Application of eucommia male flower flavone in preparation of feed additive and feed additive

Abstract

The invention discloses application of eucommia male flower flavone in preparation of a feed additive and the feed additive. The Eucommia ulmoides male flower flavone (EUMFF) is a flavone extract obtained by ethanol extraction of Eucommia ulmoides male flowers, can resist PRRSV (porcine reproductive and respiratory syndrome virus) through double ways of direct inhibition of PRRSV and immune repair, has remarkable in-vitro and in-vivo application effects, and can be used for preparing a feed additive for inhibiting PRRSV. The addition amount of the additive in a pregnant sow feed in a blue-ear disease positive field is preferably 0.06%.

Description

Technical Field

[0001] This invention relates to feed additives, and more specifically, to the application of Eucommia ulmoides male flower flavonoids in the preparation of feed additives that inhibit porcine reproductive and respiratory syndrome virus, and to feed additives in general. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS, also known as blue ear disease) is a major infectious disease of livestock and poultry caused by PRRSV. This virus is a single-stranded positive-sense RNA virus with a high rate of genetic mutation and complex serotypes. It specifically infects porcine alveolar macrophages (PAMs), causing immunosuppression and easily leading to multiple mixed infections, resulting in huge economic losses to the pig industry. Typical symptoms in infected pregnant sows include abortion, stillbirth, and weak piglets. Infected piglets exhibit severe respiratory symptoms, with a mortality rate as high as 50%–80%, making it a core disease for prevention and control in large-scale pig farms.

[0003] Currently, PRRSV control relies primarily on vaccination. However, vaccines offer limited protection against variant strains, and chemical drugs suffer from issues such as drug resistance and excessive drug residues, failing to meet the demands of green farming and food safety. Therefore, developing natural, efficient, and safe antiviral feed additives has become a hot research topic in the industry. Traditional Chinese medicine extracts offer advantages such as multi-target action, high safety, and no residues. Existing anti-PRRSV traditional Chinese medicine feed additives are mostly based on single active ingredients, such as astragalus polysaccharides, forsythosides, and hypericin. These additives either only focus on viral inhibition with weak immune-enhancing effects, or only regulate immunity without direct antiviral activity. Furthermore, some additives suffer from poor palatability, high dosage requirements, and single target sites. Astragalus polysaccharide, currently the most widely used traditional Chinese medicine feed additive for PRRSV prevention, has an in vitro comprehensive inhibition rate of approximately 55% to 60% against PRRSV. When added to feed at 0.08%, it reduces the abortion rate of PRRSV-positive sows by approximately 40% and the serum viral load by approximately 30% to 35%. Hypericin has an in vitro inhibition rate of approximately 65%, but due to its poor palatability, it can easily lead to a decrease in feed intake in sows and may have a potential slight irritation to the fetus. Forsythia extract, on the other hand, has the problem of weak replication inhibition effect and immunomodulatory effects limited to a single cytokine.

[0004] Eucommia ulmoides is a dioecious plant belonging to the Eucommia genus of the Eucommiaceae family. The male flowers of Eucommia ulmoides are unopened, tightly packed buds of male plants, with a flavonoid content of 3% to 5%, which is much higher than that of Eucommia ulmoides leaves and bark. The core active ingredients are quercetin-3-O-glucoside and kaempferol glycoside. Existing studies have only reported its inhibitory effects on influenza virus and porcine circovirus, and there is no systematic study on its anti-PRRSV and related feed control effects. Summary of the Invention

[0005] The purpose of this invention is to provide the application of Eucommia ulmoides male flower flavonoids in the preparation of feed additives and the feed additives themselves, providing a new and effective solution for the treatment and prevention of porcine reproductive and respiratory syndrome (PRRS).

[0006] To achieve the above objectives, according to one aspect of the present invention, the application of Eucommia ulmoides male flower flavonoids in the preparation of feed additives that inhibit porcine reproductive and respiratory syndrome virus is provided.

[0007] In a preferred embodiment, the flavonoids from Eucommia ulmoides male flowers are flavonoid extracts obtained by ethanol extraction of Eucommia ulmoides male flowers.

[0008] In a preferred embodiment, the effective components in the flavonoid extract, by mass fraction, include 1.25% quercetin-3-O-glucoside, 0.98% kaempferol-3-O-rutin glycoside, 0.82% rutin, and 0.76% hyperoside, with a total flavonoid content of 4.23% based on rutin.

