A novel type of α-glucan and its uses

Abstract

This invention provides a novel class of α-glucans and their uses. The α-glucans are homologous polysaccharides with the same α-1,4-glucan backbone but different degrees of branching, glucose, and glucuronic acid side chains. The glucuronic acid side chains are unique structural segments of the α-glucans and also contribute significantly to their biological activity. The α-glucans of this invention are abundant, structurally stable, and highly pure, exhibiting significant immunomodulatory and antitumor activities, and hold promise for development into drugs to improve or treat immune-related bodily dysfunctions and / or for antitumor purposes.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a novel type of α-glucan and its uses. Background Technology

[0002] Polysaccharides, especially glucans, are a typical class of immune response modifiers and regulators. Their repeating oligosaccharide structures can bind to immune cell membrane receptors with multiple valences, activating innate and adaptive immune systems, clearing foreign substances entering the body, and killing pathogens and tumor cells.

[0003] β-1,3-glucan, found in the outer layer of pathogen cell membranes, is a highly conserved pathogen-associated molecular pattern (PAMP). Animals, lacking this structure, have evolved specific pattern recognition receptors on their immune cell membranes, enabling them to recognize PAMPs and activate the immune system to kill and clear them. Therefore, β-1,3-glucan obtained from shiitake mushrooms, poria cocos, and Schizophyllum commune exhibits significant immunogenicity, upregulating immune system function and exerting anti-tumor effects. However, these exogenous polysaccharides readily trigger immune responses and also increase retention effects caused by the reticuloendothelial system and bioadhesion, making them easily cleared by the body and affecting their potency and duration.

[0004] Studies have shown that the outermost layer of some pathogens contains α-1,3-glucan, which, due to its structural similarity to endogenous glycogen (both belonging to the α-glucan family), possesses very low immunogenicity. It assists pathogens in evading immune recognition by encapsulating PAMPs, thus enhancing pathogenicity. Therefore, α-glucan has long been considered biologically inactive. Other studies have shown that in some mycobacteria, such as Mycobacterium tuberculosis, the polysaccharide encapsulating the outermost layer of the cell wall is α-1,4-glucan. Unlike the dendritic, highly branched structure of glycogen, this type of polysaccharide has low branching and long linear glycan chains. Multiple studies have shown that this type of polysaccharide can both assist Mycobacterium tuberculosis in evading immune recognition and activate dendritic cells to inhibit Mycobacterium tuberculosis, demonstrating the specificity of its immunogenicity. It has been reported that α-1,4-glucan obtained from Cordyceps sinensis, straw mushrooms, and Schisandra chinensis has significant macrophage activation effects; these also possess linear glycan chains, further confirming the biological activity of α-1,4-glucan, which is closely related to its fine structure. However, plants mainly contain β-glucan and starch; the glucan from fungi varies greatly in structure, such as molecular weight and branching degree, due to the influence of culture conditions and extraction methods, making it difficult to control its quality. This is an urgent problem to be solved in the research and application of polysaccharide drugs. Summary of the Invention

[0005] The purpose of this invention is to provide a class of α-glucans that are abundant, structurally stable, easily purified, and highly bioactive, and their use in the preparation of formulations that improve or treat immune-related bodily dysfunctions or diseases. The α-glucans involved in this invention are prepared from sea urchins, and their main chains are composed of α-glucose polymerized through (1→4) glycosidic bonds. At the 6-position of the glucose in the main chain, there are glucose side chains and glucuronic acid side chains. In particular, the glucuronic acid side chains are a unique structure not found in other α-1,4-glucans. These glucuronic acid side chains not only increase the water solubility of the α-glucans involved in this invention but also significantly enhance their bioactivity.

[0006] In a first aspect, the present invention provides a novel class of α-glucans having the basic structural unit shown in Formula I.

[0007]

[0008] Wherein, n is selected from 2.5, 2, 1.5, 1, that is, there is one side chain attached to every 6, 5, 4 or every 3 glucose residues on the main chain of the α-glucan; R is selected from glucose, glucuronic acid, mannose, ribose or galactose residues.

[0009] Preferably, the main chain of the α-glucan is polymerized from glucose via α-1,4-glycosidic bonds, and a monosaccharide side chain is attached to the 6-position of the glucose in the main chain. R is selected from glucose and glucuronic acid, as well as one or more of mannose, ribose, and galactose.

[0010] Preferably, the α-glucan has a molecular weight of 1.913 × 10⁻⁶. 7 ~3.094×10 7 According to the molar ratio, the polysaccharide structure contains 84.6-95.6% glucose, 4.4-8.0% glucuronic acid, 0.4-4.6% mannose, 0.1-2.3% ribose, and 0-1.2% galactose.

[0011] More preferably, in the α-glucan, n is selected from 2.5 or 2, R is selected from glucose and glucuronic acid, and the content of glucose is 95.6% and the content of glucuronic acid is 4.4% according to the molar ratio.

[0012] More preferably, in the α-glucan, when n is selected from 2 or 1.5, R is selected from glucose, glucuronic acid, mannose, and ribose, and the content of glucose is 85.8-93.0%, the content of glucuronic acid is 4.5-8.0%, the content of mannose is 0.4-4.6%, and the content of ribose is 0.1-2.1% according to the molar ratio.

[0013] More preferably, in the α-glucan, n is selected from 1.5 or 1, and R is selected from glucose, glucuronic acid, mannose, ribose, and galactose. Calculated by molar ratio, the content of glucose is 84.6%, the content of glucuronic acid is 7.9%, the content of mannose is 4.0%, the content of ribose is 2.3%, and the content of galactose is 1.2%.

[0014] A second aspect of the invention provides the use of one or more combinations of the α-glucan described in the first aspect of the invention in the preparation of formulations for improving or treating immune-related bodily dysfunctions or diseases.

[0015] Preferably, the one or more combinations refer to the use of any one α-glucan alone, or the use of any combination of α-glucans.

[0016] Preferably, the immune-related bodily dysfunction is an immune deficiency, low immunity, or immune damage caused by congenital deficiency, infection, malnutrition, or drugs.

[0017] Preferably, the immune-related disease is a tumor.

[0018] Preferably, the preparation is a functional food or a drug. Attached Figure Description

[0019] Figure 1 The elution curves of total polysaccharides on a DEAE-52 column are shown.

