Application of Astragalus polysaccharides in colon cancer drugs by regulating intestinal flora

Abstract

The application relates to application of Astragalus polysaccharide in colon cancer drugs by regulating intestinal flora, and extracts crude polysaccharide (ARCP) in Astragalus, which can be used for drugs for treating or preventing colon cancer, and the ARCP realizes the effect on colon cancer mainly by regulating intestinal flora; the ARCP can increase the proportion of CD8 + T cells, improve the tumor immune microenvironment, regulate T cell subgroups, the ARCP has the ability of up-regulating Tem subgroup, down-regulating Temra subgroup, making Tem the dominant subgroup, thereby increasing the cytotoxicity of CD8 + T cells, and improving the killing ability on tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drug technology, and in particular relates to the application of Astragalus polysaccharide in colon cancer drugs by regulating intestinal flora. Background Technology

[0002] Colorectal cancer (CRC) is a prevalent cancer worldwide and the second leading cause of cancer death globally, with its incidence rate increasing annually. It is projected that approximately 3.2 million new cases will be added by 2040. Current treatments for CRC primarily involve surgery, chemotherapy combined with radiotherapy, and targeted therapy. Although immunotherapy, represented by PD-1 / PD-L1 inhibitors, has improved the survival rate for some cancer patients, the response rate to immune checkpoint inhibitors in CRC patients remains less than 25%. This is because the immunosuppressive state of the tumor microenvironment (TME) limits the killing of tumor cells by immune effector cells. Therefore, targeting the tumor microenvironment and reversing its TME immunosuppressive state is an important direction for CRC immunotherapy.

[0003] Upregulation of immunosuppressive cells (such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and M2 macrophages) and immune checkpoint molecules (such as PD-L1) in the TME are key factors contributing to the inhibitory nature of the TME. These cells and molecules actively suppress the activity of cytotoxic effector T cells and natural killer (NK) cells, impairing the immune system's anti-tumor immune response. Notably, recent research evidence reveals that the gut microbiota can dynamically regulate the TME immune phenotype through the "microbiota-metabolite-immunity" axis, providing a novel perspective for overcoming resistance to immunotherapy in colorectal cancer. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an application of Astragalus polysaccharide in colon cancer drugs by regulating intestinal flora.

[0005] The technical solution adopted in this invention is: the application of Astragalus polysaccharide in the preparation of colon cancer drugs.

[0006] Preferably, the water extract of Astragalus membranaceus is taken, and after alcohol treatment, the crude polysaccharide of Astragalus membranaceus precipitate is used to prepare colorectal cancer drugs.

[0007] Preferably, the crude polysaccharide of Astragalus membranaceus is separated by ion exchange chromatography, the water elution component is neutral polysaccharide, and the sodium chloride solution elution component is acidic polysaccharide. The mixture of neutral polysaccharide and acidic polysaccharide is used to prepare colorectal cancer drugs.

[0008] Preferably, the crude polysaccharide aqueous solution of Astragalus membranaceus is loaded onto a Toyopearl DEAE 650 M column and eluted with water and 0.5 M NaCl, respectively.

[0009] Preferably, the water-soluble components of Astragalus membranaceus crude polysaccharide are used to prepare colorectal cancer drugs.

[0010] Preferably, astragalus polysaccharides exert their effect on colon cancer by regulating the intestinal flora.

[0011] A type of Astragalus polysaccharide is obtained by taking the water extract of Astragalus and the precipitate after alcohol treatment, which is the crude Astragalus polysaccharide.

[0012] Preferably, it is composed of neutral polysaccharides and / or acidic polysaccharides from Astragalus membranaceus; the crude polysaccharides from Astragalus membranaceus are separated by ion exchange chromatography, with the water elution component being neutral polysaccharides and the sodium chloride solution elution component being acidic polysaccharides.

[0013] Preferably, it is a water-soluble polysaccharide from Astragalus membranaceus crude polysaccharide.

[0014] A drug for treating or preventing colon cancer, comprising the aforementioned astragalus polysaccharide.

[0015] The advantages and positive effects of this invention are: the prepared Astragalus polysaccharide component has anti-tumor effects and can be used in colon cancer drugs; Astragalus polysaccharide can increase CD8 + Astragalus polysaccharides can improve the proportion of T cells and the tumor immune microenvironment, promote the proliferation and cytotoxicity of CD8+ T cells, and enhance the killing ability of tumor cells. In addition, astragalus polysaccharides also affect the intestinal flora and can play a role in the treatment of colon cancer by regulating the intestinal flora. Attached Figure Description

[0016] Figure 1 Physicochemical characteristics of various polysaccharide components in Astragalus membranaceus; A: Properties; B: Molecular weight distribution; C: Infrared spectrum; D: Monosaccharide composition;

[0017] Figure 2 Effects of Astragalus aqueous extract, alcohol extract and crude polysaccharide on in vivo tumors in a subcutaneously transplanted MC38 tumor-bearing mouse model; (a) Animal experimental design; (b) Tumor changes in model mice; (c) Tumor weight changes in model mice; (d) Tumor volume changes in model mice; ns, no significance; *P<0.05; **P<0.01; ***P<0.001;

[0018] Figure 3 Effects of Astragalus membranaceus crude polysaccharide ARCP on in vivo tumors in a subcutaneously transplanted MC38 tumor-bearing mouse model; (a, e) Animal experimental design; (b, f) Tumor changes in model mice; (c, g) Tumor weight changes in model mice; (d, h) Tumor volume changes in model mice; ns, no significance; *P<0.05; **P<0.01; ***P<0.001;

[0019] Figure 4 Astragalus crude polysaccharide ARCP exerts antitumor effects by remodeling gut microbiota; (a) Experimental design route; (b) Tumor changes in model mice; (c) Tumor weight changes in model mice; (d) Tumor volume changes in model mice; ns, no significance; *P<0.05; **P<0.01; ***P<0.001;

[0020] Figure 5 Flow cytometry detection of CD8 in tumor tissue + Number of T cells; (a) CD8 + Scatter plot of T cells CD8 + T(CD4 - CD8 + (b) CD8 concentration in unit tumor tissue of Model group and ARCP group + Differential analysis of T cell count; ns, no significance; *P<0.05; **P<0.01; ***P<0.001;

