Application of caesalpinia, cordyceps polysaccharide or calycosin and their combination in preparation of medicine for relieving mycotoxin toxicity damage

Abstract

The application discloses application of callilepis, cordyceps polysaccharide or calyx formosanum isoflavone or combination of callilepis, cordyceps polysaccharide and calyx formosanum isoflavone in preparation of a medicine for relieving vomitoxin toxicity. According to the application, the callilepis, cordyceps polysaccharide and calyx formosanum isoflavone are used alone and in combination as a feed additive, a medicine for relieving vomitoxin toxicity or a detoxicant, repair vomitoxin-induced cell inflammation and damage of liver, intestinal tract and kidney through single and combined action, can effectively relieve vomitoxin toxicity on animals in a safe dosage, and thus improve the health condition of animals.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of a drug combination that can relieve vomiting toxins. Background Technology

[0002] Deoxynivalenol (Vomitoxin), also known as vomitoxin or deoxy-fusarium alcohol, has the chemical name 3α,7α,15-trihydroxy-12,13-epoxy-thomyl-9-anthraphen-8-one. Vomitoxin is a type B trichothecotoxin produced by fungi such as *Fusarium graminearum*, and is widely found in grains such as wheat, corn, oats, and barley. Vomitoxin poses a serious threat to human and animal health, causing a series of toxic effects including vomiting, diarrhea, loss of appetite, decreased growth performance, and immune system disorders, leading to liver and kidney damage, decreased immune function, and intestinal inflammation. Vomitoxin activates key signaling pathways such as MAPK, NF-κB, and the NLRP3 inflammasome, resulting in the excessive release of pro-inflammatory factors (such as IL-1β and IL-6), triggering inflammatory responses and apoptosis. Meanwhile, vomitoxin can significantly induce oxidative stress, manifested as the accumulation of reactive oxygen species (ROS), an increase in the lipid peroxidation end product malondialdehyde (MDA), and a decrease in the bioactivity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), leading to cellular oxidative damage. Furthermore, vomitoxin can also disrupt mitochondrial function, exacerbate DNA damage, and consequently affect tissue homeostasis. Due to its toxicity and widespread presence, vomitoxin has become a global public health problem, posing a significant challenge to food and feed safety. Therefore, the effective degradation and control of vomitoxin is crucial for protecting animal and human health.

[0003] Although physical and chemical detoxification are the main treatment measures at the raw material stage, they still suffer from problems such as low efficiency and significant side effects. Therefore, using highly effective and low-toxicity drugs to antagonize the toxic effects of vomitoxin from the perspective of humans and animals is of great protective significance. Traditional Chinese medicine has the advantages of abundant resources, fewer toxic side effects, and relative green and safe properties; therefore, it is an important choice for antagonizing vomitoxin damage.

[0004] Cajanine, with CAS number 87402-84-4 and English name, has the molecular formula C2. 21 H 22O4, with a relative molecular mass of 338.4. Pyrenin is a natural stilbene compound isolated from the leaves of pigeon pea, a woody edible legume. As the main bioactive component of pigeon pea leaves, pyrenin has the effects of clearing heat and detoxifying, promoting blood circulation and removing blood stasis, and promoting diuresis and reducing swelling. It is often used for damp-heat jaundice, traumatic injuries, and carbuncles and boils. Studies have shown that pyrenin exhibits various biological activities, including anti-inflammatory, antibacterial, antitumor, anti-hepatitis, analgesic, anti-herpesvirus, antidepressant, learning and memory improvement, and hypoglycemic pharmacological activities. Pyrenin can prevent acetaminophen-induced liver damage by enhancing mitochondrial quality control and reducing oxidative stress. However, to date, there are no reports on pyrenin alleviating the toxicity of vomitoxin.

[0005] Calycosin, CAS number 20575-57-9, has the molecular formula C20575-57-9. 16 H 12 O5, with a relative molecular mass of 284.26, isoflavones are an important isoflavone compound, mainly found in Astragalus membranaceus, and also in Pueraria lobata, Ferns of the Armillaria thunbergii, Caragana sinica, Sophora flavescens, Millettia speciosa, and Glycyrrhiza uralensis. Astragalus membranaceus is slightly warm in nature and sweet in taste, entering the spleen and lung meridians. It has the effects of tonifying qi and raising yang, strengthening the defensive qi and consolidating the exterior, promoting diuresis and reducing swelling, generating fluids and nourishing blood, promoting blood circulation and relieving stagnation, promoting pus drainage and detoxification, and promoting tissue regeneration. It is often used for symptoms such as qi deficiency and fatigue, spontaneous sweating due to exterior deficiency, and chronic diarrhea with rectal prolapse. As one of the core active components of Astragalus membranaceus, isoflavones have various effects, including antioxidant, anti-inflammatory, anti-tumor, anti-fibrotic, antiviral, improvement of inflammatory damage in various organs, cardiovascular protection, and estrogen-like activity. Furthermore, isoflavones have low toxicity and high pharmacological activity. Studies have shown that verbascoside enhances antioxidant enzyme activity and reduces oxidative stress damage by activating the Nrf2 / ARE pathway; it also exerts anti-inflammatory and anti-organ fibrosis effects by inhibiting the NF-κB and TGF-β / Smad signaling pathways. Verascoside exerts significant protective effects against inflammatory damage in various organs, including the intestines, kidneys, liver, brain and nerves, heart, and lungs. Verascoside not only inhibits inflammatory responses but also improves intestinal structure and function. It can alleviate diabetic kidney damage by regulating the NLRP3 inflammasome. Verascoside also improves liver damage, reduces fat accumulation, alleviates liver structural damage and inflammatory responses, and improves liver fibrosis. However, there are currently no reports on the ability of verbascoside to alleviate the toxicity of vomitoxin.

[0006] Cordyceps polysaccharide, also known as cordyceps polysaccharide, is a natural active polysaccharide extracted from medicinal fungi such as Cordyceps sinensis and Cordyceps militaris. It possesses a wide range of pharmacological effects. Cordyceps polysaccharide is the core active ingredient of Cordyceps (such as Cordyceps sinensis and Cordyceps militaris). In traditional Chinese medicine theory, it is associated with the lung and kidney meridians, and is believed to tonify the lungs and kidneys, replenish essence and qi, and strengthen the body's resistance. It is commonly used to treat consumptive cough and asthma, low immunity, and chronic illness. Its core functions include: immune regulation (activating macrophages, T cells, and NK cells to enhance the body's defense capabilities), antioxidation (scavenging free radicals and inhibiting lipid peroxidation), anti-tumor adjuvant therapy (inhibiting cancer cell proliferation through immune enhancement and apoptosis induction), metabolic regulation (improving insulin sensitivity and lowering blood sugar and blood lipids), organ protection (reducing liver damage, improving kidney function, and protecting the myocardium), and anti-fatigue and neuroprotective effects (enhancing energy metabolism and delaying neurodegenerative diseases). Its mechanism of action involves the regulation of key signaling pathways such as TLR / NF-κB, PI3K / Akt, and Nrf2 / HO-1. Summary of the Invention

[0007] The purpose of this invention is to provide a drug or antidote that can alleviate the toxic effects of vomitoxin, and to provide an application of fibroin, cordyceps polysaccharide, and verbascoside as feed additives, used alone or in combination. Fibroadenoic acid, cordyceps polysaccharide, and verbascoside can repair vomitoxin-induced cellular inflammation and liver, intestinal, and kidney damage through their individual and combined effects. Based on this, the invention demonstrates that fibroin, cordyceps polysaccharide, and verbascoside, when used alone or in combination at safe dosages, can effectively alleviate the toxicity of vomitoxin to animals, thereby improving animal health.

