Application of phloretin in preparation of medicine or feed additive for treating or preventing swine envelope virus infection

Abstract

The invention discloses an application of phloretin in preparation of a medicine or a feed additive for treating or preventing swine envelope virus infection, the medicine is phloretin or oral liquid, granules, tablets and powder prepared from phloretin as an effective component and a solvent or an auxiliary material, and the use dosage is 2.5-10mg / kg of the body weight of a pig; or the phloretin is directly added into the feed, and the addition amount of the phloretin is 0.01-0.03% of the total weight of the pig feed. The phloretin or the composition containing the phloretin is used as a medicine or a feed additive, is high in safety and free of toxic and side effects, and has the effects of remarkably inhibiting replication of envelope viruses such as porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus and African swine fever virus and reducing the toxicity of the porcine envelope viruses; the defects of immune failure possibly caused by vaccination, virus variation caused by use of antiviral drugs and the like are overcome, the morbidity and mortality of the piglets infected with the swine envelope virus are effectively reduced, and the effect of preventing and treating or preventing the swine envelope virus infection diseases is achieved.

Description

Technical Field

[0001] The present application relates to the technical field of prevention and treatment of porcine envelope virus, and specifically to the application of phloretin in the preparation of drugs or feed additives for treating or preventing porcine envelope virus infection. Background Art

[0002] Porcine enveloped viruses refer to a class of swine viruses with an envelope structure that can cause a variety of diseases in pigs. Common porcine enveloped viruses include porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), and African swine fever virus (ASFV).

[0003] PEDV belongs to the Coronavirus family and the Coronavirus genus. The porcine epidemic diarrhea (PED) caused by its infection seriously affects the healthy breeding of pigs. PEDV mainly infects young piglets, especially those under 7 days old. The clinical symptoms of PED are mainly dog ​​sitting, vomiting, diarrhea, loss of appetite, etc. The watery diarrhea of ​​piglets infected with PEDV often contains milk clots. Dissection shows that the wall of the small intestine of piglets is thin and transparent, congested, and the intestinal tract is dilated. The mortality rate of newborn piglets infected with PEDV can reach 90%. PEDV is an RNA virus, so it is easy to mutate, which brings great challenges to the prevention and control of PEDV in the healthy breeding of piglets.

[0004] PRRSV infection can cause porcine reproductive and respiratory syndrome (PRRS), also known as "blue ear disease". Blue ear disease is a viral disease. The target sites of PRRSV infection are mainly the reproductive tract of sows and the respiratory tract of pigs. Therefore, blue ear disease is also called "reproductive and respiratory syndrome". The clinical manifestations of sows infected with blue ear disease are miscarriage, stillbirth, and mummified fetuses. In addition, pigs of all ages may be infected with blue ear disease and show respiratory dysfunction. Pigs infected with blue ear disease have low immunity and have basically no resistance to immune stress responses caused by pathogens, harmful factors in feed sources, abnormal environmental stimuli, etc. Therefore, blue ear disease is one of the important diseases that seriously endangers the healthy breeding and efficient reproduction of live pigs.

[0005] African swine fever (ASF) caused by ASFV infection is an acute, febrile and highly contagious disease. The clinical symptoms of ASF infection in pigs include high fever, extensive bleeding in the skin and internal organs, respiratory disorders, neurological symptoms, etc.

[0006] Currently, the most effective way to prevent and control porcine envelope virus is vaccination. However, due to the highly variable characteristics of PEDV and PRRS, the effectiveness of their vaccine immunity cannot be guaranteed. In addition, although a lot of vaccine research and development work has been carried out on ASF at home and abroad, there is currently no commercial ASFV vaccine on the market. It should also be noted that due to differences in individual immunity, vaccination cannot completely prevent and control viral infection; at the same time, for individuals who have been infected, vaccination is basically meaningless.

[0007] Therefore, it is urgent to develop new technical solutions for the prevention and control of porcine envelope virus. Summary of the invention

[0008] In view of this, the purpose of the present application is to use phloretin in the preparation of drugs or feed additives for treating or preventing porcine envelope virus infection. The phloretin has the effects of resisting porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus and African swine fever virus, and has the characteristics of high safety and convenient application. The phloretin can be used to prepare drugs or feed additives for treating or preventing porcine envelope virus infection.

