Use of a derivative of isobarbital alkaloid in the prevention and treatment of pathogens causing mastitis in dairy cows

Abstract

This invention discloses the use of any one of the derivatives of isobaiyetangine, Da-1 to Da-8, Db-1 to Db-8, Dc-1 to Dc-8, and Y-1 to Y-4, in the prevention and treatment of pathogenic bacteria causing bovine mastitis. Bioactivity tests show that these compounds exhibit certain inhibitory activity against the pathogenic bacteria of bovine mastitis, *Streptococcus agalactiae* ATCC12386, *Streptococcus agalactiae* ATCC27956, and *Streptococcus dysgalactiae* ATCC27957. The compounds involved in this invention are easy to prepare, use inexpensive and readily available raw materials, and have significant development value in the field of natural product-derived compounds for the prevention and treatment of bovine mastitis.

Description

Technical Field

[0001] This invention belongs to the field of natural medicinal chemistry and discloses a new use of isobaiye tunicine derivatives, specifically involving the use of isobaiye tunicine derivatives Da-1~Da-8, Db-1~Db-8, Dc-1~Dc-8, and Y-1~Y-4 in the prevention and treatment of mastitis in dairy cows caused by pathogenic Streptococcus agalactiae ATCC12386, Streptococcus agalactiae ATCC27956, and Streptococcus dysgalactiae ATCC27957. Background Technology

[0002] Bovine mastitis is an inflammatory disease caused by a combination of pathogenic microorganisms. It is the most common bacterial infection in dairy cows and is associated with reduced milk production and altered milk composition. For decades, the problem of bovine mastitis has remained largely unresolved, continuing to cause significant economic losses to the global dairy industry, amounting to up to $35 billion annually. Milk and dairy products are also important sources of macronutrients and micronutrients for the human diet. Good udder health is crucial not only for dairy farmers but also for the entire dairy production chain in order to obtain high-quality milk. Therefore, there is an urgent need to create green, safe, and effective compounds to address this problem.

[0003] Natural products, as a primary source for new drug development, exhibit lower toxicity and selectivity or specificity compared to traditional antibiotics. Many unique natural products, such as sesquiterpenes, diterpenes, steroids, polysaccharides, alkaloids, and other compounds, have been isolated from various organisms and found to possess diverse biological activities, including antibacterial and anti-inflammatory properties, and good environmental compatibility. From this perspective, using natural products as a strategy for the prevention and treatment of bovine mastitis is an important current research direction. Isocryptolepine is one of the natural alkaloids with a unique indole-quinoline structure extracted from the traditional African medicinal herb, *Hemiberlesia argyi*. Current reports on the biological activities of this alkaloid and its derivatives mainly focus on antimalarial, antihyperglycemic, antitumor, and antibacterial effects; however, no reports have been found on the use of this type of alkaloid and its derivatives in the prevention and treatment of bovine mastitis.

[0004]

[0005] Isocryptolepine

[0006] Therefore, we obtained a series of isobarbital-derived structures through structural optimization using isobarbital as the core framework, and conducted activity tests on them against Streptococcus agalactiae and Streptococcus faecium, showing excellent antibacterial activity. Therefore, isobarbital derivatives have potential application value in the treatment of bovine mastitis and are worthy of further research and development. Summary of the Invention

[0007] The purpose of this invention is to provide a new use of isobarbital alkaloid derivatives in the treatment of bovine mastitis, for the prevention and treatment of bovine mastitis caused by Streptococcus agalactiae and Streptococcus faecium.

[0008] To achieve the above objectives, the present invention provides the following technical method:

[0009] This invention provides a novel use of iso-white vine alkaloid derivatives in combating pathogenic bacteria causing bovine mastitis. These derivatives are administered at concentrations of 100, 50, 25, 12.5, 6.25, and 3.12 μg / mL against Streptococcus agalactiae ATCC12386, Streptococcus agalactiae ATCC27956, and Streptococcus dysgalactiae ATCC27957.

[0010] This invention synthesizes isobarbital derivatives using methods reported in the literature. Pure products were obtained through multiple silica gel column chromatography analyses and other conventional methods. The structures of the isobarbital derivatives Da-1~Da-8, Db-1~Db-8, Dc-1~Dc-8, and Y-1~Y-4 were determined using mass spectrometry and nuclear magnetic resonance spectroscopy. The compound structures are shown in Chemical Formula 1. In Chemical Formula 1, R represents 8-fluoro, 8-bromo, 8-chloro, 9-fluoro, 10-fluoro, 8-methyl, or 8-trifluoromethoxy substitutions, and R1 represents 2-fluoro substitution.

[0011]

[0012] Chemical Formula 1

[0013] Specifically, the chemical structural formula of the iso-whiteleaf alkaloid derivative is as follows:

[0014]

[0015]

[0016] The derivatives provided by this invention have the following advantages:

[0017] 1) This invention discovers that the derivative of isobarbital alkaloids exhibits excellent inhibitory effects on pathogenic bacteria causing bovine mastitis, and has great development value in the treatment of bovine mastitis.

