Extraction method of broussonetia papyrifera leaf polysaccharide and application of broussonetia papyrifera leaf polysaccharide in preparation of anti-porcine rotavirus drugs

Abstract

The invention discloses an extraction method of broussonetia papyrifera leaf polysaccharide and application of the broussonetia papyrifera leaf polysaccharide in preparation of an anti-porcine rotavirus drug. The molecular weight of the broussonetia papyrifera leaf polysaccharide is gt; and the molecular weight is 3500 Da. The extraction method of the broussonetia papyrifera leaf polysaccharide comprises the following steps: performing hot water extraction on broussonetia papyrifera leaf dry powder, performing concentration and deproteinization treatment on extract liquid to obtain supernate, performing dialysis purification on the supernate through a dialysis bag with the molecular weight cutoff of 3000-4000 Da, performing chromatography decoloration through macroporous resin, and collecting a polysaccharide elution peak; and concentrating the collected eluent, adding absolute ethyl alcohol for alcohol precipitation, centrifugally collecting the precipitate, and performing vacuum freeze drying to obtain the broussonetia papyrifera leaf polysaccharide. The extraction process of the broussonetia papyrifera leaf polysaccharide is optimized, and the broussonetia papyrifera leaf polysaccharide with the purity larger than or equal to 95 The broussonetia papyrifera leaf polysaccharide is applied to resisting porcine rotavirus infection for the first time, the growth performance of piglets is remarkably improved, diarrhea symptoms are relieved, and pigs are promoted to recover health.

Description

Technical Field

[0001] The present invention relates to a method for extracting polysaccharide, in particular to a method for extracting polysaccharide from paper mulberry leaves and application of the polysaccharide in preparing anti-porcine rotavirus drugs. Background Art

[0002] Rotavirus (RV) is one of the main pathogens causing viral diarrhea in young piglets. Globally, RV infection causes a 20%-30% annual mortality rate in piglets, accompanied by severe growth retardation and intestinal barrier dysfunction (manifested by villous atrophy and disruption of tight junction proteins). Current prevention and control measures primarily rely on vaccination and adjunctive antibiotic therapy. However, vaccines have limited serotype cross-protection and an immune gap period. Long-term antibiotic use can easily lead to drug resistance and imbalance in the intestinal microbiome, making it difficult to meet the demand for antibiotic-free farming.

[0003] In recent years, screening active ingredients with antiviral and intestinal repair functions from natural plants has become a research hotspot. Broussonetia papyrifera ) is a widely distributed medicinal plant, its leaves are rich in polysaccharides, flavonoids and alkaloid compounds. It is recorded that it has the effects of clearing heat and detoxifying, and anti-oxidation. Previous studies have shown that the polysaccharide in the leaves of the paper mulberry tree ( Broussonetia papyrifera Paper mulberry leaf polysaccharides (BPP) can alleviate intestinal inflammation by modulating immune and antioxidant pathways, but their anti-RV activity and mechanism of action have not been reported. Currently, the extraction process for BPP from paper mulberry leaves mostly uses water extraction and alcohol precipitation, but this suffers from low purity (<80%) and uneven molecular weight distribution. Furthermore, there are no systematic studies on its use in the fight against RV infection.

[0004] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute prior art known to ordinary technicians in the field. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for extracting paper mulberry leaf polysaccharide and its application in the preparation of anti-porcine rotavirus drugs, which solves the problem of low purity of paper mulberry leaf polysaccharide extracted by existing methods. The method of the present invention can improve the purity of polysaccharide (≥95%) through process optimization, and for the first time, paper mulberry leaf polysaccharide is applied to anti-RV, with very significant effect in anti-porcine rotavirus, safety, and few toxic and side effects.

[0006] In order to achieve the above object, the present invention provides the use of paper mulberry leaf polysaccharide in the preparation of an anti-porcine rotavirus drug, wherein the paper mulberry leaf polysaccharide has a molecular weight of >3500 Da.

[0007] Preferably, the application dosage of the paper mulberry leaf polysaccharide as an anti-rotavirus drug is 400-1200 mg / head, or the dosage as a functional additive in pig feed to play a relevant preventive role is 100-200 mg / kg.

[0008] The second object of the present invention is to provide an anti-porcine rotavirus drug, which comprises a paper mulberry leaf polysaccharide with a molecular weight greater than 3500 Da, and pharmaceutically acceptable excipients or auxiliary components.

[0009] Preferably, the purity of the paper mulberry leaf polysaccharide is ≥95%, and the protein residue is ≤2.5%; or / and the dosage form of the drug includes: tablets, powders, granules, premixes, oral solutions or sustained-release microcapsules.

[0010] The third object of the present invention is to provide a method for extracting polysaccharides from paper mulberry leaves, which comprises: extracting paper mulberry leaf dry powder in hot water at 55-65°C at a material-liquid ratio of 1g:30-40mL, concentrating and deproteinizing the extract to obtain a supernatant, purifying the supernatant through a dialysis bag with a molecular weight cutoff of 3000-4000 Da, decolorizing the supernatant through macroporous resin chromatography, and collecting the polysaccharide elution peak; concentrating the collected eluate, adding anhydrous ethanol for alcohol precipitation, collecting the precipitate by centrifugation, and vacuum freeze-drying to obtain paper mulberry leaf polysaccharide.

[0011] Preferably, the paper mulberry leaf powder is paper mulberry leaf powder that has passed through a 40-80 mesh sieve; or / and the hot water extraction is performed for 4-6 hours.

[0012] More preferably, the fresh paper mulberry leaves are dried at 60-70° C. and then crushed to 40-80 meshes to obtain paper mulberry leaf dry powder.

[0013] Preferably, the paper mulberry leaf dry powder is paper mulberry leaf dry powder that has passed through a 60-mesh sieve.

[0014] Preferably, the material-liquid ratio is 1 g: 35 mL; or / and, the hot water extraction temperature is 60° C.; or / and, the molecular weight cut-off of the dialysis bag is 3500 Da.

[0015] Preferably, the concentration and deproteinization treatment comprises the following steps: centrifuging the extract at 5-15° C., taking the supernatant and concentrating it by rotary evaporation to 1 / 5-1 / 4 of the original volume, adding 1.5-2.5 times the volume of Sevage reagent (chloroform: n-butanol = 4:1) for deproteinization, and taking the supernatant after centrifugation; or / and, the dialysis purification comprises placing the supernatant into a dialysis bag with a molecular weight cutoff of 3000-4000 Da, and dialyzing it with double distilled water for 48-72 hours; or / and, the macroporous resin is selected from AB-8 macroporous resin, and the eluent used is an ethanol solution; or / and, the alcohol precipitation comprises the following steps: concentrating the eluate, adding 8-10 times the volume of anhydrous ethanol, letting it stand at 4° C. for 36-48 hours, collecting the precipitate by centrifugation at 3000-4000 g, and vacuum freeze-drying it to obtain paper mulberry leaf polysaccharide.

[0016] More preferably, the centrifugation conditions of the extract are 5-15° C., 3000-4000 g centrifugation; or / and, the amount of the Sevage reagent is 2.0 times the volume of the rotary evaporation concentrate; or / and, the eluent is a 30% ethanol solution; or / and, in the Sevage reagent, the volume ratio of chloroform: n-butanol is 4:1; or / and, the amount of anhydrous ethanol in the alcohol precipitation is 9 times the volume of the eluent after concentration.

