A composition containing rutin and its use

Abstract

This invention discloses a rutin-containing composition and its applications, belonging to the field of drug development technology. Through molecular docking simulations, animal experiments, and cell experiments, this invention demonstrates that rutin exerts its detoxification effect through a dual mechanism: firstly, it antagonizes the inhibitory effect of α-amatoxins on RNA polymerase II, improving transcriptional repression induced by poisoning; secondly, it alleviates oxidative stress caused by α-amatoxins, reducing MDA content and increasing SOD activity. Rutin significantly improves the survival rate of mice poisoned by α-amatoxins, improves hepatocellular pathological damage, and reduces liver function indicators AST and ALT levels. The optimal dosage is 175 mg / kg, indicating that rutin can be considered a potential therapeutic drug for α-amatoxins poisoning. This invention provides a new treatment option for α-amatoxins poisoning. Rutin is a marketed drug with guaranteed safety and significant advantages for clinical translation.

Description

Technical Field

[0001] This invention belongs to the field of drug development technology, specifically a composition containing rutin and its application. Background Technology

[0002] Wild mushrooms are beloved for their delicious taste and rich nutritional value, but their toxicity is difficult to discern from their appearance. Over 90% of wild mushroom poisoning deaths are caused by the ingestion of poisonous Amanita mushrooms. α-Amanitin (α-AMA) is the core lethal toxin in these mushrooms. This toxin has a molecular weight of 918.97 g / mol and is highly soluble in water, resistant to drying, high temperatures, and strong acids and alkalis. Conventional cooking methods and human stomach acid cannot destroy its toxin structure, significantly increasing the risk of accidental poisoning.

[0003] The poisoning mechanism of α-amanita peptide mainly includes two core pathways: First, inhibition of cellular transcription. α-Amanita peptide entering the liver interferes with the function of the trigger loop and the bridging helix by directly forming hydrogen bonds with Glu822 of the RNA polymerase II Rpb1 bridging helix and indirectly binding to His816 of the Rpb1 bridging helix, thereby inhibiting the elongation step of the transcription process. This process is irreversible, thus reducing the level of related mRNA synthesis, which in turn blocks the synthesis of downstream proteins, ultimately leading to hepatocyte death and liver failure in poisoned animals. Second, induction of excessive oxidative stress. α-Amanita peptide can disrupt the redox balance of hepatocytes, increasing the content of malondialdehyde (MDA), a lipid peroxidation product, and decreasing the activity of the antioxidant enzyme superoxide dismutase (SOD). Excessive accumulation of reactive oxygen species (ROS) triggers the mitochondrial apoptosis pathway, further exacerbating hepatocyte apoptosis. Furthermore, α-Amanita peptides can continuously act on the body through enterohepatic circulation. The lowest lethal dose for humans is only 0.1 mg / kg, meaning that an adult can die from ingesting 50-100g of Amanita mushrooms (each gram of mushroom contains 0.02%-0.04% α-Amanita peptides), resulting in an extremely high mortality rate.

[0004] Currently, there is no specific antidote for Amanita mushroom poisoning. Clinically, treatment options include gastric lavage, catharsis, blood purification, non-specific antidotes, and symptomatic supportive care, with limited effectiveness. Existing research has only confirmed that polymyxin B can competitively bind to α-amanita peptide at the RNA polymerase II binding site, improving liver damage and survival rates in poisoned animals to some extent. However, this drug has a single mechanism of action, and if the poisoned patient has contraindications to its use, there will be no effective treatment available. Therefore, there is an urgent need to develop new, safe, and effective antidotes for α-amanita peptide poisoning to fill this clinical gap.

[0005] Rutin, also known as rutin, has the molecular formula C. 27 H 30 O16 Rutin is a flavonol glycoside compound widely found in plants. It is an FDA-approved drug and has been shown to possess various pharmacological activities, including anti-inflammatory, antioxidant, and antibacterial effects. Studies have also been conducted on its applications in diabetes, obesity, malignant tumors, liver protection, vascular protection, neuroprotection, and cardioprotection. However, there are currently no reports on the use of rutin for the treatment of α-amatoxin poisoning, nor has the detoxification effect and mechanism of rutin on α-amatoxin poisoning been disclosed. Summary of the Invention

[0006] The present invention aims to solve the technical problems mentioned in the background art and provide a composition containing rutin and its application.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a composition containing rutin, the pharmaceutical composition comprising the active ingredient rutin and a pharmaceutically acceptable carrier, wherein the therapeutically effective dose of rutin is 175 mg / kg; the carrier is composed of dimethyl sulfoxide (DMSO) and phosphate-buffered saline (PBS), and the volume percentage of DMSO in the carrier is 20%.

[0008] Preferably, the preparation method of the pharmaceutical composition includes: weighing 3.5 mg of rutin powder, dissolving it in DMSO, and diluting it with PBS to 400 μL to obtain a rutin solution containing 20% ​​DMSO, and the solution should be prepared and used immediately.

[0009] Secondly, the present invention provides the use of a rutin-containing composition in the preparation of a medicament for treating Amanita mushroom poisoning.

[0010] Furthermore, this includes the following steps: (1) Intraperitoneal injection of the composition described in the first aspect into an α-amanitin poisoning model animal, the injection dose being 175 mg / kg; (2) The first administration was performed immediately after intraperitoneal injection of α-amanita peptide, followed by administration every 24 hours until day 7 of the experiment.

[0011] Thirdly, the present invention provides the use of a rutin-containing composition in the preparation of a drug that antagonizes the inhibitory effect of RNA polymerase II.

[0012] Preferably, the inhibition of RNA polymerase II is caused by Amanita mushroom poisoning or α-amanita peptide poisoning, and rutin is used in the preparation of drugs for treating α-amanita peptide poisoning.

[0013] Fourthly, the present invention provides the use of a rutin-containing composition in the preparation of a medicament for reducing oxidative stress levels.

[0014] Preferably, the increased oxidative stress level is caused by Amanita mushroom poisoning or α-amanita peptide poisoning.

[0015] Fifthly, the present invention provides the use of a rutin-containing composition in the preparation of a medicament for treating liver injury.

[0016] Preferably, the liver injury is caused by Amanita mushroom poisoning or α-amanita peptide poisoning.

[0017] Compared with the prior art, the present invention has the following outstanding advantages: 1. This invention discloses the application of a rutin-containing composition in the preparation of a drug for treating α-amanita poisoning, filling the gap in the prior art for the lack of a specific antidote for α-amanita poisoning, and providing a brand-new treatment option for the clinical treatment of poisonous Amanita mushroom poisoning.

[0018] 2. This invention clarifies the dual mechanism of action of rutin in detoxification. On the one hand, it can competitively bind to the α-amatoxin binding site of RNA polymerase II, antagonizing the irreversible inhibition of transcription by the toxin. On the other hand, it can alleviate toxin-induced oxidative stress damage through antioxidant activity. Compared with the existing polymyxin B with only a single mechanism of action, it has a more comprehensive therapeutic target.

[0019] 3. This invention has verified the detoxification effect of rutin through complete molecular docking, animal experiments, and cell experiments, and has determined that the optimal dosage is 175 mg / kg, the administration method is intraperitoneal injection, and the administration regimen is clear and specific, which can directly guide clinical translation and application.

