Application of composite plant polysaccharide in preventing and treating porcine reproductive and respiratory syndrome

Abstract

The invention discloses application of composite plant polysaccharide in preventing and treating porcine reproductive and respiratory syndrome, and belongs to the technical field of veterinary drugs. The composite plant polysaccharide is prepared from the following raw materials: fructus arctii, euphorbia helioscopia and fructus lycii, and the feeding weight ratio of the medicinal materials is 3: 4: 3. The preparation method comprises the following steps: mixing and crushing medicinal materials, degreasing with methanol, extracting with water, precipitating with ethanol, centrifuging, washing and drying to obtain crude polysaccharide, and finally deproteinizing with a Sevag method and performing ultrafiltration to obtain the composite plant polysaccharide. Experimental results show that the compound polysaccharide composition, especially the compound polysaccharide prepared from burdock, euphorbia helioscopia and lycium barbarum according to the proportion of 3: 4: 3, has a remarkable inhibition effect on PRRSV VR-2332 standard strains and high-pathogenicity NADC30-like PRRSV strains. The antiviral activity of the compound polysaccharide is far higher than that of single medicinal material polysaccharide or compound polysaccharide in other proportions, and the compound polysaccharide shows obvious synergistic interaction and broad-spectrum antiviral characteristics.

Description

Technical Field

[0001] This invention belongs to the field of veterinary drug technology, and in particular relates to the application of a compound plant polysaccharide in the prevention and treatment of porcine reproductive and respiratory syndrome (PRRS). Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV).

[0003] Currently, the prevention and control of this disease mainly relies on vaccination and chemical intervention. However, PRRSV is characterized by rapid evolution and high-frequency gene mutation, making it highly susceptible to immune escape. This means that existing commercial vaccines often only provide limited protection against specific strains, while the cross-protection rate against emerging variant strains is generally low (usually below 40%), making it difficult to effectively cover the genetic diversity of the virus. On the other hand, the long-term and widespread use of chemical antiviral drugs and antibiotics not only easily induces drug-resistant strains and weakens the control effect, but also poses the risk of drug residues, which contradicts the increasingly stringent safety supervision requirements for livestock and poultry products. Therefore, developing new drugs to inhibit porcine reproductive and respiratory syndrome (PRRS) is an urgent problem to be solved. Summary of the Invention

[0004] The purpose of this invention is to provide an application of a compound plant polysaccharide in the prevention and treatment of porcine reproductive and respiratory syndrome (PRRS), thereby providing a novel therapeutic drug for the prevention and treatment of PRRS.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides the application of a compound plant polysaccharide in the preparation of a drug for preventing and treating porcine reproductive and respiratory syndrome (PRRS), wherein the raw materials for preparing the compound plant polysaccharide consist of 10-50 parts of burdock fruit, 20-50 parts of Euphorbia helioscopia and 10-70 parts of wolfberry.

[0006] Preferably, the preparation method of the complex plant polysaccharide includes the following steps: (1) Mix and grind burdock seeds, Euphorbia helioscopia and wolfberry in proportion, and pass through a 60-mesh sieve; (2) Degrease with methanol, reflux extract, and then evaporate the methanol to obtain defatted residue; (3) Add 10 times the amount of water to the defatted drug residue, reflux at 100°C for 3 times, 2.5 hours each time, and combine the filtrates; (4) Concentrate the filtrate to 1 / 5 of its original volume, add ethanol to a final concentration of 80% for alcohol precipitation, and let stand overnight at 4°C. (5) Centrifuge to collect the precipitate, wash it with anhydrous ethanol and acetone in sequence, and vacuum dry it to obtain crude polysaccharide; (6) After the crude polysaccharide was reconstituted, the protein was removed by the Sevag method until there was no obvious denatured protein in the intermediate layer; (7) After microfiltration, filter with a 10kDa ultrafiltration membrane, collect the retentate, and dry the retentate to obtain the complex plant polysaccharide.

[0007] Preferably, in step (1), the proportion of mixing is burdock seed: Euphorbia helioscopia: wolfberry in a ratio of 3:4:3.

[0008] Preferably, the porcine reproductive and respiratory syndrome (PRRS) is caused by the PRRSV VR-2332 strain or the NADC30-like PRRSV strain.

[0009] Preferably, the drug is a liquid drug, and the concentration of the compound plant polysaccharide in the drug is ≥50μg / mL; The drug carrier for the liquid drug is physiological saline or phosphate buffer.

[0010] Secondly, the present invention provides the application of a complex plant polysaccharide in the preparation of an inhibitory drug for porcine reproductive and respiratory syndrome virus infection, wherein the preparation method of the complex plant polysaccharide includes the following steps: (1) Mix burdock seeds, Euphorbia helioscopia and wolfberry in a ratio of 3:4:3, grind them into powder, and pass them through a 60-mesh sieve; (2) Degrease with methanol, reflux extract, and then evaporate the methanol to obtain defatted residue; (3) Add 10 times the amount of water to the defatted drug residue, reflux at 100°C for 3 times, 2.5 hours each time, and combine the filtrates; (4) Concentrate the filtrate to 1 / 5 of its original volume, add ethanol to a final concentration of 80% for alcohol precipitation, and let stand overnight at 4°C. (5) Centrifuge to collect the precipitate, wash it with anhydrous ethanol and acetone in sequence, and vacuum dry it to obtain crude polysaccharide; (6) After the crude polysaccharide was reconstituted, the protein was removed by the Sevag method until there was no obvious denatured protein in the intermediate layer; (7) After microfiltration, the retentate is collected by filtration using a 10kDa ultrafiltration membrane and dried to obtain the complex plant polysaccharide.

[0011] Preferably, the porcine reproductive and respiratory syndrome virus is the PRRSV VR-2332 strain or the NADC30-like PRRSV strain.

[0012] Thirdly, the present invention provides a drug for preventing and treating porcine reproductive and respiratory syndrome (PRRS), the drug being composed of a complex plant polysaccharide and a pharmaceutically acceptable carrier; The composite plant polysaccharide was prepared by the above-described preparation method; In the drug, the concentration of the complex plant polysaccharide is ≥50 μg / mL.

[0013] Preferably, the porcine reproductive and respiratory syndrome (PRRS) is caused by the PRRSV VR-2332 strain or the NADC30-like PRRSV strain.

[0014] Preferably, the pharmaceutically acceptable carrier is physiological saline or phosphate buffer.

