Application of Broussonetia papyrifera polysaccharide in preparing medicine for preventing or treating liver damage

Abstract

The present invention discloses a new use of paper mulberry polysaccharide, namely, its use in the preparation of drugs for preventing or treating liver damage. The inventors established a liver damage model in mice by using acetaminophen, and found that the pathological changes in the liver of mice given paper mulberry polysaccharide were significantly alleviated, and the levels of ALT, AST and inflammatory factors IL-6, IL-10 and TNF-α in the serum of mice with liver damage were significantly reduced. The levels of GSH, GSH-Px, CAT, and SOD in liver tissue were increased, and the accumulation of MDA was reduced. The content of CYP2E1 in the liver was reduced, while the content of GST and SULT1A1 was increased, thereby enhancing the liver's detoxification ability of APAP metabolites. In addition, paper mulberry polysaccharide can regulate intestinal flora and maintain intestinal homeostasis in mice with APAP liver damage. Therefore, paper mulberry polysaccharide can effectively alleviate APAP-induced drug-induced liver damage and has great potential in the development of liver-protecting drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant polysaccharides, and in particular relates to the use of paper mulberry polysaccharide in preparing a medicine for preventing or treating liver damage. Background Art

[0002] Acetaminophen (APAP) is currently the most commonly used antipyretic and analgesic drug worldwide, offering advantages such as a slow and long-lasting antipyretic effect, minimal irritation, and minimal allergic reactions. APAP is a classic drug for dose-dependent liver injury and is safe within therapeutic doses. However, overdose can cause severe liver damage and even acute liver failure.

[0003] In recent years, the incidence of APAP-induced liver injury has been increasing in my country. The main reasons are as follows: (1) The widespread use of single APAP preparations: the 2015 edition of the Chinese Pharmacopoeia includes 9 dosage forms of acetaminophen, and the 2015 edition of the Chinese Veterinary Pharmacopoeia includes 2 veterinary dosage forms. The wide range of use and individual differences have increased the possibility of APAP-induced liver injury; (2) The increase in APAP-containing preparations, including common medicines such as Quick-acting Cold Capsules, Kuai Ke, Gan Kang, and 999 Cold Relief. The mixed use of multiple medicines containing acetaminophen has resulted in the total intake exceeding the normal range, which is also the main cause of APAP liver injury. APAP-induced drug-induced liver injury (DILI) has gradually attracted everyone's attention.

[0004] The mechanism of APAP-induced DILI is complex, primarily involving the production of toxic metabolic substances, mitochondrial dysfunction, inflammation, and oxidative stress. When excessive APAP is consumed, the large amount of NAPQI produced during its metabolism exceeds the detoxification capacity of GSH, leading to GSH depletion. NAPQI continues to accumulate, forming covalent bonds with intracellular and mitochondrial proteins, causing mitochondrial damage and ultimately cell death.

[0005] Oxidative stress is a key cause of APAP-induced hepatotoxicity. During the detoxification of NAPQI, GSH depletion leads to redox imbalance in hepatocytes. The accumulation of oxidative products in hepatocytes causes oxidative stress, increasing superoxide anion production and the formation of peroxynitrite in tissues, leading to hepatocyte necrosis. Furthermore, APAP metabolism in the liver triggers oxidative stress, generating a large number of free radicals and electrophilic groups. Excessive free radicals cause peroxidation of unsaturated fatty acids in biological membranes, altering membrane fluidity and permeability and disrupting membrane integrity. Electrophilic groups bind to sulfhydryl groups on the endoplasmic reticulum membrane, leading to intracellular calcium imbalance, calcium overload, and cell death.

[0006] Inflammation is considered to be the main cause of liver tissue damage. The main characteristics of APAP-induced liver injury are hepatocellular necrosis and the release of cellular contents (including nuclear DNA fragments, high-mobility group proteins, mitochondrial DNA, uric acid and ATP). These stimulatory factors act as damage-associated molecular patterns and can transcriptionally activate proinflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-10) and chemokines (MCP-1, MIP-2 and IL-8), leading to the activation and recruitment of neutrophils and mononuclear macrophages to the damaged area of ​​the liver, causing an inflammatory response and secondary liver damage.

