CN113368141A

CN113368141A - Pharmaceutical composition for preventing and treating porcine envelope virus infectious diseases, preparation method and application of plant extract

Info

Publication number: CN113368141A
Application number: CN202010117442.XA
Authority: CN
Inventors: 胡文锋; 曹永长; 刘元; 徐志超; 胡学生; 靳改改; 朱剑锋
Original assignee: Bioforte Biotechnology Shenzhen Co ltd
Current assignee: Bioforte Biotechnology Shenzhen Co ltd
Priority date: 2020-02-25
Filing date: 2020-02-25
Publication date: 2021-09-10

Abstract

The invention relates to a pharmaceutical composition for preventing and treating porcine envelope virus infectious diseases, a preparation method and application of a plant extract. The plant extracts comprise natural plant extracts with antiviral replication, such as whiteflower peucedanum root extract, ginkgo epicarp extract, divaricate saposhnikovia root extract, fennel extract, geranium extract and the like, or are compounded with proper antibacterial agents and auxiliary materials, so that the plant extracts can be used for preventing and treating porcine enveloped virus infection and secondary infection caused by the infection of the porcine enveloped virus, building a third 'line of defense' for the pig industry, and making up the defects of vaccine immunization and 'washing and disinfecting' means. The invention has important practical significance for ensuring the normal production of the pig industry, especially for diseases caused by enveloped virus infection which can not be prevented by the vaccine at present, or diseases with low vaccine protection rate, or as a remedial measure for the immune failure and the like.

Description

Pharmaceutical composition for preventing and treating porcine envelope virus infectious diseases, preparation method and application of plant extract

Technical Field

The invention relates to the field of biotechnology application, in particular to a pharmaceutical composition for preventing and treating porcine envelope virus infectious diseases, a preparation method thereof and application of plant extracts in preparation of drugs for preventing and treating porcine envelope virus infectious diseases.

Background

Porcine Epidemic Diarrhea (PED) is an acute, highly infectious disease of pigs whose pathogenic Porcine Epidemic Diarrhea Virus (PEDV) is a single-stranded positive-strand RNA Virus with an envelope belonging to the genus Alphacoronavirus (KOCHERhANS R, BRIDGENA, ACKERMANN M, et al.2001.Complex epidemic diarrheic diarrheronavirus (PEDV) genome sequence [ J ] Virus Genes,23(2): 137-144.). PEDV can cause diarrhea, dehydration and vomiting of newborn piglets, and has high fatality rate. In recent years, PEDs have been widely spread throughout the world, and countries such as china, the united states, korea, and canada have been in outbreak of epidemic situations, which seriously jeopardize the development of the global pig industry. 29 provinces and cities of China occur and prevail PED in autonomous regions, so that the death rate of newborn piglets can reach 100 percent, and huge economic loss is caused to the pig raising industry in China.

Currently, vaccine immunization control is mainly used for controlling PED. Vaccine immunization is based on direct immunization of piglets, and vaccine immunization protects intestinal epithelial cells by inducing mucosal immunity (WANG D, FANG L, XIAO S.2016. hormone epidemic diarrhea in China [ J ]. Virus Res,226: 7-13.). For newborn piglets and lactating piglets, there are two problems with vaccine immunization: first, sows whose serum is positive for PEDV antibodies, maternal antibodies in milk, will affect the protective effect induced by oral live vaccines; secondly, the PEDV vaccine plays a role through mucosal immunity, but high-incidence groups of PED are newborn piglets and suckling-period piglets with incompletely developed mucosal immune systems, so that the vaccine immune effect of the PEDV is not ideal.

Porcine Reproductive and Respiratory Syndrome (PRRS) is an epidemic caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) characterized by Reproductive disorders in adult sows, premature birth, miscarriage and stillbirth, and respiratory disorders in piglets. PRRSV is a single-stranded positive-strand RNA, nonsegmented virus, a genus arterivirus of the arterivirus family. The PRRSV is a virus with an envelope, is similar to a sphere, has the diameter of 45 to 65 nanometers, and has a fibrous protrusion on the surface of the envelope, so that the surface of the envelope is relatively smooth.

At present, no effective measure for preventing and treating PRRS is available, and although commercial PRRS attenuated vaccines and inactivated vaccines are introduced at home and abroad and inactivated vaccines officially approved at home, the phenomenon of the virulent return of the PRRS attenuated vaccines and the safety problem increasingly cause the worry of people. Since many outbreaks of PRRS occur in swine herds due to the use of attenuated vaccines at home and abroad, live vaccines should be used with caution. The PRRS inactivated vaccine is developed in China, but the protection rate is low.

African Swine Fever (ASF) is an acute, hemorrhagic, virulent infectious disease caused by African Swine Fever Virus (ASFV) infecting domestic pigs and various wild pigs (such as African wild pigs, European wild pigs, etc.). The ASFV is a double-stranded nucleoplasmic large DNA virus, the shape is a regular icosahedron, the diameter is 175-215 nm, and the total length of the genome is 170-190 kb. The results of the research so far show that the protein contains about 151 open reading frames, can code 150-200 proteins and is a double-layer capsule membrane. The world animal health Organization (OIE) classifies the animal epidemic disease as a legal report animal epidemic disease, and the disease is also a type of animal epidemic disease which is mainly prevented in China. The clinical symptoms of African swine fever are similar to those of swine fever, and diagnosis can be confirmed only by means of laboratory monitoring.

