Application of alkaloids from Macleaya cordata in preparing products for preventing and treating bacterial pathogens in aquatic animals

Abstract

The present invention provides the use of alkaloids in Macleaya cordata in preparing products for preventing and treating bacterial pathogens of aquatic animals, and relates to the field of Macleaya cordata extracts. The use of alkaloids in Macleaya cordata in preparing products for preventing and / or treating related diseases caused by bacterial pathogens of aquatic animals; the alkaloids are at least one of the following: sanguinarine and chelerythrine. The alkaloids in Macleaya cordata (sanguinarine and chelerythrine) in the present invention show good antibacterial activity against bacterial pathogens of aquatic animals, and can effectively target related diseases caused by bacterial pathogens of aquatic animals; the alkaloids in Macleaya cordata have the advantages of being green, efficient, low toxicity, low residue, and not easy to produce resistance.

Description

Technical Field

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

[0002] Vibrio is a Gram-negative halophilic bacterium that is widely found in oceans, coasts, estuaries and other environments. As an important bacterial pathogen, infection in food mainly occurs in seafood such as fish, shrimp and shellfish. In severe cases, it can cause sepsis, necrotizing fasciitis and wound infection. Generally, it enters the human body through people's diet, causing serious harm to human health and even death, bringing great troubles and serious threats to the global aquaculture industry and public health.

[0003] With the growing scale of aquaculture, the use of antibiotics has become one of the main means of treatment and killing of pathogenic bacteria in the aquaculture process. However, its side effects are also obvious. The emergence of drug-resistant strains, drug residues in aquatic products, and water pollution will pose serious risks to human health and public health safety. In addition, the cell membrane of Gram-negative bacteria is a key component that causes pathogen resistance, which causes cells to exhibit "dormancy" and makes it difficult to treat with antibiotics. Therefore, finding safe and effective alternative antibiotics to kill pathogenic bacteria in aquatic animals is an important topic in today's green prevention and control of aquatic animal diseases.

[0004] Natural products have always been an important source in drug 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.

[0005] Macleaya cordata (Willd.) R.Br. is a perennial tall herb of the genus Macleaya in the family Papaveraceae. There are two species in the genus, namely Macleaya cordata and Macleaya microcarpa (Maxim) Fedde. Macleaya cordata is a traditional Chinese medicinal material rich in alkaloids, also known as "horn pole". It is widely distributed in my country and has abundant resources. The main biologically active substances in Macleaya cordata plants are isoquinoline alkaloids, such as sanguinarine, chelerythrine, protopine, allocryptine, etc. Modern pharmacological studies have shown that Macleaya cordata contains a variety of chemical components, the main active ingredients of which are sanguinarine and chelerythrine. It has a variety of pharmacological effects and uses such as anti-inflammatory, insecticidal, maggot killing, heat-clearing and detoxifying, enhancing human immune function and anti-tumor, and is widely used in medicine, biological pesticides, veterinary drugs, and raw materials for fine chemical products.

[0006] Therefore, the functions of alkaloids in Macleaya cordata are further developed, their application fields are expanded, and the use of alkaloids in Macleaya cordata in the preparation of products for preventing and treating bacterial pathogens of aquatic animals is provided, specifically the use of alkaloids in Macleaya cordata in preventing and / or treating related diseases caused by bacterial pathogens of aquatic animals, which has important technical significance and value. Summary of the invention

[0007] In order to solve the technical problems existing in the prior art, the present invention provides the use of alkaloids in Macleaya cordata in preparing products for preventing and treating bacterial pathogens of aquatic animals, and the use of alkaloids in Macleaya cordata in preventing and / or treating related diseases caused by bacterial pathogens of aquatic animals; in particular, the use of alkaloids in Macleaya cordata in preventing and treating bacterial hemorrhagic diseases caused by bacterial pathogens of aquatic animals.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0009] The invention discloses a use of alkaloids in Macleaya cordata for preparing products for preventing and treating bacterial pathogens of aquatic animals, and a use of alkaloids in Macleaya cordata for preparing products for preventing and / or treating related diseases caused by bacterial pathogens of aquatic animals; the alkaloids in Macleaya cordata are sanguinarine and / or chelerythrine.

[0010] The chemical structural formula of the sanguinarine / chelerythrine is:

[0011] ;

[0012] The bacterial pathogens of aquatic animals are at least one of the following: Vibrio parahaemolyticus Vibrio parahaemolyticus , Vibrio anguillarum Vibrio anguillarium , Vibrio alginolyticus Vibrio alginolyticus , Vibrio fluvialis Vibrio fluvialis。

[0013] The related diseases include: bacterial hemorrhagic diseases and bacterial infections.