[0007] Bifendate is a hepatitis treatment drug developed in my country. As an intermediate in the synthesis of Schisandrae Chinensis C, it is commonly used to treat elevated transaminases caused by viral hepatitis and drug-induced liver injury. It protects liver cells and enhances the liver's detoxification function. Recent studies have shown that various traditional Chinese medicines and their active ingredients can improve or alleviate liver damage. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide an application of paper mulberry polysaccharide in the preparation of a drug for preventing or treating liver damage.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0010] Application of paper mulberry polysaccharide in the preparation of medicines for preventing or treating liver damage.

[0011] Drugs that prevent or treat liver injury can alleviate liver damage and maintain intestinal homeostasis.

[0012] Liver injury is drug-induced liver injury.

[0013] Drug-induced liver injury is caused by acetaminophen.

[0014] Paper mulberry polysaccharide is obtained by water extraction and alcohol precipitation from paper mulberry leaves and then purification.

[0015] The purity of paper mulberry polysaccharide is not less than 75%.

[0016] Paper mulberry polysaccharide includes three molecular weight polysaccharides, which are composed of seven monosaccharides.

[0017] The three molecular weights are 2413.725 kDa, 47.590 kDa, and 10.283 kDa respectively.

[0018] The seven monosaccharides are galactose: galacturonic acid: fucose: rhamnose: glucuronic acid: glucose: arabinose, and the molar ratio is 28.2:27.9:15:12.4:9.3:3.9:3.3.

[0019] Drugs for preventing or treating liver damage are oral preparations.

[0020] Through research, the inventors discovered a new use for paper mulberry polysaccharide: its use in the preparation of drugs for the prevention or treatment of liver injury. Using acetaminophen to establish a mouse model of liver injury, the inventors found that administration of paper mulberry polysaccharide significantly alleviated liver pathological changes and significantly reduced serum levels of ALT, AST, and the inflammatory factors IL-6, IL-10, and TNF-α in mice with liver injury. It also increased liver tissue levels of GSH, GSH-Px, CAT, and SOD, reduced MDA accumulation, and reduced liver CYP2E1 content. It also increased GST and SULT1A1 levels, thereby enhancing the liver's ability to detoxify APAP metabolites. Furthermore, paper mulberry polysaccharide can modulate intestinal flora and maintain intestinal homeostasis in mice with APAP-induced liver injury. Therefore, paper mulberry polysaccharide can effectively alleviate APAP-induced drug-induced liver injury and has great potential in the development of hepatoprotective drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a structural characterization diagram of paper mulberry polysaccharide, in which: A glucose standard curve; B polysaccharide molecular weight; C monosaccharide composition; D infrared spectrum.

[0022] Figure 2 This is a diagram showing the effect of paper mulberry polysaccharide on the liver tissue morphology of mice. In the figure: CON: normal group, APAP: model group, PD: positive drug control group (bifendate), L-BPP: low-dose paper mulberry polysaccharide group, M-BPP: medium-dose paper mulberry polysaccharide group, H-BPP: high-dose paper mulberry polysaccharide group.

[0023] Figure 3 This is a diagram showing the effects of paper mulberry polysaccharide on the liver tissue structure of mice. In the figure: CON: normal group, APAP: model group, PD: positive drug control group (bifendate), L-BPP: low-dose paper mulberry polysaccharide group, M-BPP: medium-dose paper mulberry polysaccharide group, H-BPP: high-dose paper mulberry polysaccharide group.

[0024] Figure 4 This is a diagram showing the effects of paper mulberry polysaccharide on serum biochemical indicators in mice, in the figure: A alanine aminotransferase (ALT), B aspartate aminotransferase (AST); CON: normal group, APAP: model group, PD: positive drug control group (bifendate), L-BPP: low-dose paper mulberry polysaccharide group, M-BPP: medium-dose paper mulberry polysaccharide group, H-BPP: high-dose paper mulberry polysaccharide group.

[0025] Figure 5This is a diagram of the effect of paper mulberry polysaccharide on liver inflammation indicators in mice. In the figure: A interleukin 6 (IL-6), B tumor necrosis factor α (TNF-α), C interleukin 10 (IL-10); CON: normal group, APAP: model group, PD: positive drug control group (bifendate), L-BPP: low-dose paper mulberry polysaccharide group, M-BPP: medium-dose paper mulberry polysaccharide group, H-BPP: high-dose paper mulberry polysaccharide group.

