Use of hyperoside or its pharmaceutically acceptable salt as the sole active ingredient in the preparation of drugs for resisting porcine reproductive and respiratory syndrome virus infection

Abstract

The invention relates to the technical field of traditional Chinese medicine, and specifically to the use of hyperoside or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of a drug for resisting porcine reproductive and respiratory syndrome virus infection. In the invention, hyperoside is extracted by using hibiscus tiliaceus, and the obtained hyperoside is applied to the research of anti-PRRSV drugs. It is found that hyperoside plays an inhibitory role in the adsorption, internalization, replication and release of PRRSV, reduces the viremia of piglets caused by PRRSV infection, and can also significantly reduce the lung damage caused by viral infection. The drug can effectively reduce the reproductive disorders of sows and respiratory diseases of growing pigs caused by PRRSV infection, as well as the secondary infection of diseased pigs. In the invention, the extraction process of hyperoside is convenient and fast, and large-scale rapid extraction can be achieved, thereby avoiding the defect that hyperoside cannot be used on a large scale due to the difficulty of extraction. After purification and inspection during the preparation process, the anti-PRRSV application of hyperoside is further guaranteed.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and in particular to use of hyperoside or a pharmaceutically acceptable salt thereof as the sole active ingredient in preparing a drug for resisting porcine reproductive and respiratory syndrome virus infection. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease caused by infection with porcine reproductive and respiratory syndrome virus (PRRSV), which seriously endangers global pig production. It is commonly known as "blue ear disease". Its main harm is to cause reproductive disorders in sows and respiratory diseases in growing pigs, causing huge economic losses to pig farming companies. In view of the huge impact of this disease on the pig industry, it has seriously damaged the development of my country's animal husbandry economy. Therefore, effective drugs are urgently needed for the prevention and control of PRRSV.

[0003] PRRSV is a member of the Nidovirales order, Arteriviridae family, and Porartevirus genus. PRRSV includes PRRSV-1 and PRRSV-2 subtypes, of which PRRSV-1 is more common in Europe and Russia, and PRRSV-2 is mainly prevalent in North America and Asia. In 1987, PRRSV first broke out in North America. In the mid-1990s, the virus first appeared in China, and in 2006, the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) appeared. In recent years, the new variant strain NADC30-like has been prevalent in China and has gradually become the dominant strain. Given the strong variability of PRRSV, and the replication and pathogenic mechanisms are not yet fully understood. There are currently no effective vaccines and drugs, and there is an urgent need to develop economical and efficient anti-PRRSV drugs.

[0004] Hyperoside (Hyp) is a flavonoid compound extracted from traditional Chinese medicine, and its structure is quercetin-3-O-galactoside. It is mostly distributed in the fruits and whole herbs of plants such as Hypericaceae, Campanulaceae, Lamiaceae, Rosaceae, Ericaceae, Asteraceae, Berberidaceae, Garciniaaceae, Leguminosae and Celastraceae. Hyperoside has multiple effects such as antiviral, anticancer, anti-inflammatory, antidepressant, hypolipidemic, and antioxidant.

[0005] Therefore, the present invention proposes the use of hyperoside or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of a drug for resisting porcine reproductive and respiratory syndrome virus infection. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the main purpose of the present invention is to provide the use of hyperoside or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of drugs for resisting porcine reproductive and respiratory syndrome virus infection, so as to solve the problems in the above-mentioned background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] Application of hyperoside in the preparation of anti-PRRSV drugs, application of plant extracts containing hyperoside in the preparation of anti-PRRSV drugs, and application of traditional Chinese medicine compound containing hyperoside in the preparation of anti-PRRSV drugs. The structural formula of the hyperoside is shown in Formula 1.

[0009] (Formula 1)

[0010] Preferably, the maximum safe mass concentration of the hyperoside for PRRSV susceptible cells (MARC-145 and PAM) is 120 µM (µmol / L), that is, hyperoside is non-toxic to MARC-145 and PAM within this concentration range.

[0011] Preferably, the hyperoside can significantly inhibit the replication of PRRSV within a safe concentration range.