[0017] The method for extracting paper mulberry leaf polysaccharide and its application in preparing anti-porcine rotavirus drugs of the present invention solves the problem of low purity of paper mulberry leaf polysaccharide extracted by existing methods and has the following advantages: (1) The present invention optimizes the extraction process of paper mulberry leaf polysaccharide. Through hot water extraction, centrifugal concentration, deproteinization, dialysis purification and other steps, paper mulberry leaf polysaccharide with a purity of ≥95% is successfully obtained. The molecular weight of the polysaccharide is relatively large (>3500 Da) and the residual protein content is ≤2.5%. The extraction method of the present invention has a high purification efficiency. (2) This invention is the first to apply paper mulberry leaf polysaccharide to the prevention of porcine rotavirus infection. Paper mulberry leaf polysaccharide can significantly inhibit RV replication in IPEC-J2 cells by improving immune function and enhancing the antiviral immune response of cells. It also enhances the barrier function and reduces cell apoptosis by improving the antioxidant capacity of cells. (3) The present invention uses paper mulberry leaf polysaccharide as a functional feed additive to treat RV-infected piglets. In in vivo experiments on piglets, paper mulberry leaf polysaccharide significantly improved the growth performance of piglets, alleviated diarrhea symptoms, and promoted the recovery of pigs by enhancing immune response, improving intestinal barrier function, and reducing oxidative stress; (4) This invention reveals for the first time that paper mulberry leaf polysaccharide alleviates RV replication and infection in piglets by regulating immune function, and targets the PI3K / AKT / NRF2 signaling pathway to reduce RV infection-induced intestinal damage, providing a natural and efficient new strategy to solve the current RV prevention and control problems; (5) In the preparation of anti-porcine rotavirus drugs, the extracted paper mulberry leaf polysaccharide can be used as an effective antiviral component and has significant immunomodulatory and antioxidant effects. The paper mulberry leaf polysaccharide extracted by the present invention is a natural anti-porcine rotavirus drug with the advantages of high efficiency, safety, no toxic side effects and no drug residue, providing an innovative solution for antibiotic-free breeding; (6) The present invention verifies the synergistic effect of paper mulberry leaf polysaccharide in inhibiting viral infection and regulating host immune response through in vitro and in vivo models, laying a theoretical and practical foundation for the development of multi-target anti-rotavirus preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are the results of the in vitro antioxidant capacity of mulberry leaf polysaccharides.

[0019] Figure 2 These are the results of the effectiveness and safety evaluation of Broussonetia scoparia polysaccharide on IPEC-J2 cells.

[0020] Figure 3 Figure 3 is the inhibitory effect of Broussonetia scoparia polysaccharide on rotavirus-infected IPEC-J2 cells; A is the cell viability of each group at different infection times; B is the viral VP6 mRNA expression level of each group; C is the expression of cell tight junction proteins and apoptosis-related proteins detected by Western Blot.

[0021] Figure 4 Figure 3: The results of a study on the mechanism of action of mulberry leaf polysaccharide in alleviating rotavirus infection in IPEC-J2 cells; A is the inflammatory cytokines detected by ELISA; B is the mRNA expression levels of TLR3, RIG-I, MDA5, MAVS, ISG15 and interferon detected by real-time fluorescence quantitative PCR; C is the antioxidant capacity result; D is the expression analysis result of PI3K / AKT / NRF2 signaling pathway.

[0022] Figure 5 The results are for the validation of the PI3K / AKT / NRF2 signaling pathway inhibitors; A is the cell viability result; B is the protein expression level of the detected cell PI3K, p-AKT, AKT, p-NRF2, NRF2 and downstream antioxidant enzyme HO-1.

[0023] Figure 6 Effects of adding mulberry leaf polysaccharide to formula milk on the growth performance of rotavirus-infected piglets.

[0024] Figure 7 Effects of adding mulberry leaf polysaccharide to formula milk on diarrhea (A), rotavirus infection (B) and intestinal barrier function (C-D) in rotavirus-infected piglets.

[0025] Figure 8 Effects of adding mulberry leaf polysaccharide to formula milk on the immune function and antioxidant capacity of rotavirus-infected piglets. Results 1; (A) Immunoglobulin levels in serum and expression levels of inflammatory factors in jejunal mucosa; (B) mRNA expression levels of key factors in the cell pattern recognition receptor pathway and interferon in jejunal mucosa.

[0026] Figure 9 Effects of adding mulberry leaf polysaccharide to formula milk on the immune function and antioxidant capacity of rotavirus-infected piglets (result 2); (C) T-AOC, MDA content and CAT activity; (D) Western Blot analysis results and expression levels of key proteins in the PI3K / AKT / NRF2 signaling pathway. DETAILED DESCRIPTION

[0027] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0028] Note: If specific conditions are not specified in the examples, the experiments were conducted under conventional conditions or those recommended by the manufacturer. Instruments used without manufacturer information are commercially available. Raw materials and reagents used without manufacturer information are commercially available or can be prepared by known methods.

[0029] Throughout this disclosure, all features, such as values, amounts, amounts, and concentrations, specified in numerical ranges or percentage ranges are provided for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to encompass and specifically disclose all possible subranges and individual values ​​within those ranges (including integers and fractions).

[0030] The features described in this disclosure may be combined in any manner, and as long as there are no conflicts between the combinations of these features, all possible combinations should be considered within the scope of this specification. Each feature disclosed in this specification may be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the features disclosed are merely general examples of equivalent or similar features.

[0031] The test materials used in the following experimental examples are as follows: (1) The porcine RV used in the following experiments was the OSU serotype G5P9 strain (ATCC No.: VR-893), purchased from the National Veterinary Microbiology Collection Center; (2) Broussonetia papyrifera leaf polysaccharide was extracted and prepared from fresh Broussonetia papyrifera leaves collected from the Chengdu campus of Sichuan Agricultural University using the method of Experimental Example 1; (3) The IPEC-J2 cell line is maintained by our laboratory; (4) DMEM / F12 medium (Hai Kelong, China), fetal bovine serum (FBS, Gibco, USA), trypsin-EDTA digestion solution (Gibco, USA), double antibody (penicillin-streptomycin, Gibco, USA) and PBS buffer (Sevier, China) were used for cell culture; (5) CCK-8 cell viability assay kit (Biyuntian, China), BCA total protein assay kit (Biyuntian, China), and antioxidant assay kits (T-AOC, SOD, CAT: Jiancheng Biotechnology, China; GSH-Px: Biyuntian, China) were used for functional analysis; (6) ELISA kits (NSP4, RV-IgM: Nuoyuan Biotechnology, China; IFN-β, IFN-γ, IL-1β, IL-6, IL-10, TNF-α: Ruixin Biotechnology, China) were used to detect protein expression levels; (7) All Western Blot antibodies were purchased from CST, USA. The dilution ratio of all primary antibodies was 1:1000; HRP-labeled goat anti-rabbit secondary antibodies and goat anti-mouse secondary antibodies were diluted at a ratio of 1:5000. (8) Real-time fluorescence quantitative PCR primers (Sangon Biotech, China) and SYBR Green Master Mix (Dalian Takara Biotech, China) were used for gene expression analysis; (9) RNA extraction kit and reverse transcription kit (Takara Biotech, China) were used for sample processing.

