CN112089712B - Composition and application thereof in preparation of medicine for preventing and/or treating swine diseases - Google Patents
Composition and application thereof in preparation of medicine for preventing and/or treating swine diseases Download PDFInfo
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Abstract
The invention discloses a composition and application thereof in preparing a medicament for preventing and/or treating swine diseases. The composition is prepared from the following raw materials in parts by weight: 1.0-6.0 parts of chlorogenic acid, 1.0-3.0 parts of aucubin and 0.5-1.5 parts of geniposide. The experimental result shows that the composition can block the absorption and penetration of the classical swine fever virus, effectively prevent the infection of the classical swine fever virus, inhibit the replication of the classical swine fever virus, kill the classical swine fever virus and achieve the effect of treating the classical swine fever; the composition can also be used for effectively treating porcine epidemic diarrhea, and effectively inhibiting porcine Escherichia coli. Meanwhile, the composition also effectively solves the problems of large side effect and serious residue existing in the traditional antiviral drugs and antibiotic treatment. Therefore, the composition has good application prospect in preparing medicaments for resisting swine fever virus and inhibiting swine escherichia coli and medicaments for preventing and/or treating swine diseases such as swine fever, porcine epidemic diarrhea, swine edema disease and the like.
Description
Technical Field
The invention belongs to the field of pharmacy, and particularly relates to a composition and application thereof in preparing a medicament for preventing and/or treating swine diseases.
Background
In a modern large-scale animal husbandry feeding mode, the production speed is improved, a plurality of problems are caused, the quality of animal products is reduced, and various diseases are easy to occur to livestock and poultry. The common pig diseases mainly comprise white scour of piglets, yellow scour of piglets, red scour of piglets, edema disease of swine, swine plague, swine erysipelas, swine plague, transmissible gastroenteritis of swine, epidemic diarrhea of swine, blue ear disease of swine, parvovirus disease of swine, cysticercosis of swine, swine influenza, ascariasis of swine, white myopathy of piglets, nutritional anemia of piglets, nitrite poisoning of swine, salt poisoning of swine and the like.
Hog cholera is a highly infectious disease caused by hog cholera virus of the genus pestivirus of the family flaviviridae, the diseased pig is the main source of infection of hog cholera, and is mainly transmitted through the digestive tract and also transmitted through the respiratory tract, and hog cholera is characterized by high fever, hemorrhage in the eyes of internal organs and high mortality. Porcine epidemic diarrhea is a highly-contacted intestinal infectious disease caused by porcine epidemic diarrhea virus, and is characterized by vomiting, diarrhea and dehydration which are mostly generated in winter; pigs of different ages, breeds and sexes are susceptible, the incidence rate of suckling pigs, skeleton pigs and fattening pigs is generally 100%, and the incidence rate of sows is 10-90%. Swine edema disease is a disease caused by toxins produced by pathogenic escherichia coli; it mainly occurs to obese piglets with strong physique after weaning, often sudden occurrence, head edema, ataxia, rapid death, high lethality rate, and the disease is easy to occur in selenium-deficient areas.
At present, the swine diseases are mainly prevented and treated by antiviral drugs and antibiotics, and play an important role in promoting the growth of animals and improving the growth performance. However, with the abuse of antiviral drugs and antibiotics, the drug residue in pigs seriously exceeds the standard, and serious hidden danger is brought to public health and environment. In addition, the import countries such as the United states, European Union, Japan and the like continuously upgrade the requirements on drug inspection, and cause serious obstruction to the export of livestock, poultry and meat products in China.
Therefore, it is of great significance to develop a veterinary drug with high safety from the needs of human health, export needs and the like.
Chlorogenic acid, aucubin and geniposide are widely present in various medicinal plants, such as honeysuckle, eucommia leaves and the like, at present, medicinal value components in the eucommia leaves are extracted by related technologies, and patent CN104546992A provides an eucommia leaf extract, wherein the content of chlorogenic acid is not less than 9%, the content of geniposide is not less than 1%, and the content of aucubin is not less than 1%. The folium cortex eucommiae extract is a traditional Chinese medicine extract, has small side effect, can be used for a long time, does not generate toxic or side effect on organisms, is safe and effective, improves the life quality of patients, and has obvious curative effect on psoriasis. The patent CN111281921A provides a lung-shaped grass Yinqiao detoxification and anti-inflammation composition for treating cold, pneumonia, enteritis and nephritis, the composition comprises chlorogenic acid, aucubin and geniposide, and the composition has the purpose of treating cold, pneumonia, enteritis and nephritis. However, the prior art does not provide any suggestion that a composition comprising three compounds of chlorogenic acid, aucubin and geniposide as active ingredients has the effect of preventing and/or treating swine diseases (such as swine fever, porcine epidemic diarrhea, swine edema disease and the like).
Disclosure of Invention
The invention aims to provide a safe and nontoxic composition and application thereof in preparing medicaments for resisting swine fever viruses and inhibiting swine escherichia coli and medicaments for preventing and/or treating swine diseases such as swine fever, porcine epidemic diarrhea, swine edema disease and the like.
The invention provides a composition, which is prepared from the following raw materials in parts by weight: 1.0-6.0 parts of chlorogenic acid, 1.0-3.0 parts of aucubin and 0.5-1.5 parts of geniposide.
Further, the composition is prepared from the following raw materials in parts by weight: 1.3-2.5 parts of chlorogenic acid, 1.0-1.5 parts of aucubin and 1.0 part of geniposide.
Further, the composition is prepared from the following raw materials in parts by weight: 2.0-2.5 parts of chlorogenic acid, 1.0-1.5 parts of aucubin and 1.0 part of geniposide.