Before more than 100 years, the first ASF case is discovered in the east, the ASF case is introduced into China by 8 months in 2018, and no effective vaccine or medicament is developed in the industry in nearly hundred years. In the absence of an effective vaccine, the development of prophylactic and therapeutic drugs is essential. Under the influence of factors such as epidemic situation of African swine fever and environmental protection, the productivity of live pigs in China is reduced, the stock level is continuously kept low, and the price of pork in China is also high.

The development of oral antiviral products is an important idea for preventing and controlling virus infection besides vaccine immunity, so as to make up for the defects of prevention and control means such as vaccine immunity, decontamination and the like, and build a third line of defense. Antiviral products currently under study are broadly divided into two categories: one can indirectly inhibit viruses by regulating the immune function of animal organisms; another class can directly inhibit viruses. However, inhibiting the virus is not equivalent to treating the disease, and the infection of the body with the virus can cause a series of secondary infections, so that the antiviral product alone cannot completely treat the disease in practical production application, and the secondary infections and the over-reaction of the immune system can be fatal.

The body resistance strengthening and detoxifying powder, the astragalus polysaccharide, the indigowoad root and indigowoad root granules and the like in the medicines for regulating the immune function of the body are common traditional Chinese veterinary medicines in the pig industry, can improve the growth performance of piglets, reduce the diarrhea rate and regulate the immune function of the body, has broad-spectrum antibacterial and antiviral effects, and is widely applied to the pig industry as a substitute of antibiotics. The action target and mechanism of the vaccine are still directed at the natural immunity of animals, and the specificity is not strong.

The substance for directly inhibiting the virus is a target for one or more key steps in the whole process of virus invasion and replication, so that one or more steps of adhesion, invasion, release, replication, assembly, budding and shedding of the virus are effectively prevented. Currently, drugs such as Ribavirin (Ribavirin) have been found to inhibit viral proliferation on cells, in view of the early stages of viral replication, but it is an antiviral drug in humans and has been banned from veterinary clinical use; lipopeptide (Surfactin) is mainly produced by bacteria, and aiming at the invasion process of viruses, molecules of the lipopeptide have both hydrophilic ends and hydrophobic ends and can be inserted into the virus envelope to prevent the fusion of the virus envelope and cell membranes.

However, there are few additives or drug combinations that are specifically aimed at preventing and treating porcine enveloped virus infectious diseases. The development of novel medicaments with good effect of preventing and treating porcine envelope virus infectious diseases in clinic is urgently needed in the field.

Disclosure of Invention

In view of the above, the invention provides a pharmaceutical composition for preventing and treating porcine enveloped virus infectious diseases, a preparation method thereof, and application of plant extract in preparation of drugs for preventing and treating porcine enveloped virus infectious diseases, which can be used for preventing and treating porcine enveloped virus infectious diseases.

A pharmaceutical composition for preventing and treating porcine enveloped virus infectious diseases comprises plant extracts, wherein the plant extracts comprise one or more of Peucedanum praeruptorum extract, gingko biloba exocarp extract, radix Saposhnikoviae root extract, fennel extract and geranium extract.

Preferably, the pharmaceutical composition further comprises an antibacterial agent, an acidifier, an ester material and an adjuvant.

Preferably, the pharmaceutical composition consists of the following components in parts by weight: 50-200 parts of plant extract, 100-400 parts of antibacterial agent, 300-600 parts of acidifying agent, 60-150 parts of ester substance and 60-150 parts of auxiliary material.

Further preferably, the pharmaceutical composition consists of the following components in parts by weight: 100 parts of plant extract, 350 parts of antibacterial agent 250-.

Preferably, the adjuvant comprises one or more of a carrier, a diluent and a surfactant.

Preferably, the auxiliary materials comprise the following components in percentage by weight: 50-100% of carrier, 0-50% of diluent and 0-5% of surfactant.

Further preferably, the antibacterial agent is a zinc-rich inactivated lactobacillus agent.

Further preferably, the acidulant comprises a liquid acidulant comprising one or more of formic acid, acetic acid, propionic acid, lactic acid and a powder acidulant comprising one or more of citric acid or fumaric acid.

Further preferably, the ester substance comprises a liquid ester substance and a powder ester substance. Further preferably, the powder ester substances comprise one or more of lauric acid monoglyceride, oleic acid diglyceride, linoleic acid diglyceride, palmitic acid monoglyceride, palmitic acid diglyceride, lauric acid diglyceride, caprylic acid diglyceride, capric acid monoglyceride, capric acid diglyceride and linolenic acid diglyceride; the liquid ester substance comprises one or more of tributyrin, oleic acid monoglyceride, linoleic acid monoglyceride, caprylic acid monoglyceride and linolenic acid monoglyceride.

Further preferably, the carrier is one or more of silica, diatomite, montmorillonite or medical stone.

Further preferably, the diluent comprises one or more of bentonite, zeolite powder, stone powder, talcum powder, vermiculite, attapulgite, shell powder, sepiolite and kaolin.

Further preferably, the surfactant is a tween surfactant, a span surfactant or a glyceride surfactant.

The preparation method of the pharmaceutical composition for preventing and treating the porcine enveloped virus infectious diseases comprises the following steps:

s1, mixing the liquid ester substance with part of the carrier, and then compounding the mixture with the powder ester substance to form an ester mixture A;

s2, mixing the liquid acidulant with the rest of carriers, and compounding with other powder acidulants to form acidulant mixture B;

s3, at least one of the plant extracts;

s4, mixing the products obtained in the steps with an antibacterial agent to form the mixture.