[0026] Figure 6 This is a diagram of the effect of paper mulberry polysaccharide on the oxidative indicators of mouse liver, in the figure: A malondialdehyde (MDA); B reduced glutathione (GSH); C glutathione peroxidase (GSH-PX); D total superoxide dismutase (SOD); CON: normal group, APAP: model group, PD: positive drug control group (bifendate), L-BPP: low-dose paper mulberry polysaccharide group, M-BPP: medium-dose paper mulberry polysaccharide group, H-BPP: high-dose paper mulberry polysaccharide group.

[0027] Figure 7 This is a diagram showing the effects of paper mulberry polysaccharide on the main metabolic enzymes in mouse liver. In the figure: A cytochrome P450 family member 2E1 (CYP2E1), B phenol sulfotransferase (SULT1A1), C mouse glutathione S-transferase (GST); CON: normal group, APAP: model group, PD: positive drug control group (bifendate), L-BPP: low-dose paper mulberry polysaccharide group, M-BPP: medium-dose paper mulberry polysaccharide group, H-BPP: high-dose paper mulberry polysaccharide group.

[0028] Figures 4 to 7 There are significant differences between the bar graphs of different characters, P < 0.05). DETAILED DESCRIPTION

[0029] 1. Materials

[0030] 1.1 Medicinal Materials

[0031] Fresh mulberry leaves were provided by Juhe Farming and Breeding Farmers’ Professional Cooperative in Gongguan Town, Hepu County, Guangxi Zhuang Autonomous Region, and dried in a cool place for later use.

[0032] 1.2 Experimental animals

[0033] Seventy 4-week-old SPF male KM mice were purchased from Changsha Tianqin Biotechnology Co., Ltd. with license number SCXK (Xiang) 2019-0014.

[0034] 1.3 Main Reagents

[0035] Acetaminophen (AR, 99.0%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; bifendate dripping pills (H11020980) were purchased from Beijing Union Pharmaceutical Factory; anhydrous ethanol, petroleum ether, ether, acetone, phenol, and concentrated sulfuric acid were purchased from Chengdu Kelong Chemical Co., Ltd.; glucose was purchased from Tianjin Damao Chemical Reagent Factory; n-butanol and chloroform were purchased from Guangdong Chemical Reagent Engineering Technology Research and Development Center; alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were purchased from Guilin Youlite Medical Electronics Co., Ltd.; interleukin-6 (IL-6), interleukin-10 (IL-10), tumor Necrosis factor α (TNF-α), cytochrome P450 family member 2E1 (CYP2E1), mouse glutathione S-transferase (GST), and mouse phenolsulfotransferase (SULT1A1) detection kits were purchased from Jiangsu Enzyme Immunity Industry Co., Ltd.; total superoxide dismutase (SOD), malondialdehyde (MDA), reduced glutathione (GSH), and glutathione peroxidase (GSH-PX) detection kits were purchased from Nanjing Jiancheng Bioengineering Institute; D101 macroporous resin was purchased from Tianjin Guangfu Fine Chemical Research Institute, and dialysis bags (8000-14000D) were purchased from Viskase Company, USA.

[0036] 2. Methods

[0037] 2.1 Extraction and purification of paper mulberry polysaccharide

[0038] (1) Preparation of aqueous extract. Broussonetia papyrifera leaves: pure water = 1:10 (m / v). Bring to a boil over high heat, then reduce to low heat and maintain a slight boil for 1 hour. Filter the filtrate with gauze and repeat the above steps three times. Combine the three extracts and concentrate to 500 ml using a rotary evaporator at 65°C under reduced pressure. Centrifuge at 3000 rpm for 10 minutes to remove impurities in the lower layer.

[0039] (2) Alcohol precipitation. Anhydrous ethanol: concentrate = 4:1 (V / V), stand at 4°C for 24 hours; centrifuge at 3000 rpm for 10 minutes to obtain paper mulberry polysaccharide precipitate;

[0040] (3) Protein removal. Dissolve the paper mulberry polysaccharide in an appropriate amount of pure water and use the sevage method (n-butanol: chloroform = 1:4), sevage reagent: polysaccharide solution = 1:4 (V / V). After sealing, stir in a magnetic stirrer for 20 minutes, centrifuge at 3000 rpm for 10 minutes, remove the upper liquid, repeat the operation 8-10 times until the middle protein layer disappears, evaporate under reduced pressure on a rotary evaporator until there is no organic reagent smell, and repeat step (2);

[0041] (4) Defatting. The precipitate: acetone = 3:1 (m / V), stirred in a magnetic stirrer for 20 min, centrifuged at 3000 rpm for 10 min, and the supernatant liquid was removed; the operation was repeated 2-3 times, and acetone, anhydrous ethanol, and ether were added in the same order as above;

[0042] (5) Depigmentation: Static adsorption method was used: the pretreated D101 resin and polysaccharide solution (5 mg / ml) were mixed at a ratio of 1:15 (m / V) and shaken at 500 r / min for 96 h at room temperature. After the shaking, the polysaccharide solution and the resin filler were separated by filtration and the polysaccharide solution was collected.