Further, the composition is prepared from the following raw materials in parts by weight: chlorogenic acid 2.5 parts, aucubin 1.5 parts, and geniposide 1.0 part.
The invention also provides a medicament which is a preparation prepared from the composition as an active ingredient and auxiliary materials acceptable in the field of pharmacy.
Further, the preparation is an injection or a solid preparation;
preferably, the preparation is an injection, and the auxiliary material comprises one or two of a bracket agent and an antioxidant.
The invention also provides application of the composition in preparing a medicament for preventing and/or treating swine diseases.
Further, the swine disease is swine fever, porcine epidemic diarrhea or porcine edema disease.
The invention also provides the application of the composition in preparing the medicament for resisting the swine fever virus; preferably, the medicament is capable of blocking the absorption and penetration of classical swine fever virus into cells, and/or inhibiting the proliferation of classical swine fever virus, and/or killing classical swine fever virus.
The invention also provides the application of the composition in preparing a medicament for inhibiting escherichia coli; preferably, the escherichia coli is porcine escherichia coli.
The experiment result shows that the composition consisting of the chlorogenic acid, the aucubin and the geniposide in a specific ratio can block the adsorption and penetration of the classical swine fever virus, effectively prevent the classical swine fever virus from being infected, inhibit the replication of the classical swine fever virus, kill the classical swine fever virus and achieve the effect of treating the classical swine fever; the composition is also effective in treating porcine epidemic diarrhea; the composition can also effectively inhibit porcine escherichia coli, achieves a synergistic effect in inhibition effect, and provides a new choice for treatment and prevention of swine edema disease. Meanwhile, the composition also effectively solves the problems of large side effect and serious residue existing in the traditional antiviral drugs and antibiotic treatment. Therefore, the composition has good application prospect in preparing medicaments for resisting swine fever virus and inhibiting swine escherichia coli and medicaments for preventing and/or treating swine diseases such as swine fever, porcine epidemic diarrhea, swine edema disease and the like.
The composition has the advantages of easily available raw materials and simple preparation method, and is suitable for industrial production.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1, photograph of normal IBRS-2 cells.
FIG. 2 is a photograph of IBRS-2 cells after classical swine fever virus infection.
FIG. 3 is a photograph showing the prognosis of the test drug 6 stem cells of classical swine fever virus-infected IBRS-2 cells.
Detailed Description
The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.
EXAMPLE 1 preparation of a composition according to the invention
1. Composition I
Prescription: 400g of chlorogenic acid, 300g of aucubin and 300g of geniposide.
The preparation method comprises the following steps: weighing chlorogenic acid, aucubin and geniposide according to a prescription, mixing uniformly, and subpackaging into powder.
2. Composition II
Prescription: 400g of chlorogenic acid, 400g of aucubin and 200g of geniposide.
The preparation method comprises the following steps: weighing chlorogenic acid, aucubin and geniposide according to a prescription, mixing uniformly, and subpackaging into powder.
3. Composition III
Prescription: 500g of chlorogenic acid, 250g of aucubin and 250g of geniposide.
The preparation method comprises the following steps: weighing chlorogenic acid, aucubin and geniposide according to a prescription, mixing uniformly, and subpackaging into powder.
4. Composition IV
Prescription: 500g of chlorogenic acid, 300g of aucubin and 200g of geniposide.
The preparation method comprises the following steps: weighing chlorogenic acid, aucubin and geniposide according to a prescription, mixing uniformly, and subpackaging into powder.
5. Composition five
Prescription: 500g of chlorogenic acid, 350g of aucubin and 150g of geniposide.
The preparation method comprises the following steps: weighing chlorogenic acid, aucubin and geniposide according to a prescription, mixing uniformly, and subpackaging into powder.
6. Composition VI
Prescription: 600g of chlorogenic acid, 200g of aucubin and 200g of geniposide.
The preparation method comprises the following steps: weighing chlorogenic acid, aucubin and geniposide according to a prescription, mixing uniformly, and subpackaging into powder.
7. Composition seven
Prescription: 600g of chlorogenic acid, 300g of aucubin and 100g of geniposide.
The preparation method comprises the following steps: weighing chlorogenic acid, aucubin and geniposide according to a prescription, mixing uniformly, and subpackaging into powder.
EXAMPLE 2 preparation of injectable formulations of the compositions of the invention
1. Injection preparation one
Prescription: 400g of chlorogenic acid, 300g of aucubin, 300g of geniposide, 2667g of a support agent and 67g of an antioxidant.
The preparation method comprises the following steps: weighing chlorogenic acid, aucubin, geniposide, a support agent and an antioxidant according to a formula, dissolving in water for injection, filtering for sterilization, and freeze-drying to obtain the freeze-dried powder injection.
The above-mentioned support agent is mannitol, lactose or glucose; the antioxidant is sodium bisulfite, vitamin C, glutathione or folic acid.
2. Injection preparation two
Prescription: 400g of chlorogenic acid, 400g of aucubin, 200g of geniposide, 2667g of a support agent and 67g of an antioxidant.
The preparation method comprises the following steps: weighing chlorogenic acid, aucubin, geniposide, a support agent and an antioxidant according to a formula, dissolving in water for injection, filtering for sterilization, and freeze-drying to obtain the freeze-dried powder injection.
The above support agent is mannitol, lactose or glucose; the antioxidant is sodium bisulfite, vitamin C, glutathione or folic acid.
3. Injection preparation III
Prescription: 500g of chlorogenic acid, 250g of aucubin, 250g of geniposide, 2667g of a support agent and 67g of an antioxidant.
