A composite pharmaceutical preparation and its preparation method and application

Abstract

The present invention discloses a composite pharmaceutical preparation, a preparation method and an application thereof. The composite pharmaceutical preparation comprises the following components by weight: 50 to 70 parts of neomycin sulfate, 10 to 30 parts of ursolic acid and 10 to 30 parts of poplar flower extract. The composite pharmaceutical preparation has excellent delaying and blocking effects on the disease of Babylonia snails, and can significantly reduce and delay the mortality rate of Babylonia snails with the disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquatic animal breeding, and more specifically, relates to a composite pharmaceutical preparation and a preparation method and application thereof. Background Art

[0002] As a vital marine economic aquaculture shellfish in southern my country, the scale of aquaculture of Dongfeng conch has been continuously expanded in the past two decades, and the aquaculture output has continued to rise. At present, the annual output of Hainan and Fujian provinces alone has exceeded 20,000 tons. However, in recent years, due to factors such as germplasm degradation, the growth and stress resistance of Dongfeng conch have significantly declined, and problems such as frequent outbreaks of diseases have also occurred during the aquaculture process. Therefore, based on the biological research of Dongfeng conch, conducting disease research on Dongfeng conch is of great significance to the sustainable and healthy development of the Dongfeng conch aquaculture industry.

[0003] In the breeding process of Dongfeng snails, large-scale outbreaks of diseases occur repeatedly, such as kiss swelling disease, turning over disease, shelling disease, etc. In recent years, a new disease has appeared in Dongfeng snail breeding, which is called "weak muscle disease". Its symptoms are: turning over, the most prominent feature is the weakness, edema and inability of the ventral foot muscles to retract, and water will spray out from the ventral foot holes when touched. This disease has a very high infection rate and can be quickly transmitted to healthy snails. If not controlled, the diseased Dongfeng snails will die within 2 days, most of them will die completely within one day, and some will even die within a few hours. The survival time is extremely short, and the mortality rate is as high as 100%. At present, no effective treatment has been found.

[0004] At present, there have been some achievements in the research of diseases of Babylonia snails. For example, the patent with the publication number CN117618483A discloses a pharmaceutical composition for preventing and treating Babylonia spirochetis. The pharmaceutical composition includes a compound Chinese herbal medicine preparation and doxycycline hydrochloride; each portion of the compound Chinese herbal medicine preparation includes the following raw materials in parts by weight: gallnut, green fruit, scutellaria, hawthorn, honeysuckle, terminalia chebula and black plum. However, the above pharmaceutical composition is for Babylonia spirochetis, not for the "weak muscle disease" of Babylonia snails.

[0005] Therefore, developing a drug additive to target new diseases of the snail and improve the survival rate of the snail after the disease occurs is very necessary to promote the development of research on the snail disease. Summary of the invention

[0006] In view of the above-mentioned existing technical problems, the primary purpose of the present invention is to provide a compound pharmaceutical preparation, which is used in combination of three components and has excellent delaying and blocking effects on the disease of the baby snail, and can significantly reduce the mortality rate of the diseased baby snails.

[0007] The second object of the present invention is to provide a method for preparing a composite pharmaceutical preparation.

[0008] The third object of the present invention is to provide a composite pharmaceutical preparation for use in the preparation of a drug for preventing and treating Babylonia snail diseases or in the preparation of a drug for reducing the mortality rate of Babylonia snails after the occurrence of Babylonia snail diseases.

[0009] The fourth object of the present invention is to provide a method for cultivating Babylonia paniculata.

[0010] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0011] A composite pharmaceutical preparation comprises the following components by weight: 50 to 70 parts of neomycin sulfate, 10 to 30 parts of ursolic acid and 10 to 30 parts of poplar flower extract.

[0012] The present invention can play a role in synergistically improving the efficacy of drugs by adding a composite drug preparation including neomycin sulfate, ursolic acid and poplar flower extract after the onset of the disease of the babylonica, significantly preventing and treating the babylonica disease, and achieving excellent delaying effect and blocking effect.