[0009] In order to achieve or at least partially achieve the above objectives, the present application provides the following technical solutions:

[0010] Application of phloretin in the preparation of medicines or feed additives for treating or preventing porcine envelope virus infection.

[0011] Compared with the prior art, the technical solution provided by this application has at least the following advantages and effects:

[0012] 1. The phloretin or preparations containing phloretin provided in the present application can be used as feed additives for a long time to achieve the purpose of preventing epidemic diseases. Long-term use will not lead to the production of drug-resistant toxins / strains, nor will it have other toxic side effects. This is an advantage that other anti-disease drugs do not have.

[0013] 2. The phloretin or preparation containing phloretin provided in the present application can effectively prevent and control the infection of PEDV, PRRSV and ASFV by enhancing the natural immune function and antiviral and antibacterial activity of piglets.

[0014] 3. The results of in vitro experiments in this application show that phloretin has a significant inhibitory effect on the in vitro proliferation of PEDV, PRRSV and ASFV. Adding phloretin in the cell culture medium at an addition amount of 100 μg / mL can increase the TCID of PEDV, PRRSV and ASFV on the corresponding cells. 50 The titers were reduced by about 4 to 5 respectively.

[0015] 4 The results of in vivo tests in this application show that phloretin has the function of enhancing natural immunity and antiviral activity, can effectively prevent and treat PEDV, PRRSV and ASFV infections, and effectively improve the survival rate and production performance of pigs. The protection rate of phloretin against PEDV, PRRSV and ASFV infections can reach more than 80% when taken orally at a rate of 100 mg / kg pig body weight or added to piglet feed. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described here are only used to explain the present application and are not used to limit the present application.

[0017] Those skilled in the art will appreciate that, unless otherwise stated, the words "said", "the", and "the aforementioned" used in the text of the present application may also include plural forms. It should be further understood that the wording "including" used in the specification of the present application means the presence of the features, steps, and operations, but does not exclude the presence or addition of one or more other features, integers, and steps.

[0018] Those skilled in the art will appreciate that, for any specific experimental steps or conditions not specified in the examples, the experiments may be carried out according to the conventional experimental steps or conditions described in the literature in the art; any raw materials or instruments used without specifying the manufacturer are conventional products that can be purchased commercially.

[0019] Those skilled in the art will understand that, unless otherwise specified in the present application, when a numerical range is given in an embodiment, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in this application are consistent with the prior art mastery of those skilled in the art and the records of this application, and any methods, equipment and materials of the prior art similar or equivalent to the methods, equipment and materials described in the embodiments of this application can also be used to implement this application.

[0020] Phloretin (PT) was first isolated from apple bark. It belongs to a class of dihydrochalcone compounds containing two hydrogen atoms. PT is named because it is mainly found in the rhizomes and barks of most fruits and plants. The chemical name of PT is 3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)-1-propanone, which is composed of a C6-C3-C6 skeleton structure. The molecular formula of PT is C 15 H 14 O5, relative molecular mass is 274.27. Pure phloretin is light red powder, easily soluble in organic solvents such as methanol, ethanol, acetone, DMSO, insoluble in ether, chloroform and benzene, and almost insoluble in water.

[0021] The examples of the present application found through in vivo and in vitro experiments that phloretin and a composition containing phloretin can significantly inhibit the proliferation of PEDV, PRRSV and ASFV, and inhibit their infection of cells. The phloretin and a composition containing phloretin (hereinafter referred to as "phloretin composition") can be used to prepare drugs or feed additives against porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, and African swine fever virus.

[0022] Based on this, the embodiments of the present application provide the use of phloretin in the preparation of a drug or feed additive for treating or preventing porcine envelope virus infection.

[0023] In certain embodiments, the porcine virus comprises at least one of porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, and African swine fever virus.

[0024] In certain embodiments, the drug or feed additive is phloretin, which can be directly used as a drug or feed additive to treat or prevent pig diseases such as PED, PRRS and ASF.

[0025] In certain embodiments, the drug or feed additive is a phloretin composition, which includes phloretin and excipients, and the excipients include at least one of anhydrous ethanol, propylene glycol, dimethyl sulfoxide, edible oil, water, Tween-80, lactose, defatted rice bran, corn cob powder, soluble starch, bran, silicon dioxide, montmorillonite, vermiculite, medical stone, zeolite powder, and white carbon black.

