Application of myrobalan tannin chelate in preparation of drugs or feed additives for treating or preventing porcine virus infection

Abstract

The invention discloses an application of a myrobalan tannin chelate in preparation of a medicine or a feed additive for treating or preventing porcine virus infection, the medicine is the myrobalan tannin chelate, or the myrobalan tannin chelate is used as an effective component and is added with auxiliary materials to prepare oral liquid, granules, tablets and powder. The use dosage is that the content of the myrobalan tannin chelate is 5-20 mg / kg of pig body weight; or the medicine terminalia fruit tannin chelate is directly added into the feed, and the addition amount of the medicine terminalia fruit tannin chelate is 0.05-0.5% of the total weight of the pig feed. The medicine or feed additive provided by the invention is high in safety and free of toxic and side effects, can effectively prevent and treat infectious diseases caused by porcine viruses such as PEDV, PRRSV and AFSV, improves the intestinal morphological structure and absorption function of diseased pigs, and reduces the morbidity and mortality of piglets.

Description

Technical Field

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

[0002] Porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), and African swine fever virus (ASFV) are three common viruses. Porcine epidemic diarrhea virus (PEDV) is the main cause of PED. It is a coronavirus. Pigs of different ages and breeds may be infected with PEDV and become ill, but it has a high mortality rate for piglets. The mortality rate of piglets under seven days old can reach 100%. PEDV mainly infects and replicates in the villus enterocytes of the small intestine. The clinical symptoms of pigs are vomiting, diarrhea, and dehydration. PRRSV infection can cause porcine reproductive and respiratory syndrome (PRRS), also known as "blue ear disease", which is a highly contagious disease of pigs. The disease is characterized by reproductive disorders in pregnant sows (abortion, stillbirth, mummified fetuses in sows) and respiratory diseases in pigs of all ages, especially piglets. African swine fever (ASF) caused by ASFV infection is an acute, febrile and highly contagious disease characterized by high fever, extensive bleeding in the skin and internal organs, respiratory disorders, neurological symptoms and high mortality.

[0003] In addition, since vaccine immunization only prevents viral diseases from occurring, it cannot provide protection for piglets that have already been infected. Therefore, relying solely on vaccines can easily lead to immune failure, and large-scale PEDV, PRRSV, and ASFV viral infections still frequently break out.

[0004] In view of the current status of prevention and control of the above viral diseases, the development of therapeutic drugs and methods against porcine viruses such as PEDV, PRRSV and ASFV has become a market need. Summary of the invention

[0005] In view of this, the purpose of the present application is to provide an application of a terminalia tannin chelate in the preparation of a drug or feed additive for treating or preventing pig virus infection. In this application, the terminalia tannin chelate and a composition comprising the terminalia tannin chelate have resistance to a variety of pig viruses that cause pig diseases, such as PEDV, PRRSV and ASFV, and can be used to prepare a drug or feed additive for treating or preventing pig viruses. The drug or feed additive has a good preventive and therapeutic effect on diseases caused by PEDV, PRRSV and ASFV, and is highly safe and has no toxic side effects.

[0006] In order to achieve or at least partially achieve the above objectives, the present application provides the following technical solutions:

[0007] Application of terminalia chebula tannin chelate in preparing medicine or feed additive for treating or preventing pig virus infection.

[0008] Compared with the prior art, the technical solution provided by this application has at least the following advantages:

[0009] 1. The terminalia chebula tannin chelate or a composition comprising the terminalia chebula tannin chelate (hereinafter referred to as the "terminal chebula tannin chelate composition") provided in the present application is highly safe and can be used as a feed additive for a long time to prevent epidemic diseases. Long-term use will not lead to the production of drug-resistant strains and will not have other toxic side effects, which is an advantage that other antiviral drugs do not have.

[0010] 2. The terminalia chebula tannin chelate or terminalia chebula tannin chelate composition provided in the present application can effectively prevent and treat the infection of PEDV, PRRSV and AFSV by enhancing the body's antioxidant defense function, improving the natural immune function and antiviral activity of pigs, thereby reducing the incidence of the disease and the mortality rate of sick pigs. The terminalia chebula tannin chelate or terminalia chebula tannin chelate composition has significant efficacy in treating and preventing porcine viral diseases.

[0011] 3. The in vitro experimental results of the present application show that the terminalia chebula tannin chelate or terminalia chebula tannin chelate composition has the effect of significantly inhibiting the in vitro proliferation of PEDV, PRRSV and ASFV. Adding terminalia chebula tannin chelate in a dosage of 100 μg / mL to the cell culture medium can reduce the TCID50 of PEDV, PRRSV and ASFV on the corresponding cells by about 2 to 3 titers, respectively.

