Radix astragali acid low molecular weight polysaccharide, preparation method thereof and use thereof for preventing and treating intestinal injury

Abstract

The application discloses a method for preparing astragalus acidic low-molecular polysaccharide, which comprises the following steps: (1) preparing astragalus polysaccharide mother liquor; (2) performing column chromatography on the astragalus polysaccharide mother liquor through a cellulose column, eluting with water and 0.4-0.6 mol / L sodium chloride solution in sequence, retaining the elution part of the 0.4-0.6 mol / L sodium chloride solution, concentrating, dialyzing and drying the elution part of the 0.4-0.6 mol / L sodium chloride solution to obtain astragalus acidic polysaccharide freeze-dried powder; and (3) pretreating the astragalus acidic polysaccharide freeze-dried powder to obtain astragalus acidic polysaccharide solution, dialyzing and separating the astragalus acidic polysaccharide solution through a dialysis bag with a molecular weight cut-off of 50-200 kDa, collecting the solution outside the dialysis bag, concentrating and drying to obtain the astragalus acidic low-molecular polysaccharide. The astragalus acidic low-molecular polysaccharide serves as a material basis for resisting irinotecan-induced intestinal injury (IGT) and provides a reference for industrialized conversion of traditional Chinese medicine polysaccharide components.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an acidic low-molecular-weight polysaccharide of Astragalus membranaceus, its preparation method, and its use in preventing and treating intestinal damage. Background Technology

[0002] Irinotecan is a first-line chemotherapy drug for solid tumors such as colorectal cancer and pancreatic cancer, but it causes severe delayed diarrhea in up to 87% of cases, with grade 3-4 diarrhea accounting for about 40%, which is a major limiting factor in clinical application. Currently, clinical treatment mainly focuses on symptomatic relief, which is insufficient to fundamentally block the intestinal epithelial damage induced by its metabolite 7-ethyl-10-hydroxycamptothecin (SN-38). There is an urgent need to develop more targeted intervention strategies. Astragalus is a traditional Chinese medicine and food, and historical herbal texts record its use in treating dysentery. Modern clinical studies have also shown that compound preparations containing astragalus have a certain alleviating effect on chemotherapy-related diarrhea. However, its core active ingredient and mechanism of action in combating irinotecan-induced intestinal damage (IGT) remain unclear. Existing literature suggests that the occurrence of IGT is closely related to the accumulation of SN-38 in the intestine, and that gut microbiota-derived β-glucuronidase (β-GUS) plays a key role in this process. Based on this, the present invention aims to clarify the material basis and mechanism of action of Astragalus membranaceus in resisting IGT, and at the same time provide a reference for the industrial transformation of polysaccharide components of traditional Chinese medicine.

[0003] The prior art has not found the Astragalus acidic low molecular weight polysaccharide claimed in this invention, its preparation method, or its use in preventing and treating intestinal damage. Summary of the Invention

[0004] Based on this, the present invention provides a method for preparing acidic low-molecular-weight polysaccharides from Astragalus membranaceus, the method comprising the following steps: (1) Preparation of Astragalus polysaccharide mother liquor; (2) The Astragalus polysaccharide mother liquor was subjected to column chromatography using a cellulose column, eluted sequentially with water and 0.4–0.6 mol / L sodium chloride solution. The 0.4–0.6 mol / L sodium chloride solution eluent was collected, concentrated, dialyzed, and dried to obtain lyophilized Astragalus acidic polysaccharide powder; and (3) The lyophilized powder of Astragalus acidic polysaccharide was pretreated to obtain Astragalus acidic polysaccharide solution. The Astragalus acidic polysaccharide solution was dialyzed using a dialysis bag with a molecular weight cutoff of 50~200 kDa. The solution outside the dialysis bag was collected, concentrated, and dried to obtain Astragalus acidic low molecular weight polysaccharide.

[0005] Furthermore, the steps for preparing the Astragalus polysaccharide mother liquor include: weighing an appropriate amount of Astragalus polysaccharide powder, adding water, heating and stirring, dissolving with ultrasonic assistance, cooling and centrifuging, taking the supernatant and filtering to obtain the Astragalus polysaccharide mother liquor.

[0006] Furthermore, the water is distilled water.

[0007] Furthermore, the mass / volume (g / mL) ratio of the Astragalus polysaccharide powder to the water is 0.01 to 0.1, for example, about 0.04.

[0008] Furthermore, the mass of the Astragalus polysaccharide powder is 1-3 g, for example, about 2 g.

[0009] Furthermore, the volume of the water is 40 to 60 mL, for example, about 50 mL.

[0010] Furthermore, the heating and stirring is carried out using a water bath.

[0011] Furthermore, the temperature of the water bath is 50~70℃, for example, about 60℃.

[0012] Furthermore, the heating and stirring time is 20 to 40 minutes, for example, about 30 minutes.

[0013] Furthermore, the power of this ultrasound-assisted dissolution is 200~400W, for example, about 300W.

[0014] Furthermore, the time for ultrasound-assisted dissolution is 10-20 minutes, for example, about 15 minutes.

[0015] Furthermore, the cooling is to cool to room temperature.

[0016] Furthermore, the centrifuge speed is 3000~5000 r / min, for example, about 4000 r / min.

[0017] Furthermore, the centrifugation time is 10-20 minutes, for example, about 15 minutes.

[0018] Furthermore, the centrifugation temperature is 1~10℃, for example, about 4℃.

[0019] Furthermore, the filtration is a membrane vacuum filtration.

[0020] Furthermore, the pore size of the filter membrane is 0.45 μm.

[0021] Furthermore, the concentration of the Astragalus polysaccharide mother liquor is 50~200 mg / ml, for example, about 100 mg / ml.

[0022] Furthermore, the preparation method of the Astragalus polysaccharide powder includes: (i) weighing an appropriate amount of Astragalus slices and extracting them by reflux with a first solvent; (ii) extracting the residue with water, combining the extracts and concentrating them, adding a second solvent while stirring, so that the final concentration of the second solvent in the system reaches a certain concentration; and (iii) after standing overnight, centrifuging to collect the precipitate, redissolving the precipitate in water and drying it to obtain the Astragalus polysaccharide powder.

[0023] Further, in step (i), the first solvent is 80% to 100% ethanol, for example, about 95% ethanol.

[0024] Furthermore, in step (i), the extraction is performed 1 to 3 times, for example, 2 times.

[0025] Furthermore, in step (i), the extraction time for each extraction is 0.5 to 2 hours, for example, about 1 hour.

[0026] Further, in step (i), the volume / mass ratio of the first solvent to the Astragalus membranaceus slices is 5 to 15, for example, about 8 or about 10.

[0027] Further, in step (ii), the volume / mass ratio of the water to the Astragalus membranaceus slices is 4 to 10, for example, about 6.

[0028] Furthermore, in step (ii), the extraction is performed 1 to 3 times, for example, 2 times.

[0029] Furthermore, in step (ii), the extraction time for each extraction is 0.5 to 2 hours, for example, about 1.5 hours.

[0030] Further, in step (ii), the second solvent is ethanol.

[0031] Furthermore, in step (ii), the concentration is 70% to 100%, for example, about 80%.

[0032] Furthermore, in steps (ii) and (iii), the water is distilled water.

[0033] Furthermore, in step (iii), the temperature for overnight standing is 1 to 10°C, for example, about 4°C.

[0034] Furthermore, in step (iii), the centrifugation speed is 2000~4000 ×g, for example, about 3000 ×g.

[0035] Furthermore, in step (iii), the centrifugation time is 5 to 15 minutes, for example, about 10 minutes.

[0036] Furthermore, in step (iii), the drying is freeze drying.

[0037] Furthermore, the cellulose column is a DEAE Sepharose Fast Flow cellulose column.

[0038] Further, in step (2), the column chromatography steps include: (a) pre-treating the DEAE Sepharose Fast Flow packing material and packing it into a column, equilibrating it, and loading the Astragalus polysaccharide mother liquor onto the column; (b) eluting with water, collecting the eluent, and stopping elution after reaching the endpoint; and (c) eluting with approximately 0.5 mol / L sodium chloride solution, collecting the eluent, stopping elution after reaching the endpoint and collecting the eluent to obtain the eluted fraction of approximately 0.5 mol / L sodium chloride solution.

[0039] Furthermore, the water is distilled water.

[0040] Furthermore, this equilibrium is achieved by equilibrating the chromatography column to a pH of approximately 7.0 for the eluent, and / or by ensuring that the absorbance of the eluent at a wavelength of 280 nm is ≤0.02.

[0041] Furthermore, the flow rate for this equilibrium is 10–30 mL / min, for example, about 20 mL / min.

[0042] Furthermore, the loading flow rate is 5–15 mL / min, for example, about 10 mL / min.

[0043] Furthermore, the elution flow rate is 10-30 mL / min, for example, about 20 mL / min.

[0044] Furthermore, the endpoint is defined as follows: the absorbance of the eluent at 490 nm is measured using the phenol-sulfuric acid method, and the absorbance value is below 0.03 for three consecutive tubes.

[0045] Furthermore, the volume of the eluent is 1 to 20 L, for example, about 10 L.

[0046] Furthermore, in step (2), the dialysis is performed using water.

[0047] Furthermore, the water is distilled water.

[0048] Furthermore, in step (2), the dialysis time is 36 to 60 hours, for example, about 48 hours.

