Composition for resisting porcine viral diarrhea and application thereof

Abstract

The invention discloses a composition for resisting porcine viral diarrhea and application of the composition. The composition for resisting porcine viral diarrhea is prepared from the following components in parts by mass: 2 parts of phloretin, 1 part of N-acetyl-D-galactosamine and 100 parts of proline. The composition can further comprise one or more auxiliary materials of absolute ethyl alcohol, propylene glycol, dimethyl sulfoxide, edible oil, water, tween-80, lactose, defatted rice bran, corncob powder, soluble starch, bran, silicon dioxide, montmorillonite, vermiculite, medical stone, zeolite powder, white carbon black and the like. The composition can significantly inhibit the proliferation of PEDV, TGEV and PoRV, and inhibit the infection of PEDV, TGEV and PoRV on cells, and further, the composition can be used for preparing drugs or feed additives for resisting porcine diarrhea diseases caused by viruses such as PEDV, TGEV and PoRV.

Description

Technical Field

[0001] The present application relates to the technical field of pig virus prevention and treatment, and in particular to a composition for resisting pig viral diarrhea and its application. Background Art

[0002] my country is a major pig-raising country in the world, with pork consumption accounting for more than 60% of meat consumption. Therefore, the healthy breeding of pigs is related to the national economy and people's livelihood. The health status of newborn and weaned piglets seriously affects the process of healthy pig breeding. The intestine is the largest immune site in the body and the main organ for digestion and absorption of nutrients in the body. Maintaining intestinal health is the key to ensuring healthy pig breeding. However, due to the prohibition of antibiotics in feed and dairy products, pathogens in the intestine have the opportunity to proliferate in large numbers, which poses a challenge to the immune homeostasis of the intestine of young animals. The development of feed antibiotic substitutes to regulate the immune homeostasis of the intestine of young animals can resist the infection of pathogens and thus ensure the intestinal health of piglets. This is a current research hotspot and an important industrial issue.

[0003] Porcine epidemic diarrhea virus (PEDV) belongs to the Coronaviridae family and the Coronavirus genus. 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 small intestinal wall of piglets is thin and transparent, congested, and the intestinal tube 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. It is reported that PEDV has become the most important pathogen of viral diarrhea in pigs in my country, and the mutant strain is the main pathogen of PEDV infection.

[0004] Gastroenteritis in young pigs is also known as transmissible gastroenteritis of swine (TGE). It is a highly contagious viral infectious disease caused by transmissible gastroenteritis virus (TGEV). The clinical manifestations of TGE are vomiting, severe diarrhea, and dehydration. It is highly prevalent in piglets within 2 weeks of birth and has a mortality rate of over 80%. TGE causes significant harm to pigs infected for the first time. It can cause 100% disease in pigs of all ages in a short period of time, and the severity of the disease varies with age. The younger the age, the more severe the disease and the higher the mortality rate. The mortality rate of piglets within two weeks of age can reach 90% to 100%. TGEV is often mixed with rotavirus and Escherichia coli to increase the mortality rate of suckling piglets and weaned piglets. TGEV is also one of the main pathogens of diarrhea in piglets in my country, which seriously hinders the development of pig farming.

[0005] Porcine rotavirus diarrhea is an acute intestinal infectious disease caused by porcine rotavirus (Porcine Rotavirus, PoRV), which mainly affects piglets, causing symptoms such as vomiting, diarrhea, dehydration and weight loss. The virus is mainly transmitted through the fecal-oral route. The virus exists in the digestive tract of sick pigs and infected pigs and is excreted from the body with feces. Once an outbreak occurs, it may occur year after year, so sick pigs and latently infected pigs are the main sources of infection for the disease. The epidemic characteristics of rotavirus are mixed infections, which are mainly caused by poor breeding environments. In this case, it is often mistaken for piglet diarrhea caused by Escherichia coli infection, but in fact, rotavirus is also one of the important factors causing the disease. Diarrhea caused by rotavirus has not only had a serious impact on the development of animal husbandry, but also poses a threat to human health. It has become an important public health issue worldwide.

