Saposhnikovia divaricata polysaccharide and its preparation method and application

Abstract

The present invention belongs to the field of medical technology and relates to fangfeng polysaccharide and its preparation method and application. The fangfeng polysaccharide is prepared by gradient alcohol precipitation method. It is an acidic uniform polysaccharide with a molecular weight of 125.5kDa. It is composed of multiple monosaccharides connected by glycosidic bonds. The monosaccharides include arabinose, galactose, galacturonic acid, glucose, L-rhamnose, D-glucuronic acid, xylose, and mannose. The molar ratio of each monosaccharide is 27.10%, 35.35%, 21.72%, 8.97%, 3.81%, 1.67%, 0.81%, and 0.56%. The fangfeng polysaccharide forms a bidirectional regulatory network of "intestinal-liver axis" by synergistically regulating the intestinal flora structure and the downstream arachidonic acid metabolic pathway. The intestinal-liver axis homeostasis is enhanced by the "microbiome-metabolite-signaling pathway" network, and multiple pathways work synergistically to ensure the health of the intestine and liver.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a kind of Saposhnikovia divaricata A homogeneous polysaccharide (SD-1) extracted from Saccharum officinale (Turcz.) Schischk.) and a preparation method thereof, as well as an application thereof in the preparation of drugs for treating acetaminophen (APAP)-induced liver damage and drugs for regulating intestinal flora. Background Art

[0002] Acetaminophen (APAP) is a widely used antipyretic and analgesic drug worldwide, but its overdose is one of the leading causes of drug-induced liver injury (DILI) and acute liver failure (ALF), accounting for over 40% of all cases. APAP-induced acute liver injury (ALI) not only poses a serious threat to patient health but also places a heavy burden on the healthcare system. ALI patients often present with jaundice, coagulopathy, and hepatic encephalopathy, and severe cases may even require liver transplantation or face the risk of death. Therefore, in-depth research on the mechanisms of APAP-induced liver injury and the development of effective treatment strategies are of great clinical significance.

[0003] The mechanism of APAP-induced liver injury is complex, involving multiple processes, including oxidative stress, inflammation, and mitochondrial dysfunction. APAP is primarily metabolized in the liver through glucuronidation and sulfation to non-toxic products that are excreted. A small amount of APAP is metabolized by cytochrome P450 enzymes, primarily CYP2E1, to the highly reactive toxic intermediate N-acetyl-p-benzoquinoneimine (NAPQI). At normal doses, NAPQI is rapidly neutralized and detoxified by glutathione (GSH). However, excessive APAP consumption leads to significant GSH depletion, leading to NAPQI accumulation. This NAPQI then binds to proteins, lipids, and DNA in hepatocytes, triggering oxidative stress and mitochondrial dysfunction. Oxidative stress markers, such as decreased superoxide dismutase (SOD) activity and elevated malondialdehyde (MDA) levels, as well as depletion of the antioxidant GSH, are key features of APAP-induced liver injury. In addition, excessive APAP consumption can activate the nuclear factor κB (NF-κB) signaling pathway, promoting the release of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), further exacerbating liver inflammation. These pathological changes not only lead to severe liver damage but may also trigger multiple organ dysfunction syndrome (MODS), endangering the patient's life.

[0004] Furthermore, APAP-induced liver injury is often accompanied by gut microbial imbalance, which can disrupt gut-liver axis homeostasis and exacerbate liver inflammation and oxidative stress. For example, a decrease in the beneficial bacteria Akkermansia and an increase in the harmful bacteria Escherichia-Shigella are closely associated with the severity of liver injury.

[0005] As a traditional Chinese medicine extract, fangfeng polysaccharide has multiple pharmacological activities, including anti-inflammatory, antioxidant, and immunomodulatory activities. However, the effects of fangfeng polysaccharide on APAP-induced liver injury and its regulation of intestinal flora have not been reported. Summary of the Invention

[0006] The object of the present invention is to provide a fangfeng polysaccharide, which is prepared by a gradient alcohol precipitation method and differs from existing fangfeng polysaccharides in monosaccharide content. The fangfeng polysaccharide can not only prevent APAP-induced drug-induced acute liver injury, but also significantly enrich Verrucomicrobiota, Akkermansia and other anti-inflammatory properties in the intestine, thereby protecting intestinal health and reducing the risk of enteritis.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A fangfeng polysaccharide, which is an acidic homogeneous polysaccharide with a molecular weight of 125.5kDa. The polysaccharide is composed of multiple monosaccharides connected by glycosidic bonds, including arabinose, galactose, galacturonic acid, glucose, L-rhamnose, D-glucuronic acid, xylose, and mannose, and the molar ratios of the monosaccharides are 27.10%, 35.35%, 21.72%, 8.97%, 3.81%, 1.67%, 0.81%, and 0.56% respectively; the main chain is composed of β-1,4-galacturonic acid and β-1,4-glucose, and the side chains contain α-arabinose ends.

[0009] The present invention also provides a method for preparing fangfeng polysaccharide, which comprises the following steps:

[0010] Step 1. preparing a water extract of Saposhnikovia divaricata;

[0011] Step 2. The water extract of Radix Saposhnikoviae was first precipitated with 50% ethanol, the supernatant was collected, and then precipitated with 75% ethanol. The precipitate was redissolved in water, filtered, and the supernatant was concentrated under pressure to remove proteins and pigments to obtain crude Radix Saposhnikoviae polysaccharide.

[0012] Step 3. The crude polysaccharide from Radix Saposhnikoviae was subjected to gradient elution using a DEAE-52 cellulose column with a mobile phase of NaCl solution. The fractions with the highest sugar content were combined, and the NaCl solution was removed using a dialysis bag.

[0013] Step 4. Further purification was performed using Sephadex G-100 gel column chromatography to obtain fangfeng polysaccharide.

[0014] As a preferred embodiment of the present invention, the specific steps of preparing the water extract of Saposhnikovia divaricata in step 1 are: crushing the Saposhnikovia divaricata, extracting it 2-3 times with hot water at 90-95°C, with a solid-liquid ratio of 1:5, and each extraction time is 1-2 hours.

[0015] As a preferred embodiment of the present invention, step 2 uses the Sevag method to remove protein and uses D101 resin to filter and wash to remove pigment.

[0016] As a preferred embodiment of the present invention, when step 4 is purified by Sephadex G-100 gel column chromatography, distilled water is used as the mobile phase for elution, and then each fraction is detected by an evaporative light detector, and the fractions with the same peak time are combined and freeze-dried to obtain the Fangfeng polysaccharide.

[0017] The fangfeng polysaccharide provided by the present invention can significantly reduce serum ALT / AST levels and improve liver tissue pathological damage; it can inhibit the TLR4 / NF-κB signaling pathway, reduce the levels of pro-inflammatory cytokines TNF-α and IL-6 in serum and liver, and achieve anti-inflammatory effects; it can activate the Nrf2 / HO-1 pathway to exert an antioxidant effect, increase the SOD and GSH levels in the liver, and reduce the MDA level; through targeted inhibition of the overactivation of the arachidonic acid metabolic pathway, the release of inflammatory mediators is reduced, thereby alleviating APAP-induced hepatocyte oxidative stress and inflammatory outbreaks; therefore, it can be used in the preparation of drugs for preventing or treating liver damage.

[0018] As a preferred embodiment of the present invention, the liver injury is drug-induced acute liver injury induced by acetaminophen.

[0019] The fangfeng polysaccharide provided by the present invention can specifically regulate the microbial composition of mice with acetaminophen-induced liver damage, reduce the abundance of Escherichia-Shigella and Proteobacteria related to inflammation, increase the abundance of Verrucomicrobiota and Akkermansia with anti-inflammatory properties, protect intestinal health, and reduce the risk of enteritis. Therefore, it can be used in the preparation of drugs for treating or regulating intestinal flora imbalance; it can also be used in the preparation of drugs for treating or preventing enteritis.

[0020] As a preferred embodiment of the present invention, the intestinal flora imbalance refers to the intestinal flora imbalance caused by acetaminophen-induced drug-induced acute liver injury.

