Use of saponin in preparation of anti-mycoplasma bovis product

Abstract

The present application relates to the application of saponin in the preparation of anti-mycoplasma bovum products. Mycoplasma bovum can cause bovine pneumonia, arthritis and bovine mastitis and other diseases, and the drugs for preventing and treating bovine mycoplasma diseases in the art are very limited, and the effect of late treatment is not ideal, mainly through the way of strengthening feeding management for prevention and control. The present application provides the in vitro inhibition activity of dioscin and sodium aescinate on mycoplasma bovum. The prior art is blank in the related research of saponin compounds on the inhibition of mycoplasma. The above compounds not only have good inhibition effect on mycoplasma bovum, but also have less toxicity to normal cells, and have the prospect of developing into anti-mycoplasma bovum drugs.

Description

[0001] This application is a divisional application of application number 2021110227153 (application date: September 1, 2021, and invention name: Application of saponins in the preparation of products against Mycoplasma bovis). TECHNICAL FIELD

[0002] The present application belongs to the technical field of anti-Mycoplasma bovis drugs, and specifically relates to the application of saponins in the preparation of products against Mycoplasma bovis. BACKGROUND

[0003] The information disclosed in this background section is intended only to increase an understanding of the general context of the present application, and is not necessarily recognized as prior art against which the present application is configured.

[0004] Mycoplasma bovis is one of the important pathogens causing various symptoms such as pneumonia and arthritis in calves and mastitis in adult cattle. Since 1961, Hale et al. first isolated Mycoplasma bovis from milk in the United States, Mycoplasma bovis has been isolated in various parts of the world.

[0005] Mycoplasma bovis is the smallest cell-nucleated microorganism, between bacteria and viruses, but has no cell wall, and belongs to prokaryotes. Mycoplasma is widely distributed in sewage, soil, animals and humans. Mycoplasma bovis has strong resistance to the outside world, can survive in fertilizer for about 230 days, in water for about 22 days, and in sand for up to 8 months. At present, there is no effective Mycoplasma bovis vaccine at home and abroad, and the prevention and control situation is severe. Compared with bacteria, Mycoplasma bovis has no cell wall structure, which makes most commonly used broad-spectrum antibiotics ineffective, and only a few antibiotics can be used for the treatment of clinically infected cattle. Once drug resistance occurs, the ranch will face no available drugs and can only adopt the way of eliminating cattle to control the epidemic, causing great economic losses. In view of the current situation, the development and screening of compounds with inhibitory activity against Mycoplasma bovis are expected to achieve the prevention and control of Mycoplasma bovis disease and reduce the economic losses of cattle industry.

[0006] Escin sodium, with the molecular formula C 55 H 83 NaO 23 , is a white crystalline powder with a bitter and pungent taste and hygroscopicity. It has the effects of anti-inflammatory, anti-exudation, increasing venous tension, improving blood circulation, and correcting brain dysfunction. It has obvious protective effect on brain edema caused by carbon monoxide, etc. It can be used for the treatment of brain edema, swelling caused by trauma or surgery, and also for venous reflux disorder. However, no one has studied the other uses of this drug, especially its effect on Mycoplasma bovis.

[0007] Dioscin is the main component in the Dioscoreaceae plant. Traditional Chinese medicine believes that Dioscorea has the effects of eliminating phlegm, promoting digestion, water, relaxing the tendons and blood, and cutting malaria. Modern pharmacological studies show that dioscin has many pharmacological effects, especially in the anti-tumor effect. Many studies also show that dioscin can improve the symptoms of atherosclerosis, protect the function of vascular endothelium, reduce ischemia / reperfusion injury of heart, brain and kidney, reduce blood sugar, inhibit liver fibrosis, improve menopausal osteoporosis, relieve rheumatoid arthritis and ulcerative colitis, and antagonize the activity of bacteria and Mycoplasma bovis. So far, there has been no report on its use for preventing and treating Mycoplasma bovis. SUMMARY

[0008] Based on the above research background, the purpose of the present application is to screen compounds with inhibitory activity on Mycoplasma bovis, so as to provide an effective drug for treating Mycoplasma bovis disease. In order to achieve the technical purpose, the present application conceives to screen active compounds from natural medicine extracts, and for the first time verifies that many saponin components such as sodium aescinate and dioscin can effectively inhibit the in vitro proliferation of Mycoplasma bovis, and the above active ingredients have less harm to the body, and have the prospect of developing into anti-Mycoplasma bovis drugs.

