Application of Fengliao Changweikang preparation in preparing medicine for preventing or treating canine parvovirus disease

Abstract

The present invention discloses the use of Fengliao Changweikang preparation in the preparation of a medicine for preventing or treating canine parvovirus disease. The active ingredients of the Fengliao Changweikang preparation are prepared from Acer truncatum and Polygonum hydropiper, and the usage ratio of Acer truncatum and Polygonum hydropiper is (1-3):1. Through research, we found that the Fengliao Changweikang preparation can be used to prevent and treat canine parvovirus disease caused by canine parvovirus, can effectively improve the body's ability to resist canine parvovirus, improve the cure rate, and has good safety and stable effect.

Description

Technical Field

[0001] The invention relates to the field of traditional Chinese medicine compositions, and in particular to application of Fengliao Changweikang preparation in preparing medicines for preventing or treating canine parvovirus disease. Background Art

[0002] Fengliao Changweikang preparation is a classic Chinese medicine compound for preventing and treating gastrointestinal diseases. It is composed of two Chinese medicines, Acer truncatum and Polygonum hydropiper. It has the effects of regulating qi and strengthening the stomach, removing dampness and removing stagnation. It is used for acute gastroenteritis caused by poor circulation, qi stagnation and dampness, and the resulting indigestion symptoms such as abdominal distension, abdominal pain and diarrhea.

[0003] The dried whole plant of Daphniphyllum calycinum Benth. of the family Daphniphyllum (or the family Alpiniaceae) has the effects of clearing away heat and detoxifying, promoting blood circulation and relaxing muscles. It is used for colds and fever, tonsillitis, rheumatic joint pain, swelling and pain from falls, fractures, snake bites, sores and swelling, mastitis, dermatitis, and unidentified swelling.

[0004] Polygonum hydropiper L. is the dried whole herb of the Polygonum hydropiper L. plant of the Polygonaceae family. It has the effects of removing dampness and removing stagnation. It is used for diarrhea, dysentery, food stagnation, and external treatment of skin itching.

[0005] Canine parvovirus disease, also known as canine parvovirus enteritis, is a highly contagious disease caused by canine parbovirus (CPV). It is mainly characterized by acute hemorrhagic enteritis in dogs or acute myocarditis in newborn dogs. The clinical symptoms of the affected dogs are mainly fever, severe vomiting, hemorrhagic enteritis and a significant decrease in leukocytes. Canine parvovirus belongs to the Parvovirus genus of the Parvoviridae family. The virus is hexagonal in appearance, has no capsule, is icosahedral isoaxial symmetry, has a diameter of about 20nm, and the capsid consists of 32 capsomeres. The virus has strong hemagglutination properties and can agglutinate red blood cells of pigs and rhesus monkeys.

[0006] Canine parvovirus enteritis occurs all year round, especially in spring and autumn. Dogs of different species and ages can be infected with this disease, among which puppies are most vulnerable, with the highest infection rate and mortality rate. In terms of treatment, there is no effective drug and prevention method for canine parvovirus, so the cure rate of canine parvovirus is low. Finding traditional Chinese medicine for the treatment of canine parvovirus disease has the advantages of small toxic side effects and stable results. Summary of the invention

[0007] Based on this, the purpose of the present invention is to provide a new application of Fengliao Changweikang preparation to solve the problem of lack of drugs for treating canine parvovirus disease in clinical practice.

[0008] The first aspect of the present invention is to provide a Fengliao Changweikang preparation for use in the preparation of a medicament for preventing or treating canine parvovirus disease, wherein the active ingredients of the Fengliao Changweikang preparation are prepared from Acer truncatum and Polygonum hydropiper, and the mass dosage ratio of Acer truncatum and Polygonum hydropiper is (1-3):1.

[0009] In some embodiments, the mass ratio of the Herba Polygoni Multiflori to the Herba Polygoni Multiflori is 2:1.

