Process for the preparation of arabino-galactans and derivatives thereof from larch and their use

Abstract

The application belongs to the field of medicine and relates to a preparation method and application of arabinogalactan in larch; wherein the arabinogalactan derivative in larch is sulfated arabinogalactan, and the sulfated arabinogalactan can be applied in the preparation of a medicine for treating inflammatory bowel disease or colon cancer; the sulfated arabinogalactan mainly regulates NCOA4-mediated ferritin autophagy by activating the AMPK-mTOR signal pathway and increases the relative abundance of Limosilactobacillus reuteri strain HDB1243 and Lactobacillus johnsonii strain 1696 to antagonize colon cancer, thereby providing a new means for the treatment of colon cancer.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the preparation method and application of arabinogalactan and its derivatives from larch. Background Technology

[0002] Larch, belonging to the genus *Larix* of the family Pinaceae, is a major tree species in the high-altitude coniferous forests of Northeast my country. Larch has a long history of medicinal use in my country, possessing properties such as promoting blood circulation and relieving pain. Currently, it is mainly used for wood product development, inevitably leaving behind a large amount of larch waste. The content of flavonoids in larch heartwood is much higher than in spruce, making it one of the main plant sources of dihydroquercetin. Arabinogalactan (AG) is a byproduct of dihydroquercetin extraction, and research on AG has further developed and utilized larch. The abundant larch resources in Northeast China provide ample raw materials for AG.

[0003] Aggregates (AG) possess excellent biological activities. Sinha et al. extracted AG from senna leaves, which exhibited good iron-reducing antioxidant capacity and DPPH free radical scavenging ability, indicating that AG has good in vitro antioxidant activity. AG can play a therapeutic role in osteoporosis caused by hyperglycemia by inhibiting α-glucosidase activity, improving glucose tolerance, and reducing the accumulation of advanced glycation end products (AGEs). Furthermore, AG can enhance damage to macrophages and inhibit the activity of pro-inflammatory factors. De et al. extracted AG from stevia leaves and found that the crude extract, alkaline extract, and homogeneous extract of AG all had in vitro anti-herpes simplex virus type 1 (HSV-1) activity. Wang Peipei et al. isolated AG from Panax notoginseng and showed through anti-angiogenic experiments that AG could reduce the migration activity and tube-forming ability of endothelial cells on the matrix, but had no effect on endothelial cell growth; further studies found that AG can exert its anti-angiogenic effect through the BMP2 signaling pathway. AG has a promising future in the medical field due to its various biological activities, including anti-oxidation, anti-inflammation, anti-angiogenesis, anti-diabetic, and antiviral properties.

[0004] To enhance the bioactivity of herbal polysaccharides (AG), chemical modification methods are employed to alter their chemical structure and conformation. Currently, the main modification methods for herbal polysaccharides include sulfation, phosphorylation, carboxymethylation, selenization, methylation, and acetylation. Changing their structure can not only improve biological properties but sometimes also generate new biological activities. Different modification methods produce different derivatives (DS) and have varying effects on activity. Therefore, to achieve comprehensive utilization of larch, it is necessary to extract, isolate, and purify AG from larch and further study the bioactivity of AG and its derivatives. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing arabinogalactan and its derivatives from larch and its application, so as to realize the comprehensive utilization of larch resources and provide a new means for the treatment of colon cancer.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing arabinogalactan derivatives from larch, wherein the method uses arabinogalactan from larch as a raw material and modifies it by sulfation; wherein the method for preparing arabinogalactan from larch includes the following steps: (1) Add larch root powder to a beaker, add anhydrous ethanol for defatting, filter and air dry, dry the powder and extract it with distilled water at 60-80℃ with ultrasonic assistance, combine the supernatants and concentrate; add anhydrous ethanol to the concentrated solution until the ethanol concentration reaches 90%, let stand overnight, collect the precipitate by centrifugation; redissolve the precipitate in distilled water, add Sevage reagent to remove protein, and obtain crude polysaccharide by dialysis and freeze drying; (2) Weigh the crude polysaccharide, add distilled water to dissolve it completely, add anhydrous ethanol to the solution until the alcohol concentration of the solution reaches 40%, let it stand overnight, centrifuge to obtain precipitate AG-40; (3) Weigh the AG-40 sample, dilute it with distilled water, perform DEAE cellulose column chromatography, and elute with NaCl solution to obtain AG-40-I. Concentrate, dialyze and freeze-dry the obtained components. (4) Weigh the AG-40-I sample, dilute it with distilled water, perform dextran gel column chromatography, and elute with NaCl solution to obtain the final component AG-40-I-II. The AG-40-I-II is a homogeneous polysaccharide with a molecular weight of 16.8 kDa, mainly composed of galactose, arabinose and glucose in a molar ratio of 12.5: 1: 0.05. It is a neutral polysaccharide, and is an AG-II type polysaccharide with →3)-β-Gal-(1→3 and →4)-β-Glc-(1→4 as the main chain and β-Araf-(1→) as the side chain. The sulfation modification method is the chlorosulfonic acid-pyridine method, which includes the following steps: (1) Add the AG-40-I-II sample to DMF and stir at room temperature until it is completely dissolved; (2) Measure anhydrous pyridine, cool it in an ice-water bath, and add chlorosulfonic acid dropwise to obtain a light yellow esterification reagent; (3) Add the polysaccharide DMF solution to the esterification reagent, react at 50 °C for 1 h, add 2.5 mol / L NaOH solution to neutralize, and adjust the pH to 7.0; add anhydrous ethanol to precipitate the polysaccharide, centrifuge to collect the precipitate, dialyze and freeze dry to obtain the crude sulfated arabinogalactan; (4) The obtained sulfated arabinogalactan crude product was purified by Superose 12 dextran gel column and the component with the highest content was collected. The purified sulfated arabinogalactan is a homogeneous polysaccharide with a molecular weight of 17.9 kDa, mainly composed of galactose, arabinose and glucose in a molar ratio of 6.7:1:0.3.

[0007] This invention utilizes arabinogalactan and its derivatives from larch prepared using the aforementioned method to intervene in DSS (dextran sulfate sodium salt)-induced IBD mice. This significantly alleviates IBD and reduces intestinal damage caused by DSS, with SAG showing superior therapeutic effects compared to AG. Administration of AG and SAG to IBD mice reduces LPS concentration, decreases MPO secretion in colonic tissue, and lowers the level of inflammation in the colonic tissue. Furthermore, after AG and SAG treatment, goblet cell damage in the mouse colonic tissue is reduced, mucus layer thickness increases, and mucin secretion is enhanced, which is beneficial for protecting colonic tissue. Further studies show that AG and SAG can reduce the expression of the TLR4 / MyD88 / NF-κB signaling pathway, inhibit the expression of NLRP3 inflammasome-related proteins, and promote PPARγ expression, thereby effectively inhibiting inflammation. Moreover, AG and SAG can increase the metabolism of SCFAs, antagonize DSS-induced intestinal inflammation, and exert therapeutic effects on acute colitis. Therefore, the arabinogalactan derivative (SAG) from larch prepared by the above method can be used in the preparation of drugs for treating inflammatory bowel disease; specifically, in the preparation of drugs for treating acute colitis caused by DSS.

[0008] This invention also analyzed the effects of AG and SAG on the gut microbiota of mice. At the phylum level, intervention with AG and SAG increased the relative abundance of Firmicutes and Bacteroidetes. At the genus level, intervention with AG and SAG increased the relative abundance of Allobaculum and Lactobacillus. Furthermore, this invention isolated two dominant strains from the feces of mice with DSS-induced acute colitis treated with SAG: Limosilactobacillus reuteri strain HDB1243 and Lactobacillus johnsonii strain 1696. It was determined that SAG can promote the proliferation of Limosilactobacillus reuteri strain HDB1243 and Lactobacillus johnsonii strain 1696. Therefore, the arabinogalactan derivative (SAG) from larch prepared using the above method can be used in the preparation of drugs to improve intestinal flora imbalance caused by inflammatory bowel disease or colon cancer; it can also be used in the preparation of drugs that promote the proliferation of Limosilactobacillus reuteri strain HDB1243 and Lactobacillus johnsonii strain 1696 in the intestinal flora.

[0009] This invention also established a DSS / AOM co-induced CRC model and used SAG, Limosilactobacillus reuteri strain HDB1243, and Lactobacillus johnsonii strain 1696 for long-term intervention to explore their effects on colorectal cancer. The results showed that long-term intervention with these two bacteria and SAG in mice with DSS / AOM co-induced colorectal cancer could reduce damage in the colon and promote tumor cell apoptosis. Furthermore, they activated intracellular ferritin autophagy by regulating the AMPK-mTOR signaling pathway, further increasing intracellular free Fe. 2+The concentration of these strains inhibited the antioxidant activity of GSH and GPx4, increased ROS and lipid peroxidation levels, and induced ferroptosis, thereby killing tumor cells and exerting an antagonistic effect against colon cancer in mice. Importantly, the antagonistic mechanism of *Limosilactobacillus reuteri* strain HDB1243 and *Lactobacillus johnsonii* strain 1696 against colon cancer was consistent with that of *SAG*. Therefore, *Limosilactobacillus reuteri* strain HDB1243 and *Lactobacillus johnsonii* strain 1696 should be key gut bacteria in antagonizing colon cancer. Therefore, the arabinogalactan derivative (SAG) from larch prepared by the above method can further regulate NCOA4-mediated ferritin autophagy and increase the relative abundance of Limosilactobacillus reuteri strain HDB1243 and Lactobacillus johnsonii strain1696 to antagonize DSS / AOM-induced colon cancer by activating the AMPK-mTOR signaling pathway, and can be used in the preparation of drugs for the treatment of colon cancer.

[0010] Advantages and beneficial effects of the present invention: (1) The AG and SAG prepared in this invention can improve intestinal damage caused by DSS, inhibit the TLR4 / MyD88 / NF-κB and NLRP3 inflammasome signaling pathways and promote the expression of PPARγ, inhibit the occurrence of intestinal inflammation, and the metabolism of SCFAs promoted by AG and SAG can also promote the repair of colon tissue to a certain extent.

[0011] (2) This invention uses arabinogalactan from larch as raw material and obtains sulfated arabinogalactan (SAG) through sulfation modification. SAG has a significantly better therapeutic effect on inflammatory bowel disease than AG, providing a new means for the treatment of inflammatory bowel disease.

[0012] (3) The SAG prepared in this invention further regulates NCOA4-mediated ferritin autophagy by activating the AMPK-mTOR signaling pathway, thereby leading to ferroptosis and playing an antagonistic role against colon cancer.

[0013] (4) This invention is the first to discover the antagonistic effect of Limosilactobacillus reuteri strain HDB1243 and Lactobacillus johnsonii strain 1696 on colon cancer, and the prepared SAG has the same antagonistic mechanism as Limosilactobacillus reuteri strain HDB1243 and Lactobacillus johnsonii strain 1696 on colon cancer. Therefore, it is determined that SAG can exert its anti-colon cancer effect by increasing the relative abundance of Limosilactobacillus reuteristrain HDB1243 and Lactobacillus johnsonii strain 1696.