[0012] 4. The in vitro experimental results of this application show that the terminalia chebula tannin chelate or terminalia chebula tannin chelate composition has the functions of enhancing natural immune function and antiviral activity, and can effectively prevent and treat PEDV and PRRSV infections, thereby reducing the mortality rate of diseased pigs and effectively improving the survival rate and production performance of pigs. The protection rate against PEDV and PRRSV infection can reach 83.3% when the terminalia chebula tannin chelate is taken orally at 20 mg / kg body weight or added to piglet feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a graph showing the inhibitory effect of terminalia chebula tannin copper on PEDV and PRRSV observed under a fluorescence microscope, provided in an example of the present application.

[0014] Figure 2 This is a graph showing the inhibitory effect of terminalia chebula tannin zinc on PEDV and PRRSV observed under a fluorescence microscope, provided in an example of the present application. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein 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] 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.

[0018] 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.

[0019] The term "chebula tannin" is the main component of chebula, accounting for about 23.6% to 37.0% of the weight of chebula fruit. "Chbula tannin" belongs to the pyrogallol type (hydrolyzable tannin), which is a compound formed by phenolic acid and its derivatives and glucose or polyols through glycosidic bonds or ester bonds. There are many hydrolyzable tannin components in chebula fruit: chebulic acid, chebulic acid, chebulic acid, orthochebulic acid, chebulin, veratric acid, punicalagin, chebulin, corilagin, neochebulic acid, ellagic acid, 1,2,3,4,6-penta-O-galloyl glucose, 1,6-digalloyl glucose, casuarina tannin, 3,4,6-tri-O-galloyl glucose, chebula tannin, galloyl glucose, terminalenin A, terminalenin B, terminalenin C, terminalenin acid, etc.

[0020] The term "chebula tannin chelate" refers to a chelate formed by the complexation of "chebula tannin" and metal ions. The term "chebula tannin copper" refers to a chelate formed by the complexation of "chebula tannin" and divalent copper ions. The term "chebula tannin zinc" refers to a chelate formed by the complexation of "chebula tannin" and divalent zinc ions.

[0021] The examples of the present application found through in vivo and in vitro experiments that the terminalia tannin chelate can inhibit the proliferation of viruses such as PEDV, PRRSV and ASFV, and inhibit their infection of cells, that is, the terminalia tannin chelate has anti-PED, PRRS and ASF virus infection effects. The terminalia tannin chelate and the terminalia tannin chelate composition can be used to prepare anti-swine virus drugs and feed additives for the prevention and treatment of pig diseases such as PED, PRRS and ASF, and have the advantages of high safety and no toxic side effects.

[0022] Based on this, the embodiments of the present application provide the use of terminalia chebula tannin chelate in the preparation of drugs or feed additives for treating or preventing porcine viral infections.

[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 a terminalia chebula tannin chelate, which can be directly used as a drug or feed additive for treating or preventing pig diseases such as PED, PRRS and ASF.

[0025] In certain embodiments, the drug or feed additive is a terminalia chebula tannin chelate composition, which includes terminalia chebula tannin chelate and excipients, and the excipients include at least one of anhydrous ethanol, water, Tween-80, lactose, defatted rice bran, corn cob powder, soluble starch, bran, silicon dioxide, montmorillonite, vermiculite, medical stone and zeolite powder.

[0026] In certain embodiments, the terminalia chebula tannin chelate composition is a solution formed by mixing terminalia chebula tannin chelate with three solvents: anhydrous ethanol, Tween-80, and water, wherein the concentration of terminalia chebula tannin chelate is 50-100 mg / mL.

[0027] In certain embodiments, the terminalia chebula tannin chelate composition is a solid mixture of terminalia chebula tannin chelate, lactose and soluble starch, wherein the mass fraction of terminalia chebula tannin chelate in the solid mixture is 45% to 55%.

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

[0029] In certain embodiments, when the drug or feed additive is a terminalia chebula tannin chelate 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 preferred embodiments, the terminalia chebula tannin chelate is at least one of terminalia chebula tannin copper and terminalia chebula tannin zinc.

[0031] In certain embodiments, the preparation method of the terminalia chebula tannin chelate is: prepare terminalia chebula tannin extract by boiling terminalia chebula raw material, dissolve the terminalia chebula tannin extract in hot ethanol at 40-50°C, add the metal salt compound according to the mass ratio of terminalia tannin to metal salt compound of 1:1-1.5, react for 1-3 hours, and obtain the terminalia chebula tannin chelate by spray drying.