[0010] In some of the embodiments, the active ingredients of the Fengliao Changweikang preparation are obtained by water extraction.

[0011] In some embodiments, the preparation method of the Fengliao Changweikang preparation comprises:

[0012] Add water to the two medicinal materials, Polygonum multiflorum and Polygonum hydropiper, in proportion and boil them. Combine the decoctions, filter and concentrate them to obtain the product.

[0013] In some preferred embodiments, the preparation method of the Fengliao Changweikang preparation comprises:

[0014] Add water to the two medicinal materials, Polygonum multiflorum and Polygonum hydropiper, according to the proportion and boil them 1-3 times for 1-1.5 hours. Combine the decoctions, filter them, and concentrate the filtrate under reduced pressure to a thick paste.

[0015] In some of the embodiments, the Fengliao Changweikang preparation is in the form of granules, tablets, capsules (such as soft capsules), oral liquids, etc., preferably granules or tablets.

[0016] The present invention has found through research that Fengliao Changweikang preparation can be used to prevent and treat canine parvovirus disease caused by canine parvovirus, can effectively improve the body's ability to resist canine parvovirus, improve the cure rate, and has good safety and stable effect. DETAILED DESCRIPTION

[0017] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0018] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0019] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0020] The preparation method of the test product in the embodiment of the present invention can be referred to as follows.

[0021] Preparation method of Fengliao Changweikang tablets: 4000 grams of Acer truncatum and 2000 grams of Polygonum hydropiper are decocted in water twice, the first time for 1.5 hours and the second time for 1 hour, the decoctions are combined and filtered, and the filtrate is concentrated under reduced pressure to a thick paste, dried below 80°C, crushed into fine powder, added with appropriate amount of starch, mixed well, made into granules, pressed into 1000 tablets, and coated.

[0022] Preparation method of Niu Er Feng extract: Take 500 grams of Niu Er Feng and add water to boil twice, the first time for 1.5 hours and the second time for 1 hour.

[0023] Combine the decoctions, filter, and concentrate the filtrate under reduced pressure to a thick paste for later use.

[0024] Preparation method of Polygonum hydropiper extract: Take 500 grams of Polygonum hydropiper and add water to boil twice, the first time for 1.5 hours and the second time for 1 hour, combine the decoctions, filter, and concentrate the filtrate under reduced pressure to a thick paste for later use.

[0025] 1. Experimental materials:

[0026] Drug: Fengliao Changweikang Tablets (B23S004)

[0027] Compound Jiubiying Capsules (TG40008)

[0028] Fuzi Lizhong Pills (231105)

[0029] Modified Huoxiang Zhengqi Pills (B23A004)

[0030] Acer palmatum extract

[0031] Polygonum hydropiper extract

[0032] The experimental drugs were provided by Guangzhou Baiyunshan Hutchison Whampoa Chinese Medicine Co., Ltd.

[0033] Positive drug: Astragalus polysaccharide injection (Sichuan Xiongfeng Animal Pharmaceutical Co., Ltd.).

[0034] Cells and viruses: F81 cells (cat kidney cells) were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; canine parvovirus (CPV) was purchased from the China Veterinary Drug Administration, and its TCID against F81 cells was determined using the Reed-Muench method. 50 (50% cell culture infectious dose) is 10 5.0 .

[0035] Reagents: MEM liquid culture medium (Gibco); MTT reagent (Sigma); DMSO (Shanghai Biochemical Reagent Co., Ltd.).

[0036] Instruments: CO2 incubator (Thermo); BIO-RAD-680 enzyme-linked immunosorbent assay and PCR instrument (Bio-Rad); 96-well cell culture plates, 24-well cell culture plates (Nubclon).

[0037] 2. Experimental Methods

[0038] 2.1 Drug treatment

[0039] Remove the coating from Fengliao Changweikang tablets, grind into powder, and prepare into a 100 mg / ml solution for later use.