[0021] As a preferred embodiment of the present invention, the Fangfeng polysaccharide promotes competition between intestinal microorganisms, increases the abundance of beneficial bacteria (Verrucomicrobiota, Akkermansia) and competitively reduces the abundance of harmful bacteria.

[0022] When the fangfeng polysaccharide of the present invention is used as a medicine, the pharmaceutical dosage forms include oral tablets, capsules, granules or injections, and the effective dose is 50-200 mg / kg per day.

[0023] Advantages and beneficial effects of the present invention:

[0024] (1) The fangfeng polysaccharide provided by the present invention is prepared by a gradient alcohol precipitation method. The fangfeng polysaccharide is different from the existing fangfeng polysaccharide in terms of monosaccharide content. It can inhibit the TLR4 / NF-κB inflammatory pathway, reduce the expression of pro-inflammatory factors such as TNF-α and IL-6, and achieve anti-inflammatory effects; it can activate the Nrf2 / HO-1 antioxidant pathway, increase the activity of SOD and GSH and reduce the level of MDA; it can also downregulate the expression of 5-LOX and COX-2 proteins in the arachidonic acid (AA) metabolic pathway, reduce the production of pro-inflammatory mediators such as PGE2 and HETEs in the arachidonic acid metabolic pathway, alleviate APAP-induced hepatocyte oxidative stress and inflammatory outbreak, and achieve the effect of preventing or treating acute liver injury. It provides a new natural drug candidate for the treatment of acetaminophen (APAP)-induced liver injury and has important clinical application value.

[0025] (2) The Fangfeng polysaccharide provided by the present invention can reshape the intestinal flora structure, reduce the abundance of pathogenic bacteria Escherichia-Shigella and Proteobacteria, enrich the beneficial bacteria Verrucomicrobiota and Akkermansia, protect intestinal health, and reduce the risk of enteritis. It can be used in the preparation of drugs for regulating intestinal flora imbalance, and can also be used in the preparation of drugs for treating or preventing enteritis.

[0026] (3) The Fangfeng polysaccharide provided by the present invention forms a bidirectional regulatory network of the "intestine-liver axis" by synergistically regulating the intestinal flora structure and the downstream arachidonic acid metabolic pathway, and enhances the homeostasis of the intestinal-liver axis through the "flora-metabolite-signaling pathway" network. Multiple pathways work synergistically to ensure the health of the intestine and liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The following are the separation and purification of SD-1 and the analysis of its physicochemical properties. A is the elution curve of SD-1 purified by DEAE-52 cellulose column; B is the high-performance gel permeation chromatography (HPGPC) spectrum of SD-1; C is the Fourier transform infrared (FT-IR) spectrum; D is the monosaccharide composition analysis; E is the scanning electron microscope (SEM) images at magnifications of 300×, 500×, 1000×, and 2000×, respectively.

[0028] Figure 2 is the total ion chromatogram (TIC);

[0029] Figure 3 is the NMR spectrum analysis of SD-1; wherein A is 1 H NMR spectrum; B is 13C NMR spectrum; C is HH COSY spectrum; D is CH HSQC spectrum; E is HMBC spectrum; F is the predicted structure of SD-1;

[0030] Figure 4 Effects of SD-1 on serum ALT / AST levels and liver pathology in mice with APAP-induced liver injury; A is the experimental flow chart; B is the ALT level in mouse serum; C is the AST level in mouse serum; D is H&E staining of mouse liver (100×, 200×); white circles indicate necrotic areas, yellow arrows indicate inflammatory infiltration, blue arrows indicate apoptotic bodies, and red arrows indicate fat vacuoles;

[0031] Figure 5 The mechanism of action of SD-1 in inhibiting the TLR4 / NF-κB pathway and activating the Nrf2 / HO-1 pathway; A and B are the levels of IL-6 and TNF-α in mouse serum, respectively; C is the immunofluorescence imaging of TNF-α and IL-6 in mouse liver (200×); D and E are the mean fluorescence intensities of TNF-α and IL-6, respectively; FH is the protein expression of TLR4 and MyD88 in mouse liver; IK is the protein expression of NF-κB and IκB-α in mouse liver; LN is the expression levels of SOD, GSH, and MDA in mouse liver; OQ is the protein expression of Nrf2 and HO-1 in mouse liver;

[0032] Figure 6 This is a preliminary analysis of liver metabolites in acute liver injury regulated by SD-1; A is a three-dimensional plot of PCA analysis; B is an Upset plot; C is the hierarchical classification of metabolites in HMDB; D is a heat map analysis of metabolites;

[0033] Figure 7Analysis of the mechanism of action of SD-1 in inhibiting arachidonic acid metabolic pathway; A is the volcano plot of APAP vs Control; B is the volcano plot of APAP+SD-1-H vs APAP; C is the Venn diagram of increased metabolite expression in APAP vs Control and decreased metabolite expression in APAP+SD-1-H vs APAP; D is the Venn diagram of decreased metabolite expression in APAP vs Control and increased metabolite expression in APAP+SD-1-H vs APAP; E is the KEGG pathway enrichment analysis of decreased metabolite expression in APAP vs Control and increased metabolite expression in APAP+SD-1-H vs APAP; F is the KEGG pathway enrichment analysis of increased metabolite expression in APAP vs Control and decreased metabolite expression in APAP+SD-1-H vs APAP; G is the expression of metabolites in the arachidonic acid pathway regulated by SD-1; HJ are the expression of 5-LOX and COX-2 proteins in the liver regulated by SD-1;

[0034] Figure 8 Figure 3 is the differential expression analysis of arachidonic acid-related metabolites inhibited by SD-1; A is the projected variable importance (VIP) score of the control group and the APAP group; B is the VIP score of the APAP group and the APAP+SD-1-H group; C is the heat map of metabolites downregulated in the APAP group and the control group, and upregulated in the APAP+SD-1-H group and the APAP group; D is the heat map of metabolites downregulated in the APAP group and the control group, and upregulated in the APAP+SD-1-H group and the APAP group; EM is the expression level of metabolites: 5-HETE, 12-HETE, 15-HETE, 20-HETE, 5-Oxo-ETE, phosphatidylcholine, PGB2, PGA2, and PGE2;

[0035] Figure 9 Preliminary analysis of SD-1 regulation of intestinal microbial abundance Figure 1 ; Among them, A is the dilution curve; B is cluster analysis; C is PCA analysis; D is Ace index; E is Chao index; F is Shannon index;

[0036] Figure 10 Preliminary analysis of SD-1 regulation of intestinal microbial abundance Figure 2 ; Among them, A is the Venn diagram; B is the Circos analysis between each sample and microbial species at the phylum level; C is the Circos analysis between each sample and microbial species at the genus level;

[0037] Figure 11 Figure 1: SD-1 regulates the intestinal microbial community of mice at the phylum and genus levels. A represents the relative abundance of microbial communities at the phylum level in each group of mice; B represents the relative abundance of microbial communities at the genus level in each group of mice; C represents the microbial communities with significant differences between Con and APAP at the phylum level; D represents the microbial communities with significant differences between APAP and APAP+SD-1-H at the phylum level.

[0038] Figure 12 Figure 2 shows the regulation of intestinal microbial communities in mice by SD-1 at the phylum and genus levels; A is the microbial community with significant differences between Con and APAP at the genus level; B is the microbial community with significant differences between APAP and APAP+SD-1-H at the genus level; CD is the LEfSe based on LDA Through The cladogram shows the differences in the dominant microbiota among the groups;

[0039] Figure 13 Correlation between intestinal microorganisms and pathological indicators; A and B are heat maps of the correlation between pathological indicators regulated by SD-1 and intestinal microorganisms at the phylum and genus levels; C and D are heat maps of the correlation between arachidonic acid metabolites regulated by SD-1 and intestinal microorganisms at the phylum and genus levels;

[0040] Figure 14 This is a molecular ecological network analysis; A is the prediction by PICRUSt2 that SD-1 affects the AA pathway through the intestinal flora, and B is the construction of an ecological network at the phylum level to evaluate the mutual influence between SD-1 and intestinal microbial communities. DETAILED DESCRIPTION

[0041] The present invention discloses a fangfeng polysaccharide and a preparation method and application thereof. Those skilled in the art can refer to the contents of this article and appropriately improve the component parameters or process parameters to achieve it. It should be pointed out in particular that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the scope of protection of the present invention. In addition, the various chemical reagents and chemicals mentioned in the present invention are all well-known and commonly used chemical reagents and chemicals in the prior art unless otherwise specified, and the treatment methods mentioned can refer to existing methods unless otherwise specified. The following is a further detailed description of this application in conjunction with the accompanying drawings and examples.

