CN114224876B - Preparation method of sesterterpene compounds and application of sesterterpene compounds in resisting clinical drug-resistant bacteria - Google Patents

Abstract

The invention relates to a preparation method of sesterterpene compounds and application thereof in the aspect of resisting clinical drug-resistant bacteria, belonging to the technical field of antibacterial activity research. The structural formula of the sesterterpene compound is shown as a formula (I);

formula (I); the minimal inhibitory concentration MIC of the sesterterpene compound to 2 clinical drug-resistant bacteria such as methicillin-resistant staphylococcus aureus and vancomycin-resistant enterococcus faecalis is 2 microgram/mL, and the sesterterpene compound has very great application potential in the clinical drug-resistant bacteria and is easy to popularize and apply.

Description

Preparation method of sesterterpene compounds and application of sesterterpene compounds in resisting clinical drug-resistant bacteria

Technical Field

The invention belongs to the technical field of antibacterial activity research, and particularly relates to a preparation method of a sesterterpene compound quannanicalular acid and application of the sesterterpene compound quannanicalular acid in the aspect of resisting clinical drug-resistant bacteria.

Background

In recent years, with the unreasonable clinical use of antibiotic drugs, the problem of bacterial drug resistance is more and more serious, even "superbacteria", i.e. bacteria with multiple drug resistance, such as the first discovery of methicillin-resistant staphylococcus aureus (MRSA) by Je vons in the uk in 1961, and the proportion of MRSA in staphylococcus aureus increased from 35.9% to 64.4% in 1992 to 200 years, is promoted. MRSA has drug resistance to not only methicillin and most beta-lactam antibiotics, but also other antibiotics such as chloramphenicol, clindamycin, tetracycline, aminosugamides and the like, and the drug resistance rate of MRSA to most antibacterial drugs is shown to be more than 60% according to the national bacterial drug resistance monitoring network. In addition, vancomycin-resistant enterococci (VRE), since its first report at 1 980s, has spread rapidly worldwide, and VRE has become a difficult problem in the treatment of enterococcal infections due to its resistance to various antibiotics. Therefore, it is urgent to find therapeutic drugs against MRSA and VRE.

In the face of the emergence of more and more drug-resistant bacteria, the research and development of antibiotics are gradually lagged after the 'gold period of antibiotics' in the 80s, so that the health and safety of human beings are greatly threatened, the problem of drug resistance of bacteria is solved slowly, and the global attention is attracted at present. Therefore, in order to effectively prevent and treat bacterial infection diseases and solve the problem of serious shortage of the existing antibiotic drugs, the discovery and development of novel high-efficiency and low-toxicity antibacterial drugs become one of the key points in the international medicine research field at present.

The natural product of the microorganism has the characteristics of novel structure, unique activity and the like, for example, penicillin, streptomycin, erythromycin and the like are all derived from the microorganism, and the natural product of the microorganism occupies a very important position in drug discovery and development for a long time. The data of small molecule entity drugs approved to be on the market all over the world from 1981 to 2019 show that the number of anti-infective drugs approved to be on the market in the last 40 years is 401, wherein the number of the anti-infective drugs is 162, and the number of the anti-infective drugs in 162 is 90, which accounts for about 56% and is directly or indirectly derived from natural products, which indicates that the natural products have high occupancy rate in anti-infective therapy, particularly in the anti-infective drugs.

The plant endophytic fungi co-evolve in a long-term complex symbiotic relationship with host plants due to special living environment, so that the plant endophytic fungi have a unique metabolic system, can generate secondary metabolites with novel structures and diverse activities, and can generate bioactive substances similar to the host plants in some cases. A great deal of research shows that most secondary metabolites of plant endophytic fungi have remarkable antibacterial activity, and a plurality of antibacterial activity monomer compounds are separated from the secondary metabolites, so that a great deal of research space is provided for searching novel antibacterial drugs or lead compounds from the plant endophytic fungi.