[0014] (5) The SAG prepared in this invention can regulate the intestinal microbial disorder caused by DSS or DSS / AOM combination, promote the increase of the relative abundance of beneficial bacteria, and gradually restore it to the normal homeostatic level. More importantly, the regulatory trends of bacteria 1, bacteria 2 and SAG on intestinal microorganisms show consistency. Therefore, it can be preliminarily determined that SAG regulates the intestinal flora by increasing the relative abundance of Limosilactobacillus reuteri strain HDB1243 and Lactobacillus johnsonii strain1696. Attached Figure Description

[0015] Figure 1 This is a flowchart of polysaccharide methylation. Figure 2 The study aimed to isolate and purify AG and its protective effect against LPS-induced inflammatory damage to IEC-6 cells. Specifically, the study included: A. Cytotoxicity of crude polysaccharide to IEC-6 cells; B. Protection of LPS-induced IEC-6 cell inflammation by the polysaccharide component (40 μg / mL); C. Protection of LPS-induced IEC-6 cell inflammation by the polysaccharide component (80 μg / mL); and D. Separation of AG-40-Ⅰ using Superose12 dextran gel column chromatography. Figure 3 For the molecular weight and monosaccharide composition analysis of AG-40-I-II; where A. HPLC chromatogram of AG-40-I-II; B. HPLC chromatogram of the standard; C. HPLC chromatogram of the crude polysaccharide; D. HPLC chromatogram of AG-40-I-II; Figure 4 Infrared and ultraviolet spectral scans of AG-40-I-II; where A. Infrared spectrum of AG-40-I-II; B. Ultraviolet spectrum of AG-40-I-II; Figure 5 The results are GC-MS analysis of AG-40-I-II; where A. total ion chromatogram; B and C. mass spectrometry information at different time points. Figure 6 Schematic diagrams of the structures of AG-40-I-II and SAG; Figure 7 Separation of SAG using Superose 12 dextran gel column; Figure 8 The following are HPLC chromatograms for the determination of SAG monosaccharide composition; where A is the HPLC chromatogram of the standard; and B is the HPLC chromatogram of SAG. Figure 9 The DAI index and body weight of mice in different groups; where A. DAI index of mice in each group; B. Daily body weight of mice in each group; Figure 10 The values ​​represent the cytokine expression levels in different groups of mice; among them, A. IL-1β expression level; B. TNF-α expression level; C. IL-10 expression level; D. LPS expression level; E. MPO expression level; Figure 11 H&E staining of colon tissue from different groups of mice; where A. H&E staining of different groups of mice; B. Score of colon tissue damage in different groups of mice; Figure 12 The study evaluated the mucus layer thickness in the mouse colon, including: A. Alcian blue staining of the mouse colon; B. Immunofluorescence staining of the mouse colon (Muc-2); and C. Quantification of immunofluorescence intensity. Figure 13 Immunohistochemical staining of TJ protein in mouse colon; A. Immunohistochemical staining of mouse colon (ZO-1); B. Immunohistochemical staining of mouse colon (Occludin); Figure 14 To investigate the regulation of the TLR4 / MyD88 / NF-κB signaling pathway by AG and SAG; specifically, A. Western blot analysis of the TLR4 / MyD88 / NF-κB signaling pathway; BE. Protein expression levels of TLR4, MyD88, p-IκB-α / IκB-α, and p-NF-κB / NF-κB. Figure 15 To investigate the regulation of the NLRP3 inflammasome by AG and SAG; specifically, A. Western blot analysis of the NLRP3 inflammasome; BE. Protein expression levels of NLRP3, ASC, Caspase1, and IL-1β. Figure 16 To investigate the regulation of PPARγ by AG and SAG; where A. Western blot analysis of PPARγ protein; B. PPARγ protein expression level; Figure 17 The effects of AG and SAG on the relative abundance of gut microbiota (phylum and genus levels); where A. relative abundance of gut microbiota at the phylum level; B. relative abundance of Firmicutes; C. relative abundance of Bacteroidetes; D. relative abundance of gut microbiota at the genus level; E. relative abundance of Allobaculum; F. relative abundance of Lactobacillus. Figure 18 The effects of AG and SAG on gut microbiota β diversity were investigated, including: A. distance matrix and PCoA analysis; B. hierarchical cluster analysis; and C. intergroup difference analysis. Figure 19 The colons of mice in different groups in Example 5; wherein, A. colonic status of mice in different groups; B. colonic length of mice in different groups; Figure 20 The study aimed to inhibit inflammation in colon cancer tissue by inducing the growth of SAG and intestinal bacteria; among which, A. TNF-α expression level; B. IL-1β expression level; C. H&E staining; Figure 21 To investigate how SAG and gut microbiota promote apoptosis in colon tumor tissues; the methods used included: A. TUNEL staining; B. TUNEL quantification of colon tissues from different groups; C. Western blot analysis of Bcl-2 and Bax proteins; and D. Bax / Bcl-2 protein expression levels. Figure 22 To increase lipid peroxidation levels in SAG and gut microbiota; the methods used included: A. ROS staining; B. Immunohistochemical staining; C. ROS expression level; D. MDA expression level; E. GSH expression level; and F. 4-HNE expression level. Figure 23 The ultrastructure of colon tumor tissue; Figure 24 The immunohistochemical staining analysis for Example 5 includes: A. Immunohistochemical staining; B. GPx4 protein expression level; and C. ACSL4 protein expression level. Figure 25 Western blot analysis of ferroptosis protein expression was performed in Example 5; wherein, A. Western blot analysis; BD. Expression levels of ACSL4, GPx4, SLC7A11, FTH, and FTL proteins; Figure 26 The effects of SAG and gut microbiota on gut microbiota species; including A. Venn diagram; B. Species composition heatmap; Figure 27The effects of SAG and gut bacteria on the relative abundance of gut microbiota; where A. phylum-level relative abundance; B. Bacteroidetes relative abundance; C. Firmicutes relative abundance; D. genus-level relative abundance; E. Lactobacillus relative abundance; F. Bacteroides relative abundance; Figure 28 The effects of SAG and gut microbiota on β diversity were investigated, including: A. distance matrix and PCoA analysis; B. hierarchical cluster analysis; and C. inter-group difference analysis. Figure 29 The effects of SAG and gut microbiota on species differences in gut microbiota were investigated; AC. phylum-level differences between groups; DF. genus-level differences between groups; and G. LEfSe analysis. Detailed Implementation

[0016] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments of the present invention are not limited thereto. For process parameters not specifically specified, conventional techniques can be referred to. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0017] Example 1: Extraction, separation, and structural identification of arabinogalactan

[0018] 1. Materials and Methods

[0019] 1.1 Experimental Materials The larch root was provided by Changbai Mountain Forest Farm in Jilin Province and was jointly identified by Professor Liu Wencong and Professor Li Wei of the College of Traditional Chinese Medicine of Jilin Agricultural University as Changbai larch (Larix gmelinii (Rupr.) Kuzen.) of the genus Larix in the family Pinaceae.

[0020] 1.2 Main Reagents Arabicose, galactose, glucose, rhamnose, mannose, galacturonic acid, and dextran standards were purchased from Chengdu Pusi Biotechnology Co., Ltd.; 1-phenyl-3-methyl-5-pyrazolone (PMP) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; phenol and concentrated sulfuric acid were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd.; ethanol, disodium hydrogen phosphate, and sodium dihydrogen phosphate were purchased from Tianjin Huadong Reagent Factory; acetonitrile (chromatographic grade) and methanol (chromatographic grade) were purchased from Thermo Fisher Scientific, USA; and the CCK8 reagent kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0021] 1.3 Extraction and purification of arabinogalactan 1.3.1 Extraction of crude polysaccharides from larch

[0022] 500 g of larch root powder was added to a beaker, and anhydrous ethanol (1:5, w / v) was added for defatting for 12 h. After filtering and air drying, the powder was dried and extracted with distilled water (1:20, w / v) at 70℃ with ultrasonic assistance for 1 h, for a total of 3 extractions. The supernatants were combined and concentrated to about 200 mL. Anhydrous ethanol was added to the concentrated solution until the ethanol concentration reached 90%, and the mixture was allowed to stand overnight at 4℃. The precipitate was collected by centrifugation (3500 rpm, 10 min). The precipitate was redissolved in distilled water, and Sevage reagent (CHCl3 / BuOH = 4:1, v / v) was added to remove proteins. Crude polysaccharide was obtained by dialysis and lyophilization.

[0023] 1.3.2 Fractionation and precipitation of crude polysaccharides Weigh 50.0 g of crude polysaccharide, add twice the amount of distilled water to dissolve it completely, add anhydrous ethanol to the solution until the ethanol concentration reaches 30%, incubate overnight at 4 ℃, and centrifuge (3500 rpm, 10 min). Using the same method, precipitates were obtained at ethanol concentrations of 40%, 50%, 60%, 70%, and 80%, and named AG-30, AG-40, AG-50, AG-60, AG-70, and AG-80, respectively.

[0024] 1.3.3 Protective effect of crude polysaccharide against LPS-induced inflammation in IEC-6 cells

[0025] IEC-6 cells were cultured in DMEM medium containing 10% fetal bovine serum in a constant temperature incubator at 37 ℃ and 5% CO2.

[0026] CCK-8 assay: In a 96-well cell culture plate, 100 μL of cell suspension was added to each well. When cells reached the logarithmic growth phase, the drug pretreatment group was treated with the test compound. After 24 h of culture, the model group and drug pretreatment group were treated with a certain concentration of LPS, the drug treatment group was treated with the test drug, and the control group was treated with an equal volume of cell culture medium. After 24 h of combined treatment, 10% CCK-8 was added to each well, and the cells were cultured for another 1.5 h. The absorbance at 450 nm was then measured using a microplate reader. The ability of the test compound to resist the decrease in IEC-6 cell viability caused by exogenous stimulation was calculated based on the experimental results.

[0027] 1.3.4 DEAE-52 Cellulose Ion Exchange Chromatography

[0028] Accurately weigh 1.00 g of AG-40 sample and dilute to a 1.00 mL volumetric flask with distilled water. Pack a DEAE-52 cellulose column (2.8 cm × 40 cm) using the wet packing method, and equilibrate the three column volumes with distilled water before separation. Elute with NaCl solution (0–0.3 mol / L) at a flow rate of 1 mL / min (10 mL / tube). Analyze each fraction using the phenol-sulfuric acid method, measuring absorbance at 490 nm and plotting elution curves. Three components were obtained, named AG-40-I, AG-40-II, and AG-40-III. Concentrate, dialyze, and lyophilize all obtained fractions.

[0029] 1.3.5 Superose 12-dextran gel column chromatography

[0030] Accurately weigh 20.00 mg of AG-40-I sample and dilute to a 1.00 mL volumetric flask with distilled water. Pack a Superose 12 dextran gel column using a wet packing method, and equilibrate the three column volumes with 0.15 mol / L NaCl solution before separation. Elute with 0.15 mol / L NaCl solution at a flow rate of 0.15 mL / min (3 mL / tube). Detect each fraction using the phenol-sulfuric acid method, measuring absorbance at 490 nm and plotting elution curves to obtain the final fraction AG-40-I-II. AG-40-I-II is then dialyzed and lyophilized for later use.

[0031] 1.4 Structural Characterization of AG-40-I-II 1.4.1 Determination of molecular weight and uniformity

[0032] The molecular weight (Mw) of AG-40-I-II was determined by high-performance gel permeation chromatography (HPGPC), using dextran as a standard. HPGPC analysis was performed using a high-performance liquid chromatography (HPLC) system (Waters e2695, American Waters) on a TSK-Gel G3000 SWXL column (7.8 mm × 300 mm) equipped with a differential refractive index detector (RID-20A, Shimazu, Japan). The column was eluted with NaCl (0.2 mol / L) solution at a flow rate of 0.6 mL / min at 35 °C. The injection volume was 20 μL. Before injection, AG-40-I-II was filtered through a 0.22 μm water membrane.