[0006] Currently, the most effective way to prevent and control PEDV, TGEV and PoRV is vaccination. However, due to the highly variable characteristics of PEDV, TGEV and PoRV, the effectiveness of their vaccine immune effects cannot be guaranteed. Although a lot of vaccine research and development work has been carried out on these three viruses at home and abroad, there is still a lack of vaccines for these three viruses on the market and the effectiveness cannot be guaranteed. 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 infected individuals, vaccination is basically meaningless. Therefore, it is urgent to develop technical solutions to prevent and control PEDV, TGEV and PoRV. Summary of the invention

[0007] In view of this, the purpose of the present application is to provide a composition for resisting porcine viral diarrhea and its application. The composition has the advantages of high safety and convenient application, has a high protection rate against porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and porcine rotavirus infection, and can be used to prepare drugs or feed additives for resisting porcine viral diarrhea diseases.

[0008] In order to achieve or at least partially achieve the above objectives, the present application provides the following technical solutions:

[0009] In a first aspect, the present application provides a composition for preventing porcine viral diarrhea, the composition comprising phloretin, proline and N-acetyl-D-galactosamine.

[0010] In a second aspect, the present application provides the use of the aforementioned composition in the preparation of a drug or feed additive for resisting porcine viral diarrhea disease.

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

[0012] 1. The composition for preventing porcine viral diarrhea provided in the present application is prepared by compounding functional nutritional regulators proline, N-acetyl-D-galactosamine and phloretin in a mass ratio of 100:1:2. The composition has the effects of significantly inhibiting viral replication, improving the intestinal morphology and structure of piglets, and enhancing absorption and barrier function. The addition method is simple, and the above-mentioned effects can be achieved by simply adding it to the feed or taking it orally. It also overcomes the shortcomings of possible immune failure caused by vaccination and viral mutation caused by the use of antiviral drugs. It has no toxic side effects on the animal body and can be used safely.

[0013] 2. The results of in vitro experiments in this application prove that the composition prepared by proline, N-acetyl-D-galactosamine and phloretin in a mass ratio of 100:1:2 has a significant inhibitory effect on the proliferation of PEDV, TGEV and PoRV

[0014] 3. The present application proves through in vivo experimental results that the composition can effectively prevent and treat PEDV, TGEV and PoRV infections, thereby reducing the diarrhea rate and mortality rate of diseased pigs and effectively improving the survival rate of pigs. In vivo experiments also confirmed that the composition has an improving effect on the absorption and barrier function of the intestine of PEDV-infected piglets. DETAILED DESCRIPTION

[0015] 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.

[0016] 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.

[0017] In the text of this application, the terms "first", "second" and the like are used to distinguish between identical or similar items with substantially the same functions and effects. This is only for the purpose of clearly describing the technical solutions of the embodiments of this application, and shall not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features.

[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 may 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 may 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] Proline (L-Proline, Pro) is a cyclic imino acid. Among its three forms, L-proline has physiological functions. L-proline is a white crystalline powder with a slightly sweet taste. Its molecular formula is C5H9NO2, and its relative molecular mass is 115.13. It is soluble in hot water and ethanol. Proline is the second most abundant amino acid in pigs and has cell, tissue and species specificity. All mammals can synthesize proline from arginine through arginase, ornithine aminotransferase and P5C reductase.

[0022] N-acetyl-D-galactosamine belongs to the polysaccharide substance, which contains the structural skeleton of glucose and is an important nutrient in the human body. In the body, N-acetyl-D-galactosamine can be used to construct amniotic membranes and cell walls, and is one of the important raw materials for cell membrane structure.

[0023] In the embodiment of the present application, phloretin, proline, and N-acetyl-D-galactosamine are mixed in a certain mass ratio to form a composition. In vivo and in vitro experiments have shown that the composition can significantly inhibit the proliferation of PEDV, TGEV, and PoRV, as well as inhibit their infection of cells. Furthermore, the composition can be used to prepare drugs or feed additives for diarrheal diseases in pigs caused by viruses such as PEDV, TGEV, and PoRV.

[0024] Based on this, an embodiment of the present application provides a composition for preventing porcine viral diarrhea, which comprises three active ingredients: phloretin, proline and N-acetyl-D-galactosamine.

[0025] In certain preferred embodiments, the anti-swine viral diarrhea composition comprises, by mass, 2 parts of phloretin, 1 part of N-acetyl-D-galactosamine, and 100 parts of proline.

[0026] In certain embodiments, the anti-swine viral diarrhea composition also includes auxiliary materials, and 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.