[0012] Preferably, the hyperoside has an inhibitory effect on the replication of PRRSV-1 and PRRSV-2 strains GZ11-G1, P073-3, SD16, JXA1, CH-1a, and NADC30-like, indicating that hyperoside has a broad-spectrum anti-PRRSV infection effect.

[0013] Preferably, the hyperoside inhibits viral infection at multiple stages of PRRSV adsorption, internalization, replication and release.

[0014] Preferably, the hyperoside can significantly reduce piglet viremia caused by PRRSV infection.

[0015] The hyperoside can significantly reduce the lung damage of piglets caused by PRRSV infection.

[0016] Preferably, the hyperoside is extracted from hibiscus hibiscus, and includes the following raw materials: hibiscus hibiscus, naringenin, flavonol synthase, metal ions, acetate buffer, methanol solvent, stabilizer and cellulase. The specific preparation method is as follows:

[0017] S1, preparation stage: take the cleaned hibiscus, grind it and put it into a container, and spread naringenin on the bottom of the container in advance;

[0018] S2, enzymatic hydrolysis stage: adding cellulase into the container to carry out enzymatic hydrolysis reaction, and adding metal ions and flavonol synthase in sequence during the enzymatic hydrolysis process;

[0019] S3, collecting the extract: after the enzymatic hydrolysis is completed, the extract is separated from the residue by centrifugation to obtain the extract;

[0020] S4, purification: pour the extract into a filter, remove the residue and large particles of impurities, use a rotary evaporator to remove part of the solvent, reduce the volume of the extract, and then use silica gel to perform column chromatography to further purify the extract, and finally complete the purification of the extract;

[0021] S5, verification stage: using high performance liquid chromatography to verify the purity and content of the extracted hyperoside, thereby obtaining a hyperoside extract.

[0022] Preferably, in step S2, the ratio of liquid to solid in the container during enzymatic hydrolysis is 25:1, the dosage of cellulase is 5.7 mg / g, the enzymatic hydrolysis time is 105 min, the temperature in the container is 50° C., and the HP of the liquid during enzymatic hydrolysis is 4.6.

[0023] Preferably, in step S2, the solvent used to adjust HP is acetate buffer, and the addition interval of metal ions and flavonol synthase during the enzymatic hydrolysis is 10 min.

[0024] Preferably, during the extraction process of step S3, a stabilizer is added in a ratio of 20:1 to the liquid.

[0025] Preferably, during the purification process in step S4, before removing the solvent using a rotary evaporator, methanol solvent is added to the extract in a ratio of 18:3, and then other solvents are removed.

[0026] Compared with the known public technology, the technical solution provided by the present invention has the following beneficial effects:

[0027] In the present invention, hyperoside is extracted from Abelmoschus truncatus, and the obtained hyperoside is applied to the research of anti-PRRSV drugs. The results show that the drug has an inhibitory effect in multiple stages such as adsorption, internalization, replication and release of PRRSV. The drug can significantly reduce the viremia of piglets caused by PRRSV infection and significantly reduce the lung damage caused by viral infection. In the present invention, the extraction process of hyperoside is convenient and fast, and large-scale rapid extraction can be achieved, thereby solving the defect that hyperoside cannot be used on a large scale due to the difficulty of extraction. Secondly, hyperoside is purified and tested during the preparation process, which further ensures the subsequent anti-PRRSV application of hyperoside. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The figure shows the test results of the toxicity test of hyperoside on PRRSV susceptible cells MARC-145 and PAM;

[0029] Figure 2 Schematic diagram of the effect of hyperoside on the progeny virus production (A, C) and viral N protein expression (B, D) of PRRSV SD16 strain;

[0030] Figure 3 Schematic diagram of the results of hyperoside inhibiting the expression of PRRSV-1 strain (GZ11-G1, P073-3) N protein in MARC-145 and PAM and the virus infection titer;

[0031] Figure 4 Schematic diagram of the results of hyperoside inhibiting the expression of N protein of PRRSV-2 strains (JXA1, CH-1a and NADC30-like) in MARC-145 and PAM and the viral infection titer;

[0032] Figure 5 Schematic diagram of the experimental setup for different treatments of hyperoside on PRRSV infection;

[0033] Figure 6 The results of the inhibitory effects of different treatments of hyperoside on PRRSV replication (A); hyperoside has no inactivating effect on PRRSV (B).