[0043] (6) Dialysis: Pour the eluate into the treated dialysis bag (the volume does not exceed 2 / 3 of the dialysis bag volume), clamp both ends with clamps, put it into a beaker containing 5000 mL of deionized water, and place it in a refrigerator at 4°C for 96 hours. Replace the deionized water every 12 hours. The dialyzed polysaccharide solution is evaporated under reduced pressure to an appropriate volume.

[0044] (7) Freeze-drying. The concentrated polysaccharide solution was frozen in a -80°C ultra-low temperature refrigerator for 12 h and then placed in a freeze dryer for freeze-drying to obtain paper mulberry polysaccharide.

[0045] 2.2 Physicochemical properties of polysaccharides

[0046] 2.2.1 Determination of polysaccharide content and monosaccharide composition

[0047] A standard curve was established using glucose as the reference substance, and the polysaccharide content was determined by the phenol-sulfuric acid method.

[0048] (1) Polysaccharide molecular weight

[0049] Polysaccharide molecular weight and purity were determined by HPGPC: Samples and standards were accurately weighed, prepared into a 5 mg / mL solution, centrifuged at 12,000 rpm for 10 minutes, and the supernatant filtered through a 0.22 μm microporous filter membrane. The sample was then transferred to a 1.8 mL injection vial. A BRT105-104-102 tandem gel column (8 x 300 mm) was used, with 0.05 M NaCl as the mobile phase, a flow rate of 0.6 mL / min, and a column temperature of 40°C. A 20 μL injection volume was used for detection using an RI-10A differential detector.

[0050] (2) Monosaccharide composition

[0051] Seventeen monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D glucosamine, N-acetyl-D galactosamine, guluronic acid, mannuronic acid) were prepared into standard stock solutions.

[0052] Prepare a precise concentration standard from each monosaccharide standard solution as a mixed standard. Determine the mass of each monosaccharide using absolute quantitation methods, and calculate the molar ratio based on the molar mass of the monosaccharides. Accurately weigh 10 mg of sample into an ampoule, add 10 ml of 3 M trifluoroacetic acid, and hydrolyze at 120°C for 3 hours. Accurately transfer the acid hydrolysis solution to a tube and blow dry with nitrogen. Add 10 ml of water and vortex to mix thoroughly. Aspirate 100 μL and add 900 μL of deionized water. Centrifuge at 12,000 rpm for 5 minutes, and analyze the supernatant using an ion chromatograph.

[0053] (3) Polysaccharide infrared spectroscopy analysis

[0054] 2 mg of sample and 200 mg of potassium bromide were accurately weighed and compressed into tablets. A blank control was prepared by compressing potassium bromide powder into tablets. The results were scanned and recorded using a Fourier transform infrared spectrometer FT-IR650 (Tianjin Gangdong Technology Development Co., Ltd.).

[0055] 2.3 Drug administration and modeling of experimental animals

[0056] Mice were randomly divided into a normal group, a model group, a low-dose (100 mg / kg) paper mulberry polysaccharide group, a medium-dose (200 mg / kg) paper mulberry polysaccharide group, a high-dose (400 mg / kg) paper mulberry polysaccharide group, and a positive drug (bifendate) control group and housed separately. The paper mulberry polysaccharide group received daily oral gavage for 14 days according to the dose, while the control and model groups received an equal volume of distilled water. The positive drug control group received bifendate at a dose of 150 mg / kg. Except for the control group, mice were fasted for 16 hours after the last dose and then orally administered with APAP (200 mg / kg) to establish a liver injury model. Twelve hours later, blood was collected from the orbital cavity and immediately dissected. The livers were weighed and recorded. The left lobe of the liver was fixed in formaldehyde for hematoxylin and eosin staining; the remaining livers were stored at -80°C for subsequent analysis.