Disclosure of Invention

The invention mainly aims to provide a preparation method of sesterterpene compound quinanulatic acid and application thereof in the aspect of resisting clinical drug-resistant bacteria. The sesterterpene compound quinanulatii c acid can effectively inhibit the growth of 2 strains of clinical drug-resistant bacteria under the condition of low concentration, and has very large application potential.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the sesterterpene compound is used for preparing a clinical drug-resistant bacteria resistant medicament, and the structural formula of the sesterterpene compound is shown as a formula (I);

further, it is preferable that the clinically resistant bacteria are ATCC 43300 methicillin-resistant staphylococcus aureus and ATCC 51299 vancomycin-resistant enterococcus faecalis.

Further, preferably, the preparation method of the sesterterpene compound comprises the following steps:

step (1), culturing fungus Emericella sp.XL029 by using a rice solid culture medium, and standing and culturing for 28-30 days to obtain a fermentation product;

step (2), extracting the fermentation product obtained in the step (1) by using methanol, and concentrating the obtained extract under reduced pressure to obtain a crude extract;

step (3), dissolving the crude extract obtained in the step (2) with water, performing back extraction with ethyl acetate, and performing decompression concentration on the ethyl acetate to obtain an extract;

step (4), loading the extract obtained in the step (3) to a normal phase silica gel column by a dry method, then carrying out gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 7 components A-G;

step (5), loading the component B obtained in the step (4) to a normal-phase silica gel column for chromatography by a dry method, then performing gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 5 sub-components B1-B5;

and (6) purifying the subfraction B1 obtained in the step (5) to obtain the sesterterpene compound shown in the formula (I).

Further, preferably, in the step (2), the extraction mode is to add methanol with the same volume and perform ultrasonic treatment for 0.9 to 1.1 hours at room temperature, then to stand for 9 to 11 hours at room temperature, to take supernatant and to perform vacuum concentration; the extraction times are 2-4.

Further, it is preferable that, in the step (3), the volumes of water and ethyl acetate used are the same.

Further, preferably, in the step (4), the concrete method for dry-loading the extract comprises the following steps: dissolving the extract with 10 times of methanol, adsorbing and stirring the extract with 80-100 mesh silica gel with the same mass as the extract, and then loading the sample;

the silica gel in the normal phase silica gel column is 100-200 meshes of normal phase silica gel;

the mobile phase adopted by the gradient elution is petroleum ether, a mixed solvent of dichloromethane and methanol in the order of elution, the volume ratio of dichloromethane to methanol in the mixed solvent of dichloromethane and methanol is 10.

Further, preferably, in the step (5), the concrete method for dry loading the extract comprises the following steps: dissolving the component B by 10 times of methanol, adsorbing and mixing the sample by using 80-100 meshes of silica gel with the same mass as the component B, and then loading the sample;

the silica gel in the normal phase silica gel column is 100-200 mesh normal phase silica gel;

the mobile phase adopted by the gradient elution is petroleum ether and ethyl acetate, the volume ratio of the petroleum ether to the ethyl acetate is 100, 1, 60, 1, 10.

Further, preferably, in step (6), the purification is performed by gel chromatography and then by preparative liquid chromatography (HPLC).

Further, it is preferable that, in the step (6), the gel column used for gel chromatography purification is Sephadex LH-20, and the volume ratio is 1:1 as a mobile phase, and then carrying out HPLC;

for purification by gel chromatography, subfraction B1 was dissolved in 2 volumes of methanol and loaded, monitored by TLC, and the same fractions were pooled.

The HPLC chromatographic conditions are that the mobile phase is a mixture of a mobile phase and a solvent in a volume ratio of 19:1 mixed solvent of methanol and water with the flow rate of 1mL/min, collecting the component with the initial peak time of 5.6min, and adopting Agilent C ₁₈ A reverse phase chromatographic column with the specification: 4.6mmx250nm,5um; column temperature: 30 ℃, sample size per time: 20ul, ultraviolet detector, and detection wavelength of 210nm.

In the gradient elution of the invention, TLC is adopted for monitoring, and the same parts are combined.

The invention also provides an anti-clinical drug-resistant bacterial agent, wherein the active ingredient contains sesterterpene compounds, and the structural formula of the sesterterpene compounds is shown as the formula (I);

the clinical drug-resistant bacteria are methicillin-resistant staphylococcus aureus ATCC 43300 (Me thicillin-resistant staphylococcus aureus) and Vancomycin-resistant Enterococcus faecalis ATCC 51299 (Vancomycin-resistant Enterococcus faecalis). The standard strain is purchased from China Guangdong province microbial culture Collection (GDMCC), and the strain numbers are GDMCC 1.1263= ATCC 4330 and GDMCC 1.1332= ATCC 51299.

