Application of saxifraga stolonifera and product containing saxifraga stolonifera

Abstract

The invention discloses application of saxifraga stolonifera in preparation of a product for preventing and treating animal epidemic diarrhea and a product containing the saxifraga stolonifera, and belongs to the technical field of animal disease prevention and control. Tests prove that the saxifraga stolonifera aqueous extract has an inhibition effect on PEDV (Porcine Epidemic Diarrhea Virus). The action mechanism of the saxifraga stolonifera for preventing and treating the PED is disclosed from the molecular level, a theoretical basis is provided for prevention and treatment of the PED, and the PED prevention and treatment method which is safe, efficient and clear in mechanism is provided.

Description

Technical Field

[0001] The present invention relates to the technical field of animal disease prevention and control, and more specifically to application of saxifrage in preparing products for preventing and treating animal epidemic diarrhea and products containing saxifrage. Background Art

[0002] Porcine epidemic diarrhea (PED) is an acute, contagious intestinal disease caused by porcine epidemic diarrhea virus (PEDV).

[0003] PEDV is an enveloped single-stranded positive-strand RNA virus, belonging to the genus Alphacoronavirus of the Coronaviridae family. Its main structural proteins include Spike (S) protein, Membrane (M) protein, Envelope (E) protein and Nucleocapsid (N) protein. Each protein plays an important role in the reproduction of the virus. N protein is highly conserved, accounting for the largest proportion of the structural proteins of the virus. It is also a basic structural protein with the highest expression during viral replication. It is essential for inducing cellular immunity and is one of the target proteins for early and accurate detection of PEDV infection. PEDV can be transmitted through feces, breast milk, aerosols and other routes, infecting the small intestine and causing intestinal villus atrophy, spreading rapidly and causing clinical symptoms such as severe diarrhea, vomiting and dehydration. The virus has the characteristics of easy mutation and recombination and high pathogenicity, while the existing inactivated vaccine has the defect of low protection efficiency, and the attenuated vaccine has the risk of anti-strengthening, which makes the mortality rate of piglets reach more than 80%. Therefore, it is urgent to develop new products for the prevention and treatment of epidemic diarrhea. Summary of the invention

[0004] The invention provides application of saxifrage in preparing products for preventing and treating animal epidemic diarrhea, so as to solve the problem of low efficiency of PED prevention and treatment methods in the prior art.

[0005] In a first aspect, the present invention provides the use of Saxifraga in preparing a product for preventing and treating animal epidemic diarrhea.

[0006] As a possible implementation, the animal is a pig.

[0007] As a possible implementation manner, the saxifrage is saxifrage dry grass or saxifrage water extract, and the saxifrage water extract is obtained by extracting saxifrage using water as an extraction solvent.

[0008] As a possible implementation, when preparing the Saxifraga water extract, the usage ratio of Saxifraga to water is 1 g: 8 mL to 12 mL.

[0009] As a possible implementation method, the extract is soaked after adding water, boiled for 2 to 3 times, each time for 1 hour, and the decoctions are combined to obtain the water extract of Saxifraga herba.

[0010] As a possible implementation manner, the soaking time is 30 minutes to 40 minutes.

[0011] As a possible implementation, the effective dosage of the Saxifraga water extract is 200 μg / mL to 400 μg / mL.

[0012] In a second aspect, the present invention further provides a product for preventing and treating porcine epidemic diarrhea, wherein the product contains the Saxifraga described in claim 1 as the only effective active ingredient.

[0013] As a possible implementation, the product is a medicine or a feed additive.

[0014] The medicine or feed additive prepared from saxifrage can be selected in a suitable dosage form, such as granules, powders, liquid preparations or injection preparations. When preparing a specific dosage form, suitable adjuvants or carriers can be added. Exemplary, the optional adjuvants are viscosity enhancers, preservatives, flavoring agents, pH regulators, osmotic pressure regulators, etc. When prepared as a feed additive for use, the prepared feed additive can be mixed with conventional feeds, such as corn, wheat bran, soybean meal or other commercially available feeds.