[0040] Grind Fuzi Lizhong Pills and Jiawei Huoxiang Zhengqi Pills into powder respectively and prepare a 100 mg / ml solution for later use.

[0041] Open the capsule of Compound Jiubiying Capsule, pour out the powder and prepare a 100 mg / ml solution for later use.

[0042] The extract of Acer truncatum was prepared into a 100 mg / ml solution for later use.

[0043] The extract of Polygonum hydropiper was prepared into a 100 mg / ml solution for later use.

[0044] 2.2 Virus infectivity determination

[0045] Canine parvovirus (CPV) solution was diluted 10-fold into 6 concentrations using serum-free MEN medium, and added to F81 cells in a 24-well culture plate, 100 μL per well. A normal cell control was set up at the same time. After incubation at 37°C for 1 hour, 500 μL of maintenance solution was added to each well. Cell lesions were observed under a microscope for 48-72 hours. If the cells were completely lesioned, it was recorded as 4, 75% lesion was recorded as 3, 50% lesion was recorded as 2, 25% lesion was recorded as 1, and no lesion was recorded as 0. The TCID of the virus was determined by the Reed-Muench method. 50 .

[0046]

[0047] 2.3 Determination of non-toxic drug concentration

[0048] F81 cells were inoculated into 96-well cell culture plates, 100 μL per well. After the cells were basically covered with monolayer within 24 h, the cell culture medium was discarded and washed twice with Hanks solution. The drug dilution solution was filtered through a cellulose ester microporous filter membrane (pore size 0.22 μm). The drug was diluted with the maintenance solution to 10 concentrations and added to the 96-well cell culture plate. 100 μL of the drug solution was added to each well. Each concentration was repeated for 3 wells. At the same time, a cell control well without Chinese medicine solution and a blank zeroing well were set. The cells were incubated at 37 °C. After 72 h of culture, the D570 value was determined by an enzyme-linked immunosorbent assay according to the MTT method (50 μL of MTT solution was added to each well 4 h before the determination; during the determination, the culture plate was quickly turned over to shake off the culture solution in the well, 150 μL of dimethyl sulfoxide was added to each well, and the well was shaken at room temperature for 10 min. Then, the D value was determined by an enzyme-linked immunosorbent assay as an indicator of cell proliferation. The maximum non-toxic dose of the drug was determined.

[0049] 2.4 Anti-CPV infection F81 cell experiment

[0050] Set up a virus control group (without any drug) and a cell control group (with the maximum non-toxic concentration as the high concentration, and set up a medium concentration group and a low concentration group by 3-fold dilution). Take 100TCID 50 The CPV virus solution was mixed with equal volumes of drug culture medium of different concentrations and cultured at 37℃ for 2 h. The mixture was inoculated onto F81 cells and cultured at 37℃ for 24 h. The cell survival rate was detected by MTT. Blank cell group, negative control group and positive drug group were set up.

[0051] 2.5 Therapeutic effect on beagle infection

[0052] Method for parvovirus infection in beagles: Normal cat kidney cells (F81) were infected with canine parvovirus (CPV), cultured and proliferated, the culture medium was collected, and the cell residues were removed by centrifugation. Each dog was gavagely administered 2 mL 5×10 5 PFU / mL of virus liquid, and the dogs with clinical symptoms after the virus attack were detected with parvovirus test paper, and the dogs with positive test paper were selected as experimental animals. Fifteen 18-month-old beagles were selected (12 beagles were positive in the parvovirus test and 3 normal healthy beagles), 3 in each group, and a blank control group (3 healthy beagles), a negative control group (no drug treatment), Fengliao Changweikang group (200 mg / kg), Jiubiying group (60 mg / kg) and a positive drug group (15 mg / kg of Astragalus polysaccharide injection) were set up. The drugs were administered by gavage for 10 consecutive days, and the survival, feces and vomiting of the beagles were recorded. The beagles were placed in an SPF laboratory for breeding, and the test paper was tested every day after the virus attack (July 1). After the test was positive, they were randomly assigned to each group for drug administration.