[0057] 2.4 Liver tissue pathomorphological examination

[0058] The fixed liver tissue was embedded in paraffin and cut into sections with a thickness of 4-5 μm. The sections were stained with HE and observed under a microscope and photographed.

[0059] 2.5 Determination of blood biochemical indicators

[0060] ALT and AST in serum were analyzed using an automatic biochemical analyzer.

[0061] 2.6 Antioxidant capacity of the liver.

[0062] Detect liver GSH, MDA, SOD and GSH-Px levels according to the kit instructions

[0063] 2.7 Detection of liver inflammatory factors and major metabolic enzymes

[0064] The levels of liver IL-6, IL-10, TNF-α and major metabolic enzymes CYP2E1, SULT1A1, and GST were detected according to the kit instructions.

[0065] 2.8 16S sequencing of intestinal flora

[0066] 2.9 16S rDNA Sequencing of Cecal Contents

[0067] The purity and integrity of genomic DNA were tested after extraction from cecal contents. The universal primers 338F (5′-ACTCCTACGGAGGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) for 16S rRNA were used to amplify the V3-V4 hypervariable region. The PCR product was purified using an AxyPrep DNA gel recovery kit and quantified using QuantiFluor. TM -St. Petersburg (Promega, USA). PE libraries were constructed using the TruSeq™ DNA sample preparation kit according to the standard protocol of Shanghai Meiji Biopharmaceutical Technology Co., Ltd. and sequenced on the Illumina MiSeq PE300 platform. Observed taxonomic units (OTUs) were clustered at 97% similarity using UPARSE software, eliminating chimeric sequences. Principal coordinate analysis (PCoA) was performed using the R software package to visualize the beta diversity of the microbiome within the samples. The Wilcoxon rank sum test was used to compare bacterial taxonomy at the genus level between the two groups. Linear discriminant analysis (LDA = 3.5) and LDA effect size (LEfSe) were used to analyze the dominance of the bacterial communities within each group.

[0068] 3. Test results and analysis

[0069] 3.1 Structural characterization of paper mulberry polysaccharide.

[0070] With glucose as the reference substance, the content of paper mulberry polysaccharide was measured by phenol-sulfuric acid method and was 75% ( Figure 1 -A). The obtained paper mulberry polysaccharide consists of three molecular weight polysaccharides, namely 2413.725kDa, 47.590kDa, and 10.283kDa ( Figure 1 -B); Further studies have shown that the polysaccharide is composed of seven monosaccharides, and their composition and molar ratio are galactose: galacturonic acid: fucose: rhamnose: glucuronic acid: glucose: arabinose = 28.2: 27.9: 15: 12.4: 9.3: 3.9: 3.3 ( Figure 1 -C).

[0071] Infrared spectroscopy showed that the extracted paper mulberry polysaccharide had typical polysaccharide structural characteristics ( Figure 1 -D): Absorption band at 3600-3200cm -1 It is the stretching vibration absorption peak of -OH. The absorption peak in this area is the characteristic peak of sugars. -1 It is the stretching vibration absorption peak of OH, which is the characteristic peak of sugars. -1 There is an absorption peak at 1714cm, which may be attributed to CH stretching vibration. -1 There is an absorption peak at 1635m -1 There is an absorption peak at 1417cm, which may be attributed to crystal water. -1 There is an absorption peak at 1338cm, which may be attributed to CO stretching vibration. -1 There is an absorption peak at 1253cm, which may be attributed to the symmetrical stretching vibration of C=O. -1 1058cm -1 There is an absorption peak at 912cm, which may be attributed to OH angle vibration. -1 There is an absorption peak at 890cm, which may be attributed to the asymmetric ring stretching vibration of the pyran ring. -1 There is an absorption peak at , which may be attributed to the CH angle vibration of the β-end group diastereomer of the pyran ring.

[0072] 3.2 Protective effect of mulberry polysaccharide on mice with liver damage.

[0073] (1) Effect of mulberry polysaccharide on liver morphology in mice with liver injury.

[0074] The changes in liver morphology can reflect the degree of liver damage. The livers of mice in the normal group were reddish brown and soft in texture. The livers of mice in the model group were congested, uneven in appearance, and had spots. The livers of mice in the drug group showed varying degrees of recovery ( Figure 2 ).

[0075] (2) Effect of mulberry polysaccharide on the liver structure of mice with liver injury.

