Application of cryptolepine alkaloid derivative in prevention and treatment of pathogenic bacteria of cow mastitis

Abstract

The invention discloses cryptolepine alkaloid derivatives, which comprise application of cryptolepine derivatives BYT-01 to BYT-09, neocryptolepine derivatives XBYT-01 to XBYT-45, iso-cryptolepine derivatives YBYT-01 to YBYT-21 and iso-cryptolepine derivatives YXBYT-01 to YXBYT-04 in the aspect of preventing and treating pathogenic bacteria of cow mastitis, and belongs to the field of natural medicinal chemistry. The cryptolepine alkaloid derivative has the advantages that the cryptolepine alkaloid derivative has good inhibitory activity on dairy cow mastitis pathogenic bacteria such as Streptococcus agalactiae ATCC27956, Streptococcus dysgalactiae ATCC27957, Escherichia coli ATCC 25922 and Staphylococcus aureus, and the cryptolepine alkaloid derivative has great development value in the aspect of replacing antibiotics to become a source compound of a natural product for preventing and treating dairy cow mastitis.

Description

Technical Field

[0001] The invention belongs to the field of natural drug chemistry, discloses a new use of leucine alkaloid derivatives, and specifically relates to the use of the leucine alkaloid derivatives in preventing and treating cow mastitis caused by Streptococcus agalactiae ATCC 27956, Streptococcus dysgalactiae ATCC 27957, Escherichia coli ATCC 25922 and Staphylococcus aureus Newman. Background Art

[0002] Mastitis is a common disease in dairy cows, especially in the early stages of dairy farming. It is characterized by an inflammatory response in the mammary tissue, which can be triggered by physical stimulation, chemical invasion, or invasion of pathogenic microorganisms. It is estimated that about 50% of cases of mastitis in dairy cows are caused by bacteria. The most common bacterial pathogens are Streptococcus agalactiae, Streptococcus dysgalactiae, Staphylococcus aureus, and Escherichia coli. Mastitis in dairy cows poses a significant threat to health and can affect lactation as well as the quality and nutritional value of milk. Milk from sick cows is more likely to spoil, which reduces the overall value of milk and therefore its use is restricted. The economic impact of mastitis on the dairy farming industry and dairy processing industry is considerable. In addition, the potential food safety issues associated with mastitis are also a major public health issue.

[0003] The main means of treating bovine mastitis currently still relies on antibiotics, but their overuse has triggered multiple crises. On the one hand, excessive antibiotic residues in milk directly threaten human health and may induce allergic reactions or intestinal flora disorders; on the other hand, long-term abuse has led to a significant increase in the drug resistance of major pathogens such as Staphylococcus aureus and Escherichia coli, especially in high-yielding dairy cows. The rapid evolution of drug-resistant strains has caused the efficacy of antibiotics to continue to decline and the cost of treatment to rise. What is more serious is that drug-resistant genes can circulate and spread among animals, humans and ecosystems through environmental media (such as wastewater, feces) and food chains, posing a risk of cross-species transmission. According to research, about 60% of mastitis pathogens on farms carry resistance genes such as β-lactamase or mecA. These strains may be transmitted to the community through dairy products or contact, exacerbating the global public health burden. In this context, reducing dependence on antibiotics and developing natural alternatives have become an urgent need.

[0004] The leucine alkaloids (including leucine, neoleucine, isoleucine and isoneucine) are natural active ingredients isolated from the traditional African medicinal plant Leucine sanguine. Their unique indole-quinoline skeleton structure gives them diverse biological activities. Current studies have shown that this type of compound has significant potential in anti-malarial, anti-tumor, anti-inflammatory and blood sugar regulation, but its application in the antimicrobial field has not been fully explored. Our research group has conducted long-term, continuous, in-depth and comprehensive research on the application of leucine alkaloids in antibacterial and antifungal applications, and successfully developed a series of highly effective antibacterial compounds through structural modification. In response to the needs of prevention and treatment of bovine mastitis, this study focused on the main pathogens (such as Staphylococcus aureus, Streptococcus agalactiae, etc.) for evaluation. Experiments have confirmed that this type of derivatives has a significant inhibitory effect on the pathogens of bovine mastitis. This discovery not only reveals the scientific value of cephalosporin alkaloids as new antibacterial lead compounds, but also provides an important theoretical basis for the development of natural mastitis treatment agents to replace antibiotics, and has practical application prospects in solving the drug resistance crisis in animal husbandry.

