CN113248513A - Novel cytochalasin compound with function of antagonizing clinically drug-resistant bacteria and preparation method thereof - Google Patents

Novel cytochalasin compound with function of antagonizing clinically drug-resistant bacteria and preparation method thereof Download PDF

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CN113248513A
CN113248513A CN202110385287.4A CN202110385287A CN113248513A CN 113248513 A CN113248513 A CN 113248513A CN 202110385287 A CN202110385287 A CN 202110385287A CN 113248513 A CN113248513 A CN 113248513A
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杨小龙
牟青林
王文静
向婷
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South Central Minzu University
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Abstract

The invention relates to a novel cytochalasin compound with a function of antagonizing clinically drug-resistant bacteria and a preparation method thereof, belonging to the technical field of biomedicine. Firstly, carrying out fermentation culture on golden yellow shell cyst bacteria; then extracting a secondary metabolite obtained by fermenting and culturing the golden yellow shell cyst bacteria by adopting methanol, extracting by using ethyl acetate, concentrating under reduced pressure, carrying out gradient elution on the obtained crude extract to obtain six group segments, and carrying out gradient elution on the fourth group segment again to obtain four subgroup segments; separating and purifying the second subgroup section and the fourth subgroup section by using sephadex gel chromatography and high performance liquid chromatography respectively to obtain two novel cytochalasin; the two novel cytochalasin have strong antagonistic action on four clinical drug-resistant bacteria such as carbapenem-resistant pseudomonas aeruginosa, methicillin-resistant staphylococcus aureus, multi-drug-resistant enterococcus faecalis and multi-drug-resistant enterococcus faecium, and are expected to be developed into novel drugs for resisting the clinical drug-resistant bacteria.

Description

Novel cytochalasin compound with function of antagonizing clinically drug-resistant bacteria and preparation method thereof
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a novel cytochalasin compound with a function of antagonizing clinically drug-resistant bacteria and a preparation method thereof.
Background
The drug-resistant bacteria refer to bacteria with tolerance to traditional antibacterial drugs, which is a special change form in the survival process of the bacteria, when the antibiotics are used for a long time, most sensitive bacterial strains are continuously killed, drug-resistant bacterial strains are massively propagated to replace the sensitive bacterial strains, so that the drug resistance rate of the bacteria to the drugs is continuously increased, and once the drug resistance is generated, the therapeutic effect of the drugs is obviously reduced or even completely loses the therapeutic effect. In recent years, a large number of drug-resistant bacteria have been produced worldwide due to abuse of antibiotics, and it has been reported that antibiotic-resistant bacteria are spreading worldwide as early as 2014, and among these drug-resistant bacteria, relatively famous are methicillin-resistant staphylococcus aureus, streptococcus pneumoniae, multi-drug-resistant enterococcus faecalis, multi-drug-resistant enterococcus faecium, methicillin-resistant staphylococcus aureus, carbapenem-resistant pseudomonas aeruginosa, and the like.
Cytochalasin is an alkaloid secondary metabolite produced by fungi. Such compounds bind to the positive end of microfilaments within cells and cause the depolymerization of F-actin, blocking the further polymerization of subunits, and when cytochalasin is added to living cells, the actin fibrous skeleton disappears, paralyzing various activities of the animal cells, including cell migration, phagocytosis, cytokinesis, etc., hence the name cytochalasin. It has no effect on microtubules nor inhibits muscle contraction.
To prevent the emergence of drug-resistant bacteria that are continuously threatening the health of people, a large number of researchers are constantly exploring new antibiotics or new molecules that should be able to combat clinically drug-resistant bacteria. The antibiotic includes the famous Teixobactin antibiotic which is a novel antibiotic molecule discovered by Jim Rivies team of northwest university of America, has very strong antagonistic effect on MRSA bacteria and the like, and can treat various common infections such as tuberculosis, septicemia and the like. In recent years, although novel molecules capable of antagonizing clinically resistant bacteria have been reported, few scholars have reported antagonism of cytochalasin-like compounds against clinically resistant bacteria.
