A low-mid polysaccharide composition from schisandra chinensis and application thereof in anti-tumor

Abstract

The application discloses a low-metacharide composition from schisandra chinensis and application thereof in anti-tumor. The low-metacharide composition SFSC is composed of 47 kinds of polysaccharides with a polymerization degree ranging from 2 to 21, including 33 kinds of galacturonan and 14 kinds of maltotriose. The experimental results of the application show that the SFSC can specifically down-regulate the expression of CTLA-4 protein in CD8 T cells, and the immune anti-tumor effect thereof has a unique 'dendritic cell-dependent' mechanism: the SFSC can promote the maturation of dendritic cells, and down-regulate the expression of immune checkpoints PD-L1 and CTLA-4 on DC and CD8 T cells, effectively activating the CD8 T cell-mediated anti-tumor immune response. In addition, the composition can synergistically enhance the curative effect of a PD-1 / PD-L1 inhibitor, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant active polysaccharide technology, specifically, it relates to a low-to-medium polysaccharide composition derived from Schisandra chinensis and its application in anti-tumor activity. Background Technology

[0002] The core of tumor immunotherapy lies in mobilizing the body's own immune system to recognize and eliminate tumor cells, with the regulation of immune checkpoints becoming a key strategy. Cytotoxic T-lymphocyte antigen 4 (CTLA-4), as the first clinically validated immune checkpoint, has shown durable efficacy in some advanced tumors due to the fact that its inhibitors (such as ipilimumab) can restore T-cell co-stimulatory signals by blocking the binding of CTLA-4 to B7 molecules (CD80 / CD86) on the surface of antigen-presenting cells. However, the non-specific mechanism of action of this type of antibody therapy (i.e., systemic blockade of CTLA-4 function) often leads to severe immune-related adverse reactions, and the limited single-agent response rate has become a major clinical bottleneck restricting its widespread application.

[0003] To overcome this limitation, next-generation CTLA-4 regulation strategies are shifting from "functional blockade" to "expression downregulation." Theoretically, by reducing the protein level of CTLA-4 on the surface of T cells, immunosuppression can be more precisely relieved, avoiding excessive immune activation caused by persistent receptor occupancy, thereby improving safety while maintaining efficacy. Currently, research in this field mainly focuses on synthesizing small molecules or protein degradation technologies, which are difficult to develop and have uncertain drug-like properties. CTLA-4 expression downregulators derived from natural products remain a gap in the market. Furthermore, the carbohydrate components of traditional Chinese medicine possess natural immunomodulatory activity and unique structural advantages, exhibiting theoretical potential to target the immune checkpoint CTLA-4, and are expected to provide a strategic breakthrough for the development of next-generation CTLA-4 inhibitors.

[0004] Schisandra chinensis (scientific name: Schisandra chinesis ( Turcz. ) Schisandra chinensis (also known as Schisandra chinensis or Schisandra sibirica) is a plant belonging to the genus Schisandra in the family Schisandraceae. Schisandra sphenanthera RehdThe dried, ripe fruit of Schisandra chinensis et Wils. possesses astringent, qi-tonifying, fluid-generating, kidney-tonifying, and heart-calming effects. Its active ingredients, especially the sugar components, exhibit immunomodulatory potential. Chinese patent application CN116284466A discloses polysaccharide monomers isolated from Schisandra chinensis. These polysaccharide monomers are all amyloid acidic polysaccharides. One mainly contains glucose, galacturonic acid, and trace amounts of arabinose, rhamnose, and galactose, with a theoretical molar ratio of glucose to galacturonic acid of 17:3. Its absolute molecular weight is 30.5 kDa, and its polydispersity index is 1.129. The other mainly contains glucose, galacturonic acid, and trace amounts of arabinose, rhamnose, and galactose, with a theoretical molar ratio of glucose to galacturonic acid of 25:3. Its absolute molecular weight is 106.0 kDa, and its polydispersity index is 2.28. The two polysaccharide monomers provided by this patent exhibit good immunomodulatory and antitumor activities, showing promise for the preparation of antitumor or immunomodulatory drugs. Secondly, Chinese patent application CN120939044A also discloses that the polysaccharide monomer schisanan B isolated from Schisandra chinensis can effectively activate PD-L1+ TAMs cell subsets, thereby enhancing the phagocytic activity of TAMs and inhibiting the proliferation of tumor cells. This confirms that the polysaccharide monomer schisanan B isolated from Schisandra chinensis can be used as a natural PD-L1+ TAMs cell subset activator for the treatment of cancers such as non-small cell lung cancer, colorectal cancer, melanoma, urothelial carcinoma, or liver cancer. It has the advantages of being non-toxic, inexpensive, and suitable for a wide range of patients. From this patent, it can be seen that the target of the polysaccharide monomer schisanan B is tumor-associated macrophages.

[0005] While existing technologies reveal that some polysaccharides derived from Schisandra chinensis can be widely used in cancer treatment, the different compositions of these polysaccharides lead to different therapeutic targets on tumors, making it uncertain whether all polysaccharides from Schisandra chinensis possess tumor-treating properties. Furthermore, existing technologies do not disclose that Schisandra chinensis polysaccharides can enhance anti-tumor immunity by inhibiting T-cell expression of the immune checkpoint CTLA-4 and relying on dendritic cell activation, nor do they disclose that Schisandra chinensis polysaccharides can synergistically enhance anti-tumor immunity when combined with other antibodies. Therefore, providing a composition of low- and medium-polysaccharides derived from Schisandra chinensis that can exert anti-tumor immune effects through the aforementioned mechanisms and possesses synergistic drug potential has significant scientific value and clinical translational implications. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a low-molecular-weight polysaccharide composition derived from Schisandra chinensis.

[0007] A second object of the present invention is to provide the use of the above-mentioned Schisandra chinensis-derived oligosaccharide composition in the preparation of antitumor drugs.

[0008] A third objective of this invention is to provide the application of the above-mentioned Schisandra chinensis-derived low-to-medium polysaccharide composition in combination with PD-1 / PD-L1 inhibitors in the preparation of antitumor drugs.

[0009] A fourth object of the present invention is to provide the use of the above-mentioned Schisandra chinensis-derived oligosaccharide composition in the preparation of a medicament for enhancing T-cell immunity.

[0010] The fifth objective of this invention is to provide an antitumor drug.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a low-to-medium polysaccharide composition derived from Schisandra chinensis, wherein the low-to-medium polysaccharide composition comprises 33 acidic polysaccharides and 14 neutral polysaccharides with a degree of polymerization of 2 to 21; the monosaccharide composition of the low-to-medium polysaccharide composition is 9.8% rhamnose, 0.8% arabinose, 8.9% galactose, 33.1% glucose, 3.1% xylose, 3.3% mannose, and 41.0% galacturonic acid; The preparation method of the Schisandra chinensis-derived oligosaccharide composition includes the following steps: S1. Take Schisandra chinensis, use water as the extraction solvent, heat and reflux to obtain Schisandra chinensis aqueous extract; S2. Remove pigments and proteins from the Schisandra chinensis aqueous extract of step S1 and elute to obtain Schisandra chinensis aqueous eluent; S3. The Schisandra chinensis aqueous eluent from step S2 is intercepted and collected. Mw The portion <3 kDa yields the low-to-medium polysaccharide composition (SFSC) derived from Schisandra chinensis.

[0012] This invention first uses a combination of PHA-L (phytohemagglutinin-L), PMA (phorbol ester), and Ionomycin to induce Jurkat cells to express CTLA-4 protein. Flow cytometry is then used to systematically screen the CTLA-4-targeting inhibitory activity of various carbohydrate compounds. The results show that the Schisandra chinensis-derived low- and medium-saccharide composition SFSC can significantly inhibit CTLA-4 protein expression in Jurkat cells, and can serve as a candidate drug for a traditional Chinese medicine carbohydrate CTLA-4 inhibitor. To further verify whether the Schisandra chinensis-derived low- and medium-saccharide composition can act as a CTLA-4 inhibitor, this invention provides a method for preparing the Schisandra chinensis-derived low- and medium-saccharide composition, mainly including extracting Schisandra chinensis aqueous extract using water as the extraction solvent via heating and reflux extraction, removing pigments and proteins from the Schisandra chinensis aqueous extract, eluting, and collecting the extract. MwThe portion <3 kDa yielded the Schisandra chinensis low- and medium-polysaccharide composition. The Schisandra chinensis-derived low- and medium-polysaccharide composition prepared using the above extraction method was tested. This composition was a white powder, highly soluble in water, and consisted of a 2-21 sugar mixture system. It was composed of 33 highly methylated galacturonic acid polysaccharides (acidic polysaccharides) and 14 maltose-based polysaccharides (neutral polysaccharides). The monosaccharide composition of the low- and medium-polysaccharide composition was 9.8% rhamnose, 0.8% arabinose, 8.9% galactose, 33.1% glucose, 3.1% xylose, 3.3% mannose, and 41.0% galacturonic acid.

[0013] Furthermore, the acidic polysaccharide has a degree of polymerization of 2 to 21 and a highly methylated galacturonic acid polymer with a 1→4 linked structure; the neutral polysaccharide has a degree of polymerization of 2 to 15 and a maltose series polymer with a 1→4 linked structure.

[0014] Specifically, the fine chemical structure of the SFSC is shown below: .

[0015] Furthermore, the method for removing pigments and proteins in step S2 is to adsorb and elute the Schisandra chinensis aqueous extract using a macroporous resin column.

[0016] Preferably, the macroporous resin column is AB-8 type macroporous resin.

[0017] Preferably, the elution is performed with distilled water, and the elution volume is 6 times the column volume.