Preferably, the liquid ester is mixed with the carrier before the surfactant in step S1.

Preferably, the plant extract is mixed with a diluent to prepare a mixture C in the step S3.

The application of the plant extract in preparing the medicine for preventing and treating the porcine enveloped virus infectious diseases is that the plant extract is one or more of peucedanum praeruptorum dunn extract, ginkgo epicarp extract, radix sileris extract, fennel extract and geranium extract.

The invention carries out prevention and treatment drug research aiming at virus infectious diseases caused by enveloped viruses, and finds that the plant extract can achieve the purposes of inhibiting virus proliferation and controlling the occurrence and development of virus infection by blocking certain links of a virus replication process, such as prevention of adsorption, invasion, budding and shedding, and inhibition of replication in host cells.

The peucedanum praeruptorum dunn extract, the ginkgo exocarp extract, the ledebouriella root extract, the fennel extract and the geranium extract have an antiviral replication effect, can inhibit porcine epidemic diarrhea viruses, African swine fever viruses, porcine reproductive and respiratory syndrome viruses and other enveloped viruses, and can be further used for treating porcine enveloped virus infectious diseases. The plant extract has the characteristics of less toxic and side effects, simple preparation method, wide raw materials and the like.

The invention relates to a pharmaceutical composition containing plant extracts for preventing and treating porcine envelope virus infectious diseases, which takes whiteflower peucedanum root extract, ginkgo epicarp extract, divaricate saposhnikovia root extract, fennel extract and geranium extract as effective components, and is compounded with proper antibacterial agent and auxiliary materials, so that the pharmaceutical composition can be used for preventing and treating porcine envelope virus infection and diseases caused by secondary infection of the porcine envelope virus infection, builds a third 'line of defense' for the pig industry, and makes up the defects of vaccine immunity and 'washing and disinfecting' means. The preparation method of the pharmaceutical composition is simple, and the effect of the effective components is not influenced.

The invention has very important practical significance for ensuring the normal production of the pig industry, especially for preventing and treating the capsular virus disease which can not be prevented by the vaccine at present, or viral diseases with low vaccine protection rate, or remedial measures of immune failure and the like.

Drawings

FIG. 1 shows the effect of the extract of Pelargonium sidoides on Vero cell activity in example 1.

FIG. 2 shows the results of the inhibition of PEDV-induced Vero cytopathic effect of the extract of Pelargonium graveolens of example 1.

FIG. 3 shows the result of the inhibition of the expression of PEDV-N protein mRNA in Vero cells by the extract of pelargonium roseum of example 1.

FIG. 4 shows the effect of the fennel extract on Vero cell activity in example 2.

FIG. 5 shows the results of experiments in which the fennel extract of example 2 inhibited the proliferation of PEDV in Vero cells. NC in the figure is a non-drug-added control group, Zn-lac is a supernatant group of zinc-rich lactobacillus fermentation liquor, SC is a sodium caprylate group, GML is a monoglyceride laurate group, BSFLh is a hydrolyzed black water worm oil emulsion group, A is fennel extract, P is less than or equal to 0.01, and P is less than or equal to 0.0001.

FIG. 6 shows the toxicity test results of Peucedanum praeruptorum dunn extract on Vero cells in example 3.

FIG. 7 shows the results of experiments in which Peucedanum praeruptorum dunn extract inhibits the proliferation of PEDV in Vero cells in example 3.

FIG. 8 shows the results of the toxicity test of the plant extracts of example 4 on MARC-145 cells.

FIG. 9 shows the results of the experiment of the plant extracts of example 4 for inhibiting the proliferation of PRRSV in MARC-145 cells.

Fig. 10 is the results of the effect of plant extracts on the activity of PAM primary cells in example 5.

FIG. 11 is the results of experiments in example 5 in which plant extracts inhibited proliferation of AFSV in PAM cells.

FIG. 12 is the results of the third day clinical observation after challenge in example 7.

FIG. 13 is the fourth day clinical observation after challenge in example 7.

FIG. 14 is the results of the sixth day clinical observation after challenge in example 7.

FIG. 15 is the results of the ninth day clinical observation after challenge in example 7.

FIG. 16 shows the toxin expulsion test result of the anal swab in example 7.

Fig. 17 is the results of scoring the diarrhea status of piglets in example 7.

FIG. 18 shows the results of the tissue viral load test in example 7.

Detailed Description

The present invention will be described in further detail with reference to examples.

The Peucedanum praeruptorum dunn extract, gingko exocarp extract, Ledebouriella root extract, fennel extract, and Pelargonium graveolens extract can be prepared by any preparation method disclosed in the prior art, such as ultrasonic extraction, supercritical fluid extraction, semi-bionic extraction, and macroporous adsorbent resin method, or can be directly prepared from commercially available products.

The individual plant extracts used in the examples of the invention were prepared by the following general method: adding hot water into the raw materials according to the proportion of 1:30 (m: V) and carrying out ultrasonic extraction for 3-5 times, combining filtrates, carrying out vacuum concentration, carrying out alcohol precipitation on the concentrated solution by using ethanol with the mass fraction higher than 80%, carrying out centrifugation to recover precipitates, and carrying out protein removal by a Sevag method to obtain the plant extract.