[0021] Figure 6 Flow cytometry detection of CD8 in tumor tissue + T cell typing; (a) Tn subsets (CD44) - CD62L + Tcm subgroup (CD44) + CD62L + ), Temra subgroup (CD44) - CD62L + ) and Tem subgroup (CD44) + CD62L - (a) Cell typing; (b) Tn differential analysis; (c) Tcm differential analysis; (d) Tem differential analysis; (e) Temra differential analysis; ns, nosignificance; *P<0.05; **P<0.01; ***P<0.001;

[0022] Figure 7 Flow cytometry detection of CD8 + Fluorescence intensities of IFN-γ and Perforin secreted by T cells; (a) Histogram overlay of MFI for IFN-γ; (b) Differential analysis of MFI for IFN-γ between the Model group and the ARCP group; (c) Histogram overlay of MFI for Perforin; (d) Differential analysis of MFI for Perforin between the Model group and the ARCP group. ns, nosignificance; *P<0.05; **P<0.01; ***P<0.001;

[0023] Figure 8 Flow cytometry was used to detect changes in iNOS (M1 macrophages) and CD206 (M2 macrophages) signaling in tumor tissues; (a, c) iNOS and CD206 staining were used to distinguish the corresponding macrophage subsets; (b, d) M1 macrophages (iNOS) signaling expression in the Model group and ARCP group. + ) and M2 macrophages (CD206) + Difference analysis; ns, nosignificance; *P<0.05; **P<0.01; ***P<0.001;

[0024] Figure 9 Flow cytometry detection of CD4 in tumor tissue + T / CD8 + T cell percentage, CD8 + T cell typing, changes in iNOS (M1 macrophages) and CD206 (M2 macrophages) signaling expression; (a) CD4 + T(CD4) + CD8 - CD8 + T(CD4 - CD8 + (b, c, d) ABX-Model group and ABX-ARCP group CD4 + T, CD8 + T, CD4 / CD8 + Differential analysis of T cells; (e) using CD44 and CD62L staining to distinguish the corresponding T cell subsets Tn (CD44) - CD62L + ), Tcm (CD44) + CD62L + ), Temra (CD44) - CD62L + ), Tem(CD44 + CD62L - (f, g, h, i) Differential analysis of Tn, Tcm, Tem, and Temra between the ABX-Model and ABX-ARCP groups; (j, l) Differentiation of corresponding macrophage subsets using iNOS and CD206 staining; (k, m) M1 macrophages (iNOS) in the ABX-Model and ABX-ARCP groups. + ) and M2 macrophages (CD206) + Difference analysis; ns, nosignificance, *P<0.05, **P<0.01, ***P<0.001;

[0025] Figure 10. Flow cytometry detection of CD8 in unit tumor tissue of the FMT group + T cell count, CD8 + T-cell typing and CD8 + Changes in T cell secretion of IFN-γ and perforin, and changes in signal expression; (a) CD4 + T(CD4) + CD8 - CD8 + T(CD4 - CD8 + (b) CD8 concentration in unit tumor tissue of FMT-Model and FMT-ARCP groups + Differential analysis of T cell numbers; (c) Tn subsets (CD44) - CD62L + Tcm subgroup (CD44) + CD62L + ), Temra subgroup (CD44) - CD62L + ) and Tem subgroup (CD44) + CD62L - (d) Cell typing; (e) Tcm differential analysis; (f) Tem differential analysis; (g) Temra differential analysis; (h) Histogram overlay of MFI for IFN-γ and Perforin; (i) Differential analysis of MFI for IFN-γ between the FMT-Model group and the FMT-ARCP group; (k) Differential analysis of MFI for Perforin between the FMT-Model group and the FMT-ARCP group. (n, no significance; *P<0.05; **P<0.01; ***P<0.001;)

[0026] Figure 11 Flow cytometry was used to detect changes in the expression of M1 macrophage signaling iNOS and M2 macrophage signaling CD206 in tumor tissues of the FMT group; (a) Histogram overlay of MFI of iNOS and CD206; (b) Differential analysis of MFI of iNOS signal; (c) Differential analysis of MFI of CD206 signal; ns, no significance; *P<0.05; **P<0.01; ***P<0.001;

[0027] Figure 12Antitumor effects of different components in Astragalus membranaceus crude polysaccharide ARCP; (a) Animal experimental design route; (b) Tumor changes in model mice; (c) Tumor weight changes in model mice; (d) Tumor volume changes in model mice; ns, no significance; *P<0.05; **P<0.01; ***P<0.001;

[0028] Figure 13 Flow cytometry detection of the effects of different Astragalus polysaccharide components on CD8 in tumor tissue + The effect of T cell typing; (a) using CD44 and CD62L staining to distinguish the corresponding T cell subsets Tn (CD44) - CD62L + ), Tcm (CD44) + CD62L + ), Temra (CD44) - CD62L + ), Tem(CD44 + CD62L - (b, c, d, e) Analysis of differences in Tn, Tcm, Tem, and Temra among the Model group, ARCP group, ARA+ARN group, ARN group, and ARA group; ns, no significance; *P<0.05; **P<0.01; ***P<0.001;

[0029] Figure 14 Flow cytometry was used to detect the effects of different Astragalus polysaccharide components on Inos and CD206 signaling in macrophages in tumor tissues; (a, c) Inos and CD206 staining were used to distinguish the corresponding macrophage subsets; (b, d) Inos and CD206 staining were used to differentiate M1 macrophages (iNOS) in the Model group, ARCP group, ARA+ARN group, ARN group and ARA group. + ) and M2 macrophages (CD206) + () Difference analysis; ns, no significance; *P<0.05; **P<0.01; ***P<0.001;

[0030] Figure 15 Antitumor effects of Astragalus membranaceus crude polysaccharides ARCP and APCP-S; (a) Animal experimental design route; (b) Tumor changes in model mice; (c) Tumor weight changes in model mice; (d) Tumor volume changes in model mice; ns, nosignificance; *P<0.05; **P<0.01; ***P<0.001. Detailed Implementation

[0031] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0032] This invention relates to the application of Astragalus polysaccharide in colon cancer treatment by regulating intestinal flora. Astragalus is the dried root of Astragalus membranaceus and Astragalus mongholicus, belonging to the Fabaceae family. It possesses the effects of tonifying qi and raising yang, consolidating the exterior and stopping sweating, promoting diuresis and reducing swelling, generating fluids and nourishing blood, and promoting pus drainage and detoxification, making it one of the representative traditional Chinese medicines for "strengthening the body's resistance and eliminating pathogenic factors." Its active ingredients mainly include polysaccharides, saponins, alkaloids, and trace elements, among which polysaccharides, as the core active ingredient, are water-soluble heteropolysaccharides with bidirectional immune regulatory functions. Research on crude polysaccharide (ARCP) extracted from Astragalus has shown that ARCP can be used in the treatment or prevention of colon cancer. ARCP mainly achieves its effect on colon cancer by regulating intestinal flora.