[0009] In a preferred embodiment, the flavonoids from Eucommia ulmoides male flowers exert their effects by directly inhibiting porcine reproductive and respiratory syndrome virus and immune repair.

[0010] As a preferred embodiment, the direct inhibition of porcine reproductive and respiratory syndrome virus includes virus adsorption blocking, direct virus killing, and virus replication inhibition.

[0011] In a preferred embodiment, the immune repair includes increasing the secretion levels of IL-2 and IFN-γ in porcine alveolar macrophages.

[0012] According to another aspect of the present invention, a feed additive is provided, comprising Eucommia ulmoides male flower flavonoids, wherein the Eucommia ulmoides male flower flavonoids are flavonoid extracts obtained by ethanol extraction of Eucommia ulmoides male flowers.

[0013] In a preferred embodiment, the effective components in the flavonoid extract, by mass fraction, include 1.25% quercetin-3-O-glucoside, 0.98% kaempferol-3-O-rutin glycoside, 0.82% rutin, and 0.76% hyperoside, with a total flavonoid content of 4.23% based on rutin.

[0014] As a preferred embodiment, the addition amount in the feed of pregnant sows in porcine reproductive and respiratory syndrome (PRRS) positive farms is 0.04-0.07%.

[0015] As a preferred embodiment, the addition amount in the feed of pregnant sows in porcine reproductive and respiratory syndrome (PRRS) positive farms is 0.06%.

[0016] According to the experimental results, the safe concentration of Eucommia ulmoides male flower flavonoids (EUMFF) for treating porcine alveolar macrophages (PAMs) is ≤200 μg / mL. At a dose of 200 μg / mL, the overall inhibition rate against porcine reproductive and respiratory syndrome virus (PRRSV) in vitro reached 76.32%. The core inhibitory effect is achieved by blocking viral adsorption and inhibiting viral nucleic acid replication. It can also significantly increase the secretion of interleukin-2 (IL-2) and interferon-γ (IFN-γ) in porcine alveolar macrophages (P<0.05). Feed supplementation with 0.06% Eucommia ulmoides male flower flavonoids (EUMFF) showed the best effect, reducing the abortion rate of experimental sows to 3.3%, stillbirth rate to 5.1%, and serum viral load to 68.41%. Both reproductive performance and antiviral capacity were superior to the Astragalus polysaccharide control group.

[0017] Therefore, Eucommia male flower flavonoids (EUMFF) can fight porcine reproductive and respiratory syndrome virus (PRRSV) through a dual pathway of direct toxicity inhibition and immune repair. It has significant effects in both in vitro and in vivo applications and can be used as a green feed additive for the auxiliary prevention and control of porcine reproductive and respiratory syndrome virus (PRRSV) in sows. Detailed Implementation

[0018] The following embodiments of the present invention investigate the in vitro inhibitory activity, mechanism of action, and clinical control effect of Eucommia ulmoides male flower flavonoids (EUMFF) against porcine reproductive and respiratory syndrome virus (PRRSV), while simultaneously conducting in vitro cell experiments and pig farm clinical trials. In vitro, porcine alveolar macrophages (PAMs) were used as the experimental subject. The cytotoxicity of Eucommia ulmoides male flower flavonoids (EUMFF) was detected by the MTT assay. The antiviral target was identified through adsorption blocking, direct killing, and replication inhibition assays combined with indirect immunofluorescence assay (IFA) and real-time quantitative polymerase chain reaction (qRT-PCR). The secretion levels of cytokines interleukin-2 (IL-2) and interferon-γ (IFN-γ) were detected by enzyme-linked immunosorbent assay (ELISA). Clinically, 120 pregnant sows from a PRRSV-positive farm were selected and divided into graded dose experimental groups to measure the incidence of clinical symptoms, reproductive performance, and serum viral load.

[0019] In this embodiment, the male flowers of Eucommia ulmoides were collected from the Eucommia ulmoides planting base in Taiyuan, Shanxi Province. The unopened and firm flower buds were harvested in late March 2025, dried and pulverized at 60 ℃, and flavonoid extract was prepared by reflux extraction with 70% ethanol. The effective components in the flavonoid extract were 1.25% quercetin-3-O-glucoside, 0.98% kaempferol-3-O-rutin, 0.82% rutin, and 0.76% hyperoside. The total flavonoid content, calculated as rutin, was 4.23%. The extract was stored at 4 ℃ in a light-proof and sealed manner.

