gas chromatography-mass spectrometry (GC-MS) using a DB-5 capillary column. The temperature program ranged from 140 °C to 250 °C, with high-purity helium as the carrier gas, and 70 eV electron impact ionization was used for mass spectrometry.

[0143] Methylation is widely used in the structural analysis of polysaccharides because it can determine and analyze the linkage mode of glycosidic bonds in polysaccharides. In other words, it is one of the essential methods for polysaccharide structural analysis.

[0144] After methylation, acid hydrolysis, reduction, derivatization, and GC-MS analysis of the HCBP-0.1-W sample, the analytical results of methylated HCBP-0.1-W were obtained (e.g., Figure 2 (As shown in B in the figure). Figure 2B in the figure is the total ion chromatogram of HCBP-0.1-W after methylation. As can be seen from the figure, the connection mode of HCBP-0.1-W is relatively complex, with 9 peaks. Referring to the standard PMAA mass spectrometry database of the Complex Carbohydrate Research Center of the University of Georgia, USA, combined with the standard spectrum, the ionization law of PMAA and relevant literature, the analysis was carried out. The molar ratio was calculated according to the relative abundance, and the results are shown in Table 1.

[0145] Analysis results show that HCBP-0.1-W is composed of five sugar residues, namely, the branched sugar residue furanose, the terminal sugar residue furanose, the terminal sugar residue furanose, the branched sugar residue furanose, and the terminal sugar residue rhamnose, with a residue molar ratio of 59.59:34.53:5.88.

[0146] Table 1. GC-MS analysis of HCBP-0.1-W methylation

[0147]

[0148]

[0149] 1.5.3 Nuclear Magnetic Resonance Analysis

[0150] Accurately weigh 30 mg of HCBP-0.1-W polysaccharide sample and dehydrate it in a desiccator for 24 hours. Dissolve the sample in 300 μL of deuterated water (D2O). Transfer the dissolved sample to an NMR tube and perform one-dimensional and two-dimensional NMR analysis at room temperature using a nuclear magnetic resonance spectrometer. Specific detection items include: one-dimensional proton NMR (1H NMR) and carbon NMR (13C NMR), and two-dimensional NMR correlation spectroscopy (1H-1H COSY), heteronuclear multi-bond correlation spectroscopy (1H-13C HMBC), and heteronuclear single quantum coherence spectroscopy (1H-13C HSQC).

[0151] 1D NMR of HBCP-0.1-W Figure 3 As shown in A and B in HCBP-0.1-W. 1 H NMR spectrum ( Figure 3 Nine anomeric proton signals were observed at position A), located at 4.51, 4.43, 4.42, 4.68, 5.08, 5.25, 5.24, 5.09 and 4.65 ppm, respectively. The residues corresponding to these nine anomeric proton signals were labeled as residues “A” to “I”.

[0152] In HCBP-0.1-W 13 C NMR spectrum ( Figure 3Nine anomeric carbon signals were observed at B) in the figure, located at 103.49, 103.76, 103.77, 103.95, 107.79, 109.51, 109.48, 107.79 and 101.06 ppm respectively.

[0153] In HCBP-0.1-W 1H-1H COSY Atlas Figure 4 The value A) reflects the correlation between adjacent protons in the sugar residue. Combining the nine H-1 / H-2 correlation signals at δ4.51 / 3.66, δ4.43 / 3.53, δ4.42 / 3.77, δ4.68 / 3.75, δ5.08 / 4.13, δ5.25 / 4.22, δ5.24 / 4.22, δ5.09 / 4.13, and δ4.65 / 3.68 in the COSY spectrum, the H-2 of residues “A” to “I” was determined. Meanwhile, the H-3 of residues “A” to “I” was confirmed by nine additional H-2 / H-3 related signals located at δ3.66 / 3.69, δ3.53 / 3.57, δ3.77 / 3.69, δ3.75 / 3.57, δ4.13 / 3.94, δ4.22 / 4.09, δ4.22 / 4.03, δ4.13 / 4.09, and δ3.68 / 3.34 in COSY.

[0154] HSQC spectrum of HCBP-0.1-W ( Figure 4 B) indicates the correlation signal between the proton and the directly linked carbon. Further combining with HSQC, the C-1, C-2, and C-3 corresponding to H-1, H-2, and H-3 of the nine residues were assigned. The C-2 of residues “A” to “I” are located at 70.23, 72.09, 70.59, 72.08, 80.42, 81.64, 84.33, and 80.34 ppm, respectively, and the C-3 are located at 75.29, 74.70, 75.29, 74.70, 76.93, 76.93, 84.35, and 76.93 ppm, respectively.