Disclosure of Invention
In recent years, due to antibiotic abuse, various clinical drug-resistant bacteria appear worldwide, the drug-resistant bacteria threaten the physical health of people step by step, and researchers in various countries continuously develop novel molecules capable of antagonizing the drug-resistant bacteria in order to solve the worldwide public health problem. In the invention, we firstly discover novel cytochysins A and B discovered from secondary metabolites of fungi, and study the antagonistic action of the cytochysins A and B on multiple strains of clinically drug-resistant bacteria, finally discover that the two novel cytochalsins A and B have strong antagonistic action (MIC, 25 mu g/mL) on four clinically drug-resistant bacteria such as carbapenem-resistant pseudomonas aeruginosa, methicillin-resistant staphylococcus aureus, multi-drug-resistant enterococcus faecalis and multi-drug-resistant enterococcus faecium, continue to further study, and are expected to develop into novel drugs for resisting clinically drug-resistant bacteria.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a novel cytochalasin compound with the function of antagonizing clinically drug-resistant bacteria and a preparation method thereof comprise the following steps:
step (1), fermenting and culturing the golden yellow shell cyst bacteria;
step (2), extracting a secondary metabolite obtained by fermenting and culturing the golden yellow shell cyst bacteria by adopting methanol, concentrating under reduced pressure, evaporating a solvent, washing with water, extracting by using ethyl acetate, and finally concentrating under reduced pressure to obtain a crude extract;
step (3), loading the crude extract to a silica gel column by a wet method, performing gradient elution by a silica gel column chromatography method by using a dichloromethane and methanol mixed organic solvent with the volume ratio of 100: 1-1: 1, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain six component sections Fr.A-Fr.F;
and (4) loading the fourth component section Fr.D to a silica gel column by a wet method, performing gradient elution by a silica gel column chromatography method by using a dichloromethane and methanol mixed organic solvent with the volume ratio of 50: 1-1: 1, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, combining the same parts to obtain four subgroup sections Fr.D1-Fr.D4；
Step (5), segmenting Fr.D for the second subgroup2Sequentially separating and purifying by Sephadex chromatography and high performance liquid chromatography to obtain compound 2;
step (6), segmenting Fr.D of the fourth subgroup4Separating and purifying by using sephadex chromatography, collecting separated and purified liquid, standing, after crystals are separated out, cleaning impurities on the surfaces of the crystals by using dichloromethane, and finally obtaining a pure crystal compound, namely a compound 1;
wherein the structural formula of the compound 2 is as follows:
Figure BDA0003014489750000021
the structural formula of compound 1 is:
Figure BDA0003014489750000031
further, it is preferable that the specific method of step (1) is as follows:
(1.1) activating the golden yellow shell cyst bacteria on a PDA plate;
(1.2) adding the activated golden yellow shell cyst mycelium into a PDB culture medium, and carrying out constant-temperature shaking culture on a shaking table to prepare a strain seed solution;
(1.3) pouring the strain seed liquid into a solid culture medium for constant-temperature fermentation culture;
the solid culture medium is prepared by mixing MEB solution and rice according to the volume-mass ratio of 1: prepared at the ratio of 1 mL/g.
Further, it is preferable that (1.1) the activation time is 3 days; (1.2) in the step (1), the conditions of constant temperature shaking culture are 28 ℃, 160rpm and 3 days; (1.3) the conditions for constant temperature fermentation culture are 28 ℃ for 30 days.
Further, it is preferable that, in the step (2), the pressure for concentration under reduced pressure is-0.1 MPa and the temperature is-20 ℃.
Further, in the step (3), it is preferable that the volume ratio of the dichloromethane and methanol mixed organic solvent used in the gradient elution is 100: 1. 80: 1. 50: 1. 30: 1. 10: 1 and 1: 1; each gradient eluted to the TLC plate with no apparent main spot and the next concentration gradient was changed.
Further, in the step (4), it is preferable that the volume ratio of the dichloromethane and methanol mixed organic solvent used in the gradient elution is 50: 1. 20: 1. 10: 1. 1: 1; each gradient eluted to the TLC plate with no apparent main spot and the next concentration gradient was changed.
Further, it is preferable that, in the step (5), the conditions for separation and purification by high performance liquid chromatography are: acetonitrile water solution with volume concentration of 70% is taken as a mobile phase, and Agilent C is adopted18A reverse phase chromatographic column with the specification: 4.6mmx250nm, 5 um; column temperature: 30 ℃; column flow rate: 1 ml/min; sampling amount each time: 20 ul; an ultraviolet detector is adopted, and the detection wavelength is 210 nm. Collecting tRAccumulating for multiple times and evaporating to dryness;
in the step (6), the standing time is not less than 5 days.
The invention also provides a novel cytochalasin compound with the function of antagonizing clinically drug-resistant bacteria, wherein the cytochalasin compound is a compound 1, and the structural formula of the cytochalasin compound is as follows:
Figure BDA0003014489750000041
the invention also provides a novel cytochalasin compound with the function of antagonizing clinically drug-resistant bacteria, wherein the cytochalasin compound is a compound 2, and the structural formula of the cytochalasin compound is as follows:
Figure BDA0003014489750000042
the invention also provides application of the novel cytochalasin compound with the function of antagonizing clinically drug-resistant bacteria in preparation of clinically drug-resistant bacteria.
In the invention, the ascochyta chrysosporium HYQZ-931(Cytospora chrysosperma HYQZ-931) is preserved in China center for type culture collection at 3.25.2021, with the preservation number of CCTCC M2021279, and the preservation address is university of Wuhan, Wuhan.