[0049] Furthermore, in step (2), the distilled water is changed every 6 hours during the dialysis process.

[0050] Furthermore, the drying process is freeze drying.

[0051] Further, in step (3), the pretreatment step includes: taking an appropriate amount of the freeze-dried powder of Astragalus acidic polysaccharide, adding water to dissolve it by vortexing, cooling and centrifuging, taking the supernatant to obtain the Astragalus acidic polysaccharide solution.

[0052] Furthermore, the water is distilled water.

[0053] Furthermore, the cooling is to cool to room temperature.

[0054] Furthermore, the centrifuge speed is 3000~5000 r / min, for example, about 4000 r / min.

[0055] Furthermore, the centrifugation time is 10-20 minutes, for example, about 15 minutes.

[0056] Furthermore, the centrifugation temperature is 1~10℃, for example, about 4℃.

[0057] Furthermore, in step (3), the molecular weight cutoff of the dialysis bag is approximately 100 kDa.

[0058] Furthermore, in step (3), the water is distilled water.

[0059] Furthermore, in step (3), the dialysis temperature is 1~10°C, for example, about 4°C.

[0060] Furthermore, in step (3), the dialysis time is 36 to 60 hours, for example, about 48 hours.

[0061] Furthermore, in step (3), the distilled water is changed every 6 hours during the dialysis process.

[0062] Furthermore, in step (3), the drying is freeze drying.

[0063] According to another aspect of the present invention, an acidic low-molecular-weight polysaccharide of Astragalus membranaceus prepared by the above method is provided.

[0064] Furthermore, the molecular weight of the characteristic peak of this Astragalus acidic low molecular weight polysaccharide is 19.8 ± 2.0 kDa.

[0065] Furthermore, the molecular weight of the characteristic peak of this Astragalus acidic low molecular weight polysaccharide is 19.8 ± 1.0 kDa.

[0066] Furthermore, the characteristic peak of this Astragalus acidic low molecular weight polysaccharide has a molecular weight of approximately 19.8 kDa.

[0067] According to another aspect of the present invention, an astragalus acidic low molecular weight polysaccharide pharmaceutical preparation is provided, the pharmaceutical preparation comprising the above-mentioned astragalus acidic low molecular weight polysaccharide, and one or more pharmaceutically acceptable excipients.

[0068] Furthermore, the excipient is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, encapsulating agents, flavoring agents, sustained-release agents, flow aids, lubricants, dispersants, plasticizers, opacifiers, and antioxidants.

[0069] Furthermore, the dosage form of the drug preparation is tablets, pills, capsules, powders, injections, films, lozenges, granules, or oral liquids.

[0070] According to another aspect of the present invention, the use of the above-mentioned Astragalus acidic low molecular weight polysaccharide or the above-mentioned Astragalus acidic low molecular weight polysaccharide pharmaceutical preparation in the preparation of a medicament for the prevention and / or treatment of intestinal damage is provided.

[0071] Furthermore, this intestinal injury was drug-induced.

[0072] Furthermore, this drug is an antitumor chemotherapy drug.

[0073] Furthermore, the anti-tumor chemotherapy drug is irinotecan.

[0074] Furthermore, the effects of this Astragalus acidic low molecular weight polysaccharide or its pharmaceutical preparation in preventing and / or treating intestinal damage are achieved through one or more of the following mechanisms: improving general condition abnormalities, increasing colon length, improving colonic histopathological abnormalities, improving colonic mucus secretion function, restoring intestinal barrier function, promoting colonic cell proliferation, inhibiting β-GUS enzyme activity, regulating intestinal flora metabolism, inhibiting MAPK signaling pathway hyperphosphorylation, and inhibiting colonic cell apoptosis.

[0075] Furthermore, this general condition abnormality includes weight loss and / or an elevated disease activity index.

[0076] Furthermore, the histopathological abnormalities of the colon included neutrophil infiltration, goblet cell loss, epithelial barrier disruption, and / or crypt structure disorder.

[0077] Furthermore, this improvement in colonic tissue mucus secretion function resulted in an increase in the number of goblet cells.

[0078] Furthermore, this restoration of intestinal barrier function includes elevated ZO-1 mRNA levels, elevated Muc2 mRNA levels, elevated ZO-1 protein levels, elevated Occludin protein levels, elevated Muc2 protein levels, decreased plasma diamine oxidase levels, and / or decreased plasma lipopolysaccharide levels.

[0079] Furthermore, this regulation of gut microbiota metabolism includes increased levels of acetic acid, propionic acid, and butyric acid in gut microbiota metabolites, and decreased levels of adipic acid and / or lactic acid.

[0080] Furthermore, hyperphosphorylation of the MAPK signaling pathway includes elevated phosphorylation levels of P38, ERK, and / or JNK proteins.

[0081] Furthermore, this inhibition of colon cell apoptosis resulted in decreased Bax protein levels and / or increased Bcl-2 protein levels.

[0082] The beneficial effects of this invention are: The acidic low-molecular-weight polysaccharide of Astragalus membranaceus in this invention serves as the material basis for anti-IGT and provides a reference for the industrial transformation of polysaccharide components in traditional Chinese medicine. Attached Figure Description

[0083] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0084] Figure 1 This is a schematic diagram showing the characterization results of polysaccharides from different parts. (a) Molecular weight; (b) Fourier transform infrared spectrum; (c) Scanning electron microscope (×500).

[0085] Figure 2 Schematic diagram of H&E staining and semi-quantitative results of colon tissue from mice in each group. Among them, (a) pathological score of colon tissue; (b) H&E staining of colon tissue (n=6); p <0.05 (*), p <0.01 (**), p <0.001 (***), p<0.0001 (****).

[0086] Figure 3 This is a schematic diagram showing the intestinal barrier marker levels in mice of each group. (a) Tight junction protein 1 (ZO-1) mRNA; (b) Occludin mRNA; (c) Krüppel-like factor 4 (Klf4) mRNA; (d) Semi-quantitative results of ZO-1 (n=3); (e) ZO-1 immunofluorescence staining; p <0.05 (*), p <0.01 (**), p <0.001 (***), p <0.0001 (****).

[0087] Figure 4 This is a schematic diagram showing the characterization results of different polysaccharides. (a) Molecular weight; (b) Scanning electron microscopy (×500).

[0088] Figure 5 Schematic diagram of H&E staining and semi-quantitative results of colon tissue from mice in each group. Among them, (a) pathological score of colon tissue; (b) H&E staining of colon tissue (n=6); p <0.05 (*), p <0.01 (**), p <0.001 (***), p<0.0001 (****).

[0089] Figure 6 This is a schematic diagram showing the results of intestinal barrier marker levels in mice of each group. Among them, (a) Occludin mRNA (n=6); (b) Klf4 mRNA (n=6); p <0.05 (*), p <0.01 (**), p <0.001 (***), p<0.0001 (****).

[0090] Figure 7 The diagram illustrates the effect of 0.5-Out on β-GUS enzyme activity and SN-38 exposure. (a) β-GUS enzyme activity in colonic contents (n=6); (b) SN-38 exposure in colonic tissue (n=6); (c) SN-38 response intensity and in situ distribution in colonic tissue.

[0091] Figure 8 This is a schematic diagram showing the results of adjusting for acetic acid, propionic acid, butyric acid, adipic acid, and lactic acid levels with a 0.5-Out ratio. The diagram shows the levels of acetic acid, propionic acid, butyric acid, valeric acid, succinic acid, adipic acid, lactic acid, N-acetyltryptophan (NAT), indoleacetic acid (IAA), 5-hydroxyindoleacetic acid (5-HIAA), and sodium 5-methoxyindoleacetic acid (5-MIAA) in the intestinal lumen (n=6); p <0.05 (*), p <0.01 (**), p <0.001 (***).

[0092] Figure 9A schematic diagram showing the effect of 0.5-Out on irinotecan-induced hyperphosphorylation of P38, ERK, and JNK and inhibition of apoptosis. Among them, (a) Western blot results of p38 mitogen-activated protein kinase (p38), phosphorylated p38 mitogen-activated protein kinase (p-p38), extracellular signal-regulated kinase (ERK), phosphorylated extracellular signal-regulated kinase (p-ERK), c-Jun N-terminal kinase (JNK), phosphorylated c-Jun N-terminal kinase (p-JNK), Bcl-2-associated X protein (Bax), and B-cell lymphoma / leukemia-2 protein (Bcl-2) in colon tissue; (b) grayscale analysis of p-P38 / P38 protein; (c) grayscale analysis of p-ERK / ERK protein; (d) grayscale analysis of p-JNK / JNK protein; (e) grayscale analysis of Bax protein; (f) grayscale analysis of Bcl-2 protein (n=3); p <0.05 (*), p <0.01 (**), p <0.001 (***). Detailed Implementation

[0093] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0094] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0095] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0096] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0097] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0098] As described in the background section, the prior art has not found the Astragalus acidic low molecular weight polysaccharide claimed in this invention, its preparation method, or its use in preventing and treating intestinal damage. To address the above problems, this invention provides a method for preparing Astragalus acidic low molecular weight polysaccharide, comprising the following steps: (1) Preparation of Astragalus polysaccharide mother liquor; (2) The Astragalus polysaccharide mother liquor was subjected to column chromatography using a cellulose column, eluted sequentially with water and 0.4–0.6 mol / L sodium chloride solution. The 0.4–0.6 mol / L sodium chloride solution eluent was collected, concentrated, dialyzed, and dried to obtain lyophilized Astragalus acidic polysaccharide powder; and (3) The lyophilized powder of Astragalus acidic polysaccharide was pretreated to obtain Astragalus acidic polysaccharide solution. The Astragalus acidic polysaccharide solution was dialyzed using a dialysis bag with a molecular weight cutoff of 50~200 kDa. The solution outside the dialysis bag was collected, concentrated, and dried to obtain Astragalus acidic low molecular weight polysaccharide.

