Radix sophorae tonkinensis polysaccharide STGP-1, preparation method thereof and pharmaceutical use for treating lung squamous carcinoma

Abstract

The application discloses radix sophorae tonkinensis polysaccharide STGP-1, a preparation method thereof and pharmaceutical use for treating lung squamous carcinoma, and relates to the technical field of medicines. The monosaccharide composition of STGP-1 includes fucose Fuc, arabinose Ara, galactose Gal, glucose Glc, xylose Xyl and mannose Man, and the molar ratio is 0.34:12.15:25.76:57.07:1.06:3.62. The STGP-1 is obtained by extraction, separation and purification from dried roots and rhizomes of radix sophorae tonkinensis, and the preparation method comprises the steps of pretreatment and crude extraction, impurity removal treatment, anion exchange column chromatography separation, gel column chromatography purification and the like. The STGP-1 can be used alone or in combination with anti-PD-1 monoclonal antibody for preparing a medicine for treating lung squamous carcinoma. The preparation process is stable, the source is natural, and the STGP-1 can provide a new high-efficiency and low-toxicity candidate medicine for immune combined treatment of lung squamous carcinoma.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to Sophora tonkinensis polysaccharide STGP-1, its preparation method, and its pharmaceutical use in the treatment of squamous cell carcinoma of the lung. Background Technology

[0002] Lung cancer is one of the leading causes of cancer-related morbidity and mortality in China and globally, with squamous cell carcinoma accounting for approximately 30%-40%. Traditional chemotherapy, radiotherapy, and targeted therapy suffer from problems such as drug resistance, numerous adverse reactions, low response rates, and poor prognosis. In recent years, immune checkpoint inhibitors (ICIs) have ushered in a new era of immunotherapy for non-small cell lung cancer. ICIs block immune checkpoints such as PD-1 / PD-L1 or CTLA-4, relieving tumor suppression of T cells and activating the immune system to attack the tumor. They are currently recommended as the first-line standard treatment for advanced squamous cell carcinoma in domestic and international guidelines. However, most patients do not respond to ICIs or relapse after treatment, making long-term survival difficult. Therefore, improving the efficacy of immunotherapy has become a key bottleneck in the clinical treatment of squamous cell carcinoma, urgently requiring the exploration of new treatment strategies and effective drugs.

[0003] Sophora tonkinensis, a traditional Chinese medicine for clearing heat and detoxifying, was first recorded in the Song Dynasty's *Kaibao Materia Medica* and is included in the *Pharmacopoeia of the People's Republic of China* (2025 edition), which clearly identifies it as the dried root and rhizome of the legume *Sophora tonkinensis* Gagnep. According to traditional Chinese medicine theory, Sophora tonkinensis is bitter and cold in nature, and enters the lung and stomach meridians. Its core functions are clearing heat and detoxifying, reducing swelling and relieving sore throat. Clinically, it is often used to treat sore throat, swollen gums, and oral ulcers caused by the accumulation of heat toxins. Currently, traditional Chinese medicine preparations developed using Sophora tonkinensis as a raw material (such as compound Sophora tonkinensis injection and Sophora tonkinensis tablets) are used clinically for anti-tumor and sore throat treatment. Modern pharmacological studies have shown that Sophora tonkinensis has a complex chemical composition, mainly containing alkaloids, flavonoids, triterpenes, and polysaccharides, possessing various pharmacological activities such as anti-inflammatory, antibacterial, antiviral, and antitumor effects. Alkaloids (such as matrine and oxymatrine) and flavonoids are considered to be the main active ingredients, but they have certain hepatotoxicity and neurotoxicity, which limits their clinical application. In contrast, Chinese herbal polysaccharides have become a research hotspot for sensitizing strategies for immune checkpoint inhibitors (ICIs) due to their significant immunomodulatory effects, extremely low toxicity and good biocompatibility.