[0027] In certain embodiments, the anti-swine viral diarrhea composition is a solid mixture of three active ingredients, phloretin, proline, and N-acetyl-D-galactosamine, and montmorillonite, wherein the mass fraction of the three active ingredients, phloretin, proline, and N-acetyl-D-galactosamine, in the solid mixture is 40% to 60%.

[0028] In certain embodiments, the anti-swine viral diarrhea composition is a mixed solution formed by mixing three active ingredients, phloretin, proline, and N-acetyl-D-galactosamine, with acetic acid, anhydrous ethanol, Tween-80, and water, wherein the concentration of the three active ingredients, phloretin, proline, and N-acetyl-D-galactosamine, in the mixed solution is 80 to 120 mg / mL.

[0029] In certain embodiments, the anti-swine viral diarrhea composition is a powder mixture formed by mixing three active ingredients, phloretin, proline, and N-acetyl-D-galactosamine, with soluble starch or bran, wherein the mass fraction of the three active ingredients, phloretin, proline, and N-acetyl-D-galactosamine, in the powder mixture is 40% to 60%.

[0030] In certain embodiments, the anti-swine viral diarrhea composition is a flaky mixture formed by mixing three active ingredients, phloretin, proline, and N-acetyl-D-galactosamine, with soluble starch and lactose, wherein the mass fraction of the three active ingredients, phloretin, proline, and N-acetyl-D-galactosamine, in the flaky mixture is 40% to 60%.

[0031] Based on this, the embodiments of the present application provide the use of the aforementioned composition in the preparation of a drug or feed additive for resisting porcine viral diarrhea disease.

[0032] In certain embodiments, the porcine viral diarrhea disease includes at least one of porcine epidemic diarrhea caused by porcine epidemic diarrhea virus, porcine transmissible gastroenteritis caused by porcine transmissible gastroenteritis virus infection, and porcine rotavirus diarrhea caused by porcine rotavirus.

[0033] In certain embodiments, the preparation type of the drug or feed additive includes at least one of oral liquid, granules, powder, and tablets, and the preparation can be prepared by conventional pharmaceutical preparation methods.

[0034] In certain embodiments, the method of using the composition as a medicine or feed additive comprises at least one of the following methods:

[0035] (1) directly orally administering the composition to pigs at a dosage of 300 to 600 mg / kg of pig body weight of the three active ingredients of phloretin, proline, and N-acetyl-D-galactose;

[0036] (2) The composition is added to the feed at a dosage of 0.3% to 1.5% of the total weight of the pig feed of the three active ingredients of phloretin, proline and N-acetyl-D-galactose.

[0037] The technical solution of the present application and the technical effects achieved are described in detail below through more specific embodiments.

[0038] Example 1

[0039] This embodiment provides a method for preparing a composition for resisting porcine viral diarrhea, and the specific steps are as follows:

[0040] (1) Weighing 100 g of proline and 103 g of montmorillonite, mixing them evenly to obtain a first mixture;

[0041] (2) Weigh 1 g of N-acetyl-D-galactosamine and 2 g of phloretin and add them to the first mixture, mix them evenly, and obtain a second mixture; i.e., a composition G1 for treating or preventing pathogenic diarrhea in pigs.

[0042] Example 2

[0043] This embodiment provides a method for preparing a composition for resisting porcine viral diarrhea, and the specific steps are as follows:

[0044] (1) Weigh 100 g of proline, 1 g of N-acetyl-D-galactosamine, and 2 g of phloretin, mix them evenly, and obtain a solid mixture;

[0045] (2) Weigh 10 g of the solid mixture, 5 mL of 5% acetic acid, 15 mL of anhydrous ethanol, and 30 mL of Tween-80, mix them evenly, and finally add water for injection to 100 mL, mix evenly, and obtain a mixed solution, i.e., a composition G2 for treating or preventing pathogenic diarrhea in pigs.

[0046] Example 3

[0047] This embodiment provides a method for preparing a composition for resisting porcine viral diarrhea, and the specific steps are as follows:

[0048] (1) Weigh 100 g of proline, 1 g of N-acetyl-D-galactosamine, and 2 g of phloretin, mix them evenly, and obtain a solid mixture;

[0049] (2) Weigh 20 g of the solid mixture and 20 g of soluble starch, mix them evenly, and pass them through a 60-mesh sieve to obtain a powdery mixture, i.e., a composition G3 for treating or preventing pathogenic diarrhea in pigs.