[0015] The present invention provides the use of Saxifraga in resisting epidemic diarrhea virus. Experiments have shown that the Saxifraga water extract has an inhibitory effect on PEDV, and the inhibitory activity is achieved by inhibiting PEDV replication and reducing the expression of cellular proinflammatory factors stimulated by PEDV.

[0016] As an effective ingredient for preventing and treating PED, Saxifraga not only has the characteristics of multi-component, multi-pathway and multi-target effects, but also has the advantages of abundant sources, unique chemical structure, diverse activities, small toxic and side effects, and not easy to develop drug resistance. The present invention provides a safe, efficient and clear-mechanism PED prevention and treatment method. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1The results of cell activity and cytotoxicity of Vero E6 or IPEC-J2 cells affected by Sax.s concentration provided in the embodiments of the present invention; wherein A is the effect of Sax.s concentration on the cell activity of Vero E6 cells, B is the cytotoxicity of Sax.s concentration on Vero E6 cells, C is the effect of Sax.s concentration on the cell activity of IPEC-J2 cells, and D is the cytotoxicity of Sax.s concentration on IPEC-J2 cells.

[0019] Figure 2 The results of Sax.s anti-PEDVN gene expression provided in the embodiments of the present invention, wherein A is the effect of Sax.s concentration on PEDV N gene expression in Vero E6 cells, and B is the effect of Sax.s concentration on PEDVN gene expression in IPEC-J2 cells.

[0020] Figure 3 The results of Sax.s anti-PEDVN protein expression provided in the embodiments of the present invention, wherein A and C are the expression of PEDVN protein in VeroE6 and IPEC-J2 cells, respectively, and B and D are the gray value analysis of PEDVN protein expression in Vero E6 and IPEC-J2 cells, respectively.

[0021] Figure 4 This is a schematic diagram of the effect of Sax.s provided by an embodiment of the present invention on the PEDV infection stage, wherein Adsorption means adsorption, Internalization means invasion, Replication means replication, and Release means release.

[0022] Figure 5 The effect of Sax.s provided in the embodiment of the present invention on the expression of N gene in the PEDV infection stage on Vero E6 cells, wherein A is the adsorption stage, B is the invasion stage, C is the replication stage, and D is the release stage.

[0023] Figure 6 The results of Sax.s provided in the embodiments of the present invention on the N gene expression of PEDV infection stage on IPEC-J2 cells, wherein A is the adsorption stage, B is the invasion stage, C is the replication stage, and D is the release stage.

[0024] Figure 7 The results of Sax.s on the expression of N protein in the replication stage of PEDV provided in the embodiments of the present invention, wherein A and C are the expression of PEDV N protein in Vero E6 and IPEC-J2 cells, respectively, and B and D are the gray value analysis of PEDV N protein expression in Vero E6 and IPEC-J2 cells, respectively.

[0025] Figure 8 The results of Sax.s provided in the embodiments of the present invention on the expression of cytokines of Vero E6 infected with PEDV, wherein A is the expression level of TNF-α, B is the expression level of IL-1β, C is the expression level of IL-6, D is the expression level of IL-8, and E is the expression level of IL-10.

[0026] Fig. 9 The results of Sax.s provided in the embodiments of the present invention on the expression of cytokines of IPEC-J2 infected with PEDV, wherein A is the expression level of TNF-α, B is the expression level of IL-1β, C is the expression level of IL-6, D is the expression level of IL-8, and E is the expression level of IL-10.

[0027] Fig.10 The results of the interaction between PEDV N protein and p53 protein by Sax.s on Vero E6 cells provided in the embodiments of the present invention, wherein A is the Co-IP analysis result, and B is the PEDV N / p53 gray value analysis result.

[0028] Fig.11 The results of Sax.s on the expression of p53-DREAM signaling pathway related proteins in Vero E6 cells provided in an embodiment of the present invention, wherein A is the expression of p53-DREAM signaling pathway related proteins in Vero E6 cells, and B to I are gray value analyses of p53, p21, p107, p130, p-p130 (Ser672), p-p130 (Ser952), E2F4 and Cyclin, respectively.

