Application of halogenated emodin derivative in preparation of product for preventing and treating pathogenic bacteria of dairy cow

Abstract

The invention provides application of a halogenated emodin derivative in preparation of a product for preventing and treating pathogenic bacteria of dairy cows, and relates to the field of emodin derivatives. The invention also discloses application of the halogenated emodin derivative in preparation of products for preventing and treating pathogenic bacteria of dairy cows, and application of the halogenated emodin derivative in preparation of products for preventing and / or treating related dairy cow diseases caused by the pathogenic bacteria of the dairy cows. According to the application of the halogenated emodin derivative in preparing the product for preventing and treating the pathogenic bacteria of the dairy cow, the halogenated emodin derivative shows relatively good antibacterial activity on the pathogenic bacteria (streptococcus agalactiae, streptococcus dysgalactiae and staphylococcus aureus) of the dairy cow, and can effectively aim at related diseases caused by the pathogenic bacteria of the dairy cow. The halogenated emodin derivative has the advantages of being green, efficient, easy to obtain, low in toxicity, low in residue, not prone to generating resistance and the like.

Description

Technical Field

[0001] The invention relates to the field of rheumatoid arthritis derivatives, and in particular to use of a halogenated rheumatoid arthritis derivative in preparing a product for preventing and treating pathogenic bacteria in dairy cows. Background Art

[0002] Cow mastitis is an inflammatory reaction of the mammary tissue of dairy cows to pathogenic microorganisms and physical and chemical factors. It can lead to reduced milk production, reduced milk quality, slower growth and development, reduced fertility, shortened milk production years, and increased mortality rates. It seriously endangers the health and production capacity of dairy cows, causes huge economic losses to breeding farms, and seriously restricts the development of the dairy industry. Therefore, cow mastitis is a difficult problem that needs to be solved in dairy farming.

[0003] In the prior art, for the treatment of bovine mastitis, antibiotics and other drugs are generally used to act on the pathogenic bacteria of bovine mastitis; however, with the long-term and large-dose use of antibiotics, the problems of pathogenic bacteria resistance and antibiotic residues in milk have been widely concerned. Finding safe and effective alternative antibiotics to prevent and treat bovine mastitis is an important topic for green prevention and control of bovine mastitis today. In addition, bovine mastitis is mostly manifested as an inflammatory disease rather than an infectious disease; therefore, during the treatment process, attention should be paid not only to the sterilization of its pathogenic bacteria, but also to the promotion of the dissipation of its inflammation. Chinese herbal medicine is a pure natural substance with the characteristics of antibacterial, anti-inflammatory, bacteria not easy to develop drug resistance, low toxicity, etc., and has advantages that antibiotics cannot match. Therefore, the use of Chinese herbal medicine to replace antibiotics to prevent and treat bovine mastitis has increasingly attracted the attention of scholars at home and abroad.

[0004] Rhein, a macrocyclic anthraquinone compound, is the main active ingredient of traditional Chinese medicines such as rhubarb and knotweed, and has a purgative effect in clinical practice. According to modern scientific research, rhein not only has a good purgative effect, but also has good antibacterial, anti-inflammatory, antioxidant, antiviral and anti-infective protozoan pharmacological activities. Therefore, it is of great technical significance to further develop and expand the application fields of rhein and its derivatives, and to study and develop the potential application value of rhein derivatives in the treatment of bovine mastitis.

[0005] Therefore, the invention provides a use of a halogenated rhein derivative in preparing a product for preventing and treating pathogenic bacteria in dairy cows, specifically the use of the halogenated rhein derivative in preventing and / or treating related diseases caused by bacterial infection caused by pathogenic bacteria in dairy cows; in particular, the use of the halogenated rhein derivative in preventing and / or treating cow mastitis caused by pathogenic bacteria in dairy cows. Summary of the invention

[0006] In order to solve the technical problems existing in the prior art, the present invention provides a use of a halogenated rhein derivative in preparing a product for preventing and treating pathogenic bacteria in dairy cows, and the use of the halogenated rhein derivative in preventing and / or treating related dairy cow diseases caused by bacterial infection caused by pathogenic bacteria in dairy cows; in particular, the use of the halogenated rhein derivative in preventing and / or treating cow mastitis caused by pathogenic bacteria in dairy cows.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: Use of a halogenated rhein derivative in preparing a product for preventing and treating pathogenic bacteria in dairy cows, and use of the halogenated rhein derivative in preparing a product for preventing and / or treating relevant dairy cow diseases caused by pathogenic bacteria in dairy cows; The halogenated rhein derivative is at least one of the following: halogenated rhein derivative D-2, halogenated rhein derivative D-3, halogenated rhein derivative D-4, halogenated rhein derivative D-5, halogenated rhein derivative D-6; The chemical structural formula of each of the halogenated rhein derivatives is: .