[0032] In certain preferred embodiments, the metal salt compound is at least one of copper chloride and zinc chloride.

[0033] In certain embodiments, the method of using the drug or feed additive comprises at least one of the following methods:

[0034] (1) adding the terminalia chebula tannin chelate or terminalia chebula tannin chelate composition to feed at a dosage of 5 to 20 mg / kg of pig body weight;

[0035] (2) directly orally administering the terminalia chebula tannin chelate or terminalia chebula tannin chelate composition at a dosage of 5 to 20 mg / kg of pig body weight;

[0036] (3) Add the terminalia chebula tannin chelate or terminalia chebula tannin chelate combination to the feed at a dosage of 0.05% to 0.5% of the total weight of the pig feed.

[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] The present application example provides a method for preparing a terminalia chebula tannin copper oral liquid, the specific steps of which are:

[0040] (1) The terminalia chebula raw material is boiled to prepare terminalia chebula tannin extract, the terminalia chebula tannin extract is dissolved in hot ethanol at 45° C., copper chloride is added according to a mass ratio of terminalia chebula tannin to copper chloride of 1:1.2, the reaction is carried out for 2 hours, and terminalia chebula tannin copper is obtained by spray drying;

[0041] (2) Weigh 10 g of Terminalia chebula tannin copper, 20 mL of anhydrous ethanol, and 30 mL of Tween-80 for later use;

[0042] (3) Dissolve the terminalia chebula tannin copper in anhydrous ethanol, then add Tween-80, and finally add water for injection to make up to 100 mL, mix well, and obtain a terminalia chebula tannin copper solution with a concentration of 100 mg / mL, i.e., the terminalia chebula tannin copper oral solution K1.

[0043] Example 2

[0044] The present application example provides another method for preparing the terminalia chebula tannin copper oral solution, the specific steps of which are:

[0045] (1) preparing terminalia chebula tannin copper by the method of step (1) in Example 1;

[0046] (2) Weigh 5 g of terminalia chebula tannin copper, 20 mL of anhydrous ethanol, and 30 mL of Tween-80 for later use;

[0047] (3) Dissolve the terminalia chebula tannin copper in anhydrous ethanol, then add Tween-80, and finally add water for injection to make up to 100 mL, mix well, and obtain a terminalia chebula tannin copper solution with a concentration of 50 mg / mL, i.e., the terminalia chebula tannin copper oral solution K2.

[0048] Example 3

[0049] The present application example provides a method for preparing terminalia chebula tannin copper granules, the specific steps of which are:

[0050] (1) preparing terminalia chebula tannin copper by the method of step (1) in Example 1;

[0051] (2) Weigh 50 g of Terminalia chebula tannin copper, 15 g of lactose, and 35 g of soluble starch for later use;

[0052] (3) The terminalia chebula tannin copper, lactose and soluble starch are mixed and granulated, and then dried and granulated to obtain terminalia chebula tannin copper granules with a mass fraction of 50%, namely the terminalia chebula tannin copper granules K3.

[0053] Example 4

[0054] The present application embodiment provides a method for preparing terminalia chebula tannin copper powder, and the specific steps are as follows:

[0055] (1) preparing terminalia chebula tannin copper by the method of step (1) in Example 1;

[0056] (2) Weigh 50 g of Terminalia chebula tannin copper and 50 g of soluble starch and set aside;

[0057] (3) The terminalia chebula tannin copper and soluble starch are uniformly mixed and passed through a 60-mesh sieve to obtain a terminalia chebula tannin copper powder with a mass fraction of 50%, namely the terminalia chebula tannin copper powder K4.

[0058] Example 5

[0059] The present application example provides a method for preparing a terminalia chebula tannin copper tablet, the specific steps of which are:

[0060] (1) preparing terminalia chebula tannin copper by the method of step (1) in Example 1;

[0061] (2) Weigh 50 g of Terminalia chebula tannin copper, 15 g of lactose, and 35 g of soluble starch for later use;

[0062] (3) After the terminalia chebula tannin copper, lactose and soluble starch are uniformly mixed, the mixture is compressed and dried using a tablet press to obtain a tablet having a mass fraction of terminalia chebula tannin copper of 50%, namely the terminalia chebula tannin tablet K5.