The preparation method comprises the following steps: weighing chlorogenic acid, aucubin, geniposide, a support agent and an antioxidant according to a formula, dissolving in water for injection, filtering for sterilization, and freeze-drying to obtain the freeze-dried powder injection.
The above support agent is mannitol, lactose or glucose; the antioxidant is sodium bisulfite, vitamin C, glutathione or folic acid.
4. Injection preparation four
Prescription: 500g of chlorogenic acid, 300g of aucubin, 200g of geniposide, 2667g of a support agent and 67g of an antioxidant.
The preparation method comprises the following steps: weighing chlorogenic acid, aucubin, geniposide, a support agent and an antioxidant according to a formula, dissolving in water for injection, filtering for sterilization, and freeze-drying to obtain the freeze-dried powder injection.
The above-mentioned support agent is mannitol, lactose or glucose; the antioxidant is sodium bisulfite, vitamin C, glutathione or folic acid.
5. Injection preparation five
Prescription: 500g of chlorogenic acid, 350g of aucubin, 150g of geniposide, 2667g of a support agent and 67g of an antioxidant.
Prescription: the preparation method comprises the following steps: weighing chlorogenic acid, aucubin, geniposide, a support agent and an antioxidant according to a formula, dissolving in water for injection, filtering for sterilization, and freeze-drying to obtain the freeze-dried powder injection.
The above support agent is mannitol, lactose or glucose; the antioxidant is sodium bisulfite, vitamin C, glutathione or folic acid.
6. Injection preparation six
Prescription: 600g of chlorogenic acid, 200g of aucubin, 200g of geniposide, 2667g of a support agent and 67g of an antioxidant.
The preparation method comprises the following steps: weighing chlorogenic acid, aucubin, geniposide, a support agent and an antioxidant according to a formula, dissolving in water for injection, filtering for sterilization, and freeze-drying to obtain the freeze-dried powder injection.
The above-mentioned support agent is mannitol, lactose or glucose; the antioxidant is sodium bisulfite, vitamin C, glutathione or folic acid.
7. Seven injection preparation
Prescription: 600g of chlorogenic acid, 300g of aucubin, 100g of geniposide, 2667g of a support agent and 67g of an antioxidant.
The preparation method comprises the following steps: weighing chlorogenic acid, aucubin, geniposide, a support agent and an antioxidant according to a formula, dissolving in water for injection, filtering for sterilization, and freeze-drying to obtain the freeze-dried powder injection.
The above-mentioned support agent is mannitol, lactose or glucose; the antioxidant is sodium bisulfite, vitamin C, glutathione or folic acid.
The beneficial effects of the present invention are demonstrated by the following experimental examples.
Experimental example 1 in vitro tests of the compositions of the invention against classical swine fever virus
1. Test materials
The tested drugs are:
test drug 1: chlorogenic acid;
test drug 2: aucubin;
test drug 3: geniposide;
test drug 4: chlorogenic acid: aucubin: a composition comprising geniposide at a weight ratio of 40:30: 30;
test drug 5: chlorogenic acid: aucubin: a composition comprising geniposide at a weight ratio of 50:25: 25;
test drug 6: chlorogenic acid: aucubin: a composition comprising geniposide at a weight ratio of 50:30: 20.
Preparing the tested medicines according to the weight proportion by the company;
hog cholera virus strain: CSFV Shimen strain purchased from the Chinese veterinary institute.
Cell: porcine kidney passaged cells (IBRS-2) were purchased from the Chinese typical Collection.
Cell nutrient solution: prepared with Dulbecco's minimum essential medium (MEM, GIBCO BRL) containing 10% calf serum, 100IU/ml penicillin, 100IU/ml streptomycin.
Cell maintenance solution: dulbecco's MEM containing 2% calf serum, the remainder being in nutrient solution.
2. Test protocol
2.1 culture of IBRS-2 cells
The frozen IBRS-2 cells are firstly revived by taking out the freezing tube from the liquid nitrogen tank and then quickly putting the freezing tube into a 38 ℃ water bath, shaking the freezing tube without time, completely thawing the freezing tube within 1min, and then taking out the cells under aseptic condition. Centrifuging at 1000rpm for 5-10 min, discarding supernatant, adding appropriate amount of cell nutrient solution, inoculating in culture flask with inoculation concentration of 1 × 10 6 Per mL, CO addition 2 Incubator 37 deg.C, 5% CO 2 And (5) standing for culture, replacing the culture solution once the next day, continuing to culture, and observing the growth condition. The culture can be passaged when the bottom of the culture bottle is uniformly full of a monolayer of IBRS-2 cells. After IBRS-2 cells grow into a uniform monolayer at the bottom of the culture flask, the cell maintenance solution is replaced to continue the culture, and the cell state is good within one week.
2.2CSFV Shimen Virus virulence assay
Gradually diluting CSFV Shimen virus strain suspension 10 times with cell maintenance solution to obtain 10 times -1 ～10 -7 A total of 8 dilutions were added to a 96-well plate inoculated with a monolayer of IBRS-2 cells, 4 wells at 200 μ l per well, 37 ℃, 5% CO per virus dilution 2 Incubate for 2h in the incubator, discard the virus solution, wash the plate twice with PBS solution, dry-control for the last time and add 200. mu.l maintenance solution, set up normal cell control group at the same time, observe Cytopathic effect (CPE) every day under the inverted microscope. Calculation of tissue culture half-infection TCID according to Reed-Muench method 50 (Tissue culture in fectous dose 50). 100TCID for antiviral experiment 50 The amount of virus (c) was used in cellular experiments.