[0099] In this invention, when concentration, molecular weight cutoff, ratio, mass, volume, temperature, time, power, rotation speed, flow rate, molecular weight, or other values ​​or parameters are expressed as ranges, preferred ranges, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “0.4~0.6” is disclosed, the described range should be interpreted as including the ranges “0.4~0.6”, “0.4~0.56”, “0.4~0.52”, “0.4~0.48”, “0.4~0.44”, “0.44~0.6”, “0.44~0.56”, “0.44~0.52”, “0.44~0.48”, “0.48~0.6”, “0.48~0.56”, “0.48~0.52”, “0.52~0.6”, “0.52~0.56”, “0.56~0.6”, etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0100] In a preferred embodiment, the step of preparing the Astragalus polysaccharide mother liquor includes: weighing an appropriate amount of Astragalus polysaccharide powder, adding water, heating and stirring, dissolving with ultrasonic assistance, cooling and centrifuging, taking the supernatant and filtering to obtain the Astragalus polysaccharide mother liquor.

[0101] In a preferred embodiment, the water is distilled water.

[0102] In a preferred embodiment, the mass / volume (g / mL) ratio of the astragalus polysaccharide powder to the water is 0.01 to 0.1, for example, about 0.04.

[0103] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.04" includes ±5% of 0.04, or from 0.038 to 0.042.

[0104] In a preferred embodiment, the mass of the astragalus polysaccharide powder is 1 to 3 g, for example, about 2 g.

[0105] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2" includes 2 ± 5%, or from 1.9 to 2.1.

[0106] In a preferred embodiment, the volume of the water is 40 to 60 mL, for example, about 50 mL.

[0107] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50" includes 50 ± 5%, or from 47.5 to 52.5.

[0108] In a preferred embodiment, the heating and stirring is performed using a water bath.

[0109] In a preferred embodiment, the temperature of the water bath is 50~70°C, for example, about 60°C.

[0110] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0111] In a preferred embodiment, the heating and stirring time is 20 to 40 minutes, for example, about 30 minutes.

[0112] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0113] In a preferred embodiment, the power of the ultrasound-assisted dissolution is 200-400W, for example, about 300W.

[0114] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 300" includes ±5% of 300, or from 285 to 315.

[0115] In a preferred embodiment, the time for ultrasound-assisted dissolution is 10 to 20 minutes, for example, about 15 minutes.

[0116] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0117] In a preferred embodiment, the cooling is cooling to room temperature.

[0118] In a preferred embodiment, the centrifugation speed is 3000~5000 r / min, for example about 4000 r / min.

[0119] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4000" includes ±5% of 4000, or from 3800 to 4200.

[0120] In a preferred embodiment, the centrifugation time is 10 to 20 minutes, for example, about 15 minutes.

[0121] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0122] In a preferred embodiment, the centrifugation temperature is 1~10°C, for example, about 4°C.

[0123] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0124] In a preferred embodiment, the filtration is a membrane vacuum filtration.

[0125] In a preferred embodiment, the filter membrane has a pore size of 0.45 μm.

[0126] In a preferred embodiment, the concentration of the Astragalus polysaccharide mother liquor is 50-200 mg / ml, for example, about 100 mg / ml.

[0127] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 100" includes ±5% of 100, or from 95 to 105.

[0128] In a preferred embodiment, the preparation method of the Astragalus polysaccharide powder includes: (i) weighing an appropriate amount of Astragalus slices and extracting them by reflux with a first solvent; (ii) extracting the residue with water, combining the extracts and concentrating them, adding a second solvent while stirring, so that the final concentration of the second solvent in the system reaches a certain concentration; and (iii) after standing overnight, centrifuging to collect the precipitate, redissolving the precipitate in water and drying it to obtain the Astragalus polysaccharide powder.

[0129] In a preferred embodiment, in step (i), the first solvent is 80% to 100% ethanol, for example, about 95% ethanol.

[0130] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 95" includes ±5% of 95, or from 90.25 to 99.75.

[0131] In a preferred embodiment, in step (i), the extraction is performed 1 to 3 times, for example, 2 times.

[0132] In a preferred embodiment, in step (i), the extraction time for each extraction is 0.5 to 2 hours, for example, about 1 hour.

[0133] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0134] In a preferred embodiment, in step (i), the volume / mass ratio of the first solvent to the Astragalus membranaceus slices is 5 to 15, for example, about 8 or about 10.

[0135] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 8" includes ±5% of 8, or from 7.6 to 8.4; "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0136] In a preferred embodiment, in step (ii), the volume / mass ratio of the water to the Astragalus membranaceus slices is 4 to 10, for example, about 6.

[0137] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0138] In a preferred embodiment, in step (ii), the extraction is performed 1 to 3 times, for example, 2 times.

[0139] In a preferred embodiment, in step (ii), the extraction time for each extraction is 0.5 to 2 hours, for example, about 1.5 hours.

[0140] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.5" includes ±5% of 1.5, or from 1.425 to 1.575.

[0141] In a preferred embodiment, in step (ii), the second solvent is ethanol.

[0142] In a preferred embodiment, in step (ii), the concentration is 70% to 100%, for example, about 80%.

[0143] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 80" includes ±5% of 80, or from 76 to 84.

[0144] In a preferred embodiment, in steps (ii) and (iii), the water is distilled water.

[0145] In a preferred embodiment, in step (iii), the temperature for overnight standing is 1 to 10°C, for example, about 4°C.

[0146] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0147] In a preferred embodiment, in step (iii), the centrifugation speed is 2000~4000 ×g, for example about 3000 ×g.

[0148] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3000" includes ±5% of 3000, or from 2850 to 3150.

[0149] In a preferred embodiment, in step (iii), the centrifugation time is 5 to 15 minutes, for example, about 10 minutes.

[0150] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0151] In a preferred embodiment, in step (iii), the drying is freeze drying.

[0152] In a preferred embodiment, the cellulose column is a DEAE Sepharose Fast Flow cellulose column.

[0153] In a preferred embodiment, step (2) of the column chromatography includes: (a) pre-treating DEAESepharose Fast Flow packing material and packing it into a column, equilibrating it, and loading the Astragalus polysaccharide mother liquor onto the column; (b) eluting with water, collecting the eluent, and stopping elution after reaching the endpoint; and (c) eluting with approximately 0.5 mol / L sodium chloride solution, collecting the eluent, stopping elution after reaching the endpoint and collecting the eluent to obtain the eluted fraction of approximately 0.5 mol / L sodium chloride solution.

[0154] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0155] In a preferred embodiment, the water is distilled water.

[0156] In a preferred embodiment, the equilibrium is achieved by equilibrating the column to a pH of approximately 7.0 for the eluent, and / or by ensuring that the absorbance of the eluent at a wavelength of 280 nm is ≤0.02.

[0157] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 7.0" includes ±5% of 7.0, or from 6.65 to 7.35.

[0158] In a preferred embodiment, the flow rate for equilibration is 10 to 30 mL / min, for example, about 20 mL / min.

[0159] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0160] In a preferred embodiment, the loading flow rate is 5 to 15 mL / min, for example, about 10 mL / min.

[0161] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0162] In a preferred embodiment, the elution flow rate is 10 to 30 mL / min, for example, about 20 mL / min.

[0163] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0164] In a preferred embodiment, the endpoint is reached when the absorbance of the eluent at 490 nm is measured using the phenol-sulfuric acid method, and the absorbance value is below 0.03 for three consecutive tubes.

[0165] In a preferred embodiment, the volume of the eluent is 1 to 20 L, for example, about 10 L.

[0166] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0167] In a preferred embodiment, in step (2), the dialysis is performed using water.

[0168] In a preferred embodiment, the water is distilled water.

[0169] In a preferred embodiment, in step (2), the dialysis time is 36 to 60 hours, for example, about 48 hours.

[0170] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 48" includes ±5% of 48, or from 45.6 to 50.4.

[0171] In a preferred embodiment, in step (2), the distilled water is changed every 6 hours during the dialysis process.

[0172] In a preferred embodiment, the drying is freeze drying.

[0173] In a preferred embodiment, in step (3), the pretreatment step includes: taking an appropriate amount of the freeze-dried powder of Astragalus acidic polysaccharide, adding water to dissolve it by vortexing, cooling and centrifuging, taking the supernatant to obtain the Astragalus acidic polysaccharide solution.

[0174] In a preferred embodiment, the water is distilled water.

[0175] In a preferred embodiment, the cooling is cooling to room temperature.

[0176] In a preferred embodiment, the centrifugation speed is 3000~5000 r / min, for example about 4000 r / min.

[0177] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4000" includes ±5% of 4000, or from 3800 to 4200.

[0178] In a preferred embodiment, the centrifugation time is 10 to 20 minutes, for example, about 15 minutes.

[0179] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0180] In a preferred embodiment, the centrifugation temperature is 1~10°C, for example, about 4°C.

