CN117530941B - Application of 3- (4-tolyloxy) -propionic acid in bacteriostasis - Google Patents

Abstract

The invention belongs to the technical field of bacteriostasis. The invention provides an application of 3- (4-tolyloxy) -propionic acid in bacteriostasis, wherein the bacteria comprise gram-negative bacteria. The invention discovers that 3- (4-tolyloxy) -propionic acid has excellent effect on bacteriostasis to gram-negative bacteria, wherein MIC to escherichia coli and salmonella is 7.8125 mug/mL and 3.90625 mug/mL respectively, and MBC is 15.625 mug/mL and 7.8125 mug/mL respectively; 3- (4-tolyloxy) -propionic acid has simple structure and easy synthesis, and has application prospect in preparing gram-negative bacteria antibacterial preparation.

Description

Application of 3- (4-tolyloxy) -propionic acid in bacteriostasis

Technical Field

The invention belongs to the technical field of bacteriostasis, and particularly relates to application of 3- (4-tolyloxy) -propionic acid in bacteriostasis.

Background

Polyketides are a class of secondary metabolites produced by eukaryotic and prokaryotic organisms, and the biological activities of polyketides have been found to include antioxidant, antitumor, immunosuppressive, antibacterial, anti-inflammatory and antiparasitic activities, and the like.

In terms of antibacterial properties, polyketides have been found to be predominantly focused on antifungal properties, with a small portion having antibacterial activity against gram-positive bacteria, e.g., jiang Ningyu et al ^[1] isolated a polyketide 95F26A which has greater activity against gram-positive bacteria. However, the inhibitory effect of polyketides against gram-negative bacteria was found to be generally poor, such as the aforementioned compound 95F26A having a MIC of greater than 100ug/mL for E.coli, polyketides isolated as Liu Jingying et al ^[2] having a MIC of greater than 512ug/mL for E.coli, polyketides isolated as Wang Qi et al ^[3] having no inhibitory activity against E.coli, and 7 polyketides isolated as Yu Shengkai ^[4] having little inhibitory effect against E.coli.

In addition, the polyketide compounds with antibacterial activity have the defect of complex structures, and are not beneficial to synthesis and subsequent large-scale preparation.

Therefore, there is a need in the art to find a polyketide compound with a simple structure and good antibacterial activity against gram-negative bacteria, so as to complement the defects of the polyketide compound.

[1] Jiang Ningyu, peng Fei, log, et al, research on aromatic polyketide antibacterial active metabolites produced by marine source actinomyces madurae FIM95-F26 [ J ]. Journal of chinese antibiotics 2015,40 (03): 161-165.doi:10.13461/j.cnki.cja.005504.

[2] Liu Jingying, white rock, pan Huaji et al, targeted activation of the type II polyketide cluster of Streptomyces carbo Wu Ershi NA4 and identification of its products [ J ]. Microbiology report 2023,63 (10): 3891-3904.DOI:10.13343/j.cnki.wsxb.20230120.

[3] Wang Qi, yang Li, ma Qingyun et al, 'Thermology No. 1' aquilaria sinensis endophytic fungus Diaporthe sp.BMX12 secondary metabolite [ J/OL ]. Molecular plant breeding: 1-9[2023-10-18]. Http:// kns.cnki.net/kcms/detail/46.1068.S.20230508.1227.013.

[4] Yu Shengkai Aspergillus fumigatus Aspergillus fumigatu SZW01 secondary metabolite chemical composition and antibacterial Activity research [ D ]. Harbin university of commerce 2020.DOI:10.27787/d.cnki.ghrbs.2020.000692.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a polyketone compound which has a simple structure and can have better antibacterial activity on gram-negative bacteria, and provides application of the polyketone compound in the aspect of antibacterial activity on the gram-negative bacteria.

In view of the above, the present invention provides the use of 3- (4-tolyloxy) -propionic acid in bacteriostasis, including gram-negative bacteria.

Preferably, the gram negative bacteria include escherichia coli and salmonella.

More specifically, the E.coli and Salmonella are ATCC 25922 and ATCC 13076, respectively.

3- (4-Tolyloxy) -propanoic acid, chinese name 3- (4-tolyloxy) -propionic acid, CAS number 25173-37-9, was first isolated from glabrous greenbrier rhizome endogenous mango bulb holder bacteria in 2012 by Liang Faliang et al ^[5], and initially used as an antitumor active ingredient.

The inventor of the invention also separates and obtains the substance from the epicatechin smooth fungus Diaporthe sp.QX4G6 in the research process, and finds that 3- (4-tolyloxy) -propionic acid has excellent antibacterial activity for gram-negative bacteria in antibacterial experiments, MIC for escherichia coli and salmonella are 7.8125 mug/mL and 3.90625 mug/mL respectively, MBC is 15.625 mug/mL and 7.8125 mug/mL respectively, and the antibacterial effect is obviously better than that of known polyketides. Meanwhile, the compound has a simple structure and is easy to synthesize, and the synthetic method can adopt the routes described in Journal of THE AMERICAN CHEMICAL Society, vol.46, p.2863 or Journal of AMERICAN CHEMICAL Society, vol.71, p.661.

