Application of isoquinoline alkaloid in macleaya cordata to preparation of product for preventing and treating bacterial pathogenic bacteria of aquatic animals

Abstract

The invention provides application of isoquinoline alkaloid in macleaya cordata to preparation of a product for preventing and treating bacterial pathogenic bacteria of aquatic animals, and relates to the field of macleaya cordata extracts. The alkaloid in the macleaya cordata is used for preparing a product for preventing and / or treating related diseases caused by bacterial pathogenic bacteria of aquatic animals; the alkaloid is sanguinarine and / or chelerythrine; the aquatic animal bacterial pathogenic bacteria are at least one of vibrio harveyi, vibrio vulnificus, vibrio mimicus and vibrio aerogenes. Through tests, alkaloids (sanguinarine and chelerythrine) in macleaya cordata show excellent antibacterial activity on the aquatic animal bacterial pathogenic bacteria, and can be effectively applied to preparation of related products aiming at related diseases caused by the aquatic animal bacterial pathogenic bacteria; the isoquinoline alkaloid in macleaya cordata has the advantages of being green, efficient, low in toxicity, low in residue and not prone to generating resistance.

Description

Technical Field

[0001] The invention relates to the field of Macleaya cordata extract, in particular to use of isoquinoline alkaloids in Macleaya cordata in preparing products for preventing and treating bacterial pathogens of aquatic animals. Background Art

[0002] Bacterial diseases are common diseases that cause serious harm to aquatic animals. They are caused by bacterial pathogens, especially bacterial hemorrhagic disease and wound bacterial infection caused by Vibrio, which are the most serious harm to fishery production. Every year, sporadic cases are reported in coastal areas. The disease is fierce and the mortality rate of aquatic animals is high. It will enter the human body through human diet, causing serious harm to human health and even death. For bacterial diseases in aquatic animals, the most commonly used preventive and therapeutic drugs are chemical synthetic drugs such as anthrafloxacin and gentamicin sulfate and antibiotics. However, its disadvantage is that it will produce relatively serious drug resistance during long-term use, and some chemical drugs will remain and accumulate in the body of aquatic animals, enter the human body through the food chain, and endanger human health. At present, the abuse of drugs has a great impact on the healthy breeding of aquatic animals, the ecological environment and human health. Therefore, the prevention and control of aquatic animal diseases should not only focus on eliminating the hidden dangers of aquatic product quality and safety, but also avoid the adverse effects caused by the abuse of drugs. Based on this, finding safe and effective alternative antibiotics to prevent and control pathogenic bacteria in aquatic animals is an important research topic for green prevention and control of aquatic animal diseases.

[0003] Natural products have always been an important source in the field of drug research and development; in the past two decades, more than one-third of FDA-approved drugs are natural products and their derivatives. Compared with chemically synthesized compounds, natural products have some unique properties, such as higher structural complexity, more novel skeletons, better drug-like properties, etc. Therefore, natural products are a treasure trove worthy of attention and have great potential for drug acquisition.

[0004] Macleaya cordata (Willd.) R.Br. is a perennial tall herbaceous plant of the genus Macleaya in the family Papaveraceae. There are two species in the genus, namely Macleaya cordata and Macleaya microcarpa (Maxim) Fedde. According to ancient medical books, both Macleaya cordata and Macleaya microcarpa can be used as medicine. According to records, Macleaya cordata is bitter and cold in nature, and has the effects of killing insects and relieving itching, clearing away heat and detoxifying. Modern research has found that its effects are more extensive. The chemical components of Macleaya plants are mainly alkaloids, with a total alkaloid content of up to 5%. The main bioactive substances in Macleaya plants are isoquinoline alkaloids, such as sanguinarine, chelerythrine, protopine, allocryptine, etc. Modern pharmacological studies have shown that the main active ingredients in Macleaya are sanguinarine and chelerythrine, which have multiple pharmacological effects and uses, such as anti-inflammatory, insecticidal, maggot killing, inhibition of various pathogens, antiviral, heat-clearing and detoxifying, enhancement of human immune function and anti-tumor, and are widely used in medicine, biological pesticides, veterinary drugs, fine chemical products and other fields.