[0033] 1.4.2 Monosaccharide Composition Analysis Solution preparation: 2 mol / L H2SO4: Add 1.087 mL of concentrated sulfuric acid to 8.913 mL of distilled water; 2 mol / L NaOH: Dissolve 2 g of NaOH in distilled water and bring the volume to 25 mL in a volumetric flask; 0.5 mol / L PMP: Dissolve 0.87 g of PMP in methanol and bring the volume to 10 mL in a volumetric flask; 0.3 mol / L NaOH: Dissolve 1.2 g of NaOH in 100 mL of distilled water; 0.3 mol / L hydrochloric acid: Add 0.25 mL of 37% hydrochloric acid to distilled water and bring the volume to 100 mL.

[0034] Preparation of standard solution: Take appropriate amounts of mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose standards, add distilled water to make up to 1 mg / mL standard solution.

[0035] Phosphate buffer preparation: Dissolve 8.954 g Na2HPO4·12H2O and 2.496 g NaH2PO4·2H2O in 410 mL Wahaha mineral water and filter through a 0.22 μm filter membrane for later use.

[0036] Polysaccharide sample hydrolysis: 20.00 mg of accurate polysaccharide sample was added to 2 mL of 2 mol / L H2SO4 solution, and the mixture was kept under N2 protection and hydrolyzed at 100℃ for 6 h. After cooling to room temperature, 2 mol / L NaOH solution was added dropwise to adjust the pH to 7.0.

[0037] PMP standard derivatization: Take 0.5 mL of the standard solution, add 0.5 mL of PMP solution and 0.2 mol / L NaOH solution, incubate in a 70 ℃ water bath for 30 min, cool to room temperature, add 0.2 mol / L hydrochloric acid to adjust the pH to neutral, and add 1 mL of distilled water to stop the reaction. Add 1 mL of chloroform solution, shake to mix, remove the organic layer, repeat the operation 3 times, and filter the aqueous layer through a 0.22 μm filter membrane for later use.

[0038] PMP derivatization of polysaccharide samples: The derivatization method is the same as that for standards.

[0039] HPLC detection conditions: Detector: Waters 2689 UV Detector, Column: COSMOSIL C18 column (4.6×250 mm, 5 μm), Mobile phase: phosphate buffer: acetonitrile = 87:13, Column temperature: 30 ℃, Detection wavelength: 245 nm, Flow rate: 1 mL / min, Detection time: 40 min.

[0040] 1.4.3 Infrared Spectroscopic Scanning

[0041] The functional group characteristics of polysaccharides were analyzed by infrared spectroscopy. The polysaccharide samples were placed on a Fourier transform infrared spectrometer for infrared scanning. The scanning range was 4000-450 cm⁻¹. -1 .

[0042] 1.4.4 Ultraviolet Spectroscopy Scan

[0043] Ultraviolet spectral scanning was performed using an ultraviolet spectrometer in the 200-900 nm scanning mode.

[0044] 1.4.5 Polysaccharide Methylation Analysis

[0045] Methylation of polysaccharides is widely used in the analysis of polysaccharide structures. Methylation can be used to deduce the linkage patterns of glycosidic bonds in polysaccharides; the specific reaction process is as follows: Figure 1 As shown.

[0046] Preparation of NaOH-DMSO suspension: Grind 100 mg of NaOH into powder, add 10 mL of DMSO, and sonicate to make it fully mixed.

[0047] Sample solution preparation: Take 10.00 mg of polysaccharide sample, add 10 mL of DMSO solution, and sonicate to make it fully mixed.

[0048] Methylation reaction: Add NaOH-DMSO suspension to polysaccharide sample and purge with N2 for protection. After thorough mixing, add 3 mL of iodomethane. After reacting for 1 h, add 1 mL of distilled water to terminate the reaction. Extract with chloroform 3 times, and then wash with distilled water chloroform 3 times for later use.

[0049] Infrared spectroscopy detection: The methylated sample was detected using FT-IR, and the results were observed at 3400 cm⁻¹. -1 The absence of an absorption peak indicates complete methylation.

[0050] Acid hydrolysis of methylated polysaccharides: 3 mL of formic acid was added to the methylated polysaccharide sample, and the mixture was kept under N2 protection. Hydrolysis was carried out at 100 °C for 4 h. Anhydrous ethanol was then added and the mixture was repeatedly evaporated to dryness to remove all formic acid. Then, 3 mL of 2 mol / L TFA was added, and the mixture was kept under N2 protection. Hydrolysis was carried out at 100 °C for 6 h. After hydrolysis, anhydrous ethanol was added and the mixture was repeatedly evaporated to dryness to remove all TFA.

[0051] Reduction: Take the evaporated polysaccharide sample, add 1 mL of 30 mg / mL NaBH4 solution, stir at room temperature for 10 h, and after the reaction is complete, add methanol and evaporate repeatedly to remove NaBH4.

[0052] Acetylation: Take the evaporated polysaccharide sample and add 0.5 mL each of anhydrous acetic anhydride and anhydrous pyridine. Under N2 protection, react at 100 °C for 2 h, then repeatedly add methanol to evaporate to dryness to remove all acetic anhydride.

[0053] Sample testing: Dissolve the polysaccharide sample in dichloromethane, filter through a 0.22 μm filter membrane, and set aside.

[0054] 1.4.6 Nuclear Magnetic Resonance Analysis (NMR)

[0055] Weigh 20.00 mg of polysaccharide, dissolve it in 0.5 mL of D2O, and lyophilize. Add another 0.5 mL of D2O to dissolve, repeating this process three times. Then proceed with... 1 H-NMR, 13 C-NMR, HSQC, HMBC and 1 H- 1 Nuclear magnetic resonance spectroscopy detection of HCOSY.

[0056] 2. Experimental Results 2.1 Separation and purification of AG IEC-6 cells were treated with crude polysaccharide. Cell viability was assessed using a CCK8 assay kit, revealing that the crude polysaccharide was non-toxic to IEC-6 cells at concentrations ranging from 1.25 to 100 μg / mL. Notably, the highest cell viability was observed at concentrations of 40 and 80 μg / mL. Figure 2 A). Therefore, this invention selected 40 and 80 μg / mL for subsequent cell experiments.

[0057] This invention, after ethanol fractionation and precipitation, yielded six polysaccharide fractions (AG30, AG40, AG50, AG60, AG70, and AG80) comprising AG-30 (7.15%), AG-40 (65.06%), AG-50 (23.04%), AG-60 (3.26%), AG-70 (1.00%), and AG-80 (0.49%). These were then used to intervene in an LPS-induced IEC-6 cell inflammation model. CCK8 assay results showed that the AG-40 fraction, at concentrations of 40 and 80 μg / mL, reduced LPS-induced cell death in IEC-6 cells. Figure 2 (BC). Furthermore, based on the content of each component, this invention ultimately selected the AG-40 component for subsequent separation experiments.

[0058] After separation using a DEAE-52 cellulose column, three components were detected in AG-40. The two components with higher content (AG-40-I and AG-40-II) were collected. Analysis of the intervention results of the two polysaccharide components on LPS-induced IEC-6 cell inflammation showed that AG-40-I had stronger protective ability against IEC-6 cells at 40 and 80 μg / mL than AG-40-II. Therefore, AG-40-I was further separated. Finally, homogeneous components AG-40-I and AG-40-II were obtained using a Superose 12 dextran gel column. Figure 2 D).

[0059] 2.3 Structural Characterization of AG-40-I-II 2.3.1 Molecular weight and uniformity The molecular weight and homogeneity of AG-40-I-II were determined by HPGPC, confirming that AG-40-I-II is a homogeneous polysaccharide with a molecular weight of 16.8 kDa. Figure 3 A).

[0060] 2.3.2 Monosaccharide Composition The HPLC chromatographic results of the crude polysaccharide and AG-40-I-II monosaccharide composition analysis are as follows: Figure 3 As shown in BD. The above results indicate that the crude polysaccharide is mainly composed of glucose, galactose, and arabinose. AG-40-I-II is mainly composed of galactose, arabinose, and glucose, with a molar ratio of 12.5:1:0.05.

[0061] 2.3.3 Infrared and ultraviolet spectra From FT-IR analysis results ( Figure 4 A) From this perspective, 3350 cm −1 The unique broadband at this location is due to the vibration of the -OH group. 2910 cm⁻¹ −1 The weak band at this point is caused by the stretching vibration of -CH. 1650 cm -1 The weak peak at 1450 cm⁻¹ is caused by the asymmetric stretching vibration peak of C=O. −1 The strong absorption peak nearby indicates the -CH vibration. Meanwhile, at 1300 cm⁻¹... -1 Up to 1000 cm -1 The absorption peak is the stretching vibration peak of CO. 1100 cm⁻¹ -1 The weak peak at 1050 cm⁻¹ belongs to the COC stretching vibration peak. Meanwhile, the peak at 1050 cm⁻¹... -1 The absorption peak at 1744 cm⁻¹ confirms the presence of pyranose. -1 The absence of an absorption peak indicates the absence of glucuronic acid. These results also confirm that AG-40-I-II is a neutral polysaccharide.

[0062] In the ultraviolet spectrum ( Figure 4 B) The absence of nucleic acid and protein absorption peaks at 260 nm and 280 nm proves that AG-40-I-II did not bind to other substances.

[0063] 2.3.4 Methylation Analysis After methylation, the polysaccharide was further subjected to hydrolysis, reduction, and derivatization. GC-MS detected the fully methylated polysaccharide products. The test results are shown in Table 1 and... Figure 5 As shown.

[0064] Table 1. Linkage fragment analysis of polysaccharide AG-40-I-II <![CDATA[2, 3, 5-Me3-Araf]]> Araf-(1→ 81, 97, 111, 123, 151, 165 11.88 <![CDATA[2, 3, 6-Me3-Glcp]]> →4)-Glcp-(1→ 83, 101, 112, 129, 147, 178, 207 6.65 <![CDATA[2, 4, 6-Me3-Galp]]> →3)-Galp-(1→ 85, 95, 109, 124, 151 64.12 <![CDATA[2, 4-Me2-Galp]]> →3, 6)-Galp-(1→ 81, 97, 109, 123, 137, 165, 205 17.35 2.3.5 NMR Analysis according to 13 According to the C10 NMR results, no obvious uronic acid characteristic chemical shift signals were observed in the δ 160-190 ppm range, proving that the polysaccharide is a neutral polysaccharide. This result is consistent with the monosaccharide composition and FT-IR spectral analysis results. Simultaneously, in the 100-110 ppm anodic carbon signal region, three obvious characteristic signals were found at 103.36, 103.60, and 103.73 ppm, confirming the presence of three monosaccharide groups, which corresponds to the monosaccharide composition analysis results. The polysaccharide... 1 The 1H NMR spectrum contained three observable anomeric hydrogen signals at δ 4.74, 4.56, and 4.31 ppm, with the most prominent signal at 4.31 ppm. The anomeric hydrogen chemical shift values ​​were less than δ 5.00 ppm, indicating that the polysaccharide glycosyl group possesses a β-configuration glycosidic bond. Furthermore, all three signals in the combined carbon spectrum were greater than 101 ppm, further confirming that the major building blocks of the polysaccharide contain β-configuration residues.