[0033] Astragalus membranaceus raw material was decocted in water, and the resulting aqueous extract was subjected to alcohol precipitation. The precipitate was then dialyzed (molecular weight cutoff 3.5 kD) and concentrated to obtain the solid substance, which was taken as crude astragalus polysaccharide (ARCP). ARCP is a brown powder with a molecular weight mainly distributed in the range of 5.1~470.5 kD. The chemical composition of ARCP was analyzed by colorimetry. The total sugar content in ARCP was close to 100%. In addition, ARCP contained 18.9% uronic acid and a small amount of protein. The monosaccharide composition of ARCP was analyzed. ARCP mainly consisted of an absolute amount of Glc, and small amounts of Ara, GalA, Gal, and Rha.

[0034] Studies have found that polysaccharides in Astragalus membranaceus can produce anti-tumor effects. The therapeutic effect of ARCP on tumors is directly related to gut microbiota, and crude polysaccharides in Astragalus membranaceus exert their anti-tumor effects through gut microbiota dependence. ARCP can increase CD8+. + T cell ratio and improvement of the tumor immune microenvironment. Specific analysis of T cell subsets revealed that ARCP has the ability to upregulate the Tem subset and downregulate the Temra subset, making Tem the dominant subset, thereby increasing CD8+. + ARCP enhances the cytotoxicity of T cells and increases their ability to kill tumor cells. Furthermore, ARCP promotes the polarization of TAMs towards M1 macrophages, thereby increasing the proliferation and cytotoxicity of CD8+ T cells and enhancing tumor-killing capabilities. Astragalus polysaccharides can be used to prepare anti-tumor drugs, particularly suitable for treating colorectal cancer.

[0035] Further analysis of the ARCP composition was conducted. ARCP was loaded onto an ion-exchange chromatography column, and eluted with water and NaCl solutions to obtain the neutral polysaccharide ARN and the acidic polysaccharide ARA. ARN and ARA were white and pale yellow powders, respectively. The neutral polysaccharide ARN was mainly distributed in the lower range (5.1–110.9 kD), while the acidic polysaccharide ARA was distributed in a relatively higher range (17.3–470.5 kD). ARN had a high total sugar content but no uronic acid, while the acidic polysaccharide ARA had a high uronic acid content, accounting for approximately 90% of the total sugar content. Both contained small amounts of protein. Analysis of the monosaccharide composition of ARN and ARA revealed that ARN was mainly composed of a large amount of Glc and a small amount of Ara; ARA was mainly composed of GalA, with relatively high amounts of Ara and Glc, and small amounts of Gal and Rha.

[0036] Studies have found that combined administration of ARA and ARN had the same effect on CD8+ T cell typing as administration of ARCP alone, but administration of ARA and ARN alone did not show a significant effect on CD8+ T cell typing; the antitumor effect of ARCP is the synergistic effect of its neutral and acidic polysaccharides.

[0037] Based on this feature, in some embodiments of the present invention, the neutral polysaccharide ARN or the acidic polysaccharide ARA in ARCP can be extracted separately, and then the two can be mixed according to their extraction yield to obtain a composition with anti-colon cancer properties.

[0038] Astragalus crude polysaccharide also contains some water-insoluble substances. Through the extraction, removal and analysis of the water-insoluble substances, it was found that the water-soluble polysaccharide component in ARCP is the main component that exerts the anti-tumor effect, while the water-insoluble component does not show anti-tumor effect. Therefore, when preparing anti-tumor drugs, the water-insoluble precipitate component can be removed. On the one hand, this can meet the requirements of the pharmaceutical process, and on the other hand, it can also be used for liquid formulations.

[0039] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0040] Example 1: Preparation of Astragalus Extract

[0041] 1.1 Preparation of Astragalus ethanol extract (ARE), water extract (ARW) and crude Astragalus polysaccharide (ARCP)

[0042] After pulverizing the Astragalus membranaceus herb, it was soaked in 10 times the amount of ethanol for 1 hour each time, and then refluxed twice (1.5 hours each time). The residue was extracted twice with 50% ethanol (2 hours each time). The extracts were combined, concentrated under reduced pressure, and spray-dried to obtain the Astragalus membranaceus alcohol extract ARE, with a yield of 22.99% based on the herb.

[0043] After pulverizing the Astragalus membranaceus herb, it was decocted three times (1.5 hours each time) with 8-10 times the amount of water. The decoction was filtered, the extracts were combined, and spray-dried to obtain Astragalus membranaceus aqueous extract ARW, with a yield of 28.5% based on the herb.

[0044] After Astragalus membranaceus was cut into sections, it was extracted three times with water at 9, 6, and 5 times the amount of raw herb, respectively, for 1 hour, 1 hour, and 0.5 hours. The extracts were combined and concentrated to a concentration of approximately 1.5 g of raw herb per mL. Ethanol was added to achieve an alcohol content of 75%, and the extract was allowed to stand to obtain an alcohol precipitate. Further, an appropriate amount of water was added to dissolve the extract completely. After centrifugation, the extract was dialyzed for 72 hours using a dialysis bag with a molecular cutoff of 3.5 kD. The dialysate was concentrated and freeze-dried to obtain Astragalus membranaceus crude polysaccharide ARCP, with a yield of 1.71% based on the dried herb.