[0026] In certain embodiments, the phloretin composition is a solution of phloretin mixed with at least one solvent selected from anhydrous ethanol, propylene glycol, dimethyl sulfoxide, edible oil, Tween 80, and water, wherein the concentration of phloretin is 20-100 mg / mL.

[0027] In certain embodiments, the phloretin composition is a solid mixture of at least one of lactose and mannitol, phloretin, and soluble starch, wherein the mass fraction of phloretin in the solid mixture is 45% to 55%.

[0028] In certain embodiments, the phloretin composition is a powdery mixture of phloretin and soluble starch or bran, wherein the mass fraction of phloretin in the powdery mixture is 45% to 55%.

[0029] In certain embodiments, when the aforementioned drug or feed additive is a phloretin composition, its preparation type includes at least one of oral liquid, granules, tablets, and powders, and the preparation can be prepared by conventional pharmaceutical preparation methods.

[0030] In certain embodiments, the carrier added to the feed in the form of the aforementioned granules, tablets, and powders is at least one of montmorillonite, vermiculite, medical stone, vermiculite, zeolite powder, white carbon black, defatted rice bran, corn cob powder, starch, and bran.

[0031] In certain embodiments, the method of using the drug or feed additive comprises at least one of the following methods:

[0032] (1) adding phloretin or a phloretin composition to feed or drinking water at a dosage of 2.5 to 10 mg / kg of pig body weight;

[0033] (2) directly orally administering phloretin or a phloretin composition at a dosage of 2.5 to 10 mg / kg of pig body weight;

[0034] (3) Adding phloretin or the phloretin composition to the feed at a dosage of 0.01% to 0.03% of the total weight of the pig feed.

[0035] The technical solution of the present application and the technical effects achieved are described in detail below through more specific embodiments.

[0036] Example 1

[0037] This embodiment provides a method for preparing a phloretin oral liquid for resisting porcine envelope virus, and the specific steps include:

[0038] (1) Weigh 10 g of phloretin, 20 mL of anhydrous ethanol, 30 mL of Tween-80, and an appropriate amount of water for injection;

[0039] (2) First, phloretin was dissolved in anhydrous ethanol, then Tween-80 was added, and finally water for injection was slowly added to make up to 100 mL; the mixture was mixed evenly to obtain a 100 mg / mL phloretin solution, i.e., the phloretin oral solution G1.

[0040] Example 2

[0041] This embodiment provides a method for preparing phloretin granules for resisting porcine envelope virus, and the specific steps include:

[0042] (1) Weigh 50 g of phloretin, 15 g of lactose, and 35 g of soluble starch for standby use;

[0043] (2) Phloretin, lactose and soluble starch are mixed and granulated, and the granulated mixture is dried to obtain a granule with a phloretin mass fraction of 50%, i.e., phloretin granules G2.

[0044] Example 3

[0045] This embodiment provides a method for preparing phloretin powder for resisting porcine envelope virus, and the specific steps include:

[0046] (1) Weigh 50 g of phloretin and 50 g of soluble starch and set aside;

[0047] (2) The phloretin and bran were mixed evenly and passed through a 60-mesh sieve to obtain a powdery mixture with a phloretin mass fraction of 50%, i.e., phloretin powder G3.

[0048] Example 4

[0049] This embodiment provides a method for preparing a phloretin tablet for resisting porcine envelope virus, and the specific steps include:

[0050] (1) Weigh 50 g of phloretin, 15 g of lactose, and 35 g of soluble starch for standby use;

[0051] (2) After phloretin, lactose and soluble starch are uniformly mixed, the mixture is compressed and dried using a tablet press to obtain a flaky mixture having a mass fraction of phloretin of 50%, namely the phloretin tablet G4.