[0065] According to HSQC spectra, the anomeric hydrogen (H1) / anomeric carbon (C1) signals δ 4.35 / 103.12, 4.58 / 103.61, and 4.75 / 103.60 ppm corresponded to each other, with the strongest monosaccharide residue signal at δ 4.35 / 103.12, indicating its highest content and that this monosaccharide residue is the main structural component of the polysaccharide. The weakest signal was at δ 4.75 / 103.60 ppm, indicating its lowest content. Based on these results, its composition is presumed to be: →3)-β-Gal-(1→3, and small amounts of →4)-β-Glc-(1→4 and D-β-Araf-(1→(Table 2)).

[0066] Table 2. H and C NMR chemical shifts of monosaccharides →3)-β-Gal-(1 4.35 103.12 → 4)-β-Glc-(1 4.75 103.60 D-arabinose β-Araf-(1→ 4.58 103.61 By analyzing HSQC, HMBC and 1 H- 1 Two-dimensional NMR spectra of H COSY, combined with 1 H and 13 C10 NMR spectroscopy can determine chemical shifts. The chemical shifts of the anomeric hydrogen and anomeric carbon signals of the galactose residues are δ 4.35 / 103.12 ppm. 1 H- 1 In the HCl COSY spectrum, the signal at δ 4.41 / 3.46 ppm is identified as an H1 / H2 signal. Using the HSQC spectrum, relevant information at δ 3.43 / 70.34 ppm is found, thus determining the C2 chemical shift of this monosaccharide residue to be 70.34 ppm. Then, through... 1 H- 1 HCOSY spectroscopy revealed a chemical shift of 3.61 ppm for H3 associated with H2, and HSQC showed a chemical shift of 74.74 ppm for C3 corresponding to H3. Analysis revealed chemical shifts of 3.81 / 73.27, 3.55 / 72.30, and 3.67 / 60.55 for H4 / C4, H5 / C5, and H6 / C6 of the monosaccharide residues, respectively. HMBC spectroscopy showed strong interference between the anomeric hydrogen at 4.34 / 68.87 and the C-4 position, confirming that the connection between the galactose anomeric position and the C-4 position of the sugar residue is the main component. Simultaneously, a small subset was linked to the C-3 carbon of the glucose residue; the signal intensity in the HMBC spectrum confirmed that the glycoside at this position constitutes the main structure of the polysaccharide. Meanwhile, the 4.48 / 103.12 ppm in the HMBC spectrum constitutes another link between molecules. It is the side chain portion of the main chain above and has been confirmed to be a link between β-galactose C-6 and arabinose anomaly.

[0067] The structure of AG-40-I-II (hereinafter referred to as AG) is as follows: Figure 6 As shown. Based on the monosaccharide composition, FT-IR spectroscopy analysis, methylation analysis, and NMR analysis results, this polysaccharide is an AG-II type polysaccharide.

[0068] In this embodiment, a crude polysaccharide was extracted from larch, and different polysaccharide fractions were obtained through ethanol fractionation. Based on the protective effect of different polysaccharide fractions against LPS-induced inflammation in IEC-6 cells, they were separated and purified using a DEAE-52 cellulose column combined with a Superose 12 dextran gel column. Finally, the obtained polysaccharide was characterized by its physical and chemical structure, and it was determined to be an AG-II type polysaccharide.

[0069] Example 2: Preparation and structural identification of sulfated arabinogalactan There are various methods for sulfation modification of polysaccharides, such as the chlorosulfonic acid-pyridine method, the SO3-pyridine method, and the concentrated sulfuric acid method. Among them, the chlorosulfonic acid method is widely used due to its high yield and high dissolved solids (DS). DS and molecular weight (Mw) are key factors affecting the bioactivity of sulfated polysaccharides, and both are influenced by the sulfation method used. Different sulfation methods attach sulfate groups at different positions, resulting in different types of sulfated polysaccharides. The derivatization of sulfate groups alters the structure of natural polysaccharides, and these structural changes affect their bioactivity.

[0070] In this embodiment, sulfated arabinogalactan (SAG) was prepared using the chlorosulfonic acid method and the concentrated sulfuric acid method. The specific sulfation method was determined based on the different saturation concentrations (DS) and yields (DMF was selected as the solvent for the polysaccharide, and chlorosulfonic acid and pyridine were selected as the esterification reagents for the sulfonation reaction). Simultaneously, the obtained SAG was separated using a Superose 12 dextran gel column to obtain homogeneous SAG, and the structure of the sulfated arabinogalactan was characterized.

[0071] 1. Method 1.1 Specific preparation method of sulfated arabinogalactan: 300 mg of polysaccharide sample (AG-40-I-II) was dissolved completely in 25 mL of DMF at room temperature with stirring. 12 mL of anhydrous pyridine was measured and cooled in an ice-water bath. 3 mL of chlorosulfonic acid was added dropwise to obtain a pale yellow esterification reagent. The polysaccharide DMF solution was added to the esterification reagent, and the reaction was carried out at 50 °C for 1 h. The mixture was then neutralized with 2.5 mol / L NaOH solution, and the pH was adjusted to 7.0. Anhydrous ethanol was added to precipitate the polysaccharide. The precipitate was collected by centrifugation, dialyzed, and lyophilized to obtain SAG. Yield: 65.24%, DS: 0.53%.

[0072] The crude SAG was then purified using a Superose 12 dextran gel column, and the fraction with the highest content was collected for subsequent experiments (see [link]). Figure 7 ).

[0073] 1.2 The structure of sulfated arabinogalactan was characterized according to the method in Example 1.

[0074] 2. Experimental Results 2.1 Structural characterization of sulfated arabinogalactan 2.1.1 Determination of molecular weight and uniformity Based on the results of HPGPC, the molecular weight of SAG was determined to be 17.9 kDa. Furthermore, the symmetrical elution curves of SAG indicate that it is a homogeneous polysaccharide.

[0075] 2.1.2 Monosaccharide Composition Analysis

[0076] After acid hydrolysis, HPLC analysis revealed that SAG consisted of galactose, arabinose, and glucose in a molar ratio of 6.7:1:0.3. By comparing the molecular weight, monosaccharide composition, and ratio of AG and SAG, it is speculated that the sulfation modification process may have led to the decomposition of the main chain in the AG polysaccharide chain, resulting in a decrease in the proportion of galactose.

[0077] 2.1.3 Infrared and ultraviolet spectra

[0078] After sulfation, 3450 cm -1 The -OH stretching vibration peak at 2920 cm⁻¹ and the peak at 2920 cm⁻¹ -1 The -CH stretching vibration peaks at [location] all exhibit a blue shift. The -SO4 stretching vibration peak appears at 1260 cm⁻¹. -1 The above results prove that sulfation was successful.

[0079] In the ultraviolet spectrum, there are no absorption peaks for nucleic acids and proteins at 260 nm and 280 nm, proving that SAG does not bind to nucleic acids and proteins.

[0080] 2.1.4 NMR Analysis

[0081] This invention compares the NMR of SAG with that of AG. 13 Changes were observed in the C NMR spectrum. 13 NMR data for sulfated polysaccharides in some of C's papers, in 13 A signal of 81.49 ppm appeared in the C NMR spectrum, indicating that a portion of the SAG hydroxyl group was chemically converted into a sulfate group. 13 The signal intensity of C increases significantly at approximately 66-67 ppm, indicating that the primary hydroxyl group of SAG is sulfated. Based on comparative analysis, this invention found that the signals of the anomeric carbons newly appearing at 70.66, 70.24, and 69.19 ppm may also be influenced by the introduction of sulfate groups. Therefore, it is speculated that the primary hydroxyl group of SAG is chemically converted into a sulfate group (see [details of the structure]). Figure 6 ).

[0082] Example 3: Therapeutic effect of arabinogalactan and its derivatives on acute colitis In this embodiment, arabinogalactan (AG) obtained in Example 1 and sulfated arabinogalactan (SAG) obtained in Example 2 were used to intervene in DSS-induced IBD mice to preliminarily explore the effects of AG and SAG on IBD mice.

[0083] 1. Materials and Methods 1.1 Main Reagents Sodium dextran sulfate was purchased from Nanjing Dulai Biotechnology Co., Ltd.; the fecal occult blood qualitative test kit was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; the interleukin-1β kit, interleukin-10 kit, tumor necrosis factor-α kit, lipopolysaccharide kit, hematoxylin, and eosin were purchased from Nanjing Jiancheng Bioengineering Institute; Alixin Blue 8GX and Nucleotide Red were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; primary antibodies: Mucin-2, ZO-1, Occludin, TLR4, MyD88, IκBα, p-IκBα, NF-κB, p-NF-κB, NLRP3, ACS, Caspase1, IL-1β, and β-actin; secondary antibodies: rabbit anti-mouse were all purchased from Wuhan Sanying Biotechnology Co., Ltd.; the ultrasensitive ECL chemiluminescence kit was purchased from Guangzhou Saiguo Biotechnology Co., Ltd.

[0084] 1.2 Establishment of a DSS-induced acute colitis model in mice Eight-week-old male ICR mice (weighing 18-21 g) were purchased from Changchun Yisi Laboratory Animal Co., Ltd., quality certificate number: SCXK(JI)-2016-0003 (Changchun, China). The mice were acclimatized for one week under a 12-hour light-dark cycle. All animal handling and experimental procedures were conducted in accordance with the approval of the Laboratory Animal Ethics Committee of Jilin Agricultural University.

[0085] Male ICR mice were randomly divided into groups of 8 mice each. The Normal group received normal water and was administered 0.9% saline via gavage for 7 consecutive days. The DSS group received 3.5% DSS aqueous solution and was administered 0.9% saline via gavage for 7 consecutive days. The treatment group received 3.5% DSS aqueous solution, with the test compound administered in three dose groups (AG 400 mg / kg, SAG 200 mg / kg, SAG 400 mg / kg) via gavage for 7 consecutive days. The single treatment group received normal water and was administered 400 mg / kg SAG via gavage for 7 consecutive days. Fecal samples were collected from the mice for the determination of intestinal microbiota and short-chain fatty acid content. After the experiment, blood was collected via the orbital vein and the mice were dissected. The collected mouse serum was centrifuged twice at 3000 rpm for 10 minutes each time, and the supernatant was collected and stored at -80 °C until testing. Simultaneously, the colon of the mice was photographed, and the length of the colon in each group was recorded. The colon was fixed in 10% neutral formalin buffer, embedded in paraffin, wrapped in aluminum foil, rapidly frozen in liquid nitrogen, and stored at -80 °C for subsequent experimental detection.

[0086] 1.3 Disease Activity Index Score (DAI) DAI was determined based on the degree of weight loss, fecal consistency, and fecal occult blood index in each mouse. The occult blood index in mouse feces was measured using the instructions provided in the occult blood index assay kit.

[0087] 1.4 Biochemical Indicator Detection Following the instructions provided with the TNF-α, IL-1β, and IL-10 ELISA kits, the test solution, standard solution, and chromogenic reagent were added sequentially to the ELISA plate. Finally, the stop solution was added, and the OD values ​​were measured at 450 nm using a microplate reader within 15 minutes. The results were calculated to determine the levels of TNF-α, IL-1β, and IL-10. Simultaneously, the kits were used to detect the levels of LPS in serum and myeloperoxidase (MPO) in colon tissue homogenate.

[0088] 1.5 Histological staining Histopathological staining: To assess the pathological changes in colon tissue, H&E and alicin blue staining were determined for analysis. Colon tissue fixed in neutral formalin buffer was removed, and after routine processing (gradient ethanol dehydration and xylene permeation), the colon tissue was embedded in paraffin. After the paraffin block cooled, it was cut into 5 μm thick sections using a rotary microtome (Leica, RM2235, Solms, Germany). The sections were then stained with an H&E and alicin blue staining kit, and the pathological changes in the colon tissue were examined using an optical microscope (Leica, DM750, Solms, Germany).