[0045] 1.2 Preparation of acidic polysaccharide component (ARA) and neutral polysaccharide component (ARN)

[0046] ARCP was redissolved in deionized water, stirred, and centrifuged. The supernatant was then eluted sequentially with water and 0.5 M NaCl on a Toyopearl DEAE 650M column (3.6 cm × 20 cm). The eluent was collected in 20 mL tubes using an automated collector, and sugars were detected at 490 nm using the phenol-sulfuric acid method. The positive reaction fraction was collected, thus preparing neutral astragalus polysaccharide (ARN) and acidic astragalus polysaccharide (ARA) from the water elution fraction and the 0.5 M NaCl elution fraction.

[0047] 1.3 Characteristic Analysis of Crude Polysaccharides, Neutral Polysaccharides and Acidic Polysaccharides in Astragalus

[0048] First, the phenotypic characteristics of ARCP, ARN, and ARA are as follows: Figure 1 As shown in Figure A, ARCP is a brown powder, while ARN and ARA are white and pale yellow powders, respectively. The molecular weight distributions of ARCP, ARN, and ARA were determined using the HPGPC method, and the results are as follows: Figure 1 As shown in B, ARCP is mainly distributed in the range of 5.1~470.5 kD, while the neutral polysaccharide ARN is mainly distributed in the lower range (5.1~110.9 kD), and the acidic polysaccharide ARA is distributed in the relatively higher range (17.3~470.5 kD). The average molecular weight of each polysaccharide is shown in Table 1.

[0049] Table 1. Yield, molecular weight distribution, chemical composition, and monosaccharide composition of various polysaccharide components in Astragalus membranaceus.

[0050]

[0051] a. Based on dry matter; b. Based on ARCP; c. Phenol-sulfuric acid method; d. m-; e. Biored kit; *Not detected.

[0052] The chemical composition of various polysaccharide components and purified polysaccharides of Astragalus membranaceus, including total sugar content, uronic acid content, and protein content, was determined by colorimetry. The results are shown in Table 1. All polysaccharide components and purified polysaccharides were mainly composed of sugars. The total sugar content in the crude polysaccharide ARCP and the neutral polysaccharide ARN was close to 100%. ARCP contained 18.9% uronic acid and a small amount of protein. After separation by ion-exchange chromatography, ARN had a higher total sugar content but no uronic acid, while the acidic polysaccharide ARA had a higher uronic acid content, accounting for approximately 90% of the total sugar in ARA. Both contained small amounts of protein.

[0053] Infrared spectroscopy was performed on the ARCP, ARN, and ARA components of Astragalus polysaccharide using a manual tablet compression method with potassium bromide. Figure 1 As shown in C, both ARCP and ARA are at 3420 cm. -1 2929 cm -1 and 1027~1147 cm -1 The strong absorption peaks appearing nearby are attributed to the stretching vibrations of OH, methylene CH, and COC on the sugar ring, respectively, and are typical absorption peaks for polysaccharides. Additionally, in the spectra of ARCP and ARA, a peak at 1740 cm⁻¹ is observed. -1 The presence of absorption peaks nearby indicates that both ARCP and ARA contain uronic acid.

[0054] The monosaccharide compositions of Astragalus polysaccharide components ARCP, ARN, and ARA were determined by PMP-HPLC, and the results are as follows: Figure 1 As shown in D, ARCP is mainly composed of an absolute amount of Glc, and also contains small amounts of Ara, GalA, Gal, and Rha; ARN is mainly composed of a large amount of Glc and a small amount of Ara; ARA is mainly composed of GalA, and also contains a relatively high amount of Ara and Glc, as well as small amounts of Gal and Rha. The specific composition is shown in Table 1.

[0055] Example 2: Analysis of the antitumor effect of Astragalus extract

[0056] To identify the active pharmaceutical ingredients in Astragalus membranaceus that have anti-colon cancer effects, the Astragalus membranaceus alcohol extract (ARE), water extract (ARW), and crude Astragalus membranaceus polysaccharide (ARCP) prepared in Example 1 were used in MC38 tumor-bearing mouse models to determine the differences in anti-tumor effects of different Astragalus membranaceus extracts.

[0057] Female C57BL / 6 mice, weighing (20±2) g and aged 6-8 weeks, were used in the experiments, which were conducted in accordance with the guidelines of the Animal Experimentation Ethics Committee. The experimental animals were housed under specific pathogen-free (SPF) conditions with a 12-hour light-dark cycle, and provided with standard food and free access to water. After one week of acclimatization, a suspension of MC38 tumor cells in the logarithmic growth phase was prepared under ultra-clean conditions and adjusted to 1.5 × 10⁻⁶ cells / mL. 7 mL -1 For later use, the mice were anesthetized one by one, fixed on the worktable, and the hair on the back of the right hind leg of the mice was removed. The skin on the back of the C57BL / 6 mice was then disinfected with 75% alcohol. 100 μL of MC38 cell suspension was extracted and inoculated subcutaneously to obtain the MC38 mouse tumor-bearing model.

[0058] The constructed mouse model mice were randomly divided into four groups: Model group, ARW group, ARE group, and ARCP group, with eight mice in each group. For the Model group mice, 300 μL of autoclaved aspirate was administered by gavage one day after tumor inoculation, continuing for two weeks. For the ARW, ARE, and ARCP groups mice, the corresponding component (ARW, ARE, or ARCP) was dissolved in 300 μL of autoclaved aspirate and administered by gavage one day after tumor inoculation, continuing for two weeks. Based on preliminary experimental results, the ARCP group was administered at a concentration of 300 mg / kg. For the ARW and ARE groups, the dosage was adjusted according to yield equivalence in the MC38 mouse tumor-bearing model, with the ARW group at a concentration of 3100 mg / kg and the ARE group at a concentration of 2500 mg / kg.

[0059] Fourteen days after administration, tumor tissue was removed, tumor weight was recorded, and tumor growth was compared among the groups of mice to evaluate the antitumor effect of Astragalus polysaccharide. The results are as follows: Figure 2 As shown in the figure, compared with the model group, the ARCP group significantly reduced tumor volume and tumor size, the ARE group showed no significant difference, while the ARW group promoted tumor growth. This indicates that Astragalus polysaccharides play a major anti-tumor role in the MC38 xenograft mouse model, suggesting that polysaccharides are the main active substances in Astragalus's anti-tumor activity.