[0032] Experimental Example 1 Extraction method and component detection of paper mulberry leaf polysaccharide The method for extracting polysaccharides from paper mulberry leaves provided by the present invention specifically comprises the following steps: (1) Raw material pretreatment: Fresh paper mulberry leaves were dried at 60°C and then crushed through a 60-mesh sieve to prepare paper mulberry leaf powder.

[0033] (2) Hot water extraction: Add double distilled water at a material-liquid ratio of 1 g: 35 mL (W / V) and extract in a 60°C water bath for 5 hours.

[0034] (3) Centrifugal concentration and deproteinization: The extract was centrifuged at 3500g for 5 minutes at 10°C to obtain the supernatant, which was concentrated by rotary evaporation to 1 / 5 of the original volume. Two volumes of Sevage reagent (chloroform: n-butanol = 4:1) were added for deproteinization, and the clarified solution was obtained by centrifugation at 5000g for 15 minutes.

[0035] (4) Dialysis purification: The clarified liquid was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed against double distilled water for 48 hours to remove small molecule impurities.

[0036] (5) Macroporous resin chromatography decolorization: The dialysate was concentrated and then chromatographed on an ÄKTA™ pure system AB-8 macroporous resin column (eluent: 30% ethanol) to collect the main polysaccharide peak.

[0037] (6) Alcohol precipitation and drying: After the eluate is concentrated, add 9 times the volume of anhydrous ethanol and let it stand at 4°C for 48 hours. Centrifuge at 3500g for 10 minutes to collect the precipitate, and vacuum freeze-dry for 24 hours to obtain the polysaccharide of the leaves of the Broussonetia sylvestris leaf, namely BPP.

[0038] Experimental Example 2 Detection of polysaccharide components in paper mulberry leaves 1. Polysaccharide purity determination (sulfuric acid-phenol method) 5 mg of Broussonetia papyrifera polysaccharide was dissolved in 1 mL of double-distilled water. Each experiment was repeated three times (n = 3), with three replicate wells per assay. The color reaction was performed in the dark, and the absorbance at 490 nm was measured using a microplate reader. The glucose standard curve (R 2 =0.996) to calculate the polysaccharide purity. The data were expressed as mean ± standard deviation (Mean ± SD). One-way analysis of variance (ANOVA) was performed using Excel 2020. The results are shown in Table 1.

[0039] 2. Determination of protein residue (BCA method) 5 mg of Broussonetia papyrifera polysaccharide was dissolved in 1 mL of double-distilled water. Each experiment was repeated three times (n=3), and each measurement contained three parallel wells. A standard curve (R) was established using bovine serum albumin (BSA) standards (0-200 μg / mL). 2 =0.998), the absorbance at 562 nm was measured after reaction at 60°C for 30 min, and the data were expressed as mean ± standard deviation. The results are shown in Table 1.

[0040] The results are shown in Table 1 below. The purity of the paper mulberry leaf polysaccharide extracted by the present invention is 95.7±3.2%, and the residual protein content of the paper mulberry leaf polysaccharide is 25±0.65 mg / g (2.51±0.06%).

[0041] Table 1 Polysaccharide component detection

[0042] Experimental Example 3 Analysis of the Antioxidant Capacity of Broussonetia papyrifera Leaf Polysaccharides in Vitro Ascorbic acid (VC) was used as a positive control to test the antioxidant activity of Broussonetia papyrifera polysaccharides, as follows: (1) ABTS + Free radical scavenging ability: ABTS free radical working solution (absorbance 0.70±0.02, 734 nm) was prepared and reacted with different concentrations of paper mulberry leaf polysaccharide (0-3.2 mg / mL) for 10 min, and the absorbance at 734 nm was measured.

[0043] (2) DPPH free radical scavenging ability: 0.2 mM DPPH ethanol solution was reacted with mulberry leaf polysaccharide (0-3.2 mg / mL) in the dark for 30 minutes, and the absorbance at 517 nm was measured.

[0044] (3) Ferric reducing power (FRAP): Prepare FRAP working solution (0.3 mol / L acetate buffer, 10 mM TPTZ, 20 mM FeCl3), react with paper mulberry leaf polysaccharide (0-3.2 mg / mL) for 10 min, and measure the absorbance at 593 nm.

[0045] (4) Iron ion chelating ability: The polysaccharide of the leaves of the Chinese paper mulberry tree (0-3.2 mg / mL) was reacted with 0.1 mM FeCl2 and 0.25 mM phenanthroline for 10 minutes, and the absorbance at 562 nm was measured, with EDTA as the control.

[0046] Calculate the clearance rate of (1) to (4) above using the following formula: Clearance rate (%) = (A control - A sample) / A control × 100% Among them, the control used in (1) to (3) is ascorbic acid (VC), and the control used in (4) is EDTA; A control represents the absorbance of the control; A sample represents the absorbance of the sample.

[0047] The results are as follows Figure 1 As shown in the figure (activity or scavenging rate on the vertical axis), BPP exhibited excellent in vitro antioxidant activity at various concentrations. Its ABTS⁺ and DPPH free radical scavenging abilities were strong, with scavenging rates reaching a plateau at a concentration of 0.8 mg / mL, peaking at approximately 95% and 80%, respectively, comparable to the positive control (VC). Its iron reducing power significantly increased with increasing concentration, approaching the level of VC. In contrast, BPP's iron chelating ability was relatively weak, peaking at approximately 30%, significantly lower than that of the EDTA control. These results indicate that BPP possesses strong free radical scavenging and reducing abilities, but its chelating activity for metal ions is limited.

[0048] Experimental Example 4: Evaluation of the effectiveness and safety of polysaccharides from paper mulberry leaves Based on the paper mulberry leaf polysaccharide extracted from Experimental Example 1, the effectiveness and cell safety of paper mulberry leaf polysaccharide were investigated using IPEC-J2 cells. To evaluate the effectiveness and toxicity of paper mulberry leaf polysaccharide on IPEC-J2 cells, the following experiments were performed:

[0049] IPEC-J2 cells were cultured at a rate of 1 × 10 4Cells were seeded at a density of 100 μL / well in a 96-well cell culture plate. 100 μL of DMEM / F12 medium supplemented with 10% fetal bovine serum was added to each well and cultured in a 37°C, 5% CO2 incubator until the cell confluence reached approximately 70%. The medium was then replaced with serum-free medium containing varying concentrations of BPP (0, 3, 6, 12, 24, 48, 96, 192, 385, and 768 mg / L) (n = 8) and cultured for an additional 24 hours. Following incubation, 10 μL of CCK-8 reagent was added to each well, incubated in the dark for 2 hours, and absorbance at 450 nm was measured using a microplate reader.

