阅读说明：本技术 皂苷在制备抗牛支原体产品中的应用 (Application of saponin in preparation of anti-mycoplasma bovis product ) 是由 张亮 朱曼玲 张云飞 杨宏军 陈九 杨美 于 2021-09-01 设计创作，主要内容包括：本发明涉及皂苷在制备抗牛支原体产品中的应用。牛支原体能够引发牛肺炎、关节炎及牛乳腺炎等多种疾病,本领域可用于防治牛支原体疾病的药物十分有限,且晚期治疗效果并不理想,主要通过加强饲养管理的方式进行防控。本发明提供了茶皂素、薯蓣皂苷及七叶皂苷钠体外抑制牛支原体的活性,现有技术针对皂苷类化合物对支原体抑制的相关研究较为空白,上述化合物不仅对牛支原体具有良好的抑制作用,同时对正常细胞的毒性较小,具有开发成为抗牛支原体药物的前景。(The invention relates to application of saponin in preparation of an anti-mycoplasma bovis product. Mycoplasma bovis can cause various diseases such as bovine pneumonia, arthritis, bovine mastitis and the like, the medicines which can be used for preventing and treating the mycoplasma bovis disease in the field are very limited, the treatment effect in the late stage is not ideal, and the prevention and the control are mainly carried out in a mode of strengthening feeding management. The invention provides the activity of tea saponin, dioscin and aescine sodium for inhibiting mycoplasma bovis in vitro, the related research on the mycoplasma inhibition by saponin compounds in the prior art is blank, and the compounds not only have good inhibition effect on mycoplasma bovis, but also have low toxicity on normal cells, and have the prospect of being developed into anti-mycoplasma bovis medicines.)

1. Application of saponin in preparing anti-mycoplasma bovis product is provided.

2. Use of a saponin according to claim 1 in the preparation of an anti-mycoplasma bovis product, wherein the saponin comprises spirostanol and triterpene saponins;

further, the triterpenoid saponin includes but is not limited to one of glycyrrhizin, ginsenoside, notoginsenoside, tea saponin and aescin; the spirostane saponin is dioscin.

3. The use of a saponin of claim 1 in the preparation of an anti-mycoplasma bovis product, wherein the saponin is tea saponin.

4. The use of saponin according to claim 3 in the preparation of an anti-mycoplasma bovis product, wherein said tea saponin is a mixture wherein the structure of the component that predominantly exerts the pharmacological activity of tea saponin is represented by formula I:

5. use of a saponin according to claim 1 in the preparation of an anti-mycoplasma bovis product, wherein the saponin is aescin; preferably, the aescin is sodium aescin, and the structure of the aescin is shown as a formula II:

6. use of a saponin according to claim 1 in the preparation of an anti-mycoplasma bovis product, wherein the saponin is dioscin; the chemical structure is shown as the following formula III:

7. use of a saponin according to claim 1 in the preparation of an anti-mycoplasma bovis product, wherein the anti-mycoplasma bovis product is one of but not limited to veterinary medicine, feed or biochemical reagents;

preferably, the veterinary drug comprises a serum product, a vaccine, a diagnostic product, a microecological product, a Chinese medicinal material, a Chinese patent drug, a chemical, an antibiotic, a biochemical, a radiopharmaceutical, a topical insecticide or a disinfectant;

preferably, the feed comprises complete feed, concentrated feed, premix feed, concentrated feed or mixed feed;

preferably, the biochemical reagent comprises a reagent for diagnosis and biochemical research, and the specific application form comprises a diagnostic reagent and a screening reagent for resisting mycoplasma bovis active ingredients.

8. The pharmaceutical composition is characterized by at least comprising one or a combination of more of tea saponin, sodium aescinate or dioscin;

preferably, the pharmaceutical dosage of the tea saponin, the sodium aescinate or the dioscin is determined by a conventional mode in the field, and further determined by means of half effective concentration of the components and animal model experiment results;

preferably, the pharmaceutical composition further comprises a pharmaceutically necessary carrier, and the carrier comprises an excipient and/or a diluent; further, it comprises pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and non-ionic surfactants or lipids, and also pharmaceutically harmless salts, vitamins, tocopherols or antioxidants, stabilizers and/or preservatives for prolonging the use and shelf life of pharmaceutically active ingredients or formulations, or other common non-pharmaceutically active ingredients or adjuvants and additives known in the art and mixtures thereof.