[0020] 4. The rutin used in this invention is an FDA-approved drug, and its safety has been fully verified in clinical trials. Compared with newly developed detoxification drugs, it has a shorter clinical transformation cycle, lower research and development costs, and controllable drug risks, and has extremely high clinical application value and industrial promotion value. Attached Figure Description

[0021] Figure 1 This is a technical roadmap of the present invention; Figure 2 This is a schematic diagram of the hypothetical mechanism of the present invention; Figure 3 This is a schematic diagram of molecular docking according to the present invention; Figure 4 The 7-day survival rate of mice after treatment with different doses of rutin according to the present invention; Figure 5 The 7-day survival rate of mice treated with 175 mg / kg rutin according to the present invention; Figure 6 HE staining results of mouse liver (40X) according to the present invention; Figure 7The results of mouse serum AST and ALT in this invention; Figure 8 The results of mouse serum MDA and SOD in this invention; Figure 9 The fluorescence intensity of the HepG2 nascent RNA of this invention; Figure 10 The HepG2 neonatal RNA of this invention. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0023] 1. Introduction Wild mushroom poisoning incidents occur worldwide. In my country, wild mushroom poisoning has become one of the most common and fatal foodborne illnesses. [1] Currently, over 1,000 species of poisonous wild mushrooms are recorded worldwide, with approximately 520 reported in my country. These mushrooms are characterized by their diverse species and wide distribution. Wild mushrooms are often enjoyed for their delicious flavor, but relying solely on morphological identification can easily lead to the mistaken picking and ingestion of poisonous mushrooms. Every year, poisoning incidents due to the accidental ingestion of poisonous mushrooms occur frequently across various regions. [2] In my country, the southwest and central regions are high-incidence areas for wild mushroom poisoning, mainly concentrated in Yunnan, Guizhou, and Sichuan provinces, with Yunnan Province having the most cases of wild mushroom poisoning. [3] According to relevant statistics and analysis, from 2018 to 2022 alone, there were as many as 3,761 cases of wild mushroom poisoning in Yunnan Province, affecting a total of 14,254 people. [4] According to statistics from the China National Center for Food Safety Risk Assessment's foodborne disease detection and reporting system, from 2010 to 2020, there were 10,036 cases of mushroom poisoning, with the Amanita genus having the highest mortality rate. [5] Approximately 90% of deaths are caused by Amanita mushrooms. [6] According to a 2024 survey by the Chinese Center for Disease Control and Prevention, approximately 61.5% of mushroom poisoning deaths throughout the year were caused by mistakenly ingesting Amanita mushrooms. [7]It is evident that accidental ingestion of poisonous Amanita mushrooms remains the leading cause of death from mushroom poisoning. This genus mainly includes Amanita exitialis (commonly known as "White Amanita"), Amanita molliuscula, Amanita subpallidorosea, and Amanita pseudoporphyria. The main toxins in poisonous Amanita mushrooms are amatoxins, phallotoxins, and virotoxins. The most lethal toxin is α-amanitin, with a molecular weight of approximately 918.97 g / mol. It is readily soluble in water, resistant to drying, high temperatures, and strong acids and alkalis. Therefore, traditional cooking methods and stomach acid are insufficient to destroy the toxin's structure, increasing the risk of accidental poisoning. Currently, there is no specific antidote for Amanita mushroom poisoning. Clinical treatment for Amanita mushroom poisoning mainly involves non-specific antidotes and symptomatic supportive care. Previous studies by our research group have found that the median lethal dose (LD50) in mice is approximately 0.25 mg / kg. Related studies have reported that the lowest lethal dose (LD50) in humans can be as low as 0.1 mg / kg. This means that ingesting 50–100 g of mushrooms (each gram of mushroom contains 0.02%–0.04% α-amatoxins) in adults or 5–10 g of mushrooms in children can lead to death. [8,9] .

[0024] 2. Mechanism of α-Amanita poisoning 2.1. Repression of transcription Reports indicate that 58.46%–70.6% of orally ingested α-amatoxins are excreted in feces. Of the α-amatoxins absorbed into the bloodstream, 60% are excreted into the bile, allowing for enterohepatic circulation and sustaining poisoning for up to 3 days. Ultimately, over 80% is excreted through the kidneys in the urine, with less than 20% excreted in the bile.

[10] After α-amatoxins enter the bloodstream, they enter cells via organic anion transporting polypeptide 1B3 (OATP1B3) on the cell membrane surface. α-Amatoxin poisoning exhibits significant organ toxicity differences, primarily damaging organs such as the liver and kidneys, while the heart and brain are largely unaffected. Studies have reported that 0.3-100 μmol of α-amatoxins were used to incubate eight human cell lines derived from different organs (HepG2, BGC-823, HCT-8, HEK-293, AC16, A549, SH-SY5Y, and HUVEC), and CCK-8 assays were used to detect the effects. Cell viability was assessed, revealing that HepG2, BGC-823, and HEK-293 cell lines were most sensitive to α-amatoxins (IC50 values ​​of 8.01, 9.20, and 14.94 μmol, respectively), while A549, AC16, and HCT-8 cell lines showed the highest tolerance (IC50 > 100 μmol). HUVEC and SH-SY5Y cell lines exhibited moderate sensitivity, and increasing the toxin concentration did not further enhance its toxicity. Immunofluorescence and Western blot analysis of OATP1B3 expression in various cell lines showed high expression in sensitive cell lines (liver, stomach, and kidney) and low expression in tolerant cell lines (lung and heart). This indicates a positive correlation between OATP1B3 expression and cellular sensitivity to α-amatoxins, which may be closely related to the fact that α-amatoxins poisoning primarily leads to liver failure. Inhibiting OATP1B3 expression significantly improved the tolerance of HepG2 cells to α-amatoxins.

[11] .

[0025] In addition, our research group previously discovered that Na + - Taurocholic acid cotransporting polypeptide (Na + α-Amanita peptide (NAP) is also an important molecule mediating the entry of NTP into cells. Studies have shown that NTP expression is significantly upregulated in the liver of mice poisoned with NTP. HepG2 cells with lower NTP expression are more tolerant to NTP than L-02 cells with normal NTP expression. Intervention with ezetimibe, which competes with NTP on the NTCP protein, can significantly improve the survival rate of mice poisoned with NTP.

[12] .

[0026] α-Amanita peptides themselves do not have the ability to degrade proteins.

[13] The main mechanism by which α-amanitin causes poisoning is the inhibition of the activity of eukaryotic RNA polymerase II. RNA polymerase II is the most important enzyme in eukaryotes for transcription into mRNA.

[14] Brueckner & Cramer et al. isolated RNA polymerase II from Saccharomyces cerevisiae and used X-ray diffraction to capture the protein crystal structure of the interaction between α-amanitin and RNA polymerase II. They found that α-amanitin entering the liver forms hydrogen bonds directly with Glu822 of the Rpb1 bridge helix of RNA polymerase II, and indirectly binds to His816 of the Rpb1 bridge helix.

[15] This interfered with the function of the trigger ring and bridge screw.

[16] This inhibits the elongation step of transcription, a process that is irreversible. [17,18] Therefore, it reduces the level of related mRNA synthesis, thereby blocking the synthesis of downstream proteins, ultimately leading to hepatocyte death and liver failure and death in poisoned animals.

[0027] One study incubated HepG2 cells with 10 μmol and 20 μmol of α-amanitin for 6 hours and detected newly generated RNA. The results showed that the newly generated RNA in the poisoned group was reduced by 38.85% compared to the control group.

[19] Currently, there is very little research on polymyxin B competing for site occupancy with α-amanitin on RNA polymerase II. Garcia, Juliana, and others used computer simulations to discover that polymyxin B has therapeutic potential. Animal experiments showed that administering polymyxin B at 2.5 mg / kg at 4, 8, and 12 hours after modeling significantly improved liver damage and survival rate in the poisoned mice.

[20] This also indicates that the drug molecules screened through molecular docking simulation have good therapeutic potential in α-amanita peptide poisoning. However, currently only polymyxin B can antagonize the transcriptional repression effect of α-amanita peptide. If the poisoned person has contraindications to the use of polymyxin B, they will face a situation where no drug is available.

[0028] 2.2. Oxidative stress and mitochondrial apoptosis Studies have shown that α-amanita peptide poisoning leads to oxidative stress in hepatocytes. Oxidative stress is defined as the impairment of the balance between reactive oxygen species (ROS) and antioxidant capacity, and it is a common pathophysiological pathway in most types of poisoning. When α-amanita peptide enters the liver, it leads to increased SOD activity, decreased catalase activity, and promotes the production of lipid peroxidation products malondialdehyde (MDA) and tumor necrosis factor-α. [6,21,22]ROS promotes cellular oxidative stress, disrupts cellular redox balance, and produces excessive ROS. While ROS accumulation does not directly lead to cell death, it triggers the ROS / p53-mediated mitochondrial apoptosis pathway, inducing mitochondrial apoptosis in hepatocytes. Furthermore, ROS increases the Bax / Bcl-2 ratio, thereby activating downstream cysteine-aspartate specific protein families (caspase-9, caspase-3), ultimately inducing cell death. [23,24] Our previous research has shown that appropriate antioxidant therapy can improve liver damage caused by α-amanita poisoning.