[0155] In the HMBC spectrum of HCBP-0.1-W ( Figure 5In A), the signal at δ3.66 / 103.49 indicates a correlation between H-6 of β-1,6-Galp and C-1 of β-1,3,6-Galp; the signal at δ3.69 / 103.77 indicates a correlation between H-3 of β-1,3,6-Galp and C-1 of β-1,3-Galp; the signal at δ4.51 / 66.51 indicates a correlation between H-1 and C-6 of β-1,3,6-Galp; and the signal at δ4.42 / 66.51 indicates a correlation between β-1... A correlation exists between H-1 of β-3-Galp and C-6 of β-1,6-Galp; the signal at δ4.43 / 66.51 indicates a correlation between H-1 and C-6 of β-1,6-Galp; the signal at δ3.69 / 103.95 indicates a correlation between H-3 of β-1,3-Galp and C-1 of β-T-Galp; the signal at δ5.08 / 84.35 indicates a correlation between H-1 of α-T-Araf and C-3 of α-1,3-Araf; δ5 The signal at δ 0.08 / 66.51 indicates a correlation between H-1 of β-T-Galp and C-6 of β-1,3,6-Galp; the signal at δ 5.25 / 75.29 indicates a correlation between H-1 of α-1,5-Araf and C-3 of β-1,3-Galp; the signal at δ 5.25 / 75.29 indicates a correlation between H-1 of α-1,5-Araf and C-3 of β-1,3,6-Galp; the signal at δ 5.24 / 66.62 indicates a correlation between α-1,3... A correlation exists between H-1 of α-Araf and C-5 of α-1,5-Araf; the signal at δ3.81 / 109.51 indicates a correlation between H-5 of α-1,2,5-Araf and C-1 of α-1,5-Araf; the signal at δ3.86 / 109.51 indicates a correlation between H-5 and C-1 of α-1,5-Araf; the signal at δ5.08 / 66.62 indicates a correlation between H-1 of α-T-Araf and C-5 of α-1,5-Araf.

[0156] HMBC maps provide connectivity information between adjacent sugar residues, reflecting long-range coupling of approximately two to three bond lengths between protons and carbons. In addition to reflecting adjacent carbon-hydrogen correlations within the same sugar ring, HMBC can also provide signals related to the linkages between adjacent residues.

[0157] In the NOESY spectrum of HCBP-0.1-W ( Figure 5The 14 signals at δ5.25 / 3.69, δ5.08 / 3.86, δ5.25 / 3.81, δ5.24 / 3.86, δ4.03 / 5.08, δ3.69 / 4.68, δ4.51 / 3.66, δ4.43 / 3.66, δ4.68 / 3.66, δ4.51 / 3.66, δ3.69 / 4.42, δ4.42 / 3.66, δ5.25 / 3.86, and δ3.69 / 5.25 further confirm these connections. The NOESY spectrum simultaneously reflects spatial crossover signals within and between sugar residues. Generally, HMBC is often used in conjunction with NOESY for resolving sugar residue linkage sequences.

[0158] Based on the above information, the possible structure of HCBP-0.1-W can be deduced as follows: Figure 6 As shown, the polysaccharide backbone consists of 12 galactose molecules linked together via the following pathways: →6-β-Galp-1→3,6-β-Galp-1→, →3,6-β-Galp-1→3,6-β-Galp-1→, →3,6-β-Galp-1→6-β-Galp-1→, and →6-β-Galp-1→6-β-Galp-1→.

[0159] From left to right on the main chain, these are branch chains 1 to 8:

[0160] Branch 1 is connected to the main chain by two galactoses (including the terminal galactose) via →3,6-β-Galp-1→3-β-Galp-1→.

[0161] Branch 2 consists of 3 galactoses (including the terminal galactose) connected to the main chain via →3,6-β-Galp-1→6-β-Galp-1→, and 2 galactoses connected via →6-β-Galp-1→3-β-Galp-1→.

[0162] Branch 3 is connected to the main chain by a galactose and a terminal rhamnose via →3,6-β-Galp-1→3-β-Galp-1→.