[0015] The beneficial effects of this invention are as follows: This invention provides a compound polysaccharide composed of burdock seed, Euphorbia helioscopia, and wolfberry, and its application in the prevention and treatment of porcine reproductive and respiratory syndrome (PRRS). Extensive experimental verification has demonstrated its significant antiviral activity and excellent clinical application potential. The beneficial effects of this invention are mainly reflected in: First, this invention reveals that three plant polysaccharides, in a specific ratio, exhibit significant synergistic antiviral effects. Experimental results show that, compared with single plant polysaccharides (comparative example), the complex polysaccharide group can greatly improve the survival rate of PRRSV-infected cells and significantly reduce viral titers. The compound ratio of burdock seed: Euphorbia helioscopia: wolfberry in 3:4:3 (Example 1) is particularly effective, exhibiting a significantly greater inhibitory effect on the PRRSV VR-2332 standard strain than all other single and compound ratios, demonstrating unexpected and excellent efficacy. This synergistic effect not only improves antiviral efficiency but also provides a safe and efficient natural drug resource for the prevention and control of porcine reproductive and respiratory syndrome (PRRS).

[0016] Secondly, the compound polysaccharide of this invention exhibits broad-spectrum and highly efficient inhibitory activity against different pathogenic PRRSV strains. Test results against the more pathogenic NADC30-like PRRSV strain show that the optimal formulation (Example 1) still significantly increases cell viability to 78.5% while substantially reducing viral titer. This effect is not only superior to all control groups and the second-best formulation, but also demonstrates that the compound polysaccharide possesses broad-spectrum antiviral properties, effectively combating currently prevalent variant strains in the aquaculture industry and overcoming the limitations of traditional vaccines and drugs in providing low protection against variant strains. Attached Figure Description

[0017] Figure 1 The image shows the cell morphology of the control group Marc-145 cells after 48 hours of culture. Figure 2 This is a morphological image of Marc-145 virome cells after 48 hours of culture. Figure 3 This is a cell morphology diagram of Marc-145 cells cultured for 48 hours in Example 1. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; other experimental methods and technical means not specifically mentioned herein refer to experimental methods and technical means commonly used by one of ordinary skill in the art.

[0020] Example 1 (1) Weigh 30g of burdock seeds, 40g of Euphorbia helioscopia and 30g of wolfberry and put them into a grinder to grind for 2 minutes. Pass them through a 60-mesh standard inspection sieve and collect the powder that passes through the sieve. (2) Place the powder into a round-bottom flask, add analytical grade methanol at a ratio of 1:2 (v / w, i.e., 2 mL methanol per 1 g of medicinal material), and stir thoroughly to suspend the powder; (3) Place the round-bottom flask in a constant temperature water bath, set the temperature to 80°C, connect the spherical condenser, and heat under reflux for 1.5 hours, stirring once every 15 minutes during the process; (4) Filter with qualitative filter paper, collect the residue, spread the residue on a petri dish, and place it in a fume hood to evaporate the methanol naturally to obtain defatted residue; (5) Add 10 times the volume (v / w) of distilled water to the defatted medicinal residue, stir well, and pre-soak at room temperature for 30 minutes to allow the medicinal material to fully absorb water; (6) Place the flask in an electric heating mantle, set the temperature to 100°C, connect the condenser, and reflux for extraction 3 times, each time for 2.5 hours. After each extraction, filter with 4 layers of gauze, collect the filtrate, and use the residue for the next extraction. (7) Combine the three filtrates and concentrate them to 1 / 5 of the original volume using a rotary evaporator (temperature 50℃, speed 50rpm) to obtain polysaccharide concentrate; (8) Slowly add 95% analytical grade ethanol to the concentrate while stirring until the final ethanol concentration reaches 80%. After sealing, place it in a refrigerator at 4°C and let it precipitate overnight to obtain the precipitate. (9) Transfer the alcohol precipitate to a centrifuge tube, centrifuge at 4000 r / min for 15 minutes, discard the supernatant, and collect the crude polysaccharide precipitate at the bottom; (10) The crude polysaccharide precipitate was washed twice with anhydrous ethanol and acetone, respectively, and then placed in a vacuum drying oven and dried for 8 hours to obtain crude polysaccharide powder. (11) Dissolve the crude polysaccharide powder in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL; add 20% chloroform by volume, then add n-butanol at 20% chloroform volume, shake vigorously for 20 minutes, and perform deproteinization treatment; (12) Centrifuge at 3000 r / min for 10 minutes, collect the upper polysaccharide aqueous solution, repeat the deproteinization treatment and centrifugation until there is no obvious denatured protein in the middle layer; (13) After deproteinizing, the polysaccharide aqueous solution was filtered through a 0.45 μm microfiltration membrane and then filtered through a 10 kDa ultrafiltration membrane, and the retentate was collected. (14) The retentate was concentrated to 1 / 5 of its original volume using a rotary evaporator and then dried in a vacuum drying oven to obtain deproteinized complex polysaccharide a.

[0021] Example 2 (1) Weigh 10g of burdock seeds, 20g of Euphorbia helioscopia and 70g of wolfberry and put them into a grinder to grind for 2 minutes. Pass them through a 60-mesh standard inspection sieve and collect the powder that passes through the sieve. (2) Place the powder into a round-bottom flask, add analytical grade methanol at a ratio of 1:2 (v / w, i.e., 2 mL methanol per 1 g of medicinal material), and stir thoroughly to suspend the powder; (3) Place the round-bottom flask in a constant temperature water bath, set the temperature to 80°C, connect the spherical condenser, and heat under reflux for 1.5 hours, stirring once every 15 minutes during the process; (4) Filter with qualitative filter paper, collect the residue, spread the residue on a petri dish, and place it in a fume hood to evaporate the methanol naturally to obtain defatted residue; (5) Add 10 times the volume (v / w) of distilled water to the defatted medicinal residue, stir well, and pre-soak at room temperature for 30 minutes to allow the medicinal material to fully absorb water; (6) Place the flask in an electric heating mantle, set the temperature to 100°C, connect the condenser, and reflux for extraction 3 times, each time for 2.5 hours. After each extraction, filter with 4 layers of gauze, collect the filtrate, and use the residue for the next extraction. (7) Combine the three filtrates and concentrate them to 1 / 5 of the original volume using a rotary evaporator (temperature 50℃, speed 50rpm) to obtain polysaccharide concentrate; (8) Slowly add 95% analytical grade ethanol to the concentrate while stirring until the final ethanol concentration reaches 80%. After sealing, place it in a refrigerator at 4°C and let it precipitate overnight to obtain the precipitate. (9) Transfer the alcohol precipitate to a centrifuge tube, centrifuge at 4000 r / min for 15 minutes, discard the supernatant, and collect the crude polysaccharide precipitate at the bottom; (10) The crude polysaccharide precipitate was washed twice with anhydrous ethanol and acetone, respectively, and then placed in a vacuum drying oven and dried for 8 hours to obtain crude polysaccharide powder. (11) Dissolve the crude polysaccharide powder in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL; add 20% chloroform by volume, then add n-butanol at 20% chloroform volume, shake vigorously for 20 minutes, and perform deproteinization treatment; (12) Centrifuge at 3000 r / min for 10 minutes, collect the upper polysaccharide aqueous solution, repeat the deproteinization treatment and centrifugation until there is no obvious denatured protein in the middle layer; (13) After deproteinizing, the polysaccharide aqueous solution was filtered through a 0.45 μm microfiltration membrane and then filtered through a 10 kDa ultrafiltration membrane, and the retentate was collected. (14) The retentate was concentrated to 1 / 5 of its original volume using a rotary evaporator and then dried in a vacuum drying oven to obtain deproteinized complex polysaccharide b.