[0004] Gut microbiota and their metabolites play a crucial role in regulating the body's anti-tumor immune response. Studies have shown that polysaccharides from traditional Chinese medicine can promote the maturation of antigen-presenting cells and regulate T lymphocyte subsets (such as enhancing CD8). +It enhances anti-tumor immunity through mechanisms such as T-cell activity and synergistic effects with ICIs, improving tumor suppression rates and immune cell infiltration. Furthermore, the immunomodulatory effects of traditional Chinese medicine polysaccharides are closely related to the gut microbiota. Numerous studies have found that polysaccharides promote the proliferation of bacteria such as Bifidobacteria and Lactobacillus, thereby regulating the balance of the gut microbiota. These bacteria can further regulate the function of immune cells and enhance anti-tumor immune responses by producing metabolites such as orotic acid, short-chain fatty acids, and inosine. Although *Sophora tonkinensis* polysaccharides possess various biological activities including anti-inflammatory, antioxidant, and immunomodulatory effects, and have shown potential in regulating tumor immunity... However, the following problems still exist: (1) Unclear structural characterization: Existing technologies lack fine structural analysis of single-component polysaccharides with definite sensitizing activity in Sophora tonkinensis; (2) Unclear sensitizing mechanism: Existing studies focus on the direct effect of polysaccharides on immune cells, but lack in-depth exploration of the systemic mechanism by which Sophora tonkinensis polysaccharides can synergistically enhance the efficacy of α-PD-1 through the 'intestinal flora-metabolites-immune signaling pathway' axis; (3) Lack of targeted application data: There are no clear records of the application of Sophora tonkinensis polysaccharides with specific structural parameters in the combined treatment of squamous cell carcinoma of the lung with immunosuppressants.

[0005] Therefore, further exploring the active components in Sophora tonkinensis polysaccharides that have the potential to enhance ICIs and elucidating their structure and mechanism of action is of great significance for developing novel tumor immunotherapy strategies. Summary of the Invention

[0006] The main objective of this invention is to overcome the shortcomings of the prior art and provide a Sophora tonkinensis polysaccharide STGP-1 with a well-defined structure, as well as a method for preparing the polysaccharide and its pharmaceutical use in the treatment of squamous cell carcinoma of the lung.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a novel traditional Chinese medicine monomer, Sophora tonkinensis polysaccharide STGP-1, the chemical structural formula of which is shown below:

[0009] .

[0010] like Figure 1 As shown, the Sophora tonkinensis polysaccharide STGP-1 provided by this invention is a homogeneous polysaccharide with a weight-average molecular weight (MW) of 7.726 kDa, a number-average molecular weight (Mn) of 7.045 kDa, a polydispersity (Mw / Mn) of 1.097, and a molecular conformation mainly in the form of an expanded coil.

[0011] This polysaccharide is composed of fucose (Fuc), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), and mannose (Man), with a molar ratio of 0.34:12.15:25.76:57.07:1.06:3.62. Its main chain is formed by interconnected linkages of →4)-α-D-Glcp-(1→, →4)-β-D-Galp-(1→, →4,6)-α-D-Glcp-(1→, →4,6)-β-D-Galp-(1→ and →3,6)-β-D-Galp-(1→), while the side chains are composed of α-D-Glcp-(1→ linked to the O-6 positions of the sugar residue →4,6)-α-D-Glcp-(1→).

[0012] Secondly, the present invention provides a method for preparing the above-mentioned Sophora tonkinensis polysaccharide STGP-1, comprising the following steps:

[0013] (1) Grind the dried roots of Sophora tonkinensis into powder, defatted with anhydrous ethanol, collected the precipitate by centrifugation, added water to the precipitate for water bath extraction, collected the supernatant extract, concentrated the supernatant extract, added anhydrous ethanol to the concentrated solution for alcohol precipitation, collected the precipitate, and dried to obtain crude polysaccharide extract.