The preparation method of the zinc-rich inactivated lactobacillus agent comprises the following steps: 1) the lactobacillus acidophilus is firstly prepared into strains and screenedSelecting solid MRS culture medium as basic culture medium, adding 0.1% CaCO₃Sterilizing the culture medium at 121 ℃ for 20-30 minutes, and cooling to 37 ℃ for later use, wherein the pH value is 6.4; 2) fermenting and culturing lactobacillus acidophilus, wherein the fermentation culture medium is liquid MRS culture medium as basic culture medium, pH value is 6.4, the culture medium is sterilized for 20-30 min at 121 ℃, cooled to 37 ℃, and added with 0.12% ZnSO₄Uniformly mixing the components for standby, wherein the fungus age of the seeds is 12-18 hours, the inoculation amount is 3% -5% of V/V or V/W, the fermentation time is 36-48 hours, the fermentation temperature is 37 ℃, and the pH value is maintained at about 5.5; 3) adding 0.1-0.5% (w/v) of protective agent into the fermentation liquor after the fermentation is finished, wherein the protective agent is skimmed milk; 4) inactivation: heat inactivating, heating the fermentation liquid to 60-80 deg.C, maintaining for 30-60 min, centrifuging the fermentation liquid at 3000rpm for 15 min, removing supernatant, and collecting thallus; 5) adding an excipient into the thallus, adding 650 g of an excipient into 350 g of a zinc-rich lactobacillus acidophilus fermentation product, wherein the excipient is a mixture of 80-mesh corn starch and precipitated silicon dioxide, and mixing and stirring for 20-30 minutes to fully mix and adsorb; 6) the zinc-rich inactivated lactobacillus acidophilus which is fully mixed with the excipient is dried at the temperature of 90-105 ℃ to ensure that the residual water content is less than or equal to 15 percent.

The assay methods used in the present invention are those conventionally used in the art, and the assay reagents, cells, etc. used therein are commercially available or prepared according to the conventional methods in the art.

Example 1 in vitro inhibition of PEDV assay by Pelargonium extract

1.1 toxicity test of Pelargonium extract on Vero cells

Inoculation of 6X 10 in 96-well plates⁵cells/mL cell suspension (100. mu.L/well) leaving a column without seeding cells. At 37 ℃ with 5% CO₂Culturing under the condition until the cells are completely confluent. The medium was aspirated, washed 3 times with PBS, and the geranium extract (hereinafter abbreviated as PE9) was diluted with DMEM to various concentrations and added to a 96-well plate (100. mu.L/well) at each dilution concentration for 8 replicates. At 37 ℃ with 5% CO₂Culturing under the conditions of 24 hr and 48 hr, removing the medicinal liquid, washing with PBS for 3 times, adding 10% CCK-8 solution into 96-well plate (100 μ L/well) with DMEM, and culturing at 37 deg.C with 5% CO₂Incubating for 1h under the condition, and detecting by an enzyme-linked immunosorbent assayAnd (3) determining an absorbance value (lambda is 450nm) and calculating the cell viability after the liquid medicine treatment for 24h and 48h at different concentrations.

The results are shown in FIG. 1. Figure 1A shows that the concentration of geranium extract (PE9) after 48h treatment had no significant effect on Vero cell activity at no greater than 10mg/ml (n-8). FIG. 1B shows that PE9 completely inhibited virus proliferation on Vero cells at a concentration of 10mg/ml for the same treatment time by the blank control.

1.2 assay of Pelargonium extract (PE9) for inhibition of proliferation of PEDV in Vero cells

From example 1.1, the tolerance concentration of Vero cells to the geranium extract can be known, and the maximum tolerance concentration of model cells with normal growth is obtained; the maximum tolerated concentration was taken as the concentration at which the pelargonium extract was subsequently tested for Vero cell inhibition.

1. In 3 6-well plates by 6X 10⁵cell concentration of cells/mL 2mL cells per well were plated in 3 wells per plate, 5% CO at 37 ℃₂Culturing under the condition until the cells are completely confluent.

2. The medium was discarded, washed three times with PBS, DMEM mixed with PEDV (MOI 0.2) was added to the wells of the control group, and DMEM mixed with PEDV virus solution (MOI 0.2) at Fe concentrations of 500 μ g/mL and 1000 μ g/mL, 2mL per well, was added to the experimental group, respectively. Incubating at 37 deg.C for 1h, discarding the incubation solution, washing with PBS for three times, adding 2mL DMEM containing Pelargonium extract into each well, incubating at 37 deg.C with 5% CO₂Culturing for 3h, 6h and 9h under the condition.

3. Observing cytopathic conditions of the experimental group and the control group under a microscope, photographing, removing a culture medium, washing for 3 times by PBS (phosphate buffer solution), adding 600 mu L of RLT Plus lysate (tissue/cell RNA rapid extraction kit-Edele biotechnology Co., Ltd.) into each well, splitting for 3min at room temperature, blowing and uniformly mixing, transferring into 1.5mL EP tubes of RNase free respectively, carrying out vortex oscillation for 20s, adding all the lysis mixtures onto a DNA removal column, immediately centrifuging for 60s at 13000rpm, and keeping the filtrate.

4. Accurately measuring the volume of the filtrate by using a pipette, transferring the filtrate into a 1.5mL EP tube of RNase free, adding equal volume of 70% ethanol, immediately blowing and uniformly mixing, adding the mixture into an RNA adsorption column, centrifuging at 13000rpm for 30s, and discarding waste liquid.