[0018] Specifically, in step S2, the method for removing pigments and proteins is to adsorb the aqueous extract through AB-8 type macroporous resin for 12 hours and then elute with distilled water until the eluent is pale yellow as detected by the phenol-sulfuric acid method.

[0019] Furthermore, in step S3, the throttling is performed by using a 3 kDa ultrafiltration column to throttle the water eluent of Schisandra chinensis.

[0020] Preferably, the sample concentration during interception is 7:1 (water volume mL: medicinal material weight g), and the ultrafiltration pressure is 0.06 MPa.

[0021] Furthermore, the preparation method also includes dissolving the Schisandra chinensis low-molecular-weight polysaccharide composition obtained in step S3, followed by elution, concentration, and lyophilization using a HILIC chromatographic column.

[0022] This invention further investigates the antitumor activity and immune-dependent properties of the aforementioned Schisandra chinensis-derived oligosaccharide composition. In in vivo efficacy evaluation, mouse lung cancer xenograft models were established using immunocompetent C57BL / 6 mice and T-cell-deficient Balb / cNude nude mice, respectively. SFSC was administered orally for intervention. Experimental results showed that SFSC exhibited significant antitumor activity in immunocompetent C57BL / 6 mice, but the tumor-suppressive effect of SFSC was completely absent in T-cell-deficient nude mice. This indicates that the antitumor effect of SFSC depends on T-cell-mediated adaptive immunity. Furthermore, this invention used flow cytometry to detect the effect of SFSC on CTLA-4 expression in mouse tumor-infiltrating T cells in vivo. The results showed that SFSC treatment could reduce CTLA-4 expression in T cells in vivo, and this reduction was specific to CD8 T cell subtypes. Subsequently, B16-F10 melanoma and Hepa1-6 hepatocellular carcinoma xenograft models were established using immunocompetent C57BL / 6 mice to further evaluate the broad-spectrum antitumor activity of SFSC and verify the universality of its core mechanism (downregulation of CTLA-4). The results showed that SFSC exhibited broad-spectrum antitumor activity and a common core molecular event directly related to the efficacy was discovered: SFSC can specifically and significantly downregulate the protein expression level of CTLA-4, a key immune checkpoint on the surface of tumor-infiltrating CD8T cells.

[0023] To elucidate the specific mechanism by which the above-mentioned Schisandra chinensis oligosaccharide composition enhances T-cell immunity and is used for tumor immunotherapy, this invention utilizes mass spectrometry combined with conventional flow cytometry to conduct in-depth analysis of the tumor microenvironment in mice before and after drug administration. The results showed that SFSC significantly increased the infiltration of immune cells within tumors, reduced the proportion of the exhausted subset (CD8Tex) of tumor-infiltrating CD8T cells, downregulated CTLA-4 levels on CD8T cells, and simultaneously increased the tumor-killing ability of effector CD8T cell subsets, manifested by increased expression of Granzyme B and Perforin. Furthermore, SFSC significantly increased the level of CD103+ dendritic cells (CD103+DCs) and promoted DC maturation (manifested as upregulated CD80 expression), while reducing PD-L1 levels on DCs. These results suggest that SFSCs may activate anti-tumor immune responses by promoting dendritic cell maturation and inhibiting the expression of immune checkpoints PD-L1 and CTLA-4 on tumor-associated DCs and CD8T cells, respectively, thereby increasing DC-mediated CD8T cell activation (DC / T cell crosstalk).

[0024] Further, after eliminating CD8T cells in the body through intraperitoneal injection of CD8a neutralizing antibodies, the antitumor effect of the drug was completely eliminated. In addition, utilizing... Batf3A lung cancer model was established using gene knockout (Batf3-KO) mice (which specifically lack CD103+ dendritic cells). In this model, the SFSCs described in this invention also completely lost their tumor-suppressive effect. CD103+ dendritic cells are key upstream regulatory cells that initiate and activate the anti-tumor immune response of CD8T cells, thus confirming that its pharmacological pathway is: SFSC → CD103+ dendritic cells → CD8T cells → tumor cells.

[0025] In in vitro co-culture experiments, the composition was first applied to DC2.4 cells (a mouse-derived dendritic cell line), effectively promoting their maturation (increased CD80 expression). SFSC-pretreated DC2.4 cells, when co-cultured with CTLL-2 T cells (a mouse-derived T cell line), significantly promoted CTLL-2 cell proliferation. Finally, these activated CTLL-2 cells, co-cultured with LLC lung cancer cells, exhibited significant tumor cell killing ability. This experiment visually replicated and confirmed the conclusions of the in vivo mechanism studies.

[0026] Therefore, the present invention provides the use of the above-mentioned Schisandra chinensis-derived low- and medium-polysaccharide composition in the preparation of antitumor drugs.

[0027] This invention also combines the aforementioned Schisandra chinensis-derived low- and medium-polysaccharide composition with existing standard immune checkpoint inhibitors (anti-PD-L1 antibody and anti-CTLA-4 antibody). Compared with the individual monotherapy groups, the combination with the anti-PD-L1 monoclonal antibody produced a significant synergistic effect. This phenomenon can be perfectly explained mechanistically: the composition of this invention downregulates CTLA-4 and activates T cells in a DC-dependent manner, which is equivalent to "starting and strengthening the accelerator signal of immune attack"; while the anti-PD-L1 antibody removes the "brake signal" of the tumor microenvironment on T cells. The two work together to reshape the immune microenvironment from different dimensions and through complementary mechanisms, opening up the entire process of anti-tumor immunity, thereby achieving excellent synergistic therapeutic effects and providing core technical support for the development of next-generation highly effective tumor immunotherapy combination regimens.

[0028] Notably, in combination therapy experiments with different immune checkpoint inhibitors, SFSC combined with anti-PD-L1 antibodies exhibited a significantly superior synergistic effect compared to its combination with anti-CTLA-4 antibodies. This is presumably because SFSC, as a "downregulator" of CTLA-4 expression, overlaps with CTLA-4 "functional blockers" (monoclonal antibodies) in terms of target and action phase, while forming a spatiotemporal complementarity with antibodies acting on the downstream effector phase of the PD-1 / PD-L1 pathway. This phenomenon not only further confirms the core mechanism by which the composition of this invention exerts its effect through downregulating CTLA-4 expression, but also provides clear guidance for its optimal clinical combination therapy strategy—namely, preferential combination with PD-1 / PD-L1 inhibitors. Therefore, this invention not only confirms the superior monotherapy efficacy of SFSC, but also reveals its broad clinical application prospects through combination therapy studies.

[0029] More importantly, the SFSC described in this invention, while exhibiting excellent efficacy, also demonstrates superior safety not found in traditional CTLA-4 targeted therapies. Systematic toxicological evaluation showed that in long-term animal administration experiments across different batches, the weight gain of experimental animals was unaffected after administration of effective therapeutic doses of this composition, and their behavior, feeding, and physiological state remained normal. Comprehensive analysis of key serum biochemical indicators further confirmed that all core indicators, including ALT and AST reflecting liver function, CRE and BUN reflecting kidney function, CK reflecting myocardial injury, and total protein TP, fluctuated within normal physiological ranges and showed no statistically significant differences compared to the control group. This series of data consistently indicates that SFSC did not exhibit any drug-related toxicity to major parenchymal organs such as the liver, kidneys, and heart. It shows significant synergy with existing therapies and possesses good intrinsic safety, circumventing the clinical bottleneck of the high toxicity of existing CTLA-4 antibody therapies, demonstrating extremely high clinical development value and market application prospects.

[0030] Therefore, the present invention also provides the application of the above-mentioned Schisandra chinensis-derived low-to-medium polysaccharide composition in combination with a PD-1 / PD-L1 inhibitor in the preparation of an anti-tumor drug, wherein the PD-1 / PD-L1 inhibitor is an anti-PD-L1 antibody.

[0031] Furthermore, the ratio (mass ratio) of the low-to-medium polysaccharide composition derived from Schisandra chinensis to the PD-1 / PD-L1 inhibitor is 18 to 22:1.

[0032] Furthermore, the aforementioned tumors include, but are not limited to, lung cancer, melanoma, or liver cancer.

[0033] Furthermore, the lung cancer in question is non-small cell lung cancer.

[0034] Furthermore, the aforementioned drugs exert their anti-tumor effects by enhancing CD8T cell-mediated adaptive immunity.

[0035] Furthermore, the drug achieves therapeutic effects by downregulating the expression of the immune checkpoint CTLA-4 on CD8T cells.

[0036] Furthermore, the drug achieves treatment by reducing the proportion of the exhaustion subset (CD8Tex) in CD8T cells.

[0037] Furthermore, the drug achieves therapeutic effects by upregulating the expression of Granzyme B and Perforin in CD8T cells.

[0038] Furthermore, the drug achieves therapeutic effects by increasing the level of CD103+ dendritic cells (CD103+DCs) and promoting the maturation of DCs (manifested as upregulated CD80 expression).

[0039] Furthermore, the drug achieves treatment by reducing the level of PD-L1 on DC cells.

[0040] Furthermore, the drug achieves therapeutic effects by activating dendritic cells and downregulating the expression of the immune checkpoint CTLA-4 on CD8T cells.

[0041] Preferably, the drug promotes the maturation of CD103+ dendritic cells (CD103+DCs) by increasing the level of these cells, inhibiting the expression of the immune checkpoint PD-L1 on tumor-associated DCs, increasing DC-mediated CD8T cell activation (DC / T cell crosstalk), downregulating the expression of the immune checkpoint CTLA-4 on CD8T cells, and upregulating the expression of Granzyme B and Perforin on CD8T cells, thereby activating an anti-tumor immune response to achieve therapeutic effects. Therefore, the pharmacodynamic pathway of the Schisandra chinensis-derived oligosaccharides provided by this invention is: SFSC → CD103+ dendritic cells → CD8T cells → tumor cells.

