Application of polydatin in preparation of medicine for relieving hepatotoxicity caused by zearalenone

Abstract

The invention discloses application of polydatin in preparation of a medicine for relieving hepatotoxicity caused by zearalenone, and belongs to the technical field of biological medicine. The invention reveals that polydatin inhibits hepatotoxicity caused by zearalenone through targeted xanthine oxidase for the first time, so that the toxicity of zearalenone to an organism is relieved. Polydatin can be directly combined with xanthine oxidase to inhibit the catalytic activity of xanthine oxidase, so that the oxidative stress injury of the liver after ZEA exposure is reduced, meanwhile, the content of malondialdehyde (MDA) in the liver can be remarkably reduced, the activity of superoxide dismutase (SOD) is improved, the liver oxidation-antioxidant balance is recovered, and liver cells are protected from oxidative injury of ZEA. The invention provides a new scientific basis and development direction for application of polydatin in the fields of mycotoxin detoxification, oxidation resistance, liver protection and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of polygalactosin in the preparation of drugs that alleviate hepatotoxicity caused by zearalenone. Background Technology

[0002] Zearalenone (ZEN) is a mycotoxin produced by Fusarium spp., which widely contaminates grains such as corn, wheat, and barley, especially under high temperature and humidity storage conditions. ZEN exhibits significant estrogen-like activity, competitively binding to estrogen receptors (ER), interfering with the endocrine systems of animals and humans, leading to reproductive dysfunction, immunosuppression, and metabolic disorders. Furthermore, ZEN's toxicity is also reflected in its ability to induce oxidative stress, promoting the excessive production of reactive oxygen species (ROS), triggering lipid peroxidation, protein damage, and DNA breaks, ultimately leading to dysfunction of vital organs such as the liver and kidneys. Due to the widespread contamination of food and feed by ZEN and its stable chemical properties, its food safety risks are increasingly concerning, and effective intervention strategies to mitigate its toxic effects are urgently needed.

[0003] Xanthine oxidase (XOD) is a key enzyme in the purine metabolism pathway, catalyzing the oxidation of hypoxanthine and xanthine to uric acid, accompanied by the generation of reactive oxygen species (ROS). Under pathological conditions, excessive activation of XOD leads to exacerbated oxidative stress, which is closely related to the development of various diseases, such as liver damage, cardiovascular disease, and inflammatory diseases. Studies have shown that ZEN exposure can upregulate XOD expression and activity, further promoting ROS accumulation and exacerbating tissue oxidative damage. Therefore, regulating XOD activity may be one of the potential targets for alleviating ZEN toxicity.

[0004] Polydatin (PD) is a flavonoid compound extracted from the traditional Chinese medicine Polygonum cuspidatum, possessing various biological activities including antioxidant, anti-inflammatory, and cytoprotective effects. Its antioxidant activity is mainly achieved by activating endogenous antioxidant systems (such as SOD and GSH-Px) and directly scavenging free radicals. In recent years, the role of PD in liver protection has received widespread attention, with studies showing that it can alleviate liver damage caused by chemical toxins or metabolic diseases by regulating oxidative stress and inflammatory responses. However, whether PD alleviates ZEN-induced liver injury by affecting XOD activity is currently poorly studied, and its specific mechanism of action requires further investigation.

[0005] In summary, ZEN damages organs such as the liver by inducing oxidative stress and inflammatory responses, while XOD may play an important role in ZEN-mediated ROS generation. PD, as a natural antioxidant, has potential hepatoprotective effects, but whether it antagonizes ZEN toxicity by regulating the XOD pathway requires further investigation. In-depth exploration of the mechanism of action of PD will provide a theoretical basis for developing novel ZEN detoxification strategies and offer new intervention ideas for food safety and animal health. Summary of the Invention

[0006] The purpose of this invention is to provide the application of polygalactosin in the preparation of drugs that alleviate hepatotoxicity caused by zearalenone, in order to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides the application of polygalactoside in the preparation of zearalenone antidote.

[0009] Optionally, the zearalenone antidote is used to alleviate liver toxicity caused by zearalenone.

[0010] Optionally, the hepatotoxicity refers to liver damage.