[0063] Example 6

[0064] The present application example provides a method for preparing a terminalia chebula tannin zinc oral liquid, the specific steps of which are:

[0065] (1) preparing a terminalia chebula tannin extract from terminalia chebula raw material by a water boiling method, dissolving the terminalia chebula tannin extract in hot ethanol at 45° C., adding zinc chloride in a mass ratio of terminalia chebula tannin to zinc chloride of 1:1.2, reacting for 2 hours, and spray drying to obtain terminalia chebula tannin zinc;

[0066] (2) Weigh 10 g of terminalia chebula tannin zinc, 20 mL of anhydrous ethanol, and 30 mL of Tween-80 for later use;

[0067] (3) Dissolve the terminalia chebula tannin zinc in anhydrous ethanol, then add Tween-80, and finally add water for injection to make up to 100 mL, mix well, and obtain a terminalia chebula tannin zinc solution with a concentration of 100 mg / mL, i.e., the terminalia chebula tannin zinc oral solution K6.

[0068] Example 7

[0069] The present application example provides a method for preparing a terminalia chebula tannin zinc oral liquid, the specific steps of which are:

[0070] (1) preparing terminalia chebula tannin zinc by the method of step (1) in Example 6;

[0071] (2) Weigh 5 g of terminalia chebula tannin zinc, 20 mL of anhydrous ethanol, and 30 mL of Tween-80 for later use;

[0072] (3) Dissolve the terminalia chebula tannin zinc in anhydrous ethanol, then add Tween-80, and finally add water for injection to make up to 100 mL, mix well, and obtain a terminalia chebula tannin zinc solution with a concentration of 50 mg / mL, i.e., the terminalia chebula tannin zinc oral solution K7.

[0073] Example 8

[0074] The present application example provides a method for preparing terminalia chebula tannin zinc granules, the specific steps of which are:

[0075] (1) preparing terminalia chebula tannin zinc by the method of step (1) in Example 6;

[0076] (2) Weigh 50 g of terminalia chebula tannin zinc, 15 g of lactose, and 35 g of soluble starch and set aside;

[0077] (3) The terminalia chebula tannin zinc, lactose and soluble starch are mixed and granulated, and the granules are dried to obtain granules with a mass fraction of terminalia chebula tannin zinc of 50%, namely the terminalia chebula tannin zinc granules K8.

[0078] Example 9

[0079] The present application example provides a method for preparing terminalia chebula tannin zinc powder, the specific steps of which are:

[0080] (1) preparing terminalia chebula tannin zinc by the method of step (1) in Example 6;

[0081] (2) Weigh 50 g of terminalia chebula tannin zinc and 50 g of soluble starch and set aside;

[0082] (3) The terminalia chebula tannin zinc and soluble starch are uniformly mixed and passed through a 60-mesh sieve to obtain a powder with a mass fraction of terminalia chebula tannin zinc of 50%, namely the terminalia chebula tannin zinc powder K9.

[0083] Example 10

[0084] The present application example provides a method for preparing a terminalia chebula tannin zinc tablet, the specific steps of which are:

[0085] (1) preparing terminalia chebula tannin zinc by the method of step (1) in Example 6;

[0086] (2) Weigh 50 g of terminalia chebula tannin zinc, 15 g of lactose, and 35 g of soluble starch and set aside;

[0087] (3) After the terminalia chebula tannin zinc, lactose and soluble starch are uniformly mixed, the mixture is compressed and dried using a tablet press to obtain a tablet with a mass fraction of terminalia chebula tannin zinc of 50%, namely the terminalia chebula tannin zinc tablet K10.

[0088] The formula and parameters of the terminalia chebula tannin chelate preparation prepared in the above example are shown in Table 1 below:

[0089] Table 1

[0090] Example No. Preparation No. Formulation type Components Terminalia chebula tannin chelate concentration Example 1 K1 Oral solution Terminalia chebula tannin copper, anhydrous ethanol, Tween-80 100mg / mL Example 2 K2 Oral solution Terminalia chebula tannin copper, anhydrous ethanol, Tween-80 50mg / mL Example 3 K3 Granules Terminalia chebula tannin copper, lactose, soluble starch 50% (mass fraction) Example 4 K4 powder Terminalia chebula tannin copper, soluble starch 50% (mass fraction) Example 5 K5 tablet Terminalia chebula tannin copper, lactose, soluble starch 50% (mass fraction) Example 6 K6 Oral solution Terminalia chebula tannin zinc, anhydrous ethanol, Tween-80 100mg / mL Example 7 K7 Oral solution Terminalia chebula tannin zinc, anhydrous ethanol, Tween-80 50mg / mL Example 8 K8 Granules Terminalia chebula tannin zinc, lactose, soluble starch 50% (mass fraction) Example 9 K9 powder Terminalia chebula tannin zinc, soluble starch 50% (mass fraction) Example 10 K10 tablet Terminalia chebula tannin zinc, lactose, soluble starch 50% (mass fraction)