[0029] Fig.12 The results of the interaction between Sax.s on PEDVN protein and p53 protein on IPEC-J2 cells provided in the embodiments of the present invention, wherein A is the Co-IP analysis result, and B is the PEDV N / p53 gray value analysis result.

[0030] Fig.13 The results of Sax.s on the expression of p53-DREAM signaling pathway related proteins in IPEC-J2 cells provided in the embodiments of the present invention, wherein A is the expression of p53-DREAM signaling pathway related proteins in Vero E6 cells, and B to I are the gray value analysis of p53, p21, p107, p130, p-p130 (Ser672), p-p130 (Ser952), E2F4 and Cyclin, respectively. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In order to solve the problem of low efficiency of PED prevention and control methods in the prior art, the present invention provides the use of Saxifraga in resisting epidemic diarrhea virus. Experiments have shown that the water extract of Saxifraga has an inhibitory effect on PEDV, which achieves the inhibitory activity by inhibiting PEDV replication and reducing the expression of cellular proinflammatory factors stimulated by PEDV.

[0033] The present invention reveals the mechanism of action of Saxifraga in preventing and controlling PED at the molecular level, and provides a theoretical basis for the prevention and control of PED. As an effective ingredient for preventing and controlling PED, Saxifraga not only has the characteristics of multi-component, multi-pathway, and multi-target action, but also has the advantages of abundant sources, unique chemical structure, diverse activities, low toxicity and side effects, and not easy to produce drug resistance, etc., providing a safe, efficient, and clear mechanism of PED prevention and control method.

[0034] The saxifrage hay used in the examples of the present invention was purchased from Beijing Tong Ren Tang; Vero E6 cells, IPEC-J2 cells and PEDV LW / L strains were all stored in Beijing Key Laboratory of Veterinary Medicine (Traditional Chinese Medicine). The main reagents used are shown in Table 1, and the main instruments used are shown in Table 2.

[0035] Table 1 Reagents and sources used in the examples of the present invention

[0036]

[0037]

[0038] Table 2 Instruments and sources used in the examples of the present invention

[0039]

[0040]

[0041] The primer sequences used in PCR in the examples of the present invention are shown in Table 3.

[0042] Table 3 Primer sequences

[0043]

[0044] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0045] Example 1

[0046] This example provides a cytotoxicity assay of the water extract of Saxifraga heracleifolia.

[0047] Cut the dried Saxifrage into pieces, add deionized water at a mass ratio of 1:10, soak for 30 minutes, boil over high heat, simmer for 1 hour over low heat, filter, and retain the filtrate; add deionized water to the residue again, repeat the decocting once according to the above steps, combine the two filtrates, dry with a vacuum rotary evaporator, and obtain the Saxifrage water extract, which is marked as Sax.s.

[0048] Preheat the complete medium, which is composed of DMEM + 10% FBS + 1% double antibody. Take out the Vero E6 cells frozen in liquid nitrogen, quickly place them in a 37% constant temperature water bath, and shake them gently for 1 to 2 minutes. Add 2 mL of complete medium to a clean 15 mL centrifuge tube in the clean bench, slowly add the melted Vero E6 cell mixture to the wall, mix well by blowing and aspirating, and centrifuge at 1000r / min for 5 minutes. Discard the supernatant, add complete medium again to fully resuspend the cells accumulated by centrifugation, mix well, transfer to a T25 cell culture flask, and culture in a cell culture incubator at 37°C and 5% CO2.

[0049] 100 μL Vero E6 cell suspension was evenly inoculated into a 96-well plate at a density of 1 × 10 cells per well. 4 After mixing, place in a 5% CO2 incubator at 37°C and culture until monolayer; set up cell control wells and blank wells without cells at the same time. Dilute the prepared Sax.s with DMEM culture medium, filter with a 0.22μm filter, discard the culture medium, add different concentrations of Sax.s (3200, 1600, 800, 400, 200, 100, 50, 25μg / mL) to the 96-well plate at a dose of 100μL / well, repeat 3 times for each concentration, discard the culture medium after incubation for 12h, rinse twice with D-Hanks, add DMEM culture medium (containing 10% CCK-8 solution) at a dose of 100μL / well, incubate in the incubator in the dark for 2h, and measure its absorbance with a 450nm microplate reader. Perform the same operation using IPEC-J2 cells instead of Vero E6 cells.

