Application of soybean saponin in preparing a medicament for preventing and / or treating porcine reproductive and respiratory syndrome

Abstract

The application of soybean saponins in the preparation of drugs for preventing and / or treating porcine reproductive and respiratory syndrome belongs to the technical field of research on the biological functions of phytochemicals. To solve the technical problem that there is no effective drug for treating porcine reproductive and respiratory syndrome caused by PRRSV infection, the present invention studied the inhibitory effect of soybean saponins on the proliferation of PRRSV. Through indirect immunofluorescence assay and immunoblotting assay, it was found that administering soybean saponins simultaneously with PRRSV infection, before PRRSV infection, and after PRRSV infection could all inhibit the proliferation of PRRSV. Moreover, the inhibitory effect of soybean saponins on the proliferation of PRRSV has a time-dependent effect and a dose-dependent effect. Therefore, soybean saponins have the prospect of being developed into drugs against porcine reproductive and respiratory syndrome virus.

Description

Technical Field

[0001] The present invention belongs to the technical field of research on the biological functions of phytochemicals, and specifically relates to the application of soyasaponins in the preparation of drugs for preventing and / or treating porcine reproductive and respiratory syndrome. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a major disease currently harming the pig industry. The disease has an acute onset, a fast transmission speed, and a high mortality rate, and is mainly characterized by causing respiratory disorders in piglets and reproductive disorders in sows. The pathogen of the disease is porcine reproductive and respiratory syndrome virus (PRRSV). Porcine reproductive and respiratory syndrome is very similar in clinical manifestations globally. However, despite this, their respective pathogens have great differences in virulence, antigenicity, and genetic characteristics. According to these differences, PRRSV is divided into two subtypes, namely the North American type and the European type. Since China first isolated PRRSV from aborted fetuses in 1996, North American epidemic strains such as CH-1a, HuN4, and NADC30-like have been widely prevalent in Chinese pig farms. Currently, there is no effective drug reported for the pig diseases caused by this virus infection. Therefore, there is an urgent need for a method that can rapidly and efficiently inhibit PRRSV infection, laying a foundation for the research on the anti-PRRSV mechanism and the prevention and treatment of PRRS in pig farms.

[0003] Soyasaponins are a type of saponins widely present in leguminous plants such as soybeans and their products. They are a class of phytochemicals formed by the condensation of soyasapogenins and oligosaccharides, belonging to pentacyclic triterpenoid oleanane-type compounds. Legumes are the main dietary source of soyasaponins, and soybeans are one of the most important food and oil crops in the world. The raw material source of soyasaponins is rich, cheap, and easily available. Therefore, it is of great significance to study and develop the biological activities of soyasaponins.

[0004] Soyasaponins have various biological activities such as anti-cancer, anti-mutagenic, hypoglycemic, cholesterol-lowering, liver-protecting, immunomodulatory, neuroprotective, anticoagulant, anti-inflammatory, and antioxidant. However, there is currently no report on the use of soyasaponins for the treatment of porcine reproductive and respiratory syndrome. Summary of the Invention

[0005] To solve the technical problem of the lack of effective drugs for treating porcine reproductive and respiratory syndrome caused by PRRSV infection, the present invention studied the inhibitory effect of soyasaponins on PRRSV proliferation. Through indirect immunofluorescence experiments and immunoblotting experiments, it was found that administering soyasaponins simultaneously with, before, and after PRRSV infection can all inhibit the proliferation of PRRSV.

[0006] To solve the above technical problems and achieve the corresponding technical effects, the present invention provides the following technical solutions:

[0007] The first object of the present invention is to provide the use of soyasaponin in the preparation of animal drugs for anti-porcine reproductive and respiratory syndrome virus.

[0008] The second object of the present invention is to provide the use of soyasaponin in the preparation of animal drugs for preventing porcine reproductive and respiratory syndrome.

[0009] The third object of the present invention is to provide the use of soyasaponin in the preparation of animal drugs for treating porcine reproductive and respiratory syndrome.

[0010] The fourth object of the present invention is to provide an animal drug for anti-porcine reproductive and respiratory syndrome virus, characterized in that the animal drug takes soyasaponin as an active ingredient.

[0011] The fifth object of the present invention is to provide an animal drug for preventing porcine reproductive and respiratory syndrome, characterized in that the animal drug takes soyasaponin as an active ingredient.

[0012] The sixth object of the present invention is to provide an animal drug for treating porcine reproductive and respiratory syndrome, characterized in that the animal drug takes soyasaponin as an active ingredient.

[0013] In one embodiment of the present invention, the animal drug contains excipients acceptable in animal pharmacy.