[0009] In view of the above technical effects, the present application mainly provides the application of saponin in the preparation of anti-Mycoplasma bovis products.

[0010] Specifically, the present application specifically verifies the application of sodium aescinate and dioscin in the preparation of the anti-Mycoplasma bovis products.

[0011] Sodium aescinate is a commonly used drug for treating cerebral vascular disease in clinic, which is used for cerebral vascular dilation, improving venous tension, and accelerating venous return. The present application first confirms that the compound sodium aescinate can effectively inhibit the proliferation of Mycoplasma bovis. Experiments show that the half toxicity concentration (CC 50 ) of sodium aescinate to MDBK cells is 703.13 μM, and the half effective concentration (EC 50 ) of sodium aescinate to Mycoplasma bovis is 24.41 μM; the therapeutic index of sodium aescinate to Mycoplasma bovis is 28.80. The results show that sodium aescinate has the prospect of developing into an anti-Mycoplasma bovis drug, and provides a new medical use for sodium aescinate.

[0012] Dioscin is an important basic raw material for producing steroid hormone drugs. Steroid hormones have strong pharmacological effects of anti-infection, anti-allergy, anti-virus and anti-shock, and are important drugs for treating rheumatism, cardiovascular disease, lymphatic leukemia, encephalitis, skin disease, anti-tumor and rescuing critical patients. In addition, dioscin has many pharmacological activities, but mainly focuses on the research of anti-tumor activity. The application provides the activity of dioscin in mycoplasma inhibition for the first time, and provides the application of dioscin in the field of veterinary drugs. It is verified that the half toxicity concentration (CC 50 ) of dioscin to MDBK cells is 31.25 muM, the half effective concentration (EC 50 ) of dioscin to bovine mycoplasma is 3.25 muM, and the therapeutic index of dioscin to bovine mycoplasma is 9.62.

[0013] The application verifies the inhibition of dioscin to bovine mycoplasma, and the toxicity of dioscin to cells is small, so that the dioscin opens up a new drug use.

[0014] The beneficial effects of the one or more technical solutions above are as follows:

[0015] The application provides the activity of sodium aescinate and dioscin in inhibiting the proliferation of bovine mycoplasma in vitro for the first time, based on the research results, the medical use of the above compounds is further expanded. In addition, the means for preventing and treating bovine mycoplasma in the field are very limited, and the research results of the application can provide certain technical guidance for the development of anti-bovine mycoplasma active ingredients. The toxicity of the above compounds to normal cells is small, and it is of great significance to develop into an anti-bovine mycoplasma drug. BRIEF DESCRIPTION OF DRAWINGS

[0016] The drawings accompanying the specification of the application form part of the application and serve to provide further understanding of the application, the illustrative embodiments of the application and the description thereof serve to explain the application, and do not constitute an improper limitation on the application.

[0017] Figure 1 It is a diagram of the effect of sodium aescinate on bovine mycoplasma damaged cells in example 2;

[0018] Among them Figure 1 The left graph is the MDBK normal cell group; Figure 1 The middle graph is the MDBK infected bovine mycoplasma control group; Figure 1 The right graph is the infected cell drug test group (50 muM sodium aescinate is applied);

[0019] Figure 2 It is a diagram of the half cytotoxicity concentration (CC 50 ) of sodium aescinate to MDBK cells in example 2;

[0020] Figure 3The half-maximal effective concentration (EC50) of sodium aescinate against bovine mycoplasma in Example 2. 50 )picture;

[0021] Figure 4 This is a diagram illustrating the effect of diosgenin on bovine mycoplasma-damaged cells in Example 3.

[0022] in, Figure 4 The left side shows the MDBK normal cell group; Figure 4 The middle group is the control group infected with Mycoplasma bovis (MDBK). Figure 4 The right side shows the cell infection drug test group (administered with 40 μM diosgenin);

[0023] Figure 5 The half-maximal cytotoxic concentration (CC) of diosgenin against MDBK cells in Example 3. 50 )picture;

[0024] Figure 6 The half-maximal effective concentration (EC50) of diosgenin against bovine mycoplasma in Example 3. 50 )picture. Detailed Implementation

[0025] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, 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 pertains.