9. A medicament for the control of mycoplasma bovis disease, comprising the composition of claim 8;

preferably, the medicament is in the form of an oral preparation, an external medicament or an injection; further, the oral preparation comprises an oral solid preparation or an oral liquid preparation;

specifically, the oral solid preparation comprises but is not limited to one of powder, soluble powder, premix, tablet, granule, capsule, pill, paste and paste;

specifically, the oral liquid preparation comprises but is not limited to one of water agent, tincture, decoction and mixture;

specifically, the external medicine includes but is not limited to aerosol, drench or drop spray;

further, the injection includes, but is not limited to, powder injection, infusion solution, suspension, emulsion, hydro-acupuncture or oil-acupuncture, and the injection form includes, but is not limited to, intramuscular, subcutaneous, intradermal or intravenous injection.

10. A method for controlling a disease associated with mycoplasma bovis, comprising administering to a subject in need thereof the pharmaceutical composition of claim 8 or the medicament of claim 9;

preferably, the subject in need thereof comprises a mammal, preferably a mouse, rat or other rodent, rabbit, dog, cat, pig, cow, sheep, horse or primate; further preferably bovine;

preferably, the administration mode includes, but is not limited to, adding the pharmaceutical composition or the drug to the drinking water of the cattle for administration, and further includes killing the breeding environment of the cattle by using the pharmaceutical composition or the drug.

Technical Field

The invention belongs to the technical field of anti-mycoplasma bovis medicines, and particularly relates to an application of saponin compounds in preparation of anti-mycoplasma bovis products.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Mycoplasma bovis (Mycoplasma bovis) is one of the important pathogens causing many symptoms such as pneumonia and arthritis in calves and mastitis in adults. Since 1961, Hale et al isolated Mycoplasma bovis from the milk of the United states for the first time, Mycoplasma bovis was isolated in succession around the world. In 2008, the mycoplasma bovis pneumonia epidemic situation is firstly discovered in beef cattle in China, and then the disease is discovered in transferred calves in areas such as Hubei, Guizhou, Ningxia, inner Mongolia, Guangxi, Chongqing and the like successively, the morbidity is 50% -100%, the fatality rate is as high as 10% -50%, and huge economic loss is caused to cattle raising industry in China.

Mycoplasma bovis is a minimal microorganism with cell tuberculosis, which is intermediate between bacteria and viruses, but has no cell wall, and belongs to prokaryotes. Mycoplasma is widely distributed in sewage, soil, animals and humans. The mycoplasma bovis has strong resistance to the outside, can survive for about 230 days in the fertilizer, can survive for about 22 days in water, and can survive for 8 months in sand. At present, no effective mycoplasma bovis vaccine exists at home and abroad, and the prevention and control situation is severe. In contrast to bacteria, mycoplasma bovis has no cell wall structure, which makes most of the commonly used broad-spectrum antibiotics ineffective against them, and only a few antibiotics are available for the treatment of clinically infected cattle. Once drug resistance is generated, a pasture is faced with no drug, and epidemic diseases can be controlled only by adopting a mode of eliminating cattle, so that great economic loss is caused. Aiming at the current situation, the development and screening of the compound with the activity of inhibiting the mycoplasma bovis are expected to realize the prevention and treatment of the mycoplasma bovis and reduce the economic loss of the cattle industry.

Tea saponin (Tea saponin) is a saccharide-type compound extracted from seeds of Theaceae plants, belongs to saponin class, and is a natural nonionic surfactant. The tea saponin has good emulsifying, dispersing, foaming, moistening, antiinflammatory, analgesic, and anti-osmosis effects. The tea saponin product is faint yellow fine powder and is widely applied to the production of washing, wool spinning, knitting, medicine, daily chemical industry and the like. The compound can be used as wetting agent and suspending agent in solid pesticide, as synergist and spreader in emulsifiable pesticide, or as biological pesticide. However, the effect of the drug on other uses, particularly on Mycoplasma bovis, has not been studied.