[0053] 2.6 Statistical analysis

[0054] All experimental data are expressed as Mean ± SD. SPSS 22.0 was used for statistical analysis, and Prism version 8.0 (GraphPad) was used for plotting and analysis. One-way ANOVA and Bonferroni multiple comparison test were used for analysis among multiple groups. P A value of <0.05 was defined as a significant difference.

[0055] 3. Results

[0056] 3.1 Determination of Maximum Non-Toxic Concentration

[0057] The maximum non-toxic concentration of Fengliao Changweikang Tablets and Fufang Jiubiying Capsules to F81 cells was detected by MTT experiment:

[0058] 3.1.1 Fengliao Changweikang Tablets

[0059] Table 1. Toxic effects of Fengliao Changweikang tablets on F81 cells

[0060]

[0061] 3.1.2 Compound Jiubiying Capsules

[0062] Table 2. Toxic effects of compound Jiubiying capsule on F81 cells

[0063]

[0064] 3.1.3 Fuzi Lizhong Pills

[0065] Table 3. Toxicity of Fuzilizhong Pills to F81 cells

[0066]

[0067] 3.1.4 Modified Huoxiang Zhengqi Pills

[0068] Table 4. Toxic effects of Jiawei Huoxiang Zhengqi Pills on F81 cells

[0069]

[0070] 3.1.5 Acer palmatum extract

[0071] Table 5. Toxic effects of Acer truncatum extract on F81 cells

[0072]

[0073] 3.1.6 Polygonum hydropiper extract

[0074] Table 6. Toxic effects of Polygonum hydropiper extract on F81 cells

[0075]

[0076] The above data showed that 1 mg / mL Fengliao Changweikang Tablets, 1 mg / mL Compound Jiubiying Capsules, 1 mg / mL Fuzi Lizhong Pills and Jiawei Huoxiang Zhengqi Pills showed no toxicity to F81 cells, and 0.01 mg / ml Niuerfeng extract and 0.1 mg / ml Polygonum hydropiper extract showed no toxicity to F81 cells. Therefore, in subsequent experiments, 1 mg / mL Fengliao Changweikang Tablets, 1 mg / mL Compound Jiubiying Capsules, 1 mg / mL Fuzi Lizhong Pills, 1 mg / mL Jiawei Huoxiang Zhengqi Pills, 0.01 mg / ml Niuerfeng extract and 0.1 mg / ml Polygonum hydropiper extract were selected as high concentration doses, and 3-fold proportional dilutions were performed to resist CPV infection of F81 cells.

[0077] 3.2 Anti-CPV infection of F81 cells

[0078] F81 cells were infected with CPV and treated with low concentration (0.11 mg / ml), medium concentration (0.33 mg / ml) and high concentration (1 mg / ml) of Fengliao Changweikang Tablets, Fufang Jiubiying Capsules, Fuzi Lizhong Pills, Jiawei Huoxiang Zhengqi Pills, Niuerfeng and Polygonum hydropiper extracts. The results are shown in the following table: The results indicate that infection of F81 cells with CPV can significantly reduce the proliferation rate of cells ( P <0.01), after drug treatment, Fengliao Changweikang Tablets and Fufang Jiubiying Capsules in the medium-dose and high-dose groups showed obvious anti-CPV-infected F81 cell effects, and the cell proliferation rate increased significantly ( P <0.01). Fuzilizhong Pills and Jiawei Huoxiangzhengqi Pills had no significant effect on the anti-CPV infection of F81 cells ( P >0.05). High doses of Acer truncatum extract ( P <0.01) and Polygonum hydropiper extract ( P <0.05) had a significant effect on F81 cells infected with CPV, and the cell proliferation rate was significantly increased.