[0011] Optionally, the liver injury is oxidative damage to the liver.

[0012] Optionally, the polygalactoside can alleviate liver damage caused by zearalenone and reduce the toxicity of zearalenone to the body by inhibiting the activity of xanthine oxidase.

[0013] Optionally, the zearalenone antidote may also include a pharmaceutically acceptable carrier or excipient.

[0014] Optionally, the zearalenone antidote may be available in oral formulation.

[0015] Optionally, the oral dosage form includes tablets, pills, capsules, granules, and mixtures.

[0016] This invention also provides the application of polygalactosin in the preparation of feed additives that alleviate the toxicity of zearalenone to the body.

[0017] Optionally, the toxicity of zearalenone to the body includes liver toxicity caused by zearalenone.

[0018] Optionally, the liver toxicity includes oxidative damage to the liver.

[0019] The present invention discloses the following technical effects:

[0020] This invention reveals for the first time that polygalactoside inhibits the hepatotoxicity induced by zearalenone by targeting xanthine oxidase, thereby alleviating the toxicity of zearalenone to the body. Polygalactoside can directly bind to xanthine oxidase, inhibiting its catalytic activity, thereby reducing oxidative stress damage to the liver after ZEA exposure. Simultaneously, it can significantly reduce the content of malondialdehyde (MDA) in the liver, increase the activity of superoxide dismutase (SOD), restore the hepatic oxidative-antioxidant balance, and protect hepatocytes from oxidative damage caused by ZEA.

[0021] Polygonum cuspidatum glycoside, as a natural active ingredient, has the characteristics of low toxicity and high biocompatibility compared with chemically synthesized drugs. It can be safely used in feed additives or drug development, providing a green and efficient solution for liver toxicity in livestock, poultry and humans caused by zearalenone contamination.

[0022] This invention reveals for the first time the molecular mechanism by which resveratrol inhibits the hepatotoxicity of zearalenone by targeting xanthine oxidase, providing new scientific basis and development direction for the application of resveratrol in the fields of mycotoxin detoxification, antioxidant liver protection and other fields. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The images show the GO enrichment analysis of the liver transcriptome and the quantitative fluorescence detection of oxidative damage indicators in the ZEN and Con groups. Specifically, A is a bar chart showing the significant differences in GO enrichment between the ZEN treatment group and the control group (Con) during biological processes; B is a GO enrichment chord plot showing the association between functional terms and differentially expressed genes; C is a cassette diagram of XDH gene expression; and D is a quantitative fluorescence expression map of oxidative stress-related genes such as SOD1, SOD2, CAT, NQO1, and GSH-PX.

[0025] Figure 2To verify the molecular mechanism and function of polygalactosyl in regulating xanthine oxidase; A shows the molecular docking results and parameters of polygalactosyl and xanthine oxidase; B shows the Gibbs free energy landscape of the polygalactosyl-xanthine oxidase system (left) and the febuxostat-xanthine oxidase system (right); C shows the root mean square deviation (RMSD) of xanthine oxidase under the action of febuxostat (blue) and polygalactosyl (red); D shows the root mean square fluctuation (RMSF) distribution of xanthine oxidase residues under the action of febuxostat (blue) and polygalactosyl (red); E shows the change of radius of gyration (Rg) of xanthine oxidase under the action of febuxostat (blue) and polygalactosyl (red); F shows the inhibition of xanthine oxidase (XOD) activity by polygalactosyl (top) and zearalenone (ZEN, bottom).

[0026] Figure 3 The results show the molecular docking of five main active ingredients in Polygonum cuspidatum extract—citric acid (A), allopurinol (B), resveratrol (C), phenacetin (D), and emodin (E)—with the target protein. The band diagram shows the overall three-dimensional structure of the target protein (blue) and the binding sites of the ligands (rod-shaped model), while the magnified diagram highlights hydrogen bonds and hydrophobic interactions (dashed lines) and key residues.

