A homogeneous dendrobium huoshanense acidic polysaccharide, a preparation method and application thereof

Abstract

The application relates to the technical field of polysaccharides, and discloses a uniform Dendrobium huoshanense acidic polysaccharide as well as a preparation method and application thereof; the main chain of the chemical structure of the uniform Dendrobium huoshanense acidic polysaccharide comprises ->3,6)-beta-D-Galp-(1->, ->3)-beta-D-Galp-(1-> and ->3)-beta-D-Glcp-(1-> residues, side chains comprise alpha-L-Araf-(1->4)-beta-D-Xylp-(1->, alpha-L-Arap-(1->3)-alpha-L-Araf->, alpha-L-Araf-(1->4)-beta-D-Manp-> and 4-OMe-beta-D-Galp-(1->4)-beta-D-Galp->, and the side chains are all connected to O-6 sites of the ->3,6)-beta-D-Galp-(1-> residues; the uniform Dendrobium huoshanense acidic polysaccharide is a polysaccharide with a novel structure and can be used for preparing immunomodulatory products.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide technology, specifically to a homogeneous acidic polysaccharide from Dendrobium huoshanense, its preparation method, and its application. Background Technology

[0002] The immune system is crucial for maintaining physiological homeostasis, as it defends against pathogens, clears abnormal cells, and regulates inflammatory responses. Immune system dysfunction can lead to a range of diseases, including autoimmune diseases, chronic inflammatory diseases, and decreased anti-tumor defense capabilities. Therefore, the search for safe and effective immunomodulators has become a key focus of biomedical and pharmaceutical research. Currently, commonly used immunosuppressants and immunostimulants in clinical practice are often accompanied by significant adverse reactions. Therefore, developing low-toxicity, high-efficiency immunomodulators from natural sources is considered a highly promising strategy.

[0003] Polysaccharides, as the main active ingredients of traditional Chinese medicine, are known for their immunomodulatory properties, low toxicity, and good biocompatibility. Studies have shown that polysaccharides derived from plants, animals, and microorganisms can regulate immune responses by activating immune cells, enhancing cytokine release, and influencing signaling pathways. Their bioactivity is closely related to their physicochemical properties, such as molecular weight, monosaccharide composition, glycosidic bonds, and branching patterns.

[0004] Dendrobium huoshanense CZ Tang et SJ Cheng is a perennial herb belonging to the genus Dendrobium in the family Orchidaceae. It originates from Huoshan County, Anhui Province, and has important medicinal value. Its stems contain various compounds such as polysaccharides, bibenzyl groups, phenylpropanoids, and flavonoids. Among them, polysaccharides are the main active ingredients. However, the structure of Dendrobium huoshanense polysaccharides is complex. In the current technology, research on Dendrobium huoshanense polysaccharides in wild simulated environments is still relatively limited. In particular, the efficient extraction and structural study of polysaccharides with good immunomodulatory properties in Dendrobium huoshanense still need further exploration. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a homogeneous Dendrobium huoshanense acidic polysaccharide. The main chain of the homogeneous Dendrobium huoshanense acidic polysaccharide comprises →3,6)-β-D-Galp-(1→, →3)-β-D-Galp-(1→ and →3)-β-D-Glcp-(1→) residues, and the side chains comprise α-L-Araf-(1→4)-β-D-Xylp-(1→, α-L-Arap-(1→3)-α-L-Araf→, α-L-Araf-(1→4)-β-D-Manp→ and 4-OMe-β-D-Galp-(1→4)-β-D-Galp→. All side chains are attached to the O-6 sites of the →3,6)-β-D-Galp-(1→) residues.

[0006] As can be seen from the above, the homogeneous Dendrobium huoshanense acidic polysaccharide is an acidic polysaccharide with a novel structure that is different from existing technologies.

[0007] Preferably, the homogeneous Dendrobium huoshanense acidic polysaccharide has a molecular weight of 83.637 kDa, a number-average molecular weight of 52.439 kDa, and a polydispersity index of 1.595.

[0008] Preferably, the homogeneous Dendrobium huoshanense acidic polysaccharide has a triple helix conformation.

[0009] Preferably, the homogeneous Dendrobium huoshanense acidic polysaccharide includes arabinose, rhamnose, xylose, glucose, mannose, and galactose.

[0010] Preferably, the molar ratio of arabinose, rhamnose, xylose, glucose, mannose and galactose is 29.03:36.51:10.03:8.69:7.49:4.32.

[0011] This invention also discloses a method for preparing uniform acidic polysaccharides from Dendrobium huoshanense, comprising the following steps: The refined polysaccharides of Dendrobium huoshanense were separated by DEAE-52 cellulose column elution, eluted with NaCl aqueous solution, and the eluent was collected, concentrated and freeze-dried to obtain the acidic polysaccharide fraction of Dendrobium huoshanense. The acidic polysaccharide fraction of Dendrobium huoshanense was separated and purified multiple times by Sephadex G-200 gel column elution to obtain a homogeneous acidic polysaccharide of Dendrobium huoshanense, denoted as DHPS1Ah.

[0012] Preferably, the Dendrobium huoshanense includes Dendrobium huoshanense cultivated in a semi-wild environment.

[0013] Preferably, the method for preparing the refined polysaccharide of Dendrobium huoshanense is as follows: Dendrobium huoshanense crude polysaccharide is mixed with water at a mass ratio of 1:5 and dissolved to obtain an aqueous solution of Dendrobium huoshanense crude polysaccharide; a n-butanol-chloroform solution is prepared at a volume ratio of 1:4, and the n-butanol-chloroform solution and the Dendrobium huoshanense crude polysaccharide aqueous solution are thoroughly mixed at a volume ratio of 4:1, centrifuged, and the aqueous phase is collected. The above steps are repeated until no denatured proteins appear at the interface and no ultraviolet light absorption occurs in the wavelength range of 250-280 nm. The resulting supernatant is concentrated by rotary evaporation and then freeze-dried.

