Application of naringenin in preparation of anti-EHV-8 medicine

Abstract

The invention relates to the technical field of preparation of veterinary antiviral traditional Chinese medicines, in particular to application of naringenin in preparation of an anti-EHV-8 medicine, including application of a naringenin monomer, a plant extract containing the naringenin monomer and a traditional Chinese medicine compound in preparation of an EHV-8 replication inhibiting medicine. The naringenin disclosed by the invention plays an antiviral role in an EHV-8 replication stage, cannot directly inactivate viruses, has the maximum safe concentration of 100 mu M, has a remarkable inhibition effect on a plurality of strains, can reduce lung injury of a mouse model caused by EHV-8 infection, is natural in source and low in toxicity, has various pharmacological activities such as anti-inflammation and the like, and provides a reference for anti-EHV-8 clinical medication.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of veterinary antiviral Chinese medicines, and in particular to application of naringenin in the preparation of anti-EHV-8 medicines. Background Art

[0002] Equine herpesvirus 8 (EHV-8) belongs to the alphaherpesvirus subfamily and has a double-stranded DNA genome. It infects horses, donkeys, and other equine species, primarily causing diseases such as rhinopneumonia, abortion, and viral encephalitis. In recent years, with the growth of intensive equine farming, EHV-8 infection has led to an increasing incidence of abortions and respiratory illnesses in donkeys. However, vaccines and medications for EHV-8 prevention and control are very limited, and clinical treatment primarily relies on symptomatic treatment. There is an urgent need to develop effective drugs to prevent and control EHV-8.

[0003] Naringenin is a dihydroflavonoid compound found widely in citrus fruits (such as grapefruit and orange) and traditional Chinese medicines (such as immature bitter orange and dried tangerine peel). Its molecular formula is C15H12O5 and its molecular weight is 272.25. It exhibits multiple pharmacological activities, including antiviral, anti-inflammatory, antioxidant, and immunomodulatory activities. Previous studies have shown that naringenin can combat infections from various viruses (such as influenza, dengue, and porcine epidemic diarrhea). This drug class boasts high safety, good bioavailability, and is not prone to inducing drug resistance.

[0004] Chinese Patent Publication (Announcement) No. CN119326754A discloses the use of naringenin in the preparation of a drug for the prevention and treatment of diseases caused by pseudorabies virus (PRV). This patent discloses the use of naringenin in the preparation of a drug for the prevention and treatment of pseudorabies virus (PRV) disease, demonstrating that naringenin exerts its effects by inhibiting viral replication, reducing gB protein expression, and regulating the TLR4 / NF-κB pathway. However, although PRV belongs to the Herpesviridae family, its host range and pathogenicity (primarily infecting pigs and causing neurological diseases) differ significantly from those of EHV-8. EHV-8 specifically infects equines, primarily targeting the respiratory tract and reproductive system. Furthermore, the two viruses differ significantly in terms of viral genome sequence, key functional proteins (such as differences in gB protein homology), and host immune response mechanisms. Therefore, the mechanism of action model established for PRV cannot be directly extrapolated to EHV-8. In particular, key parameters such as drug targeting, effective concentration, and safety evaluation in equine cell models need to be revalidated. This patent does not involve any research on the effects of naringenin on EHV-8 and cannot solve the technical problems of EHV-8 infection prevention and treatment.