[0027] Figure 4 Figure 1 shows the docking results and docking score statistics of five other major extracts from Polygonum cuspidatum: apigenin (A), epicatechin (B), epicatechin-3-O-gallate (C), 8-glucosyltoracryloylone (D), and polyglycoside (E) with xanthine oxidase (XOD). The figure shows the binding sites of the banded protein structure and the rod-shaped ligand, and the magnified view shows the key hydrogen bonds and hydrophobic interactions. Figure F is the XOD enzyme activity inhibition rate curve, which evaluates the effects of PD and ZEN on XOD activity.

[0028] Figure 5 Bar charts showing the distribution of XOD enzyme activity (A) and its mRNA expression in mouse liver tissue in the ZEN-treated and Polygonum cuspidatum extract combined treatment groups (BJ) and the content of antioxidant enzyme activity and lipid peroxidation products (KM) are provided. In BJ, the mRNA expression distribution of antioxidant-related genes XDH, HO-1, NQO1, Keap1, Nrf2, SOD1, SOD2, CAT and GPX1 are shown in order. K and L represent the activities of antioxidant enzymes CAT and SOD, and M represents the content of lipid peroxidation product MDA.

[0029] Figure 6 To detect cell viability using the CCK-8 assay; A is a bar chart of cell viability under different concentrations of ZEN treatment; B is a bar chart of cell viability under combined treatment with ZEN and different concentrations of PD; C is a bar chart of cell viability under PD treatment alone; D and E are dot plot analyses of cell XOD enzyme activity under ZEN treatment alone and ZEN combined with PD treatment, respectively.

[0030] Figure 7 The data includes a bar chart of XDH gene expression (A); an intracellular ROS immunofluorescence map (B), grouped as control, PD, ZEN, and ZEN+PD); a bar chart of ROS relative fluorescence intensity quantification (C); a bar chart of Nrf2 and its downstream antioxidant-related genes (HO-1, Keap1, SOD1, NQO1, Nrf2, SOD2) mRNA expression (D–I); and a bar chart of antioxidant enzymes GSH-Px and CAT gene expression (JK), systematically demonstrating the regulatory mechanism of PD on ZEN-induced oxidative stress. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] Example 1: Study on the mechanism of ZEN's effect on oxidative damage in mouse liver

[0037] 1. Constructing a mouse in vivo ZEN exposure model

[0038] This invention establishes a ZEN (zearalenone)-induced in vivo oxidative stress model in mice via gavage to simulate its hepatotoxic effects. SPF-grade healthy male ICR mice (28±2g) were used in the experiment. After 7 days of acclimatization, they were randomly assigned to different groups. During the experiment, mice had free access to food and water, and were kept at a constant temperature and humidity (22±2℃, relative humidity 55±10%) with a 12-hour light / dark cycle.

[0039] Mice were randomly divided into a control group (Con) and a ZEN treatment group (n=6 in each group). The control group was given an equal volume of physiological saline solution by gavage; the ZEN group was given ZEN (25 mg / kg bw) by gavage for 14 days, with a gavage frequency of once daily and a gavage volume of no more than 10 mL / kg each time. ZEN was dissolved in a small amount of dimethyl sulfoxide (DMSO) and then diluted with 0.9% sodium chloride injection (physiological saline) to the target concentration, ensuring that the final DMSO concentration was controlled below 0.5%, and was prepared and used immediately.

[0040] The criteria for successful modeling include: mild behavioral changes in mice after ZEN treatment, such as reduced activity and decreased appetite; slight increase in liver volume or darkening of liver color observed by the naked eye; biochemical indicators showing typical oxidative stress characteristics such as increased XOD enzyme activity, decreased SOD / CAT activity, and increased MDA content; and quantitative fluorescence detection results indicating significant differences in the expression levels of antioxidant-related genes (such as XDH, HO-1, NQO1, GSH-Px, etc.), which are statistically significant.

[0041] 2. Transcriptomics detection and analysis

[0042] To further explore the molecular regulatory mechanism of ZEN exposure on mouse liver tissue, a ZEN exposure model was constructed and transcriptomic analysis was performed. The specific steps are as follows:

[0043] Total RNA was extracted from the liver tissues of ZEN-exposed and control mice.

[0044] RNA was extracted using the TRIzol reagent method, and RNA quality was assessed using NanoDrop and agarose gel electrophoresis.