5. Adding 700 μ L deproteinizing solution RW1, standing at room temperature for 1min, 12000rpm, centrifuging for 30s, and discarding waste liquid.

6. Adding 500 μ L of rinsing solution RW, centrifuging at 12000rpm for 30s, discarding waste liquid, repeating, centrifuging at 13000rpm for 3min, and removing rinsing solution as much as possible.

7. The RNA adsorption column was transferred to a new 1.5mL EP tube of RNase free, 50. mu.L of RNase free water was added to the middle of the adsorption membrane, and the mixture was left at room temperature for 2min and centrifuged at 12000rpm for 1 min.

8. RNA concentration and quality was determined as ReverTra

The method of qPCR RT Master Mix with gDNA Remover kit is used for cDNA synthesis, and after centrifugation and uniform mixing, reverse transcription is carried out on a PCR instrument.

TABLE 1 reverse transcription System and procedure

9. Add 40. mu.L sterile double distilled water to the genomically removed cDNA product, vortex, mix well and store at-20 ℃ for future use.

10. real-time-PCR reaction system was prepared by performing real-time fluorescent quantitative PCR according to the instructions of 2 XSSYBR Green qPCR Master Mix kit from bimake.

TABLE 2 qPCR reaction System

3 replicates of each sample were added to an 8-plex PCR tube, centrifuged to collect the liquid at the bottom of the tube, and the reaction was performed on an ABI 7500Fast instrument. Reaction procedure: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, extension at 70 ℃ for 30s, and 40 cycles; dissolution curve: 95 ℃, 15s, 60 ℃, 1min, 95 ℃, 15 s. GAPDH was set as an internal reference gene, and the mRNA level of each gene was analyzed by the 2-rho. Ct method.

TABLE 3 real-time fluorescent quantitative PCR primers

The proliferation of PEDV in Vero cells after treatment with the geranium extract (PE9) was judged by cytopathic effect and relative fluorescence quantification. The micrograph (fig. 2) shows: after the drug treatment for 12 hours, the pathological changes of the cells in the experimental group are obviously less than those in the control group; the relative fluorescence quantification results (fig. 3) show: the PEDV-N protein gene expression level of the drug group (PE9) at 1000. mu.g/mL after 6h and 9h treatment is obviously lower than that of the control group (N is 3, and P is less than or equal to 0.0001).

Example 2 Fennel extract inhibits proliferation of PEDV in vitro

2.1 toxicity test of Fennel extract on Vero cells

The experimental procedure is as described in 1.1 of example 1. As shown in FIG. 4, the concentration of fennel extract (abbreviated as A) was 1000. mu.g/mL or less, which did not significantly affect the activity of Vero cells by comparative experiments.

2.2 test of the Fennel extract for inhibition of the proliferation of PEDV in Vero cells

The experimental procedure is as described in 1.2 of example 1. This example compares the fennel extract (a) with other drugs. And (3) respectively taking two concentration gradients under the highest safe concentration of each drug for 24h, adding drugs in the whole process of PEDV proliferation, harvesting an RNA sample at the end of the first replication cycle of PEDV, namely 6h.p.i., and detecting the level of PEDV N gene by real-time quantitative fluorescence PCR to obtain a drug test result.

As shown in fig. 5, the level of PEDV N gene was reduced by adding 250ug/mL of supernatant of zinc-rich lactobacillus fermentation broth (Zn-lac), but not by adding 125ug/mL of supernatant of zinc-rich lactobacillus fermentation broth, compared to the control group without drug; 500ug/mL hydrolyzed Hermetia illucens oil emulsion (BSFLh) can reduce the level of PEDV N gene, but 250ug/mL hydrolyzed Hermetia illucens oil emulsion cannot reduce; fennel extract (A) reduced the level of the PEDV N gene at both 1000ug/mL and 500 ug/mL. Therefore, the supernatant of the zinc-rich lactobacillus fermentation liquor, the hydrolyzed hermetia illucens oil emulsion and the fennel extract have the capacity of inhibiting the proliferation of PEDV; the addition of Sodium Caprylate (SC) and lauric acid monoglyceride (GML) did not reduce the level of PEDV N gene, indicating that it did not have the ability to inhibit PEDV proliferation.

Example 3 Peucedanum praeruptorum dunn extract inhibits proliferation of PEDV in vitro

3.1 toxicity test of Peucedanum praeruptorum dunn extract (PE6) on Vero cells

The experimental procedure is as described in 1.1 of example 1. The results are shown in fig. 6, where the peucedanum praeruptorum dunn extract (PE6) below 10% (v/v) had no significant effect on the viability of Vero cells; the capric acid monoglyceride (PO4) below 0.03mg/ml had no significant effect on Vero cell viability.

3.2 Peucedanum praeruptorum dunn extract (PE6) test for inhibiting proliferation of PEDV in Vero cells

The experimental procedure is as described in 1.2 of example 1. As shown in fig. 7, it was found that peucedanum praeruptorum dunn extract (PE6) and monoglycerol decanoate (PO4) significantly reduced the expression level of PEDV mRNA and had the ability to inhibit PEDV proliferation in Vero cells under the condition of MOI of 0.2 and 12 hpi.