[0029] 2.3. Other Mechanisms In 2023, Chinese researchers Wang and his team, through genome-wide screening of clustered regularly spaced short palindromic repeats, discovered that the N-glycan biosynthesis pathway catalyzed by the STT3 oligosaccharyltransferase complex catalytic subunit B (STT3B) plays a crucial role in α-amanita poisoning. Knocking out STT3B significantly reduced the amount of α-amanita entering cells without affecting the expression of OATP1B3 and NTCP. Through molecular docking simulation screening of FDA-approved drug molecules, indocyanine green (ICG) was identified as an STT3B inhibitor. Injections of ICG into mice at 4, 6, and 8 hours after α-amanita poisoning reduced liver and kidney damage and improved survival rates.

[25] Although many studies have been conducted on the mechanisms of α-amanita poisoning, the mechanisms by which α-amanita poisoning leads to acute liver injury and death are still not fully understood.

[0030] 3. Molecular docking simulation like Figure 3As shown, computer molecular docking simulation refers to the use of computer programs to predict the optimal binding mode of two or more molecules in three-dimensional space, as well as the interaction forces between them. This typically involves a larger molecule (receptor) and a smaller molecule (ligand), such as the docking between a protein and a drug molecule. The aim is to predict the binding modes and affinities of small molecule drugs with biomacromolecule targets (such as proteins), understand the mechanisms of intermolecular interactions, assist in drug design and structure optimization, and conduct virtual screening, i.e., identifying potential active molecules from a large number of compounds. Researchers such as Brueckner & Cramer isolated RNA polymerase II from Saccharomyces cerevisiae and used X-ray diffraction to capture the protein crystal structure of the interaction between α-amanita peptide and RNA polymerase II, explaining how α-amanita peptide inhibits the activity of RNA polymerase II to interfere with the elongation step of transcription.

[16] This embodiment utilizes the protein crystal structure constructed by Brueckner & Cramer et al. (https: / / www.rcsb.org (2VUM)), using the binding site of α-amanita peptide to RNA polymerase II as the docking active pocket (X: 104.07, Y: -67.52, Z: -17.8). Using the Schrodinger Maestro 13.5 molecular docking simulation program, FDA-approved drugs were screened (https: / / pubchem.ncbi.nlm.nih.gov / ), and polymyxin B, currently the only reported drug with a locating effect on α-amanita peptide, was used as a reference to evaluate and search for drugs that may play a detoxifying role, providing more options for the treatment of α-amanita peptide poisoning.

[0031] 4. Pharmacological effects of rutin The drug rutin was ultimately selected. Rutin, also known as rutin glycoside, has the molecular formula C. 27 H 30 O 16 With a molecular weight of 610.52 g / mol, rutin is a yellow crystalline powder with a bitter taste. It is a flavonol glycoside widely found in plants, primarily citrus fruits, onions, apples, tea, and buckwheat. After oral ingestion, rutin cannot be absorbed naturally by the animal's intestines. It must be broken down into quercetin by intestinal microorganisms before being absorbed into the bloodstream through the small intestine to exert its biological activity.

[26] Rutin possesses a variety of pharmacological activities, including anti-inflammatory, antioxidant, and antibacterial effects. Its applications in diabetes, obesity, malignant tumors, liver protection, vascular protection, neuroprotection, and cardioprotection have also been studied. [27-39]Rutin exhibits significant antioxidant activity, primarily due to the ortho-dihydroxy group on ring B, the 2,3-double bond in ring C forming a conjugated system with the 4-oxofunctional group, and the 7-hydroxy and 5-hydroxy groups in rings A and C connected to the 4-oxofunctional group.

[40] Rutin can scavenge oxygen free radicals, inhibit the peroxidation of polyunsaturated fatty acids on biological membranes, remove lipid peroxidation products, and protect the integrity of biological membranes and subcellular structures.

[41] Studies have reported that rutin can regulate hepatic redox homeostasis through multiple pathways by activating the Nrf2 / HO-1 axis: on the one hand, it can upregulate HO-1 expression, increase the activity of antioxidant enzymes such as SOD, CAT, and GPX, repair the GSH system to clear ROS, reduce oxidative damage markers such as MDA, NO, and LOOH, and block lipid peroxide damage to hepatocytes; on the other hand, it can inhibit downstream pathways such as MAPK / NF-κB and JNK / P38, reduce hepatocyte apoptosis, and can target and regulate hepatic oxidative stress and liver damage caused by drugs such as acetaminophen and tetracycline, as well as chemical toxins such as carbon tetrachloride and mercuric chloride.

[42] .

[0032] 5. Research Content and Technical Principles 5.1. Main Research Content Using molecular docking simulation software, FDA-approved drugs were screened to identify those that compete with α-amanitin for site occupancy on RNA polymerase II. These drugs were then compared with polymyxin B to evaluate their therapeutic potential.

[0033] Using male KM mice as the research subjects, α-amanita peptide was injected intraperitoneally to induce a model, followed by treatment with different doses of rutin. The 7-day survival rate of KM mice was observed and recorded to explore and determine the optimal therapeutic dose of rutin.

[0034] After determining the optimal therapeutic dose of rutin, the experiment was repeated. Liver damage and oxidative stress were assessed 30 hours after successful modeling to explore the therapeutic mechanism of rutin.

[0035] like Figure 9-10 As shown, using HepG2 cells as the research object, different doses of α-amanita peptide were used to successfully establish the cell model, followed by different doses of rutin intervention. The amount of newly generated RNA was detected to determine whether rutin could antagonize the inhibitory effect of α-amanita peptide on cell transcription.

[0036] 5.2. Research Hypothesis and Technology Roadmap Research hypothesis Experimental technique roadmap as follows Figure 1 As shown. The research hypothesis is as follows: Figure 2 As shown.

[0037] 1. Rutin reduces the inhibitory effect of α-amanita peptide on cell transcription by competitive site occupancy, thus playing a protective role against α-amanita peptide poisoning.

[0038] 2. Rutin can protect against α-amatoxin poisoning by reducing oxidative stress in hepatocytes induced by α-amatoxin.

[0039] Materials and Methods 1. Main Instruments Table 1 Main Experimental Instruments

[0040] 2. Main reagents Table 2. Main experimental reagents and manufacturers

[0041] 3. Main Experimental Methods 3.1. Molecular docking simulation First, download the protein crystal structure constructed by Brueckner & Cramer et al. (https: / / www.rcsb.org (2VUM)), and download the FDA-approved drug (https: / / pubchem.ncbi.nlm.nih.gov / ) and polymyxin B (https: / / www.rcsb.org (5L3F)). Second, prepare the downloaded protein and small molecules for docking using the Schrodinger Maestro 13.5 molecular docking simulation program. The Protein Preparation module is used for pre-docking protein molecule preparation, including water removal, hydrogen atom addition, and structure optimization. Third, the LigPrep module is used for small molecule drug ligand preparation. Fourth, the Receptor Grid Generation module is used to construct a docking activity pocket (X: 104.07, Y: -67.52, Z: -17.8) based on the binding site of α-amanita peptide and RNA polymerase II. Fifth, molecular docking is performed using the Ligand Docking module, followed by SP (Standard)... The drug molecules were screened in the Precision mode and selected from the top 10%. Then, in vitro molecular docking simulation was performed in the Extra Precision mode. Finally, drugs with therapeutic potential were identified based on docking scores. Drugs with severe liver damage or inability to enter the cell nucleus were excluded. Finally, the binding free energy was calculated using the Prime MM / GBSA module, and the docking score and binding free energy of polymyxin B were used as a reference to evaluate the therapeutic potential of the final docked drug molecules.

[0042] 3.2. Animal Experiments 3.2.1. Establishment of a poisoned mouse model Animal model creation: A poisoning animal model was created by intraperitoneal injection of 0.327 mg / kg α-amanita peptide into mice. A 1 ml syringe was used to draw the prepared α-amanita peptide solution, and all air was expelled. Holding the mouse's tail with the right hand, the index finger and thumb of the left hand gently slid upwards from the mouse's back, then grasped the head and back fur. The mouse was held belly-up with its head lowered, and the movements were gentle throughout the grasping process to avoid injury. The puncture point was selected approximately 1.5 cm to the left of the line connecting the base of the mouse's left hind leg and the midline of the abdomen. The needle was slowly inserted at a 45° angle to the skin. A breakthrough sensation indicated that the needle had entered the abdominal cavity. The insertion depth was generally less than 1 cm to avoid damaging abdominal organs. After successful puncture, the drug was slowly injected, and the needle was withdrawn. After successful injection, the mouse's condition and any bleeding or drug leakage at the puncture site were observed.