[0013] Specifically, the inventors have found through long-term research that neomycin sulfate is an aminoglycoside antibiotic that has a good antibacterial effect on Enterobacteriaceae. It mainly acts on the bacterial protein synthesis process, causing abnormally synthesized proteins to hinder the release of synthesized proteins, increasing the permeability of bacterial cell membranes and leading to the leakage of some important physiological substances, which can inhibit the growth and reproduction of pathogens and even cause bacterial death. Ursolic acid can inhibit the release of inflammatory mediators and the activation of inflammatory cells, and reduce inflammatory reactions. By scavenging free radicals, it reduces the damage of oxidative stress to cells and protects cell structure and function. It has a certain regulatory effect on the immune system and enhances the body's immune function. Poplar flower extract mainly contains flavonoids, organic acids, amino acids, sugars and alkaloids. Poplar flower extract can directly inhibit and kill some pathogens; it can improve the resistance of the body of Dongfeng snails, counteract immunosuppressants, regulate the body's immune function, and effectively alleviate various stress reactions during the onset of Dongfeng snails; it can increase the tension of the gastrointestinal muscles and sphincters of Dongfeng snails, inhibit secretion function, and show astringent and anti-diarrhea effects. Promote digestion and growth.

[0014] The inventors found through research that the combination of neomycin sulfate, ursolic acid and poplar flower extract has a synergistic effect, which complements each other and expands the therapeutic effect. The composite pharmaceutical preparation provided by the present invention can significantly reduce the mortality rate of Babylonia snails, effectively improve the survival rate and survival time of diseased snails after the disease occurs during the breeding process of Babylonia snails, and improve the timeliness of the survival of diseased snails.

[0015] Preferably, the compound pharmaceutical preparation comprises the following components by weight: 50-60 parts of neomycin sulfate, 20-30 parts of ursolic acid and 20-30 parts of poplar flower extract. Further preferably, the compound pharmaceutical preparation comprises the following components by weight: 50 parts of neomycin sulfate, 20 parts of ursolic acid and 30 parts of poplar flower extract.

[0016] Preferably, the poplar flower extract is a poplar flower alcohol extract.

[0017] Preferably, the preparation method of the poplar flower extract is: pre-treated poplar flowers are mixed and soaked with an ethanol aqueous solution, reflux extracted, and post-treated to obtain the poplar flower extract.

[0018] Preferably, the post-treatment is one or more of filtration, concentration and drying.

[0019] Preferably, the volume concentration of ethanol in the ethanol aqueous solution is 50-70%.

[0020] Preferably, the number of reflux extractions is 3 to 5. Further preferably, the time for each reflux extraction is 2 to 3 hours.

[0021] Preferably, the temperature of the reflux extraction is 50-60°C.

[0022] More preferably, the pore size of the filtration is 20 to 60 meshes.

[0023] Preferably, the particle size of the pretreated poplar flowers is ≤0.5 mm.

[0024] Furthermore, the present invention also claims protection for a method for preparing a compound pharmaceutical preparation, wherein neomycin sulfate, ursolic acid and poplar flower extract are uniformly mixed to obtain the compound pharmaceutical preparation.

[0025] Furthermore, the present invention also seeks to protect the use of a composite pharmaceutical preparation in the preparation of a drug for preventing and treating Babylonia snail diseases or in the preparation of a drug for reducing the mortality rate of Babylonia snails after the occurrence of Babylonia snail diseases.

[0026] Specifically, the disease of the baby snail described in the present invention is a new disease of the baby snail, which is called "weak myopathy". More specifically, the symptoms of "weak myopathy" are: back rolling, the most prominent feature is that the ventral foot muscles are weak, edematous, and unable to retract, and water will spray out from the ventral foot holes when touched.