[0181] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0182] In a preferred embodiment, in step (3), the molecular weight cutoff of the dialysis bag is about 100 kDa.

[0183] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 100" includes ±5% of 100, or from 95 to 105.

[0184] In a preferred embodiment, in step (3), the water is distilled water.

[0185] In a preferred embodiment, in step (3), the dialysis temperature is 1 to 10°C, for example, about 4°C.

[0186] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0187] In a preferred embodiment, the dialysis time in step (3) is 36 to 60 hours, for example, about 48 hours.

[0188] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 48" includes ±5% of 48, or from 45.6 to 50.4.

[0189] In a preferred embodiment, in step (3), the distilled water is changed every 6 hours during the dialysis process.

[0190] In a preferred embodiment, in step (3), the drying is freeze drying.

[0191] According to another aspect of the present invention, an acidic low-molecular-weight polysaccharide of Astragalus membranaceus prepared by the above method is provided.

[0192] In a preferred embodiment, the characteristic peak of the Astragalus acidic low molecular weight polysaccharide has a molecular weight of 19.8 ± 2.0 kDa.

[0193] In a preferred embodiment, the characteristic peak of the Astragalus acidic low molecular weight polysaccharide has a molecular weight of 19.8 ± 1.0 kDa.

[0194] In a preferred embodiment, the characteristic peak of the Astragalus acidic low molecular weight polysaccharide has a molecular weight of approximately 19.8 kDa.

[0195] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 19.8" includes ±5% of 19.8, or from 18.81 to 20.79.

[0196] According to another aspect of the present invention, an astragalus acidic low molecular weight polysaccharide pharmaceutical preparation is provided, the pharmaceutical preparation comprising the above-mentioned astragalus acidic low molecular weight polysaccharide, and one or more pharmaceutically acceptable excipients.

[0197] In a preferred embodiment, the excipient is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, encapsulating agents, flavoring agents, sustained-release agents, flow aids, lubricants, dispersants, plasticizers, light-blocking agents, and antioxidants.

[0198] In a preferred embodiment, the dosage form of the pharmaceutical preparation is tablets, pills, capsules, powders, injections, films, lozenges, granules, or oral liquids.

[0199] According to another aspect of the present invention, the use of the above-mentioned Astragalus acidic low molecular weight polysaccharide or the above-mentioned Astragalus acidic low molecular weight polysaccharide pharmaceutical preparation in the preparation of a medicament for the prevention and / or treatment of intestinal damage is provided.

[0200] In a preferred embodiment, the intestinal injury is drug-induced intestinal injury.

[0201] In a preferred embodiment, the drug is an antitumor chemotherapy drug.

[0202] In a preferred embodiment, the antitumor chemotherapy drug is irinotecan.

[0203] In a preferred embodiment, the Astragalus acidic low molecular weight polysaccharide or the Astragalus acidic low molecular weight polysaccharide pharmaceutical preparation prevents and / or treats intestinal damage through one or more of the following mechanisms: improving general condition abnormalities, increasing colon length, improving colonic histopathological abnormalities, improving colonic tissue mucus secretion function, restoring intestinal barrier function, promoting colonic cell proliferation, inhibiting β-GUS enzyme activity, regulating intestinal flora metabolism, inhibiting MAPK signaling pathway hyperphosphorylation, and inhibiting colonic cell apoptosis.

[0204] In a preferred embodiment, the general condition abnormality includes weight loss and / or an elevated disease activity index.

[0205] In a preferred embodiment, the histopathological abnormality of the colon includes neutrophil infiltration, goblet cell loss, epithelial barrier disruption, and / or crypt structure disorder.

[0206] In a preferred embodiment, the improvement in colonic tissue mucus secretion function is achieved by an increase in the number of goblet cells.

[0207] In a preferred embodiment, the restoration of intestinal barrier function includes elevated ZO-1 mRNA levels, elevated Muc2 mRNA levels, elevated ZO-1 protein levels, elevated Occludin protein levels, elevated Muc2 protein levels, decreased plasma diamine oxidase levels, and / or decreased plasma lipopolysaccharide levels.

[0208] In a preferred embodiment, the regulation of gut microbiota metabolism includes increasing the levels of acetic acid, propionic acid, butyric acid, adipic acid, and / or lactic acid in gut microbiota metabolites.

[0209] In a preferred embodiment, the hyperphosphorylation of the MAPK signaling pathway includes elevated phosphorylation levels of P38, ERK, and / or JNK proteins.

[0210] In a preferred embodiment, the inhibition of colon cell apoptosis is achieved by a decrease in Bax protein levels and / or an increase in Bcl-2 protein levels.

[0211] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0212] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0213] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0214] Example 1 1. Materials and Methods 1.1 Experimental Materials 1.1.1. Experimental Instruments The experimental instruments are shown in Table 1.

[0215] Table 1 Experimental Instruments

[0216] 1.1.2. Experimental Reagents The experimental reagents are shown in Table 2.

[0217] Table 2 Experimental Reagents

[0218] 1.1.3. Experimental Animals A total of 36 mice were used in this experiment, provided by Shanghai Jihui Experimental Animal Breeding Co., Ltd. (Shanghai, China). The animals were housed in a specific pathogen-free (SPF) environment at the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine (License No.: SCXK (Hu) 2022-0004). The environmental conditions were controlled as follows: temperature of 22±2°C, relative humidity of 40%, and a 12 h light / dark cycle. The mice had free access to standard rodent feed and water. All experimental operations were strictly performed in accordance with the guidelines approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (Approval No.: PZSHUTCM2406170006).

[0219] 1.2. Experimental Methods 1.2.1. Preparation of Acidic Polysaccharides and Neutral Polysaccharides 2.0 kg of Astragalus membranaceus decoction pieces were taken, first subjected to reflux extraction with 10 times the volume of 95% ethanol for 1 h, followed by a second extraction (8 times the volume, 1 h). The two ethanol extracts were combined, concentrated under reduced pressure, and then freeze-dried to obtain the small molecular fraction (SM, mass 492.0 g, yield 24.6%). The extraction residue was then extracted twice with distilled water, with 6 times the volume of distilled water added each time, for 1.5 h per extraction. The extracts were combined and concentrated, and ethanol was added under stirring to make the final ethanol concentration of the system reach 80% (v / v). After standing overnight at 4°C, the precipitate was collected by centrifugation at 3000×g for 10 min. The precipitate was redissolved in distilled water and then lyophilized to obtain the total polysaccharide fraction (APS, mass 171.3 g, yield 8.6%).

[0220] 10 g of APS powder was accurately weighed, 100 mL of distilled water was added, the mixture was heated and stirred in a 60°C water bath for 30 min, and dissolved with ultrasonic assistance (power 300 W, time 15 min). After cooling to room temperature, the mixture was centrifuged (4000 rpm, 15 min, 4°C), the supernatant was taken, and vacuum filtered through a 0.45 μm filter membrane to remove insoluble impurities, so as to obtain an APS sample stock solution (100 mg / mL), which was stored in a 4°C refrigerator for later use.

[0221] The pretreated DEAE Sepharose Fast Flow packing material was uniformly packed into the chromatography column. The column was equilibrated with distilled water until the pH of the eluent was 7.0. The equilibration flow rate was controlled at 20 mL / min until the absorbance of the eluent at 280 nm was ≤0.02 (no absorption of contaminating proteins).

[0222] The prepared APS sample stock solution was slowly loaded onto a pre-equilibrated DEAE-cellulose column at a flow rate of 10 mL / min. After loading, elution was continued with distilled water at a flow rate of 20 mL / min. 5 mL of eluent was collected from each tube. The absorbance of each tube of eluent was measured at 490 nm using the phenol-sulfuric acid method, and an elution curve was plotted. When the absorbance value was below 0.03 for three consecutive tubes, distilled water elution was stopped, and all eluent from this stage was collected to obtain the aqueous polysaccharide (APS-W)—i.e., neutral polysaccharide—with a yield of 2.75%.

[0223] After elution of the neutral polysaccharide, elution was carried out using 0.5 mol / L NaCl solution at a flow rate of 20 mL / min. 5 mL of eluent was collected from each tube, and the absorbance at 490 nm was measured using the phenol-sulfuric acid method. Elution curves were plotted. Elution was stopped when the absorbance value was below 0.03 for three consecutive tubes. All eluent from this stage was collected to obtain the 0.5 fraction polysaccharide (APS-0.5)—i.e., acidic polysaccharide—with a yield of 18.02%.

[0224] The collected neutral polysaccharide eluent and acidic polysaccharide eluent were combined and placed in ordinary dialysis bags. Dialysis was performed with distilled water for 48 hours, with the distilled water being replaced every 6 hours to remove NaCl and small molecule impurities from the solution.

[0225] After dialysis, the two polysaccharide solutions were centrifuged separately (4000 rpm, 15 min, 4℃), and the supernatant was collected, concentrated, and lyophilized to obtain white powdered neutral polysaccharide and acidic polysaccharide, respectively. They were sealed and stored in a desiccator for later use.

[0226] 1.2.2 Characterization of acidic and neutral polysaccharides 1.2.2.1 Homogeneity and Molecular Weight Determination The molecular weight and homogeneity of neutral and acidic polysaccharides separated by APS were determined by high-performance gel permeation chromatography (HPGPC). A TSK-GEL G-4000 PWXL (300 × 7.8 mm) and a TSK-GEL G-2500 PWXL (300 × 7.8 mm) column were used in series. The column temperature was 40℃, the injection volume was 100 μL, the mobile phase was A (0.02% NaN3, 0.1 mol / L NaNO3), the flow rate was 0.8 mL / min, and the elution gradient was isocratic for 80 min.