[0008] To achieve the above objectives, the present invention provides the application of genistein, cordyceps polysaccharide, or verbascoside, or a combination of genistein, cordyceps polysaccharide, and verbascoside in the preparation of drugs to alleviate the toxicity of vomitoxin.

[0009] Preferably, a drug for relieving cellular oxidative stress induced by vomitoxin is prepared using either genistein, cordyceps polysaccharide, or verbascoside alone, or in combination of any two or three of genistein, cordyceps polysaccharide, or verbascoside. A combination of genistein, cordyceps polysaccharide, and verbascoside is more preferred.

[0010] One or a combination of douchosin, cordyceps polysaccharide, or verbascoside isoflavones can alleviate cellular oxidative stress caused by vomitoxin by regulating the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and reactive oxygen species (ROS).

[0011] Preferably, any one or any two or three of the above-mentioned ingredients, such as fibroin, cordyceps polysaccharide, or verbascoside isoflavone, are used to prepare a drug for relieving cellular inflammation caused by vomitoxin.

[0012] One or a combination of douchosin, cordyceps polysaccharide, or verbascoside alleviates cellular inflammation caused by vomitoxin by regulating the relative expression levels of the inflammatory genes IL-6 and IL-1β.

[0013] Preferably, the concentration of the fibroin at the cellular level is 0.01–250 μg / mL.

[0014] Preferably, the cellular level concentration of the Cordyceps polysaccharide is 0.02–500 μg / mL.

[0015] Preferably, the concentration of the verrucous isoflavone at the cellular level is 0.01–225 μg / mL.

[0016] The preferred embodiment of the above is the combined use of any two of the following: 0.01–250 μg / mL fibroin, 0.02–500 μg / mL cordyceps polysaccharide, and 0.01–225 μg / mL verbascoside isoflavone.

[0017] The preferred embodiment of any of the above is the combined use of three drugs: 0.01–250 μg / mL fibroin, 0.02–500 μg / mL cordyceps polysaccharide, and 0.01–225 μg / mL verbascoside isoflavone.

[0018] Preferably, the combination of 0.01–250 μg / mL of genistein, 0.02–500 μg / mL of cordyceps polysaccharide, and 0.01–225 μg / mL of verbascoside isoflavone is used, and within this combined use concentration range, the molar ratio of genistein:cordyceps polysaccharide:verascoside isoflavone is 1:2:5 to 5:2:1; that is, the molar ratio of genistein:cordyceps polysaccharide:verascoside isoflavone is (1–5):2:(5–1). More preferably, the combination at a ratio of 1:1:1 yields the best effect.

[0019] Preferably, the cells are CEF cells or the porcine small intestinal epithelial cell line IPEC-J2.

[0020] Preferably, when used alone, the concentration of the fibroblastin at the cellular level is 0.01–250 μg / mL. Preferably, fibroblastin concentrations of 0.01 μg / mL, 0.025 μg / mL, 0.075 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 125 μg / mL, 150 μg / mL, 175 μg / mL, 200 μg / mL, 225 μg / mL, and 250 μg / mL, and within the range thereof, effectively alleviate the reduction in cell activity caused by vomitoxin, while having no toxic side effects on the cells themselves. Preferably, the concentration of ligustrobin is greater than 0.01 μg / mL, and a concentration of ligustrobin greater than 0.01 μg / mL can significantly increase cell viability. More preferably, the concentration of ligustrobin is greater than 5 μg / mL. Preferably, when the concentration reaches 5 μg / mL, it can essentially completely alleviate the damage to cells caused by vomitoxin, and within this concentration range, the drug does not cause significant damage to cells.

[0021] Preferably, when used alone, the concentration of Cordyceps polysaccharide at the cellular level is 0.02–500 μg / mL. Preferably, concentrations of Cordyceps polysaccharide ranging from 0.02 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, and 500 μg / mL effectively alleviate the reduction in cell activity caused by vomitoxin, while having no toxic side effects on the cells themselves. Preferably, the concentration of Cordyceps polysaccharide is greater than 0.02 μg / mL, and a concentration greater than 0.02 μg / mL can significantly increase cell viability. More preferably, the concentration of Cordyceps polysaccharide is greater than 7.5 μg / mL. Preferably, when the concentration reaches 7.5 μg / mL, it can essentially completely alleviate the damage to cells caused by vomitoxin, and within this concentration range, the drug does not cause significant damage to cells.

[0022] Preferably, when used alone, the concentration of verbascoside at the cellular level is 0.01–225 μg / mL. Preferably, concentrations of verbascoside within the range of 0.01 μg / mL, 0.025 μg / mL, 0.075 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 125 μg / mL, 150 μg / mL, 175 μg / mL, 200 μg / mL, and 225 μg / mL effectively alleviate the reduction in cell activity caused by vomitoxin, while having no toxic side effects on the cells themselves. Preferably, the concentration of verbascoside isoflavone is greater than 0.01 μg / mL, and a concentration greater than 0.01 μg / mL can significantly increase cell viability. More preferably, the concentration of verbascoside isoflavone is greater than 6 μg / mL. Preferably, when the concentration reaches 6 μg / mL, it can essentially completely alleviate the damage to cells caused by vomitoxin, and within this concentration range, the drug does not cause significant damage to cells.

[0023] The present invention also provides the application of genistein, cordyceps polysaccharide or verbascoside, or a combination of genistein, cordyceps polysaccharide and verbascoside, in the preparation of drugs for the repair of vomitoxin-induced cellular inflammation and liver, intestine and kidney.

[0024] The effects of fibroin, cordyceps polysaccharide, or verbascoside alone or in combination can repair vomitoxin-induced cellular inflammation and liver, intestinal, and kidney damage. At safe dosages, they can effectively alleviate the toxicity of vomitoxin to animals, thereby improving their health.

[0025] One or a combination of douchosin, cordyceps polysaccharide, or verbascoside isoflavone can alleviate the elevated levels of aspartate transferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (γ-GT), and lactate dehydrogenase (LDH) in chick serum caused by liver damage due to vomitoxin.