2.3 determination of IBRS-2 cytotoxicity of the inventive drugs
Diluting each test drug with purified water to 10mg/ml initial concentration, and adding cell maintenance solution to make the test drug 2 -1 ～2 -9 Diluted in multiples and inoculated monolayers of IBRS-2 cells (1X 10 concentration) were added 5 Cell suspension 100. mu.1/ml) in a 96-well plate, while a normal cell control is set up. The cell culture plate was placed at 37 ℃ in 5% CO 2 The cells were cultured in an incubator for four days, and the growth of the cells was observed with an inverted microscope 2 times a day. Finally, the highest dilution concentration capable of maintaining normal cell growth is taken as the safe concentration of the drug on the cells. And calculating 50% toxicity concentration TD by Reed-Muench method 50 (Toxic dose 50) and maximum nontoxic concentration TD 0 (Toxic dose 0) in TD 0 As the initial concentration of drug in the cell experiments.
2.4 Effect of the inventive Agents on Swine fever Virus in vitro
Setting of drug concentration: all tested drug groups were tested from TD 0 Ten-fold dilution of each 3 concentrations was started. Each concentration was 4 wells, 50. mu.l per well. Setting a normal control group, a virus control group, adding maintenance liquid only in the normal control group, and adding 100TCID only in the virus control group 50 50. mu.l of the virus solution (4) per well. When the IBRS-2 cells in a 96-well cell culture plate basically grow into a monolayer, the experiment is divided into three groups according to three different modes of the effects of drugs, viruses and cells:
2.4.1 therapeutic Effect of drugs on viruses (therapeutic administration mode)
That is, the cell is infected with virus first and then the medicine is added to research the inhibition effect of the medicine on the virus replication in the cell.
Taking the cell culture plate with grown monolayer cells, pouring out the culture solution, and inoculating 100TCID to each well 50 50 μ l of the virus solution (4) was placed at 37 ℃ in 5% CO 2 Adsorbing lh infected cells in an incubator, discarding virus solution, washing the plate twice with PBS solution, and draining. Then, the drug with safe concentration and the maintenance solution are respectively added into 50 mul/hole of the liquor with corresponding dilution degree by 2 times, and 4 holes are added into each concentration. At 37 ℃ with 5% CO 2 Culturing in an incubator. The culture medium in the plate was discarded, the plate was washed twice with PBS solution, drained, and 100. mu.l of maintenance medium was added to each well to observe cytopathic effect. The cell growth condition was observed every 12h, and a virus control group and a normal control group were set. When the cells of the cell control group grow normally, the experimental result is recorded when the CPE of the virus control group reaches +++ the cells of the cell control group grow normally.
2.4.2 direct Effect of drugs with viruses (direct action mode of administration):
that is, the drug and the virus act for a period of time before infecting the cells, and the direct killing effect of the drug on the virus is researched.
Will 100TCID 50 50 μ l of the virus solution was mixed with the same amount of the drug of the present invention at different concentrations, and acted at room temperature for lh, and then inoculated into a cell culture plate full of monolayer cells, 4 wells per concentration, at 37 ℃ with 5% CO 2 And (4) culturing. The cell growth condition was observed every 12h, and a virus control group and a normal control group were set. When the cells of the cell control group grow normally, the experimental result is recorded when the CPE of the virus control group reaches +++ the cells of the cell control group grow normally.
2.4.3 prophylactic Effect of drugs on viruses (prophylactic administration)
That is, the cell is firstly acted with the drug for a period of time and then infected with the virus, and the blocking effect of the drug on the virus-adsorbed cell is researched.
The drug of the present invention was added to the cell culture plate, which had been grown to a monolayer of cells, at each concentration of 50. mu.l per well, at 37 ℃ and 5% CO 2 Culturing in medium. The culture medium in the plate was discarded,washing the plate twice with PBS, draining, diluting 50 μ l virus solution with 100TCID50 concentration with maintenance solution, adding into the inoculated cell culture plate, and incubating at 37 deg.C and 5% CO 2 Adsorbing infected cells for 1h in an incubator, discarding virus solution, washing the plate twice with PBS solution, and draining. The maintenance solution was added in an amount of 100. mu.l per well, and cytopathic effect was observed. Observing the cell growth condition every 12h, and setting a virus control group and a normal control group at the same time. When the cells of the cell control group grow normally, the experimental result is recorded when the CPE of the virus control group reaches +++ the cells of the cell control group grow normally.
2.5 Observation index
2.5.1 Observation Standard for cytopathic Effect (CPE)
Cytopathic effect: the cell morphology was observed by an inverted biomicroscope and divided into five stages.
"-": the cells are polygonal, are uniformly distributed and are tightly arranged;
"+": the cells contract and become round, and the number of diseased cells is less than about 25 percent of the total number of cells;
"++": the cells contract and become round, and have aggregation tendency, and the number of the pathological cells is 25 to 50 percent;
"+++": a large amount of cells are aggregated into a cluster, and the number of pathological cells is less than 50-75% of the total number;
"++++": the cells are aggregated in pieces, and the number of the pathological cells exceeds 75 percent of the total number of the cells.
2.5.2MTT method
The cultured cell plates were carefully discarded, washed 3 times with PBS, and the culture medium containing 0.5% MTT was added to each well at 37 ℃ with 5% CO 2 After further culturing for 4h in the incubator, the culture medium in the wells was carefully aspirated, 150. mu.l of DMSO was added to each well, and the mixture was shaken on a shaker at a low speed for 10min to dissolve the crystals sufficiently. Microplate reader for determining D of each well 492nm Values, analysis of values using SPSS 18.0, P<A difference of 0.05 is statistically significant. D 492nm The value indicates that the sample to be tested has the maximum absorbance value at the wavelength of 492nm, and the DMSO can dissolve formazan in cells, and the number of living cells can be indirectly reflected by the measured absorbance value.