Sodium aescinate (Escin) with molecular formula of C₅₅H₈₃NaO₂₃It is a white crystalline powder, bitter and spicy in taste, and has hygroscopicity. Has antiinflammatory, exudation resisting, venous tension increasing, blood circulation improving, and brain function disorder correcting effects. Has obvious protective effect on cerebral edema caused by carbon monoxide and the like. Can be used for treating swelling caused by cerebral edema, wound or operation, and venous reflux disorder diseases. However, the effect of the drug on other uses, particularly on Mycoplasma bovis, has not been studied.

Dioscin (Dioscin) is the major component in dioscoreaceae. The traditional Chinese medicine holds that the yam has the effects of eliminating phlegm, promoting digestion and diuresis, relaxing muscles and tendons, activating blood circulation, preventing malaria and the like. Modern pharmacological studies show that dioscin has a plurality of pharmacological effects, and particularly, the research on the anti-tumor effect is more. Many studies have also shown that dioscin has activity in ameliorating atherosclerotic symptoms and protecting vascular endothelial function, reducing ischemia/reperfusion injury of heart, brain and kidney, lowering blood glucose, inhibiting liver fibrosis, improving climacteric osteoporosis, alleviating rheumatoid arthritis and ulcerative colitis, and antagonizing pathogenic bacteria and mycoplasma bovis. So far, no report about the application of the compound in preventing and treating mycoplasma bovis exists.

Disclosure of Invention

Based on the above-mentioned research background, the present invention aims to screen compounds having inhibitory activity against mycoplasma bovis, thereby providing effective drugs for the treatment of mycoplasma bovis diseases. In order to realize the technical purpose, the invention conjectures active compounds screened from natural medicine extracts, and verifies that a plurality of saponin components such as tea saponin, sodium aescinate, dioscin and the like can effectively inhibit the in vitro proliferation of mycoplasma bovis for the first time, and the active components have small harm degree to organisms and have the prospect of being developed into the anti-mycoplasma bovis medicine.

Aiming at the technical effects, the invention mainly provides the application of the saponin in preparing the anti-mycoplasma bovis product.

Specifically, the invention specifically verifies the application of the tea saponin, the sodium aescinate and the dioscin in the preparation of the mycoplasma bovis resistant product.

In the prior art, tea saponin is usually added into biological pesticides as a surfactant, and in the aspect of medicinal value, the tea saponin is mainly used for improving capillary permeability and can also be used for improving the conditions of blood sugar and blood pressure. The invention provides application of a compound tea saponin in inhibiting proliferation of mycoplasma bovis, and experiments prove that the tea saponin has half toxicity concentration (CC) to MDBK cells₅₀) 937.5 μ M, and half the Effective Concentration (EC) against M.bovis₅₀) 97.66. mu.M; the therapeutic index of tea saponin to mycoplasma bovis is 9.60.

Sodium aescinate is a clinically common medicament for treating cerebrovascular diseases, and is used for expanding cerebral vessels, improving venous tension, accelerating venous return and the like. The invention proves that the compound sodium aescinate can effectively inhibit the proliferation of mycoplasma bovis for the first time, and experiments prove that the sodium aescinate has half toxic concentration (CC) to MDBK cells₅₀) 703.13 μ M, and half the Effective Concentration (EC) against M.bovis₅₀) 24.41 μ M; the therapeutic index of sodium aescinate on mycoplasma bovis is 28.80. The result shows that the sodium aescinate has the prospect of being developed into the anti-mycoplasma bovis medicine, and provides a new medical application for the sodium aescinate.