[0014] (2) The crude polysaccharide extract was dissolved in water, hydrolyzed by protease, and then the protein was removed by Sevag reagent. It was then extracted with petroleum ether, and then small molecule impurities were removed by adsorption with macroporous resin. After dialysis and alcohol precipitation and drying, the impurity-removed polysaccharide was obtained.

[0015] (3) The purified polysaccharide was loaded onto an anion exchange column and eluted sequentially with pure water and NaCl solution of increasing concentration, and the main components were collected.

[0016] (4) The main component obtained in step (3) was loaded onto a gel purification column, eluted with water, dialyzed and freeze-dried to obtain Sophora tonkinensis polysaccharide STGP-1.

[0017] As a priority, in step (1):

[0018] The material-to-liquid ratio during degreasing is 1:10 (w / v), and the degreasing time is 30 minutes.

[0019] The water bath extraction was performed at a temperature of 60°C for 4 hours.

[0020] The volume of anhydrous ethanol added during the alcohol precipitation is 4 times the volume of the concentrated liquid, and the alcohol precipitation time is 12 hours.

[0021] As a priority, in step (2):

[0022] The proteases used in the enzymatic hydrolysis were papain and a complex protease, and the hydrolysis time was 12 hours.

[0023] The volume ratio of chloroform to n-butanol in the Sevag reagent is 4:1;

[0024] The macroporous resin is of type AB-8;

[0025] The dialysis bag used in the dialysis had a molecular weight cutoff of 3000 Da, and the dialysis time was 48 hours.

[0026] As a priority, in step (3):

[0027] The anion exchange column is a DEAE seplife FF anion exchange column;

[0028] The concentration gradient of the NaCl solution is 0.1M, 0.2M and 0.3M respectively, and the elution flow rate is 4mL / min.

[0029] As a priority, in step (4):

[0030] The gel purification column was a Sephacryl S-400HR gel column;

[0031] The elution flow rate is 1 mL / min.

[0032] Thirdly, the present invention provides the application of the above-mentioned Sophora tonkinensis polysaccharide STGP-1 in the preparation of anti-lung squamous cell carcinoma drugs.

[0033] Preferably, the present invention provides the application of Sophora tonkinensis polysaccharide STGP-1 in combination with anti-PD-1 monoclonal antibody in the preparation of anti-lung squamous cell carcinoma drugs.

[0034] Preferably, the anti-PD-1 monoclonal antibody includes one or more of pembrolizumab (Keytruda), nivolumab (Opdivo), sintilimab (Daboshu), camrelizumab (Airika), and tislelizumab (Baizean).

[0035] Fourthly, the present invention provides an anti-squamous cell carcinoma drug for lung, comprising the above-mentioned Sophora tonkinensis polysaccharide STGP-1.

[0036] As a preferred option, the present invention provides a combination drug for treating squamous cell carcinoma of the lung, comprising the above-mentioned Sophora tonkinensis polysaccharide STGP-1 and an anti-PD-1 monoclonal antibody (α-PD-1).

[0037] Preferably, the Sophora tonkinensis polysaccharide STGP-1 and the anti-PD-1 monoclonal antibody are separate pharmaceutical formulations.

[0038] Preferably, the pharmaceutical preparation, in addition to including Sophora tonkinensis polysaccharide STGP-1 or anti-PD-1 monoclonal antibody, also contains pharmaceutically acceptable excipients. The dosage form of the pharmaceutical preparation includes, but is not limited to, oral administration, granules, capsules, tablets, etc.

[0039] Compared with the prior art, the present invention can achieve at least the following beneficial effects:

[0040] 1. The Sophora tonkinensis polysaccharide STGP-1 obtained by this invention is a homogeneous polysaccharide with a well-defined structure. Compared with the traditional alkaloids and flavonoids in Sophora tonkinensis, STGP-1 did not show obvious liver, kidney and myocardial toxicity at the same efficacy dose, which significantly improved the clinical safety of Sophora tonkinensis in medicinal development.