2.6TCID 50 And TD 0 Test results
2.6.1 assay for CSFV Shimen Virus virulence assay
As the dilution of the virus increases, the amount of virus decreases, cytopathic effects caused by the virus gradually decrease, and the number of diseased wells decreases. Calculating half of infection amount TCID according to Reed-Muench method 50 TCID of CSFV Shimen Strain 50 ＝10 -5.5 /0.2ml。
2.6.2 toxicity test of the drug of the invention against IBRS-2 cells
The experimental results show that different drug concentrations have different effects on cells, and the higher the mass concentration is, the higher the toxicity on the cells is. When the mass concentration of the test drug 1>5mg/ml, mass concentration of test drug 2>2.5mg/ml, mass concentration of drug under test 3>2.5mg/ml, mass concentration of drug 4 tested>2.5mg/ml, mass concentration of drug 5 tested>5mg/ml, mass concentration of test drug 6>When the concentration is 5mg/ml, the cells can be flaked and shed after contacting with a monolayer of IBRS-2 cells for 2 hours; when the mass concentration of the components is reduced to: test drug 1:2 -4 X10 mg/ml, test drug 2: 2 -4 X10 mg/ml, test drug 3: 2 -4 X10 mg/ml, test drug 4: 2 -3 X10 mg/ml, test drug 5:2 -3 X10 mg/ml, test drug 6: 2 -3 At x10 mg/ml, no abnormal expression of IBRS-2 cell growth was observed, consistent with the growth of the control cells without drug. Therefore, the concentration can be determined as the safe concentration of each drug group to IBRS-2 cells, i.e., the maximum nontoxic concentration TD 0 。
Calculated TD for each test drug 50 And TD 0 (see Table 1). With a non-pathological drug concentration TD 0 As the initial drug concentration against the virus.
TABLE 1 toxic Effect of the drugs of the present invention on IBRS-2 cells
| Medicine | TD 50 (*10mg/ml) | TD 0 (*10mg/ml) |
| Test drug 1 | 2 -1.5 | 2 -3 |
| Test drug 2 | 2 -2.5 | 2 -4 |
| Test drug 3 | 2 -2.5 | 2 -4 |
| Test drug 4 | 2 -2.5 | 2 -4 |
| Test drug 5 | 2 -1.5 | 2 -3 |
| Test drug 6 | 2 -1.5 | 2 -3 |
3. The effect of the inventive drug on classical swine fever virus in vitro
3.1 the in vitro therapeutic action of the inventive drug on hog cholera
The scheme evaluates the viral load indexes of cytopathic effect (CPE) and D 492nm The value is obtained.
The normal cell control group has clear cell culture fluid, round or oval cells, orderly and tightly arranged cells, a lining shape and a clear cytoplasm (see figure 1). Following classical swine fever virus infection, the virus control group showed CPE for IBRS-2 cells as: cells appear vacuolated, cells morphologically swell, round, intercellular spaces increase, nuclei round and burst, and in severe cases, cells are partially or totally necrotized and exfoliated (see fig. 2). For dry drug prognosis, test drug 5 is at a concentration of 1.25mg/ml, and test drug 6 is at a concentration of 1.25X 10 -2 At a concentration range of 1.25mg/ml, the cell morphology is obviously deviated from normal, only part of cells have pathological characteristics, are in a shriveling shape, or are in a string shape or are overlapped or shed, most of cells have a normal epithelial shape, and the drug inhibits the replication and proliferation of the virus in the cells to a certain extent (see figure 3), and the tested drug groups at other concentrations have no significant difference compared with the virus control group.
Also, according to the MTT method (Table 2), the test drug 5 was at a concentration of 1.25mg/ml, and the test drug 6 was at 1.25X 10 -2 At the concentration range of 1.25mg/ml, the difference is significant compared with a virus control group (P)<0.05), which shows that the tested drugs 5 and 6 have the function of blocking virus from adsorbing cells under certain concentration conditions.
The experimental results show that 1.25mg/ml of the tested drug 5, 1.25X 10 -1 The tested medicine 6 of 1.25mg/ml can effectively inhibit the multiplication of the classical swine fever virus in cells and has obvious treatment effect on virus infection.
3.2 Effect of the inventive drug in vitro direct killing of classical swine fever virus
Test drug 6 at 1.25X 10 -1 1.25mg/ml, and at the same time, the tested drug 6 in this concentration range was significantly different (P) from the virus control group according to the MTT method (Table 2)<0.05), description 1.25X 10 -1 About 1.25mg/ml of test drug 6 the drug of the present invention can directly kill hog cholera virus and increase the number of living cells.
3.3 preventive action of the drug of the present invention on hog cholera
The monolayer of IBRS-2 cells at a concentration of 1.25mg/ml of the test drug 1, 1.25X 10 -1 Tested drug 5, 1.25X 10 under the concentration range of 1.25mg/ml -2 The test drug 6 adsorbed at a concentration range of about 1.25mg/ml to block or reduce CPE formation in cells caused by the virus, and at the same time, according to the MTT method (Table 2), the drug groups in this concentration range were significantly different from the virus control group (P)<0.05), indicating a test drug 1, 1.25X 10 at a concentration of 1.25mg/ml -1 Tested drug 5, 1.25X 10 under the concentration range of 1.25mg/ml -2 The tested drug 6 can effectively prevent virus infection in vitro under the concentration range of 1.25 mg/ml.