[0020] Figure 2 Elution curves of total polysaccharides on a Cellufine A-500 column are shown.

[0021] Figure 3 The high-performance gel permeation chromatography chromatogram of the α-glucan of the present invention is shown.

[0022] Figure 4 The monosaccharide composition of the α-glucan of the present invention is shown. ManA is mannuronic acid, Man is mannose, Rib is ribose, Rha is rhamnose, GlcA is glucuronic acid, GalA is galacturonic acid, GlcNAc is acetylglucosamine, Glc is glucose, Gal is galactose, Xyl is xylose, Ara is arabinose, and Fuc is fuc.

[0023] Figure 5 The methylation analysis results of the α-glucan of the present invention are shown. The fully methylated polysaccharide product was hydrolyzed at 110°C for 3 h.

[0024] Figure 6 The methylation analysis results of the α-glucan (HPP-6S and HPP-7S) of the present invention are shown. The fully methylated products of the polysaccharides were hydrolyzed at 110°C for 3 h.

[0025] Figure 7 The methylation analysis results of the α-glucan of the present invention are shown. The fully methylated polysaccharide product was hydrolyzed at 120°C for 4 h.

[0026] Figure 8 The infrared spectrum of the α-glucan of the present invention is shown.

[0027] Figure 9 The 1H and 1C NMR spectra of the α-glucan of the present invention are shown.

[0028] Figure 10 The effect of the α-glucan of the present invention on macrophage survival is shown.

[0029] Figure 11 The effect of the α-glucan of the present invention on promoting the release of NO from macrophages is demonstrated.

[0030] Figure 12 The invention demonstrates how α-glucan activates macrophages to secrete TNF-α and IL-6.

[0031] Figure 13 The α-glucan of the present invention is shown to enhance the phagocytic capacity of macrophages.

[0032] Figure 14 The effect of the α-glucan of the present invention on the proliferation of spleen lymphocytes isolated from mice is shown.

[0033] Figure 15 The protective effect of the α-glucan of the present invention on the spleen of mice with cyclophosphamide-induced immunosuppression is demonstrated.

[0034] Figure 16 This invention demonstrates the protective effect of α-glucan on the thymus of cyclophosphamide-induced immunosuppressed mice.

[0035] Figure 17 The α-glucan of the present invention is shown to have an effect on the CD4 subset of peripheral blood T lymphocytes in cyclophosphamide-induced immunosuppressed mice. + / CD8 + The regulatory effect; A. Blank control group, B. Model control group, C. Low-dose polysaccharide group, D. Medium-dose polysaccharide group, E. High-dose polysaccharide group.

[0036] Figure 18 The invention demonstrates that the α-glucan increases the expression levels of IL-2 and TNF-α in the serum of cyclophosphamide-induced immunosuppressed mice.

[0037] Figure 19 The invention demonstrates that the α-glucan of the present invention increases the expression levels of IL-2 and TNF-α in the serum of H22 tumor-bearing mice.

[0038] Figure 20 The inhibitory effect of the α-glucan of the present invention on S180 sarcoma in mice is shown in (A), and the images of mouse spleen (B) and thymus (C) are also shown.

[0039] Figure 21 The invention demonstrates that the α-glucan of the present invention increases the expression level of IL-2 in the serum of mice bearing S180 sarcoma.

[0040] Figure 22 The inhibitory effect of the α-glucan of the present invention on B16 melanoma in mice is shown in (A), and the images of mouse spleen (B) and thymus (C) are also shown.

[0041] Figure 23 The invention demonstrates that the α-glucan increases the expression levels of IL-2 and TNF-α in the serum of mice carrying B16 melanoma. Detailed Implementation

[0042] The following specific embodiments further illustrate the α-glucan structure and its uses of the present invention. These examples are intended to explain the technical content of the invention and make its advantages clearer, and are not intended to limit the scope of the invention. All changes or equivalent substitutions made based on the substance of the disclosure below should fall within the protection scope of the present invention.

[0043] Example 1: Isolation of α-glucan

[0044] The gonads of sea urchins were homogenized and then defatted by soaking in anhydrous ethanol to obtain defatted powder. The defatted powder was extracted three times with 10 times its volume of water at 50°C. The extract was concentrated under reduced pressure and then enzymatically hydrolyzed with papain at 55°C, followed by inactivation at 90°C. The supernatant was collected by centrifugation. The supernatant was extracted with Sevage reagent (chloroform:n-butanol = 4:1) to remove proteins. The deproteinized polysaccharide aqueous solution was dialyzed with flowing distilled water for 24 hours using a dialysis bag with a molecular weight cutoff of 6000 Da. Four times its volume of anhydrous ethanol was added to the dialyzed polysaccharide aqueous solution, the precipitate was collected by centrifugation, and dried to obtain sea urchin total polysaccharide powder, named HPP.

[0045] HPP was separated using a DEAE-52 ion-exchange column, eluted with water and gradient concentrations of NaCl solution, and collected in one test tube for every 10 mL. Figure 1 As shown, HPP-1D was obtained by elution with water, HPP-2D by elution with 1% NaCl solution, and HPP-3D by elution with 5% NaCl solution. HPP-1D, HPP-2D, and HPP-3D were purified by Sepharose CL-2B agarose gel column chromatography, and the polysaccharide products were obtained by elution with distilled water. After concentration under reduced pressure and freeze-drying, the products were stored at -20°C for testing their macrophage activation activity.

[0046] The separation results of Example 1 showed that *Hippophae rhamnoides* mainly contained HPP-1D, while the contents of HPP-2D and HPP-3D were low. Furthermore, HPP-2D and HPP-3D were distributed in multiple test tubes, indicating that HPP-2D and HPP-3D contained various polysaccharides and had low purity. The structure of HPP-1D has been previously analyzed; it is an α-1,4-glucan with a glucose side chain or glucuronic acid side chain attached to the 6-position of every 5 glucose molecules in the main chain.

[0047] Example 2: Preparation of the homologous series α-glucan of the present invention

[0048] This invention further optimizes the ion chromatography purification method by using Cellufine A-500 ion exchange resin as the stationary phase. Elution is performed sequentially with water and NaCl solutions of 0.017, 0.034, 0.051, 0.068, 0.085, 0.103, and 0.171 mol / L to obtain HPP-1S, HPP-2S, HPP-3S, HPP-4S, HPP-5S, HPP-6S, HPP-7S, and HPP-8S, respectively. These are then purified by Sepharose CL-2B agarose gel column chromatography, with distilled water elution yielding high-purity polysaccharides.