[0034] Figure 7 The protective effect of hyperoside on PRRSV-infected piglets: (A) Detection of virus copy number in pig blood on the 7th day of virus infection; (B) Appearance of lungs on the 28th day of virus infection; (C) HE staining of lungs in each experimental group.

[0035] Beneficial Effects

[0036] Compared with the known public technology, the technical solution provided by the present invention has the following beneficial effects:

[0037] In the present invention, hyperoside is extracted from hibiscus tiliaceus, and then the obtained hyperoside is applied to anti-PRRSV drugs, which can effectively prevent and treat diseases such as abortion in pregnant pigs and respiratory diseases in growing pigs caused by PRRSV infection. The extraction process of hyperoside in the present invention is convenient and fast, and large-scale and rapid extraction can be achieved, thereby avoiding the defect that hyperoside cannot be used on a large scale due to extraction difficulties. Secondly, hyperoside is purified and tested during the preparation process, which further ensures the application of hyperoside. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The present invention will be further described below in conjunction with the embodiments.

[0040] The hyperoside is extracted from hibiscus hibiscus, and includes the following raw materials: hibiscus hibiscus, naringenin, flavonol synthase, metal ions, acetate buffer, methanol solvent, stabilizer and cellulase. The specific preparation method is as follows:

[0041] S1, preparation stage: take the cleaned hibiscus, grind it and put it into a container, and spread naringenin on the bottom of the container in advance;

[0042] S2, enzymatic hydrolysis stage: adding cellulase into the container to carry out enzymatic hydrolysis reaction, and adding metal ions and flavonol synthase in sequence during the enzymatic hydrolysis process;

[0043] S3, collecting the extract: after the enzymatic hydrolysis is completed, the extract is separated from the residue by centrifugation to obtain the extract;

[0044] S4, purification: pour the extract into a filter, remove the residue and large particles of impurities, use a rotary evaporator to remove part of the solvent, reduce the volume of the extract, and then use silica gel to perform column chromatography to further purify the extract, and finally complete the purification of the extract;

[0045] S5, verification stage: using high performance liquid chromatography to verify the purity and content of the extracted hyperoside, thereby obtaining a hyperoside extract.

[0046] Wherein, in step S2, the ratio of liquid to solid in the container during the enzymatic hydrolysis process is 25:1, the dosage of cellulase is 5.7 mg / g, the enzymatic hydrolysis time is 105 min, the temperature in the container is 50°C, and the HP of the liquid during the enzymatic hydrolysis process is 4.6.

[0047] Wherein, in step S2, the solvent used to adjust HP is acetate buffer, and the addition interval of metal ions and flavonol synthase during the enzymatic hydrolysis is 10 min.

[0048] During the extraction process of step S3, a stabilizer is added in a ratio of 20:1 to the liquid.

[0049] In the purification process of step S4, before removing the solvent by a rotary evaporator, methanol solvent is added to the extract in a ratio of 18:3, and then other solvents are removed.

[0050] Example 1

[0051] African green monkey kidney cells (MARC-145) and porcine alveolar macrophages (PAM) were inoculated into 96-well culture plates (1×10 4 / well), when the cell confluence reached 80%, 100 μL of hyperoside solution (DMSO was the solvent) of different concentrations (0, 10 μM, 20 μM, 40 μM, 80 μM, 120 μM and 160 μM) was added to each well of the experimental group. At the same time, a negative control (DMSO treatment group) and a blank control were set up, and 3 replicate wells were set up in each group. After incubation in a 37°C, 5% CO2 incubator for 24 h, 10 μL of CCK-8 solution was added to each well and cultured for another 2 h. The absorbance at 450 nm was read using an ELISA reader, and the following formula was used to calculate the effect of different concentrations of hyperoside on the activity of MARC-145 and PAM cells, and the maximum safe mass concentration of hyperoside was estimated:

[0052] Cell viability (%) = [A1-A2] / [A3-A2] × 100%

[0053] A1: Absorbance value of the test group (wells with cells, culture medium, CCK-8 solution and drug solution of corresponding concentration)

[0054] A2: Absorbance value of blank control (wells with culture medium and CCK-8 solution but no cells, no drug solution added)

[0055] A3: Absorbance value of negative control (wells with cells, culture medium, CCK-8 solution but no drug)

[0056] The results are as follows Figure 2 As shown, different concentrations of hyperoside had no significant effect on the activity of MARC-145 and PAM, and 120 μM was the maximum safe mass concentration of hyperoside.