[0041] 2. The extraction and separation process established in this invention is stable and has clearly defined parameters. Through specific gradient elution and gel purification steps, high-purity components can be stably obtained, with a purity of up to 92.4%, good reproducibility, and is conducive to industrial-scale production.

[0042] 3. STGP-1 is stable and can be used as a single active ingredient to prepare immunomodulatory drugs, or as an adjuvant ingredient in combination with existing chemotherapy or immunotherapy drugs, showing good prospects for clinical application. Experiments have shown that the combination of STGP-1 and anti-PD-1 monoclonal antibody has a significant synergistic therapeutic effect on squamous cell carcinoma of the lung, providing experimental and technical basis for the development of novel immune enhancers and adjuvant drugs for squamous cell carcinoma of the lung. Attached Figure Description

[0043] Figure 1 The structure of STGP-1, a polysaccharide from Sophora tonkinensis (from top to bottom: the chemical structural formula, symbol diagram, and linear sequence of the sugar chain of STGP-1).

[0044] Figure 2 This diagram illustrates the extraction, separation, and purification of STGP-1, a polysaccharide from Sophora tonkinensis. A: Flowchart of STGP-1 extraction and purification process; B: DEAE Sepharose FF ion exchange chromatography pattern; C: Sephacryl S-400HR gel filtration chromatography pattern; D: Ultraviolet (UV) scanning spectrum.

[0045] Figure 3 The diagram shows the physicochemical properties of STGP-1, a polysaccharide from Sophora tonkinensis. A: SEC-MALS (size exclusion chromatography-multi-angle light scattering) chromatogram of STGP-1; B: Molecular conformation of STGP-1.

[0046] Figure 4 Infrared spectrum of STGP-1, a polysaccharide from Sophora tonkinensis.

[0047] Figure 5X-ray image of STGP-1, a polysaccharide from Sophora tonkinensis.

[0048] Figure 6 This is a scanning electron microscope image of STGP-1, a polysaccharide from Sophora tonkinensis. The magnifications of images A, B, and C are 2000×, 5000×, and 10000×, respectively.

[0049] Figure 7 This diagram illustrates the effect of STGP-1, a polysaccharide from Sophora tonkinensis, on enhancing the antitumor activity of α-PD-1. In the diagram, A represents the experimental design and grouping; B represents the macroscopic morphology of the tumor; C represents the tumor growth curve; D represents tumor weight statistics; E represents changes in body weight; and F represents biochemical indicators.

[0050] Figure 8 STGP-1, a polysaccharide from Sophora tonkinensis, is effective against CD4+ in mouse tumor tissue. + and CD8 + Immunohistochemical results of T cells. A: CD4+ in tumor tissue. + / CD8 + Representative immunohistochemical image of cells; B: CD4 + T cell count; C:CD8 + T cell statistics.

[0051] Figure 9 The results of flow cytometry analysis of the effects of Sophora tonkinensis polysaccharide STGP-1 on T cells in mouse tumor tissue and on serum TNF-α, IFN-γ, and GZMB were presented. A: CD4+ in tumor tissue. + Representative flow cytometry plot of T cells; B: CD8+ in tumor tissue. + Representative flow cytometry plot of T cells; C: CD4 + T cell count; D: CD8 + T cell statistics; E: TNF-α statistics; F: IFN-γ statistics; G: GZMB (granzyme B) statistics. Detailed Implementation

[0052] This invention will describe in detail the preparation method, structure and uses of Sophora tonkinensis polysaccharide STGP-1 through several embodiments and experimental examples.

