Application of herba epimedii extract in preparation of medicine for preventing and / or treating infectious bronchitis virus infection

Abstract

The invention relates to the field of prevention and control of poultry viral diseases, in particular to application of herba epimedii extract in preparation of a medicine for preventing and / or treating infectious bronchitis virus infection. The herba epimedii extract is a herba epimedii ethanol extract. The biological safety and antiviral activity of the extract are evaluated by constructing a chick embryo infection model, and the epimedium extract does not generate obvious toxic influence on chick embryos; after infectious bronchitis virus infection, the survival rate of chick embryos can be increased. Furthermore, tissue immunofluorescence detection, western blot analysis and probe method fluorescent quantitative PCR detection results show that the extract can reduce the virus antigen expression level and virus nucleic acid copy number in chick embryo liver tissue and allantoic fluid, and therefore the effect of preventing or treating IBV infection is achieved. The herba epimedii extract is stable in raw material source, simple and convenient in preparation process and relatively low in production cost, and has a good popularization and application prospect.

Description

Technical Field

[0001] This invention relates to the field of avian viral disease prevention and control, specifically to the use of an epimedium extract in the preparation of a drug for the prevention and / or treatment of infectious bronchitis virus infection in chickens. Background Technology

[0002] Infectious bronchitis (IB) is an acute, highly contagious avian viral disease caused by infectious bronchitis virus (IBV), belonging to the Coronaviridae family. This virus primarily attacks the respiratory system of chickens, but can also affect multiple organs and tissues, including the kidneys and reproductive system. Clinically, it often manifests as abnormal breathing, sneezing, coughing, decreased feed intake, and restricted growth and development. In laying hens, infection frequently results in reduced egg production and decreased eggshell quality. Inadequate control measures can easily lead to high morbidity and mortality rates, causing significant economic losses to poultry production.

[0003] Currently, IBV prevention and control mainly rely on vaccination and enhanced biosafety management. However, IBV is characterized by frequent genetic mutations and complex serotypes, resulting in limited cross-immune protection between different strains and fluctuations in vaccine efficacy during actual production. Vaccination failure or latent infection can still occur in some farms, making it difficult to completely block virus transmission. For infected flocks, there is currently a lack of safe and effective antiviral drugs specifically targeting IBV; clinical treatment mainly focuses on supportive care or adjunctive medication, which is insufficient to fundamentally inhibit viral replication and control disease progression.

[0004] With the deepening research on the active ingredients of natural plants, the application of plant-derived extracts in the prevention and control of viral diseases in animals has gradually attracted attention. Plant extracts generally have the characteristics of stable source, high safety and low residue risk. Some plant components have been proven to have antiviral, anti-inflammatory and immunomodulatory biological activities, showing certain potential in the field of poultry disease prevention and control. Epimedium ( Epimedium spp As a traditional medicinal plant, its extract contains various active substances such as icariin, flavonoids, and phenolic compounds. Currently, there is a lack of systematic research and public reports on the application of epimedium extract in the prevention and treatment of infectious bronchitis virus (IBV) in chickens, and the number of natural antiviral candidates targeting IBV remains relatively limited. Summary of the Invention

[0005] To address the problems of rapid spread, frequent strain mutation, limited cross-protection capabilities of vaccines, and lack of specific antiviral treatments associated with IBV in existing technologies, this invention proposes an anti-IBV technology derived from natural plants. This method uses Epimedium extract as the active ingredient to prepare a drug for the prevention and treatment of IBV infection.

[0006] The Epimedium extract is obtained from the aerial stems and leaves of Epimedium using ethanol as the extraction solvent, through extraction, separation, concentration, and drying. The safety and antiviral effects of this extract were evaluated using a chicken embryo infection model. Results showed that within a certain dosage range, the Epimedium extract had no significant toxic effects on chicken embryos. Treatment with different doses after IBV infection improved embryo survival and reduced tissue damage. Further analysis using tissue immunofluorescence, Western blotting, and probe-based quantitative PCR showed that the Epimedium extract could reduce viral antigen expression levels and viral nucleic acid copy numbers in chicken embryo liver tissue and allantoic fluid, thereby inhibiting IBV replication in vivo and exhibiting preventative and / or therapeutic effects against IBV infection.