[0043] Preparation of α-Amanita peptide stock solution: Commercially available α-Amanita peptide is typically supplied as a white powder, usually in 1 mg / vial form. First, dissolve it in 1 mL of 20% DMSO to prepare a 1 mg / mL stock solution. Further dilute to the required concentration according to experimental needs. The solution should be stored at -20°C and prepared fresh whenever possible to ensure its stability and activity.

[0044] Rutin Dosage Selection and Preparation: Commercially available rutin is supplied as a yellow powder, and the standard specification used in research is 5g / bottle. Dissolve 6mg, 20mg, 30mg, 35mg, and 40mg of rutin powder in 20% DMSO. To ensure its activity, it should be prepared and used immediately.

[0045] 3.2.2. Exploring the optimal therapeutic dose of rutin Animal experiment groups: control group (CON), α-amanita peptide poisoning group (α-AMA), and rutin treatment group (α-AMA + different doses of rutin), with 10 mice in each group.

[0046] (1) CON group: Day 1: 400 μL of 20% DMSO was injected intraperitoneally. Days 2 to 7: 400 μL of 20% DMSO was injected intraperitoneally once a day.

[0047] (2) α-AMA group: Day 1: 0.327 mg / kg α-Amanita peptide (dissolved in 400 μL of 20% DMSO) was injected intraperitoneally. Days 2 to 7: 400 μL of 20% DMSO was injected intraperitoneally once a day.

[0048] (3) α-AMA + 30mg / kg Rutin group: 0.327mg / kg α-Amanita peptide (dissolved in 20% DMSO) was injected intraperitoneally. Immediately after the α-Amanita peptide injection model was established, 30mg / kg Rutin (dissolved in 20% DMSO) was injected intraperitoneally. The total volume of α-Amanita peptide and Rutin was 400μL. From the second to the seventh day, 30mg / kg Rutin (dissolved in 20% DMSO) was injected intraperitoneally. The total volume of Rutin was 400μL. Once a day.

[0049] (4) α-AMA + 100mg / kg Rutin group: 0.327mg / kg α-Amanita peptide (dissolved in 20% DMSO) was injected intraperitoneally. Immediately after the α-Amanita peptide injection model was established, 100mg / kg Rutin (dissolved in 20% DMSO) was injected intraperitoneally. The total volume of α-Amanita peptide and Rutin was 400μL. From the second to the seventh day, 100mg / kg Rutin (dissolved in 20% DMSO) was injected intraperitoneally. The total volume of Rutin was 400μL. Once a day.

[0050] (5) α-AMA + 150mg / kg Rutin group: 0.327mg / kg α-Amanita peptide (dissolved in 20% DMSO) was injected intraperitoneally. Immediately after the α-Amanita peptide injection model was established, 150mg / kg Rutin (dissolved in 20% DMSO) was injected intraperitoneally. The total volume of α-Amanita peptide and Rutin was 400μL. From the second to the seventh day, 150mg / kg Rutin (dissolved in 20% DMSO) was injected intraperitoneally. The total volume of Rutin was 400μL. Once a day.

[0051] (6) α-AMA + 175mg / kg Rutin group: 0.327mg / kg α-Amanita peptide (dissolved in 20% DMSO) was injected intraperitoneally. Immediately after the α-Amanita peptide injection model was established, 175mg / kg Rutin (dissolved in 20% DMSO) was injected intraperitoneally. The total volume of α-Amanita peptide and Rutin was 400μL. From the second to the seventh day, 175mg / kg Rutin (dissolved in 20% DMSO) was injected intraperitoneally. The total volume of Rutin was 400μL. Once a day.

[0052] (7) α-AMA + 200mg / kg Rutin group: 0.327mg / kg α-Amanita peptide (dissolved in 20% DMSO) was injected intraperitoneally. Immediately after the α-Amanita peptide injection model was established, 200mg / kg Rutin (dissolved in 20% DMSO) was injected intraperitoneally. The total volume of α-Amanita peptide and Rutin was 400μL. From the second to the seventh day, 200mg / kg Rutin (dissolved in 20% DMSO) was injected intraperitoneally. The total volume of Rutin was 400μL. Once a day.

[0053] 3.2.3. Repeat the experiment to determine the optimal therapeutic dose of rutin. Animal experiment groups: control group (CON), α-amanita peptide poisoning group (α-AMA), and rutin treatment group (α-AMA + 175 mg / kg Rutin), with 7 mice in each group.

[0054] (1) CON group: Day 1: 400 μL of 20% DMSO was injected intraperitoneally. Days 2 to 7: 400 μL of 20% DMSO was injected intraperitoneally once a day.

[0055] (2) α-AMA group: Day 1: 0.327 mg / kg α-Amanita peptide (dissolved in 400 μL of 20% DMSO) was injected intraperitoneally. Days 2 to 7: 400 μL of 20% DMSO was injected intraperitoneally once a day.

[0056] (3) α-AMA + 175mg / kg Rutin group: 0.327mg / kg α-Amanita peptide (dissolved in 20% DMSO) was injected intraperitoneally. Immediately after the α-Amanita peptide injection model was established, 175mg / kg Rutin (dissolved in 20% DMSO) was injected intraperitoneally. The total volume of α-Amanita peptide and Rutin was 400μL. From the second to the seventh day, 175mg / kg Rutin (dissolved in 20% DMSO) was injected intraperitoneally. The total volume of Rutin was 400μL. Once a day.

[0057] like Figure 4-5 As shown, the optimal therapeutic dose of rutin was finally determined to be 175 mg / kg, and all subsequent experiments were conducted at this concentration.

[0058] 3.2.4. Tissue Sampling from Laboratory Animals In the survival rate experiment, mice were observed to die 48-120 hours after modeling. Therefore, sample collection was performed 30 hours after successful modeling. All surgical instruments used for sample collection, including forceps and scissors, were sterilized at high temperatures. Aseptic technique and protection from light were observed during sample collection to ensure sample stability and experimental accuracy.

[0059] Blood was collected from the eyeballs of mice while they were still alive. The blood was placed in EP tubes containing EDTA and immediately centrifuged (1000 rpm, 10 min) to separate the plasma. The plasma samples were stored in a freezer at -80°C for subsequent testing.

[0060] After the mice were euthanized, their livers were quickly removed, rinsed with sterile PBS, and placed in clean, high-temperature sterilized EP tubes. Finally, they were stored in a -80°C freezer for subsequent experiments.

[0061] 3.3. Detection of liver tissue damage like Figure 6As shown, the pathological changes in mouse liver tissue were observed using the hematoxylin-eosin (HE) staining method. (1) Tissue fixation: 30 hours after successful modeling, the mice were dissected and liver tissues of each group were collected. The fresh liver tissues were fixed in 4% paraformaldehyde fixative for 24 hours and then rinsed under running water for 12 hours after appropriate trimming.

[0062] (2) Dehydration: After rinsing, liver tissue was placed in different concentrations of gradient ethanol solutions (70%, 85%, 95%, 100%) for dehydration, with each gradient treatment lasting 40 min.

[0063] (3) Transparency: The dehydrated tissue was sequentially transferred to a xylene-ethanol mixture, xylene I, and xylene II, with each step lasting 10 minutes, to enhance tissue transparency.

[0064] (4) Embedding in paraffin: After the tissue has been transparent, it is embedded in liquid paraffin to allow it to fully penetrate. After adjusting the orientation of the tissue, it is quickly cooled and solidified to form a wax block to fix the liver tissue.

[0065] (5) Sectioning: Using a paraffin microtome, trim the paraffin block and cut it into continuous tissue sections 3 μm thick. After flattening the sections, spread them in a water bath at 45°C and then attach them to two-thirds of the glass slide.