[0091] Inhibitory Effect of Terminalia chebula Tannin Chelate on Swine Virus

[0092] In the present application, the above prepared terminalia chebula tannin copper and terminalia chebula tannin zinc were used to verify the inhibitory effect on the in vitro proliferation of three porcine viruses, PEDV, PRRSV and ASFV. The verification method is as follows:

[0093] 1. Materials and methods:

[0094] 1.1 Viruses:

[0095] PEDV YN strain and DR13 strain, PRRSV, and ASFV were isolated and preserved by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0096] 1.2 Cells:

[0097] African green monkey kidney cells (Vero), used for the proliferation of PEDV DR13 and YN strains;

[0098] Monkey embryonic kidney epithelial cells (Marc-145) were used for the proliferation of PRRSV;

[0099] Porcine alveolar macrophages (primary cells) were used for the proliferation of ASFV;

[0100] Vero and Marc-145 cells are laboratory-maintained cell lines, and porcine alveolar macrophages were isolated from the lungs of healthy piglets.

[0101] 1.3 Trial drugs:

[0102] The chebula tannin copper and chebula tannin zinc were prepared according to the above method;

[0103] 1.4 Reagents: DMEM medium, 1640 medium, trypsin, fetal bovine serum and double antibody (penicillin-streptomycin) were purchased from GIBCO;

[0104] 1.5 In vitro proliferation inhibition:

[0105] (1) PEDV in vitro proliferation inhibition test: All in vitro tests were set up with three test groups and one blank group. Different doses of terminalia tannin chelate (termia tannin copper or terminalia tannin zinc) were added to the culture medium in the test groups, including a low-dose group (added at 25 μg / mL culture medium), a medium-dose group (added at 50 μg / mL culture medium), a high-dose group (added at 100 μg / mL culture medium) and a blank group (termia tannin copper and terminalia tannin zinc were not added to the culture medium); Vero cells were infected with the PEDVDR13 strain (MOI = 0.001), and terminalia tannin chelate of different concentrations was added at the same time of infection. 24 hours after infection, the effect of terminalia tannin chelate on the in vitro proliferation of the PEDV DR13 strain was investigated by indirect immunofluorescence assay; at the same time, the virus liquid of the PEDV YN strain was serially diluted 10 times with DMEM maintenance medium, and 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Six dilutions were inoculated into 96-well plates with cultured Vero cell monolayers, 100 μL / well, and DMEM maintenance solution containing different concentrations of terminalia chebula tannin chelate was added to each group. The cells were placed in a 37°C incubator with 5% CO2 for 48 hours and the cell lesions were observed. The number of lesion-positive wells in each dilution was counted, and the TCID of each group was calculated using the Reed-Muench method. 50 , compare different groups of TCID 50 The inhibitory effect of terminalia chebula tannin chelate on the proliferation of PEDV in vitro was judged by the value of , and three replicates were performed in each group.

[0106] (2) PRRSV in vitro proliferation inhibition test: PRRSV was maintained in DMEM and serially diluted 10-fold. -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Six dilutions were inoculated into 96-well plates with cultured Marc-145 cell monolayers, 100 μL / well, and DMEM maintenance solution containing different concentrations of terminalia chebula tannin chelate was added to each group. The cells were placed in a 37°C incubator with 5% CO2 for 72 hours and the cell lesions were observed. The number of lesion-positive wells in each dilution was counted, and the TCID of each group was calculated using the Reed-Muench method. 50 , compare different groups of TCID50 The inhibitory effect of terminalia chebula tannin chelate on the proliferation of PRRSV in vitro was judged by the value of , and three replicates were performed in each group.

[0107] (3) ASFV in vitro proliferation inhibition test: ASFV virus liquid was diluted 10 times with 1640 maintenance liquid, and 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Six dilutions were inoculated into the cultured alveolar macrophage monolayer of 96-well plates, 100 μL / well, and 1640 maintenance solution containing different concentrations of terminalia chebula tannin chelate was added to each group, and the cells were cultured for 72 hours in a 37°C incubator containing 5% CO2 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 Poria polysaccharide group was calculated using the Reed-Muench method. 50 , compare different groups of TCID 50 The inhibitory effect of Terminalia chebula tannin chelate on the proliferation of ASFV in vitro was judged by the value of , and three replicates were performed in each group.

[0108] 1.6 Data analysis: The experimental data were analyzed using one-way ANOVA and Duncan's multiple comparison method in SPSS13.0 statistical software, with P < 0.05 as the standard of significant difference and P < 0.01 as the standard of extremely significant difference. The results are expressed as mean values.