[0005] Therefore, the functions of the isoquinoline alkaloids in Macleaya cordata are further developed, their application fields are expanded, and the use of the isoquinoline alkaloids in Macleaya cordata in the preparation of products for preventing and treating bacterial pathogens of aquatic animals is provided, specifically the use of the isoquinoline alkaloids in Macleaya cordata in preventing and / or treating related diseases caused by bacterial pathogens of aquatic animals, which has important technical significance and research value. Summary of the invention

[0006] In order to solve the technical problems existing in the prior art, the present invention provides the use of isoquinoline alkaloids in Macleaya cordata in preparing products for preventing and treating bacterial pathogens of aquatic animals, especially the use of isoquinoline alkaloids in Macleaya cordata in preventing and / or treating related diseases caused by bacterial pathogens of aquatic animals.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: Use of isoquinoline alkaloids in Macleaya cordata in preparing products for preventing and treating bacterial pathogens of aquatic animals, and use of isoquinoline alkaloids in Macleaya cordata in preparing products for preventing and / or treating related diseases caused by bacterial pathogens of aquatic animals; The isoquinoline alkaloids in the Macleaya cordata are sanguinarine and / or chelerythrine.

[0008] The chemical structural formula of the sanguinarine and chelerythrine is: .

[0009] The bacterial pathogens of aquatic animals are at least one of the following: Vibrio harveyi Vibrio harveyi , Vibrio vulnificus Vibrio vulnificus , Vibrio mimicus Vibrio mimicus , Vibrio aerogenes Vibrio aerogenes .

[0010] The related diseases include: bacterial hemorrhagic diseases and bacterial infections.

[0011] Furthermore, the product is selected from: a pharmaceutical composition, a feed additive.

[0012] The pharmaceutical composition adopts at least one isoquinoline alkaloid or its salt in Macleaya cordata as an effective component or a main effective component, and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable excipient.

[0013] The feed additive uses at least one isoquinoline alkaloid or its salt in Macleaya cordata as an effective component or a main effective component, and is prepared into a nutritionally acceptable dosage form with a nutritionally acceptable excipient.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention uses isoquinoline alkaloids (sanguinarine and chelerythrine) in Macleaya cordata to target bacterial pathogens of aquatic animals (Vibrio harveyi Vibrio harveyi BNCC 336937, Vibrio vulnificus Vibrio vulnificus BNCC 186281, Vibrio mimicus Vibrio mimicus BNCC 195661, Vibrio aerogenes Vibrio aerogenes BNCC 362836), were screened for inhibitory activity, and all showed excellent antibacterial inhibition, which is of great application value in preventing and treating bacterial pathogens of aquatic animals. The purpose of using isoquinoline alkaloids (sanguinarine, chelerythrine) in Macleaya cordata in preventing and / or treating related diseases caused by bacterial pathogens of aquatic animals is provided; the application field of isoquinoline alkaloids in Macleaya cordata is further expanded, which has broad application prospects.

[0015] (2) The isoquinoline alkaloids (sanguinarine and chelerythrine) in the Macleaya cordata of the present invention are used to prevent and treat related diseases (especially bacterial hemorrhagic diseases and bacterial infections caused by them) caused by bacterial pathogens of aquatic animals (Vibrio harveyi, Vibrio vulnificus, Vibrio mimicus and Vibrio aerogenes). The isoquinoline alkaloid components (sanguinarine and chelerythrine) in Macleaya cordata are natural plant-derived antibacterial agents obtained from natural plants, and have the advantages of low toxicity, low residue and low resistance. They are unique and irreplaceable in preventing and treating bacterial pathogens of aquatic animals. In addition, Macleaya cordata is widely distributed in my country and has abundant resources. It is green, efficient and not easy to develop drug resistance. It has high application value in preventing and treating related diseases caused by bacterial pathogens of aquatic animals and is worthy of further research and development.

[0016] (3) Using the isoquinoline alkaloid (sanguinarine) in Macleaya cordata described in the present invention, at a dosage concentration of 100 μg / mL, the activity of sanguinarine against Vibrio harveyi Vibrio harveyi BNCC 336937, Vibrio vulnificus Vibrio vulnificus BNCC 186281, Vibrio mimicus Vibrio mimicus BNCC 195661, Vibrio aerogenes Vibrio aerogenes The inhibition rate of BNCC 362836 can reach 100%; further, sanguinarine has an inhibitory effect on Vibrio harveyi Vibrio Harvey BNCC 336937, Vibrio vulnificus Vibrio vulnificus BNCC 186281, Vibrio mimicus Vibrio mimic BNCC 195661, Vibrio aerogenes Vibrio aerogenes The minimum inhibitory concentration (MIC) of BNCC 362836 is 25-100 μg / mL, and it can be effectively used in related products to prevent and / or treat related diseases caused by the aforementioned bacterial pathogens of aquatic animals.