[0163] Branch 4 consists of 4 galactoses (including terminal galactose) and 1 terminal rhamnose connected to the main chain via →3,6-β-Galp-1→3-β-Galp-1→, and 3 galactoses connected via →3-β-Galp-1→6-β-Galp-1→3,6-β-Galp-1→.

[0164] Branch 5 consists of one galactose and five arabinoses (including the terminal arabinose) connected to the main chain via →3,6-β-Galp-1→3-β-Galp-1→. The galactose and arabinose are connected via →3-β-Galp-1→2,5-α-Araf-1→, and the five arabinoses are connected via →2,5-α-Araf-1→5-α-Araf-1→.

[0165] Branch 6 consists of three arabinoses (including the terminal arabinose) connected to the main chain via →3,6-β-Galp-1→5-α-Araf-1→, and the three arabinoses are connected via →5-α-Araf-1→5-α-Araf-1→.

[0166] Branch 7 consists of two arabinoses (including the terminal arabinose) connected to the main chain via →3,6-β-Galp-1→5-α-Araf-1→.

[0167] Branch 8 consists of three arabinoses (including the terminal arabinose) connected to the main chain via →3,6-β-Galp-1→5-α-Araf-1→, and the three arabinoses are connected via →5-α-Araf-1→3-α-Araf-1→.

[0168] 1.6 Bone marrow-derived dendritic cell experiment

[0169] 1.6.1 Flow cytometry analysis and enzyme-linked immunosorbent assay

[0170] Bone marrow-derived dendritic cells (BM-DCs) were obtained using an induced culture method. Cells were collected and counted on day 7. Different concentrations of the sample polysaccharide HCBP-0.1-W were then added to cell culture plates. The experimental group used 40 ng / mL LPS (lipopolysaccharide) as a positive control. After 12 hours of culture, the expression levels of CD40 and CD86 were analyzed by flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was performed using a double-antibody sandwich method to detect cytokines (IL-6, TNF-α, IL-12p40, and IL-1β) to confirm the effect of polysaccharides on cytokine release.

[0171] Flow cytometry results showed that the polysaccharide significantly promoted DC maturation after stimulation with gradient concentrations of HCBP-0.1-W (20-100 μg / ml), as evidenced by the presence of the surface co-stimulatory molecule CD40. Figure 7 A in CD86 Figure 7 B) and MHC II ( Figure 7 The expression of C) in the LPS was upregulated in a dose-dependent manner, and the expression level in the 100 μg / ml concentration group was comparable to that in the LPS positive control group.

[0172] Meanwhile, ELISA detection showed the pro-inflammatory cytokine IL-6 ( Figure 8 A) TNF-α Figure 8 B), IL-12p40 ( Figure 8 C) and IL-1β ( Figure 8 The secretion of D) in the body was significantly increased, especially the high expression of IL-12p40, suggesting that it promoted Th1 immune polarization.

[0173] Functional aspects ( Figure 9 HCBP-0.1-W inhibited the phagocytic ability of DCs, and this inhibitory effect increased with increasing concentration, which is consistent with the transition from antigen capture to antigen presentation function during DC maturation.

[0174] Migration capability detection shows ( Figure 10 In both groups A and B, HCBP-0.1-W significantly upregulated CCR7 expression and enhanced DC migration in the Transwell system, comparable to the LPS group. Further T cell co-culture experiments demonstrated (…). Figure 11 (A and B in the sample), HCBP-0.1-W significantly promoted the proliferation of CD4+ and CD8+ T cells, especially CD8+ T cells.

[0175] In addition, intracellular IFN-γ detection showed ( Figure 11 In the C and D sections, HCBP-0.1-W treatment enhanced the cytotoxic T lymphocyte (CTL) response of CD8+ T cells, suggesting that it exerts an immune activation effect by promoting Th1-type immune effector function against pathogens. These results indicate that HCBP-0.1-W successfully mimics the activation characteristics of pathogen-associated molecules by synergistically regulating the phenotypic maturation and functional transition of DCs, prompting DCs to transform from highly efficient antigen capture to producing pro-inflammatory cytokines and initiating specific T cell responses, demonstrating its potential as an immunomodulator.