[0022] Example 3 (1) Weigh 50g of burdock seeds, 40g of Euphorbia helioscopia and 10g of wolfberry and put them into a grinder to grind for 2 minutes. Pass them through a 60-mesh standard inspection sieve and collect the powder that passes through the sieve. (2) Place the powder into a round-bottom flask, add analytical grade methanol at a ratio of 1:2 (v / w, i.e., 2 mL methanol per 1 g of medicinal material), and stir thoroughly to suspend the powder; (3) Place the round-bottom flask in a constant temperature water bath, set the temperature to 80°C, connect the spherical condenser, and heat under reflux for 1.5 hours, stirring once every 15 minutes during the process; (4) Filter with qualitative filter paper, collect the residue, spread the residue on a petri dish, and place it in a fume hood to evaporate the methanol naturally to obtain defatted residue; (5) Add 10 times the volume (v / w) of distilled water to the defatted medicinal residue, stir well, and pre-soak at room temperature for 30 minutes to allow the medicinal material to fully absorb water; (6) Place the flask in an electric heating mantle, set the temperature to 100°C, connect the condenser, and reflux for extraction 3 times, each time for 2.5 hours. After each extraction, filter with 4 layers of gauze, collect the filtrate, and use the residue for the next extraction. (7) Combine the three filtrates and concentrate them to 1 / 5 of the original volume using a rotary evaporator (temperature 50℃, speed 50rpm) to obtain polysaccharide concentrate; (8) Slowly add 95% analytical grade ethanol to the concentrate while stirring until the final ethanol concentration reaches 80%. After sealing, place it in a refrigerator at 4°C and let it precipitate overnight to obtain the precipitate. (9) Transfer the alcohol precipitate to a centrifuge tube, centrifuge at 4000 r / min for 15 minutes, discard the supernatant, and collect the crude polysaccharide precipitate at the bottom; (10) The crude polysaccharide precipitate was washed twice with anhydrous ethanol and acetone, respectively, and then placed in a vacuum drying oven and dried for 8 hours to obtain crude polysaccharide powder. (11) Dissolve the crude polysaccharide powder in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL; add 20% chloroform by volume, then add n-butanol at 20% chloroform volume, shake vigorously for 20 minutes, and perform deproteinization treatment; (12) Centrifuge at 3000 r / min for 10 minutes, collect the upper polysaccharide aqueous solution, repeat the deproteinization treatment and centrifugation until there is no obvious denatured protein in the middle layer; (13) After deproteinizing, the polysaccharide aqueous solution was filtered through a 0.45 μm microfiltration membrane and then filtered through a 10 kDa ultrafiltration membrane, and the retentate was collected. (14) The retentate was concentrated to 1 / 5 of its original volume using a rotary evaporator and then dried in a vacuum drying oven to obtain deproteinized complex polysaccharide c.

[0023] Example 4 (1) Weigh 30g of burdock seeds, 30g of Euphorbia helioscopia and 40g of wolfberry and put them into a grinder to grind for 2 minutes. Pass them through a 60-mesh standard inspection sieve and collect the powder that passes through the sieve. (2) Place the powder into a round-bottom flask, add analytical grade methanol at a ratio of 1:2 (v / w, i.e., 2 mL methanol per 1 g of medicinal material), and stir thoroughly to suspend the powder; (3) Place the round-bottom flask in a constant temperature water bath, set the temperature to 80°C, connect the spherical condenser, and heat under reflux for 1.5 hours, stirring once every 15 minutes during the process; (4) Filter with qualitative filter paper, collect the residue, spread the residue on a petri dish, and place it in a fume hood to evaporate the methanol naturally to obtain defatted residue; (5) Add 10 times the volume (v / w) of distilled water to the defatted medicinal residue, stir well, and pre-soak at room temperature for 30 minutes to allow the medicinal material to fully absorb water; (6) Place the flask in an electric heating mantle, set the temperature to 100°C, connect the condenser, and reflux for extraction 3 times, each time for 2.5 hours. After each extraction, filter with 4 layers of gauze, collect the filtrate, and use the residue for the next extraction. (7) Combine the three filtrates and concentrate them to 1 / 5 of the original volume using a rotary evaporator (temperature 50℃, speed 50rpm) to obtain polysaccharide concentrate; (8) Slowly add 95% analytical grade ethanol to the concentrate while stirring until the final ethanol concentration reaches 80%. After sealing, place it in a refrigerator at 4°C and let it precipitate overnight to obtain the precipitate. (9) Transfer the alcohol precipitate to a centrifuge tube, centrifuge at 4000 r / min for 15 minutes, discard the supernatant, and collect the crude polysaccharide precipitate at the bottom; (10) The crude polysaccharide precipitate was washed twice with anhydrous ethanol and acetone, respectively, and then placed in a vacuum drying oven and dried for 8 hours to obtain crude polysaccharide powder. (11) Dissolve the crude polysaccharide powder in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL; add 20% chloroform by volume, then add n-butanol at 20% chloroform volume, shake vigorously for 20 minutes, and perform deproteinization treatment; (12) Centrifuge at 3000 r / min for 10 minutes, collect the upper polysaccharide aqueous solution, repeat the deproteinization treatment and centrifugation until there is no obvious denatured protein in the middle layer; (13) After deproteinizing, the polysaccharide aqueous solution was filtered through a 0.45 μm microfiltration membrane and then filtered through a 10 kDa ultrafiltration membrane, and the retentate was collected. (14) The retentate was concentrated to 1 / 5 of its original volume using a rotary evaporator and then dried in a vacuum drying oven to obtain deproteinized complex polysaccharide d.