[0014] Preferably, the method for preparing the acidic polysaccharide component of Dendrobium huoshanense is as follows: Refined polysaccharide from Dendrobium huoshanense is mixed with water at a mass ratio of 1:5, separated using a DEAE-52 cellulose column, and eluted with a gradient of distilled water, 0.1 mol / L NaCl aqueous solution, 0.2 mol / L NaCl aqueous solution, and 0.3 mol / L NaCl aqueous solution at a flow rate of 5 mL / min. The eluted portion from the 0.1 mol / L NaCl aqueous solution is collected, concentrated, and then freeze-dried.

[0015] Preferably, the Sephadex G-200 gel column separation and purification method involves using distilled water as the mobile phase at a flow rate of 0.5 mL / min and collecting single-peak components.

[0016] Preferably, the Sephadex G-200 gel column separation and purification is performed twice.

[0017] The homogeneous Dendrobium huoshanense acidic polysaccharide of the present invention can be used in immunomodulatory products.

[0018] Furthermore, the immunomodulatory products include immunomodulatory drugs or immunomodulatory functional foods.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention obtains a homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) from Dendrobium huoshanense cultivated in a semi-wild environment. The chemical structure of the Dendrobium huoshanense acidic polysaccharide includes a main chain composed of →3,6)-β-D-Galp-(1→, →3)-β-D-Galp-(1→ and →3)-β-D-Glcp-(1→) residues, and side chains composed of α-L-Araf-(1→4)-β-D-Xylp-(1→, α-L-A Composed of rap-(1→3)-α-L-Araf→, α-L-Araf-(1→4)-β-D-Manp→, and 4-OMe-β-D-Galp-(1→4)-β-D-Galp→, with side chains attached to the O-6 sites of the →3,6)-β-D-Galp-(1→) residues, this polysaccharide exhibits a highly branched arabinogalactan structure and a triple helix conformation; furthermore, its molecular weight is 83.637. The homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) has a number-average molecular weight of 52.439 kDa and a polydispersity index of 1.595. Furthermore, the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) is composed of arabinose (29.03%), rhamnose (36.51%), xylose (10.03%), glucose (8.69%), mannose (7.49%) and galactose (4.32%). Therefore, the DHPS1Ah of the present invention is different from the polysaccharides of the prior art and is an acidic polysaccharide with a novel structure.

[0020] 2. The homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) of the present invention possesses excellent immunomodulatory properties due to the uniqueness of its monosaccharide types, composition, and structure. Specifically, DHPS1Ah contains six monosaccharides: arabinose (Ara), rhamnose (Rha), galactose (Gal), glucose (Glc), xylose (Xyl), and mannose (Man). Among them, arabinose can serve as a TLR4 receptor recognition site, constructing arabinogalactan side chains, improving water solubility, and bidirectionally regulating the immune response; rhamnose has the highest proportion, giving the polysaccharide acidic characteristics and enhancing the interaction between the polysaccharide and the immune system. The binding capacity of the TLR4-MD2 complex stabilizes the triple helix conformation and prevents excessive activation of inflammation; xylose elongates the polysaccharide side chain structure, enabling multivalent binding to immune receptors to amplify activation effects and regulate intestinal mucosal immunity; glucose participates in the backbone construction via β-1,3 glycosidic bonds, supporting the higher-order conformation of the triple helix and synergistically amplifying immune signals with multiple receptors; mannose can specifically bind to the macrophage mannose receptor, synergistically promoting lymphocyte proliferation with the TLR4 pathway and maintaining immune homeostasis; galactose is the core unit of the backbone, relying on the O-6 branch site to connect all active side chains, and is the key backbone for the formation of the triple helix structure of polysaccharides; Secondly, the differentiated monosaccharide molar ratio of DHPS1Ah in this invention differs from existing polysaccharides. The high ratio of rhamnose and arabinose generates numerous branched side chains, resulting in a polysaccharide molecular weight of 83.637 kDa. Unlike existing low-molecular-weight linear neutral polysaccharides from Dendrobium huoshanense, this moderate molecular weight offers significant advantages. On one hand, it can multivalently cross-link the TLR4 receptor, efficiently initiating the MAPK / NF-κB signaling pathway with ERK, JNK, p38, and p65 phosphorylation. On the other hand, the large molecule is not easily degraded by gastric acid and digestive enzymes, allowing it to reach the intestines and exert a long-lasting effect. Simultaneously, the hydrophilic side chains compensate for the poor solubility of the large molecule, and DHPS1Ah only needs to bind to cell membrane surface receptors to take effect, eliminating the problem of insufficient cell penetration. Furthermore… A special monosaccharide ratio forms an α / β mixed glycosidic bond system. The β-(1→3) type galactose and glucose glycosidic bonds in the main chain maintain molecular rigidity and ensure the integrity of the triple helix conformation. Once the conformation is unwound, the immune activity will be significantly reduced. The main chain →3,6)-β-D-Galp-(1→) serves as a branching point, carrying various active side chains. The side chains are derived from arabinose, xylose, and mannose, providing a large number of immune recognition sites. They can promote the secretion of NO, IL-6, and TNF-α in a dose-dependent manner and enhance the proliferation capacity of splenic lymphocytes. Therefore, relying on the multi-level synergistic effect of monosaccharide composition, molecular weight, glycosidic bonds and higher conformations, DHPS1Ah exhibits superior immunomodulatory activity compared to similar polysaccharides under non-cytotoxic conditions.

