A composition for resisting PRRSv and improving sow reproductive performance and its application

Abstract

The present application relates to the field of traditional Chinese medicinal materials, in particular to a composition for resisting PRRSv and improving the reproductive performance of sows and application thereof. The present application formulates a composition composed of 10 plant extracts (Angelica sinensis extract, Atractylodes macrocephala Koidz extract, Glycyrrhiza uralensis Fisch extract, Eucommia ulmoides Oliver extract, Eucommia ulmoides leaf extract, Populus davidiana Dode extract, Perilla frutescens leaf extract, Gynostemma pentaphyllum Makino extract, Leonurus japonicus extract and Astragalus membranaceus Bge extract) for sows in late pregnancy, and collects sow blood and placenta samples at the time of delivery, studies the influence of the composition on sow reproduction, and explores the mechanism of action. The experimental results show that the composition can improve the reproductive performance of sows by increasing the total number of piglets and other indicators; meanwhile, the composition can effectively inhibit the replication of PRRSv.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, and in particular to a composition for resisting PRRSv and improving the reproductive performance of sows and its application. Background Technology

[0002] Sow breed, feeding method, growth environment, reproductive technology, dietary metabolism level, and nutrient supply all affect sow reproduction. During late gestation (70-114 days), sows require more nutrition to support fetal bone and muscle development. Poor pigsty conditions, improper feeding, rough handling during early mating, and mechanical damage during regrouping can all lead to bacterial infections in the uterus of sows in late gestation. These infections can produce endotoxins or lipopolysaccharides, inducing a pro-inflammatory environment that causes endometritis. The presence of bacterial endotoxins or lipopolysaccharides induces a pro-inflammatory environment, which then induces the production of secondary inflammatory mediators through Toll-like receptors in the peritoneal cavity. These uterine lesions alter the sow's uterine environment, leading to endometrial and vaginal microbiota imbalance, oxidative stress, increased cell apoptosis, and adverse effects on placental function.

[0003] The porcine placenta, composed of the trophoblast (connective tissue and blood vessels), amnion (ectoderm and endoderm), urothelial membrane, and amniotic fluid, is a diffuse placental structure. Serving as the link between the mother and fetus in mammals, it maintains maternal pregnancy, regulates fetal growth and development, facilitates the exchange of substances and gases, and possesses secretory and defensive functions. As an immune barrier, it protects the health of the fetus. Therefore, placental nutritional regulation is crucial for maintaining pregnancy and healthy fetal development. Studies have shown that changes in the uterine environment can disrupt placental development and function, affecting angiogenesis, structural integrity, and the exchange of substances between the mother and fetus. Impaired placental angiogenesis leads to intrauterine growth retardation (IUGR) and severe pregnancy complications in pigs, even resulting in fetal loss and pregnancy arrest, ultimately causing a decline in maternal reproductive performance.

[0004] Traditional Chinese medicine theory analyzes this as a disorder of the sow's Qi (vital energy) and an imbalance of Yin, Yang, and Qi and blood in the internal organs, resulting in deficiency of both Qi and blood. This manifests as abortion, stillbirth, weak piglets, and mummified fetuses. Given the reproductive problems faced by sows in late pregnancy, clinical treatment primarily focuses on nutritional regulation. Plant extracts, used as feed additives, possess medicinal and edible properties, antibacterial and antioxidant effects, enhance immunity, regulate reproductive hormone levels, and improve sow reproductive performance. Therefore, this invention aims to find a composition that possesses antiviral activity against porcine reproductive and respiratory syndrome virus (PRRSv) and improves sow reproductive performance. Summary of the Invention

[0005] The purpose of this invention is to provide a composition for resisting PRRSv and improving sow reproductive performance, and its application, to solve the problems existing in the prior art. The composition of this invention can significantly improve the reproductive performance of sows and effectively resist PRRSv.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a composition for resisting PRRSv and improving the reproductive performance of sows, the composition comprising the following components: Angelica sinensis extract, Atractylodes macrocephala extract, Glycyrrhiza uralensis extract, Eucommia ulmoides extract, Eucommia ulmoides leaf extract, Populus tomentosa flower extract, Perilla frutescens leaf extract, Gynostemma pentaphyllum extract, Leonurus japonicus extract and Astragalus membranaceus extract;

[0008] The mass ratio of the Angelica sinensis extract, the Atractylodes macrocephala extract, the Glycyrrhiza uralensis extract, the Eucommia ulmoides extract, the Eucommia ulmoides leaf extract, the Populus tomentosa flower extract, the Perilla frutescens leaf extract, the Gynostemma pentaphyllum extract, the Leonurus japonicus extract, and the Astragalus membranaceus extract is 4:2:2:1:1:1:1:1:1:1.

[0009] Preferably, the method for preparing the Angelica extract includes the steps of mixing Angelica with water, and then sequentially performing hot water extraction, impurity removal, concentration, and drying to obtain the Angelica extract;

[0010] The preparation method of the Atractylodes macrocephala extract includes the steps of mixing Atractylodes macrocephala with water, and then sequentially performing reflux extraction, alcohol extraction, impurity removal and purification to obtain the Atractylodes macrocephala extract;

[0011] The method for preparing the licorice extract includes the steps of mixing licorice with ethanol, followed by impregnation, vacuum concentration and drying to obtain the licorice extract.

[0012] The preparation method of the Eucommia ulmoides extract includes the following steps: mixing Eucommia ulmoides with ethanol, and then sequentially performing reflux extraction, vacuum concentration, centrifugation, n-butanol extraction, methanol extraction and drying to obtain the Eucommia ulmoides extract;

[0013] The preparation method of the Eucommia ulmoides leaf extract includes the steps of mixing Eucommia ulmoides leaves with ethanol, and then performing reflux extraction and filtration sequentially to obtain the Eucommia ulmoides leaf extract.

[0014] The preparation method of the poplar flower extract includes the steps of mixing poplar flowers with ethanol, then sequentially performing alcohol extraction, filtration, mixing and concentration to obtain the poplar flower extract.

[0015] The preparation method of the perilla leaf extract includes the steps of mixing perilla leaves with ethanol, and then sequentially performing reflux extraction, filtration and vacuum concentration to obtain the perilla leaf extract.

[0016] The preparation method of the Gynostemma pentaphyllum extract includes the steps of mixing Gynostemma pentaphyllum with ethanol, and then sequentially performing alcohol extraction, filtration and drying to obtain the Gynostemma pentaphyllum extract.

[0017] The preparation method of the motherwort extract includes the steps of mixing motherwort with ethanol, and then performing reflux extraction and filtration sequentially to obtain the motherwort extract.

[0018] The preparation method of the Astragalus extract includes the steps of mixing Astragalus with ethanol, followed by ethanol extraction, filtration and drying to obtain the Astragalus extract.

[0019] Preferably, when preparing the Angelica sinensis extract, the material-to-liquid ratio is 1g:12mL; the hot water extraction is performed twice at a temperature of 95℃, and each hot water extraction lasts for 2 hours.

[0020] When preparing the Atractylodes macrocephala extract, the mass-to-volume ratio of Atractylodes macrocephala to water is 2g:60mL; the reflux extraction temperature is 75℃ and the time is 2h.

[0021] In preparing the licorice extract, the impregnation includes a first impregnation and a second impregnation; during the first impregnation, the material-to-liquid ratio is 1g:10mL, and during the second impregnation, the material-to-liquid ratio is 1g:5mL.

[0022] When preparing the Eucommia ulmoides leaf extract, the material-to-liquid ratio was 1g:8mL; the reflux extraction was performed 3 times at a temperature of 65℃ for 2.5h.

[0023] When preparing the Eucommia ulmoides leaf extract, the material-to-liquid ratio was 1g:11mL; the reflux extraction temperature was 75℃ and the time was 1.5h.

[0024] When preparing the poplar flower extract, the material-to-liquid ratio is 50g:1mL; the alcohol extraction temperature is 85℃ and the time is 3h.

[0025] When preparing the perilla leaf extract, the material-to-liquid ratio was 1g:11mL; the reflux extraction was performed 3 times at a temperature of 65℃, and the reflux extraction time was 3 days each time.

[0026] When preparing the Gynostemma pentaphyllum extract, the material-to-liquid ratio is 1g:20mL; the alcohol extraction temperature is 90℃ and the time is 2h.