[0005] Chinese Patent Publication (Announcement) No. CN112843046A discloses the use of naringenin and its pharmaceutically acceptable salts in the preparation of drugs for treating or preventing toxoplasmosis. The effective concentration of naringenin is 50-120 μg / mL. Also disclosed is the use of a pharmaceutical composition containing naringenin and its pharmaceutically acceptable salts in the preparation of drugs for treating or preventing toxoplasmosis. This patent discloses the use of naringenin in the preparation of drugs for treating toxoplasmosis, determining its effective concentration to be 50-120 μg / mL and confirming its low toxicity. However, Toxoplasma gondii belongs to the phylum Apicomplexa and differs fundamentally from EHV-8 (a double-stranded DNA virus) in terms of biological classification, pathogenic mechanism, and drug targets. The prevention and treatment of toxoplasmosis relies on inhibiting parasite invasion and proliferation, while the prevention and treatment of EHV-8 requires inhibiting viral replication. The mechanisms of action of the two drugs are not directly related. In addition, the research object of this patent is monkey kidney cells (Vero), and it does not involve the toxicity evaluation and antiviral effect verification of primary cells or related cell lines of equine animals, and no research is conducted on members of the Herpesviridae family, and it cannot provide technical inspiration for the prevention and treatment of EHV-8 infection.

[0006] In summary, although existing technologies have revealed the potential for the application of naringenin in the fight against herpes simplex virus (PRV) and protozoa (Toxoplasma gondii), there are no relevant research reports on its antiviral activity, targets, effective doses, and safety against EHV-8, a specific equine pathogen. This represents a clear technological gap in the prevention and treatment of equine viral diseases. Targeted research on the use of naringenin against EHV-8 is urgently needed to fill the gaps in existing technologies and promote the development of new antiviral drugs. Summary of the Invention

[0007] The object of the present invention is to provide an application of naringenin in the preparation of an anti-EHV-8 drug to solve the problems raised in the above background technology.

[0008] To achieve the above objectives, the present invention provides the use of naringenin in the preparation of anti-EHV-8 drugs, including the use of naringenin in the preparation of drugs for inhibiting EHV-8 replication, the use of plant extracts containing naringenin in the preparation of anti-EHV-8 drugs, and the use of traditional Chinese medicine compounds containing naringenin in the preparation of anti-EHV-8 drugs;

[0009] The naringenin plays a role in the replication stage of the EHV-8 virus, but has no significant inhibitory effect in the adsorption and entry stages. However, the naringenin cannot directly inactivate the EHV-8 virus.

[0010] Preferably, the naringenin, the plant extract containing naringenin, and the traditional Chinese medicine compound containing naringenin are used for preventing and treating respiratory diseases caused by EHV-8 infection in equine animals, for preventing and treating abortion caused by EHV-8 infection in equine animals, and for treating viral encephalitis caused by EHV-8 infection. At the level of susceptible cells, the maximum safe mass concentration of naringenin is 100 μM. This provides the use of naringenin and related products in preventing and treating various diseases caused by EHV-8 infection, and provides the maximum safe mass concentration at the level of susceptible cells, providing an important reference for clinical application.

[0011] Preferably, the naringenin, the plant extract containing naringenin, and the traditional Chinese medicine compound containing naringenin have significant inhibitory effects on multiple strains of EHV-8 SDLC66, SD2020113, and donkey / Shandong / 10 / 2021, demonstrating the inhibitory effect of naringenin and its related products on multiple EHV-8 strains, showing their broad-spectrum antiviral activity, and providing the possibility for the development of drugs against multiple EHV-8 strains.

[0012] Preferably, the naringenin, the plant extract containing naringenin, and the traditional Chinese medicine compound containing naringenin are used to reduce lung damage in a mouse model caused by EHV-8 infection. The mouse model experiment verifies the effect of naringenin and its related products in reducing lung damage caused by EHV-8 infection, providing an animal experimental basis for further clinical research.

[0013] Preferably, the naringenin is used to prepare a drug for inhibiting EHV-8 genome replication and gD protein expression. The drug exerts an antiviral effect by inhibiting the transcription and translation processes of EHV-8 in susceptible cells (RK-13, NBL-6). This deeply reveals the specific mechanism by which naringenin inhibits EHV-8, namely, exerting its antiviral effect by inhibiting the viral replication stage, providing a deeper understanding of the pharmacological effects of naringenin.