[0020] 1. Cytotoxicity of Eucommia male flower flavonoids to PAMs The cytotoxicity of flavonoids from Eucommia ulmoides male flowers to PAMs was detected using the MTT assay. PAMs in the logarithmic growth phase were seeded in 96-well cell culture plates at a density of 1 × 10⁶ cells per well. 5 Cells were cultured at 37 ℃ and 5% CO2 for 24 h, then gradient concentrations of EUMFF (final concentrations of 50, 100, 150, 200, 250, and 300 μg / mL) were added. A blank control group (only cell culture medium was added) was included. Each group was repeated in triplicate, and the cells were cultured for another 36 h. 20 μL of MTT solution (5 mg / mL) was added to each well, and after incubation for 4 h, the supernatant was discarded. 150 μL of dimethyl sulfoxide (DMSO) was added, and the mixture was shaken for 10 min to dissolve the formazan crystals. The absorbance (OD value) was measured at 490 nm using a microplate reader, and cell viability was calculated. The concentration corresponding to a cell viability ≥ 80% was considered the safe concentration. Cell viability (%) = (OD value of experimental wells - OD value of blank wells) / (OD value of control group - OD value of blank wells) × 100%.

[0021] The raw data from the MTT assay are shown in Table 1. When the EUMFF concentration was ≤200 μg / mL, the cell viability of PAMs was ≥84.14%, with the 200 μg / mL dose group showing a cell viability of 84.62%. Cells exhibited intact morphology and good growth, with no significant cytotoxicity. However, when the concentration was ≥250 μg / mL, the cell viability plummeted to below 62.71%, with cells shrinking and detaching, indicating extremely significant toxicity (P < 0.01). The safe concentration range for EUMFF to act on PAMs was determined to be 50–200 μg / mL, and this concentration range was used in subsequent in vitro experiments.

[0022] Table 1. Cytotoxicity of Eucommia ulmoides male flower flavonoids to PAMs (MTT method, n=3, OD 490 nm)

[0023] 2. In vitro inhibitory effect of Eucommia ulmoides male flower flavonoids on PRRSV The experimental doses were selected at EUMFF concentrations of 50, 100, and 200 μg / mL within the safe concentration range. A virus control group (inoculated with PRRSV only) and a cell control group (cultured PAMs only) were established, with three replicates per group. Three types of targeted experiments were conducted: ① Adsorption blocking assay: PAMs were incubated with different concentrations of EUMFF for 4 h, the drug solution was discarded, and PRRSV (multiple of infection MOI=0.1) was inoculated. The virus solution was discarded after adsorption at 37 ℃ for 1 h, and cell maintenance medium was added for 36 h of culture. ② Direct killing test: EUMF of different concentrations and PRRSV (MOI=0.1) were co-incubated at room temperature for 2 h, the mixture was inoculated with PAMs, the mixture was discarded after 1 h of adsorption, and maintenance medium was added for 36 h of incubation; ③ Replication inhibition assay: PAMs were first inoculated with PRRSV (MOI=0.1), and after 1 h of adsorption, the virus solution was discarded. Maintenance solution containing different concentrations of EUMFF was added, and the mixture was cultured for 36 h.

[0024] The PRRSV inhibition effect was detected using: ① Indirect immunofluorescence assay (IFA): After culture, cells were fixed with 4% paraformaldehyde for 30 min, incubated with PRRSV N protein-specific antibody at 37 ℃ for 1 h, incubated with fluorescently labeled secondary antibody for 30 min, and fluorescent positive cells were observed under a fluorescence microscope. The positive cell rate was counted. ② Real-time quantitative PCR (qRT-PCR): Total RNA was extracted from cells in each group and reverse transcribed to synthesize cDNA. The PRRSV N gene was used as the detection target and β-actin was used as the internal reference gene. The viral nucleic acid copy number was amplified and detected by real-time quantitative PCR, and the viral inhibition rate was calculated.

[0025] Virus inhibition rate (%) = (Virus copy number in control group - Virus copy number in experimental group) / Virus copy number in control group × 100%.

[0026] 2.1 Results of indirect immunofluorescence (IFA) detection The original data from IFA observation are shown in Table 2. There were no fluorescent positive cells in the cell control group; the fluorescent positive cell rate in the virus control group was as high as 92.4%, with dense and strong cell fluorescence; the fluorescent positive cell rate in each EUMFF dose group decreased significantly with increasing concentration, showing a clear dose-dependent effect. The average positive rate in the 200 μg / mL dose group was only 8.9%, and the antiviral effect was obvious and significant.