[0008] The dairy cattle pathogenic bacteria are at least one of the following: Streptococcus agalactiae Streptococcus agalactiae , Streptococcus dysgalactiae Streptococcus dysgalactiae , Escherichia coli Escherichia coli , Staphylococcus aureus Staphylococcus aureus .

[0009] Furthermore, the related cow diseases include: cow mastitis and bacterial infection.

[0010] Furthermore, the products include: pharmaceutical compositions and feed additives.

[0011] A pharmaceutical composition for treating bovine mastitis caused by bovine pathogenic bacteria uses at least one of the above-mentioned halogenated rhein derivatives as an effective component or a main effective component and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable excipient.

[0012] A feed additive for preventing cow mastitis caused by cow pathogenic bacteria uses at least one of the above-mentioned halogenated rhein derivatives as an effective component or a main effective component, and is prepared into a nutritionally acceptable dosage form with a nutritionally acceptable excipient.

[0013] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention uses rhein as a lead compound to synthesize a series of halogenated rhein derivatives, and determines the antibacterial activity of each halogenated rhein derivative against the following pathogens of bovine mastitis, including: Streptococcus agalactiae Streptococcus agalactiaeATCC27956, Streptococcus dysgalactiae Streptococcus dysgalactia ATCC27957, Escherichia coli Escherichia coli ATCC 25922 and Staphylococcus aureus Staphylococcus aureus Newman The invention discloses a method for preparing a novel halogenated rhein derivative for preventing and treating bovine mastitis caused by pathogenic bacteria of dairy cows, and provides a method for preparing a halogenated rhein derivative for preventing and treating bovine mastitis caused by pathogenic bacteria of dairy cows, and provides a method for preparing a halogenated rhein derivative for preventing and treating bovine mastitis caused by pathogenic bacteria of dairy cows, and further expands the application field of the rhein derivatives, and has broad application prospects and application value.

[0014] (2) The halogenated rhein derivatives of the present invention are natural antibacterial agents, which are easy to prepare and have the advantages of being green, highly effective, low toxicity, having a broad antibacterial spectrum, low residue and not prone to resistance. They have the potential to be further developed into new fungicides and are unique and irreplaceable in preventing and treating bovine mastitis caused by pathogenic bacteria, and are of great application value.

[0015] (3) Using each of the halogenated rhein derivatives of the present invention, at a dosage concentration of 100 μg / mL, the halogenated rhein derivatives have an inhibitory effect on Streptococcus agalactiae. Streptococcus agalactiae ATCC27956, Streptococcus dysgalactiae Streptococcus dysgalactiae ATCC27957, Staphylococcus aureus Staphylococcus aureus Newman The inhibition rate can reach 100%; and for Streptococcus agalactiae Streptococcus agalactiae The MIC value of ATCC27956 can reach 6.25μg / mL, which is effective against Streptococcus dysgalactiae. Streptococcus dysgalactiae The MIC value of ATCC27957 can reach 3.12 μg / mL, and can be used in related products to inhibit the aforementioned dairy cow pathogens to achieve preventive and / or therapeutic effects on related diseases caused by them; in particular, it can be used to prevent and treat dairy cow mastitis caused by the aforementioned dairy cow pathogens. DETAILED DESCRIPTION

[0016] 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.

[0017] This embodiment provides a use of a halogenated rhein derivative in preparing a product for preventing and treating pathogenic bacteria in dairy cows, and the use of the halogenated rhein derivative in preparing a product for preventing and / or treating related dairy cow diseases caused by pathogenic bacteria in dairy cows.

[0018] The halogenated rhein derivative is at least one of the following: halogenated rhein derivative D-2, halogenated rhein derivative D-3, halogenated rhein derivative D-4, halogenated rhein derivative D-5, halogenated rhein derivative D-6; The chemical structural formulas of the halogenated rhein derivatives are: .

[0019] The dairy cattle pathogenic bacteria are at least one of the following: Streptococcus agalactiae Streptococcus agalactiae , Streptococcus dysgalactiae Streptococcus dysgalactiae , Escherichia coli Escherichia coli , Staphylococcus aureus Staphylococcus aureus .

[0020] Furthermore, the related cow diseases include: cow mastitis and bacterial infection.

[0021] Furthermore, the products include: pharmaceutical compositions and feed additives.

