CN110664881A - Medicine for resisting aquatic pathogenic Citrobacter freundii and preparation method thereof - Google Patents

Medicine for resisting aquatic pathogenic Citrobacter freundii and preparation method thereof Download PDF

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CN110664881A
CN110664881A CN201810714884.5A CN201810714884A CN110664881A CN 110664881 A CN110664881 A CN 110664881A CN 201810714884 A CN201810714884 A CN 201810714884A CN 110664881 A CN110664881 A CN 110664881A
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extract
dark plum
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曹海鹏
陈思佳
叶伟东
郭成
顾颖
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Shanghai Ocean University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
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    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
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    • A—HUMAN NECESSITIES
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    • A61K31/14—Quaternary ammonium compounds, e.g. edrophonium, choline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps

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Abstract

The invention relates to a medicament for resisting aquatic pathogenic citrobacter freundii, which is a Chinese and western medicine compound preparation and contains a traditional Chinese medicine extract smoked plum extract, a garden burnet extract and a western medicine didecyl dimethyl ammonium chloride, not only can scientifically combine the advantages of high-efficiency and quick-acting antibacterial of the western medicine with the characteristics of difficult drug resistance generation and small toxic and side effects of the traditional Chinese medicine preparation, but also saves the cost.

Description

Medicine for resisting aquatic pathogenic Citrobacter freundii and preparation method thereof
Technical Field
The invention relates to a medicament for resisting aquatic pathogenic citrobacter freundii and a preparation method thereof, belonging to the technical field of aquaculture medicaments.
Background
The citrobacter freundii is a typical opportunistic pathogenic bacterium of human-animal-fish comorbidity, and can cause the death of aquaculture species such as giant salamanders, procambarus clarkii, megalobrama amblycephala and the like.
In aquaculture production, cephalosporins antibacterial drugs such as ceftriaxone, cefotaxime and the like are usually used for controlling the citrobacter freundii, but the antibacterial drugs have high production cost, large side effect, unsatisfactory antibacterial effect and universal drug resistance spectrum, and bring difficulty to the wide application of the drugs in the control of the citrobacter freundii (separation and identification of citrobacter freundii in diseased giant salamanders [ J ] microbiological report 2012, 52(2):169 and 176. separation and identification of citrobacter freundii as a pathogen of procambarus clarkii and susceptibility and adhesion characteristics [ J ] aquatic report 2016, 40 (6): 946 and 955).
Therefore, it is highly necessary to develop a new formulation against the pathogenic bacterium citrobacter freundii.
The compound preparation of Chinese and western medicines is a new medicine, is a compound preparation after the Chinese and western medicines are combined (trial and review Chinese and western medicine compound preparation [ J ], Chinese veterinary medicine journal, 1999(02):49-53.), scientifically combines the advantages of high-efficiency and quick-acting antibacterial of the western medicines and the characteristics of difficult generation of drug resistance and small toxic and side effects of the Chinese medicine preparation, and has the advantages that the single use of the western medicines and the single use of the Chinese medicines are difficult to compare.
Therefore, the development of a novel preparation of the pathogenic citrobacter freundii for resisting aquatic production from the perspective of the compound of the Chinese and western medicines is also a new idea, and the development of a novel preparation of the pathogenic citrobacter freundii for resisting aquatic production scientifically combines the advantages of high efficiency and quick-acting antibiosis of the western medicines and the characteristics of difficult generation of drug resistance and small toxic and side effects of the Chinese medicinal preparation together on one hand, more effectively controls the pathogenic citrobacter freundii for aquatic production, and reduces the occurrence rate of aquatic animal diseases caused by the pathogenic citrobacter freundii; on the other hand, the cost of the medicine is reduced.
Disclosure of Invention
In order to solve the technical problems, the invention provides a medicament for resisting aquatic pathogenic citrobacter freundii, which is a Chinese and western medicine compound preparation, and is an aqueous solution containing dark plum extract, garden burnet extract and didecyl dimethyl ammonium chloride, wherein the concentration of crude drugs contained in the dark plum extract is 0.4-0.6 g/ml; the radix Sangusorbae extract contains crude drug with concentration of 0.08-0.12 g/ml; the concentration of the didecyl dimethyl ammonium chloride is 0.28-0.36 g/ml.