[0027] Table 2. Effect of Eucommia ulmoides male flower flavonoids on the IFA-positive cell rate of PRRSV-infected PAMs (n=3, %)

[0028] 2.2 Results of viral copy number and inhibition rate in targeted assays Raw data from qRT-PCR showed that the inhibitory effect of EUMF on PRRSV was concentration-dependent, with significant differences in antiviral efficacy across different targeting assays. In the adsorption blocking assay (Table 3), the average viral copy number in the 200 μg / mL dose group was 2.30 × 10⁻⁶. 6 The copy number / μL showed an inhibition rate of 73.32%; in the replication inhibition assay (Table 4), the average viral copy number in the same dose group was 2.73 × 10⁻⁶. 6 The inhibition rate was 68.44% at a copy number / μL; the direct killing test showed relatively weak antiviral effect (Table 5), with an inhibition rate of 35.27% at a dose of 200 μg / mL.

[0029] Table 3 Results of the anti-PRRSV adsorption blocking assay of flavonoids from Eucommia ulmoides male flowers (qRT-PCR, n=3, copy number / μL)

[0030] Table 4 Results of direct killing assay of PRRSV by flavonoids from Eucommia ulmoides male flowers (qRT-PCR, n=3, copy number / μL)

[0031] Table 5 Results of the assay for PRRSV replication inhibition by flavonoids from Eucommia ulmoides male flowers (qRT-PCR, n=3, copy number / μL)

[0032] 2.3 Comprehensive inhibitory effect against PRRSV in vitro Based on the results of the three types of targeted experiments (Table 6), the overall antiviral effect of EUMFF against PRRSV increased significantly with increasing concentration, with the 200 μg / mL dose group showing the highest overall inhibition rate of 76.32%. Combined with the targeted experiment data, it can be seen that the core targets of EUMFF against PRRSV are the viral adsorption and replication stages, and its direct inactivation effect on free viruses is relatively weak.

[0033] Table 6 Summary of the overall inhibitory rate of Eucommia ulmoides male flower flavonoids against PRRSV in vitro (n=3, %)

[0034] 3. Effects of Eucommia ulmoides male flower flavonoids on PAMs secreted cytokines Collect cell culture supernatant from each group and strictly follow the instructions for the IL-2 and IFN-γ ELISA assay kits. Detect OD values ​​using a microplate reader at a wavelength of 450 nm and calculate cytokine levels with reference to a standard curve. The assay method has been validated and the results are accurate and reliable.

[0035] Raw data from ELISA (Table 7) showed that, compared with the cell control group, the levels of IL-2 and IFN-γ secreted by PAMs in the virus control group were significantly decreased (P < 0.05), indicating that PRRSV infection can significantly inhibit the secretion of immune cytokines. Compared with the virus control group, all EUMFF dose groups significantly increased the secretion levels of IL-2 and IFN-γ (P < 0.05), and the concentration was positively correlated with the amount of cytokine secreted. The average IL-2 content in the 200 μg / mL dose group reached 126.35 pg / mL, which was 63.21% higher than that in the virus control group, and the average IFN-γ content reached 98.72 pg / mL, which was 58.34% higher than that in the virus control group. This indicates that EUMFF can effectively repair the damage to immune cells caused by PRRSV and enhance the body's antiviral immune response.

[0036] Table 7 Effects of Eucommia ulmoides male flower flavonoids on the secretion of IL-2 and IFN-γ by PAMs (ELISA, n=3, pg / mL)

[0037] 4. Clinical effect of Eucommia ulmoides male flower flavonoids on the prevention and control of porcine reproductive and respiratory syndrome (PRRS) in sows in farms. Clinical efficacy trial of feed for disease prevention and control: ① Experimental grouping: 120 pregnant sows (60-70 days gestation age) with positive porcine reproductive and respiratory syndrome (PRRS) were randomly divided into 4 groups of 30 sows each. The control group (Group I) was fed a basal diet; the low-dose group (Group II) was fed a basal diet plus 0.04% EUMFF; the medium-dose group (Group III) was fed a basal diet plus 0.06% EUMFF; and the positive control group (Group IV) was fed a basal diet plus 0.08% Astragalus polysaccharide. The experimental period was 30 days.