[0007] The raw materials used in this invention are stable, the extraction process is simple, the production cost is low, and the biosafety is good, making it suitable for large-scale production and practical application.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The use of an epimedium extract in the preparation of a medicament for the prevention and / or treatment of infectious bronchitis virus infection in chickens; wherein the epimedium extract is an ethanol extract of epimedium.

[0009] Preferably, the Epimedium extract can effectively improve the survival rate of infected chicken embryos, reduce the expression level of viral antigens in the liver, and reduce the viral nucleic acid copy number in the liver and allantoic fluid.

[0010] Preferably, the Epimedium ethanol extract is prepared by using Epimedium as raw material and ethanol as extraction solvent, through extraction, filtration and concentration; the ethanol is an aqueous solution with a volume fraction of 60% to 95%.

[0011] Preferably, the extraction is performed by one or more of ultrasonic extraction, reflux extraction, or immersion extraction.

[0012] Preferably, the Epimedium extract is used with pharmaceutically acceptable excipients to prepare a pharmaceutical formulation for combating infectious bronchitis virus in chickens. The formulation is further preferably one of an oral liquid, granules, injection, or spray.

[0013] Preferably, the preparation method of the Epimedium ethanol extract includes the following steps: (1) Dry the Epimedium and then pulverize it through a 40-80 mesh sieve; (2) Add Epimedium powder to a 60-95% ethanol solution at a solid-liquid ratio of 1:8-20, mix thoroughly, and then extract by ultrasonication 3 times; (3) After extraction, centrifuge the extract, take the supernatant, filter it through medium-speed filter paper, place the filtrate in a rotary evaporator, and recover ethanol and concentrate it to a thick state under the conditions of 40~50℃ temperature, -0.085~-0.095 MPa vacuum degree and 80~120 rpm rotation speed. (4) The obtained concentrate was vacuum dried to constant weight and then pulverized to obtain the ethanol extract of Epimedium.

[0014] Preferably, the Epimedium powder and an 80% ethanol solution are mixed at a solid-liquid ratio of 1:9.

[0015] Preferably, the ultrasonic extraction conditions are: frequency 40 kHz, output power 300 W, and extraction time 30 min per extraction.

[0016] Preferably, the centrifugation conditions are centrifugation at 3500 rpm for 10 min; the drying conditions are drying at a temperature of 40~50℃ and a vacuum of -0.085~-0.095 MPa.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: (1) Within a certain dosage range, no obvious toxic reaction was observed in chicken embryos by the Epimedium extract of the present invention, indicating that it has high biosafety in practical applications and is suitable for poultry control.

[0018] (2) The present invention verified through a chicken embryo infection model that Epimedium extract can significantly improve the survival rate of infected chicken embryos, reduce the expression level of viral antigens in the liver, and reduce the viral nucleic acid copy number in the liver and allantoic fluid, showing a strong anti-IBV effect and a good viral inhibition effect, with a clear antiviral effect.

[0019] (3) The present invention uses ethanol extraction to extract Epimedium. The process steps are simple, easy to operate, the raw material source is stable, and the process is mature. It is suitable for large-scale production and has good potential for industrialization and promotion. The process is highly feasible.

[0020] (4) The Epimedium extract of the present invention, as an antiviral drug of natural plant source, can not only provide a new option for the prevention and treatment of infectious bronchitis virus in chickens, but also conforms to the trend of green breeding and antibiotic-free development, has good market application prospects, and can meet the needs of modern animal husbandry for safe and environmentally friendly drugs. Attached Figure Description

[0021] Figure 1 Effect of EE on chicken embryo weight changes Figure 2 Effect of EE on weight changes in IBV-infected chicken embryos Figure 3 Indirect immunofluorescence analysis of the effect of EE on IBV distribution in chicken embryo liver Figure 4 Effect of EE on IBV N protein expression in chicken embryo liver Figure 5 Effect of EE on IBV copy number in chicken embryo liver and allantoic fluid (1) Quantitative analysis of IBV copy number in chicken embryo liver; (2) Quantitative analysis of IBV copy number in chicken embryo allantoic fluid Note: Figure 1 , 2 In cases 4 and 5, the same letter or no letter in the shoulder inscription indicates that the difference is not significant. P >0.05), different letters indicate significant differences ( P <0.05). Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to preferred embodiments and accompanying drawings. The listed embodiments are intended to specifically illustrate and demonstrate the technical content of the present invention so that those skilled in the art can understand and implement the present invention, but should not be construed as limiting the scope of protection of the present invention.