Preferably, the crude drug concentration of the dark plum extract is 0.5 g/ml; the radix Sangusorbae extract contains crude drug with concentration of 0.1 g/ml; the concentration of the didecyl dimethyl ammonium chloride is 0.32 g/ml.
The invention also provides a preparation method of the drug for resisting the aquatic pathogenic Citrobacter freundii, which comprises the steps of preparing a dark plum extracting solution, preparing a sanguisorba extracting solution and mixing the obtained dark plum extracting solution, the sanguisorba extracting solution and a didecyldimethylammonium chloride solution.
Preferably, the step of preparing the dark plum extract comprises: according to the proportion of adding 6-15 g of dark plum into every 100ml of water, soaking the dark plum in the water for 20-60 minutes, then decocting, wherein the decocting process is to boil the water and then boil the water for 20-60 minutes, and filtering out filtrate; decocting for 2-5 times, mixing the filtrates, and concentrating until the crude drug concentration of the dark plum extract is 0.8-1.2 g/ml, to obtain the dark plum extract; the preparation method of the garden burnet extract comprises the following steps: soaking radix Sangusorbae in water for 20-60 min at a ratio of 8-12g radix Sangusorbae per 100ml water, decocting, wherein the decocting process is boiling water, decocting with slow fire for 20-60 min, and filtering to obtain filtrate; decocting for 2-5 times, mixing the filtrates, and concentrating until the concentration of the crude drug in the sanguisorba extract is 0.8-1.2 g/ml, thereby obtaining the sanguisorba extract.
Preferably, the dark plum extract and the garden burnet extract are mixed and uniformly stirred for 0.5 to 1.0 hour at room temperature under the condition of 30 to 50r/min, and then a didecyl dimethyl ammonium chloride solution with the concentration of 0.7 to 0.9g/ml is added, and the uniform stirring is continuously carried out for 0.5 to 1.0 hour at room temperature under the condition of 30 to 50 r/min.
The medicament for resisting the aquatic pathogenic citrobacter freundii is a Chinese and western medicine compound preparation, is an aqueous solution containing traditional Chinese medicine extracts (dark plum extract and garden burnet extract) and western medicine (didecyl dimethyl ammonium chloride), can scientifically combine the advantages of high-efficiency and quick-acting antibacterial of the western medicine and the characteristics of difficult drug resistance generation and small toxic and side effects of the traditional Chinese medicine preparation, saves the cost, and more importantly, the inventor unexpectedly discovers that when the dark plum extract, the garden burnet extract and the didecyl dimethyl ammonium chloride with specific concentrations are adopted, the compounding of the dark plum extract, the garden burnet extract and the didecyl dimethyl ammonium chloride can be synergistically promoted, and the aquatic pathogenic citrobacter freundii can be effectively resisted.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to these specific embodiments.
The explanation about the terms is as follows:
the term "crude drug-containing concentration" refers to the concentration obtained by dividing the weight of all natural unprocessed or simply processed Chinese medicinal materials (plants, animals and minerals) in a prescription by the volume of a decoction.
In a specific embodiment of the invention, the invention provides a medicament for resisting aquatic pathogenic Citrobacter freundii, wherein the medicament is a Chinese and western medicine compound preparation, and is an aqueous solution containing a dark plum extract, a garden burnet extract and didecyl dimethyl ammonium chloride, and the concentration of crude drugs contained in the dark plum extract is 0.4-0.6 g/ml; the radix Sangusorbae extract contains crude drug with concentration of 0.08-0.12 g/ml; the concentration of the didecyl dimethyl ammonium chloride is 0.28-0.36 g/ml.
In a preferred embodiment of the present invention, the concentration of the crude drug contained in the ume extract is 0.5 g/ml; the radix Sangusorbae extract contains crude drug with concentration of 0.1 g/ml; the concentration of the didecyl dimethyl ammonium chloride is 0.32 g/ml.
Regarding the research and development of the drug for resisting the pathogenic aquatic product Citrobacter freundii, the inventor screens out a plurality of traditional Chinese medicine candidates with better effects including a dark plum extract and a sanguisorba officinalis extract from a large number of traditional Chinese medicine candidates, and screens out a plurality of western medicine candidates with better effects including a didecyl dimethyl ammonium chloride solution from a large number of western medicine candidates.