[0050] Example 4

[0051] This embodiment provides a method for preparing a composition for resisting porcine viral diarrhea, and the specific steps are as follows:

[0052] (1) Weigh 100 g of proline, 1 g of N-acetyl-D-galactosamine, and 2 g of phloretin, mix them evenly, and obtain a solid mixture;

[0053] (2) Weigh 50 g of the solid mixture, 15 g of lactose, and 35 g of soluble starch, mix them evenly, press them into tablets using a tablet press, and dry them to obtain a tablet-like mixture, i.e., a composition G4 for treating or preventing pathogenic diarrhea in pigs.

[0054] The formula and parameters of the composition preparation prepared in the above embodiment are shown in Table 1 below:

[0055] Table 1

[0056]

[0057] The above composition can be used as a medicine or feed additive to prevent or treat pathogenic diarrhea diseases in pigs. When used for the prevention of PEDV, TGEV and PoRV infections, the composition can be orally administered to pigs at a content of 300 mg / kg of pig body weight of the three active ingredients, and used throughout the entire feeding cycle; when used for the clinical treatment of PED, TGE or acute intestinal infectious diseases, the composition can be orally administered to pigs at a content of 600 mg / kg of pig body weight, or the oral liquid can be mixed with the daily diet at a content of 600 mg / kg of pig body weight of the three active ingredients, added to the feed three times a day in the morning, noon and evening, and used continuously for one week; the dosage of the composition can also be adjusted according to the severity of the pig disease, and the dosage range is controlled within a content of 300 to 600 mg / kg of pig body weight of the three active ingredients.

[0058] Effect of the composition on inhibiting the proliferation of PEDV, TGEV and PoRV in vitro

[0059] In the present application example, the above-prepared anti-swine viral diarrhea composition was used to verify the inhibitory effect of the in vitro proliferation of three swine viruses, PEDV, TGEV, and PoRV. The verification method is as follows:

[0060] 1. Materials and methods:

[0061] 1.1 Viruses: PEDV YN strain, TGEV WH1 strain, and PoRV TM-α strain were isolated, preserved, and donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0062] 1.2 Cells: African green monkey kidney cells (Vero cells) were used for the proliferation of PEDV YN strain; porcine kidney cells (PK-15) were used for the proliferation of TGEV WH1 strain; rhesus monkey embryonic kidney cell line (MA-104 cells) were used for the proliferation of PoRV TM-α strain. All cells were donated by Huazhong Agricultural University.

[0063] 1.3 Experimental drugs: Proline, N-acetyl-D-galactosamine, and phloretin were purchased from Maclean Company with a purity of >98%.

[0064] 1.4 Reagents: DMEM culture medium, EDTA, trypsin, fetal bovine serum and double antibody (penicillin-streptomycin) were purchased from GIBCO.

[0065] 1.5 In vitro proliferation inhibition:

[0066] First, 1 g of proline, 0.01 g of N-acetyl-D-galactosamine, and 0.02 g of phloretin were weighed, placed in 10.03 mL of phosphate buffer solution, fully dissolved, and mixed evenly to obtain a mother solution of a composition with proline, N-acetyl-D-galactosamine and phloretin in a mass ratio of 100:1:2, and then the concentration of the composition was adjusted to 100 μg / mL for subsequent experiments.

[0067] (1) PEDV in vitro proliferation inhibition test: PEDV YN strain virus liquid was diluted 10 times with DMEM culture medium, and 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Six dilutions were inoculated in 96-well plates that were covered with Vero cell monolayers, 100 μL / well, and DMEM culture medium containing different concentrations of the composition (0, 25, 50, 100 μg / mL) was added to each group, and the cells were placed in a 37°C incubator with 5% CO2 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 concentration composition group was calculated using the Reed-Muench method. 50 , compare different groups of TCID 50 The inhibitory effect of the composition on the proliferation of PEDV in vitro was judged based on the value of , and three replicates were performed for each group.

[0068] (2) TGEV in vitro proliferation inhibition test: The (TGEV) WH1 strain virus liquid was diluted 10 times with DMEM culture medium, and 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Six dilutions were inoculated in 96-well plates that were covered with PK-15 cell monolayers, 100 μL / well, and DMEM culture medium containing different concentrations of the composition (0, 25, 50, 100 μg / mL) was added to each group, and the cells were placed in a 37°C incubator with 5% CO2 for 48 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 concentration composition group was calculated using the Reed-Muench method. 50 , compare different groups of TCID 50The inhibitory effect of the composition on the proliferation of TGEV in vitro was judged by the value of , and three replicates were performed in each group.