[0026] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0027] Terminology Explanation:

[0028] "Prevention and / or treatment": refers to any measure applicable to the treatment of bovine mycoplasma-related diseases, or preventive treatment of the disease or its symptoms, or prevention of recurrence of the disease, such as recurrence after the end of a treatment period or treatment of symptoms of an existing disease.

[0029] "Anti-Bovine Mycoplasma drug" refers to a substance that has a significant inhibitory effect on bovine mycoplasma. It acts directly on the early stages of bovine mycoplasma, and its direct killing effect on bovine mycoplasma is better than its adsorption blocking effect and replication blocking effect.

[0030] As introduced in the background, Mycoplasma bovis is an important factor threatening the cattle breeding industry, and currently there are very limited drugs available in the art for Mycoplasma bovis, in order to solve the above technical problems, the application provides application of saponins in preparation of products against Mycoplasma bovis.

[0031] In a first aspect, the application provides application of saponins in preparation of products against Mycoplasma bovis.

[0032] The saponins in the first aspect are classified according to the structure of saponin aglycone, including spirostane and triterpenoid saponins.

[0033] In a preferred scheme of the application, the triterpenoid saponins include but are not limited to one of glycyrrhizin, ginsenoside, panax notoginseng saponin and aescine; and the specific examples of spirostane saponins include dioscin.

[0034] In one embodiment of the application, application of aescine in preparation of products against Mycoplasma bovis is provided.

[0035] Aescine is the main active ingredient in the extract of Sterculia lychnophora Hance, and since aescine is insoluble in water, the sodium salt form of aescine, i.e., sodium aescinate (CAS No.: 6805-41-0), is commonly used in the art, and the structure is shown in formula I below. Therefore, in a more specific embodiment of the application, application of sodium aescinate in preparation of products against Mycoplasma bovis is further provided.

[0036]

[0037] In another embodiment of the application, application of dioscin in preparation of products against Mycoplasma bovis is provided.

[0038] Dioscin is the main component in the Dioscoreaceae plant, and the chemical structure is shown in formula II below.

[0039]

[0040] In the above embodiments, the aescine and dioscin further include pharmaceutically acceptable salts thereof, the pharmaceutically acceptable salts include salts formed by aescine or dioscin and inorganic acids or organic acids; in specific examples, the inorganic acid is hydrochloric acid, sulfuric acid, nitric acid or hydrobromic acid; and the organic acid is methanesulfonic acid, toluenesulfonic acid or trifluoroacetic acid.

[0041] It should also be understood that the products against Mycoplasma bovis in the first aspect are used for preventing and / or treating diseases related to Mycoplasma bovis, and the product forms include but are not limited to one of veterinary drugs, feed or biochemical reagents.

[0042] Further, the veterinary drugs include serum products, vaccines, diagnostic products, microecological products, Chinese herbal medicines, Chinese patent medicines, chemical medicines, antibiotics, biochemical medicines, radioactive medicines, external insecticides or disinfectants.

[0043] Further, the feed includes complete feed, concentrated feed, premixed feed, fine feed or mixed feed.

[0044] Further, the biochemical reagent includes reagents for diagnosis and biochemical research, and the specific application forms include diagnostic reagents, screening reagents for active ingredients against Mycoplasma bovis, etc.

[0045] In the second aspect of the present application, a pharmaceutical composition is provided, which comprises at least one or a combination of several esculin sodium or dioscin.

[0046] In the pharmaceutical composition of the first aspect, the pharmaceutical dosage of the esculin sodium or dioscin can be determined by conventional methods in the art, which can be determined by means of the half maximal effective concentration of the above-mentioned components and the experimental results of animal models.

[0047] Preferably, the pharmaceutical composition further comprises a pharmaceutically necessary carrier, which includes excipients and / or diluents. For example, pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and non-ionic surfactants or lipids, and pharmacologically harmless salts (such as sodium chloride, flavoring agents), vitamins (such as vitamin A or vitamin E), tocopherols or antioxidants (such as ascorbic acid), stabilizers and / or preservatives for extending the use and storage time of the pharmaceutical active ingredients or formulations, or other commonly used non-pharmaceutical active ingredients or adjuvants and additives known in the prior art and mixtures thereof.

[0048] In the third aspect of the present application, a medicine for preventing and treating Mycoplasma bovis disease is provided, which comprises the composition of the second aspect.

[0049] Preferably, the medicine is in the form of oral preparation, external medicine or injection; further, the oral preparation includes oral solid preparation or oral liquid preparation.