[0045] Constructing transcriptome sequencing libraries, including poly-A enrichment, RNA fragmentation, cDNA synthesis, adapter ligation, and library amplification;

[0046] Paired-end high-throughput sequencing was performed using the Illumina NovaSeq 6000 platform (PE150).

[0047] Quality control (QC), connector and low-quality sequence removal are performed on the raw data;

[0048] Clean reads were aligned to the reference genome (Mus musculus GRCm39) using HISAT2.

[0049] Quantitative analysis was performed using FeatureCounts to obtain the expression levels of each gene;

[0050] Differentially expressed genes (DEGs) were screened using DESeq2 software, with the screening criteria being |log2FC|>1 and p<0.05;

[0051] GO functional enrichment analysis and KEGG pathway enrichment analysis were performed on the selected DEGs, focusing on pathway changes related to oxidative stress, inflammatory response and xanthine oxidase activity.

[0052] Cross-validation and pathway construction were performed by combining the results of quantitative real-time PCR.

[0053] The results are as follows Figure 1 As shown in Figures AB, the functions of oxidoreductases related to XOD enzyme activity were significantly enriched, including "oxidoreductase activity," "heme binding," and "iron ion binding," indicating that the proteins encoded by the relevant genes possess redox catalytic activity and metal ion binding capacity, which are key characteristics of XOD enzyme activity. These results support the significant changes in XOD enzyme activity and related redox responses after ZEN exposure, revealing the important role of XOD in hepatic oxidative stress. This invention is the first to reveal an important molecular mechanism of ZEN-induced hepatotoxicity at the transcriptomic level, providing theoretical support for subsequent functional validation and target screening.

[0054] 3. Detection of oxidative damage indicators

[0055] The specific steps for detecting changes in antioxidant enzymes and oxidative damage markers in the liver of infected mice using real-time quantitative PCR are as follows:

[0056] Total RNA was extracted from mouse liver tissue;

[0057] Reverse transcription to synthesize cDNA;

[0058] Real-time quantitative PCR amplification was performed using specific primers;

[0059] The expression levels of SOD1, SOD2, CAT, GSH-PX, KEAP1, Nrf2, HO-1, and NQO1 in mouse liver tissue were detected.

[0060] To analyze the effects of ZEN exposure on liver oxidative damage and antioxidant response.

[0061] The primers for real-time quantitative PCR amplification are shown in Table 1, and the reaction system is shown in Table 2. A 2... -△△CT The relative content of the target gene is calculated using this method.

[0062] Table 1 Primers used for qRT-PCR detection

[0063]

[0064] Table 2 qPCR reaction system

[0065]

[0066] The results are as follows Figure 1 Following exposure to CH and ZEN, the activity of antioxidant enzymes in the liver of mice decreased significantly, specifically manifested as reduced expression levels of antioxidant enzymes such as SOD1, SOD2, CAT, and GSH-PX. Furthermore, the expression levels of oxidative damage markers KEAP1, Nrf2, HO-1, and NQO1 were significantly altered, indicating that ZEN may exacerbate oxidative damage in the liver by regulating these key molecules. Real-time quantitative PCR results further demonstrated that ZEN induced an imbalance in the antioxidant response in liver tissue, leading to an exacerbation of oxidative stress, suggesting that ZEN has a certain pathogenic effect on oxidative damage in the liver.

[0067] Data analysis in this invention revealed that ZEN significantly alters the oxidative damage state of mouse liver, primarily through regulating the activity of antioxidant enzymes and changes in oxidative damage markers. Following ZEN exposure, the expression of antioxidant enzymes such as SOD1, SOD2, CAT, and GSH-PX in mouse liver significantly decreased, demonstrating its inhibitory effect on the antioxidant system. Simultaneously, the expression levels of oxidative damage markers KEAP1, Nrf2, HO-1, and NQO1 changed significantly, indicating that ZEN may exacerbate hepatic oxidative stress by inhibiting these key molecules. Based on the above analysis, XOD inhibitors can be further explored as potential therapeutic agents, analyzing their alleviating effect on ZEN-induced hepatic oxidative damage, and conducting a series of related therapeutic efficacy evaluations and statistical analyses.