[0060] Example 3: Antitumor effects of Astragalus polysaccharides and their mediating role in gut microbiota

[0061] 3.1 Effects of gut microbiota on the antitumor effect of Astragalus polysaccharides

[0062] A mouse model of colorectal cancer was constructed using MC38 cells (as in Example 2). Based on this, some mouse models were constructed as colorectal cancer mouse models with gut microbiota cleared by an antibiotic mixture (ABX). The gut microbiota of mice was cleared by the antibiotic mixture (ABX) to evaluate the antitumor effect of Astragalus polysaccharide and the mediating role of gut microbiota.

[0063] C57BL / 6 mice were used, and the feeding conditions before model construction were as shown in Example 2. The mice were randomly divided into 4 groups: Model group, ARCP group, ABX-Model group, and ABX-ARCP group, with 8 mice in each group. In the gut microbiota elimination group (including the ABX treatment process), each mouse was administered 500 μL of ABX by gavage daily one week before tumor inoculation. ABX consisted of vancomycin (500 mg / L), ampicillin (1 g / L), neomycin (1 g / L), and metronidazole (1 g / L). The mice's drinking water was replaced with the antibiotic mixture to eliminate the intestinal microbiota of the mice in advance.

[0064] Model group / ABX-Model group: One day after tumor inoculation, 300 μL of autoclaved sterile water was administered by gavage for two weeks; ARCP group / ABX-ARCP group: One day after tumor inoculation, ARCP was dissolved in autoclaved sterile water and administered by gavage at a concentration of 300 mg / kg-300 μL for two weeks.

[0065] Fourteen days after drug administration, tumor tissue was removed, tumor weight was recorded, and tumor growth was compared among the groups of mice. The results are as follows: Figure 3 As shown, compared with the Model group, the tumor volume and weight in the ARCP group were significantly reduced, indicating that ARCP has a significant anti-tumor effect on colon tumors. After bacterial clearance, there was no significant difference in tumor volume and weight between the ABX-MODEL and ABX-ARCP groups. These results suggest that the therapeutic effect of ARCP on tumors is directly related to the bacterial community, and that Astragalus crude polysaccharide exerts its anti-tumor effect through the bacterial community.

[0066] 3.2 Verification of the influence of gut microbiota on the antitumor effect of ARCP using fecal microbiota transplantation (FMT) technique

[0067] To further confirm whether the anti-tumor effect of ARCP is mediated by gut microbiota, a fecal microbiota transplantation (FMT) experiment was conducted. The gut microbiota was investigated using a mixture of antibiotics to clear bacteria.

[0068] Mice in the Model group and ARCP group constructed by the aforementioned method were placed in empty cages that had been sterilized by ultraviolet light beforehand. Feces were collected 4 days after drug administration. 500 mg of feces was picked up with tweezers and crushed in 2 mL of sterile PBS. The mixture was filtered through a 70 μm filter membrane and resuspended in 2 mL of sterile PBS to prepare fecal suspensions.

[0069] C57BL / 6 mice were randomly divided into two groups: the FMT-Model group and the FMT-ARCP group, with 8 mice in each group. One week before tumor inoculation, each mouse was administered 500 μL of ABX by gavage daily, and the mice's drinking water was replaced with an antibiotic mixture to preemptively eliminate the intestinal flora. One day after tumor inoculation, mice in the FMT-Model group were given a fecal suspension prepared from the aforementioned Model group mice, and each mouse was administered 50 mg of feces-200 μL of sterile PBS by gavage for 20 days; mice in the FMT-ARCP group were given a fecal suspension prepared from the aforementioned ARCP group mice, and each mouse was administered 50 mg of feces-200 μL of sterile PBS by gavage for 20 days.

[0070] The results are as follows Figure 4 As shown, when feces from mice in the drug-treated group were given to model mice with gut microbiota clearance, the tumor volume and weight in the FMT-ARCP group were significantly reduced compared with the FMT-MODEL group, further clarifying that the anti-tumor effect of ARCP depends on the mediation of gut microbiota.

[0071] Example 4: Analysis of the antitumor mechanism of Astragalus crude polysaccharide ARCP

[0072] 4.1 ARCP on CD8 in the tumor microenvironment of model mice + T cell regulation

[0073] CD8+ levels in unit tumor tissue were detected by flow cytometry. + Changes in the number of T cells are used to illustrate the regulatory role of ARCP in the tumor microenvironment.

[0074] Tumor tissues from the Model group and ARCP group mice were collected separately. The tumor tissues, placed in culture medium, were transferred to 3 mL of digestion solution and then minced. Digestion was performed at 37°C and 180 rpm for 30 min on a shaker. After digestion, the minced tumor tissue was poured onto a 70 μm filter membrane and gently ground in a circular motion with a grinding rod until the tissue became a cell suspension. The tissue was then thoroughly agitated with PBS until no obvious tissue fragments were observed in the suspension. The cell suspension was filtered through a filter and then transferred to centrifuge tubes. Centrifugation was performed at 4°C and 1400 rpm for 5 min. The supernatant was discarded, and the tubes were washed once with PBS. 3 mL of 30% Percoll was added to each centrifuge tube, and the mixture was thoroughly mixed, inverted, and then equilibrated before being placed in a centrifuge. The centrifugation speed was set to 1400 rpm, and slow heating and cooling were performed for 20 min. The supernatant was discarded, and the tubes were washed once with 3 mL of PBS. Add 5 mL of erythrocyte lysis buffer and lyse at room temperature for 15 minutes. Then, add PBS to terminate the lysis. Centrifuge at 1400 rpm for 5 minutes at 4°C and discard the supernatant. Stain L / D (1:1000) at room temperature in the dark for 30 minutes, centrifuge at 1400 rpm for 5 minutes at 4°C and discard the supernatant. Wash once with 200 μL of PBS. Stain CD45-Bv421 (1:800), CD4-PE-Cy7 (1:800), CD8-Percp-Cy5.5 (1:800), CD44-FITC (1:400), and CD62L (1:800) in the dark at 4°C for 30 minutes. Centrifuge at 1400 rpm for 5 minutes at 4°C and discard the supernatant. Wash once with 200 μL of PBS, resuspend the cells in 400 μL of PBS, and transfer the cell suspension through a nylon sieve to a flow cytometer for analysis.