The minimum inhibitory concentration MIC of the sesterterpene compound to 2 clinical drug-resistant bacteria such as methicillin-resistant staphylococcus aureus and vancomycin-resistant enterococcus faecalis is 2 microgram/mL.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention provides an application of sesterterpene compounds quinanulatic acid in 2 strains of clinical drug-resistant bacteria, wherein the sesterterpene compounds can inhibit the growth of 2 strains of clinical drug-resistant bacteria under the condition of low concentration; the minimum inhibitory concentration MIC of the compound to 2 clinical drug-resistant bacteria of methicillin-resistant staphylococcus aureus and vancomycin-resistant enterococcus faecalis is 2 mug/mL. Therefore, the sesterterpene compounds have great application potential in clinical drug-resistant bacteria.

(2) The invention also provides a preparation method of the compound, and the sesterterpene compound can be quickly, accurately and efficiently prepared from the fungus E meriella sp.XL029.

Drawings

FIG. 1 is a chemical structural formula of sesterterpene compounds of the present invention;

FIG. 2 shows sesterterpenes of the present invention ¹ H NMR chart;

FIG. 3 shows the sesterterpene compounds of the present invention ¹³ C NMR chart.

The fungus Emericella sp.XL029 is preserved in China center for type culture collection (CCTCC M20211154) at 9.9.2021, wherein the preservation address is No. 299 eighth way in Wuchang district, wuhan university, hubei province.

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. Those skilled in the art will recognize that the specific techniques or conditions, not specified in the examples, are according to the techniques or conditions described in the literature of the art or according to the product specification. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.

The formula of the rice solid culture medium provided by the invention is as follows: rice and water by mass 1:1, and sterilizing at 121 ℃ for 25min to obtain the product.

Example 1

The application of sesterterpene compounds in preparing medicaments for resisting clinical drug-resistant bacteria is disclosed, wherein the structural formula of the sesterterpene compounds is shown as a formula (I);

the clinical drug-resistant bacteria are ATCC 43300 methicillin-resistant staphylococcus aureus and ATCC 51299 vancomycin-resistant enterococcus faecalis.

Example 2

The preparation method of the sesterterpene compound comprises the following steps:

step (1), culturing fungus Emericella sp.XL029 by using a rice solid culture medium, and standing for 29 days to obtain a fermentation product;

step (2), extracting the fermentation product obtained in the step (1) by using methanol, and concentrating the obtained extract under reduced pressure to obtain a crude extract;

step (3), dissolving the crude extract obtained in the step (2) with water, performing back extraction with ethyl acetate, and performing decompression concentration on the ethyl acetate to obtain an extract;

step (4), loading the extract obtained in the step (3) to a normal phase silica gel column by a dry method, then carrying out gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 7 components A-G;

step (5), loading the component B obtained in the step (4) to a normal phase silica gel column for chromatography by a dry method, then carrying out gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 5 sub-components B1-B5;

and (6) purifying the subfraction B1 obtained in the step (5) to obtain the sesterterpene compound shown in the formula (I).

Example 3

The preparation method of the sesterterpene compound comprises the following steps:

culturing fungus Emericella sp.XL029 by using a rice solid culture medium, and performing static culture for 28 days to obtain a fermentation product;

step (2), extracting the fermentation product obtained in the step (1) by using methanol, and concentrating the obtained extract under reduced pressure to obtain a crude extract;

step (3), dissolving the crude extract obtained in the step (2) with water, back-extracting with ethyl acetate, and concentrating the ethyl acetate phase under reduced pressure to obtain an extract;

step (4), loading the extract obtained in the step (3) to a normal phase silica gel column by a dry method, then carrying out gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 7 components A-G;

step (5), loading the component B obtained in the step (4) to a normal phase silica gel column for chromatography by a dry method, then carrying out gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 5 sub-components B1-B5;

and (6) purifying the subfraction B1 obtained in the step (5) to obtain the sesterterpene compound shown in the formula (I).

