Cyclic lipopeptide compound as well as preparation method and application thereof

Abstract

The invention discloses a cyclic lipopeptide compound as well as a preparation method and application thereof. The cyclic lipopeptide compound is a compound C76H135N17O17, and the structural formula of the cyclic lipopeptide compound is shown in the description. The preparation method comprises the following steps: fermenting seawater bacillus miller in a liquid culture medium, centrifuging the fermented liquid culture to obtain a cell culture, soaking the cell culture in acetone, ultrasonically crushing, repeatedly soaking and extracting by using a mixed solution of methanol and dichloromethane, merging extract liquor, and concentrating to obtain a fermented crude extract; and repeatedly extracting the crude extract by using a dichloromethane-water solution, collecting a dichloromethane component, purifying by using semi-preparative liquid chromatography by using an isocratic acetonitrile-water solution as a mobile phase, and collecting a specific absorption peak. The compound extracted from the fermentation product of the seawater bacillus miller has remarkable inhibitory activity on gram-positive bacteria, gram-negative bacteria and fungi, and has important value in research and development of antibacterial and fungal drugs.

Description

Technical Field

[0001] The present invention relates to the technical fields of medicinal chemistry and microbial medicine technology, and more specifically, to a cyclic lipopeptide compound obtained from the fermentation product of Aquimarina muelleri, and its preparation method and application. Background Art

[0002] Cyclic lipopeptides are a class of compounds composed of a hydrophilic polypeptide core and at least one hydrophobic fatty acyl chain that are linked to form a cyclic structure, and can be produced by a variety of bacteria. Cyclic lipopeptides with diverse shapes, sizes, and chemical compositions have been discovered in nature, demonstrating the rich structural diversity of this class of molecules. Many cyclic peptides exhibit good biological activities, such as antibacterial, antiviral, antitumor, and immunosuppressive activities, and have significant potential for drug development. Summary of the Invention

[0003] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a method for obtaining a cyclic lipopeptide compound from the fermentation product of Aquimarina muelleri, its separation and purification method, and its application in inhibiting the activities of Gram-positive bacteria, Gram-negative bacteria, and fungi.

[0004] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] I. A cyclic lipopeptide compound:

[0006] The cyclic lipopeptide compound has the molecular formula C 76 H 135 N 17 O 17 , and the structural formula is Formula I (the Arabic numerals in the structural formula are the position markers of carbon atoms in the chemical structure, FA is fatty acid, L-Orn is L-ornithine, L-Phe is L-phenylalanine, D-β-OH-Val is D-β-hydroxyvaline, D-allo-Thr is D-allothreonine, γ-OH-Orn is γ-hydroxyornithine, D-Val is D-valine, L-Ile is L-isoleucine, L-Leu is L-leucine, D-Orn is D-ornithine, L-Thr is L-threonine, Gly is glycine):

[0007]

[0008]

[0009] II. A preparation method of the cyclic lipopeptide compound according to claim 1:

[0010] 1) Ferment Aquimarina muelleri in a liquid medium. After fermentation, centrifuge the liquid culture to obtain a cell culture. Soak it with acetone and ultrasonically disrupt it. Then repeatedly soak and extract it with a mixed solution of methanol and dichloromethane, and combine the extracts and concentrate to obtain a crude fermentation extract;

[0011] 2) Repeatedly extract the crude fermentation extract 2 - 4 times with a dichloromethane - aqueous solution;

[0012] 3) Collect the components obtained by extraction in the above steps and perform semi - preparative high - performance liquid chromatography (HPLC) purification. Use an isocratic acetonitrile - aqueous solution as the mobile phase, and collect the absorption peak to obtain the target compound, that is, a cyclic lipopeptide compound.

[0013] In the mixed solution of methanol and dichloromethane in step 1), the volume ratio of methanol to dichloromethane v / v is 1:1.