[0052] The formula and parameters of the phloretin preparation prepared in the above example are shown in Table 1 below:

[0053] Table 1

[0054] Example No. Preparation No. Formulation type Components Terminalia chebula tannin chelate concentration Example 1 G1 Oral solution Phloretin, anhydrous ethanol, Tween-80 100mg / mL Example 2 G2 Granules Phloretin, lactose, soluble starch 50% (mass fraction) Example 3 G3 powder Phloretin, soluble starch 50% (mass fraction) Example 4 G4 tablet Phloretin, lactose, soluble starch 50% (mass fraction)

[0055] The above-mentioned phloretin preparation can be used as a medicine or feed additive to prevent or treat porcine envelope virus infection diseases. When used for routine prevention of pig diseases, the phloretin preparation can be added to the feed at a dosage of 2.5 mg / kg pig body weight and used throughout the entire feeding cycle; when used for clinical treatment of pig diseases, the phloretin preparation can be orally administered at a dosage of 10 mg / kg pig body weight; or the phloretin can be dissolved in water and administered or mixed with the daily diet at a dosage of 10 mg / kg pig body weight, and added to the feed three times a day in the morning, noon and evening for continuous use for one week; the dosage of the phloretin preparation can also be adjusted according to the severity of the pig disease, and the dosage range is controlled within a phloretin content of 2.5 to 10 mg / kg pig body weight.

[0056] Inhibitory effect of phloretin on proliferation of PEDV, PRRSV and ASFV in vitro

[0057] The present application example verifies the inhibitory effect of phloretin on the in vitro proliferation of three porcine viruses, PEDV, PRRSV and ASFV, and the verification method is as follows:

[0058] 1. Materials and methods:

[0059] 1.1 Viruses: PEDV YN strain, PRRSV, and ASFV were isolated and preserved by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0060] 1.2 Cells: African green monkey kidney cells (Vero) are used for the proliferation of PEDV YN strain; monkey embryonic kidney epithelial cells (Marc-145) are used for the proliferation of PRRSV; porcine alveolar macrophages (primary cells) are used for the proliferation of ASFV. Vero and Marc-145 cells are cell lines preserved by the Animal Nutrition and Intestinal Health Science and Technology Innovation Team of Wuhan University of Technology, and porcine alveolar macrophages are isolated from the lungs of healthy piglets.

[0061] 1.3 Experimental drugs: Phloretin (purity > 99.9%) was purchased from Maclean Company.

[0062] 1.4 Reagents: DMEM medium, 1640 medium, trypsin, fetal bovine serum and double antibody (penicillin-streptomycin) were purchased from GIBCO.

[0063] 1.5 In vitro proliferation inhibition:

[0064] (1) PEDV in vitro proliferation inhibition test: PEDV YN strain virus liquid was diluted 10 times with DMEM medium, and 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Six dilutions were inoculated in 96-well plates covered with Vero cell monolayers, 100 μL / well, and the PEDV YN strain virus solution was divided into three test groups and one blank group. DMEM culture medium containing different concentrations of phloretin (25, 50, 100 μg / mL) was added to the three groups, and DMEM culture medium without phloretin was added to the blank group. The cells were placed in a 37°C incubator with 5% CO2 and cultured for 36 hours to observe the cell lesions. The number of lesion-positive wells in the cell wells of each dilution was counted, and the TCID of each phloretin concentration group was calculated using the Reed-Muench method. 50 , compare different groups of TCID 50 The inhibitory effect of phloretin on the proliferation of PEDV in vitro was determined by the value of , and three replicates were performed in each group.

[0065] (2) PRRSV in vitro proliferation inhibition test: PRRSV virus solution was diluted 10-fold using DMEM medium to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7Six dilutions were inoculated into 96-well plates with cultured Marc-145 cell monolayers, 100 μL / well, and the PRRSV virus solution was divided into three test groups and one blank group. DMEM culture medium containing different concentrations of phloretin (25, 50, 100 μg / mL) was added to the three groups, and DMEM culture medium without phloretin was added to the blank group. The cells were placed in a 37°C incubator containing 5% CO2 and cultured for 72 hours to observe the cell lesions. The number of lesion-positive wells in the cell wells of each dilution was counted, and the TCID of each phloretin concentration group was calculated using the Reed-Muench method. 50 , compare different groups of TCID 50 The inhibitory effect of phloretin on the proliferation of PRRSV in vitro was determined by the value of , and three replicates were performed in each group.