In the step (2), the extraction mode is to add methanol with the same volume for ultrasonic treatment for 0.9h at room temperature, then to stand for 9h at room temperature, to take supernatant and to concentrate under reduced pressure; the number of extractions was 2.

In step (3), the volume of water and ethyl acetate used was the same.

In the step (4), the concrete method for dry loading the extract comprises the following steps: dissolving the extract by 10 times of methanol, adsorbing and mixing the extract by 80-mesh silica gel with the same mass as the extract, and then loading the sample;

the silica gel in the normal phase silica gel column is 100 meshes of normal phase silica gel;

the mobile phase adopted by the gradient elution is sequentially petroleum ether, a mixed solvent of petroleum ether and ethyl acetate and methanol according to the elution order, the volume ratio of the petroleum ether to the ethyl acetate in the mixed solvent of the petroleum ether and the ethyl acetate is 1, 80, 1, 20 and 1, and after each gradient elution is completed to no point on a TLC point plate, the next gradient elution is replaced, and 7 components A-G are obtained.

In the step (5), the concrete method for dry loading the extract comprises the following steps: dissolving the component B by 10 times of methanol, adsorbing and mixing the sample by using 80-mesh silica gel with the same mass as the component B, and then loading the sample;

the silica gel in the normal phase silica gel column is 100 meshes of normal phase silica gel;

the mobile phases adopted in the gradient elution are petroleum ether and ethyl acetate, the volume ratio of the petroleum ether to the ethyl acetate is 100.

In the step (6), the purification is performed by gel chromatography and HPLC.

In the step (6), a gel column adopted for gel chromatography purification is Sephadex LH-20, and the volume ratio is 1:1 as a mobile phase, and then carrying out HPLC;

the HPLC chromatographic conditions are that the mobile phase is a mixture of a mobile phase and a solvent in a volume ratio of 19:1 mixed solvent of methanol and water with the flow rate of 1mL/min, collecting the component with the peak starting time of 5.6min, and adopting Agilent C ₁₈ A reverse phase chromatographic column with the specification: 4.6mmx250nm,5um; column temperature: 30 ℃, sample amount per time: 20ul, ultraviolet detector, and detection wavelength of 210nm.

Example 4

The preparation method of the sesterterpene compound comprises the following steps:

culturing fungus Emericella sp.XL029 by using a rice solid culture medium, and standing and culturing for 30 days to obtain a fermentation product;

step (2), extracting the fermentation product obtained in the step (1) by using methanol, and concentrating the obtained extract under reduced pressure to obtain a crude extract;

step (3), dissolving the crude extract obtained in the step (2) with water, back-extracting with ethyl acetate, and concentrating the ethyl acetate phase under reduced pressure to obtain an extract;

step (4), loading the extract obtained in the step (3) to a normal phase silica gel column by a dry method, then carrying out gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 7 components A-G;

step (5), loading the component B obtained in the step (4) to a normal-phase silica gel column for chromatography by a dry method, then performing gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 5 sub-components B1-B5;

and (6) purifying the subfraction B1 obtained in the step (5) to obtain the sesterterpene compound shown in the formula (I).

In the step (2), the extraction mode is to add methanol with the same volume for ultrasonic treatment for 1.1h at room temperature, then to stand for 11h at room temperature, to take supernatant and to concentrate under reduced pressure; the number of extractions was 4.

In step (3), the volume of water and ethyl acetate used was the same.

In the step (4), the concrete method for dry loading the extract comprises the following steps: dissolving the extract by 10 times of methanol, adsorbing and stirring the extract by using 100-mesh silica gel with the same mass as the extract, and then loading the sample;

the silica gel in the normal phase silica gel column is 200 meshes of normal phase silica gel;

the mobile phase adopted by the gradient elution is petroleum ether, a mixed solvent of petroleum ether and ethyl acetate and methanol in the following order of elution, the volume ratio of the petroleum ether to the ethyl acetate in the mixed solvent of the petroleum ether and the ethyl acetate is 1, 80, 1, 50, 1, 20 and 1.