[0176] 1.6.2 Immunoblotting assay

[0177] Collect induced dendritic cells (DCs), wash twice with 5 mL PBS (1200 rpm, 7 min), and discard the supernatant. Add RIPA lysis buffer containing 1 / 100 volume of protease inhibitor, incubate on ice for 20 min, vortexing for 30 seconds every 5 min. Centrifuge at 12000 rpm for 15 min at 4 °C, collect the supernatant as total protein, and store at -20 °C. Determine protein concentration using a BSA standard curve according to the Thermo Fisher Protein Quantification Kit instructions. Adjust protein volume, add sample buffer, and boil in a water bath for 10 min to denature. Prepare a 10% SDS-PAGE gel, perform electrophoresis at 50 mA for 90 min, transfer the protein to a PVDF membrane, and block with 5% skim milk powder for 1 hour. Incubate with primary antibody overnight at 4 °C, wash three times with PBST the next day, and incubate with HRP secondary antibody at 37 °C for 1 hour. Detect protein bands using chemiluminescence staining and LAS4000 imaging.

[0178] To investigate the signal transduction pathway by which HCBP-0.1-W promotes DC maturation, DCs were pretreated with the TLR4 inhibitor TAK-242 and the NF-κB inhibitor BAY11-7082. The results showed ( Figure 12 In the absence of inhibitors, both HCBP-0.1-W and LPS significantly increased the expression of CD86 and CD40; however, this upregulation effect was significantly inhibited in the presence of inhibitors, indicating that the TLR4 and NF-κB signaling pathways play a key role in the regulation of DC maturation by HCBP-0.1-W.

[0179] Combined with Western Blot analysis ( Figure 13 and Figure 15 Treatment with HCBP-0.1-W led to upregulation of MyD88 protein expression, along with a significant increase in the phosphorylation levels of IKKα / β, p65, and p38 proteins, reflecting activation of the NF-κB and MAPK signaling pathways. These data suggest that HCBP-0.1-W promotes DC maturation and immune function by activating the TLR4 signaling pathway, which uses MyD88 as an aptamer, thereby inducing the activation of downstream key transcription factors.

[0180] In summary, HCBP-0.1-W relies on the TLR4 / NF-κB pathway to activate its immunomodulatory effects (see...). Figure 14 The precise mechanism of action and potential cross-regulation of this pathway should be further clarified through molecular biology methods such as gene silencing and overexpression, so as to provide a theoretical basis for understanding the immune activation mechanism of this polysaccharide and its development.

[0181] 1.7 Mouse experimental protocol and metabolomics analysis

[0182] 1.7.1 Experimental Design

[0183] Healthy male BALB / c mice weighing 18-22g were randomly divided into four groups: a control group, a model group, a positive control group (LEX), and HCBP-0.1-W treatment groups (10, 20, and 30 mg / kg), with 10 mice in each group. During the experiment, mice in the model and treatment groups received intraperitoneal injections of CTX to induce immunosuppression, followed by gavage administration of different doses of HCBP-0.1-W starting from day 4 for 13 days. Mouse weight was monitored, and blood samples and tissue samples from multiple organs were collected on day 17 for subsequent analysis, including serum cytokine levels, complete blood count, liver and kidney function, and organ indices.

[0184] 1.7.2 Metabolomics Analysis

[0185] After collecting serum samples, metabolite analysis was performed using ultra-high performance liquid chromatography (UHPLC) and high-resolution mass spectrometry (HMS). Sample processing included metabolite extraction using methanol and acetonitrile, followed by two-step mass spectrometry detection using nitrogen splitting. After processing and mass spectrometry identification of the raw data, 27,925 stable metabolites were ultimately screened. Of these, 1,624 identified metabolites underwent principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). KEGG pathway annotation and enrichment analysis identified significant metabolic pathways, and the regulatory relationships between metabolites and pathways were visualized using bubble diagrams and heatmaps, providing clues to the immunomodulatory effects and potential mechanisms of HCBP-0.1-W.

[0186] 1.7.3HCBP-0.1-W restores immune organ function

[0187] HCBP-0.1-W significantly enhanced the immunosuppressive effect of CTX-induced mice. In the experiment, after continuous gavage intervention with HCBP-0.1-W (10-30 mg / kg) or the positive control drug LEX (20 mg / kg), the body weight of mice in the CTX group decreased significantly, while HCBP-0.1-W intervention showed a dose-dependent protective effect. The high-dose group (30 mg / kg) showed a significantly higher rate of body weight recovery from day 7 than the CTX group. Figure 17 Post-anatomical organ index assessment showed that CTX treatment resulted in a significant decrease in spleen and thymus indices (p<0.001). Figure 16 (This is accompanied by visible organ atrophy. However, after HCBP-0.1-W intervention, the spleen index and thymus index recovered in a dose-dependent manner, and the organ volume and tissue structure in the medium and high dose groups were close to normal levels.)