[0066] (3) ASFV in vitro proliferation inhibition test: ASFV virus solution was diluted 10-fold using 1640 medium to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Six dilutions were inoculated into 96-well plates of cultured alveolar macrophage monolayers, 100 μL / well, and the ASFV virus solution was divided into three test groups and one blank group. The three groups were added with 1640 medium containing different concentrations of phloretin (25, 50, 100 μg / mL), and the blank group was added with 1640 medium without phloretin. The cells were placed in a 37°C incubator with 5% CO2 and cultured for 72 hours to observe cell lesions. The number of lesion-positive wells in the cell wells of each dilution was counted, and the TCID of each phloretin concentration group was calculated using the Reed-Muench method. 50 , compare different groups of TCID 50 The inhibitory effect of phloretin on the proliferation of ASFV in vitro was judged by the value of , and three replicates were performed in each group.

[0067] 1.6 Data analysis: The experimental data were analyzed by one-way ANOVA in SPSS23.0 statistical software, and Duncan's method was used for multiple comparisons between groups. P < 0.05 was considered a significant difference, and P < 0.01 was considered an extremely significant difference. The results were expressed as mean ± standard deviation.

[0068] 2. Test results:

[0069] Table 2 shows the TCID of different concentrations of phloretin against different porcine envelope viruses 50 Influence results, where a, b, c, and d are marks of significant difference.

[0070] Table 2

[0071] Group PEDV PPRSV ASFV Blank Group <![CDATA[10 7.83±0.25a ]]> <![CDATA[10 8.08±0.36a ]]> <![CDATA[10 8.42±0.24a ]]> 25 μg / mL group <![CDATA[10 7.16±0.31b ]]> <![CDATA[10 6.97±0.21b ]]> <![CDATA[10 6.77±0.31b ]]> 50 μg / mL group <![CDATA[10 5.33±0.42c ]]> <![CDATA[10 5.11±0.19c ]]> <![CDATA[10 4.37±0.27c ]]> 100 μg / mL group <![CDATA[10 3.21±0.07d ]]> <![CDATA[10 3.01±0.23d ]]> <![CDATA[10 2.77±0.12d ]]>

[0072] The results in Table 2 show that as the concentration of phloretin increases, the TCID 50 The results showed that phloretin could inhibit the proliferation of PEDV in Vero cells cultured in vitro. With the increase of phloretin concentration, the TCID 50 The results showed that phloretin had an inhibitory effect on the proliferation of PRRSV in Marc-145 cells. Similarly, with the increase of phloretin concentration, the TCID 50 It decreased significantly, indicating that phloretin also has an inhibitory effect on the proliferation of ASFV in alveolar macrophages. When the added concentration of phloretin is 100 μg / mL, the inhibitory effect of phloretin on the proliferation of three porcine envelope viruses, PEDV, PRRSV and ASFV, in vitro reached the strongest level.

[0073] Protective effect of phloretin preparation on piglets infected with PEDV

[0074] The present application example verifies the protective effect of the phloretin preparation on piglets infected with PEDV, and the verification method is as follows:

[0075] 1. Materials and methods:

[0076] 1.1 Experimental virus: PEDV YN strain was donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0077] 1.2 Experimental drug: Phloretin granules G2.

[0078] 1.3 Experimental animals: 60 healthy 3-day-old newborn piglets of the same genetic background, uniform body weight and without maternal antibodies to PEDV were evenly divided into 4 treatment groups, including three experimental groups (low-, medium- and high-dose groups of phloretin) and a blank group, each with 15 pigs. Phloretin granules G2 were dissolved in artificial milk and diluted to 3 mL for oral administration, including a low-dose group of phloretin (added at a phloretin content of 2.5 mg / kg pig body weight), a medium-dose group of phloretin (added at a phloretin content of 5 mg / kg pig body weight), and a high-dose group of phloretin (added at a phloretin content of 10 mg / kg pig body weight).

[0079] 1.4 Challenge test: After feeding for one week, the piglets in each group were fed with 10 6 TCID 50 / mL of PEDV (YN strain) was used for challenge. Each piglet was orally infused with 3.0 mL at one time. After challenge, the piglets were observed for 3 consecutive days until the end of the experiment.

[0080] 1.5 Observation indicators: The diarrhea of ​​the experimental piglets was observed every day. The experimental piglets were slaughtered at the end of the experiment to observe intestinal lesions, and the number of dead pigs was counted, and the diarrhea rate and mortality rate were calculated. The expression of PEDV structural gene N in the intestinal tissue of piglets was detected by real-time quantitative PCR to explore the in vivo inhibition of phloretin on PEDV virus.