[0014] Preferably, the drug includes: naringenin monomer, naringenin-containing plant extracts, naringenin-containing traditional Chinese medicine compounds, and naringenin-containing pharmaceutical compositions. This clarifies the various forms of naringenin-related drugs, providing more options for drug development and production. Different drug forms can meet the needs of different patients. For example, monomeric drugs may be more suitable for precise control of dosage and efficacy, while plant extracts and traditional Chinese medicine compounds may have a wider range of biological activities and better synergistic effects.

[0015] Preferably, the plant extract is derived from citrus fruits or traditional Chinese medicines, the citrus fruits including grapefruit and orange, and the traditional Chinese medicines including immature bitter orange and dried tangerine peel. The traditional Chinese medicine compound comprises naringenin as one of its active ingredients and is combined with the traditional Chinese medicine. This provides the specific source of the naringenin plant extract and the composition of the traditional Chinese medicine compound, providing clear guidance for the acquisition of pharmaceutical raw materials and the development of traditional Chinese medicine compounds. This helps reduce drug R&D costs, improves drug accessibility, and also provides new ideas for the modernization and internationalization of traditional Chinese medicine.

[0016] Preferably, the pharmaceutical composition containing naringenin is in the form of tablets, capsules, granules, injections or oral liquids, and the pharmaceutically acceptable excipients include one or more of fillers, disintegrants, lubricants, binders, flavoring agents or preservatives. This clarifies the various dosage forms and pharmaceutically acceptable excipients of the naringenin pharmaceutical composition, providing specific guidance for the development and production of pharmaceutical formulations. Drugs in different dosage forms can meet the medication needs of different patients. For example, tablets and capsules are easy to carry and take, while injections are suitable for situations where rapid onset of action is required. At the same time, reasonable excipient selection can improve the stability and bioavailability of the drug, ensuring the safety and effectiveness of the drug.

[0017] Preferably, the method for preparing naringenin with anti-EHV-8 viral activity comprises the following steps: extracting crude naringenin from citrus fruits or traditional Chinese medicine, purifying the crude naringenin by chromatography to obtain naringenin with a purity greater than 95%, and drying and pulverizing the purified naringenin to prepare the pharmaceutical composition containing naringenin. This method provides a detailed preparation method for naringenin with anti-EHV-8 viral activity, offering a feasible technical route for industrial production of the drug. This method has the advantages of simple operation, low cost, and high purity, helping to ensure the quality and stability of the drug and improve its efficacy and safety.

[0018] Preferably, the extraction step uses ethanol or methanol as the extraction solvent, and the purification step uses high-performance liquid chromatography or preparative liquid chromatography. This clarifies the specific solvents and methods for naringenin extraction and purification, providing a detailed operational guide for the extraction and purification steps in drug development and production. Ethanol and methanol as extraction solvents offer advantages such as high extraction efficiency and low cost; high-performance liquid chromatography and preparative liquid chromatography offer advantages such as good separation, high purity, and ease of operation. The use of these specific methods helps improve the extraction efficiency and purity of the drug, ensuring its quality and stability.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. Inhibit viral replication

[0021] The maximum safe concentration of naringenin in susceptible cells (RK-13 and NBL-6) is 100 μM, effectively inhibiting viral replication within this concentration range. In vitro studies on susceptible RK-13 and NBL-6 cells revealed that naringenin significantly inhibited the replication of EHV-8 SDLC66. Quantitative polymerase chain reaction (qPCR) and Western blot analysis demonstrated that naringenin significantly inhibited EHV-8 gD gene copy number and gD protein expression. qPCR and Western blot analyses confirmed that naringenin significantly inhibited the replication of multiple EHV-8 strains, including SD2020113 and donkey / Shandong / 10 / 2021. qPCR and Western blot analyses confirmed that naringenin exerts an antiviral effect during the EHV-8 replication phase but does not directly inactivate EHV-8.