[0042] The present invention also provides the application of the above-mentioned Schisandra chinensis-derived oligosaccharides in the preparation of drugs that enhance T-cell immunity.

[0043] This invention provides the application of the above-mentioned Schisandra chinensis-derived oligosaccharides in the preparation of antitumor drugs that have both dendritic cell activation and CTLA-4 expression downregulation or T cell immune enhancement effects.

[0044] The present invention also provides an antitumor drug comprising the above-mentioned low-to-medium polysaccharide composition derived from Schisandra chinensis.

[0045] Furthermore, the drug also contains a PD-1 / PD-L1 inhibitor; the PD-1 / PD-L1 inhibitor is an anti-PD-L1 antibody.

[0046] Furthermore, the drug also contains pharmaceutically acceptable excipients.

[0047] Furthermore, the dosage forms of the aforementioned drugs include, but are not limited to, oral formulations or injections. Those skilled in the art can prepare the aforementioned drugs into conventional pharmaceutical formulations using conventional methods in the field of pharmaceutical formulation.

[0048] The excipients mentioned in this invention refer to conventional excipients or carriers in the pharmaceutical field, such as diluents, disintegrants, lubricants, excipients, binders, flow aids, fillers, surfactants, etc. Additionally, other excipients, such as flavoring agents and sweeteners, may be added to the composition. For example: The diluent can be one or more ingredients that increase the weight and volume of the tablet. Commonly used diluents include lactose starch, pregelatinized starch, microcrystalline cellulose, sorbitol, mannitol, and inorganic calcium salts, etc.; among which lactose, starch, and microcrystalline cellulose are the most commonly used. The disintegrant may be one or a mixture of several of the following: cross-linked polyvinylpyrrolidone (2-6% by weight), cross-linked sodium methyl cellulose (2-6% by weight), alginate (2-5% by weight), and microcrystalline cellulose (5-15% by weight). The lubricant includes one or a mixture of several of the following: stearic acid, sodium stearate, magnesium stearate, calcium stearate, polyethylene glycol, talc, and hydrogenated vegetable oil. The dosage range of the lubricant (0.10–1% of the total weight) is generally 0.25–0.75%. The binder can be one or more components that are beneficial to granulation, such as starch paste (10-30%, by weight of the binder), hydroxypropyl methylcellulose (2-5%, by weight of the binder), or polyvinylpyrrolidone (2-20%, by weight of the binder), preferably an ethanol-water solution of polyvinylpyrrolidone. The flow aid can be one or a mixture of several of the following: micronized silica gel, talc, and magnesium trisilicate. The surfactant can be one or more components that can improve wettability and increase drug dissolution, commonly sodium dodecyl sulfate (common range is 0.2-6% by weight).

[0049] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a low-to-medium polysaccharide composition derived from Schisandra chinensis and its application in antitumor activity. The invention uses water as the extraction solvent and employs a heating reflux extraction method to obtain an aqueous extract of Schisandra chinensis, which is then collected. MwThe <3 kDa fraction yields the Schisandra chinensis low-to-medium polysaccharide composition (SFSC). This SFSC composition consists of 47 polysaccharides with a degree of polymerization ranging from 2 to 21, including 33 highly methylated galacturonic acid polysaccharides and 14 maltose-based polysaccharides. Experimental results of this invention show that the SFSC can specifically downregulate CTLA-4 protein expression in CD8T cells, and its immuno-antitumor effect has a unique "dendritic cell-dependent" mechanism: it can promote dendritic cell (DC) maturation while downregulating the expression of immune checkpoints PD-L1 and CTLA-4 on DCs and CD8T cells, effectively activating CD8T cell-mediated antitumor immune responses. Furthermore, this composition can synergistically enhance PD-L1 and CTLA-4 expression. 1 / PD The efficacy of L1 inhibitors, and the fact that they are non-toxic to major organs such as the liver, kidneys, and heart at effective doses, overcomes the limitations of existing CTLA inhibitors. 4. Despite the high toxicity and side effects of targeted therapy, it has promising prospects for clinical application. Attached Figure Description

[0050] Figure 1 The screening of CTLA-4 inhibitors derived from traditional Chinese medicine carbohydrates. Among them, Figure 1 In the figure, A is the histogram of CTLA-4 protein expression in Jurkat cells detected by flow cytometry; B is the quantitative graph of CTLA-4 protein expression in Jurkat cells induced by the combined use of PHA-L, PMA, and Ionomycin; C is the normalized result of the inhibition of CTLA-4 protein expression in Jurkat cells by 30 kinds of traditional Chinese medicine carbohydrate combinations. The experimental results were statistically analyzed, n=3; among them, ### P <0.001, * P <0.05,** P <0.01, *** P <0.001.

[0051] Figure 2 This is a chemical composition study for SFSC. Among them, Figure 2 In the diagram, A represents the extraction process of SFSC; B represents the separation process of SFSC monomers; C represents the monosaccharide composition of SFSC; and D represents the total ion chromatogram of SFSC.

[0052] Figure 3 This is a mass spectrometry characterization diagram of SFSC. Among them, Figure 3 In the diagram, A is the secondary mass spectrum of SFSC peak 22; B is a schematic diagram of the fragmentation of peak 22; C is the secondary mass spectrum of SFSC peak 26; and D is a schematic diagram of the fragmentation of peak 26.

[0053] Figure 4 The image shows the mass spectrometry characterization of SFSC monomer 6. Among them, Figure 4In the diagram, A is the total ion chromatogram of SFSC monomer 6; B is the primary mass spectrum of SFSC monomer 6; C is the secondary mass spectrum of SFSC monomer 6; and D is a schematic diagram of the fragmentation of SFSC monomer 6.

[0054] Figure 5 The 1D-NMR characterization of SFSC monomer 6 is as follows. Figure 5 In the diagram, A represents the 1H NMR spectrum of SFSC monomer 6; B represents the 1C NMR spectrum of SFSC monomer 6.

[0055] Figure 6 2D-NMR characterization of SFSC monomer 6. Figure 6 In the diagram, A is the 1H-1H COSY spectrum of SFSC monomer 6; B is the HSQC spectrum of SFSC monomer 6.

[0056] Figure 7 2D-NMR characterization of SFSC monomer 6. Figure 7 In the diagram, A is the HMBC spectrum of SFSC monomer 6; B is the TOCSY spectrum of SFSC monomer 6.

[0057] Figure 8 The effects of SFSC on lung cancer xenograft mice. Figure 8 In the diagram, A represents the experimental flowchart of the C57BL / 6 mouse model; B represents the anatomical diagram of the xenograft at the end of drug administration in each group of mice (C57BL / 6 mice), n=9; C represents the weight of the xenograft at the end of drug administration in each group of mice (C57BL / 6 mice), n=9; D represents the change in xenograft volume in mice (C57BL / 6 mice), n=9; E represents the change in body weight in mice (C57BL / 6 mice), n=9; F represents the survival time of mice (C57BL / 6 mice), n=10; and G represents the CT values ​​in tumor-infiltrating T cells in mice. Histogram of CTLA-4 protein expression; H represents the quantitative result of CTLA-4 protein expression in mouse tumor-infiltrating T cells, n=5; I represents the histogram of CTLA-4 protein expression in mouse tumor-infiltrating CD4T cells; J represents the quantitative result of CTLA-4 protein expression in mouse tumor-infiltrating CD4T cells, n=5; K represents the histogram of CTLA-4 protein expression in mouse tumor-infiltrating CD8T cells; L represents the quantitative result of CTLA-4 protein expression in mouse tumor-infiltrating CD8T cells, n=5; M is the flowchart of the Balb / c Nude nude mouse model experiment; N is the anatomical diagram of the transplanted tumor at the end of drug administration in each group of mice (Balb / c Nude nude mice), n=7; O is the weight of the transplanted tumor at the end of drug administration in each group of mice (Balb / c Nude nude mice), n=7; P is the change in the growth volume of the transplanted tumor in mice (Balb / c Nude nude mice), n=7. The experimental results were statistically analyzed, where * P <0.05;** P <0.01; ***P <0.001; ns, no significant change.

[0058] Figure 9 The effects of SFSC on mice with melanoma and liver cancer xenografts. Figure 9 In this diagram, A represents the experimental flowchart of the B16-F10 melanoma model; B represents the anatomical diagram of the transplanted tumor at the end of drug administration in each group of mice (B16-F10 melanoma model), n=8; C represents the weight of the transplanted tumor at the end of drug administration in each group of mice (B16-F10 melanoma model), n=8; D represents the change in the growth volume of the transplanted tumor in mice (B16-F10 melanoma model), n=8; E represents the change in the body weight of mice (B16-F10 melanoma model), n=8; F represents the histogram of CTLA-4 expression on tumor-infiltrating CD8T cells in each group of mice (B16-F10 melanoma model); and G represents the quantitative results of CTLA-4 expression on tumor-infiltrating CD8T cells in each group of mice (B16-F10 melanoma). Model), n=6; H is the experimental flowchart of the Hepa1-6 liver cancer model; I is the anatomical diagram of the transplanted tumor at the end of drug administration in each group of mice (Hepa1-6 liver cancer model), n=8; J is the weight of the transplanted tumor at the end of drug administration in each group of mice (Hepa1-6 liver cancer model), n=8; K is the change in the growth volume of the transplanted tumor in mice (Hepa1-6 liver cancer model), n=8; L is the change in the body weight of mice (Hepa1-6 liver cancer model), n=8; M is the histogram of CTLA-4 expression on tumor-infiltrating CD8T cells in each group of mice (Hepa1-6 liver cancer model); N is the quantitative result of CTLA-4 expression on tumor-infiltrating CD8T cells in each group of mice (Hepa1-6 liver cancer model), n=6. The experimental results were statistically analyzed, where * P <0.05;** P <0.01; *** P <0.001; ns, no significant change.