[0027] Furthermore, the present invention seeks to protect a method for breeding Dongfeng snails, comprising: putting the above-mentioned composite drug preparation into a breeding water body of the diseased Dongfeng snails for soaking; or mixing the above-mentioned composite drug preparation with bait, and putting it into a breeding water body of the diseased Dongfeng snails for soaking.

[0028] Preferably, 10 to 20 g of the composite pharmaceutical preparation is added into every cubic meter of aquaculture water.

[0029] Preferably, the compound pharmaceutical preparation is added ≥ 1 time per day, and each soaking is for 1 to 2 hours. More specifically, the compound pharmaceutical preparation is added at least 2 times per day.

[0030] Preferably, after soaking, the water is changed. The flow rate of the water change is controlled to be 0.5 to 1 m 3 / h, and add oxygen to keep the dissolved oxygen in the aquaculture water ≥5mg / L.

[0031] Specifically, before adding the compound drug preparation, the completely dead baby snails are screened and removed. More specifically, the completely dead baby snails show stiff gastropod muscles and a pointed triangle at the end.

[0032] Specifically, the Babylonia snail is of the genus Babylonia (Babylonia), including but not limited to Babylonia areolata, Babylonia lutosa, Babylonia formosae, etc.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention provides a composite pharmaceutical preparation, which effectively reduces the mortality rate and survival time of diseased snails after the disease occurs during the breeding period of the baby snails, enhances the timeliness of the survival of the diseased snails, is conducive to further exploring the pathogen types, pathogen culture and vaccine research of the diseased snails, has great application value, and opens up a new disease resistance idea for the prevention and control of baby snail diseases.

[0035] Instruction Manual

[0036] Figure 1 This is a schematic diagram of the survival status of the diseased snails of the Babylonia concha collected in Example 7 on the first day.

[0037] Figure 2 This is a schematic diagram of the status of the diseased snails of the Babylonia concha collected in Example 7 on the 7th day of survival.

[0038] Figure 3 This is a schematic diagram of the status of diseased snails of the Babylonia coli collected in Comparative Example 1. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0040] Example 1 Preparation of a composite pharmaceutical preparation

[0041] (1) Preparation of poplar flower extract: Select poplar flowers with vigorous growth and no pests and diseases as raw materials, remove stems, leaves and impurities, wash with clean water for 4 times, dry until there is no moisture on the surface, put into a grinder and grind to a particle size (i.e., particle diameter) ≤ 0.5 mm to obtain pretreated poplar flowers.

[0042] The pretreated poplar flowers are mixed with a 70% ethanol aqueous solution by volume and soaked for 48 hours. The soaked mixture is placed in a reflux extractor, heated to 55°C, and reflux extracted 4 times, each time for 2 hours. The extract is preliminarily filtered with multiple layers of 40-mesh gauze to obtain a crude extract. The crude extract is concentrated by a vacuum concentrator to about 1 / 10 of the original volume, and then placed in an oven at 50°C for 24 hours to obtain a poplar flower extract.

[0043] (2) By weight, 50 parts of neomycin sulfate (source: Shanghai MacLean Biochemical Technology Co., Ltd.), 20 parts of ursolic acid (source: Xi'an Tianguangyuan Biotechnology Co., Ltd.) and 30 parts of poplar flower extract prepared in step (1) were mixed evenly to obtain a composite pharmaceutical preparation.

[0044] Example 2 Preparation of a composite pharmaceutical preparation

[0045] The difference between this embodiment and embodiment 1 is that 60 parts of neomycin sulfate, 30 parts of ursolic acid and 10 parts of poplar flower extract prepared in step (1) are evenly mixed to obtain a composite pharmaceutical preparation.

[0046] Example 3 Preparation of a composite pharmaceutical preparation

[0047] The difference between this embodiment and embodiment 1 is that 70 parts of neomycin sulfate, 10 parts of ursolic acid and 20 parts of poplar flower extract prepared in step (1) are evenly mixed to obtain a composite pharmaceutical preparation.