[0022] 2. Anti-EHV-8 effect in vivo

[0023] In the mouse model, naringenin can effectively reduce the replication level of EHV-8 in the lungs of BALB / c mice. Compared with the control group, the histopathological HE staining and TCID 50 The results showed that the naringenin-treated group could significantly reduce the replication of EHV-8.

[0024] 3. Drug advantages

[0025] Naringenin can be used as an effective drug ingredient for preventing and treating EHV-8 infection. Naringenin is widely present in citrus fruits and is natural and low in toxicity.

[0026] Naringenin has anti-inflammatory effects and can reduce the damage caused by inflammatory responses to host cells. After infection with EHV-8, host cells release a variety of inflammatory factors. Naringenin can protect the host from the dual damage of the virus and inflammation by inhibiting the release of inflammatory factors.

[0027] Naringenin has multiple pharmacological activities such as anti-inflammatory, antibacterial, antiviral, antioxidant, anti-tumor, and liver protection, which provides it with more potential advantages as an anti-EHV-8 drug.

[0028] In summary, the application of naringenin in the preparation of anti-EHV-8 drugs of the present invention has been systematically studied in vitro and in vivo in mouse models to investigate the anti-EHV-8 effect of naringenin, revealing that naringenin can significantly inhibit the replication of EHV-8, and can be used as an effective ingredient in drugs for preventing and treating EHV-8 infection. It also gives full play to the advantages of natural compounds with low toxicity and side effects, providing a valuable theoretical basis and reference for the clinical use of EHV-8 drugs, including antiviral and anti-inflammatory effects, which can protect the host from the dual damage of virus and inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they provide further detailed explanations but do not constitute a limitation of the present invention.

[0030] Figure 1 This is the molecular formula diagram of naringenin;

[0031] Figure 2 The toxicity test results of naringenin on EHV-8 susceptible cells are shown in the figure.

[0032] Figure 3 The naringenin of the present invention significantly inhibits the infection of EHV-8 SDLC66; the gD gene copy number (A) and the expression level of gD protein (B) are reduced;

[0033] Figure 4 The naringenin of the present invention has an inhibitory effect on different strains of EHV-8, and the gD gene transcription (A) and the expression of gD protein (B) are reduced;

[0034] Figure 5 Schematic diagram of the experimental setup of different naringenin treatment methods and EHV-8 infection in the present invention;

[0035] Figure 6 This is a graph showing the results of the present invention showing that naringenin exerts an inhibitory effect on the EHV-8 replication stage;

[0036] Figure 7 The results show that naringenin of the present invention cannot directly inactivate EHV-8; gD gene transcription (A) and gD protein (B) expression levels;

[0037] Figure 8 HE staining of naringenin in the present invention to reduce the damage caused by EHV-8 in mouse lungs;

[0038] Figure 9 This is a graph showing the results of naringenin of the present invention reducing the replication level of EHV-8 in mouse lungs. DETAILED DESCRIPTION

[0039] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0040] Example 1 Toxicity test of naringenin on EHV-8 susceptible cells

[0041] Rabbit kidney cells RK-13 and horse dermal cells NBL-6 were seeded into 96-well plates (1×10 4 / well), when the cells grew to about 80%, 100 μL of naringenin dilutions of different concentrations (0, 25 μM, 50 μM, 75 μM, 100 μM, 150 μM, and 200 μM) were added to each well. At the same time, negative controls and blank controls were set up, and 4 replicate wells were set up for each concentration. After incubation in a 37°C, 5% CO2 incubator for 24 hours, 10 μL of CCK-8 solution was added to each well and cultured for another 2 hours. The absorbance value at OD450 nm was read with the help of a microplate reader, and the following formula was used to calculate the effect of naringenin on the activity of different cells, and the maximum safe mass concentration of naringenin was evaluated (the results are shown in the figure). Figure 2 shown).