[0026] Preferably, the dosage of the drug used in animals is 0.2–420 mg / (kg·d) of fibroin.

[0027] Preferably, the dosage of the drug used in animals is 0.6–800 mg / (kg·d) of Cordyceps polysaccharide.

[0028] Preferably, the dosage of the drug used in animals is 1–350 mg / (kg·d) of verbascoflavonoid.

[0029] Preferably, the drug is used in animals at a dose of 0.2–420 mg / (kg·d) of fibroin, 0.6–800 mg / (kg·d) of cordyceps polysaccharide, and 1–350 mg / (kg·d) of verbascoside isoflavone in combination of any two of these.

[0030] Preferably, the drug is used in animals at a combined dose of three of the following: 0.2–420 mg / (kg·d) of fibroin, 0.6–800 mg / (kg·d) of cordyceps polysaccharide, and 1–350 mg / (kg·d) of verbascoside isoflavone.

[0031] Preferably, the drug is administered in vivo in animals at a combined dose of 0.2–420 mg / (kg·d) of fibroin, 0.6–800 mg / (kg·d) of cordyceps polysaccharide, and 1–350 mg / (kg·d) of verbascoside. Furthermore, within this combined administration range, the molar ratio of fibroin:cordyceps polysaccharide:verascoside is (1–5):2:(5–1); more preferably, a combined administration ratio of 1:1:1 yields the best effect.

[0032] This invention also provides the application of genistein, cordyceps polysaccharide, or verbascoside, or a combination of genistein, cordyceps polysaccharide, and verbascoside, as a feed additive. The combination of genistein, cordyceps polysaccharide, or verbascoside, or a combination of genistein, cordyceps polysaccharide, and verbascoside, alleviates vomitoxin toxicity.

[0033] The present invention also provides a vomitoxin antidote, preferably, the vomitoxin antidote is used as a vomitoxin antidote drug or feed additive.

[0034] The present invention provides a drug or feed additive for alleviating the toxic effects of vomitoxin, including at least one of fibroin, cordyceps polysaccharide, or verbascoside isoflavone.

[0035] Preferably, the drug is xylem extract.

[0036] Preferably, the drug is cordyceps polysaccharide.

[0037] Preferably, the drug is verbascoside isoflavone.

[0038] Preferably, the drug is a combination of genistein, cordyceps polysaccharide, and verbascoside, and the combined use of genistein, cordyceps polysaccharide, and verbascoside greatly enhances the drug's effect in relieving the toxicity of vomitoxin.

[0039] One or a combination of douchosin, cordyceps polysaccharide, or verbascoside isoflavone can alleviate the cell-activating effect of vomitoxin by enhancing the cell activity of animal cells.

[0040] The results of this invention indicate that stimulation with 0.5 μM vomitoxin for 9 hours can induce cellular oxidative stress and inflammation. Compared with the vomitoxin-treated group, the addition of 0.01–250 μg / mL of fibroin, 0.02–500 μg / mL of cordyceps polysaccharide, or 0.01–225 μg / mL of verbascoside, or a combination thereof, while simultaneously adding vomitoxin, can significantly improve cell viability and superoxide dismutase (SOD) levels, and reduce reactive oxygen species (ROS) and malondialdehyde (MDA) levels. Fibroadenoic acid, cordyceps polysaccharide, or verbascoside, alone or in combination, can also significantly downregulate the relative expression levels of inflammatory genes such as IL-6 and IL-1β, alleviating vomitoxin-induced oxidative stress, inflammatory responses, and cytotoxicity.

[0041] The results of this invention also show that adding 0.2–420 mg / (kg·d) of fibroin, or 0.6–800 mg / (kg·d) of cordyceps polysaccharide, or 1–350 mg / (kg·d) of verbenafil isoflavone to the diet of chicks, or a combination of 0.2–420 mg / (kg·d) of fibroin, 0.6–800 mg / (kg·d) of cordyceps polysaccharide, and 1–350 mg / (kg·d) of verbenafil isoflavone, can effectively alleviate the damage of vomitoxin to the liver, intestines, and kidneys of chicks, and reduce the elevation of serum aspartate transferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (γ-GT), and lactate dehydrogenase (LDH) caused by liver damage. This invention provides a feed additive for chickens, pigs, and other livestock and poultry, using fibroin, cordyceps polysaccharide, and verbascoside as a means to alleviate the toxicity of vomitoxin, thereby laying the foundation for improving the health of livestock and poultry. Attached Figure Description

[0042] Figure 1 The figure shows the results of the combined use of fibroin, cordyceps polysaccharide and verbascoflavonoids in the preferred embodiment of the present invention on the cytotoxicity of CEF cells.

[0043] Figure 2 The figure shows the results of the combined use of fibroin, cordyceps polysaccharide and verbascoside in the preferred embodiment 2 of the present invention to alleviate the reduction in CEF cell viability caused by vomitoxin.

[0044] Figure 3 The figure shows the results of the relief of SOD reduction in CEF cells caused by vomitoxin when the following drugs, namely, fibroin, cordyceps polysaccharide and verbascoside, were used alone and in combination, according to preferred embodiment 3 of the present invention.

[0045] Figure 4 The figure shows the results of the relief of increased reactive oxygen species in CEF cells caused by vomitoxin using fibroin, cordyceps polysaccharide and verbascoside alone and in combination, as described in the preferred embodiment 3 of the present invention.

[0046] Figure 5 The figure shows the relief effect of fibroin, cordyceps polysaccharide and verbascoside alone and in combination on MDA accumulation caused by vomitoxin in the preferred embodiment 4 of the present invention.

[0047] Figure 6 The image shows the qPCR results of the preferred embodiment 5 of this invention, specifically the effects of fibroin, cordyceps polysaccharide, and verbascoside, alone and in combination, on inflammatory factors induced by vomitoxin. A. mRNA expression level of the inflammation-related gene IL-6; B. mRNA expression level of the inflammation-related gene IL-1β.

[0048] Figure 7 The figure shows the effect of the combined use of fibroin, cordyceps polysaccharide and verbascoside in the preferred embodiment 6 of the present invention on the relief of liver tissue damage caused by vomitoxin.

[0049] Figure 8 The figure shows the effect of the combined use of fibroin, cordyceps polysaccharide and verbascoside in the preferred embodiment 6 of the present invention on alleviating kidney tissue damage caused by vomitoxin.

[0050] Figure 9 The figure shows the effect of the combined use of fibroin, cordyceps polysaccharide and verbascoside in the preferred embodiment of the present invention (Example 6) on alleviating intestinal tissue damage caused by vomitoxin.

[0051] Figure 10 The figure shows the relief effect of the combined use of fibroin, cordyceps polysaccharide and verbascoside in the preferred embodiment 6 of the present invention on the increase in serum AST, ALT, γ-GT and LDH levels caused by vomitoxin.