[0027] When preparing the motherwort extract, the material-to-liquid ratio is 1g:25mL; the reflux extraction temperature is 65℃ and the time is 2h.

[0028] When preparing the Scutellaria baicalensis extract, the alcohol extraction includes a first alcohol extraction and a second alcohol extraction; when performing the first alcohol extraction, the material-to-liquid ratio is 1g:13mL; when performing the second alcohol extraction, the material-to-liquid ratio is 1g:10mL.

[0029] Preferably, the improvement of sow performance includes improving the physiological and biochemical indicators of sow blood, reducing the progesterone content in sow serum, promoting estrus in sows after weaning, increasing the level of immunoglobulin IgA in sow serum, increasing the levels of reactive oxygen species and superoxide dismutase in sows, reducing the levels of LA and TBA in sow umbilical cord blood, increasing the level of IL-10 (interleukin-10) in sow placenta, increasing the expression of tight junction factors in sow placental tissue, inhibiting sow placental cell apoptosis, promoting the expression of angiogenesis factors and growth factors in sow placental tissue, increasing the expression of PLET1 in sow placenta, and improving the immunity of pregnant sows.

[0030] More preferably, in preparing the licorice extract, the first impregnation uses ethanol with a volume percentage of 95%; the second impregnation uses ethanol with a volume percentage of 10%.

[0031] When preparing the Eucommia ulmoides extract, the volume percentage of ethanol is 60%;

[0032] When preparing the Eucommia ulmoides leaf extract, the volume percentage of ethanol is 20%;

[0033] When preparing the poplar flower extract, the volume percentage of ethanol is 40%;

[0034] When preparing the perilla leaf extract, the volume percentage of ethanol is 50%;

[0035] When preparing the Gynostemma pentaphyllum extract, the volume percentage of ethanol is 80%;

[0036] When preparing the motherwort extract, the volume percentage of ethanol is 70%;

[0037] In preparing the Scutellaria baicalensis extract, the volume percentage of ethanol used in both the first and second alcohol extractions is 80%.

[0038] This invention provides the use of the above-described composition in the preparation of formulations for anti-PRRSv and for improving sow reproductive performance.

[0039] Preferably, the improvement of sow performance includes improving the physiological and biochemical indicators of sow blood, reducing the progesterone content in sow serum, promoting estrus in sows after weaning, increasing the level of immunoglobulin IgA in sow serum, increasing the levels of reactive oxygen species and superoxide dismutase in sows, reducing the levels of LA and TBA in sow umbilical cord blood, increasing the IL-10 level in sow placenta, increasing the expression of tight junction factors in sow placental tissue, inhibiting sow placental cell apoptosis, promoting the expression of angiogenesis factors and growth factors in sow placental tissue, increasing the expression of PLET1 in sow placenta, and improving the immunity of pregnant sows.

[0040] This invention provides a formulation for resisting PRRSv and improving the reproductive performance of sows, wherein the active ingredient of the formulation comprises the above-described composition.

[0041] Further preferably, the improvement of sow performance includes improving the physiological and biochemical indicators of sow blood, reducing the progesterone content in sow serum, promoting estrus in sows after weaning, increasing the level of immunoglobulin IgA in sow serum, increasing the levels of reactive oxygen species and superoxide dismutase in sows, reducing the levels of LA and TBA in sow umbilical cord blood, increasing the IL-10 level in sow placenta, increasing the expression of tight junction factors in sow placental tissue, inhibiting sow placental cell apoptosis, promoting the expression of angiogenesis factors and growth factors in sow placental tissue, increasing the expression of PLET1 in sow placenta, and improving the immunity of pregnant sows.

[0042] This invention provides a method for preparing the above-mentioned formulation, comprising the following steps:

[0043] The preparation is obtained by mixing the Angelica sinensis extract, Atractylodes macrocephala extract, Glycyrrhiza uralensis extract, Eucommia ulmoides extract, Eucommia ulmoides leaf extract, Populus tomentosa flower extract, Perilla frutescens leaf extract, Gynostemma pentaphyllum extract, Leonurus japonicus extract and Astragalus membranaceus extract in a mass ratio of 4:2:2:1:1:1:1:1:1:1 to the above ratio.

[0044] Preferably, the method for preparing the Angelica extract includes the steps of mixing Angelica with water, and then sequentially performing hot water extraction, impurity removal, concentration, and drying to obtain the Angelica extract;

[0045] The preparation method of the Atractylodes macrocephala extract includes the steps of mixing Atractylodes macrocephala with water, and then sequentially performing reflux extraction, alcohol extraction, impurity removal and purification to obtain the Atractylodes macrocephala extract;

[0046] The method for preparing the licorice extract includes the steps of mixing licorice with ethanol, followed by impregnation, vacuum concentration and drying to obtain the licorice extract.

[0047] The preparation method of the Eucommia ulmoides extract includes the following steps: mixing Eucommia ulmoides with ethanol, and then sequentially performing reflux extraction, vacuum concentration, centrifugation, n-butanol extraction, methanol extraction and drying to obtain the Eucommia ulmoides extract;

[0048] The preparation method of the Eucommia ulmoides leaf extract includes the steps of mixing Eucommia ulmoides leaves with ethanol, and then performing reflux extraction and filtration sequentially to obtain the Eucommia ulmoides leaf extract.

[0049] The preparation method of the poplar flower extract includes the steps of mixing poplar flowers with ethanol, then sequentially performing alcohol extraction, filtration, mixing and concentration to obtain the poplar flower extract.

[0050] The preparation method of the perilla leaf extract includes the steps of mixing perilla leaves with ethanol, and then sequentially performing reflux extraction, filtration and vacuum concentration to obtain the perilla leaf extract.

[0051] The preparation method of the Gynostemma pentaphyllum extract includes the steps of mixing Gynostemma pentaphyllum with ethanol, and then sequentially performing alcohol extraction, filtration and drying to obtain the Gynostemma pentaphyllum extract.

[0052] The preparation method of the motherwort extract includes the steps of mixing motherwort with ethanol, and then performing reflux extraction and filtration sequentially to obtain the motherwort extract.

[0053] The preparation method of the Astragalus extract includes the steps of mixing Astragalus with ethanol, followed by ethanol extraction, filtration and drying to obtain the Astragalus extract.

[0054] More preferably, in preparing the licorice extract, the first impregnation uses ethanol with a volume percentage of 95%; the second impregnation uses ethanol with a volume percentage of 10%.

[0055] When preparing the Eucommia ulmoides extract, the volume percentage of ethanol is 60%;

[0056] When preparing the Eucommia ulmoides leaf extract, the volume percentage of ethanol is 20%;

[0057] When preparing the poplar flower extract, the volume percentage of ethanol is 40%;

[0058] When preparing the perilla leaves, the volume percentage of ethanol is 50%;

[0059] When preparing the Gynostemma pentaphyllum extract, the volume percentage of ethanol is 80%;

[0060] When preparing the motherwort extract, the volume percentage of ethanol is 70%;

[0061] In preparing the Scutellaria baicalensis extract, the volume percentage of ethanol used in both the first and second alcohol extractions is 80%.

[0062] This invention provides the use of the above-described composition or formulation in the preparation of products having anti-PRRSv and sow reproductive performance-enhancing effects.

[0063] More preferably, the product includes feed, feed additives, and drugs.

[0064] Further preferably, the improvement of sow performance includes improving the physiological and biochemical indicators of sow blood, reducing the progesterone content in sow serum, promoting estrus in sows after weaning, increasing the level of immunoglobulin IgA in sow serum, increasing the levels of reactive oxygen species and superoxide dismutase in sows, reducing the levels of LA and TBA in sow umbilical cord blood, increasing the IL-10 level in sow placenta, increasing the expression of tight junction factors in sow placental tissue, inhibiting sow placental cell apoptosis, promoting the expression of angiogenesis factors and growth factors in sow placental tissue, increasing the expression of PLET1 in sow placenta, and improving the immunity of pregnant sows.

[0065] The present invention discloses the following technical effects:

[0066] In the composition provided by the present invention, Angelica sinensis extract is the principal drug, Atractylodes macrocephala extract, Eucommia ulmoides extract, Eucommia ulmoides leaf extract, and Perilla frutescens leaf extract are the assistant drugs, Populus tomentosa flower extract, Gynostemma pentaphyllum extract, and Leonurus japonicus extract are the adjuvant drugs, and Glycyrrhiza uralensis extract is the guiding drug.