[0069] (3) PoRV in vitro proliferation inhibition test: The PoRV TM-α strain virus solution was diluted 10 times with DMEM culture medium, and 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Six dilutions were inoculated in 96-well plates that were covered with MA-104 cell monolayers, 100 μL / well, and DMEM culture medium containing different concentrations of the composition (0, 25, 50, 100 μg / mL) was added to each group, and the cells were placed in a 37°C incubator with 5% CO2 for 48 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 concentration composition group was calculated using the Reed-Muench method. 50 , compare different groups of TCID 50 The inhibitory effect of the composition on the proliferation of PoRV in vitro was judged based on the value of , and three replicates were performed for each group.

[0070] 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.

[0071] 2. Test results:

[0072] Table 2 shows the TCID of different viruses against different concentrations of the composition 50 Influence results, where a, b, c, and d are marks of significant difference.

[0073] Table 2

[0074] Group PEDV TGEV PoRV Blank Group <![CDATA[10 8.37±0.42a ]]> <![CDATA[10 7.21±0.96a ]]> <![CDATA[10 8.01±0.89a ]]> 25 μg / mL group <![CDATA[10 7.76±0.61a ]]> <![CDATA[10 6.91±0.51a ]]> <![CDATA[10 5.77±0.49b ]]> 50 μg / mL group <![CDATA[10 6.32±0.41b ]]> <![CDATA[10 4.91±0.33b ]]> <![CDATA[10 4.02±0.27c ]]> 100 μg / mL group <![CDATA[10 4.27±0.42c ]]> <![CDATA[10 4.01±0.63c ]]> <![CDATA[10 3.71±0.63d ]]>

[0075] It can be seen from Table 2 that the composition with a concentration of 25 μg / mL has basically no inhibitory effect on PEDV and TGEV, but can inhibit the proliferation of PoRV; the compositions with concentrations of 50 μg / mL and 100 μg / mL show significant inhibitory effects on PEDV, TGEV, and PoRV; and as the concentration of the composition increases, the inhibitory effect becomes more obvious.

[0076] Protective effect of the composition on piglets infected with PEDV

[0077] The present application example verifies the protective effect of the composition on piglets infected with PEDV, and the verification method is as follows:

[0078] 1. Materials and methods:

[0079] 1.1 Experimental virus: PEDV YN strain was donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0080] 1.2 Test drug: Composition G2.

[0081] 1.3 Experimental animals: A 2×2 factorial design was used to divide 60 healthy 3-day-old newborn piglets of the same genetic background, uniform body weight, and without maternal antibodies to PEDV into 4 treatment groups, with 15 pigs in each treatment group. The treatment groups included a control group, a composition treatment group (the composition was orally administered to pigs at an active ingredient content of 300 mg / kg pig body weight), a PEDV treatment group, and a composition treatment + PEDV group. The piglets in the composition treatment group and the composition treatment + PEDV group were mixed with the composition in artificial milk at a dose of 300 mg / kg pig body weight for one week; the artificial milk fed to the other two groups was supplemented with an equal amount of phosphate buffer solution.

[0082] 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, and each pig was orally infused with 3.0 mL. After challenge, the pigs were observed for 3 consecutive days until the end of the experiment.

[0083] 1.5 Observation indicators: Observe the diarrhea of ​​the experimental piglets every day. Slaughter the experimental piglets at the end of the test period to observe intestinal lesions, count the number of dead pigs, and calculate the diarrhea rate and mortality rate. The expression of PEDV structural genes N, M, and S in the intestinal tissue of piglets was detected by real-time quantitative PCR to explore the in vivo inhibition of the composition on PEDV virus. The intestinal morphology was observed through tissue sections, and the absorption function of the intestine was evaluated by detecting the content of D-xylose and diamine oxidase (DAO) in serum.

[0084] 1.6 Data analysis: The experimental data were analyzed by two-way ANOVA using SPSS 26.0 statistical software. The main effects of the model included composition, PEDV, and the interaction between the two. Duncan's multiple comparisons were used in the case of interaction. The experimental results were expressed as mean and SEM values, and P < 0.05 indicated significant differences.