[0017] (4) Using the isoquinoline alkaloid (chelerythrine) in Macleaya cordata described in the present invention, at a dosage concentration of 100 μg / mL, chelerythrine had an inhibitory effect on Vibrio harveyi. Vibrio harveyi BNCC 336937, Vibrio vulnificus Vibrio vulnificus BNCC 186281, Vibrio mimicus Vibrio mimicus BNCC 195661, Vibrio aerogenes Vibrio aerogenes The inhibition rate of BNCC 362836 can reach 100%; further, chelerythrine has an inhibitory effect on Vibrio harveyi Vibrio Harvey BNCC 336937, Vibrio vulnificus Vibrio vulnificus BNCC 186281, Vibrio mimicus Vibrio mimic BNCC 195661, Vibrio aerogenes Vibrio aerogenes The minimum inhibitory concentration (MIC) of BNCC 362836 is 25-100 μg / mL, and it can be effectively used in related products to prevent and / or treat related diseases caused by the aforementioned bacterial pathogens of aquatic animals. DETAILED DESCRIPTION

[0018] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific embodiments of the present invention are now described. It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0019] The embodiment of the present invention provides the use of isoquinoline alkaloids in Macleaya cordata in preparing products for preventing and treating bacterial pathogens of aquatic animals, and the use of isoquinoline alkaloids in Macleaya cordata in preparing products for preventing and / or treating related diseases caused by bacterial pathogens of aquatic animals; The isoquinoline alkaloids in the Macleaya cordata are sanguinarine and / or chelerythrine.

[0020] The chemical structural formula of the sanguinarine and chelerythrine is: .

[0021] The bacterial pathogens of aquatic animals are at least one of the following: Vibrio harveyi Vibrio harveyi , Vibrio vulnificus Vibrio vulnificus , Vibrio mimicus Vibrio mimicus , Vibrio aerogenes Vibrio aerogenes .

[0022] The related diseases include: bacterial hemorrhagic diseases and bacterial infections.

[0023] Furthermore, the product is selected from: a pharmaceutical composition, a feed additive.

[0024] The pharmaceutical composition adopts at least one isoquinoline alkaloid or its salt in Macleaya cordata as an effective component or a main effective component, and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable excipient.

[0025] The feed additive uses at least one isoquinoline alkaloid or its salt in Macleaya cordata as an effective component or a main effective component, and is prepared into a nutritionally acceptable dosage form with a nutritionally acceptable excipient.

[0026] The aforementioned alkaloid salt refers to a derivative of sanguinarine or chelerythrine; the parent compound is modified by making an acid salt or an alkaline salt of the parent compound. Examples of compound salts include, but are not limited to: inorganic acid salts or organic acid salts of basic residues (such as amine groups); alkaline salts or organic salts of acidic residues (such as carboxylic acids, etc.). The alkaloid salts of the present invention can be synthesized from a parent compound containing a basic part or an acidic part by conventional chemical methods. In addition, the aforementioned alkaloid salts can be prepared in situ during the final separation and purification of the compound.

[0027] The aforementioned excipients refer to any excipients, solvents, dispersion media, absorption blockers, diluents or adjuvants that do not cause secondary reactions such as allergic reactions in humans or animals; such as preservatives or antioxidants, fillers, binders, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersion media, coating agents, antibacterial agents, isotonic agents and absorption delay agents. Typical examples of excipients include, but are not limited to, mannitol, lactose, magnesium stearate, sodium saccharide, talc, cellulose, cross-linked sodium carboxymethyl cellulose, glucose, gelatin, starch, lactose, dicalcium phosphate, sucrose, kaolin, magnesium carbonate, wetting agents, emulsifiers, solubilizers, sterile water, physiological saline, pH buffers, nonionic surfactants, lubricants, stabilizers, binders and edible oils (such as peanut oil, sesame oil, etc.). At the same time, the use of the aforementioned excipients in the pharmaceutical composition or feed additives is foreseeable.

[0028] The present invention is further described below in conjunction with some specific embodiments.

[0029] Example 1 This example provides an activity determination of isoquinoline alkaloids (sanguinarine / chelerythrine) in Macleaya cordata against bacterial pathogens of aquatic animals, specifically: 1) Test agents: sanguinarine, chelerythrine; 2) Test bacteria: Vibrio harveyi Vibrio harveyi BNCC 336937, Vibrio vulnificus Vibrio vulnificus BNCC 186281, Vibrio mimicus Vibrio mimicus BNCC 195661, Vibrio aerogenes Vibrio aerogenes BNCC 362836.

[0030] The test strains were obtained by purchasing freeze-dried powder from Beina Biotech (Beijing Beina Chuanglian Biotechnology Research Institute) through commercial channels.