[0089] Immunofluorescence staining: 5 μm thick colon tissue sections were deparaffinized and rehydrated. The sections were placed in citrate buffer (0.01 mol / L, pH 6.0) for antigen retrieval for 20 min, followed by washing three times with PBS buffer. Then, they were incubated with 3% bovine serum albumin (BSA) for 30 min to prevent nonspecific staining. After aspirating excess liquid, primary antibody was applied to the colon tissue sections and incubated overnight. The next day, after washing three times with PBS, the sections were incubated with fluorescent secondary antibody in the dark for 1 h. Subsequently, the secondary antibody was removed, and the sections were washed three times with PBS for 10 min each time. Finally, images were taken using a Leica microscope (Leica, TCS SP8, Solms, Germany).

[0090] Immunohistochemical staining: Paraffin was removed from 5 μm thick colon tissue sections and the sections were rehydrated. The sections were placed in citrate buffer (0.01 mol / L, pH 6.0) for antigen retrieval for 20 min, followed by washing three times with PBS buffer. Then, they were incubated with 3% bovine serum albumin (BSA) for 30 min to prevent nonspecific staining. After aspirating excess liquid, primary antibody was applied to the colon tissue sections and incubated overnight. The next day, after washing three times with PBS, the sections were incubated with secondary antibody for 1 h in the dark. Afterward, the secondary antibody was removed, and the sections were washed three times with PBS for 10 min each time. Finally, images were taken using a Leica microscope (Leica, TCSSP8, Solms, Germany).

[0091] 1.6 Western blot analysis Colon tissue was dissolved in RIPA Lysis Buffer, and equal amounts of protein were separated on a vertical electrophoresis apparatus using 10% or 12% SDS-PAGE gels and transferred to PVDF membranes (0.22 μm). The target bands were blocked with 5% skim milk for 2.5 h and incubated with specific primary antibody for 2 h. The membrane was then washed three times with TBS·T (TBS + 0.4% Tween-20) and incubated with HRP-labeled secondary antibody at room temperature for 1.5 h. Proteins were visualized using a gel imaging system, and their grayscale values ​​were calculated using Image-Pro plus 6.0 software.

[0092] 1.7 Determination of Short-Chain Fatty Acids Take an appropriate amount of cecal contents, add 2 mL of solution (1:3 phosphate aqueous solution), mix well, and extract with 2 mL of ether. Centrifuge at low temperature for 20 min (4000 rpm / min). Extract again with ether, combine and dilute to 2 mL, and perform GC-MS analysis. Calculate W using the formula W=(C-C0)×V×N / m. (W: short-chain fatty acid content; C: short-chain fatty acid concentration in the test solution; C0: target substance concentration in the reference standard; V: final volume; N: dilution factor; m: sample weight).

[0093] 1.8 Intestinal Microbiome Assay Cecal contents were collected, merged, and processed using 16S rRNA sequencing. This invention used Illumina MiSeq to sequence the V3-V4 regions of the species' 16S ribosomal RNA genes. Cluster analysis was performed on the raw data, and sequences were ordered according to abundance. OUT units with a similarity of 97% were considered as belonging to a single species. After classification and annotation in the Silva (Release132, https: / / www.arb-silva.de / ) database, the results were analyzed using the GenesCloud platform (https: / / www.genescloud.cn / home).

[0094] 1.9 Statistical Analysis All data are expressed as mean ± standard deviation (mean ± SD) established across different experiments and analyzed using one-way ANOVA and Bonferroni post-hoc tests. Statistical graphs were generated using GraphPad Prism 8.0.2 software (GraphPad Software, Inc., San Diego, USA). p < 0.05 or 0.01 was considered statistically significant.

[0095] 2. Experimental Results 2.1 AG and SAG alleviate DSS-induced colitis in mice DAI score and colon length directly reflect the severity of IBD in animal models and are often used for preliminary assessment of colitis severity. ICR mice were given AG (400 mg / kg) and SAG (200 mg / kg and 400 mg / kg).

[0096] The DAI scores and body weights of mice in each group from days 8 to 17 are as follows: Figure 9 As shown in AB. The DAI scoring criteria were as per Table 3. After mice drank DSS, the body weight of the model group mice showed a significant decreasing trend, which continued to decrease over the next few days, accompanied by severe diarrhea and fecal hemorrhage. Compared with the DSS group, the body weight and DAI index of mice in other groups were significantly different. After administering AG and different doses of SAG to the mice, the symptoms of weight loss, diarrhea, and fecal hemorrhage were all alleviated to varying degrees.

[0097] Table 3 DAI Scoring Criteria 0 0% normal Negative 1 1%-5% normal Negative 2 5%-10% loose Positive 3 10%-20% loose Positive 4 ＞20% diarrhea Severe bleeding This invention also observed the colonic condition of mice in different groups. No significant edema was observed in the colons of the control group and the SAG400 group, but the colon length of the model group mice was significantly shortened and edema was observed. Injection of mice with different doses of AG increased colon length and reduced edema. These results indicate that AG and SAG can significantly alleviate IBD, and that SAG has a better therapeutic effect than AG.

[0098] 2.2 AG and SAG reduce the levels of pro-inflammatory cytokines and inhibit the infiltration of inflammatory cells into the colon. Cytokines are mainly produced by lymphocytes, monocytes, macrophages, and epithelial cells. They have pro-inflammatory effects, such as TNF-α and IL-1β, or anti-inflammatory effects, such as IL-10. Cytokines in healthy individuals are in homeostasis and play crucial roles in immune regulation, tissue repair, intestinal barrier function, and intestinal homeostasis. In mice, DSS intervention significantly increased the expression levels of IL-1β, TNF-α, and IL-10, and AG and SAG significantly inhibited the production of pro-inflammatory cytokines IL-1β and TNF-α. When inflammation occurs, the body produces the anti-inflammatory cytokine IL-10 to combat the inflammatory response. Compared with the control group, the DSS group showed increased IL-10 expression, and treatment with AG and SAG significantly upregulated IL-10 levels to inhibit inflammation. Figure 10 AC).

[0099] When mice develop colitis, blood LPS (lipopolysaccharide) levels increase. This increased LPS continuously stimulates the immune system, exacerbating the inflammatory response. To further investigate the effects of AG and SAG on DSS-induced colitis, the concentration of LPS in mouse blood was measured. Compared to the control group, the LPS concentration was significantly increased in the DSS group, and decreased after administration of AG and SAG. Figure 10 D).

[0100] The level of MPO (peroxidase) reflects the infiltration of neutrophils in colonic tissue after colitis induction. Compared with the control group, the MPO content in the colonic tissue homogenate of mice in the DSS group was increased, and the MPO content decreased after administration of AG and SAG. Figure 10 E).

[0101] In mice treated with DSS, intestinal epithelial cells were eroded in the colonic tissue, thereby increasing colonic mucosal permeability. To visually represent the colonic damage in each group of mice, this invention used H&E staining for scoring (Table 4). H&E staining showed that the colonic wall edema was more severe in the model group mice, crypt structures were deformed or disappeared, and goblet cells were destroyed or disappeared. Figure 11(AB). Compared with the DSS group, the damage to the colonic tissues was reduced after treatment with AG and SAG. The colonic tissue condition of mice in the SAG400 group was similar to that of the control group, which demonstrates that SAG did not cause damage to the colonic tissues of mice.

[0102] Based on the expression of cytokines and the extent of damage to colonic tissue, it can be seen that both AG and SAG can alleviate colonic damage caused by DSS, and at the same dose, SAG has a better therapeutic effect than AG.

[0103] Table 4 Scoring criteria for H&E staining Severity of inflammation 0 none Severity of inflammation 1 Slightly Severity of inflammation 2 generally Severity of inflammation 3 serious Degree of inflammatory damage 0 No inflammation Degree of inflammatory damage 1 mucous membrane Degree of inflammatory damage 2 Submucosal layer Degree of inflammatory damage 3 Transmural crypt injury 0 Undamaged crypts crypt injury 1 One-third of the crypt was damaged. crypt injury 2 Two-thirds of the crypt was damaged. crypt injury 3 The crypts are missing, but the surface epithelium remains. crypt injury 4 The crypts disappeared, and the surface epithelium disappeared. Damage percentage 0 0% Damage percentage 1 1%-25% Damage percentage 2 26%-50% Damage percentage 3 51%-75% Damage percentage 4 76%-100% 2.3 AG and SAG increase the thickness of the intestinal mucosa in colonic tissue and promote Mucin-2 protein expression. Alcian blue (AB) staining was used to observe the colonic mucosa to determine the thickness of the mucus layer from the proximal colon to the rectum. Figure 12 In group A, compared with the control group, a decrease in the number of colonic goblet cells and a thinner mucus layer were observed in the DSS group. Compared with the DSS group, AG and SAG promoted the differentiation and formation of colonic goblet cells, promoted mucus synthesis by goblet cells, increased the thickness of the colonic mucus layer, and increased the coverage of colonic epithelial cells. Meanwhile, SAG treatment alone had little effect on colonic tissue, and the mucus layer thickness was consistent with the control group. Mucin-2 is the main mucin secreted by goblet cells into the large intestine lumen. Mucin-2 immunofluorescence staining was used to assess mucus layer thickness, goblet cell number, and mucus secretion function. Figure 12 BC). AG and SAG can significantly increase mucus layer thickness and improve the reduction in goblet cell number caused by DSS.

[0104] Evaluation of colonic mucus layer thickness revealed that both AG and SAG improved the reduction in mucus layer thickness caused by DSS and promoted Muc-2 protein expression. SAG showed superior therapeutic efficacy compared to AG at the same dosage. Furthermore, the SAG400 group did not affect mucus layer thickness, indicating that SAG had no adverse effects on the mouse colon.

[0105] 2.4 AG and SAG promote the expression of TJ protein in colon tissue TJ proteins, as an important component of the intestinal barrier, reduce intestinal permeability and prevent the absorption of exogenous substances such as LPS by the intestinal mucosa. During DSS-induced colitis in mice, intestinal permeability is significantly altered, making TJ proteins particularly important. In DSS-treated colonic tissue, the expression of TJ proteins ZO-1 and Occludin was significantly reduced. However, intervention with AG and SAG in mice showed improved expression of ZO-1 and Occludin, with an increasing trend. Figure 13AB).

[0106] 2.5 Effects of AG and SAG on the TLR4 / MyD88 / NF-κB signaling pathway, NLPR3 inflammasome, and PPARγ in colonic tissue The expression of proteins related to the NF-κB signaling pathway was detected by Western blot to explore the mechanism of changes in inflammation levels. In mice treated with DSS, MyD88 was stimulated by TLR4 recognition, thereby increasing the expression of its downstream proteins p-IκB-α / IκB-α and p-NF-κB / NF-κB. Figure 14 (AE). Administration of AG and SAG to mice inhibited the expression of TLR4 and MyD88, thereby reducing the expression of p-IκB-α / IκB-α and p-NF-κB / NF-κB. Compared with the control group, the protein expression levels of TLR4, MyD88, p-IκB-α / IκB-α, and p-NF-κB / NF-κB in the SAG 400 group showed almost no significant change.