[0068] Example 2: Molecular docking and molecular dynamics simulation screening of the binding stability of Polygonum cuspidatum extract to XOD.

[0069] Molecular docking experiments were conducted to screen the binding stability of the active ingredients in Polygonum cuspidatum extract with XOD. The specific implementation steps are as follows:

[0070] The three-dimensional structures of the main compounds in the extract of Polygonum cuspidatum were obtained from the relevant database (Protein Data Bank (PDB));

[0071] Molecular docking was performed using molecular docking websites such as CD dock2 to compare the binding energies of various compounds with XOD.

[0072] Based on the binding energy, the components that bind most stably to XOD were screened, especially the compounds that exhibited the lowest binding energy, and their binding stability was further verified by molecular dynamics simulation.

[0073] In docking analysis, the activity of compounds was determined by evaluating binding energy and binding sites with XOD, and the best candidate molecules were selected. Subsequently, molecular dynamics simulations were used to analyze the interaction between the compound and XOD, assessing whether the compound could inhibit the enzymatic activity of XOD and whether the binding was stable.

[0074] The results are as follows Figures 2-4 Based on Table 1 and the docking results, it can be concluded that PD, the main component of Polygonum cuspidatum, may inhibit the enzyme activity by directly binding to XOD, providing a theoretical basis for subsequent biological experiments.

[0075] Example 3: Screening and Biochemical Experiments of XOD Inhibitors

[0076] XOD inhibitor screening experiment: Experimental groups were set up according to different PD concentrations (0μM, 1μM, 5μM, 10μM, 15μM, 20μM, 35μM, 50μM, 75μM, 100μM), and the XOD inhibitor screening kit (Beyotime) was used for the experiment. The specific steps are as follows:

[0077] According to the kit instructions, xanthine reacts with oxygen under XOD catalysis to produce uric acid and H2O2.

[0078] In the presence of HRP, XO Probe reacts with H2O2 to produce the red fluorescent substance Resorufin, and the fluorescence intensity is proportional to the amount of H2O2 produced.

[0079] Different concentrations of PD solution were added to react with XOD substrate, and the changes in fluorescence intensity were monitored to plot XOD inhibition curves.

[0080] Add a positive control inhibitor, observe changes in fluorescence intensity, and evaluate the inhibitory effect on PD.

[0081] The experimental results, shown in Table 1, obtained by treating XOD with different concentrations of PD, demonstrate that PD significantly inhibited XOD activity, and the decrease in fluorescence intensity showed a clear dose-dependent relationship with PD concentration. This is consistent with the binding site and affinity predicted in molecular docking, further proving PD's potential as an XOD inhibitor. The inhibitory effect of PD was confirmed by comparison with the positive control group, suggesting that PD may exert its potential biological role by binding to XOD and inhibiting its catalytic activity, thereby reducing H2O2 production.

[0082] Table 1. Docking data of XOD with ten Polygonum cuspidatum extract derivatives.

[0083]

[0084]

[0085] Example 4: Effect of PD on intracellular XOD enzyme activity

[0086] 1. Cell culture and processing

[0087] The AML12 cell line was cultured in 1640 medium containing 10% FBS at 37°C and 5% CO2 until the logarithmic growth phase.

[0088] Cells were seeded in 96-well plates. After cell adhesion, different concentrations of PD solution (0.1, 0.5, 1, 5, 10, 20, 30, 40 and 50 μM) were added after infection. Negative control group and positive control group were set up and cultured for 24 hours.

[0089] After processing, cells are collected to facilitate subsequent sample processing.

[0090] 2. Sample processing

[0091] The xanthine oxidase (XOD) assay kit (A002-1-1) was used, with a cell count of 0.5-1 × 10⁻⁶ cells / cells. 4 Add reagent 1 at a ratio of 1 mL and sonicate; centrifuge at 8000 g, 4℃ for 10 min, collect the supernatant and place on ice for testing.

[0092] 3. XOD enzyme activity detection (colorimetric method)

[0093] Preheat the spectrophotometer for at least 30 minutes, adjust the wavelength to 530 nm, and zero the instrument with distilled water.