According to the components and action mechanism of the substances, the inventor can conclude that organic acids and antioxidant substances in the dark plum extract, saponin, chrysophanol and other active substances in the garden burnet extract can inhibit the growth of bacteria, and didecyl dimethyl ammonium chloride can change the permeability of the bacterial cell serosa, so that bacterial substances are infiltrated outwards to kill the bacteria. However, when the smoked plum extract and the sanguisorba officinalis extract are used independently, the bacteriostatic action on aquatic pathogenic citrobacter freundii is weak, the dosage is large, the curative effect is not obvious or the effect is slow, when the didecyl dimethyl ammonium chloride serving as a western medicine is used independently, the toxic and side effects are large, the medicine residue is large, the environmental pollution is large, the aquatic pathogenic citrobacter freundii is easy to generate the medicine resistance to cause the sensitivity to be reduced, and the antibacterial effect is easy to be reduced due to the influence of organic matters in the water environment.
The inventor carries out any compound combination of the traditional Chinese medicine and western medicine candidates, and makes a large number of combination experiments, and unexpectedly discovers that when the traditional Chinese medicine and western medicine compound preparation prepared by compounding the dark plum extract, the garden burnet extract and the didecyl dimethyl ammonium chloride solution with specific concentrations is adopted, the compound of the three can be synergistically promoted, and the aquatic pathogenic Citrobacter freundii can be effectively resisted. After the inventor obtains the unexpected finding, the inventor further conducts the research of the drug mechanism, and speculates that according to the research result: the active ingredients in the dark plum and garden burnet extracts can enable the active ingredients of didecyl dimethyl ammonium chloride to be easily gathered on the surfaces of cells, the capability of destroying the cell walls and cell membranes of aquatic pathogenic citrobacter freundii thalli is enhanced, the activity of enzyme which is generated in the aquatic pathogenic citrobacter freundii and inactivates western medicines is inhibited, the sensitivity of the aquatic pathogenic citrobacter freundii to the western medicines is improved, the antibacterial activity to the aquatic pathogenic citrobacter freundii is obviously enhanced, and the interference capability to organic matters in aquaculture water is enhanced; therefore, the combination of the Chinese medicine and the western medicine reduces the using amount of the western medicine, reduces the pollution to the environment and the toxic and side effect, and is favorable for avoiding the generation of drug resistance of the aquatic pathogenic Citrobacter freundii to the medicine.
Example 1
The medicine for resisting the aquatic pathogenic citrobacter freundii is a Chinese and western medicine compound preparation, and is an aqueous solution containing a dark plum extract, a sanguisorba extract and didecyl dimethyl ammonium chloride, wherein the concentration of crude drugs contained in the dark plum extract is 0.5g/ml, the concentration of crude drugs contained in the sanguisorba extract is 0.1g/ml, and the concentration of didecyl dimethyl ammonium chloride is 0.32 g/ml.
The preparation method comprises the following steps:
firstly, respectively preparing dark plum extract and sanguisorba extract, and then mixing the obtained dark plum extract, sanguisorba extract and didecyl dimethyl ammonium chloride solution, which is as follows.
The preparation method of the dark plum extract comprises the following steps: according to the proportion that 10g of dark plum is added into each 100ml of water, the dark plum is soaked in the water for 30 minutes and then decocted, the decocting process is that the water is boiled and then decocted for 30 minutes by slow fire, and the filtrate is filtered; decocting for 3 times, mixing filtrates, and concentrating to obtain mume fructus extract with crude drug concentration of 1g/ml to obtain mume fructus extractive solution with crude drug concentration of 1 g/ml;
the preparation method of the garden burnet extract comprises the following steps: soaking sanguisorba officinalis in water for 30 minutes according to the proportion that 10g of sanguisorba officinalis is added into every 100ml of water, decocting, wherein the decocting process is to boil the water and then boil the water for 30 minutes with slow fire, and filtering out filtrate; decocting for 3 times, mixing filtrates, and concentrating until the concentration of radix Sangusorbae extract containing crude drug is 1g/ml to obtain radix Sangusorbae extractive solution containing crude drug at 1 g/ml;
mixing the obtained mume fructus extract 50ml and radix Sangusorbae extract 10ml, stirring at room temperature under 30-50r/min for 0.5-1.0 hr, adding 0.8g/ml didecyl dimethyl ammonium chloride solution 40ml, and stirring at room temperature under 30-50r/min for 0.5-1.0 hr to obtain 100ml medicine for resisting aquatic pathogenic Citrobacter freundii.
Effect data