In the step (5), the concrete method for dry loading the extract comprises the following steps: dissolving the component B by 10 times of methanol, adsorbing and mixing the sample by 100-mesh silica gel with the same mass as the component B, and then loading the sample;

the silica gel in the normal phase silica gel column is 200 meshes of normal phase silica gel;

the mobile phase adopted by the gradient elution is petroleum ether and ethyl acetate, the volume ratio of the petroleum ether to the ethyl acetate is 100, 1, 60, 1, 10.

In the step (6), the purification is performed by gel chromatography and then HPLC.

In the step (6), a gel column adopted for gel chromatography purification is Sephadex LH-20, and the volume ratio is 1:1 as a mobile phase, and then carrying out HPLC;

the HPLC chromatographic conditions are that the mobile phase is a mixture of a mobile phase and a solvent in a volume ratio of 19:1 mixed solvent of methanol and water with the flow rate of 1mL/min, collecting the component with the peak starting time of 5.6min, and adopting Agilent C ₁₈ A reverse phase chromatographic column with the specification: 4.6mmx250nm,5um; column temperature: 30 ℃, sample amount per time: 20ul, ultraviolet detector, and detection wavelength of 210nm.

Example 5

The preparation method of the sesterterpene compound comprises the following steps:

step (1), culturing fungus Emericella sp.XL029 by using a rice solid culture medium, and standing for 29 days to obtain a fermentation product;

step (2), extracting the fermentation product obtained in the step (1) by using methanol, and concentrating the obtained extract under reduced pressure to obtain a crude extract;

step (3), dissolving the crude extract obtained in the step (2) with water, back-extracting with ethyl acetate, and concentrating the ethyl acetate phase under reduced pressure to obtain an extract;

step (4), loading the extract obtained in the step (3) to a normal phase silica gel column by a dry method, then carrying out gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 7 components A-G;

step (5), loading the component B obtained in the step (4) to a normal phase silica gel column for chromatography by a dry method, then carrying out gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 5 sub-components B1-B5;

and (6) purifying the subfraction B1 obtained in the step (5) to obtain the sesterterpene compound shown in the formula (I).

In the step (2), the extraction mode is that methanol with the same volume is added for ultrasonic treatment for 1h at room temperature, then the mixture is kept stand for 10h at room temperature, and supernate is taken and concentrated under reduced pressure; the number of extractions was 3.

In step (3), the volume of water and ethyl acetate used was the same.

In the step (4), the concrete method for dry loading the extract comprises the following steps: dissolving the extract by 10 times of methanol, adsorbing and stirring the extract by using 90-mesh silica gel with equal mass of the extract, and then loading the sample;

the silica gel in the normal phase silica gel column is 160 meshes of normal phase silica gel;

the mobile phase adopted by the gradient elution is petroleum ether, a mixed solvent of petroleum ether and ethyl acetate and methanol in the following order of elution, the volume ratio of the petroleum ether to the ethyl acetate in the mixed solvent of the petroleum ether and the ethyl acetate is 1, 80, 1, 50, 1, 20 and 1.

In the step (5), the concrete method for dry loading the extract comprises the following steps: dissolving the component B by 10 times of methanol, adsorbing and mixing the sample by 90-mesh silica gel with the same mass as the component B, and then loading the sample;

the silica gel in the normal phase silica gel column is 160 meshes of normal phase silica gel;

the mobile phase adopted by the gradient elution is petroleum ether and ethyl acetate, the volume ratio of the petroleum ether to the ethyl acetate is 100, 1, 60, 1, 10.

In the step (6), the purification is performed by gel chromatography and HPLC.

In the step (6), a gel column adopted for gel chromatography purification is Sephadex LH-20, and the volume ratio is 1:1 as a mobile phase, and then carrying out HPLC;

the HPLC chromatographic conditions are that the mobile phase is a mixture of a mobile phase and a solvent in a volume ratio of 19:1 mixed solvent of methanol and water with the flow rate of 1mL/min, collecting the component with the peak starting time of 5.6min, and adopting Agilent C ₁₈ A reverse phase chromatographic column with the specification: 4.6mmx250nm,5um; column temperature: 30 ℃, sample amount per time: 20ul, ultraviolet detector, and detection wavelength of 210nm.