[0042] Example 1 Preparation and structural characterization of fangfeng polysaccharide (SD-1):

[0043] 1. Materials and Methods

[0044] 1.1. Materials:

[0045] Saposhnikovia divaricata was purchased from Jilin City (Jilin Province, China); DEAE cellulose-52, Sephadex G-100, glucose, mannose, rhamnose, galactose, glucuronic acid, and galacturonic acid standards were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Coomassie Brilliant Blue G250 and 1-phenyl-3-methyl-5-pyrazolone (PMP) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0046] 1.2. Extraction, separation and purification of SD-1 polysaccharide:

[0047] After the Radix Saposhnikoviae was crushed, it was extracted with hot water three times (90-95°C, solid-liquid ratio 1:5, each extraction time was 2 hours), and the filtrate was concentrated under reduced pressure to obtain the Radix Saposhnikoviae water extract; the Radix Saposhnikoviae water extract was first precipitated with 50% ethanol for 24 hours, and the supernatant was taken; it was then precipitated with 75% ethanol for 24 hours; the precipitate was redissolved in hot water, filtered, centrifuged at 7000 rpm for 20 minutes, and the supernatant was taken and concentrated under pressure; the protein was removed by Sevag method, and the pigment was removed by filtration and elution with D101 resin. The eluate was freeze-dried to obtain the Radix Saposhnikoviae crude polysaccharide; the Radix Saposhnikoviae crude polysaccharide was gradient eluted with DEAE-52 cellulose column chromatography, and the mobile phase was NaCl solution (NaCl concentration was 0, 0.1, 0.3, 0.5, 0.7, 1.0 mol / L); the fractions with the highest sugar content were combined, and the NaCl solution was removed with a dialysis bag; and the elution was continued with Sephadex. Further purification was performed by G-100 gel column chromatography; specifically, the crude fangfeng polysaccharide eluted from DEAE-52 was added to a chromatography column filled with Sephadex G-100 and eluted using distilled water as the mobile phase; each fraction was then detected by an evaporative light detector, and the fractions with consistent peak elution times were combined and freeze-dried to obtain a uniform polysaccharide, namely, fangfeng polysaccharide (SD-1).

[0048] 1.3. Determination of total sugar, protein and uronic acid content of SD-1:

[0049] The total sugar content of SD-1 was determined by the phenol-sulfuric acid method, the protein content was determined by the Bradford method, and the uronic acid content was determined by the m-hydroxybenzene method.

[0050] 1.4. Fourier transform infrared spectroscopy (FT-IR) detection:

[0051] Take an appropriate amount of SD-1, place it in the detection hole of FT-IR, cover it with KBr glass, and detect at 4000-400cm −1 Infrared spectrum.

[0052] 1.5.SD-1 molecular weight detection:

[0053] The molecular weight of purified polysaccharides from each fraction of Saposhnikovia divaricata was determined using high-performance gel permeation chromatography (HPGPC). Dextran molecular weight standards of varying molecular weights were prepared into 1 mg / ml solutions. Detection was performed using a Waters evaporative light scattering detector (ELSD), and a standard curve was constructed. The molecular weight was calculated based on the retention time of SD-1.

[0054] 1.6. Monosaccharide composition detection:

[0055] SD-1 (5 mg) was hydrolyzed with trifluoroacetic acid (2 M) in a sealed tube at 121°C for 2 hours. The sample was blown dry with nitrogen. The sample was washed with methanol, then blown dry, and the methanol wash was repeated two to three times. The residue was redissolved in deionized water and filtered through a 0.22 μm filter membrane before analysis. The sample extract was analyzed by high-performance anion exchange chromatography (HPAEC) on a Dionex CarboPac PA-20 anion exchange column (3 × 150 mm) using a pulsed amperometric detector (PAD; Dionex ICS 5000+ system). Flow rate: 0.5 ml / min; injection volume: 5 μl; solvent system A: (deionized water), solvent system B: (0.1 M sodium hydroxide), solvent system C: (0.1 M sodium hydroxide, 0.2 M sodium acetate); gradient program: volume ratio of solutions A, B, and C: 95:5:0 at 0 min, 85:5:10 at 26 min, 85:5:10 at 42 min, 60:0:40 at 42.1 min, 60:40:0 at 52 min, 95:5:0 at 52.1 min, and 95:5:0 at 60 min. Data were acquired on an ICS5000+ (Thermo Fisher Scientific) and processed using Chromeleon 7.2 CDS (Thermo Fisher Scientific).

[0056] 1.7. SD-1 methylation test:

[0057] Add 10 mg of SD-1 to 1 mL of a NaOH-DMSO suspension. Slowly add 1 mL of CHI to 31 mL in an ice-water bath and continue stirring for 30 minutes. Terminate the reaction with 1 mL of distilled water, place the reaction solution in a dialysis bag, dialyze for 48 hours, and then freeze-dry. Repeat the above steps for secondary methylation. Add an equal volume of dichloromethane for extraction three times, and pool the organic phases. Drain under nitrogen, reconstitute with 1 mL of distilled water, and freeze-dry. Add 1 mL of 2 mol / L TFA to LT-1 and hydrolyze at 120°C for 3 hours. Then, add anhydrous ethanol and repeatedly evaporate to remove all TFA. Add 1 mL of 30 mg / mL NaBH₄ to the hydrolyzed and evaporated polysaccharide sample and stir under magnetic stirring at room temperature for 12 hours. After the reaction is complete, add methanol several times and repeatedly evaporate to remove all NaBH₄. Add 0.5 mL each of acetic anhydride and anhydrous pyridine to the reduced polysaccharide sample and react at 100°C for 2 hours. The samples were freeze-dried and dissolved in CHCl3 for GC-MS analysis.

[0058] 1.8. Nuclear Magnetic Resonance (NMR) Detection of SD-1:

[0059] 20 mg of SD-1 was dissolved in 500 μL of D2O in a NMR tube and the NMR spectrometer (Bruker Avance III 600 MHz) was used to record the 1 H NMR, 13 C NMR, HSQC, 1 H- 1 HCOSY, HMBC.

[0060] 1.9. Scanning electron microscope (SEM) detection:

[0061] A small amount of SD-1 was placed on the sample stage. After being gold-sprayed in a vacuum sputtering apparatus, scanning electron microscopy (SEM, JSM-6490LV, Japan) was used at an accelerating voltage of 20 kV to capture SEM images of the surface morphology of each sample.

[0062] 2. Results:

[0063] 2.1. Analysis of the physical and chemical properties and spatial structure of SD-1:

[0064] After the crude polysaccharide of Radix Saposhnikoviae was eluted with DEAE-52, it was detected by phenol-sulfuric acid method. Figure 1 As shown in A, the results showed that the total sugar content in the eluate under 0.1 mol / L NaCl conditions was the highest. The subsequent purification was further performed by Sephadex G-100 chromatography. The high performance gel permeation chromatography (HPGPC) spectrum of SD-1 is shown in Figure 1 As shown in B, through Figure 1As can be seen from B, the homogeneous polysaccharide is SD-1. The chemical property test results show that the total sugar content of SD-1 is 70.19%, the protein content is 1.51%, the uronic acid content is 21.58%, and the molecular weight is 125.5kDa. Figure 1 As shown in C, SD-1 shows a classic polysaccharide infrared scanning spectrum, 3271 cm -1 The broad peak at 2928 cm is the -OH stretching vibration absorption peak in the polysaccharide sample. -1 The peak attributed to CH stretching vibration, 1736 cm -1 It is the carbonyl carbon-oxygen stretching vibration absorption peak, corresponding to the result of chemical detection of uronic acid in SD-1, 1071 cm -1 、1018 cm -1 The absorption peak at 762 cm is the stretching vibration absorption peak of COC. -1 The absorption band at indicates that SD-1 has a pyran ring; in addition, the monosaccharide composition test results are as follows Figure 1 As shown in Figure D, SD-1 is mainly composed of Ara (arabinose), Gal (galactose), GalA (galacturonic acid), Glc (glucose), Rha (L-rhamnose), GlcA (D-glucuronic acid), Xyl (xylose), and Man (mannose), with a molar ratio of 27.10%, 35.35%, 21.72%, 8.97%, 3.81%, 1.67%, 0.81%, and 0.56%; among them, the content of GalA is consistent with the uronic acid test results. The above results preliminarily determine that SD-1 is an acidic homogeneous polysaccharide mainly composed of Ara, Gal, GalA, and Glc. The present invention also performed SEM examination on SD-1 to observe its surface morphology, and the results are as follows. Figure 1 As shown in Figure E, SDP-1 presents lamellar and porous structures, which are common surface morphologies of polysaccharides, indicating that there is an attractive effect between the functional groups on the polysaccharide surface, which can cause the polysaccharide chains to aggregate.