[0022] This embodiment also provides a pharmaceutical composition for treating bovine mastitis caused by bovine pathogenic bacteria, which uses at least one of the aforementioned halogenated rhein derivatives as an effective ingredient or a main effective ingredient and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable excipient.

[0023] A feed additive for preventing cow mastitis caused by cow pathogenic bacteria uses at least one of the above-mentioned halogenated rhein derivatives as an effective component or a main effective component, and is prepared into a nutritionally acceptable dosage form with a nutritionally acceptable excipient.

[0024] Among them, 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. 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, 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.

[0025] The specific molecular structures of the aforementioned halogenated rhein derivative D-2, halogenated rhein derivative D-3, halogenated rhein derivative D-4, halogenated rhein derivative D-5, and halogenated rhein derivative D-6 have been disclosed in the prior art. The specific preparation methods of the halogenated rhein derivatives used in the subsequent activity assay of the present invention are shown in Examples 1-5.

[0026] Example 1 The preparation method of the halogenated rhein derivative D-2 is specifically as follows: Take rheumatoid artemisia (2g, 7.4mmol, 1.0 equivalent), 40mL anhydrous acetic acid, 4mL concentrated hydrochloric acid and stir in an oil bath at 85°C for 10min, then add 30wt% hydrogen peroxide (0.816mL, 8mmol, 1.08 equivalent) dropwise (hydrogen peroxide is added 8 times, once every 5min, and 102μL is slowly added each time); after the hydrogen peroxide is added, continue to react for 20min and terminate the reaction, add to 200mL distilled water after cooling, and immediately a yellow precipitate is precipitated, and the precipitate is filtered under reduced pressure, and the filter cake is washed with water several times, and then dried; pass through a silica gel column with an eluent of petroleum ether: dichloromethane = 2:1 to obtain a yellow solid product, i.e., a halogenated rheumatoid artemisia derivative D-2 (1.3g, yield 58%). The mass spectrum and nuclear magnetic resonance data of the halogenated rheumatoid artemisia derivative D-2 prepared in this example are consistent with those reported in existing literature.

[0027] Example 2 The preparation method of the halogenated rhein derivative D-3 is specifically as follows: Take rheumatoid artemisia (2g, 7.4mmol, 1.0 equivalent), 40mL anhydrous acetic acid, and 4mL concentrated hydrochloric acid and stir them in an oil bath at 85°C for 10min, then add 30wt% hydrogen peroxide (0.816mL, 8mmol, 1.08 equivalent) all at once, continue stirring for 20min after the addition is complete, and terminate the reaction; after cooling, add it to 200mL of distilled water, and a yellow precipitate will immediately precipitate, and filter it under reduced pressure, wash the filter cake with water several times, and then dry it; pass it through a silica gel column with an eluent of dichloromethane: methanol = 300:1 to obtain a yellow solid product, i.e., a halogenated rheumatoid artemisia derivative D-3 (0.9g, yield 36%). The mass spectrum and nuclear magnetic resonance data of the halogenated rheumatoid artemisia derivative D-3 prepared in this example are consistent with those reported in the literature.

[0028] Example 3 The preparation method of the halogenated rhein derivative D-4 is specifically as follows: At 0°C, N-bromosuccinimide (1650 mg, 9.2 mmol, 2.5 equivalents) was added to a tetrahydrofuran (100 mL) solution of rhein (1 g, 3.7 mmol, 1.0 equivalent); the reaction mixture was stirred at room temperature overnight; then, the reaction mixture was extracted with dichloromethane (3x200 mL), and the organic layers were combined; the combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated; the concentrated residue was passed through a silica gel column using dichloromethane as an eluent to obtain an orange solid product, i.e., a halogenated rhein derivative D-4 (850 mg, yield 54%). The mass spectrum and NMR data of the halogenated rhein derivative D-4 prepared in this example are consistent with those reported in the literature.

[0029] Example 4 The preparation method of the halogenated rhein derivative D-5 is specifically as follows: At 0°C, a solution of NaHCO3 (933 mg, 11.1 mmol, 3.0 equivalent) in tetrahydrofuran (65 mL) was added with a water (90 mL) solution and iodine (1409 mg, 5.5 mmol, 1.5 equivalent); the reaction mixture was stirred at room temperature overnight; then, the reaction mixture was extracted with dichloromethane (3x200 mL), and the organic layers were combined; the combined organic layers were washed with sodium thiosulfate, dried with anhydrous Na2SO4, filtered and concentrated; the concentrated residue was purified by column chromatography (dichloromethane: ethyl acetate = 4:5) to obtain a yellow solid product, i.e., a halogenated rhein derivative D-5 (880 mg, yield 60%). The mass spectrum and nuclear magnetic resonance data of the halogenated rhein derivative D-5 prepared in this example are consistent with those reported in the literature.