[0023] Unless otherwise explicitly stated, the experimental methods involved in the embodiments are all conventional techniques known in the art, and can be performed with reference to relevant literature or according to the instructions of reagents and instruments. All raw materials, reagents, and equipment used can be obtained through conventional commercial channels.

[0024] Example 1 (Preparation of Epimedium Extract)

[0025] (1) Raw material processing The above-ground parts of Epimedium (including leaves and stems) are selected, dried, pulverized, and passed through a 60-mesh sieve to obtain Epimedium powder for later use.

[0026] (2) Ultrasonic extraction Weigh the Epimedium powder at a solid-liquid ratio of 1:9 (g:mL), add an 80% ethanol solution, mix thoroughly, and place in an ultrasonic extraction device. Extract three times at a frequency of 40 kHz and a power of 300 W, each time for 30 min.

[0027] (3) Separation and concentration The extract was centrifuged at 3500 rpm for 10 min, the supernatant was collected and filtered through medium-speed filter paper, and the filtrates were combined. The filtrate was placed in a rotary evaporator and the ethanol was recovered and concentrated to a viscous state at 45 ℃, -0.09 MPa, and 100 rpm.

[0028] (4) Drying treatment The concentrate was placed in a vacuum drying oven and dried to constant weight at 45 °C and -0.09 MPa.

[0029] (5) Obtaining the finished product The dried product was pulverized to obtain Epimedium extract ( Epimedium spp . extract, EE).

[0030] Example 2 (Preparation of Epimedium Extract)

[0031] (1) Raw material processing The above-ground parts of Epimedium (including leaves and stems) are selected, dried, pulverized, and passed through a 40-mesh sieve to obtain Epimedium powder for later use.

[0032] (2) Ultrasonic extraction Weigh the Epimedium powder at a solid-liquid ratio of 1:20 (g:mL), add an 80% ethanol solution, mix thoroughly, and place in an ultrasonic extraction device. Extract three times at a frequency of 40 kHz and a power of 300 W, each time for 30 min.

[0033] (3) Separation and concentration The extract was centrifuged at 3500 rpm for 10 min, the supernatant was collected and filtered through medium-speed filter paper, and the filtrates were combined. The filtrate was placed in a rotary evaporator, and ethanol was recovered and concentrated to a viscous state at 40℃, -0.09 MPa, and 120 rpm.

[0034] (4) Drying treatment The concentrate was placed in a vacuum drying oven and dried to constant weight at 40 °C and -0.09 MPa.

[0035] (5) Obtaining the finished product The dried product was pulverized to obtain Epimedium extract ( Epimedium spp . extract, EE).

[0036] Experimental Example 1 (Toxicity evaluation of Epimedium extract on chicken embryos) This embodiment aims to evaluate the safety of EE in specific pathogen-free (SPF) fertilized chicken embryos, providing a reference for subsequent dose selection for anti-IBV action. The specific operating steps are as follows: (1) Chicken embryo selection and grouping SPF fertilized chicken embryos with normal development, intact shells, and no cracks were selected, and all embryos came from the same batch. After incubation under standard incubation conditions until 10 days of age, they were randomly divided into 6 treatment groups, with 5 embryos in each group. A control group (CON) and treatment groups with different doses of Epimedium extract (EE) were established.

[0037] (2) Sample preparation and processing The EE obtained in Example 1 was prepared into a stock solution using dimethyl sulfoxide (DMSO) as a co-solvent. This stock solution was then serially diluted with sterile physiological saline to prepare working solutions of different concentrations: 40 mg / mL, 20 mg / mL, 10 mg / mL, 5 mg / mL, and 2.5 mg / mL. Following the allantoic cavity inoculation route, each chicken embryo was injected with 100 μL of the corresponding concentration of sample solution; the CON group was injected with 100 μL of sterile physiological saline in the same manner.

[0038] (3) Observation and Recording After treatment, the chicken embryos were placed under standard incubation conditions for further culture. The survival rate, time of death, and embryonic development status of the chicken embryos were observed and recorded within 96 hours. At the end of the experiment, the weight of the chicken embryos in each group was measured, and the survival rate and growth and development of each group were recorded.

[0039] (4) Toxicity evaluation Based on the survival rate, mortality time distribution, and presence of toxicity symptoms such as developmental delay or malformation in chicken embryos of each treatment group, the safe dose range of EE in SPF fertilized chicken embryos was evaluated, providing a reference for the selection of dosage in subsequent anti-IBV protection trials.