The invention has the beneficial effects that:

the invention discovers that 3- (4-tolyloxy) -propionic acid has excellent effect on bacteriostasis to gram-negative bacteria, wherein MIC to escherichia coli and salmonella is 7.8125 mug/mL and 3.90625 mug/mL respectively, and MBC is 15.625 mug/mL and 7.8125 mug/mL respectively; 3- (4-tolyloxy) -propionic acid has simple structure and easy synthesis, and has application prospect in preparing gram-negative bacteria antibacterial preparation.

[5] Liang Faliang, li Dongli, chen Yuchan, etc. A secondary metabolite of Mangifera Indicae and its anti-tumor activity is obtained by researching (J) Chinese herbal medicines 2012,43 (05): 856-860.

Drawings

FIG. 1 is a graph of the MIC test results for 3- (4-tolyloxy) -propionic acid, wherein the well plate labeled "3" behaves as 3- (4-tolyloxy) -propionic acid, "5" as positive control and "6" as vehicle control; "-" is a negative control;

FIG. 2 is a graph showing experimental results of MBC of 3- (4-tolyloxy) -propionic acid.

Detailed Description

The present invention is described in detail below by way of examples, which are necessary to be pointed out herein for further illustration of the invention and are not to be construed as limiting the scope of the invention, since numerous insubstantial modifications and adaptations of the invention will occur to those skilled in the art in light of the foregoing disclosure.

Example 1

1.1 Test materials

1.1.1 Main test consumables and the reagents Potato Dextrose (PDB) and Potato Dextrose Agar (PDA) media, both purchased from Shanghai Bo microorganism technologies Co., ltd; column chromatography silica gel (100-200 mesh, 200-300 mesh), thin layer silica gel plate (GF ₂₅₄) and semi-prepared silica gel thin layer plate (GF ₂₅₄) are all purchased from Qingdao ocean chemical Co., ltd; hydrolyzed casein (MH) medium, available from south-access kefir biotechnology development limited; organic microporous filter (0.22 μm), available from MerckMillipore, germany; petroleum ether (boiling range 60-90), chloroform, ethyl acetate and methanol, all analytically pure, all available from Tianjin Fuyu fine chemical Co., ltd; deuterated methanol and deuterated chloroform, both available from ala Ding Shiji (Shanghai) limited; levofloxacin, lot BA043G1, available from first co-pharmaceutical (beijing) inc; color-developing agent: 10% concentrated sulfuric acid ethanol is self-made in a laboratory.

1.1.2 Main test instruments Water-proof constant temperature incubator, model GSP-9160MBE, purchased from Shanghai BoXie medical biological instruments Co., ltd; a rotary evaporator, model N-1300D-W, available from Shanghai Ailang instruments Co., ltd; the camera-type ultraviolet analyzer, model WFH-203B, is available from Hangzhou Qigou impressive and dignified manner; electronic balance, model BSA223S, available from Sartorius company, germany; nuclear magnetic resonance and mass spectrometry were performed by the covalent bond boeing technology, ltd.

1.1.3 Test strains the epicatechin smooth fungus Diaporthe sp.QX4G6 was isolated from the earlier work of the project group, and the standard strains E.coli ATCC 25922 and Salmonella ATCC 13076 were all from the animal husbandry and veterinary institute of Guizhou province.

1.2 Test content and method

1.2.1 Extraction and separation of Compounds

Resuscitating the seed fungus Diaporthe sp.QX4G6 of the epicatechin-light, inoculating the seed fungus to 300mL of PDB culture medium sterilized by high pressure steam under aseptic condition, inoculating 36 bottles altogether, culturing at room temperature of 27 ℃ for 30 days, leaching 3 times with ethyl acetate for 24 hours each time, combining and filtering the extracting solutions, and concentrating by rotary evaporation to obtain 19.15g of extract.

Crude separation of the total extract was performed by silica gel column chromatography, gradient elution with petroleum ether-ethyl acetate, from 1：0(1500mL)、50：1(1000mL)、30：1(3000mL)、10：1(1000mL)、8：1(2500mL)、5：1(1500mL)、3：1(4000mL)、2：1(2000mL)、1：1(3000mL) to 0:1 (2000 mL) and finally column flushed with methanol (1500 mL). Concentrating each bottle of eluent, adding a silica gel thin layer plate, developing with concentrated sulfuric acid-ethanol, mixing similar fractions, and respectively obtaining components 1-14 according to low-to-high polarity, wherein component 6 (196 mg) is subjected to petroleum ether-ethyl acetate (8:1) silica gel column chromatography, and then 6 components are obtained according to low-to-high polarity; component 6-3 (101 mg) via petroleum ether: after ethyl acetate (7:1) silica gel column chromatography, 3 components are obtained according to the polarity from low to high; component 6-3-2 (80 mg) via petroleum ether: after ethyl acetate (7:1) silica gel column chromatography, 3 components are obtained according to the polarity from low to high; 6-3-2-3 (63 mg) via petroleum ether: ethyl acetate (7:1) silica gel column chromatography to obtain 3 components; component 6-3-2-3-2 (46 mg) was purified by petroleum ether: after ethyl acetate (8:1) silica gel column chromatography, 1 monomer compound 3 (31 mg) was obtained therefrom and designated GEC-I-3.