[0059] Figure 10 This study aimed to analyze the effects of SFSCs on the tumor microenvironment of lung cancer mice using a combination of mass spectrometry and conventional flow cytometry. Figure 10 In the diagram, A represents the experimental flowchart; B represents the total cell clustering tSNE plot; C represents the total cell subset identification tSNE plot; D represents the distribution of non-immune and immune cells in mouse tumors; and E represents CD45. + Immune cell subset identification tSNE plot; F represents CD45 +Analysis of the proportion of each immune cell subset (n=5); G represents the change in the proportion of CD8Tex in CD8T cells (n=5); H represents the change in CTLA-4 expression level on total CD8T cells (n=5); I represents the change in CTLA-4 expression level on CD8Tex cells (n=5); J represents the change in Granzyme B expression level in the effector CD8T subset CD8Teff1 (n=5); K represents the change in Perforin expression level in the effector CD8T subset CD8Teff1 (n=5); L represents the tSNE representation of PD-L1 expression changes in myeloid subsets; M represents the mean expression intensity of PD-L1 in myeloid subsets (n=5); N represents the change in CD80 expression level on DC cells (n=6). Statistical analysis was performed on the experimental results, where * P <0.05;** P <0.01; *** P <0.001.

[0060] Figure 11 This study validated the efficacy of SFSC in a CD8T cell clearance model. Figure 11 In the figures, A represents the experimental flowchart; B represents the anatomical diagram of the transplanted tumor in each group of mice at the end of drug administration (n=7); C represents the weight of the transplanted tumor in each group of mice at the end of drug administration (n=7); D represents the change in tumor growth volume in mice (n=7); E represents the change in mouse body weight (n=7); F represents the histogram of CD80 expression on tumor-infiltrating DC cells in each group of mice; G represents the quantitative results of CD80 expression on tumor-infiltrating DC cells in each group of mice (n=6); H represents the scatter plot of CD8T cell infiltration in each group of mice; and I represents the quantitative results of CD8T cell infiltration in each group of mice (n=6). The experimental results were statistically analyzed, where * P <0.05;** P <0.01; *** P <0.001; ns, no significant change.

[0061] Figure 12 This study validates the efficacy of SFSC in the Batf3-KO model. Figure 12In the diagram, A represents the experimental flowchart; B represents the anatomical diagram of the transplanted tumor at the end of drug administration in each group of mice (n=7); C represents the weight of the transplanted tumor at the end of drug administration in each group of mice (n=7); D represents the change in tumor growth volume in mice (n=7); E represents the change in mouse body weight (n=7); F represents a scatter plot of CD8T and CD8Tex cell infiltration in each group of mice; G represents the quantitative results of CD8T cell infiltration in each group of mice (n=4); H represents the quantitative results of CD8Tex cell infiltration in each group of mice (n=4); I represents a scatter plot of Perforin expression in CD8T cells infiltrating tumors in each group of mice; J represents the quantitative results of Perforin expression in CD8T cells infiltrating tumors in each group of mice (n=4); and K represents the expression of Granzyme in CD8T cells infiltrating tumors in each group of mice. B is a scatter plot; L represents the quantitative results of Granzyme B expression in tumor-infiltrating CD8T cells of mice in each group, n=4; M is a histogram of CTLA-4 expression in tumor-infiltrating CD8T cells of mice in each group; N represents the quantitative results of CTLA-4 expression in tumor-infiltrating CD8T cells of mice in each group, n=4; O represents a histogram of CTLA-4 expression in tumor-infiltrating CD8Tex cells of mice in each group; P represents the quantitative results of CTLA-4 expression in tumor-infiltrating CD8Tex cells of mice in each group, n=4. The experimental results were statistically analyzed, where * P <0.05;** P <0.01; *** P <0.001; ns, no significant change.

[0062] Figure 13 This is an in vitro cell co-culture experiment. Among them, Figure 13 In the diagram, A represents the histogram of CD80 expression on the surface of DC2.4 cells; B represents the quantitative map of CD80 expression on the surface of DC2.4 cells (n=4); C represents the histogram of CD86 expression on the surface of DC2.4 cells; D represents the quantitative map of CD86 expression on the surface of DC2.4 cells (n=4); E represents the change in TNF-α mRNA level in DC2.4 cells (n=4); F represents the change in IL-6 mRNA level in DC2.4 cells (n=4); G represents the change in IL-1β mRNA level in DC2.4 cells (n=4); H represents the change in IL-12B mRNA level in DC2.4 cells (n=4); I represents the schematic diagram of the co-culture steps of DC2.4 / CTLL-2 / LLC cells; J represents the scatter plot of CTLL-2 cell proliferation; K represents the quantitative map of CTLL-2 cell proliferation (n=4); L represents the fluorescence map of LLC cell survival; and M represents the quantitative map of LLC cell survival (n=4). The experimental results were statistically analyzed, where * P <0.05;** P <0.01; *** P <0.001.

[0063] Figure 14 This study evaluates the in vivo efficacy of SFSC in combination with anti-PD-L1 and anti-CTLA-4 antibodies. Figure 14 In the figures, A represents the experimental flowchart; B represents the tumor weight of each group of mice at the end of drug administration (n=7); C represents the change in tumor growth volume in mice (n=7); D represents the anatomical diagram of the tumor in each group of mice at the end of drug administration (n=7); E represents the scatter plot of MDSCs cell infiltration in the tumors of each group of mice; F represents the quantitative map of MDSCs cell infiltration in the tumors of each group of mice (n=5); G represents the scatter plot of the proportion of Treg cells in tumor-infiltrating CD4T cells in each group of mice; H represents the quantitative map of the proportion of Treg cells in tumor-infiltrating CD4T cells in each group of mice (n=5); I represents the scatter plot of Perforin expression in tumor-infiltrating CD8T cells in each group of mice; J represents the quantitative map of Perforin expression in tumor-infiltrating CD8T cells in each group of mice (n=5); K represents the scatter plot of Granzyme B expression in tumor-infiltrating CD8T cells in each group of mice; L represents the quantitative map of Granzyme B expression in tumor-infiltrating CD8T cells in each group of mice (n=5). The experimental results were statistically analyzed, where * P <0.05;** P <0.01; *** P <0.001; ns, no significant change.

[0064] Figure 15 This is for the SFSC's drug safety evaluation. Among them, Figure 15 In the table, A represents the serum ALT level of each group of mice, n=6; B represents the serum AST level of each group of mice, n=6; C represents the serum URE level of each group of mice, n=6; D represents the serum BUN level of each group of mice, n=6; E represents the serum CK level of each group of mice, n=6; and F represents the serum TP level of each group of mice, n=6. Detailed Implementation

[0065] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0066] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0067] Example 1: Screening of CTLA-4 inhibitors derived from traditional Chinese medicine I. Experimental Methods To screen for CTLA-4 inhibitors derived from traditional Chinese medicine (TCM) carbohydrates, this invention induced Jurkat cells to express CTLA-4 protein in vitro using a combination of PHA-L (phytohemagglutinin-L, 1 g / mL), PMA (phorbol ester, 20 ng / mL), and Ionomycin (ionomycin, 500 ng / mL). Simultaneously, different TCM carbohydrates (from the inventors' previously constructed TCM carbohydrate resource library, which includes Schisandra chinensis low-to-medium polysaccharide compositions (SFSC, see...)) were administered. Figure 1 The drug was treated with A)). After 24 h, flow cytometry was used to systematically screen the CTLA-4 targeting activity of various traditional Chinese medicine carbohydrate compounds in order to identify candidate drugs for CTLA-4 inhibitors of traditional Chinese medicine carbohydrates.

[0068] II. Experimental Results Experimental results are as follows Figure 1 As shown, compared with the control (CON) group, the combined induction of PHA-L, PMA and Ionomycin significantly induced the expression of CTLA-4 protein in Jurkat cells. When different sugars were administered, some sugars could inhibit the expression of CTLA-4 protein to varying degrees, among which SFSC had the most significant inhibitory effect.

[0069] Example 2 Preparation and characterization of Schisandra chinensis low-mesopolysaccharide composition (SFSC) I. Experimental Methods 1. Preparation of Schisandra chinensis low-molecular-weight polysaccharide composition Take 5 kg of Schisandra chinensis, and extract 500 g of each time. Add 5 L of distilled water and extract at 100℃ for 3 hours each time. Filter the residue through gauze. Repeat the extraction three times with the residue and combine the Schisandra chinensis aqueous extracts. Adsorb the aqueous extract through AB-8 macroporous resin for 12 hours and elute with distilled water until the eluent is pale yellow according to the phenol-sulfuric acid method (this method can remove some pigments, proteins, and other substances from Schisandra chinensis). Separate the macroporous adsorption resin eluent through a 3000 Da molecular weight ultrafiltration column to obtain... Mw The low-to-medium polysaccharide SFSC (<3000 Da) is a completely different substance from the polysaccharide monomer schisanan B isolated from Schisandra chinensis disclosed in Chinese patent CN120939044A, and its preparation process is similar to that of SFSC. Figure 2 (As shown in A in the diagram).