To demonstrate the efficacy of the anti-pathogenic bacterial strain of citrobacter freundii of example 1, the inventors selected three conventional pathogenic bacterial strains of citrobacter freundii, namely, pathogenic bacterial strain LGB1 of snakehead, pathogenic bacterial strain HD0103 of sturgeon, and pathogenic bacterial strain MINA freundii of eel, all of which were provided by the aquatic animal pathogen pool of Shanghai ocean university.
Three bacterial suspensions of the above-mentioned citrobacter freundii were first prepared: respectively picking up pathogenic Citrobacter freundii from solid inclined planeStreaking and inoculating a nutritional agar plate with bacterial lawn of LGB1, eel pathogenic Citrobacter freundii MINA and sturgeon pathogenic Citrobacter freundii HD0103, placing in a constant-temperature incubator at 28 ℃, and culturing for 24 h; selecting single colony in nutrient broth liquid culture medium, shake culturing at 28 deg.C for 18 hr at 200r/min, and adjusting bacteria liquid concentration to 10 with sterile normal saline7cfu/mL, stored in a refrigerator at 4 ℃ for later use.
Control group 1: extracting mume fructus;
control group 2: extracting solution of garden burnet;
control group 3: aqueous solution of didecyl dimethyl ammonium chloride;
control group 4: an aqueous solution of cefotaxime sodium;
control group 5: an aqueous solution of gentamicin sulfate;
wherein, the control groups 4 and 5 are both conventional antibacterial western medicines; in addition, blank sterile nutrient broth of the same culture conditions was used as a negative control;
the minimum inhibitory concentrations of the drugs of the control groups 1 to 5 and example 1 against the three conventional pathogenic citrobacter freundii for aquatic products were measured, respectively, and the mass concentration (or crude drug-containing concentration) of the drug that grew aseptically and was diluted to the maximum was taken as the minimum inhibitory concentration (mg/L) of the drug against the pathogenic citrobacter freundii for aquatic products.
See table 1 below for results;
TABLE 1
Figure BDA0001717236280000051
Figure BDA0001717236280000061
As can be seen from the results in table 1, the minimum inhibitory concentration of the drug against pathogenic aquatic pathogenic citrobacter freundii of the present invention in example 1 is much lower than that of the conventional western drug (cefotaxime sodium or gentamicin sulfate), which proves that the drug of the present invention in example 1 has excellent effect against pathogenic aquatic pathogenic citrobacter freundii;
in addition, the minimum inhibitory concentration of the drug against the pathogenic aquatic citrobacter freundii in the embodiment 1 of the present invention is not only lower than that of a single western medicine medicament (aqueous solution of didecyldimethylammonium chloride), but also far lower than that of a single traditional Chinese medicine medicament (dark plum and sanguisorba officinalis extract), so that the synergistic effect between the three components in the drug in the embodiment 1 of the present invention is proved; taking the data of the pathogenic Citrobacter freundii LGB1 of the snakehead as an example, the synergistic action index (FIC index) among the three components in the medicine of the embodiment 1 of the invention is calculated by the following formula:
FIC index ═ MIC (example 1 drug)/MIC (dark plum alone) + MIC (example 1 drug)/MIC (garden burnet alone) + MIC (example 1 drug)/MIC (didecyldimethylammonium chloride alone)
The FIC value is calculated to be 0.307 (less than 0.5), which shows that the compounding of the dark plum extract, the garden burnet extract and the didecyl dimethyl ammonium chloride solution has synergistic promotion effect on resisting aquatic pathogenicity Citrobacter freundii.
In conclusion, the medicine for resisting the aquatic pathogenic citrobacter freundii is a Chinese and western medicine compound preparation, is an aqueous solution containing traditional Chinese medicine extracts (dark plum extract and garden burnet extract) and western medicine (didecyl dimethyl ammonium chloride), can scientifically combine the advantages of high-efficiency and quick-acting antibacterial of the western medicine and the characteristics of difficult drug resistance generation and small toxic and side effects of the traditional Chinese medicine preparation, saves the cost, and more importantly, the inventor unexpectedly discovers that when the dark plum extract, the garden burnet extract and the didecyl dimethyl ammonium chloride with specific concentrations are adopted, the compounding of the dark plum extract, the garden burnet extract and the didecyl dimethyl ammonium chloride can be synergistically promoted, and the aquatic pathogenic citrobacter freundii can be effectively resisted.
It should be understood that although the present description refers to embodiments, not every embodiment contains only a single technical solution, and such description is for clarity only, and those skilled in the art should make the description as a whole, and the technical solutions in the embodiments can also be combined appropriately to form other embodiments understood by those skilled in the art.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.