[0021] 3. This invention uses *Dendrobium huoshanense* cultivated in a semi-wild environment as raw material. The purification process involves ethanol extraction, water extraction, Sevag deproteinization, DEAE cellulose column separation, and two Sephadex G-200 gel column separations to obtain DHPS1Ah. During the DEAE cellulose column separation, a 0.1M NaCl aqueous solution was chosen as the eluent. This is because DHPS1Ah is a highly branched acidic arabinogalactan with a specific density of negative charge, forming a moderate electrostatic bond with the DEAE cellulose column packing material. Water elution only collects neutral polysaccharides, while higher salt concentrations (0.2 mol / L NaCl or higher) simultaneously elute heteropolysaccharides with higher charges. However, 0.1 mol / L NaCl... The ionic strength of NaCl is just right to neutralize the negative charge on the surface of DHPS1Ah, accurately dissociating the target component and achieving single enrichment. This compatibility is determined by the unique structure of DHPS1Ah, which does not match the charge characteristics of conventional Dendrobium huoshanense polysaccharides. Using general elution conditions, it is impossible to specifically separate and obtain the homogeneous polysaccharide of this invention. Therefore, based on the special structure and properties of DHPS1Ah, this invention obtains homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) by designing and optimizing the extraction process. Furthermore, the present invention employs a multiple Sephadex G-200 gel column purification strategy, which is more targeted and reproducible in operation compared with the interception method in the prior art. Therefore, the polysaccharide extraction yield is higher according to the method of the present invention. In addition, the chromatography column separation technology used in the present invention can select and assemble preparation columns of different specifications according to the sample amount, which is suitable for the preparation of large-scale homogenized sugars. Attached Figure Description

[0022] Figure 1 This is a flowchart of the preparation process of homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) in Example 1 of the present invention; Figure 2 The DEAE-52 cellulose column elution curve of the refined polysaccharide of Dendrobium huoshanense prepared in Example 1 of this invention; Figure 3 This is the second Sephadex G-200 gel column elution curve of the acidic polysaccharide component (DHPS1) of Dendrobium huoshanense prepared in Example 1 of this invention; Figure 4 This is the ultraviolet spectrum of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention; Figure 5 This is the HPSEC-MALLS-RI chromatogram of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention; Figure 6 This refers to the molar mass of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention; Figure 7 This is a chromatogram of the monosaccharide composition of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention; Figure 8 This is the infrared spectrum of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention; Figure 9 The results of the triple helix structure analysis of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention; Figure 10 This is the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention. 1 H NMR spectrum; Figure 11 This is the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention. 13 C NMR spectrum; Figure 12 This is the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention. 1 H- 13 C HSQC chart; Figure 13 This is the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention. 1 H- 13 C HMBC map; Figure 14 This is the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention. 1 H- 1 H NOSEY map; Figure 15 This is a schematic diagram of the chemical structure of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) of the present invention; Figure 16 This is the chemical structural formula of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention; Figure 17 This describes the effect of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention on the cell viability of Raw 264.7 cells at different concentrations. Figure 18 This is the effect of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention on the proliferation of splenic lymphocytes at different concentrations; Figure 19 The results show the effect of different concentrations of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention on the NO level in Raw264.7 cells; Figure 20 The results show the effect of different concentrations of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention on the IL-6 concentration in Raw264.7 cells; Figure 21 The results show the effect of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention on the TNF-α concentration in Raw264.7 cells at different concentrations. Figure 22 The effect of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention on the expression of TLR4 / MAPK signaling pathway-related proteins in RAW264.7 macrophages; Figure 23 This invention relates to a quantitative analysis of the expression of TLR4 / MAPK signaling pathway-related proteins in RAW264.7 macrophages using homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention. Figure 23 Figure a shows the relative expression level of TLR4 protein, Figure b shows the relative phosphorylation intensity of p-ERK / ERK protein, Figure c shows the relative phosphorylation intensity of p-JNK / JNK protein, Figure d shows the relative phosphorylation intensity of p-p65 / p65 protein, and Figure e shows the relative phosphorylation intensity of p-p38 / p38 protein. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0024] Example 1 This embodiment discloses a method for preparing uniform acidic polysaccharides from Dendrobium huoshanense, comprising the following steps: Step S1: Pulverize the dried, semi-wild cultivated Dendrobium huoshanense stems, then extract them with a 95% ethanol aqueous solution for 3 hours to obtain the residue. Step S2: Evaporate the residue from Step S1 to dryness, add water, the mass ratio of residue to water is 1:10, extract at 70 ℃ for 4 h, repeat 3 times, combine the filtrates and concentrate, add 4 times the volume of ethanol to the filtrate, precipitate at 4 ℃ for 24 h to obtain crude polysaccharide of Dendrobium huoshanense, denoted as CDHP. Step S3: Mix the crude polysaccharide of Dendrobium huoshanense with water at a mass ratio of 1:5 and dissolve to obtain an aqueous solution of crude polysaccharide of Dendrobium huoshanense; prepare a n-butanol-chloroform solution at a volume ratio of 1:4, and mix the n-butanol-chloroform solution with the aqueous solution of crude polysaccharide of Dendrobium huoshanense at a volume ratio of 4:1. Centrifuge at 5000 r / min for 5 min, collect the aqueous phase and repeat the above steps until no denatured proteins appear at the interface and no ultraviolet light absorption occurs in the wavelength range of 250-280 nm. The supernatant obtained is concentrated by rotary evaporation and then freeze-dried to obtain refined polysaccharide of Dendrobium huoshanense, denoted as RDHP. Step S4: The refined polysaccharide of Dendrobium huoshanense was mixed with water at a mass ratio of 1:5, dissolved, and separated using a DEAE-52 cellulose column (16×600mm). Gradient elution was performed with distilled water, 0.1 mol / L NaCl aqueous solution, 0.2 mol / L NaCl aqueous solution, and 0.3 mol / L NaCl aqueous solution at a flow rate of 5 mL / min. The eluted fraction of 0.1 mol / L NaCl aqueous solution was collected, concentrated, and freeze-dried to obtain the acidic polysaccharide component of Dendrobium huoshanense, denoted as DHPS1. Step S5: The acidic polysaccharide component of Dendrobium huoshanense was further separated and purified using a Sephadex G-200 gel column (20×1000 mm). Distilled water was used as the mobile phase and the flow rate was 0.5 mL / min. The single peak component was collected to obtain DHPS1A. DHPS1A was purified by Sephadex G-200 column purification in the same way as above to obtain homogeneous Dendrobium huoshanense acidic polysaccharide, denoted as DHPS1Ah.