Application example 1

The preparation method of the sesterterpene compound quinanulatic acid comprises the following steps:

1) Culturing the fungus Emericella sp.XL029 by using a rice solid culture medium, and standing for 30 days, wherein a fermentation product is a material used by the invention;

2) Extracting the fermentation product with methanol, and concentrating under reduced pressure to obtain crude extract about 0.4kg; the extraction method comprises the steps of adding methanol with the same volume, performing ultrasonic treatment for 1h at room temperature, standing for 3h at room temperature, taking supernatant, and performing reduced pressure concentration; the number of extractions was 3.

3) Dissolving the crude extract by using water, and then performing back extraction by using ethyl acetate, wherein the volume ratio of the water to the ethyl acetate is 1:1, carrying out decompression concentration on ethyl acetate to obtain about 0.1kg of extract;

4) Dissolving the extract with methanol, adsorbing and mixing with silica gel (ca.0.2kg), and performing coarse gradient elution by normal phase silica gel column chromatography to obtain components A-G, wherein the stationary phase of the silica gel column chromatography is 100-200 mesh normal phase silica gel, and the mobile phase sequentially comprises petroleum ether, dichloromethane/methanol (100 → 0;

the method comprises the following specific steps: dissolving the extract by 10 times of methanol, adsorbing and stirring the extract by using 100-mesh silica gel with the same mass as the extract, and then loading the sample;

the mobile phase adopted by the gradient elution is petroleum ether, a mixed solvent of dichloromethane and methanol in the order of elution, the volume ratio of dichloromethane to methanol in the mixed solvent of dichloromethane and methanol is 10.

5) The component B was separated into 5 subfractions (B1-B5) by normal phase silica gel column chromatography, the stationary phase of the silica gel column chromatography was 100-200 mesh normal phase silica gel, and the mobile phase was petroleum ether/ethyl acetate (100: 1) (ii) a

The method specifically comprises the following steps: dissolving the component B by 10 times of methanol, adsorbing and mixing the component B by 100-mesh silica gel with the same mass as the component B, and then loading the sample; the mobile phase adopted by the gradient elution is petroleum ether and ethyl acetate, the volume ratio of the petroleum ether to the ethyl acetate is 100, 1, 60, 1, 10.

6) B1 was purified by gel column chromatography Sephadex LH-20 (MeOH) HPLC with the mobile phase of the gel being dichloromethane/methanol (1: 1) The mobile phase of HPLC was methanol/water (19: 1) The flow rate was 1ml/min, and fractions having a peak start time of 5.6min were collected to give the compound quinanulatic acid (1.2 mg).

For purification by gel chromatography, subfraction B1 was dissolved in 2 volumes of methanol and loaded.

Collecting with 10ml test tube, collecting 6ml in each tube, collecting 60 tubes in this time, analyzing by TLC (thin layer chromatography), monitoring by TLC, combining the same parts, combining 10-20 tubes, concentrating under reduced pressure, and purifying by HPLC.

The HPLC chromatographic conditions are that the mobile phase is a mixture of a mobile phase and a solvent in a volume ratio of 19:1 mixed solvent of methanol and water with the flow rate of 1mL/min, collecting the component with the peak starting time of 5.6min, and adopting Agilent C ₁₈ A reverse phase chromatographic column with the specification: 4.6mmx250nm,5um; column temperature: 30 ℃, sample size per time: 20ul, ultraviolet detector, and detection wavelength of 210nm.

The structure of the obtained monomeric compound was determined by nuclear magnetism, and the properties and spectral data of the obtained compound were as follows:

the structural formula of the sesterterpene compound is shown as a formula (I);

how the names of Chinese characters are named, wherein the English name is quinanutic acid, the light yellow amorphous powder has a molecular formula of C ₂₅ H ₃₈ O ₂ The molecular weight is: 370.2872.