[0053] I. Preparation method and structural characterization of polysaccharide STGP-1

[0054] (I) Preparation method

[0055] refer to Figure 2 AC, the Sophora tonkinensis polysaccharide STGP-1 of the present invention was extracted, isolated and purified from dried roots and rhizomes of Sophora tonkinensis, and the specific steps are as follows:

[0056] 1. Pretreatment and crude extraction

[0057] like Figure 2As shown in Figure A, the Sophora tonkinensis root was dried, ground into powder, and passed through a 60-mesh sieve. Anhydrous ethanol (1:10 ratio of material to liquid) was added to the Sophora tonkinensis root powder, and the mixture was stirred at room temperature for 30 minutes to extract fat-soluble pigments and some impurities. The precipitate was collected by centrifugation. Pure water (1:20 ratio of material to liquid) was added to the precipitate, and the mixture was extracted in a 60°C water bath for 4 hours. The supernatant was collected. The precipitate residue was extracted again. The two extracts were combined, concentrated to 1 / 10 of the original volume by rotary evaporation, and then precipitated with four times the volume of anhydrous ethanol for 12 hours. The precipitate solid was collected and dried to obtain the crude polysaccharide extract.

[0058] 2. Impurity removal treatment

[0059] Continue to refer to Figure 2 A. The crude polysaccharide was dissolved thoroughly in pure water. Papain and a complex protease were added to the solution for enzymatic hydrolysis for 12 hours. The supernatant was collected, and 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1) was added to remove proteins. 1 / 4 volume of petroleum ether was added to the aqueous phase, and the mixture was collected. The lower layer was then added, and 1 / 2 volume of macroporous resin AB-8 was added, and the mixture was mixed and adsorbed for 12 hours. The liquid was collected and dialyzed through a 3000 Da dialysis bag for 48 hours to remove small molecule components. The polysaccharide solution was then precipitated with alcohol and dried to obtain the purified polysaccharide.

[0060] 3. Anion exchange column chromatography separation

[0061] Also refer to Figure 2 B. The polysaccharides were further purified using a DEAE Seplife FF anion exchange column. The purified polysaccharides were dissolved in pure water (flow rate 4 mL / min) and eluted sequentially with pure water and 0.1 M, 0.2 M, and 0.3 M NaCl solutions, collecting one tube of eluent every 15 mL. Different polysaccharide fractions were collected from tubes with different serial numbers, resulting in a total of four polysaccharide fractions (STGP-1, STGP-2, STGP-3, and STGP-4). The main fraction, STGP-1, was selected.

[0062] 4. Gel column chromatography purification

[0063] Also refer to Figure 2 C. The above STGP-1 fraction was loaded onto a Sephacryl S-400HR gel purification column (2.6×100cm), eluted with pure water at a flow rate of 1mL / min, and one tube of eluent was collected every 10mL. The polysaccharide was collected from tubes 43-51, dialyzed and freeze-dried to obtain purified Sophora tonkinensis polysaccharide STGP-1.

[0064] In one specific embodiment, 2.0 kg of *Sophora tonkinensis* root was used as raw material. Crude polysaccharide STGP was obtained by extraction in a 60℃ water bath followed by ethanol precipitation, with a purity of 33.4% (yield 3.6%, 72 g). After impurity removal, the polysaccharide purity was 75.0% (yield 9.7%, 5 g). Fractionation was performed using a DEAE Seplife FF anion exchange column (2.6 × 40 cm), yielding four fractions: STGP-1, STGP-2, STGP-3, and STGP-4 from the polysaccharide eluent. Fraction STGP-1 had the highest purity and yield (82.6% purity, 20.0% yield, 1.0 g) and was used for subsequent experiments. The final polysaccharide purity was determined by Sephacryl S-400HR (2.6 × 100 cm) gel chromatography using the phenol-sulfuric acid method, reaching 92.4% (yield 11.6%, 0.116 g).

[0065] (ii) Structural characterization

[0066] The structure of STGP-1 prepared in the above embodiments was analyzed, and the results are as follows:

[0067] 1. Ultraviolet spectroscopy analysis

[0068] The purity of STGP-1 was determined to be 92.4% using the phenol-sulfuric acid method. Ultraviolet spectroscopy showed no absorption peaks at 260 nm and 280 nm, indicating the absence of nucleic acids and proteins. Specific results are as follows... Figure 2 As shown in D.