[0024] Example 5 (1) Weigh 33.3g of burdock seeds, 33.3g of Euphorbia helioscopia and 33.3g of wolfberry and put them into a grinder and grind for 2 minutes. Then pass them through a 60-mesh standard inspection sieve and collect the powder that passes through the sieve. (2) Place the powder into a round-bottom flask, add analytical grade methanol at a ratio of 1:2 (v / w, i.e., 2 mL methanol per 1 g of medicinal material), and stir thoroughly to suspend the powder; (3) Place the round-bottom flask in a constant temperature water bath, set the temperature to 80°C, connect the spherical condenser, and heat under reflux for 1.5 hours, stirring once every 15 minutes during the process; (4) Filter with qualitative filter paper, collect the residue, spread the residue on a petri dish, and place it in a fume hood to evaporate the methanol naturally to obtain defatted residue; (5) Add 10 times the volume (v / w) of distilled water to the defatted medicinal residue, stir well, and pre-soak at room temperature for 30 minutes to allow the medicinal material to fully absorb water; (6) Place the flask in an electric heating mantle, set the temperature to 100°C, connect the condenser, and reflux for extraction 3 times, each time for 2.5 hours. After each extraction, filter with 4 layers of gauze, collect the filtrate, and use the residue for the next extraction. (7) Combine the three filtrates and concentrate them to 1 / 5 of the original volume using a rotary evaporator (temperature 50℃, speed 50rpm) to obtain polysaccharide concentrate; (8) Slowly add 95% analytical grade ethanol to the concentrate while stirring until the final ethanol concentration reaches 80%. After sealing, place it in a refrigerator at 4°C and let it precipitate overnight to obtain the precipitate. (9) Transfer the alcohol precipitate to a centrifuge tube, centrifuge at 4000 r / min for 15 minutes, discard the supernatant, and collect the crude polysaccharide precipitate at the bottom; (10) The crude polysaccharide precipitate was washed twice with anhydrous ethanol and acetone, respectively, and then placed in a vacuum drying oven and dried for 8 hours to obtain crude polysaccharide powder. (11) Dissolve the crude polysaccharide powder in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL; add 20% chloroform by volume, then add n-butanol at 20% chloroform volume, shake vigorously for 20 minutes, and perform deproteinization treatment; (12) Centrifuge at 3000 r / min for 10 minutes, collect the upper polysaccharide aqueous solution, repeat the deproteinization treatment and centrifugation until there is no obvious denatured protein in the middle layer; (13) After deproteinizing, the polysaccharide aqueous solution was filtered through a 0.45 μm microfiltration membrane and then filtered through a 10 kDa ultrafiltration membrane, and the retentate was collected. (14) The retentate was concentrated to 1 / 5 of its original volume using a rotary evaporator and then dried in a vacuum drying oven to obtain deproteinized complex polysaccharide e.

[0025] Example 6 (1) Weigh 50g of burdock seeds, 25g of Euphorbia helioscopia and 25g of wolfberry and put them into a grinder to grind for 2 minutes. Pass them through a 60-mesh standard inspection sieve and collect the powder that passes through the sieve. (2) Place the powder into a round-bottom flask, add analytical grade methanol at a ratio of 1:2 (v / w, i.e., 2 mL methanol per 1 g of medicinal material), and stir thoroughly to suspend the powder; (3) Place the round-bottom flask in a constant temperature water bath, set the temperature to 80°C, connect the spherical condenser, and heat under reflux for 1.5 hours, stirring once every 15 minutes during the process; (4) Filter with qualitative filter paper, collect the residue, spread the residue on a petri dish, and place it in a fume hood to evaporate the methanol naturally to obtain defatted residue; (5) Add 10 times the volume (v / w) of distilled water to the defatted medicinal residue, stir well, and pre-soak at room temperature for 30 minutes to allow the medicinal material to fully absorb water; (6) Place the flask in an electric heating mantle, set the temperature to 100°C, connect the condenser, and reflux for extraction 3 times, each time for 2.5 hours. After each extraction, filter with 4 layers of gauze, collect the filtrate, and use the residue for the next extraction. (7) Combine the three filtrates and concentrate them to 1 / 5 of the original volume using a rotary evaporator (temperature 50℃, speed 50rpm) to obtain polysaccharide concentrate; (8) Slowly add 95% analytical grade ethanol to the concentrate while stirring until the final ethanol concentration reaches 80%. After sealing, place it in a refrigerator at 4°C and let it precipitate overnight to obtain the precipitate. (9) Transfer the alcohol precipitate to a centrifuge tube, centrifuge at 4000 r / min for 15 minutes, discard the supernatant, and collect the crude polysaccharide precipitate at the bottom; (10) The crude polysaccharide precipitate was washed twice with anhydrous ethanol and acetone, respectively, and then placed in a vacuum drying oven and dried for 8 hours to obtain crude polysaccharide powder. (11) Dissolve the crude polysaccharide powder in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL; add 20% chloroform by volume, then add n-butanol at 20% chloroform volume, shake vigorously for 20 minutes, and perform deproteinization treatment; (12) Centrifuge at 3000 r / min for 10 minutes, collect the upper polysaccharide aqueous solution, repeat the deproteinization treatment and centrifugation until there is no obvious denatured protein in the middle layer; (13) After deproteinizing, the polysaccharide aqueous solution was filtered through a 0.45 μm microfiltration membrane and then filtered through a 10 kDa ultrafiltration membrane, and the retentate was collected. (14) The retentate was concentrated to 1 / 5 of its original volume using a rotary evaporator and then dried in a vacuum drying oven to obtain deproteinized complex polysaccharide e.

[0026] Example 7 (1) Weigh 25g of burdock seeds, 50g of Euphorbia helioscopia and 25g of wolfberry and put them into a grinder to grind for 2 minutes. Pass them through a 60-mesh standard inspection sieve and collect the powder that passes through the sieve. (2) Place the powder into a round-bottom flask, add analytical grade methanol at a ratio of 1:2 (v / w, i.e., 2 mL methanol per 1 g of medicinal material), and stir thoroughly to suspend the powder; (3) Place the round-bottom flask in a constant temperature water bath, set the temperature to 80°C, connect the spherical condenser, and heat under reflux for 1.5 hours, stirring once every 15 minutes during the process; (4) Filter with qualitative filter paper, collect the residue, spread the residue on a petri dish, and place it in a fume hood to evaporate the methanol naturally to obtain defatted residue; (5) Add 10 times the volume (v / w) of distilled water to the defatted medicinal residue, stir well, and pre-soak at room temperature for 30 minutes to allow the medicinal material to fully absorb water; (6) Place the flask in an electric heating mantle, set the temperature to 100°C, connect the condenser, and reflux for extraction 3 times, each time for 2.5 hours. After each extraction, filter with 4 layers of gauze, collect the filtrate, and use the residue for the next extraction. (7) Combine the three filtrates and concentrate them to 1 / 5 of the original volume using a rotary evaporator (temperature 50℃, speed 50rpm) to obtain polysaccharide concentrate; (8) Slowly add 95% analytical grade ethanol to the concentrate while stirring until the final ethanol concentration reaches 80%. After sealing, place it in a refrigerator at 4°C and let it precipitate overnight to obtain the precipitate. (9) Transfer the alcohol precipitate to a centrifuge tube, centrifuge at 4000 r / min for 15 minutes, discard the supernatant, and collect the crude polysaccharide precipitate at the bottom; (10) The crude polysaccharide precipitate was washed twice with anhydrous ethanol and acetone, respectively, and then placed in a vacuum drying oven and dried for 8 hours to obtain crude polysaccharide powder. (11) Dissolve the crude polysaccharide powder in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL; add 20% chloroform by volume, then add n-butanol at 20% chloroform volume, shake vigorously for 20 minutes, and perform deproteinization treatment; (12) Centrifuge at 3000 r / min for 10 minutes, collect the upper polysaccharide aqueous solution, repeat the deproteinization treatment and centrifugation until there is no obvious denatured protein in the middle layer; (13) After deproteinizing, the polysaccharide aqueous solution was filtered through a 0.45 μm microfiltration membrane and then filtered through a 10 kDa ultrafiltration membrane, and the retentate was collected. (14) The retentate was concentrated to 1 / 5 of its original volume using a rotary evaporator and then dried in a vacuum drying oven to obtain deproteinized complex polysaccharide f.