TABLE 2MTT assay D 492nm Value of
Note: p < 0.05 compared to virus control group.
4 conclusion
In the experiment, whether the medicine has the effect of inhibiting the proliferation of the virus in the cell is observed in a mode of firstly infecting the virus and then adding the medicine; observing whether the medicine has the effect of directly contacting and inhibiting the virus or not in a mode of infecting the virus and adding the medicine; whether the medicament can prevent virus infection or not is observed in a mode of adding the medicament first and then infecting the virus. The results of the experiment showed that 1.25mg/ml of the test drug 5, 1.25X 10 against the CSFV Shimen strain -1 The tested drug 6 of 1.25mg/ml can inhibit the multiplication of the classical swine fever virus in cells and has obvious treatment effect on virus infection; 1.25X 10 -1 The tested medicine 6 of 1.25mg/ml can directly kill the classical swine fever virus and increase the number of living cells; test drug 1, 1.25X 10 at a concentration of 1.25mg/ml -1 Tested drug 5, 1.25X 10 under the concentration range of 1.25mg/ml -2 The test drug 6 had the effect of preventing viral infection at a concentration range of about 1.25 mg/ml.
Therefore, the pharmaceutical composition consisting of the chlorogenic acid, the aucubin and the geniposide in a specific weight ratio, in particular to the pharmaceutical composition with the weight ratio of the chlorogenic acid, the aucubin and the geniposide being 50:30:20, can effectively block the adsorption and penetration of the classical swine fever virus, effectively prevent the classical swine fever virus infection, inhibit the replication of the classical swine fever virus, kill the classical swine fever virus and treat the classical swine fever virus infection.
Experimental example 2 in vivo treatment of porcine epidemic diarrhea test with the composition of the present invention
1. Test materials
1.1 test drugs
Test drug 1: chlorogenic acid;
test drug 2: aucubin;
test drug 3: geniposide;
test drug 4: the preparation method comprises the following steps of: aucubin: geniposide: mannitol: the weight ratio of sodium bisulfite is 40:30: 30: 26.7: 0.7 is an injection preparation prepared by the prescription;
test drug 5: the method comprises the following steps of: aucubin: geniposide: mannitol: the weight ratio of sodium bisulfite is 50:25: 25: 26.7: 0.7 is an injection preparation prepared by the prescription;
test drug 6: the method comprises the following steps of: aucubin: geniposide: mannitol: the weight ratio of sodium bisulfite is 50:30: 20: 26.7: 0.7 is an injection preparation prepared by the prescription;
test drug 7: the preparation method comprises the following steps of: aucubin: 50 parts of geniposide acid by weight: 30:20 is oral preparation prepared by prescription;
positive drug: gentamicin.
The preparation method of the injection preparation comprises the following steps: weighing chlorogenic acid, aucubin, geniposide, mannitol and sodium bisulfite according to the proportion of the prescription, dissolving in water for injection, filtering, sterilizing, and freeze drying to obtain the freeze-dried powder injection with the labeled amount of 30 mg/powder.
The preparation method of the oral preparation comprises the following steps: weighing chlorogenic acid, aucubin and geniposide according to the prescription, mixing well, and packaging into powder.
Before use, each tested drug and positive drug are diluted with normal saline and dissolved to the required concentration.
1.2 animals
Selecting 65 diarrhea piglets with similar body weights in 12-24 days old like a pine pig farm from the Mongolia market in Hunan province.
2. Test protocol
2.1 test grouping
According to the administration mode and the dosage, the diarrhea piglets are divided into 13 groups according to the principle of similar body weight, and the group weight is respectively as follows:
(1) negative control group: intramuscular injection of physiological saline 2 times daily for 7 days;
(2) test drug 1 group: intramuscular injection of the test drug 1, 3 mg/kg/time, 2 times daily for 7 days;
(3) test drug 2 group: intramuscular injection of 2, 3 mg/kg/time of the test drug 2 times a day for 7 days;
(4) test drug 3 groups: intramuscular injection of the test drug 3, 3 mg/kg/time, 2 times daily, for 7 days;
(5) test drug 4 low dose group: intramuscular injection of 4, 3 mg/kg/time of the test drug 2 times a day for 7 days;
(6) test drug 4 high dose group: intramuscular injection of 4, 6 mg/kg/time of the test drug 2 times a day for 7 days;
(7) test drug 5 low dose group: intramuscular injection of 5, 3 mg/kg/time of the test drug 2 times a day for 7 days;
(8) test drug 5 high dose group: intramuscular injection of 5, 6 mg/kg/time of the test drug 2 times a day for 7 days;
(9) test drug 6 low dose group: intramuscular injection of the test drug 6, 3 mg/kg/time, 2 times daily for 7 days;
(10) test drug 6 high dose group: intramuscular injection of 6, 6 mg/kg/time of the test drug 2 times a day for 7 days;
(11) test drug 7 low dose group: the medicine to be tested is injected orally for 7 times of 3 mg/kg/time, 3 times per day and is continuously administrated for 7 days;
(12) test drug 7 high dose group: the medicine to be tested is orally injected for 7, 6 mg/kg/time, 3 times a day and is continuously administrated for 7 days;
(13) positive drug group: gentamicin was administered intramuscularly at 3 mg/kg/dose 2 times daily for 7 days.
2.2 evaluation index
All piglets were observed daily for body temperature, mental, appetite, drinking, bowel movement, exercise, relapse of diarrhea, toxic side effects and death, and piglet quality was measured and recorded.