[0038] ② Indicator detection: Daily records of clinical symptoms such as fever (body temperature ≥39.5 ℃) and cough in sows; postpartum statistics of abortion rate, stillbirth rate, and healthy piglet rate; serum samples were collected on days 1, 15, and 30 of the experiment, and PRRSV load was detected by qRT-PCR and PRRSV-specific antibody titer was detected by ELISA; survival rate of piglets was recorded at 21 days of age.

[0039] 4.1 Clinical symptom improvement effect

[0040] The original data of clinical symptoms (Table 8) showed that the incidence of typical symptoms of porcine reproductive and respiratory syndrome (PRRS) in sows in the control group was 33.3%, including a fever incidence of 20.0% and a cough and depression incidence of 13.3%. The incidence of symptoms was significantly reduced in the low- and medium-dose EUMFF groups. The incidence of symptoms in the medium-dose group (0.06% addition) was only 6.7%, which was significantly lower than that in the control group (P<0.05) and slightly better than that in the Astragalus polysaccharide positive control group (symptom incidence of 10.0%). It can effectively alleviate the discomfort symptoms of sows after infection.

[0041] Table 8 Effects of Eucommia ulmoides male flower flavonoids on clinical symptoms of porcine reproductive and respiratory syndrome (PRRS) positive sows (experimental period 30 days, n=30)

[0042] 4.2 Effect on improving reproductive performance EUMFF significantly improved the reproductive performance of sows in porcine reproductive and respiratory syndrome (PRRS)-positive farms, with the medium-dose group showing the best effect. Abortion statistics (Table 9) showed that the abortion rate in the medium-dose group was only 3.3%, a decrease of 80.2% compared to the control group (16.7%). Piglet performance data (Table 10) showed that the stillbirth rate in the medium-dose group was 5.1%, the healthy piglet rate was 92.3%, and the 21-day survival rate of piglets reached 93.5% (Table 11), all significantly better than the control group and the Astragalus polysaccharide-positive control group (P < 0.05), indicating the best core reproductive performance indicators.

[0043] Table 9. Effect of Eucommia male flower flavonoids on abortion rate in porcine reproductive and respiratory syndrome (PRRS) positive sows (n=30)

[0044] Table 10. Effects of Eucommia ulmoides male flower flavonoids on farrowing performance of piglets from sows positive for porcine reproductive and respiratory syndrome (PRRS) (normal farrowing sows)

[0045] Table 11 Effects of Eucommia ulmoides male flower flavonoids on the survival rate of piglets at 21 days of age

[0046] 4.3 Changes in serum viral load and antibody levels Raw data from serum viral load testing (Table 12) showed no significant difference in PRRSV nucleic acid copy numbers among the sows in the initial stage of the experiment; on day 15, the viral load in the medium-dose EUMFF group decreased significantly first; on day 30, the viral load in the medium-dose group decreased to 2.47 × 10⁻⁶. 5 The copy number / μL decreased by 68.41% compared to the initial level, a significantly greater decrease than in other groups. Serum-specific antibody detection showed (Table 13) that on day 30 of the experiment, the average PRRSV antibody titer in the medium-dose group reached 1:1280, significantly higher than that in the control group (1:640) and the positive control group (1:960), indicating that EUMFF can effectively reduce viral load and increase the level of specific antiviral antibodies in vivo.

[0047] Table 12 Effect of Eucommia male flower flavonoids on serum PRRSV load in porcine reproductive and respiratory syndrome (PRRS) positive sows (qRT-PCR, 10 sows randomly assigned to each group, copy number / μL)

[0048] Table 13 Effect of Eucommia ulmoides male flower flavonoids on serum PRRSV-specific antibody titers in porcine reproductive and respiratory syndrome (PRRS) positive sows (day 30 of the experiment, ELISA, 10 sows randomly assigned to each group).

[0049] Furthermore, a parallel comparative experiment was conducted between Eucommia ulmoides male flower flavonoids and existing anti-PRRSV traditional Chinese medicine feed additives (Astragalus polysaccharide, hypericin, and Forsythia suspensa extract). The results showed that under the same in vitro concentration (200 μg / mL), Eucommia ulmoides male flower flavonoids achieved a comprehensive inhibition rate of 76.32% against PRRSV, which was 17.82% higher than Astragalus polysaccharide (58.25%), 11.45% higher than hypericin (64.87%), and 24.19% higher than Forsythia suspensa extract (52.13%). Compared with existing anti-PRRSV traditional Chinese medicine feed additives, Eucommia ulmoides male flower flavonoids achieved a dual synergistic effect of direct toxicity inhibition and immune repair, with substantial improvements in toxicity inhibition efficiency, prevention and control effect, dosage, palatability, and safety. The core advantage lies in the fact that its multiple active ingredients can act on multiple key links of PRRSV infection simultaneously and can comprehensively regulate the body's antiviral immune response, solving the shortcomings of existing additives with single function and limited effect.

