Application of soyasaponin in preparation of medicine for preventing and / or treating porcine reproductive and respiratory syndrome
By studying the inhibitory effect of soy saponins on pig reproduction and respiratory syndrome virus (PRRSV), it was found that soy saponins can effectively inhibit viral proliferation at different times and doses, solving the problem of lack of effective drugs in the existing technology to inhibit PRRSV infection, and providing new drug development directions and clinical application prospects.
Patent Information
- Application Number
- CN202510034867.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-03-14
- Estimated Expiration
- 2045-01-09
AI Technical Summary
There is currently no effective drug that can quickly and efficiently inhibit pig breeding and respiratory syndrome caused by pig breeding and respiratory syndrome virus (PRRSV) infection.
The inhibitory effect of soy saponin on PRRSV proliferation was studied. Through indirect immunofluorescence experiments and immunoblotting experiments, it was found that administration of soy saponin on PRRSV infection, before and after infection, respectively, can effectively inhibit the proliferation of PRRSV.
Soybean saponin can significantly inhibit the proliferation of PRRSV in cells, and its inhibitory effect is time- and dose-dependent, providing the prospect of developing anti-pig reproduction and respiratory syndrome virus drugs, and has important clinical application value in the prevention and treatment of diseases.
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Figure CN119607002A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biological function research of phytochemicals, and specifically relates to the application of soybean saponin in preparing medicines for preventing and / or treating porcine reproductive and respiratory syndrome. Background Art
[0002] Porcine reproductive and respiratory syndrome (PRRS) is a major disease that currently threatens the pig industry. The disease has an acute onset, rapid spread, and high mortality rate. It is mainly characterized by respiratory disorders in piglets and reproductive disorders in sows. The pathogen of the disease is porcine reproductive and respiratory syndrome virus (PRRSV). The clinical manifestations of porcine reproductive and respiratory syndrome around the world are very similar, but despite this, their respective pathogens have great differences in virulence, antigenicity, and genetic characteristics. Based on these differences, PRRSV is divided into two subtypes, namely North American and European. 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. At present, there is no effective drug reported for pig diseases caused by this virus infection. Therefore, there is an urgent need for a method that can quickly and efficiently inhibit PRRSV infection to lay a foundation for the study of anti-PRRSV mechanisms and the prevention and control of PRRS disease in pig farms.
[0003] Soy saponin is a kind of saponin widely found in soybean and its products of the legume family. It is a kind of phytochemical formed by the condensation of soybean sapogenin and oligosaccharide, and belongs to the pentacyclic triterpenoid oleanane type compound. Beans are the main source of soy saponin in the diet, and soybean is one of the most important food and oil crops in the world. The raw material of soy saponin is abundant, cheap and easy to obtain, so it is of great significance to study and develop the biological activity of soy saponin.
[0004] Soybean saponins have multiple biological activities such as anti-cancer, anti-mutagenic, hypoglycemic, hypocholesterolemic, hepatoprotective, immunomodulatory, neuroprotective, anticoagulant, anti-inflammatory, and antioxidant. However, there is no report on the use of soybean saponins in the treatment of porcine reproductive and respiratory syndrome. Summary of the invention
[0005] In order 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 studies the inhibitory effect of soybean saponins on the proliferation of PRRSV. Through indirect immunofluorescence experiments and immunoblotting experiments, it is found that the administration of soybean saponins at the same time as PRRSV infection, before PRRSV infection and after PRRSV infection can inhibit the proliferation of PRRSV.
[0006] In order to solve the above technical problems and achieve 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 soybean saponin in preparing animal medicines against porcine reproductive and respiratory syndrome virus.
[0008] The second object of the present invention is to provide the use of soybean saponin in the preparation of animal medicines for preventing porcine reproductive and respiratory syndrome.
[0009] The third object of the present invention is to provide the use of soybean saponin in the preparation of animal medicines for treating porcine reproductive and respiratory syndrome.
[0010] The fourth object of the present invention is to provide an animal drug for resisting porcine reproductive and respiratory syndrome virus, characterized in that the animal drug uses soybean saponin as an active ingredient.
[0011] A fifth object of the present invention is to provide an animal medicine for preventing porcine reproductive and respiratory syndrome, characterized in that the animal medicine uses soybean saponin as an active ingredient.
[0012] The sixth object of the present invention is to provide an animal medicine for treating porcine reproductive and respiratory syndrome, characterized in that the animal medicine uses soybean saponin as an active ingredient.
[0013] In one embodiment of the present invention, the animal drug contains excipients that are pharmaceutically acceptable to animals.