[0188] 1.7.4HCBP-0.1-W regulates immune cell subsets

[0189] Flow cytometry results showed that HCBP-0.1-W exhibited significant dose heterogeneity in restoring immune cell subsets. The repair of T cells (including CD4+ T cells and CD8+ T cells) and DCs (CD11c-positive cells) was optimal in the medium-dose group (20 mg / kg); the ratio of DCs to macrophages almost recovered to levels close to the control group (p>0.05), showing superior effects compared to the high- and low-dose groups. In contrast, B cells and NK cells showed an inverse dose-dependent effect, with the low-dose group (10 mg / kg) showing the most significant effect on B cell ratio restoration and NK cell ratio recovery, while the high-dose group (30 mg / kg) showed weaker effects. These results suggest that HCBP-0.1-W can target and reconstruct immune homeostasis through precise dose regulation, and its mechanism of action may involve the multi-level dynamic balance of the TLR4 pathway.

[0190] 1.7.5 Spleen Microenvironment Restoration

[0191] Immunohistochemical results from spleen tissue indicated that HCBP-0.1-W had a significant repairing effect on CTX-induced spleen immune cell damage. Figure 19 In the CTX model group, the density of dendritic cells (DCs) and T cells in the spleen of mice was significantly reduced (p<0.001 vs. control group), with white pulp structure disintegration and sparse lymphocyte distribution. After intervention with medium-dose HCBP-0.1-W, the density of DCs significantly increased; CD4+ T cells and CD8+ T cells recovered synchronously, with their densities reaching 41% and 43% of the control group, respectively. Figure 18 The above results suggest that HCBP-0.1-W reverses CTX-induced immunosuppression by regulating the proliferation and localization of key immune cells in the spleen microenvironment.

[0192] 1.7.6 Metabolic Regulation Mechanisms

[0193] To further elucidate the metabolic mechanism of HCBP-0.1-W, KEGG was used for pathway enrichment analysis to screen differential metabolites and explore the metabolic pathways through which HCBP-0.1-W intervenes in CTX. Characteristic differential metabolites of HCBP-0.1-W were used to screen metabolic pathways with p < 0.05 and impact > 0.01. The metabolic regulatory pathways of each intervention group on CTX mice were investigated, such as... Figure 20 As shown.

[0194] Differential metabolites from CTX and HCBP-0.1-W were mainly annotated and enriched in 10 metabolic pathways, with 7 being the most significant (p<0.05). CTX / HCBP-0.1-W differential metabolites were enriched in pyrimidine metabolism, choline metabolism, arginine biosynthesis, and glycerophospholipid metabolism. Correlation analysis of 17 differential metabolites with cytokines and immune cells showed that HCBP-0.1-W intervention increased the levels of arginine, γ-glutamate, lysine, and dimethylglycine. These metabolites may promote the activation of monocytes, lymphocytes, and neutrophils through some mechanism, affecting the secretion of pro-inflammatory cytokines such as IL-6, IL-12, and IL-1β. This positive correlation suggests that *Hedyotis diffusa* polysaccharide mainly regulates immune homeostasis through amino acid metabolism, such as... Figure 21 As shown.

[0195] 1.8 Statistical Analysis of Data

[0196] Experimental data are expressed as mean ± standard deviation (mean ± SD) and were analyzed using GraphPad Prism 5.0 software for one-way ANOVA. For comparisons among multiple groups, if the ANOVA result was significant (p < 0.05), Tukey's multiple comparison test was further used to determine which groups had significant differences. All experiments were repeated at least three times independently to ensure the reliability and reproducibility of the results. A p-value less than 0.05 was considered statistically significant (p < 0.05; p < 0.01; p < 0.001).