[0027] Comparative Example 1 (1) Weigh 100g of burdock seeds and put them in a grinder to grind for 2 minutes. Pass the powder through a 60-mesh standard inspection sieve and collect the powder that passes through the sieve. (2) Place the powder into a round-bottom flask, add analytical grade methanol at a ratio of 1:2 (v / w, i.e., 2 mL methanol per 1 g of medicinal material), and stir thoroughly to suspend the powder; (3) Place the round-bottom flask in a constant temperature water bath, set the temperature to 80°C, connect the spherical condenser, and heat under reflux for 1.5 hours, stirring once every 15 minutes during the process; (4) Filter with qualitative filter paper, collect the residue, spread the residue on a petri dish, and place it in a fume hood to evaporate the methanol naturally to obtain defatted residue; (5) Add 10 times the volume (v / w) of distilled water to the defatted medicinal residue, stir well, and pre-soak at room temperature for 30 minutes to allow the medicinal material to fully absorb water; (6) Place the flask in an electric heating mantle, set the temperature to 100°C, connect the condenser, and reflux for extraction 3 times, each time for 2.5 hours. After each extraction, filter with 4 layers of gauze, collect the filtrate, and use the residue for the next extraction. (7) Combine the three filtrates and concentrate them to 1 / 5 of the original volume using a rotary evaporator (temperature 50℃, speed 50rpm) to obtain polysaccharide concentrate; (8) Slowly add 95% analytical grade ethanol to the concentrate while stirring until the final ethanol concentration reaches 80%. After sealing, place it in a refrigerator at 4°C and let it precipitate overnight to obtain the precipitate. (9) Transfer the alcohol precipitate to a centrifuge tube, centrifuge at 4000 r / min for 15 minutes, discard the supernatant, and collect the crude polysaccharide precipitate at the bottom; (10) The crude polysaccharide precipitate was washed twice with anhydrous ethanol and acetone, respectively, and then placed in a vacuum drying oven and dried for 8 hours to obtain crude polysaccharide powder. (11) Dissolve the crude polysaccharide powder in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL; add 20% chloroform by volume, then add n-butanol at 20% chloroform volume, shake vigorously for 20 minutes, and perform deproteinization treatment; (12) Centrifuge at 3000 r / min for 10 minutes, collect the upper polysaccharide aqueous solution, repeat the deproteinization treatment and centrifugation until there is no obvious denatured protein in the middle layer; (13) After deproteinizing, the polysaccharide aqueous solution was filtered through a 0.45 μm microfiltration membrane and then filtered through a 10 kDa ultrafiltration membrane, and the retentate was collected. (14) The retentate was concentrated to 1 / 5 of its original volume using a rotary evaporator and then dried in a vacuum drying oven to obtain deproteinized burdock polysaccharide.

[0028] Comparative Example 2 (1) Weigh 100g of Euphorbia helioscopia and put it in a pulverizer to grind for 2 minutes. Pass it through a 60-mesh standard inspection sieve and collect the powder that passes through the sieve. (2) Place the powder into a round-bottom flask, add analytical grade methanol at a ratio of 1:2 (v / w, i.e., 2 mL methanol per 1 g of medicinal material), and stir thoroughly to suspend the powder; (3) Place the round-bottom flask in a constant temperature water bath, set the temperature to 80°C, connect the spherical condenser, and heat under reflux for 1.5 hours, stirring once every 15 minutes during the process; (4) Filter with qualitative filter paper, collect the residue, spread the residue on a petri dish, and place it in a fume hood to evaporate the methanol naturally to obtain defatted residue; (5) Add 10 times the volume (v / w) of distilled water to the defatted medicinal residue, stir well, and pre-soak at room temperature for 30 minutes to allow the medicinal material to fully absorb water; (6) Place the flask in an electric heating mantle, set the temperature to 100°C, connect the condenser, and reflux for extraction 3 times, each time for 2.5 hours. After each extraction, filter with 4 layers of gauze, collect the filtrate, and use the residue for the next extraction. (7) Combine the three filtrates and concentrate them to 1 / 5 of the original volume using a rotary evaporator (temperature 50℃, speed 50rpm) to obtain polysaccharide concentrate; (8) Slowly add 95% analytical grade ethanol to the concentrate while stirring until the final ethanol concentration reaches 80%. After sealing, place it in a refrigerator at 4°C and let it precipitate overnight to obtain the precipitate. (9) Transfer the alcohol precipitate to a centrifuge tube, centrifuge at 4000 r / min for 15 minutes, discard the supernatant, and collect the crude polysaccharide precipitate at the bottom; (10) The crude polysaccharide precipitate was washed twice with anhydrous ethanol and acetone, respectively, and then placed in a vacuum drying oven and dried for 8 hours to obtain crude polysaccharide powder. (11) Dissolve the crude polysaccharide powder in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL; add 20% chloroform by volume, then add n-butanol at 20% chloroform volume, shake vigorously for 20 minutes, and perform deproteinization treatment; (12) Centrifuge at 3000 r / min for 10 minutes, collect the upper polysaccharide aqueous solution, repeat the deproteinization treatment and centrifugation until there is no obvious denatured protein in the middle layer; (13) After deproteinizing, the polysaccharide aqueous solution was filtered through a 0.45 μm microfiltration membrane and then filtered through a 10 kDa ultrafiltration membrane, and the retentate was collected. (14) The retentate was concentrated to 1 / 5 of its original volume using a rotary evaporator and then dried in a vacuum drying oven to obtain deproteinized Euphorbia helioscopia polysaccharide.