[0070] Dissolve 50 g of SFSC in a 55% acetonitrile-water solution, sonicate for 30 min to ensure complete dissolution, then centrifuge at 5000 rpm for 15 min. Collect the supernatant and slowly pass it into a HILIC column (12 μm particle size, 5.0 × 50 cm) at a flow rate of 2 mL / min. Elute sequentially with 90%, 75%, 65%, and 55% acetonitrile-water solutions, collecting 1.5 L of each solution and 4 L of the 55% solution every 500 mL. Concentrate and lyophilize the eluents. Dissolve the lyophilized fraction directly in 50% acetonitrile to prepare a concentration of 100.0 mg / mL, then centrifuge (12,000 rpm, 10 min) and filter through a 0.22 μm filter into a vial.

[0071] 2. HPLC-ELSD Detection Conditions: An Agilent Infinity 1260 high-performance liquid chromatograph connected to an evaporative light detector was used for detection. A Waters BEH Amide preparative column (9×250 mm, 4.6 μm) was used at a flow rate of 2.3 mL / min. Mobile phase A was an aqueous solution, and mobile phase B was an acetonitrile solution. The HPLC elution program was as follows: 0–10 min, 90%–50% B; 10–30 min, 50%–50% B; 30–40 min, 50%–90% B; column temperature was set at 30℃, and sample chamber temperature was set at 10℃. The injection volume was 30 μL.

[0072] 3. HPAEC-PAD Monosaccharide Composition Detection Conditions: Schisandra chinensis low-polymerization sugars (5 mg) were hydrolyzed with 2 mol / L TFA (5 mg) at 110℃ for 4 h, followed by washing three times with methanol and evaporation to remove TFA. After complete acid hydrolysis, the monosaccharide composition was identified and quantified using HPAEC-PAD (ICS 6000, Thermo Fisher Scientific, USA). AgCl was used as the reference electrode, and detection was performed on a gold working electrode using a PAD. Dionex CarboPac was employed. TM The determination was performed using a PA20 guard column (3 × 30 mm) and an analytical column (3 × 150 mm, 10 μm). Mobile phase A was water, mobile phase B was 150 mm NaOH, and mobile phase C was 100 mm sodium acetate. The flow rate was 0.5 mL / min, the column temperature was 25℃, and the injection volume was 1 μL.

[0073] 4. UPLC-QTOF-MS EDetection conditions: A Waters ACQUITY UPLC / SYNAPT G2-SI QTOF system in negative ion mode was used, employing a Waters BEH Amide guard column (2.1 × 5 mm, 1.8 μm) and analytical column (2.1 × 100 mm, 1.8 μm). Mobile phase A was 0.1% formic acid (v / v) aqueous solution, and mobile phase B was 0.1% formic acid (v / v) acetonitrile solution. Gradient elution was used: 0–25 min, 90%–50% B; 25–28 min, 50%–50% B; 28–30 min, 50%–90% B. The flow rate was 0.3 mL / min, the injection volume was 3 μL, and the column temperature was 35℃. All samples were analyzed by MS. E The detection was performed in a mode with a resolution of 20,000 and a scan quality range of 100-3000 Da. Key parameters included an ion source temperature of 100℃, a desolvation temperature of 250℃, a cone gas flow rate of 50.0 L / h, and a desolvation gas flow rate of 600.0 L / h.

[0074] 5. NMR Detection Conditions: Weigh 5 mg of the sample and dissolve it in 1 mL of D₂O. The sample was subjected to three freeze-thaw cycles. Analysis was performed on a Bruker Avance III 400 MHz NMR spectrometer (Bruker, Rheinstetten, Germany). At 25 °C, various spectral data based on 400 MHz 1H-NMR and 100 MHz 13C-NMR were collected using the Avance III 400 MHz spectrometer, including 1H-1H COSY, 1H / 13C HSQC, 1H / 13C HMBC, 1H-1H NOESY, and TOSY 1H. For 1H-NMR acquisition, the following parameters were set: pulse program Zg 30, spectral width 8.22 kHz, acquisition time 4 s, relaxation delay 1 s, and 64 scans. For 13C-NMR acquisition, the parameters were set as follows: pulse program ZgPg 30, spectral width 24.0 kHz, acquisition time 1.36 s, relaxation delay 1 s, and 73,728 scans. The data were processed and analyzed in depth using Topspin 4.0.4 and MestReNova software.

[0075] II. Experimental Results 1. Chromatographic characterization of Schisandra chinensis low-mesopolysaccharide composition An Agilent Infinity 1260 high-performance liquid chromatograph connected to an evaporative light detector (ELD) and equipped with a Waters BEH Amide preparative column was used for detection and preparation. Since the ELD would damage the sugar structure, the detector was not connected during preparation. A single chromatographic peak was collected using an automated fraction collector, concentrated, and lyophilized to obtain SFSC monomers 1-8. The separation process is described in [details omitted]. Figure 2 B in the middle.

[0076] SFSC is a white powder, highly soluble in water. Monosaccharide composition was analyzed by high-performance anion exchange chromatography (HPAEC-PAD). Figure 2 Using the direct hydrolysis of trifluoroacetic acid, the monosaccharide composition of SFSC was determined to be rhamnose, arabinose, galactose, glucose, xylose, mannose, and galacturonic acid, with experimental monosaccharide contents of 9.8%, 0.8%, 8.9%, 33.1%, 3.1%, 3.3%, and 41.0%, respectively.

[0077] 2. LC-MS characterization of Schisandra chinensis low-mesopolysaccharide composition To further determine the fine chemical structure of SFSC, UPLC-QTOF-MS was used for detection, and the total ion chromatogram was obtained. Figure 2 (D in the table). As shown in Table 1, a total of 47 sugar fragments were identified, and the corresponding [MH] fragments were then analyzed using non-targeted LC-QTOF-MSE and targeted LC-QTOF-MS / MS techniques. - and [M-2H] 2- The chemical structures of oligosaccharides and small polysaccharides were determined, revealing that they are primarily acidic and neutral polysaccharides. Based on the monosaccharide composition, the acidic polysaccharides contain galacturonic acid. Analysis was conducted using the acidic polysaccharide at peak 22 and the neutral polysaccharide at peak 26 as examples.

[0078] Table 1. Mass Spectrometry Analysis of SFSC Fragments

[0079] The retention time of peak 22 was 22.76 min, according to [MH]. - Its molecular weight was determined to be 1495, implying a degree of polymerization of 8, composed of 3 galacturonic acid molecules and 5 methyl galacturonates (GalA3-GalAOMe5). According to... Figure 3 As shown in A, the mass spectrum at a collision energy of 30 eV shows a series of B, C, Y, and Z ions, revealing the main fragmentation pathway ( Figure 3(B in the original text), that is: m / z 1495→1319→1129→939→749→559→383→193 ( Δm (176→190→190→190→190→176→190), simultaneously visible 2,4 A2 and 0,2 Fragments 235 and 323 of A2 were identified. Their structures were determined by neutral loss and transcyclic fragmentation, as shown in Table 1 (peak 22).

[0080] In addition, the retention time of peak 26 was 23.84 min, according to [MH]. - Its molecular weight was determined to be 1475, implying a degree of polymerization of 9, composed of 9 hexose molecules (Hex9). According to... Figure 3 As shown in C, the mass spectrum at a collision energy of 30 eV shows a series of B, C, Y, and Z ions, revealing the main fragmentation pathway ( Figure 3 D in the middle), that is: m / z 1475→1313→1151→989→827→665→503→341→179 ( Δm (162→162→162→162→162→162→162→162), and a series of... 2,4 A(1355, 1193, 1031, 869, 707, 545, 383 and 221) and 0,2 Fragments of A (1091, 929, 767, 605, 443, and 281). Their structures were determined by neutral loss and transcyclic fragmentation, as shown in Table 1, peak 26.

[0081] 3. NMR characterization of Schisandra chinensis low-mesopolysaccharide composition Taking SFCS monomer 6 as an example, its total ion chromatogram, primary and secondary mass spectra, and fragmentation pathway diagram are shown in Figure 4. The main chromatographic peak at a retention time of 28.58 min is [MH]. Ions are m / z 2447, its second order [M-2H] 2 [M+HCOOH-2H] 2 The ions are respectively m / z 1223 and 1246; based on a series of C-class fragment ions, it is inferred that monomer 6 is a 1→4 glycosidic bonded 15Glc polysaccharide.

[0082] First, the structure of SFSC monomer 6 was characterized by 1D-NMR, while 1H NMR provides anomeric hydrogen and other hydrogen signals. For example... Figure 5As shown in Figure A, the terminal proton (anomeric hydrogen) region of carbohydrate compounds is δ 4.3–5.8 ppm, while the cyclic proton signal is δ 4.2–3.4 ppm. Three distinct signals were observed in the terminal proton region: δ(H) 5.24, 5.07, and 4.49, indicating the presence of three sugar residues, named A, B, and C. The signal shift at δ 5.24 ppm is assigned to →4)-α-Glcp-(1→(A), with a coupling constant JH-1,H-2 = 4.0 Hz; the signal shift at δ 5.07 ppm is assigned to α-Glcp-(1→(B), with a coupling constant JH-1,H-2 = 3.6 Hz, indicating that sugar residues A and B are α-configured. The coupling constant JH-1,H-2 = 8.0 Hz at δ 4.49 ppm indicates that sugar residue C is β-configured.

[0083] 13C NMR was used to observe the carbon signal of SFSC monomer 6. For example... Figure 5 As shown in B, the relatively few signals in the carbon spectrum indicate that there are few glycoside linkages in this monomer. A relatively obvious carbon resonance peak at δ 99.6 ppm was observed in the anomeric carbon region, which was assigned to C-1 of →4)-α-Glcp-(1→ (A) and α-Glcp-(1→ (B). The weak resonance peak at δ 95.7 ppm was assigned to →4)-β-Glcp (C).