[0025] Experimental Example I. The method according to Example 1 of the present invention (preparation process as follows) Figure 1 (As shown) Homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) was prepared, and the extraction yield was tested. The extraction yield (R) was calculated using the following formula: R(%)=(W 1 / W)×100； in, W 1 represents the dry weight of refined polysaccharide (RDHP), acidic polysaccharide component (DHPS1), and homogeneous acidic polysaccharide (DHPS1Ah) from Dendrobium huoshanense. W The dry weight of Dendrobium huoshanense stems cultivated in a semi-wild environment.

[0026] The extraction yields of refined polysaccharide (RDHP) from Dendrobium huoshanense were 12.75%; the extraction yield of acidic polysaccharide component (DHPS1) from Dendrobium huoshanense was 1.66%; the extraction yield of DHPS1A was 46.5%; and the extraction yield of homogeneous acidic polysaccharide (DHPS1Ah) from Dendrobium huoshanense was 88.6% (relative to DHPS1A). As can be seen from the above, the extraction efficiency of homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) according to the method of Example 1 of the present invention is relatively high. First, the present invention selects 0.1M NaCl aqueous solution as the eluent. This is because DHPS1Ah is a highly branched acidic arabinogalactan with a specific density of negative charge on its molecules, which forms a moderate electrostatic bond with DEAE cellulose column packing. Water elution only collects neutral polysaccharides, while high salts such as 0.2 mol / L NaCl aqueous solution will simultaneously elute heteropolysaccharides with higher charges. The ionic strength of 0.1 mol / L NaCl is just enough to neutralize the negative charge on the surface of DHPS1Ah, accurately dissociate the target component, and achieve single enrichment. This compatibility is determined by the unique structure of DHPS1Ah. The charge characteristics of conventional Dendrobium huoshanense polysaccharides do not match it, and the homogeneous polysaccharide of the present invention cannot be specifically separated using general elution conditions. Furthermore, the present invention further purifies the elution fraction from 0.1M NaCl aqueous solution using a Sephadex G-200 gel column to obtain DHPS1Ah. The present invention employs a multiple Sephadex G-200 gel column purification strategy, which is more targeted and reproducible in operation compared with the interception method in the prior art. Therefore, the polysaccharide extraction yield is higher according to the method of the present invention. In addition, the chromatography column separation technology used in the present invention allows for the assembly of different specifications of preparation columns according to the sample amount, which is suitable for the preparation of large-scale homogenized sugars.

[0027] II. The structure of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention was characterized by the following method: 1. In Example 1 of this invention, the refined polysaccharide of *Dendrobium huoshanense* was purified using a DEAE-52 cellulose column to obtain the acidic polysaccharide component (DHPS1). During the DEAE-52 cellulose column purification process, the absorbance was measured using the anthrone-sulfuric acid assay to plot the elution curve. Then, the acidic polysaccharide component of *Dendrobium huoshanense* was separated and purified twice using a Sephadex G-200 gel column to obtain a homogeneous acidic polysaccharide of *Dendrobium huoshanense* (DHPS1Ah). During the Sephadex G-200 gel column purification process, the absorbance was measured using the anthrone-sulfuric acid assay to plot the elution curve. The test results are as follows: Figures 2-3 As shown: Figure 2 The DEAE-52 cellulose column elution curve of the refined polysaccharide of Dendrobium huoshanense prepared in Example 1 of this invention; Figure 3 This is the second Sephadex G-200 gel column elution curve of the acidic polysaccharide component (DHPS1) of Dendrobium huoshanense prepared in Example 1 of this invention; Depend on Figures 2-3It can be seen that after preliminary separation of the refined polysaccharide of Dendrobium huoshanense using a DEAE-52 cellulose column, DHPS1 was obtained from the 0.1 M NaCl eluent, accounting for 1.66% of the total product. Subsequently, DHPS1 was separated by size exclusion chromatography using a Sephadex G-200 gel column to obtain DHPS1A (accounting for 46.5%). Then, DHPS1A was purified twice using Sephadex G-200 gel columns using the same method to obtain homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah).

[0028] 3. Purity testing The homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) sample prepared in Example 1 of this invention was scanned at all wavelengths using a UV-Vis spectrophotometer (model: TU-1950) to verify that there were no nucleic acids or residual proteins in the sample. Test results are as follows Figure 4 As shown: Figure 4 This is the UV spectrum of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention; (The text abruptly ends here, likely due to an incomplete sentence or missing information.) Figure 4 It can be seen that no obvious absorption peaks were observed at 260 nm and 280 nm, indicating that the DHPS1Ah sample has been purified to remove protein and nucleic acid biomolecules, and the sample has high purity.

[0029] 4. Molecular weight determination The molecular weight of DHPS1Ah was determined by high performance size exclusion chromatography-multi-angle laser light scattering-differential refractive index detection (HPSEC-MALLS-RI). The specific detection method was as follows: the molecular weight and uniformity of DHPS1Ah were determined by size exclusion chromatography (SEC-MALLS-RI) coupled with multi-angle laser light scattering and refractive index detection; the analysis was performed using a Thermo U3000 high-performance liquid chromatography (HPLC) platform equipped with a DAWN HELEOS II light scattering detector and an Optilab T-rEX refractive index detector; chromatographic separation was performed at 45 °C, with Ohpak SB-805 HQ and SB-803 HQ columns (300×8 mm) connected in series; an aqueous buffer solution containing 0.1 M NaNO3 and 0.02 wt% NaN3 was used as the mobile phase, and elution was performed at a constant flow rate of 0.6 mL / min.

[0030] Test results are as follows Figures 5-6 As shown; Figure 5 This is the HPSEC-MALLS-RI chromatogram of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention; Figure 6 This refers to the molar mass of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention; Depend on Figures 5-6 It can be seen that the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 exhibits a single and symmetrical elution peak, indicating that the sample is a homogeneous polysaccharide. A homogeneous polysaccharide refers to a polysaccharide composed of fixed repeating units, and is not limited to a polysaccharide composed of only one monosaccharide. Its weight-average molecular weight (Mw) was calculated to be 83.637 kDa, number-average molecular weight (Mn) was 52.439 kDa, polydispersity index (PDI) was 1.595, Z-average molecular weight (Mz) was 228.731 kDa, and peak molecular weight (Mp) was 69.104 kDa.