[0049] Separation results as follows Figure 2 As shown. The structural characterization in Example 3 below confirms that the HPP-1S structure is identical to the HPP-1D structure, and provides a more accurate analysis of the side chain distribution. The optimized method of this invention separates HPP-2D and HPP-3D into seven high-purity polysaccharides: HPP-2S, HPP-3S, HPP-4S, HPP-5S, HPP-6S, HPP-7S, and HPP-8S.

[0050] Example 3: Structural characterization of homologous α-glucan series

[0051] (1) Testing the purity and molecular weight of polysaccharides

[0052] The sugar contents of HPP-1S, HPP-2S, HPP-3S, HPP-4S, HPP-5S, HPP-6S, HPP-7S, and HPP-8S, determined by the phenol-sulfuric acid method, were 99.91%, 99.84%, 99.43%, 98.21%, 93.52%, 94.08%, 93.30%, and 93.82%, respectively.

[0053] No protein residue was detected in any of the polysaccharides using the Bradford method.

[0054] Analysis using high-performance gel permeation chromatography revealed that each polysaccharide exhibited a single, symmetrical chromatographic peak. Figure 3This indicates that the molecular weight distribution of each polysaccharide is relatively narrow and the uniformity is good.

[0055] Based on HPP-1S (molecular weight determined to be 2.996 × 10⁻⁶ using a multi-angle laser light scattering instrument), 7 Molecular weight standard curves were plotted using Da and standard molecular weight dextran (T-20, T-50, T-1000, and T-2000) (lgMw = -1.713T + 18.38, R...). 2 =0.994) The molecular weights of HPP-2S, HPP-3S, HPP-4S, HPP-5S, HPP-6S, HPP-7S, and HPP-8S are calculated to be 2.153 × 10⁻⁶. 7 Da, 2.928×10 7 Da, 2.311×10 7 Da, 1.913×10 7 Da, 2.975×10 7 Da, 3.094×10 7 Da and 2.987×10 7 Da.

[0056] (2) Analysis of monosaccharide composition

[0057] HPP-1S to HPP-8S (5 mg) were hydrolyzed with trifluoroacetic acid and evaporated to dryness under reduced pressure. Then, 0.5 mol / L of 1-(3'-sulfonylphenyl)-3-methyl-5-pyrazolone (PMP) and 0.3 mol / L of NaOH were added, and the mixture was reacted at 70 °C for 1 h. The mixture was then neutralized with HCl, and excess PMP was removed by extraction with dichloromethane. The aqueous layer was analyzed by high-performance liquid chromatography (HPLC). The detector wavelength was 245 nm; the column temperature was 35 °C; and the mobile phase was phosphate buffer (pH 6.7) / CH3CN (85:15, V:V).

[0058] like Figure 4 As shown, HPP-1S displays a glucose signal, while HPP-2S, HPP-3S, HPP-4S, HPP-5S, HPP-6S, and HPP-7S display signals for glucose, mannose, and ribose, and HPP-8S displays signals for glucose, mannose, ribose, and galactose. Furthermore, the peak areas indicate that the content of mannose and ribose generally increases from HPP-2S to HPP-7S.

[0059] Based on the separation principle of ion chromatography, as the salt concentration of the eluent increases, polysaccharides with increasing ionic strength are eluted from the column sequentially. Previous studies have confirmed that HPP-1S contains uronic acid, indicating that the uronic acid content of HPP-2S to HPP-7S increases sequentially. The absence of a uronic acid signal in liquid chromatography may be due to the destruction of uronic acid dissociated at high temperatures. This is consistent with previous results showing no uronic acid signal detected in HPP-1S after complete acid hydrolysis using high-performance anion exchange chromatography.

[0060] (3) Methylation analysis

[0061] To further analyze the accurate content of uronic acid, polysaccharides HPP-2S to HPP-8S (4 mg) were methylated using the box-and-mortise method with NaOH as a catalyst and iodomethane as a methylating agent. The fully methylated product was hydrolyzed with trifluoroacetic acid at 110 °C for 3 h, then reduced with sodium borohydride and acetylated with acetic anhydride to obtain partially methylated aldose acetate (PMAA). Finally, the product was analyzed by GC-MS. The results are as follows: Figure 5 As shown, the GC-MS total ion chromatogram reveals four main structural fragments identical to HPP-1S: α-glucose (1→(a), α-glucuronic acid (1→(b), →4)-α-glucose (1→(c); →4,6)-α-glucose (1→(d)). The difference is that, in addition to these four peaks, several impurity peaks of non-monosaccharide derivatization products are also observed. Furthermore, the total amount of terminal sugars (fragments a and b) is significantly higher than that at the branch point (fragment d). This may be because HPP-2S~HP The increased branching degree of P-8S and incomplete hydrolysis of the main chain led to a decrease in the peak size at the branching point and the appearance of multiple impurity peaks. However, under these conditions, glucose (fragment a) and glucuronic acid (fragment b) on the side chains were more completely dissociated, and the b / a (t-GlcA / t-Glc) ratio was observed to increase sequentially (the ratio in HPP-1S is 2.5:1). This indicates that the uronic acid content in the structure increases sequentially from HPP-1S to HPP-6S. The b / a ratio decreased in HPP-7S and HPP-8S. Figure 6 This may be due to experimental error, and it is speculated that its b / a ratio is higher than that of HPP-6S (1.6:1).