[0075] The results are as follows Figure 5 As shown in the comparison, ARCP can increase CD8 concentration per unit of tumor tissue. + The number of T cells indicates that ARCP increases CD8+. + It plays a role in increasing the number of T cells and improving the tumor immune microenvironment, thereby enhancing the ability to kill tumor cells.

[0076] 4.2 ARCP on CD8 in the tumor microenvironment of model mice + Regulation of T cell subtyping

[0077] The experimental results above show that ARCP increases CD8. +The proportion of T cells plays a crucial role, and CD8⁺ T cell typing holds a central position in the tumor microenvironment (TME), with its functional heterogeneity directly determining the efficacy of anti-tumor immune responses. CD8⁺ T cell abundance alone cannot distinguish effector memory subsets with potent killing capabilities. Flow cytometry staining to detect the proportions and marker expression of these subsets can provide insights into the immune status within the tumor microenvironment. Furthermore, flow cytometry was used to detect the composition of Tn, Tcm, Tem, and Temra subsets in different groups of tumor tissues, and CD44 and CD62L staining was used to differentiate the corresponding T cell subsets.

[0078] The results are as follows Figure 6 As shown, ARCP has the ability to upregulate the Tem subpopulation and downregulate the Temra subpopulation, making Tem the dominant subpopulation. (CD8) + Increased T cell cytotoxicity and enhanced anti-tumor activity indicate that ARCP enhances CD8+. + The role of T cells in killing tumor cells.

[0079] 4.3 ARCP on CD8 in the tumor microenvironment of model mice + Regulation of T-secretion of interferon (IFN-γ) and perforin.

[0080] To assess the impact of ARCP on CD8⁺ T cell function, we used flow cytometry to detect the intracellular levels of key effector molecules. CD8 T cells play a crucial role in the immune response, secreting various effector molecules such as interferon-γ (IFN-γ) and perforin to combat viral infections and tumor cells. Upon activation, CD8 T cells secrete large amounts of interferon, especially IFN-γ. This cytokine possesses potent antiviral and immunomodulatory functions, exerting its effects through direct killing (inhibiting proliferation, promoting apoptosis), indirect regulation (activating macrophages and NK cells), and altering the tumor environment (anti-angiogenesis, recruiting immune cells). Perforin is a key molecule for CD8 T cell killing of target cells. It creates pores in the target cell membrane, allowing other toxic molecules such as granzymes to enter the cell and induce apoptosis. After recognition and activation, CD8 T cells rapidly release perforin, effectively clearing infected or abnormal cells.

[0081] Tumor tissues from the Model group and ARCP group mice were collected separately. The tumor tissues, placed in culture medium, were transferred to 3 mL of digestion solution and then minced. Digestion was performed at 37°C and 180 rpm for 30 min on a shaker. After digestion, the minced tumor tissue was poured onto a 70 μm filter membrane and gently ground in a circular motion with a grinding rod until the tissue became a cell suspension. The tissue was then thoroughly agitated with PBS until no obvious tissue fragments were observed in the suspension. The cell suspension was filtered through a filter and then transferred to centrifuge tubes. Centrifugation was performed at 4°C and 1400 rpm for 5 min. The supernatant was discarded, and the tubes were washed once with PBS. 3 mL of 30% Percoll was added to each centrifuge tube, and the mixture was thoroughly mixed, inverted, and then equilibrated before being placed in a centrifuge. The centrifugation speed was set to 1400 rpm, and slow heating and cooling were performed for 20 min. The supernatant was discarded, and the tubes were washed once with 3 mL of PBS. Add 5 mL of erythrocyte lysis buffer and lyse at room temperature for 15 minutes. Then add PBS to terminate the lysis. Centrifuge at 1400 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend cells in 2 ml PBS, add 100 µl to a 96-well plate, and add 100 µl of CAC. Stimulate at 37°C, 5% CO2 for 6 h. After stimulation, centrifuge at 4°C, 1400 rpm for 5 min, discard the supernatant, and wash once with PBS. Stain L / D (1:1000) at room temperature in the dark for 30 min, centrifuge at 4°C, 1400 rpm for 5 min, discard the supernatant, and wash once with 200 µl PBS. Stain CD3-PE-Cy7 (1:800), CD4-FITC (1:800), and CD8-Percp-Cy5.5 (1:800) in the dark, incubate at 4°C in the dark for 30 min, centrifuge at 4°C, 1400 rpm for 5 min, discard the supernatant, and wash once with 200 µl PBS. Add 200 µl of 4% paraformaldehyde and fix overnight at 4°C in the dark. After fixation, centrifuge at 4°C, 1400 rpm for 5 min. Centrifuge at 1400 rpm for 5 min at room temperature, discard the supernatant, wash once with PBS, add 1× permeabilization buffer, and permeabilize for 5 min at room temperature. Centrifuge at 1400 rpm for 5 min at 4℃, discard the supernatant, and stain with IFN-γ-APC (1:800) and Perforin-PE (1:800) in the dark. Wash once with 200 µl PBS, resuspend the cells in 200 µl PBS, and transfer the cell suspension through a nylon sieve to a flow cytometer for analysis.

[0082] The mean fluorescence intensity (MFI) of IFN-γ and perforin secreted by CD8+ T cells was detected by flow cytometry. The results are as follows: Figure 7As shown, compared with the Model group, ARCP enhanced the MFI of IFN-γ and Perforin. The enhanced MFI of IFN-γ indicates an expansion of the breadth of the immune response. IFN-γ can enhance antigen presentation by upregulating MHC-I expression in tumor cells, activate innate immune cells such as macrophages and NK cells, and exert anti-angiogenic effects within the tumor microenvironment. The increased MFI level of Perforin indicates that ARCP enhances the cytotoxicity of CD8⁺ T cells, enabling them to more effectively initiate apoptosis recognition of tumor cells through the perforin-granzyme pathway. These results demonstrate that the effector function of CD8⁺ T cells is enhanced under ARCP intervention.

[0083] 4.4 Regulation of tumor-associated macrophage (TAM) polarization in the tumor microenvironment of model mice by ARCP

[0084] In the tumor microenvironment, tumor-associated macrophages (TAMs) are the most abundant immune cells. Macrophages in the tumor microenvironment have the ability to polarize into M1 macrophages, which possess anti-tumor and pro-inflammatory capabilities, and M2 macrophages, which possess pro-tumor and anti-inflammatory capabilities. Flow cytometry was used to detect the expression of iNOS (a marker of M1 macrophages) and CD206 (a marker of M2 macrophages) in macrophages from different tumor tissue groups.