[0014] In one embodiment of the present invention, the dosage form of the animal drug is injection, oral solution, pill, granule, capsule or tablet.
[0015] In one embodiment of the present invention, the animal drug is administered by injection or oral administration.
[0016] In one embodiment of the present invention, the auxiliary material is a liquid auxiliary material or a solid auxiliary material.
[0017] In one embodiment of the present invention, the effective concentration of soybean saponin 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 multiple times continuously.
[0020] Beneficial effects of the present invention:
[0021] The present invention studies the inhibitory effect of soybean saponin on the proliferation of PRRSV. Through indirect immunofluorescence experiments and immunoblotting experiments, it is found for the first time that administration of soybean saponin at the same time of PRRSV infection, before PRRSV infection and after PRRSV infection can inhibit the proliferation of different epidemic strains of PRRSV, can play a role in the body's antiviral response, and the inhibitory effect of soybean saponin on the proliferation of PRRSV has time-dependent effects and dose-dependent effects. Therefore, soybean saponin has the prospect of being developed into an anti-porcine reproductive and respiratory syndrome virus drug, and has important clinical application prospects in the prevention and treatment of PRRSV infection diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 The chemical structure diagram of soybean saponin;
[0023] Figure 2 The results of indirect immunofluorescence assay to detect the effect of different concentrations of soybean saponin on the proliferation ability of PRRSV when soybean saponin and PRRSV were added to Marc-145 cells at the same time;
[0024] Figure 3 The results of indirect immunofluorescence assay to detect the effect of adding soybean saponin in advance on the proliferation ability of PRRSV after inoculation of PRRSV in Marc-145 cells at different culture times;
[0025] Figure 4 The results of indirect immunofluorescence detection of the effect of adding soybean saponin on the proliferation ability of PRRSV after inoculation of PRRSV in Marc-145 cells for different infection time;
[0026] Figure 5 This is the result of indirect immunofluorescence assay to detect the effect of soybean saponin and PRRSV on the proliferation ability of PRRSV after adding Marc-145 cells after different in vitro exposure time;
[0027] Figure 6 The results of Western blotting to detect the effect of adding soybean saponin in advance on the proliferation ability of PRRSV after inoculation of PRRSV in Marc-145 cells at different time periods;
[0028] Figure 7 The results of Western blotting to detect the effect of adding soybean saponin on the proliferation ability of PRRSV after inoculating PRRSV into Marc-145 cells for different infection time;
[0029] Figure 8 This is a graph showing the inhibitory effect of soybean saponins on the proliferation of different epidemic strains of PRRSV on Marc-145 cells detected by indirect immunofluorescence method. DETAILED DESCRIPTION
[0030] The present invention is further described in detail below in conjunction with specific examples and accompanying drawings, and the embodiments are intended to illustrate the present invention only, rather than to limit the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements are within the protection scope of the present invention. The process, conditions, experimental methods and reagents for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and conventional products in the market in the art, and the present invention does not particularly limit the contents.
[0031] The chemical structural formula of soybean saponin (SS) used in the present invention is as follows Figure 1 As shown, the molecular formula is C 47 H 76 O 17 , the molecular weight is 913.1g / mol, the purity is not less than 98%, and the storage concentration solution is prepared for standby use;
[0032] The Marc-145 cell line used in the present invention was cryopreserved in liquid nitrogen in this laboratory;
[0033] The RPMI-1640 culture medium used in the present invention was purchased from Gibco, and the fetal bovine serum was purchased from Procell;
[0034] The PRRSV used in the present invention includes HuN4 strain, VR2332 strain, NADC 30-like strain and CH-1a strain, and the above PRRSV epidemic strains are all preserved by Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences;
[0035] The penicillin, streptomycin, PMSF, PVDF, and BAC protein detection kit used in the present invention were purchased from Beijing Bio-Tech.
[0036] The CCK-8 cell proliferation toxicity detection kit used in the present invention was purchased from Tongren Chemical;
[0037] The cell culture flask, 12-well cell culture plate and 96-well cell culture plate used in the present invention were purchased from NEST Company.
[0038] The present invention adopts the following method to culture Marc-145 cell line:
[0039] The Marc-145 cell line frozen in liquid nitrogen was revived according to conventional methods and cultured in RPMI-1640 culture medium containing 8% fetal bovine serum, 100U / mL penicillin, and 0.1mg / mL streptomycin in a constant temperature incubator at 37°C and 5% CO2. The cells were passaged every 2-3 days, and the cells in the logarithmic growth phase were used for experiments.