[0050] The results are as follows Figure 2 As shown in the results, compared with the control group, the concentration of paper mulberry leaf polysaccharide in the range of 3~768 mg / L could significantly improve the survival rate of IPEC-J2 cells without showing obvious cytotoxic effect ( P <0.05). Taking into account the cost and practical application requirements of paper mulberry leaf polysaccharide, 48 mg / L paper mulberry leaf polysaccharide was selected as the optimal concentration for subsequent in vitro cell tests.

[0051] Experimental Example 5 In vitro study on the anti-rotavirus effect of Broussonetia papyrifera polysaccharide on porcine intestinal epithelial cells Based on the paper mulberry leaf polysaccharide extracted from Experimental Example 1, IPEC-J2 cells were used as the subject to explore the antiviral effect and mechanism of paper mulberry leaf polysaccharide in a rotavirus (RV) infection model. The specific implementation process is as follows: 1. Cell culture and treatment IPEC-J2 cells were seeded into cell culture plates and cultured in a 37°C, 5% CO2 incubator. When the cells grew to about 70%, the old culture medium was discarded, washed twice with phosphate buffer, and replaced with serum-free culture medium containing mulberry leaf polysaccharide at a concentration of 48 mg / L. The cells were incubated until the optimal treatment time. RV (MOI = 3, 1 × 10 6 TCID 50 / mL), discard the old culture medium after 1 hour, replace it with the culture medium containing paper mulberry leaf polysaccharide, and collect the cells after 24 hours.

[0052] In the validation study of PI3K / AKT / NRF2 signaling pathway inhibitors, cells were treated with Broussonetia papyrifera polysaccharide and then treated with an NRF2 inhibitor (ML385, 5 μM, pretreatment for 5 hours) and an AKT inhibitor (MK-2206 2HCl, 10 μM, pretreatment for 1 hour). Cells were then challenged with RV and harvested 24 hours later. The experimental treatments and groupings are shown in Table 2.

[0053] Table 2 Cell experiment design

[0054] 2. Viral infection Activate the RV virus solution using DMEM / F12 medium containing 20 μg / mL trypsin (1:1 volume ratio). Mix thoroughly and incubate at 37°C, 5% CO2 for 30 minutes. After treatment, rinse the cells twice with PBS buffer, discard the remaining solution, add the activated virus solution, and incubate at 37°C, 5% CO2 for 1 hour. Gently shake the cell culture plate every 20 minutes to ensure uniform viral adsorption. After adsorption, rinse the cells once with phosphate buffered saline, discard the remaining solution, add DMEM / F12 medium containing 1 μg / mL trypsin, and continue incubating at 37°C, 5% CO2 for 24 hours before collecting cell samples.

[0055] 3. Sample collection After the cell culture is completed, collect the cell samples using the following method: (1) Collect cells with a cell scraper into a 1.5 mL centrifuge tube, centrifuge at 1000-2000 g for 1-2 minutes, and collect the cell supernatant for detection with an ELISA kit; (2) Discard the residual liquid, rinse the cells once with phosphate buffer, digest the cells with EDTA-free trypsin, centrifuge at 1000-2000 g for 1-2 minutes, discard the supernatant, collect the cells in physiological saline, and use them for antioxidant enzyme activity determination; (3) Discard the residual liquid, lyse the cells with RIPA lysis buffer (containing 1% protease phosphatase inhibitors), extract the total cell protein, determine the total protein concentration by BCA method, use GAPDH as an internal reference, and detect the protein expression level by Western Blot; (4) Discard the residual liquid, treat the cells with Trizol, extract RNA, and reverse transcribe it into cDNA. Use the GAPDH gene as an internal reference and detect the mRNA expression level by real-time fluorescence quantitative PCR.

[0056] The data obtained from the above experiments were preliminarily organized by Microsoft Excel 2020 and statistically analyzed by SPSS 26.0. The effects of different concentrations of paper mulberry leaf polysaccharide on cell viability were analyzed by one-way analysis of variance. The inhibitor group and paper mulberry leaf polysaccharide treatment group were used to verify the effect of PI3K / AKT / NRF2 signaling pathway inhibitors. t Test, other groups of data Duncan Multiple comparisons were performed. The other data were subjected to two-way ANOVA, and the main effects of the model included RV challenge and paper mulberry leaf polysaccharide treatment and the interaction effect between the two. Duncan Multiple comparisons. P The difference was considered significant when the difference was less than 0.05. All data were expressed as mean ± standard error.

[0057] 4. Experimental results In the above experiments, in order to study the antiviral effect and mechanism of paper mulberry leaf polysaccharide, different treatments were set up. The specific treatments and results are as follows: 1) Effect of Broussonetia papyrifera polysaccharides on the inhibition of RV infection of IPEC-J2 cells (1) Improve cell survival rate: To investigate the protective effect of Broussonetia papyrifera polysaccharide on the survival rate of IPEC-J2 cells infected with RV, IPEC-J2 cells were cultured at a rate of 1×10 4 The cells were seeded at a density of 100 cells / well in a 96-well plate. A control group (CON), RV infection group (CON+RV) and paper mulberry leaf polysaccharide intervention group (BPP+RV) were set up (see Table 2). Cell viability was detected by CCK-8 assay 12, 24, 36, 48, 60 and 72 h after infection.

[0058] The results are as follows Figure 3 As shown in Figure A, compared with the control group, the cell survival rate in the RV-infected group decreased significantly to 28.0±1.7% ( P <0.05, while under the intervention of 48 mg / L paper mulberry leaf polysaccharide, the survival rate of infected cells was significantly restored to 71.5±4.4% ( P <0.05). The above results indicate that Broussonetia papyrifera polysaccharide has a significant inhibitory effect on RV-induced cell damage and can effectively improve the survival rate of infected cells.

[0059] (2) Inhibit RV replication, improve intestinal barrier function and cell apoptosis: To further study the inhibitory effect of Broussonetia papyrifera polysaccharide on RV replication, IPEC-J2 cells were cultured at a density of 5×10 5 Cells were seeded in 6-well plates at a density of 100 cells / well. The control group (CON), RV infection group (CON+RV), paper mulberry leaf polysaccharide treatment group (BPP), and paper mulberry leaf polysaccharide intervention group (BPP+RV) were set up (see Table 2). After infection, the expression levels of RV structural protein 6 (VP6) mRNA and protein were determined by real-time quantitative PCR and Western blotting, respectively. To determine the effects of paper mulberry leaf polysaccharide on intestinal barrier function and apoptosis in IPEC-J2 cells under RV infection, the expression of tight junction proteins (ZO-1 and OCCLUDIN) and apoptosis-related proteins (BAX, BCL-2, and CASPASE-3) were examined by Western blotting.

[0060] The results are as follows Figure 3 As shown in Figures B and C, compared with the RV infection group, 48 mg / L Broussonetia papyrifera polysaccharide intervention significantly reduced the viral VP6 mRNA expression level and protein expression abundance (P <0.05), indicating that Broussonetia papyrifera polysaccharide can effectively inhibit the replication of RV in IPEC-J2 cells and has potential antiviral activity.