[0057] Example 2

[0058] 1. qPCR detection of PRRSV progeny in culture supernatant

[0059] MARC-145 and PAM cells were seeded in 6-well culture plates (2×10 5 / well), when the cells grew to 80% confluence, the experimental group was pretreated with different concentrations of hyperoside (0, 15 μM, 30 μM, 60 μM and 120 μM) for 2 h, placed in a 37°C, 5% CO2 incubator for incubation, and then infected with PRRSV SD16 (MOI = 0.1) for 1 h, the drug- and virus-containing supernatant was discarded, and replaced with 3% FBS DMEM (containing the corresponding drug concentration of hyperoside, refer to pretreatment), and continued to be placed in a 37°C, 5% CO2 incubator; at the same time, the DMSO-treated group was set as a negative control and the normal cell untreated group (not pretreated, not infected with PRRSV) was set as a blank control. After 24 h, the cell supernatant was collected, the total cell RNA was extracted and reverse transcribed using a kit, and the supernatant progeny virus copy number was detected by absolute quantitative PCR (qPCR). The results are shown in Figure 2 As shown in A and 2C, hyperoside significantly inhibited the production of PRRSV in progeny in a dose-dependent manner.

[0060] The specific process of the above absolute quantitative PCR determination method is as follows:

[0061] The PRRSV-1 and PRRSV-2 ORF7 genes were cloned into the pMD-18T vector and named pMD-18T-ORF7-1 and pMD-18T-ORF7-2, respectively. The above recombinant plasmids were diluted 10 times. The above diluted plasmids were used as standards, and the samples to be tested in Example 2 (i.e., MARC-145 and PAM) were collected, RNA was extracted, and cDNA was obtained with the help of invertase. The fluorescent quantitative PCR instrument detects the CT values ​​of the standard and the samples to be tested. A standard curve is drawn with the logarithm of the copy number of the standard as the horizontal axis and the measured Ct value as the vertical axis. When the sample to be tested is quantified, the copy number of the sample can be obtained in the standard curve according to the Ct value of the sample.

[0062] 2. Western blot analysis of PRRSV N protein expression

[0063] MARC-145 and PAM cells were seeded into 6-well culture plates (2×10 5 / well), when the cells grew to 80% confluence, different concentrations of hyperoside solution (0, 15 μM, 30 μM, 60 μM, and 120 μM) were added for pretreatment for 2 h, and then PRRSV SD16 0.1 MOI was added. After 1 h of infection, the supernatant containing the drug and virus was discarded and replaced with 3% FBS DMEM (containing the corresponding concentration of hyperoside), and placed in a 37°C, 5% CO2 incubator; at the same time, the DMSO treatment group was set as a negative control and the normal cell untreated group was set as a blank control. After 24 h, the cells were collected, the total cell protein was extracted using a kit, and the changes in the expression level of PRRSV N protein were detected by Western blot. The results are shown in Figure 2 As shown in B and 2D, hyperoside significantly inhibited the expression of PRRSV N protein in a dose-dependent manner.

[0064] 3. Experimental study on the inhibition of hyperoside on infection of multiple PRRSV-1 and PRRSV-2 strains in vitro

[0065] MARC-145 and PAM cells were seeded in 6-well culture plates (2×10 5 / well), when the cells grew to 80% confluence, they were pretreated with hyperoside (120 μM) for 2 h, and infected with 0.1 MOI PRRSV-1 strains GZ11-01 (GenBank: KF001144.1) and P073-3 (GenBank: MK214314.1) or PRRSV-2 strains JXA1 (GenBank: EF112445.1), CH-1a (GenBank: AY032626), NADC30-like (GenBank: KX766379) for 1 h, respectively. After discarding the liquid, they were replaced with 3% FBS DMEM (containing 120 μM hyperoside) and placed in a 37°C, 5% CO2 incubator; a DMSO treatment group was set as a negative control. After 24 h, the cells were harvested and the expression of viral N protein was detected by Western blot. At the same time, the cell supernatant was collected and the expression of viral N protein was detected by TCID 50 The titer of progeny virus was determined by the assay method, and the results showed that hyperoside significantly reduced the infection titer of different strains of PRRSV-1 and PRRSV-2 ( Figure 3 and Figure 4 ).