Dioscin is an important basic raw material for producing steroid hormone medicines. The steroid hormone has strong pharmacological actions of resisting infection, allergy, virus and shock, and is an important medicine for treating rheumatism, cardiovascular diseases, lympholeukemia, cellular encephalitis, skin diseases, tumors and critical patients. In addition, the pharmacological activities of dioscin are numerous, but the research on the antitumor activity is mainly focused on. The invention provides the activity of dioscin in mycoplasma inhibition for the first time, provides the application of dioscin in the field of veterinary drugs, and verifies that the dioscin has half toxicity concentration (CC) to MDBK cells₅₀) 31.25. mu.M, and a half-Effective Concentration (EC) against M.bovis₅₀) 3.25. mu.M; the therapeutic index of dioscin to mycoplasma bovis is 9.62.

The invention verifies the inhibition effect of the dioscin on mycoplasma bovis and has low toxicity on cells, and the result opens up a new medicinal application for the dioscin.

The beneficial effects of one or more technical schemes are as follows:

the invention provides the activity of tea saponin, sodium aescinate and dioscin for inhibiting the proliferation of mycoplasma bovis in vitro for the first time, and based on the research result, the invention further expands the medical application of the compounds. In addition, the control means aiming at the mycoplasma bovis in the field is very limited, and the research result of the invention can provide certain technical guidance for the development of the active ingredients for resisting the mycoplasma bovis; the compound has small toxicity to normal cells, and has important significance in developing anti-mycoplasma bovis medicines.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a graph showing the effect of tea saponin against M.bovis injured cells in example 1;

wherein, the left part of the figure 1 is an MDBK normal cell group; FIG. 1 shows a control group of MDBK-infected Mycoplasma bovis; FIG. 1 shows the right panel of the drug test group for infected cells (using 200. mu.M tea saponin);

FIG. 2 is the half Cytotoxic Concentration (CC) of theasaponin against MDBK cells in example 1₅₀) A drawing;

FIG. 3 is a graph showing the half-maximal Effective Concentration (EC) of tea saponin against M.bovis in example 1₅₀) A drawing;

FIG. 4 is a graph showing the effect of sodium aescinate on M.bovis-damaged cells in example 2;

wherein: wherein the left panel of FIG. 4 is a MDBK normal cell group; FIG. 4 is a control group of MDBK-infected Mycoplasma bovis; FIG. 4 right panel shows drug test group of infected cells (administered 50 μ M sodium aescinate);

FIG. 5 is the half-Cytotoxic Concentration (CC) of sodium aescinate on MDBK cells in example 2₅₀) A drawing;

FIG. 6 is the half-Effective Concentration (EC) of sodium aescinate against M.bovis in example 2₅₀) A drawing;

FIG. 7 is a graph showing the effect of dioscin against damaged cells of Mycoplasma bovis in example 3;

wherein, the left part of the FIG. 7 is MDBK normal cell group; FIG. 7 shows a control group of MDBK-infected Mycoplasma bovis; FIG. 7 right is the drug test group of infected cells (40 μ M dioscin administration);

FIG. 8 is the half Cytotoxic Concentration (CC) of dioscin against MDBK cells in example 3₅₀) A drawing;

FIG. 9 shows the half-Effective Concentration (EC) of dioscin against Mycoplasma bovis in example 3₅₀) Figure (a).

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

Interpretation of terms:

"prevention and/or treatment": means any measure suitable for the treatment of mycoplasma bovis-related diseases, or the prophylactic treatment of such manifested diseases or manifested symptoms, or the avoidance of recurrence of such diseases, e.g. recurrence after the end of a treatment period or treatment of symptoms of already-onset diseases.

"anti-mycoplasma bovis medicine" means a substance which has a significant inhibitory effect on mycoplasma bovis, which acts directly on the early stage of mycoplasma bovis, and whose direct killing effect on mycoplasma bovis is better than the adsorption blocking effect and the replication blocking effect.

As described in the background art, the mycoplasma bovis is an important factor threatening the cattle breeding industry, the medicines which can be used for the mycoplasma bovis in the field are quite limited at present, and in order to solve the technical problems, the invention provides the application of the saponin compound in preparing the anti-mycoplasma bovis product.

In a first aspect of the invention, the application of saponin in the preparation of an anti-mycoplasma bovis product is provided.

