CN113651677B - Compound eutyscoparin G, preparation method thereof and application thereof in preparation of antibacterial drugs - Google Patents

Abstract

The invention discloses a compound eutyscoparins G, a preparation method thereof and application thereof in preparing antibacterial drugs. A compound, eutyscoparins G, of formula (I): the compound, namely the teutyspropalins G, is separated and prepared from endophytic fungi Eutypthus scoparia SCBG-8, has relatively obvious antibacterial activity, can be used for preparing antibacterial drugs, provides candidate compounds for researching and developing new antibacterial drugs, and provides scientific basis for developing and utilizing natural active substances from plant endophytic bacteria.

Description

Compound eutyscoparin G, preparation method thereof and application thereof in preparation of antibacterial drugs

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to a compound eutyscoparin G, a preparation method thereof and application thereof in preparation of antibacterial drugs.

Background

Eutypella fungi are widely distributed, and secondary metabolites are abundant and diverse. The main metabolites include terpenes, cytochalasin, steroids and lactones, wherein the terpenes mainly comprise pimarane diterpenoids and sesquiterpenes. Moreover, the secondary metabolite shows various biological activities such as antitumor, antibacterial, anti-inflammatory and antiproliferative activities. The Sesquiterpenes compounds are metabolites in fungi, and have the structural characteristics that the molecules contain three isoprene units consisting of 15 carbon atoms and have various framework structures such as chain structures, ring structures and the like. Molecules of this type have been reported to possess a variety of biological activities. We found 1 novel compound eutyscoparin G with antibacterial activity in the study of endophytic fungus SCGB-8.

The invention content is as follows:

it is a first object of the present invention to provide a compound eutyscoparins G having antibacterial activity.

The structure of the compound eutyscoparin G is shown as the formula (I):

the second purpose of the invention is to provide a preparation method of a compound, eutyscoparin G, in the preparation method, the compound, eutyscoparin G, is separated and prepared from a fermentation culture of a plant endophytic fungus, eutypella scoparia SCBG-8, and specifically comprises the following steps:

(1) Preparing solid fermentation culture of endophytic fungus Eutyphylla scoparia SCBG-8, extracting the solid fermentation culture with ethyl acetate, and concentrating ethyl acetate extractive solution to obtain extract;

subjecting the extract to silica gel column chromatography, and eluting with petroleum ether-ethyl acetate in a volume ratio of 100; the product obtained by eluting with n-hexane-ethyl acetate in a volume ratio of 10: ethyl acetate =5, 1v/v develops a component fr.3 with Rf = 0.5-0.6;

subjecting fraction fr.3 to reverse phase column chromatography with an acetone gradient elution with a methanol-water volume ratio of 80, 85, 15,90, 10,95, 5,100, to give 6 subfractions fr.3.1-fr.3.6, wherein fraction fr.3.2 is purified on Sephadex LH-20 column chromatography to give 4 fractions fr.3.2.1-fr3.2.4.Fr.3.2.2 was passed through a further normal phase silica gel column, purified with n-hexane: ethyl acetate 20. Fr.3.2.2.3 was isolated and purified by semi-preparative HPLC to give the compound eutyscoparin G.

The preparation of the solid fermentation culture of the endophytic fungus Eutypella scoparia SCBG-8 comprises the following specific steps: selecting mycelium of the SCBG-8, inoculating the mycelium into a potato glucose liquid culture medium, culturing for 5 days at the temperature of 28 ℃ and the speed of 120r/min to prepare a seed solution, then inoculating the seed solution into a rice culture medium according to the inoculation amount of 0.1mL/g, and culturing for 30 days at the temperature of 28 ℃ to prepare a solid fermentation culture of the SCBG-8, wherein each liter of the potato glucose liquid culture medium is prepared by the following method: boiling 200g of potato in 500mL of pure water for 20min, filtering to obtain potato juice, adding glucose 20g and KH ₂ PO ₄ 3g、MgSO ₄ 1.5g, vitamin B ₁ 10mg, supplementing water to 1000mL, and sterilizing; the rice culture medium is prepared by the following method: mixing 300g rice with 360mL water, and sterilizing.

Experiments show that the compound eutyscoparin G has IC (integrated Circuit) effect on staphylococcus aureus and methicillin-resistant staphylococcus aureus ₅₀ The value was 6.25. Mu.g/mL. IC of positive control vancomycin against the above two gram-positive bacteria ₅₀ The value was 1.25. Mu.g/mL. This result shows that: the compound eutyscoprins G of the invention has relatively obvious antibacterial activity.