[0065] 2.2. Analysis of monosaccharide residues in SD-1:

[0066] The present invention carried out methylation treatment on the SD-1 sample and then analyzed it by gas chromatography-mass spectrometry (GC-MS). The analysis included total ion chromatography (TIC) and secondary mass spectrometry (MS / MS) identification. The total ion chromatogram was as follows: Figure 2 The specific results are shown in Table 1.

[0067] Table 1. Methylation data of SD-1

[0068] Linkage types Name of derivative Mass fragments (m / z) Molar ratio t-Rha(p) 1,5-di-O-acetyl-6-deoxy-2,3,4-tri-O-methyl mannitol 59,72,89,102,115,118,131,145,162,175 1.12 t-Ara(f) 1,4-di-O-acetyl-2,3,5-tri-O-methyl arabinitol 71,87,102,118,129,145,161 15.85 1, 2-Rha(p) 1,2,5-tri-O-acetyl-6-deoxy-3,4-di-O-methyl mannitol 89,100,115,130,131,175,190 1.26 t-Glc(p)-UA 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol 73,89,102,118,147,162,163,207 1.04 t-Glc(p) 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol 87,102,118,129,145,161,162,205 1.01 t-Gal(p) 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol 87,102,118,129,145,161,162,205 3.31 1,5-Ara(f) 1,4,5-tri-O-acetyl-2,3-di-O-methyl arabinitol 87,102,118,129,162,189 6.29 1,2,4-Rha(p) 1,2,4,5-tetra-O-acetyl-6-deoxy-3-O-methyl mannitol 88,101,117,130,143,190,203 1.80 1,3-Gal(p) 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl galactitol 87,101,118,129,161,202,234 9.75 1,4-Gal(p)-UA 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol 87,99,102,115,118,131,162,175,235 24.19 1,4-Glc(p) 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol 87,102,113,118,129,162,233 14.92 1,6-Gal(p) 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl galactitol 87,99,102,118,129,162,189,233 7.31 1,3,4-Gal(p)-UA 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl galactitol 87,118,131,143,185,205,307 1.27 1,4,6-Gal(p) 1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl galactitol 85,102,118,127,159,162,201,261 1.17 1,3,6-Gal(p) 1,3,5,6-tetra-O-acetyl-2,4-di-O-methyl galactitol 87,101,118,129,160,189,234 8.61 1,3,4,6-Gal(p) 1,3,4,5,6-penta-O-acetyl-2-O-methylgalactitol 97,118,129,139,160,333 1.10

[0069] From the composition of the methylation test in Table 1, it was found that the polysaccharide was composed of various residues of various monosaccharides, among which 1,4-GalA was the most abundant, and it was judged to be an acidic heteropolysaccharide, which confirmed the results of the monosaccharide composition, among which galacturonic acid, galactose, arabinose and glucose accounted for a relatively high proportion.

[0070] 2.3. NMR and structural characterization of SD-1:

[0071] Comprehensive NMR spectrum analysis shows that 1,4-GalA has the highest proportion among all monosaccharide residues, which is judged to be the main part of the main chain. In addition, it contains a large number of galactose residues, forming glycosidic bonds at positions 3 and 6 of the residues. The methylation results show that there are four terminal sugars, most of which are t-Ara, indicating that arabinose forms the main branch end of the polysaccharide. The composition and structure of the heteropolysaccharide were confirmed by one-dimensional and two-dimensional nuclear magnetic resonance spectra. In the nuclear magnetic hydrogen spectrum 1 Multiple anomeric hydrogen H1 signals were detected within the anomeric region of the H NMR spectrum (4.3-5.8 ppm), with the primary peaks identified at 5.33, 5.18, 5.09, 5.02, 4.90, 4.62, and 4.42 ppm. This confirms the presence of both α- and β-configured residual sugars in this polysaccharide, with a rich variety of residues, consistent with the methylation results in Table 1. The 3.0-4.3 ppm range of the H NMR spectrum represents alkyl proton signals from residual sugar structures, excluding anomeric hydrogens. The signals in this region are numerous and overlap significantly, a typical characteristic of H NMR spectra of polysaccharides.

[0072] C-NMR 13 In C NMR, uronic acid carbonyl signals were detected in the low-field region of 170-200 ppm, at 175.08 and 170.65 ppm, respectively. The anomeric region of the C-NMR spectrum (90-110 ppm) exhibited multiple primary anomeric carbon C1 signals, at 109.24, 107.41, 107.09, 104.08, 100.40, and 99.55 ppm, respectively. The high number of anomeric carbon peaks may be due to the high number of residual sugars. Multiple carbon peaks were observed in the 60-80 ppm range, excluding the anomeric carbon. Among these, multiple signals near 61.2 ppm were attributed to C6 or C5 methylene -CH2- secondary carbons outside the residual sugar ring.

[0073] Two-dimensional NMR spectroscopy was used to analyze the composition and structural details of this polysaccharide. In the HSQC 2D NMR spectrum, multiple major H1 / C1-related signals were identified in the anomeric region at 5.34 / 99.53, 5.18 / 109.20, 5.08 / 107.04, 5.05 / 107.04, 5.02 / 107.39, 4.89 / 100.12, 4.61 / 103.22, 4.46 / 103.21, 4.41 / 102.75, and 4.40 / 103.22 ppm, respectively. By integrating signals from multiple NMR spectra and combining them with peak signals from research literature, the major anomeric positions and residues were comprehensively identified, as shown in Table 2.

[0074] By combining multiple two-dimensional NMR spectra, the positions and structural correspondence of the carbon-hydrogen signals of various residual sugars in the polysaccharide structure were assigned in sequence. For example, the relatively low-field proton signal of 5.18 ppm was determined to be assigned to the anomeric H1 of the arabinose residue α-t-Araf-1→. By identifying the hydrogen-hydrogen related COSY two-dimensional spectrum, a strongly related signal at 4.15 ppm was found, confirming the positions of H1 and H2 at 5.18 and 4.15 ppm, respectively. The HSQC spectrum verified the signals at 4.15 / 81.18 and the C2 signal at 81.18 ppm. The COSY two-dimensional spectrum further found the related signals at 4.15 / 3.87, and the HSQC confirmed the H3 / C3 at 3.87 / 76.41. The same method was used to determine the position information of H4 / C4 and H5 / C5 at 4.02 / 83.81 and 3.76(3.82) / 61.12. A similar assignment approach was applied to the other residue assignments to form the corresponding residue table 2.