The saponins of the first aspect are classified by the structure of sapogenins, including spirostanol and triterpene saponins.

In a preferred embodiment of the present invention, the triterpenoid saponin includes, but is not limited to, one of glycyrrhizin, ginsenoside, notoginsenoside, tea saponin and aescin; specific examples of the spirostane saponin include dioscin.

In one embodiment of the invention, the application of tea saponin in preparing a mycoplasma bovis resistant product is provided.

The tea saponin comprises tea saponin and tea seed saponin (CAS No.: 8047-15-2), and it should be clear that the tea saponin called in the field is obtained by extracting tea, tea seeds, soap bark and the like as raw materials in a water extraction and alcohol precipitation mode, and the like, and is a mixture, wherein the component structure mainly exerting the pharmacological activity of the tea saponin is shown as the following formula I:

it should be noted that the tea saponin is not limited to the pure compounds represented by the above structures, and the tea saponin (with the purity of 10-70%) commercially available can achieve the effect of the invention, and can be used for development of related mycoplasma bovis resistant products.

In another embodiment of the invention, the application of the aescin in preparing the anti-mycoplasma bovis product is provided.

Aescin is the main active component in buckeye seed extract, and because aescin is insoluble in water, the sodium salt form of aescin, namely aescin sodium (CAS No.: 6805-41-0), is commonly used in the field, and the structure is shown in the following formula II. Therefore, in a more specific embodiment of the invention, the application of sodium aescinate in preparing the anti-mycoplasma bovis product is also provided.

In another embodiment of the invention, the application of dioscin in preparation of mycoplasma bovis resistant products is provided.

Dioscin is the main component of Dioscoreaceae plant, and its chemical structural formula is shown in formula III below.

In the above embodiments, the tea saponin, aescin and dioscin further comprise pharmaceutically acceptable salts thereof, wherein the pharmaceutically acceptable salts comprise salts of tea saponin, aescin or dioscin with inorganic acids or organic acids; in specific examples, the inorganic acid is hydrochloric acid, sulfuric acid, nitric acid, or hydrobromic acid; the organic acid is methanesulfonic acid, toluenesulfonic acid or trifluoroacetic acid.

It will also be appreciated that the anti-mycoplasma bovis product of the first aspect, for use in the prevention and/or treatment of disease associated with mycoplasma bovis, comprises, but is not limited to, one of veterinary medicine, feed or biochemical.

Further, the veterinary drug comprises a serum product, a vaccine, a diagnostic product, a microecological product, a Chinese medicinal material, a Chinese patent drug, a chemical drug, an antibiotic, a biochemical drug, a radiopharmaceutical, an external insecticide or a disinfectant.

Further, the feed comprises complete feed, concentrated feed, premix feed, concentrated feed or mixed feed.

Furthermore, the biochemical reagent comprises a reagent for diagnosis and biochemical research, and the specific application form comprises a diagnostic reagent, a screening reagent for resisting mycoplasma bovis active ingredients and the like.

In a second aspect of the present invention, a pharmaceutical composition is provided, wherein the pharmaceutical composition at least comprises one or a combination of several of tea saponin, sodium aescinate or dioscin.

In the pharmaceutical composition of the first aspect, the pharmaceutical dosage of the tea saponin, sodium aescinate or dioscin can be determined by the conventional methods in the art, and the conventional methods can be determined by the half effective concentration of the above components and the experimental results of animal models.

Preferably, the pharmaceutical composition further comprises a pharmaceutically necessary carrier, and the carrier comprises an excipient and/or a diluent. Such as pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and non-ionic surfactants or lipids, and also pharmaceutically harmless salts (e.g. sodium chloride, flavourings), vitamins (e.g. vitamin a or vitamin E), tocopherols or antioxidants (e.g. ascorbic acid), stabilizers and/or preservatives for prolonging the use and shelf life of the pharmaceutically active ingredient or formulation, or other usual non-pharmaceutically active ingredients or auxiliaries and additives well known in the art and mixtures thereof.