[0048] Example 4 Preparation of a composite pharmaceutical preparation

[0049] The difference between this embodiment and embodiment 1 is that 50 parts of neomycin sulfate, 25 parts of ursolic acid and 25 parts of poplar flower extract prepared in step (1) are evenly mixed to obtain a composite pharmaceutical preparation.

[0050] Example 5 Preparation of a composite pharmaceutical preparation

[0051] The difference between this embodiment and embodiment 1 is that the volume concentration of ethanol in step (1) is 50%; reflux extraction is performed 5 times, each time for 3 hours.

[0052] Example 6 Preparation of a composite pharmaceutical preparation

[0053] The difference between this embodiment and embodiment 1 is that the volume concentration of ethanol in step (1) is 60%; reflux extraction is performed 4 times, each time for 3 hours.

[0054] Example 7 Babylonia snail farming

[0055] Collect 100 Babylonia square-shaped snails that are diseased but not dead. The symptoms of Babylonia square-shaped snail disease are: turning over, the most prominent feature is that the ventral foot muscles are weak, edematous, and unable to retract. When touched, water will spray out from the ventral foot hole.

[0056] The composite pharmaceutical preparation prepared in Example 1 was used for drug bathing, and the specific operation was as follows:

[0057] The collected Babylonia areolata were placed in a square frame without sand or feeding. The dissolved oxygen was maintained at ≥ 5 mg / L, the temperature was 26-27°C, the pH was 8.0-8.1, and the salinity was 28-29. 20 g of the composite pharmaceutical preparation prepared in Example 1 was added to 1 cubic meter of aquaculture water. The water was used twice a day. After soaking for 1 hour, the water valve was opened to flow water (the flow rate was 0.5-1 m 3 / h), inspect every morning, noon and evening, and remove dead and sick snails in time. The total experiment period is 10 days.

[0058] Figure 1 This is a schematic diagram of the status of the diseased snails of the Babylonia snails collected in Example 7 on the first day of survival. The diseased snails are turned over and cannot return to normal on their own. The ventral foot muscles are weak and accompanied by edema and cannot retract into the shell. When the ventral foot is touched, water will spray out from the ventral foot hole. Figure 2 This is a schematic diagram of the status of diseased Babylonia snails collected in Example 7 on the 7th day of survival, among which a small number of Babylonia snails have died, while most are still in a diseased state, but can maintain this state and survive.

[0059] Example 8 Babylonia snail farming

[0060] The difference between this embodiment and embodiment 7 is that the composite pharmaceutical preparation prepared in embodiment 2 is adopted.

[0061] Example 9: Babylonia snail farming

[0062] The difference between this embodiment and embodiment 7 is that the composite pharmaceutical preparation prepared in embodiment 3 is adopted.

[0063] Example 10 Babylonia concha farming

[0064] The difference between this embodiment and embodiment 7 is that the composite pharmaceutical preparation prepared in embodiment 4 is adopted.

[0065] Comparative Example 1

[0066] The difference between this comparative example and Example 7 is that the composite pharmaceutical preparation of Example 1 is not added.

[0067] Figure 3 This is a schematic diagram of the state of diseased snails collected from Babylonia ovale in Comparative Example 1. Figure 3 As shown, all the diseased snails died after 6 hours without any treatment measures.

[0068] Comparative Example 2

[0069] The difference between this comparative example and Example 7 is that the composite pharmaceutical preparation of Example 1 contains only neomycin sulfate and ursolic acid.

[0070] Comparative Example 3

[0071] The difference between this comparative example and Example 7 is that the composite pharmaceutical preparation of Example 1 contains only neomycin sulfate and poplar flower extract.

[0072] Comparative Example 4

[0073] The difference between this comparative example and Example 7 is that the composite pharmaceutical preparation of Example 1 contains only ursolic acid and poplar flower extract.

[0074] Comparative Example 5

[0075] The difference between this comparative example and Example 7 is that the composite pharmaceutical preparation of Example 1 contains only poplar flower extract.