[0062] To further accurately analyze its branching degree, the fully methylated products of HPP-1S, HPP-2S, HPP-3S, HPP-4S, HPP-6S, and HPP-8S were hydrolyzed at a higher temperature (120℃) for a longer time (4 h), followed by reduction with sodium borohydride and acetylation with acetic anhydride to obtain PMAA, which was then analyzed by GC-MS. The results are as follows: Figure 7As shown, the total ion chromatogram only shows four main peaks (a, b, c, d), and the impurity peaks disappear. The peak of α-glucuronic acid (1→(b)) is significantly reduced, possibly because some uronic acid is destroyed after the reaction temperature and time are increased, which is consistent with the results of monosaccharide analysis. This condition gives a more accurate ratio of main chain sugar to branch point sugar. The c / d (1,4-Glc / 1,4,6-Glc) ratio of HPP-1S is 4.5:1 (the ratio obtained under the reaction conditions of 110℃ and 3h is 4:1), indicating that every 5 or 6 sugars on the main chain of HPP-1S are attached to a side chain. Moreover, from HPP-2S to HPP-8S, the proportion of branch point sugars gradually increases, indicating an increase in branching degree. Every 5 sugars on the main chain of HPP-2S are attached to a side chain. P-3S and HPP-4S have side chains attached to every four sugars in their main chain, while HPP-6S and HPP-8S have side chains attached to every three or four sugars in their main chain. HPP-5S and HPP-7S were not analyzed, but their branching degree should be similar to that of HPP-6S. Additionally, the chromatogram of HPP-8S also showed signals for terminal mannose and terminal galactose, indicating that these are on the side chains. The variation in main chain branching degree and the content of monosaccharides such as uronic acid and mannose indicates that HPP-1S to HPP-8S are homologous α-glucans with the same main chain. The mannose, ribose, and galactose, which are present in very small amounts in their structure, are all side chains attached to the C-6 position of the main chain.

[0063] Based on the results of monosaccharide PMP derivatization and methylation analysis, the content of each monosaccharide in the α-glucan involved in this invention, calculated according to molar ratio, is listed in Table 1. HPP-1S contains glucose and glucuronic acid, with contents of 95.6% and 4.4%, respectively. HPP-2S to HPP-7S contain glucose, glucuronic acid, mannose, and ribose, with glucose contents of 85.8% to 93.0%, glucuronic acid contents of 4.5% to 8.0%, mannose contents of 0.4% to 4.6%, and ribose contents of 0.1% to 2.1%. HPP-8S contains glucose, glucuronic acid, mannose, ribose, and galactose, with glucose contents of 84.6%, glucuronic acid contents of 7.9%, mannose contents of 4.0%, ribose contents of 2.3%, and galactose contents of 1.2%.

[0064] Table 1

[0065]

[0066]

[0067] It should be noted that the α-1,4-glucan involved in this invention obtained by eluting with water after changing the type of ion exchange resin (DEAE-52 or Cellufine A-500) has the same structure. It is a polysaccharide mainly contained in sea urchins and has the advantages of abundant source, stable structure, high purity and easy water solubility.

[0068] It is particularly important to emphasize that the results of Examples 1, 2, and 3 show that the contents of uronic acid, mannose, etc., in the α-glucan structure involved in this invention vary within a fixed range, and their specific contents are related to the salt concentration of the eluent. The specific structure of the α-glucan shown in this invention was obtained by elution using the NaCl concentration of this invention. Based on these results, the following conclusion can be drawn: if other concentrations of NaCl solution are used, similar polysaccharides with different proportions of branching degree, uronic acid, and mannose content within this range can be obtained, and all of these should be within the protection scope of this invention.

[0069] (4) Infrared spectroscopy

[0070] Take 1 mg of polysaccharide, add an appropriate amount of potassium bromide, grind evenly, compress into tablets, and then analyze using a Fourier transform infrared spectrometer. Infrared spectra of HPP-1S~HPP-8S ( Figure 8 They show the same characteristic peaks. Taking HPP-2S as an example, 3397 cm⁻¹ -1 The absorption peak is due to the stretching vibration of the hydroxyl group, at 2928 cm⁻¹. -1 The stretching vibration absorption peaks are for saturated alkyl groups, at 1643 and 1414 cm⁻¹. -1 These are characteristic peaks of the carboxyl group, at 1152, 1079, and 1023 cm⁻¹. -1 The absorption peak is at 844 cm⁻¹, which is the absorption peak of the glycosidic bond. -1 The peak is a characteristic peak of α-pyranose, indicating that it is α-glucan.

[0071] (5) Nuclear magnetic resonance spectroscopy analysis

[0072] HPP-2S to HPP-8S were dissolved in heavy water and tested. 1 H NMR and 13 C NMR. (e.g.) Figure 9 As shown, HPP-2S to HPP-8S exhibit characteristic proton and carbon signals that are essentially the same as those of HPP-1S, further confirming the homology of the α-glucan involved in this invention. Due to the low content of mannose, ribose, and galactose, no obvious NMR signals were observed, but the characteristic signals of uronic acid were evident. Taking HPP-4S as an example, its NMR data are listed in Table 2.

[0073] Table 2

[0074]

[0075] Example 4: Activating macrophages to release nitric oxide (NO)

[0076] RAW264.7 macrophages were selected as the cell model. Cells in the logarithmic growth phase were pipetted from the culture dish, and the cell density was adjusted to 2 × 10⁻⁶ cells / mL. 4 Cells were cultured at a concentration of 100 μL / mL in each well of a 96-well plate and incubated at 37°C in a 5% CO2 incubator for 24 h. The activation effects of sea urchin total polysaccharides (HPP), HPP-1D, HPP-2D, and HPP-3D on macrophages were tested at concentrations of 30, 150, and 500 μg / mL. Culture medium and lipopolysaccharide (LPS, 1 μg / mL) served as blank and positive controls, respectively. After 24 h of culture, the 96-well plates were removed, and 100 μL of cell supernatant was transferred from each well to a new 96-well plate. 100 μL of Griess reagent was added under light-protected conditions, and the plates were shaken for 10 min. The OD value of each well was measured at 540 nm. The results are shown in Table 3. HPP, HPP-1D, HPP-2D, and HPP-3D showed significant NO-releasing effects at a concentration of 30 μg / mL, and their activation effects were dose-dependent.

[0077] Table 3

[0078]

[0079] Compared with the control group, *P<0.05, **P<0.01.

[0080] The effect of HPP-1S to HPP-8S on activating NO release from macrophages was tested using the same method. The dosage concentrations were: HPP-1S to HPP-3S: 15.6, 31.3, 62.5, 125, 250, and 500 μg / mL; HPP-4S to HPP-8S: 1.95, 3.9, 7.8, 15.6, 31.3, 62.5, 125, and 250 μg / mL. The effect of the α-glucan of this invention on macrophage survival was first evaluated, and the results are as follows: Figure 10 As shown, the polysaccharides of the present invention did not significantly inhibit macrophages within the administered concentration range, indicating that the α-glucan of the present invention has good safety. The α-glucan of the present invention significantly stimulated macrophages to release NO, showing a dose-response relationship, and significant differences in activation ability were observed. Figure 11For example, HPP-1S to HPP-4S show progressively increasing activity. HPP-4S exhibits significant activation at a very low concentration of 3.9 μg / mL, and reaches its maximum NO release at 15.6 μg / mL. Their structures contain extremely low levels of mannose and ribose, contributing little to activity. The main structural change is the gradual increase in uronic acid content from HPP-1S to HPP-4S, resulting in a very significant increase in activity. This demonstrates the specificity of uronic acid as a structural unit and its significant contribution to activity.