[0052] Figure 11 The figure shows the results of using fibroin, cordyceps polysaccharide and verbascoside alone and in combination, as described in the preferred embodiment 7 of the present invention, to alleviate the reduction in the viability of porcine small intestinal epithelial cells IPEC-J2 caused by vomitoxin.

[0053] Figure 12 The figure shows the results of alleviating the reduction of SOD in porcine small intestinal epithelial cells IPEC-J2 caused by vomitoxin, using the following preferred embodiment 8 of the present invention, alone and in combination. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0055] Unless otherwise specified, the drugs and reagents involved in this invention are all commercially available reagents or mixtures of artificially synthesized reagents. The preparation or extraction methods of artificial synthesis are existing technologies and are not the subject matter protected by this invention.

[0056] Example 1

[0057] Preparation of CEF cells:

[0058] 1. Take a 10-day-old SPF chicken embryo and wipe it with an alcohol swab, starting from the air cell and working outwards to disinfect it. Then place the embryo on a clean bench with the air cell facing upwards. Use sterile tweezers to break the air cell and remove the eggshell.

[0059] 2. Using new sterile forceps, peel off the allantoic chorion membrane and discard the eggshell and yolk membrane. Hold the head of the chicken embryo with sterile forceps and gently move it to a petri dish.

[0060] 3. Secure the head with sterile forceps, cut off the head, limbs, and organs with sterile scissors, and rinse repeatedly with sterile PBS until colorless.

[0061] 4. Use sterile scissors to cut the tissue block into small pieces until there are no large tissue particles. Add 8 mL of sterile PBS to wash the tissue, let it stand for 1 minute, and discard the suspension containing red blood cells and tissue fragments with a pipette.

[0062] 5. Add 0.25% trypsin at 3 to 5 times the volume of the tissue block, place in a 37°C water bath for 15 minutes, shaking continuously during digestion. Once the tissue turns white and becomes loose, add FBS to a final concentration of 10% to stop digestion, and repeatedly pipette the cells to disperse them into single cells.

[0063] 6. After filtering the cell suspension with sterile gauze, add the filtrate to a centrifuge tube, centrifuge at 1000 rpm for 8 minutes at room temperature, and discard the supernatant.

[0064] 7. Resuspend the cells in DMEM medium containing 10% FBS, dilute to an appropriate concentration, add to a cell culture flask, and incubate at 37°C in a 5% CO2 incubator until the cells grow into a monolayer and reach 80% abundance.

[0065] CCK-8 assay for the cytotoxicity of fibroin, cordyceps polysaccharide, or verbascoflavonoids, and their combination, to CEF:

[0066] 1. Treatment of CEF cells with genistein, cordyceps polysaccharide, or verbascoside: The specific steps are as follows: Seed 8000 CEF cells into each well of a 96-well plate. Dilute genistein, cordyceps polysaccharide, or verbascoside into a concentration gradient using culture medium. Add 100 μL of the culture medium containing the concentration gradient of genistein, cordyceps polysaccharide, or verbascoside to the 96-well plate and incubate at 37°C in a 5% CO2 incubator for 12 hours.

[0067] 2. Cell viability was assessed using the CCK-8 assay. The specific procedure was as follows: 10 μL of CCK-8 solution was added to each well of a 96-well plate. The plate was then incubated at 37°C in a 5% CO2 incubator for 2 hours in the dark. The absorbance (OD value) at 450 nm was measured using a microplate reader, and the average OD value of each parallel well was taken. Cell viability % = (OD450 of drug-treated cells - OD450 of blank cells) / (OD450 of control cells - OD450 of blank cells) × 100%. The experiment was repeated three times. A cell viability of at least 95% of the drug-free group was determined as the safe concentration of xanthocarne, cordyceps polysaccharide, or verbascoside for CEF cells. Results are as follows: Figure 1 Cell viability was above 95% when treated individually with 275 μg / mL of genistein, 550 μg / mL of cordyceps polysaccharide, and 250 μg / mL of verbenafil isoflavone, but cell viability was below 95% when used in combination at these concentrations. However, cell viability remained above 95% when treated with 250 μg / mL of genistein, 500 μg / mL of cordyceps polysaccharide, and 225 μg / mL of verbenafil isoflavone, regardless of whether used alone or in combination. Therefore, the safe concentrations for genistein in CEF cells are <250 μg / mL, for cordyceps polysaccharide in CEF cells are <500 μg / mL, and for verbenafil isoflavone in CEF cells are <225 μg / mL.

[0068] Example 2

[0069] The use of ligustiol, cordyceps polysaccharide, and verbascoside, alone and in combination, can alleviate the decrease in CEF cell viability caused by vomitoxin.

[0070] The specific procedure for drug treatment of CEF cells is as follows: 8000 CEF cells are seeded into each well of a 96-well plate and cultured for 12 hours. Then, the culture medium is replaced with one of the following solutions: 0.5 μM vomitoxin and 1 μg / mL fibrocin; 0.5 μM vomitoxin and 1 μg / mL cordyceps polysaccharide; 0.5 μM vomitoxin and 1 μg / mL verbascoflavonoids; or 0.5 μM vomitoxin and a medium containing 1 μg / mL fibrocin, 1 μg / mL cordyceps polysaccharide, and 1 μg / mL verbascoflavonoids. Cell viability is then measured using the CCK8 assay. Figure 2As shown, the experiment was repeated three times. The results showed that compared with the vomitoxin treatment group without the addition of genistein, cordyceps polysaccharide, or verbascoside, the cell viability of CEF cells increased with treatments of genistein, cordyceps polysaccharide, and verbascoside alone and in combination. Specifically, the genistein group increased by 32.00%, the cordyceps polysaccharide group by 35.39%, the verbascoside group by 39.90%, and the combined treatment group (genistein, cordyceps polysaccharide, and verbascoside in a mass ratio of 1:1:1) increased by 77.06%. The Combination Index (CI) was analyzed using CompuSyn (v1.0.1). As shown in Table 1, the combined treatment showed the best effect at a dosage ratio of genistein:cordyceps polysaccharide:verascoside = 1:1:1, which was superior to the single treatment, indicating a synergistic effect (CI < 0.4). The results indicate that the individual and combined use of fibroin, cordyceps polysaccharide and verbascoside can alleviate the cytotoxicity caused by vomitoxin. In particular, when the concentration of cordyceps polysaccharide is 100 μg / mL, the combined use of fibroin:cordyceps polysaccharide:verascoside in the mass ratio of (1-5):2:(5-1) significantly alleviates the cytotoxicity caused by vomitoxin. Moreover, the combined use of fibroin at a ratio of 1:1:1 is more effective than the individual use or other ratios. Table 1 shows the combined use index.

[0071] Table 1.