[0061] The results are as follows Figure 3 As shown in Figure C, compared with the control group, RV infection reduced the expression levels of tight junction proteins (ZO-1, OCCLUDIN) and anti-apoptotic proteins (BCL-2) in IPEC-J2 cells, while increasing the expression levels of pro-apoptotic proteins (BAX, CASPASE-3), indicating that RV infection can severely damage the cell barrier function and induce cell apoptosis. Compared with the RV infection group, 48 mg / L paper mulberry leaf polysaccharide intervention increased the expression levels of ZO-1, OCCLUDIN, and BCL-2 proteins, while reducing the expression levels of pro-apoptotic proteins BAX and CASPASE-3, indicating that paper mulberry leaf polysaccharide can effectively protect the integrity of the cell intestinal barrier under RV infection conditions and significantly inhibit virus-induced cell apoptosis.

[0062] 2) Effects of Broussonetia papyrifera polysaccharides on inflammatory cytokine levels and pattern recognition receptors in IPEC-J2 cells infected with RV To clarify the immunoregulatory mechanism of mulberry leaf polysaccharide on RV-infected IPEC-J2 cells, ELISA kits were used to detect the expression levels of pro-inflammatory cytokines (IL-6, IL-1β) and anti-inflammatory cytokines (IL-10), and real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of key factors in the cell pattern recognition receptor pathway (TLR3, RIG-I, MDA5, MAVS, ISG15) and interferons (IFN-β, IFN-γ, IFN-λ). IPEC-J2 cells were seeded in 6-well plates (5×10 5 The control group (CON), RV infection group (CON+RV), paper mulberry leaf polysaccharide treatment group (BPP) and paper mulberry leaf polysaccharide intervention group (BPP+RV) were set up (see Table 2).

[0063] (1) Reduce the expression of inflammatory cytokines: ELISA test results Figure 4 As shown in A, compared with the control group, the expression levels of pro-inflammatory factors IL-6 and IL-1β in the RV infection group were significantly increased ( P <0.05), and the expression level of the anti-inflammatory factor IL-10 was significantly decreased ( P <0.05), indicating that RV infection significantly induced cellular inflammatory response. In contrast, the paper mulberry leaf polysaccharide intervention group significantly reduced the expression levels of IL-6 and IL-1β, while significantly increasing the expression level of the anti-inflammatory factor IL-10 ( P <0.05), indicating that paper mulberry leaf polysaccharide can effectively inhibit the pro-inflammatory response caused by RV infection and reduce cell inflammatory damage.

[0064] (2) Activation of pattern recognition receptor pathway and interferon expression: Real-time fluorescence quantitative PCR detection results Figure 4 As shown in Figure B, compared with the control group, RV infection significantly increased the mRNA expression levels of TLR3, RIG-I, MDA5, MAVS and ISG15 ( P <0.05), and significantly increased the mRNA expression of interferon IFN-β, IFN-γ, and IFN-λ ( P <0.05), indicating that RV infection activated the body's innate immune antiviral pathway. The BPP intervention group further significantly enhanced the expression levels of TLR3, RIG-I, MDA5 and MAVS ( P <0.05, and significantly increased the expression level of IFN-λ ( P <0.05).

[0065] The above results show that paper mulberry leaf polysaccharides can further enhance the cell's virus recognition ability and antiviral immune response by effectively activating the pattern recognition receptor pathway and promoting interferon expression, thereby accelerating virus clearance and reducing the damage caused by viral infection.

[0066] 3) Effects of Broussonetia papyrifera polysaccharides on the antioxidant capacity and PI3K / AKT / NRF2 pathway of RV-infected IPEC-J2 cells To further explore the antioxidant protection mechanism of paper mulberry leaf polysaccharide, the total antioxidant capacity (T-AOC), catalase (CAT) activity and protein carbonyl content of IPEC-J2 cells were measured using kits, and the expression of proteins related to the PI3K / AKT / NRF2 signaling pathway was detected by Western Blot. IPEC-J2 cells were seeded in 6-well plates (5×10 5 The control group (CON), RV infection group (CON+RV), paper mulberry leaf polysaccharide treatment group (BPP) and paper mulberry leaf polysaccharide intervention group (BPP+RV) were set up (see Table 2).

[0067] (1) Evaluation of antioxidant capacity: The results are as follows Figure 4 As shown in C, RV infection significantly reduced the T-AOC and CAT activities of cells compared with the control group ( P <0.05), and significantly increased the protein carbonyl content ( P <0.05), indicating that viral infection induced cellular oxidative stress damage. The intervention group of paper mulberry leaf polysaccharide significantly increased the T-AOC and CAT activities of cells and significantly reduced the protein carbonyl content ( P<0.05). This indicates that Broussonetia papyrifera polysaccharides can effectively alleviate cellular oxidative stress damage caused by RV infection by enhancing antioxidant enzyme activity and reducing oxidative damage levels.

[0068] (2) Expression analysis of PI3K / AKT / NRF2 signaling pathway: The results are as follows Figure 4 As shown in Figure D, compared with the control group, RV infection suppressed the expression of p-NRF2, PI3K, and p-AKT proteins, while upregulating the expression of the pathway's negative regulator, GSK-3β. However, the mulberry leaf polysaccharide-treated group increased the expression of PI3K and p-NRF2 proteins. Under RV infection, mulberry leaf polysaccharide effectively activated the PI3K / AKT signaling pathway, promoting the phosphorylation of AKT and NRF2 proteins, thereby exerting an antioxidant defense effect.

[0069] 4) Validation of PI3K / AKT / NRF2 signaling pathway inhibitors To further confirm that the protective effect of paper mulberry leaf polysaccharide on RV-infected IPEC-J2 cells is mediated through the PI3K / AKT / NRF2 signaling pathway, the AKT-specific inhibitor MK-2206 2HCl and the NRF2-specific inhibitor ML385 were introduced into the cell experiment for verification.

[0070] (1) Cell viability: IPEC-J2 cells were cultured at 1×10 4 Cells were seeded into 96-well plates at 100 μL per well. The experiments were divided into the control group (CON), RV-infected group (CON+RV), paper mulberry polysaccharide-treated group (BPP), NRF2 inhibitor group (ML385), and AKT inhibitor group (MK-2206 2HCl) (see Table 2). After cell treatment, 10 μL of CCK-8 reagent was added to each well. The cells were incubated in the dark for 2 hours, and the absorbance was measured at 450 nm using a microplate reader.

[0071] The results are as follows Figure 5 As shown in A, compared with the group treated with mulberry leaf polysaccharide, the cell viability of the AKT inhibitor group and the NRF2 inhibitor group was significantly reduced ( P <0.05), indicating that the protective effect of mulberry leaf polysaccharide on RV-infected cells is related to the presence of NRF2 and AKT pathways.

[0072] (2) Expression of PI3K / AKT / NRF2 signaling pathway: IPEC-J2 cells were seeded in 6-well plates (5 × 10 5The experiments were divided into control group (CON), RV infection group (CON+RV), paper mulberry leaf polysaccharide treatment group (BPP), AKT inhibitor group (MK-2206 2HCl) and NRF2 inhibitor group (ML385) (see Table 2). Western Blot was used to detect the protein expression levels of PI3K, p-AKT, AKT, p-NRF2, NRF2 and downstream antioxidant enzyme HO-1 in cells.