[0081] Based on the above separation and bioactivity test results, HPP is a mixture of polysaccharides involved in this invention, and HPP-2D and HPP-3D are mixtures of HPP-2S to HPP-8S. These polysaccharide mixtures and pure products can activate macrophages, indicating that one or more of the series of α-glucans of this invention, used alone or in combination, have macrophage activation effects.

[0082] Example 5: Promotes macrophage secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6).

[0083] TNF-α and IL-6 are inflammatory cytokines secreted by activated immune cells. They can kill tumor cells and also act as specific antigens to activate the body's adaptive immunity, making them important indicators of immune function. After culturing RAW264.7 cells for 24 hours, HPP-1S, HPP-3S, and HPP-6S were administered at the concentrations set in Example 4, and the cells were grouped and cultured for 24 hours. Cell supernatant was collected into 1.5 mL centrifuge tubes, and after centrifugation, the supernatant was transferred to new centrifuge tubes. Using an ELISA kit, the levels of TNF-α and IL-6 in the supernatant of HPP-1S, HPP-3S, and HPP-6S were detected according to the manufacturer's instructions. The results are as follows: Figure 12As shown, HPP-1S significantly promoted the secretion of TNF-α and IL-6 at concentrations of 31.3 and 15.6 μg / mL, respectively. HPP-3S significantly promoted the secretion of TNF-α and IL-6 at concentrations of 1.95 and 3.9 μg / mL, and HPP-6S at concentrations of 1.95 and 7.8 μg / mL, respectively. The activity intensity was HPP-6S > HPP-3S > HPP-1S, consistent with the trend observed in the nitric oxide promotion experiment. Compared with the control group's TNF-α secretion (30.3 pg / mL), the highest concentration groups of HPP-1S, HPP-3S, and HPP-6S showed increases of 15, 81, and 124 times, respectively, while IL-6 secretion increased by 51, 173, and 154 times, respectively (the control group's IL-6 secretion was 99.0 pg / mL). These bioactivity data demonstrate that the α-1,4-main chain, glucose and glucuronic acid side chains, and high branching degree of the α-glucan of the present invention are the structural basis for its strong immunomodulatory activity, and its activity is significantly enhanced when the glucuronic acid content increases.

[0084] Example 6: Enhancing Macrophage Phagocytic Capacity

[0085] RAW264.7 cells were co-cultured with HPP-1S (12.5, 31.25, 62.5, 125, 250, and 500 μg / mL) for 24 h. The 96-well plates were then removed, and the supernatant was discarded. 100 μL of 0.1% neutral red solution was added to each well, and the cells were incubated for another 3 h. After discarding the supernatant, the cells were washed with PBS, and 100 μL of lysis buffer was added. The cells were incubated overnight at room temperature. After cell lysis, the OD value at 540 nm was measured. Results are as follows: Figure 13 As shown, HPP-1S can significantly promote the phagocytic ability of macrophages and enhance their activity.

[0086] Example 7: Effects on mouse splenic lymphocytes

[0087] Spleens were aseptically harvested from male Kunming mice, and splenic lymphocyte suspensions were prepared and seeded into 96-well plates. 100 μL of the appropriate drug concentration was added, and the plates were incubated for 48 h. Then, 20 μL of CCK-8 enhancement solution was added, and the plates were incubated for 4 h. The OD value was measured at 450 nm, and lymphocyte viability was calculated. The following groups were included: a blank control group, a concanavalin A (ConA) control group (final concentration 5 μg / mL), a ConA (5 μg / mL) + HPP-1S (31.25, 62.5, 125, 250, 500, 1000 μg / mL) treatment group, and an HPP-1S treatment group (31.25, 62.5, 125, 250, 500, 1000 μg / mL).

[0088] The results are as follows Figure 14As shown, after ConA induction, the T lymphocyte proliferation rate increased by 64%–77% in each group compared with the blank control group. Compared with the ConA model group, the ConA+HPP-1S combined administration group had no significant effect on the T lymphocyte proliferation rate. When only HPP-1S was added without ConA induction, compared with the blank group, HPP-1S had no significant effect on spleen lymphocyte proliferation in the concentration range of 31.25–62.5 μg / mL, but significantly promoted its proliferation in the concentration range of 125–1000 μg / mL. This indicates that HPP-1S is non-toxic to spleen lymphocytes in vitro in the concentration range of 31.25–1000 μg / mL, and can also promote spleen lymphocyte proliferation at high concentrations. This demonstrates that the α-glucan of the present invention has high safety and can activate spleen lymphocytes.

[0089] Example 8: Protective effect of α-glucan against cyclophosphamide (CTX)-induced immune damage

[0090] Ninety-six healthy SPF-grade male Kunming mice weighing 20±2g were randomly divided into six groups of 16 mice each. There were no statistically significant differences in mouse weight within or between groups.

[0091] Grouping: 1) Normal control group: Mice were injected with saline for 1-10 days; 2) Model control group: Mice were injected intraperitoneally with saline for 1-10 days, and injected with cyclophosphamide (40 mg / kg / day) for 9-10 days; 3) Low-dose HPP-1S group: Mice were injected with HPP-1S (20 mg / kg / day) for 1-10 days, and injected with cyclophosphamide (40 mg / kg / day) for 9-10 days; 4) Medium-dose HPP-1S group: Mice were injected with HPP-1S (40 mg / kg / day) for 1-10 days. 5) High-dose HPP-1S group: HPP-1S (80mg / Kg / day) injected for 1-10 days, followed by cyclophosphamide (40mg / Kg / day) injected for 9-10 days; 6) High-dose HPP-1S gavage group: HPP-1S (80mg / Kg / day) administered by gavage for 1-10 days, followed by cyclophosphamide (40mg / Kg / day) injected for 9-10 days.