The endophytic fungi is ascochyta aurantiacus, is a microorganism of Cytospora, has white filamentous hypha, is black brown and irregular in conidiophore, is multi-chambered, is buried in a stroma, has a common hole extending out of the stroma and protrudes out of the host epidermis, and has a shape similar to that of ascospore, colorless and single cell. The strain can grow at 4-35 deg.C, but the growth at 25 deg.C is most suitable. The pH value most suitable for hypha growth is 4, and the proper temperature for germination of conidia and ascospores is 25-30 ℃.
The invention firstly discovers novel cytochrysins A and B from secondary metabolites of fungi, researches the antagonism of the cytochrysins A and B on multiple strains of clinical drug-resistant bacteria, finally discovers that the two novel cytochrysins A and B have strong antagonism (MIC, 25 mu g/mL) on four clinical drug-resistant bacteria such as carbapenem-resistant pseudomonas aeruginosa, methicillin-resistant staphylococcus aureus, multi-drug-resistant enterococcus faecalis and multi-drug-resistant enterococcus faecium, continues further research, and is expected to develop novel drugs for resisting the clinical drug-resistant bacteria.
Compared with the prior art, the invention has the beneficial effects that:
1. two cytochrysins A and B reported in the invention are novel compounds discovered for the first time (see the structure of the compound in figure 1).
2. The invention also reports the inhibitory activity of the two compounds to four clinical drug-resistant bacteria (carbapenem-resistant pseudomonas aeruginosa, methicillin-resistant staphylococcus aureus, multi-drug-resistant enterococcus faecalis and multi-drug-resistant enterococcus faecium) for the first time (the activity results are shown in the attached table 3), and the two compounds continue to be further studied and are expected to be developed into novel drugs for resisting the clinical drug-resistant bacteria.
Drawings
FIG. 1 is the structural formula of Compound 1;
FIG. 2 is the structural formula of Compound 2;
FIG. 3 is the 1H NMR spectrum of Compound 1 (deuterated methanol, 600 MHz);
FIG. 4 is a 13C and DEPT nuclear magnetic spectrum (deuterated methanol, 150MHz) of Compound 1;
FIG. 5 is a high resolution mass spectrum of Compound 1;
FIG. 6 is a graph showing an ultraviolet absorption spectrum of Compound 1;
FIG. 7 is the 1H nuclear magnetic spectrum of Compound 2 (deuterated methanol, 600 MHz);
FIG. 8 is a 13C and DEPT nuclear magnetic spectrum (deuterated methanol, 150MHz) of Compound 2;
FIG. 9 is a high resolution mass spectrum of Compound 2;
FIG. 10 is a graph showing an ultraviolet absorption spectrum of Compound No. 2.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.
The percentage numbers represent percent by mass unless otherwise specified herein.
Example 1
The preparation method of the novel cytochalasin compound with the function of antagonizing clinically drug-resistant bacteria comprises the following steps:
step (1), fermenting and culturing the golden yellow shell cyst bacteria;
step (2), extracting a secondary metabolite obtained by fermenting and culturing the golden yellow shell cyst bacteria by adopting methanol, concentrating under reduced pressure, evaporating a solvent, washing with water, extracting by using ethyl acetate, and finally concentrating under reduced pressure to obtain a crude extract;
step (3), loading the crude extract to a silica gel column by a wet method, performing gradient elution by a silica gel column chromatography method by using a dichloromethane and methanol mixed organic solvent with the volume ratio of 100: 1-1: 1, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain six component sections Fr.A-Fr.F;
and (4) loading the fourth component section Fr.D to a silica gel column by a wet method, performing gradient elution by a silica gel column chromatography method by using a dichloromethane and methanol mixed organic solvent with the volume ratio of 50: 1-1: 1, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, combining the same parts to obtain four subgroup sections Fr.D1-Fr.D4；
Step (5), segmenting Fr.D for the second subgroup2Sequentially separating and purifying by Sephadex chromatography and high performance liquid chromatography to obtain compound 2;
step (6), segmenting Fr.D of the fourth subgroup4Separating and purifying by using sephadex chromatography, collecting separated and purified liquid, standing, after crystals are separated out, cleaning impurities on the surfaces of the crystals by using dichloromethane, and finally obtaining a pure crystal compound, namely a compound 1;
wherein the structural formula of the compound 2 is as follows:
Figure BDA0003014489750000061
the structural formula of compound 1 is:
Figure BDA0003014489750000062
example 2
The preparation method of the novel cytochalasin compound with the function of antagonizing clinically drug-resistant bacteria comprises the following steps:
step (1), fermenting and culturing the golden yellow shell cyst bacteria;
step (2), extracting a secondary metabolite obtained by fermenting and culturing the golden yellow shell cyst bacteria by adopting methanol, concentrating under reduced pressure, evaporating a solvent, washing with water, extracting by using ethyl acetate, and finally concentrating under reduced pressure to obtain a crude extract;