[0014] In one embodiment of the present invention, the dosage form of the animal drug is injection, oral liquid, pill, granule, capsule or tablet.

[0015] In one embodiment of the present invention, the administration method of the animal drug is injection administration or oral administration.

[0016] In one embodiment of the present invention, the excipient is a liquid excipient or a solid excipient.

[0017] In one embodiment of the present invention, the effective concentration of soyasaponin in the animal drug is greater than 20 μg / mL.

[0018] In one embodiment of the present invention, the animal drug can be used alone or in combination with other drugs.

[0019] In one embodiment of the present invention, the animal drug can be administered once or continuously for multiple times.

[0020] The beneficial effects of the present invention:

[0021] The present invention studied the inhibitory effect of soyasaponins on the proliferation of PRRSV. Through indirect immunofluorescence assay and immunoblotting assay, it was first found that administering soyasaponins simultaneously with PRRSV infection, before PRRSV infection, and after PRRSV infection could all inhibit the proliferation of different epidemic strains of PRRSV, play a role in the antiviral response of the body, and the inhibitory effect of soyasaponins on the proliferation of PRRSV had a time-dependent effect and a dose-dependent effect. Therefore, soyasaponins have the prospect of being developed into drugs against porcine reproductive and respiratory syndrome virus and have important clinical application prospects in the prevention and treatment of PRRSV-infected diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the chemical structural formula diagram of soyasaponin;

[0023] Figure 2 is the result diagram of the effect of different concentrations of soyasaponins on the proliferation ability of PRRSV detected by indirect immunofluorescence assay when soyasaponins and PRRSV are simultaneously added to Marc-145 cells;

[0024] Figure 3 is the result diagram of the effect of adding soyasaponins in advance on the proliferation ability of PRRSV after inoculating PRRSV at different culture times of Marc-145 cells detected by indirect immunofluorescence assay;

[0025] Figure 4 is the result diagram of the effect of adding soyasaponins after different infection times of inoculating PRRSV into Marc-145 cells on the proliferation ability of PRRSV detected by indirect immunofluorescence assay;

[0026] Figure 5 is the result diagram of the effect of adding Marc-145 cells after soyasaponins and PRRSV act on each other in vitro for different times on the proliferation ability of PRRSV detected by indirect immunofluorescence assay;

[0027] Figure 6 is the result diagram of the effect of adding soyasaponins in advance on the proliferation ability of PRRSV after inoculating PRRSV at different culture times of Marc-145 cells detected by immunoblotting assay;

[0028] Figure 7 is the result diagram of the effect of adding soyasaponins after different infection times of inoculating PRRSV into Marc-145 cells on the proliferation ability of PRRSV detected by immunoblotting assay;

[0029] Figure 8 is the result diagram of the inhibitory effect of soyasaponins on the proliferation of different epidemic strains of PRRSV on Marc-145 cells detected by indirect immunofluorescence assay. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention will be further described in detail below in conjunction with specific embodiments and the accompanying drawings. The embodiments are only intended to illustrate the present invention by way of example, rather than to limit the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements are within the protection scope of the present invention. The processes, conditions, experimental methods, reagents, etc. for implementing the present invention are all common knowledge in the art and conventional products on the market, except for the specifically mentioned content below. The present invention has no special restrictive content.

[0031] The chemical structural formula of soyasaponin (SS) used in the present invention is as Figure 1 shown, and the molecular formula is C 47 H 76 O 17 , with a molecular weight of 913.1 g / mol, a purity of not less than 98%, and a stock solution with a storage concentration is prepared for use;

[0032] The Marc-145 cell line used in the present invention is cryopreserved in liquid nitrogen in this laboratory;

[0033] The RPMI-1640 culture medium used in the present invention is purchased from Gibco, and fetal bovine serum is purchased from Procell;

[0034] The PRRSV used in the present invention includes HuN4 strain, VR2332 strain, NADC 30-like strain and CH-1a strain. The above-mentioned PRRSV epidemic strains are all preserved by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences;

[0035] The penicillin, streptomycin, PMSF, PVDF, and BAC protein detection kits used in the present invention are all purchased from Beijing Biocytogen;

[0036] The CCK-8 cell proliferation toxicity detection kit used in the present invention is purchased from Dojindo;

[0037] The cell culture flasks, 12-well cell culture plates, and 96-well cell culture plates used in the present invention are all purchased from NEST Company.

[0038] The present invention adopts the following method to culture the Marc-145 cell line:

[0039] The Marc-145 cell line cryopreserved in liquid nitrogen is thawed according to the conventional method and cultured in RPMI-1640 culture medium containing 8% fetal bovine serum, 100 U / mL penicillin, and 0.1 mg / mL streptomycin, and cultured in a constant temperature incubator at 37 °C and 5% CO2. Passage is carried out every 2-3 days, and cells in the logarithmic growth phase are taken for experiments.