[0085] 2. Experimental results:

[0086] The results of the effect of composition G2 on the diarrhea rate and mortality of piglets infected with PEDV are shown in Table 3 below.

[0087] Table 3

[0088]

[0089] It can be seen from Table 3 that the diarrhea rate and mortality rate of piglets in the PEDV group were high, while the diarrhea rate and mortality rate of piglets in the composition treatment + PEDV group were significantly reduced, indicating that the composition can significantly reduce the diarrhea rate and mortality rate of PEDV-infected piglets, further indicating that the composition can have a protective effect on PEDV-infected piglets.

[0090] The expression levels of PEDV structural genes M, N, and S in the intestinal mucosa of the jejunum and ileum tissues of piglets were detected respectively. The test results are shown in Table 4 below, where a and b are significant differences.

[0091] Table 3

[0092]

[0093] As shown in Table 4, the composition can significantly down-regulate the transcription levels of N and S genes in PEDV in the jejunum; in addition, the composition significantly down-regulates the transcription levels of M, N, and S genes in PEDV in the ileum. The results show that the composition can inhibit the replication of PEDV in piglets.

[0094] Table 5 shows the effect of composition G2 on serum D-xylose and DAO levels in piglets infected with PEDV.

[0095] Table 5

[0096]

[0097] As shown in Table 5, PEDV infection increases the content of diamine oxidase (DAO) in the serum of piglets and reduces the level of D-xylose in the serum. DAO is normally present in the intestinal tissue and is released into the blood when the intestine is damaged. Therefore, the higher the DAO content in the blood, the more obvious the degree of intestinal damage; D-xylose can be absorbed by the intestine but cannot be used by the body. The intestinal absorption function can be evaluated by evaluating the level of D-xylose in the serum. The results in the table show that the composition can alleviate the decrease in the level of D-xylose in the serum of piglets caused by PEDV infection, indicating that the composition can improve the intestinal absorption function of piglets infected with PEDV.

[0098] Table 6 shows the effects of composition G2 on the intestinal morphology of piglets infected with PEDV.

[0099] Table 6

[0100]

[0101] As can be seen from Table 6, PEDV infection reduces the villus height and the ratio of villus height to crypt depth of piglets. Composition G2 can alleviate the reduction of ileal villus height caused by PEDV infection, and alleviate the reduction of the ratio of villus height to crypt depth in the jejunum and ileum, further indicating that the composition can improve the morphological structure of the intestine of PEDV-infected piglets.

[0102] Protective effect of the composition on piglets infected with TGEV

[0103] The present application example verifies the protective effect of the composition on piglets infected with PEDV, and the verification method is as follows:

[0104] 1. Materials and methods:

[0105] 1.1 Experimental virus: TGEV WH1 strain was donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0106] 1.2 Test drug: Composition G3.

[0107] 1.3 Experimental animals: A 2×2 factorial design was used to divide 60 healthy 3-day-old newborn piglets of the same genetic background, uniform body weight, and without TGEV maternal antibodies into 4 treatment groups, with 15 pigs in each treatment group. The treatment groups were a control group, a composition treatment group (the composition was orally administered to pigs at an active ingredient content of 300 mg / kg pig body weight), a TGEV treatment group, and a composition treatment + TGEV group. The piglets in the composition treatment group and the composition treatment + TGEV group were mixed with the composition in artificial milk at a dose of 300 mg / kg pig body weight for one week; the other two groups were fed with an equal amount of phosphate buffer solution in the artificial milk.

[0108] 1.4 Challenge test: After feeding for one week, the piglets in each group were fed with 10 5.5 TCID 50 / mL of TGEV WH1 strain was used for infection, and each head was orally infused with 3.0mL. After infection, the animals were observed for 7 consecutive days until the end of the experiment.

[0109] 1.5 Observation indicators: Observe the diarrhea of ​​the experimental piglets every day. Slaughter the experimental piglets at the end of the experiment to observe intestinal lesions, count the number of dead pigs, and calculate the diarrhea rate and mortality rate. The immune function of the piglets is evaluated by detecting the level of pro-inflammatory cytokines in the serum, and the antiviral ability of the body is evaluated by detecting the transcription level of antiviral genes.

[0110] 1.6 Data analysis: The experimental data were analyzed by two-way ANOVA using SPSS 26.0 statistical software. The main effects of the model included composition, TGEV, and the interaction between the two. Duncan's multiple comparisons were used in the case of interaction. The experimental results were expressed as mean and SEM values, and P < 0.05 indicated significant differences.