[0067] Based on the theory of traditional Chinese medicine compatibility and combined with the TCM syndrome differentiation theory of qi and blood deficiency in clinical sows, this invention formulates a composition consisting of 10 plant extracts (Angelica sinensis extract, Atractylodes macrocephala extract, Glycyrrhiza uralensis extract, Eucommia ulmoides extract, Eucommia ulmoides leaf extract, Populus tomentosa flower extract, Perilla frutescens leaf extract, Gynostemma pentaphyllum extract, Leonurus japonicus extract, and Astragalus membranaceus extract). This composition is used on sows in late pregnancy, and blood and placental samples are collected from the sows at farrowing to study the effect of this composition on sow reproduction and explore its mechanism of action. Experimental results show that this composition can improve the reproductive performance of sows by increasing the total number of piglets born, specifically by: improving the physiological and biochemical indicators of sow blood, reducing the progesterone content in sow serum, promoting estrus after weaning, increasing the level of immunoglobulin IgA in serum, increasing the levels of reactive oxygen species and superoxide dismutase in sows, reducing the levels of LA and TBA in umbilical cord blood, increasing the level of IL-10 in the placenta, increasing the expression of tight junction factors in placental tissue, inhibiting placental cell apoptosis, promoting the expression of angiogenesis factors and growth factors in sow placental tissue, increasing the expression of PLET1 in sow placenta, and improving the immunity of pregnant sows; at the same time, this composition can also effectively inhibit the replication of PRRSV. Attached Figure Description

[0068] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0069] Figure 1 The graph shows the percentage of litters born; where A represents the percentage of litters born in the control group and B represents the percentage of litters born in the experimental group.

[0070] Figure 2 The graph shows the results of detecting the inhibitory effect of the formulation prepared in Example 1 on the expression of N protein in Marc145 cells; where A is the result of real-time immunofluorescence quantitative PCR experiment; PRRSV is the control group treated only with the virus; PRRSV + formulation 200 μg / mL means that Marc145 cells were first challenged with PRRSV, and then treated with the formulation prepared in Example 1, with a dosage of 200 μg / mL; PRRSV + formulation 400 μg / mL means that Marc145 cells were first challenged with PRRSV, and then treated with the formulation prepared in Example 1, with a dosage of 400 μg / mL; formulation + PRRSV 200 μg / mL was used when Marc145 cells were first treated with the formulation prepared in Example 1, and then challenged with PRRSV. The dosage of the formulation prepared in Example 1 was 200 μg / mL. 400 μg / mL of formulation + PRRSV was used when Marc145 cells were first treated with the formulation prepared in Example 1, and then challenged with PRRSV. The dosage of the formulation prepared in Example 1 was 400 μg / mL. B shows the results of the indirect immunofluorescence experiment. PRRSV + 200 μg / mL formulation was used when treating with the 200 μg / mL formulation prepared in Example 1. PRRSV + 400 μg / mL formulation was used when treating with the 400 μg / mL formulation prepared in Example 1. Mock was the blank group. PRRSV was the control group that only received the virus.

[0071] Figure 3 The graph shows the results of PRRSV virus infection detection in sows at a pig farm; line 1 is the automatic baseline, line 2 represents the amplification curve of positive samples, and line 3 represents the amplification curve of the sample.

[0072] Figure 4This is a graph showing the results of physiological and biochemical analysis of sow blood; where A and C represent the detection results of red blood cells, white blood cells, and platelets, respectively; the control group represents normal sows, and the experimental group represents sows that received the treatment; data are expressed as mean ± standard deviation. "*" indicates a significant difference (P < 0.05); "**" indicates a highly significant difference (P < 0.01); "***" indicates an extremely significant difference (P < 0.001).

[0073] Figure 5 The graph shows the results of blood biochemical analysis of sows; where AF represents the results of total protein, albumin, globulin, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase, respectively; the control group represents normal sows, and the experimental group represents sows that received the treatment; data are expressed as mean ± standard deviation. "*" indicates a significant difference (P < 0.05); "**" indicates a highly significant difference (P < 0.01); "***" indicates an extremely significant difference (P < 0.001).

[0074] Figure 6 The following graphs show the effects of the formulation prepared in Example 1 on progesterone and immune levels in sows; where A represents the progesterone levels in sows; B represents the serum levels of immunoglobulins IgG, IgA, and IgM; and C represents the levels of immunoglobulins IgG, IgA, and IgM in sow colostrum. Data are expressed as mean ± standard deviation. "; The control group represents normal sows, and the experimental group represents sows that received the treatment; "*" indicates a significant difference, P<0.05; "**" indicates a highly significant difference, P<0.01; "***" indicates an extremely significant difference, P<0.001;

[0075] Figure 7 Figure A shows the results of detecting the effect of the formulation prepared in Example 1 on the antioxidant function of sows; Figure B shows the results of the investigation of reactive oxygen species in sow serum and placenta; Figure C shows the results of the detection of superoxide dismutase levels in sow serum and placenta; Figure D shows the results of the detection of placental antioxidant factors in umbilical cord blood; Figure D shows the results of the detection of TBA in placental tissue; Figure E shows the results of the detection of LA in umbilical cord blood; The control group represents normal sows, and the experimental group represents sows that received the formulation; Data are expressed as mean ± standard deviation. "*" indicates a significant difference (P < 0.05); "**" indicates a highly significant difference (P < 0.01); "***" indicates an extremely significant difference (P < 0.001).

[0076] Figure 8 The image shows the results of interleukin-6 levels in sow serum; the control group represents normal sows, and the experimental group represents sows that received the preparation.

[0077] Figure 9 This image shows the expression levels of the inflammatory factors interleukin-6, interleukin-10, tumor necrosis factor-α, and interleukin-1β in sow placental tissue. The control group represents normal sows, and the experimental group represents sows treated with the prescribed formulation. Data are expressed as mean ± standard deviation. "*" indicates a significant difference (P < 0.05); "**" indicates a highly significant difference (P < 0.01); "***" indicates an extremely significant difference (P < 0.001).

[0078] Figure 10 This image shows the expression of tight junction factors CDH1, TJP1, ZO-1, claudin1, claudin3, and occludin in sow placental tissue. The control group represents normal sows, and the experimental group represents sows treated with the formulation. Data are expressed as mean ± standard deviation. "*" indicates a significant difference (P < 0.05); "**" indicates a highly significant difference (P < 0.01); "***" indicates an extremely significant difference (P < 0.001).

[0079] Figure 11 The figure shows the effect of the formulation prepared in Example 1 on apoptosis in sow placenta; the control group represents normal sows, and the experimental group represents sows that received the formulation; the data are expressed as mean ± standard deviation. "*" indicates a significant difference (P < 0.05); "**" indicates a highly significant difference (P < 0.01); "***" indicates an extremely significant difference (P < 0.001).

[0080] Figure 12 The figure shows the results of the investigation on the effect of the formulation prepared in Example 1 on the expression level of placental growth factor in sows; where the control group represents normal sows and the experimental group represents sows that took the formulation.

[0081] Figure 13 The effect of the preparation obtained in Example 1 on the expression level of placental growth factor in sows is shown in the figure; where the control group represents normal sows and the experimental group represents sows that received the preparation; the data are expressed as mean ± standard deviation. "*" indicates a significant difference (P < 0.05); "**" indicates a highly significant difference (P < 0.01); "***" indicates an extremely significant difference (P < 0.001).

[0082] Figure 14The following graph shows the effects of the formulation prepared in Example 1 on the levels of progesterone, interleukin-6, and immunoglobulins in the serum of high-producing sows. In this graph, A represents the progesterone level in the serum of high-producing sows; B represents the interleukin-6 level; and C represents the immunoglobulin level. The control group represents normal sows, and the experimental group represents sows treated with the formulation. Data are expressed as mean ± standard deviation. "*" indicates a significant difference (P < 0.05); "**" indicates a highly significant difference (P < 0.01); "***" indicates an extremely significant difference (P < 0.001).

[0083] Figure 15 The following graph shows the effects of the formulation prepared in Example 1 on the levels of progesterone, interleukin-6, and immunoglobulins in the serum of low-producing sows. In this graph, A represents the progesterone level in the serum of low-producing sows; B represents the interleukin-6 level; and C represents the immunoglobulin level. The control group represents normal sows, and the experimental group represents sows treated with the formulation. Data are expressed as mean ± standard deviation. "*" indicates a significant difference (P<0.05); "**" indicates a highly significant difference (P<0.01); "***" indicates an extremely significant difference (P<0.001). Detailed Implementation

[0084] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0085] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0086] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0087] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0088] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0089] Unless otherwise specified, the components used in the examples are all readily available to those skilled in the art.