[0031] 5. Monosaccharide composition analysis The monosaccharide composition of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention was tested using acid hydrolysis combined with ion chromatography: The specific detection method was as follows: Monosaccharide components were detected by ICS 5000+ high-performance anion exchange chromatography-pulse amperometric method (HPAEC-PAD) using a CarboPac PA-20 column for quantitative analysis. Before injection, DHPS1Ah (5 mg) was mixed with 2 mL of 2 mol / L trifluoroacetic acid aqueous solution and hydrolyzed at 120 °C for 2 h. Acid was then removed by multiple methanol evaporations under nitrogen atmosphere. The resulting dried hydrolysis product was dissolved in deionized water and eluted at a flow rate of 0.5 mL / min. The mobile phase included water (A), 0.1 M NaOH aqueous solution (B), and a mixture containing 0.1 M NaOH and 0.2 M NaOH. The aqueous solution of sodium acetate (C) was prepared using the following gradient program: 0 minutes was 95:5:0 (volume ratio of A:B:C, the same below), 26-42 minutes was 85:5:10, 42.1 minutes was 60:0:40, 52 minutes was 60:40:0, and 52.1-60 minutes was 95:5:0.

[0032] Test results are as follows Figure 7 As shown: Figure 7 This is a chromatogram of the monosaccharide composition of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention; [The remaining text appears to be incomplete and requires further context.] Figure 7 It is known that DHPS1Ah is mainly composed of arabinose (Ara), rhamnose (Rha), galactose (Gal), glucose (Glc), xylose (Xyl), and mannose (Man), with a molar ratio of 29.03:36.51:4.32:8.69:10.03:7.49; in addition, it also contains small amounts of fucose (Fuc) and glucuronic acid (Glc-UA).

[0033] 6. Infrared spectroscopy analysis Fourier transform infrared spectroscopy was used to analyze the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention at 4000-400 cm⁻¹ using a Thermo iS50 infrared spectrometer. -1 The Fourier transform infrared spectrum (FT-IR) within the range is used for detection; Test results are as follows Figure 8 As shown: Figure 8 This is the infrared spectrum of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention; as shown... Figure 8 As shown, at 3325 cm -1 and 2935 cm -1 The absorption peaks at 1735 cm⁻¹ correspond to the stretching vibrations of the OH and CH bonds, respectively; -1 and 1630 cm -1 The stretching signal at this location confirmed the presence of uronic acid; 1141 cm -1 and 1030 cm -1 The peak at 875 cm⁻¹ indicates the presence of pyranose and furanose structures; -1 and 810 cm -1 The peaks at these locations correspond to the β- and α-terminal group configurations, respectively.

[0034] 7. Triple Helix Structure Analysis The triple helix conformation of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention was evaluated using the Congo red test. The specific detection method is as follows: DHPS1Ah solution with a concentration of 2.0 mg / mL (solvent is PBS buffered salt solution) and Congo red solution with a concentration of 80 μM (solvent is PBS buffered salt solution) are mixed at a volume ratio of 1:1. Then, NaOH aqueous solutions of different concentrations are added to make the final NaOH concentration between 0 and 1.0 M. After incubation for 5 min, the maximum absorption wavelength (λmax) is measured in the wavelength range of 400-700 ns. DHPS1Ah solution without Congo red is used as a blank control.

[0035] Test results are as follows Figure 9 As shown: Figure 9 This is the result of the triple helix structure analysis of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention; as... Figure 9 As shown, the maximum absorption wavelength of the DHPS1Ah-Congo red complex exhibits a significant red shift, confirming the presence of a triple helix conformation in this polysaccharide.

[0036] 8. Methylation analysis The homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention was subjected to methylation derivatization analysis, and the results were detected by GC-MS.

[0037] The specific detection method is as follows: Dissolve DHPS1Ah (2 mg) in 500 μL DMSO, add 1 mg NaOH, incubate at 30 ℃ for 30 min, add 50 μL iodomethane, react at 25 ℃ for 1 h, add 1 mL water and 3 mL dichloromethane, vortex extract, centrifuge to separate layers, discard the upper aqueous phase, repeat the extraction step 3 times; combine the organic phases, blow dry with nitrogen, add 100 μL 2 mol / L TFA aqueous solution, acid hydrolyze at 121 ℃ for 90 min, evaporate dry at 30 ℃, add ammonia aqueous solution (2 mol / L, 50 μL) and NaBD4 aqueous solution (1 mol / L, 50 μL) in sequence, mix well, reduce at 25 ℃ for 2.5 h, add 20 μL acetic acid to terminate the reaction, blow dry with nitrogen, then wash twice with 250 μL methanol, blow dry again, add 250 μL acetic anhydride, vortex, acetylate at 100 ℃ for 2.5 h, cool, add 1 mL water mL, let stand for 10 min, add 500 μL of dichloromethane, vortex, centrifuge, discard the aqueous phase, wash three times, collect the lower dichloromethane phase for GC-MS detection; an Agilent 6890A 5977B GC-MS system equipped with a G4567A autosampler was used, with a TG200 column (30 m × 0.25 mm × 0.25 μm); injection volume 1 μL, split ratio 10:1, carrier gas high-purity helium, flow rate 1.5 mL / min; column temperature program: 150 ℃ for 1 min, ramp to 210 ℃ at 2 ℃ / min and hold for 2 min, then ramp to 240 ℃ at 2 ℃ / min and hold for 3 min; mass spectrometer equipped with EI ion source, run in SCAN mode; operating parameters included: ion source temperature 200 ℃, ionization voltage 50 eV, quadrupole temperature 110 ℃, transfer interface temperature 210 eV. ℃, scan range set to m / z 50-350.