[0111] 2. Experimental results:

[0112] The results of the effect of composition G3 on the diarrhea rate and mortality of TGEV-infected piglets are shown in Table 7 below.

[0113] Table 7

[0114]

[0115] It can be seen from Table 7 that the diarrhea rate and mortality rate of piglets in the TGEV group were high, while the diarrhea rate and mortality rate of piglets in the composition treatment + TGEV group were significantly reduced, indicating that the composition can significantly reduce the diarrhea rate and mortality rate of TGEV-infected piglets, further indicating that the composition can have a protective effect on TGEV-infected piglets.

[0116] Table 8 shows the effects of composition G3 on the levels of serum pro-inflammatory cytokines in TGEV-infected piglets, where a, b, c, and d are significant differences.

[0117] Table 8

[0118]

[0119] As shown in Table 8, TGEV infection significantly increased the levels of IL-1β and TNF-α in piglet serum and reduced the level of IL-10. Composition G3 alleviated the increase in serum IL-1β and TNF-α levels in piglets caused by TGEV infection, indicating that the composition can alleviate the inflammatory response of piglets caused by TGEV infection.

[0120] The expression levels of TGEV genes MX1, OASL, IFN-β, and IRF7 in the intestinal mucosa of the jejunum, ileum, and colon tissues of piglets were detected respectively. The test results are shown in Table 9 below, where a, b, and c are significant differences.

[0121] Table 9

[0122]

[0123]

[0124] As shown in Table 9, TGEV infection upregulated the transcription levels of IFN-β, antiviral-related genes (MX1, OASL) and interferon regulatory factor 7 (IRF-7). Composition G3 can induce the expression of IFN-β in the intestine of piglets, and downregulate the upregulation of the transcription levels of antiviral-related genes (MX1, OASL) and interferon regulatory factor 7 (IRF-7) caused by TGEV infection, indicating that the composition can regulate the expression of antiviral genes in the body by inducing the expression of interferon, thereby alleviating the negative impact of TGEV on the intestine of piglets.

[0125] Protective effect of the composition on piglets infected with PoRV

[0126] The present application example verifies the protective effect of the composition on piglets infected with PoRV, and the verification method is as follows:

[0127] 1. Materials and methods:

[0128] 1.1 Experimental virus: PoRV TM-a strain was donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University;

[0129] 1.2 Test drug: Composition G4.

[0130] 1.3 Experimental animals: A 2×2 factorial design was used to divide 60 healthy 3-day-old newborn piglets of the same genetic background, uniform body weight, and without PoRV maternal antibodies into 4 treatment groups, with 15 pigs in each treatment group. The treatment groups included a control group, a composition treatment group (the composition was orally administered to pigs at an active ingredient content of 300 mg / kg pig body weight), a PoRV treatment group, and a composition treatment + PoRV group. The piglets in the composition treatment group and the composition treatment + PoRV group were mixed with the composition in artificial milk at a dose of 300 mg / kg pig body weight, and fed continuously for one week; the artificial milk fed to the other two groups was supplemented with an equal amount of phosphate buffer solution.

[0131] 1.4 Challenge test: After feeding for one week, the piglets in each group were fed with 10 7.5 TCID 50 / mL of PoRV TM-a strain was used for infection, with 3.0mL injected orally to each head. The animals were observed for 7 consecutive days after infection until the end of the experiment.

[0132] 1.5 Observation indicators: Observe the diarrhea of ​​the experimental piglets every day. Slaughter the experimental piglets at the end of the experiment to observe intestinal lesions, count the number of dead pigs, and calculate the diarrhea rate and mortality rate. The intestinal absorption function is evaluated by detecting the content of D-xylose in serum, and the degree of intestinal damage of piglets is evaluated by detecting the intestinal morphology and serum DAO level.

[0133] 1.6 Data analysis: The experimental data were analyzed by two-way ANOVA using SPSS 26.0 statistical software. The main effects of the model included composition, PoRV and the interaction between the two. Duncan's multiple comparison was used in the case of interaction. The test results were expressed as mean and SEM values, and P < 0.05 indicated significant differences.

[0134] 2. Experimental results:

[0135] The results of the effect of composition G4 on the diarrhea rate and mortality of piglets infected with PoRV are shown in Table 10 below.