[0090] Unless otherwise specified, "%" in the examples represents the meaning of volume percentage.

[0091] Example 1

[0092] The formulation of an anti-PRRSv and sow reproductive performance enhancement agent is as follows: Angelica sinensis extract, Atractylodes macrocephala extract, Glycyrrhiza uralensis extract, Eucommia ulmoides extract, Eucommia ulmoides leaf extract, Populus tomentosa flower extract, Perilla frutescens leaf extract, Gynostemma pentaphyllum extract, Leonurus japonicus extract, and Astragalus membranaceus extract; the mass ratio of Angelica sinensis extract, Atractylodes macrocephala extract, Glycyrrhiza uralensis extract, Eucommia ulmoides extract, Eucommia ulmoides leaf extract, Populus tomentosa flower extract, Perilla frutescens leaf extract, Gynostemma pentaphyllum extract, Leonurus japonicus extract, and Astragalus membranaceus extract is 4:2:2:1:1:1:1:1:1:1.

[0093] The preparation method is as follows:

[0094] (1) After crushing the Angelica sinensis slices, sieve them to obtain Angelica sinensis powder; mix the Angelica sinensis powder with water and extract twice with hot water at 95℃, each extraction time is 2h. Combine the two water extracts to obtain a water extract mixture. Each time, the Angelica sinensis powder and water are mixed at a mass-volume ratio of 1g:12mL. Then, use a D4006 macroporous adsorption resin chromatographic column to remove impurities from the water extract mixture to obtain the effluent. Concentrate the effluent to a relative density of 1.2 to obtain a concentrate. The concentration time is 30min and the temperature is 65℃. Mix the concentrate with β-cyclodextrin at a volume-mass ratio of 1mL:1g and dry at 75℃ for 3h to obtain Angelica sinensis extract (polysaccharide).

[0095] (2) Atractylodes macrocephala extract was prepared by hot water extraction. Water and Atractylodes macrocephala were mixed at a material-to-liquid ratio of 60 mL: 2 g, heated to reflux at 75 °C for 2 h, and filtered while hot (using a 0.45 μm filter membrane) to obtain the first filtrate and the first filter residue. 40 mL of water was added to the first filter residue, and the mixture was heated to reflux at 75 °C for 30 min and filtered (using a 0.45 μm filter membrane) to obtain the second filtrate and the second filter residue. The two filtrates were combined and diluted to 100 mL to obtain the Atractylodes macrocephala aqueous extract. The Atractylodes macrocephala aqueous extract was then mixed with 95% ethanol to make the alcohol concentration in the mixture 75%. The mixture was precipitated twice with alcohol, the solution was discarded, and the precipitate was collected. Water was added to the precipitate to a final volume of 25 mL. The solution was dissolved to obtain a crude Atractylodes macrocephala solution. A combination of Sevag method and enzymatic method was used to remove proteins and purify the solution by dialysis to prepare the Atractylodes macrocephala extract. Specifically, the crude Atractylodes macrocephala solution was mixed with papain (0.1% of the volume of the crude Atractylodes macrocephala solution), hydrolyzed at 60℃ and pH 5 for 1 hour, centrifuged at 5000 rpm for 3 minutes, and the supernatant was collected. The solution was then deproteinized once with Sevag reagent for 5 minutes with shaking. Afterward, it was dialyzed with 0.5% acetic acid at 4℃ (dialysis bag molecular weight cutoff 12000) for 5 hours and stored at 4℃ to obtain the Atractylodes macrocephala extract (polysaccharide).

[0096] (3) Grind licorice into coarse powder; mix the coarse licorice powder with 95% ethanol at a mass-volume ratio of 1g:10mL, soak at room temperature for 2h, then add 10% ethanol and soak at room temperature for 2h. The mass-volume ratio of 10% ethanol to coarse licorice powder is 5mL:1g. Then concentrate under reduced pressure (vacuum degree is 0.085MPa, temperature is 70℃) to obtain a concentrated solution; mix the concentrated solution with β-cyclodextrin at a mass-volume ratio of 1mL:1g, and dry at 75℃ for 3h to obtain licorice extract (total flavonoids);

[0097] (4) The dried bark of Eucommia ulmoides was cut into shreds to obtain Eucommia ulmoides bark shreds. The shreds were extracted three times by refluxing with 60% ethanol to obtain combined extracts. The ratio of 60% ethanol to Eucommia ulmoides bark shreds was 8 mL: 1 g for each refluxing extraction. The refluxing temperature was 65℃ and the extraction time was 2.5 h. After that, the extract was concentrated under reduced pressure (vacuum degree 0.085 MPa, temperature 75℃) to recover the solvent and obtain an extract. The extract was mixed with water at a mass ratio of 1:5 and centrifuged at 2000 rpm for 3 min to obtain the supernatant. The supernatant was mixed with n-butanol at a ratio of 1:3 and extracted at room temperature for 1 hour. The upper layer was collected and concentrated (vacuum degree 0.085 MPa, temperature 70℃) to obtain an extract. The extract was mixed with 50% methanol at a mass-to-volume ratio of 1 g:1 mL and filtered (using a 0.45 μm filter membrane) to obtain a filtrate. The filtrate was mixed with β-cyclodextrin at a volume-to-mass ratio of 1 mL:1 g and dried at 75℃ for 3 hours to obtain Eucommia ulmoides extract (lignin).

[0098] (5) Accurately weigh 5.0g of dried Eucommia ulmoides leaf powder sample and prepare Eucommia ulmoides leaf extract using ethanol-hydrothermal reflux extraction process. The specific steps are as follows: according to the material-liquid ratio of 1g:11mL, mix Eucommia ulmoides leaf powder sample with 20% ethanol (pH controlled at 3.5), heat and reflux at 75℃ for 1.5h, take the supernatant, filter (0.45μm filter membrane) to obtain Eucommia ulmoides leaf extract (chlorogenic acid);

[0099] (6) After crushing poplar flowers through a 60-mesh sieve, poplar flower powder is obtained. 18g of poplar flower powder is accurately weighed and 40% ethanol is added at a material-to-liquid ratio of 1mL:50g. The mixture is extracted at 85℃ for 3h to obtain crude poplar flower extract. After filtering the crude poplar flower extract (using a 0.45μm filter membrane), it is concentrated by rotary evaporation until the relative density is 1.2 to obtain a concentrated solution. The concentrated solution is then transferred to a volumetric flask and diluted to 500mL for later use. This is recorded as poplar flower extract (total flavonoids).

[0100] (7) Accurately weigh 200g of perilla leaf slices, add 50% ethanol and reflux at 65℃ for 3 times, each time for 3 days, filter (0.45μm filter membrane) the residue, and mix according to the ratio of 1g:11mL for each reflux extraction; then, combine the extracts and concentrate under reduced pressure (vacuum degree of 0.085MPa, temperature of 75℃) to obtain perilla leaf extract;

[0101] (8) After crushing a certain amount of Gynostemma pentaphyllum herb and passing it through a 100-mesh sieve, add 80% ethanol solution at a material-to-liquid ratio of 1g:20mL, extract at 90℃ for 2h, filter (0.45μm filter membrane) to obtain crude extract of Gynostemma pentaphyllum; then dry at 85℃ for 3h to obtain extract of Gynostemma pentaphyllum (flavonoids).

[0102] (9) Weigh a certain amount of motherwort, crush it and pass it through a 50-mesh sieve. Accurately weigh 1g of motherwort powder, add 25mL of 70% ethanol, weigh it, heat it at 65℃ under reflux for 2h, cool it at room temperature, weigh it again and use 70% ethanol to make up the lost weight, shake it well, filter it (0.45μm filter membrane), and obtain the filtrate, which is the motherwort extract.