[0040] As shown in Table 1, at 24 h, the survival rate of chicken embryos in all treatment groups was 100%, with no significant difference. At 48 h, except for the 40 mg / mL EE treatment group, the survival rate of chicken embryos in all other groups remained at 100%; the survival rate of the 40 mg / mL EE group decreased to 80%. When the observation time was extended to 72 h and 96 h, the survival rate of chicken embryos in the 2.5–20 mg / mL EE groups and the CON group remained at 100%, while the survival rate of the 40 mg / mL EE group further decreased to 60%. In addition, at the end of the experiment, statistical analysis of chicken embryo weight revealed that within the dosage range of 2.5–10 mg / mL, there was no significant difference in chicken embryo weight between the treatment groups and the CON group. P>0.05), overall growth was good. The embryo weight in the 20 mg / mL EE group showed a certain decreasing trend, but the difference compared to the control group was not statistically significant. The embryo weight in the 40 mg / mL EE group was significantly reduced, with greater intra-group variability, and the difference compared to the control group was statistically significant. P <0.05).

[0041] In summary, under the conditions of this invention, Epimedium extract did not exhibit significant acute toxicity or growth inhibition in SPF chicken embryos within a dose range of 2.5–20 mg / mL. However, when the dose increased to 40 mg / mL, the survival rate of chicken embryos decreased, accompanied by a reduction in body weight, suggesting that high-dose treatment may have adverse effects on chicken embryo growth and development. These results provide experimental basis for determining the dosage in subsequent anti-IBV protection experiments.

[0042]

[0043] Experimental Example 2 (Verification of the protective effect of Epimedium extract against IBV-infected chicken embryos) This experimental example was used to verify the protective effect of EE prepared by the method in Example 1 against IBV infection and its effect on inhibiting viral replication. The specific steps are as follows: (1) Chicken embryo selection and grouping SPF fertilized chicken embryos from the same batch with normal development, intact shells, no cracks, and normal egg development were selected and incubated under standard incubation conditions until 10 days of age. They were then randomly divided into 5 treatment groups, with 5 embryos in each group. A control group (CON), an IBV-infected group (IBV), and co-intervention groups with different doses of IBV+EE (including IBV+2.5 mg / mL EE, IBV+5 mg / mL EE, and IBV+10 mg / mL EE) were established.

[0044] (2) Viral infection and drug administration The IBV strain isolated and purified in this laboratory was selected, and a concentration of [missing information] was used. Virus solution of copies / μL. IBV infection group: 100 μL of virus solution was thoroughly mixed with 100 μL of sterile physiological saline and inoculated into chicken embryos via the allantoic cavity to establish an IBV infection model; EE intervention group: 100 μL of virus solution was mixed with 100 μL of EE solution of different concentrations and inoculated via the same route; Control group: only 200 μL of sterile physiological saline was injected. The above virus solution, sterile physiological saline, and mixtures of virus solution and different doses of EE were pre-incubated in a 37℃ incubator for 1 hour before inoculation.

[0045] (3) Observation and Recording After processing, the chicken embryos were kept in incubation conditions and their survival, mortality time distribution and embryonic development status were observed and recorded regularly within 96 hours. At the end of the experiment, the weight of the chicken embryos was measured and the survival rate and growth and development of each group were statistically analyzed.

[0046] (4) Sample collection Upon observation of embryonic death, allantoic fluid was immediately collected, and necropsy was performed to extract liver tissue. The obtained samples were used for subsequent pathological observation, immunofluorescence analysis, and viral nucleic acid detection.

[0047] (5) Immunofluorescence detection Immunofluorescence assay (IFA) was performed on chicken embryo liver tissue sections to detect the distribution and enrichment of IBV antigen in the liver tissue, so as to assess the level of viral infection in the tissue. The primary antibody used was a monoclonal antibody against IBV nucleocapsid protein (N protein) prepared in our laboratory.