[0085] Different groups of mouse tumor tissues were collected from culture medium and transferred to 3 mL of digestion solution, then chopped. Digestion was performed at 37°C and 180 rpm for 30 min. After digestion, the chopped tumor tissue was poured onto a 70 μm filter membrane and gently ground in a circular motion with a grinding rod until the tissue became a cell suspension. The tissue was then thoroughly agitated with PBS until no obvious tissue fragments were observed in the suspension. The cell suspension was filtered and transferred to centrifuge tubes. Centrifuged at 4°C and 1400 rpm for 5 min, the supernatant was discarded, and the tubes were washed once with PBS. 3 mL of 30% Percoll was added to each centrifuge tube, mixed thoroughly, inverted, and then equilibrated before being placed in a centrifuge. The centrifugation speed was set to 1400 rpm, and slow heating and cooling were performed for 20 min. The supernatant was discarded, and the tubes were washed once with PBS. 5 mL of erythrocyte lysis buffer was added, and lysis was performed at room temperature for 15 min, followed by PBS to terminate the lysis. The tubes were then centrifuged at 4°C and 1400 rpm for 5 min, and the supernatant was discarded.

[0086] Stain with LD (1:1000) at room temperature in the dark for 30 min, centrifuge at 1400 rpm at 4℃ for 5 min, discard the supernatant, and wash once with PBS. Stain with F4 / 80-FITC (1:400), CD11b-APC (1:400), and CD45-PE-Cy7 (1:800) in the dark and incubate on ice for 30 min; centrifuge at 1400 rpm at 4℃ for 5 min, discard the supernatant, and wash once with PBS. Fix and perforate the membrane with 150 μl of paraformaldehyde fixative and incubate on ice in the dark for 30 min; centrifuge at 1400 rpm at 4℃ for 5 min, discard the supernatant, and wash once with PBS; add 150 μL of perforation buffer, perforate the membrane at room temperature for 5 min, centrifuge at 1400 rpm at 4℃ for 5 min, discard the supernatant, and wash once with PBS. The cells were stained with CD206-PerCp-Cy5.5 (1:200) and iNOS-PE (1:400); the cell suspension was then transferred to flow cytometry tubes for analysis.

[0087] Test results as follows Figure 8 As shown in (a) and (b), ARCP upregulates M1 macrophages, while (c) and (d) show that ARCP does not exhibit a significant regulatory effect on M2 macrophages. Based on these results, it can be concluded that ARCP promotes the polarization of TAMs towards M1 macrophages, thereby enhancing the proliferation and cytotoxicity of CD8+ T cells and increasing tumor-killing ability.

[0088] 4.5 ARCP on CD4 in the tumor microenvironment of mice after knockdown of gut microbiota + T / CD8 + T cell percentage, CD8 + T cell subtyping and regulation of TAM polarization

[0089] The results of Example 3 showed that transplanted ARCP feces inhibited tumor growth, while ARCP did not regulate tumor weight or volume in mice after ABX knockdown of the intestinal flora. Further analysis was conducted by measuring the CD4+T / CD8+T cell ratio, CD8+T cell typing, and TAM polarization in the tumor tissue.

[0090] Tumor tissues were collected from mice in the ABX-Model group and ABX-ARCP group, and CD4 levels in the tumor tissues were detected using the aforementioned method. + T / CD8 + T cell percentage. Results as follows: Figure 9 As shown in Figure (ai), CD4 in tumor tissue was detected by flow cytometry. + T cells and CD8 +T cells showed no significant differences between ABX-ARCP and ABX-Model in terms of CD4, CD8, CD4 / CD8 ratio, and CD8 typing, indicating that after knocking down the gut microbiota in mice, ARCP lost its due regulatory effect on T cells in the tumor microenvironment.

[0091] The differentiation of M1 and M2 macrophages in tumor tissues of mice in the ABX-Model and ABX-ARCP groups was then examined, and the results are as follows: Figure 9 As shown in Figure (jm), flow cytometry analysis of the mean fluorescence intensity of M1 and M2 macrophages in tumor tissue revealed no significant difference between ABX-ARCP and ABX-Model in M1 macrophages. This indicates that knocking down the mouse gut microbiota also diminishes the regulatory effect of ARCP on M1 macrophages in the tumor microenvironment.

[0092] The above results show that after knocking down the gut microbiota, ARCP loses its immunomodulatory effect on the tumor microenvironment, further proving that ARCP exerts its anti-tumor effect through the gut microbiota.

[0093] 4.6 Effects of FMT-ARCP on CD8+ in the tumor microenvironment of model mice after fecal microbiota transplantation + Regulation of T cells and regulation of TAM polarization

[0094] Using tumor tissues from the FMT-Model group and the FMT-ARCP group as the detection subjects, the CD8 concentration per unit tumor tissue was measured. + Changes in T cell count, CD8 + Changes in T cell typing, changes in IFN-γ and perforin secreted by CD8+ T cells, and TAM polarization. Results are as follows: Figure 10-11 As shown, the FMT-ARCP group administered ARCP feces compared to the FMT-Model group administered Model feces showed CD8 concentrations per unit tumor tissue. + The number of T cells increased significantly, showing the same trend as the comparison results between the ARCP and Model groups mentioned above.

[0095] CD8 values ​​of FMT-ARCP group given ARCP feces compared to FMT-Model group given Model group feces + The trend of T cell subtyping is that the Tem subset is upregulated and the Temra subset is downregulated, making Tem the dominant subset, which is consistent with the regulatory effect of ARCP on CD8+ T cell subtyping mentioned earlier.

[0096] Compared to the FMT-Model group, which received fecal samples from the Model group, the FMT-ARCP group, which received fecal samples from ARCP, showed a significant enhancement in the MFI of IFN-γ and Perforin. This is consistent with the regulatory effect of ARCP on the MFI of IFN-γ and Perforin secreted by CD8+ T cells described earlier.