[0050] GraphPad Prism 9.0 software was used to calculate the 50% inhibition concentration (IC 50 ), cell viability (%) = (OD administration - OD blank) / (OD control - OD blank) × 100%. Figure 1 Results shown.

[0051] Depend on Figure 1As shown in A and C, as the drug concentration increases, the cytotoxicity also increases, indicating that the cytotoxicity is positively correlated with the drug concentration; when the Sax.s concentration is less than 400 μg / mL, the cell morphology of Vero E6 and IPEC-J2 does not change significantly, and the cell viability remains above 80%.

[0052] Depend on Figure 1 As shown in Figures B and D, the IC values ​​of Sax.s on Vero E6 and IPEC-J2 cells 50 The concentrations of Sax.s were 1091 and 1015 μg / mL respectively. Sax.s at a concentration less than 400 μg / mL had no cytotoxicity to Vero E6 and IPEC-J2 cells.

[0053] Therefore, the concentration selected was IC 20 (400 μg / mL) of Sax.s was used as the subsequent antiviral activity study with different administration methods.

[0054] Example 2

[0055] This example provides the effect of Sax.s on the expression level of PEDV N.

[0056] Vero E6 cells were cultured at 2 × 10 5 Each well was inoculated in a 6-well plate and cultured in an incubator at 37°C and 5% CO2 for 24 hours. The culture wells were divided into a negative control group, a positive control group, and a drug treatment group; the positive control group and the drug treatment group were inoculated with PEDV LW / L (MOI = 0.1 / 1), and the negative control group was added with an equal amount of virus maintenance solution and cultured under the same conditions for 1 hour; the culture medium was discarded, and D-Hanks was added to wash 3 times, and Sax.s was added to the drug treatment group at different concentrations (25, 50, 100, 200, 400 μg / mL), and the negative control group and the positive control group were added with an equal amount of DMEM and cultured under the same conditions for 12 hours. 4×10 5 IPEC-J2 cells / well replace 2×10 5 The same operation was performed on Vero E6 cells per well, and the cell extracts were collected. The relative expression level of PEDV N mRNA was detected by fluorescence quantitative PCR, and the results were as follows: Figure 2 The results shown in the figure are as follows. The relative expression of PEDVN protein was detected by Western Blot method. Figure 3 Results shown.

[0057] Depend on Figure 2 As shown in A, compared with the positive control group, 200 and 400 μg / mL of Sax.s significantly inhibited the expression of PEDVN gene (p<0.01); Figure 2As shown in Figure B, 100, 200 and 400 μg / mL of Sax.s significantly inhibited the expression of PEDV N gene (p<0.05). In summary, Sax.s at a concentration of 200-400 μg / mL had a significant inhibitory effect on PEDV and reduced the expression level of PEDV N gene in a concentration-dependent manner.

[0058] Depend on Figure 3 As shown in A and B, in Vero E6 cells, compared with the positive control group, 200 and 400 μg / mL Sal.s treatment significantly inhibited the expression of PEDVN protein (p<0.05); Figure 3 As shown in C and D, in IPEC-J2 cells, 200 and 400 μg / mL Sax.s treatment significantly inhibited PEDV replication (p<0.05). In summary, Sax.s inhibited the expression of PEDVN protein in a concentration-dependent manner and had a significant antagonistic effect on PEDV. Therefore, in subsequent experiments, Sax.s at a concentration of 200 μg / mL was selected to study its antiviral effect.

[0059] Example 3

[0060] This example provides the effects of Sax.s on the PEDV infection life cycle and cytokines.