[0031] 3) Test method: The freeze-dried powder of Vibrio perfringens was opened in a safety cabinet, the top was burned with alcohol, sterile water was dripped on it to break it, and then it was broken with tweezers and 0.5 mL was drawn out. 2216E marine liquid medium (peptone: 5.0 g, yeast extract: 1.0 g, ferric citrate: 0.1 g, potassium bromide: 0.08 g, NaCl: 19.45 g, MgCl2: 5.98 g, Na2SO4: 3.24 g, CaCl2: 1.8 g, KCl: 0.55 g, Na2CO3: 0.16 g, NaNO3: 0.0016 g, Na2HPO4: 0.008 g, strontium chloride: 0.034 g, boric acid: 0.022 g, sodium silicate: 0.004 g, sodium fluoride: 0.0024 g, distilled water: 1 L; sterilized at 121 °C for 20 min) was poured into a freeze-dried tube, fully dissolved and poured back into the liquid test tube, mixed well; then 0.2 mL of bacterial suspension was poured into the plate and spread evenly. Repeat twice to obtain two 2216E seawater agar plates (peptone: 5.0 g, yeast extract: 1.0 g, ferric citrate: 0.1 g, potassium bromide: 0.08 g, NaCl: 19.45 g, MgCl2: 5.98 g, Na2SO4: 3.24 g, CaCl2: 1.8 g, KCl: 0.55 g, Na2CO3: 0.16 g, NaNO3: 0.0016 g, Na2HPO4: 0.008 g, strontium chloride: 0.034 g, boric acid: 0.022 g, sodium silicate: 0.004 g, sodium fluoride: 0.0024 g, agar: 15.0 g, distilled water: 1 L; sterilize at 121°C for 20 min). Cultivate at 30°C until bacteria grow out; then streak on 2216E seawater agar plates and culture at 30°C until single colonies grow out. Pick single colonies and culture them in 2216E liquid culture medium at 30°C, 150rpm constant temperature shaker until logarithmic growth phase.

[0032] Similarly, the freeze-dried powders of Vibrio harveyi, Vibrio vulnificus, and Vibrio mimicus were taken out and incubated in BHI solid medium (peptone: 5.0 g, yeast extract powder: 1.0 g, ferric citrate: 0.1 g, NaCl: 19.45 g, potassium bromide: 0.08 g, MgCl2: 5.98 g, Na2SO4: 3.24 g, CaCl2: 1.8 g, KCl: 0.55 g, Na2CO3: 0.16 g, NH 4NO3: 0.00116g, Na2HPO4: 0.008g, strontium chloride: 0.034g, boric acid: 0.022g, sodium silicate: 0.004g, sodium fluoride: 0.0024g, agar: 20.0g, distilled water: 1L; sterilize at 121℃ for 20min), and culture at a constant temperature of 30℃ until bacteria grow; then streak on BHI solid plates respectively, and culture at a constant temperature of 30℃ until single colonies grow. Single colonies were picked and placed into BHI liquid medium (peptone: 5.0 g, yeast extract powder: 1.0 g, ferric citrate: 0.1 g, NaCl: 19.45 g, potassium bromide: 0.08 g, MgCl2: 5.98 g, Na2SO4: 3.24 g, CaCl2: 1.8 g, KCl: 0.55 g, Na2CO3: 0.16 g, NH4NO3: 0.00116 g, Na2HPO4: 0.008 g, strontium chloride: 0.034 g, boric acid: 0.022 g, sodium silicate: 0.004 g, sodium fluoride: 0.0024 g, distilled water: 1 L; sterilized at 121°C for 20 min) and cultured in a constant temperature shaker at 30°C and 150 rpm until the logarithmic growth phase.

[0033] Then, each strain in the logarithmic growth phase was diluted with the corresponding liquid culture medium to a bacterial concentration of about 1×10 6 CFU / mL for later use. Dissolve the isoquinoline alkaloids (sanguinarine / chelerythrine) in Macleaya cordata with DMSO, add them to the liquid culture medium, mix them evenly, and prepare sanguinarine and chelerythrine into a drug-containing liquid culture medium with a concentration of 200 μg / mL. Take 50 μL of the drug-containing culture medium and the same volume of about 1×10 6 CFU / mL bacterial culture was added to the wells of a 96-well plate, and the final administration concentration of isoquinoline alkaloids (sanguinarine or chelerythrine) in Macleaya cordata was controlled to be 100μg / mL. A 100μL bacterial solution of the same concentration containing an equal amount of DMSO was used as a control. The 96-well plate was cultured in a 30℃ constant temperature incubator for 12-24h until the bacterial solution of the control group grew, and the OD value (OD 600 ). In addition, the OD values ​​of 100 μL of liquid culture medium and the drug with a concentration of 100 μg / mL were measured to correct the OD values ​​caused by the culture medium and the drug itself. The calculation formula for the corrected OD value and inhibition rate is as follows: Corrected OD value = OD value of bacterial culture medium - OD value of sterile culture medium; Inhibition rate = [(corrected OD value of bacterial solution in control culture medium - corrected OD value of drug-containing culture medium) / corrected OD value of bacterial solution in control culture medium] × 100%.