[0103] (10) After pulverizing the Astragalus membranaceus slices, sieve them to obtain Astragalus membranaceus powder; mix the Astragalus membranaceus powder with 85% ethanol at a mass-volume ratio of 1g:13mL, extract with ethanol at room temperature for 1.5h, filter (0.45μm filter membrane) to obtain the first filtrate and the first filter residue; mix the first filter residue with 85% ethanol at a mass-volume ratio of 1g:10mL, extract with ethanol at room temperature for 1h to obtain the second filtrate and the second filter residue; combine the first filtrate and the second filter residue to obtain the mixed filtrate; mix the mixed filtrate with β-cyclodextrin at a volume-volume ratio of 1mL:1g, dry at 75℃ for 3h to obtain Astragalus membranaceus extract (polysaccharide);

[0104] (11) The extracts of Angelica sinensis, Atractylodes macrocephala, Glycyrrhiza uralensis, Eucommia ulmoides, Eucommia ulmoides leaf, Populus tomentosa flower, Perilla frutescens leaf, Gynostemma pentaphyllum, Leonurus japonicus and Astragalus membranaceus were mixed in a mass ratio of 4:2:2:1:1:1:1:1:1:1 to obtain the preparation.

[0105] Experiment 1 aimed to improve the reproductive performance of sows by increasing indicators such as the total number of piglets born.

[0106] Two hundred sows were randomly selected and randomly divided into an experimental group and a control group, with 100 sows in each group and one replicate. The experimental design started on day 74 of gestation. The experimental group was fed the basal diet supplemented with 0.2% of the formulation prepared in Example 1 by weight of the basal diet, and continued until day 114 of gestation. The control group was fed the basal diet normally. The experimental diet of the sows met the nutritional requirements of sows in late gestation recommended by the National Research Council (2012). The gestation diet formula and nutritional standards were provided by Chia Tai Kangdi Group and manufactured by Guangxi Liyuan Jinling Feed Co., Ltd. Within 24 hours of farrowing, the litter size, number of live piglets, number of healthy piglets, number of weak piglets, number of stillborn piglets, number of mummified piglets, birth weight of piglets, and number of piglets weaned at 21 days and weaning weight were recorded. The results are shown in Table 1. 。

[0107] Table 1. Effects of the formulation prepared in Example 1 on the reproductive performance of sows.

[0108] index Control group (n=100 animals) Experimental group (n=100 heads) p-value Total number of piglets born (heads) 11.32±0.31 13.06±0.31 <0.001*** Number of live offspring (heads) 10.42±0.32 11.96±0.28 0.001*** Number of Kenzai (heads) 10.13±0.33 11.74±0.28 <0.001*** Number of weak offspring (heads) 0.29±0.07 0.21±0.06 0.517 Number of birth defects (heads) 0.26±0.06 0.2±0.05 0.555 Number of stillbirths (heads) 0.46±0.1 0.44±0.09 0.595 Number of mummies (heads) 0.18±0.06 0.46±0.09 0.005 Newborn litter weight (kg) 15.86±0.50 18.13±0.40 0.001** Number of breasts weaned (next breast) 10.43±0.19 11.59±0.15 <0.001*** Total weight of the litter after weaning (kg) 76.72±1.42 83.77±0.93 <0.001*** Weaned piglets weight (kg) 7.36±0.01 7.25±0.04 0.637

[0109] Note: Criteria for determining the number of healthy piglets: newborn piglets weighing ≥ 0.75 kg, "***" indicates a p-value ≤ 0.001, and "**" indicates a p-value ≤ 0.01.

[0110] As shown in Table 1, feeding the sows with the formulation prepared in Example 1 significantly increased the total number of piglets born, the number of live piglets, the number of healthy piglets, the initial litter weight, and the number of weaned piglets and the weaning litter weight after 21 days (P≤0.001).

[0111] The percentages of sows with a total litter size of ≥14, ≤11, and 13 piglets were statistically analyzed, and the results are as follows: Figure 1 As shown, in the control group, 31% of sows had a total litter size of ≥14 piglets, and 43% had a total litter size of ≤11 piglets. In the experimental group, the proportions of sows with a total litter size of ≥14 piglets and ≤11 piglets were 46% and 25%, respectively. This indicates that the formulation prepared in Example 1 improved the total litter size of low-producing sows.

[0112] The reproductive performance of sows with different total litters was then analyzed, and the results are shown in Tables 2 and 3.

[0113] Table 2. Effects of the formulation prepared in Example 1 on the reproductive performance of sows (sows with a total litter size ≤ 11 piglets).

[0114]

[0115]

[0116] Note: Criteria for determining the number of healthy piglets: newborn piglets weighing ≥ 0.75 kg.

[0117] Table 3. Effects of the formulation prepared in Example 1 on the reproductive performance of sows (sows with a total litter size > 11 piglets).

[0118] index Control group (n=53 animals) Experimental group (n=53 heads) p-value Total number of piglets born (heads) 13.53±0.17 14.62±0.29 0.006 Number of live offspring (heads) 12.49±0.23 13.49±0.26 0.007 Number of Kenzai (heads) 12.15±0.26 13.17±0.26 0.005 Number of weak offspring (heads) 0.34±0.10 0.32±0.09 0.772 Number of birth defects (heads) 0.3±0.09 0.25±0.08 0.75 Number of stillbirths (heads) 0.51±0.14 0.4±0.10 0.9 Number of mummies (heads) 0.23±0.09 0.49±0.12 0.017 Newborn litter weight (kg) 18.72±0.40 19.98±0.35 0.024 Number of breasts weaned (next breast) 10.73±0.66 11.87±0.15 <0.001*** Total weight of the litter after weaning (kg) 78.98±0.42 85.02±0.54 <0.001***

[0119] Note: Criteria for determining the number of healthy piglets: newborn piglets weighing ≥ 0.75 kg, "***" indicates a p-value ≤ 0.001.

[0120] The data recorded in Tables 2 and 3 show that, compared with the control group, the experimental group had a highly significant increase in the total number of piglets born, the number of live piglets, the number of healthy piglets, the birth litter weight, the number of piglets weaned, and the total weaning litter weight for low-producing (total number of piglets ≤ 11) sows and high-producing (total number of piglets > 11) sows.

[0121] Experiment Example 2: Suppressing PRRSv Replication

[0122] Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious disease caused by PRRSV. Its main characteristics include fever and anorexia in infected pigs, late-term abortion, premature birth, stillbirth, weak piglets, and mummified fetuses in pregnant sows, and respiratory disorders in pigs of all ages (especially piglets). The nucleocapsid protein (N protein) is one of the most antigenic structural proteins of PRRSV and plays a crucial role in viral packaging. Early immune responses after infection primarily target the N protein, and it can be detected as early as one week after infection; therefore, this protein has been used as a diagnostic antigen for detecting serum antibodies. MARC145 (African green monkey embryonic kidney cells) are mainly used for virus culture, and PRRSV is particularly sensitive to these cells. The formulation prepared in Example 1 was used to treat PRRSV-infected Marc145 cells. The expression level of the N protein in PRRSV-infected Marc145 cells after drug treatment was detected using real-time quantitative PCR and indirect immunofluorescence assay (IFA).

[0123] 1. Real-time immunofluorescence quantitative PCR experiment

[0124] (1) Dilution of drug: The preparations prepared in Example 1 were diluted with PBS to 200 and 400 μg / mL, respectively.

[0125] (2) Seed Marc145 cells in 24-well plates and wait for the cells to grow into a monolayer.

[0126] (3) Drug-to-poison combination: 500 μL of the formulations prepared in Example 1 at concentrations of 200 μg / mL and 400 μg / mL were added to each well of a 24-well plate. A blank control group and a poison-only control group were set up. The plates were incubated at 37°C for 1 h. The liquid was then discarded, washed twice with PBS, and then 100 TCID60 was added. 50 PRRSV virus virulence, 200 μL per well, incubated at 37°C for 2 h, with each incubator performing a 15 min incubation cycle. The incubator was then discarded, washed twice with PBS, and 500 μL of 2% FBS maintenance medium was added. The mixture was then incubated at 37°C for 48 h.

[0127] (4) Poison first, then medicine: Add 100 TCID 50 PRRSV virus virulence, 200 μL per well, incubated at 37°C for 2 h, with incubation every 15 min, discarding the solution, washing twice with PBS, adding 200 and 400 μg / mL of the formulations prepared in Example 1 to 24-well plates, 500 μL per well, respectively, setting up blank and virus-only control groups, and incubating at 37°C for 48 h.

[0128] (5) After culturing for 48 hours, the 24-well plate was placed in a freezer at -80℃ and repeatedly frozen and thawed between room temperature 3 times, and RNA was extracted using the Trizol method.