1.2.2 In vitro bacteriostasis test of monomeric Compounds

1.2.2.1 Preparation of test sample and reference solution respectively accurately weighing 1.00mg of GEC-I-3 monomer compound and norfloxacin as a positive control drug in a PE tube, adding a proper amount of 10% methanol physiological saline solution to prepare a solution with initial concentration of 1.00 mg.mL ^-1, vortex mixing uniformly, filtering with a 0.22 μm filter head under aseptic operation to prepare a test solution, and simultaneously preparing a solvent solution test (10% methanol-physiological saline) for later use. The final concentrations of some of the test solutions in the 96-well plates are shown in Table 1.

TABLE 1 final sample solution concentration

1.2.2.2 Preparation of bacterial liquid, inoculating Escherichia coli and salmonella onto MH solid culture medium, streaking and activating, placing the culture medium at 37 ℃ for 12 hours, picking single bacterial colony into a test tube containing MH liquid culture medium, placing the test tube into the culture medium at 37 ℃ for shake culture at 200 r.min ^-1 for 12 hours. The concentration of the bacterial liquid is regulated to be about 10 ⁵～10⁶cfu·mL^-1 by MH liquid culture medium for standby.

1.2.2.3 Determination of Minimum Inhibitory Concentration (MIC) A micro broth double dilution method was used, 50. Mu.L broth medium was added to each well of a sterile 96 well plate, 50. Mu.L of the prepared drug solution was added to the first well, and after mixing, the mixture was diluted in sequential double ratios until the last well was mixed, and 50. Mu.L of the mixture was discarded. Each well was filled with 50. Mu.L of the above-prepared bacterial liquid. Each plate was set with positive control (levofloxacin), negative control (no drug added with bacteria and no drug added with bacteria) and vehicle (10% methanol physiological saline solution) control, and after incubation at 37 ℃ for 16-18 h, the plates were taken out and observed for bacterial growth. 10 mu L of 0.5% TTC (2, 3, 5-triphenyltetrazolium chloride) is added into each well of the 96-well plate after 18h of culture, the light is prevented from being used for 0.5h at 37 ℃, the color change of bacterial liquid is observed, and the MIC result is judged by adopting a TTC method. The lowest drug mass concentration at which the solution is clear and the color is unchanged is visually observed as the MIC of the drug.

1.2.2.4 Determination of Minimum Bactericidal Concentration (MBC) 10. Mu.L of liquid from the last 3 wells of the above test group, which were not developed, were plated onto MH solid medium and incubated at 37℃for 16-18 h for observation. The lowest drug mass concentration at which no colony grew was observed visually was its MBC.

2 Results

2.1 Identification of monomeric Compounds

The specific structure, spectral data and designations of GEC-I-3 are as follows:

GEC-I-3: pale yellow oily matter, is easy to dissolve in chloroform and ethyl acetate, and is developed by 10% concentrated sulfuric acid ethanol to be light pink, and ESI-MS m/z:179.26[ M-H ] ^-, with the chemical formula C ₁₀H₁₂O₃. ¹H-NMR、¹³ C-NMR spectrum data of this compound is shown below ：¹H-NMR(CDCl₃,400MHz)：δ7.06(2H,d,J＝8.46Hz,H-2',6'),6.77(2H,d,J＝8.53Hz,H-3',5'),4.24(2H,t,J＝7.12Hz,H-2),2.86(2H,tJ＝7.11Hz,H-3),2.04(3H,s,H-7').¹³C-NMR(CDCl₃,400MHz)：δ171.86(C-1),154.74(C-1'),130.18(C-4'),129.71(C-3',5'),115.59(C-2',6'),65.60(C-3),34.35(C-2),21.21(C-7'). this compound is 3- (4-tolyloxy) -propionic acid (3- (4-tolyloxy) -propanoic acid) and the structure is shown below:

2.2 in vitro bacteriostasis test of monomer Compounds

As shown in FIGS. 1 and 2, the Minimum Inhibitory Concentrations (MIC) of GEC-I-3 for E.coli and Salmonella were 7.8125 μg/mL and 3.90625 μg/mL, respectively; the Minimum Bactericidal Concentration (MBC) for E.coli and Salmonella was 15.625. Mu.g/mL and 7.8125. Mu.g/mL, respectively.

Claims (1)

1. Use of 3- (4-tolyloxy) -propionic acid for the manufacture of a medicament for inhibiting gram-negative bacteria, wherein the gram-negative bacteria comprise escherichia coli and salmonella.