[0038] The results are shown in Table 1: Table 1

[0039] As shown in Table 1, this invention analyzed and identified 10 sugar alcohol acetate derivatives, mainly including terminal arabinose (t-Araf, t-Arap), 1,3-linked arabinose (1,3-Araf), terminal galactose (t-Galp), 1,4-linked xylose (1,4-Xylp), 1,3-linked glucose (1,3-Glcp), 1,3-linked galactose (1,3-Galp), 1,4-linked mannose (1,4-Manp), 1,4-linked galactose (1,4-Galp), and 1,3,6-linked galactose (1,3,6-Galp).

[0040] 9. Nuclear magnetic resonance spectroscopy analysis One-dimensional and two-dimensional nuclear magnetic resonance spectroscopy analysis was performed on the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention. 1 H NMR, 13 C NMR, 1 H- 1 H COSY、 1 H- 13 C HSQC, 1 H- 13 C HMBC, 1 H- 1 H NOSEY); The specific detection method is as follows: DHPS1Ah (10 mg) is dissolved in D2O (0.5 mL) and detected using a 600 MHz nuclear magnetic resonance (NMR) instrument at 298 K.

[0041] The results are shown in Table 2 and Figures 10-16 As shown, Table 2

[0042] Figure 10 This is the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention. 1 H NMR spectrum; Figure 11 This is the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention. 13 C NMR spectrum; Figure 12 This is the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention. 1 H- 13 C HSQC chart; Figure 13 This is the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention. 1 H- 13C HMBC map; Figure 14 This is the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention. 1 H- 1 H NOSEY map; Figure 15 This is a schematic diagram of the structure of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) of the present invention; Figure 16 This is the chemical structural formula of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention; From Table 2 and Figure 10-16 It can be known that: The ¹H NMR spectrum showed a dominant signal in the δ 3.0–5.5 ppm range, with multiple coupling peaks detected in the terminal region (δ 4.3–5.4 ppm), confirming the presence of various sugar residues and a complex structure. Specific terminal hydrogen chemical shifts were observed at δ 4.41 ppm, 4.42 ppm, 4.43 ppm, 4.44 ppm, 4.57 ppm, 4.62 ppm, 4.67 ppm, 5.02 ppm, 5.17 ppm, and 5.19 ppm, respectively. The internal solvent signal was located at δ 4.71 ppm, while the O-CH3 proton signal appeared at δ 3.42 ppm. 13 The C10 NMR spectrum also exhibits multiple resonance peaks in the terminal carbon region. The HSQC spectrum clearly shows the signals of these terminal hydrogens and their corresponding carbon atoms. The heteronuclear correlation cross-peaks are located at δ 4.43 / 103.16, 5.19 / 109.24, 4.42 / 103.7, 5.02 / 107.39, 5.17 / 107.07, 4.44 / 103.47, 4.41 / 101.63, 4.58 / 104.35, 4.67 / 100.72, and 4.63 / 103.85. ppm; these typical signals are labeled as residues of type A, B, C, D, E, F, G, H and J, respectively; the following structural units were inferred by combining methylation data, terminal chemical shifts and relevant literature data: →3,6)-β-D-Galp-(1→, α-L-Araf-(1→, →3)-β-D-Galp-(1→, α-L-Arap-(1→, →3)-α-L-Araf-(1→, 4-OMe-β-D-Galp-(1→, →4)-β-D-Xylp-(1→, →4)-β-D-Galp-(1→, →4)-β-D-Manp-(1→ and →3)-β-D-Glcp-(1→); the complete identification of H2-H6 signals within each residue was completed by combining HSQC spectroscopy with known NMR databases (Table 3); in addition, δ 59.98 The carbon signal at ppm indicates the presence of O-CH3 groups; To clearly define the specific glycosidic linkages, the following was performed: 1 H- 13 C HMBC and 1 H- 1 H NOSEY analysis, 1 H- 13 The CHMBC spectrum revealed key inter-residue cross-peaks, specifically including: the correlation between C-1 of residue A and H-3 of residue C (δ 103.16 / 3.61), H-1 of residue D and C-3 of residue E (δ 5.02 / 83.31), H-1 of residue G and C-6 of residue A (δ 4.41 / 69.48), and H-1 of residue I and C-6 of residue A (δ 4.67 / 69.48); through 1 H- 1 H NOSEY spectra verified spatial connectivity, thus defining the polysaccharide sequence. The major intra- and inter-residual proton correlations (H1→Hx) identified are as follows: A-H1 / A-H3 (δ 4.43 / 3.65 ppm) and A-H1 / C-H3 (δ 4.43 / 3.61 ppm); B-H1 and G-H4 (δ 5.19 / 3.70 ppm) and I-H4 (δ 5.19 / 3.61 ppm); C-H1 / J-H3 (δ 4.42 / 3.81 ppm), E-H1 / A-H6 (δ 5.17 / 3.86 ppm), and F-H1 / H-H4 (δ 4.43 / 4.06 ppm); Additionally, G-H1 / A-H6 (δ 4.41 / 3.99 ppm) and H-H1 / A-H6 (δ 4.41 / 3.99 ppm) were also identified. The correlation between 4.58 / 3.86 ppm and J-H1 / A-H3 (δ4.63 / 3.65 ppm); In summary, the structural diagram of DHPS1Ah is as follows: Figure 15 As shown, the main chain consists of →3,6)-β-D-Galp-(1→, →3)-β-D-Galp-(1→ and →3)-β-D-Glcp-(1→) residues, and the side chains consist of α-L-Araf-(1→4)-β-D-Xylp-(1→, α-L-Arap-(1→3)-α-L-Araf→, α-L-Araf-(1→4)-β-D-Manp→ and 4-OMe-β-D-Galp-(1→4)-β-D-Galp→, all linked to the O-6 sites of the →3,6)-β-D-Galp-(1→) residue. Its chemical structure is shown in the figure. Figure 16 As shown, this polysaccharide has the structural characteristics of arabinogalactan.

[0043] III. Evaluation of the immunomodulatory activity of homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) 1. Cell Culture Mouse spleen lymphocyte isolation: The spleen was aseptically removed, minced in RPMI-1640 medium, filtered through a 200-mesh sieve, and the red blood cells were lysed. The lymphocytes were resuspended in RPMI-1640 medium containing 10% fetal bovine serum (FBS). Raw264.7 macrophages were cultured in DMEM medium containing 10% heat-inactivated fetal bovine serum at 37 ℃ in a 5% CO2 incubator.