[0129] (6) RNA concentration was measured and volume was adjusted.

[0130] (7) The CT value was obtained by performing a real-time quantitative PCR experiment using the Aibotec one-step fluorescence quantitative PCR reagent (RK20404) and a real-time fluorescence quantitative PCR instrument.

[0131] 2. Indirect immunofluorescence assay

[0132] (1) Dilution of drug: The preparations prepared in Example 1 were diluted to 200 and 400 μg / mL using PBS, respectively.

[0133] (2) Seed Marc145 cells in 24-well plates and wait for the cells to grow into a monolayer.

[0134] (3) Drug-before-poisoning: 200 and 400 μg / mL of the formulations prepared in Example 1 were added to 24-well plates, 500 μL per well, and incubated at 37°C for 1 h. The solution was then discarded, washed twice with PBS, and then 100 TCID10 was added. 50PRRSV virus virulence, 200 μL per well, incubated at 37°C for 2 h, with each incubator performing a 15 min incubation cycle. The incubator was then discarded, washed twice with PBS, and 500 μL of 2% FBS maintenance medium was added. The mixture was then incubated at 37°C for 48 h.

[0135] (4) Poison first, then medicine: Add 100 TCID 50 PRRSV virus solution, 200 μL per well, was incubated at 37°C for 2 h, with incubation every 15 min. The solution was discarded, and the plates were washed twice with PBS. 200 μg / mL of the formulations prepared in Example 1 were added to 24-well plates, 500 μL per well, and incubated at 37°C for 48 h. A negative control group and a virus-only control group were also included.

[0136] (5) Indirect immunofluorescence: Add 150 μL of tissue cell fixative to each well and fix for 8-10 min; wash 3 times with PBS, shaking for 5 min each time; add Triton (PBS, 1:1000 dilution, 400 μL per well) and shake for 10 min; add 2% BSA to block for 30 min, 400 μL per well; add primary antibody and shake overnight at 4℃, 400 μL per well; wash 3 times with PBS, shaking for 5 min each time; add secondary antibody (PBS, 1:200 dilution, 200 μL per well) and shake for 1 h; wash 3 times with PBS, shaking for 5 min each time.

[0137] (6) Observe fluorescence under a fluorescence microscope.

[0138] 3. Test Results

[0139] The results are as follows Figure 2 As shown in the figure. The results of real-time immunofluorescence quantitative PCR and indirect immunofluorescence experiments both showed that, compared with the control group that was only inoculated with virus, both the treatment of inoculating with virus first and then drug and the treatment of drug first and then virus could reduce the expression level of N protein in Marc145 cells. Among them, the preparation prepared in Example 1, which was first inoculated with PRRSV virus venom and then added with 200 μg / mL, had the strongest inhibitory effect on the expression of N protein in Marc145 cells.

[0140] Experimental Example 3

[0141] 1. Investigation of PRRSV antigen infection status in the experimental pig farm

[0142] In Experiment 1, 15 mL of blood was collected from the anterior vena cava of sows on the day of farrowing under fasting conditions. The blood was then divided into 10 mL of a vacuum blood collection tube containing EDTA and 5 mL of a vacuum blood collection tube. After the vacuum coagulation tube was left to stand at room temperature for 30 min, the precipitated serum was aspirated into a centrifuge tube, centrifuged at 3000 r / min for 15 min, and the serum samples were extracted, placed in sterile EP tubes, numbered, and stored in a -20℃ refrigerator.

[0143] After parturition, 10 sows with successful parturition were selected from each group, and their placental tissues were collected, packed in sampling bags, quickly frozen in liquid nitrogen, and transferred to -80 °C for storage pending testing. The viral RNA of serum and placental samples was extracted using an RNA extraction kit, and after reverse transcription, the PRRSV antigen was detected using a real-time fluorescence RT-PCR detection kit. The judgment criteria were as follows: positive: CT ≤ 38 and showing an S-shaped amplification curve; negative: no CT and no S-shaped amplification curve; recheck: 38 < CT < 40; after recheck, if CT < 40 and showing an S-shaped amplification curve, it was positive, otherwise it was negative. The results were as Figure 3 shown. The results showed that there were no CT values and no S-shaped amplification curves in the detected serum and placental samples, indicating that there was no PRRSV infection in the samples selected from this pig farm, and the impact of PRRS virus on the reproductive performance of sows had been excluded.

[0144] 2. Improve the physiological and biochemical indexes of sows' blood

[0145] Analysis of blood physiological indexes: The blood of sows in a vacuum centrifuge tube containing EDTA was counted hematologically using a three-part automatic hematology analyzer (Guilin Uritt Medical Electronics Co., Ltd., URIT-5180). After standing for 30 min, the numbers of white blood cells, red blood cells, and platelets were counted.

[0146] Analysis of blood biochemical indexes: It was measured using an automatic biochemical analyzer (Shenzhen Rayto Life Science, Chemray 240, 420, 800), including total protein, albumin, globulin, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase. The above detection methods were strictly measured according to the methods provided in the analyzer instruction manual, and the reagent kits were purchased from Rayto / Changchun Huili.

[0147] The survey results were as Figure 4 and Figure 5 shown. The results showed that the red blood cells in the experimental group increased; the low white blood cells in the control group indicated a decline in the immunity of sows, and there was a tendency for the white blood cells in the experimental group to be higher than those in the control group, indicating enhanced immunity. The low platelets in the experimental group indicated that the accumulation of platelets could be reduced and the probability of thrombosis formation could be decreased.

[0148] Analysis of the biochemical results showed that the indexes of three enzymes reflecting liver function had an upward trend, but there was no significance, indicating that the liver function of the experimental group had been improved to a certain extent.

[0149] 4. Reduce the content of progesterone in sows' serum, promote estrus after weaning, and increase the level of immunoglobulin IgA in serum

[0150] Serum samples were analyzed using enzyme-linked immunosorbent assay (ELISA) to determine the level of hormone P in serum and the contents of immunoglobulins IgA, IgM, and IgG in serum and breast milk. All procedures were performed according to the ELISA kit instructions. All kits were purchased from Jiangsu Enzyme Immunoassay Co., Ltd. Detailed operating procedures can be found in the kit instructions. Results are as follows: Figure 6 As shown in the figure. The results indicated that the P level decreased after farrowing in the experimental group sows, which could promote estrus after weaning. The levels of immunoglobulins IgG, IgA, and IgM in serum were measured, and the results showed that the IgA level was significantly increased (P<0.05), and the IgM level showed an increasing trend, but the effect was not significant. There was no effect on the immunoglobulin level in sow milk.

[0151] 5. Design of relevant primers

[0152] Primer synthesis was based on sequences of porcine placental functional genes (GLUT1 (glucose transporter 1), PLET1 (placental transcription factor 1), SNATI (sodium-coupled neutral amino acid transporter 1), SNAT2 (sodium-coupled neutral amino acid transporter 2), Flt1 (vascular endothelial growth factor receptor 1)), placental growth factors (IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor), VEGFA (vascular endothelial growth factor A), TGFBR1 (transforming growth factor β receptor 1), PGF (placental growth factor), FGFβ1 (transforming growth factor β receptor 1), FGFβ2 (transforming growth factor β receptor 2) genes), antioxidant enzymes (HO-1 (heme oxygenase 1), CAT (catalase)), and apoptosis-related factors (BAX, caspase-3, Fasl genes) and the reference gene β-actin, all registered in GenBank. Premier was used to synthesize these primers. 5.0 software was used to design amplification primers, which were then synthesized by Sangon Biotech (Shanghai) Co., Ltd. Detailed primer information is shown in Table 4.

[0153] Table 4 Primer Information

[0154]

[0155] 6. Increases the levels of reactive oxygen species and superoxide dismutase in sows, and reduces the levels of LA and TBA in umbilical cord blood.

[0156] The levels of reactive oxygen species (ROS) in sow serum and placenta were detected using an ELISA kit, and the results showed a significant increase (P<0.01), along with an increased level of superoxide dismutase (SOD) (P<0.05). Figure 7 (A and B in the text).