3. Test results
The change of the piglet weight in each test group is shown in table 3, and the treatment effect of each test group on the porcine epidemic diarrhea is shown in table 4.
TABLE 3 weight change of piglets for each drug group
Note: p < 0.01 compared to negative group; compared with the positive drug group, the delta p is less than 0.01.
TABLE 4 therapeutic effect of each drug group on porcine epidemic diarrhea
As can be seen from Table 3, chlorogenic acid, aucubin and geniposide can be used alone, and the injection and oral preparation of the composition in each proportion can effectively reduce the weight loss of diarrhea pigs, and have significant difference with a negative control group; compared with the positive control group, the low and high doses of the test drug 6 and the high dose of the test drug 7 are significantly different, and the body weight of the sick pig gradually increases.
As can be seen from Table 4, when the chlorogenic acid, aucubin and geniposide are used alone, the treatment effect on the porcine epidemic diarrhea is not good, but the composition of the chlorogenic acid, aucubin and geniposide can effectively treat the porcine epidemic diarrhea, wherein the injection preparation of the tested drug 6 has the most remarkable effect, namely when the weight ratio of the chlorogenic acid, aucubin and geniposide is 50:30:20, the injection preparation of the composition can remarkably improve the treatment effect on the porcine epidemic diarrhea.
4. Conclusion
The above experiments show that the composition of chlorogenic acid, aucubin and geniposide can be used for preventing and treating porcine epidemic diarrhea, wherein the chlorogenic acid: aucubin: the medicinal composition of geniposide 50:30:20 has the best effect.
Experimental example 3 in vitro bacteriostatic (porcine Escherichia coli) test for the composition of the invention
The main pathogenic bacteria of the swine edema disease is swine escherichia coli, so the swine escherichia coli is selected for the in vitro test of the scheme.
1. Test materials
1.1 test drugs
Test drug 1: chlorogenic acid;
test drug 2: aucubin;
test drug 3: geniposide;
test drug 4: chlorogenic acid: aucubin: geniposide composition at a weight ratio of 40:30: 30;
test drug 5: chlorogenic acid: aucubin: geniposide composition at a weight ratio of 50:25: 25;
test drug 6: chlorogenic acid: aucubin: a composition of geniposide acid at a weight ratio of 50:30: 20;
positive drug: enrofloxacin, available from bayer (sichuan) animal health, llc.
Preparing the tested medicines according to the proportion.
1.2 strains
Porcine Escherichia coli virulent strain K88ac, purchased from veterinary microorganism collection of Chinese veterinary drug laboratory.
1.3 culture Medium
Fresh blood agar medium: purchased from zhengzhou bossai biotechnology limited;
LB broth culture medium: purchased from biotechnology responsibility of ozoocin, beijing, ltd.
2. Test protocol
2.1 cultivation of the Strain
The preserved strain is inoculated in a fresh blood agar culture medium in advance, cultured for 18h at 37 ℃, taken out and inoculated in an LB broth culture medium, and cultured for 14h at 37 ℃. Taking out and measuring the bacterial liquid concentration by using a turbidimetric method to be about 9 multiplied by 10 8 CFU/ml, diluting the bacterial liquid with physiological saline at a ratio of 1:10 to 9 × 10 5 CFU/m1, as bacteriostatic test concentration.
2.2 grouping of test drugs
The test drugs are divided into 8 groups according to the concentration and the type of the test drugs, and the groups comprise: a negative control group, 1-6 groups of tested medicines and a positive medicine group; wherein the negative drug group is normal saline, and each group of drugs is dissolved by ultrapure water to make the final concentration 20 mg/ml.
2.3 in vitro inhibition of bacteria
2.3.1 cup and dish method
Taking two flat-bottom dishes with the diameter of 90mm and the height of 16-17 mm, sterilizing at high pressure, respectively injecting 20ml of melted 2% agar culture medium under aseptic operation, uniformly spreading the bottoms of the dishes, placing a horizontal table, adding 4-5 ml of cultured strains after solidification, then placing 3 Oxford cups with the inner diameter of 6.0 +/-0.1 mm, the height of 10.0 +/-0.1 mm and the outer diameter of 7.8 +/-0.1 mm into each flat-bottom double-dish, respectively dropping each test solution into each group of Oxford cups, culturing for 24 hours in a 37 ℃ water-proof electric heating thermostat based on the fact that the cups are full (can not overflow out of the cups), and measuring the diameter of a bacteriostatic ring by using a vernier caliper.
2.3.2 criterion for determining bacteriostatic intensity
According to the judgment action standard of 'inspection of antibiotic medicine' and 'empirical record for traditional Chinese medicine preparation'. Namely the diameter of the bacteriostatic ring is less than or equal to 8.00mm, and the bacteriostatic ring is insensitive to the first step; the diameter of the antibacterial ring is 8.10-13.00 mm, and the low sensitivity (+); the diameter of the antibacterial ring is 13.10-19.00 mm, and the antibacterial ring is moderate sensitive (++); the diameter of the antibacterial ring is 19.10-25.00 mm, and the antibacterial ring is highly sensitive (+++).