[0066] (6) Baking the slides: Place the glass slides in a 65℃ baking machine and bake for 1 hour, then transfer them to an oven and bake at 60℃ for 24 hours. After that, store them at 4℃ for later use.

[0067] (7) Dewaxing: The slides were placed in xylene I and II for 10 min each, then dehydrated in a gradient of 100%, 95%, 85% and 70% ethanol for 3 min each, and finally soaked in distilled water for 2 min to remove residual alcohol.

[0068] (8) Staining: The slide was stained in hematoxylin solution for 10 min, rinsed with running water for 15 min to make the tissue turn blue. Differentiate with 1% hydrochloric acid alcohol for 3 seconds, and then rinsed with running water to promote blueing.

[0069] (9) Eosin counterstaining and dehydration clearing: The sections were placed in 50%, 70%, and 80% ethanol for dehydration, 3 min for each gradient. Then, they were stained with eosin ethanol solution for 2 min and quickly washed with anhydrous ethanol for 1 min. After that, they were placed in anhydrous ethanol for 5 min, and finally transferred to xylene I and II for 5 min each for clearing.

[0070] (10) Mounting: After air drying, use neutral resin to mount the slide. After air drying, observe it using an optical microscope.

[0071] 3.4. Detection of liver function damage in mice like Figure 7 As shown, in this embodiment, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were used as biomarkers to detect the level of liver damage in mice. First, a suitable amount of blood (approximately 0.5-1 mL) was collected from mice via ocular blood collection. The blood was placed in a clean EP tube, and then the plasma was separated by centrifugation (1000 rpm, 10 min). The plasma was transferred to a clean EP tube and finally stored at -80°C to prevent degradation of the test components. The plasma sample was then used for subsequent liver function level testing. Next, different kits were used to detect the activities of AST and ALT.

[0072] 3.4.1. AST Activity Detection (1) Dilute the serum sample 10 times with distilled water and keep it away from light throughout the detection process.

[0073] (2) Take 5 μL of the prepared serum sample (two replicates per sample) and react with reagent 1 (provided in the kit) at 37°C for 30 min.

[0074] (3) Add 25 μL of reagent 2 (provided in the kit) and react at 37℃ for 20 min.

[0075] (4) Finally, add 240 μL of reagent 3 (provided in the kit) and let stand at room temperature for 10 min.

[0076] (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 505 nm.

[0077] (6) Finally, a standard curve was plotted using the standard, and the activity units of AST in each group of serum were calculated in U / L.

[0078] 3.4.2. ALT Activity Detection (1) Dilute the serum sample 10 times with distilled water and keep it away from light throughout the detection process.

[0079] (2) Take 5 μL of the prepared serum sample (two replicates per sample) and react with reagent 1 (provided in the kit) at 37°C for 30 min.

[0080] (3) Add 25 μL of reagent 2 (provided in the kit) and react at 37℃ for 20 min.

[0081] (4) Finally, add 240 μL of reagent 3 (provided in the kit) and let stand at room temperature for 10 min.

[0082] (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 505 nm.

[0083] (6) Finally, a standard curve was plotted using the standard, and the activity units of AST in each group of serum were calculated in U / L.

[0084] 3.5. Detection of oxidative stress levels in mouse peripheral blood In this embodiment, malondialdehyde (MDA) and superoxide dismutase (SOD), two biomarkers of oxidative stress, were used to detect the level of oxidative stress in mouse peripheral blood. First, a suitable amount of blood (approximately 0.5-1 mL) was collected from mice via ocular smear. The blood was placed in a clean EP tube, and then the plasma was separated by centrifugation (1000 rpm, 10 min). The plasma was transferred to a clean EP tube and finally stored at -80°C to prevent degradation of the components to be tested. The plasma sample was then used for subsequent oxidative stress level detection. Next, different kits were used to detect the activities of MDA and SOD.

[0085] 3.5.1. MDA Content Detection (1) Take 100 μL of plasma sample and add an appropriate amount of MDA working solution (prepared from TBA diluent, TBA stock solution and antioxidant provided by the kit), heat the sample to 100°C and keep it for 15 min.

[0086] (2) Cool the sample to room temperature and remove the precipitate by centrifugation, and take the supernatant.

[0087] (3) Use a multi-functional microplate reader to detect the photometric value at 532 nm, and calculate the MDA concentration in the sample (unit: nmol / mL) through the standard MDA curve.

[0088] 3.5.2. SOD Activity Detection (1) Incubate 20 μL of plasma sample with 20 μL of enzyme working solution (provided in the kit) and 200 μL of substrate application solution (provided in the kit) at 37°C for 20 min.

[0089] (2) The absorbance at 450 nm was detected by a multi-functional microplate reader. The inhibition rate of the reaction was calculated by the calculation formula (provided by the kit). The inhibition rate was controlled at about 50% by adjusting the dilution ratio. The experiment was repeated after determining the dilution ratio.

[0090] (3) Finally, the activity units of SOD in each group (unit: U / mL) are calculated using the calculation formula (provided by the kit).

[0091] 3.6. Transcriptional Activity Detection In this embodiment, the experimental method of Rodrigues, DF is referenced.

[19] Using HepG2 cells as the research subject, the experiment was divided into a control group (CON), a poisoning group (α-AMA), and a rutin treatment group (α-AMA+Rutin).

[0092] 3.6.1. Experimental Grouping (1)CON: Normal HepG2 cell group, without any intervention.

[0093] (2) 10 μmol α-AMA group: HepG2 cells were incubated with 10 μmol α-Amanita peptide for 6 h.

[0094] (3) 20 μmol α-AMA group: HepG2 cells were incubated with 20 μmol α-Amanita peptide for 6 h.

[0095] (4) 10 μmol α-AMA + 50 μmol Rutin group: HepG2 cells were incubated with 10 μmol α-Amanita peptide + 50 μmol rutin for 6 h.

[0096] (5) 10μmol α-AMA + 100μmol Rutin group: HepG2 cells were incubated with 10μmol α-Amanita peptide + 100μmol rutin for 6h.

[0097] (6) 10 μmol α-AMA + 200 μmol Rutin group: HepG2 cells were incubated with 10 μmol α-Amanita peptide + 200 μmol rutin for 6 h.

[0098] (7) 10 μmol α-AMA + 250 μmol Rutin group: HepG2 cells were incubated with 10 μmol α-Amanita peptide + 250 μmol rutin for 6 h.

[0099] (8) 20μmol α-AMA + 50μmol Rutin group: HepG2 cells were incubated with 20μmol α-Amanita peptide + 50μmol rutin for 6h.

[0100] (9) 20μmol α-AMA + 100μmol Rutin group: HepG2 cells were incubated with 20μmol α-Amanita peptide + 100μmol rutin for 6h.

[0101] (10) 20μmol α-AMA + 200μmol Rutin group: HepG2 cells were incubated with 20μmol α-Amanita peptide + 200μmol rutin for 6h.

[0102] (11) 20μmol α-AMA + 250μmol Rutin group: HepG2 cells were incubated with 20μmol α-Amanita peptide + 250μmol rutin for 6h.

[0103] 3.6.2. Detection of newly generated RNA (1) Each group was incubated at 37℃ for 6 hours.

[0104] (2) After washing the plate twice, change the culture medium and then add 1 mmol of EU-488 (provided by the kit) and incubate at 37°C for 2 hours.

[0105] (3) Remove the culture medium, and after washing and permeation, add Click reaction solution (provided by the kit) and incubate at room temperature in the dark for 30 min.

[0106] (4) After washing with washing solution, add 1X Hoechst 33342 (provided by the kit) for nuclear staining and incubate at room temperature in the dark for 10 min.

[0107] (5) The approximate treatment dose was determined by detecting the fluorescence intensity using a multifunctional microplate reader. The maximum excitation wavelength was 495 nm and the maximum emission wavelength was 519 nm. The fluorescence intensity of Azide 488 (representing the amount of newly generated RNA) was detected to assess transcriptional activity.

[0108] (6) Repeat the experiment at a determined dose, observe with a fluorescence microscope, observe and count the green fluorescence intensity, and evaluate transcriptional activity.

[0109] 3.7. Statistical Analysis All data in this experiment were statistically analyzed and plotted using GraphPad Prism 9.0. For experimental data with only two groups, a two-sample t-test was used for comparison and analysis; for experimental data with three or more groups, one-way ANOVA was used for statistical testing. A p-value < 0.05 was used as the criterion for statistical significance.