[0042] Cell viability (%) = [A1-A2] / [A3-A2] × 100%

[0043] A1 test group: absorbance values of wells with cells, CCK-8 solution, and naringenin solution

[0044] A2 Blank control: absorbance value of the well containing only culture medium and CCK-8 solution but no cells

[0045] A3 Negative control: absorbance value of the well containing cells and CCK-8 solution but no naringenin solution

[0046] The results are as follows Figure 2 As shown in the results, different concentrations of naringenin had no significant effect on the viability of RK-13 and NBL-6 cells, and the maximum safe concentration was 100 μM.

[0047] Example 2 Naringenin inhibits EHV-8 SDLC66 infection in vitro

[0048] 1. Analysis of EHV-8 gD gene expression by qPCR and Western blot

[0049] RK-13 and NBL-6 cells were seeded into 12-well cell plates (1×10 5 Cells were pretreated with naringenin at different concentrations (0 μM, 25 μM, 50 μM, 100 μM, and 150 μM) for 2 hours after reaching 80% confluency. One hour after inoculation with EHV-8 SDLC66 (0.1 MOI), the virus solution was removed and cell maintenance medium containing naringenin was added. DMSO-treated and untreated normal cells served as controls. 24 hours after infection, cell supernatants were collected, and total DNA was extracted using a kit. Changes in EHV-8 gene copy number and gD protein expression were determined by qPCR and Western blot.

[0050] 2. Naringenin has an inhibitory effect on multiple EHV-8 strains

[0051] RK-13 cells were seeded into 12-well cell culture plates (1 × 10 5 Cells were pretreated with 100 μM naringenin when they reached 80% growth. DMSO-treated cells served as a negative control, and the drug solution was removed after 2 hours. Subsequently, cells were infected with EHV-8 EHV-8SD2020113 and EHV-8 donkey / Shandong / 10 / 2021 for 1 hour. The supernatant containing the drug and virus was removed and replaced with MEM containing 3% FBS. 48 hours after infection, the cell plates were repeatedly frozen and thawed, and the supernatant was centrifuged and collected. The effects of different drug treatment doses on EHV-8 gD transcription and protein expression were analyzed by qPCR and Western blot.

[0052] 3. Determine the Effect of Naringenin on EHV-8 Replication Cycle

[0053] To further explore the mechanism by which naringenin affects EHV-8 infection, RK-13 and NBL-6 cells were seeded in 12-well plates (1×10 5 After the cells grew to 80%, the virus-only control group, naringenin pretreatment group (Pre), naringenin and EHV-8 co-treatment group (Co), EHV-8 infection followed by naringenin incubation group (Post), and naringenin all-stage treatment group (All stages-treatment) were set up, and EHV-8 SDLC66 was inoculated (MOI = 0.1). After 24 hours, the cells were collected and the expression of EHV-8 gD protein was evaluated by Western blot.

[0054] 4. Determine whether naringenin has the activity to inactivate EHV-8

[0055] To further explore the mechanism of naringenin on EHV-8 infection, RK-13 and NBL-6 cells were seeded in 12-well plates (1×10 5 After overnight culture, naringenin (100 μM) was incubated with different doses of EHV-8 SDLC66 (0.1 MOI, 0.5 MOI) at 37°C for 2 hours. After infection for 1 hour, the cells were replaced with 3% FBS MEM for 24 hours. The cells were then harvested and the transcription and protein expression levels of EHV-8 gD were analyzed by qPCR and western blot, respectively.

[0056] 5. Naringenin inhibits EHV-8 infection in vivo

[0057] Twelve SPF-grade BALB / c mice were randomly divided into four groups, with three mice in each group. A negative control group (Mock); a DMSO-treated control group plus EHV-8; and a naringenin plus EHV-8 group were set up. Naringenin-treated mice were intraperitoneally injected one day before infection, while the control group was injected with an equal dose of DMSO solution. Except for the negative control group, mice in each group were intranasally infected with 100 μL (1 × 10 5 PFU / mouse) EHV-8 SDLC66 was administered once at the set time points on the first day after infection (1dpi) and the third day after infection (3dpi), and the weight changes and health status of the mice were monitored daily. On the seventh day after virus infection (7dpi), the mice were killed by cervical dislocation, and the lung tissues were collected and fixed for histopathological examination. The results showed that naringenin can significantly reduce the pathological damage caused by EHV-8. At the same time, lung tissues were collected on the 7th dpi for grinding and tissue TCID 50 Determination.