[0227] 1.2.2.2 Scanning electron microscopy analysis The morphology of neutral and acidic polysaccharides separated by APS was observed using high-resolution field emission scanning electron microscopy. The two polysaccharide samples were sieved using a 100-mesh sieve, coated with a thin layer of gold, and observed under high vacuum at an accelerating voltage of 3.0 kV, with photographs taken for recording.

[0228] 1.2.2.3 Determination of total sugar content Accurately weigh 10.0 mg of glucose standard and place it in a 100 mL volumetric flask. Dissolve the solution in distilled water and dilute to the mark. Shake well to obtain a glucose standard stock solution with a concentration of 0.1 mg / mL. Accurately pipette 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of the glucose standard stock solution into stoppered test tubes, respectively. Add distilled water to each tube to a final volume of 2.0 mL, then add 1.0 mL of 5% phenol solution. Shake well and quickly add 5.0 mL of concentrated sulfuric acid. Mix thoroughly and allow to stand at room temperature for 10 min. Then heat in a boiling water bath for 15 min and cool to room temperature. Using distilled water as a blank control, measure the absorbance of each standard solution at a wavelength of 490 nm. Plot a standard curve with glucose concentration as the x-axis and absorbance as the y-axis, and calculate the regression equation.

[0229] Accurately weigh 10.0 mg of each of the two polysaccharide samples and place them in 100 mL volumetric flasks. Add distilled water to dissolve and dilute to the mark. Shake well to obtain sample stock solutions. Then accurately pipette 1.0 mL of the sample stock solution into a stoppered test tube and operate according to the above standard curve preparation method. Measure its absorbance value and substitute it into the regression equation to calculate the total sugar content in the sample.

[0230] 1.2.2.4 Determination of uronic acid content Accurately weigh 10.0 mg of D-glucuronic acid standard and place it in a 100 mL volumetric flask. Dissolve the standard in distilled water and dilute to the mark. Shake well to obtain a 0.1 mg / mL D-glucuronic acid standard stock solution. Accurately pipette 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of the standard stock solution into stoppered test tubes, respectively. Add distilled water to each tube to a final volume of 2.0 mL, then add 0.2 mL of 0.1% carbazole ethanol solution. Shake well and slowly add 5.0 mL of concentrated sulfuric acid. Mix thoroughly and heat in a boiling water bath for 15 min. Remove from the heat and cool rapidly to room temperature. Using distilled water as a blank control, measure the absorbance of each standard solution at a wavelength of 530 nm. Plot a standard curve with D-glucuronic acid concentration as the x-axis and absorbance as the y-axis, and calculate the regression equation.

[0231] Accurately weigh 10.0 mg of each of the two polysaccharide samples and place them in 100 mL volumetric flasks. Add distilled water to dissolve and dilute to the mark. Shake well to obtain sample stock solutions. Then accurately pipette 1.0 mL of the sample stock solution into a stoppered test tube and operate according to the above standard curve preparation method. Measure its absorbance value and substitute it into the regression equation to calculate the uronic acid content in the sample.

[0232] 1.2.2.5 Fourier Infrared Spectroscopy Analysis Neutral and acidic polysaccharides separated by APS were recorded at 4000-4000 cm⁻¹ using a Nicolet iZ-10 FTIR spectrometer. -1 Infrared spectra within the range. Small amounts of the two polysaccharide samples were weighed and mixed with 200 mg KBr, then pressed into 1 mm thick sheets and analyzed.

[0233] 1.2.3. Construction of the mouse IGT model and administration methods of acidic and neutral polysaccharides. In the experiment on the administration of polysaccharides from different parts of Astragalus membranaceus, the sample size for each group was n=6, and the following groups were set up: control group, irinotecan group (75 mg / kg / day), low-dose group of aqueous part (30 mg / kg / day), high-dose group of aqueous part (60 mg / kg / day), low-dose group of 0.5 part (200 mg / kg / day), and high-dose group of 0.5 part (400 mg / kg / day). The dosage of each part was calculated according to its extraction yield. The crude drug concentration corresponding to the low dose was equivalent to 60 g / day for an adult (60 kg), and 120 g / day for the high dose.

[0234] Mice were randomly assigned to groups (n=6) after 7 days of acclimatization. The test drug was administered by gavage once daily for 14 consecutive days. Starting from day 5, irinotecan was administered intraperitoneally at a dose of 75 mg / kg / day for 10 consecutive days. From the first day of administration, the Disease Activity Index (DAI) was assessed every 48 hours according to the established scoring criteria.

[0235] 1.2.4 Preparation of acidic and neutral polysaccharides The lyophilized acidic polysaccharide (APS-0.5) powder obtained by DEAE Sepharose Fast Flow column chromatography was first dissolved in an appropriate amount of distilled water by vortexing. After cooling to room temperature, the supernatant was collected by centrifugation (4000 rpm, 15 min, 4℃) to obtain the acidic polysaccharide solution. Dialysis was performed using a dialysis bag with a molecular weight cutoff of 100 kDa to obtain the low molecular weight fraction (0.5-Out) and the high molecular weight fraction (0.5-In) refined polysaccharide. The specific operation was as follows: the dissolved acidic polysaccharide solution was slowly added to the dialysis bag, the two ends of the dialysis bag were tied tightly, and the bag was placed in sufficient distilled water. Dialysis was performed at 4℃ for 48 h, and the distilled water was changed every 6 h to ensure complete dialysis. After dialysis, the solution outside the dialysis bag (low molecular weight fraction, 0.5-Out) and the solution inside the dialysis bag (high molecular weight fraction, 0.5-In) were collected separately. The two solutions were centrifuged separately (4000 rpm, 15 min, 4℃), and the supernatant was collected. After lyophilization, 0.5-Out and 0.5-In were obtained, sealed and stored in a desiccator for later use.

[0236] 1.2.5 Characterization of acidic and neutral polysaccharides 1.2.5.1 Homogeneity and Molecular Weight Determination The molecular weight and homogeneity of neutral and acidic polysaccharides separated by APS were determined by high-performance gel permeation chromatography (HPGPC). A TSK-GEL G-4000 PWXL (300 × 7.8 mm) and a TSK-GEL G-2500 PWXL (300 × 7.8 mm) column were used in series. The column temperature was 40℃, the injection volume was 100 μL, the mobile phase was A (0.02% NaN3, 0.1 mol / L NaNO3), the flow rate was 0.8 mL / min, and the elution gradient was isocratic for 80 min.

[0237] 1.2.5.2 Scanning Electron Microscopy Analysis The morphology of neutral and acidic polysaccharides separated by APS was observed using high-resolution field emission scanning electron microscopy. The two polysaccharide samples were sieved using a 100-mesh sieve, coated with a thin layer of gold, and observed under high vacuum at an accelerating voltage of 3.0 kV, with photographs taken for recording.

[0238] 1.2.6. Construction of a mouse IGT model and administration methods for polysaccharides of different molecular weights In the experiment on the administration of different polysaccharides from Astragalus membranaceus, the sample size for each group was n=6, and the following groups were set up: control group, irinotecan group (75 mg / kg / day), high molecular weight low-dose group (65 mg / kg / day), high molecular weight high-dose group (130 mg / kg / day), low molecular weight low-dose group (55 mg / kg / day), and low molecular weight high-dose group (110 mg / kg / day). The dosage of different polysaccharides was calculated according to their extraction yield. The crude drug concentration corresponding to the low dose was equivalent to 60 g / day for an adult (60 kg), and 120 g / day for the high dose.

[0239] Mice were randomly assigned to groups (n=6) after 7 days of acclimatization. The test drug was administered by gavage once daily for 14 consecutive days. Starting from day 5, irinotecan was administered intraperitoneally at a dose of 75 mg / kg / day for 10 consecutive days. DAI was assessed every 48 hours from the first day of administration according to the established scoring criteria.

[0240] 1.2.7 Mouse Dissection and Tissue Collection After the last administration, mice were fasted for 12 hours but allowed free access to water. They were then anesthetized, blood was collected from the abdominal aorta, and the neck was dislocated. The cecum, colon, and rectum were completely removed for photography. Subsequently, the contents of the cecum and colon were removed separately. One cm of colon tissue was placed in an EP tube containing 4% paraformaldehyde and fixed. The remaining colon tissue was placed in an EP tube, frozen in liquid nitrogen, and stored at -80°C.

[0241] Blood samples were left to stand at room temperature for 4 hours, then centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant was collected and stored at -80°C.

[0242] 1.2.8. H&E staining of colon tissue Prepared paraffin sections were baked at 70°C for 30 minutes, dewaxed, and then stained with hematoxylin for 5 minutes. Hematoxylin, as a basic dye, binds to nucleic acids in the cell nucleus, staining the nucleus. After staining, the sections were rinsed three times with tap water and then treated with 0.4% hydrochloric acid-alcohol solution for 1 second. The hydrochloric acid-alcohol solution dissolves some of the hematoxylin that is not bound to the nucleus, making the nuclear staining more prominent. The sections were then rinsed with running water for 10 minutes to achieve a blue stain. Under a microscope, the cell nuclei appeared blue. Cytoplasmic staining was then performed: eosin staining for 30 seconds, followed by rinsing with tap water once. After drying, the sections were mounted with neutral resin. The sections were observed and images were acquired under an optical microscope, and pathological scoring was performed using ImageJ software.