[0197] From the above description, it can be seen that the embodiments of the present invention achieve the following technical effects: This study isolated and purified a homogeneous polysaccharide HCBP-0.1-W (molecular weight 149 kDa) from *Hedyotis diffusa*. Structural analysis (FTIR, GC-MS, NMR) showed that HCBP-0.1-W is a highly branched arabinogalactan, mainly composed of arabinose and galactose, with a complex three-dimensional structure. In vitro experiments showed that HCBP-0.1-W significantly promoted the maturation of bone marrow-derived dendritic cells (BM-DCs), upregulated the expression of co-stimulatory molecules (CD40, CD86, MHC II), and enhanced the secretion of pro-inflammatory cytokines (IL-6, TNF-α, IL-12p40, IL-1β), its effects depending on the TLR4 / NF-κB signaling pathway. In vivo experiments showed that in a cyclophosphamide (CTX)-induced immunosuppressed mouse model, HCBP-0.1-W could reverse weight loss, immune organ atrophy, and leukopenia, and restore the balance of immune cell subsets. Metabolomics analysis further revealed that HCBP-0.1-W enhances immune function by regulating amino acid metabolism pathways.

[0198] This study elucidates for the first time that HCBP-0.1-W is an arabinogalactan targeting TLR4, which can activate dendritic cells and improve CTX-induced immunosuppression, providing a theoretical basis for its clinical application as an immunoadjuvant. Furthermore, in-depth research into the immunomodulatory effects of *Hedyotis diffusa* polysaccharides can not only elucidate the material basis of its water extract in treating related diseases, but also provide new insights for the development and utilization of *Hedyotis diffusa*.

[0199] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polysaccharide of *Hedyotis diffusa*, characterized in that, The hard-tip vanilla polysaccharide is composed of galactose, arabinose, and rhamnose; the hard-tip vanilla polysaccharide has the following structure; the molecular weight of the hard-tip vanilla polysaccharide is 149 kDa, and the molar ratio of galactose, arabinose, and rhamnose is 59.59:34.53:5.88; 2. The method for preparing the polysaccharide of *Hedyotis diffusa* according to claim 1, characterized in that, The preparation method includes: Degreasing and impurity removal were performed on the hard-tip angelica to obtain pretreated hard-tip angelica; The pretreated vanilla buds were subjected to water extraction and alcohol precipitation to obtain crude polysaccharide; The crude polysaccharide was subjected to decolorization and protein removal treatment to obtain a polysaccharide mixture; The polysaccharide mixture was purified to obtain the hard-tipped vanilla polysaccharide.

3. The preparation method according to claim 2, characterized in that, The steps of defatting and removing impurities from the hard-tipped angelica include: The herb *Hedyotis diffusa* was soaked in an organic reagent, filtered, and dried to obtain the pretreated herb *Hedyotis diffusa*. Preferably, the organic reagent is selected from any one or more of the following: petroleum ether or 80-95% ethanol; Preferably, the mass-to-volume ratio of the herb *Hedyotis diffusa* to the organic reagent is 1:3 to 6; Preferably, the soaking time is 1 to 2 hours; Preferably, the soaking temperature is 50–60°C.

4. The preparation method according to claim 2, characterized in that, The steps of water extraction and alcohol precipitation include: Water was mixed with the pretreated hard-tipped angelica to extract polysaccharides, resulting in a solution containing polysaccharides; The polysaccharide-containing solution was mixed with ethanol to precipitate the polysaccharide, thereby obtaining the crude polysaccharide. Preferably, the mass-to-volume ratio of the pretreated hard-tip angelica to the water is 1:20-45; Preferably, the polysaccharide extraction temperature is 75–100°C; the polysaccharide extraction time is 0.5–2.5 h. Preferably, the final concentration of the ethanol is 60-80%; Preferably, the polysaccharide precipitation time is 20-26 hours.

5. The preparation method according to claim 2, characterized in that, Choose any one of the following decolorizing agents for the decolorization: polyamide, activated carbon, or hydrogen peroxide; Preferably, the mass ratio of the decolorizing agent to the crude polysaccharide is 2-3:1; Preferably, the decolorization time is 2 to 6 hours; Preferably, the deproteinization process is performed using the sevage method.

6. The preparation method according to any one of claims 2-5, characterized in that, The purification process includes performing anion exchange chromatography and gel filtration chromatography on the polysaccharide mixture to obtain the hard-tip vanilla polysaccharide.

7. The preparation method according to claim 6, characterized in that, The packing material for the anion exchange chromatography is selected from any of the following: DEAE SFF.

8. The preparation method according to claim 6, characterized in that, The packing material for the gel filtration chromatography is selected from any of the following: Sephadex G-75.

9. Hard-tipped vanilla polysaccharide prepared by any one of claims 2-8.

10. The use of the senna polysaccharide of claim 1, or the preparation method of any one of claims 2-8, or the senna polysaccharide of claim 9 in the preparation of a medicament for immunomodulation.

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