[0005] Summary of the invention

[0006] The purpose of the present invention is to provide a new use of leucophylline alkaloid derivatives in treating cow mastitis, which is used for preventing and treating cow mastitis caused by pathogenic bacteria such as Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli and Staphylococcus aureus.

[0007] In order to achieve the above objectives, the present invention provides the following technical methods:

[0008] The invention provides novel uses of leucine derivatives, neoleucine derivatives, isoleucine derivatives and isoneucine derivatives in combating pathogenic bacteria of bovine mastitis. The dosage concentrations of the leucine derivatives for Streptococcus agalactiae ATCC 27956, Streptococcus dysgalactiae ATCC 27957, Escherichia coli ATCC 25922 and Staphylococcus aureus Newman are 100, 50, 25, 12.5, 6.25, 3.12 μg / mL and the like.

[0009] The chemical structural formula of the leucophylline derivatives BYT-01 to BYT-09 of the present invention is shown in Chemical Formula 1:

[0010]

[0011] Chemical formula 1

[0012] The chemical structural formula of the new cephalaenopsis alkaloid derivatives XBYT-01 to XBYT-45 of the present invention is shown in Chemical Formula 2:

[0013]

[0014]

[0015] Chemical formula 2

[0016] The chemical structural formula of the isobenzamine derivatives YBYT-01 to YBYT-21 of the present invention is shown in Chemical Formula 3:

[0017]

[0018] Chemical formula 3

[0019] The chemical structural formula of the isoneurobacterine derivatives YXBYT-01 to YXBYT-04 of the present invention is shown in Chemical Formula 4:

[0020]

[0021] Chemical formula 4

[0022] The invention adopts the method reported in the literature to synthesize leucine derivatives, neoleucine derivatives, isoleucine derivatives and isoneucine derivatives, separates and obtains pure products by conventional methods such as multiple silica gel column chromatography, and determines the structures of leucine derivatives BYT-01 to BYT-09, neoleucine derivatives XBYT-01 to XBYT-45, isoleucine derivatives YBYT-01 to YBYT-21 and isoneucine derivatives YXBYT-01 to YXBYT-04 by spectral techniques such as mass spectrometry and nuclear magnetic resonance, and the structural formulas are shown in chemical formulas 1 to 4. The activity screening results show that the compounds described in the invention exhibit a certain degree of inhibitory effect on the pathogenic bacteria of bovine mastitis. DETAILED DESCRIPTION

[0023] In order to better understand the present invention, the above content of the present invention is further described in detail below through specific embodiments. However, this should not be construed as limiting the present invention. The experimental methods described in the following examples are conventional methods unless otherwise specified.

[0024] Example 1: Determination of the activity of leucophylline alkaloid derivatives against pathogenic bacteria of bovine mastitis

[0025] 1) Test agents: cephalaenine derivatives BYT-01 to BYT-09, neocephalaenine derivatives XBYT-01 to XBYT-45, isocephalaenine derivatives YBYT-01 to YBYT-21 and isoneocephalaenine derivatives YXBYT-01 to YXBYT-04.

[0026] 2) Test bacteria: Streptococcus agalactiae ATCC 27956, Streptococcus dysgalactiae ATCC 27957, Escherichia coli ATCC 25922 and Staphylococcus aureus Newman.

[0027] 3) Test method:

[0028] The strains used in this experiment were frozen in the laboratory at -80℃ with 30% glycerol. The frozen strains of Streptococcus agalactiae and Streptococcus dysgalactiae were taken out and streaked on TSA (tryptic soy peptone agar medium) + 5% sheep blood agar plates, and cultured at 37℃ until single colonies grew. Single colonies were picked and added to brain heart infusion broth (BHI) liquid culture medium (tryptone: 10.0g, beef heart extract powder: 17.5g, sodium chloride: 5.0g, glucose: 2.0g, disodium hydrogen phosphate (12H2O): 2.5g, distilled water: 1L; sterilized at 121℃ for 20min), and cultured at 37℃, 140rpm constant temperature shaker until the logarithmic growth phase. Similarly, the frozen strains of Escherichia coli and Staphylococcus aureus were taken out and streaked on MH solid culture medium (beef powder: 2.0g, soluble starch: 1.5g, acid hydrolyzed casein: 17.5g, agar: 20g, distilled water: 1L; sterilized at 121℃ for 20min), and cultured at 37℃ until single colonies grew. Single colonies were picked and added to MH broth medium (beef powder: 2.0g, soluble starch: 1.5g, acid hydrolyzed casein: 17.5g, distilled water: 1L; sterilized at 121℃ for 20min). Then, the strains in the logarithmic growth phase were diluted to about 10 with the corresponding liquid culture medium. 6 Dissolve the compounds in DMSO, add them to the liquid culture medium, mix them evenly, and prepare the compounds 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 the drug-containing medium containing about 10 6CFU / mL bacterial culture was added to the wells of a 96-well plate, and the final compound concentration was 100μg / mL. 100μL of 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 control bacterial solution 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:

[0029] Corrected OD value = OD value of bacterial culture medium - OD value of sterile culture;

[0030] Inhibition rate = (corrected control culture medium bacterial solution OD value - corrected drug-containing culture medium OD value) / corrected control culture medium bacterial solution OD value × 100%

[0031] All experiments were repeated three times, and the inhibition rates of the compounds were shown in Tables 1-4.

[0032] Table 1. Inhibition rate of leucophylline derivatives at 100 μg / mL on pathogenic bacteria of bovine mastitis (%)

[0033]

[0034] Table 2. Inhibition rate of new leucophylla alkaloid derivatives at 100 μg / mL on pathogenic bacteria of bovine mastitis (%)

[0035]

[0036]

[0037] Table 3. Inhibition rate of isoflavone derivatives at 100 μg / mL on pathogenic bacteria of bovine mastitis (%)

[0038]

[0039]

[0040] Table 4. Inhibition rate of isoneurospermum officinale derivatives at 100 μg / mL on pathogenic bacteria of bovine mastitis (%)

[0041]

[0042] It can be seen from the test results in Tables 1-4 that the compounds involved in the present invention show good antibacterial activity against the tested strains.

[0043] The drug-containing liquid culture medium of the active compound was diluted by two-fold dilution 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 test method in Example 1. The lowest concentration with an inhibition rate greater than 90% was defined as MIC, and the activity data obtained by the determination are shown in Tables 5-8.

[0044] Table 5. MIC values ​​of leucophylline derivatives against pathogenic bacteria of bovine mastitis

[0045]

[0046] Table 6. MIC values ​​of new leucophylline derivatives against pathogenic bacteria of bovine mastitis

[0047]

[0048]

[0049]

[0050] Table 7. MIC values ​​of isoflavone derivatives against pathogenic bacteria of bovine mastitis

[0051]

[0052]

[0053] Table 8. MIC values ​​of isoneurospermum officinale derivatives against pathogenic bacteria of bovine mastitis

[0054]

[0055] The experimental results show that the leucine alkaloid derivatives of the present invention show excellent bactericidal activity against the test strains, among which BYT-03 among the leucine derivatives has the best antibacterial activity against Streptococcus agalactiae and Streptococcus dysgalactiae, with a MIC value of 3.12 μg / mL; XBYT-18 among the neoleucine derivatives shows the best antibacterial activity, with a MIC value of 3.12-12.5 μg / mL; YBYT-01 and YBYT-13 among the isoleucine derivatives have broad-spectrum antibacterial activity, with MIC values ​​of 12.5-100 μg / mL and 25-100 μg / mL, respectively; isoneoleucine derivatives have broad-spectrum antibacterial activity and YXBYT-03 has the best activity, with a MIC value of 6.25-12.5 μg / mL. All of the above compounds show significant antibacterial activity.

[0056] In summary, the leucoderma alkaloid derivatives described in the present invention show significant antibacterial activity against Streptococcus agalactiae ATCC 27956, Streptococcus dysgalactiae ATCC27957, Escherichia coli ATCC 25922, and Staphylococcus aureus Newman, and can be further studied and developed in the prevention and treatment of bovine mastitis.

Claims

1. The present invention relates to the use of leucophylline alkaloid derivatives in preventing and treating pathogenic bacteria of cow mastitis.

2. The use according to claim 1, wherein The structural formula of the compound involved is as follows:

3. The use of the leucine derivatives BYT-01 to BYT-09, the neoleucine derivatives XBYT-01 to XBYT-45, the isoleucine derivatives YBYT-01 to YBYT-21 and the isoneucine derivatives YXBYT-01 to YXBYT-04 according to claims 1-2 in preventing and treating bovine mastitis caused by Streptococcus agalactiae ATCC 27956, Streptococcus dysgalactiae ATCC 27957, Escherichia coli ATCC 25922 and Staphylococcus aureus Newman.

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