[0014] In step 1), the liquid medium is a liquid medium containing 5.0 g of peptone, 1.0 g of yeast extract powder, 0.1 g of ferric citrate, 19.45 g of sodium chloride, 5.98 g of magnesium chloride, 3.24 g of sodium sulfate, 0.008 g of disodium hydrogen phosphate, 1.8 g of calcium chloride, 0.55 g of potassium chloride, 0.16 g of sodium carbonate, 0.08 g of potassium bromide, 0.034 g of strontium chloride, 0.004 g of sodium silicate, 0.022 g of boric acid, 0.0024 g of sodium fluoride, 0.0016 g of ammonium nitrate in every 1000 mL of deionized water, and the pH is 7.6.

[0015] In step 1), the specific process conditions for fermenting Aquimarina muelleri in the liquid medium are as follows:

[0016] Inoculate Aquimarina muelleri into a 2216E liquid medium, and culture it at 28 °C on a shaker at 200 rpm for 3 days as a seed liquid; Take 100 μL of the above - mentioned seed liquid and inoculate it into a 500 mL conical flask containing 100 mL of sterilized 2216E liquid medium, and culture it at 28 °C on a shaker at 200 rpm for 5 days.

[0017] In the mixed solution of dichloromethane and water in step 2), the volume ratio of dichloromethane to water v / v is 1:1.

[0018] In step 3), the treatment with the isocratic acetonitrile - aqueous solution as the mobile phase is specifically set in the 0 - 10 min stage, which contains 80% (volume fraction) of acetonitrile (ACN).

[0019] In the above-mentioned acetonitrile-aqueous solution, 0.1% (by volume) of trifluoroacetic acid (TFA) is added to acetonitrile (ACN) and H2O. The addition of trifluoroacetic acid (TFA) is used to reduce the absorption peak tailing and improve the separation effect.

[0020] In step 3), the absorption peak at 210 nm in the range of 5.9 - 6.3 min is collected to obtain the target compound.

[0021] Use of the cyclic lipopeptide compound in inhibiting bacteria and fungi and in the preparation of antibacterial and antifungal drugs.

[0022] The bacteria are Gram-positive bacteria and Gram-negative bacteria, and the fungi are yeasts. Specifically, the Gram-positive bacteria include Bacillus subtilis, Rhodococcus erythropolis, and Enterococcus faecium. The Gram-negative bacteria include Escherichia coli DH5α. The fungus is Saccharomyces cerevisiae.

[0023] According to literature research, the cyclic lipopeptide compound involved in the present invention is a new compound, which has not been reported in the literature before and its antibacterial activity has not been studied. The prior experiments of the present invention show that its inhibitory activities against Gram-positive bacteria, Gram-negative bacteria, and fungi make it a potential active molecule for antibacterial and antifungal applications.

[0024] Advantages of the present invention:

[0025] The present invention discovers a cyclic lipopeptide compound with a novel structure and significant antibacterial and antifungal activities from the fermentation product of the liquid medium of the bacterium Aquimarina muelleri belonging to the genus Aquimarina. There is currently no report on the chemical structure and antibacterial activity of this compound, and thus there are no related drugs on the market.

[0026] Through inhibitory activity tests, the compound has significant inhibitory activities against the Gram-positive bacteria Bacillus subtilis, Rhodococcus erythropolis, Enterococcus faecium, the Gram-negative bacterium Escherichia coli DH5α, and the fungus Saccharomyces cerevisiae, and can be used as a new drug ingredient or lead compound for antibacterial and antifungal applications. Detailed implementation manners

[0027] The following specific examples are used to further illustrate the present invention, but the present invention is by no means limited to these examples.

[0028] The compounds referred to in the following examples of the present invention were isolated from Aquimarina muelleri, and the chemical structures of the compounds are shown in Formula I (the Arabic numerals in the structural formula are the positions of carbon atoms in the chemical structure):

[0029]

[0030] Aquimarina muelleri is characterized by orange-yellow colonies on 2216E medium, without obvious spores, and the colonies are without folds.