[0109] 2. Experimental results:

[0110] 2.1 Evaluation of the inhibitory effect of terminalia chebula tannin chelate on PEDV and PRRSV by indirect immunofluorescence assay

[0111] Figure 1 The figure shows the inhibitory effect of terminalia chebula tannin copper on PEDV and PRRSV observed by fluorescence microscopy. Figure 2 The figure shows the inhibitory effect of terminalia chebula tannin zinc on PEDV and PRRSV observed by fluorescence microscopy. Figure 1 , 2 It can be seen that with the increase of the amount of terminalia tannin copper or terminalia tannin zinc additives, the weaker the fluorescence signal, indicating that the proliferation of PEDV and PRRSV is less, indicating that terminalia tannin copper or terminalia tannin zinc has a significant inhibitory effect on the in vitro proliferation of PEDV and PRRSV, terminalia tannin copper or terminalia tannin zinc has an effective antiviral effect in vitro, and its antiviral effect shows obvious dosage dependence.

[0112] 2.2 TCID of Terminalia chebula tannin chelate against different viruses 50 Impact

[0113] Table 2 shows the TCID of different doses of Terminalia chebula tannin copper and Terminalia chebula tannin zinc against different viruses 50 Influence results, where a, b, c, and d are marks of significant difference.

[0114] Table 2

[0115]

[0116] The results in Table 2 show that the addition of different concentrations of terminalia tannin copper or terminalia tannin zinc to the cell culture medium significantly affected the TCID of PEDV on Vero cells. 50 The TCID values ​​of PEDV on Vero cells increased with the addition of copper or zinc tannins from Terminalia chebula. 50 The addition of different concentrations of chebula tannin copper or chebula tannin zinc to the cell culture medium significantly affected the TCID of PRRSV on marc-145 cells. 50 The TCID values ​​of PRRSV on Marc-145 cells increased with the addition of copper or zinc tannins from Terminalia chebula. 50 The results showed that the copper or zinc tannins of terminalia chebula significantly decreased, indicating that the copper or zinc tannins of terminalia chebula significantly inhibited the proliferation of PRRSV in Marc-145 cells cultured in vitro. Adding different concentrations of copper or zinc tannins of terminalia chebula to the cell culture medium significantly affected the TCID of ASFV on alveolar macrophages. 50 The TCID values ​​of ASFV on alveolar macrophages increased with the addition of copper or zinc tannins from Terminalia chebula. 50 The results showed that the content of chebula tannin copper or chebula tannin zinc had a significant inhibitory effect on the proliferation of ASFV in vitro.

[0117] Protective effect of terminalia chebula tannin chelate compound on piglets infected with PEDV

[0118] The present application example verifies the protective effect of the terminalia chebula tannin chelate preparation on piglets infected with PEDV, and the verification method is as follows:

[0119] 1. Materials and methods:

[0120] 1.1 Experimental virus: PEDV YN strain, donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0121] 1.2 Experimental drugs: Terminalia chebula tannin copper oral solution K1, Terminalia chebula tannin zinc granules K8.

[0122] 1.3 Experimental animals: 3-day-old healthy piglets of the same breed and origin, similar body weight and without maternal antibodies were selected for the challenge test. The experimental piglets were divided into three experimental groups and one blank group. The terminalia tannin copper oral solution K1 or terminalia tannin zinc granules K8 were added to the feed of the experimental groups, including the low-dose group (addition according to the terminalia tannin copper or terminalia tannin zinc content of 5 mg / kg pig body weight), the medium-dose group (addition according to the terminalia tannin copper or terminalia tannin zinc content of 10 mg / kg pig body weight), the high-dose group (addition according to the terminalia tannin copper or terminalia tannin zinc content of 20 mg / kg pig body weight) and the blank group (no terminalia tannin chelate or its preparation was added to the feed), each group had 12 piglets.

[0123] 1.4 Challenge test: After feeding for one week, the piglets in each group were fed with 10 6 TCID 50 / mL PEDV (YN strain) was used for virus challenge. Each head was orally infused with 3.0mL of virus solution. The control group was infused with the same dose of normal saline. Then, the animals were isolated and reared and observed for 7 consecutive days until the end of the experiment.

[0124] 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. The number of dead pigs was counted and the diarrhea rate, mortality rate and protection rate were calculated. The inhibitory effect of the terminalia chebula tannin chelate preparation on PEDV was determined by detecting the expression of the PEDV structural gene N in the intestinal mucosa of piglets through real-time quantitative PCR.