[0081] 1.6 Data analysis: The experimental data were analyzed by one-way ANOVA in SPSS23.0 statistical software, and Duncan's method was used for multiple comparisons between groups. P < 0.05 was considered a significant difference, and P < 0.01 was considered an extremely significant difference. The results were expressed as mean ± standard deviation.

[0082] 2. Experimental results:

[0083] After different doses of phloretin granules G2 were added to artificial milk and fed to piglets infected with PEDV, the morbidity and mortality of piglets in each group are shown in Table 3.

[0084] Table 3

[0085] Group Diarrhea rate (%) mortality rate(%) Blank Group 86.7 66.7 2.5mg / kg group 53.3 33.3 5mg / kg group 33.3 26.7 10mg / kg group 13.3 6.7

[0086] The results in Table 3 show that different doses of phloretin granules G2 can significantly reduce the diarrhea rate and mortality rate of PEDV-infected piglets. Compared with the blank group, the diarrhea rate of PEDV-infected piglets decreased to 13.3% to 53.3%; and the mortality rate of diseased pigs decreased to 6.7% to 33.3%. The experimental results fully demonstrate that the addition of phloretin preparations can significantly improve the protection rate of piglets against PEDV infection.

[0087] The expression level of PEDV structural gene N in the intestinal mucosa of the duodenum, jejunum and ileum tissues of each group of piglets was detected respectively. The test results are shown in Table 4 below, where a, b, c, and d are significant differences.

[0088] Table 4

[0089] Sampling tissue Detection of genes Blank Group 2.5mg / kg group 5mg / kg group 10mg / kg group P-value Duodenum N <![CDATA[1.000±0.221 a ]]> <![CDATA[0.791±0.258 ab ]]> <![CDATA[0.305±0.077 c ]]> <![CDATA[0.053±0.001 d ]]> 0.001 jejunum N <![CDATA[1.000±0.213 a ]]> <![CDATA[0.743±0.122 ab ]]> <![CDATA[0.487±0.113 c ]]> <![CDATA[0.136±0.009 d ]]> 0.001 Ileum N <![CDATA[1.000±0.191 a ]]> <![CDATA[0.823±0.176 ab ]]> <![CDATA[0.328±0.015 c ]]> <![CDATA[0.159±0.003 d ]]> 0.001

[0090] As shown in Table 4, compared with the blank group, different doses of phloretin granules G2 can significantly reduce the N gene expression of PEDV, indicating that phloretin can inhibit the replication of PEDV in the intestine of piglets.

[0091] Protective effect of phloretin preparation on piglets infected with PRRSV

[0092] The present application example verifies the protective effect of the phloretin preparation on piglets infected with PRRSV, and the verification method is as follows:

[0093] 1. Materials and methods:

[0094] 1.1 Experimental virus: PRRSV was donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0095] 1.2 Experimental drug: phloretin powder G3.

[0096] 1.3 Experimental animals: 60 healthy 3-day-old newborn piglets of the same genetic background, uniform body weight and without PRRSV maternal antibodies were evenly divided into 4 treatment groups, including three experimental groups (low-, medium- and high-dose groups of phloretin) and a blank group, each with 15 pigs. Phloretin powder G3 was dissolved in artificial milk and diluted to 3 mL for oral administration, including a low-dose group of phloretin (added at a phloretin content of 2.5 mg / kg pig body weight), a medium-dose group of phloretin (added at a phloretin content of 5 mg / kg pig body weight), and a high-dose group of phloretin (added at a phloretin content of 10 mg / kg pig body weight).

[0097] 1.4 Challenge test: After feeding for one week, the piglets in each group were fed with 10 6.5 TCID 50 / mL PRRSV was used for challenge, and each piglet was orally infused with 3.0mL at one time. After challenge, the piglets were observed for 7 consecutive days until the end of the experiment.

[0098] 1.5 Observation indicators: After the experiment, the piglets were slaughtered to observe the lung lesions, and the number of dead pigs was counted to calculate the morbidity and mortality. The expression of PRRSV ORF7 gene in the brain and lung of piglets was detected by real-time quantitative PCR to determine whether phloretin has an inhibitory effect on PRRSV.