[0110] result 1. Molecular docking simulation results After molecular docking using the Ligand Docking module, some hepatotoxic drug molecules and those unable to enter the cell nucleus were excluded, ultimately resulting in the selection of rutin (Docking Score: -11.631 kcal / mol). Polymyxin B, currently the only reported drug with a competitive occupancy effect against α-amanita peptide, has a Docking Score of -12.408 kcal / mol, suggesting that their matching with the docking pocket is almost identical. MM / GBSA binding free energy calculations showed rutin ΔG bind: -51.37 kcal / mol, while polymyxin B ΔG bind: -49.13 kcal / mol, indicating that rutin's overall binding affinity is higher than that of polymyxin B. Using the polymyxin B result as a reference, rutin demonstrates good therapeutic potential for α-amanita peptide poisoning. Molecular docking scoring and binding free energy results are presented.

[0111]

[0112] 2. Efficacy and mechanism of action of rutin 2.1. Exploring the optimal therapeutic dose of rutin Mice were injected intraperitoneally with 0.327 mg / kg of α-amanita peptide, followed immediately by different doses of rutin. The corresponding dose of rutin was then injected intraperitoneally every 24 hours until day 7 or when all mice died. Survival observation over 7 days revealed a significant decrease in survival rate after poisoning. In the treatment groups, those ranging from 30 mg / kg to 200 mg / kg showed some protective effect and improved mouse survival rates, but the differences were not statistically significant (P>0.05). The protective effect was best at 175 mg / kg.

[0113] 2.2. Repeated verification of the protective effect of rutin Repeated experiments revealed that after intraperitoneal injection of 0.327 mg / kg α-amatoxins into mice, immediate intervention with 175 mg / kg rutin followed by intraperitoneal injection of rutin every 24 hours until day 7 or the death of all mice, the 7-day survival rate observation showed that 175 mg / kg rutin had a protective effect against α-amatoxins poisoning in mice, and the difference was statistically significant (P<0.05).

[0114] 2.3. Rutin treatment improves liver damage Thirty hours after the model was established, mice in the control group (CON), poisoned group (α-AMA), and treatment group (Rutin) were dissected, and their livers were harvested for histopathological examination.

[0115] Pathological results showed that in HE-stained sections of liver tissue, compared with the control group, mice in the α-amatoxin poisoning group showed obvious damage to the lobular region of the liver, specifically manifested as disordered hepatocyte structure, necrosis of hepatocytes around the central vein, and the appearance of a large number of vacuoles after cell necrosis. In contrast, the hepatocytes of mice in the rutin treatment group were arranged in an orderly manner, and the morphology of the hepatocytes was more intact than that of the poisoning group.

[0116] 2.4. Rutin treatment reduced serum liver function indicators. Peripheral blood was collected from mice in each group 30 hours after successful modeling, and the serum AST and ALT levels in peripheral blood were detected using AST and ALT detection kits.

[0117] The results showed that at 30 hours after poisoning, AST and ALT levels in mice in the α-amanita poisoning group were significantly higher than those in the control group (P<0.05). AST and ALT levels in the rutin treatment group were lower than those in the poisoning group, but the difference was not statistically significant (P>0.05). Considering the insufficient treatment duration, peripheral blood samples were collected from mice at day 7. The results showed that AST and ALT levels in the rutin treatment group were significantly lower at day 7 compared to 30 hours (P<0.05).

[0118] 2.5. Rutin treatment reduces serum oxidative stress levels. To assess oxidative stress levels and treatment efficacy, peripheral blood was collected from mice in each group 30 hours after successful modeling. Serum oxidative stress levels, including MDA and SOD, were measured using a kit.

[0119] like Figure 8 As shown, the results indicated that in peripheral blood, the serum MDA (dimethylaminopropyl hydroxyl) content in the poisoned group was significantly higher than that in the healthy control group (P < 0.05), and the MDA content significantly decreased after rutin treatment (P < 0.05). The SOD activity in the blood of the poisoned group was lower than that in the healthy control group, but the difference was not statistically significant (P > 0.05), while the SOD activity significantly increased after rutin treatment (P < 0.05). Independent samples t-tests were performed between the healthy control group and the poisoned group, and between the poisoned group and the treatment group. The results showed that the SOD activity in the poisoned group was lower than that in the healthy control group (P < 0.05), while the SOD activity in the treatment group was higher than that in the poisoned group (P < 0.05). This is likely due to the large intragroup variation in the treatment group and the small sample size.

[0120] 2.6. Rutin treatment improves transcriptional repression To assess cellular transcription levels, HepG2 liver cancer cells were selected as the research subject. After plating and adhesion, the control group was incubated with an equal volume and concentration of DMSO, the poisoning group was incubated with 10 and 20 μmol α-amatoxins, and the treatment group was co-incubated with 10 and 20 μmol α-amatoxins plus 50, 100, 200, and 250 μmol rutin. After 6 hours, the amount of newly generated RNA was detected using an RNA synthesis assay kit to assess cellular transcription. The results showed that the therapeutic effect was best with 10 μmol α-amatoxins plus 200 μmol rutin, and the difference was statistically significant (P < 0.05).

[0121] Based on the above experimental results, the experiment was repeated using 10 μmol α-amatoxin + 200 μmol rutin. The results showed that the amount of newly generated RNA in the α-amatoxin poisoning group was significantly lower than that in the control group after 6 hours, while the amount of newly generated RNA in the rutin treatment group was higher than that in the poisoning group, indicating that the inhibition of cell transcription was improved.

[0122] According to official statistics, Yunnan Province ranks first in China in terms of suitable growing area, yield, and output value of edible wild mushrooms. It possesses 91% of China's edible wild mushroom resources and 43% of the world's, with commercialized edible wild mushrooms accounting for over 70% of the national total. According to statistics from the Yunnan Provincial Edible Mushroom Industry Upgrading and Development Office in 2024, the province's edible mushroom production reached 1.1941 million tons, with a total output value of 47.25 billion yuan, and a comprehensive output value exceeding 110 billion yuan. Data released by "Yunnan Release" in 2025 shows that Yunnan's wild mushroom industry will continue to lead the country, with a total edible mushroom production of 1.2386 million tons and a total output value of 49.605 billion yuan. This will directly create 200,000 jobs and indirectly benefit 3 million people, with 10 million person-times of employment, and will help farmers increase their annual income by an average of 3,000 yuan. Edible wild mushrooms are a specialty of Yunnan Province, an important manifestation of the province's ecological and cultural diversity, and are of great significance to Yunnan's economic development, cultural dissemination, and tourism industry. However, due to the growth characteristics of wild mushrooms, it is difficult to identify poisonous wild mushrooms by their appearance. Therefore, poisoning incidents caused by mistakenly eating poisonous wild mushrooms often occur, with Yunnan Province being a high-incidence area for wild mushroom poisoning. [3] According to relevant statistics and analysis, from 2018 to 2022 alone, there were as many as 3,761 cases of wild mushroom poisoning in Yunnan Province, affecting a total of 14,254 people. [4] Among all mushroom poisoning incidents, poisoning by Amanita mushrooms is a significant cause of death. The most potent Amanita toxin is α-amatoxin, but currently there is no specific antidote for α-amatoxin. Clinical treatment primarily involves non-specific drug therapy, symptomatic relief, and supportive care.

[43] The main mechanism of α-amanita peptide poisoning is the inhibition of RNA polymerase II activity, which reduces the synthesis of related mRNAs, leading to a decrease in the synthesis of cell-related proteins and ultimately cell death. This study investigated the mechanism of α-amanita peptide poisoning and found that intraperitoneal injection of 0.327 mg / kg α-amanita peptide into male KM mice significantly increased the mortality rate, with peak mortality occurring between 48 and 120 hours, and a final mortality rate of 80%–90%. Liver tissue and blood samples taken 30 hours later revealed extensive necrosis of hepatocytes around the interlobular veins in the poisoned group, with visible vacuoles after cell necrosis and lysis. In contrast, the healthy control group showed no obvious pathological damage. Blood tests of liver function indicators AST and ALT showed significantly higher levels in the poisoned group compared to the healthy control group. Therefore, the liver is the main target organ for α-amanita peptide poisoning, and liver failure is the primary cause of death in animals poisoned by α-amanita peptide. Further investigation into the mechanism of α-amanita peptide poisoning revealed that the poisoned mice had increased levels of lipid peroxidation product MDA and decreased levels of antioxidant enzyme SOD compared to the healthy control group, indicating that the poisoned mice experienced severe oxidative stress. Simultaneously, a significant decrease in newly generated RNA in HepG2 cells was found in the poisoned group, suggesting that α-amanita peptide inhibited the transcriptional process in HepG2 cells.