[0073] The results are as follows Figure 5 As shown in Figure B, after the addition of AKT inhibitors (MK-2206 2HCl) and NRF2 inhibitors (ML385), the activation of AKT and NRF2 protein phosphorylation and its promotion of downstream antioxidant enzyme HO-1 expression by Broussonetia papyrifera polysaccharides were significantly inhibited. This result further confirms that Broussonetia papyrifera polysaccharides exert antioxidant protection by specifically activating the PI3K / AKT / NRF2 signaling pathway, effectively alleviating cell damage caused by viral infection.

[0074] Experimental Example 6 Study on the in vivo effects of Broussonetia papyrifera polysaccharides on rotavirus-infected piglets Based on the in vitro mechanism studies in Experimental Example 4, the in vivo antiviral effects of paper mulberry leaf polysaccharide were verified by constructing a piglet model infected with RV. Based on the in vitro antiviral concentration (48 mg / L), combined with parameters such as pig weight, feed intake, and in vivo digestion and metabolism, a dietary dosage of 110 mg / kg of paper mulberry leaf polysaccharide was selected. This dose ensured dose equivalence between in vitro and in vivo pharmacological effects and accounted for the subtherapeutic dose of the drug as a feed additive. The in vivo study was conducted as follows:

[0075] 1. RV propagation and testing IPEC-J2 cells were used for RV culture. IPEC-J2 cells were cultured in DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% double-antibody. RV was inoculated when the cells reached approximately 80% confluency. RV virus was mixed with 20 μg / mL trypsin (EDTA-free) in a 1:1 volume ratio and incubated at 37°C in a water bath for 30 minutes to activate RV. After inoculation, the cells were allowed to adsorb for 1 hour in a 37°C, 5% CO2 incubator, with the cell culture flask shaken every 20 minutes to ensure uniform viral adsorption. After adsorption, the cells were rinsed once with phosphate buffered saline, the residual solution discarded, and DMEM / F12 medium supplemented with 1 μg / mL trypsin (EDTA-free) was added. Culture was continued for 3–5 days until more than 70% of the cells showed a significant cytopathic effect (CPE) after virus inoculation. The virus was then harvested, titered, and stored at -80°C until further use.

[0076] The TCID50 ) method to determine the virus titer. The virus was dissolved in DMEM / F12 and diluted 10 times in a row, starting from 10 -1 Dilute to 10 -10 The diluted virus was inoculated into IPEC-J2 cells in a 96-well plate, with each dilution inoculating one column of 6 wells, and 100 μL was inoculated into each well. A column of uninoculated normal cells was set up as a negative control. CPE was observed and recorded 24 hours after virus inoculation, and the virus titer was calculated according to the Reed-Muench method. The calculation method is as follows:

[0077] Virus titer (-1g TCID 50 / mL) = (lesion rate above 50% - 50%) / (lesion rate above 50% - lesion rate below 50%) × logarithm difference of dilution + logarithm of dilution rate above 50% 2. Experimental Animals and Experimental Design Twenty-four healthy, 7-day-old, newborn DLY (Durcus × ​​Long × Da) barrows (4 per litter) were selected from six healthy sows. Their weights were close to the average litter weight (initial weight 2.46 ± 0.06 kg). A 2×2 two-factor design was used, with the experimental factors being formula supplemented with BPP (0 or 110 mg / kg) and RV challenge (gavage feeding with RV or homologous culture medium). The 24-day experimental period was divided into two phases: pre-RV challenge (experimental days 1–21) and post-RV challenge (experimental days 22–24). On day 1, pigs were randomly assigned to two treatments, each containing 12 replicates (1 pig per replicate) based on similar body weights. Both treatments were fed formula supplemented with or without Broussonetia papyrifera polysaccharide. On the 22nd day of the experiment, all piglets were weighed on an empty stomach and gavage administered 5 mL of 100 mmol / L sterile NaHCO3. 15 minutes later, 6 piglets were randomly selected from each treatment and gavage administered 25 mL of RV virus solution (1.6×10 7 TCID 50 / mL), and the remaining piglets were gavage-administered 25 mL of homologous DMEM / F12 medium. The experimental design and treatments are shown in Table 3.

[0078] Table 3 Experimental design

[0079] 3. Sample collection and processing The dry matter content of the formula consumed by the piglets was recorded daily. All piglets were weighed on an empty stomach on days 1, 22, and 25 of the experiment. On the morning of day 25 of the experiment, venous blood was collected from all piglets to obtain serum. All piglets were slaughtered and samples were collected. The sample collection and processing were as follows:

[0080] (1) Growth performance and diarrhea status: Average daily feed intake (total feed intake per stage / number of days per stage), average daily weight gain ([weight at the end of the stage - weight at the beginning of the stage] / days), and feed-to-gain ratio (total feed intake per stage / total weight gain per stage) were calculated for each stage. Following RV challenge, diarrhea was scored four times daily, and the diarrhea rate (number of diarrhea episodes / total number of diarrhea episodes × 100) and average diarrhea index (total diarrhea score / [total number of pigs × total days]) were calculated. Fecal scoring criteria are shown in Table 4; a score ≥2 was defined as diarrhea.

[0081] Table 4 Evaluation criteria for diarrhea

[0082] (2) Serum and intestinal tissue indicators: On the evening of the 24th day of the experimental period, all experimental piglets were settled for residual feed. On the morning of the 25th day, 20 mL of blood was collected from all experimental piglets and placed in ordinary blood collection tubes. The tubes were allowed to stand at room temperature for 30 minutes and centrifuged at 3500 rpm for 15 minutes to absorb the upper serum. The serum was aliquoted and stored at -20°C for later use. After blood collection, all experimental pigs were slaughtered, the intestinal segments were quickly separated, and the jejunal mucosa was carefully scraped and placed in cryopreservation tubes and transferred to a -80°C ultra-low temperature freezer for storage until testing. Jejunal segments (approximately 2 cm) were collected and fixed in 4% paraformaldehyde solution. Paraffin sections were made and stained with hematoxylin-eosin for morphological structure observation.

[0083] (3) Jejunal mucosal sample processing: The jejunal mucosa was ground into a fine powder in a clean liquid nitrogen pre-cooled mortar. Three ground jejunal mucosa samples (j, k, and l) were prepared and subjected to the following treatments: j The cells were treated with Trizol, RNA was extracted, and reverse transcribed to synthesize cDNA. The mRNA expression level was detected by real-time fluorescence quantitative PCR using the ACTIN gene as an internal reference. k was treated with pre-cooled physiological saline, homogenized by ultrasonication, centrifuged at 2000 rpm for 15 min at 4°C, and the supernatant was collected for the detection of antioxidant indicators and related indicators of ELISA kits; l Lyse the cells with RIPA lysis buffer (containing 1% protease phosphatase inhibitors) to extract total protein. Determine the total protein concentration using the BCA assay. Add SDS-PAGE protein loading buffer and boil in boiling water for 10 minutes to denature the protein. Use ACTIN as an internal control, and detect the expression levels of related proteins in the samples by Western blotting.

[0084] The data obtained from the above experiments were preliminarily calculated and sorted using Microsoft Excel 2020, and the growth performance of piglets in each treatment group before challenge was analyzed using SPSS 26.0. tThe chi-square test was performed on the piglet diarrhea data, and the other data were subjected to two-way analysis of variance. The main effects of the model included RV challenge and paper mulberry leaf polysaccharide treatment and the interaction effect between the two. Duncan Multiple comparisons. P The difference was considered significant when the difference was less than 0.05. All data were expressed as mean ± standard error.