[0061] Vero E6 cells were cultured at 2 × 10 5 Inoculate 1000 cells / well in a 6-well plate and culture for 24 hours until a monolayer is formed. Figure 3 The experiment was carried out in the groups and treatments shown in the figure. Adsorption stage: precooling at 4°C for 30 min, rinsing with virus maintenance solution 3 times, adding a mixture of PEDV LW / L (MOI=0.1 / 1) and 200 μg / mL Sax.s, and adsorbing for 2 h at 4°C; invasion stage: precooling at 4°C for 30 min, rinsing with virus maintenance solution 3 times, adding PEDV LW / L (MOI=0.1 / 1) and incubating at 4°C for 2 h, washing with D-Hanks 3 times, adding 200 μg / mL Sax.s and incubating at 37°C for 3 h; replication stage: rinsing with virus maintenance solution 3 times, inoculating with PEDV LW / L (MOI=0.1 / 1) for 1 h, washing with D-Hanks 3 times, culturing the virus maintenance solution for 3 h and discarding it, and then adding 200 μg / mL Sax.s and incubating for 12 h; release stage: rinsing with virus maintenance solution 3 times, inoculating with PEDV After 1 hour of LW / L (MOI = 0.1 / 1), the cells were washed with D-Hanks for 3 times, the virus maintenance solution was discarded after 5 hours of incubation, and 200 μg / mL Sax.s was added and incubated for 2 hours. At the same time, a negative control group and a positive control group were set up for each group. 5 IPEC-J2 cells / well replace 2×10 5The same operation was performed on Vero E6 cells per well, and the cells were collected after the treatment. The relative expression of PEDVN mRNA was detected by fluorescent quantitative PCR, and the relative expression of PEDV N protein during the replication phase was detected by Western Blot. Figures 5 to 7 Results shown.

[0062] Depend on Figure 5 It can be seen that 200 μg / mL Sax.s has the best inhibitory effect on PEDV in the virus replication stage, and significantly reduces the N gene expression of the virus in Vero E6 cells (p<0.01). The inhibitory effect on PEDV in the other three stages (adsorption, invasion, and release) is not significant (p>0.05).

[0063] Depend on Figure 6 It can be seen that 200 μg / mL Sax.s also significantly inhibited the proliferation of PEDV in the viral replication stage (p<0.05); the effect was not significant in the other three stages (adsorption, invasion, and release) (p>0.05). The above results show that Sax.s has significant anti-PEDV activity and inhibits PEDV in the replication stage.

[0064] Depend on Figure 7 As shown in A and B, 200 μg / mL Sax.s significantly reduced the expression of viral N protein in Vero E6 cells (p<0.01). Figure 7 As shown in C and D, 200 μg / mL of Sax.s also significantly reduced the expression of PEDV N protein in IPEC-J2 cells during the viral replication stage (p<0.01).

[0065] The above results indicate that Sax.s can play an inhibitory role in the viral replication stage.

[0066] Vero E6 cells were collected at 2 × 10 5 100 μg / well were inoculated in 6-well plates and cultured for 24 hours until monolayer was formed. The virus maintenance solution was rinsed 3 times, PEDV LW / L (MOI = 0.1 / 1) was inoculated for 1 hour, and D-Hanks was washed 3 times. The virus maintenance solution was cultured for 3 hours and then discarded. 200 μg / mL Sax.s was added and incubated for 12 hours. 4×10 5 IPEC-J2 cells / well replace 2×10 5 The same operation was performed on Vero E6 cells per well and the cells were collected. The relative expression of TNF-α, IL-1β, IL-6, and IL-10 mRNA levels in Vero E6 and IPEC-J2 cells after virus infection was detected by fluorescence quantitative PCR. Figure 8 The results shown and Fig. 9The interaction between PEDVN protein and p53 protein was detected by Co-IP experimental method, and the expression of proteins related to p53-DREAM signaling pathway was detected by Western Blot. Figure 10 to Figure 13 Results shown.

[0067] Depend on Figure 8 As shown in A to D, after PEDV infection, the expression of TNF-α, IL-1β, IL-6 and IL-8 mRNA levels in Vero E6 cells was significantly upregulated. After treatment with 200 μg / mL Shax.s, the upregulation of TNF-α, IL-1β, IL-6 and IL-8 caused by PEDV was significantly reduced (p<0.01). Figure 8 As shown in E, 200 μg / mL of Sax.s can significantly increase the expression of IL-10 mRNA level.