[0072]

[0073] Example 3

[0074] The alleviating effects of genistein, cordyceps polysaccharide, and verbascoside, alone and in combination, on the reduction of antioxidant capacity in CEF cells by vomitoxin.

[0075] 1. Determination of total SOD content in cells (WST-8 assay), the specific operating steps are as follows:

[0076] The specific steps for drug treatment of CEF cells are as follows: Seed 600,000 CEF cells into each well of a 6-well plate and culture for 12 hours. Then, replace the culture medium with one of the following: 0.5 μM vomitoxin and 1 μg / mL fibroin; 0.5 μM vomitoxin and 1 μg / mL cordyceps polysaccharide; 0.5 μM vomitoxin and 1 μg / mL verbascoflavonoids; or 0.5 μM vomitoxin and a medium containing 1 μg / mL fibroin, 1 μg / mL cordyceps polysaccharide, and 1 μg / mL verbascoflavonoids. Culture for another 12 hours. Discard the supernatant and collect the cell pellet.

[0077] The SOD content was detected according to the kit instructions. The specific steps were as follows: Add 60 μL of SOD sample preparation solution to each tube of cells, gently pipette to lyse the cells, centrifuge at 12,000 × g for 3–5 minutes at 4°C, and collect the supernatant as the test sample. Then add freshly prepared WST-8 / enzyme working solution and reaction start-up working solution, transfer to a 96-well plate, and incubate at 37°C in the dark for 30 minutes. Measure the absorbance (OD value) at 450 nm using a microplate reader, and average the OD values ​​of all parallel wells. Inhibition rate % = (OD450 of drug-treated cells - OD450 of blank cells) / (OD450 of control cells - OD450 of blank cells) × 100%. The experiment was repeated three times. The corresponding SOD activity (U / mL) was determined from the standard curve based on the inhibition rate. Total SOD activity of the sample = corresponding value on the standard curve × dilution factor / sample protein concentration (mg / mL). Figure 3 As shown, the results indicated that, compared with the vomitoxin treatment group without the addition of genistein, cordyceps polysaccharide, or verbascoside, treatment with genistein, cordyceps polysaccharide, and verbascoside alone or in combination increased the SOD activity of CEF cells. Specifically, the genistein group increased by 55.28%, the cordyceps polysaccharide group by 50.73%, the verbascoside group by 56.21%, and the combination treatment group by 65.26%. This suggests that genistein, cordyceps polysaccharide, and verbascoside, whether alone or in combination, can alleviate the decrease in SOD activity caused by vomitoxin, and the combination treatment is more effective than the single treatment.

[0078] The present invention also tested the combined use of 0.01–250 μg / mL of pachymansin, 0.02–500 μg / mL of cordyceps polysaccharide, and 0.01–225 μg / mL of verbascoside. Compared with the control group of vomitoxin without the addition of pachymansin, cordyceps polysaccharide, and verbascoside, the combined use of pachymansin, cordyceps polysaccharide, and verbascoside within this concentration range increased SOD activity by 91.36%. To further investigate the therapeutic effects of combined use of different proportions of pamoatein, cordyceps polysaccharide, and verbascoside, the concentration of cordyceps polysaccharide was set at 100 μg / mL. When the mass ratio of pamoatein, cordyceps polysaccharide, and verbascoside was in the range of (1–5):2:(5–1), the composition significantly inhibited the decrease in superoxide dismutase (SOD) activity in CEF cells induced by vomitoxin, increasing SOD activity by 78.2% to 91.36%. It should be noted that the mass ratio of pamoatein, cordyceps polysaccharide, and verbascoside in the combined use of this invention (1–5):2:(5–1) is not limited to 100 μg / mL cordyceps polysaccharide, but is applicable to combined use of pamoatein at concentrations of 0.01–250 μg / mL, cordyceps polysaccharide at concentrations of 0.02–500 μg / mL, and verbascoside at concentrations of 0.01–225 μg / mL.

[0079] 2. Intracellular reactive oxygen species (ROS) detection (DCFH-DA method)

[0080] The specific steps for drug treatment of CEF cells are as follows: 600,000 CEF cells are seeded into each well of a 6-well plate and cultured for 12 hours. Then, the culture medium is replaced with a medium containing 0.5 μM vomitoxin and 1 μg / mL genistein, or a medium containing 0.5 μM vomitoxin and 1 μg / mL cordyceps polysaccharide, or a medium containing 0.5 μM vomitoxin and 1 μg / mL verbascoside, or a medium containing 0.5 μM vomitoxin and 1 μg / mL genistein, 1 μg / mL cordyceps polysaccharide, and 1 μg / mL verbascoside, and cultured for 12 hours.

[0081] Intracellular ROS were detected according to the instructions of the reactive oxygen species (ROS) detection kit. The specific steps were as follows: 1 mL of 10 μM DCFH-DA probe was added to each tube of cells, and the cells were incubated at 37°C in the dark for 20 minutes. The cells were then washed three times with PBS to remove any probe that had not entered the cells. The cells were observed and photographed under a fluorescence microscope. The fluorescence intensity of each group was analyzed and calculated using ImageJ: Mean fluorescence intensity = Total fluorescence intensity of the region (IntDen) / Area of ​​the region (Area). The experiment was repeated three times to determine the content of reactive oxygen species in CEF cells. Results are as follows: Figure 4 As shown, compared with the vomitoxin treatment group without the addition of genistein, cordyceps polysaccharide, or verbascoside, treatment with genistein, cordyceps polysaccharide, and verbascoside alone or in combination reduced ROS in CEF cells and decreased intracellular ROS fluorescence intensity. The mean fluorescence intensity was calculated using ImageJ, showing a 49.34% reduction in the genistein group, a 54.41% reduction in the cordyceps polysaccharide group, a 55.89% reduction in the verbascoside group, and a 66.06% reduction in the combination treatment group. This indicates that genistein, cordyceps polysaccharide, and verbascoside, whether alone or in combination, can alleviate the ROS elevation caused by vomitoxin, with combination treatment being more effective than single treatment.

[0082] This invention tested the combined use of 0.01–250 μg / mL of pamoatein, 0.02–500 μg / mL of cordyceps polysaccharide, and 0.01–225 μg / mL of verbascoside. Compared with a control group of vomitoxin without the addition of pamoatein, cordyceps polysaccharide, and verbascoside, the combined use of pamoatein, cordyceps polysaccharide, and verbascoside within this concentration range reduced ROS levels by 92.77%. Furthermore, to study the therapeutic effect of different ratios of pamoatein, cordyceps polysaccharide, and verbascoside, the concentration of cordyceps polysaccharide was set at 100 μg / mL. When the mass ratio of pamoatein, cordyceps polysaccharide, and verbascoside was in the range of (1–5):2:(5–1), the composition significantly inhibited the increase in reactive oxygen species (ROS) in CEF cells caused by vomitoxin, reducing ROS levels by 69.58% to 92.77%. It should be noted that the mass ratio of genistein, cordyceps polysaccharide and verbascoflavonoids in the combined drug of the present invention is (1-5):2:(5-1), and is not limited to cordyceps polysaccharide at 100 μg / mL, but is applicable to the combined drug use of genistein at a concentration of 0.01-250 μg / mL, cordyceps polysaccharide at 0.02-500 μg / mL, and verbascoflavonoids at 0.01-225 μg / mL.