[0029] Comparative Example 3 (1) Weigh 100g of wolfberries and put them in a grinder to grind for 2 minutes. Pass them through a 60-mesh standard inspection sieve and collect the powder that passes through the sieve. (2) Place the powder into a round-bottom flask, add analytical grade methanol at a ratio of 1:2 (v / w, i.e., 2 mL methanol per 1 g of medicinal material), and stir thoroughly to suspend the powder; (3) Place the round-bottom flask in a constant temperature water bath, set the temperature to 80°C, connect the spherical condenser, and heat under reflux for 1.5 hours, stirring once every 15 minutes during the process; (4) Filter with qualitative filter paper, collect the residue, spread the residue on a petri dish, and place it in a fume hood to evaporate the methanol naturally to obtain defatted residue; (5) Add 10 times the volume (v / w) of distilled water to the defatted medicinal residue, stir well, and pre-soak at room temperature for 30 minutes to allow the medicinal material to fully absorb water; (6) Place the flask in an electric heating mantle, set the temperature to 100°C, connect the condenser, and reflux for extraction 3 times, each time for 2.5 hours. After each extraction, filter with 4 layers of gauze, collect the filtrate, and use the residue for the next extraction. (7) Combine the three filtrates and concentrate them to 1 / 5 of the original volume using a rotary evaporator (temperature 50℃, speed 50rpm) to obtain polysaccharide concentrate; (8) Slowly add 95% analytical grade ethanol to the concentrate while stirring until the final ethanol concentration reaches 80%. After sealing, place it in a refrigerator at 4°C and let it precipitate overnight to obtain the precipitate. (9) Transfer the alcohol precipitate to a centrifuge tube, centrifuge at 4000 r / min for 15 minutes, discard the supernatant, and collect the crude polysaccharide precipitate at the bottom; (10) The crude polysaccharide precipitate was washed twice with anhydrous ethanol and acetone, respectively, and then placed in a vacuum drying oven and dried for 8 hours to obtain crude polysaccharide powder. (11) Dissolve the crude polysaccharide powder in distilled water to prepare a polysaccharide solution with a concentration of 10 mg / mL; add 20% chloroform by volume, then add n-butanol at 20% chloroform volume, shake vigorously for 20 minutes, and perform deproteinization treatment; (12) Centrifuge at 3000 r / min for 10 minutes, collect the upper polysaccharide aqueous solution, repeat the deproteinization treatment and centrifugation until there is no obvious denatured protein in the middle layer; (13) After deproteinizing, the polysaccharide aqueous solution was filtered through a 0.45 μm microfiltration membrane and then filtered through a 10 kDa ultrafiltration membrane, and the retentate was collected. (14) The retentate was concentrated to 1 / 5 of its original volume using a rotary evaporator and then dried in a vacuum drying oven to obtain deproteinized wolfberry polysaccharide.

[0030] Example 5 Effects of different polysaccharide pretreatments on cell viability of PRRSV-infected cells (1) Take the Marc-145 cell cryopreservation tube out of liquid nitrogen, thaw it quickly in a 37°C water bath, transfer it to a 15mL centrifuge tube, and add 5mL of preheated DMEM complete medium (10% FBS, 1% antibiotics). (2) Centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend in 5 mL of fresh culture medium, inoculate into T25 culture flask, and incubate at 37℃ and 5% CO2 for 24 h before changing the medium; (3) When the cell confluence reaches 90%, the culture medium is aspirated, 2 mL of PBS is added to wash once, 1 mL of 0.25% trypsin (containing 0.02% EDTA) is added, and the cells are digested at 37℃ for 2-3 min. 3 mL of complete culture medium is added to stop the digestion. The cells are gently pipetted to form a single-cell suspension. The cells are passaged at a ratio of 1:3 every 2-3 days. The 5th generation cells are used for experiments. (4) Seed the cells into 96-well cell culture plates, 1×10 4 Cells / well, 100μL / well, cultured until cell density is greater than 85%; (5) Dissolve 10 mg of the polysaccharides obtained in Examples 1-7 and Comparative Examples 1-3 in 1 mL of sterile PBS to prepare a stock solution with a final concentration of 10 mg / mL; (6) After filtration through a 22 μm filter membrane, dilute to a concentration of 50 μg / mL using maintenance medium (2% FBS); (7) The cells were treated according to the following grouping and design: Blank group: Blank wells containing no cells, with only maintenance medium added; Control group: After treatment with maintenance medium for 2 hours, the medium was replaced and cultured for another 48 hours. Virome: After treatment with maintenance medium for 2 hours, the virus was infected with PRRSV VR-2332 standard strain with MOI=0.01 for 1 hour, and then cultured in maintenance medium for another 48 hours. Example 1 group: After treatment with compound polysaccharide a for 2 hours, the PRRSV VR-2332 standard strain with MOI=0.01 was used for infection for 1 hour, and maintenance medium was added for continued culture for 48 hours; Example 2 group: After treatment with compound polysaccharide b for 2 hours, the PRRSV VR-2332 standard strain with MOI=0.01 was used for infection for 1 hour, and maintenance medium was added for continued culture for 48 hours; Example 3 group: After treatment with compound polysaccharide c for 2 hours, the PRRSV VR-2332 standard strain with MOI=0.01 was used for infection for 1 hour, and maintenance medium was added for continued culture for 48 hours; Example 4 group: After treatment with compound polysaccharide d for 2 hours, the PRRSV VR-2332 standard strain with MOI=0.01 was used for infection for 1 hour, and maintenance medium was added for continued culture for 48 hours; Example 5 group: After treatment with compound polysaccharide e for 2 hours, the PRRSV VR-2332 standard strain with MOI=0.01 was used for infection for 1 hour, and maintenance medium was added for continued culture for 48 hours; Example 6 group: After treatment with compound polysaccharide f for 2 hours, the PRRSV VR-2332 standard strain with MOI=0.01 was used for infection for 1 hour, and maintenance medium was added for continued culture for 48 hours; Example 7 group: After treatment with compound polysaccharide g for 2 hours, the PRRSV VR-2332 standard strain with MOI=0.01 was used for infection for 1 hour, and maintenance medium was added for continued culture for 48 hours; Comparative Example 1: After treatment with burdock polysaccharide for 2 hours, the samples were infected with PRRSV VR-2332 standard strain with MOI=0.01 for 1 hour, and then cultured in maintenance medium for 48 hours. Comparative Example 2: After treatment with Euphorbia helioscopia polysaccharide for 2 hours, the cells were infected with PRRSV VR-2332 standard strain with MOI=0.01 for 1 hour, and then cultured in maintenance medium for 48 hours. Comparative Example 3: After treatment with Lycium barbarum polysaccharide for 2 hours, the cells were infected with PRRSV VR-2332 standard strain with MOI=0.01 for 1 hour, and then cultured in maintenance medium for 48 hours.

[0031] 48 hours after infection, 10 μL of CCK-8 reagent was added to each well of a 96-well plate and incubated at 37°C in the dark for 2 hours. The absorbance at 450 nm was measured using a microplate reader and the cell viability was calculated.

[0032] Table 1 Cell viability after different polysaccharide pretreatments

[0033] As shown in Table 1, compared with the control group, the cell survival rate was significantly reduced after PRRSV virus infection, indicating that the infection was successful. Compared with the virus group, the cell survival rates of Comparative Examples 1, 2, and 3 were improved, but the improvement was relatively limited, indicating that the ability of the burdock polysaccharide, Euphorbia helioscopia polysaccharide, or Lycium barbarum polysaccharide prepared in this invention to inhibit PRRSV VR-2332 standard strain infection was relatively limited.