[0094] Dilute the standard solution to 0.25 μmol / mL, and add 50 μL of standard solution to each standard tube;

[0095] According to the kit instructions, add the corresponding reagents to the blank tube, test tube, and standard tube, mix well, and place in a 37°C water bath or constant temperature incubator for a specific time.

[0096] Measure the absorbance (A) at 530 nm in a 1 mL glass cuvette and calculate ΔA;

[0097] Calculate the XOD enzyme activity in the cell sample according to the XOD activity calculation formula. The XOD activity calculation formula is:

[0098] XOD activity in tissue (U / gprot) = ((A assay - A blank) / ε) × (V total / V sample) × (1 / (T×d)) ÷ Cpr

[0099] Note: ε is the extinction molar coefficient of the chromogenic substance, 12.6 × 10⁻⁶ -3 Vreactiontotal is the total volume of the reaction solution, (2.27+a) mL; Vsample is the sample volume, mL; T is the reaction time, 20 minutes; d is the colorimetric path length, 1 cm; CHb is the hemoglobin content, gHb / L; Cpr is the homogenate protein content, gprot / L.

[0100] The results are as follows Figure 6 , Figure 6 This indicates that PD treatment significantly inhibited the intracellular XOD enzymatic activity. Colorimetric assay results showed that XOD activity in the PD group was significantly lower than that in the control group, and this inhibitory effect was concentration-dependent. Figure 6 (DE). This finding indicates that PD can exert a potential biological role by inhibiting XOD enzyme activity, reducing xanthine oxidative metabolism, and thus decreasing H2O2 production. Compared with the positive control group, the XOD activity in the PD-treated group was significantly different, suggesting that PD may inhibit XOD activity by binding to or affecting its catalytic mechanism, further validating the potential of PD as an XOD inhibitor.

[0101] Example 5: Establishment of a PD-induced model to mitigate ZEN-induced AML12 oxidative damage

[0102] AML12 cells were resuscitated, mixed thoroughly with complete culture medium, and transferred to culture flasks. The cells were cultured at 37°C in a 5% CO2 incubator until stable. Stable-growing AML12 cells were seeded into 96-well plates and exposed to different concentrations of ZEN standard solution (0, 10, 20, 40, 50, 60, 70, 80, 90, and 100 μM) for CCK8 assays. The concentration at which a 25% biological effect was observed (IC50) was determined. 25 Meanwhile, the non-toxic concentration of PD and the concentration at which PD is significantly relieved were determined.

[0103] First, AML12 cells were treated with different concentrations of PD (0.5, 1, 5, 10, 20, 50, 100, 200, and 400 μM) to test its cytotoxicity. The non-toxic concentration of PD (CCK8 absorbance close to the control group) and the concentration that significantly alleviated ZEN-induced oxidative damage were determined using the CCK8 assay (CCK8 absorbance significantly higher than the ZEN group, close to the normal group).

[0104] Total RNA was then extracted from AML12 cells using TRIzol reagent according to the manufacturer's instructions in a clean bench. 1 μL of the RNA extract was diluted 100-fold with RNAase water, and the RNA concentration was determined using UV spectrophotometry. cDNA was synthesized using reagents provided by GenStar; the reverse transcription system and reaction conditions are shown in Table 3.

[0105] Table 3 Reverse transcription system and conditions

[0106]

[0107] The expression levels of SOD1, SOD2, CAT, GSH-PX, KEAP1, Nrf2, HO-1, and NQO1 were verified by qPCR detection of cDNA samples. The specific method is the same as in Example 1.

[0108] Example 6: Detection of ROS accumulation levels in AML12 cells

[0109] After exposing AML12 cells to ZEN standard solution for 6–24 hours, the culture medium was removed, the cells were washed three times with PBS, and DCFH-DA (10 μM) was added and incubated at 37°C for 30 minutes. The cells were then washed twice with PBS to remove unreacted dye, and images were captured using a fluorescence microscope. Simultaneously, a PD group was set up as the intervention group, and an allopurinol group as the positive control group, with AML12 cells treated at the corresponding concentrations.

[0110] ImageJ software was used to calculate the average fluorescence intensity of cells to assess the level of ROS accumulation.