Example 4 plant extracts inhibit PRRSV proliferation in vitro

4.1 toxicity test of plant extracts on MARC-145 cells

The experimental procedure is as described in 1.1 of example 1. Results As shown in FIG. 8, panel, Ledebouriella root extract (PE5) had no significant effect on MARC-145 cell viability below 10% (v/v); peucedanum praeruptorum dunn extract (PE6) has no significant effect on MARC-145 cell viability below 10% (v/v); less than 10% (v/v) of the geranium extract (PE9) had no significant effect on the viability of MARC-145 cells; capric acid monoglyceride (PO4) below 0.125mg/ml had no significant effect on MARC-145 cell viability.

4.2 plant extracts test for inhibition of PRRSV proliferation in MARC-145 cells

The experimental procedure is as described in 1.2 of example 1. As shown in fig. 9, by comparing the results of experiments on different drugs for inhibiting PRRSV proliferation in MARC-145 cells, it was found that ledebouriella root extract (PE5), peucedanum praeruptorum dunnii extract (PE6), geranium extract (PE9) and monoglycerol decanoate (PO4) significantly reduced PPRSV mRNA expression levels and had the ability to inhibit PPRSV proliferation in MARC-145 cells under the conditions of MOI of 0.5 and 24 hpi.

Example 5 in vitro inhibition of ASFV proliferation by plant extracts

5.1 Effect of plant extracts on PAM Primary cells

The experimental procedure is as described in 1.1 of example 1. The results are shown in FIG. 10, where PAM primary cells were resistant to Peucedanum praeruptorum dunn extract (PE6) at a maximum of 5mg/mL, to Ginkgo biloba exocarp extract (PE8) at a maximum of 10mg/mL, and to Pelargonium extract (PE9) at a maximum of 5 mg/mL.

5.2 plant extracts test for inhibiting AFSV proliferation in PAM cells

The experimental procedure is as described in 1.2 of example 1. As shown in fig. 11, by setting up the negative control and the positive control, after 48h of the challenge treatment, from the viewpoint of cytopathic effect and fluorescence phenomenon, the peucedanum praeruptorum dunn extract (PE6), ginkgo epicarp extract (PE8) and geranium extract (PE9) had significant inhibitory effect on ASFV proliferation, and the cytopathic effect was less compared to the positive control group; compared with the positive control group, the PE9 group has the least fluorescence and the best virus inhibition effect, and the number of PE is 6. In the lower figure, a positive control group has a large amount of ASFV antigen, a negative control group has no ASFV antigen, and PE9 group has almost no ASFV antigen, which proves that the pelargonium extract has a significant inhibitory effect on ASFV proliferation.

EXAMPLE 6 preparation of pharmaceutical composition for prevention and treatment of porcine enveloped virus infectious diseases

6.1 pharmaceutical composition for preventing and treating porcine enveloped virus infectious diseases containing Fennel extract

The pharmaceutical composition for preventing and treating the porcine envelope virus infectious diseases is prepared by the following steps:

s1, 25 g of tributyrin was mixed uniformly with 25 g of silica and then with 50 g of lauric monoglyceride to give 100 g of an ester mixture.

S2, 200 g of lactic acid and 140 g of silica were uniformly mixed, and then 100 g of fumaric acid and 60 g of citric acid were uniformly mixed to obtain 500g of an acidulant mixture.

S3, fennel extract 50 g.

S4, uniformly mixing the three mixtures with 350 g of zinc-rich inactivated lactobacillus to obtain 1000g of the pharmaceutical composition for preventing and treating the porcine enveloped virus infectious diseases.

6.2 pharmaceutical composition for preventing and treating porcine enveloped virus infectious diseases containing Pelargonium extract

S1, 40 g of tributyrin was mixed uniformly with 25 g of silica and then with 35 g of lauric monoglyceride to give 100 g of an ester mixture.

S2, 150 g of lactic acid was uniformly mixed with 100 g of silica, and then uniformly mixed with 125 g of fumaric acid and 125 g of citric acid to obtain 500g of an acidulant mixture.

S3, 100 g of geranium extract.

S4, uniformly mixing the three mixtures with 300 g of zinc-rich inactivated lactobacillus to obtain 1000g of the pharmaceutical composition for preventing and treating the porcine enveloped virus infectious diseases.

6.3 pharmaceutical composition containing plant extract for preventing and treating porcine envelope virus infectious diseases

S3, fennel extract, ledebouriella root extract each 25 g, geranium extract 50 g, etc., to obtain 100 g of plant extract mixture.

6.4 pharmaceutical composition containing plant extract for preventing and treating porcine envelope virus infectious diseases

S1, 15 g of caprylic acid monoglyceride and 15 g of tributyrin are mixed, 1 g of Tween is added, the mixture is uniformly mixed with 20 g of silicon dioxide, and then the mixture is mixed with 50 g of lauric acid monoglyceride, so that 100 g of ester mixture is obtained.

S2, 100 g of formic acid was mixed homogeneously with 80 g of silica and 20 g of bentonite, and then homogeneously mixed with 150 g of fumaric acid and 150 g of citric acid to obtain 500g of an acidulant mixture.

S3, 50 g of radix sileris extract, 25 g of ginkgo epicarp extract and 25 g of whiteflower hogfennel extract are evenly mixed to obtain 100 g of plant extract mixture.

Example 7 challenge protection of combinations of Fennel extracts on PEDV pigs

A challenge protection test was performed on pigs with the drug prepared in example 6.1. A virus solution PEDV and fennel extract combination (fennel, denoted by PE) was prepared in the early stage. Selecting 23-day-old piglets (negative pigs for detecting PDCoV, PEDV, TGEV and PRoV), and dividing into five groups with 8 heads each.

