Use of phloretin in the preparation of a medicament or feed additive for the treatment or prevention of porcine paratuberculosis

Abstract

The application discloses application of phloretin in preparation of a medicine or a feed additive for treating or preventing porcine capsid virus infection, the medicine being phloretin or oral liquid, granules, tablets, powder prepared by adding a solvent or excipient with phloretin as an effective component, and a dosage being 2.5-10 mg / kg of pig body weight; or the phloretin being directly added into feed, and an adding amount being 0.01%-0.03% of the total weight of pig feed. The application takes phloretin or a composition containing phloretin as the medicine or the feed additive, is high in safety, free of toxic side effects, has a significant effect of inhibiting replication of capsid viruses such as porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus and African swine fever virus, reducing the virulence of porcine capsid virus, overcoming the disadvantages of possible immunization failure of vaccination and virus variation caused by use of antiviral drugs, effectively reducing the morbidity and mortality of piglets infected with porcine capsid virus, and having the effect of preventing and treating or preventing porcine capsid virus infection diseases.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of prevention and treatment of porcine cecum membrane virus, and particularly relates to application of phloretin in preparation of a medicine or a feed additive for treating or preventing porcine cecum membrane virus infection. BACKGROUND

[0002] Porcine cecum membrane virus refers to a kind of porcine virus with a cecum membrane structure, which can cause various diseases of pigs. At present, common porcine cecum membrane viruses include porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), African swine fever virus (ASFV) and the like.

[0003] PEDV belongs to the family of Coronaviridae and the genus of Coronavirus, and the infection caused by PEDV seriously affects the healthy breeding of pigs. PEDV mainly infects young piglets, especially piglets before 7 days old. The clinical symptoms of PED mainly include piglet crouching, vomiting, diarrhea, loss of appetite and the like. The watery diarrhea of piglets infected with PEDV often contains milk clots, and the piglets can be dissected to show thin and transparent small intestine wall, hyperemia, intestinal expansion and the like. The mortality rate of newborn piglets infected with PEDV can reach 90%. PEDV is an RNA virus, and thus it is easy to mutate, which brings great challenges to the prevention and control of PEDV in piglet healthy breeding. According to reports, PEDV has become the most important pathogen of viral diarrhea in pigs in China, and the mutant strain is the main pathogen in PEDV infection.

[0004] PRRSV infection can cause porcine reproductive and respiratory syndrome (PRRS), which is also called “porcine blue ear disease”. Porcine blue ear disease is a viral disease occurring in developed countries in the late 1980s, and the target site of PRRSV infection mainly reflects 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 include abortion, stillbirth, production of mummy fetus and the like. In addition, pigs of all ages can be infected with blue ear disease and show respiratory dysfunction and the like. Pigs infected with blue ear disease have low immunity, and they basically have no resistance to immune stress response caused by pathogenic bacteria, harmful factors from feed sources and abnormal environmental stimulation. Therefore, blue ear disease is one of the important diseases that seriously endanger the healthy breeding and efficient reproduction of pigs.

[0005] African swine fever (ASF) caused by ASFV infection is an acute, febrile and highly contagious disease. The clinical symptoms of pigs infected with ASF include high fever, extensive bleeding in the skin and internal organs, respiratory dysfunction, neurological symptoms, etc. ASF was first reported in Kenya, then spread to Europe and Asia. Since 2018, the mortality rate of pigs caused by ASF infection in China has been high, and effective prevention and control measures are still lacking, so ASF has become one of the diseases with great harm to the current pig breeding in China.

[0006] Currently, the most effective method to prevent and control porcine cuniculitis virus is vaccination, but due to the highly variable characteristics of PEDV and PRRS, the effectiveness of the vaccine immune effect cannot be guaranteed. In addition, although a large number of vaccine research and development work has been carried out on ASF at home and abroad, there is still no commercial ASFV vaccine on the market. It should be noted that due to the difference 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 a new technical solution to prevent and control porcine cuniculitis virus. SUMMARY

[0008] Therefore, the purpose of the present application is to use phloretin in the preparation of a drug or feed additive for treating or preventing porcine cuniculitis virus infection. The phloretin has the effects of resisting porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus and African swine fever virus, is safe and convenient to use. The phloretin can be used to prepare a drug or feed additive for treating or preventing porcine cuniculitis virus infection.