[0083] Example 4

[0084] The alleviating effect of douchosin, cordyceps polysaccharide or verbascoside alone or in combination on the accumulation of MDA in CEF cells induced by vomitoxin.

[0085] The specific steps for drug treatment of CEF cells are as follows: 600,000 CEF cells are seeded into each well of a 6-well plate and cultured for 12 hours. Then, the culture medium is replaced with a medium containing 0.5 μM vomitoxin and 1 μg / mL genistein, or a medium containing 0.5 μM vomitoxin and 1 μg / mL cordyceps polysaccharide, or a medium containing 0.5 μM vomitoxin and 1 μg / mL verbascoside, or a medium containing 0.5 μM vomitoxin and 1 μg / mL genistein, 1 μg / mL cordyceps polysaccharide, and 1 μg / mL verbascoside, and cultured for 12 hours. The supernatant is then discarded and the cell pellet is collected.

[0086] The MDA content was detected according to the kit instructions. The specific steps were as follows: Add 0.1 mL of reagent extraction buffer to each tube of cell pellet, and vortex to disrupt the cells. Centrifuge at 12,000 × g for 3–5 minutes, and collect the supernatant as the test sample. Then add freshly prepared working solution, mix well, heat at 95°C for 1 hour, cool on ice, and centrifuge at 4000 × g for 10 minutes. Collect the supernatant in a 96-well plate and measure the absorbance (OD value) at 532 nm using a microplate reader. The OD values ​​of all parallel wells were averaged. Calculate the MDA concentration (nmol / mL) in the sample based on the standard curve. Sample MDA content (nmol / mg prot) = MDA concentration / sample protein concentration (mg / mL). The experiment was performed in triplicate. The MDA content in CEF cells was determined using a cell malondialdehyde (MDA) detection kit. Figure 5 As shown, the results indicated that, compared with the vomitoxin treatment group, treatment with genistein, cordyceps polysaccharide, and verbascoside, both individually and in combination, reduced MDA in CEF cells. Specifically, the genistein group saw a reduction of 38.35%, the cordyceps polysaccharide group a reduction of 49.52%, the verbascoside group a reduction of 63.81%, and the combination treatment group a reduction of 72.89%. This suggests that genistein, cordyceps polysaccharide, and verbascoside, both individually and in combination, can alleviate MDA accumulation induced by vomitoxin, with the combination treatment showing better efficacy than the individual treatments.

[0087] This invention tested the combined use of 0.01–250 μg / mL pachyman, 0.02–500 μg / mL cordyceps polysaccharide, and 0.01–225 μg / mL verbascoside. Compared with a control group of vomitoxin without the addition of pachyman, cordyceps polysaccharide, and verbascoside, the combined use of pachyman, cordyceps polysaccharide, and verbascoside within this concentration range reduced MDA by 94.18%. Furthermore, to study the therapeutic effect of different ratios of pachyman, cordyceps polysaccharide, and verbascoside, the concentration of cordyceps polysaccharide was set at 100 μg / mL. When the mass ratio of pachyman, cordyceps polysaccharide, and verbascoside was in the range of (1–5):2:(5–1), the composition significantly inhibited the accumulation of malondialdehyde (MDA) in CEF cells induced by vomitoxin, reducing MDA by 75.43% to 94.18%. It should be noted that the mass ratio of genistein, cordyceps polysaccharide and verbascoflavonoids in the combined drug of the present invention is (1-5):2:(5-1), and is not limited to cordyceps polysaccharide at 100 μg / mL, but is applicable to the combined drug use of genistein at a concentration of 0.01-250 μg / mL, cordyceps polysaccharide at 0.02-500 μg / mL, and verbascoflavonoids at 0.01-225 μg / mL.

[0088] Example 5

[0089] The alleviating effects of douchosin, cordyceps polysaccharide and verbascoside alone and in combination on CEF cell inflammatory damage induced by vomitoxin.

[0090] The specific steps for drug treatment of CEF cells are as follows: 600,000 CEF cells are seeded into each well of a 6-well plate and cultured for 12 hours. Then, the culture medium is replaced with a medium containing 0.5 μM vomitoxin and 1 μg / mL genistein, or a medium containing 0.5 μM vomitoxin and 1 μg / mL cordyceps polysaccharide, or a medium containing 0.5 μM vomitoxin and 1 μg / mL verbascoside, or a medium containing 0.5 μM vomitoxin and 1 μg / mL genistein, 1 μg / mL cordyceps polysaccharide, and 1 μg / mL verbascoside, and cultured for 12 hours. The supernatant is then discarded and the cell pellet is collected.

[0091] Total RNA was extracted from cells per well. Real-time quantitative PCR primers were designed based on the sequences of the inflammatory genes IL-6 and IL-1β; the upstream and downstream primers are shown in Table 2. Total RNA was reverse transcribed, followed by Q-PCR. Quantitative analysis was performed based on the results. Figure 6 As shown, the experiment was repeated three times. The results showed that compared with the vomitoxin treatment group, the expression of IL-6 and IL-1β at the mRNA level was decreased in both the individual and combined treatments of fibroin, cordyceps polysaccharide, and verbascoside (p<0.05). Specifically, the fibroin group showed a decrease of 38.43% and 65.79%, respectively; the cordyceps polysaccharide group showed a decrease of 40.77% and 65.67%, respectively; the verbascoside group showed a decrease of 50.21% and 68.21%, respectively; and the combined treatment group showed a decrease of 64.66% and 74.37%, respectively. This indicates that the individual and combined treatments of fibroin, cordyceps polysaccharide, and verbascoside can alleviate the inflammatory damage caused by vomitoxin, and the combined treatment is more effective than the individual treatment.

[0092] This invention investigated the combined use of 0.01–250 μg / mL fibrocin, 0.02–500 μg / mL cordyceps polysaccharide, and 0.01–225 μg / mL verbascoside. Compared with a control group of vomitoxin without the addition of fibrocin, cordyceps polysaccharide, and verbascoside, the combined use of fibrocin, cordyceps polysaccharide, and verbascoside within this concentration range reduced IL-6 mRNA levels by 84.96% and IL-1β mRNA levels by 92.17%. Further research was conducted... The therapeutic effects of combined use of different proportions of genistein, cordyceps polysaccharide, and verbascoside were investigated. When the concentration of cordyceps polysaccharide was 100 μg / mL, and the mass ratio of genistein, cordyceps polysaccharide, and verbascoside was in the range of (1-5):2:(5-1), the composition significantly inhibited the increase in IL-6 and IL-1β mRNA levels caused by vomitoxin, reducing IL-6 mRNA levels by 63.85% to 84.96% and IL-1β mRNA levels by 72.14% to 92.17%. It should be noted that the mass ratio of genistein, cordyceps polysaccharide and verbascoflavonoids in the combined drug of the present invention is (1-5):2:(5-1), and is not limited to cordyceps polysaccharide at 100 μg / mL, but is applicable to the combined drug use of genistein at a concentration of 0.01-250 μg / mL, cordyceps polysaccharide at 0.02-500 μg / mL, and verbascoflavonoids at 0.01-225 μg / mL.