step (3), loading the crude extract to a silica gel column by a wet method, performing gradient elution by a silica gel column chromatography method by using a dichloromethane and methanol mixed organic solvent with the volume ratio of 100: 1-1: 1, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain six component sections Fr.A-Fr.F;
and (4) loading the fourth component section Fr.D to a silica gel column by a wet method, performing gradient elution by a silica gel column chromatography method by using a dichloromethane and methanol mixed organic solvent with the volume ratio of 50: 1-1: 1, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, combining the same parts to obtain four subgroup sections Fr.D1-Fr.D4；
Step (5), segmenting Fr.D for the second subgroup2Sequentially separating and purifying by Sephadex chromatography and high performance liquid chromatography to obtain compound 2;
step (6), segmenting Fr.D of the fourth subgroup4Separating and purifying by using sephadex chromatography, collecting separated and purified liquid, standing, after crystals are separated out, cleaning impurities on the surfaces of the crystals by using dichloromethane, and finally obtaining a pure crystal compound, namely a compound 1;
wherein the structural formula of the compound 2 is as follows:
Figure BDA0003014489750000071
the structural formula of compound 1 is:
Figure BDA0003014489750000072
the specific method of the step (1) is as follows:
(1.1) activating the golden yellow shell cyst bacteria on a PDA plate;
(1.2) adding the activated golden yellow shell cyst mycelium into a PDB culture medium, and carrying out constant-temperature shaking culture on a shaking table to prepare a strain seed solution;
(1.3) pouring the strain seed liquid into a solid culture medium for constant-temperature fermentation culture;
the solid culture medium is prepared by mixing MEB solution and rice according to the volume-mass ratio of 1: prepared at the ratio of 1 mL/g.
Example 3
The preparation method of the novel cytochalasin compound with the function of antagonizing clinically drug-resistant bacteria comprises the following steps:
step (1), fermenting and culturing the golden yellow shell cyst bacteria;
step (2), extracting a secondary metabolite obtained by fermenting and culturing the golden yellow shell cyst bacteria by adopting methanol, concentrating under reduced pressure, evaporating a solvent, washing with water, extracting by using ethyl acetate, and finally concentrating under reduced pressure to obtain a crude extract;
step (3), loading the crude extract to a silica gel column by a wet method, performing gradient elution by a silica gel column chromatography method by using a dichloromethane and methanol mixed organic solvent with the volume ratio of 100: 1-1: 1, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain six component sections Fr.A-Fr.F;
step (4), the fourth component section Fr.D is loaded to a silica gel column by a wet method, and dichloromethane and methanol with the volume ratio of 50: 1-1: 1 are carried out by a silica gel column chromatography methodMixing with organic solvent, performing gradient elution, collecting gradient eluents of each gradient, concentrating, monitoring by TLC, and mixing identical parts to obtain four subgroups segmented Fr.D1-Fr.D4；
Step (5), segmenting Fr.D for the second subgroup2Sequentially separating and purifying by Sephadex chromatography and high performance liquid chromatography to obtain compound 2;
step (6), segmenting Fr.D of the fourth subgroup4Separating and purifying by using sephadex chromatography, collecting separated and purified liquid, standing, after crystals are separated out, cleaning impurities on the surfaces of the crystals by using dichloromethane, and finally obtaining a pure crystal compound, namely a compound 1;
wherein the structural formula of the compound 2 is as follows:
Figure BDA0003014489750000081
the structural formula of compound 1 is:
Figure BDA0003014489750000082
the specific method of the step (1) is as follows:
(1.1) activating the golden yellow shell cyst bacteria on a PDA plate;
(1.2) adding the activated golden yellow shell cyst mycelium into a PDB culture medium, and carrying out constant-temperature shaking culture on a shaking table to prepare a strain seed solution;
(1.3) pouring the strain seed liquid into a solid culture medium for constant-temperature fermentation culture;
the solid culture medium is prepared by mixing MEB solution and rice according to the volume-mass ratio of 1: prepared at the ratio of 1 mL/g.
In the step (2), the pressure of the reduced pressure concentration is-0.1 Mpa, and the temperature is-20 ℃.
In the step (3), during gradient elution, the volume ratio of the dichloromethane and methanol mixed organic solvent is 100: 1. 80: 1. 50: 1. 30: 1. 10: 1 and 1: 1; each gradient eluted to the TLC plate with no apparent main spot and the next concentration gradient was changed.
In the step (4), during gradient elution, the volume ratio of the dichloromethane and methanol mixed organic solvent is 50: 1. 20: 1. 10: 1. 1: 1; each gradient eluted to the TLC plate with no apparent main spot and the next concentration gradient was changed.