[0050] Based on the above experimental results, in in vitro experiments, the safe concentration of EUMFF is ≤200 μg / mL, with no cytotoxicity, and it can safely act on PAMs. The overall inhibition rate at a dose of 200 μg / mL is 76.32%. It works by blocking viral adsorption and inhibiting nucleic acid replication. It is speculated that its active ingredients, quercetin-3-O-glucoside and kaempferol, can competitively bind to the CD163 receptor on the surface of PAMs (a key receptor for PRRSV infection), occupying the binding site to block invasion. At the same time, it inhibits viral RNA polymerase activity, interfering with nucleic acid replication and protein expression. However, its ability to directly inactivate free viruses is limited, suggesting that it should be added in advance in actual feeding to preferentially block the viral infection process.

[0051] Immune repair is another core advantage of EUMFF's anti-PRRSV effect in this invention. PRRSV infection significantly inhibits the secretion of IL-2 and IFN-γ by PAMs. IL-2 can promote lymphocyte proliferation and differentiation, while IFN-γ can activate macrophage phagocytosis and induce antiviral immunity. Insufficient secretion of both exacerbates immunosuppression. In this invention, EUMFF can significantly increase the levels of both cytokines, repair immune damage, break immunosuppression, and reduce the risk of secondary infections by regulating immune cell signaling pathways.

[0052] In clinical trials for feed use, 0.06% was the preferred dosage of EUMFF, which reduced sow abortion rate to 3.3% and stillbirth rate to 5.1%, and increased piglet survival rate to 93.5% at 21 days of age. This effect was superior to conventional astragalus polysaccharide additives, without any risk of anorexia or additional abortion, making it suitable for use in the entire physiological stage of pregnant sows. Its mechanism of improving reproductive performance may be as follows: flavonoid components regulate sow reproductive endocrine function, increase progesterone levels, protect the placental barrier, and reduce viral invasion of the fetus; simultaneously, it improves mammary gland microcirculation, ensures milk production, provides sufficient maternal antibodies for piglets, and improves piglet survival rate.

Claims

1. Application of Eucommia ulmoides male flower flavonoids in the preparation of feed additives that inhibit porcine reproductive and respiratory syndrome virus.

2. The application according to claim 1, characterized in that: The flavonoids from Eucommia ulmoides male flowers are flavonoid extracts obtained by ethanol extraction of Eucommia ulmoides male flowers.

3. The application according to claim 2, characterized in that: The active ingredients in the flavonoid extract, by mass fraction, include 1.25% quercetin-3-O-glucoside, 0.98% kaempferol-3-O-rutin glycoside, 0.82% rutin, and 0.76% hyperoside, with a total flavonoid content of 4.23% based on rutin.

4. The application according to claim 1, 2 or 3, characterized in that: The flavonoids from Eucommia ulmoides male flowers exert their effects by directly inhibiting porcine reproductive and respiratory syndrome virus and promoting immune repair.

5. The application according to claim 4, characterized in that: The direct inhibition of porcine reproductive and respiratory syndrome virus includes blocking virus adsorption, direct killing of the virus, and inhibition of virus replication.

6. The application according to claim 4, characterized in that: The immune repair includes increasing the secretion levels of IL-2 and IFN-γ in porcine alveolar macrophages.

7. A feed additive, characterized in that: It includes Eucommia ulmoides male flower flavonoids, which are flavonoid extracts obtained by ethanol extraction of Eucommia ulmoides male flowers.

8. The feed additive according to claim 7, characterized in that: The active ingredients in the flavonoid extract, by mass fraction, include 1.25% quercetin-3-O-glucoside, 0.98% kaempferol-3-O-rutin glycoside, 0.82% rutin, and 0.76% hyperoside, with a total flavonoid content of 4.23% based on rutin.

9. The feed additive according to claims 7 and 8, characterized in that: The addition amount of this substance to the feed of pregnant sows in porcine reproductive and respiratory syndrome (PRRS) positive farms is 0.04-0.07%.

10. The feed additive according to claim 9, characterized in that: The addition amount of this substance to the feed of pregnant sows in porcine reproductive and respiratory syndrome (PRRS) positive farms was 0.06%.

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