[0075] It is worth noting that careful observation of the NMR spectrum and combination with the data can also reveal the presence of some methylated galacturonic acid residue information, with the COOMe methyl ester group formed by the carboxyl group at position 6. In the HSQC spectrum, signals of 5.08 / 70.36 and 4.70 / 71.47 were found, which were judged to be the hydrogen-carbon signal information at position 5 of the methylated and unmethylated galacturonic acid. The HMBC two-dimensional spectrum found hydrogen-carbon related signals of 5.08 / 170.73 and 3.73 / 170.68, confirming that the methyl proton signal of the methylated galacturonic acid was at 3.73 ppm, while the carbonyl carbon position signal of the residue was 170.6 ppm. In addition, combined with the carbon spectrum information, relevant signal information of the unmethylated galacturonic acid can also be found, with the carbonyl carbon position at 175.6 ppm. Notably, a small amount of methylated hydroxyl signals can also be detected in the NMR. Based on the HSQC signals at positions 3.42 / 59.94 and 3.22 / 81.95, combined with literature information, it is speculated that these signals represent the methyl and H4 / C4 positions of the terminal 4-MeO-GlcA residue, which is present in small quantities. Considering the low abundance of this methylated residue (1.04%), it is not labeled in the NMR spectrum or residue table. Based on the literature, it is speculated that its likely location is the terminal group of the galactoarabinose polysaccharide, a small acidic terminal sugar. Furthermore, combined with the common arabinose signal region and analysis of multiple NMR sites and the 5.18 ppm anomeric proton of the arabinose residue α-t-Araf-1→, two major anomeric NMR sites for this terminal group were identified, generating signals at positions E and E'. These signals were confirmed by HMBC signals as the 3-position of the galactose linkage and the 5-position of the arabinose linkage, respectively.

[0076] Table 2 Main residues of SD-1 1 H and 13 C NMR chemical shift.

[0077]

[0078] Example 2 Protective Effect of SD-1 on APAP Liver Injury in Mice

[0079] 1.1. Animals and Experimental Design

[0080] Fifty C57BL / 6J male mice (8 weeks old, 18-22 g) were purchased from Changchun Yisi Laboratory Animal Technology Co., Ltd. and housed in an SPF-grade environment at 25 ± 2°C with a 12 h / 12 ​​h light / dark cycle. Mice had free access to drinking water and feed. After one week of acclimatization, the mice were randomly divided into five groups (n = 8). The mice were divided into two groups: the control group (control group), which was given the same dose of normal saline; the APAP group (400 mg / kg), which was given the same dose of normal saline; the APAP+N-acetylcysteine ​​(NAC, 150 mg / kg); the APAP+SD-1-L (100 mg / kg); and the APAP+SD-1-H group (200 mg / kg). From day 1 to day 7, the control and APAP groups were given 0.01 ml / g normal saline by gavage; the APAP+NAC group was given 150 mg / kg NAC by intraperitoneal injection; the APAP+SD-1-L group and the APAP+SD-1-H group were given 100 mg / kg or 200 mg / kg SD-1 by gavage, respectively. On day 8, all groups except the control group were given APAP 400 mg / kg. After 24 hours, the mice were euthanized, and their serum, liver tissue, and feces were collected, and the relevant data were recorded and analyzed.

[0081] 1.2. H&E staining:

[0082] Liver tissue from each mouse group was fixed in 4% paraformaldehyde and then dehydrated. After embedding, serial sections were sectioned at 5 μm thickness. Paraffin sections were stained with hematoxylin for 10 minutes and then with eosin for 2 minutes to prepare H&E-stained sections.

[0083] 1.3. Immunohistofluorescence staining (IF):

[0084] Paraffin sections were deparaffinized and then dehydrated using a gradient process. Endogenous peroxidases were blocked with 3% hydrogen peroxide. After antigen retrieval, sections were permeabilized with 0.3% Triton for 15 minutes at room temperature. Sections were blocked with 10% NGS and incubated with antibodies overnight at 4°C. Fluorescent secondary antibodies were then added and incubated at room temperature in the dark for 1 hour. Cell nuclei were labeled with DAPI fluorescent dye and observed and photographed under a microscope. Antibodies for TNF-α and IL-6 were purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0085] 1.4. Liver metabolomics analysis

[0086] Six randomized liver samples were collected from each group for metabolite extraction. Analysis was performed using a UHPLC-Q Exactive system. Raw data were imported into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) to generate a data matrix of retention time, mass-to-charge ratio, and peak intensity. MS and MSMS mass spectral information was then matched to the public metabolic databases HMDB (http: / / www.hmdb.ca / ) and Metlin (https: / / metlin.scripps.edu / ), as well as Metlin's own database, to obtain metabolite information. The preprocessed matrix files were then subjected to differential analysis. In addition, student's t-tests and fold-difference analyses were performed. Differential metabolites were selected based on the variable weight (VIP) obtained from the OPLS-DA model and the student's t-test p-value. Metabolites with a VIP > 1 and a P < 0.05 were considered differential metabolites. The differential metabolites were annotated with metabolic pathways using the KEGG database (https: / / www.kegg.jp / kegg / pathway.html) to obtain the pathways in which the differential metabolites participated. Data were analyzed on the MajorBio cloud platform (www.majorbio.com).

[0087] 1.5. Intestinal microbial analysis:

[0088] Six stool samples were randomly collected from each group to verify the purity and concentration of the extracted DNA. The 16S ribosomal RNA gene was amplified using primers and sequenced using the Illumina MiSeq platform. After splicing and alignment using FLASH 1.211 and Qiime 1.9.1 software, highly similar sequences (>97% similarity) were grouped into operational taxonomic units (OTUs) using Uparse 7.0 software and species annotation was performed using the SILVA database. Data were analyzed online on the MajorBio cloud platform (www.majorbio.com).

[0089] 1.6. Biochemical tests of serum and liver:

[0090] Blood was collected from the ocular vein of mice and centrifuged at 3000 rpm for 15 minutes at 4°C. Serum was collected and stored at -80°C. Mouse liver tissue was minced, homogenized on ice with 1 mL of pre-chilled lysis buffer, and centrifuged at 12000 g for 10 minutes at 4°C. The supernatant was collected and protein concentration was determined using the BCA assay and adjusted to a consistent concentration. Serum levels of TNF-α, IL-6, ALT, and AST were assayed according to the corresponding kit instructions. Superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) were assayed in liver tissue. ALT and AST were purchased from Nanjing Jiancheng Biological Co., Ltd. TNF-α, IL-6, SOD, MDA, and GSH were purchased from Beyotime Biological Co., Ltd.

[0091] 1.7. Western Blot Analysis

[0092] Mouse liver tissue was collected and ground in RIPA lysis buffer. The ground sample was centrifuged at 5000 rpm for 10 minutes at 4°C, and the supernatant was collected. The protein concentration in the supernatant was determined using a BCA assay kit. 5× SDS loading buffer was then added to the supernatant, boiled at 100°C for 10 minutes, and stored at -80°C until further use. Protein electrophoresis was performed at 80 V for 30 minutes, then the voltage was adjusted to 120 V for another 100 minutes to separate the proteins. After electrophoresis, the proteins were transferred to a PVDF membrane. After transfer, the membrane was blocked with 5% skim milk for 2 hours at room temperature. After blocking, the membrane was incubated with the corresponding primary antibody overnight at 4°C, followed by incubation with the secondary antibody for 2 hours at room temperature. Finally, the membrane was developed using ECL chemiluminescent solution and images were captured using a chemiluminescent gel imager. The grayscale values ​​of the protein bands were analyzed using Image J software. Antibodies include: anti-NF-κB p65, anti-IκB, anti-phosphor-IκB, anti-TLR4, anti-MyD88, anti-Nrf2, anti-HO-1, anti-5-LOX, anti-COX-2, anti-GAPDH, and anti-Lamin B. All antibodies were purchased from Wuhan Proteintech Biotechnology Co., Ltd.

[0093] 1.8. Data Analysis:

[0094] All figures were generated using GraphPad Prism 6 software. Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison post hoc test. Data are expressed as mean ± SEM. (* or #) p<0.05 indicates The difference is significant, ( ** or ##) p < 0.01 indicates extremely significant differences.

[0095] 2. Results:

[0096] 2.1. SD-1 alleviates APAP-induced liver injury in mice:

[0097] The experimental process is as follows Figure 4 A. Then the ALT and AST levels in the serum of each group of mice were detected, as shown in Figure 4 As shown in B and C, compared with the control group, the levels of ALT and AST in the serum of APAP-induced mice were significantly increased (P<0.001). The levels of ALT and AST in the NAC group were significantly decreased (P<0.001). Pretreatment with SD-1 significantly inhibited the levels of ALT and AST (P<0.001). Figure 4 H&E pathological examination in D showed that the hepatocytes around the central vein of the liver in the APAP group mice showed extensive necrosis, efflorescence of the cell nucleus, and increased cytoplasmic eosinophilia. Inflammatory cell infiltration was observed in and around the necrotic area. Round apoptotic bodies appeared in the hepatocytes, indicating apoptosis in the liver. A large number of round or oval fat vacuoles appeared in the hepatocytes around the central vein, suggesting that APAP-induced liver injury is accompanied by significant lipid metabolism disorders. However, the liver injury of mice in the APAP+SD-1-H group was improved, including a decrease in inflammatory infiltration, apoptotic bodies, and fat vacuoles. These results indicate that oral administration of SD-1 can help prevent APAP-induced drug-induced acute liver injury.