[0031] The said strain is documented in the literature and can also be obtained from public circulation channels, such as the literature Int.J.Syst.Evol.Microbiol., 55, 225 - 229, 2005, DOI: 10.1099 / ijs.0.63349 - 0. Description of Aquimarina muelleri gen.nov., sp.nov., and proposal of the reclassification of [Cytophaga] latercula Lewin 1969 as Stanierella latercula gen.nov., comb.nov. Nedashkovskaya OI, Kim SB, Lysenko AM, Frolova GM, Mikhailov VV, Lee KH, Bae KS.

[0032] Example 1. Fermentation production, separation and purification of the compound shown in Formula I:

[0033] Inoculate Aquimarina muelleri used in the present invention into 2216E liquid medium and culture it on a shaker at 28 °C and 200 rpm for 3 days as the seed solution.

[0034] Take 100 μL of the above seed solution and inoculate it into a sterilized 500 mL conical flask containing 100 mL of 2216E liquid medium, and culture it on a shaker at 28 °C and 200 rpm for 5 days.

[0035] The liquid medium is a liquid medium containing 5.0 g of peptone, 1.0 g of yeast extract powder, 0.1 g of ferric citrate, 19.45 g of sodium chloride, 5.98 g of magnesium chloride, 3.24 g of sodium sulfate, 0.008 g of disodium hydrogen phosphate, 1.8 g of calcium chloride, 0.55 g of potassium chloride, 0.16 g of sodium carbonate, 0.08 g of potassium bromide, 0.034 g of strontium chloride, 0.004 g of sodium silicate, 0.022 g of boric acid, 0.0024 g of sodium fluoride, 0.0016 g of ammonium nitrate in 1000 mL of deionized water, with a pH of 7.6.

[0036] The liquid culture obtained by fermenting the above-mentioned Marinobacter milleri in a liquid medium was centrifuged to obtain a cell culture, which was soaked in acetone and ultrasonically disrupted, and then soaked and extracted 3 times with methanol:dichloromethane = 1:1 (v / v). The extraction solutions were combined and concentrated to obtain a crude fermentation extract.

[0037] The crude fermentation extract was repeatedly extracted with a dichloromethane-aqueous solution, and the fractions obtained by dichloromethane extraction were collected and purified by semi-preparative high performance liquid chromatography (HPLC). An isocratic acetonitrile-aqueous solution (in the 0 - 10 min stage, set at a volume concentration of 80% ACN) containing 0.1% (v / v) trifluoroacetic acid (TFA) was used as the mobile phase, and the absorption peaks were collected:

[0038] The absorption peak at 5.9 - 6.3 min at a wavelength of 210 nm was collected to obtain the target compound;

[0039] The structural identification result is shown in Formula I:

[0040]

[0041] The compound has the following physicochemical and spectral properties:

[0042] Compound: gray amorphous powder, 1H NMR and 13C NMR are shown in Table I; high-resolution ESI mass spectrometry positive ion mode m / z 1559.0237 [M+H] + , C 76 H 136 N 17 O 17 The calculated value is 1559.0295.

[0043] Table I Data of 1H NMR (600 MHz, solvent DMSO-d6) and 13C NMR (150 MHz, solvent DMSO-d6) of the compound

[0044]

[0045]

[0046] The signal assignment in this table is based on1 H, 13 C, DEPT, 1 H- 1 The analytical results of H-H COSY, TCOSY, HSQC and HMBC spectra, and the multiplicity of carbon signals were determined by the DEPT method.

[0047] Test: Gram-positive bacteria and Gram-positive bacteria and fungal inhibitory activities:

[0048] The antibacterial activity of the compound shown in Formula I was detected by the minimum inhibitory concentration method. Three strains of Gram-positive bacteria, Bacillus subtilis, Rhodococcus erythropolis, Enterococcus faecium, one strain of Gram-negative bacteria, Escherichia coli, and one strain of fungus, Saccharomyces cerevisiae, were selected for antibacterial activity testing.