In a third aspect of the invention, a medicament for preventing and treating mycoplasma bovis diseases is provided, wherein the medicament comprises the composition of the second aspect.

Preferably, the medicament is in the form of an oral preparation, an external medicament or an injection; further, the oral preparation comprises an oral solid preparation or an oral liquid preparation;

specifically, the oral solid preparation includes, but is not limited to, powders, soluble powders, premixes, tablets, granules, capsules, pills, pastes and the like;

specifically, the oral liquid preparation comprises but is not limited to one of water agent, tincture, decoction and mixture;

specifically, the external medicine includes, but is not limited to, an aerosol, a drench or a drip spray.

Further, the injection includes, but is not limited to, powder injection, infusion solution, suspension, emulsion, hydro-acupuncture or oil-acupuncture, and the injection form includes, but is not limited to, intramuscular, subcutaneous, intradermal or intravenous injection.

In a fourth aspect of the present invention, there is provided a method for controlling a disease associated with mycoplasma bovis, said method comprising administering the pharmaceutical composition of the second aspect or the medicament of the third aspect to an individual in need thereof.

In the fourth aspect, the term "subject in need thereof" includes mammals, preferably mice, rats or other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses or primates, and preferably cattle.

In one embodiment, the administration includes, but is not limited to, adding the pharmaceutical composition or medicament to the drinking water of the cattle for administration, and further includes killing the cattle breeding environment with the pharmaceutical composition or medicament.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example 1 validation of the Effect of tea saponin on inhibiting Mycoplasma bovis

Firstly, toxicity test of tea saponin on MDBK cells:

MDBK cells are susceptible cells to Mycoplasma bovis. Therefore, the cytotoxicity of tea saponin to MDBK cells is firstly detected, and the specific experimental steps are as follows:

(1) 100 μ L of cells (MDBK 5000 per well) were seeded in 96-well plates.

(2) After about 12h of incubation, the next dosing analysis was performed. Media was discarded and 100 μ L of 2% FBS DMEM containing different drug concentrations were added to each well, 3 replicates for each concentration. At the same time, control wells: 100 μ L of 2% FBS DMEM medium containing 0.9% DMSO was added. Zero setting hole: cells were not plated.

(3) At 37 ℃ 5% CO₂After culturing for 48h under the conditions, the medium in the wells was discarded. The effect of the drug on the CCK8 response was excluded by washing twice with 100 μ L PBS. Each well was supplemented with 100. mu.L of DMEM medium plus 10. mu.L of CCK8 solution.

(4)37℃，5％CO₂After further incubation for 4h under these conditions, the absorbance was measured at 450 nm. 450nm of untreated cells was set as 100% cell control.

(5) The above experiment was repeated three times, and the half Cytotoxic Concentration (CC) of tea saponin was calculated by analyzing the data using GraphPad Prism5 software₅₀) The value is obtained. The test result shows that the tea saponin has a dose-dependent relationship, namely, the tea saponin shows that cytopathic effect is obvious along with the increase of the concentration of the medicine. Determining half poisoning concentration (CC) of tea saponin by statistical analysis₅₀) 937.5 μ M.

Secondly, the inhibition experiment of tea saponin on mycoplasma bovis is as follows:

(1) 1X 10 inoculations in each well of a 96-well plate⁴MDBK cells, 37 ℃, 5% CO₂Culturing overnight in an incubator until the cells grow full;

(2) the medium was discarded and 100. mu.L of 100TCID was added to each well₅₀In a dilution of Mycoplasma bovis (Mycoplasma bovis dilution prepared with 2% FBS DMEM), at an initial concentration of 50. mu.M, twice concentratedGradient dilution with 5% CO₂Culturing in an incubator;

(3) after 48 hours, operating according to the instruction of a CCK-8 kit, and measuring the OD value at 450nm by using an enzyme-labeling instrument;

(4) the above test was repeated three times and statistical analysis of data was performed. The inhibition rate (%) of mycoplasma bovis (OD value 450nm in the drug-treated group-OD value 450nm in the mycoplasma bovis control group)/(OD value 450nm in the normal cell control group-OD value 450nm in the mycoplasma bovis control group) × 100%, and the half Effective Concentration (EC) of tea saponin was obtained by GraphPad Prism5 software₅₀) The value is obtained. The results are shown in FIG. 3. Then according to the formula TI ═ CC₅₀/EC₅₀And calculating the corresponding therapeutic index TI value.