[0076] Normal tissue structure is the basis for ensuring organ function. The liver lobule structure of mice in the normal group was intact, the hepatic cords were neatly arranged, the liver cells were round and plump, and no degeneration or necrosis was observed. The liver lobule structure of mice in the model group was destroyed, the hepatic cords were almost invisible, the liver cell degeneration, the liver cell necrosis around the central vein and the liver cell nuclear dissolution disappeared, accompanied by a certain amount of bleeding. Different doses of paper mulberry polysaccharide groups had a certain recovery effect on the liver damage caused by APAP, especially the high-dose group (H-BPP) group, the liver tissue structure was basically normal ( Figure 3 ).

[0077] (3) Effects of mulberry polysaccharide on liver blood biochemistry in mice with liver injury.

[0078] The main feature of APAP-induced liver damage is liver cell necrosis. After liver cell necrosis, the cell contents will be released into the blood, causing changes in blood-related biochemical indicators. The levels of ALT, AST, ALP, TP, and ALB in the serum can directly reflect liver function. Compared with the control group, the serum ALT, AST, and ALP levels in the model group increased significantly, indicating that the model was successful, while the decrease in TP and ALB indicated that the ability of liver protein synthesis was affected; compared with the model group, the BPP-administered group significantly decreased the serum AST, ALT, and ALP levels, reaching a level comparable to that of bifendate. Moreover, high-dose mulberry polysaccharide (H-BPP) is better than bifendate ( Figure 4 ).

[0079] (4) Effects of mulberry polysaccharide on liver inflammation indicators in mice with liver damage.

[0080] IL-6, TNF-α, and IL-10 play an important role in the development of APAP-induced liver injury. APAP-induced IL-6 and TNF-α can lead to aggravated liver cell damage and even death. Compared with the control group, the IL-6, IL-10, and TNF-α factors in the model group were significantly increased. Compared with the model group, L-BPP and H-BPP can reduce the IL-6 content in the liver, but the effect is weaker than that of bifendate. M-BPP can reduce TNF-α in the liver of mice and restore it to normal levels. Different doses of BPP can reduce the IL-10 level in the liver of mice with liver injury, among which H-BPP can restore IL-10 to normal levels ( Figure 5 ). The above results indicate that paper mulberry polysaccharide has certain anti-inflammatory effects.

[0081] (5) Effects of Broussonetia papyrifera polysaccharide on liver oxidative indices in mice

[0082] Dysregulation of the body's oxidative and antioxidant systems is a key characteristic of APAP-induced liver injury. During oxidative stress, the enzymes most susceptible to changes in the enzymatic defense system are superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Catalase (CAT) is also abundant in the liver. Among non-enzymatic defense systems, changes in reduced glutathione (GSH) and malondialdehyde (MDA) are the most common.

[0083] Compared with the normal group, the MDA content in the model group increased significantly, while the content of various substances with antioxidant function in the main enzyme defense system and non-enzyme defense system decreased significantly, indicating that the liver has undergone severe oxidative stress. Compared with the model group, the MDA level in the BPP group decreased significantly, and H-BPP can restore the levels of GSH, GSH-Px, CAT, and SOD to the levels of the normal group ( Figure 6 ). This shows that paper mulberry polysaccharide has good antioxidant capacity.

[0084] (6) Effects of Broussonetia papyrifera polysaccharides on major metabolic enzymes in mouse liver

[0085] Chemical metabolism is an important detoxification mechanism for exogenous toxic compounds and can be divided into two sequential phases, primarily relying on Phase I and Phase II metabolic enzymes. In the first phase of APAP metabolism, cytochrome P450 (primarily CYP2E1) activates the exogenous compound through initial oxidation, forming a highly reactive toxic substance. The second phase, primarily the detoxification phase, involves Phase II enzymes such as glutathione transferase (GST) and sulfotransferase (SULT1A1), catalyzing the conjugation of the metabolites from the Phase I reaction with endogenous substrates (such as glutathione).

[0086] APAP generates the toxic substance NAPQI under the action of the liver CYP450 system (mainly CYP2E1). Compared with the normal group, the CYP2E1 content in the liver of the model group mice increased, while the SULT1A1 and GST contents decreased, resulting in a large accumulation of toxic NAPQI in the liver, inducing and aggravating liver damage in mice. BPP can reduce the activity of CYP2E1 enzyme and reduce the accumulation of NAPQI; at the same time, BPP can also increase the content of SULT1A1 and GST, enhancing the liver's detoxification ability of APAP ( Figure 7 ). This indicates that paper mulberry polysaccharide can alleviate liver damage in mice by enhancing the liver's detoxification ability of APAP and has good antioxidant capacity.