[0050] Specifically, the oral solid preparation includes but is not limited to powder, soluble powder, premix, tablet, granule, capsule, pill, paste, paste type, etc.

[0051] Specifically, the oral liquid preparation includes but is not limited to one of water preparation, tincture, decoction, mixture.

[0052] Specifically, the external medicine includes but is not limited to aerosol, shower or drop spray.

[0053] Further, the injection includes but is not limited to powder injection, large infusion, suspension, emulsion, water injection or oil injection preparation, and injection forms include but are not limited to intramuscular, subcutaneous, intradermal or intravenous injection.

[0054] In a fourth aspect of the present application, a method for preventing and treating Mycoplasma bovis related diseases is provided, wherein the prevention method comprises administering the pharmaceutical composition of the second aspect or the drug of the third aspect to an individual in need thereof.

[0055] In the above fourth aspect, the "individual in need thereof" includes mammals, preferably mice, rats or other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses or primates, and preferably cows.

[0056] In an embodiment, the administration method includes but is not limited to adding the pharmaceutical composition or drug to the diet or drinking water of the cow, and also includes using the pharmaceutical composition or drug to disinfect the breeding environment of the cow.

[0057] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with specific examples.

[0058] Example 1: Effect verification of sodium aescinate on inhibiting Mycoplasma bovis

[0059] I. Toxicity experiment of sodium aescinate on MDBK cells:

[0060] First, the cytotoxicity of sodium aescinate on MDBK cells was detected, and the specific experimental steps are as follows:

[0061] (1) Inoculate 100 μL of cells (MDBK 5000 cells / well) in a 96-well plate.

[0062] (2) After about 12 h of culture, the next step of drug addition analysis was performed. Discard the culture medium, add 100 μL of 2% FBS DMEM containing different drug concentrations to each well, and make 3 parallel samples for each concentration. The control well: add 100 μL of 2% FBS DMEM containing 0.9% DMSO medium. Zero adjustment well: no cells are plated.

[0063] (3) After 48 h of culture at 37℃, 5% CO2, discard the culture medium in the well. Wash twice with 100 μL of PBS to exclude the influence of drugs on CCK8 reaction. Add 100 μL of DMEM medium + 10 μL of CCK8 solution to each well.

[0064] (4) Continue to culture at 37℃, 5% CO2 for 4 h, and then measure the absorbance at 450 nm. Set the 450 nm of untreated cells as 100% cell control.

[0065] (5) The above test was repeated three times, and the data was analyzed by GraphPad Prism5 software to calculate the half cytotoxic concentration (CC 50 ) value of sodium aescinate. The test results showed that sodium aescinate showed a dose-dependent relationship, that is, as the drug concentration increased, the cytopathic effect was more obvious. Statistical analysis showed that the half toxic concentration (CC 50 ) of sodium aescinate was 703.13 μM.

[0066] II. Inhibition experiment of sodium aescinate on Mycoplasma bovis:

[0067] (1) 1×10 4 MDBK cells were inoculated in each well of a 96-well plate, and cultured overnight at 37°C in a 5% CO2 incubator until the cells were full;

[0068] (2) Discard the culture medium, add 100 μL of 100 TCID 50 Mycoplasma bovis diluent (Mycoplasma bovis diluent prepared with 2% FBS DMEM) to each well, and add the drug with a two-fold concentration gradient according to the initial concentration of 50 μM, and culture in a 5% CO2 incubator;

[0069] (3) After 48 h, follow the CCK-8 kit instructions, and measure the OD value at 450 nm with a microplate reader;

[0070] (4) The above test was repeated three times, and the data was statistically analyzed. The Mycoplasma bovis inhibition rate (%) = (drug treatment group 450 nm OD value - Mycoplasma bovis control group 450 nm OD value) / (normal cell control group 450 nm OD value - Mycoplasma bovis control group 450 nm OD value) x 100%, and the half effective concentration (EC 50 ) value of sodium aescinate was obtained by GraphPad Prism5 software. The results are shown in Figure 3 . Then calculate the corresponding therapeutic index TI value according to the formula TI = CC 50 / EC 50 .