[0066] The above TCID 50 The specific process of the assay is as follows: New MARC-145 cells or PAM cells were inoculated into 96-well plates (1×10 4 When the cells grew to 80% confluence, the collected PRRSV infected supernatant was diluted 10-fold to 10-8 Gradient (other gradients are 10 -7 , 10 -6 , 10 -5 , 10 -4 , 10 -3 , 10 -2 , 10 -1 ), set up 8 parallel wells for each dilution, add the corresponding supernatant to the cells 1 hour later, discard the supernatant, add DMEM containing 3% FBS, and continue to culture in a 37℃, 5% CO2 incubator for 3-5 days, observe the cell lesions every day, and calculate TCID using the Reed-Muench formula 50 value:

[0067] lgTCID 50 = ratio distance × difference between dilution logarithms + logarithm of dilution with higher than 50% lesion rate

[0068] Wherein, ratio = (percentage of lesion rates above 50% - 50%) / (percentage of lesion rates above 50% - percentage of lesion rates below 50%).

[0069] 4. Determine the effect of hyperoside on the PRRSV replication cycle

[0070] See also Figure 5 To explore the mechanism by which hyperoside affects PRRSV infection, MARC-145 cells were inoculated into 6-well culture plates (2 × 10 5 / well), when the cells grew to 80% confluence, they were infected with PRRSV SD16 0.1 MOI according to the set hyperoside pretreatment group (Pre), hyperoside and PRRSV co-treatment group (Co), PRRSV infection and hyperoside incubation group (Post), and hyperoside all-stage treatment group (All stages-treatment). After 24 hours, the cells were collected and the expression changes of PRRSV N protein were analyzed by Western Blot. The results showed that the expression levels of PRRSV N protein in the hyperoside drug treatment groups Pre, Co, and Post were significantly lower than those in the DMSO treatment group, indicating that hyperoside can play an antiviral role in multiple stages of PRRSV replication ( Figure 6 A).

[0071] To test whether hyperoside has the effect of inactivating PRRSV, MARC-145 cells were inoculated into 6-well culture plates (2×10 5 / well), when the cells grew to 80% confluence, hyperoside (120 μM) was mixed with PRRSV SD16 (0.1 MOI and 1 MOI) and placed at 37℃ for 2 h, and the cells were infected for 1 h. The original solution was discarded and replaced with 3% FBS DMEM for 24 h. The cells were collected and analyzed for the expression changes of PRRSV N protein by Western Blot. The results showed that there was no significant change in the expression of PRRSV N protein compared with the control group, indicating that hyperoside had no direct inactivation effect on PRRSV ( Figure 6 B).

[0072] 4. Hyperoside in vivo anti-PRRSV infection test:

[0073] Twelve blue ear disease double negative weaned piglets were purchased and randomly divided into 3 groups, 4 pigs / group. Hyperoside + PRRSV treatment group, DMSO + PRRSV treatment group and Mock group were set up. 5 TCID 50 / head), oral administration of 150 mg / kg was started 1 day before the virus attack. The drugs were administered on days 1, 3, 5 and 8 after virus infection. Clinical symptoms were observed every day. Blood was collected from different groups of pigs 7 days after virus infection, and the number of virus copies was detected by absolute quantitative PCR. The pigs were euthanized on day 28 after virus infection, and lung tissues were taken for formaldehyde fixation and HE staining to evaluate the protective effect of hyperoside against PRRSV.