[0136] Table 10

[0137]

[0138] As can be seen from Table 10, the diarrhea rate and mortality rate of piglets in the PoRV group were high, while the diarrhea rate and mortality rate of piglets in the composition treatment + PoRV group were significantly reduced, indicating that the composition can significantly reduce the diarrhea rate and mortality rate of PoRV-infected piglets, further indicating that the composition can have a protective effect on PoRV-infected piglets.

[0139] Table 11 shows the effect of composition G4 on the levels of D-xylose and DAO in the serum of piglets infected with PoRV.

[0140] Table 11

[0141]

[0142] As can be seen from Table 11, PoRV infection significantly increased the serum DAO content in piglets and reduced the level of D-xylose in serum; composition G4 can alleviate the decrease in serum D-xylose content and the increase in DAO activity in piglets caused by PoRV infection, indicating that the composition can improve the intestinal absorption function of PoRV-infected piglets and has a protective effect on intestinal damage caused by PoRV infection.

[0143] Table 12 shows the effects of composition G4 on the intestinal morphology of piglets infected with PoRV.

[0144] Table 12

[0145]

[0146]

[0147] As can be seen from Table 12, PoRV infection significantly reduces the villus height and the ratio of villus height to crypt depth in the jejunum and ileum of piglets. Composition G4 can alleviate the negative impact of PoRV infection on the morphological structure of the jejunum and ileum of piglets. The results further confirm the protective effect of the composition on the intestine of PoRV-infected piglets.

[0148] 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. A composition for resisting porcine viral diarrhea, characterized in that: The composition comprises three active ingredients: phloretin, proline and N-acetyl-D-galactosamine.

2. The composition according to claim 1, characterized in that The composition for resisting porcine viral diarrhea comprises, by weight, 2 parts of phloretin, 1 part of N-acetyl-D-galactosamine, and 100 parts of proline.

3. The composition according to claim 1, characterized in that The composition for resisting porcine viral diarrhea also includes auxiliary materials, which 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.

4. The composition according to claim 3, characterized in that The composition for preventing porcine viral diarrhea is at least one of the following (1) to (4): (1) a solid mixture of three active ingredients, phloretin, proline and N-acetyl-D-galactosamine, and montmorillonite, wherein the mass fraction of the three active ingredients, phloretin, proline and N-acetyl-D-galactosamine, in the solid mixture is 40% to 60%; (2) a mixed solution formed by mixing three active ingredients, phloretin, proline and N-acetyl-D-galactosamine, with acetic acid, anhydrous ethanol, Tween-80 and water, wherein the concentrations of the three active ingredients, phloretin, proline and N-acetyl-D-galactosamine, in the mixed solution are 80 to 120 mg / mL; (3) a powdery mixture of three active ingredients, phloretin, proline and N-acetyl-D-galactosamine, and soluble starch or bran, wherein the mass fraction of the three active ingredients, phloretin, proline and N-acetyl-D-galactosamine, in the powdery mixture is 40% to 60%; (4) is a flake mixture formed by mixing three active ingredients, phloretin, proline and N-acetyl-D-galactosamine, with soluble starch and lactose, wherein the mass fraction of the three active ingredients, phloretin, proline and N-acetyl-D-galactosamine, in the flake mixture is 40% to 60%.

5. Use of the composition according to any one of claims 1 to 4 in the preparation of a drug or feed additive for resisting porcine viral diarrhea.

6. The use according to claim 5, characterized in that: The porcine viral diarrhea disease includes at least one of porcine epidemic diarrhea caused by porcine epidemic diarrhea virus, porcine transmissible gastroenteritis caused by porcine transmissible gastroenteritis virus infection, and porcine rotavirus diarrhea caused by porcine rotavirus.

7. The use according to claim 5, characterized in that: The preparation types of the medicine or feed additive include at least one of oral liquid, granules, powders and tablets, and the preparation can be prepared by conventional pharmaceutical preparation methods.

8. The use according to claim 5, characterized in that: The method of using the composition as a medicine or feed additive includes at least one of the following methods: (1) directly orally administering the composition to pigs at a dosage of 300 to 600 mg / kg of pig body weight of the three active ingredients of phloretin, proline, and N-acetyl-D-galactose; (2) The composition is added to the feed at a dosage of 0.3% to 1.5% of the total weight of the pig feed of the three active ingredients of phloretin, proline and N-acetyl-D-galactose.

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