[0014] Furthermore, the products include: pharmaceutical compositions and feed additives.

[0015] A pharmaceutical composition for preventing and treating diseases caused by bacterial pathogens of aquatic animals, which uses the alkaloids in the above-mentioned Macleaya cordata or its salts, tautomers, stereoisomers, precursor compounds as active ingredients or main active ingredients, and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable excipient.

[0016] A feed additive for preventing diseases caused by bacterial pathogens of aquatic animals, which uses the alkaloids in the above-mentioned Macleaya cordata or its salts, tautomers, stereoisomers, precursor compounds as effective ingredients or main effective ingredients, and is prepared into a nutritionally acceptable dosage form with nutritionally acceptable excipients.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention uses alkaloids (sanguinarine and chelerythrine) from Macleaya cordata to target the following bacterial pathogens of aquatic animals, including: Vibrio parahaemolyticus Vibrio parahaemolyticus BNCC 138541, Vibrio anguillarum Vibrio anguillarium BNCC 354817, Vibrio alginolyticus Vibrio alginolyticus BNCC 337013, Vibrio fluviatilis Vibrio fluvialis BNCC 337051, after screening for inhibitory activity, showed excellent antibacterial inhibition, and provided the use of alkaloids (sanguinarine, chelerythrine) in Macleaya cordata in preventing and / or treating related diseases caused by bacterial pathogens of aquatic animals; in particular, the use of alkaloids (sanguinarine, chelerythrine) in Macleaya cordata in preventing and treating bacterial hemorrhagic diseases caused by bacterial pathogens of aquatic animals, further expanding the application field of alkaloids in Macleaya cordata, and having broad application prospects.

[0019] (2) The alkaloids (sanguinarine and chelerythrine) in the Macleaya cordata of the present invention are used to prevent and treat related diseases (especially bacterial hemorrhagic diseases) caused by bacterial pathogens of aquatic animals. Since the alkaloid components (sanguinarine and chelerythrine) in Macleaya cordata are natural plant-derived antibacterial agents obtained from natural plants, they have the advantages of low toxicity, low residue and low resistance, and are 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.

[0020] (3) Using the alkaloid (sanguinarine) in Macleaya cordata described in the present invention, under the condition of a dosage concentration of 100 μg / mL, the inhibition rate of sanguinarine on Vibrio parahaemolyticus BNCC 138541 is 100%, the inhibition rate on Vibrio anguillarum BNCC 354817 is 100%, the inhibition rate on Vibrio fluvii BNCC 337051 is 100%, and the inhibition rate on Vibrio alginolyticus BNCC 337013 is 70.62±0.76%; further, the minimum inhibitory concentration (MIC) of sanguinarine on Vibrio parahaemolyticus BNCC 138541, Vibrio anguillarum BNCC 354817, and Vibrio fluvii BNCC 337051 is 25-100 μg / mL, and the use of sanguinarine in related products can achieve a preventive and / or therapeutic effect on related diseases caused by the aforementioned bacterial pathogens of aquatic animals by inhibiting them.

[0021] (4) When the alkaloid (chelerythrine) in Macleaya cordata described in the present invention is used, at a dosage concentration of 100 μg / mL, the inhibition rate of chelerythrine on Vibrio parahaemolyticus BNCC 138541 is 100%, the inhibition rate of chelerythrine on Vibrio anguillarum BNCC 354817 is 100%, and the inhibition rate of chelerythrine on Vibrio fluvii BNCC 337051 is 100%; further, the minimum inhibitory concentration (MIC) of chelerythrine on Vibrio parahaemolyticus BNCC 138541, Vibrio anguillarum BNCC 354817, and Vibrio fluvii BNCC 337051 is 25-100 μg / mL, and the use of chelerythrine in related products can achieve a preventive and / or therapeutic effect on related diseases caused by the aforementioned bacterial pathogens of aquatic animals by inhibiting them. DETAILED DESCRIPTION

[0022] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific implementation methods of the present invention are now described.

[0023] This embodiment provides the use of alkaloids in Macleaya cordata in preparing products for preventing and treating bacterial pathogens of aquatic animals, and the use of alkaloids in Macleaya cordata in preparing products for preventing and / or treating related diseases caused by bacterial pathogens of aquatic animals; the alkaloids in Macleaya cordata are sanguinarine and / or chelerythrine.