2.3.3 two-fold dilution method for determining bacteriostatic potency of drug
11 tubes of 13X100mm sterilized cotton plugs were used in each group and numbered on a tube rack, and 2.0ml of sterile LB broth was added to each tube under aseptic conditions, and then the sterilized test tubes were added to branch 12.0ml of liquid medicine is evenly mixed, 2.0ml of the liquid medicine is taken out and put into the 2 nd tube, and the rest is done in the same way until the 9 th tube is taken out and 2.0ml is discarded, so that the ratio of 1: 2. 1: 4. 1: 8. 1: 16. 1: 32 … …, etc. No drug plus bacteria was added as a control in the 10 th tube to see if the medium was suitable for bacterial growth. The 11 th tube is added with the tested medicine, mixed evenly, taken out for 2.0ml and discarded, and the tested bacteria are not added, so as to observe whether the tested medicine is polluted or not. Then diluting the test bacterium liquid to 10 with sterile broth -3 And (3) adding 0.1m of the mixture into the tubes l to 10 respectively, uniformly mixing, and culturing in a 37 ℃ water-proof electric heating thermostat for 16 to 24 hours to observe the result. Turbid broth indicates bacterial growth (++, +++); if the broth is completely clear, no bacteria are growing (one).
3. Test results
3.1 cup and dish method
The results of the bacteriostatic experiments of the tested drugs on the escherichia coli of the pigs are shown in table 5, the negative control group has no bacteriostatic effect, the chlorogenic acid, aucubin and geniposide are separately used and are sensitive to low degrees of bacteria, and the composition of the chlorogenic acid, aucubin and geniposide has a remarkable bacteriostatic effect on the bacteria; in the composition, the tested 5 groups have the same effect as the positive groups, and the tested 6 groups have the better effect than the positive groups, so the bacteriostatic effect is the best.
TABLE 5 bacteriostatic test results of tested drugs on porcine escherichia coli
Therefore, the pharmaceutical composition of chlorogenic acid, aucubin and geniposide has a strong inhibiting effect on porcine escherichia coli, wherein the chlorogenic acid: aucubin: geniposide is 50:30:20 with the best effect.
3.2 test for determining drug bacteriostatic potency by double dilution method in test tube
The result of the test drug on the antibacterial potency of the escherichia coli of the pigs is shown in table 6, the negative control group has no antibacterial effect, the titers of the chlorogenic acid, the aucubin and the genipin in single use are all 1:2, the titer of the composition with the ratio of 40:30:30 is 1:8, the titer of the composition with the ratio of 50:25:25 is equivalent to the titer of the enrofloxacin and is 1:16, the titer of the composition with the ratio of 50:30:20 is 1:64, and the antibacterial effect is strongest.
TABLE 6 results of the determination of the bacteriostatic potency of each test drug against Escherichia coli in swine
Therefore, the pharmaceutical composition of chlorogenic acid, aucubin and geniposide has a remarkably enhanced inhibition effect on porcine escherichia coli, and a synergistic effect is achieved, wherein the chlorogenic acid: aucubin: genipin-50: 30:20 is the most effective.
4. Conclusion
When the chlorogenic acid, the aucubin and the geniposide are used independently, the effect of inhibiting the pig escherichia coli is weak, but the bacteriostatic action of the composition of the chlorogenic acid, the aucubin and the geniposide is obviously enhanced, and the synergistic effect is obtained, wherein the chlorogenic acid: aucubin: the genipin acid has the strongest bacteriostatic action of 50:30:20, and provides a new choice for treating and preventing the swine edema disease.
In conclusion, the invention provides a composition consisting of chlorogenic acid, aucubin and geniposide in a specific ratio and application thereof in preparing a medicament for preventing and/or treating swine diseases. The experimental result shows that the composition can block the absorption and penetration of the classical swine fever virus, effectively prevent the infection of the classical swine fever virus, inhibit the replication of the classical swine fever virus, kill the classical swine fever virus and achieve the effect of treating the classical swine fever; the composition is also effective in treating porcine epidemic diarrhea; the composition can also effectively inhibit porcine escherichia coli, achieves a synergistic effect of the inhibition effect, and provides a new choice for treating and preventing the swine edema disease. Meanwhile, the composition also effectively solves the problems of large side effect and serious residue existing in the traditional antiviral drugs and antibiotic treatment. Therefore, the composition has good application prospect in preparing medicaments for resisting swine fever virus and inhibiting swine escherichia coli and medicaments for preventing and/or treating swine diseases such as swine fever, porcine epidemic diarrhea, swine edema disease and the like.
Claims (8)
1. The application of a composition in preparing a medicament for resisting swine fever virus; the composition is prepared from the following raw materials in parts by weight: 1.0-6.0 parts of chlorogenic acid, 1.0-3.0 parts of aucubin and 0.5-1.5 parts of geniposide.
2. Use according to claim 1, characterized in that: the composition is prepared from the following raw materials in parts by weight: 1.3-2.5 parts of chlorogenic acid, 1.0-1.5 parts of aucubin and 1.0 part of geniposide.
3. Use according to claim 2, characterized in that: the composition is prepared from the following raw materials in parts by weight: 2.0-2.5 parts of chlorogenic acid, 1.0-1.5 parts of aucubin and 1.0 part of geniposide.
4. Use according to claim 3, characterized in that: the composition is prepared from the following raw materials in parts by weight: chlorogenic acid 2.5 parts, aucubin 1.5 parts, and geniposide 1.0 part.
5. Use according to any one of claims 1 to 4, characterized in that: the medicament can block the absorption and penetration of the classical swine fever virus to cells, and/or inhibit the proliferation of the classical swine fever virus, and/or kill the classical swine fever virus.
6. Use according to any one of claims 1 to 4, characterized in that: the medicine is a preparation prepared by taking the composition as an active ingredient and adding auxiliary materials acceptable in the field of pharmacy.
7. Use according to claim 6, characterized in that: the preparation is an injection or a solid preparation.
8. Use according to claim 7, characterized in that: the preparation is an injection, and the auxiliary material comprises one or two of a bracket agent and an antioxidant.
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