[0069] 2. Molecular weight analysis

[0070] The weight-average molecular weight (MW) of STGP-1 was determined to be 7.726 kDa, the number-average molecular weight (Mn) to be 7.045 kDa, and the polydispersity (Mw / Mn) to be 1.097 using gel permeation chromatography-differential laser light scattering (GPC-RI-MALS) system. Specific results are as follows: Figure 3 As shown in Figure A.

[0071] 3. Conformational Analysis

[0072] In a double logarithmic coordinate graph of radius of gyration versus molar mass (a Mark-Houwink type molecular conformation diagram), the slope can be used as a reference for molecular conformation. Generally, a slope of 1 indicates a rod-like shape, a slope of 0.5-0.6 indicates a random coil, and a slope of 1 / 3 indicates a spherical shape. The slope of the fitted line for STGP-1 is approximately 0.70, indicating a conformation primarily of an expanded coil (more extended than a typical random coil). Specific results are as follows... Figure 3 As shown in B.

[0073] 4. Monosaccharide composition analysis

[0074] The monosaccharide composition of STGP-1 was analyzed and detected using a high-performance ion chromatography (HPIC) system. STGP-1 consists of fucose (Fuc), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), and mannose (Man), with a molar ratio of 0.34:12.15:25.76:57.07:1.06:3.62. The specific peak times are shown in Table 1.

[0075] Table 1. Peak time of STGP-1 monosaccharide

[0076]

[0077] 5. FT-IR spectral analysis

[0078] See Figure 4 At 3274.76cm -1 A strong and broad absorption band is observed at 2926.24 cm⁻¹, indicating the stretching vibration of the hydroxyl group. -1 The absorption peak at 1652.19 cm⁻¹ corresponds to the CH stretching vibration, including -CH, -CH₂, and -CH₃. -1 The absorption at this point is related to the CO stretching vibration. At 1647 cm⁻¹ -1 The peak value at that location corresponds to bound water. 1361.98 cm -1 The absorption peak at 1020.21 cm⁻¹ was assigned to the carbonyl CH deformation vibration. -1 A characteristic absorption peak of the pyran ring appears at 857.75 cm⁻¹. Additionally, a peak is observed at 857.75 cm⁻¹. -1 The weak peak at that point suggests the presence of a β-glycosidic bond.

[0079] 6. X-ray diffraction (XRD) analysis

[0080] Typical amorphous polysaccharides exhibit broad, diffuse peaks in XRD patterns, while crystalline materials show sharp diffraction peaks. See also... Figure 5 The spectrum of STGP-1 shows typical amorphous diffraction characteristics, indicating that its molecular chains are arranged randomly and have no obvious crystal structure, which is consistent with the typical crystal form characteristics of polysaccharides.

[0081] 7. Electron microscopy

[0082] Scanning electron microscopy (SEM) offers high-precision characterization of microstructures. See also... Figure 6 AC analysis revealed that STGP-1 does not exist as completely isolated single chains, but rather forms irregular clusters of aggregates. These are multi-scale, irregular particle aggregates with rough surfaces and exhibiting a loose, porous, amorphous morphology.

[0083] 8. Methylation and Nuclear Magnetic Resonance (NMR) Analysis

[0084] See Table 2, which uses methylation analysis combined with one-dimensional / two-dimensional NMR to infer:

[0085] Sugar residue types: STGP-1 contains 11 major sugar residues: sugar residue A is →4)-α-D-Glcp-(1→); sugar residue B is α-D-Glcp-(1→); sugar residue C is →4)-β-D-Galp-(1→; sugar residue D is →4,6)-α-D-Glcp-(1→; sugar residue E is →5)-α-L-Araf-(1→; sugar residue F is β-D-Galf-(1→; sugar residue G is →4)-α-D-Glcp; sugar residue H is β-D-Glcp-(1→; sugar residue I is →4,6)-β-D-Galp-(1→; sugar residue J is α-L-Araf-(1→; sugar residue J' is α-L-Araf-(1→; sugar residue K is →3,6)-β-D-Galp-(1→).