[0085] 4. Experimental results 1) Effects of adding Broussonetia scoparia polysaccharide to formula milk on growth performance of piglets infected with RV The average daily feed intake, average daily weight gain and feed-to-weight ratio of piglets at each stage were calculated to evaluate the growth performance of piglets; serum urea nitrogen levels were measured using a serum biochemical analyzer to evaluate the protein metabolism efficiency of piglets. Figure 6 As shown in the results, from day 1 to day 21 of the experiment, the addition of mulberry leaf polysaccharide to formula milk could significantly increase the average daily weight gain of piglets ( P <0.05), but had no significant effect on the average daily feed intake; after the challenge (experimental period 22-24 days), the average daily weight gain of piglets in the challenge group (CON+RV) was significantly reduced compared with the control group (CON) ( P <0.05), and the average daily feed intake showed a downward trend ( P = 0.08), serum urea nitrogen level was significantly increased ( P <0.05); the average daily weight gain of the paper mulberry leaf polysaccharide treatment group (BPP) was significantly higher than that of the control group ( P <0.05), serum urea nitrogen levels were significantly decreased ( P <0.05); Under RV infection, compared with the RV challenge group, the paper mulberry leaf polysaccharide intervention group (BPP+RV) significantly alleviated the growth inhibition caused by RV infection ( P <0.05), and improved the serum urea nitrogen level of piglets ( P <0.05).

[0086] The above results indicate that adding mulberry leaf polysaccharide to formula milk can effectively alleviate the growth inhibition and reduced protein metabolism efficiency caused by RV infection, and play a positive role in the growth performance of RV-infected piglets.

[0087] 2) Effects of adding mulberry leaf polysaccharide to formula milk on diarrhea and viral infection in RV-infected piglets The diarrhea rate and diarrhea index of piglets were calculated, and the levels of RV-IgM antibodies and non-structural protein NSP4 in serum and jejunal mucosa were detected to evaluate the effects of paper mulberry leaf polysaccharide on diarrhea and viral infection in RV-infected piglets. Figure 7As shown in Figure A, the diarrhea rate and diarrhea index of piglets began to gradually increase 12 hours after RV infection; 36 hours after infection, the diarrhea index of the paper mulberry leaf polysaccharide intervention group began to decline, and the diarrhea rate also began to decline 48 hours later, while the diarrhea index and diarrhea rate of piglets in the control group continued to increase, indicating that the addition of paper mulberry leaf polysaccharide to formula milk can effectively alleviate the diarrhea symptoms caused by RV infection and shorten the course of diarrhea.

[0088] like Figure 7 As shown in Figure B, the ELISA test results showed that compared with the control group, RV challenge significantly increased the levels of RV-IgM antibodies and jejunal virus non-structural protein NSP4 in serum and jejunal mucosa ( P <0.05, confirming that the virus successfully infected and stimulated the host immune response. The paper mulberry leaf polysaccharide intervention group showed an obvious dual regulatory effect, on the one hand, promoting the secretion of RV-IgM antibodies ( P <0.05), and improved the humoral immunity level; on the other hand, it significantly reduced the expression of NSP4 protein in the jejunum ( P <0.05), inhibiting viral replication and reducing viral enterotoxin damage. The addition of mulberry leaf polysaccharide to formula milk effectively alleviates the negative effects of RV infection on piglets by simultaneously promoting immune response and inhibiting viral proliferation.

[0089] 3) Effects of adding Broussonetia scoparia polysaccharide to formula milk on intestinal barrier function in piglets infected with RV The jejunal tissue morphology was observed by hematoxylin-eosin staining, and the expressions of mucin (MUC2), tight junction proteins (ZO-1, OCCLUDIN), and apoptosis-related molecules (BAX, BCL-2, CASPASE-3) in the jejunal mucosa were detected by ELISA kits and Western Blot to evaluate the intestinal barrier function. Figure 7 As shown in C-D, compared with the control group, the jejunal villus height and villus-crypt ratio of piglets in the RV challenge group were significantly reduced ( P <0.05), the expression levels of MUC2, tight junction proteins and BCL-2 proteins in the jejunal mucosa were significantly decreased ( P <0.05), and the protein expression levels of pro-apoptotic molecules (BAX, CASPASE-3) were significantly increased ( P <0.05, indicating that RV infection leads to impaired intestinal barrier function. The jejunal villus height and villus-crypt ratio were significantly increased in the mulberry leaf polysaccharide treatment group ( P <0.05, and effectively increased the expression levels of MUC2, tight junction proteins, and anti-apoptotic protein BCL-2, while significantly reducing the expression levels of pro-apoptotic proteins BAX and CASPASE-3 ( P<0.05). Under RV infection, compared with the RV challenge group, the jejunal villus height and villus-crypt ratio were further increased in the mulberry leaf polysaccharide intervention group ( P <0.05), significantly improved the expression of tight junction proteins and reduced apoptosis, significantly alleviating intestinal barrier damage caused by RV infection. This suggests that the addition of Broussonetia scoparia polysaccharide to formula milk can effectively protect the intestinal barrier function of RV-infected piglets, improve intestinal mucosal integrity, and reduce intestinal damage caused by viral infection.

[0090] 4) Effects of adding Broussonetia scoparia polysaccharide to formula milk on immune function and expression of cellular pattern recognition receptor pathways in piglets infected with RV ELISA kits were used to detect the levels of immunoglobulins (IgA, IgM, IgG) in serum and the expression levels of inflammatory factors (IL-6, IL-1β, IL-10) in the jejunal mucosa to evaluate the effect of paper mulberry leaf polysaccharide on the immune function of piglets infected with RV. Figure 8 As shown in A, compared with the control group, the levels of IgA and IgG in the serum of piglets in the RV challenge group were significantly reduced ( P <0.05, while the IgM content was significantly increased ( P <0.05); in contrast, the Broussonetia papyrifera polysaccharide-treated group significantly increased the levels of serum IgA, IgM, and IgG ( P <0.05). Under RV infection, the Broussonetia papyrifera polysaccharide intervention group further increased the levels of IgA, IgM, and IgG compared with the RV challenge group ( P <0.05). This indicates that Broussonetia papyrifera polysaccharides can significantly enhance the humoral immune function of piglets infected with RV.

[0091] To further explore the effect of paper mulberry leaf polysaccharide on the innate immune signaling pathway in piglets infected with RV, real-time fluorescence quantitative PCR was used to analyze the mRNA expression levels of key factors in the cell pattern recognition receptor pathway (TLR3, RIG-I, MDA5, MAVS) and interferons (IFN-β, IFN-γ, IFN-λ) in the jejunal mucosa. Figure 8 As shown in Figure B, RV challenge significantly upregulated the expression of TLR3, RIG-I, MDA5, and MAVS ( P <0.05), and also increased the mRNA expression levels of IFN-β, IFN-γ and IFN-λ ( P <0.05), indicating that RV infection stimulated the body's innate immune antiviral response. Treatment with paper mulberry leaf polysaccharide significantly enhanced the expression of RIG-I, MAVS and ISG15 in the non-infected state ( P<0.05), suggesting that paper mulberry leaf polysaccharide helps to improve the body's innate immune recognition and antiviral ability. Under RV infection conditions, paper mulberry leaf polysaccharide intervention further promoted the activation of the above antiviral pathways ( P <0.05, and significantly increased the expression level of IFN-λ ( P <0.05), thereby enhancing viral recognition and antiviral signal transduction, accelerating viral clearance, and reducing damage caused by infection.