[0092] (1) Protective effect of α-glucan on spleen and thymus

[0093] Twenty-four hours after the last administration, mice were anesthetized, sacrificed, and dissected. Spleen and thymus tissues were removed, connective and adipose tissues were removed, and the tissues were washed with physiological saline and fixed overnight in 4% paraformaldehyde solution. The tissues were then dehydrated using an automated dehydrator, embedded in paraffin, and sectioned (5 μm thick). HE staining was used to observe the pathological changes in the spleen and thymus tissues. Results are as follows: Figure 15As shown, the spleen capsule of normal mice was thicker, and the red and white pulp were clearly defined in the splenic parenchyma, with a higher density of lymphocytes in the white pulp. Compared with the normal group, the white pulp in the spleen parenchyma of model mice had a disordered structure and sparse lymphocytes, the boundary between the red and white pulp was blurred, and the capsule was thinner. The low, medium, and high doses of HPP-1S significantly improved the tissue structure of the spleen parenchyma, gradually clarifying the boundary between the red and white pulp and thickening the capsule. Figure 16 The results showed that in the control group, the thymus cortex of mice was thinner and more disorganized, with a relative increase in medulla and blurred boundaries between the cortex and medulla. In the HPP-1S-treated group, the thymus cortex of mice was relatively thickened, and the corticomedullary boundary was more clearly defined. This indicates that HPP-1S can significantly protect the spleen and thymus, reduce cyclophosphamide-induced immune organ damage, and has a significant immunoprotective effect.

[0094] (2) Increased phagocytic function of mouse mononuclear macrophages

[0095] Twenty-four hours after the last administration, diluted ink was injected via the tail vein. Timing began immediately after injection. Blood was collected from the canthal venous plexus at 2 min (t1) and 10 min (t2) and transferred to centrifuge tubes. 20 μL of each sample was pipetted into 2 mL of 0.1% Na2CO3 solution and mixed thoroughly. The OD value at 600 nm was measured. Mice were euthanized, and their spleens and livers were dissected. Surrounding fat and connective tissue were carefully removed, and the livers were weighed. The clearance rate (K) and phagocytic index (α) were calculated using the following formulas.

[0096] K = (lgOD1 - lgOD2) / (t2 - t1)

[0097] α = Body weight / (Liver weight + Spleen weight) × K 1 / 3

[0098] Where OD1 is the OD value at time t1, and OD2 is the OD value at time t2.

[0099] The results are shown in Table 4. Compared with the blank group, the clearance rate K and phagocytic index α of the model group were significantly reduced, indicating that cyclophosphamide inhibited the phagocytic capacity of monocytes and macrophages. Compared with the model control group, the clearance rate K and phagocytic index α of the HPP-1S-treated group were significantly increased. The experimental results show that HPP-1S can reverse the decrease in macrophage activity in mice induced by cyclophosphamide and reactivate macrophage function under immune damage.

[0100] Table 4

[0101] Group Dosage (mg / Kg / d) Clearance rate K Phagocytosis Index α normal control group — 0.017±0.003 3.612±0.545 Model control group — <![CDATA[0.002±0.001 ### ]]> <![CDATA[2.045±0.137 ### ]]> HPP-1S low-dose group 20 <![CDATA[0.006±0.003 * ]]> <![CDATA[2.829±0.242 *** ]]> HPP-1S medium dose group 40 <![CDATA[0.005±0.002 * ]]> <![CDATA[2.749±0.439 *** ]]> HPP-1S high-dose group 80 <![CDATA[0.005±0.001 ** ]]> <![CDATA[2.851±0.282 *** ]]>

[0102] Values ​​are expressed as mean ± SD (n = 10). Compared with the control group, #p < 0.05, ##p < 0.01, ###p < 0.001; compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0103] (3) Blood routine indicators

[0104] Twenty-four hours after the last administration, 50 μL of blood was collected from the canthus of the mouse and placed into an EDTA-2K anticoagulant tube for blood routine tests.

[0105] The results are shown in Table 5. Compared with the blank control group, the model control group showed a significant decrease in white blood cell (WBC), neutrophil (NE#), and lymphocyte (LY#) counts, as well as a reduction in platelet (PLT) count. Compared with the model group, the HPP-1S administration group significantly improved the decrease in WBC and PLT counts induced by cyclophosphamide. The low-dose HPP-1S group increased the number of neutrophils (NE#) in peripheral blood, while the high-dose HPP-1S group increased the number of lymphocytes (LY#). High-dose oral administration also significantly increased the number of white blood cells and platelets, but its overall effect was lower than that of the intraperitoneal injection group. These experimental results indicate that the α-glucan of the present invention has a good therapeutic effect on the hematologic and lymphatic system toxicity caused by cyclophosphamide, and demonstrates the effectiveness of oral administration of the α-glucan of the present invention.

[0106] Table 5

[0107]

[0108] Values ​​are expressed as mean ± SD (n = 10). Compared with the normal control group, #p < 0.05, ##p < 0.01, ###p < 0.001; compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0109] (4) Measurement of CD4+ in mouse peripheral blood T lymphocytes + / CD8 + ratio

[0110] Blood was collected from the eyeballs of mice before intraperitoneal injection of cyclophosphamide for modeling and 24 hours after the last administration. The blood samples were placed in 2.5 mL EDTA·2K anticoagulant tubes and diluted with an equal volume of whole blood diluent. The cells were lysed with erythrocyte lysis buffer at room temperature, centrifuged, washed with PBS, resuspended in 100 μL PBS, and analyzed by flow cytometry to measure CD4 count. + / CD8 + ratio.

[0111] The proliferation and differentiation of peripheral blood T lymphocytes play a crucial role in the body's response to external stimuli and in the immune response. The level of lymphocyte function in the body can be indirectly evaluated by the levels of T lymphocyte subsets. CD4 + Subgroups can regulate the body's immune function; a decrease in their numbers can lead to suppression of immune regulation. + Subgroups have direct killing effects; excessive expression can lead to immunodeficiency. CD4 + / CD8 + The ratio serves as an indicator of immune system dysregulation; an increase in the ratio suggests a positive regulatory effect on the body's immune function.