In the step (5), the conditions for separating and purifying the high performance liquid chromatography are as follows: acetonitrile water solution with volume concentration of 70% is taken as a mobile phase, and Agilent C is adopted18A reverse phase chromatographic column with the specification: 4.6mmx250nm, 5u m; column temperature: 30 ℃; column flow rate: 1 ml/min; sampling amount each time: 20 ul; an ultraviolet detector is adopted, and the detection wavelength is 210 nm. Collecting tRAccumulating for multiple times and evaporating to dryness;
in the step (6), the standing time is not less than 5 days.
Example 4
The preparation method of the novel cytochalasin compound with the function of antagonizing clinically drug-resistant bacteria comprises the following steps:
step (1), fermenting and culturing the golden yellow shell cyst bacteria;
step (2), extracting a secondary metabolite obtained by fermenting and culturing the golden yellow shell cyst bacteria by adopting methanol, concentrating under reduced pressure, evaporating a solvent, washing with water, extracting by using ethyl acetate, and finally concentrating under reduced pressure to obtain a crude extract;
step (3), loading the crude extract to a silica gel column by a wet method, performing gradient elution by a silica gel column chromatography method by using a dichloromethane and methanol mixed organic solvent with the volume ratio of 100: 1-1: 1, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain six component sections Fr.A-Fr.F;
and (4) loading the fourth component section Fr.D to a silica gel column by a wet method, performing gradient elution by a silica gel column chromatography method by using a dichloromethane and methanol mixed organic solvent with the volume ratio of 50: 1-1: 1, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, combining the same parts to obtain four subgroup sections Fr.D1-Fr.D4；
Step (ii) of(5) Segmenting Fr.D. for the second subgroup2Sequentially separating and purifying by Sephadex chromatography and high performance liquid chromatography to obtain compound 2;
step (6), segmenting Fr.D of the fourth subgroup4Separating and purifying by using sephadex chromatography, collecting separated and purified liquid, standing, after crystals are separated out, cleaning impurities on the surfaces of the crystals by using dichloromethane, and finally obtaining a pure crystal compound, namely a compound 1;
wherein the structural formula of the compound 2 is as follows:
Figure BDA0003014489750000101
the structural formula of compound 1 is:
Figure BDA0003014489750000102
the specific method of the step (1) is as follows:
(1.1) activating the golden yellow shell cyst bacteria on a PDA plate;
(1.2) adding the activated golden yellow shell cyst mycelium into a PDB culture medium, and carrying out constant-temperature shaking culture on a shaking table to prepare a strain seed solution;
(1.3) pouring the strain seed liquid into a solid culture medium for constant-temperature fermentation culture;
the solid culture medium is prepared by mixing MEB solution and rice according to the volume-mass ratio of 1: prepared at the ratio of 1 mL/g.
(1.1), the activation time is 3 days; (1.2) in the step (1), the conditions of constant temperature shaking culture are 28 ℃, 160rpm and 3 days; (1.3) the conditions for constant temperature fermentation culture are 28 ℃ for 30 days.
In the step (2), the pressure of the reduced pressure concentration is-0.1 Mpa, and the temperature is-20 ℃.
In the step (3), during gradient elution, the volume ratio of the dichloromethane and methanol mixed organic solvent is 100: 1. 80: 1. 50: 1. 30: 1. 10: 1 and 1: 1; each gradient eluted to the TLC plate with no apparent main spot and the next concentration gradient was changed.
In the step (4), during gradient elution, the volume ratio of the dichloromethane and methanol mixed organic solvent is 50: 1. 20: 1. 10: 1. 1: 1; each gradient eluted to the TLC plate with no apparent main spot and the next concentration gradient was changed.
In the step (5), the conditions for separating and purifying the high performance liquid chromatography are as follows: acetonitrile water solution with volume concentration of 70% is taken as a mobile phase, and Agilent C is adopted18A reverse phase chromatographic column with the specification: 4.6mmx250nm, 5 um; column temperature: 30 ℃; column flow rate: 1 ml/min; sampling amount each time: 20 ul; an ultraviolet detector is adopted, and the detection wavelength is 210 nm. Collecting tRAccumulating for multiple times and evaporating to dryness;
in the step (6), the standing time is not less than 5 days.
Examples of the applications
The two cytochalasin molecules disclosed by the invention are obtained by separating and purifying secondary metabolites of plant endophytic fungus-phaeocystis aurantiaca HYQZ-931(Cytospora chrysosperma HYQZ-931). The plant endophytic fungi is isolated from desert plants, namely sea buckthorn (Hippophae rhamnoides), and is finally identified as ascosphaera aurantiaca (Cytospora chrysosperma, Genbank accession number is KU 143710.1.