[0098] 2.2. SD-1 inhibits inflammatory factors and oxidative stress:

[0099] TNF-α and IL-6 are both pro-inflammatory cytokines and usually play an important role in inflammatory responses. Figure 5 A and B show that after APAP induction, the levels of TNF-α and IL-6 in the mouse serum were significantly increased (P<0.001). After NAC or SD-1 pretreatment, the inflammation of the mice was significantly alleviated, and the levels of TNF-α and IL-6 were significantly suppressed (P<0.001). Subsequently, the present invention also examined the expression of TNF-α and IL-6 in the liver by immunofluorescence. The results are as follows Figure 5 As shown in Figures C, D, and E, compared with the control group, the fluorescence intensities of TNF-α and IL-6 were significantly increased in the APAP group (P < 0.001). In contrast, the fluorescence intensities of TNF-α and IL-6 were significantly decreased in the APAP + SD-1-H and APAP + SD-1-L groups (P < 0.01, P < 0.05, respectively). These results suggest that SD-1 has significant anti-inflammatory effects. Therefore, the present invention further examined inflammatory pathways using Western blot, among which the TLR4 / NF-κB signaling pathway plays a central role in the inflammatory response. Figure 5The results of F, G, and H showed that the expression of TLR4 and MyD88 proteins in the APAP group was significantly increased (P<0.05, P<0.001), and SD-1-H significantly inhibited the expression of TLR4 and MyD88 proteins (P<0.05, P<0.001). Figure 5 In Figures I, J, and K, the APAP group showed a significant increase in IκB-α phosphorylation and NF-κBp65 translocation into cells (P<0.05, P<0.01). SD-1-H not only significantly inhibited IκB-α phosphorylation in the mouse liver but also inhibited NF-κBp65 nuclear translocation (P<0.05, P<0.01). These results suggest that APAP can activate the TLR4 / NF-κB signaling pathway, inducing an outbreak of inflammatory factors in the liver, and that SD-1 achieves its anti-inflammatory effects by inhibiting this pathway.

[0100] Inflammation and oxidative stress are two closely related biological processes. APAP induces an outbreak of inflammatory factors, which further aggravates oxidative stress damage in the liver. Figure 5 Results from L, M, and N studies showed that APAP-induced liver damage significantly decreased SOD and GSH levels (P<0.001), while MDA levels significantly increased (P<0.001). Treatment with NAC and SD-1-H significantly enhanced liver antioxidant capacity, with SOD and GSH levels significantly increased (P<0.001) and MDA levels decreased (P<0.001). These results suggest that APAP-induced liver damage increases oxidative stress, and that SD-1 also has antioxidant effects. Subsequently, the present invention further explored the underlying mechanism by which SD-1 regulates oxidative stress. The Nrf2 / HO-1 pathway plays a key role in cellular antioxidant defense. Under oxidative stress, Nrf2 is activated and translocated to the nucleus, where it forms a heterodimer with Maf proteins and binds to the antioxidant response element (ARE), exerting its antioxidant effects. Furthermore, Nrf2 activation promotes transcription of the HO-1 gene. HO-1 catalyzes the degradation of heme into biliverdin, carbon monoxide (CO), and free iron, which have antioxidant and anti-inflammatory effects. Figure 5 O, P, and Q results showed that APAP-induced Nrf2 was activated, with its protein expression increased (P < 0.05). Pretreatment with SD-1-H further increased Nrf2 and HO-1 protein expression compared to the APAP group (P < 0.05, P < 0.01). These results suggest that SD-1 can exert its antioxidant effects by activating the Nrf2 / HO-1 pathway.

[0101] 2.3. Effects of SD-1 on metabolites in APAP-induced liver injury in mice:

[0102] Metabolomics sequencing of the liver was used to screen for differential metabolites in the regulation of APAP-induced acute liver injury in mice by SD-1. The results of principal component analysis showed that ( Figure 6 A), the control group, APAP group, and APAP+SD-1-H group showed obvious clustering, indicating that there were differences in metabolite expression between the groups. According to Upset analysis ( Figure 6 B) shows that the number of metabolites regulated by the Control group, APAP group, and APAP+SD-1-H group were 1281, 1249, and 1294, respectively. There were 1222 metabolites regulated in common by the three groups. Based on the comparison with the HMDB database, the present invention classified the identified metabolites. The results showed that the metabolites were mainly divided into the following categories ( Figure 6 C), among which Lipids and lipid-like molecules accounted for the highest proportion, with a total of 314 metabolites identified, accounting for 29.79% of the total metabolites. The second largest group was Organic acids and derivatives, with a total of 269 metabolites identified, accounting for 25.52% of the total metabolites. The metabolite heatmap results of each group are shown in Figure 2. Figure 6 As shown in Figure D, the APAP+SD-1-H group essentially reversed the APAP-induced metabolite changes. These results demonstrate that SD-1 can regulate the changes in liver metabolites in APAP-induced acute liver injury in mice.

[0103] 2.4.SD-1 inhibits acute liver injury by regulating the arachidonic acid metabolic pathway:

[0104] Through further analysis, according to the volcano map, Venn diagram results show that ( Figure 7 AD), there were 204 upregulated metabolites and 217 downregulated metabolites in APAP vs Control. There were 191 upregulated metabolites and 183 downregulated metabolites in APAP+SD-1-H vs APAP. Subsequently, the present invention constructed a dataset of metabolites downregulated in APAP vs Con and metabolites upregulated in APAP vs APAP+SD-1-H, and performed KEGG metabolic pathway enrichment analysis ( Figure 7 E), the results showed that the enriched metabolic pathways mainly included biosynthesis of cofactors, purine metabolism and other metabolic pathways. The present invention also constructed a data set of metabolites upregulated in APAP vs Con and metabolites downregulated in APAP vs APAP+SD-1-H, and performed KEGG metabolic pathway enrichment analysis ( Figure 7F), the results showed that APPA-activated arachidonic acid metabolism, pyrimidine metabolism and other metabolic pathways were downregulated by SD-1-H. Studies have shown that arachidonic acid metabolism (AA) plays an important role in the pathogenesis of acute liver injury (ALI). Figure 7 G The results showed that the relevant metabolites in the APAP+SD-1-H group were significantly downregulated, including PGE2, PGA2, PGB2, 5-HETE, 5-OxoETE, 12-HETE, 15-HETE, 20-HETE, and Lecithin. AA is released from cell membrane phospholipids through phospholipase A2 (PLA2) under the stimulation of oxidative stress, inflammation, and toxins. The released AA promotes the production of various bioactive lipid mediators through cyclooxygenase (COX) and lipoxygenase (LOX), including prostaglandins (PGs) and epoxyeicosatrienoic acids (EETs), which are involved in inflammation, oxidative stress, and cell death, thereby aggravating the inflammatory response of ALI. The results are as follows Figure 7 HJ showed that APAP-induced liver protein expression of 5-LOX and COX-2 was significantly increased (P<0.01, P<0.001), while SD-1-H significantly inhibited their protein expression (P<0.05, P<0.01). These results suggest that SD-1 blocks AA signaling by reducing 5-LOX and COX-2 protein expression and simultaneously downregulates AA pathway metabolites associated with inflammation and oxidative stress (such as prostaglandins and hydroxyeicosatetraenoic acid).