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

[0010] The application of phloretin in the preparation of a drug or feed additive for treating or preventing porcine cuniculitis virus infection.

[0011] The technical solution provided by the present application has at least the following advantages and effects compared with the prior art:

[0012] 1. The phloretin or the preparation containing phloretin provided by the present application can be used as a feed additive for a long time to achieve the purpose of preventing epidemic diseases. Long-term use will not lead to the generation of drug-resistant strains or other toxic side effects, which is an advantage that other anti-disease drugs do not have.

[0013] 2. The phloretin or the preparation containing phloretin provided by 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 present application shows that by in vitro experimental results: phloretin has a significant inhibitory effect on the in vitro proliferation of PEDV, PRRSV and ASFV, and the addition of phloretin in the cell culture medium at an addition amount of 100 μg / mL can reduce the TCID 50 of PEDV, PRRSV and ASFV on the corresponding cells by about 4-5 titers, respectively.

[0015] 4. The present application shows that by in vivo test results: phloretin has the effects of enhancing natural immune function and antiviral activity, and can effectively prevent and treat PEDV, PRRSV and ASFV infection, and effectively improve the survival rate and production performance of pigs. The protection rate of PEDV, PRRSV and ASFV infection can reach more than 80% by adding phloretin at 100 mg / kg of pig body weight orally or in piglet feed. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical scheme and advantages of the present application more clear and apparent, the present application will be further described in detail below in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0017] Those skilled in the art can understand that, unless specifically stated otherwise, the use of "said", "the", "the foregoing" in the text of the present application can also include the plural form. It should be further understood that the use of the phrase "comprising" in the specification of the present application means that the features, steps, operations exist, but does not exclude the existence or addition of one or more other features, integers, steps.

[0018] Those skilled in the art can understand that, unless the specific experimental steps or conditions are indicated in the embodiments, the operation or conditions can be carried out according to the conventional experimental steps described in the literature in the art; and the raw materials or instruments and equipment not indicated by the manufacturer are all conventional products that can be obtained by market purchase.

[0019] Those skilled in the art can understand that, unless otherwise stated in the present application, when the embodiments give numerical ranges, each numerical range and any number between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present application are used by those skilled in the art in the present technical field and the description of the present application, and any method, equipment and material of the prior art similar or equivalent to the methods, equipment and materials described in the embodiments of the present application can be used to realize the present application.

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

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

[0022] Based on this, the present application provides an application of phloretin in preparing a medicine or a feed additive for treating or preventing porcine cyst membrane virus infection.

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

[0024] In some embodiments, the medicine or the feed additive is phloretin, which can be directly used as a medicine or a feed additive for treating or preventing PED, PRRS and ASF and other porcine diseases.

[0025] In some embodiments, the medicine or the feed additive is a phloretin composition, which includes phloretin and excipients. 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 some embodiments, the aforementioned phloretin composition is a solution of phloretin mixed with at least one solvent of anhydrous ethanol, propylene glycol, dimethyl sulfoxide, edible oil, Tween 80, and water, wherein the concentration of phloretin is 20-100 mg / mL.

[0027] In some embodiments, the aforementioned 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 some embodiments, the aforementioned phloretin composition is a powder mixture of phloretin and soluble starch or bran, wherein the mass fraction of phloretin in the powder mixture is 45% to 55%.

[0029] In some embodiments, when the aforementioned pharmaceutical or feed additive is a phloretin composition, the preparation type includes at least one of oral liquid, granules, tablets, and powder, and the preparation can be prepared by using a conventional pharmaceutical preparation method.