[0111] The results are as follows Figure 7 As shown, by Figure 7 It is known that polygalactoside (PD) can regulate ZEN-induced cellular oxidative stress: PD downregulates XDH gene expression ( Figure 7 A), reducing ZEN-induced intracellular ROS accumulation ( Figure 7 (BC); by activating the Nrf2 pathway, it upregulates downstream antioxidant genes such as HO-1 and SOD1 (BC); Figure 7 DI), and restore the expression of antioxidant enzymes such as GSH-Px and CAT ( Figure 7The JK (a type of PD) synergistically alleviates ZEN-induced oxidative damage, clarifying the protective mechanism of PD against ZEN-induced oxidative stress.

[0112] Example 7: Validation of PD-mediated mitigation of ZEN oxidative damage in mouse in vivo model

[0113] 1. Grouping of experimental animals

[0114] Healthy male ICR mice were selected and, after one week of acclimatization, were randomly divided into six groups, with at least six mice in each group (n≥6). All animals were housed under the same environmental conditions, with free access to food and water. The specific grouping method is shown in Table 5.

[0115] Table 5 Grouping Processing

[0116] Group Handling method Con Control group (administered an equal volume of PBS) PD 200mg / kgb.wPD ZEN 25mg / kgb.wZEN ZP1 25mg / kgb.w ZEN + 50mg / kgb.w PD ZP2 25mg / kgb.w ZEN + 100mg / kgb.w PD ZP3 25mg / kgb.w ZEN + 200mg / kgb.w PD

[0117] ZEN (25 mg / kg b.w) and PD (50, 100, 200 mg / kg bw) were administered by gavage, twice daily (morning and evening), for 21 consecutive days. Control group mice received an equal volume of physiological saline. During the experiment, the mice's general behavior, weight changes, and food intake were observed. After administration, the mice were fasted for 12 hours, weighed, and anesthetized with isoflurane. Blood was collected, serum was separated, and liver tissue was rapidly dissected.

[0118] 2. Real-time quantitative PCR detection of changes in antioxidant-related genes in the liver of ZEN-exposed mice

[0119] (1) The mRNA expression levels of HO-1, NQO1, KEAP1 and Nrf2 in mouse liver tissue were detected. The specific implementation steps were the same as in Example 1. The effects of ZEN exposure on the antioxidant stress-related signaling pathways in mouse liver and the alleviating effect of PD on ZEN-induced oxidative damage were analyzed.

[0120] 3. XOD enzyme activity detection (colorimetric method)

[0121] Preheat the spectrophotometer for at least 30 minutes, adjust the wavelength to 530 nm, and zero the instrument with distilled water.

[0122] Dilute the standard solution to 0.25 μmol / mL, and add 50 μL of standard solution to each standard tube;

[0123] According to the kit instructions, add the corresponding reagents to the blank tube, test tube, and standard tube, mix well, and place in a 37°C water bath or constant temperature incubator for a specific time.

[0124] Measure the absorbance (A) at 530 nm in a 1 mL glass cuvette and calculate ΔA;

[0125] The XOD activity in the tissue sample was calculated using the XOD activity calculation formula. The XOD activity calculation formula is as follows:

[0126] XOD activity in tissue (U / gprot) = ((A assay - A blank) / ε) × (V total / V sample) × (1 / (T×d)) ÷ Cpr

[0127] Note: ε is the extinction molar coefficient of the chromogenic substance, 12.6 × 10⁻⁶ -3 Vreactiontotal is the total volume of the reaction solution, (2.27+a) mL; Vsample is the sample volume, mL; T is the reaction time, 20 minutes; d is the colorimetric path length, 1 cm; CHb is the hemoglobin content, gHb / L; Cpr is the homogenate protein content, gprot / L.

[0128] The results are as follows Figure 5 , Figure 5 The results showed that PD treatment significantly inhibited the enzymatic activity of XOD in cells and tissues. Colorimetric assays indicated that XOD activity in the PD group was significantly lower than that in the control group, and this inhibition was concentration-dependent. This suggests that PD can exert a potential biological effect by inhibiting XOD activity, reducing xanthine oxidative metabolism, and thus decreasing H2O2 production. Compared with the positive control group, the PD-treated group also showed a significant difference in XOD activity, further demonstrating that PD may inhibit XOD activity by directly binding to it or affecting its catalytic process, thereby validating PD's potential as an XOD inhibitor.