[0243] 1.2.9 Immunohistochemistry / fluorescence of colon tissue After dewaxing the slides to water, they were immersed in sodium citrate solution and microwaved at 100°C for 8 min, then at 60°C for 25 min for repair, and allowed to cool naturally. The slides were then washed with PBS buffer for 5 min with shaking, repeated three times. They were then immersed in a prepared 3% hydrogen peroxide solution in the dark for 10 min to inactivate peroxidase. The slides were washed three times with PBS using the same procedure, blocked with 5% BSA for 30 min, and then incubated overnight at 4°C with the prepared primary antibody. The slides were washed three times with PBS, incubated with the corresponding secondary antibody for 30 min (immunofluorescence should be performed in the dark during this step and subsequent operations), and then washed three more times with PBS.

[0244] Immunohistochemical staining: DAB reagent was used for staining, and the slides were observed under a microscope during the process. Once the staining was complete, the slides were immediately immersed in water to stop the staining. Hematoxylin was used to stain the nuclei for 2 minutes, followed by washing three times with tap water and differentiation with 0.4% hydrochloric acid alcohol. The slides were then rinsed with running water to regain their blue color, dried, and mounted. Images were observed and acquired under an optical microscope, and semi-quantitative analysis was performed using ImageJ software.

[0245] Immunofluorescence staining: DAPI was incubated in the dark for 2 min, washed 3 times with PBS, and then mounted with anti-fluorescence quenching mounting solution. Data were collected and analyzed using a laser confocal microscope, and semi-quantitative analysis was performed using ImageJ software.

[0246] 1.2.10 RNA Extraction and qPCR Total RNA was extracted from mouse colon tissue using TRIzol reagent. RNA purity and concentration were determined using a microplate reader. cDNA was synthesized using a reverse transcription kit. Quantitative PCR was performed on a ViiA™7 real-time quantitative PCR system using TB Green® Premix Ex Taq™ as the fluorescent dye. The expression levels of the target gene were normalized using β-actin and GAPDH as internal reference genes, and relative quantification was performed using the -2ΔΔCt method.

[0247] 2. Experimental Results 2.1 Characterization of acidic and neutral polysaccharides Characterization results of polysaccharides from different parts are as follows: Figure 1As shown, APS-W (water fraction) has a total sugar content of 74.59% and a uronic acid content of only 4.41%, indicating that it is mainly composed of neutral polysaccharides. In contrast, APS-0.5 (0.5 fraction) has a total sugar content of 40.24% and a uronic acid content as high as 52.55%, indicating that this fraction contains more acidic polysaccharides. Uric acid residues are one of its main structural features, consistent with the separation pattern of DEAE cellulose column chromatography—acidic polysaccharides are eluted at higher salt concentrations. HPGPC results show that APS-W mainly exhibits a relatively flat peak with a molecular weight of 97.2 kDa; APS-0.5 has two main polysaccharide components, a high molecular weight component of 670 kDa and a low molecular weight component of 30.9 kDa, indicating a wider molecular weight distribution and significant polydispersity, suggesting a potentially more complex structure. FTIR spectra further confirm the structural differences between the two polysaccharides: both APS-W and APS-0.5 show peaks at 3400 cm⁻¹. -1 A broad and strong O–H stretching vibration peak appears nearby, at 2930 cm⁻¹. -1 The presence of C–H stretching vibration peaks nearby is consistent with the typical infrared characteristics of polysaccharides; among them, APS-0.5 has a peak at 1746 cm⁻¹. -1 The presence of a distinct ν(C=O) stretching vibration peak nearby further confirms the presence of a large amount of uronic acid structures, while no such characteristic peak was observed in APS-W, consistent with its low uronic acid content. SEM results show that APS-W exhibits a porous, wrinkled network structure with a rough surface and abundant pores, which may be related to its higher molecular weight and more uniform chain structure; while APS-0.5 exhibits a sheet-like, irregular blocky morphology with a relatively smooth surface but local aggregation. This difference may stem from the charge effect and intermolecular interactions brought about by uronic acid residues in acidic polysaccharides, resulting in a significantly different aggregation state compared to neutral polysaccharides.

[0248] In summary, APS-W is a neutral polysaccharide with a high total sugar content, low uronic acid content, relatively uniform medium molecular weight, and porous network morphology; APS-0.5 is an acidic polysaccharide with a wider molecular weight distribution and exhibits obvious acidic sugar characteristic peaks in infrared spectroscopy, with a microstructure mainly consisting of sheet-like aggregates. These results demonstrate that DEAE cellulose column chromatography can effectively separate neutral and acidic polysaccharides with significant differences in structure and properties, laying the foundation for subsequent activity studies.

[0249] 2.2 Study on the effects of acidic and neutral polysaccharides on restoring the structural integrity of intestinal tissue like Figure 2As shown in the images, H&E staining and histopathological observation revealed significant neutrophil infiltration, reduced goblet cells, epithelial barrier damage, and crypt structure disruption in the colon tissue of mice in the irinotecan group. Both representative images and H&E pathological scoring results indicate that the high-dose group at the 0.5 site showed the most significant remission effect.

[0250] 2.3 Study on the effects of acidic and neutral polysaccharides on restoring intestinal barrier function in IGT mice Intestinal barrier markers such as Figure 3 As shown, among intestinal barrier-related markers, only the high-dose group at the 0.5 fraction significantly upregulated ZO-1 mRNA expression, while Occludin and Klf4 mRNA expression were significantly increased only in the low / high-dose groups at the 0.5 fraction. Subsequently, this study examined ZO-1 expression at the protein level, and the results showed that irinotecan significantly downregulated the expression of this intestinal barrier protein. Similar to the PCR results, the recovery effect of the high-dose group at the 0.5 fraction was significantly better than that of the high-dose group at the water fraction.

[0251] 2.4 Characterization of polysaccharides with different molecular weights To date, the laboratory preparation of homogeneous polysaccharides typically involves separating fractional polysaccharides using a DEAE column, followed by further separation using a dextran gel column to obtain homogeneous polysaccharides. Compared to this traditional purification method, dialysis bags offer significant advantages for large-scale production and practical applications: the method operates under mild conditions, purification can be completed in aqueous solution, the process is simple, low-cost, and easy to scale up; moreover, it does not use organic solvents throughout the process, ensuring high safety and better meeting the requirements for large-scale production in food, health products, and pharmaceuticals, thus showing promising application prospects in the industrial purification of polysaccharides. Therefore, this study primarily aims to obtain astragalus polysaccharides of different molecular weight ranges through separation using dialysis bags.

[0252] The inner (0.5-In) and outer (0.5-Out) portions of the dialysis bag corresponded to the high and low molecular weight polysaccharides, respectively. We determined the molecular weight of the two types of polysaccharides using HPGPC. The results are as follows: Figure 4 As shown, 0.5-In mainly contains characteristic peaks of 485 kDa and 52.2 kDa, while 0.5-Out has a characteristic peak of 19.8 kDa, indicating that the two polysaccharides can be effectively distinguished by molecular weight. SEM results show that both refined polysaccharides exhibit a sheet-like, irregular blocky morphology with a relatively smooth surface but localized aggregation, possibly due to the charge effect and intermolecular interactions of uronic acid residues in the acidic polysaccharide at the 0.5 position.

[0253] 2.5 Study on the effects of polysaccharides of different molecular weights on restoring the structural integrity of intestinal tissue like Figure 5As shown, H&E staining and histopathological observation revealed significant inflammatory damage in the colon tissue of irinotecan-treated mice, including neutrophil infiltration, goblet cell loss, epithelial barrier disruption, and crypt structure disorder. Representative pathological images and H&E pathological scores indicate that the low molecular weight high-dose group showed the most significant reduction in pathological damage, with a significantly lower score than the high molecular weight high-dose group. Therefore, the low molecular weight high-dose group was more effective at alleviating intestinal damage in IGT mice compared to the high molecular weight high-dose group.

[0254] 2.6 Study on the effect of polysaccharides of different molecular weights on restoring intestinal barrier function in IGT mice The detection results of intestinal barrier-related markers are as follows: Figure 6 As shown, the expression of Occludin and Klf4 mRNA was significantly increased only in the low molecular weight high-dose group. Overall, the low molecular weight high-dose group exhibited the most significant regulatory effect on intestinal barrier mRNA.

[0255] Example 2 1. Materials and Methods 1.1 Experimental Materials 1.1.1 Experimental Apparatus The experimental instruments were the same as in Example 1, and were additionally equipped with a high-resolution mass spectrometer (Thermo UHPLC-Q Exactive, Thermo Fisher Scientific (China) Co., Ltd.) and a SCIEX Triple Quad 4500 (Shanghai Aibocaisi Analytical Instruments Trading Co., Ltd.).