[0084] 2D-NMR was used to further determine the signal shifts of each sugar residue in SFSC monomer 6. Specifically, 1H-1H COSY provides the coupling correlation of adjacent hydrogen nuclei in the sugar ring, TOCSY spectrum provides the full correlation of hydrogen nuclei, and HSQC reflects the coupling relationship between directly linked 1H and 13C atoms. Figure 6 The 1H-1H COSY spectrum of A in the figure shows that the cross peak at the position is the cross signal between H-1 and H-2 on the sugar ring of (A), indicating that the signal shift of H-2 of sugar residue A is δ 3.44 ppm. Furthermore, TOCSY ( Figure 7 In B), the crossover signal with δ(H / H) 5.24 / 3.79 is →4)-α-Glcp-(1→ (A) the crossover signal between H-1 and H-3 on the sugar ring. Further, from HSQC ( Figure 6From B), we know that the C-2 signal shift of sugar residue A is δ 71.4 ppm, and the C-3 signal shift is δ 73.4 ppm. Therefore, the H-2 / C-2 of →4)-α-Glcp-(1→ (A) can be assigned as δ(H / C) 3.44 / 71.4, H-3 / C-3 as δ(H / C) 3.79 / 73.4, H-4 / C-4 as δ(H / C) 3.49 / 76.8, H-5 / C-5 as δ(H / C) 3.68 / 71.2, and H-6 / C-6 as δ(H / C) 3.69 / 60.2. And so on. The detailed signal shifts of each sugar residue are shown in Table 2.

[0085] Table 2. Chemical shifts of 1H and 13C in SFSC monomer 6

[0086] HMBC long-range correlation spectroscopy was used to further analyze the structure of Schisandra chinensis oligosaccharide monomers. For example... Figure 7 As shown in Figure A, the cross-peak of H-1 of →4)-α-Glcp-(1→ (A) and C-1 of →4)-α-Glcp-(1→ (A) at δ 5.25 / 76.8 ppm confirms the existence of the →4)-α-Glcp-(1→4)-α-Glcp-(1→) structural domain. Simultaneously, the cross-peak of H-1 of α-Glcp-(1→ (B) and C-1 of →4)-α-Glcp-(1→ (A) at δ 5.08 / 76.8 ppm confirms that the reducing end of α-Glcp is directly connected to →4)-α-Glcp-(1→). The structure of Schisandra chinensis oligosaccharide monomer 6 can be determined to be α-Glcp-(1→4)-α-Glcp-(1→[4)-α-Glcp-(1)] 12 →4)-β-Glcp is maltodecyl sugar.

[0087] Example 3: In vivo pharmacodynamic evaluation of Schisandra chinensis oligosaccharide composition as a tumor immunotherapy drug 1. Efficacy evaluation of the drug in lung cancer models in immunocompetent and immunodeficient mice. To investigate the antitumor activity and immune dependence of SFSC, C57BL / 6 mice and Balb / c Nude mice were used to establish in vivo lung cancer (LLC lung cancer) xenograft models to study the in vivo antitumor efficacy of SFSC.

[0088] (1) Collect Lewis cells in the logarithmic growth phase by centrifugation and adjust the cell density to 1×10⁻⁶ with physiological saline. 7Lewis cell suspension was implanted subcutaneously into the right axilla of C57BL / 6 mice or Balb / c Nude mice, with 200 μL injected into each mouse. Animals were observed daily after modeling; successful modeling was indicated by palpable subcutaneous masses.

[0089] (2) Successfully modeled C57BL / 6 mice were randomly divided into 5 groups: control group, anti-CTLA-4 monoclonal antibody treatment group (αCTLA-4, 100 μg / mouse), low-dose SFSC group (50 mg / kg), medium-dose SFSC group (100 mg / kg), and high-dose SFSC group (200 mg / kg), with 10 mice in each group. The SFSC treatment group was administered SFSC by gavage once a day for 15 consecutive days; the anti-CTLA-4 monoclonal antibody treatment group was injected intraperitoneally with αCTLA-4, 100 µg / mouse, 0.2 mL / time every 3 days; the control group was administered an equal volume of physiological saline by gavage daily. The general condition of the mice was observed daily, and the body weight and tumor volume were recorded regularly. After 15 consecutive days of administration, the subcutaneous xenografts of each group of mice were carefully dissected, weighed, and photographed. The expression level of CTLA-4 protein in tumor-infiltrating T lymphocytes was further analyzed by flow cytometry. In addition, the Balb / c Nude nude mouse xenograft model was divided into a control group and an SFSC treatment group (SFSC, 200 mg / kg), with 10 mice in each group.

[0090] The experimental flowchart for the C57BL / 6 mouse model is as follows: Figure 8 As shown in Figure A, SFSC exhibits significant antitumor activity in an immunocompetent C57BL / 6 mouse model. Figure 8 As shown in Figure BD, compared with the control group, SFSC treatment effectively inhibited tumor growth, and the average tumor weight at the end of treatment was significantly reduced. Throughout the treatment period, the weight gain of mice in the SFSC treatment group remained synchronized with that in the control group, without any abnormal fluctuations, indicating that the drug was well-tolerated at this dosage range. Figure 8 E in the text). Further survival analysis showed that SFSC treatment significantly prolonged the survival of tumor-bearing mice (E). Figure 8 The F in the figure reflects its clear therapeutic benefit.

[0091] To validate the findings of Example 1 in vivo, flow cytometry analysis was performed on tumor-infiltrating T lymphocytes. The results showed that SFSC treatment significantly reduced the protein expression level of CTLA-4 on T cell subsets in the tumor microenvironment. Figure 8(GH in the figure). Importantly, this downregulation showed high specificity for CD8 T cell subtypes (8I-L in the figure). This result is the first in vivo demonstration that SFSCs can specifically downregulate the key immune checkpoint CTLA-4 on tumor-killing CD8 T cells.

[0092] To clarify whether this antitumor effect depends on T cell-mediated adaptive immunity, parallel validation was performed in a T cell-deficient Balb / cNude nude mouse model. The experimental procedure is as follows: Figure 8 The results showed that SFSC completely lost its anti-tumor effect. For example... Figure 8 As shown in NP, in the nude mouse model, there were no statistically significant differences in tumor growth curves and final tumor weight between the SFSC treatment group and the solvent control group.

[0093] This experiment demonstrates that the anti-tumor efficacy of SFSC provided by this invention strictly depends on the body's intact T-cell immune function and can specifically downregulate CTLA-4 expression on tumor-infiltrating CD8T cells.

[0094] 2. Validation of the efficacy and universality of CTLA-4 inhibition in different tumor models. To further verify the broad spectrum of SFSC's antitumor effects and the universality of its core mechanism (downregulation of CTLA-4), it was evaluated in two different tumor models: melanoma and hepatocellular carcinoma.

[0095] (1) Using B16-F10 melanoma cells and Hepa1-6 liver cancer cells, respectively, xenograft models were established subcutaneously in C57BL / 6 mice. The experimental procedure is as follows: Figure 9 As shown in A and H in the figure. Successfully modeled C57BL / 6 mice were randomly divided into 5 groups: control group, anti-CTLA-4 monoclonal antibody positive drug group (αCTLA-4, 100 μg / mouse), low-dose SFSC group (Low, 50 mg / kg), medium-dose SFSC group (Middle, 100 mg / kg), and high-dose SFSC group (High, 200 mg / kg), with 10 mice in each group. Each SFSC dose group was administered by gavage once daily for 13 / 15 days; the αCTLA-4 group was administered by intraperitoneal injection once every 3 days, 100 μg / mouse (0.2 mL injection volume); the control group was administered an equal volume of physiological saline by gavage daily. During the treatment period, the general condition of the mice was observed daily, and body weight and tumor volume were recorded every 3 days. After the administration was completed, the mice were sacrificed, the subcutaneous xenografts were carefully dissected, weighed, and photographed. The xenografts were further analyzed by flow cytometry to determine the expression level of CTLA-4 protein in tumor-infiltrating CD8T cells.

[0096] Experimental results are as follows Figure 9As shown, SFSC exhibited significant dose-dependent antitumor activity in both B16-F10 melanoma and Hepa1-6 liver cancer models. Figure 9 BD and IK in the model. In both models, the weight gain of mice in the SFSC treatment group was basically consistent with that in the control group (BD, IK). Figure 9 The presence of E and L in the data indicates good tolerability. Flow cytometry analysis showed that the therapeutic effect of SFSC was closely related to its immunomodulatory effect on the tumor microenvironment. In both models, SFSC treatment significantly downregulated the protein expression level of CTLA-4 on the surface of tumor-infiltrating CD8T cells (E, L), indicating good tolerability. Figure 9 FG, MN). This result is consistent with findings in an LLC lung cancer model ( Figure 8 The K and L values ​​in the model are highly consistent.

[0097] This part of the study confirms that SFSC has a broad-spectrum anti-tumor effect, exhibiting clear inhibitory effects against LLC lung cancer, B16-F10 melanoma, and Hepa1-6 liver cancer. More importantly, the core mechanism of SFSC specifically downregulating CTLA-4 expression on tumor-infiltrating CD8 T cells was reproduced in all three tumor models with different immunogenicity, demonstrating the universality of this mechanism of action.

[0098] Example 4: Analysis of the Tumor Immune Microenvironment in Mice I. Experimental Methods The effects of SFSC on the tumor microenvironment of mouse lung cancer xenografts were analyzed using mass spectrometry combined with conventional flow cytometry.