[0030] Example 5 The preparation method of the halogenated rhein derivative D-6 is specifically as follows: At 0°C, sodium bicarbonate (1554 mg, 18.5 mmol, 5.0 equivalent) in water (65 mL) and iodine (7513 mg, 29.6 mmol, 8.0 equivalent) were added to a tetrahydrofuran (65 mL) solution of rhein (1 g, 3.7 mmol, 1.0 equivalent); the reaction mixture was stirred at room temperature overnight; then, the reaction compound was extracted with dichloromethane (3x200 mL), and the organic layers were combined; the combined organic layers were washed with sodium thiosulfate, dried with anhydrous Na2SO4, filtered and concentrated; the concentrated residue was purified by column chromatography (dichloromethane: ethyl acetate = 4:5) to obtain a red solid product, i.e., a halogenated rhein derivative D-6 (940 mg, yield 49%). The mass spectrum and nuclear magnetic resonance data of the halogenated rhein derivative D-6 prepared in this example are consistent with those reported in the literature.

[0031] Example 6 The activity of halogenated rhein derivatives against the pathogenic bacteria of bovine mastitis is determined as follows: 1) Test agents: halogenated rhein derivative D-2, halogenated rhein derivative D-3, halogenated rhein derivative D-4, halogenated rhein derivative D-5, halogenated rhein derivative D-6, rhein (control), rhein methyl ether (control), norfloxacin (control); The chemical structural formulas of the halogenated rhein derivatives are: .

[0032] 2) Test strains: The pathogenic bacteria used in dairy cow mastitis is Streptococcus agalactiae Streptococcus agalactiae , deposit number ATCC27956; Streptococcus dysgalactiae Streptococcus dysgalactiae , deposit number ATCC27957; Escherichia coli Escherichia coli , deposit number ATCC25922; Staphylococcus aureus Staphylococcus aureus Newman The aforementioned test strains are all existing strains, and are purchased from commercial sources.

[0033] 3) Test method: The strain used in the test experiment of this embodiment is a strain frozen in 30% glycerol at a temperature of -80°C in the laboratory.

[0034] The frozen strains of Streptococcus agalactiae and Streptococcus dysgalactiae were taken out respectively, streaked on TSA (tryptic soy peptone agar medium) + 5% sheep blood agar plates, and cultured at a constant temperature of 37°C until single colonies grew; single colonies were picked out and added to brain heart infusion broth (BHI) liquid culture medium (tryptone: 10.0 g, beef heart extract powder: 17.5 g, sodium chloride: 5.0 g, glucose: 2.0 g, disodium hydrogen phosphate (12H2O): 2.5 g, distilled water: 1 L; sterilized at 121°C for 20 min), and cultured in a constant temperature shaker at 37°C and 140 rpm until the logarithmic growth phase.

[0035] Similarly, the frozen strains of Escherichia coli and Staphylococcus aureus were taken out, streaked on MH solid culture medium (beef powder: 2.0 g, soluble starch: 1.5 g, acid hydrolyzed casein: 17.5 g, agar: 20 g, distilled water: 1 L; sterilized at 121°C for 20 min), and cultured at 37°C until single colonies grew; single colonies were picked up and transferred to MH broth culture medium (beef powder: 2.0 g, soluble starch: 1.5 g, acid hydrolyzed casein: 17.5 g, distilled water: 1 L; sterilized at 121°C for 20 min) liquid culture medium, and cultured in a constant temperature shaker at 37°C and 140 rpm until the logarithmic growth phase.

[0036] Then, each strain in the logarithmic growth phase was diluted to 1.0×10 6 CFU / mL, for future use.

[0037] Dissolve the above-mentioned test agents in DMSO, add them to the liquid culture medium, and mix them evenly. Specifically, prepare rhein and rhein methyl ether into a drug-containing liquid culture medium with a concentration of 1600 μg / mL; prepare each halogenated rhein derivative 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 10 6 CFU / mL of bacterial culture was added to the wells of a 96-well plate, and the final dosing concentration of rhein and rhein methyl ether was controlled to be 800μg / mL, and the final dosing concentration of each halogenated rhein derivative was 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 37°C 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%.