¹ H NMR(CDCl ₃ ,600MHz)δ _H :2.90(1H,d,J＝15.80Hz,H-18a)； 2.40(1H,t,J＝9.10Hz,H-7)；2.19(1H,d,J＝15.80Hz,H-18b)； 2.09(1H,m,H-2a)；2.04(1H,m,H-6a)；1.93(1H,m,H-20)；1.88(1H,m,H-5)；1.84(1H,m,H-3a)；1.72(1H,m,H-9a),1.66(2H,m,H-10 a)；1.57(1H,m,H-14a)；1.52(1H,m,H-3b),1.45(1H,m,H-6b)；1.4 1(3H,s,H-25)；1.30(6H,m,H-2b,H-9b,H-11,H-12,H-13a,H-14b,), 1.11(2H,m,H-10b,H-13b)；0.94(3H,d,J＝6.20Hz,H-23)；0.91(3 H,d,J＝6.50Hz,H-22)；0.90(3H,s,H-24)；0.81(3H,d,J＝6.60H z,H-21)。

¹³ C NMR(CDCl ₃ ,150MHz)δc:184.0(C-19),135.8(C-16),133.8(C- 17),65.1(C-12)62.7(C-8),60.6(C-15),54.6(C-1),53.2(C-7),46.0(C-5),45.7(C-4),43.1(C-11),39.0(C-2),36.8(C-18),35.7(C-10),34.8(C-14),31. 2(C-3),29.2(C-13),29.1(C-9),29.0(C-20),28.2(C-6),24.2(C-22),21.6(C-21),21.5(C-24),20.0(C-23),9.5(C-25).

application example 2

The sesterterpene compound quinanulatic acid obtained in the example is used for carrying out MIC (MIC) determination on 2 strains of clinical drug-resistant bacteria by adopting a two-fold dilution method, and the specific steps comprise:

1) Determination of clinical standard drug-resistant bacteria MIC: the resulting compounds were each prepared in 10mM stock solutions in DMSO.

500mL of MH broth (specific procedure: 10.5g of MH (Mueller-Hinton) broth powder (Haibo, qingdao) was prepared, and distilled water was added to 500mL to adjust pH to 7.4. + -. 0.2, and the mixture was pressurized at 121 ℃ for 15min for use.

ATCC 43300, ATCC 51299 as test strains; recovering the test strain on a PDA plate, inoculating a blood agar plate (Baibo, jinan), and picking out a single colony to obtain pure bacteria. Preparing bacterial suspension with McLeod turbidimeter (Merrier, france), and diluting pure bacteria with 0.9% physiological saline solution to McLeod of 0.5Mc (equivalent to 1.5x10) ⁸ cells/mL), then diluted 1:10 times with MH broth and then 1.

The compound stock solution was diluted 10-fold with DMSO (dimethyl sulfoxide) to obtain an antibacterial solution having a concentration of 1 mM. Based on the compound's relative molecular weight of 370, a series of concentration gradients between 8, 4, 2, 1, 0.5, 0.125 μ g/mL were set and diluted with MH broth. Taking a sterile 96-well plate, adding 100 mu L of compound with the concentration of 8, 4, 2, 1, 0.5 and 0.125 mu g/mL into the first to the sixth wells respectively, then adding 100 mu L of prepared bacterial suspension (namely 2-time final concentration inoculation bacterial liquid), adding 100 mu L of MH broth and 100 mu L of sterilized distilled water into the seventh well as a negative control, and adding 100 mu L of MH broth and 100 mu L of bacterial suspension (namely 2-time final concentration inoculation bacterial liquid) into the eighth well as a positive control. Setting 3 times for each well, placing the processed 96-well plate in a constant temperature incubator at 37 ℃ and culturing the plate on tinfoil paper in a dark place, and observing the plate for 20-24 h. If a concentration of the compound is active, the corresponding well at that concentration is clear, whereas it is turbid. The compound group at 4-8. Mu.g/mL was found to be completely inhibited at 24h, with the negative control filling the bottom of the well and the positive control growing no longer. The working concentration of the compound was 2. Mu.g/mL, i.e., the complete inhibitory concentration of the compound against drug-resistant bacteria was 2. Mu.g/mL.

The minimum inhibitory concentration MIC of the compound quinanulatic acid obtained in example 1 to 2 clinical drug-resistant bacteria, namely methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococcus faecalis, is 2 mug/mL. Therefore, the sesterterpene compound quinanulatic acid has great application potential in the aspect of resisting clinical drug-resistant bacteria.