Accordingly, a third object of the present invention is to provide the use of the compound eutyscoparin G, or a pharmaceutically acceptable salt thereof, for the preparation of antibacterial agents. The antibacterial drugs are preferably anti-staphylococcus aureus drugs and anti-methicillin-resistant staphylococcus aureus drugs.

It is a fourth object of the present invention to provide an antibacterial agent comprising the compound, deutscoparins G, or a pharmaceutically acceptable salt thereof as an active ingredient. Preferably, the antibacterial agent is an anti-staphylococcus aureus and anti-methicillin-resistant staphylococcus aureus drug.

A fifth object of the present invention is to provide the use of the endophytic fungus Eutypella scoparia SCBG-8 for the preparation of the compound teutyscoparins G.

Compared with the prior art, the invention has the advantages that:

the compound, namely the teutyspropalins G, is separated and prepared from endophytic fungi Eutypthus scoparia SCBG-8, has relatively obvious antibacterial activity, can be used for preparing antibacterial drugs, provides candidate compounds for researching and developing new antibacterial drugs, and provides scientific basis for developing and utilizing natural active substances from plant endophytic bacteria.

The fungus Eutypella scoparia SCBG-8 of the present invention has been disclosed in Wenge Zhang, miaomiao Wang, sha ZHa, kangping Xu, guishan Tan, shengxiang Qiu, zhenxing Zou, haibo Tan Eutypopanols A-G, polyketide derivatives from endenoptic funguses Eutypella scoparia SCBG-8, fitoterapia,2020, 146,104681.

Drawings

FIG. 1 is a schematic representation of the compound, teutyscoparins G ¹ H NMR spectrum;

FIG. 2 is a schematic representation of the compound deutyscoparins G ¹³ C NMR spectrum;

FIG. 3 is a schematic representation of the compound teutyscoparins G ¹ H- ¹ H COSY spectrum;

FIG. 4 is the HSQC spectra of compound deutyscoparins G;

FIG. 5 is an HMBC spectrum of compound deutyscoparins G;

FIG. 6 is a NOESY spectrum of the compound, teutyscaprins G;

FIG. 7 is an HRESIMS spectrum of compound deutyscoparins G;

FIG. 8 is a UV spectrum of compound deutyscoparins G;

FIG. 9 is a CD spectrum of compound deutyscoparins G;

FIG. 10 is an IR spectrum of the compound deutyscoparins G;

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1

1. Isolation, purification and characterization of the endophytic fungus Eutypella scoparia SCBG-8

The endophytic fungus Eutypella scoparia SCBG-8 of the invention is separated from leaves of Melittleria excelsa (Leptospermum brachyanum) in the Australian plant park (SCBG) of the Chinese academy of sciences at 2016 (9 months), is further identified by ITS sequence analysis, has a GenBank accession number of MN788605, and belongs to Eutypella genus fungus through blast comparison and homology analysis, and is named as Eutypella scoparia SCBG-8.

2. Solid fermentation of Eutypella scoparia SCBG-8

Inoculating activated mycelia of Eutyphylla scoparia SCBG-8 to potato glucose liquid culture medium (per liter of culture medium is prepared by boiling 200g of potato in 500mL of pure water, boiling for 20min, filtering to obtain potato juice, adding 20g of glucose and KH ₂ PO ₄ 3g、MgSO ₄ 1.5g, vitamin B ₁ 10mg, supplemented to 1000mL with water, sterilized), cultured at 28 ℃ for 5 days at 120r/min to obtain a seed solution, and then the seed solution was inoculated into a rice medium at an inoculum size of 0.1mL/g rice medium (the medium was prepared by the following method: mixing rice 300g with water 360mL, sterilizing, mixing, autoclaving at 121 deg.C for 20min, and cooling) and culturing at 28 deg.C for 30 days to obtain solid fermentation culture of SCBG.

3. Preparation of compound deutyscoparins G

(1) Adding ethyl acetate into the solid fermentation culture of SCBG, soaking and extracting for 24 hr, repeating the extraction for 3 times, mixing the extractive solutions, and concentrating to obtain extract (45 g).