[0099] 1.6 Data analysis: The experimental data were analyzed by one-way ANOVA in SPSS23.0 statistical software, and Duncan's method was used for multiple comparisons between groups. P < 0.05 was considered a significant difference, and P < 0.01 was considered an extremely significant difference. The results were expressed as mean ± standard deviation.

[0100] 2. Experimental results:

[0101] After adding different doses of phloretin powder G3 into artificial milk and feeding it to piglets infected with PRRSV, the mortality rate of piglets in each group is shown in Table 5.

[0102] Table 5

[0103] Group mortality rate(%) Blank Group 73.3 2.5mg / kg group 53.3 5mg / kg group 33.3 10mg / kg group 20

[0104] The results in Table 5 show that different doses of phloretin powder G3 can significantly reduce the mortality of piglets infected with PRRSV. Compared with the blank group, the mortality of piglets infected with PRRSV decreased to 20% to 73.3%. The test results fully demonstrate that the addition of phloretin preparations can significantly improve the protection rate of piglets against PRRSV infection.

[0105] The expression levels of ORF7 gene of PRRSV virus in the lung and hypothalamus of each group of piglets were detected respectively. The detection results are shown in Table 6 below, where a, b, c, and d are marks of significant difference.

[0106] Table 6

[0107] Sampling tissue Detection of genes Blank Group 2.5mg / kg group 5mg / kg group 10mg / kg group P-value lung ORF7 <![CDATA[1.000±0.293 a ]]> <![CDATA[0.601±0.132 ab ]]> <![CDATA[0.287±0.013 c ]]> <![CDATA[0.021±0.001 d ]]> 0.001 Hypothalamus ORF7 <![CDATA[1.000±0.101 a ]]> <![CDATA[0.543±0.112 ab ]]> <![CDATA[0.121±0.002 c ]]> <![CDATA[0.009±0.001 d ]]> 0.001

[0108] As shown in Table 6, compared with the blank group, different doses of phloretin powder G3 can significantly reduce the expression level of ORF7 gene of PRRSV virus, indicating that phloretin can inhibit the replication of PRRSV virus in the intestine of piglets.

[0109] Therapeutic effect of phloretin preparation on piglets infected with PEDV

[0110] The present application example also verifies the therapeutic effect of the phloretin preparation on PEDV-infected piglets, and the verification method is as follows:

[0111] 1. Materials and methods:

[0112] 1.1 Experimental virus: PEDV YN strain was donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0113] 1.2 Experimental drug: Phloretin tablets G4.

[0114] 1.3 Experimental animals: 60 healthy 3-day-old newborn piglets of the same genetic background, uniform body weight and without PEDV maternal antibodies were evenly divided into 3 treatment groups, including a treatment group, a blank group and a control group, with 15 pigs in each treatment. Phloretin tablets G4 were dissolved in artificial milk and added at a dose of 2.5 mg / kg pig body weight. The volume was adjusted to 3 mL and administered to the piglets in the treatment group. The blank group and the control group were administered with artificial milk without the addition of phloretin tablets G4.

[0115] 1.4 Challenge test: After one week of feeding, piglets in the treatment group and the blank group were fed with 10 6 TCID 50 / mL PEDV (YN strain) was used for challenge, and the challenge dose was: 3.0mL was orally infused once per piglet; the control group was instilled with an equal volume of normal saline. At this time, the piglets in the treatment group were continuously fed with a phloretin content of 10 mg / kg pig body weight until the end of the experiment.

[0116] 1.5 Observation indicators: The diarrhea of ​​each group of experimental piglets was observed every day. The experimental piglets were slaughtered at the end of the experiment to observe intestinal lesions. The number of deaths of piglets in each group was counted, and the diarrhea rate and mortality rate were calculated. The expression of PEDV structural gene N in the intestinal tissue of piglets was detected by real-time quantitative PCR to explore the in vivo inhibition of phloretin on PEDV virus.

[0117] 1.6 Data analysis: The experimental data were analyzed by one-way ANOVA in SPSS23.0 statistical software, and Duncan's method was used for multiple comparisons between groups. P < 0.05 was considered a significant difference, and P < 0.01 was considered an extremely significant difference. The results were expressed as mean ± standard deviation.

[0118] 2. Experimental results:

[0119] After different doses of phloretin tablets G4 were added to artificial milk and fed to piglets infected with PEDV, the morbidity and mortality of piglets in each group are shown in Table 7.