The general steps of the experiment are that firstly, the plant endophytic fungus-golden yellow shell cyst is fermented and cultured in a solid culture medium of rice and MEB (malt extract) for 30 days at constant temperature, then methanol is used for extracting secondary metabolite, ethyl acetate is used for extracting the secondary metabolite extracted by the methanol, decompression and concentration are carried out to obtain crude extract, the crude extract is subjected to separation and purification means of silica gel column chromatography, gel chromatography analysis and high performance liquid phase preparation to obtain two novel cytochrysins A, B, the molecular structure of the compound is shown in figure 1, and finally, the activity experiment of resisting clinical drug-resistant bacteria is carried out on the obtained two novel cytochrysin compounds (the result of the bacteriostatic activity experiment is shown in figure 3). I will now explain the whole experimental process in detail.
1. Fermentation culture of plant endophytic fungus-golden yellow shell cyst
1.1 the cells of Chaetomium aurantiacum were removed and activated on PDA (Potato dextrose agar) plates for 3 days.
Manufacturing a PDA flat plate: 38g of PDA powder (Beijing Oborstar) was added with 1000ml of distilled water, autoclaved at 121 ℃ for 20min, and the PDA solution was poured into a sterile petri dish (the amount poured was about one-half of the volume of the petri dish) while it was hot, and allowed to cool to obtain a PDA plate. (the PDA powder produced by Beijing Omboxing company already contains agar, no additional need exists, the plate has no special requirement for pH, and the plate is neutral)
1.2 preparing PDB (potato dextrose broth) culture medium and autoclaving for 30 minutes at high temperature (121 ℃, 30min), then picking out a little activated theca aurantiaca mycelium and adding into the PDB culture medium, and carrying out constant temperature shaking culture on a shaking table for 3 days (28 ℃, 160rpm), thereby preparing the strain seed liquid.
Preparing a PDB culture medium: the PDB medium was prepared by adding 1000ml of distilled water to 38g of PDB powder (Oboxing, Beijing), and autoclaving at 121 deg.C for 20 min.
1.3 preparing a MEB (malt extract) solution, then according to MEB: 1: 1, preparing a solid culture medium required for fermentation, which comprises the specific steps of preparing 200 bottles of the solid culture medium containing 100g of rice and 100ml of MEB solution in each bottle, sterilizing at high temperature and high pressure (121 ℃, 0.3Mpa) for 30 minutes, and finally pouring a little prepared strain seed liquid into the sterilized solid culture medium for constant-temperature fermentation culture for 30 days (28 ℃).
Preparing an MEB solution: 2% of malt extract (2% means that 2g of malt extract is added to 100ml of distilled water), 2% of sucrose and 0.1% of peptone.
2. Preparation of crude extract
After 30 days of fermentation, secondary metabolites of G.chrysosporium were extracted with methanol (40L/time, 3 times). The mixture was concentrated under reduced pressure to evaporate the solvent, washed with water, extracted with ethyl acetate, and finally concentrated under reduced pressure to evaporate the solvent to obtain a crude extract (109 g).
3. Separation and purification of compounds
Dissolving the crude extract with small amount of ethyl acetate, mixing with 80 mesh silica gel,loading by wet method, performing silica gel column chromatography (loading silica gel column of 200-300 mesh), and selecting dichloromethane-methanol (CH)2Cl2MeOH) gradient elution (100: 1-1: 1) the crude extract was subjected to gradient elution (follow-up by TLC) to give six group fractions (fr.a-F), where fr.a group fraction was fractionated by a 100: 1, fr.b-fr.f by a gradient elution of 80: 1. 50: 1. 30: 1. 10: 1. 1: gradient elution of 1.
Fr.D (8g), mixing with 80 mesh silica gel, wet loading, and separating with silica gel column chromatography (loading silica gel column of 200 meshes and 300 meshes) to obtain CH2Cl2MeOH gradient elution (50: 1-1: 1) gradient elution again for Fr.D group fractions (follow-up by TLC) to give four subgroup fractions (Fr.D)1-D4，50：1、20：1、10：1、1：1)。
Wherein Fr.D2(324mg) sub-groups are segmented and then sequentially analyzed by sephadex chromatography, an automatic fraction collector is adopted to collect samples, the volume of each tube is 3ml, the same components are combined by a TLC spot plate, the 14 th tube to the 25 th tube are collected, and then the high performance liquid chromatography is used for separation and purification, so that a compound 2(cytochrysins B, 12mg) is obtained; the high performance liquid chromatography separation and purification conditions are as follows: acetonitrile water solution with volume concentration of 70% is taken as a mobile phase, and Agilent C is adopted18A reverse phase chromatographic column with the specification: 4.6mmx250nm, 5 um; column temperature: 30 ℃; column flow rate: 1 ml/min; sampling amount each time: 20 ul; an ultraviolet detector is adopted, and the detection wavelength is 210 nm. Collecting tRAccumulating for multiple times and evaporating to dryness;
Fr.D4(190mg) subgroup segmentation is analyzed by adopting sephadex chromatography, an automatic fraction collector is used for sample collection, the volume of each tube is 3ml, the 10 th tube and the 21 st tube are dotted and combined and kept still for 7d, crystals are separated out in a bottle, and then dichloromethane is used for cleaning impurities on the surfaces of the crystals, and finally a pure crystal compound, namely compound 1(cytochrysins A, 6mg) is obtained;
4. physical and chemical properties and structural analysis of two compounds
4.1 physicochemical Properties and structural analysis of Compound 1(cytochrysins A)
Compound 1 is colorless crystal, melting point is 241.2-243.7 deg.C, and is dissolved in 1mg/ml methanol solutionThe optical rotation is +72 ℃, the maximum ultraviolet absorption wavelength can be seen to be 205nm in an ultraviolet spectrum (figure 6), and the molecular ion peak M/z 416.2794[ M + H ] is measured by high-resolution mass spectrum]+ (FIG. 5) molecular formula is C25H37NO4. For nuclear magnetic data required for structural analysis of compound 1, see fig. 3-4, table 1 and table 2. In addition, compound 1 was successfully crystallized in a methanol + water ((0.1%) system, and the absolute configuration of compound 1 was determined by X-ray single crystal diffraction, wherein CCDC (Cambridge Crystal data center) number is 2053963.