Claims (5)

1. A medicament against pathogenic aquatic citrobacter freundii, characterized in that:
the medicine is a compound preparation of Chinese and western medicines, which is an aqueous solution containing dark plum extract, garden burnet extract and didecyl dimethyl ammonium chloride,
the crude drug concentration of the mume fructus extract is 0.4-0.6 g/ml;
the radix Sangusorbae extract contains crude drug with concentration of 0.08-0.12 g/ml;
the concentration of the didecyl dimethyl ammonium chloride is 0.28-0.36 g/ml.
2. The anti-aquatic pathogenic Citrobacter freundii medicament of claim 1, wherein:
the crude drug concentration of the dark plum extract is 0.5 g/ml;
the radix Sangusorbae extract contains crude drug with concentration of 0.1 g/ml;
the concentration of the didecyl dimethyl ammonium chloride is 0.32 g/ml.
3. The method of claim 1 for the preparation of a medicament against aquapathogenic Citrobacter freundii, characterized in that:
the preparation method comprises the steps of preparing dark plum extract, preparing sanguisorba extract and mixing the obtained dark plum extract, the obtained sanguisorba extract and didecyl dimethyl ammonium chloride solution.
4. The method of claim 3, wherein:
the preparation method of the dark plum extract comprises the following steps: according to the proportion of adding 6-15 g of dark plum into every 100ml of water, soaking the dark plum in the water for 20-60 minutes, then decocting, wherein the decocting process is to boil the water and then boil the water for 20-60 minutes, and filtering out filtrate; decocting for 2-5 times, mixing the filtrates, and concentrating until the crude drug concentration of the dark plum extract is 0.8-1.2 g/ml, to obtain the dark plum extract;
the preparation method of the garden burnet extract comprises the following steps: soaking radix Sangusorbae in water for 20-60 min at a ratio of 8-12g radix Sangusorbae per 100ml water, decocting, wherein the decocting process is boiling water, decocting with slow fire for 20-60 min, and filtering to obtain filtrate; decocting for 2-5 times, mixing the filtrates, and concentrating until the concentration of the crude drug in the sanguisorba extract is 0.8-1.2 g/ml, thereby obtaining the sanguisorba extract.
5. The method of claim 4, wherein:
and mixing the dark plum extract and the garden burnet extract, uniformly stirring for 0.5-1.0 hour at room temperature under the condition of 30-50r/min, then adding a didecyl dimethyl ammonium chloride solution with the concentration of 0.7-0.9 g/ml, and continuously uniformly stirring for 0.5-1.0 hour at room temperature under the condition of 30-50 r/min.
CN201810714884.5A 2018-07-03 2018-07-03 Medicine for resisting aquatic pathogenic Citrobacter freundii and preparation method thereof Pending CN110664881A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017049242A2 (en) * 2015-09-17 2017-03-23 Contrafect Corporation Use of lysin to restore/augment antibacterial activity in the presence of pulmonary surfactant of antibiotics inhibited thereby

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017049242A2 (en) * 2015-09-17 2017-03-23 Contrafect Corporation Use of lysin to restore/augment antibacterial activity in the presence of pulmonary surfactant of antibiotics inhibited thereby

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
国家食品药品监督管理总局执业药师资格认证中心: "《中药学综合知识与技能》", 31 January 2016, 中国医药科技出版社 *
杨移斌等: "41种中草药对3种鲟源病原菌的体外抑菌效果", 《淡水渔业》 *

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