[0049] 1) Antibacterial activity test (MIC method):

[0050] The minimum inhibitory concentration (MIC) is the lowest drug concentration that can inhibit bacterial growth in vitro. In a 96-well microplate, by adding different concentrations of the drug to the bacterial suspension of the test bacteria and observing after incubation, if the indicator bacteria grow in a certain well, it means that the drug concentration in that well cannot inhibit the growth of the bacteria, and the liquid in that well is turbid and the light transmittance decreases significantly. On the contrary, the liquid in that well is clear and the light transmittance decreases insignificantly. The lowest sample concentration that completely inhibits the growth of the indicator bacteria in the small well is the MIC of the compound.

[0051] 2) Preparation of bacterial suspension

[0052] The above-mentioned test bacteria were cultured overnight at 37 °C (Bacillus subtilis, Escherichia coli, Enterococcus faecium using LB medium) and 28 °C (Rhodococcus using LB medium, Saccharomyces cerevisiae using PDB medium), and the bacterial solution concentration was adjusted to OD 600 = 0.08 - 0.1.

[0053] 3) Preparation of samples

[0054] About 1 mg of the test sample (the above-obtained compound) and positive controls (chloramphenicol, kanamycin) were taken, and DMSO was used as the solvent to prepare a sample solution of 4 mg / mL.

[0055] 4) Blank control: Select the pure solvent that does not dissolve the test sample as the blank control.

[0056] 5) MIC determination process

[0057] 5.1) Under aseptic conditions, add 5 μL of the sample solution to the sterile 96-well plate (rows A-H, columns 1-12) at A1.

[0058] 5.2) Take 195 μL of the prepared bacterial suspension and add it to A1 of the 96-well plate. At the same time, add 100 μL of the bacterial suspension to A2 - A10. Gently mix the liquid in well A1 with a micropipette. After ensuring that the sample and the bacterial suspension are fully mixed, take 100 μL of the mixed solution from A1 and transfer it to A2. Repeat the above steps, successively taking 100 μL of the mixed solution from well A2 and transferring it to well A3 until well A10, so that the final concentrations of the sample are 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195 μg / mL in sequence. After gently shaking and mixing evenly, seal the 96-well plate and place it in an incubator at 37 °C or 28 °C. Start observing after culturing for 12 h.

[0059] 5.3) If the indicator bacteria grow in a certain well, it means that the drug concentration in that well cannot inhibit the growth of the bacteria, and the liquid in that well becomes turbid and the light transmittance decreases significantly. On the contrary, the liquid in that well is clear and the decrease in light transmittance is not significant. The lowest sample concentration that completely inhibits the growth of the indicator bacteria in the small well is the MIC of this compound.

[0060] The experimental results show that the compound has inhibitory activities against Gram-positive bacteria Bacillus subtilis, Rhodococcus erythropolis, Enterococcus faecium, and its minimum inhibitory concentrations (MICs) are 3.125 μg / mL, 25 μg / mL, 12.5 μg / mL respectively; the compound has antibacterial activity against Gram-negative bacterium Escherichia coli, and its minimum inhibitory concentration (MIC) is 3.125 μg / mL; the compound also has antibacterial activity against the fungus Saccharomyces cerevisiae, and its minimum inhibitory concentration (MIC) is 3.125 μg / mL.

[0061] The above experimental results prove that the compound involved in the present invention has significant inhibitory activities against Gram-positive bacteria Bacillus subtilis, Rhodococcus erythropolis, Enterococcus faecium, Gram-negative bacterium Escherichia coli DH5α, and fungus Saccharomyces cerevisiae, and they may be used to prepare new drugs against Gram-positive bacteria, Gram-negative bacteria, and fungi.

[0062] As can be seen from the above implementation, the compound extracted from the fermentation product of Marinobacter millerae of the present invention has significant inhibitory activities against Gram-positive bacteria (Bacillus subtilis, Rhodococcus sp., Enterococcus faecalis), Gram-negative bacteria (Escherichia coli), and fungi (Saccharomyces cerevisiae), showing its important value in the research and development of antibacterial and antifungal drugs.