[0107] The Nod-like receptor protein 3 (NLRP3) inflammasome is an intracellular protein complex that regulates the body's inflammatory response. Results showed that DSS intervention stimulated the expression of NLRP3 and ASC, significantly increasing the expression of their downstream proteins Caspase 1 and IL-1β, thus inducing an inflammatory response. Treatment with AG and SAG in mice inhibited the expression of NLRP3 and ASC, thereby reducing stimulation of Caspase 1 and IL-1β and decreasing the incidence of inflammatory response. SAG 400 intervention in normal mice did not affect the expression of NLRP3, ASC, Caspase 1, or IL-1β proteins (see [link to relevant documentation]). Figure 15 ).

[0108] PPARγ has been reported to exert a good anti-inflammatory effect in an acute colitis model. Results showed that PPARγ expression was significantly reduced in the DSS group compared to the control group, and administration of AG and SAG significantly increased PPARγ expression levels. Furthermore, this invention found that the SAG 400 group showed increased PPARγ expression levels compared to the control group. Figure 16 AB).

[0109] 2.6 AG and SAG promote the metabolism of short-chain fatty acids It is well known that short-chain fatty acids (especially acetate, butyrate, and propionate) are major metabolites of gut microbiota. Currently, a growing body of reports confirms the role of SCFAs in immune regulation. This invention uses GC-MS to detect the content of SCFAs in mouse feces. The results showed that SCFA metabolism in the cecal contents of mice decreased after DSS intervention. Treatment with AG and SAG increased the amount of SCFAs in the cecal contents of mice. In the SAG 400 group, mice were able to produce more acetate and butyrate. Importantly, studies have shown that butyrate helps treat acute colitis. Therefore, AG and SAG can improve the metabolism of SCFAs in mice, thereby improving DSS-induced acute colitis in mice.

[0110] 2.7 AG and SAG altered the species classification and abundance of mouse gut microbiota. After DSS intervention, the number of OUTs showed a decreasing trend, proving that DSS reduced gut microbiota diversity to some extent. Further analysis of the gut microbiota composition of each group using species composition heatmaps showed that DSS intervention altered the gut microbiota of normal mice, and under the regulation of AG and SAG, gradually converged towards the gut microbiota of normal mice.

[0111] To more clearly analyze the effects of AG and SAG on the gut microbiota of mice, this invention evaluated the relative abundance of gut microbiota at the phylum and genus levels. At the phylum level, DSS intervention altered the gut microbiota of mice, decreasing the relative abundance of Firmicutes and Bacteroidetes. Intervention with AG and SAG increased the relative abundance of Firmicutes and Bacteroidetes. Figure 17 At the genus level, DSS intervention decreased the relative abundance of Allobaculum and Lactobacillus. AG and SAG interventions reversed this trend, increasing the relative abundance of Allobaculum and Lactobacillus. Figure 17 DF).

[0112] 2.8 Effects of AG and SAG on DSS-induced gut microbial diversity in mice The α-diversity index of gut microbiota in each group was analyzed, and corresponding dilution curves were generated. The results showed that the sparse curves of each group initially increased and then flattened with increasing measurement depth, indicating good diversity, richness, and evenness of the measured samples. The Simpson and Shannon indices were used to evaluate microbial diversity; the higher the index, the higher the diversity. The Chao1 index estimated the number of OTUs in the sample. A higher Chao1 index indicates more species in the sample, while the Goods-Coverage index represents the true microbial composition of the sample. The results of this invention show that the Simpson, Shannon, and Chao1 indices of the gut microbiota in the control group mice changed compared to the DSS group mice, indicating that the diversity and abundance of gut microbiota in the DSS group mice had been affected. After treatment with AG and SAG, all indicators were affected, indicating that AG and SAG can influence gut microbial diversity. Furthermore, the increase in the Goods-Coverage index after AG and SAG intervention also strongly supports the reliability of the data.

[0113] To further reveal the differences in microbial diversity among samples, principal coordinate analysis (PCoA) was used to compare the differences between samples. The first principal coordinate (PCoA1) and the second principal coordinate (PCoA2) were 15.5% and 9.3%, respectively. Figure 18 A). PCA analysis revealed a discrepancy between the gut microbiota and the control group after DSS intervention. After AG and SAG treatment, the gut microbiota showed a discrepancy compared to the DSS group, demonstrating that AG and SAG can modulate the DSS-induced changes in gut microbiota. Importantly, the changes in gut microbiota resulting from AG and SAG treatment were not consistent with those in normal mice. Furthermore, the SAG 400 group did not show the same changes as the control group, indicating that SAG did not alter the gut microbiota composition of normal mice.

[0114] UPGMA hierarchical clustering can visually display the degree of difference in microorganisms among different samples. Figure 18 B). The distance between the DSS group and the control group indicated a significant difference in gut microbiota between the DSS intervention group and the control group, and changes in gut microbiota structure were also observed. Separation from the DSS group occurred after administration of AG and SAG, suggesting that AG and SAG help regulate changes in gut microbiota structure and composition in DSS-induced colitis mice. Through intergroup difference analysis ( Figure 18 The gut microbiota of mice treated with DSS (diethyltoluene-associated glutathione) differed from that of the control group. AG (a type of glutathione) and SAG (a type of glutathione) also showed differences compared to the DSS group after treatment.

[0115] The above results demonstrate that AG and SAG can modulate the changes in gut microbiota caused by DSS. Compared with the control group, the SAG400 group showed altered gut microbiota. This indicates that SAG can modulate the gut microbiota of normal mice.

[0116] 2.9 Species Difference Analysis of Gut Microbiota in DSS-Induced Colitis Mice by AG and SAG To further analyze the effects of AG and SAG on gut microbiota, this invention analyzed the differences between different groups at the phylum and genus levels, finding that the gut microbiota of mice in the DSS group separated from that in the Control group after DSS intervention. Under low-dose SAG intervention, the gut microbiota of mice was partially similar to that of the DSS group, but a trend of divergence had already emerged. Under high-dose AG and SAG intervention, there was almost no similarity between the gut microbiota of mice and those in the DSS group, indicating significant changes in the gut microbiota. The intervention results of SAG in normal mice showed that SAG could induce slight changes in the gut microbiota, consistent with the results of β-diversity analysis.

[0117] Analysis of the gut microbiota results showed that DSS intervention altered the composition and abundance of the mouse gut microbiota. Furthermore, intervention using AG and SAG could reverse the DSS-induced changes in gut microbiota, moving towards restoring normal gut microbiota homeostasis.

[0118] Example 4: Regulation and isolation of gut microbiota by SAG 1. Materials and Methods 1.1 Mouse feces were obtained from the mouse feces in Example 3 (feces of mice with DSS-induced acute colitis treated with SAG).

[0119] 1.2 Main Reagents MRS solid and liquid culture media were purchased from Nanjing Dulai Biotechnology Co., Ltd.; Gram staining solution was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; and artificial gastric and intestinal fluids were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0120] 1.3 The steps for isolating gut microbiota are as follows: MRS medium was used to isolate Lactobacillus. MRS liquid and solid media were prepared and autoclaved. Mouse feces were then placed in the MRS liquid medium. The MRS liquid medium was incubated at 37 °C for 24 h, and then transferred to larger Erlenmeyer flasks for further culture. The culture was taken and serially diluted, and then streaked onto MRS solid medium. The plates were inverted and incubated at 37 °C for 24 h. Single colonies were picked using an inoculation loop and re-inoculated onto MRS liquid medium. After incubation at 37 °C for 24 h, the colonies were streaked onto MRS solid medium until only single colonies were observed on the MRS solid medium. The final single colonies were then cultured.

[0121] 1.4 Identification of bacterial strains 1.4.1 Flat Plate Shape Straw the bacterial culture from the liquid culture medium onto the surface of the solid culture medium, invert the plate, and incubate at 37 ℃ for 24 h before observing the colony morphology.

[0122] 1.4.2 Gram staining

[0123] Gram staining was performed on the liquid bacterial culture after 24 hours of incubation, following the instructions from the Haibo Gram staining manual. After staining, observation was performed using an oil immersion microscope.

[0124] 1.4.3 Scanning electron microscopy observation

[0125] The liquid bacterial culture after 24 h of culture was centrifuged at 6000 r / min for 5 min, washed three times with sterile PBS, and the bacterial sludge was collected. 2.5% glutaraldehyde fixative was added, and the mixture was fixed at 4 ℃ in the dark for 12 h. After removing the glutaraldehyde, the mixture was washed three times with sterile PBS, and eluted with 30%, 50%, 70%, 90% and 100% ethanol for 15 min each. The eluted bacterial sludge was freeze-dried and observed under a scanning electron microscope.

[0126] 1.4.4 16S rDNA Sequencing

[0127] An appropriate amount of bacterial cells was dissolved in 50 μL of TaKaRa Lysis Buffer for Microorganism to DirectPCR (Code No. D304), denatured, and centrifuged. The supernatant was used as template DNA. The target fragment was amplified by PCR using a 2×TransTaq® High Fidelity (HiFi) PCR SuperMix I (TransGen Biotech, Code No: AS131). Sequencing was performed using a DNA sequencer. The obtained gene sequence was compared for homology with 16S rDNA sequences in the Genebank database using the BLAST functional component.

[0128] 1.5 Determination of gastric juice resistance The bacterial suspension was spread onto a solid culture medium and incubated upside down for 24 h. The number of colonies was then counted. Alternatively, the bacterial suspension was incubated with simulated gastric juice at 37 °C for 3 h, then spread onto a solid culture medium and incubated upside down for 24 h. The gastric juice resistance of the strain was calculated based on the number of colonies.

[0129] 1.6 Determination of resistance to intestinal fluid Bacterial culture in gastric fluid for three hours was co-cultured with artificial intestinal fluid. The culture was then spread onto solid culture medium at 4 and 8 hours and incubated upside down for 24 hours. The intestinal fluid tolerance of the bacterial strain was calculated based on the number of colonies.

[0130] 2. Experimental Results 2.1 Identification of fungal strains This invention isolated two strains of bacteria using MRS medium. Based on 16S rDNA sequencing results, they were identified as *Limosilactobacillus reuteri* strain HDB1243 and *Lactobacillus johnsonii* strain 1696, both Lactobacillus species. Morphological observation on agar plates revealed that *Limosilactobacillus reuteristrain* HDB1243 formed smooth, round, milky-white colonies with regular edges and a raised center, while *Lactobacillus johnsonii* strain 1696 formed smooth, round, translucent colonies with regular edges and a raised periphery. Gram staining confirmed that both bacteria are Gram-negative. Scanning electron microscopy revealed that Limosilactobacillus reuteri strain HDB1243 has a short rod-like structure, without flagella or spores; Lactobacillus johnsonii strain 1696 has a rod-like structure, longer than Limosilactobacillus reuteristrain HDB1243, without flagella or spores.

[0131] 2.2 Resistance to intestinal fluid The drug enters the digestive system and spends approximately 8 hours in the intestines. Bacterial cultures cultured in simulated gastric fluid are transferred to simulated intestinal fluid and cultured further at 37 °C. Both *Limosilactobacillus reuteri* strain HDB1243 and *Lactobacillus johnsonii* strain 1696 survived after 4 and 8 hours of culture in simulated intestinal fluid before plating. At 4 hours, the survival rate of *Limosilactobacillus reuteri* strain HDB1243 was 96.9%, and the survival rate of *Lactobacillus johnsonii* strain 1696 was 78.4%. At 8 hours, the survival rate of *Limosilactobacillus reuteri* strain HDB1243 was 28.9%, and the survival rate of *Lactobacillus johnsonii* strain 1696 was 42.5% (demonstrating that both strains can successfully pass through the gastrointestinal system and reach the colon). It is evident that both strains of bacteria have the potential to tolerate the environment of gastric and intestinal fluids, enter the colon, and exert their effects in the colon.