[0087] (7) Effect of mulberry polysaccharide on the intestinal flora of mice.

[0088] To evaluate the effects of BPP treatment on the species richness and diversity of the intestinal bacterial community in mice with APAP liver injury, the inventors analyzed the species and Shannon index observed in each sample. Rarefaction curves indicated that the sequencing data were reasonably reliable. The results showed that the bacterial richness and diversity of the intestinal microbiota in the APAP group were lower than those in the CON and BPP groups. The PCoA plot showed that the bacterial community in the feces of APAP-treated mice was significantly different from that of the untreated group, while the bacterial community in the BPP group was close to that of the untreated group. These results indicate that APAP causes an imbalance in the intestinal microbiota and that BPP restores APAP-induced changes in bacterial communities.

[0089] In addition, the distribution of bacteria at the phylum level showed that the relative abundance of Deferribacterota was significantly increased and the relative abundance of Firmicutes was significantly decreased after APAP treatment (P<0.05), while the relative abundance of Desulfobacterota and Deferribacterota in the fecal microbiota of mice in the BPP treatment group was significantly lower than that in the APAP group, and the abundance of Bdellovibrionota was significantly increased (P<0.01 or P<0.05).

[0090] At the genus level, the two major bacterial genera in APAP-treated mice showed a significant decrease in Lactobacillus and Odoribacter, but the relative abundance of Enterococcus, Bacteroides, norank_f__norank_o__Clostridia_UCG-014, Erysipelatoclostridium, Blautia, Colidextribacter, Gordonibacter, Eubacterium_fissicatena_group, norank_f__Eubacterium_coprostanoligenes_group, Eubacterium_nodatum_group, Family_XIII_AD3011_group, and Eubacterium_brachy_group Oscillibacter was significantly increased (P<0.01). Compared with the APAP group, the abundance of Corynebacterium, Prevotellaceae_UCG-001, Alloprevotella, Jeotgalicoccus, and Paenochrobactrum in the intestine of BPP-treated mice was significantly increased. Enterorhabdus, norank_f__norank_o__Clostridia_UCG-014, Erysipelatoclostridium, Gordonibacter, norank_f__Eubacterium_coprostanoligenes_group, Eubacterium_nodatum_group, Family_XIII_AD3011_group, Eubacterium_brachy_group, Candidatus_Stoquefichusnorank_f__Eggerthellaceae were significantly decreased (P<0.01 or P<0.05). However, BPP could reverse this change. Sequence Listing <110> Guangxi University <120> Application of Broussonetia papyrifera polysaccharide in preparing medicine for preventing or treating liver damage <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 actcctacgg agggcagcag 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 ggactachvg ggtwtctaat 20

Claims

1. Use of paper mulberry polysaccharide in the preparation of a drug for preventing or treating liver damage, characterized in that: The paper mulberry polysaccharide is obtained by extracting paper mulberry leaves with water, precipitating with alcohol and then purifying the leaves.

2. The use according to claim 1, characterized in that: The drug for preventing or treating liver damage can alleviate liver damage and maintain intestinal homeostasis.

3. The use according to claim 2, characterized in that: The liver injury is drug-induced liver injury.

4. The use according to claim 3, characterized in that: The drug-induced liver injury was caused by acetaminophen.

5. The use according to claim 4, characterized in that: The purity of the paper mulberry polysaccharide is not less than 75%.

6. The use according to claim 5, characterized in that: The paper mulberry polysaccharide includes three types of polysaccharides of molecular weight, which are composed of seven monosaccharides.

7. The use according to claim 6, characterized in that: The three molecular weights are 2413.725 kDa, 47.590 kDa, and 10.283 kDa, respectively.

8. The use according to claim 7, characterized in that: The seven monosaccharides are galactose: galacturonic acid: fucose: rhamnose: glucuronic acid: glucose: arabinose, and the molar ratio is 28.2: 27.9: 15：12.4：9.3：3.9：3.3。 9. The use according to claim 1, characterized in that: The medicine for preventing or treating liver injury is an oral preparation.

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