[0074] In short, the present invention takes traditional Chinese medicine technology as a starting point to find effective drug ingredients for resisting PRRSV infection. In vitro susceptible cells and pig in vivo experiments have confirmed that hyperoside has a significant inhibitory effect on the replication of PRRSV SD16. The drug has a good inhibitory effect on multiple strains of PRRSV-1 and PRRSV-2. In-depth research has found that hyperoside can play an antiviral role in multiple stages of PRRSV replication. At the same time, pig body test results confirm that hyperoside has a significant inhibitory effect on PRRSV infection. It is proved that hyperoside can be used to prepare drugs for preventing and treating PRRSV infection.

[0075] Through the above-mentioned Example 1 and Example 2, it can be known that:

[0076] The maximum safe mass concentration of hyperoside for PRRSV susceptible cells (MARC-145 and PAM) is 120 µM (µmol / L), that is, hyperoside is non-toxic within this concentration range.

[0077] The hyperoside significantly inhibits the replication of PRRSV.

[0078] The hyperoside has an inhibitory effect on the replication of PRRSV-1 and PRRSV-2 strains GZ11-G1, P073-3, SD16, JXA1, CH-1a and NADC30-like, indicating that hyperoside has the characteristics of broad-spectrum resistance to PRRSV infection.

[0079] The hyperoside exerts an inhibitory effect in multiple stages of PRRSV adsorption, internalization, replication and release.

[0080] The hyperoside is used for preventing and treating piglet viremia caused by PRRSV infection.

[0081] The hyperoside can significantly reduce the lung damage of piglets caused by PRRSV infection.

[0082] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of hyperoside or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of a drug for resisting porcine reproductive and respiratory syndrome virus infection, characterized in that: The structural formula of the hyperoside is shown in Formula 1: (Formula 1) The disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) infection is porcine reproductive and respiratory syndrome (PRRS), and the maximum safe mass concentration of the hyperoside for PRRSV susceptible cells MARC-145 and PAM is 120 µM.

2. Use of hyperoside or a pharmaceutically acceptable salt thereof according to claim 1 as the sole active ingredient in the preparation of a drug for resisting porcine reproductive and respiratory syndrome virus infection, characterized in that: The hyperoside has a significant effect of inhibiting the replication of PRRSV SD16 and exhibits a dose-dependent characteristic.

3. Use of hyperoside or a pharmaceutically acceptable salt thereof according to claim 2 as the sole active ingredient in the preparation of a drug for resisting porcine reproductive and respiratory syndrome virus infection, characterized in that: The hyperoside has an inhibitory effect on the replication of both PRRSV-1 and PRRSV-2 strains.

4. Use of hyperoside or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of a drug for resisting porcine reproductive and respiratory syndrome virus infection according to claim 3, characterized in that: The hyperoside exerts a blocking effect at multiple stages of PRRSV adsorption, internalization, replication and release.

5. Use of hyperoside or a pharmaceutically acceptable salt thereof according to claim 4 as the sole active ingredient in the preparation of a drug for resisting porcine reproductive and respiratory syndrome virus infection, characterized in that: The hyperoside has the effect of reducing the viremia of piglets caused by PRRSV infection, and the hyperoside can significantly reduce the lung damage of piglets caused by PRRSV infection.

6. Use of hyperoside or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 as the sole active ingredient in the preparation of a drug for resisting porcine reproductive and respiratory syndrome virus infection, characterized in that: The hyperoside is extracted from hibiscus tiliaceus, and the specific preparation method is as follows: S1, preparation stage: take the cleaned hibiscus tiliaceus, grind it and put it into a container, and spread naringenin on the bottom of the container in advance; S2, enzymatic hydrolysis stage: adding cellulase into the container to carry out enzymatic hydrolysis reaction, and adding metal ions and flavonol synthase in sequence during the enzymatic hydrolysis process; S3, collecting the extract: after the enzymatic hydrolysis is completed, the extract is separated from the residue by centrifugation to obtain the extract; S4, purification: pour the extract into a filter, remove the residue and large particles of impurities, use a rotary evaporator to remove part of the solvent, reduce the volume of the extract, and then use silica gel to perform column chromatography to further purify the extract, and finally complete the purification of the extract; S5, verification stage: using high performance liquid chromatography to verify the purity and content of the extracted hyperoside, thereby obtaining a hyperoside extract.

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