[0034] The results of Examples 1-7 showed that the compound polysaccharide of burdock seed, Euphorbia helioscopia, and wolfberry was significantly better than that of a single polysaccharide in inhibiting PRRSV VR-2332 standard strain infection. Among them, the effect of Example 1 was significantly better than that of Examples 2-7, demonstrating excellent anti-infection effect.

[0035] Example 6 Detecting the effect of different polysaccharide pretreatments on PRRSV viral titers (1) Take the Marc-145 cell cryopreservation tube out of liquid nitrogen, thaw it quickly in a 37°C water bath, transfer it to a 15mL centrifuge tube, and add 5mL of preheated DMEM complete medium (10% FBS, 1% antibiotics). (2) Centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend in 5 mL of fresh culture medium, inoculate into T25 culture flask, and incubate at 37℃ and 5% CO2 for 24 h before changing the medium; (3) When the cell confluence reaches 90%, the culture medium is aspirated, 2 mL of PBS is added to wash once, 1 mL of 0.25% trypsin (containing 0.02% EDTA) is added, and the cells are digested at 37℃ for 2-3 min. 3 mL of complete culture medium is added to stop the digestion. The cells are gently pipetted to form a single-cell suspension. The cells are passaged at a ratio of 1:3 every 2-3 days. The 5th generation cells are used for experiments. (4) Seed the cells into 96-well cell culture plates, 1×10 4 Cells / well, 100μL / well, cultured until cell density is greater than 85%; (5) Dissolve 10 mg of the polysaccharides obtained in Examples 1-7 and Comparative Examples 1-3 in 1 mL of sterile PBS to prepare a stock solution with a final concentration of 10 mg / mL; (6) After filtration through a 22 μm filter membrane, dilute to a concentration of 50 μg / mL using maintenance medium (2% FBS); (7) The cells were treated according to the following grouping and design: Control group: After treatment with maintenance medium for 2 hours, the medium was replaced and cultured for another 48 hours. Virome: After treatment with maintenance medium for 2 hours, the virus was infected with PRRSV VR-2332 standard strain with MOI=0.01 for 1 hour, and then cultured in maintenance medium for another 48 hours. Example 1 group: After treatment with compound polysaccharide a for 2 hours, the PRRSV VR-2332 standard strain with MOI=0.01 was used for infection for 1 hour, and maintenance medium was added for continued culture for 48 hours; Example 2 group: After treatment with compound polysaccharide b for 2 hours, the PRRSV VR-2332 standard strain with MOI=0.01 was used for infection for 1 hour, and maintenance medium was added for continued culture for 48 hours; Example 3 group: After treatment with compound polysaccharide c for 2 hours, the PRRSV VR-2332 standard strain with MOI=0.01 was used for infection for 1 hour, and maintenance medium was added for continued culture for 48 hours; Example 4 group: After treatment with compound polysaccharide d for 2 hours, the PRRSV VR-2332 standard strain with MOI=0.01 was used for infection for 1 hour, and maintenance medium was added for continued culture for 48 hours; Example 5 group: After treatment with compound polysaccharide e for 2 hours, the PRRSV VR-2332 standard strain with MOI=0.01 was used for infection for 1 hour, and maintenance medium was added for continued culture for 48 hours; Example 6 group: After treatment with compound polysaccharide f for 2 hours, the PRRSV VR-2332 standard strain with MOI=0.01 was used for infection for 1 hour, and maintenance medium was added for continued culture for 48 hours; Example 7 group: After treatment with compound polysaccharide g for 2 hours, the PRRSV VR-2332 standard strain with MOI=0.01 was used for infection for 1 hour, and maintenance medium was added for continued culture for 48 hours; Comparative Example 1: After treatment with burdock polysaccharide for 2 hours, the samples were infected with PRRSV VR-2332 standard strain with MOI=0.01 for 1 hour, and then cultured in maintenance medium for 48 hours. Comparative Example 2: After treatment with Euphorbia helioscopia polysaccharide for 2 hours, the cells were infected with PRRSV VR-2332 standard strain with MOI=0.01 for 1 hour, and then cultured in maintenance medium for 48 hours. Comparative Example 3: After treatment with Lycium barbarum polysaccharide for 2 hours, the cells were infected with PRRSV VR-2332 standard strain with MOI=0.01 for 1 hour, and then cultured in maintenance medium for 48 hours. (8) Collect the culture supernatant of each group, perform a 10-fold serial dilution, take 100 μL of virus solution of different dilutions and inoculate them into Marc-145 cell monolayers in 96-well plates, set 3 replicates for each dilution, and incubate in a 37℃, 5%CO2 incubator for 72 h. (9) After culture, observe the cytopathic effects in each well under an inverted microscope and record the number of wells showing cytopathic effects. Calculate the viral titer using the Reed-Muench method, expressed as log... 10 TCID 50 / mL represents the volume of water.

[0036] Table 2. Effects of different polysaccharide pretreatments on PRRSV viral titer

[0037] As shown in Table 2, the viral titers in Comparative Examples 1-3, which used burdock seed polysaccharide, Euphorbia helioscopia polysaccharide, or Lycium barbarum polysaccharide, decreased, but the effect was limited. Examples 1-7 all showed better results than the comparative examples, indicating that the combined polysaccharide of burdock seed, Euphorbia helioscopia, and Lycium barbarum had a better inhibitory effect on the replication of the PRRSV VR-2332 standard strain than the individual effects of the three polysaccharides. In particular, Example 1 showed an unexpectedly significant decrease in viral titer, far exceeding any other single or combined ratio, demonstrating a remarkable effect.

[0038] Example 7 Effect of polysaccharide pretreatment on cell viability of NADC30-like PRRSV-infected cells (1) Cell treatment was the same as in Example 5, and the cells were grouped as follows: Blank group: Blank wells containing no cells, with only maintenance medium added; Control group: After treatment with maintenance medium for 2 hours, the culture medium was replaced and the cells were cultured for another 48 hours (cell morphology after culture is shown in the figure). Figure 1 ); Virome: After treatment with maintenance medium for 2 hours, cells were infected with NADC30-like PRRSV at MOI=0.01 for 1 hour, and then cultured in maintenance medium for another 48 hours (cell morphology after culture is shown in the figure). Figure 2 ); Example 1 group: After treatment with compound polysaccharide a for 2 hours, cells were infected with NADC30-like PRRSV with MOI=0.01 for 1 hour, and then cultured in maintenance medium for 48 hours (cell morphology after culture is shown in the figure). Figure 3 ); Example 4 group: After treatment with compound polysaccharide d for 2 hours, the cells were infected with NADC30-like PRRSV with MOI=0.01 for 1 hour, and then cultured in maintenance medium for 48 hours. Example 5 group: After treatment with compound polysaccharide e for 2 hours, the cells were infected with NADC30-like PRRSV with MOI=0.01 for 1 hour, and then cultured in maintenance medium for 48 hours. 48 hours after infection, 10 μL of CCK-8 reagent was added to each well of a 96-well plate and incubated at 37°C in the dark for 2 hours. The absorbance at 450 nm was measured using a microplate reader and the cell viability was calculated.