[0030] In some embodiments, the carrier for adding the aforementioned granules, tablets, and powder to the feed is at least one of montmorillonite, vermiculite, rhyolite, zeolite powder, white carbon black, defatted rice bran, corn cob powder, starch, and bran.

[0031] In some embodiments, the method for using the aforementioned pharmaceutical or feed additive includes at least one of the following methods:

[0032] (1) Adding phloretin or a phloretin composition to feed or drinking water for use, according to a dosage of 2.5 to 10 mg / kg of pig body weight in terms of phloretin content;

[0033] (2) Directly orally taking phloretin or a phloretin composition, according to a dosage of 2.5 to 10 mg / kg of pig body weight in terms of phloretin content;

[0034] (3) Adding phloretin or a phloretin composition to feed for use, according to a dosage of 0.01% to 0.03% of the total weight of the pig feed in terms of phloretin content.

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

[0036] Embodiment 1

[0037] This embodiment provides a preparation method of a phloretin oral liquid for resisting porcine cystic disease, and the specific steps include:

[0038] (1) Weighing 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, dissolve the phloretin in anhydrous ethanol, then add Tween-80, and finally slowly add water for injection to make up to 100 mL; mix well to obtain a 100 mg / mL phloretin solution, i.e., the phloretin oral liquid G1.

[0040] Example 2

[0041] The present example provides a preparation method of phloretin granules against porcine cystic virus, and the specific steps include:

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

[0043] (2) Mix phloretin, lactose and soluble starch to granulate, and dry the granules with a phloretin mass fraction of 50% to obtain a granular mixture, i.e. phloretin granules G2.

[0044] Example 3

[0045] The present example provides a preparation method of phloretin powder against porcine cystic virus, and the specific steps include:

[0046] (1) Take 50 g of phloretin and 50 g of soluble starch, and wait for use;

[0047] (2) Mix phloretin and bran evenly, and pass 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] The present example provides a preparation method of phloretin tablets against porcine cystic virus, and the specific steps include:

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

[0051] (2) Mix phloretin, lactose and soluble starch evenly, and then press and dry the tablets with a tablet press to obtain a tablet-like mixture with a phloretin mass fraction of 50%, i.e. the phloretin tablets G4.

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

[0053] Table 1

[0054]

[0055] The above phloretin preparation can be used as a medicine or a feed additive for preventing or treating porcine virus infection diseases. When used for preventing porcine diseases in general, the phloretin preparation can be added to feed at an additive amount of 2.5 mg / kg of pig body weight, and used throughout the feeding period; when used for clinically treating porcine diseases, the phloretin preparation can be orally poured into the oral cavity at a dose of 10 mg / kg of pig body weight; or the phloretin can be dissolved in water and poured into the oral cavity or mixed with daily feed and added to feed, and used three times a day, for one week continuously; or the dose of the phloretin preparation can be adjusted according to the severity of the porcine diseases, and the dose range is controlled to be 2.5-10 mg / kg of pig body weight.

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

[0057] The embodiments of the present application verify the inhibitory effect of phloretin on the in-vitro proliferation of PEDV, PRRSV and ASFV, and the verification method is as follows:

[0058] 1. Materials and methods:

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

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

[0061] 1.3 Test drug: phloretin (purity > 99.9%) was purchased from Macklin Company.

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

[0063] 1.5 In-vitro proliferation inhibition:

[0064] (1) PEDV in-vitro proliferation inhibition test: the 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, respectively, inoculated in Vero cell monolayer has grown in 96-well plates, 100 μL / well, PEDV YN strain virus liquid into three test groups and a blank group, three groups were added to the DMEM medium containing different concentrations of phloretin (25, 50, 100 μg / mL), the blank group was added to the DMEM medium without phloretin, placed in a 37°C 5% CO2 incubator for 36 h, and observed for cytopathic effect. The number of positive wells in each dilution was counted, and the TCID 50 of each concentration of phloretin group was calculated by Reed-Muench method 50 , and the inhibitory effect of phloretin on PEDV in vitro proliferation was compared by comparing the values of different groups of TCID -2 , each group was repeated three times.