[0157] After farrowing, 10 sows from each group that successfully farrowed were selected, and placental tissue was collected, aliquoted into sampling bags, and rapidly frozen in liquid nitrogen. The tissue was then stored at -80℃ for later analysis. 0.10g of placental tissue was placed in a 1.5mL enzyme-free centrifuge tube and ground. 0.2mL of chloroform solution was added to each tube, mixed well, and allowed to stand at room temperature for 15 minutes. After standing, the tube was centrifuged at 12000 rpm for 15 minutes at 4℃. The clear supernatant was transferred to another enzyme-free centrifuge tube, discarding the middle and lower layers. Care was taken to avoid contact with the middle layer when collecting the supernatant to prevent contamination. 500mL of isopropanol was added to each centrifuge tube, and the mixture was thoroughly shaken and allowed to stand at room temperature. After standing for 10 minutes, the tube was centrifuged at 12000 rpm for 10 minutes at 4℃, and the supernatant was discarded, retaining the precipitate. The precipitate was gently rinsed 3-4 times with 75% ethanol and centrifuged at 3500 rpm for 5 minutes at room temperature. Remove the ethanol solution, let the centrifuge tube stand until the precipitate becomes slightly transparent, then add a certain amount of enzyme-free water according to the amount of precipitate, mix thoroughly to dissolve the precipitate, and the RNA solution is obtained. Use an OD-1000 to determine the extracted RNA concentration, 260 / 280, 260 / 230, etc. Samples with 260 / 280 between 1.8 and 2.0 can still be used.

[0158] Total RNA was reverse-transcribed into cDNA: The RNA obtained in the previous step was processed according to the instructions of the Lamborghini Reverse Transcription Kit (All in One First-Strand Synthesis MasterMix (with ds DNase)) to obtain cDNA. Then, qPCR was performed using Lamborghini reagent (Taq SYBR Green qPCR Premix) to detect the expression levels of antioxidant enzymes superoxide dismutase, catalase, and heme oxygenase 1 in placental tissue. The results showed that the levels of catalase and heme oxygenase 1 were significantly increased (P<0.01), indicating that the preparation obtained in Example 1 increased the production of reactive oxygen species in sows by increasing the number of piglets born, but also significantly improved the antioxidant levels of sows after administration. Figure 7 (C in the middle).

[0159] During farrowing, 10 sows from each group were selected, and umbilical cord tissue was collected from their fetuses. The tissue was aliquoted into sampling bags, rapidly frozen in liquid nitrogen, and then stored at -80°C for later analysis. Umbilical cord blood was collected from the venous vein according to the method described by Dennis (2014). Umbilical cord blood samples were collected from piglets immediately after detachment from the placenta to avoid interfering with blood flow. The umbilical cord was then clamped at the placental end at the rupture point, and again at the piglet end approximately 7.5 cm from the piglet. Blood samples were collected between the two clamps. Whole blood samples were immediately transferred to 10 mL vacuum blood collection tubes containing EDTA and placed in a -20°C freezer for 2-4 hours, until transferred to a -80°C freezer. Umbilical cord lactate concentration was determined using an ELISA kit (purchased from Jiangsu Enzyme Immunoassay Co., Ltd.). Detailed operating instructions can be found in the kit's manual. Serum total bile acid levels were detected using an automated biochemical analyzer (Shenzhen Leidu Life Science & Technology Co., Ltd., Chemray 240, 420, 800). Results are as follows: Figure 7 As shown in D and E, the formulation prepared in Example 1 reduced the levels of lactate (LA) and bile acid (TBA) in umbilical cord blood, indicating that it can alleviate oxidative stress and promote fetal growth.

[0160] 7. It exhibits anti-inflammatory effects by increasing interleukin-10 levels in the placenta.

[0161] The levels of IL-6 (interleukin-6) in sow serum were detected using an ELISA kit, and the results showed no significant effect. Figure 8 ) qPCR was used to detect the expression levels of inflammatory factors interleukin-6, interleukin-10, tumor necrosis factor-α, and interleukin-1β in placental tissue, such as Figure 9 As shown in the figure. The results showed that the levels of pro-inflammatory factors IL-6, TNFα, and IL-1β did not change significantly, but the level of anti-inflammatory factor interleukin-10 increased significantly (P<0.05), indicating that the medication did not have a significant effect on the inflammation level of sows, but it could improve the anti-inflammatory level of sows to some extent.

[0162] 8. Increase the expression of tight junction factors in placental tissue

[0163] The expression of tight junction factors CDH1, TJP1, ZO-1, claudin1, claudin3, and occludin in placental tissue was detected using qPCR. Figure 10 As shown. The results indicate that the formulation prepared in Example 1 can increase the expression levels of tight junction factors occludin (P<0.05), claudin1 (P<0.05), claudin3 (P<0.01) and CDH1 (P<0.01), while having no significant effect on the expression levels of ZO-1 and TJP1, but showing a tendency to promote their expression.

[0164] 9. Inhibits placental cell apoptosis

[0165] The expression of apoptosis genes Caspase-3, BAX, and Fasl was detected using qPCR, such as... Figure 11 As shown in the figure. The results indicate that the formulation prepared in Example 1 has the function of regulating cell apoptosis.

[0166] 10. Effects on the expression of angiogenesis factors and growth factors in sow placental tissue

[0167] The expression levels of placental growth factor, insulin-like growth factor 1, epidermal growth factor, vascular endothelial growth factor A, transforming growth factor β receptor 1, placental growth factor, transforming growth factor β receptor 1, and transforming growth factor β receptor 2 genes were detected using qPCR. Figure 12 As shown in the figure. The results indicated that all growth factors showed an increasing trend. Among them, the expression of vascular endothelial growth factor A (VEGF A) mRNA was significantly increased after feeding the formulation prepared in Example 1. It can stimulate placental angiogenesis and growth, better meeting the growth needs of the fetus. FGFβR1 belongs to the FGF family, whose members have extensive mitotic and cell survival activities and can effectively participate in various biological processes such as embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion. After feeding the formulation prepared in Example 1, its receptor FGFβR1 was significantly increased.

[0168] 12. Effects on the expression of functional genes in the sow placenta

[0169] The expression of placental functional genes GLUT1, SNATI, SNAT2, PLET1, and Flt1 was detected using qPCR. The results are as follows: Figure 13As shown in the figure. The results indicate that GLUT1 is responsible for transporting glucose from the mother to the fetus, which is particularly important for fetal growth and development. After using the formulation prepared in Example 1, the GLUT1 level in the sow placenta showed a decreasing trend, which is consistent with the trend of glucose content in the serum. This indicates that due to the increased number of piglets born and healthy piglets, more glucose in the sow's body is transferred to the piglets through the placenta, thus increasing the glucose content in the piglets. SNAT1 and SNAT2 are two important neutral amino acid transporters in mammals, mainly mediating the transport of neutral amino acids (such as glutamine, alanine, and proline). Placental amino acid transport is crucial for fetal growth and development. Amino acid transport between the mother and fetus mainly depends on amino acid transport carriers present in the placenta. Abnormal expression of amino acid transport carriers will directly affect the intrauterine fetal absorption of amino acids, thereby affecting fetal growth and development. After using the formulation prepared in Example 1, the SNAT1 level in the sow placenta did not show an increasing trend, indicating that the formulation has a small effect on amino acid transport. PLET1 affects the interaction between trophoblast cells and maternal endometrial cells. After using the formulation prepared in Example 1, PLET1 levels in the sow placenta increased significantly (P<0.01), which is beneficial for fetal development. Flt1 levels increased in tumor cells, but the Flt1 content in the sow placenta decreased after using the formulation prepared in Example 1, indicating that the formulation prepared in Example 1 can improve the immunity of sows and piglets.

[0170] 13. Effects on serum progesterone, IL-6 and immunoglobulin levels in high-producing sows

[0171] The levels of progesterone P, interleukin-6, and immunoglobulins in the serum of high-producing sows (total litter size > 11 piglets) were detected using an ELISA kit. The results are as follows: Figure 14 As shown, the progesterone content in high-producing sows decreased, which was beneficial for post-weaning sows. The level of interleukin-6 pro-inflammatory factor showed a downward trend, indicating that the preparation obtained in Example 1 alleviated the inflammatory response in sows. At the same time, the immunoglobulin IgA content increased significantly (P<0.05), which improved the immunity of pregnant sows.