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 (8)

1. The application of sesterterpene compounds in preparing clinical drug-resistant bacteria resisting agents is characterized in that the structural formula of the sesterterpene compounds is shown as a formula (I);

formula (I);

the clinical drug-resistant bacteria are ATCC 43300 methicillin-resistant staphylococcus aureus and ATCC 51299 vancomycin-resistant enterococcus faecalis.

2. The use of sesterterpenoids according to claim 1 for the preparation of a medicament against clinically resistant bacteria, wherein the method for the preparation of sesterterpenoids comprises the steps of:

step (1) of subjecting a fungus toEmericellaCulturing sp, XL029 with a rice solid culture medium, and performing static culture for 28-30 days to obtain a fermentation product;

step (2), extracting the fermentation product obtained in the step (1) by using methanol, and concentrating the obtained extract under reduced pressure to obtain a crude extract;

step (3), dissolving the crude extract obtained in the step (2) with water, back-extracting with ethyl acetate, and concentrating the ethyl acetate phase under reduced pressure to obtain an extract;

step (4), loading the extract obtained in the step (3) to a normal phase silica gel column by a dry method, then carrying out gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 7 components A-G;

step (5), loading the component B obtained in the step (4) to a normal phase silica gel column for chromatography by a dry method, then carrying out gradient elution, collecting gradient eluents of all gradients, concentrating, monitoring by TLC, and combining the same parts to obtain 5 sub-components B1-B5;

and (6) purifying the subfraction B1 obtained in the step (5) to obtain the sesterterpene compound shown in the formula (I).

3. The use of sesterterpenoids according to claim 2 for the preparation of a medicament against clinically resistant bacteria, wherein in step (2) the extraction is carried out by adding methanol of the same volume under ultrasound at room temperature for 0.9-1.1h, then standing at room temperature for 9-11h, collecting the supernatant, and concentrating under reduced pressure; the extraction times are 2-4.

4. Use of sesterterpenoids according to claim 2 for the preparation of a medicament against clinically resistant bacteria, wherein in step (3) the volume of water and ethyl acetate used is the same.

5. The application of sesterterpene compounds as claimed in claim 2 for preparing anti-clinically drug-resistant bacteria agent, wherein in step (4), the concrete method for dry loading the extract comprises: dissolving the extract by 10 times of methanol, adsorbing and mixing the extract by 80-100 mesh silica gel with equal mass of the extract, and then loading the sample;

the silica gel in the normal phase silica gel column is 100-200 mesh normal phase silica gel;

the mobile phase adopted by the gradient elution is petroleum ether, a mixed solvent of dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol in the mixed solvent of the dichloromethane and the methanol is 100, 80, 1, 50, 1, 20 and 10 in turn, and after each gradient is eluted to a TLC point plate without points, the next gradient elution is replaced, and 7 components A-G are obtained in total.

6. The application of sesterterpene compounds as claimed in claim 2 for preparing anti-clinically-drug-resistant bacteria agent, wherein in step (5), the concrete method for dry-loading the extract comprises: dissolving the component B by 10 times of methanol, adsorbing and mixing the sample by using 80-100 meshes of silica gel with the same mass as the component B, and then loading the sample;

the silica gel in the normal phase silica gel column is 100-200 mesh normal phase silica gel;

the mobile phases adopted in the gradient elution are petroleum ether and ethyl acetate, the volume ratio of the petroleum ether to the ethyl acetate is 100.

7. The use of sesterterpenoids according to claim 2 for the preparation of a medicament against clinically resistant bacteria, wherein in step (6) said purification is performed by gel chromatography followed by HPLC.

8. The use of sesterterpenoids according to claim 7 for the preparation of a medicament against clinically resistant bacteria, wherein in step (6), gel column Sephadex LH-20 is used for gel chromatography purification, and the volume ratio of Sephadex LH-20 is 1:1 as a mobile phase, and then carrying out HPLC;

the HPLC chromatographic conditions are that the mobile phase is a mixture of a mobile phase and a solvent in a volume ratio of 19:1 mixed solvent of methanol and water with the flow rate of 1mL/min, collecting the component with the peak starting time of 5.6min, and adopting Agilent C ₁₈ A reverse phase chromatographic column with the specification: 4.6mmx250nm,5um; column temperature: 30 ℃, sample amount per time: 20ul, ultraviolet detector, and detection wavelength of 210nm.

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