[0018] 2) The derivatives of isobaiye teng alkaloids are easy to synthesize, have simple structures, and are green and efficient, and are worthy of further research and development. Detailed Implementation

[0019] To better understand the present invention, the following specific embodiments further illustrate the above-described content of the present invention. However, this should not be construed as a limitation of the present invention. The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention. However, the content of the present invention is not limited to the scope described in the embodiments. All changes or equivalent substitutions that do not depart from the concept of the present invention are included within the protection scope of the present invention.

[0020] Example 1: Activity assay of iso-Baiyetangine derivatives against pathogenic bacteria causing bovine mastitis

[0021] 1) Test reagent: Iso-white vine alkaloid derivative.

[0022] 2) Test strains: Streptococcus agalactiae ATCC12386, Streptococcus agalactiae ATCC27956, Streptococcus dysgalactiae ATCC27957.

[0023] 3) Testing methods:

[0024] The bacterial strains used in this experiment were cryopreserved in the laboratory at -80℃ with 30% glycerol. The cryopreserved strains were removed and streaked onto sheep blood agar plates, then incubated at 37℃ until single colonies appeared. Single colonies were then transferred to brain heart broth (BHI) liquid medium (tryptone: 10.0g, beef heart extract: 17.5g, sodium chloride: 5.0g, glucose: 2.0g, disodium hydrogen phosphate (12H2O): 2.5g, distilled water: 1L; sterilized at 121℃ for 20min) and incubated at 37℃ with shaking at 140rpm until the logarithmic growth phase. The strains in the logarithmic growth phase were diluted with the appropriate liquid medium to approximately 10... 6 CFU / mL is prepared for use. Dissolve the compounds separately in DMSO, add them to the liquid culture medium, mix thoroughly, and prepare 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 approximately 10... 6CFU / mL bacterial culture was added to the wells of a 96-well plate, resulting in a final drug concentration of 100 μg / mL. A control of 100 μL of bacterial culture containing an equal amount of DMSO was used. The 96-well plates were incubated at 37°C for 12–24 h until bacterial growth was observed in the control group. The OD value (OD) of the bacterial culture in each well was measured using a microplate reader. 600 In addition, the OD values ​​of 100 μL of liquid culture medium and a 100 μg / mL drug concentration were measured to correct for the OD values ​​caused by the culture medium and the drug itself. The formulas for calculating the corrected OD value and inhibition rate are as follows:

[0025] Corrected OD value = OD value of bacterial culture medium - OD value of sterile culture medium;

[0026] Inhibition rate = (OD value of bacterial suspension in the control medium after correction - OD value of bacterial suspension in the drug-containing medium after correction) / OD value of bacterial suspension in the control medium after correction × 100%

[0027] All experiments were conducted in triplicate, and the inhibition rates of the compounds were measured and are shown in Table 1.

[0028] Table 1. Inhibition rate (%) of isobarbital derivatives against pathogenic bacteria causing bovine mastitis at 100 μg / mL

[0029]

[0030]

[0031] As shown in Table 1, the iso-white leaf alkaloid derivatives involved in this invention all exhibited certain antibacterial activity against the tested strains, with most compounds showing high activity.

[0032] The drug-containing liquid culture medium of the active compound was diluted in 96-well plates using a two-fold dilution method to obtain a series of 50 μL drug-containing culture media of various concentrations. Then, the inhibition rate corresponding to the series of concentrations was determined according to the same test method as in Example 1. The lowest concentration with an inhibition rate greater than 90% was defined as the MIC. The measured activity data are shown in Table 2.

[0033] Table 2. MIC values ​​of highly active compounds against pathogenic bacteria causing bovine mastitis

[0034]

[0035]

[0036] As shown in Tables 1 and 2, the iso-Baiye Teng alkaloid derivatives involved in this invention exhibit good antibacterial activity against the test strains. At a test concentration of 100 μg / mL, the inhibition rate of most compounds was greater than 90%. Among them, compounds Y-2, Y-3, and Y-4 showed the best activity, with MIC values ​​ranging from 3.12 to 12.5 μg / mL, demonstrating excellent antibacterial activity.

[0037] In summary, the iso-white vine alkaloid derivatives described in this invention exhibit good antibacterial activity against the pathogenic bacteria Streptococcus agalactiae ATCC12386, Streptococcus agalactiae ATCC27956, and Streptococcus dysgalactiae ATCC27957, and can be further studied and developed for the prevention and treatment of mastitis in dairy cows.

Claims

1. The use of a alkaloid derivative of leucocephala, Da-1~Da-8, Db-1~Db-8, Dc-1~Dc-8, Y-1~Y-4, in the preparation of a drug for the prevention and treatment of mastitis in dairy cows, characterized in that... The derivative has the following molecular structure, and the pathogens causing the mastitis in dairy cows are *Streptococcus agalactiae* ATCC12386, *Streptococcus agalactiae* ATCC27956, and *Streptococcus dysgalactiae* ATCC27957. .

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