[0040] Example 1: Application of soyasaponin in inhibiting the proliferation of PRRSV

[0041] 1. Determination of the cytotoxic effect of soyasaponin on Marc-145 cells by CCK-8 method

[0042] Marc-145 cells in the logarithmic growth phase were digested and resuspended to prepare a cell suspension with a density of 5×10 5 / mL. The cell suspension was inoculated into a 96-well plate at an inoculation volume of 200 μL per well. After the cells grew to 90% confluence, the culture medium was replaced with complete medium containing soyasaponin at concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, and 320 μg / mL. Each concentration was set with 3 replicates, and the complete medium without soyasaponin was set as the blank group. The cells were further cultured in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for 24, 48, and 72 h. Then, 10 μL of CCK-8 reagent was added to each well, and the OD 450 value was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent detector. The experimental results showed that soyasaponin had no toxicity to Marc-145 cells when the concentration was less than or equal to 20 μg / mL.

[0043] 2. Detection of the effect of soyasaponin on the proliferation of PRRSV by indirect immunofluorescence assay

[0044] In this experiment, a simultaneous drug addition group, an early drug addition group, a late drug addition group, and an in vitro co-treatment group were set up.

[0045] Simultaneous drug addition group: Marc-145 cells in the logarithmic growth phase were digested and resuspended to prepare a cell suspension, which was inoculated into a 12-well plate at a volume of 1 mL per well. After the cells grew to 90% confluence, the culture medium was replaced with complete medium containing 5 μg / mL, 10 μg / mL, and 20 μg / mL soyasaponin, and the cells cultured without soyasaponin were set as the control group. At the same time, 0.5 MOI of PRRSV was added, and the cells were further cultured in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for 48 h. The cells were fixed with 75% ethanol solution by volume, washed with PBS, and PRRSV monoclonal antibody and the corresponding secondary antibody were added, and then observed under a fluorescence microscope.

[0046] Early drug addition group: Marc-145 cells in the logarithmic growth phase were digested and resuspended to prepare a cell suspension, which was inoculated into a 12-well plate at a volume of 1 mL per well. After the cells grew to 90% confluence, the culture medium was replaced with a complete medium containing 20 μg / mL soyasaponin. After culturing for 8 h, 6 h, 4 h, 2 h, and 0 h respectively, 0.5 MOI of PRRSV was added, and the cells were further cultured in a constant temperature incubator at 37 °C, 5% CO2, and saturated humidity for 48 h. Then the cells were fixed with 75% ethanol solution by volume, washed with PBS, PRRSV monoclonal antibody and the corresponding secondary antibody were added, and observed under a fluorescence microscope.

[0047] Late drug addition experimental group: Marc-145 cells in the logarithmic growth phase were digested and resuspended to prepare a cell suspension, which was inoculated into a 12-well plate at a volume of 1 mL per well. After the cells grew to 90% confluence, 0.5 MOI of PRRSV was inoculated. At 2 h, 4 h, 6 h, and 8 h after infection, 20 μg / mL soyasaponin was added respectively. The cells were further cultured in a constant temperature incubator at 37 °C, 5% CO2, and saturated humidity for 48 h. Then the cells were fixed with 75% ethanol solution by volume, washed with PBS, PRRSV monoclonal antibody and the corresponding secondary antibody were added, and observed under a fluorescence microscope.

[0048] In vitro co-treatment group: 20 μg / mL soyasaponin and 1 MOI of PRRSV were mixed in vitro in DMEM medium containing 2% serum, and placed in a 37 °C constant temperature incubator for 0.5 h, 1 h, and 2 h respectively. Then it was inoculated into a 12-well plate with Marc-145 cells that had grown to 90% confluence, and further cultured in a constant temperature incubator at 37 °C, 5% CO2, and saturated humidity for 48 h. Then the cells were fixed with 75% ethanol solution by volume, washed with PBS, PRRSV monoclonal antibody and the corresponding secondary antibody were added, and observed under a fluorescence microscope.