[0034] All the above experiments were repeated three times to determine the inhibition rate (%) of each test agent on each aquatic animal bacterial pathogen. The specific test results are shown in the following table:

[0035] It can be seen from the above test results that the isoquinoline alkaloids (sanguinarine / chelerythrine) in the Macleaya cordata of the present invention show excellent antibacterial activity against Vibrio harveyi, Vibrio vulnificus, Vibrio mimicus and Vibrio aerogenes.

[0036] Example 2 On the basis of Example 1, the drug-containing liquid culture medium of isoquinoline alkaloids (sanguinarine or chelerythrine) in Macleaya cordata was diluted by a two-fold dilution method in a 96-well plate to obtain 50 μL of drug-containing culture medium of a series of concentrations, and then the inhibition rate corresponding to the series of concentrations was determined according to the same test method in Example 1; the lowest concentration with an inhibition rate greater than 90% was defined as the minimum inhibitory concentration MIC, and the MIC value of each test agent for each aquatic animal bacterial pathogen was determined. The specific measurement results are shown in the following table:

[0037] From the above test results, it can be seen that the MIC values ​​of isoquinoline alkaloids (sanguinarine / chelerythrine) in Macleaya cordata against the test strains (Vibrio harveyi, Vibrio vulnificus, Vibrio mimics, Vibrio aerogenes) are all between 25-100μg / mL, and they all show excellent antibacterial activity; among them, the MIC value against Vibrio mimics is 25μg / mL, with excellent antibacterial activity.

[0038] In summary, the isoquinoline alkaloids (sanguinarine / chelerythrine) in the present invention have an effective effect on bacterial pathogens of aquatic animals (Vibrio harveyi Vibrio harveyi BNCC 336937, Vibrio vulnificus Vibrio vulnificus BNCC186281, Vibrio mimicus Vibrio mimicus BNCC 195661, Vibrio aerogenes Vibrio aerogenes BNCC 362836) all show significant antibacterial activity, and the isoquinoline alkaloids or their salts in the Macleaya cordata can be used as the effective ingredient or the main effective ingredient to prepare a pharmaceutical composition or a feed additive product for preventing and treating related diseases caused by the above-mentioned bacterial pathogens of aquatic animals; and provide a reference for the research and development of related products for preventing and treating bacterial pathogens of aquatic animals.

[0039] Unless otherwise specified, all percentages used in the present invention are by mass.

[0040] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of isoquinoline alkaloids in Macleaya cordata in preparing products for preventing and treating bacterial pathogens in aquatic animals, characterized in that: Use of the isoquinoline alkaloids in Macleaya cordata in preparing products for preventing and / or treating related diseases caused by bacterial pathogens of aquatic animals; The isoquinoline alkaloids in the Macleaya cordata are sanguinarine and / or chelerythrine; The bacterial pathogens of aquatic animals are at least one of the following: Vibrio harveyi, Vibrio vulnificus, Vibrio mimicus, and Vibrio aerogenes.

2. The use of isoquinoline alkaloids in Macleaya cordata according to claim 1 in preparing products for preventing and treating bacterial pathogens of aquatic animals, characterized in that: The related diseases include: bacterial hemorrhagic disease and bacterial infection.

3. The use of isoquinoline alkaloids in Macleaya cordata according to claim 1 in preparing products for preventing and treating bacterial pathogens of aquatic animals, characterized in that: The product is selected from: a pharmaceutical composition and a feed additive.

4. The use of isoquinoline alkaloids in Macleaya cordata according to claim 3 in preparing products for preventing and treating bacterial pathogens of aquatic animals, characterized in that: The pharmaceutical composition adopts at least one isoquinoline alkaloid or its salt in Macleaya cordata as an effective component or a main effective component, and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable excipient.

5. The use of isoquinoline alkaloids in Macleaya cordata according to claim 3 in preparing products for preventing and treating bacterial pathogens of aquatic animals, characterized in that: The feed additive uses at least one isoquinoline alkaloid or its salt in Macleaya cordata as an effective component or a main effective component, and is prepared into a nutritionally acceptable dosage form with a nutritionally acceptable excipient.

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