[0068] Depend on Fig. 9 As shown in A to D, in IPEC-J2 cells, PEDV infection also significantly increased the expression of TNF-α, IL-1β, IL-6 and IL-8 mRNA levels (p<0.05), and 200μg / mL Sal.s treatment significantly reduced the expression of TNF-α and IL-6 (p<0.01). Fig. 9 As shown in E, 200 μg / mL of Sax.s also significantly increased the expression of IL-10 (p<0.01).

[0069] The above results indicate that PEDV can stimulate the expression of pro-inflammatory factors in cells, and Sax.s treatment can significantly reduce the expression of inflammatory factors and increase the expression of anti-inflammatory factors, indicating that Sax.s has a certain anti-inflammatory effect.

[0070] Depend on Fig.10 It can be seen that in Vero E6 cells, compared with the PEDV infection positive control group, 200 μg / mL of Sax.s can significantly inhibit the interaction between PEDV N and p53 protein (p<0.01), effectively downregulating the replication of PEDV.

[0071] Depend on Fig.11 As shown in A to C, the expression of p53 and p21 proteins was significantly upregulated after PEDV infected Vero E6 cells. Fig.11 As shown in A, F, H and I, p130 phosphorylation (Ser672), E2F4 and CyclinA protein expression were significantly downregulated (p<0.05). Fig.11As shown in D, E and G, there was no significant effect on the expression of p107, p130 and p130 phosphorylation (Ser952) proteins (p>0.05). After treatment with 200 μg / mL of Sax.s, the expression of p53 and p21 proteins was significantly reduced, and the expression of p130 phosphorylation, E2F4 and CyclinA proteins was significantly increased (p<0.01).

[0072] In summary, Sax.s can play an anti-PEDV effect in Vero E6 cells by inhibiting the interaction between PEDV N and p53 protein.

[0073] Depend on Fig.12 It can be seen that in IPEC-J2 cells, compared with the PEDV infection positive control group, 200 μg / mL Sax.s can also significantly inhibit the interaction between PEDVN and p53 protein (p<0.01), which is consistent with the experimental results of Vero E6 cells.

[0074] Depend on Fig.13 It can be seen that after treatment with 200 μg / mL Sax.s, the expression of p53 and p21 proteins was also significantly downregulated, and the expression of p130 phosphorylation, E2F4 and CyclinA proteins was significantly upregulated (p<0.01), but there was no significant effect on the expression of p107, p130 and p130 phosphorylation (Ser952) proteins (p>0.05). Fig.11 The results shown that Sax.s regulates the expression of proteins related to the Vero E6 cell signaling pathway are consistent.

[0075] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0076] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of saxifrage in the preparation of products for preventing and treating animal epidemic diarrhea.

2. The use according to claim 1, characterized in that: The animal is a pig.

3. The use according to claim 1, characterized in that: The saxifrage is saxifrage dry grass or saxifrage water extract, and the saxifrage water extract is obtained by extracting saxifrage using water as an extraction solvent.

4. The use according to claim 3, characterized in that: When the water extract of Saxifraga herba is prepared, the usage ratio of Saxifraga herba to water is 1 g: 8 mL to 12 mL.

5. The use according to claim 4, characterized in that: When preparing the water extract of Saxifraga herba, water is added and then soaked, and the mixture is decocted for 2 to 3 times, each time for 1 hour, and the decoctions are combined to obtain the water extract of Saxifraga herba.

6. The use according to claim 5, characterized in that: The soaking time is 30min to 40min.

7. The use according to claim 5, characterized in that: The effective dosage of the Saxifraga water extract is 200 μg / mL to 400 μg / mL.

8. A product for preventing and treating porcine epidemic diarrhea, characterized in that: The product contains the saxifrage described in claim 1 as the only effective active ingredient.

9. The product according to claim 8, characterized in that The product is a medicine or a feed additive.

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