[0076] Comparative Example 6

[0077] The difference between this comparative example and Example 7 is that the composite pharmaceutical preparation of Example 1 contains only neomycin sulfate.

[0078] Comparative Example 7

[0079] The difference between this comparative example and Example 7 is that the composite pharmaceutical preparation of Example 1 contains only ursolic acid.

[0080] Comparative Example 8

[0081] The difference between this comparative example and Example 7 is that enrofloxacin (source: Hefei Qianfang Animal Health Technology Co., Ltd.) is used instead of neomycin sulfate.

[0082] Comparative Example 9

[0083] The difference between this comparative example and Example 7 is that florfenicol (source: Henan Nanhua Qianmu Biotechnology Co., Ltd.) is used instead of neomycin sulfate.

[0084] Test Case

[0085] Inspections were conducted three times a day, morning, noon and evening, and dead individuals were removed and the number of deaths on the day was recorded. Death judgment criteria: if the ventral foot did not move after multiple touches, it was considered dead. The test results are shown in Table 1.

[0086] Table 1

[0087]

[0088] It can be seen from the above Examples 5 to 10 that the composite pharmaceutical preparation provided by the present invention has excellent delaying effect and blocking effect on the disease of Babylonia snails, and can significantly reduce the mortality rate of Babylonia snails suffering from the disease. After using the composite pharmaceutical preparation, the number of deaths of Babylonia snails on the 10th day was reduced to less than 50. Furthermore, in Example 7, the number of deaths of Babylonia snails on the 10th day was reduced to less than 44.

[0089] In addition, the culture conditions of the snails in Examples 7 to 10 were continuously observed, and no dead snails were found after 10 days.

[0090] Comparative Examples 2 to 4 are comparative examples using any two components of the present invention, and Comparative Example 1 is a comparative example using only one component. The mortality rate of the cultured Babylonia snails reached 100% on the 5th day.

[0091] In addition, it can be seen from Example 7, Comparative Example 8 and Comparative Example 9 that when other antibacterial agents in the field are used, it is difficult to have a significant therapeutic effect on the disease of the Babylonia snail, and it is difficult to achieve the technical effect of the present invention. The mortality rate of the cultured Babylonia snails reached 100% on the 5th day.

[0092] The foregoing examples are merely illustrative and are used to explain some features of the method of the present invention. The appended claims are intended to require the widest possible range that can be imagined, and the embodiments presented herein are demonstrated by the applicant's actual test results. Therefore, the applicant's intention is that the appended claims are not limited by the selection of examples that illustrate the features of the present invention. Some numerical ranges used in the claims also include sub-ranges therein, and changes in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. A composite pharmaceutical preparation, characterized in that: The invention is composed of the following components by weight: 50 to 70 parts of neomycin sulfate, 10 to 30 parts of ursolic acid and 10 to 30 parts of poplar flower extract; The preparation method of the poplar flower extract comprises: mixing and soaking the pretreated poplar flowers with an ethanol aqueous solution, refluxing and extracting, and post-treating to obtain the poplar flower extract; In the ethanol aqueous solution, the volume concentration of ethanol is 50-70%; The temperature of reflux extraction is 50-60°C.

2. The composite pharmaceutical preparation according to claim 1, characterized in that: The post-treatment is one or more of filtration, concentration and drying.

3. The method for preparing the composite pharmaceutical preparation according to any one of claims 1 to 2, characterized in that: The compound pharmaceutical preparation is obtained by uniformly mixing neomycin sulfate, ursolic acid and poplar flower extract.

4. Use of the composite pharmaceutical preparation according to any one of claims 1 to 2 in the preparation of a drug for preventing and treating Babylonia snail diseases or in the preparation of a drug for reducing the mortality rate of Babylonia snails after the occurrence of Babylonia snail diseases, characterized in that: The disease of the Babylonia snail is weak muscle disease, and the symptoms of the weak muscle disease are: turning over, weakness and edema of the ventral foot muscles, and inability to retract, and water will spray out from the ventral foot holes when touched.

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