[0079] Table 7. Treatment results of Fengliao Changweikang Tablets

[0080]

[0081] Table 8. Treatment results of compound Jiubiying capsule

[0082]

[0083] Table 9. Treatment results of Fuzi Lizhong Pills

[0084]

[0085] Table 10. Treatment results of Jiawei Huoxiang Zhengqi Pills

[0086]

[0087] Table 11. Treatment results of Acer truncatum extract

[0088]

[0089] Table 12. Treatment results of Polygonum hydropiper extract

[0090]

[0091] 3.3 Therapeutic effect on beagle infection

[0092] The following are the treatment results of beagles in each experimental group:

[0093] Experimental description: Mental state refers to the daily activities and diet of the beagle. The criteria are:

[0094] Good: active in daily activities, good appetite, and loud voice.

[0095] General: Daily activity level is moderate, appetite is decreased, and voice is weak or muffled.

[0096] Poor: Inactive in daily activities, poor appetite or not eating, extremely weak or muffled voice.

[0097] Vomiting: Observe the cleanliness of the beagle's breeding room and record the incidence of vomiting.

[0098] Diarrhea: Observe the cleanliness of the beagle's breeding room and record the incidence of diarrhea.

[0099] Table 13. Treatment results of blank group

[0100]

[0101] Table 14. Treatment results of negative control group

[0102]

[0103] Table 15. Treatment results of Fengliao Changweikang group

[0104]

[0105] Table 16. Treatment results of the compound Jiubiying group

[0106]

[0107] Table 17. Treatment results of positive drug control group

[0108]

[0109] Table 18. Statistics of treatment results of each group

[0110]

[0111] The CPV infection model of beagles was constructed. We found that beagles in the negative control group developed diarrhea, excreted a large amount of tomato-like feces, had vomiting symptoms, were mentally depressed, and died within 10 days after CPV infection, with a mortality rate of 100%. The mental state of beagles was significantly improved after treatment with Fengliao Changweikang Tablets. One dog vomited during the experiment, and continued medication stopped vomiting and recovered. After 10 days of treatment, no beagles died in the Fengliao Changweikang group. One dog in the Fufang Jiubiying Capsule group had vomiting and diarrhea during the experiment. Diarrhea can cause rapid loss of body fluids, especially when diarrhea is severe and persistent, which may lead to severe dehydration and electrolyte imbalance in dogs. Dehydration and electrolyte imbalance are one of the main causes of death in parvovirus infection. The above results show that Fengliao Changweikang Tablets have a good effect in resisting CPV infection and curing canine parvovirus disease caused by canine parvovirus, and the overall effect is better than Fufang Jiubiying Capsule.

[0112] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of Fengliao Changweikang preparation as the sole active ingredient in the preparation of a drug for preventing or treating canine parvovirus disease, wherein the active ingredients of the Fengliao Changweikang preparation are prepared from Acer truncatum and Polygonum hydropiper, and the mass dosage ratio of Acer truncatum and Polygonum hydropiper is (1-3):

1.

2. The use according to claim 1, wherein: The mass dosage ratio of the Acer truncatum and the Polygonum hydropiper is 2:

1.

3. The use according to claim 1, wherein: The active ingredient of the Fengliao Changweikang preparation is a water extract.

4. The use according to claim 3, wherein: The preparation method of the Fengliao Changweikang preparation comprises: Add two medicinal materials, Polygonum multiflorum and Polygonum hydropiper, into water according to the proportion, and boil them for 2-3 times; combine, filter and concentrate the liquid from each boiling.

5. The use according to any one of claims 1 to 4, wherein: The Fengliao Changweikang preparation is in the form of granules, tablets, capsules, oral liquids and decoctions.

6. The use according to claim 5, wherein: The Fengliao Changweikang preparation is a granule or a tablet.

7. The use according to claim 5, wherein: The Fengliao Changweikang preparation is a soft capsule.