TABLE 4 Table of protection test groups of piglets against PEDV challenge by combination of fennel extracts

Grouping	Number of heads	Dosage/counteracting toxic substance
			Group A: PEDV positive group	8	0/10ml
Group B: 5% PE	8	4g/10ml
			Group C: 6.7% PE	8	4g/10ml
Group D: 10% PE	8	4g/10ml
			Group E: negative blank group	8	0/0

After grouping, each pig was given an ear number. B, C, D three groups were fed with the fennel extract combination according to Table 4 one day before challenge and challenged. Group E was a negative blank. And (5) observing the diarrhea condition of the piglets after the challenge, collecting anus swabs, and scoring the diarrhea. Then, a piglet anus swab is collected every day, and diarrhea scoring, clinical symptom observation and the like are carried out.

1. Clinical observations

On the third day after challenge (fig. 12), the experimental groups all showed significant diarrhea and loose stool, and the positive group a was slightly more severe. On day four after challenge (fig. 13), the positive control group a pigs still had more severe diarrhea; the test group B and the test group C have a small amount of diarrhea, and the diarrhea conditions are similar; the diarrhea was the least severe in test group D. On the fifth day after challenge, one dead pig appeared in group A, and no dead pigs were present in the other groups. And (3) taking the intestinal tract of the dead pig in the positive group A for virus load detection, determining the cause of death, and displaying that the virus load is extremely high and the cause of death is caused by PEDV. Sixth day after challenge (fig. 14), the positive control group a pigs suffered the most severe diarrhea and did not improve; test groups B, C and D all had only mild diarrhea, with most of the swine feces being formed.

The ninth day after challenge (fig. 15), diarrhea was better in each group. As can be seen from the panel of FIG. 15, the swine waste of each group was formed and the diarrhea condition was not severe, indicating that the diarrhea condition was significantly improved. The diarrhea of the control group A pigs is improved, the pigs only rely on the autoimmunity to overcome the diarrhea caused by PEDV, but 1 pig is dead; the diarrhea condition of the test group is stable overall and only slight diarrhea shows that the medicament plays a role in counteracting and protecting the pig and reduces the harm of virus to the pig.

In the whole test period, the negative blank control group is managed and fed according to daily procedures, and health abnormal conditions such as diarrhea of pigs are not found.

2. Anal swab detoxification detection

From the analysis of the anal swab detoxification detection result (fig. 16), the piglet detoxification amount is higher at the 2 nd to 6 th days, and the D group detoxification amount is obviously lower than that of the other three groups in the period. Compared with the positive group A, the B, C group also has higher toxin expelling amount (the lower the Ct value is, the higher the toxin expelling amount is).

3. Diarrhea scoring

The diarrhea score criteria in this example are shown in table 5 below.

TABLE 5 diarrhea score criteria

Score of	Clinical symptoms
		0	Normal and no diarrhea
1	Mild and fluidized diarrhea
		2	Moderate watery diarrhea
3	Severe watery diarrhea
		4	Death was caused by death

Each group was scored for diarrhea and the results are shown in fig. 17. In the experimental process, the positive group A has obvious and serious diarrhea, the administered group B and the administered group C have the same serious diarrhea, and the administered group D has mild condition.

4. Survival rate

In the whole test process, 1 dead pig appears in the positive group A on 5 th and 6 th days respectively, and the survival rate is 75%; the survival rate of the non-dead pigs in other groups was 100%.

5. Tissue viral load detection

Taking one end of each group for killing after the sixth day of virus attack, and taking the intestinal tract to detect the virus load; the reason for the dead pigs in the positive group A was also examined. The results are shown in FIG. 18, the dead piglets in group A have extremely high intestinal tract toxic load, the pigs killed by group ABC have higher toxic load, and the pigs killed by group D have lower toxic load (the lower the Ct value is, the higher the viral load is)

From clinical symptoms, diarrhea scores and toxin expelling tests, the combination of the fennel extract with high concentration has lighter diarrhea in the D group, and the medicament in the BC group has partial effect. Two dead pigs appeared in the positive group A during the experiment, and no dead pigs appeared in the other groups. In general, the low-concentration fennel extract group has the effect of relieving diarrhea to a certain extent, the high-concentration group has better effect, and the effect of the fennel extract pharmaceutical composition on preventing and treating PEDV infection is drug concentration dependence.

Example 8 plant extract combination control test on porcine in Lancet Virus-Positive farm

198 basal piggery sows in a certain pig farm are subjected to virus detection of African swine fever, measures such as pig disinfection and accurate tooth extraction are implemented in the pig farm, the diseases are always developed, the sows exist in all stages, and the diseases are developed after weaning sows are bred. Aiming at epidemic situation pressure in a pig farm, protection of threatened swinery is expected to be implemented by using the plant extract combination, the breeding swinery is kept, the production of the swinery is recovered, and the pressure of blue ear disease of the sow group is reduced.

Starting in 2019, 9 months, the pharmaceutical composition of fennel extract, saposhnikovia divaricata root extract and geranium extract of the present invention (prepared as in example 6.3) was used in the pig farm, when 156 gilts were present. The application time is about 9 weeks, and 4kg of the plant extract composition is added to every ton of sow feed and the sow feed is fed normally. No other antibiotics and traditional Chinese medicines are added to the whole basal sow for health care, and no blue-ear vaccine is applied.