[0105] In metabolomics research, VIP analysis can effectively identify metabolites with significant differences between different groups, and then further explore their potential biological significance. The present invention uses the OPLS-DA model to analyze the two groups of data and extract representative metabolites and their corresponding VIP values. In the comparison between the APAP and Con groups ( Figure 8A), many metabolites also had VIP_Oplsda values ​​exceeding 2, such as Enterolactone 3'-Sulfate with a VIP_Oplsda value of 3.1941; Cephalosporin C with a VIP_Oplsda value of 3.0549; and Panose with a VIP_Oplsda value of 2.8487. In terms of the arachidonic acid pathway, the VIP_Oplsda value of 5-Oxo-Eicosa-tetraenoic acid (5-Oxo-ETE) was 2.3402. Its significant VIP value indicated that this metabolite was significantly different between the APAP and Con groups, suggesting that the arachidonic acid pathway may play an important role in the metabolic changes between the two groups. When comparing the data of the APAP+SD-1-H and APAP groups ( Figure 8 B) found that several metabolites had VIP_Oplsda values ​​exceeding 2, indicating that these metabolites were highly significant in the inter-group differences. Further observation revealed that metabolites related to the arachidonic acid pathway, such as 5-Oxo-Eicosa-tetraenoic acid (5-Oxo-ETE), play a key role in the arachidonic acid pathway. 5-Oxo-ETE had a VIP_Oplsda value of 2.3402, indicating significant differences between the APAP+SD-1-H and APAP groups, further supporting the importance of the arachidonic acid pathway.

[0106] Subsequently, the present invention further established two metabolic sets, Figure 8 C indicates metabolites that were downregulated in APAP vs Con and upregulated in APAP+SD-1-H vs APAP. Figure 8 D represents metabolites that were upregulated in APAP vs Con and downregulated in APAP+SD-1-H vs APAP. Through in-depth analysis of the two groups of metabolites, it was found that in the APAP-induced liver injury model, the relevant metabolites in the arachidonic acid pathway were significantly activated, while the pathway was significantly inhibited after SD-1-H treatment. The present invention independently displays the metabolites in the arachidonic acid metabolic pathway that are significantly regulated by SD-1, and the results are as follows: Figure 8EM showed that prostaglandin E2 (PGE2), the terminal inflammatory factor in arachidonic acid metabolism, was also significantly decreased in the APAP + SD-1-H group (P < 0.001), further confirming the biological effects of SD-1 inhibition of this pathway. Furthermore, within the arachidonic acid metabolic pathway, different HETE (hydroxyeicosatetraenoic acid) derivatives are produced through specific enzymatic pathways and exhibit significant differences in their biological functions, some of which play a proinflammatory role. 5-HETE is produced by the oxidation of arachidonic acid catalyzed by 5-lipoxygenase (5-LOX) and is a precursor for the synthesis of leukotrienes (such as LTB4). It directly promotes neutrophil chemotaxis, degranulation, and the release of reactive oxygen species (ROS), exacerbating oxidative stress and hepatocellular necrosis. 5-OxoETE, produced by the oxidation of 5-HETE by 5-hydroxydehydrogenase (5-HEDH), is a potent chemotactic factor for eosinophils and neutrophils. It promotes inflammatory cell infiltration and increased vascular permeability by activating the OXER1 receptor. 12-HETE and 15-HETE play a key role in chronic inflammatory diseases such as asthma and liver fibrosis. In liver injury, 12-HETE and 15-HETE may exacerbate the inflammatory response by activating the NF-κB pathway. 20-HETE increases ROS production by activating NADPH oxidase, inducing endothelial cell damage and vasoconstriction, and exacerbating ischemic injury. In liver injury, elevated levels of 20-HETE are associated with disrupted sinusoidal blood flow and oxidative stress. SD-1 inhibits arachidonic acid-related HETEs to varying degrees (including 5-HETE, 5-oxoETE, 12-HETE, 15-HETE, and 20-HETE; P < 0.001). For example, 5-HETE, a key product of arachidonic acid metabolism, was significantly upregulated in the APAP-treated group (P < 0.001), while its expression was significantly decreased in the APAP + SD-1-H group (P < 0.001), suggesting that SD-1-H reduces the production of the proinflammatory mediator 5-HETE by regulating the activity of the rate-limiting enzyme in arachidonic acid metabolism. Notably, anti-inflammatory metabolites such as epoxyeicosatrienoic acids (EETs) did not show significant changes in the APAP+SD-1-H group, suggesting that SD-1-H may specifically inhibit proinflammatory branch pathways rather than the overall metabolic flux. These results suggest that APAP+SD-1-H alleviates APAP-induced hepatocyte oxidative stress and inflammatory flare by targeting the overactivation of the arachidonic acid metabolic pathway and reducing the release of inflammatory mediators. This mechanism is highly consistent with previous pathological observations showing reduced inflammatory infiltration in liver tissue and decreased serum ALT / AST levels, providing a molecular explanation for the hepatoprotective effects of SD-1-H.

[0107] Effects of SD-1 on intestinal microbiota in APAP-induced acute liver injury in mice:

[0108] The present invention uses 16S rRNA sequencing technology to explore the effect of SD-1 on the structure of intestinal flora. Figure 9 A) shows that the sequencing depth coverage is sufficient and the data reliability is good. Based on β diversity analysis, hierarchical clustering ( Figure 9 B) showed that the microbial communities in each group were highly similar, while principal component analysis (PCA, Figure 9 C) confirmed that there were significant differences in the bacterial community composition among the Control group, APAP group, and APAP+SD-1-H group, suggesting that SD-1 intervention can reshape the intestinal microbial community structure. α diversity analysis ( Figure 9 DF) showed that there was no statistically significant difference in species richness and evenness among the groups, but the Venn diagram ( Figure 10 A) The number of unique operational taxonomic units (OTUs) in the APAP+SD-1-H group (276) was significantly higher than that in the Control group (206) and the APAP group (182). At the phylum level, Firmicutes, Bacteroidota, and Actinobacteriota were the core bacterial communities in each group, while at the genus level, norank_f__Muribaculaceae, Lactobacillus, and Allobaculum were the main bacterial communities ( Figure 10 B, C). In summary, although SD-1 did not significantly change the overall diversity of the intestinal microbial community, β-diversity analysis and changes in the number of unique OTUs suggested that it could specifically regulate the microbial composition of mice with APAP-induced liver injury.

[0109] 2.6.SD-1 regulates the intestinal microbial community at the phylum and genus levels:

[0110] like Figure 11 The phylum classification statistics and comparison results of A showed that, overall, the dominant microbial communities of the three groups of samples were Firmicutes, Bacteroidota, Actinobacteriota, and Proteobacteria. Figure 11 The genus classification statistics and comparison results of B showed that, overall, the dominant microbial communities of the three groups of samples included norank_f__Muribaculaceae, Lactobacillus, etc. Subsequently, the present invention further analyzed the differential expression between the microbial communities of each group, and the results were as follows Figure 11C, Compared with the Control group, the relative abundance of Proteobacteria in the APAP group increased (P<0.01). In addition, among other non-dominant microbial communities, the relative abundance of Verrucomicrobiota in the APAP group decreased significantly (P<0.01). Figure 11 D analysis revealed a significant decrease in the abundance of Proteobacteria (P<0.01) and a significant increase in the abundance of Verrucomicrobiota (P<0.01) in the APAP+SD-1-H group. Related studies have shown that Verrucomicrobiota, which possess anti-inflammatory properties, are abundant in the intestinal mucosal lining of healthy individuals, and its Akkermansia subfamily, in particular, is known as an important probiotic for protecting intestinal function.

[0111] The present invention continues to investigate the regulation of mouse intestinal microbial communities by SD-1 at the genus level. Figure 12 As shown in A, the significantly increased microbial communities in the APAP group compared with the Control group mainly included Escherichia-Shigella (P < 0.01). Figure 12 In Figure B, the richness of the Akkermansia microbial community in the APAP+SD-1-H group was significantly increased (P<0.01), while the richness of the Escherichia-Shigella microbial community was significantly decreased (P<0.01). In addition, the LEfSe multi-level species hierarchical tree diagram showed the microbial community structure from the phylum to the genus level and its differences between groups ( Figure 12 C, D, LDA score>4.0). Inflammation-related Proteobacteria and Escherichia-Shigella were significantly enriched in the APAP group, while anti-inflammatory Verrucomicrobiota and Akkermansia were significantly enriched in the APAP+SD-1-H group.