[0097] Compared to the model group, the FMT-ARCP group treated with ARCP feces showed upregulated expression of M1 macrophages, but no significant change was observed in M2 macrophages, consistent with the results of the ARCP-Model group comparison above. This further demonstrates that ARCP primarily exerts its anti-tumor effect through gut microbiota.

[0098] Example 5: Analysis of the components responsible for the anti-colon cancer effect of Astragalus membranaceus crude polysaccharide ARCP

[0099] Since the ratio of neutral polysaccharide ARN to acidic polysaccharide ARA in Astragalus crude polysaccharide ARCP is approximately 1, structural analysis revealed that ARN is amyloid α-glucan and ARA is pectin-like polysaccharide. It is generally believed that amyloid α-glucan is an energy-providing substance and does not have activity. In this example, the effects of neutral polysaccharide ARN and acidic polysaccharide ARA in antitumor activity were analyzed.

[0100] 5.1 Comparison of the antitumor effects of neutral polysaccharide ARN and acidic polysaccharide ARA

[0101] The MC38 mouse tumor-bearing model was constructed according to the method in Example 2. The mice were randomly divided into 5 groups: Model group, ARCP group, ARN+ARA group, ARN group and ARA group. According to the method in Example 2, the drugs were administered one day after tumor inoculation. The ARCP group, ARN+ARA group, ARN group and ARA group were administered equivalent drugs to the model mice according to the yield.

[0102] Fourteen days after drug administration, tumor tissue was removed, tumor weight was recorded, and tumor growth was compared among the groups of mice. The results are as follows: Figure 12 As shown, compared with the Model group, the ARCP group showed a significant reduction in both tumor volume and weight, consistent with the aforementioned results. Surprisingly, the antitumor effects disappeared after ARN and ARA were administered alone, but their antitumor effects were restored after combining ARN and ARA. These results clearly suggest that the antitumor effect of ARCP is due to the synergistic effect of its neutral and acidic polysaccharides.

[0103] 5.2 Regulation of CD8+ T cell subtyping in the tumor microenvironment by different Astragalus polysaccharide components

[0104] The regulatory effects of different Astragalus polysaccharide components on CD8+ T cell typing in the tumor microenvironment were detected by flow cytometry. T cell typing in tumor tissues of mice in the Model group, ARCP group, ARN+ARA group, ARN group, and ARA group were detected according to the method in Example 4. The results are as follows: Figure 13 As shown, combined administration of ARA and ARN had the same effect on CD8+ T cell typing as administration of ARCP alone, but administration of ARA and ARN alone did not show a significant effect on CD8+ T cell typing.

[0105] 5.3 Effects of different Astragalus polysaccharide components on TAM polarization in the tumor microenvironment

[0106] The expression of iNOS (a marker of M1 macrophages) and CD206 (a marker of M2 macrophages) in macrophages from tumor tissues of mice in the Model group, ARCP group, ARN+ARA group, ARN group, and ARA group was detected by flow cytometry. Results are as follows: Figure 14 As shown, the combination of ARA and ARN and ARCP alone had the same effect on tumor-associated macrophage cell polarization. ARA and ARN also upregulated M1 macrophages. The combination of ARA and ARN showed a significant difference from ARN, while ARCP and ARA showed a certain trend. Meanwhile, ARCP, ARN+ARA, ARN and ARA had no regulatory effect on M2 macrophages.

[0107] Example 6: Preparation of water-soluble polysaccharides from Astragalus membranaceus crude polysaccharide extract and their antitumor effects

[0108] The ARCP prepared in Example 1 contained a high amount of water-insoluble components (39.3%). The study investigated whether removing the water-insoluble components would affect the antitumor effect of ARCP.

[0109] C57BL / 6 mice were randomly divided into three groups: the Model group, the ARCP group, and the ARCP-S group, with eight mice in each group. The two groups of MC38 mouse tumor-bearing models were administered drugs according to the method in Example 2. Based on the product yield, ARCP and ARCP-S (water-soluble crude polysaccharide) were administered at equivalent doses.

[0110] Two weeks after feeding, the tumor growth of mice in each group was examined, and the results were as follows: Figure 15 As shown, compared with the model group, the tumor volume and weight of both the ARCP and ARCP-S groups were significantly reduced, suggesting that the water-soluble polysaccharides in Astragalus crude polysaccharide are the active substances that exert anti-tumor effects, and the water-insoluble precipitates can be removed.

[0111] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. Application of Astragalus polysaccharide in the preparation of colon cancer drugs.

2. Use according to claim 1, characterized in that: The water extract of Astragalus membranaceus was treated with alcohol, and the crude polysaccharide of the precipitate was used to prepare a drug for colorectal cancer.

3. Use according to claim 1, characterized in that: Astragalus crude polysaccharides were separated by ion exchange chromatography. The water elution component was neutral polysaccharide, and the sodium chloride solution elution component was acidic polysaccharide. The mixture of neutral and acidic polysaccharides was used to prepare colorectal cancer drugs.

4. Use according to claim 3, characterized in that: The crude polysaccharide aqueous solution of Astragalus membranaceus was loaded onto a ToyopearlDEAE 650 M column and eluted with water and 0.5 M NaCl, respectively.

5. Use according to claim 2, characterized in that: The water-soluble components of crude polysaccharide from Astragalus membranaceus were used to prepare colorectal cancer drugs.

6. Use according to any one of claims 1 to 5, characterized in that: Astragalus polysaccharides exert their effects on colon cancer by regulating the gut microbiota.

7. A polysaccharide of Astragalus membranaceus, characterized in that: The precipitate obtained after alcohol treatment of the aqueous extract of Astragalus membranaceus is crude polysaccharide.

8. The Astragalus polysaccharide according to claim 7, characterized in that: It is composed of neutral polysaccharides and / or acidic polysaccharides from Astragalus membranaceus; the crude polysaccharides from Astragalus membranaceus are separated by ion exchange chromatography, with the water elution component being neutral polysaccharides and the sodium chloride solution elution component being acidic polysaccharides.

9. The Astragalus polysaccharide according to claim 7, characterized in that: Specifically, it refers to the water-soluble polysaccharides in Astragalus membranaceus crude polysaccharide.

10. A medicament for treating or preventing colon cancer, characterized by comprising the compound of claim 1. Includes the astragalus polysaccharide described in any one of claims 7-9.

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