[0099] Tumor single-cell suspensions were obtained using enzymatic digestion, blocked, and then stained with extracellular and intracellular antigens (the antibodies used for mass cytometry were pre-labeled with metal isotopes). Data were subsequently acquired using a Helios mass cytometer. After FlowJo preprocessing, the data were further subjected to dimensionality reduction visualization and cluster analysis. Figure 10 (A in the middle).

[0100] II. Experimental Results The results showed that SFSC significantly increased the infiltration of immune cells within the tumor. Figure 10 (BD in the text), especially CD103 was added. + Dendritic cells (CD103) + The level of DCs was reduced, and the level of tumor-associated macrophages (TAMs) was decreased. Figure 10 In addition, SFSC can significantly reduce the proportion of the depletion subset (CD8Tex) in CD8T and the level of CTLA-4 on total CD8T and CD8Tex. Figure 10GI), increases the expression of tumor-killing molecules Granzyme B and Perforin in effector CD8 T cell subsets ( Figure 10 JK in the middle), and can broadly reduce PD-L1 levels on myeloid cells ( Figure 10 LM in DC cells increases the level of CD80 in DC cells. Figure 10 The N in the text suggests that SFSCs may increase DC-mediated CD8 T cell activation (DC / T cell crosstalk) and activate anti-tumor immune responses by inhibiting the expression of immune checkpoints PD-L1 and CTLA-4 on tumor-associated DCs and CD8 T cells, respectively.

[0101] Example 5: Validation of CD8T cell clearance in vivo I. Experimental Methods To verify whether the anti-tumor efficacy of the SFSC described in this invention depends on CD8T cells, the ultimate effector cells, CD8T cells in mice were eliminated by intraperitoneal injection of CD8a neutralizing antibodies.

[0102] Immunocompromised C57BL / 6 mice were selected, and LLC lung cancer cells were subcutaneously inoculated to establish a xenograft model as described in Example 3.1. The experimental procedure is as follows: Figure 11 As shown in A in the figure. After successful modeling, mice were randomly divided into the following 4 groups: WT control group (injected with 250 μg / mouse of Rat IgG2b isotype control antibody), αCD8a control group (injected with 250 μg / mouse of anti-mouse CD8a neutralizing antibody), SFSC single drug group (injected with 250 μg / mouse of Rat IgG2b isotype control antibody + 200 mg / kg SFSC), and SFSC+αCD8a group (injected with 250 μg / mouse of anti-mouse CD8a neutralizing antibody + 200 mg / kg SFSC). The SFSC treatment group received SFSC via gavage once daily for 15 consecutive days. The anti-mouse CD8a neutralizing antibody group received intraperitoneal injections of αCD8a (250 µg / mouse, 0.2 mL / injection) every 3 days. The WT control group and the SFSC monotherapy group received intraperitoneal injections of isotype control antibody (250 µg / mouse, 0.2 mL / injection) every 3 days. The WT control group and the αCD8a control group received an equal volume of physiological saline via gavage daily. Mouse weight and tumor volume were recorded regularly. After the experiment, subcutaneous xenografts were carefully dissected from each group of mice, and tumor weight was recorded to assess drug efficacy and photographed. The xenografts were further analyzed by flow cytometry to examine changes in tumor-infiltrating immune cells.

[0103] II. Experimental Results Experimental results are as follows Figure 11As shown, in the SFSC monotherapy group without CD8T cell clearance, tumor growth was significantly inhibited. However, in the "SFSC+αCD8a group" using antibodies to clear CD8T cells, the tumor-suppressive effect of the drug was completely eliminated, and the tumor growth curve and tumor weight were not significantly different from the control group. Figure 11 (BD in the middle). Throughout the experiment, the body weight of mice in all groups remained stable, indicating good treatment tolerance. Figure 11 (E in the text).

[0104] Analysis of the tumor microenvironment further clarified the upstream and downstream relationships of drug action. First, flow cytometry confirmed that αCD8a treatment effectively eliminated CD8 T cells within the tumor (…). Figure 11 (HI in the middle). A key and insightful finding is that, despite the elimination of CD8 T cells, SFSC treatment can still significantly upregulate the expression level of CD80, a key co-stimulatory molecule on the surface of intratumoral dendritic cells (DCs). Figure 11 (FG in the image). This demonstrates that the activation and regulation of DCs by SFSCs is an independent upstream event, independent of feedback from CD8T cells.

[0105] This experiment demonstrates that CD8T cells are the effector cells of the antitumor effect of the carbohydrate composition described in this invention; their activation of upstream dendritic cells is independent, but must be achieved through CD8T cells to ultimately translate into effective tumor suppression.

[0106] Example 6: Validation of the efficacy of the Batf3-KO gene defect model I. Experimental Methods This embodiment aims to verify from a genetic perspective whether the anti-tumor effect of SFSC described in this invention strictly depends on CD103, a key upstream cell that initiates the anti-tumor immune response. + Dendritic cells.

[0107] Batf3 gene knockout (Batf3-KO) mice and their littermate wild-type (WT) control mice were selected. LLC lung cancer cells were subcutaneously inoculated using the method described in Example 3.1 to establish a xenograft model. The experimental procedure is as follows: Figure 12 As shown in A, mice were divided into four groups: WT control group, WT-SFSC group (200 mg / kg SFSC), and... Batf3 Knockout control group Batf3 Knockout-SFSC group (200 mg / kg SFSC). The SFSC treatment group received SFSC via gavage once daily for 15 consecutive days; WT control group and... Batf3The knockout control group was administered an equal volume of physiological saline via gavage daily. Mouse weight and tumor volume were recorded regularly. After the experiment, subcutaneous xenografts were carefully dissected from each group of mice, tumor weight was recorded to assess drug efficacy, and photographs were taken. The xenografts were further analyzed by flow cytometry to examine changes in tumor-infiltrating immune cells.

[0108] II. Experimental Results Experimental results are as follows Figure 12 As shown, in WT mice, SFSC treatment significantly inhibited tumor growth and reduced tumor weight; however, in Batf3-KO mice, the tumor-suppressive effect of SFSC was completely lost, and their tumor growth curves and tumor weights were not different from those of the genotype control group. Figure 12 The BD in the middle). The body weight of mice in all groups was stable, indicating that the drug was well tolerated in both genotypes of mice. Figure 12 (E in the text).

[0109] In-depth analysis of the tumor immune microenvironment revealed the cellular and molecular basis for drug efficacy loss. First, the tumor microenvironment in Batf3-KO mice exhibited an ineffectively activated state: compared to the WT control group, the overall infiltration level of CD8 T cells within the tumor was significantly reduced, and almost no exhausted CD8 T cells (CD8Tex) were detected. Figure 12 (FH in CD103). This confirms CD103. + Dendritic cells (DCs) are essential for initiating antigen-specific T cell responses and recruiting and differentiating them at tumor sites. In this context, the regulatory function of SFSCs is completely ineffective due to the lack of a suitable substrate. On the one hand, in Batf3-KO mice, SFSCs failed to increase the expression levels of the CD8 T cell killing molecules perforin and granzyme B. Figure 12 IL in the middle). On the other hand, SFSC cannot downregulate the expression of CTLA-4 protein on the surface of tumor-infiltrating CD8T cells and CD8Tex (IL). Figure 12 (MP in). This contrasts directly with the significant downregulation observed in WT mice.

[0110] This embodiment provides decisive evidence from both positive and negative dimensions: in the positive dimension, the effectiveness of SFSC in WT mice demonstrates the existence of its therapeutic pathway; in the negative dimension, complete inactivation in Batf3-KO mice precisely locates the upstream switch of this pathway. In summary, this indicates that the antitumor activity of SFSC and its downregulation of CTLA-4 expression are strictly dependent on CD103. + The presence of dendritic cells (Batf3-dependent DCs), the "unactivated" state of CD8 T cell responses in Batf3-KO mice, and the complete failure of drug regulation together reveal a precise causal relationship: SFSCs must rely on CD103...+ DCs initiate and guide the subsequent CD8T cell immune response; without this crucial step, the entire cascade reaction cannot be initiated.

[0111] Example 7: Functional Validation of In Vitro DC-T-LLC Cell Co-culture I. Experimental Methods This embodiment aims to directly verify, at the in vitro cellular level, the core pathway by which the SFSC described in this invention enhances T cell-mediated anti-tumor immunity by regulating dendritic cell (DC) function. A co-culture system was constructed using mouse dendritic cell line DC2.4, cytotoxic T lymphocyte line CTLL-2, and mouse lung cancer cells LLC.

[0112] First, the direct effects of SFSC on DC2.4 cells were evaluated. Flow cytometry analysis showed that, at concentrations of 10–2000 μg / mL, SFSC treatment specifically and significantly upregulated the expression of the key co-stimulatory molecule CD80 on the surface of DC2.4 cells. Figure 13 The expression of AB in the sample had no significant effect on the expression of its homolog CD86. Figure 13 The presence of CD in the data indicates that SFSCs exhibit molecular selectivity for DC activation. Further qPCR analysis revealed that SFSC treatment also significantly increased the mRNA transcription levels of various pro-inflammatory and immune-activation-related cytokines (including TNF-α, IL-6, IL-1β, and IL-12B) in DC2.4 cells. Figure 13 (EH in the middle). The above results together demonstrate that SFSC can directly induce the maturation and functional activation of DCs.