[0044] 2. Cell viability assay The cytotoxicity of DHPS1Ah against Raw 264.7 cells was evaluated using the CCK-8 assay. Raw 264.7 cells were seeded in 96-well plates (1×10⁶ cells / wells). 5 Different concentrations (50-400 μg / mL) of DHPS1Ah solution were prepared using PBS buffer and Raw 264.7 cells were treated for 24 hours. After incubation with CCK-8 solution for 4 hours, the absorbance was measured at 450 nm. A blank control group was also included, using an equal volume of PBS buffer instead of DHPS1Ah solution, with all other conditions remaining the same. Results are as follows: Figure 17 As shown; Figure 17 This describes the effect of homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention on the cell viability of Raw 264.7 cells at different concentrations; Figure 17 It can be seen that DHPS1Ah has no significant effect on cell viability within the concentration range of 50-400 μg / mL, indicating that this polysaccharide has no cytotoxicity within this concentration range.

[0045] 3. Spleen lymphocyte proliferation experiment Splenic lymphocytes (1×10) 5 Cells / well were incubated with different concentrations (50-400 μg / mL) of DHPS1Ah solution (using PBS buffer as solvent) for 24 hours, and cell proliferation was detected by CCK-8 assay. In addition, a blank control group and a positive control group were set up. The blank control group was replaced with an equal volume of PBS buffer instead of DHPS1Ah solution, and the positive control group was replaced with an equal volume of 5 μg / mL Concanavalin A instead of DHPS1Ah solution, with other conditions remaining unchanged. The results are as follows Figure 18 As shown: Figure 18This describes the effect of homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention on the proliferation of splenic lymphocytes at different concentrations; [The remaining text appears to be a fragmented and incomplete sentence, possibly due to OCR errors. A more accurate translation would require the full context.] Figure 18 It can be seen that DHPS1Ah can significantly stimulate lymphocyte proliferation at 50 μg / mL, and the stimulatory effect is enhanced with increasing concentration.

[0046] 4. NO and cytokine secretion detection Raw264.7 cells (1×10⁻⁶) 5 Cells / well were co-incubated with DHPS1Ah solutions of different concentrations (50-400 μg / mL) for 24 hours. NO levels in the supernatant were detected using the Griess method, and TNF-α and IL-6 concentrations were detected using an ELISA kit. In addition, a blank control group and a positive control group were set up. The blank control group was replaced with an equal volume of PBS buffer instead of DHPS1Ah solution, and the positive control group was replaced with an equal volume of lipopolysaccharide at a concentration of 1 μg / mL instead of DHPS1Ah solution. All other conditions remained unchanged. The results are as follows Figures 19-21 As shown: Figure 19 The results show the effect of different concentrations of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention on the NO production level of Raw264.7 cells; Figure 20 The results show the effect of different concentrations of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention on the IL-6 concentration in Raw264.7 cells; Figure 21 The results show the effect of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention on the TNF-α concentration in Raw264.7 cells at different concentrations. Depend on Figures 19-21 It was found that, compared with the blank control group, the release of NO, TNF-α and IL-6 in the treatment group was significantly increased in a dose-dependent manner (p<0.05), indicating that DHPS1Ah can effectively activate macrophages.

[0047] 5. Western blot analysis Raw264.7 cells (1×10⁻⁶) 6Cells (cells / well) were seeded in 6-well plates and treated with different concentrations (50, 100, 200 μg / mL) of DHPS1Ah solution for 24 hours. Total protein was extracted, and the phosphorylation levels of TLR4, NF-κB p65, ERK, p38, and JNK were detected by Western blot. A blank control group and a positive control group were also set up. The blank control group was treated with an equal volume of PBS buffer instead of DHPS1Ah solution, and the positive control group was treated with an equal volume of lipopolysaccharide (1 μg / mL) instead of DHPS1Ah solution. All other conditions remained unchanged. Results are as follows: Figures 22-23 As shown: Figure 22 The effect of the homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention on the expression of TLR4 / MAPK signaling pathway-related proteins in RAW264.7 macrophages; Figure 23 This invention relates to a quantitative analysis of the expression of TLR4 / MAPK signaling pathway-related proteins in RAW264.7 macrophages using homogeneous Dendrobium huoshanense acidic polysaccharide (DHPS1Ah) prepared in Example 1 of this invention. Figure 23 Figure a shows the relative expression level of TLR4 protein, Figure b shows the relative phosphorylation intensity of p-ERK / ERK protein, Figure c shows the relative phosphorylation intensity of p-JNK / JNK protein, Figure d shows the relative phosphorylation intensity of p-p65 / p65 protein, and Figure e shows the relative phosphorylation intensity of p-p38 / p38 protein. Depend on Figures 22-23 It was found that at concentrations of 100 μg / mL and 200 μg / mL, TLR4 protein expression was significantly upregulated (p<0.01), and the phosphorylation levels of NF-κB p65, ERK, p38 and JNK were significantly increased, indicating that DHPS1Ah exerts an immunomodulatory effect by activating the TLR4 / MAPK / NF-κB signaling pathway.