[0123] Studies have revealed very few reports on the competition for site space between RNA polymerase II and α-amanita peptide. Only one study reported that polymyxin B can antagonize the inhibitory effect of α-amanita peptide on RNA polymerase II activity, but its mechanism of action is singular, and there are currently no other drugs of this type. If patients with Amanita mushroom poisoning have contraindications to this drug, there are no other similar drugs available. Therefore, this embodiment uses computer molecular docking simulations to screen FDA-approved drugs, aiming to find new drugs that can exert a protective effect by antagonizing the transcriptional inhibition of α-amanita peptide. Rutin was ultimately selected. Molecular docking simulation evaluation and binding free energy calculations showed that rutin and polymyxin B have almost identical binding site matching, but rutin's binding capacity is stronger than that of polymyxin B. Rutin is a flavonol glycoside compound with various effects such as antioxidation, antibacterial, and anti-inflammatory properties, and excessive oxidative stress is one of the mechanisms of α-amanita peptide poisoning. Therefore, it is hypothesized that rutin can exert a protective effect by antagonizing the inhibitory effect of α-amanitin on cell transcription and reducing oxidative stress caused by α-amanitin poisoning.

[0124] In this embodiment, it was found that rutin treatment significantly improved the survival rate of mice poisoned by α-amatoxins. Further investigation of the rutin treatment dosage revealed that 175 mg / kg showed the best therapeutic effect. Pathological observation of liver sections showed that rutin treatment improved liver damage in mice; hepatocytes in the rutin-treated group were arranged in an orderly manner, and their morphology was relatively intact. Liver function indicators AST and ALT were measured. The AST and ALT levels in the α-amatoxins poisoning group were significantly higher than those in the control group. While AST and ALT levels decreased slightly after 30 hours of rutin treatment compared to the poisoning group, the difference was not statistically significant, suggesting that the treatment duration might be insufficient. Therefore, the treatment course was extended to 7 days, and peripheral blood was collected from the mice on day 7. The results showed that AST and ALT levels in the rutin-treated group were significantly lower on day 7 compared to 30 hours, with a statistically significant difference. The effects of rutin on oxidative stress induced by α-amatoxins were also evaluated. Measurements of SOD and MDA showed that rutin treatment reduced MDA levels and increased SOD activity in the treatment group. However, there was no statistically significant difference in SOD levels between the control and treatment groups, likely due to large individual sample differences and a small sample size within the treatment group. Finally, the antagonistic effect of rutin on the inhibition of α-amatoxins on cellular transcription was explored. Rutin treatment revealed an increase in newly generated RNA in HepG2 cells compared to the poisoning group, indicating an improvement in transcriptional inhibition.

[0125] This embodiment explored the poisoning mechanism of α-amanita peptide and confirmed the effectiveness and mechanism of action of rutin in treating α-amanita peptide poisoning. However, this embodiment still has limitations. First, clinical treatment of Amanita mushroom poisoning often involves multiple methods and combined medications. This embodiment only explored the therapeutic effect of rutin alone. Future research may explore the therapeutic effects of rutin combined with multiple routes, methods, and medications. Second, the research subjects in this embodiment were the KM mouse, a commonly used animal model for toxicology, and the HepG2 cell, a commonly used model for hepatocyte research. However, these models still differ from humans. Therefore, future research should further investigate the safety and efficacy in more representative animal models (such as primates).

[0126] In summary, this embodiment demonstrates that rutin can exert a protective effect by antagonizing the inhibitory effect of α-amatoxins on cellular transcription and alleviating oxidative stress induced by α-amatoxins, providing new support for the clinical application of rutin and offering a new direction for the treatment of Amanita mushroom poisoning.

[0127] This embodiment delves into the mechanism of α-amanita poisoning, revealing that α-amanita poisoning inhibits cellular transcription, leading to increased mortality in mice, oxidative stress, and severe liver damage. Furthermore, it demonstrates that rutin, a drug discovered through molecular docking simulations, can antagonize the inhibitory effect of α-amanita on cellular transcription and alleviate oxidative stress induced by α-amanita, thereby reducing liver damage, decreasing liver failure, and ultimately improving mouse survival rates. As an FDA-approved drug, rutin has a assured safety profile and offers advantages in clinical translation.

[0128] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features of the invention herein.

[0129] References [1] Yu Chengmin, Li Haijiao. Expert consensus on clinical diagnosis and treatment of poisoning by mushrooms containing amatoxins in China [J]. Chinese Journal of Critical Care Medicine (Electronic Edition), 2020, 13(01): 20-8. [2]LI H, TIAN Y, MENOLLI N, JR., et al. Reviewing the world's ediblemushroom species: A new evidence-based classification system [J]. Compr RevFood Sci Food Saf, 2021, 20(2): 1982-2014. [3] Fan Penghui, Li Hongqiu, Chu Zunhua, et al. Analysis of the results of foodborne disease outbreak surveillance in China in 2023 [J]. Chinese Journal of Food Hygiene, 2024, 36(10): 1199-208. [4] Xiao Weijie, Su Weiwei, Ruan Yuan, et al. Epidemiological characteristics and spatial correlation analysis of wild mushroom poisoning in Yunnan Province from 2018 to 2022 [J]. Practical Preventive Medicine, 2025, 32(08): 902-6. [5]LI W, PIRES SM, LIU Z, et al. Mushroom Poisoning Outbreaks -China, 2010-2020 [J]. China CDC Wkly, 2021, 3(24): 518-22. [6]GARCIA J, COSTA VM, CARVALHO A, et al. Amanita phalloidespoisoning: Mechanisms of toxicity and treatment [J]. Food Chem Toxicol, 2015,86: 41-55. [7]LI H, ZHANG Y, ZHANG H, et al. Mushroom Poisoning Outbreaks -China, 2024 [J]. China CDC Wkly, 2025, 7(19): 645-9. [8] Sun Chengye. Prevention and control of mushroom poisoning urgently needs to be strengthened [J]. Chinese Journal of Emergency Medicine, 2016, 25(8): 981-4. [9]YILMAZ I, ERMIS F, AKATA I, et al. A Case Study: What Doses ofAmanita phalloides and Amatoxins Are Lethal to Humans? [J]. WildernessEnviron Med, 2015, 26(4): 491-6.

[10] ESCODA O, REVERTER E, TO-FIGUERAS J, et al. Potential value of urinary amatoxin quantification in patients with hepatotoxic mushroompoisoning [J]. Liver Int, 2019, 39(6): 1128-35.

[11] GONG M, LI Z, XU H, et al. Amanitin-induced variable cytotoxicity in various cell lines is mediated by the different expression levels ofOATP1B3 [J]. Food Chem Toxicol, 2024, 188: 114665.

[12] XUE J, LOU

[13] TODOROVIC M, RIVOLLIER P, WONG A, et al. Rationally DesignedAmanitins Achieve Enhanced Cytotoxicity [J]. J Med Chem, 2022, 65(15): 10357-76.

[14] Xu Yue, Wang Chan, Lin Shucheng, et al. Research progress on the mechanism of hepatotoxicity induced by α-amanitin [J]. Journal of Kunming Medical University, 2022, 43(12): 153-8.

[15] BUSHNELL DA, CRAMER P, KORNBERG R D. Structural basis oftranscription: alpha-amanitin-RNA polymerase II cocrystal at 2.8 A resolution[J]. Proc Natl Acad Sci USA, 2002, 99(3): 1218-22.

[16] BRUECKNER F, CRAMER P. Structural basis of transcriptioninhibition by alpha-amanitin and implications for RNA polymerase IItranslocation [J]. Nat Struct Mol Biol, 2008, 15(8): 811-8.