[0071] Results: The CCK-8 kit was used to detect cell viability, and the effective inhibition rate of the drug on Mycoplasma bovis was calculated. As can be seen from the results, within the safe concentration range of sodium aescinate, the effective inhibition rate increased with the increase of drug concentration, showing a certain dose-effect relationship. Through analysis software, the half effective concentration (EC 50 ) of Mycoplasma bovis was 24.41 μM. Combined with the toxicity experiment results of MDBK cells, the half toxic concentration (CC 50The concentration was 703.13 μM, and the therapeutic index for bovine mycoplasma was calculated to be 28.80. Example 2: Verification of the inhibitory effect of diosgenin on bovine mycoplasma.

[0072] I. Toxicity experiment of diosgenin on MDBK cells:

[0073] First, the cytotoxicity of diosgenin on MDBK cells was tested. The specific experimental steps are as follows:

[0074] (1) Seed 100 μL of cells (MDBK 5000 cells / well) into a 96-well plate.

[0075] (2) After approximately 12 hours of culture, proceed with the next step of drug addition analysis. Discard the culture medium and add 100 μL of 2% FBS DMEM containing different drug concentrations to each well, performing three replicates for each concentration. Simultaneously, add 100 μL of 2% FBS DMEM containing 0.9% DMSO to the control wells. Do not seed cells in the zeroing wells.

[0076] (3) After culturing at 37℃ and 5% CO2 for 48 h, discard the culture medium in the wells. Wash twice with 100 μL PBS to eliminate the influence of the drug on the CCK8 reaction. Add 100 μL DMEM medium + 10 μL CCK8 solution to each well.

[0077] (4) After culturing at 37℃ and 5% CO2 for another 4 hours, the absorbance was measured at 450nm. The absorbance of untreated cells at 450nm was set as 100% cell control.

[0078] (6) The above experiment was repeated three times. The data were analyzed using GraphPad Prism5 software to calculate the half-maximal cytotoxic concentration (CMC) of diosgenin. 50 The value is as follows. Figure 5 As shown.

[0079] Results: Diosgenin showed a dose-dependent effect, meaning that the cytopathic effect became more pronounced with increasing drug concentration. Statistical analysis determined the median toxic concentration (LC50) of diosgenin. 50 The concentration was 31.25 μM. II. Inhibition experiment of diosgenin on bovine mycoplasma:

[0080] (1) Inoculate each well of the 96-well plate with 1×10 4 One MDBK cell was cultured overnight at 37°C in a 5% CO2 incubator until the cells reached confluence.

[0081] (2) Discard the culture medium and add 100 μL of 100 TCID to each well. 50The bovine mycoplasma diluent (bovine mycoplasma diluent configured with 2% FBS DMEM) was diluted in a two-fold concentration gradient with an initial concentration of 50 μM, and was cultured in a 5% CO2 incubator.

[0082] (3) After 48 h, the OD value at 450 nm was measured by CCK-8 kit according to the instructions, and was measured by an enzyme-labeled instrument;

[0083] (4) The above test was repeated three times, and data statistical analysis was performed. The inhibition rate of bovine mycoplasma (%) = (OD value at 450 nm of the drug treatment group-OD value at 450 nm of the bovine mycoplasma control group) / (OD value at 450 nm of the normal cell control group-OD value at 450 nm of the bovine mycoplasma control group) x 100%, and the half effective concentration (EC 50 ) value of dioscin was obtained by GraphPad Prism 5 software. The results are shown in Figure 6 . Then, the corresponding therapeutic index TI value was calculated according to the formula TI = CC 50 / EC 50 .

[0084] Results: The effective inhibition rate of the drug on bovine mycoplasma can be calculated by detecting the cell viability by CCK-8 kit. It can be seen from the results that the effective inhibition rate of dioscin increases with the increase of the drug concentration in the safe concentration range, and shows a certain dose-effect relationship. The half effective concentration (EC 50 ) of bovine mycoplasma is 3.25 μM by analyzing the software. Combined with the results of MDBK cell toxicity experiment, the half toxic concentration (CC 50 ) of dioscin is 31.25 μM, and the therapeutic index of bovine mycoplasma is 9.62.

[0085] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A use of sodium aescinate or dioscin in the preparation of an anti-mycoplasmal bovine product; The anti-mycoplasmal bovine product is one of a veterinary drug and a feed; The structure of the sodium aescinate is shown in formula I: Formula I.

2. Use of sodium aescinate or dioscin according to claim 1 for the preparation of a product against Mycoplasma bovis, characterized in that, The veterinary drug is selected from Chinese patent medicines and chemical drugs; The feed is selected from complete feed, concentrated feed, premixed feed, fine feed or mixed feed.

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