[0048] In summary, DHPS1Ah of the present invention is an acidic polysaccharide with excellent immunomodulatory effects. As is well known, monosaccharide composition is one of the fundamental structural components of polysaccharide bioactivity; the types and molar ratios of the component monosaccharides, as well as their synergistic effects with other structural parameters, jointly determine the functional properties of polysaccharides. Due to the uniqueness of its monosaccharide types, composition, and structure, DHPS1Ah of the present invention possesses the aforementioned immunomodulatory properties, specifically manifested in: First, the DHPS1Ah of this invention contains six monosaccharides: arabinose (Ara), rhamnose (Rha), galactose (Gal), glucose (Glc), xylose (Xyl), and mannose (Man). Among them, arabinose can serve as a TLR4 receptor recognition site, constructing arabinogalactan side chains, improving water solubility and bidirectionally regulating immune responses; rhamnose has the highest proportion, giving the polysaccharide acidic characteristics, enhancing the binding ability of the polysaccharide to the TLR4-MD2 complex, stabilizing the triple helix conformation, and avoiding excessive activation of inflammation; xylose extends the polysaccharide side chain structure, realizing multivalent binding to immune receptors to amplify the activation effect, and can also regulate intestinal mucosal immunity; glucose participates in the main chain construction through β-1,3 glycosidic bonds, supporting the triple helix higher-order conformation, and synergistically amplifying immune signals with multiple receptors; mannose can specifically bind to macrophage mannose receptors, synergistically promoting lymphocyte proliferation with the TLR4 pathway, and maintaining immune homeostasis; galactose is the core unit of the main chain, relying on the O-6 branch site to connect all active side chains, and is the key backbone for the formation of the triple helix structure of the polysaccharide. Secondly, the differentiated monosaccharide molar ratio of DHPS1Ah in this invention differs from existing polysaccharides. The high rhamnose and arabinose ratio generates numerous branched side chains, resulting in a polysaccharide molecular weight of 83.637 kDa. Unlike existing low-molecular-weight linear neutral polysaccharides from Dendrobium huoshanense, this moderate molecular weight offers significant advantages. On one hand, it can multivalently cross-link the TLR4 receptor, efficiently initiating the MAPK / NF-κB signaling pathway with ERK, JNK, p38, and p65 phosphorylation. On the other hand, the large molecule is not easily degraded by gastric acid and digestive enzymes, allowing it to reach the intestines and exert a long-lasting effect. Simultaneously, the hydrophilic side chains compensate for the poor solubility of the large molecule, and the polysaccharide only needs to bind to cell membrane surface receptors to take effect, eliminating the problem of insufficient cell penetration. Furthermore, the special… The monosaccharide ratio forms an α / β mixed glycosidic bond system. The β-(1→3) type galactose and glucose glycosidic bonds in the main chain maintain molecular rigidity and ensure the integrity of the triple helix conformation. Once the conformation is unwound, the immune activity will be significantly reduced. The main chain →3,6)-β-D-Galp-(1→) serves as a branching point, carrying various active side chains. The side chains, derived from arabinose, xylose, and mannose, provide a large number of immune recognition sites, which can promote the secretion of NO, IL-6, and TNF-α in a dose-dependent manner and enhance the proliferation capacity of splenic lymphocytes. Therefore, relying on the multi-level synergistic effect of monosaccharides, molecular weight, glycosidic bonds and higher conformations, DHPS1Ah exhibits superior immunomodulatory activity compared to similar polysaccharides under non-cytotoxic conditions.

[0049] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A homogeneous acidic polysaccharide from Dendrobium huoshanense, characterized in that, The uniform chemical structure of Dendrobium huoshanense acidic polysaccharide The main chain includes →3,6)-β-D-Galp-(1→, →3)-β-D-Galp-(1→ and →3)-β-D-Glcp-(1→) residues, and the side chains include α-L-Araf-(1→4)-β-D-Xylp-(1→, α-L-Arap-(1→3)-α-L-Araf→, α-L-Araf-(1→4)-β-D-Manp→ and 4-OMe-β-D-Galp-(1→4)-β-D-Galp→. All side chains are attached to the O-6 sites of the →3,6)-β-D-Galp-(1→) residues.

2. The homogeneous Dendrobium huoshanense acidic polysaccharide according to claim 1, characterized in that, The homogeneous Dendrobium huoshanense acidic polysaccharide has a molecular weight of 83.637 kDa, a number-average molecular weight of 52.439 kDa, and a polydispersity index of 1.

595.

3. The homogeneous Dendrobium huoshanense acidic polysaccharide according to claim 1, characterized in that, The homogeneous Dendrobium huoshanense acidic polysaccharide has a triple helix conformation.

4. The homogeneous Dendrobium huoshanense acidic polysaccharide according to claim 1, characterized in that, The homogeneous Dendrobium huoshanense acidic polysaccharide includes arabinose, rhamnose, xylose, glucose, mannose, and galactose.

5. The homogeneous Dendrobium huoshanense acidic polysaccharide according to claim 4, characterized in that, The molar ratio of arabinose, rhamnose, xylose, glucose, mannose and galactose is 29.03:36.51:10.03:8.69:7.49:4.

32.

6. A method for preparing homogeneous Dendrobium huoshanense acidic polysaccharides as described in any one of claims 1-5, characterized in that, Includes the following steps: The refined polysaccharides of Dendrobium huoshanense were separated by DEAE-52 cellulose column, eluted with NaCl aqueous solution, the eluent was collected, concentrated and freeze-dried to obtain the acidic polysaccharide component of Dendrobium huoshanense. The acidic polysaccharide components of Dendrobium huoshanense were purified multiple times using a Sephadex G-200 gel column to obtain homogeneous Dendrobium huoshanense acidic polysaccharides.

7. The method according to claim 6, characterized in that, The preparation method of the acidic polysaccharide component of Dendrobium huoshanense is as follows: refined polysaccharide of Dendrobium huoshanense is mixed with water at a mass ratio of 1:5, separated by a DEAE-52 cellulose column, and eluted with distilled water, 0.1 mol / L NaCl aqueous solution, 0.2 mol / L NaCl aqueous solution and 0.3 mol / L NaCl aqueous solution respectively at a flow rate of 5 mL / min. The eluted fraction of 0.1 mol / L NaCl aqueous solution is collected, concentrated and then freeze-dried.

8. The method according to claim 6, characterized in that, The Sephadex G-200 gel column separation and purification method is as follows: distilled water is used as the mobile phase, the flow rate is 0.5 mL / min, and single-peak components are collected.

9. The application of the homogeneous Dendrobium huoshanense acidic polysaccharide according to claim 1 in the preparation of immunomodulatory products.

10. The application according to claim 9, characterized in that, The immunomodulatory products include immunomodulatory drugs or immunomodulatory functional foods.

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