[0125] 1.6 Data analysis: The experimental data were analyzed using one-way ANOVA and Duncan's multiple comparison method in SPSS13.0 statistical software, with P < 0.05 as the standard of significant difference and P < 0.01 as the standard of extremely significant difference. The results are expressed as mean values.

[0126] 2. Experimental results:

[0127] After adding different doses of Terminalia chebula tannin copper oral solution K1 and Terminalia chebula tannin zinc granules K8 to the feed, the morbidity and mortality of piglets in each group are shown in Table 3.

[0128] Table 3

[0129]

[0130] The results in Table 3 show that adding different doses of terminalia tannin copper oral solution K1 to piglet feed can significantly reduce the diarrhea rate and mortality of piglets infected with PEDV. Compared with the blank group, the diarrhea rate of piglets infected with PEDV decreased to 33.3%-91.7%, while the mortality of diseased pigs decreased to 16.7%-75%; adding different doses of terminalia tannin zinc granules K8 to piglet feed can significantly reduce the diarrhea rate and mortality of piglets infected with PEDV. Compared with the blank group, the diarrhea rate of piglets infected with PEDV decreased to 25%-83.3%; while the mortality of diseased pigs decreased to 8.3%-66.7%. The experimental results fully show that the addition of terminalia tannin copper preparation or terminalia tannin zinc preparation can significantly improve the protection rate of piglets against PEDV infection.

[0131] The PEDV structural gene N was detected in the intestinal mucosa of the duodenum, jejunum, and ileum tissues of piglets, and the test results are shown in Table 4 below.

[0132] Table 4

[0133]

[0134] The results in Table 4 show that the addition of Terminalia chebula tannin copper oral liquid K1 or Terminalia chebula tannin copper granules K8 can significantly reduce the N gene expression of PEDV. The results fully demonstrate that the addition of Terminalia chebula tannin copper oral liquid K1 or Terminalia chebula tannin copper granules K8 can significantly reduce the replication of PEDV virus in the small intestine.

[0135] Protective effect of terminalia chebula tannin chelate on piglets infected with PRRSV

[0136] The present application example verifies the protective effect of the terminalia chebula tannin chelate preparation on piglets infected with PRRSV, and the verification method is as follows:

[0137] 1 Materials and methods

[0138] 1.1 Experimental virus: PRRSV, donated by the National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0139] 1.2 Experimental drugs: Terminalia chebula tannin copper tablets K5 and Terminalia chebula tannin zinc powder K9.

[0140] 1.3 Experimental animals: 25-day-old healthy piglets of the same breed and origin, similar body weight and without maternal antibodies were selected for the challenge test. The experimental piglets were divided into three experimental groups and one blank group. Terminalia chebula tannin copper tablets K5 or terminalia tannin zinc powder K9 were added to the feed of the experimental groups, including the low-dose group (addition according to the dosage of terminalia tannin copper or terminalia tannin zinc content of 5 mg / kg pig body weight), the medium-dose group (addition according to the dosage of terminalia tannin copper or terminalia tannin zinc content of 10 mg / kg pig body weight), the high-dose group (addition according to the dosage of terminalia tannin copper or terminalia tannin zinc content of 20 mg / kg pig body weight) and the blank group (no terminalia tannin chelate or its preparation was added to the feed), each group had 12 piglets.

[0141] 1.4 Challenge test: After feeding for 1 week, the piglets in each group were fed with 10 7 TCID 50 / mL PRRSV was used for infection. Each piglet was orally infused with 5.0mL of virus solution. The control group was infused with the same dose of normal saline. Then, they were raised in isolation and observed for 10 to 15 days until the end of the experiment.

[0142] 1.5 Observation index: The status of the experimental piglets was observed every day. The experimental piglets were slaughtered at the end of the experiment to observe the lung lesions. The number of dead pigs was counted and the incidence and mortality were calculated. The inhibitory effect of the terminalia chebula tannin chelate preparation on PRRSV was determined by detecting the expression of PRRSV ORF7 gene in the brain and lung of piglets through real-time quantitative PCR.

[0143] 1.6 Data analysis: The experimental data were analyzed using one-way ANOVA and Duncan's multiple comparison method in SPSS13.0 statistical software, with P < 0.05 as the standard of significant difference and P < 0.01 as the standard of extremely significant difference. The results are expressed as mean values.

[0144] 2. Experimental results:

[0145] After adding different doses of Terminalia chebula tannin copper tablets K5 or Terminalia chebula tannin zinc powder K9 to the feed, the morbidity and mortality of piglets in each group are shown in Table 5.