4.2 physicochemical Properties and structural analysis of Compound 2(cytochrysins B)
Compound 3 is white powder, has optical rotation of +122 deg.C in 1mg/ml methanol solution, has maximum ultraviolet absorption wavelength of 210nm in ultraviolet spectrum (FIG. 10), and has molecular ion peak M/z 448.3057[ M + H ] measured by high resolution mass spectrometry]+(FIG. 9) molecular formula C26H41NO5. See fig. 7-8, table 1 and table 2 for all nuclear magnetic data required for structural analysis of compound 2.
TABLE 1 of Compounds 1 and 21H nuclear magnetic data (deuterated methanol, 600MHz)
Figure BDA0003014489750000131
Figure BDA0003014489750000141
TABLE 2 preparation of Compounds 1 and 213C NMR Nuclear magnetic data (deuterated methanol, 150MHz)
Figure BDA0003014489750000142
Figure BDA0003014489750000151
5. And (3) performing anti-clinical drug-resistant bacterial activity tests on the two compounds.
5.1 two compounds of interest and ciprofloxacin (positive control) were formulated at a concentration of 1 mg/ml.
5.2 activating target drug-resistant bacteria (carbapenem-resistant pseudomonas aeruginosa, methicillin-resistant staphylococcus aureus, multi-drug-resistant enterococcus faecalis and multi-drug-resistant enterococcus faecium) in an LB (lysis broth) culture medium for 8 hours, and adding 50ul of the activated bacteria into 50ml of the LB culture medium to obtain diluted bacteria liquid.
Preparing an LB culture medium: 25g of LB powder (Beijing Ku Laibobu Co.), 1000ml of distilled water was added and autoclaved at 121 ℃ for 20 min. (the medium has no special requirement for pH, and is neutral)
And 5.3, taking ciprofloxacin as a positive control, adding 2ul of target compound into 198ul of target bacterial liquid, and testing the antagonistic activity of the two compounds on the four clinical drug-resistant bacteria by adopting a two-fold dilution method.
(the results of the activity are shown in Table 3)
Table 3: inhibitory Activity of four clinically resistant bacteria of Compounds 1 and 2 (ciprofloxacin as Positive control)
Figure BDA0003014489750000152
6. Conclusion
The experimental result shows that two cytochrysins A and B which are found in the experiment are novel compounds which are found for the first time, and the compounds have good antagonistic activity (MIC, 25 mu g/mL) on four clinical drug-resistant bacteria such as carbapenem-resistant pseudomonas aeruginosa, methicillin-resistant staphylococcus aureus, multi-drug-resistant enterococcus faecalis and multi-drug-resistant enterococcus faecium.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (10)

1. The preparation method of the novel cytochalasin compound with the function of antagonizing clinically drug-resistant bacteria is characterized by comprising the following steps:
step (1), fermenting and culturing the golden yellow shell cyst bacteria;
step (2), extracting a secondary metabolite obtained by fermenting and culturing the golden yellow shell cyst bacteria by adopting methanol, concentrating under reduced pressure, evaporating a solvent, washing with water, extracting by using ethyl acetate, and finally concentrating under reduced pressure to obtain a crude extract;
step (3), loading the crude extract to a silica gel column by a wet method, performing gradient elution by a silica gel column chromatography method by using a dichloromethane and methanol mixed organic solvent with the volume ratio of 100: 1-1: 1, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain six component sections Fr.A-Fr.F;
and (4) loading the fourth component section Fr.D to a silica gel column by a wet method, performing gradient elution by a silica gel column chromatography method by using a dichloromethane and methanol mixed organic solvent with the volume ratio of 50: 1-1: 1, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, combining the same parts to obtain four subgroup sections Fr.D1-Fr.D4；
Step (5), segmenting Fr.D for the second subgroup2Sequentially separating and purifying by Sephadex chromatography and high performance liquid chromatography to obtain compound 2;
step (6), segmenting Fr.D of the fourth subgroup4Separating and purifying by using sephadex chromatography, collecting separated and purified liquid, standing, after crystals are separated out, cleaning impurities on the surfaces of the crystals by using dichloromethane, and finally obtaining a pure crystal compound, namely a compound 1;
wherein the structural formula of the compound 2 is as follows:
Figure FDA0003014489740000011
the structural formula of compound 1 is:
Figure FDA0003014489740000012
2. the method for preparing a novel cytochalasin compound with antagonistic action against clinically drug-resistant bacteria as claimed in claim 1, wherein the specific method in step (1) is as follows:
(1.1) activating the golden yellow shell cyst bacteria on a PDA plate;
(1.2) adding the activated golden yellow shell cyst mycelium into a PDB culture medium, and carrying out constant-temperature shaking culture on a shaking table to prepare a strain seed solution;
(1.3) pouring the strain seed liquid into a solid culture medium for constant-temperature fermentation culture;
the solid culture medium is prepared by mixing MEB solution and rice according to the volume-mass ratio of 1: prepared at the ratio of 1 mL/g.