[0256] 1.1.2 Experimental Reagents The experimental reagents are the same as those in Example 1, additionally including β-GUS kit (JL-T0729, Shanghai Jianglai Biotechnology Co., Ltd.), 3-nitrophenylhydrazine hydrochloride (N21804, Merck (China) Co., Ltd.), EDC hydrochloride (E7750, Merck (China) Co., Ltd.), Shim-pack GIST-HP C18 chromatographic column (2.1×50 mm, 3 μm, Shimadzu Corporation, Japan), metabolite standards (3-indolepropionic acid, 5-hydroxyindoleacetic acid, 5-methoxyindole-3-acetic acid, adipic acid, succinic acid, butyric acid, 3-indoleacetic acid, lactic acid, N-acetyltryptophan, valeric acid, propionic acid, purchased from Shanghai Standard Technology Co., Ltd., Merck (China) Co., Ltd., Chengdu Aifa Biotechnology Co., Ltd., etc.) and antibodies against p38 mitogen-activated protein kinase (p38), phosphorylated p38 mitogen-activated protein kinase (p-p38), extracellular signal-regulated kinase (ERK), phosphorylated extracellular signal-regulated kinase (p-ERK), c-Jun N-terminal kinase (JNK), phosphorylated c-Jun N-terminal kinase (p-JNK), Bcl-2-associated X protein (Bax), B-cell lymphoma / leukemia-2 protein (Bcl-2), etc. (purchased from Cell Signaling Technology, Wuhan Sanying Biotechnology Co., Ltd., Abcam (Shanghai) Trading Co., Ltd., Cell Signaling Technology, Selleck Chemicals).

[0257] 1.1.3. Experimental animals A total of 36 mice were used in this experiment, which were provided by Shanghai Jihui Experimental Animal Feeding Co., Ltd. (Shanghai, China). The animals were housed in a specific pathogen free (SPF) environment in the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine (license number: SCXK (Shanghai) 2022-0004). The environmental conditions were controlled as follows: temperature 22±2°C, relative humidity 40%, 12 h light / dark cycle, and the mice had free access to standard rodent feed and water. All experimental operations strictly followed the guidelines approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (approval number: PZSHUTCM2406170006).

[0258] 1.2. Experimental methods 1.2.1. Determination of β-GUS enzyme activity β-GUS activity in colon contents samples was determined using the microplate method. Approximately 0.1 g of colon contents and extract were homogenized in an ice bath, centrifuged at 15,000 rpm for 10 min at 4 °C, and the supernatant was collected and placed on ice for testing. The microplate reader was preheated and set to 405 nm wavelength. The sample supernatant and relevant reagents from the kit were added to the test tube and control tube, respectively. The reaction was incubated at 37 °C for 30 min, and then the stop reagent was added. After mixing, if precipitation occurred, the sample was centrifuged at room temperature. The supernatant was collected and added to a 96-well plate. The absorbance was measured at 405 nm and ΔA was calculated. Finally, the β-GUS enzyme activity in the sample was calculated according to the kit standard curve and the corresponding formula.

[0259] 1.2.2 Determination of SN-38 content Approximately 50 mg of mouse colon tissue was extracted by homogenizing it in pre-cooled methanol-water solution. After vortexing and mixing, the mixture was centrifuged at 12700 rpm for 15 min. The supernatant was dried under nitrogen and reconstituted with the initial mobile phase. After filtration through a 0.22 μm filter membrane, the sample was detected by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS).

[0260] Chromatographic separation using C 18 The chromatographic column was maintained at 35°C. The mobile phase consisted of 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution, using a gradient elution program at a flow rate of 0.3 mL / min. Mass spectrometry was performed in positive ion electrospray ionization mode with multiple reaction monitoring (MRM) mode for quantitative analysis, and the content of SN-38 in colon tissue was calculated using the internal standard method.

[0261] 1.2.3. Colonic microbial metabolomics analysis Liquid chromatography-mass spectrometry (LC-MS) was used to detect tryptophan derivatives, fatty acids and other acidic substances in mouse feces, with indolepropionic acid-d6 as an internal standard for quantitative analysis.

[0262] For sample pretreatment, take an appropriate amount of mouse fecal sample, add water and homogenize it, add methanol-acetonitrile mixed extractant containing internal standard and vortex mix, sonicate on ice and centrifuge, take the supernatant and add derivatization catalyst, pyridine methanol solution and NPH-3 reagent and mix thoroughly, after derivatization reaction at 40℃, add pre-cooled ultrapure water and vortex, centrifuge again and take the supernatant for testing.

[0263] Prepare a mixed standard solution and add an internal standard of the same concentration. Then, gradually dilute the solution with methanol to obtain a series of standard solutions with different concentration gradients. Perform derivatization according to the sample pretreatment method described above to prepare a working curve.

[0264] The instrument used a C18 column for detection, with gradient elution using an aqueous solution containing 0.1% formic acid and acetonitrile as the mobile phase. The mass spectrometry employed multiple reaction monitoring mode, with corresponding mass spectrometry parameters set. After derivatization with NPH-3, qualitative and quantitative analyses were performed based on the corresponding mass spectrometry detection parameters. Finally, the content of each target substance in the sample was calculated by combining the working curve and the internal standard method.

[0265] 1.2.4 Western blot method Western blot (WB) was used to detect the expression of relevant proteins. Samples were homogenized in ice bath with lysis buffer, centrifuged, and the supernatant was collected. Protein concentration was determined using the BCA method, and the loading amount was standardized. After protein denaturation, SDS-PAGE electrophoresis was performed, followed by wet transfer to a PVDF membrane. After blocking with skim milk at room temperature, primary antibody was added and incubated overnight at 4°C. After washing with TBST, secondary antibody was added and incubated at room temperature. After washing again, chemiluminescence was used for color development. Bands were acquired using an imaging system, and quantification was performed using image analysis software. The grayscale ratio of the target protein to the internal control protein was calculated to represent its relative expression level.

[0266] 1.2.5. Mass spectrometry imaging technology shows the in-situ distribution of SN-38. Frozen sections were dried in a vacuum desiccator at -20°C for half an hour. DESI-MSI was run in resolution mode (in... m / z With a resolution of 20,000 FWHM at 500, it is equipped with a two-dimensional DESI XS spray source and includes a high-performance DESI sprayer. Methanol / water (95:5, v / v DESI-MSI analysis was performed at a flow rate of 5 μL / min. The spray voltage was 3 kV, the nebulized nitrogen pressure was 0.4 MPa, and the spatial resolution was 100 μm. Data were acquired and mined using MassLynx™ software version 4.2 (Waters Corporation, Manchester, UK). Imaging data acquisition setup, processing, and visualization were performed using High Resolution Imaging (HDI) 1.8 software (Waters Corporation, Manchester, UK).

[0267] 2. Experimental Results 2.1 Acidic low molecular weight polysaccharides inhibit β-GUS enzyme activity, thereby reducing SN-38 exposure. The results of the determination of intestinal β-GUS enzyme activity and colon tissue SN-38 exposure are as follows: Figure 7 As shown, 0.5-Out can reduce the accumulation of SN-38 in colonic tissue by inhibiting β-GUS enzyme activity, thereby directly alleviating the stimulation of colonic epithelial cells by SN-38.

[0268] 2.2. Acidic low molecular weight polysaccharides regulate the metabolic profile of intestinal flora The results are as follows Figure 8 As shown, this experiment detected 11 enterobiotic metabolites, of which 5 showed significant metabolic changes after administration of low molecular weight polysaccharides. The results showed that irinotecan treatment significantly increased lactate levels, while significantly decreasing levels of metabolites such as acetic acid, propionic acid, and butyric acid. This suggests that irinotecan may inhibit the utilization of lactate by the gut microbiota, thereby reducing the release of acetic acid, propionic acid, and butyric acid. This situation was significantly alleviated after administration of low molecular weight polysaccharides.

[0269] 2.3. Acidic low molecular weight polysaccharides inhibit the over-activated MAPK signaling pathway in IGT mice and alleviate cell apoptosis in colon tissue. We then characterized the expression of key proteins in the MAPK signaling pathway and its downstream apoptosis proteins, and the results are as follows: Figure 9 As shown, irinotecan treatment significantly upregulated phosphorylation levels in all branches of the MAPK pathway (P38, ERK, JNK), consistent with the trend of enhanced inflammatory response and apoptotic activity. Notably, acidic low-molecular-weight polysaccharides effectively inhibited the phosphorylation of these proteins, reduced apoptosis, and ultimately exerted a protective effect against IGT.

[0270] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for preparing acidic low-molecular-weight polysaccharides from Astragalus membranaceus, characterized in that, The method includes the following steps: (1) Preparation of Astragalus polysaccharide mother liquor; (2) The Astragalus polysaccharide mother liquor was subjected to column chromatography through a cellulose column, and eluted sequentially with water and 0.4~0.6 mol / L sodium chloride solution. The eluted fraction of 0.4~0.6 mol / L sodium chloride solution was collected, concentrated, dialyzed, and dried to obtain Astragalus acidic polysaccharide lyophilized powder; (3) The lyophilized powder of Astragalus acidic polysaccharide is pretreated to obtain Astragalus acidic polysaccharide solution. The Astragalus acidic polysaccharide solution is dialyzed using a dialysis bag with a molecular weight cutoff of 50~200 kDa. The solution outside the dialysis bag is collected, concentrated, and dried to obtain Astragalus acidic low molecular weight polysaccharide.