At weeks 1-4 of administration, the number of ASFV-positive pigs increased and then decreased, and no positive pigs were found at and after week 5 (see Table 6).

TABLE 6 statistics of disease onset and epidemic points

Date	Number of suspected epidemic spots	Number of suspected positive pigs	Number of eliminated pigs
				9 month, 1 week	4	4	4
9 month, 2 nd week	6	6	6
				9 month, 3 rd week	7	7	7
9 month, 4 th week	4	4	4
				10 months and 1 week	/	/	/
10 month, 2 nd week	/	/	/
				10 month, 3 rd week	/	/	/

In the test process, the sows, either in first birth or in menstruation, need to be bred as long as the oestrus of the sows is normal. As can be seen from Table 7, the sows had recovered productivity after the use of the pharmaceutical composition of the present invention. Before the experiment begins, 7 months 20 days to 8 months 30 days, the sow has 43 fetuses in total; after the start of the experiment, no sow miscarriage occurred. In addition, in the test process, compared with the test in the month of 10 and the test in the month of 9, the production data is comprehensively improved, which shows that after the pharmaceutical composition disclosed by the invention is used, the productivity of sows can be comprehensively improved, and the productivity of sows is improved and gradually recovered.

TABLE 7 statistical table of sow production performance

The basal sows are only used for the whole field of basal sows, and other antibiotics and traditional Chinese medicines are not added for health care, and blue fungus vaccines are not applied. The test results show that the pharmaceutical composition can block virus replication, eliminate endotoxin in pigs, obviously reduce tear spots (bright eyes of sows), improve the health condition of sows, improve the production performance (reduction of dead mummy), and also has the effect of improving the weaning survival rate of piglets; no health-care medicine is added to the weaned piglets in the delivery room in 10 months, and the health condition is good and the activity is strong at present.

The above examples are merely representative of a few embodiments of the present invention, and although the description is specific and detailed, the present invention should not be construed as limited the scope of the claims. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Claims

1. A pharmaceutical composition for preventing and treating porcine enveloped virus infectious diseases is characterized in that: the pharmaceutical composition comprises a plant extract; the plant extract is one or more of Peucedanum praeruptorum dunn extract, Ginkgo biloba exocarp extract, radix Saposhnikoviae root extract, fructus Foeniculi extract, and flos Pelargonii Hortori extract.

2. The pharmaceutical composition of claim 1, wherein: the pharmaceutical composition also comprises an antibacterial agent, an acidifier, an ester substance and an auxiliary material.

3. The pharmaceutical composition of claim 2, wherein: the pharmaceutical composition comprises the following components in parts by weight: 50-200 parts of plant extract, 100-400 parts of antibacterial agent, 300-600 parts of acidifying agent, 60-150 parts of ester substance and 60-150 parts of auxiliary material.

4. The pharmaceutical composition of claim 3, wherein: the pharmaceutical composition comprises the following components in parts by weight: 100 parts of plant extract, 350 parts of antibacterial agent 250-.

5. The pharmaceutical composition according to any one of claims 2-4, wherein: the antibacterial agent is a zinc-rich inactivated lactobacillus agent; the acidulant comprises a liquid acidulant comprising one or more of formic acid, acetic acid, propionic acid, lactic acid, and a powder acidulant comprising one or more of citric acid or fumaric acid; the ester substance comprises a liquid ester substance and a powder ester substance, wherein the powder ester substance comprises one or more of lauric acid monoglyceride, oleic acid diglyceride, linoleic acid diglyceride, palmitic acid monoglyceride, palmitic acid diglyceride, lauric acid diglyceride, caprylic acid diglyceride, capric acid monoglyceride, capric acid diglyceride and linolenic acid diglyceride; the liquid ester substance comprises one or more of tributyrin, oleic acid monoglyceride, linoleic acid monoglyceride, caprylic acid monoglyceride and linolenic acid monoglyceride.

6. The pharmaceutical composition according to claims 2-4, wherein: the auxiliary material comprises one or more of a carrier, a diluent and a surfactant, and the auxiliary material comprises the following components in percentage by weight: 50-100% of carrier, 0-50% of diluent and 0-5% of surfactant.

7. The pharmaceutical composition of claim 6, wherein: the carrier is one or more of silicon dioxide, diatomite, montmorillonite or medical stone, the diluent comprises one or more of bentonite, zeolite powder, stone powder, talcum powder, vermiculite, attapulgite, shell powder, sepiolite and kaolin, and the surfactant is a Tween surfactant, a span surfactant or a glyceride surfactant.

8. The preparation method of the pharmaceutical composition for preventing and treating the porcine enveloped virus infectious diseases as claimed in any one of claims 1 to 7 comprises the following steps:

s3, at least one of the plant extracts;

9. The preparation method of the pharmaceutical composition for preventing and treating porcine enveloped virus infectious diseases according to claim 8, characterized in that: in the step S1, the liquid ester substance is mixed with the surfactant and then mixed with the carrier; the plant extract is mixed with a diluent in the step S3 to prepare a mixture C.

10. The application of plant extracts in preparing medicines for preventing and treating porcine enveloped virus infectious diseases comprises one or more of whiteflower hogfennel root extract, ginkgo epicarp extract, divaricate saposhnikovia root extract, fennel extract and geranium extract.