[0048] (6) Detection of protein by Western blotting Chicken embryo liver tissue was collected, homogenized with lysis buffer containing protease inhibitors, and the supernatant was collected after centrifugation as the total protein extract. Protein concentration was determined using the BCA method, and the loading amount for each group of samples was standardized. Equal amounts of protein samples were denatured and then separated by SDS-PAGE electrophoresis. After electrophoresis, the samples were transferred to an NC membrane. The membrane was blocked, and primary and secondary antibodies against IBV N protein were added sequentially. After incubation at room temperature, chemiluminescence staining was performed, and band signals were acquired using an imaging system. β-actin was used as an internal control protein, and semi-quantitative analysis of the band gray values ​​was performed to calculate the relative expression level of IBV N protein.

[0049] (7) Probe-based quantitative PCR detection Specific primers and probes were designed based on the target gene sequence, and a series of graded dilutions of standards were constructed to generate a standard curve. Absolute quantitative analysis of IBV nucleic acid copy numbers in chicken embryo liver tissue and allantoic fluid was performed to compare viral load differences among different treatment groups, thereby evaluating the inhibitory effect of EE on IBV replication.

[0050]

[0051] The results are shown in Table 3. At 24 h and 48 h, the survival rate of chicken embryos in all groups was 100%, with no significant difference. At 72 h, the survival rate of the IBV-infected group decreased to 80%, while the IBV+2.5 mg / mL EE group and the IBV+5 mg / mL EE group maintained 100%, and the IBV+10 mg / mL EE group was 80%. When the observation time was extended to 96 h, the survival rate of the IBV-infected group further decreased to 40%; in contrast, the survival rate of chicken embryos in all EE intervention groups was 80%. These results indicate that under the conditions of this embodiment, EE intervention can improve the survival rate of IBV-infected chicken embryos to a certain extent, showing a protective trend. Furthermore, the chicken embryo weight at the end of the experiment was statistically analyzed (…). Figure 2 The results showed that the body weight of the IBV group was significantly lower than that of the CON group. P <0.05), indicating that viral infection has a significant inhibitory effect on chicken embryo growth. Compared with the IBV group, the body weight of all EE intervention groups increased, with the 10 mg / mL EE group showing a body weight level close to the control group and no significant difference from the CON group. P The concentration of IBV (>0.05) indicates a certain trend of improvement. Considering both survival rate and weight changes, it is evident that under the conditions of this embodiment, Epimedium extract can alleviate the adverse effects of IBV infection on chicken embryo survival and growth to a certain extent, especially showing a more significant protective effect within an appropriate dosage range.

[0052]

[0053] Figure 3 The results showed that the overall positive signal of IBV N protein in the CON group was weak, while the IBV group showed significantly enhanced positive signal of IBV N protein. Green fluorescence was multifocally distributed in the tissue, accompanied by local diffuse diffusion; some areas formed relatively concentrated high-intensity expression foci. In the IBV + 2.5 mg / mL EE treatment group, green fluorescence was mainly distributed in small foci, with mild aggregation in local areas, but no large-area continuous diffusion was observed. In the IBV + 5 mg / mL EE treatment group, obvious high-intensity green fluorescent clumps appeared in local areas, showing focal concentrated distribution, with a small amount of scattered signal around them, but no widespread diffuse diffusion was observed. In the IBV + 10 mg / mL EE treatment group, only a few sporadic green fluorescent signals were observed, without obvious aggregation or focal distribution, and no obvious antigen-enriched areas were observed in the tissue. Overall, compared with the IBV group, the positive signal of IBV N protein in liver tissue of each dose group of EE treatment showed an overall weakening trend, especially under higher dose treatment conditions, the antigen expression level was significantly reduced. The results suggest that, under the conditions of this experiment, EE treatment may affect the antigen expression level of IBV in liver tissue to some extent.

[0054] Figure 4 The results showed that the expression level of N protein in the liver of chicken embryos in the IBV group was significantly higher than that in the CON group, indicating that the virus was expressed in large quantities in the liver tissue. Compared with the IBV infection group, the expression level of N protein in the EE intervention group showed an overall decreasing trend, with the expression levels in the IBV+5 mg / mL EE treatment group and the IBV+10 mg / mL EE treatment group being close to those in the CON group. These results indicate that under the conditions of this embodiment, EE can reduce the expression level of IBV N protein to a certain extent, reflecting its inhibitory effect on viral replication at the protein expression level.