[17] IVLEVA E A, BOGOMOLOV D V, PUTINTSEV V A, et al. [The peculiarfeatures of the morphological manifestations of the damage to the liverassociated with the death cap mushroom poisoning][J]. Sud Med Ekspert, 2017,60(3): 23-6.

[18] LIU X, FARNUNG L, WIGGE C, et al. Cryo-EM structure of amammalian RNA polymerase II elongation complex inhibited by α-amanitin [J]. JBiol Chem, 2018, 293(19): 7189-94.

[19] RODRIGUES D F, PIRES DAS NEVES R, CARVALHO A T P, et al. In vitromechanistic studies on α-amanitin and its putative antidotes [J]. ArchToxicol, 2020, 94(6): 2061-78.

[20] GARCIA J, COSTA V M, CARVALHO A T, et al. A breakthrough onAmanita phalloides poisoning: an effective antidotal effect by polymyxin B[J]. Arch Toxicol, 2015, 89(12): 2305-23.

[21] LE DARé B, FERRON PJ, GICQUEL T. Toxic Effects of Amanitins:Repurposing Toxicities toward New Therapeutics [J]. Toxins (Basel), 2021, 13(6).

[22] Lu Zhongqiu, Hong Guangliang, Sun Chengye, et al. Clinical consensus of Chinese experts on the diagnosis and treatment of mushroom poisoning[J]. Chinese Journal of Emergency Medicine, 2019, 39(08): 717-25.

[23] MAGDALAN J, OSTROWSKA A, PIOTROWSKA A, et al. alpha-Amanitin induced apoptosis in primary cultured dog hepatocytes [J]. Folia HistochemCytobiol, 2010, 48(1): 58-62.

[24] MAGDALAN J, PIOTROWSKA A, GOMUŁKIEWICZ A, et al. Influence of commonly used clinical antidotes on antioxidant systems in human hepatocyteculture intoxicated with alpha-amanitin [J]. Hum Exp Toxicol, 2011, 30(1):38-43.

[25] WANG B, WAN AH, XU Y, et al. Identification of indocyanine greenas a STT3B inhibitor against mushroom α-amanitin cytotoxicity [J]. NatCommun, 2023, 14(1): 2241.

[26] LI Q, QIN M, LI T, et al. Rutin protects against pirarubicin-induced cardiotoxicity by adjusting microRNA-125b-1-3p-mediated JunDsignaling pathway [J]. Mol Cell Biochem, 2020, 466(1-2): 139-48.

[27] Mao Yajun, Feng Yali, Wang Mengjiao, et al. Research progress on rutin derivatives [J]. Chinese Journal of Traditional Chinese Medicine, 2021, 46(18): 4654-65.

[28] HOSSEINZADEH H, NASSIRI-ASL M. Review of the protective effects of rutin on the metabolic function as an important dietary flavonoid [J]. JEndocrinol Invest, 2014, 37(9): 783-8.

[29] SUZUKI T, KUROKOH R, MURAKAMI S, et al. Rutin Concentration and Characterization of Rutinosidase in Perennial Buckwheat (Fagopyrum cymosum) and Its Application in Foods [J]. Foods, 2023, 12(7).

[30] SELLOUM L, BOURICHE H, TIGRINE C, et al. Anti-inflammatory effect of rutin on rat paw oedema, and on neutrophils chemotaxis and degranulation[J]. Exp Toxicol Pathol, 2003, 54(4): 313-8.

[31] YANG J, GUO J, YUAN J. In vitro antioxidant properties of rutin[J]. LWT-Food Science&Technology, 2008, (6): 41.

[32] JANBAZ K H, SAEED S A, GILANI A H. Protective effect of rutin onparacetamol- and CCl4-induced hepatotoxicity in rodents [J]. Fitoterapia,2002, 73(7-8): 557-63.

[33] ALINEJAD B, GHORBANI A, SADEGHNIA H R. Effects of combinations ofcurcumin, linalool, rutin, safranal, and thymoquinone on glucose / serumdeprivation-induced cell death [J]. Avicenna J Phytomed, 2013, 3(4): 321-8.

[34] SADEGHNIA H R, YOUSEFSANI B S, RASHIDFAR M, et al. Protectiveeffect of rutin on hexachlorobutadiene-induced nephrotoxicity [J]. Ren Fail,2013, 35(8): 1151-5.

[35] BENAVENTE-GARCíA O, CASTILLO J. Update on uses and properties ofcitrus flavonoids: new findings in anticancer, cardiovascular, and anti-inflammatory activity [J]. J Agric Food Chem, 2008, 56(15): 6185-205.

[36] LI Y, QIN R, YAN H, et al. Inhibition of vascular smooth musclecells premature senescence with rutin attenuates and stabilizes diabeticatherosclerosis [J]. J Nutr Biochem, 2018, 51: 91-8.

[37] BEN SGHAIER M, PAGANO A, MOUSSLIM M, et al. Rutin inhibitsproliferation, attenuates superoxide production and decreases adhesion andmigration of human cancerous cells [J]. Biomed Pharmacother, 2016, 84: 1972-8.

[38] SONG H L, ZHANG X, WANG W Z, et al. Neuroprotective mechanisms ofrutin for spinal cord injury through anti-oxidation and anti-inflammation andinhibition of p38 mitogen activated protein kinase pathway [J]. Neural RegenRes, 2018, 13(1): 128-34.

[39] LIU Y, SUN Z, DONG R, et al. Rutin ameliorated lipid metabolismdysfunction of diabetic NAFLD via AMPK / SREBP1 pathway [J]. Phytomedicine,2024, 126: 155437.

[40] OLIVEIRA CA, PERES DD, GRAZIOLA F, et al. Cutaneousbiocompatible rutin-loaded gelatin-based nanoparticles increase the SPF of the association of UVA and UVB filters [J]. Eur J Pharm Sci, 2016, 81: 1-9.

[41] Gu Hongyan, Zhang Yuanying, Zhai Jing, et al. Effects of vitamin C and rutin on free radical metabolism in tissues of exhausted mice [J]. Chinese Journal of Clinical Rehabilitation, 2005, (44): 132-4.

[42] FENG Y, PENG L, LIU

[43] XUE J, LOU X, NING D, et al. Mechanism and treatment of α-amanitin poisoning [J]. Arch Toxicol, 2023, 97(1): 121-31.

Claims

1. A composition containing rutin, characterized in that, The pharmaceutical composition comprises the active ingredient rutin and a pharmaceutically acceptable carrier, wherein the therapeutically effective dose of rutin is 175 mg / kg; the carrier is composed of dimethyl sulfoxide (DMSO) and phosphate-buffered saline (PBS), and the volume percentage of DMSO in the carrier is 20%.

2. The composition according to claim 1, characterized in that: The preparation method of the pharmaceutical composition includes: weighing 3.5 mg of rutin powder, dissolving it in DMSO, and then diluting it with PBS to 400 μL to obtain a rutin solution containing 20% ​​DMSO, and the solution should be prepared and used immediately.

3. The use of a rutin-containing composition in the preparation of a medicament for treating Amanita mushroom poisoning.

4. The application as described in claim 3, characterized in that, Includes the following steps: In an α-amanitin poisoning model animal, the composition of claim 1 was injected intraperitoneally at a dose of 175 mg / kg. The first administration was performed immediately after intraperitoneal injection of α-amanita peptide, followed by administration every 24 hours until day 7 of the experiment.

5. The use of a rutin-containing composition in the preparation of a drug that antagonizes the inhibitory effect of RNA polymerase II.

6. The application according to claim 5, characterized in that, The inhibition of RNA polymerase II is caused by Amanita mushroom poisoning or α-amanita peptide poisoning. Rutin is used in the preparation of α-amanita peptide (… α-amanitin, Application in α-AMA poisoning drugs.

7. The use of a rutin-containing composition in the preparation of a medicament for reducing oxidative stress levels.

8. The application according to claim 7, characterized in that, The increased oxidative stress level was caused by Amanita mushroom poisoning or α-amanita peptide poisoning.

9. The use of a rutin-containing composition in the preparation of a medicament for treating liver injury.

10. The application according to claim 9, characterized in that, The liver damage was caused by Amanita mushroom poisoning or α-amanita peptide poisoning.

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