[0040] Example 1: Application of soybean saponin in inhibiting the proliferation of PRRSV
[0041] 1. CCK-8 method to determine the toxic effect of soybean saponins on Marc-145 cells
[0042] Marc-145 cells in the logarithmic growth phase were taken, digested and resuspended to a density of 5×10 5 / mL of cell suspension was inoculated into a 96-well plate at an inoculum volume of 200 μL per well. After the cells grew to 90% of the monolayer, the culture medium was replaced with complete culture medium containing 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL and 320 μg / mL of soybean saponin, respectively. Three replicate wells were set for each concentration, and a complete culture medium without soybean saponin was set as a blank group. The plates were placed in a constant temperature incubator at 37°C, 5% CO2 and saturated humidity for 24, 48 and 72 h, and then 10 μL of CCK-8 reagent was added to each well. After incubation for 4 h, the OD was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay. 450 The experimental results showed that soybean saponin had no toxicity to Marc-145 cells when the concentration was less than or equal to 20 μg / mL.
[0043] 2. Indirect immunofluorescence assay to detect the effect of soybean saponins on PRRSV proliferation
[0044] This experiment set up a simultaneous drug addition group, an advance drug addition group, a post-drug addition group and an in vitro co-treatment group.
[0045] Simultaneous drug addition group: Marc-145 cells in the logarithmic growth phase were taken, digested and resuspended to make a cell suspension, and 1 mL per well was inoculated in a 12-well plate. After the cells grew to 90% of the monolayer, the culture medium was replaced with complete culture medium containing 5 μg / mL, 10 μg / mL, and 20 μg / mL soybean saponin, respectively. Cells cultured without soybean saponin were set as the control group, and 0.5 MOI of PRRSV was added at the same time. The cells were placed in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for further culture for 48 h. The cells were fixed with 75% volume fraction of ethanol solution, washed with PBS, and PRRSV monoclonal antibody and corresponding secondary antibody were added, and observed under a fluorescence microscope.
[0046] Pre-drug addition group: Marc-145 cells in the logarithmic growth phase were taken, digested and resuspended to make a cell suspension, and inoculated into a 12-well plate at a volume of 1 mL per well. After the cells grew to 90% of the monolayer, the culture medium was replaced with a complete culture medium containing 20 μg / mL soybean saponin. After culturing for 8 h, 6 h, 4 h, 2 h and 0 h, 0.5 MOI of PRRSV was added, and the cells were placed in a constant temperature incubator at 37 ° C, 5% CO2, and saturated humidity for a further 48 h. The cells were fixed with a volume fraction of 75% ethanol solution, washed with PBS, and PRRSV monoclonal antibody and corresponding secondary antibody were added, and observed under a fluorescence microscope.
[0047] Post-drug treatment experimental group: Marc-145 cells in the logarithmic growth phase were taken, digested and resuspended to make a cell suspension, and inoculated into a 12-well plate at a volume of 1 mL per well. After the cells grew to 90% of the monolayer, 0.5 MOI of PRRSV was inoculated, and 20 μg / mL soybean saponin was added at 2h, 4h, 6h, and 8h after infection. The cells were placed in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for further culture for 48h, and then the cells were fixed with a volume fraction of 75% ethanol solution, washed with PBS, and PRRSV monoclonal antibody and corresponding secondary antibody were added, and observed under a fluorescence microscope.
[0048] In vitro co-treatment group: 20 μg / mL soybean saponin and 1 MOI of PRRSV were mixed in vitro in DMEM medium containing 2% serum, placed in a 37°C constant temperature incubator for 0.5 h, 1 h, and 2 h, respectively, and then inoculated into a 12-well plate with 90% monolayer Marc-145 cells, placed in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for further culture for 48 h, and then the cells were fixed with 75% volume fraction of ethanol solution, washed with PBS, added with PRRSV monoclonal antibody and corresponding secondary antibody, and observed under a fluorescence microscope.
[0049] Figure 2 The figure shows the inhibitory effect of soybean saponin at different concentrations (5μg / mL, 10μg / mL, 20μg / mL) on PRRSV proliferation in the simultaneous drug addition group. It can be seen from the figure that the inhibitory effect of soybean saponin is dose-dependent, and it can completely inhibit viral proliferation at 20μg / mL; Figure 3 The figure shows the inhibitory effect of soybean saponin added at a concentration of 20 μg / mL in the pre-drug addition group on the proliferation of PRRSV after inoculation of PRRSV in Marc-145 cells at different culture times (8h, 6h, 4h, 2h and 0h). It can be seen from the figure that the longer the pre-drug addition time, the better the inhibitory effect; Figure 4The figure shows the inhibitory effect of soybean saponin at a concentration of 20 μg / mL on the proliferation of PRRSV in Marc-145 cells inoculated with PRRSV at different infection times (2h, 4h, 6h, 8h) in the post-drug addition experimental group. It can be seen from the figure that the earlier the drug is administered after infection, the better the inhibitory effect, and the best inhibitory effect is achieved 2h after virus infection. Figure 5 The figure shows the inhibitory effect on PRRSV proliferation of soybean saponins at a concentration of 20 μg / mL in the in vitro co-treatment group after being added to Marc-145 cells with PRRSV for different in vitro action times. It can be seen from the figure that the longer the in vitro co-treatment time of soybean saponins and PRRSV, the better the inhibitory effect on PRRSV.