2. The method according to claim 1, characterized in that, The steps for preparing Astragalus polysaccharide mother liquor include: weighing an appropriate amount of Astragalus polysaccharide powder, adding water, heating and stirring, dissolving with ultrasonic assistance, cooling and centrifuging, taking the supernatant and filtering to obtain the Astragalus polysaccharide mother liquor. Preferably, the water is distilled water; Preferably, the mass / volume (g / mL) ratio of the astragalus polysaccharide powder to the water is 0.01 to 0.2, for example, about 0.1; Preferably, the mass of the astragalus polysaccharide powder is 1-20 g, for example, about 10 g; Preferably, the volume of the water is 20 to 200 mL, for example, about 100 mL; Preferably, the heating and stirring is performed using a water bath. More preferably, the temperature of the water bath is 50~70°C, for example, about 60°C; More preferably, the heating and stirring time is 20 to 40 minutes, for example, about 30 minutes; More preferably, the power of the ultrasound-assisted dissolution is 200~400W, for example, about 300W; More preferably, the time for ultrasound-assisted dissolution is 10-20 minutes, for example, about 15 minutes; Preferably, the cooling is cooling to room temperature; Preferably, the centrifugation speed is 3000~5000 r / min, for example, about 4000 r / min; Preferably, the centrifugation time is 10-20 minutes, for example, about 15 minutes; Preferably, the centrifugation temperature is 1~10°C, for example, about 4°C; More preferably, the filtration is a membrane vacuum filtration; Particularly preferred is that the pore size of the filter membrane is 0.45 μm; Particularly preferred is that the concentration of the Astragalus polysaccharide mother liquor is 50-200 mg / ml, for example, about 100 mg / ml.

3. The method according to claim 1 or 2, characterized in that, The preparation method of the Astragalus polysaccharide powder includes: (i) weighing an appropriate amount of Astragalus slices and extracting them by reflux with a first solvent; (ii) extracting the residue with water, combining the extracts and concentrating them, adding a second solvent while stirring, so that the final concentration of the second solvent in the system reaches a certain concentration; and (iii) after standing overnight, centrifuging to collect the precipitate, redissolving the precipitate in water and drying it to obtain the Astragalus polysaccharide powder. Preferably, in step (i), the first solvent is 80% to 100% ethanol, for example, about 95% ethanol; Preferably, in step (i), the extraction is performed 1 to 3 times, for example, 2 times; Preferably, in step (i), the extraction time for each extraction is 0.5 to 2 hours, for example, about 1 hour; Preferably, in step (i), the volume / mass ratio of the first solvent to the Astragalus membranaceus slices is 5 to 15, for example, about 8 or about 10; Preferably, in step (ii), the volume / mass ratio of the water to the Astragalus membranaceus slices is 4 to 10, for example, about 6; Preferably, in step (ii), the extraction is performed 1 to 3 times, for example, 2 times; Preferably, in step (ii), the extraction time for each extraction is 0.5 to 2 hours, for example, about 1.5 hours; Preferably, in step (ii), the second solvent is ethanol; Preferably, in step (ii), the concentration is 70% to 100%, for example, about 80%; Preferably, in steps (ii) and (iii), the water is distilled water; Preferably, in step (iii), the temperature for overnight standing is 1~10°C, for example, about 4°C; Preferably, in step (iii), the centrifugation speed is 2000~4000 ×g, for example about 3000 ×g; Preferably, in step (iii), the centrifugation time is 5 to 15 minutes, for example, about 10 minutes; Preferably, in step (iii), the drying is freeze-drying; Preferably, the cellulose column is a DEAE Sepharose Fast Flow cellulose column; Preferably, in step (2), the column chromatography steps include: (a) pre-treating the DEAE Sepharose Fast Flow packing material and packing it into a column, equilibrating it, and loading the Astragalus polysaccharide mother liquor onto the column; (b) eluting with water, collecting the eluent, and stopping elution after reaching the endpoint; and (c) eluting with approximately 0.5 mol / L sodium chloride solution, collecting the eluent, stopping elution after reaching the endpoint and collecting the eluent to obtain the eluted portion of approximately 0.5 mol / L sodium chloride solution. Preferably, the water is distilled water; Preferably, the equilibrium is achieved by equilibrating the chromatography column until the pH of the eluent is approximately 7.0, and / or the absorbance of the eluent at a wavelength of 280 nm is ≤0.02; Preferably, the equilibrium flow rate is 10-30 mL / min, for example, about 20 mL / min; Preferably, the sample loading flow rate is 5-15 mL / min, for example, about 10 mL / min; More preferably, the elution flow rate is 10-30 mL / min, for example, about 20 mL / min; More preferably, the endpoint is reached when the absorbance of the eluent at 490 nm is measured using the phenol-sulfuric acid method, and the absorbance value is below 0.03 for three consecutive tubes. More preferably, the volume of the eluent is 1 to 20 L, for example, about 10 L.

4. The method according to claim 1, characterized in that, In step (2), the dialysis is performed using water; Preferably, the water is distilled water; Preferably, in step (2), the dialysis time is 36-60 hours, for example, about 48 hours; Preferably, in step (2), the distilled water is changed every 6 hours during the dialysis process; Preferably, the drying is freeze-drying.

5. The method according to claim 1, characterized in that, In step (3), the pretreatment step includes: taking an appropriate amount of the freeze-dried Astragalus acidic polysaccharide powder, adding water to dissolve it by vortexing, cooling and centrifuging, taking the supernatant to obtain the Astragalus acidic polysaccharide solution; Preferably, the water is distilled water; Preferably, the cooling is cooling to room temperature; Preferably, the centrifugation speed is 3000~5000 r / min, for example, about 4000 r / min; Preferably, the centrifugation time is 10-20 minutes, for example, about 15 minutes; Preferably, the centrifugation temperature is 1~10°C, for example, about 4°C; More preferably, in step (3), the molecular weight cutoff of the dialysis bag is about 100 kDa; More preferably, in step (3), the water is distilled water; More preferably, in step (3), the dialysis temperature is 1~10°C, for example, about 4°C; More preferably, in step (3), the dialysis time is 36 to 60 hours, for example, about 48 hours; More preferably, in step (3), the distilled water is replaced every 6 hours during the dialysis process; More preferably, in step (3), the drying is freeze drying.

6. An acidic low-molecular-weight polysaccharide of Astragalus membranaceus prepared by any one of claims 1 to 5; Preferably, the molecular weight of the characteristic peak of the Astragalus acidic low molecular weight polysaccharide is 19.8 ± 2.0 kDa; More preferably, the molecular weight of the characteristic peak of the Astragalus acidic low molecular weight polysaccharide is 19.8 ± 1.0 kDa; Preferably, the characteristic peak of the astragalus acidic low molecular weight polysaccharide has a molecular weight of about 19.8 kDa.

7. A pharmaceutical preparation of Astragalus acidic low molecular weight polysaccharide, characterized in that, The pharmaceutical preparation comprises the Astragalus acidic low molecular weight polysaccharide of claim 6, and one or more pharmaceutically acceptable excipients; Preferably, the excipients are selected from one or more of the following: diluents, wetting agents, binders, disintegrants, encapsulating agents, flavoring agents, sustained-release agents, flow aids, lubricants, dispersants, plasticizers, light-blocking agents, and antioxidants; More preferably, the dosage form of the pharmaceutical preparation is tablets, pills, capsules, powders, injections, films, lozenges, granules, or oral liquids.

8. Use of the Astragalus acidic low molecular weight polysaccharide of claim 6 or the Astragalus acidic low molecular weight polysaccharide pharmaceutical preparation of claim 7 in the preparation of a medicament for the prevention and / or treatment of intestinal injury.

9. The use according to claim 8, characterized in that, The intestinal injury mentioned is drug-induced intestinal injury; Preferably, the drug is an antitumor chemotherapy drug; More preferably, the antitumor chemotherapy drug is irinotecan.

10. The use according to claim 8, characterized in that, The effects of the Astragalus acidic low molecular weight polysaccharide or the Astragalus acidic low molecular weight polysaccharide pharmaceutical preparation in preventing and / or treating intestinal injury are achieved through one or more of the following mechanisms: improving general condition abnormalities, increasing colon length, improving colonic histopathological abnormalities, improving colonic mucus secretion function, restoring intestinal barrier function, promoting colonic cell proliferation, inhibiting β-GUS enzyme activity, regulating intestinal flora metabolism, inhibiting MAPK signaling pathway hyperphosphorylation, and inhibiting colonic cell apoptosis. Preferably, the general condition abnormality includes weight loss and / or an increased disease activity index; Preferably, the histopathological abnormalities of the colon include neutrophil infiltration, goblet cell loss, epithelial barrier damage, and / or crypt structure disorder. Preferably, the improvement in colonic tissue mucus secretion function is characterized by an increase in the number of positive goblet cells; Preferably, the restoration of intestinal barrier function includes increased ZO-1 mRNA levels, increased Muc2 mRNA levels, increased ZO-1 protein levels, increased Occludin protein levels, increased Muc2 protein levels, decreased plasma diamine oxidase levels, and / or decreased plasma lipopolysaccharide levels. Preferably, the regulation of gut microbiota metabolism includes increasing the levels of acetic acid, propionic acid, and butyric acid in gut microbiota metabolites, and decreasing the levels of adipic acid and / or lactic acid. Preferably, the hyperphosphorylation of the MAPK signaling pathway includes elevated phosphorylation levels of P38 protein, ERK protein, and / or JNK protein; Preferably, the inhibition of colon cell apoptosis is characterized by a decrease in Bax protein levels and / or an increase in Bcl-2 protein levels.

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