As a result: the CCK-8 kit is used for detecting the cell viability, so that the effective inhibition rate of the medicament on the mycoplasma bovis can be calculated. The results show that the effective inhibition rate of the tea saponin is increased along with the increase of the concentration of the medicine within the safe concentration range, and the effective inhibition rate is in a certain dose-effect relationship. Half the Effective Concentration (EC) of M.bovis by analytical software₅₀) The concentration was 97.66. mu.M. Combined with the toxicity test result of MDBK cells, the median toxic concentration (CC) of theasaponin₅₀) At 937.5. mu.M, the therapeutic index of M.bovis was calculated to be 9.60.

Example 2 validation of the Effect of sodium aescinate on inhibiting Mycoplasma bovis

Toxicity test of sodium aescinate on MDBK cells:

firstly, detecting the cytotoxicity of sodium aescinate on MDBK cells, wherein the specific experimental steps are as follows:

(1) 100 μ L of cells (MDBK 5000 per well) were seeded in 96-well plates.

(2) After about 12h of incubation, the next dosing analysis was performed. Media was discarded and 100 μ L of 2% FBS DMEM containing different drug concentrations were added to each well, 3 replicates for each concentration. At the same time, control wells: 100 μ L of 2% FBS DMEM medium containing 0.9% DMSO was added. Zero setting hole: cells were not plated.

(3) At 37 ℃ 5% CO₂After culturing for 48h under the conditions, the medium in the wells was discarded. The effect of the drug on the CCK8 response was excluded by washing twice with 100 μ L PBS. Each well is supplemented with 100. mu.L of DMEM medium+ 10. mu.L of CCK8 solution.

(4)37℃，5％CO₂After further incubation for 4h under these conditions, the absorbance was measured at 450 nm. 450nm of untreated cells was set as 100% cell control.

(6) The above experiment was repeated three times, and the half Cytotoxic Concentration (CC) of sodium aescinate was calculated by analyzing the data using GraphPad Prism5 software₅₀) The value is obtained. The test result shows that the aescin sodium has a dose-dependent relationship, namely, the aescin sodium shows that the cytopathic effect is obvious along with the increase of the concentration of the medicine. Determining sodium aescinate half poisoning concentration (CC) by statistical analysis₅₀) 703.13 μ M.

Secondly, the test of inhibiting the mycoplasma bovis by the sodium aescinate comprises the following steps:

(1) 1X 10 inoculations in each well of a 96-well plate⁴MDBK cells, 37 ℃, 5% CO₂Culturing overnight in an incubator until the cells grow full;

(2) the medium was discarded and 100. mu.L of 100TCID was added to each well₅₀The mycoplasma bovis diluent (mycoplasma bovis diluent prepared by 2% FBS DMEM) is diluted and added with medicine according to 50 mu M initial concentration and double concentration gradient, and 5% CO₂Culturing in an incubator;

(3) after 48 hours, operating according to the instruction of a CCK-8 kit, and measuring the OD value at 450nm by using an enzyme-labeling instrument;

(4) the above test was repeated three times and statistical analysis of data was performed. The percent (%) inhibition of m.bovis (drug-treated group 450nm OD value-m.bovis control group 450nm OD value)/(normal cell control group 450nm OD value-m.bovis control group 450nm OD value) × 100%, and the half effective concentration of escin sodium (EC) was obtained using GraphPad Prism5 software₅₀) The value is obtained. The results are shown in FIG. 6. Then according to the formula TI ═ CC₅₀/EC₅₀And calculating the corresponding therapeutic index TI value.