[0092] In summary, the addition of mulberry leaf polysaccharide to formula milk can enhance the immune defense ability of RV-infected piglets by enhancing humoral immunity and activation of cellular pattern recognition receptor pathways, effectively reduce the immunosuppressive effect caused by viral infection, and promote the body's antiviral response.

[0093] 5) Effects of adding Broussonetia scoparia polysaccharide to formula milk on intestinal antioxidant capacity and PI3K / AKT / NRF2 signaling pathway expression in piglets infected with RV Kits were used to measure the total antioxidant capacity (T-AOC), malondialdehyde (MDA) content, and catalase (CAT) activity of the piglet jejunal mucosa. In addition, the expression levels of key proteins in the PI3K / AKT / NRF2 signaling pathway (NRF2, p-NRF2, KEAP1, HO-1, PI3K, p-AKT, and GSK-3β) were analyzed by Western Blot. Figure 9 As shown in C, compared with the control group, the MDA level in the jejunal mucosa of piglets in the RV challenge group was significantly increased ( P <0.05, while T-AOC and CAT activities were significantly decreased ( P <0.05). In contrast, the paper mulberry leaf polysaccharide treatment group effectively reduced the MDA level and significantly increased the T-AOC and CAT activities ( P <0.05). Under RV infection, the mulberry leaf polysaccharide intervention group significantly reduced the levels of RV-induced oxidative damage markers compared with the RV challenge group ( P <0.05, and significantly restored the activities of T-AOC and antioxidant enzymes ( P <0.05). Western Blot analysis results ( Figure 9D) showed that RV challenge inhibited the activation of NRF2 and AKT and their downstream antioxidant signaling pathways, as manifested by decreased expression of p-NRF2, HO-1, PI3K and p-AKT proteins, while increased expression of KEAP1 and GSK-3β proteins. Treatment with mulberry leaf polysaccharide can increase the protein expression levels of p-NRF2, HO-1, PI3K and p-AKT, and reduce the expression of KEAP1 and GSK-3β. Under RV infection conditions, intervention with mulberry leaf polysaccharide further enhanced the expression of p-NRF2, HO-1, PI3K and p-AKT, and inhibited the expression of KEAP1 and GSK-3β. Gray value analysis also showed that RV challenge significantly reduced the phosphorylation levels of AKT and NRF2 ( P <0.05, while the treatment with paper mulberry leaf polysaccharide significantly increased the phosphorylation levels of the above proteins ( P <0.05), also showing significant effects under RV infection. This suggests that the addition of mulberry leaf polysaccharide to formula milk significantly enhances the intestinal antioxidant defense capacity of RV-infected piglets by activating the PI3K / AKT / NRF2 signaling pathway, effectively alleviating the oxidative stress damage caused by RV infection.

[0094] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. Application of paper mulberry leaf polysaccharide in the preparation of anti-porcine rotavirus drugs, characterized in that: The molecular weight of the paper mulberry leaf polysaccharide is greater than 3500 Da.

2. The use according to claim 1, characterized in that The application dosage of the paper mulberry leaf polysaccharide as an anti-rotavirus drug is 400-1200 mg / head, or the dosage of the paper mulberry leaf polysaccharide as a functional additive in pig feed to play a relevant preventive role is 100-200 mg / kg.

3. An anti-porcine rotavirus drug, characterized in that: The medicine comprises paper mulberry leaf polysaccharide with a molecular weight greater than 3500 Da, and pharmaceutically acceptable excipients or auxiliary components.

4. The drug according to claim 3, characterized in that The purity of the paper mulberry leaf polysaccharide is ≥95%, and the protein residue is ≤2.5%; Or / and, the dosage form of the drug includes: tablets, powders, granules, premixes, oral solutions or sustained-release microcapsules.

5. A method for extracting polysaccharides from paper mulberry leaves as claimed in claim 1, characterized in that: The method includes: The paper mulberry leaf powder was extracted with hot water at a material-liquid ratio of 1 g: 30-40 mL at 55-65°C, and the extract was concentrated and deproteinized to obtain a supernatant. The supernatant was purified by dialyzing through a dialysis bag with a molecular weight cutoff of 3000-4000 Da, and then decolorized by macroporous resin chromatography to collect the polysaccharide elution peak; The collected eluate was concentrated and added with anhydrous ethanol for alcohol precipitation. The precipitate was collected by centrifugation and freeze-dried in vacuum to obtain Broussonetia sylvestris polysaccharide.

6. The method for extracting polysaccharides from paper mulberry leaves according to claim 5, characterized in that: The paper mulberry leaf powder is paper mulberry leaf powder that has passed through a 40-80 mesh sieve; or / and, the hot water extraction is performed for 4 to 6 hours.

7. The method for extracting polysaccharides from paper mulberry leaves according to claim 6, characterized in that: The paper mulberry leaf dry powder is paper mulberry leaf dry powder that has passed through a 60-mesh sieve.

8. The method for extracting polysaccharides from paper mulberry leaves according to claim 5, characterized in that: The material-liquid ratio is 1g:35mL; or / and, the hot water extraction temperature is 60°C; Or / and, the molecular weight cut-off of the dialysis bag is 3500Da.

9. The method for extracting polysaccharides from paper mulberry leaves according to claim 5, characterized in that: The concentration and deproteinization treatment comprises centrifuging the extract at 5-15° C., taking the supernatant and concentrating it by rotary evaporation to 1 / 5-1 / 4 of the original volume, adding 1.5-2.5 times the volume of Sevage reagent for deproteinization, and taking the supernatant after centrifugation; or / and, the dialysis purification, placing the supernatant into a dialysis bag with a molecular weight cut-off of 3000-4000 Da, and dialyzing against double distilled water for 48-72 hours; Or / and, the macroporous resin is selected from AB-8 macroporous resin, and the eluent used is ethanol solution; or / and, the alcohol precipitation, after the eluate is concentrated, 8 to 10 times the volume of anhydrous ethanol is added, and the mixture is allowed to stand at 4° C. for 36 to 48 hours, and the precipitate is collected by centrifugation at 3000 to 4000 g, and vacuum freeze-dried to obtain paper mulberry leaf polysaccharide.

10. The method for extracting polysaccharides from paper mulberry leaves according to claim 9, characterized in that: The centrifugation conditions of the extract are 5-15° C. and 3000-4000 g; or / and, the amount of the Sevage reagent is 2.0 times the volume of the rotary evaporation concentrate; Or / and, in the Sevage reagent, the volume ratio of chloroform to n-butanol is 4:1; Or / and, the eluent is 30% ethanol solution; Or / and, the amount of anhydrous ethanol used in the alcohol precipitation is 9 times the volume of the concentrated eluent.

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