[0112] The results are as follows Figure 17 As shown, compared with the blank control group, the CD4 count in the model control group after cyclophosphamide modeling was significantly higher. + / CD8 + The ratio was significantly reduced. Compared with the model control group, all HPP-1S-treated groups showed an increase, especially the low-dose HPP-1S group, which showed a significant increase in CD4 compared with the model control group. + / CD8 + The ratio increased by 1.4 times, indicating that HPP-1S can reverse the damage to T lymphocyte immune function caused by cyclophosphamide.

[0113] (5) Measurement of cytokines in mouse serum

[0114] Twenty-four hours after the last administration, blood was collected from mice under anesthesia into 1.5 mL centrifuge tubes. After standing for 4 hours, the tubes were centrifuged at 3000 rpm / min for 10 minutes at 4°C. The supernatant serum was transferred to a new 1.5 mL centrifuge tube and stored at -80°C for later use. The levels of IL-2 and TNF-α were determined using an ELISA kit.

[0115] The results are as follows Figure 18 As shown, cyclophosphamide significantly reduced the levels of IL-2 and TNF-α in mouse serum. Administration of HPP-1S significantly reversed the cyclophosphamide-induced decrease in IL-2 and TNF-α levels in a dose-dependent manner. The low-dose HPP-1S group showed 60% higher levels of IL-2 and TNF-α compared to the model group, while the high-dose HPP-1S group showed 86% of the TNF-α level in the control group. These results indicate that HPP-1S can promote the increase of immune factor expression, reduce the cyclophosphamide-induced decrease in immunity, and enhance the body's immune function.

[0116] Data from Examples 4, 5, 6, 7, and 8 show that the α-glucan of the present invention has no toxic side effects on macrophages and mouse spleen lymphocytes, and also has a certain proliferative effect, demonstrating high safety. In cell experiments, it can significantly activate macrophages, promote phagocytosis, and increase the expression levels of NO, TNF-α, and IL-6. Further animal experiments confirmed the in vivo and oral efficacy of the α-glucan of the present invention, which can improve various immunosuppressive effects induced by cyclophosphamide, protect immune organs, reduce damage, increase white blood cell and platelet levels, and enhance immune cell function and immune factor expression. It is expected to be developed into a functional food or drug to improve or treat immune-related bodily dysfunctions.

[0117] Example 9: α-Glucan's Inhibition of H22 Hepatocellular Carcinoma Solid Tumors and Immunomodulatory Activity in Mice

[0118] Eighty tumor-bearing mice were randomly divided into eight groups of ten each. There were no statistically significant differences in body weight and tumor volume within or between groups. Saline, HPP-1S, and cyclophosphamide were administered via intraperitoneal injection.

[0119] 1) Model control group: injected with physiological saline for 1-10 days;

[0120] 2) Positive control group: Cyclophosphamide (20 mg / kg / day) was injected for 1–10 days;

[0121] 3) Low-dose HPP-1S group: HPP-1S (20mg / Kg / day) was injected for 1–10 days;

[0122] 4) HPP-1S medium-dose group: HPP-1S (40mg / Kg / day) was injected for 1–10 days;

[0123] 5) High-dose HPP-1S group: HPP-1S (80mg / Kg / day) was injected for 1–10 days;

[0124] 6) Combined low-dose group: HPP-1S (20mg / Kg / day) + cyclophosphamide (20mg / Kg / day) injected for 1-10 days;

[0125] 7) Combined medium-dose group: HPP-1S (40mg / Kg / day) + cyclophosphamide (20mg / Kg / day) injected for 1–10 days;

[0126] 8) Combined high-dose group: HPP-1S (80mg / Kg / day) + cyclophosphamide (20mg / Kg / day) injected for 1-10 days.

[0127] Twenty-four hours after the last administration, mice were weighed, anesthetized, and euthanized. Tumors were removed, and connective and adipose tissue were carefully removed before weighing. The tumor growth inhibition rate was calculated using the formula: Tumor inhibition rate (%) = (Mean tumor mass of the model control group - Mean tumor mass of the administered group) / Mean tumor mass of the model control group × 100%.

[0128] After carefully removing connective and adipose tissue from the spleen and thymus, they were weighed, and the spleen index and thymus index were calculated using the formula: Spleen (thymus) index = Spleen (thymus) weight / Mouse body weight × 100%.

[0129] The results are shown in Table 6. The tumor inhibition rates of the low, medium, and high dose HPP-1S groups were 43.70%, 43.75%, and 52.88%, respectively, while the tumor inhibition rate of the positive control group was 70.83%. The tumor inhibition rates of the combined low, medium, and high dose groups were 69.41%, 67.79%, and 74.82%, respectively. Among them, the tumor inhibition rate of the high dose HPP-1S group reached 52.88%, and the tumor inhibition rate was as high as 74.82% when used in combination with cyclophosphamide. The experimental results show that HPP-1S has a significant anti-tumor effect, and its high dose combined with cyclophosphamide can improve the tumor inhibition rate to a certain extent.

[0130] Table 6

[0131]

[0132] Values ​​are expressed as mean ± SD (n = 10). Compared with the normal control group, #p < 0.05, ##p < 0.01, ###p < 0.001; compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0133] The levels of IL-2 and TNF-α in mouse serum were detected. Twenty-four hours after the last administration, mice were anesthetized, and blood was collected into 1.5 mL centrifuge tubes. After standing at room temperature for 4 hours, the tubes were centrifuged at 3000 rpm / min for 10 minutes at 4°C. The supernatant serum was then transferred to 1.5 mL centrifuge tubes. The levels of IL-2 and TNF-α were measured using an ELISA kit. The results are as follows: Figure 19 As shown, compared with the model group, the TNF-α levels in the HPP-1S monotherapy groups were increased, with the high-dose group showing a significant increase. Compared with the positive control group, the TNF-α levels in the combined medium and high-dose groups were significantly increased, and the effect of HPP-1S on TNF-α secretion in mice was dose-dependent. This indicates that HPP-1S can effectively improve the decreased IL-2 and TNF-α secretion levels caused by decreased immunity, and enhance the body's immune capacity.