[0038] All the above experiments were repeated three times. The specific results of the inhibition rates of physalis (800 μg / mL) and physalis methyl ether (800 μg / mL) on the pathogenic bacteria of bovine mastitis are shown in the following table:

[0039] The specific results of the inhibition rate of each halogenated rhein derivative (100 μg / mL) on each bovine mastitis pathogen are shown in the following table:

[0040] It can be seen that the halogenated rhamnolide derivatives of the present invention exhibit good antibacterial activity against the tested strains except Escherichia coli, among which the antibacterial activity against Streptococcus agalactiae is 、 Streptococcus dysgalactiae 、 Staphylococcus aureus showed high antibacterial activity. At the same time, compared with rhein (800μg / mL) and rhein methyl ether (800μg / mL), halogenated rhein derivatives can significantly improve the antibacterial activity against each tested strain.

[0041] Example 7 On the basis of Example 6, the drug-containing liquid culture medium of each halogenated rhein derivative, rhein, and rhein methyl ether was diluted in a 96-well plate by a two-fold dilution method 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 6; the lowest concentration with an inhibition rate greater than 90% was defined as MIC, and the MIC value of each test agent for each bovine mastitis pathogen was determined. The specific measurement results are shown in the following table:

[0042] It can be seen that the halogenated rhein derivatives of the present invention exhibit good antibacterial activity against the test strains except Escherichia coli; among them, the antibacterial activity of the halogenated rhein derivative D-2 (1-chloro substitution) is the most prominent, and the MIC value against Streptococcus agalactiae and Streptococcus dysgalactiae is 3.12-6.25 μg / mL, showing better antibacterial activity than the positive control antibiotic (norfloxacin). In addition, from the overall antibacterial properties of the halogenated rhein derivatives, the introduction of halogen atoms in rhein can greatly enhance the antibacterial activity of rhein and further expand the application of rhein.

[0043] In summary, the halogenated rhubarb derivatives of the present invention have an inhibitory effect on Streptococcus agalactiae. Streptococcus agalactia ATCC27956, Streptococcus dysgalactiae Streptococcus dysgalactiae ATCC27957, Staphylococcus aureus Staphylococcus aureus Newman All showed significant antibacterial activity, but against Escherichia coli ATCC Escherichia coli The antibacterial activity of 25922 needs to be optimized; each of the halogenated rhubarb derivatives can effectively inhibit the pathogenic bacteria of bovine mastitis (Streptococcus agalactiae Streptococcus agalactiae ATCC27956, Streptococcus dysgalactiae Streptococcus dysgalactiae ATCC27957, Staphylococcus aureus Staphylococcus aureus Newman ), the halogenated rhein derivative can be used as an effective ingredient or main effective ingredient to prepare a pharmaceutical composition or feed additive for preventing and treating diseases such as cow mastitis or bacterial infection caused by the above-mentioned cow pathogenic bacteria.

[0044] Unless otherwise specified, all percentages used in the present invention are by mass.

[0045] 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 a halogenated rhein derivative in preparing a product for preventing and treating pathogenic bacteria in dairy cows, characterized in that: Use of the halogenated rhein derivatives in preparing products for preventing and / or treating relevant cow diseases caused by cow pathogenic bacteria; The halogenated rhein derivative is at least one of the following: halogenated rhein derivative D-2, halogenated rhein derivative D-3, halogenated rhein derivative D-4, halogenated rhein derivative D-5, halogenated rhein derivative D-6; The chemical structural formula of each of the halogenated rhein derivatives is: ； The dairy cattle pathogenic bacteria are at least one of the following: Streptococcus agalactiae Streptococcus agalactiae , Streptococcus dysgalactiae Streptococcus dysgalactiae , Staphylococcus aureus Staphylococcus aureus .

2. Use of the halogenated rhein derivative according to claim 1 in preparing a product for preventing and treating pathogenic bacteria in dairy cows, characterized in that: The related dairy cow diseases include: dairy cow mastitis and bacterial infection.

3. Use of the halogenated rhein derivative according to claim 1 in preparing a product for preventing and treating pathogenic bacteria in dairy cows, characterized in that: The products include: pharmaceutical compositions and feed additives.

4. A pharmaceutical composition for treating bovine mastitis caused by bovine pathogenic bacteria, characterized in that: At least one halogenated rhein derivative as claimed in claim 1 is used as an effective ingredient or a main effective ingredient, and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable excipient.

5. A feed additive for preventing cow mastitis caused by cow pathogenic bacteria, characterized in that: At least one halogenated rhein derivative as claimed in claim 1 is used as an effective ingredient or a main effective ingredient, and is prepared into a nutritionally acceptable dosage form with a nutritionally acceptable excipient.

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