[0172] 14. Effects on serum progesterone, interleukin-6, and immunoglobulin levels in low-producing sows

[0173] The levels of progesterone P, interleukin-6, and immunoglobulins in the serum of low-producing sows (total litter size ≤ 11 piglets) were detected using an ELISA kit. The results are as follows: Figure 15As shown, the progesterone content in low-producing sows decreased, which was beneficial for post-weaning sows. The level of interleukin-6 pro-inflammatory factor showed a downward trend, indicating that the preparation obtained in Example 1 alleviated the inflammatory response in sows. At the same time, the levels of immunoglobulins IgG (P<0.01), IgM (P<0.01), and IgA (P<0.05) increased significantly or substantially, which improved the immunity of pregnant sows.

[0174] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A composition for resisting PRRSv and improving sow reproductive performance, characterized in that, The composition comprises the following components: Angelica sinensis extract, Atractylodes macrocephala extract, Glycyrrhiza uralensis extract, Eucommia ulmoides extract, Eucommia ulmoides leaf extract, Populus tomentosa flower extract, Perilla frutescens leaf extract, Gynostemma pentaphyllum extract, Leonurus japonicus extract and Astragalus membranaceus extract. The mass ratio of the Angelica sinensis extract, the Atractylodes macrocephala extract, the Glycyrrhiza uralensis extract, the Eucommia ulmoides extract, the Eucommia ulmoides leaf extract, the Populus tomentosa flower extract, the Perilla frutescens leaf extract, the Gynostemma pentaphyllum extract, the Leonurus japonicus extract, and the Astragalus membranaceus extract is 4:2:2:1:1:1:1:1:1:1; The preparation method of the Angelica sinensis extract includes the steps of mixing Angelica sinensis with water, and then sequentially performing hot water extraction, impurity removal, concentration and drying to obtain the Angelica sinensis extract; The preparation method of the Atractylodes macrocephala extract includes the steps of mixing Atractylodes macrocephala with water, and then sequentially performing reflux extraction, alcohol extraction, impurity removal and purification to obtain the Atractylodes macrocephala extract; The method for preparing the licorice extract includes the steps of mixing licorice with ethanol, followed by impregnation, vacuum concentration and drying to obtain the licorice extract. The preparation method of the Eucommia ulmoides extract includes the following steps: mixing Eucommia ulmoides with ethanol, and then sequentially performing reflux extraction, vacuum concentration, centrifugation, n-butanol extraction, methanol extraction and drying to obtain the Eucommia ulmoides extract; The preparation method of the Eucommia ulmoides leaf extract includes the steps of mixing Eucommia ulmoides leaves with ethanol, and then performing reflux extraction and filtration sequentially to obtain the Eucommia ulmoides leaf extract. The preparation method of the poplar flower extract includes the steps of mixing poplar flowers with ethanol, then sequentially performing alcohol extraction, filtration, mixing and concentration to obtain the poplar flower extract. The preparation method of the perilla leaf extract includes the steps of mixing perilla leaves with ethanol, and then sequentially performing reflux extraction, filtration and vacuum concentration to obtain the perilla leaf extract. The preparation method of the Gynostemma pentaphyllum extract includes the steps of mixing Gynostemma pentaphyllum with ethanol, and then sequentially performing alcohol extraction, filtration and drying to obtain the Gynostemma pentaphyllum extract. The preparation method of the motherwort extract includes the steps of mixing motherwort with ethanol, and then performing reflux extraction and filtration sequentially to obtain the motherwort extract. The preparation method of the Astragalus extract includes the steps of mixing Astragalus with ethanol, followed by ethanol extraction, filtration and drying to obtain the Astragalus extract.

2. The composition according to claim 1, characterized in that, When preparing the Angelica sinensis extract, the material-to-liquid ratio is 1g:12mL; the hot water extraction is performed twice at a temperature of 95℃, and each hot water extraction lasts for 2 hours. When preparing the Atractylodes macrocephala extract, the mass-to-volume ratio of Atractylodes macrocephala to water is 2g:60mL; the reflux extraction temperature is 75℃ and the time is 2h. In preparing the licorice extract, the impregnation includes a first impregnation and a second impregnation; during the first impregnation, the material-to-liquid ratio is 1g:10mL, and during the second impregnation, the material-to-liquid ratio is 1g:5mL. When preparing the Eucommia ulmoides leaf extract, the material-to-liquid ratio was 1g:8mL; the reflux extraction was performed 3 times at a temperature of 65℃ for 2.5h. When preparing the Eucommia ulmoides leaf extract, the material-to-liquid ratio was 1g:11mL; the reflux extraction temperature was 75℃ and the time was 1.5h. When preparing the poplar flower extract, the material-to-liquid ratio is 50g:1mL; the alcohol extraction temperature is 85℃ and the time is 3h. When preparing the perilla leaf extract, the material-to-liquid ratio was 1g:11mL; the reflux extraction was performed 3 times at a temperature of 65℃, and the reflux extraction time was 3 days each time. When preparing the Gynostemma pentaphyllum extract, the material-to-liquid ratio is 1g:20mL; the alcohol extraction temperature is 90℃ and the time is 2h. When preparing the motherwort extract, the material-to-liquid ratio is 1g:25mL; the reflux extraction temperature is 65℃ and the time is 2h. When preparing the Astragalus extract, the alcohol extraction includes a first alcohol extraction and a second alcohol extraction; when performing the first alcohol extraction, the material-to-liquid ratio is 1g:13mL; when performing the second alcohol extraction, the material-to-liquid ratio is 1g:10mL.

3. The use of the composition according to claim 1 or 2 in the preparation of formulations for anti-PRRSv and improving sow reproductive performance.

4. A formulation for inhibiting PRRSv and improving sow reproductive performance, characterized in that, The active ingredient of the preparation is the composition described in claim 1 or 2.

5. The method for preparing the formulation according to claim 4, characterized in that, Includes the following steps: The preparation is obtained by mixing the Angelica sinensis extract, Atractylodes macrocephala extract, Glycyrrhiza uralensis extract, Eucommia ulmoides extract, Eucommia ulmoides leaf extract, Populus tomentosa flower extract, Perilla frutescens leaf extract, Gynostemma pentaphyllum extract, Leonurus japonicus extract and Astragalus membranaceus extract in a mass ratio of 4:2:2:1:1:1:1:1:1:1 to the above ratio.

6. The preparation method according to claim 5, characterized in that, The preparation method of the Angelica sinensis extract includes the steps of mixing Angelica sinensis with water, and then sequentially performing hot water extraction, impurity removal, concentration and drying to obtain the Angelica sinensis extract; The preparation method of the Atractylodes macrocephala extract includes the steps of mixing Atractylodes macrocephala with water, and then sequentially performing reflux extraction, alcohol extraction, impurity removal and purification to obtain the Atractylodes macrocephala extract; The method for preparing the licorice extract includes the steps of mixing licorice with ethanol, followed by impregnation, vacuum concentration and drying to obtain the licorice extract. The preparation method of the Eucommia ulmoides extract includes the following steps: mixing Eucommia ulmoides with ethanol, and then sequentially performing reflux extraction, vacuum concentration, centrifugation, n-butanol extraction, methanol extraction and drying to obtain the Eucommia ulmoides extract; The preparation method of the Eucommia ulmoides leaf extract includes the steps of mixing Eucommia ulmoides leaves with ethanol, and then performing reflux extraction and filtration sequentially to obtain the Eucommia ulmoides leaf extract. The preparation method of the poplar flower extract includes the steps of mixing poplar flowers with ethanol, then sequentially performing alcohol extraction, filtration, mixing and concentration to obtain the poplar flower extract. The preparation method of the perilla leaf extract includes the steps of mixing perilla leaves with ethanol, and then sequentially performing reflux extraction, filtration and vacuum concentration to obtain the perilla leaf extract. The preparation method of the Gynostemma pentaphyllum extract includes the steps of mixing Gynostemma pentaphyllum with ethanol, and then sequentially performing alcohol extraction, filtration and drying to obtain the Gynostemma pentaphyllum extract. The preparation method of the motherwort extract includes the steps of mixing motherwort with ethanol, and then performing reflux extraction and filtration sequentially to obtain the motherwort extract. The preparation method of the Astragalus extract includes the steps of mixing Astragalus with ethanol, followed by ethanol extraction, filtration and drying to obtain the Astragalus extract.

7. Use of the composition of claim 1 or 2 or the formulation of claim 4 in the preparation of products for treating PRRSv.

8. The use of the composition of claim 1 or 2 or the formulation of claim 4 in the preparation of a product having the effect of improving the reproductive performance of sows.

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