[0120] Table 7

[0121] Group Diarrhea rate (%) mortality rate(%) Control group 13.3 0 Blank Group 93.3 60 Treatment Group 26.7 20

[0122] The results in Table 7 show that the diarrhea rate and mortality rate of piglets in the treatment group taking phloretin tablets G4 were reduced to 26.7% and 20%, respectively, compared with the blank group.

[0123] The expression level of PEDV structural gene N in the intestinal mucosa of the duodenum, jejunum and ileum tissues of each group of piglets was detected respectively. The test results are shown in Table 8 below, where a and b are significant differences.

[0124] Table 8

[0125] Sampling tissue Control group Blank Group Treatment Group P-value Duodenum / <![CDATA[1.000±0.301 a ]]> <![CDATA[0.218±0.012 b ]]> 0.001 jejunum / <![CDATA[1.000±0.132 a ]]> <![CDATA[0.372±0.183 b ]]> 0.001 Ileum / <![CDATA[1.000±0.103 a ]]> <![CDATA[0.183±0.001 b ]]> 0.001

[0126] As shown in Table 8, compared with the blank group, the piglets in the treatment group taking phloretin tablets G4 can significantly reduce the N gene expression of PEDV in the intestinal tissue. The test results in Tables 7 and 8 fully demonstrate that phloretin preparations can play a therapeutic role in piglets infected with PEDV.

[0127] In summary, the phloretin and phloretin preparations provided in the embodiments of the present application have the effect of resisting porcine envelope viruses such as PEDV, PRRSV and ASFV, and can be used to prepare drugs and feed additives for treating and preventing pig diseases infected by porcine envelope viruses such as PEDV, PRRSV and ASFV. The dosage forms of the drugs and feed additives include oral liquids, granules, powders, tablets and other dosage forms.

[0128] The present application is described in detail above. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the present application and its core ideas. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. Application of phloretin in the preparation of drugs or feed additives for treating or preventing porcine envelope virus infection.

2. The use according to claim 1, characterized in that: The porcine enveloped virus includes at least one of porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, and African swine fever virus.

3. The use according to claim 1, characterized in that: The medicine or feed additive is phloretin.

4. The use according to claim 1, characterized in that: The medicine or feed additive is a phloretin composition comprising phloretin and auxiliary materials, wherein the auxiliary materials include at least one of anhydrous ethanol, propylene glycol, dimethyl sulfoxide, edible oil, water, Tween-80, lactose, defatted rice bran, corn cob powder, soluble starch, bran, silicon dioxide, montmorillonite, vermiculite, medical stone, zeolite powder and white carbon black.

5. The use according to claim 4, characterized in that: The phloretin composition is a solution of phloretin mixed with at least one solvent selected from anhydrous ethanol, propylene glycol, dimethyl sulfoxide, edible oil, Tween 80 and water, wherein the concentration of phloretin is 20-100 mg / mL.

6. The use according to claim 4, characterized in that: The phloretin composition is a solid mixture of at least one of lactose and mannitol, phloretin and soluble starch, wherein the mass fraction of phloretin in the solid mixture is 45% to 55%.

7. The use according to claim 4, characterized in that: The phloretin composition is a powdery mixture of phloretin and soluble starch or bran, wherein the mass fraction of phloretin in the powdery mixture is 45% to 55%.

8. The use according to claim 4, characterized in that: The preparation types of the medicine or feed additive include at least one of oral liquid, granules, tablets and powders, and the preparation can be prepared by conventional pharmaceutical preparation methods.

9. The use according to claim 8, characterized in that: The carrier added to the feed by the granules, tablets and powders is at least one of montmorillonite, vermiculite, medical stone, vermiculite, zeolite powder, white carbon black, defatted rice bran, corn cob powder, starch and bran.

10. The use according to claim 1, characterized in that: The method for using the drug or feed additive includes at least one of (1) to (3): (1) adding phloretin or a phloretin composition to feed or drinking water at a dosage of 2.5 to 10 mg / kg of pig body weight; (2) directly orally administering phloretin or a phloretin composition at a dosage of 2.5 to 10 mg / kg of pig body weight; (3) Adding phloretin or the phloretin composition to the feed at a dosage of 0.01% to 0.03% of the total weight of the pig feed.