[0134] Example 10: α-Glucan's Inhibitory Effects on S180 Sarcoma in Mice and its Immunomodulatory Effects

[0135] Twenty-four hours after inoculation, Kunming mice were randomly divided into seven groups: normal (not inoculated with S180), model, positive control group (CTX, 20 mg / kg / d), low-dose HPP-1S (20 mg / kg / d), medium-dose HPP-1S (40 mg / kg / d), high-dose HPP-1S (80 mg / kg / d), and combined administration group (CTX 20 mg / kg / d + HPP-1S 80 mg / kg / d). The drugs were administered intraperitoneally for 12 consecutive days. Mice were then euthanized by anesthesia and dislocation, and the tumor, spleen, and thymus tissue were dissected and weighed.

[0136] The results are as follows Figure 20 As shown in Table 7, the α-glucan of this invention significantly inhibited the growth of S180 sarcoma. The tumor inhibition rate increased significantly with increasing polysaccharide dosage, reaching 55.49% at a high dose of 80 mg / kg / d, slightly lower than the positive control group (CTX). The inhibition rate of the polysaccharide combined with CTX was slightly higher than that of CTX alone. In mice treated with different doses of polysaccharide, the spleen index was greater than that of the positive control group, especially in the medium and high dose groups, where the spleen index reached 22.38, far exceeding the 4.42 of the cyclophosphamide group. The spleen index of the combined treatment group was 6.76, also higher than the 4.42 of the CTX group. These experimental results indicate that HPP-1S exhibits significant anti-tumor activity, immune organ protection, and a certain degree of synergistic effect and significant toxicity reduction.

[0137] Table 7

[0138]

[0139] Values ​​are expressed as mean ± SD (n = 12). Compared with the model group, #p < 0.05, ##p < 0.01, ###p < 0.001.

[0140] The expression level of IL-2 in mouse serum was detected by ELISA. The results are as follows: Figure 21 As shown, the concentration of IL-2 in the tumor model group and the CTX-positive control group was significantly lower than that in the normal group. This indicates that the immune system of the experimental animals in this group was suppressed, resulting in a decrease in the expression level of immune factors and interfering with the body's normal function of killing tumor cells. In contrast, the expression level of IL-2 was significantly improved in the polysaccharide administration group, as well as the polysaccharide combined with CTX group, especially the high-dose group. This suggests that HPP-1S can treat the decrease in the expression level of immune factors in the body caused by tumors and chemotherapy drugs, and can play an anti-tumor role through immune regulation.

[0141] Example 11: α-Glucan's Inhibitory Effects on B16 Melanoma and Immunomodulatory Effects in Mice

[0142] Twenty-four hours after inoculation, mice were randomly divided into six groups: a model group, a positive control group (CTX, 20 mg / kg / d), a low-dose HPP-1S group (20 mg / kg / d), a medium-dose HPP-1S group (40 mg / kg / d), a high-dose HPP-1S group (80 mg / kg / d), and a combined administration group (CTX 20 mg / kg / d + HPP-1S 80 mg / kg / d). The drugs were administered intraperitoneally for 12 consecutive days. Mice were then euthanized by anesthesia and dislocation, and the tumor, spleen, and thymus tissue were dissected and weighed.

[0143] The results are as follows Figure 22 As shown in Table 8, the α-glucan of this invention significantly inhibits the growth of melanoma in a dose-dependent manner, with an inhibition rate of up to 47.60% at the highest dose. The spleen index of mice in different polysaccharide administration groups was higher than that of the positive control group (CTX) and also higher than that of the blank model group. The high-dose (80 mg / kg / d) HPP-1S administration group reached 10.72, far exceeding the 4.3 of the CTX positive control group. The spleen index of the combined treatment group was 10.38, comparable to that of the high-dose administration group, indicating that the polysaccharide can reduce CTX-induced immune damage, protect immune organs, and exert anti-tumor effects by upregulating immune function.

[0144] Table 8

[0145]

[0146]

[0147] Values ​​are expressed as mean ± SD (n = 10). Compared with the model group, *p < 0.05, **p < 0.01.

[0148] The levels of IL-2 and TNF-α in mouse serum were detected by ELISA. The results are as follows: Figure 23 As shown, HPP-1S can increase the expression levels of IL-2 and TNF-α in serum.

[0149] Animal experiments have shown that the α-glucan of the present invention can activate the body's immune system function, reverse the immunosuppressive effects of tumors and chemotherapy drugs, enhance the immune system's ability to kill tumor cells, and has tumor immunotherapy effects. It can be used to develop anti-tumor drugs.

Claims

1. A class of sea urchin gonad alpha-glucans, characterized in that, having a structure shown in Formula I: wherein n is selected from 2.5, 2, 1.5, 1, and R is selected from glucose, glucuronic acid, mannose, ribose, and galactose residues; the main chain in the structure of Formula I is polymerized by glucose through α-1,4-glycosidic bond, and R in the structure is a monosaccharide side chain, R is selected from glucose, glucuronic acid, mannose, ribose, and galactose, the content of glucose is 84.6-93.0%, the content of glucuronic acid is 4.5-8.0%, the content of mannose is 0.4-4.6%, the content of ribose is 0.1-2.3%, and the content of galactose is 0-1.2%.

2. The alpha-glucan of claim 1, wherein, The molecular weight of the polysaccharide is 1.913 x 10 7 ~ 3.094 x 10 7 Da.

3. Use of one or several combinations of sea urchin gonad α-glucan having a structure shown in Formula I in preparation of a preparation for improving or treating immune-related body dysfunction or disease; wherein n is selected from 2.5, 2, 1.5, 1, and R is selected from glucose, glucuronic acid, mannose, ribose, and galactose residues; the main chain in the structure of Formula I is polymerized by glucose through α-1,4-glycosidic bond, and R in the structure is a monosaccharide side chain, R is selected from glucose and glucuronic acid, and the contents of glucose and glucuronic acid are 95.6% and 4.4% respectively; R is selected from glucose, glucuronic acid, mannose, ribose, and galactose, the content of glucose is 84.6-93.0%, the content of glucuronic acid is 4.5-8.0%, the content of mannose is 0.4-4.6%, the content of ribose is 0.1-2.3%, and the content of galactose is 0-1.2%.

4. Use according to claim 3, characterized in that, The molecular weight of the polysaccharide is 1.913 x 10 7 ~ 3.094 x 10 7 Da.

5. The use according to claim 3, characterized in that, The immune-related body dysfunction is immune function deficiency, low immunity, or immune damage caused by cyclophosphamide.

6. The use according to claim 3, characterized in that, The preparation is a functional food or a medicine.

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