[0086] Linkage: STGP-1 is mainly composed of interconnected main chains such as →4)-α-D-Glcp-(1→, →4)-β-D-Galp-(1→, →4,6)-α-D-Glcp-(1→, →4,6)-β-D-Galp-(1→ and →3,6)-β-D-Galp-(1→), while the side chains are composed of α-D-Glcp-(1→ linked to the O-6 positions of the sugar residue →4,6)-α-D-Glcp-(1→).

[0087] Table 2, STGP-1 NMR 1 H and 13 C displacement

[0088]

[0089] II. Application of polysaccharide STGP-1

[0090] The Sophora tonkinensis polysaccharide STGP-1 of the present invention can be used to prepare anti-lung squamous cell carcinoma drugs that enhance the immunotherapy effect of anti-PD-1 monoclonal antibodies. When used in combination with anti-PD-1 monoclonal antibodies, both should be formulated into separate drug dosage forms. Sophora tonkinensis polysaccharide STGP-1 can also be used alone or in combination with other pharmaceutically acceptable excipients to prepare immunomodulators that regulate intestinal flora and activate anti-tumor T cell immunity, thereby also playing an anti-lung squamous cell carcinoma role.

[0091] (I) Overview of Drugs

[0092] 1. Basic Uses

[0093] The *Sophora tonkinensis* polysaccharide STGP-1 of this invention is used to prepare drugs or adjuvants that enhance the therapeutic effect of immune checkpoint inhibitors (preferably anti-PD-1 monoclonal antibodies) on squamous cell carcinoma of the lung. Furthermore, STGP-1 can act as an immunomodulator, activating anti-tumor T-cell immunity (CD4+). + CD8 + Drugs targeting T cells.

[0094] 2. Drug dosage form

[0095] This drug uses STGP-1, a polysaccharide from Sophora tonkinensis, as its main active ingredient, supplemented with pharmaceutically acceptable carriers or excipients. Based on the loose and porous microstructure and good gastrointestinal stability of STGP-1, this dosage form is preferably prepared as an oral dosage form, including but not limited to oral boluses, granules, capsules, and tablets.

[0096] 3. Dosing regimen

[0097] Route of administration: Daily oral administration is preferred.

[0098] Dosage: The effective dosage for mice is 300 mg / kg / day. The human dosage can be initially estimated based on the interspecies body surface area conversion ratio (e.g., the conversion factor between mice and humans is approximately 12.33), and further adjusted based on the results of clinical trials.

[0099] (II) Experimental verification data

[0100] 1. In vivo synergistic effect against lung squamous cell carcinoma experiment

[0101] See Figure 7 A. Seven-week-old male C57BL / 6J mice were selected to establish a subcutaneous xenograft model of KLN205 lung squamous cell carcinoma cells. The mice were randomly divided into PBS group, α-PD-1 group (200 μg / mouse), STGP-1 group (300 mg / kg / d), and STGP-1 + α-PD-1 combination group. STGP-1 group and combination group were administered the drug by gavage daily. α-PD-1 was injected intraperitoneally every 3 days after tumor formation, for a total of 4 times, for 20 days of intervention.

[0102] See Figure 7 The results showed that compared with the PBS group, tumor growth was significantly slowed in the STGP-1 group, α-PD-1 group, and combination group. The tumor volume and weight in the combination group were significantly lower than those in the single-drug group, indicating the best anti-tumor effect. During the experiment, there was no significant change in the body weight of mice in each group. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea (UREA), creatinine (CREA), and creatine kinase isoenzyme MB (CK-MB) were all within the normal range, confirming that STGP-1 has no obvious toxicity.