[0024] The chemical structural formula of the sanguinarine / chelerythrine is:

[0025] ;

[0026] The bacterial pathogens of aquatic animals are at least one of the following ： Vibrio parahaemolyticus Vibrio parahaemolyticus , Vibrio anguillarum Vibrio anguillarium , Vibrio alginolyticus Vibrio alginolyticus , Vibrio fluvialis Vibrio fluvialis。

[0027] Furthermore, the related diseases include: bacterial hemorrhagic diseases and bacterial infections.

[0028] Furthermore, the products include: pharmaceutical compositions and feed additives.

[0029] This embodiment also provides a pharmaceutical composition for preventing and treating related diseases caused by bacterial pathogens of aquatic animals, which uses the alkaloids in Macleaya cordata or its salts, tautomers, stereoisomers, and precursor compounds as active ingredients or main active ingredients, and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

[0030] This embodiment also provides a feed additive for preventing related diseases caused by bacterial pathogens of aquatic animals, which uses the alkaloids in Macleaya cordata or its salts, tautomers, stereoisomers, and precursor compounds as active ingredients or main active ingredients, and is prepared into a nutritionally acceptable dosage form with nutritionally acceptable excipients.

[0031] Wherein, the aforementioned compound salt refers to the 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 compound 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 compound salts can be prepared in situ during the final separation and purification of the compound.

[0032] The aforementioned excipients refer to any excipients, solvents, dispersion media, absorption retardants, 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. Examples of typical 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.

[0033] Example 1

[0034] The activity of alkaloids (sanguinarine / chelerythrine) in Macleaya cordata against bacterial pathogens of aquatic animals is determined as follows:

[0035] 1) Test agents: sanguinarine, chelerythrine;

[0036] The chemical structural formula of the sanguinarine / chelerythrine is:

[0037] ;

[0038] 2) Test strains:

[0039] Use bacterial pathogens from aquatic animals, specifically Vibrio parahaemolyticus Vibrio parahaemolyticus BNCC 138541, Vibrio anguillarum Vibrio anguillarium BNCC 354817, Vibrio alginolyticus Vibrio alginolyticus BNCC 337013, Vibrio fluviatilis Vibrio fluvialis BNCC 337051.

[0040] 3) Test method:

[0041] The strains used in the test experiments of this example were all purchased from Beina Biotech Co., Ltd. as freeze-dried powders.

[0042] The freeze-dried powders of Vibrio anguillarum, Vibrio alginolyticus, and Vibrio fluvialis were opened in a safety cabinet, the tops were burned with alcohol, sterile water was dripped on them to break them, and then they were broken with tweezers. 0.5 mL of 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, MgCl 2 :5.98g,Na2 SO 4 :3.24g, CaCl 2 :1.8g, KCl:0.55g, Na 2 CO 3 :0.16g, NaNO 3 :0.0016g, Na 2 HPO 4 :0.008g, strontium chloride: 0.034g, boric acid: 0.022g, sodium silicate: 0.004g, sodium fluoride: 0.0024g, distilled water: 1L; sterilized at 121℃ for 20min) into a freeze-dried tube, and after fully dissolved, return it to the liquid test tube and mix well; then draw 0.2mL of bacterial suspension into the plate and spread it evenly. Repeat twice to obtain two 2216E seawater agar plates (peptone: 5.0g, yeast extract: 1.0g, ferric citrate: 0.1g, potassium bromide: 0.08g, NaCl: 19.45g, MgCl 2 :5.98g,Na 2 SO 4 :3.24g, CaCl 2 :1.8g, KCl:0.55g, Na 2 CO 3 :0.16g, NaNO 3 :0.0016g, Na 2 HPO 4 :0.008g, strontium chloride: 0.034g, boric acid: 0.022g, sodium silicate: 0.004g, sodium fluoride: 0.0024g, agar: 15.0g, distilled water: 1L; sterilize at 121℃ for 20min). Cultivate at 30℃ until bacteria grow; then streak on 2216E seawater agar plates and culture at 30℃ until single colonies grow. Pick single colonies and culture them in 2216E liquid culture medium at 30℃, 150rpm constant temperature shaker until logarithmic growth phase.