[0113] To clarify whether this DC activation can effectively transmit and empower T cells, a model was established... Figure 13 The co-culture model is shown in Figure I. In this system, to examine the effect of SFSC-pretreated DC2.4 cells on CTLL-2 cell proliferation, DC2.4 cells pretreated with SFSC (50 μg / mL or 100 μg / mL) or solvent were co-incubated with CFSE-labeled CTLL-2 cells. The results showed that, compared with co-culture of solvent-treated DCs, SFSC-pretreated DC2.4 cells significantly promoted the proliferation of CTLL-2 cells. Figure 13(JK in the text). To investigate the tumor-killing effect of SFSC on DC-mediated T cell in vitro, DC2.4 cells pretreated with SFSC or solvent were co-incubated with CTLL-2 cells. The co-culture supernatant and activated CTLL-2 cells were then co-cultured with CFSE-labeled LLC tumor cells. The results showed that CTLL-2 cells co-cultured with SFSC-treated DC2.4 cells significantly enhanced their ability to kill LLC tumor cells, resulting in a substantial decrease in LLC cell survival (JK in the text). Figure 13 (LM in the middle).

[0114] This result provides direct and robust cellular functional evidence for the "dendritic cell-dependent" antitumor immune activation mechanism observed in in vivo studies in this invention.

[0115] Example 8: Experiment on the combination of Schisandra chinensis oligosaccharide composition with CTLA-4 monoclonal antibody and PD-L1 monoclonal antibody I. Experimental Methods To evaluate the therapeutic potential of the SFSC described in this invention in combination with existing clinical immune checkpoint inhibitors, this embodiment conducted a systematic in vivo pharmacodynamic and mechanistic study.

[0116] C57BL / 6 mice were used, and LLC lung cancer cells were subcutaneously inoculated to establish a xenograft model according to the method described in Example 3.1. The experimental procedure is as follows: Figure 14 As shown in A, mice were divided into 7 groups: control group, anti-CTLA-4 monoclonal antibody group (αCTLA-4, 100 μg / mouse), anti-PD-L1 monoclonal antibody group (αPD-L1, 100 μg / mouse (10 mg / kg)), SFSC monotherapy group (SFSC, 200 mg / kg), anti-CTLA-4 monoclonal antibody combined with anti-PD-L1 monoclonal antibody group (α-α, αCTLA-4 and αPD-L1, 100 μg / mouse each), SFSC combined with anti-PD-L1 monoclonal antibody group (SFSC 200 mg / kg + αPD-L1 100 μg / mouse), and SFSC combined with anti-CTLA-4 monoclonal antibody group (SFSC 200 mg / kg + αCTLA-4 100 μg / mouse). SFSC was administered daily by gavage, and monoclonal antibodies were administered intraperitoneally every three days for a 15-day treatment period. Mouse body weight and tumor volume were recorded regularly. After the experiment, the subcutaneous transplanted tumors of each group of mice were carefully dissected, the tumor weight was recorded to assess the drug efficacy, and photographs were taken. The transplanted tumors were further analyzed by flow cytometry to analyze changes in tumor-infiltrating immune cells.

[0117] II. Experimental Results Experimental results are as follows Figure 14 As shown, the combination therapy of SFSC and αPD-L1 produced a significant synergistic anti-tumor effect. Figure 14As shown in BD, tumor growth was most strongly inhibited in the combination therapy group, with a final tumor weight significantly lower than either single-agent therapy group, and efficacy comparable to the current clinical standard combination regimen (αCTLA-4 combined with αPD-L1), with no statistically significant difference in tumor weight between the two groups. However, the combination of SFSC and αCTLA-4 did not show a similar synergistic enhancement effect, and its efficacy was similar to that of each single-agent group. Figure 14 (BD in the middle).

[0118] In-depth analysis of the tumor immune microenvironment elucidates the mechanistic basis of the aforementioned differences in efficacy. Myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) are considered to be two major immunosuppressive populations in the tumor microenvironment, which can lead to tumor immune escape by suppressing T lymphocyte immunity. Regarding the regulation of MDSCs, both single-agent and combination therapy groups effectively reduced the infiltration ratio of MDSCs within the tumor; the combination of SFSC with αPD-L1 or αCTLA-4 significantly enhanced the inhibitory ability of the corresponding monoclonal antibody against MDSCs. Figure 14 (EF in the middle). Regarding Treg regulation, flow cytometry was used to detect CD25 in CD4T cells. + FoxP3 + Cell proportions showed that both αCTLA-4 and αPD-L1 treatments significantly reduced Treg frequencies, especially αCTLA-4; while SFSC monotherapy had no significant effect on Tregs, indicating that the mechanism of action of SFSC differs from that of traditional CTLA-4 antibodies. Since SFSC itself does not act on Tregs, its combination with αCTLA-4 or αPD-L1 did not further enhance the effect of the respective monoclonal antibodies on Treg proportions. However, compared with SFSC monotherapy, both combination therapy groups showed significantly enhanced Treg inhibition, which is attributed to the effect of the combined monoclonal antibodies (…). Figure 14 (GH in the middle).

[0119] In terms of core effector cell function, the combination therapy of SFSC and αPD-L1 has shown remarkable efficacy. Figure 14 As shown in the IL data, the combination of SFSC and αPD-L1 most effectively promoted the proportion of functional effector cells expressing perforin and granzyme B in CD8T cells, with effects comparable to the standard bispecific antibody combination (α-α) regimen. On this key endpoint, the SFSC plus αPD-L1 regimen showed comparable enhancement to the standard bispecific antibody combination regimen, and both were significantly superior to the single-agent groups. This result is perfectly consistent with the tumor growth inhibition phenotype.

[0120] The results of this embodiment demonstrate that the SFSC provided by this invention possesses a unique immunomodulatory spectrum: it can specifically activate the killing function of CD8T cells and inhibit MDSCs, but has no direct effect on Tregs. When SFSC is used in combination with αPD-L1, the two work synergistically in inhibiting MDSCs and activating the killing function of CD8T cells, thereby exerting a superior anti-tumor effect. However, no synergistic effect was observed when SFSC was used in combination with αCTLA-4, further confirming that the mechanism of action of SFSC—namely, downregulating CTLA-4 expression in CD8T cells through a "dendritic cell-dependent" pathway—is fundamentally different from the mechanism of traditional αCTLA-4, which directly blocks function and affects Tregs. This highlights the value of the combination of SFSC and αPD-L1 as a novel combination therapy strategy with complementary mechanisms and superior potential.

[0121] Example 9 Drug Safety Evaluation I. Experimental Methods The changes in biochemical indicators such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea (URE), blood urea nitrogen (BUN), creatine kinase (CK), and total protein (TP) in the serum of the experimental and control groups of the above batches of animal experiments were measured using a fully automated biochemical analyzer.

[0122] II. Experimental Results Experimental results are as follows Figure 15 As shown, no significant damage to liver, kidney, or heart function was observed after in vivo administration of SFSC, further demonstrating the biosafety of SFSC at the selected dosage.

Claims

1. A low-mesopolysaccharide composition derived from Schisandra chinensis, characterized in that, The low-to-medium polysaccharide composition consists of 33 acidic polysaccharides and 14 neutral polysaccharides with a degree of polymerization of 2 to 21; the monosaccharide composition of the low-to-medium polysaccharide composition is 9.8% rhamnose, 0.8% arabinose, 8.9% galactose, 33.1% glucose, 3.1% xylose, 3.3% mannose, and 41.0% galacturonic acid; The acidic polysaccharide has a degree of polymerization of 2 to 21 and a highly methylated galacturonic acid polymer with a 1→4 linked structure; the neutral polysaccharide has a degree of polymerization of 2 to 15 and a maltose series polymer with a 1→4 linked structure. The preparation method of the Schisandra chinensis-derived oligosaccharide composition includes the following steps: S1. Take Schisandra chinensis, use water as the extraction solvent, heat and reflux to obtain Schisandra chinensis aqueous extract; S2. Remove pigments and proteins from the Schisandra chinensis aqueous extract of step S1 and elute to obtain Schisandra chinensis aqueous eluent; the method for removing pigments and proteins is to adsorb and elute the Schisandra chinensis aqueous extract using a macroporous resin column. S3. The Schisandra chinensis aqueous eluent from step S2 is intercepted and collected. Mw The portion with less than 3 kDa yields the Schisandra chinensis low-molecular-weight polysaccharide composition.

2. The use of the Schisandra chinensis-derived oligosaccharide composition according to claim 1 in the preparation of an antitumor drug, characterized in that, The drug achieves therapeutic effects by activating dendritic cells and downregulating the expression of the immune checkpoint CTLA-4 on CD8T cells; the tumors include lung cancer, melanoma, or liver cancer.

3. The use of the Schisandra chinensis-derived oligosaccharide composition of claim 1 in combination with a PD-1 / PD-L1 inhibitor in the preparation of an antitumor drug, characterized in that... The PD-1 / PD-L1 inhibitor is an anti-PD-L1 antibody; the tumor includes lung cancer, melanoma, or liver cancer.

4. The application according to claim 2 or 3, characterized in that, The drug exerts its anti-tumor effect by enhancing CD8T cell-mediated adaptive immunity.

5. The use of the Schisandra chinensis-derived low-to-medium polysaccharide composition according to claim 1 in the preparation of a medicament for enhancing T-cell immunity, characterized in that, The drug achieves therapeutic effects by activating dendritic cells and downregulating the expression of the immune checkpoint CTLA-4 on CD8T cells.

6. An antitumor drug, characterized in that, The drug comprises the low-molecular-weight polysaccharide composition derived from Schisandra chinensis as described in claim 1.

7. The drug according to claim 6, characterized in that, The drug also contains a PD-1 / PD-L1 inhibitor; the PD-1 / PD-L1 inhibitor is an anti-PD-L1 antibody.

8. The drug according to claim 6 or 7, characterized in that, The drug also contains pharmaceutically acceptable excipients.

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