[0146] Table 5

[0147]

[0148] The results in Table 5 show that oral administration of different doses of terminalia tannin copper tablets K5 by piglets can significantly reduce the morbidity and mortality of piglets infected with PRRSV. Compared with the blank group, with the increase of dose, the morbidity of piglets infected with PRRSV decreased to 25%-75%; and the mortality of diseased pigs decreased to 16.7%-50%; oral administration of different doses of terminalia tannin zinc powder K9 by piglets can significantly reduce the morbidity and mortality of piglets infected with PRRSV. Compared with the blank group, with the increase of dose, the morbidity of piglets infected with PRRSV decreased to 33.3%-83.3%; and the mortality of diseased pigs decreased to 16.7%-33.3%; the experimental results fully show that the addition of terminalia tannin copper preparations or terminalia tannin zinc preparations can significantly improve the protection rate of piglets against PRRSV infection.

[0149] The expression levels of PRRSV ORF7 gene in the brain and lung of piglets were detected respectively. The detection results are shown in Table 6 below.

[0150] Table 6

[0151]

[0152] The results in Table 6 show that the addition of terminalia tannin copper tablets K5 or terminalia tannin zinc powder K9 can significantly reduce the expression level of ORF7 gene of PRRSV. The results fully demonstrate that the addition of terminalia tannin copper tablets K5 or terminalia tannin zinc powder K9 can significantly reduce the replication of PRRSV in the brain and lungs.

[0153] In summary, the terminalia chebula tannin chelate and terminalia chebula tannin chelate preparation provided in the embodiments of the present application have the effect of resisting porcine 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 viruses such as PEDV, PRRSV and ASFV. The dosage forms of the drugs and feed additives include oral liquids, granules, powders, tablets and the like.

[0154] 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 terminalia chebula tannin chelate in the preparation of medicines or feed additives for treating or preventing pig viral infections.

2. The use according to claim 1, characterized in that: The terminalia chebula tannin chelate is at least one of terminalia chebula tannin copper and terminalia chebula tannin zinc.

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

4. The use according to claim 1, characterized in that: The medicine or feed additive is terminalia chebula tannin chelate.

5. The use according to claim 1, characterized in that: The medicine or feed additive is a terminalia chebula tannin chelate composition, which comprises terminalia chebula tannin chelate and auxiliary materials, wherein the auxiliary materials comprise at least one of anhydrous ethanol, water, Tween-80, lactose, defatted rice bran, corn cob powder, soluble starch, bran, silicon dioxide, montmorillonite, vermiculite, medical stone and zeolite powder.

6. The use according to claim 5, characterized in that: The terminalia chebula tannin chelate composition is at least one of the following (1) to (3): (1) a solution prepared by mixing the terminalia chebula tannin chelate with anhydrous ethanol, Tween-80 and water, wherein the concentration of the terminalia chebula tannin chelate is 50 to 100 mg / mL; (2) a solid mixture of terminalia chebula tannin chelate, lactose and soluble starch, wherein the mass fraction of terminalia chebula tannin chelate in the solid mixture is 45% to 55%; (3) A powdery mixture of terminalia chebula tannin chelate and soluble starch or bran, wherein the mass fraction of terminalia chebula tannin chelate in the powdery mixture is 45% to 55%.

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, tablets and powders, and the preparation can be prepared by conventional pharmaceutical preparation methods.

8. The use according to claim 1, characterized in that: The preparation method of the terminalia chebula tannin chelate is: The terminalia chebula tannin extract is prepared from terminalia chebula raw material by a water boiling method, the terminalia chebula tannin extract is dissolved in hot ethanol at 40-50°C, a metal salt compound is added according to a mass ratio of terminalia tannin to metal salt compound of 1:1-1.5, the reaction is carried out for 1-3 hours, and the terminalia tannin chelate is obtained by spray drying.

9. The use according to claim 8, characterized in that: The metal salt compound is at least one of copper chloride and zinc chloride.

10. The use according to claim 1, characterized in that: The method of using the drug or feed additive includes at least one of the following methods: (1) adding the terminalia chebula tannin chelate or terminalia chebula tannin chelate composition to feed at a dosage of 5 to 20 mg / kg of pig body weight; (2) directly orally administering the terminalia chebula tannin chelate or terminalia chebula tannin chelate combination at a dosage of 5 to 20 mg / kg of pig body weight; (3) Add the terminalia chebula tannin chelate or terminalia chebula tannin chelate composition to the feed at a dosage of 0.05% to 0.5% of the total weight of the pig feed.

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