[0132] Example 5: Therapeutic effect of sulfated arabinogalactan on colon cancer 1. Materials and Methods 1.1 Experimental Materials SAG was selected from the polysaccharide prepared in Example 2, and Limosilactobacillus reuteri strain HDB1243 and Lactobacillus johnsonii strain 1696 were selected from the intestinal bacteria isolated in Example 4.

[0133] 1.2 Establishment of a mouse colon cancer model induced by DSS combined with AOM Establishment of a mouse colon cancer model: Male ICR mice were randomly divided into three groups of 14 mice each. Control group: 0.9% saline was administered via gavage, followed by normal water. DSS / AOM group: 10 mg / mL AOM was injected intraperitoneally, followed by 2% DSS aqueous solution for 7 days, and normal water for 14 days as one cycle, for a total of 3 cycles. 0.9% saline was administered via gavage continuously. Drug treatment group: Continuous administration of drug (SAG / intestinal bacteria) via gavage for three cycles. Fecal samples were collected from mice for intestinal microbiota analysis. After the experiment, blood was collected via the orbital vein and the mice were dissected. The collected mouse blood was centrifuged twice at 3000 rpm / min, 10 min each time, and the supernatant was collected. Serum was used for biochemical index detection. Simultaneously, the mouse colon was harvested and photographed. The colon was fixed in 10% neutral formalin buffer and embedded in paraffin, or wrapped in aluminum foil and rapidly frozen in liquid nitrogen and stored at -80 °C.

[0134] 1.3 Biochemical indicator detection The levels of TNF-α, IL-1β, GSH, MDA, and iron in mice were measured. The specific procedures were performed according to the method described in Example 3.

[0135] 1.4 Histological staining Histopathological staining and immunohistochemical staining are performed according to the methods in Example 3.

[0136] TUNEL staining: After dewaxing the tissue sections, add proteinase K solution and hydrolyze for 15 min at room temperature to remove tissue proteins. Incubate with PBS containing 2% hydrogen peroxide for 5 min at room temperature. Wash twice with PBS, 5 min each time. Remove excess liquid from the slide, add 2 drops of TdT enzyme buffer, and incubate at room temperature for 1–5 min. Blot away excess liquid with filter paper, add 54 μL of TdT enzyme reaction solution to the section, and incubate in a humidified chamber at 37 °C for 1 h. Add wash and stop reaction buffer, and incubate at 37 °C for 30 min to stop the reaction. Add two drops of peroxidase-labeled anti-digoxigenin antibody, and incubate in a humidified chamber at room temperature for 30 min. Add freshly prepared 0.05% DAB solution, and develop the color at room temperature for 3–6 min. Counterstain with methyl green at room temperature for 10 min. Mount and dry the slides, then observe under an optical microscope.

[0137] ROS staining: Paraffin was removed from 5 μm thick colon tissue sections and the sections were rehydrated. DCFH-DA was diluted 1:1000 to a final concentration of 10 μmol / L, and an appropriate volume of diluted DCFH-DA working solution was added. Washing was performed to remove any DCFH-DA that had not penetrated the tissue. After mounting and drying, the sections were observed directly using a laser confocal microscope.

[0138] 1.5 Transmission Electron Microscopy Analysis Fresh mouse colon tissue was collected, minimizing mechanical damage such as traction, contusion, and compression. The tissue volume was generally no more than 2 mm × 2 mm × 2 mm. The tissue was quickly fixed in electron microscopy fixative at 4 °C for 2–4 h. The tissue was then rinsed three times with 0.1 mol / L PBS (pH=7.4), 15 min each time. Next, it was fixed in 0.1 mol / L PBS (1% osmium tetroxide) at room temperature for 2 h, followed by three more rinses in 0.1 mol / L PBS buffer, 15 min each time. Finally, the tissue was sequentially dehydrated in 50%-70%-80%-90%-95%-100%-100% ethanol-100% acetone-100% acetone solutions, 15 min each time. After dehydration, the tissue was infiltrated in an acetone:812 embedding medium solution of 1:1 for 2–4 h. After the initial infiltration, the tissue was infiltrated overnight in an acetone:812 embedding medium solution of 1:2. Tissues were then embedded using 812 embedding medium. After 5–8 hours, the medium was poured into embedding plates, and the samples were inserted and incubated overnight at 37 °C. The next day, the samples were polymerized at 60 °C for 48 hours. After embedding, ultrathin sections of 60–80 nm were prepared using an ultramicrotome and stained with 2% uranium acetate saturated alcohol solution and lead citrate for 15 minutes each. After staining, the sections were dried at room temperature overnight. Finally, the sections were observed under a transmission electron microscope, and images were acquired and analyzed.

[0139] 2. Experimental Results 2.1 SAG and gut bacteria alleviate DSS / AOM combined-induced colon cancer in mice In the case of combined DSS / AOM intervention, tumors appeared in the colon of mice in the DSS / AOM group. The colons of mice in different groups were photographed to compare their condition. Long-term intervention with the gut bacteria *Limosilactobacillus reuteri* strain HDB1243, *Lactobacillus johnsonii* strain 1696 (hereinafter referred to as bacteria 1 and 2), and SAG reduced the number of tumors in the colon. Furthermore, intervention with bacteria 1, bacteria 2, and SAG alleviated colon shortening to some extent. Figure 19 ).

[0140] 2.2 SAG and gut microbiota reduce the levels of pro-inflammatory cytokines and inhibit the infiltration of inflammatory cells into the colon. Under the intervention of DSS combined with AOM, the levels of inflammatory factors TNF-α and IL-1β in mouse serum were increased. After long-term intervention with bacteria 1, bacteria 2 and SAG, the levels of inflammatory factors were reduced. Figure 20 AB). Simultaneously, H&E staining revealed that in the DSS / AOM group, the intestinal villi were shorter and the crypts less distinct. Figure 20 C). Intervention with bacteria 1, bacteria 2, and SAG can alter the condition of the colon and restore it to a normal state. By detecting the levels of inflammatory factors and the extent of colonic damage, bacteria 1 and bacteria 2 can work with SAG to inhibit inflammation and reduce inflammatory damage.

[0141] 2.3 SAG and gut bacteria promote apoptosis in colon tumor tissues To investigate how bacteria 1, bacteria 2, and SAG reduce the number of tumors in the colon, this invention performed TUNEL staining on colonic tumor sites (… Figure 21 AB). The results showed that, compared with the DSS / AOM group, intervention with bacteria 1, bacteria 2, and SAG all promoted apoptosis in colon tumor cells. Simultaneously, Western blotting was used to detect the protein expression levels of Bcl-2 and Bax in colon tumor cells; under the intervention of bacteria 1, bacteria 2, and SAG, the expression levels of Bcl-2 / Bax increased ( ). Figure 21 CD). It is evident that bacteria 1, bacteria 2, and SAG may reduce the number of tumors in the colon by promoting apoptosis in colon tumor sites.

[0142] 2.4 SAG and gut bacteria activate AMPK-mTOR, thereby inducing autophagy in colon tumor tissue. This invention uses Western blotting to detect proteins related to AMPK-mTOR-induced autophagy in the colon. Results showed that bacteria 1, 2, and SAG activated AMPK phosphorylation and upregulated p-AMPK expression, inhibiting mTOR phosphorylation via AMPK-mTOR, thereby activating autophagy. During autophagy, bacteria 1, 2, and SAG upregulated the expression levels of autophagy marker proteins LC3, ATG7, ATG3, and Beclin-1, while the levels of the autophagy substrate p62 and the autophagy-specific carrier NCOA4 gradually decreased. These results demonstrate that bacteria 1, 2, and SAG can induce autophagy in colon tumor tissue and may potentially regulate free Fe in colon tumor tissue through NCOA4-mediated ferritin autophagy. 2+ The levels of bacteria have an impact. Therefore, bacteria 1, bacteria 2, and SAG may reduce tumor number by promoting autophagy at the site of colonic tumors.

[0143] 2.5 SAG and intestinal bacteria induce ferroptosis in colon tumor tissues Preliminary experimental results indicate that bacteria 1, 2, and SAG can promote autophagy in colon tumor tissues and reduce tumor number. Simultaneously, they may regulate free Fe within colon tumor tissues through NCOA4-mediated ferritin autophagy. 2+ The content of [certain substances] is affected. Therefore, this invention detects ferroptosis in colon tumor tissue.

[0144] 2.5.1 SAG and gut bacteria increase the degree of lipid peroxidation This invention involves ROS staining of colon tumor tissue, and observation and comparison of fluorescence intensity in the colon of mice in different groups using fluorescence microscopy. The ROS fluorescence was significantly enhanced after intervention with bacteria 1, bacteria 2, and SAG. Figure 22 A, C). Notably, after intervention by bacteria 1, bacteria 2, and SAG, the levels of MDA and GSH, as well as the expression level of 4-HNE in the colonic tissue, were significantly enhanced. Figure 22 B, DF).

[0145] Intracellular ROS can be converted into oxygen free radicals, which react with polyunsaturated fatty acids and lipoproteins on the cell membrane to undergo lipid peroxidation, leading to DNA damage. The main final product of this lipid peroxidation is malondialdehyde (MDA), which can cause changes in protein structure and function and exhibits certain biotoxicity. MDA levels can reflect the degree of cellular lipid peroxidation. GSH can synergistically convert lipid oxides into lipid alcohols with GPx4, reducing the accumulation of intracellular lipid oxides and affecting the development of ferroptosis. Therefore, a decrease in GSH promotes ferroptosis. 4-HNE is a harmful metabolite produced by fatty acid peroxidation. When cells experience oxidative stress, lipid peroxidation leads to the production of 4-HNE from fatty acids. Therefore, long-term intervention with bacteria 1, bacteria 2, and SAG can increase the degree of lipid peroxidation in colon tumor sites.

[0146] 2.5.2 SAG and intestinal bacteria can increase Fe in colon tumor tissue. 2+ content Compared with the DSS / AOM group, intervention with bacteria 1, bacteria 2, and SAG significantly increased Fe in mouse colon tumor tissue. 2+ The content of [unclear - possibly referring to a specific substance or component]. Bacteria 1, Bacteria 2, and SAG can reduce the number of tumors in colon tumor tissue, increase the degree of lipid peroxidation, and [unclear - possibly referring to a specific substance or component]. 2+ The content is consistent with the characteristics of ferrodegeneration.

[0147] 2.5.3 SAG and gut microbiota alter the ultrastructure of colon tumor tissue Previous studies have demonstrated that bacteria 1, bacteria 2, and SAG can induce ferroptosis in colon tumor tissues. Transmission electron microscopy observation of the ultrastructure of colon tumor sites showed that intervention with bacteria 1, bacteria 2, and SAG could cause mitochondrial shrinkage, mitochondrial membrane rupture, and a reduction or disappearance of cristae in cells. Figure 23 This result also proves that bacteria 1, bacteria 2, and SAG can induce ferroptosis.

[0148] 2.5.4 SAG and gut microbiota promote the expression of iron metabolism-related proteins in colon tumor tissue This invention describes immunohistochemical staining of GPx4 and ASCL4 proteins in colon tumor tissues from different groups of mice. GPx4 is an intracellular selenoprotein antioxidant enzyme and an important regulator of ferroptosis, capable of scavenging lipid peroxidation products and preventing oxidative stress. GPx4 prevents ferroptosis by eliminating intracellular lipid peroxidation. ASCL4 is a key enzyme regulating lipid composition and can promote lipid peroxidation. Simultaneously, ASCL4 can promote the synthesis of unsaturated fatty acids, thereby inducing ferroptosis. Under the intervention of bacteria 1, bacteria 2, and SAG, the expression of GPx4 in colon tumor tissues was inhibited, while the protein expression of ASCL4 was upregulated. Figure 24 AC). It is evident that bacteria 1, bacteria 2, and SAG can regulate ferroptosis induced by GPx4 and ASCL4 proteins.