[0112] It can be seen that SD-1 can regulate the structure of intestinal flora and significantly enrich Verrucomicrobiota, Akkermansia and other anti-inflammatory properties. It may form a bidirectional regulatory network of "intestine-liver axis" by synergistically regulating the structure of intestinal flora and downstream arachidonic acid metabolic pathways, thereby reducing hepatocyte oxidative damage and inflammatory cascade reactions.

[0113] 2.7. Correlation between intestinal microorganisms and pathological indicators and ecological network analysis:

[0114] Further analysis of the correlation between pathological indicators and intestinal microorganisms, such as Figure 13Results of A and B showed that at the phylum and genus levels, SOD and GSH were negatively correlated with Proteobacteria and Escherichia-Shigella (P<0.01, P<0.001), and positively correlated with Verrucomicroboita and Akkermansia (P<0.01). ALT, AST, TNF-α, IL-6, and MDA were positively correlated with Proteobacteria and Escherichia-Shigella (P<0.01, P<0.001), and negatively correlated with Verrucomicroboita and Akkermansia (P<0.05, P<0.01). Correlation between SD-1-regulated metabolites in AA and intestinal microorganisms ( Figure 13 C, D). At the phylum and genus levels, PGE2, PGA2, PGB2, 5-HETE, 5-OxoETE, 12-HETE, 15-HETE, 20-HETE, and Lecithin were positively correlated with Proteobacteria and Escherichia-Shigella (P<0.05 or P<0.001), and negatively correlated with Verrucomicroboita and Akkermansia (P<0.05 or P<0.01 or P<0.001). In addition, the present invention performed PICRUSt2 functional prediction analysis on intestinal microorganisms, showing that SD-1 affects many metabolic pathways through the intestinal flora, including the AA pathway. The results are as follows: Figure 14 As shown in A, compared with the Con group, the AA pathway in the APAP group was significantly increased (P<0.001), while the AA pathway in the APAP+SD-1-H group was significantly inhibited (P<0.05). This result was consistent with the results of metabolomics, and also confirmed that SD-1 inhibits liver damage based on the AA pathway.

[0115] Subsequently, the present invention simulated the intestinal microorganisms of mice as a whole ecological environment and analyzed the interaction between each group of microorganisms at the phylum level ( Figure 14B) Comparison of the topological coefficients with those of random networks revealed significant differences in the generated empirical networks. Comparison of the number of nodes and edges in each network revealed that the APAP group had higher numbers of nodes and edges than both the control and APAP+SD-1-H groups. This seems counterintuitive, as the complexity of the gut microbiome is positively correlated with stability. Analysis suggests this may be due to an outbreak of pathogenic bacteria within the gut microbiome during the short period of APAP-induced acute liver injury in mice. In particular, the increased complexity of harmful bacteria further exacerbates intestinal inflammation. Further analysis of the dynamics of microbial competition / cooperation revealed that the APAP+SD-1-H group had a decrease in positive links (from 59.3% to 50.0%) and an increase in negative links (from 40.7% to 50.0%) compared to the APAP group. It is speculated that SD-1 may promote competition among gut microbes, increasing the abundance of beneficial bacteria to competitively reduce the abundance of harmful bacteria, achieving a microbial community similar to that of the control group.

[0116] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Non-essential technical features can be added or removed according to actual needs to meet the requirements of different situations.

Claims

1. The use of fangfeng polysaccharide in the preparation of a medicament for preventing or treating liver damage, wherein the preparation method of the fangfeng polysaccharide comprises the following steps: Step 1. preparing a water extract of Saposhnikovia divaricata; Step 2. The water extract of Radix Saposhnikoviae was first precipitated with 50% ethanol, the supernatant was collected, and then precipitated with 75% ethanol. The precipitate was redissolved in water, filtered, and the supernatant was concentrated under pressure to remove proteins and pigments to obtain crude Radix Saposhnikoviae polysaccharide. Step 3. The crude polysaccharide from Radix Saposhnikoviae was subjected to gradient elution using a DEAE-52 cellulose column with a mobile phase of NaCl solution. The fractions with the highest sugar content were combined, and the NaCl solution was removed using a dialysis bag. Step 4. Further purification using Sephadex G-100 gel column chromatography to obtain fangfeng polysaccharide; The fangfeng polysaccharide is an acidic homogeneous polysaccharide with a molecular weight of 125.5 kDa. It is composed of multiple monosaccharides connected by glycosidic bonds. The monosaccharides include arabinose, galactose, galacturonic acid, glucose, L-rhamnose, D-glucuronic acid, xylose, and mannose. The molar ratios of each monosaccharide are 27.10%, 35.35%, 21.72%, 8.97%, 3.81%, 1.67%, 0.81%, and 0.56%; its main chain is composed of β-1,4-galacturonic acid and β-1,4-glucose, and the side chains contain α-arabinose ends.

2. The use according to claim 1, characterized in that Step 1: The steps of preparing the water extract of Saposhnikovia divaricata are as follows: crushing the Saposhnikovia divaricata, extracting it 2-3 times with hot water at 90-95° C., with a material-liquid ratio of 1:5, and each extraction time is 1-2 hours.

3. The use according to claim 1, characterized in that In step 2, the protein was removed by Sevag method and the pigment was removed by filtration using D101 resin.

4. The use according to claim 1, characterized in that In step 4, when purifying by Sephadex G-100 gel column chromatography, distilled water is used as the mobile phase for elution, and then each fraction is detected by an evaporative light detector. The fractions with the same peak time are combined and freeze-dried to obtain the Fangfeng polysaccharide.

5. The use according to claim 1, characterized in that The liver injury is drug-induced acute liver injury induced by acetaminophen.

6. The polysaccharide of Radix Saposhnikoviae is used in the preparation of a drug for treating or regulating intestinal flora imbalance, or in the preparation of a drug for treating or preventing enteritis; the preparation method of the polysaccharide of Radix Saposhnikoviae comprises the following steps: Step 1. preparing a water extract of Saposhnikovia divaricata; Step 2. The water extract of Radix Saposhnikoviae was first precipitated with 50% ethanol, the supernatant was collected, and then precipitated with 75% ethanol. The precipitate was redissolved in water, filtered, and the supernatant was concentrated under pressure to remove proteins and pigments to obtain crude Radix Saposhnikoviae polysaccharide. Step 3. The crude polysaccharide from Radix Saposhnikoviae was subjected to gradient elution using a DEAE-52 cellulose column with a mobile phase of NaCl solution. The fractions with the highest sugar content were combined, and the NaCl solution was removed using a dialysis bag. Step 4. Further purification using Sephadex G-100 gel column chromatography to obtain fangfeng polysaccharide; The fangfeng polysaccharide is an acidic homogeneous polysaccharide with a molecular weight of 125.5 kDa. It is composed of multiple monosaccharides connected by glycosidic bonds. The monosaccharides include arabinose, galactose, galacturonic acid, glucose, L-rhamnose, D-glucuronic acid, xylose, and mannose. The molar ratios of each monosaccharide are 27.10%, 35.35%, 21.72%, 8.97%, 3.81%, 1.67%, 0.81%, and 0.56%; its main chain is composed of β-1,4-galacturonic acid and β-1,4-glucose, and the side chains contain α-arabinose ends.

7. The use according to claim 6, characterized in that Step 1: preparing the water extract of Saposhnikovia divaricata is as follows: crushing the Saposhnikovia divaricata, extracting it 2-3 times with hot water at 90-95° C., with a solid-liquid ratio of 1:5, and each extraction time being 1-2 hours; step 2: removing protein by the Sevag method, and removing pigment by filtration and washing with D101 resin.

8. The use according to claim 6, characterized in that In step 4, when purifying by Sephadex G-100 gel column chromatography, distilled water is used as the mobile phase for elution, and then each fraction is detected by an evaporative light detector. The fractions with the same peak time are combined and freeze-dried to obtain the Fangfeng polysaccharide.

9. The use according to claim 6, characterized in that The intestinal flora imbalance refers to the intestinal flora imbalance caused by acetaminophen-induced drug-induced acute liver injury.

10. The use according to claim 6, characterized in that The fangfeng polysaccharide competitively reduces the abundance of harmful bacteria by promoting competition between intestinal microorganisms and increasing the abundance of Verrucomicrobiota and Akkermansia.

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