[0129] 4. The kit was used to detect oxidative stress levels in mouse livers.

[0130] Take 0.1g of liver tissue sample, add physiological saline, homogenize thoroughly under ice bath conditions, then centrifuge at 4℃ and 12000r / min for 15 minutes, and take the supernatant as the sample to be tested.

[0131] According to the CAT kit instructions, an appropriate amount of reagent was added to each well, and the absorbance change of the enzyme reaction was measured at a wavelength of 405 nm using an ELISA reader. The CAT activity (U / mgprot) in mouse liver tissue was calculated according to the formula. The effect of ZEN exposure on CAT activity in mouse liver was analyzed, and the PD alleviation effect was evaluated.

[0132] According to the kit instructions, add appropriate amounts of reagent to each well and measure the absorbance change at 532 nm using a microplate reader. Calculate the MDA content (nmol / mgprot) in mouse liver tissue using a formula, analyze the effect of ZEN exposure on MDA content in mouse liver, and evaluate the PD-relieving effect.

[0133] According to the T-SOD kit instructions, add an appropriate amount of reagent to each well and measure the absorbance change at 550 nm using an ELISA reader to calculate the total SOD activity (U / mgprot) in mouse liver tissue.

[0134] The results are as follows Figure 5 As shown, Figure 5 BM analysis showed that the MDA content in the liver of mice treated with ZEA was significantly increased, while the activities of SOD and CAT were significantly decreased, indicating that ZEA induced severe oxidative damage. After PD treatment, the MDA content in the liver of mice was significantly reduced, and the activities of SOD and CAT recovered to near normal levels, demonstrating the antioxidant effect of PD. Quantitative real-time PCR analysis showed that PD significantly inhibited the upregulation of HO-1, NQO1, and Nrf2 expression induced by ZEA, suggesting that PD may exert its effects by regulating antioxidant pathways. Western blot results showed that PD significantly inhibited the increase in XOD activity induced by ZEA, thereby alleviating oxidative stress and liver damage, further confirming the role of PD in alleviating ZEA-induced oxidative damage. This indicates that PD alleviates ZEA-induced oxidative damage by inhibiting XOD activity.

[0135] In summary, this invention proposes that the toxic mechanism of ZEA primarily involves inducing oxidative damage and impairing liver function. Polydatin (PD) directly interacts with XOD enzymes, inhibiting their activity, thereby alleviating ZEA-induced oxidative damage and improving liver function. PD reduces the amount of free radicals generated by XOD catalysis through direct binding, thus decreasing oxidative stress and mitigating ZEA-induced liver damage. Therefore, Polydatin exerts a protective effect against ZEA-induced oxidative damage through direct interaction with XOD enzymes and inhibition of their activity.

[0136] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of Polygonin in the preparation of zearalenone antidote.

2. The application according to claim 1, characterized in that, The zearalenone antidote is used to alleviate liver toxicity caused by zearalenone.

3. The application according to claim 2, characterized in that, The hepatotoxicity refers to liver damage.

4. The application according to claim 3, characterized in that, The liver injury mentioned is oxidative damage to the liver.

5. The application according to claim 2, characterized in that, The polygalactoside alleviates liver damage caused by zearalenone and reduces the toxicity of zearalenone to the body by inhibiting the activity of xanthine oxidase.

6. The application according to claim 1, characterized in that, The zearalenone antidote also includes pharmaceutically acceptable carriers or excipients.

7. The application according to claim 1, characterized in that, The dosage forms of the zearalenone antidote include tablets, pills, capsules, granules, and mixtures.

8. Application of Polygonin in the preparation of feed additives that alleviate the toxicity of zearalenone to the body.

9. The application according to claim 8, characterized in that, The toxicity of zearalenone to the body includes liver toxicity caused by zearalenone.

10. The application according to claim 9, characterized in that, The liver toxicity includes oxidative damage to the liver.

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