Subjecting the extract to silica gel column chromatography, and eluting with petroleum ether-ethyl acetate in a volume ratio of (1, 50; collecting n-hexane-ethyl acetate obtained by elution in a volume ratio of 10 and performing TLC thin layer chromatography with n-hexane: ethyl acetate =5, 1v/v develops a component fr.3 with Rf = 0.5-0.6;

subjecting fraction fr.3 to reverse phase column chromatography with a methanol-water volume ratio of 80, 85, 15,90, 10,95, 5,100, acetone gradient elution yielding in order 6 subfractions fr.3.1-fr.3.6, wherein fraction fr.3.2 is purified on a Sephadex LH-20 column chromatograph yielding 4 fractions fr.3.2.1-fr3.2.4.Fr.3.2.2 (polarity: rf value 0.2-0.8 by TLC chromatography on a hexane: acetone =5/1v/v chromatography creeper, with uv, vanillin dark blue) through a further normal phase silica gel column, purified by n-hexane: ethyl acetate 20. Fr.3.2.2.3 using semi-preparative HPLC with column parameters of YMC-Pack ODS-A, elution conditions of volume fraction of 85% methanol water, flow rate of 2ml/min, and collection of 34min retention time fraction to obtain compound eutyscoparin G.

4. Structure identification of compound eutyscoparin G

¹ H-NMR、 ¹³ C-NMR and HMBC nuclear magnetic resonance spectrograms are measured by a Bruker Advance-600 nuclear magnetic resonance spectrometer, and Tetramethylsilane (TMS) is used as an internal standard; ESI-MS data were measured with VG Autospec-3000 type mass spectrometer; the ultraviolet spectrum is measured by using an Shimadzu UV-2600 spectrophotometer, and the structure identification is as follows:

as shown in FIGS. 1 to 10, FIG. 1 is a schematic representation of the compound eutyscoparin G ¹ H NMR spectrum; FIG. 2 is a schematic representation of the compound eutyscoparin G ¹³ C NMR spectrum; FIG. 3 is a schematic representation of compound eutyscoparins G ¹ H- ¹ H COSY spectra; FIG. 4 is an HSQC spectrum of compound eutyscoparin G; FIG. 5 is an HMBC spectrum of compound eutyscoparin G; FIG. 6 is a NOESY spectrum of compound eutyscoparin G; FIG. 7 is an HRESIMS spectrum of compound eutyscoparin G; FIG. 8 is a UV spectrum of the compound eutyscoparins G; FIG. 9 is a CD spectrum of compound eutyscoparins G; FIG. 10 is an IR spectrum of compound eutyscoparin G;

compound 1 is a yellow powderEnd (nuclear magnetic data shown in table 1); according to HRESIMS [ M + H ]] ⁺ m/z 223.2054， C ₁₅ H ₂₇ O, calculated value is 223.2056, and the molecular formula of the compound is determined to be C ₁₅ H ₂₆ O, unsaturation is 3; ¹ the H-NMR spectrum showed an olefin proton signal [ delta ] _H 5.31(s,H-3)](ii) a 3 methyl signals [ delta ] _H 0.93(d,J＝6.9Hz,H ₃ -13),1.6 (s,H ₃ -14),0.77(s,H ₃ -15)]。 ¹³ The C-NMR spectrum and the HSQC spectrum showed 15 carbon signals in Compound 1, including 2 quaternary carbons (including 1 alkenyl carbon), 4 methines, 6 methylenes, and 3 methyl groups. The planar structure of the compound eutyscoparin G is determined by further analyzing two-dimensional spectrograms such as COSY, HSQC, HMBC and the like, and the absolute configuration of the compound I is determined by calculating an ECD method.

The structural formula of the target compound eutyscoparins G separated by the method is shown as the formula (I):

TABLE 1 Nuclear magnetic data for Compound 1 (500 MHz/125MHz, Δ in ppm, J in Hz)

Example 2

The antibacterial activity of compound eutyscoparin G was tested by MHB (Microbroth dilution in Mueller-Hinton broth) method.

1. Test reagents: the compound eutyscoparin G prepared by the invention is dissolved in dimethyl sulfoxide (DMSO) to obtain a mother solution with the concentration of 1mg/mL, and then the mother solution is subjected to gradient dilution by using a 96-well plate. The positive control was vancomycin.

The strains used in this experiment were s.aureus (26003) and MRSA (JCSC 3063).

2. The experimental method comprises the following steps: compounds were evaluated according to the CLSI guidelines for MIC values determined by the MHB (Microbroth in Mueller-Hinton broth) method with Resazurin as the visual indicatorThe antibacterial activity of (1). Fresh HB medium was diluted to OD using a UV-6000 spectrophotometer using S.aureus (26003) and MRSA (JCSC 3063) bacteria at logarithmic growth phase ₆₀₀ =0.07, the above bacterial suspension and resazurin are mixed and inoculated into 96-well plates in a volume ratio of 6 ₂ Culturing in an incubator, and observing color change after 16-20 h. Each experiment was repeated 3 times and its MIC value was calculated.