[0043] Similarly, the lyophilized powder of Vibrio parahaemolyticus was taken out and incubated in BHI solid medium (peptone: 5.0 g, yeast extract: 1.0 g, ferric citrate: 0.1 g, NaCl: 19.45 g, potassium bromide: 0.08 g, MgCl 2 :5.98g,Na 2 SO 4 :3.24g, CaCl 2 :1.8g, KCl:0.55g, Na 2 CO 3 :0.16g, NH 4 NO 3:0.00116g, Na 2 HPO 4 :0.008g, strontium chloride: 0.034g, boric acid: 0.022g, sodium silicate: 0.004g, sodium fluoride: 0.0024g, agar: 20.0g, distilled water: 1L; sterilized at 121℃ for 20min) and cultured at 30℃ until bacteria grew out; then streaked on BHI solid plates and cultured at 30℃ until single colonies grew out. Pick single colonies and add them to BHI liquid culture medium (peptone: 5.0g, yeast extract: 1.0g, ferric citrate: 0.1g, NaCl: 19.45g, potassium bromide: 0.08g, MgCl 2 :5.98g,Na 2 SO 4 :3.24g, CaCl 2 :1.8g, KCl:0.55g, Na 2 CO 3 :0.16g, NH 4 NO 3 :0.00116g, Na 2 HPO 4 : 0.008g, strontium chloride: 0.034g, boric acid: 0.022g, sodium silicate: 0.004g, sodium fluoride: 0.0024g, distilled water: 1L; sterilized at 121℃ for 20min) and cultured in a constant temperature shaker at 30℃ and 150rpm until the logarithmic growth phase.

[0044] 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 alkaloids (sanguinarine / chelerythrine) in Macleaya cordata separately with DMSO, add them to the liquid culture medium, mix them evenly, and prepare the sanguinarine and chelerythrine into a drug-containing liquid culture medium with a concentration of 200 μg / mL.

[0045] Take 50 μL of drug-containing culture medium and the same volume of medium containing about 1×10 6 CFU / mL bacterial culture was added to the wells of a 96-well plate, and the final administration concentration of the 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:

[0046] Corrected OD value = OD value of bacterial culture medium - OD value of sterile culture medium;

[0047] 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%.

[0048] 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:

[0049]

[0050] From the above test results, it can be seen that the alkaloids (sanguinarine or chelerythrine) in the Macleaya cordata of the present invention show excellent antibacterial activity against the test strains Vibrio parahaemolyticus, Vibrio anguillarum, and Vibrio fluvialis, and sanguinarine shows good antibacterial activity against Vibrio alginolyticus.

[0051] Example 2

[0052] On the basis of Example 1, the drug-containing liquid culture medium of 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:

[0053]

[0054] From the above test results, it can be seen that the MIC values ​​of alkaloids (sanguinarine or chelerythrine) in Macleaya cordata against the test strains (Vibrio parahaemolyticus, Vibrio anguillarum, Vibrio fluvialis) are all between 25 and 100 μg / mL, showing excellent antibacterial activity.

[0055] In summary, the alkaloids (sanguinarine or chelerythrine) in the Macleaya cordata of the present invention have an effect on bacterial pathogens of aquatic animals (especially Vibrio parahaemolyticus Vibrio parahaemolyticus BNCC 138541, Vibrio anguillarum Vibrio anguillarium BNCC 354817) exhibits significant antibacterial activity, and the alkaloids or their salts, tautomers, stereoisomers, and precursor compounds in the Macleaya cordata can be used as effective ingredients or main effective ingredients to prepare a pharmaceutical composition or feed additive product for preventing and treating related diseases caused by the above-mentioned bacterial pathogens of aquatic animals.

[0056] Unless otherwise specified, all percentages used in the present invention are by mass.

[0057] 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 alkaloids from Macleaya cordata in the preparation of products for preventing and / or treating bacterial infections caused by bacterial pathogens of aquatic animals; The alkaloid is at least one of the following: sanguinarine and chelerythrine; The bacterial pathogen of aquatic animals is Vibrio fluvibrios Vibrio fluvialis; The product is selected from: pharmaceutical compositions, feed additives; The pharmaceutical composition adopts the alkaloid or its salt as an effective ingredient or a main effective ingredient and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable excipient; The feed additive adopts the alkaloid or its salt as an effective component or a main effective component, and is prepared into a nutritionally acceptable dosage form with a nutritionally acceptable excipient.

2. Use of alkaloids in Macleaya cordata in the preparation of products for preventing and / or treating bacterial hemorrhagic diseases caused by bacterial pathogens of aquatic animals; The alkaloid is at least one of the following: sanguinarine and chelerythrine; The bacterial pathogen of aquatic animals is Vibrio fluvibrios Vibrio fluvialis; The product is selected from: pharmaceutical compositions, feed additives; The pharmaceutical composition adopts the alkaloid or its salt as an effective ingredient or a main effective ingredient and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable excipient; The feed additive adopts the alkaloid or its salt 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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