As a result: the CCK-8 kit is used for detecting the cell viability, so that the effective inhibition rate of the medicament on the mycoplasma bovis can be calculated. The results show that the effective inhibition rate of the sodium aescinate is increased along with the increase of the concentration of the medicament within the safe concentration range, and the effective inhibition rate is in a certain dose-effect relationship. By analytical software, it was effective on half of mycoplasma bovisConcentration (EC)₅₀) The concentration was 24.41. mu.M. Toxicity test results of MDBK cells in combination with sodium aescinate half toxic concentration (CC)₅₀) At 703.13. mu.M, the therapeutic index of M.bovis was calculated to be 28.80.

Example 3 verification of the Effect of dioscin on inhibiting Mycoplasma bovis

Firstly, toxicity test of dioscin on MDBK cells:

firstly, detecting the cytotoxicity of dioscin on MDBK cells, wherein the specific experimental steps are as follows:

(1) 100 μ L of cells (MDBK 5000 per well) were seeded in 96-well plates.

(2) After about 12h of incubation, the next dosing analysis was performed. Media was discarded and 100 μ L of 2% FBS DMEM containing different drug concentrations were added to each well, 3 replicates for each concentration. At the same time, control wells: 100 μ L of 2% FBS DMEM medium containing 0.9% DMSO was added. Zero setting hole: cells were not plated.

(3) At 37 ℃ 5% CO₂After culturing for 48h under the conditions, the medium in the wells was discarded. The effect of the drug on the CCK8 response was excluded by washing twice with 100 μ L PBS. Each well was supplemented with 100. mu.L of DMEM medium plus 10. mu.L of CCK8 solution.

(4)37℃，5％CO₂After further incubation for 4h under these conditions, the absorbance was measured at 450 nm. 450nm of untreated cells was set as 100% cell control.

(7) The above experiment was repeated three times, and the half Cytotoxic Concentration (CC) of dioscin was calculated by analyzing the data using GraphPad Prism5 software₅₀) The value is obtained. The results are shown in FIG. 8.

As a result: the dioscin has a dose-dependent relationship, namely, the cell pathological changes are more obvious along with the increase of the concentration of the medicine. Determining dioscin half poisoning concentration (CC) by statistical analysis₅₀) The concentration was 31.25. mu.M.

Secondly, an inhibition experiment of dioscin on mycoplasma bovis:

(1) 1X 10 inoculations in each well of a 96-well plate⁴MDBK cells, 37 ℃, 5% CO₂Culturing overnight in an incubator until the cells grow full;

(2) the medium was discarded and 100. mu.l of medium was added to each wellL 100TCID₅₀The mycoplasma bovis diluent (mycoplasma bovis diluent prepared by 2% FBS DMEM) is diluted and added with medicine according to 50 mu M initial concentration and double concentration gradient, and 5% CO₂Culturing in an incubator;

(3) after 48 hours, operating according to the instruction of a CCK-8 kit, and measuring the OD value at 450nm by using an enzyme-labeling instrument;

(4) the above test was repeated three times and statistical analysis of data was performed. The inhibition rate (%) of mycoplasma bovis (OD value 450nm of drug-treated group-OD value 450nm of mycoplasma bovis control group)/(OD value 450nm of normal cell control group-OD value 450nm of mycoplasma bovis control group) × 100%, and the half Effective Concentration (EC) of dioscin was obtained by GraphPad Prism5 software₅₀) The value is obtained. The results are shown in FIG. 9. Then according to the formula TI ═ CC₅₀/EC₅₀And calculating the corresponding therapeutic index TI value.

As a result: the CCK-8 kit is used for detecting the cell viability, so that the effective inhibition rate of the medicament on the mycoplasma bovis can be calculated. The results show that the effective inhibition rate of the dioscin is increased along with the increase of the concentration of the medicine within the safe concentration range, and the effective inhibition rate is in a certain dose-effect relationship. Half the Effective Concentration (EC) of M.bovis by analytical software₅₀) It was 3.25. mu.M. Combined with the MDBK cytotoxic test results, the median toxic concentration (CC) of dioscin₅₀) At 31.25. mu.M, the therapeutic index of M.bovis was found to be 9.62 by calculation.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.