[0055] Figure 5 The results showed that no IBV nucleic acid signal was detected in the CON group samples. High viral copy numbers were detected in the liver and allantoic fluid of chicken embryos in the IBV group, indicating extensive viral replication in vivo. Compared with IBV, the IBV + 10 mg / mL EE treatment group showed significantly lower viral copy numbers in both liver tissue and allantoic fluid, and the difference between groups was statistically significant. P <0.05). The above results indicate that under the conditions of this embodiment, EE can inhibit the replication level of IBV in chicken embryos to a certain extent and reduce the viral nucleic acid content in liver tissue and allantoic fluid. This result is corroborated by the survival rate trend and N protein expression results, supporting the inhibitory effect of Epimedium extract on IBV infection from multiple indicator levels.

[0056] In summary, this invention provides the use of Epimedium extract in the preparation of a drug for the prevention and treatment of infectious bronchitis virus (IBV) infection in chickens. A systematic evaluation of the biosafety and antiviral activity of the Epimedium extract was conducted using a chicken embryo infection model. The results showed that, within an appropriate dosage range, the extract did not produce significant toxic effects on SPF chicken embryos, demonstrating a good biosafety basis. Under IBV infection conditions, the extract could improve chicken embryo survival rate and alleviate infection-induced growth inhibition. Further analysis using tissue immunofluorescence, Western blotting, and probe-based quantitative PCR verified the inhibitory effect of the Epimedium extract on IBV replication at both the antigen expression level and viral nucleic acid copy number levels. The results of multiple indicators corroborate each other, indicating that the technical solution described in this invention can exert preventive or therapeutic effects against IBV infection in chickens under certain conditions.

[0057] The Epimedium extract used in this invention has a stable source, a well-defined and simple preparation process, and is suitable for large-scale production, demonstrating good industrialization feasibility and promising application prospects. This technical solution provides a novel natural plant-based candidate for the prevention and control of infectious bronchitis virus infection in chickens, and has certain application value.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Without departing from the spirit and essence of the present invention, those skilled in the art can make various modifications or equivalent substitutions to the present invention. All improvements falling within the scope of the inventive concept of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of an epimedium extract in the preparation of a medicament for the prevention and / or treatment of infectious bronchitis virus infection in chickens; wherein the epimedium extract is an ethanol extract of epimedium.

2. The use according to claim 1, characterized in that, The use of the Epimedium extract in the preparation of drugs that improve the survival rate of infected chicken embryos, reduce the expression level of viral antigens in the liver, and reduce the viral nucleic acid copy number in the liver and allantoic fluid.

3. The use according to claim 1, characterized in that, The Epimedium ethanol extract is prepared by using Epimedium as raw material and ethanol as extraction solvent, through extraction, filtration and concentration; the ethanol is an aqueous solution with a volume fraction of 60% to 95%.

4. The use according to claim 3, characterized in that, The extraction method is one or more of ultrasonic extraction, reflux extraction, or immersion extraction.

5. The use according to any one of claims 1 to 4, characterized in that, The epimedium extract, combined with pharmaceutically acceptable excipients, is used to prepare a pharmaceutical formulation for combating infectious bronchitis virus in chickens.

6. The use according to claim 5, characterized in that, The preparation is one of oral liquid, granules, injection, or spray.

7. The use according to claim 1, characterized in that, The preparation method of the Epimedium ethanol extract includes the following steps: (1) Dry the Epimedium and then pulverize it through a 40-80 mesh sieve; (2) Add Epimedium powder to a 60-95% ethanol solution at a solid-liquid ratio of 1:8-20, mix thoroughly, and then extract by ultrasonication 3 times; (3) After extraction, centrifuge the extract, take the supernatant, filter it through medium-speed filter paper, place the filtrate in a rotary evaporator, and recover ethanol and concentrate it to a thick state under the conditions of 40~50℃ temperature, -0.085~-0.095 MPa vacuum degree and 80~120 rpm rotation speed. (4) The obtained concentrate was vacuum dried to constant weight and then pulverized to obtain the ethanol extract of Epimedium.

8. The use according to claim 7, characterized in that... The Epimedium powder and an 80% ethanol solution were mixed at a solid-liquid ratio of 1:

9.

9. The use according to claim 7, characterized in that... The ultrasonic extraction conditions are: frequency 40 kHz, output power 300 W, and extraction time 30 min per extraction.

10. The use according to claim 7, characterized in that... The centrifugation conditions are centrifugation at 3500 rpm for 10 min; the drying conditions are drying at a temperature of 40~50℃ and a vacuum of -0.085~-0.095 MPa.

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