[0065] (2) PRRSV in vitro proliferation inhibition test: DMEM medium was used to dilute PRRSV virus liquid by 10 times, and 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Six dilutions, respectively, inoculated in Marc-145 cell monolayer has been cultured in 96-well plates, 100 μL / well, PRRSV virus liquid into three test groups and a blank group, three groups were added to the DMEM medium containing different concentrations of phloretin (25, 50, 100 μg / mL), the blank group was added to the DMEM medium without phloretin, placed in a 37°C 5% CO2 incubator for 72 h, and observed for cytopathic effect. The number of positive wells in each dilution was counted, and the TCID 50 of each concentration of phloretin group was calculated by Reed-Muench method 50 , and the inhibitory effect of phloretin on PRRSV in vitro proliferation was compared by comparing the values of different groups of TCID -2 , each group was repeated three times.

[0066] (3) ASFV in vitro proliferation inhibition test: 1640 medium was used to dilute ASFV virus liquid by 10 times, and 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7Six dilutions, respectively, inoculated in the 96-well plate of the cultured alveolar macrophage monolayer, 100 μL / well, the ASFV virus liquid was divided into three test groups and a blank group, three groups were added to 1640 culture medium containing different concentrations of phloretin (25, 50, 100 μg / mL), the blank group was added to 1640 culture medium without phloretin, and placed in a 37℃ CO2 incubator containing 5% for 72 h, and the cytopathic effect was observed. The number of positive wells in each dilution was counted, and the TCID of each concentration of phloretin group was calculated by Reed-Muench method 50 , and the values of TCID 50 of different groups were compared to judge the inhibitory effect of phloretin on ASFV proliferation in vitro, and each group was repeated three times.

[0067] 1.6 Data analysis: The experimental data was analyzed by One-way ANOVA in SPSS 23.0 statistical software, and Duncan's method was used for multiple comparisons between groups, with P<0.05 as the significant difference standard, and P<0.01 as the extremely significant difference standard. The results were expressed as mean ± standard deviation.

[0068] 2. Test results:

[0069] Table 2 shows the effect of different concentrations of phloretin on the TCID 50 of different porcine membrane viruses, where a, b, c, and d represent significant difference.

[0070] Table 2

[0071]

[0072] The results of Table 2 show that as the concentration of phloretin increases, the TCID 50 of PEDV on Vero cells decreases significantly, indicating that phloretin has an inhibitory effect on the proliferation of PEDV in Vero cells in vitro; as the concentration of phloretin increases, the TCID 50 of PRRSV on Marc-145 cells decreases significantly, indicating that phloretin has an inhibitory effect on the proliferation of PRRSV in Marc-145 cells; similarly, as the concentration of phloretin increases, the TCID 50 of ASFV on alveolar macrophages decreases significantly, indicating that phloretin also has an inhibitory effect on the proliferation of ASFV on alveolar macrophages; when the concentration of phloretin added is 100 μg / mL, the inhibitory effect of phloretin on the proliferation of PEDV, PRRSV, and ASFV in vitro reaches the strongest.

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

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

[0075] 1. Materials and methods:

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

[0077] 1.2 Test drug: phloretin granules G2.

[0078] 1.3 Test animals: 60 3-day-old Du Changda newborn piglets with the same genetic background, uniform body weight, health and no PEDV maternal antibody were evenly divided into 4 treatment groups, including three test groups (low, medium and high dose phloretin groups) and one blank group, each group with 15 pigs. Phloretin granules G2 were dissolved in artificial milk, and the volume was adjusted to 3 mL for oral administration. The low dose phloretin group (added according to the phloretin content of 2.5 mg / kg pig body weight), the medium dose phloretin group (added according to the phloretin content of 5 mg / kg pig body weight), and the high dose phloretin group (added according to the phloretin content of 10 mg / kg pig body weight).