[0063] The above specific implementation manners are used to explain and illustrate the present invention, rather than to limit the present invention. Any modification and change made to the present invention within the spirit and scope of the claims of the present invention fall within the protection scope of the present invention.

[0064] The above are only the preferred implementation manners of the present invention. Therefore, all equivalent changes or modifications made according to the structures, features, and principles described in the scope of the patent application of the present invention are included in the scope of the patent application of the present invention.

Claims

1. A cyclic lipopeptide compound, characterized in that: The cyclic lipopeptide compound has a molecular formula of C 76 H 135 N 17 O 17 , and its structural formula is Formula I:

2. A preparation method of the cyclic lipopeptide compound according to claim 1, characterized in that: 1) Ferment Aquimarina muelleri in a liquid medium, centrifuge the fermented liquid culture to obtain a cell culture, soak it with acetone and ultrasonically disrupt it, then repeatedly soak and extract it with a mixed solution of methanol and dichloromethane, and combine and concentrate the extract to obtain a crude fermentation extract; 2) Repeatedly extract the crude fermentation extract with a dichloromethane - aqueous solution; 3) Collect the components obtained by extraction in the above steps and perform semi - preparative high - performance liquid chromatography (HPLC) purification. Use an isocratic acetonitrile - aqueous solution as the mobile phase, and collect the absorption peak to obtain the target compound.

3. According to the preparation method described in claim 2, characterized in that: In the mixed solution of methanol and dichloromethane in step 1), the volume ratio of methanol: dichloromethane v / v is 1:

1.

4. According to the preparation method described in claim 2, characterized in that: In step 1), the liquid medium contains 5.0 g of peptone, 1.0 g of yeast extract powder, 0.1 g of ferric citrate, 19.45 g of sodium chloride, 5.98 g of magnesium chloride, 3.24 g of sodium sulfate, 0.008 g of disodium hydrogen phosphate, 1.8 g of calcium chloride, 0.55 g of potassium chloride, 0.16 g of sodium carbonate, 0.08 g of potassium bromide, 0.034 g of strontium chloride, 0.004 g of sodium silicate, 0.022 g of boric acid, 0.0024 g of sodium fluoride, 0.0016 g of ammonium nitrate in every 1000 mL of deionized water, and the pH is 7.6 liquid medium.

5. According to the preparation method described in claim 2, characterized in that: In step 1), the specific process conditions for fermenting Aquimarina muelleri in the liquid medium are: Inoculate Aquimarina muelleri into 2216E liquid medium, culture it at 200 rpm on a shaker at 28 °C for 3 days as the seed liquid; take 100 μL of the above seed liquid and inoculate it into a 500 mL conical flask containing 100 mL of sterilized 2216E liquid medium, and culture it at 200 rpm on a shaker at 28 °C for 5 days.

6. According to the preparation method described in claim 2, characterized in that: In the mixed solution of dichloromethane and water in step 2), the volume ratio of dichloromethane: water v / v is 1:

1.

7. According to the preparation method described in claim 2, characterized in that: In step 3), the treatment with the isocratic acetonitrile - aqueous solution as the mobile phase is specifically set in the 0 - 10 min stage, which contains 80% (volume fraction) of acetonitrile (ACN).

8. According to the preparation method described in claim 2, characterized in that: In step 3), collect the absorption peak at 210 nm wavelength from 5.9 - 6.3 min to obtain the target compound.

9. Use of the cyclic lipopeptide compound according to claim 1 or the cyclic lipopeptide compound prepared by the preparation method according to any one of claims 2-8, characterized in that: The application of the cyclic lipopeptide compound in inhibiting bacteria and fungi and in the preparation of antibacterial and antifungal drugs.

10. An application of the cyclic lipopeptide compound according to claim 9, characterized in that: The bacteria are Gram-positive bacteria and Gram-negative bacteria, and the fungi are yeasts.

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