[0149] Western blot analysis was performed to analyze the expression of ASCL4 and GPx4 proteins in colon tumor tissues. Figure 25 AC). Intervention by bacteria 1, 2, and SAG upregulated ACSL4 expression and inhibited GPx4 protein expression, thereby promoting ferroptosis. This result is consistent with immunohistochemical analysis. SLC7A11 can exchange extracellular cysteine ​​and intracellular glutamate, ensuring sufficient intracellular cysteine ​​for GSH synthesis. GSH, in conjunction with GPx4, converts lipid oxides into lipid alcohols, reducing intracellular lipid oxide accumulation and influencing the development of ferroptosis. The cellular transport system maintains intracellular iron levels at homeostasis; transferrin and its receptor transport extracellular iron into the cell. Intracellular iron is primarily stored in the form of ferritin, which is divided into heavy chain ferritin (FTH) and light chain ferritin (FTL). Intervention by bacteria 1, 2, and SAG inhibited SLC7A11 expression in colon tumor tissues, accelerating lipid oxide accumulation and promoting ferroptosis. Meanwhile, during NCOA4-mediated ferritin autophagy, the expression of FTL and FTH, which store iron ions in the cell, was reduced, indicating that bacteria 1, bacteria 2, and SAG can increase the intracellular iron content and thus promote ferroptosis. Figure 25 DF).

[0150] 2.6 Regulation of gut microbiota by SAG and gut bacteria 2.6.1 SAG and gut microbiota altered the species classification and abundance of the mouse gut microbiota. Intervention with bacteria 1, bacteria 2, and gut microbiota altered the types and diversity of gut microbiota in mice. A species composition heatmap revealed a tendency towards consistency in their regulatory effects on gut microbiota. Figure 26 It is evident that bacteria 1, bacteria 2, and SAG have consistent regulatory effects on gut microbiota.

[0151] Interventions with bacteria 1, 2, and SAG altered the relative abundance of dominant species at both the phylum and genus levels in the gut microbiota. DSS / AOM intervention decreased the relative abundance of Bacteroidetes and Firmicutes. Bacterium 1 intervention increased the relative abundance of both Bacteroidetes and Firmicutes. Interventions with bacteria 2 and SAG increased the relative abundance of both Bacteroidetes and Firmicutes. This indicates that at the phylum level, bacteria 1, 2, and SAG can increase the relative abundance of beneficial bacteria in the gut microbiota.

[0152] At the genus level, the relative abundance of Lactobacillus and Bacteroides decreased after DSS / AOM intervention. Intervention with bacteria 1, 2, and SAG reversed the decreasing trend induced by DSS / AOM, resulting in varying degrees of increase in the relative abundance of both Lactobacillus and Bacteroides (see [link to relevant documentation]). Figure 27 ).

[0153] Therefore, intervention by bacteria 1, bacteria 2 and SAG can increase the number of beneficial bacteria at the phylum and genus levels of the gut microbiota, and the regulatory trends of bacteria 1 and bacteria 2 are roughly consistent with those of SAG.

[0154] 2.6.2 Effects of SAG and gut microbiota on gut microbial diversity in DSS / AOM-induced colon cancer mice The alpha diversity index of gut microbiota in each group was analyzed, and corresponding dilution curves were constructed. The Simpson and Shannon indices were used to evaluate microbial diversity; higher indices indicated greater diversity. A higher Chao1 index indicated a greater number of species in the sample, while the Goods-Coverage index represented the true microbial diversity of the sample. Our results showed that the Simpson, Shannon, and Chao1 indices of the gut microbiota in the Control group mice changed compared to the DSS / AOM group mice, indicating that the diversity and abundance of gut microbiota in the DSS / AOM group mice had been affected. After intervention with bacteria 1, bacteria 2, and SAG, all indicators changed, indicating that bacteria 1, bacteria 2, and SAG can influence gut microbial diversity. Furthermore, the increase in the Goods-Coverage index after intervention with bacteria 1, bacteria 2, and SAG strongly supports the reliability of the data.

[0155] To further reveal the differences in microbial diversity among samples, principal coordinate analysis (PCoA) was used to compare the differences between samples. The first principal coordinate (PCoA1) and the second principal coordinate (PCoA2) were 15.3% and 9.8%, respectively. Figure 28A). PCoA analysis revealed a discrepancy between the gut microbiota and the control group mice after DSS / AOM intervention. After intervention with bacteria 1, bacteria 2, and SAG, the gut microbiota showed a discrepancy compared to the DSS / AOM group, demonstrating that bacteria 1, bacteria 2, and SAG can regulate the gut microbiota changes induced by DSS / AOM. Furthermore, bacteria 2 and SAG brought the gut microbiota closer to that of normal mice, indicating their ability to regulate the gut microbiota to normal levels.

[0156] Hierarchical clustering can visually display the degree of difference in microorganisms among different samples. Figure 28 B). In this invention, the Control group and the DSS / AOM group exhibited two extreme trends, indicating that DSS / AOM intervention can alter the gut microbiota composition of normal mice. After administration of bacteria 1, bacteria 2, and SAG, the mice separated from the DSS / AOM group and gradually approached the Control group, suggesting that bacteria 1, bacteria 2, and SAG help regulate the structure and composition of the gut microbiota and restore normal gut microbiota homeostasis. Through inter-group difference analysis (… Figure 28 (C) It was found that the gut microbiota of mice treated with DSS / AOM differed from that of the Control group. After intervention with bacteria 1, bacteria 2, and SAG, the gut microbiota of mice diverged from the DSS / AOM group and approached that of the Control group, and their regulatory trends were consistent.

[0157] The above results demonstrate that bacteria 1, bacteria 2, and SAG can regulate the gut microbiota dysbiosis caused by DSS / AOM. Furthermore, under long-term intervention, they can restore the gut microbiota to normal homeostasis, and their regulatory effects show consistency.

[0158] 2.6.3 Species Difference Analysis of SAG and Intestinal Bacteria on Gut Microbiota in DSS / AOM Co-induced Colon Cancer Mice To further analyze the effects of bacteria 1, bacteria 2, and SAG on gut microbiota, this study analyzed the differences between different groups at the phylum and genus levels. Figure 29 (AF). The results showed that intervention with bacteria 1, bacteria 2, and SAG significantly altered the gut microbiota of mice compared to the DSS / AOM group, and made them similar to the gut microbiota of the Control group. Furthermore, the regulatory effects of bacteria 1, bacteria 2, and SAG on gut microbiota were consistent.

[0159] LEfSe analysis was performed when LDA > 2 to analyze differences between groups. Under the influence of DSS / AOM, the differences in gut microbiota were significant. Figure 29G). c_Verrucomicrobiae, p_Verrucomicrobia, o_Verrucomicrobiales, g_Akkermansia, and f_Verrucomicrobiaceae were enriched in the DSS / AOM group, with the highest abundance. Intervention with bacteria 1, bacteria 2, and SAG reduced the differentiality of the mouse gut microbiota and decreased the relative abundance of harmful bacteria.

[0160] The above studies revealed that long-term intervention with bacteria 1, 2, and SAG in mice with DSS / AOM-induced colon cancer reduced colonic damage and tumor number. Limosilactobacillus reuteristrain HDB1243 and Lactobacillus johnsonii strain 1696 are considered key gut bacteria antagonizing colon cancer. SAG further antagonized DSS / AOM-induced colon cancer by activating the AMPK-mTOR signaling pathway, regulating NCOA4-mediated ferritin autophagy, and increasing the relative abundance of Limosilactobacillus reuteri strain HDB1243 and Lactobacillus johnsonii strain 1696. Simultaneously, they induced AMPK activation, promoting intracellular Fe... 2+ Excessive accumulation of lipid peroxidation metabolites leads to ferroptosis. Furthermore, *Limosilactobacillus reuteristrain* HDB1243 and *Lactobacillus johnsonii* strain 1696 exhibit antagonistic effects against DSS / AOM-induced colon cancer in mice, similar to SAG, and their regulatory trends on gut microbiota are also consistent with SAG. Therefore, SAG primarily exerts its antagonistic effect against colon cancer by increasing the relative abundance of *Limosilactobacillus reuteri* strain HDB1243 and *Lactobacillus johnsonii* strain 1696; thus, it can be used in the preparation of drugs for colon cancer.

[0161] The above description is a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing arabinogalactan derivatives from larch, wherein the method uses arabinogalactan from larch as raw material and modifies it by sulfation; characterized in that, The method for preparing arabinogalactan from larch includes the following steps: (1) Add larch root powder to a beaker, add anhydrous ethanol for defatting, filter and air dry, dry the powder and extract it with distilled water at 60-80℃ with ultrasonic assistance, combine the supernatants and concentrate; add anhydrous ethanol to the concentrated solution until the ethanol concentration reaches 90%, let stand overnight, collect the precipitate by centrifugation; redissolve the precipitate in distilled water, add Sevage reagent to remove protein, and obtain crude polysaccharide by dialysis and freeze drying; (2) Weigh the crude polysaccharide, add distilled water to dissolve it completely, add anhydrous ethanol to the solution until the alcohol concentration of the solution reaches 40%, let it stand overnight, centrifuge to obtain precipitate AG-40; (3) Weigh the AG-40 sample, dilute it with distilled water, perform DEAE cellulose column chromatography, and elute with NaCl solution to obtain AG-40-I. Concentrate, dialyze and freeze-dry the obtained components. (4) Weigh the AG-40-I sample, dilute it with distilled water, perform dextran gel column chromatography, and elute with NaCl solution to obtain the final component AG-40-I-II. The AG-40-I-II is a homogeneous polysaccharide with a molecular weight of 16.8 kDa, mainly composed of galactose, arabinose and glucose in a molar ratio of 12.5: 1: 0.

05. It is a neutral polysaccharide, and is an AG-II type polysaccharide with →3)-β-Gal-(1→3 and →4)-β-Glc-(1→4 as the main chain and β-Araf-(1→) as the side chain. The sulfation modification method is the chlorosulfonic acid-pyridine method, which includes the following steps: (1) Add the AG-40-I-II sample to DMF and stir at room temperature until it is completely dissolved; (2) Measure anhydrous pyridine, cool it in an ice-water bath, and add chlorosulfonic acid dropwise to obtain a light yellow esterification reagent; (3) Add the polysaccharide DMF solution to the esterification reagent, react at 50 °C for 1 h, add 2.5 mol / L NaOH solution to neutralize, and adjust the pH to 7.0; add anhydrous ethanol to precipitate the polysaccharide, centrifuge to collect the precipitate, dialyze and freeze dry to obtain the crude sulfated arabinogalactan; (4) The obtained sulfated arabinogalactan crude product was purified using a Superose 12 dextran gel column. The component with the highest content was collected. The purified sulfated arabinogalactan was a homogeneous polysaccharide with a molecular weight of 17.9 kDa, mainly composed of galactose, arabinose and glucose in a molar ratio of 6.7:1:0.

3. The structural formula is as follows: ； The sulfated arabinogalactan antagonizes colon cancer by activating the AMPK-mTOR signaling pathway and regulating NCOA4-mediated ferritin autophagy, thereby leading to ferroptosis.

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