[0093] Table 2:

[0094]

[0095] Note: F represents the upstream primer, and R represents the downstream primer.

[0096] Example 6

[0097] The alleviating effects of genistein, cordyceps polysaccharide, and verbascoside, alone and in combination, on liver, kidney, and intestinal damage in chickens caused by vomitoxin.

[0098] The specific procedure was as follows: One-day-old White Leghorn SPF chickens were used. The control group was not given any drug, while the vomitoxin group was given moldy feed (CP chicken feed and moldy wheat in a 1:1 ratio). The drug-treated groups were also given moldy feed (CP chicken feed and moldy wheat in a 1:1 ratio), a genistein group (0.2–420 mg / (kg·d) genistein, preferably 420 mg / (kg·d)), a cordyceps polysaccharide group (0.6–800 mg / (kg·d) cordyceps polysaccharide, preferably 800 mg / (kg·d)), a verbascoside group (1–350 mg / (kg·d) verbascoside, preferably 350 mg / (kg·d) verbascoside), and a combined drug treatment group (genistein: cordyceps polysaccharide: verbascoside = 1:1:1, all at a dose of 150 mg / (kg·d)) by gavage. Eight chickens were in each group. After 7 days of continuous administration, phenobarbital was used to anesthetize the patient and euthanize her. Immediately afterward, a systematic dissection was performed, and blood, liver, kidneys, and intestines were collected.

[0099] Specific procedures for tissue sectioning: Randomly cut a portion of tissue and immerse it in a 4% paraformaldehyde solution for 24 hours for fixation. Prepare 5μm thick paraffin sections, stain with standard hematoxylin and eosin (HE), mount the stained sections with neutral resin, and then scan them using a tissue scanner.

[0100] Liver tissue section results as follows Figure 7 As shown, compared with the control group, the hepatocytes in the vomitoxin group were disordered, the hepatic lamina structure was unclear, the hepatic sinusoids were narrowed, fibrosis was observed in the portal areas, and patchy necrosis of hepatocytes was visible, with large areas of focal inflammatory cell infiltration. In contrast, the combined medication group alleviated liver inflammation, with regular hepatocyte arrangement and clear hepatic lamina structure. Although focal necrosis was still visible in hepatocytes, the area of ​​necrotic foci was significantly smaller than in the injury group, and the degree of inflammatory cell infiltration within the necrotic foci was significantly less than in the vomitoxin group. This indicates that the combined use of xanthomasin, cordyceps polysaccharide, and verbascoside effectively alleviated liver damage induced by vomitoxin.

[0101] Kidney tissue section results as follows Figure 8 As shown, compared with the control group, although the glomerular structure and number were normal in the vomitoxin group, the renal tubular epithelial cells showed more significant damage, and the renal tubules exhibited atrophy and atresia, with slightly more severe interstitial edema and inflammatory cell infiltration. The combined medication group significantly alleviated the kidney inflammation caused by the vomitoxin group. This indicates that the combined use of xanthomaspin, cordyceps polysaccharide, and verbascoside effectively alleviated kidney damage caused by vomitoxin.

[0102] Results of intestinal tissue sections Figure 9 As shown, compared with the control group, the vomitoxin group exhibited focal loose edema in the lamina propria of the mucosa. The combined medication group alleviated the intestinal damage caused by the vomitoxin group, and its tissue characteristics were similar to those of the control group. This indicates that the combined use of xanthomasin, cordyceps polysaccharide, and verbascoside effectively alleviated intestinal damage caused by vomitoxin.

[0103] Specific procedures for detecting liver biochemical indicators in serum: Inject blood into sterilized centrifuge tubes and incubate at room temperature for 30 minutes. After observing blood clotting, centrifuge at 3000 rpm for 10 minutes, and collect the serum after centrifugation. Use an automated animal biochemical analyzer to detect aspartate transferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (γ-GT), and lactate dehydrogenase (LDH) levels in the serum. Figure 10 The figure shows the results of the combined treatment of pamoatein, cordyceps polysaccharide, and verbascoside in Example 6 to alleviate the elevated serum AST, ALT, γ-GT, and LDH levels caused by vomitoxin. A represents AST level; B represents ALT level; C represents γ-GT level; and D represents LDH level. The experiment was repeated three times. The results showed that compared with the control group, the vomitoxin group had elevated serum aspartate transferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (γ-GT), and lactate dehydrogenase (LDH) levels due to liver damage. However, compared with the vomitoxin treatment group, the combined treatment of pamoatein, cordyceps polysaccharide, and verbascoside reduced the serum aspartate transferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (γ-GT), and lactate dehydrogenase (LDH) levels by 39.79%, 53.51%, 56.31%, and 66.23%, respectively. The above results indicate that the combined use of fibroin, cordyceps polysaccharide and verbascoside can alleviate liver damage caused by vomitoxin.

[0104] This invention investigated the combined use of 0.2–420 mg / (kg·d) of fibroin, 0.6–800 mg / (kg·d) of cordyceps polysaccharide, and 1–350 mg / (kg·d) of verbenafil. Compared with a control group of vomitoxin without the addition of fibroin, cordyceps polysaccharide, and verbenafil, at these drug dosages, the levels of aspartate aminotransferase (AST) decreased to 87.06%; alanine aminotransferase (ALT) decreased to 88.15%; gamma-glutamyl transferase (γ-GT) decreased to 89.14%; and lactate dehydrogenase (LDH) decreased to 93.76%. Furthermore, to study the therapeutic effects of different ratios of fibroin, cordyceps polysaccharide, and verbenafil in combination, the dosage of cordyceps polysaccharide was... When the dosage is 240 mg / (kg·d), and the mass ratio of fibroin, cordyceps polysaccharide, and verbascoside isoflavone is in the range of (1-5):2:(5-1), the composition can significantly inhibit the increase in serum aspartate transferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (γ-GT), and lactate dehydrogenase (LDH) levels caused by vomitoxin. The reduction range of AST level is 54.16% to 87.06%; the reduction range of ALT level is 67.75% to 88.15%; the reduction range of γ-glutamyl transferase (γ-GT) level is 69.13% to 89.14%; and the reduction range of LDH level is 73.54% to 93.76%. It should be noted that the mass ratio of fibroin, cordyceps polysaccharide and verbascoflavonoids in the combined drug of the present invention is (1-5):2:(5-1), and is not limited to 240 mg / (kg·d) of cordyceps polysaccharide, but is applicable to the combined drug use of 0.2-420 mg / (kg·d) of fibroin, 0.6-800 mg / (kg·d) of cordyceps polysaccharide and 1-350 mg / (kg·d) of verbascoflavonoids.