[0058] In summary, this invention, based on natural medicines, is dedicated to discovering pharmaceutical ingredients with potential for combating EHV-8 infection. By treating EHV-8-susceptible cells (RK-13 and NBL-6) with naringenin and conducting in vivo experiments, we confirmed that naringenin significantly inhibits EHV-8 replication. Further studies demonstrated that this pharmaceutical ingredient exhibited inhibitory effects against multiple EHV-8 strains and further explored how naringenin affects the replication stages of EHV-8. These findings demonstrate the potential of naringenin as a drug for the prevention and treatment of EHV-8 infection.

[0059] The application of naringenin in the preparation of anti-EHV-8 drugs has the following advantages:

[0060] 1. Inhibit viral replication

[0061] The maximum safe concentration of naringenin in susceptible cells is 100 μM. Quantitative polymerase chain reaction (qPCR) and Western blot analysis demonstrated that naringenin significantly inhibited EHV-8 gD gene copy number and protein expression, effectively inhibiting EHV-8 replication at this concentration. In vitro studies in EHV-8-susceptible cells (RK-13 and NBL-6) revealed that naringenin significantly inhibited the replication of multiple EHV-8 strains, including SD2020113 and donkey / Shandong / 10 / 2021. This confirms that naringenin exerts antiviral effects during the EHV-8 replication phase but does not directly inactivate EHV-8.

[0062] 2. Anti-EHV-8 effect in vivo

[0063] In a mouse model, naringenin can effectively reduce the level of EHV-8 replication in the lungs. Through a BALB / c mouse challenge test, different treatment groups were set up, and the results showed that the amount of EHV-8 replication in the lungs of mice treated with naringenin was significantly lower than that of the control group. In addition, naringenin significantly reduced the pathological damage caused by EHV-8, which was confirmed by formaldehyde fixation of mouse lung tissue, histopathological examination, and tissue TCID 50 The determination was confirmed.

[0064] 3. Drug advantages

[0065] Naringenin can be used as an effective drug ingredient for preventing and treating EHV-8 infection. Naringenin is widely present in citrus fruits and is natural and low in toxicity.

[0066] Naringenin has anti-inflammatory effects and can reduce the damage caused by inflammatory responses to host cells. After infection with EHV-8, host cells release a variety of inflammatory factors. Naringenin can protect the host from the dual damage of the virus and inflammation by inhibiting the release of inflammatory factors.

[0067] Naringenin has multiple pharmacological activities such as anti-inflammatory, antibacterial, antiviral, antioxidant, anti-tumor, and liver protection, which provides it with more potential advantages as an anti-EHV-8 drug.

[0068] Naringenin exhibits significant antiviral activity against a variety of viruses, including influenza, dengue, and porcine epidemic diarrhea. EHV-8 is a herpesvirus that is highly pathogenic to equines. Therefore, naringenin could be a potential anti-EHV-8 drug candidate, potentially protecting the host from viral damage by inhibiting viral replication and spread and mitigating damage to host cells.

[0069] In summary, the application of naringenin in the preparation of anti-EHV-8 drugs of the present invention has been systematically studied in vitro and in vivo in mouse models to investigate the anti-EHV-8 effect of naringenin, revealing that naringenin can significantly inhibit the replication of EHV-8, and can be used as an effective ingredient in drugs for preventing and treating EHV-8 infection. It also gives full play to the advantages of natural compounds with low toxicity and side effects, providing a valuable theoretical basis and reference for the clinical use of EHV-8 drugs, including antiviral and anti-inflammatory effects, which can protect the host from the dual damage of virus and inflammation.