[0103] 2. Experiment on activating anti-tumor T-cell immunity

[0104] See Figure 8 AC and Figure 9 AD, flow cytometry and immunohistochemical detection showed that CD8 in the spleen and tumor tissue of mice in the STGP-1+α-PD-1 combination group was high. + T cells / CD4 + The proportion of T cells was significantly higher in the group receiving the drug alone than in the group receiving the drug alone. Figure 9 Serum ELISA testing of EG showed that the levels of effector cytokines such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and granzyme B (GZMB) were significantly increased in the combination group.

Claims

1. Sophora tonkinensis polysaccharide STGP-1, characterized in that, Its chemical structural formula is shown below: 。 2. A method for preparing the Sophora tonkinensis polysaccharide STGP-1 as described in claim 1, characterized in that, Includes the following steps: (1) Grind the dried roots of Sophora tonkinensis into powder, defatted with anhydrous ethanol, collected the precipitate by centrifugation, added water to the precipitate for water bath extraction, collected the supernatant extract, concentrated the supernatant extract, added anhydrous ethanol to the concentrated solution for alcohol precipitation, collected the precipitate, and dried to obtain crude polysaccharide extract. (2) The crude polysaccharide extract was dissolved in water, hydrolyzed by protease, and then the protein was removed by Sevag reagent. It was then extracted with petroleum ether, and then small molecule impurities were removed by adsorption with macroporous resin. After dialysis and alcohol precipitation and drying, the impurity-removed polysaccharide was obtained. (3) The purified polysaccharide was loaded onto an anion exchange column and eluted sequentially with pure water and NaCl solution of increasing concentration, and the main components were collected. (4) The main component obtained in step (3) was loaded onto a gel purification column, eluted with water, dialyzed and freeze-dried to obtain Sophora tonkinensis polysaccharide STGP-1.

3. The preparation method according to claim 2, characterized in that, In step (1): The material-to-liquid ratio during degreasing is 1:10 (w / v), and the degreasing time is 30 minutes. The water bath extraction was performed at a temperature of 60°C for 4 hours. The volume of anhydrous ethanol added during the alcohol precipitation is 4 times the volume of the concentrated liquid, and the alcohol precipitation time is 12 hours.

4. The preparation method according to claim 2, characterized in that, In step (2): The proteases used in the enzymatic hydrolysis were papain and a complex protease, and the hydrolysis time was 12 hours. The volume ratio of chloroform to n-butanol in the Sevag reagent is 4:1; The macroporous resin is of type AB-8; The dialysis bag used in the dialysis had a molecular weight cutoff of 3000 Da, and the dialysis time was 48 hours.

5. The preparation method according to claim 2, characterized in that, In step (3): The anion exchange column is a DEAE seplife FF anion exchange column; The concentration gradient of the NaCl solution is 0.1 M, 0.2 M and 0.3 M respectively, and the elution flow rate is 4 mL / min.

6. The preparation method according to claim 2, characterized in that, In step (4): The gel purification column was a Sephacryl S-400HR gel column; The elution flow rate is 1 mL / min.

7. The use of the Sophora tonkinensis polysaccharide STGP-1 as described in claim 1 in the preparation of drugs for treating lung squamous cell carcinoma.

8. The application of the Sophora tonkinensis polysaccharide STGP-1 combined with anti-PD-1 monoclonal antibody as described in claim 1 in the preparation of anti-lung squamous cell carcinoma drugs.

9. A drug for treating squamous cell carcinoma of the lung, characterized in that, Includes the Sophora tonkinensis polysaccharide STGP-1 as described in claim 1.

10. A combination drug for treating squamous cell carcinoma of the lung, characterized in that, Includes the Sophora tonkinensis polysaccharide STGP-1 as described in claim 1 and the anti-PD-1 monoclonal antibody.

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