3. The experimental results are as follows: the MIC value of the compound eutyscoparin G prepared by the invention to gram-positive bacteria S.aureus (26003) and MRSA (JCSC 3063) is 6.25 mu G/mL. The MIC value for the positive control vancomycin for S.aureus (26003) and MRSA (JCSC 3063) was 1.25. Mu.g/mL. This result shows that: the compound eutyscoparin G has relatively obvious antibacterial activity, so that the compound provided by the invention provides a candidate compound for researching and developing a new antibacterial drug, and provides a scientific basis for developing and utilizing natural active substances from plant endophytes.

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.

The invention discloses a compound eutyscoparin G, a preparation method thereof and application thereof in preparing antibacterial drugs. The compound eutyscoparin G is prepared by separating and preparing fermentation culture of endophytic fungus Eutypella scoparia SCBG-8. Experiments prove that the MIC value of the compound eutyscoparin G on gram-positive bacteria S.aureus (26003) and MRSA (JCSC 3063) is 6.25 mu G/Ml, the compound eutyscoparin G shows remarkable antibacterial activity and can be used for preparing antibacterial drugs.

Claims (5)

1. A compound, eutyscoparins G, of formula (I):

2. a method for preparing eutyscoparins G compound according to claim 1, comprising the following steps:

(1) Preparing solid fermentation culture of endophytic fungus Eutypella scoparia SCBG-8, extracting the solid fermentation culture with ethyl acetate, and concentrating ethyl acetate extract to obtain extract;

(2) Subjecting the extract to silica gel column chromatography, and eluting with petroleum ether-ethyl acetate in a volume ratio of 1, 50; the product obtained by eluting with n-hexane-ethyl acetate in a volume ratio of 10: ethyl acetate =5, 1v/v develops a component fr.3 with Rf = 0.5-0.6;

subjecting fraction fr.3 to reverse phase column chromatography, eluting with a methanol-water volume ratio of 80, 85, 15,90, 10,95, 5,100, acetone gradient, yielding 6 subfractions fr.3.1-fr.3.6, wherein fraction fr.3.2 is purified on Sephadex LH-20 column chromatography yielding 4 fractions fr.3.2.1-fr3.2.4; fr.3.2.2 was passed through a further normal phase silica gel column, purified with n-hexane: ethyl acetate 20; fr.3.2.2.3 using semi-preparative HPLC to obtain compound eutyscoparin G;

the separation and purification of the component Fr.3.2.2.3 by HPLC specifically comprises the following steps: subjecting the fraction Fr.3.2.2.3 to semi-preparative HPLC using se:Sup>A YMC-ODS-A/AQ column with se:Sup>A mobile phase of methanol/water at se:Sup>A volume ratio of 85 to 15 and se:Sup>A flow rate of 3mL/min, and collecting the eluted fraction with se:Sup>A retention time of 34min to obtain se:Sup>A compound eutyscoparin G;

the step (1) of preparing the solid fermentation culture of the endophytic fungus Eutypella scoparia SCBG-8 comprises the following specific steps: selecting mycelium of SCBG-8, inoculating to potato glucose liquid culture medium, culturing at 28 deg.C and 120r/min for 5 days to obtain seed solution, and inoculating the seed solution at an inoculum size of 0.1mL/gInoculating the mixture into a rice culture medium, and culturing at 28 ℃ for 30 days to prepare a solid fermentation culture of SCBG-8, wherein each liter of the potato glucose liquid culture medium is prepared by the following method: decocting 200g of potato in 500mL of pure water, boiling for 20min, filtering to obtain potato juice, and adding glucose 20g and KH ₂ PO ₄ 3g、MgSO ₄ 1.5g, vitamin B ₁ 10mg, supplementing water to 1000mL, and sterilizing; the rice culture medium is prepared by the following method: mixing 300g rice with 360mL water, and sterilizing.

3. The use of the compound eutyscoparin G or its pharmaceutically acceptable salt of claim 1 in the preparation of antibacterial agents, wherein the antibacterial agents are anti-Staphylococcus aureus and methicillin-resistant Staphylococcus aureus agents.

4. An antibacterial agent comprising the compound eutyscoparin G or a pharmaceutically acceptable salt thereof according to claim 1 as an active ingredient.

5. Use of the endophytic fungus Eutypella scoparia SCBG-8 for the preparation of the compound eutyscoparins G according to claim 1, which is prepared according to the preparation method of claim 2.

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