3. The method for producing a novel cytochalasin compound as claimed in claim 2, which is effective in antagonizing clinically drug-resistant bacteria, wherein (1.1) the activation time is 3 days; (1.2) in the step (1), the conditions of constant temperature shaking culture are 28 ℃, 160rpm and 3 days; (1.3) the conditions for constant temperature fermentation culture are 28 ℃ for 30 days.
4. The method for preparing a novel cytochalasin compound as claimed in claim 1, which is effective in antagonizing clinically drug-resistant bacteria at-20 ℃ under-0.1 MPa in step (2).
5. The method for producing a novel cytochalasin compound as claimed in claim 1, which is characterized in that in step (3), the volume ratio of the mixed organic solvent of dichloromethane and methanol used in the gradient elution is 100: 1. 80: 1. 50: 1. 30: 1. 10: 1 and 1: 1; each gradient is eluted until no obvious main point exists on a TLC point plate, and then the next concentration gradient can be replaced;
in the step (4), during gradient elution, the volume ratio of the dichloromethane and methanol mixed organic solvent is 50: 1. 20: 1. 10: 1. 1: 1; each gradient eluted to the TLC plate with no apparent main spot and the next concentration gradient was changed.
6. The method for producing a novel cytochalasin compound as claimed in claim 1, which is characterized in that in step (5), the conditions for separation and purification by HPLC are as follows: acetonitrile water solution with volume concentration of 70% is taken as a mobile phase, and Agilent C is adopted18A reverse phase chromatographic column with the specification: 4.6mmx250nm, 5 um; column temperature: 30 ℃; column flow rate: 1 ml/min; sampling amount each time: 20 ul; an ultraviolet detector is adopted, and the detection wavelength is 210 nm. Collecting tRAccumulating for multiple times and evaporating to dryness;
in the step (6), the standing time is not less than 5 days.
7. The novel cytochalasin compound as claimed in claim 1, which is a compound 1 with a structural formula:
Figure FDA0003014489740000031
8. the novel cytochalasin compound as claimed in claim 1, which is a compound 2, having the formula:
Figure FDA0003014489740000032
9. use of the novel cytochalasin compound as claimed in claim 1 with antagonistic effect on clinically drug-resistant bacteria for the preparation of clinically drug-resistant bacteria.
10. The ascochyta aurantiaca HYQZ-931(Cytospora chrysosper ma HYQZ-931) as claimed in claim 1, which has been deposited at the chinese type culture collection on 25 months and 3 months in 2021 under the deposition number CCTCC M2021279.
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Publication number Priority date Publication date Assignee Title
CN116969878A (en) * 2023-03-15 2023-10-31 广东医科大学附属医院 Cytochalasin compound and preparation method and application thereof

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* Cited by examiner, † Cited by third party
Title
CAI-JUAN ZHENG ET AL.: ""Bioactive Phenylalanine Derivatives and Cytochalasins from the Soft Coral-Derived Fungus, Aspergillus elegans"", 《MAR. DRUGS》 *
QING-LIN MOU ET AL.: ""New cytochalasan alkaloids and cyclobutane dimer from an endophytic fungus Cytospora chrysosperma in Hippophae rhamnoides and their antimicrobial activities"", 《TETRAHEDRON LETTERS》 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116969878A (en) * 2023-03-15 2023-10-31 广东医科大学附属医院 Cytochalasin compound and preparation method and application thereof
CN116969878B (en) * 2023-03-15 2024-01-19 广东医科大学附属医院 Cytochalasin compound and preparation method and application thereof

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