[0049] Figure 2 The inhibitory effects of different concentrations of soyasaponin (5 μg / mL, 10 μg / mL, 20 μg / mL) on PRRSV proliferation in the simultaneous drug addition group were shown. It can be seen from this figure that the inhibitory effect of soyasaponin is dose-dependent, and the virus proliferation can be completely inhibited at 20 μg / mL; Figure 3 The inhibitory effects of adding soyasaponin with a concentration of 20 μg / mL in advance on PRRSV proliferation after inoculating PRRSV at different times (8 h, 6 h, 4 h, 2 h, and 0 h) in the early drug addition group were shown. It can be seen from this figure that the longer the time of adding the drug in advance, the better the inhibitory effect; Figure 4It shows the inhibitory effect of adding soyasaponin at a concentration of 20 μg / mL on the proliferation of PRRSV after Marc-145 cells were inoculated with PRRSV at different infection times (2 h, 4 h, 6 h, 8 h) in the post-drug addition experimental group. It can be seen from this figure that the earlier the drug is administered after infection, the better the inhibitory effect, and the inhibitory effect is the best when the drug is administered 2 h after virus infection. Figure 5 It shows the inhibitory effect of adding soyasaponin at a concentration of 20 μg / mL and PRRSV on the proliferation of PRRSV after different incubation times in vitro and then adding them to Marc-145 cells in the in vitro co-treatment group. It can be seen from this figure that the longer the co-treatment time of soyasaponin and PRRSV in vitro, the better the inhibitory effect on PRRSV.

[0050] Based on the above results, it can be found that soyasaponin administered in different ways can significantly inhibit the proliferation of PRRSV in Marc-145 cells. The experimental results show that the inhibitory effect of soyasaponin on the proliferation of PRRSV has a dose-dependent effect and a time-dependent effect.

[0051] 3. Detection of the effect of soyasaponin on the proliferation of PRRSV by Western blotting (WB)

[0052] Repeat the experiment in 2 above, with the difference that after the cells were cultured for another 48 h, the cells were collected, washed with PBS, lysed with cell lysis buffer containing PMSF, the protein concentration was determined using a BCA protein assay kit, 20 μg of protein from each group was electrophoresed on 12% SDS-PAGE, then transferred to a PVDF membrane, the PVDF membrane was blocked with 5% skim milk powder, washed with PBST, incubated with PRRSV monoclonal antibody as the primary antibody at room temperature for 2 h, the membrane was washed, incubated with horseradish peroxidase-labeled goat anti-mouse IgG at room temperature for 1 h, the reaction bands were exposed in an ECL system, and gray-scale analysis was performed using Image J software.

[0053] Figure 6 It shows the inhibitory effect of adding soyasaponin at a concentration of 20 μg / mL in advance on the proliferation of PRRSV after Marc-145 cells were inoculated with PRRSV at different culture times (8 h, 6 h, 4 h, 2 h, and 0 h) in the pre-drug addition group. Figure 7 It shows the inhibitory effect of adding soyasaponin at a concentration of 20 μg / mL after Marc-145 cells were inoculated with PRRSV at different infection times (2 h, 4 h, 6 h, 8 h) in the post-drug addition experimental group on the proliferation of PRRSV. Figure 6 and Figure 7 The results shown are consistent with those obtained by indirect immunofluorescence assay, and it can also indicate that soyasaponin administered in different ways can significantly inhibit the proliferation of PRRSV in Marc-145 cells. The inhibitory effect of soyasaponin on the proliferation of PRRSV has a dose-dependent effect and a time-dependent effect.

[0054] 4. Detection of the inhibitory effect of soybean saponins on the proliferation of different prevalent strains of PRRSV by indirect immunofluorescence assay

[0055] Marc-145 cells in the logarithmic growth phase were digested, resuspended to prepare a cell suspension, and inoculated into a 12-well plate at a volume of 1 mL per well. After the cells grew to 90% confluence, 0.5 MOI of PRRSV was added. After 2 hours of infection, soybean saponins with concentrations of 5 μg / mL, 10 μg / mL, and 20 μg / mL were added respectively, and the cells were further cultured in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for 48 hours. Then the cells were fixed with 75% ethanol solution by volume, washed with PBS, and PRRSV monoclonal antibody and the corresponding secondary antibody were added, and observed under a fluorescence microscope.

[0056] The results are as Figure 8 shown. Soybean saponins can inhibit the proliferation of different prevalent strains of PRRSV, and the inhibitory effect is dose-dependent. When the concentration of soybean saponins is 20 μg / mL, the inhibitory effect on the proliferation of different prevalent strains of PRRSV is the best.

[0057] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Use of soyasaponin in the preparation of animal drugs for anti-porcine reproductive and respiratory syndrome virus, characterized in that, The structural formula of the soyasaponin is as follows:

2. Use of soyasaponin in the preparation of animal drugs for preventing porcine reproductive and respiratory syndrome, characterized in that, The structural formula of the soyasaponin is as follows:

3. Use of soyasaponin in the preparation of animal drugs for treating porcine reproductive and respiratory syndrome, characterized in that, The structural formula of the soyasaponin is as follows:

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