[0105] Example 7

[0106] The use of ligustiol, cordyceps polysaccharide, and verbascoside alone and in combination can alleviate the reduction in IPEC-J2 cell viability caused by vomitoxin.

[0107] The specific steps for drug treatment of IPEC-J2 cells are as follows: 8000 IPEC-J2 cells are seeded into each well of a 96-well plate and cultured for 12 hours. Then, the culture medium is replaced with one of the following solutions: 0.5 μM vomitoxin and 1 μg / mL fibroin; 0.5 μM vomitoxin and 1 μg / mL cordyceps polysaccharide; 0.5 μM vomitoxin and 1 μg / mL verbascoflavonoids; or 0.5 μM vomitoxin and a medium containing 1 μg / mL fibroin, 1 μg / mL cordyceps polysaccharide, and 1 μg / mL verbascoflavonoids. Cell viability is then measured using the CCK8 assay. Figure 11 As shown, the experiment was repeated three times. The results showed that, compared with the vomitoxin treatment group without the addition of genistein, cordyceps polysaccharide, or verbascoside, the cell viability of CEF cells increased with treatment alone and in combination with genistein, cordyceps polysaccharide, and verbascoside. Specifically, the genistein group increased by 26.37%, the cordyceps polysaccharide group by 29.35%, the verbascoside group by 33.90%, and the combined treatment group (genistein, cordyceps polysaccharide, and verbascoside in a 1:1:1 mass ratio) increased by 50.81%.

[0108] Example 8

[0109] The specific steps for determining the total SOD content in cells (WST-8 assay) are as follows:

[0110] The specific steps for drug treatment of IPEC-J2 cells are as follows: Seed 600,000 IPEC-J2 cells into each well of a 6-well plate and culture for 12 hours. Then, replace the culture medium with one of the following: 0.5 μM vomitoxin and 1 μg / mL genistein; 0.5 μM vomitoxin and 1 μg / mL cordyceps polysaccharide; 0.5 μM vomitoxin and 1 μg / mL verbascoside; or 0.5 μM vomitoxin and 1 μg / mL genistein, 1 μg / mL cordyceps polysaccharide, and 1 μg / mL verbascoside. Culture for another 12 hours. Discard the supernatant and collect the cell pellet.

[0111] The SOD content was detected according to the kit instructions. The specific steps were as follows: Add 60 μL of SOD sample preparation solution to each tube of cells, gently pipette to lyse the cells, centrifuge at 12,000 × g for 3–5 minutes at 4°C, and collect the supernatant as the test sample. Then add freshly prepared WST-8 / enzyme working solution and reaction start-up working solution, transfer to a 96-well plate, and incubate at 37°C in the dark for 30 minutes. Measure the absorbance (OD value) at 450 nm using a microplate reader, and average the OD values ​​of all parallel wells. Inhibition rate % = (OD450 of drug-treated cells - OD450 of blank cells) / (OD450 of control cells - OD450 of blank cells) × 100%. The experiment was repeated three times. The corresponding SOD activity (U / mL) was determined from the standard curve based on the inhibition rate. Total SOD activity of the sample = corresponding value on the standard curve × dilution factor / sample protein concentration (mg / mL). Figure 12 As shown, the results indicated that, compared with the vomitoxin treatment group without the addition of genistein, cordyceps polysaccharide, or verbascoside, treatment with genistein, cordyceps polysaccharide, and verbascoside alone or in combination increased the SOD activity of CEF cells. Specifically, the genistein group increased by 47.70%, the cordyceps polysaccharide group by 43.28%, the verbascoside group by 55.38%, and the combination treatment group by 65.18%. This suggests that genistein, cordyceps polysaccharide, and verbascoside, whether alone or in combination, can alleviate the decrease in SOD activity caused by vomitoxin, and the combination treatment is more effective than the single treatment.

[0112] The above embodiments are merely illustrative examples and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of the combination of fibroin, cordyceps polysaccharide, and verbascoside in the preparation of drugs to alleviate the toxicity of vomitoxin, characterized in that, The combined use of 0.01–250 μg / mL genistein, 0.02–500 μg / mL cordyceps polysaccharide and 0.01–225 μg / mL verbascoside isoflavone; the molar ratio of genistein: cordyceps polysaccharide: verbascoside isoflavone is (1–5): 2: (5–1).

2. The application as described in claim 1, characterized in that, The combination of pamoatein, cordyceps polysaccharide, and verbascoside isoflavone is used to prepare a drug that alleviates cellular oxidative stress caused by vomitoxin. The drug regulates the levels of malondialdehyde, superoxide dismutase, and reactive oxygen species to alleviate cellular oxidative stress caused by vomitoxin.

3. The application as described in claim 1, characterized in that, The combination of genistein, cordyceps polysaccharide, and verbascoside isoflavone is used to prepare a drug that alleviates cellular inflammation caused by vomitoxin. The drug alleviates cellular inflammation caused by vomitoxin by regulating the relative expression levels of the inflammatory genes IL-6 and IL-1β.

4. The application as described in any one of claims 1 to 3, characterized in that, The combined use of 0.2–420 mg / (kg·d) of genistein, 0.6–800 mg / (kg·d) of cordyceps polysaccharide and 1–350 mg / (kg·d) of verbenafil isoflavone, within this range of combined use, the dosage ratio of genistein:cordyceps polysaccharide:verenafil isoflavone is (1–5):2:(5–1).

5. The application of the combination of fibroin, cordyceps polysaccharide, and verbascoside in the preparation of feed additives to alleviate vomitoxin toxicity, characterized in that... The combined use of 0.01–250 μg / mL genistein, 0.02–500 μg / mL cordyceps polysaccharide and 0.01–225 μg / mL verbascoside isoflavone, with a molar concentration ratio of genistein: cordyceps polysaccharide: verbascoside isoflavone of (1–5): 2: (5–1).

6. The application of the combination of fibroin, cordyceps polysaccharide, and verbascoside in the preparation of a drug for alleviating vomitoxin-induced liver, intestinal, and kidney damage, characterized in that... The combined use of 0.01–250 μg / mL genistein, 0.02–500 μg / mL cordyceps polysaccharide and 0.01–225 μg / mL verbascoside isoflavone, with a molar concentration ratio of genistein: cordyceps polysaccharide: verbascoside isoflavone of (1–5): 2: (5–1).

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