[0039] Table 3. Effects of polysaccharide pretreatment on cell viability of NADC30-like PRRSV-infected cells.

[0040] As shown in Table 3, compared to the PRRSV VR-2332 standard strain, NADC30-like PRRSV infection resulted in a more significant decrease in cell viability. Compared to the virus group, the cell viability of the Example 1 group increased to 78.5%, a 141.5% increase, still demonstrating excellent inhibitory effects on infection. In contrast, the effects of Example 4 and Example 5, which showed good efficacy against the PRRSV VR-2332 standard strain infection, were relatively poor. These results indicate that the complex polysaccharide prepared in Example 1 is not only more effective but also exhibits excellent inhibitory effects against highly infectious strains.

[0041] Example 8 Effect of polysaccharide pretreatment on NADC30-like PRRSV virus titers (1) Cell treatment was the same as in Example 5, and the cells were grouped as follows: Control group: After treatment with maintenance medium for 2 hours, the medium was replaced and cultured for another 48 hours. Virome: After treatment with maintenance medium for 2 hours, NADC30-like PRRSV with MOI=0.01 was used for infection for 1 hour, and maintenance medium was added for continued culture for 48 hours; Example 1 group: After treatment with compound polysaccharide a for 2 hours, the cells were infected with NADC30-like PRRSV with MOI=0.01 for 1 hour, and then cultured in maintenance medium for 48 hours. Example 4 group: After treatment with compound polysaccharide d for 2 hours, the cells were infected with NADC30-like PRRSV with MOI=0.01 for 1 hour, and then cultured in maintenance medium for 48 hours. Example 5 group: After treatment with compound polysaccharide e for 2 hours, the cells were infected with NADC30-like PRRSV with MOI=0.01 for 1 hour, and then cultured in maintenance medium for 48 hours.

[0042] Table 4. Effects of polysaccharide pretreatment on NADC30-like PRRSV virus titers

[0043] As shown in Table 4, the viral titer of the virus group infected with NADC30-like PRRSV was significantly higher than that of the PRRSV VR-2332 standard strain, indicating that NADC30-like PRRSV infection has a stronger replication capacity. Compared with the virus, the viral titer of the Example 1 group was significantly reduced, while the decrease in viral titer of the Example 4 and Example 5 groups was relatively limited. Corresponding to the cell viability results, this fully demonstrates that the complex polysaccharide a prepared in Example 1 has extremely strong broad-spectrum and highly efficient antiviral activity against highly pathogenic NADC30-like strains.

Claims

1. The application of a compound plant polysaccharide in the preparation of a drug for preventing and treating porcine reproductive and respiratory syndrome (PRRS), characterized in that, The raw materials for preparing the complex plant polysaccharide consist of 10-50 parts of burdock fruit, 20-50 parts of Euphorbia helioscopia, and 10-70 parts of wolfberry.

2. The application according to claim 1, characterized in that, The preparation method of the complex plant polysaccharide includes the following steps: (1) Mix and grind burdock seeds, Euphorbia helioscopia and wolfberry in proportion, and pass through a 60-mesh sieve; (2) Degrease with methanol, reflux extract, and then evaporate the methanol to obtain defatted residue; (3) Add 10 times the amount of water to the defatted drug residue, reflux at 100°C for 3 times, 2.5 hours each time, and combine the filtrates; (4) Concentrate the filtrate to 1 / 5 of its original volume, add ethanol to a final concentration of 80% for alcohol precipitation, and let stand overnight at 4°C. (5) Centrifuge to collect the precipitate, wash it with anhydrous ethanol and acetone in sequence, and vacuum dry it to obtain crude polysaccharide; (6) After the crude polysaccharide was reconstituted, the protein was removed by the Sevag method until there was no obvious denatured protein in the intermediate layer; (7) After microfiltration, the retentate is collected by filtration using a 10kDa ultrafiltration membrane and dried to obtain the complex plant polysaccharide.

3. The application according to claim 2, characterized in that, In step (1), the proportion of mixing is burdock seed, eupatorium fortunei and wolfberry in a ratio of 3:4:

3.

4. The application according to claim 3, characterized in that, The porcine reproductive and respiratory syndrome (PRRS) is caused by the PRRSV VR-2332 strain or the NADC30-like PRRSV strain.

5. The application according to claim 4, characterized in that, The drug is a liquid drug, and the concentration of the compound plant polysaccharide in the drug is ≥50μg / mL; The drug carrier for the liquid drug is physiological saline or phosphate buffer.

6. The application of a complex plant polysaccharide in the preparation of an inhibitory drug for porcine reproductive and respiratory syndrome virus infection, characterized in that, The preparation method of the complex plant polysaccharide includes the following steps: (1) Mix burdock seeds, Euphorbia helioscopia and wolfberry in a ratio of 3:4:3, grind them into powder, and pass them through a 60-mesh sieve; (2) Degrease with methanol, reflux extract, and then evaporate the methanol to obtain defatted residue; (3) Add 10 times the amount of water to the defatted drug residue, reflux at 100°C for 3 times, 2.5 hours each time, and combine the filtrates; (4) Concentrate the filtrate to 1 / 5 of its original volume, add ethanol to a final concentration of 80% for alcohol precipitation, and let stand overnight at 4°C. (5) Centrifuge to collect the precipitate, wash it with anhydrous ethanol and acetone in sequence, and vacuum dry it to obtain crude polysaccharide; (6) After the crude polysaccharide was reconstituted, the protein was removed by the Sevag method until there was no obvious denatured protein in the intermediate layer; (7) After microfiltration, the retentate is collected by filtration using a 10kDa ultrafiltration membrane and dried to obtain the complex plant polysaccharide.

7. The application according to claim 6, characterized in that, The porcine reproductive and respiratory syndrome virus is either the PRRSVVR-2332 strain or the NADC30-like PRRSV strain.

8. A drug for preventing and treating porcine reproductive and respiratory syndrome (PRRS), characterized in that, The drug is composed of a complex plant polysaccharide and a pharmaceutically acceptable carrier; The composite plant polysaccharide is prepared by the preparation method described in claim 6; In the drug, the concentration of the complex plant polysaccharide is ≥50 μg / mL.

9. The medicament according to claim 8, characterized in that, The porcine reproductive and respiratory syndrome (PRRS) is caused by the PRRSV VR-2332 strain or the NADC30-like PRRSV strain.

10. The medicament according to claim 9, characterized in that, The pharmaceutically acceptable carrier is physiological saline or phosphate buffer.

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