[0079] 1.4 Challenge test: After one week of feeding, each group of piglets was challenged with PEDV (YN strain) at a concentration of 10 6 TCID 50 / mL, each piglet was orally perfused with 3.0 mL at one time, and observed continuously for 3 days after challenge until the end of the test.

[0080] 1.5 Observation index: The diarrhea of the test piglets was observed every day, and the intestinal lesions of the test piglets were observed after the test period. 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 test data was analyzed by One-way ANOVA in SPSS 23.0 statistical software, and Duncan's method was used for multiple comparisons between groups. P<0.05 was the significant difference standard, and P<0.01 was the extremely significant difference standard. The results were expressed as mean ± standard deviation.

[0082] 2. Experimental results:

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

[0084] Table 3

[0085]

[0086] The results of Table 3 show that the phloretin granules G2 of different doses 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 is reduced to 13.3%~53.3%; and the mortality rate of sick pigs is reduced to 6.7%~33.3%; the test results fully show that the phloretin preparation after adding can significantly improve the protection rate of piglets to PEDV infection.

[0087] The expression amount of PEDV structural gene N in the intestinal mucosa of duodenum, jejunum and ileum of piglets in each group was detected, and the detection results are shown in Table 4, wherein a, b, c and d are significant difference identifiers.

[0088] Table 4

[0089]

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

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

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

[0093] 1. Materials and methods:

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

[0095] 1.2 Test drug: phloretin powder G3.

[0096] 1.3 Test animals: 60 three-day-old Duliangda new born piglets with the same genetic background, uniform weight, health and no PRRSV maternal antibody were evenly divided into four treatment groups, including three test groups (low, medium and high dose phloretin groups) and one blank group, each treatment group had 15 pigs, phloretin powder G3 was dissolved in artificial milk, and the volume was adjusted to 3 mL for oral administration, wherein the low dose phloretin group (added according to the phloretin content of 2.5 mg / kg pig body weight dose), the medium dose phloretin group (added according to the phloretin content of 5 mg / kg pig body weight dose), and the high dose phloretin group (added according to the phloretin content of 10 mg / kg pig body weight dose).

[0097] 1.4 Challenge test: each group of piglets was fed for one week, and then 10 6.5 TCID50 The piglets were orally infected with PRRSV at a dose of 3.0 mL per piglet, and observed for 7 days after the infection until the end of the experiment.

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

[0099] 1.6 Data analysis: The data was analyzed by One-way ANOVA in SPSS 23.0 statistical software, and the multiple comparisons between groups were performed by Duncan's method. P<0.05 was considered as a significant difference, and P<0.01 was considered as a highly significant difference. The results were expressed as mean ± standard deviation.

[0100] 2. Experimental results:

[0101] After the piglets infected with PRRSV were fed with artificial milk containing different doses of phloretin powder G3, the mortality of the piglets in each group was shown in Table 5.

[0102] Table 5

[0103]

[0104] Table 5 shows that different doses of phloretin powder G3 can significantly reduce the mortality of piglets infected with PRRSV, and compared with the blank group, the mortality of piglets infected with PRRSV is reduced to 20%~73.3%; the test results fully show that the addition of phloretin preparation can significantly improve the protection rate of piglets against PRRSV infection.

[0105] The expression of ORF7 gene of PRRSV virus in the lung and hypothalamus of the piglets in each group was detected, and the detection results were shown in Table 6, in which a, b, c, and d were significant difference identifiers.

[0106] Table 6

[0107]

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

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

[0110] The embodiments of the present application also verify the effect of the treatment of phloretin preparation on PEDV infected piglets, and the verification method is as follows:

[0111] 1. Materials and methods:

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

[0113] 1.2 Test drug: phloretin tablet G4.