[0070] The basic principles, main features, and advantages of the present invention are shown and described above. It should be understood by those skilled in the art that the present invention is not limited to the above-described embodiments. The above-described embodiments and descriptions are merely preferred examples of the present invention and are not intended to limit the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention claimed.

Claims

1. An application of naringenin in the preparation of an anti-EHV-8 drug, characterized in that: Including the use of naringenin in preparing drugs for inhibiting EHV-8 replication, the use of plant extracts containing naringenin in preparing anti-EHV-8 drugs, and the use of traditional Chinese medicine compounds containing naringenin in preparing anti-EHV-8 drugs; The naringenin exerts an antiviral effect during the EHV-8 virus replication stage, but has no effect during the virus adsorption and entry stages. However, the naringenin cannot directly inactivate the EHV-8 virus.

2. The use of naringenin in the preparation of anti-EHV-8 drugs according to claim 1, characterized in that The naringenin, the plant extract containing naringenin, and the traditional Chinese medicine compound containing naringenin are used for preventing and treating respiratory diseases caused by EHV-8 infection in equine animals, for preventing and treating abortion caused by EHV-8 infection in equine animals, and for treating viral encephalitis caused by EHV-8 infection. At the level of susceptible cells, the maximum safe mass concentration of the naringenin is 100 μM.

3. The use of naringenin in the preparation of anti-EHV-8 drugs according to claim 1, characterized in that The naringenin, the plant extract containing naringenin, and the traditional Chinese medicine compound containing naringenin all have significant inhibitory effects on multiple strains of EHV-8 SDLC66, SD2020113, and donkey / Shandong / 10 / 2021.

4. The use of naringenin in the preparation of anti-EHV-8 drugs according to claim 1, characterized in that The naringenin, the plant extract containing naringenin and the traditional Chinese medicine compound containing naringenin are used for reducing lung damage in a mouse model caused by EHV-8 infection.

5. The use of naringenin in the preparation of anti-EHV-8 drugs according to claim 1, characterized in that The naringenin is used to prepare a drug for inhibiting EHV-8 genome replication and gD protein expression. The drug exerts an antiviral effect by inhibiting the transcription and translation processes of EHV-8 in susceptible cells RK-13 and NBL-6.

6. The use of naringenin in the preparation of anti-EHV-8 drugs according to claim 1, characterized in that The medicine includes: naringenin monomer, plant extract containing naringenin, traditional Chinese medicine compound containing naringenin and pharmaceutical composition containing naringenin.

7. The use of naringenin in the preparation of anti-EHV-8 drugs according to claim 6, characterized in that The plant extract is derived from citrus fruits or traditional Chinese medicines. The citrus fruits include grapefruit and orange. The traditional Chinese medicines include immature bitter orange and dried tangerine peel. The traditional Chinese medicine compound uses naringenin as one of the active ingredients and is prepared in combination with the traditional Chinese medicines.

8. The use of naringenin in the preparation of anti-EHV-8 drugs according to claim 6, characterized in that The pharmaceutical composition containing naringenin is in the form of tablets, capsules, granules, injections or oral liquids, and the pharmaceutically acceptable excipients include one or more of fillers, disintegrants, lubricants, binders, flavoring agents or preservatives.

9. The use of naringenin in the preparation of anti-EHV-8 drugs according to claim 1, characterized in that The method for preparing naringenin with anti-EHV-8 viral activity comprises the following steps: extracting crude naringenin from citrus fruits or traditional Chinese medicine, purifying the crude naringenin by chromatography to obtain naringenin with a purity greater than 95%, and drying and pulverizing the purified naringenin to prepare the pharmaceutical composition containing naringenin.

10. The use of naringenin in the preparation of anti-EHV-8 drugs according to claim 9, characterized in that: The extraction step uses ethanol or methanol as the extraction solvent, and the purification step uses high performance liquid chromatography or preparative liquid chromatography.

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