[0050] Based on the above results, it can be found that different ways of administering soybean saponins can significantly inhibit the proliferation of PRRSV in Marc-145 cells. The experimental results show that the inhibitory effect of soybean saponins on PRRSV proliferation has a dose-dependent effect and a time-dependent effect.
[0051] 3. Western blotting (WB) to detect the effect of soybean saponins on PRRSV proliferation
[0052] The experiment in 2 above was repeated, except that after the cells were cultured for another 48 h, the cells were collected, washed with PBS, lysed with cell lysis buffer containing PMSF, and the protein concentration was determined using a BCA protein detection kit. 20 μg of protein from each group was electrophoresed on 12% SDS-PAGE and 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, washed, and incubated with horseradish peroxidase-labeled goat anti-mouse IgG at room temperature for 1 h. The reaction bands were exposed using an ECL system and grayscale analysis was performed using Image J software.
[0053] Figure 6 The results show that the addition of 20 μg / mL soybean saponin in advance in the pre-drug group has an inhibitory effect on the proliferation of PRRSV inoculated after different culture times (8h, 6h, 4h, 2h and 0h) of Marc-145 cells. Figure 7 The results show the inhibitory effect of soybean saponin at a concentration of 20 μg / mL on the proliferation of PRRSV in Marc-145 cells in the post-drug addition experimental group after different infection times (2h, 4h, 6h, 8h). Figure 6 and Figure 7 The results shown are consistent with those obtained by indirect immunofluorescence analysis, and also indicate that different administration methods of soybean saponins can significantly inhibit the proliferation of PRRSV in Marc-145 cells, and that the inhibitory effect of soybean saponins on PRRSV proliferation is dose-dependent and time-dependent.
[0054] 4. Indirect immunofluorescence assay to detect the inhibitory effect of soybean saponins on the proliferation of different epidemic strains of PRRSV
[0055] Marc-145 cells in the logarithmic growth phase were taken, digested and resuspended to make a cell suspension, and inoculated into a 12-well plate at a volume of 1 mL per well. After the cells grew to 90% of the monolayer, 0.5 MOI of PRRSV was inoculated. 2 hours after infection, soybean saponin with concentrations of 5 μg / mL, 10 μg / mL, and 20 μg / mL were added respectively. The cells were placed in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for further culture for 48 hours, and then the cells were fixed with a volume fraction of 75% ethanol solution, washed with PBS, and PRRSV monoclonal antibody and corresponding secondary antibody were added, and observed under a fluorescence microscope.
[0056] The results are as follows Figure 8 As shown, soybean saponins can inhibit the proliferation of different epidemic 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 epidemic strains of PRRSV is the best.
[0057] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.
Claims
1. Application of soybean saponins in the preparation of animal medicines against porcine reproductive and respiratory syndrome virus.
2. Application of soybean saponins in the preparation of animal medicines for preventing porcine reproductive and respiratory syndrome.
3. Application of soybean saponins in the preparation of animal medicines for the treatment of porcine reproductive and respiratory syndrome.
4. An animal drug for resisting porcine reproductive and respiratory syndrome virus, characterized in that: The animal medicine uses soybean saponin as an active ingredient.
5. An animal drug for preventing porcine reproductive and respiratory syndrome, characterized in that: The animal medicine uses soybean saponin as an active ingredient.
6. An animal drug for treating porcine reproductive and respiratory syndrome, characterized in that: The animal medicine uses soybean saponin as an active ingredient.
7. The animal drug according to any one of claims 4 to 6, characterized in that: The animal medicine contains auxiliary materials acceptable to animal pharmacy.
8. The animal drug according to any one of claims 4 to 6, characterized in that: The dosage form of the animal medicine is injection, oral solution, pill, granule, capsule or tablet.
9. The animal drug according to any one of claims 4 to 6, characterized in that: The administration method of the animal drug is injection or oral administration.
10. The animal drug according to claim 7, characterized in that: The auxiliary material is a liquid auxiliary material or a solid auxiliary material.