[0114] 1.3 Test animals: 60 three-day-old Du Changda newborn piglets with the same genetic background, uniform body weight, health and no PEDV maternal antibody were evenly divided into three treatment groups, including a treatment group, a blank group and a control group, 15 pigs in each treatment. Phloretin tablet G4 was dissolved in artificial milk, and was added according to the dosage of 2.5 mg / kg pig body weight, and was poured into the treatment group piglets, and the blank group and the control group were poured into artificial milk without phloretin tablet G4.

[0115] 1.4 Challenge test: The piglets in the treatment group and the blank group were challenged with PEDV (YN strain) at a concentration of 10 6 TCID 50 / mL after one week of feeding, and the challenge dose was 3.0 mL of oral perfusion per piglet at one time; the control group was poured into the same volume of normal saline. At this time, the piglets in the treatment group were continuously fed according to the dosage of 10 mg / kg pig body weight until the end of the test.

[0116] 1.5 Observation index: The diarrhea of the test piglets in each group was observed every day, and the intestinal lesions of the test piglets were observed after slaughter at the end of the test, and the number of deaths of the 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 test data was analyzed by One-way ANOVA in SPSS 23.0 statistical software, and Duncan's method was used for multiple comparisons between groups, and P<0.05 was the significant difference standard, and P<0.01 was the extremely significant difference standard. The results were expressed as mean ± standard deviation.

[0118] 2. Experimental results:

[0119] After adding different doses of phloretin tablet G4 in artificial milk and feeding to PEDV infected piglets, the morbidity and mortality of piglets in each group were shown in Table 7.

[0120] Table 7

[0121]

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

[0123] The expression amount of PEDV structural gene N in the intestinal mucosa of the duodenum, jejunum and ileum of the piglets in each group is detected respectively, and the detection results are shown in Table 8, wherein a and b are significant difference identifiers.

[0124] Table 8

[0125]

[0126] As can be seen from Table 8, compared with the blank group, the piglets in the treatment group taking the phloretin tablet G4 can significantly reduce the expression amount of N gene of PEDV in the intestinal tissue. The test results of Table 7 and Table 8 fully show that the phloretin preparation can play a therapeutic role on the piglets infected with PEDV.

[0127] In summary, the phloretin and the phloretin preparation provided by the embodiments of the present application have the effect of resisting PEDV, PRRSV and ASFV and other porcine cyst membrane viruses, and can be used for preparing a medicine and a feed additive for treating and preventing the diseases of pigs infected with PEDV, PRRSV and ASFV and other porcine cyst membrane viruses. The dosage form of the medicine and the feed additive includes oral liquid, granules, powder, tablets and other dosage forms.

[0128] The above has introduced the present application in detail, and the principle and implementation mode of the present application are described by applying specific examples. The above description of the embodiments is only for helping to understand the present application and the core idea. It should be pointed out that, for ordinary skilled in the art, without departing from the principle of the present application, the present application can be improved and modified in several ways, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. Use of phloretin in the preparation of a medicine or feed additive for treating or preventing porcine paratuberculosis infection. The porcine paratuberculosis virus is at least one of porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, and African swine fever virus.

2. Use according to claim 1, characterized in that, The medicine or feed additive is a phloretin composition comprising phloretin and an excipient, wherein the excipient comprises 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.

3. Use according to claim 2, characterized in that, The aforementioned phloretin composition is a solution of phloretin mixed with at least one of anhydrous ethanol, propylene glycol, dimethyl sulfoxide, edible oil, Tween-80, and water, wherein the concentration of phloretin is 20-100 mg / mL.

4. Use according to claim 2, 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 45-55%.

5. Use according to claim 2, 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 45-55%.

6. Use according to claim 2, characterized in that, The preparation type of the medicine or feed additive comprises at least one of oral liquid, granules, tablets, and powder, and the preparation is prepared by using a conventional pharmaceutical preparation method.

7. Use according to claim 6, characterized in that, The carrier of the granules, tablets, and powder added to the feed is at least one of montmorillonite, vermiculite, medical stone, zeolite powder, white carbon black, defatted rice bran, corn cob powder, starch, and bran.

Images