Monoterpene naphthoquinone compound from sponge coexisting streptomyces and preparation method thereof

Abstract

The application discloses a monoterpenoid naphthoquinone compound from a sponge co-parasitic Streptomyces, and the structure is shown as formula I: the monoterpenoid naphthoquinone compound is derived from a fermentation product of Streptomyces diacarni LHW51701.The monoterpenoid naphthoquinone compound from the sponge co-parasitic Streptomyces has simple preparation method, and has the growth inhibiting effect on methicillin-resistant Staphylococcus aureus.The application provides a new compound for marine drug research, and provides a scientific basis for developing and utilizing marine antibacterial drugs in China.

Description

Technical Field

[0001] The present invention belongs to the field of marine biology and medical technology, and specifically relates to a monoterpene naphthoquinone compound obtained by extraction, separation and purification from the fermentation product of sponge-associated Streptomyces diacarni LHW51701, a preparation method thereof, and application thereof in antibacterial drugs. Background Art

[0002] Streptomyces, belonging to the phylum Actinobacteria, is widely distributed in nature. Currently, a variety of structural secondary metabolites have been discovered from Streptomyces, including polyketides, alkaloids, terpenes, peptides, glycosides, macrolides, and quinones. Some of these secondary metabolites exhibit excellent antimicrobial activity. Currently, approximately two-thirds of antibiotics used in clinical practice are produced by Streptomyces, demonstrating the promising potential of Streptomyces to produce antimicrobial secondary metabolites. Summary of the Invention

[0003] The invention aims to provide a monoterpene naphthoquinone compound derived from sponge-associated Streptomyces.

[0004] Another object of the present invention is to provide a method for preparing the monoterpene naphthoquinone compounds derived from the sponge-associated Streptomyces.

[0005] Another object of the present invention is to provide a use of the monoterpene naphthoquinone compound derived from the sponge-associated Streptomyces in the preparation of antibacterial drugs.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a monoterpene naphthoquinone compound derived from Streptomyces symbiotically attached to a sponge, the structure of which is shown in Formula I:

[0008]

[0009] The molecular formula of the compound described in Formula I is C 21 H 24 O6.

[0010] The monoterpene naphthoquinone compound derived from sponge-associated Streptomyces is derived from the fermentation product of sponge-associated Streptomyces diacarni LHW51701.

[0011] The second aspect of the present invention provides a method for preparing the monoterpene naphthoquinone compound derived from the sponge-associated Streptomyces, comprising the following steps:

[0012] The first step is to prepare the total extract

[0013] Streptomyces diacarni LHW51701, a sponge-associated bacterium, was inoculated into TSBY medium and then into MYGS medium for further culture. The extract was extracted with an equal volume of ethyl acetate to obtain an ethyl acetate extract, which was then dissolved in 95% methanol-water, defatted with n-hexane, and concentrated under reduced pressure to obtain a methanol total extract.

[0014] The second step is separation and purification

[0015] 1) The methanol total extract was separated by normal phase silica gel vacuum column chromatography using a dichloromethane-methanol solution as the eluent for gradient elution. Similar fractions were combined according to the thin layer chromatography color to obtain 8 components A1-A8;

[0016] 2) Fraction A3 was separated by ODS medium-pressure column chromatography using an acetonitrile-water gradient elution. Similar fractions were combined based on TLC color development to obtain 12 fractions A3A-A3L.

[0017] 3) Component A3F is separated by reverse-phase high performance liquid chromatography to obtain the monoterpene naphthoquinone compounds derived from the sponge-associated Streptomyces.

[0018] The culture conditions in the TSBY medium in the first step are: 220 rpm, temperature 30° C., and culture for six days.

[0019] The conditions for inoculating into MYGS medium and continuing to culture in the first step are: 220 rpm, temperature 30° C., and culture for one week.

[0020] In the first step, TSBY medium: sucrose 100 g / L, tryptone soy broth 30 g / L, yeast extract 5 g / L, defoamer 1 mL / L.

[0021] In the first step, MYGS culture medium: 30 g / L sea salt, 10 g / L malt extract, 4 g / L glucose, 4 g / L yeast extract, and 1 mL / L defoaming agent.

[0022] In the second step, in 1), the conditions for gradient elution using dichloromethane-methanol solution as the eluent are: elution with dichloromethane-methanol solution with a volume ratio of 100:1, 75:1, 50:1, 30:1, 15:1, 5:1, 1:1, and 0:1 in sequence.

[0023] In the second step, in 2), the volume ratio of acetonitrile-water solution in gradient elution is 10%→100%, and the time is 180 min.

[0024] In the second step, in 3), the conditions for reversed-phase high-performance liquid separation are: the eluent is acetonitrile-water solution (10%→100%, 20 min) gradient elution, the chromatographic column is YMC-Pack Pro C18 RS (10×250 mm, 5 μm), the flow rate is 2.0 mL / min, the detection wavelengths are 227 and 261 nm, and the retention time is 17 minutes.

[0025] The third aspect of the present invention provides a use of the monoterpene naphthoquinone compound derived from sponge-associated Streptomyces in the preparation of antibacterial drugs.

[0026] The bacteria is methicillin-resistant Staphylococcus aureus.

[0027] In vitro experiments show that the MIC value of the compound of the present invention against methicillin-resistant Staphylococcus aureus is 256 μg / mL, so the compound of the present invention can be used to prepare antibacterial drugs.

[0028] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0029] The compound of the present invention has a simple preparation method and good antibacterial activity, and can provide a basis for the development of new marine antibacterial drugs.

[0030] The monoterpene naphthoquinone compounds of the present invention are derived from the fermentation products of Streptomyces diacarni LHW51701, a sponge-associated bacterium. The monoterpene naphthoquinone compounds derived from Streptomyces diacarni LHW51701 are simple to prepare and exhibit growth inhibition against methicillin-resistant Staphylococcus aureus. This invention provides new compounds for marine drug research and a scientific basis for the development and utilization of marine antimicrobial drugs in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the DP4+ probability analysis of the monoterpene naphthoquinone compounds of the present invention.

[0032] Figure 2 Schematic diagram of the calculated and measured ECD of the monoterpene naphthoquinone compounds of the present invention. DETAILED DESCRIPTION

[0033] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0034] The present invention is a monoterpene naphthoquinone compound isolated from the fermentation product of Streptomyces diacarni LHW51701 (purchased from the German Collection of Microorganisms, accession number: DSM 106126) (abbreviated as S.diacarni LHW51701), a sponge-associated bacterium. The compound can also be prepared using other bacteria or artificial synthesis methods.

[0035] Example 1

[0036] Preparation of the compounds of the present invention

[0037] The first step is to prepare the total extract

[0038] The sponge-associated Streptomyces diacarni LHW51701 was inoculated into TSBY medium (100 g / L sucrose, 30 g / L tryptone soy broth, 5 g / L yeast extract, 1 mL / L defoamer) at 220 rpm and 30°C for six days. The inoculation was then carried out into MYGS medium (30 g / L sea salt, 10 g / L malt extract, 4 g / L glucose, 4 g / L yeast extract, 1 mL / L defoamer) at 220 rpm and 30°C for one week. The extract was extracted with an equal volume of ethyl acetate to obtain an ethyl acetate layer, which was then dissolved in 95% methanol in water, defatted with n-hexane, and concentrated under reduced pressure to obtain a methanol total extract.

[0039] The second step is separation and purification

[0040] 1) The total extract of the methanol layer was separated by normal phase silica gel vacuum column chromatography using a dichloromethane-methanol solution (100:1, 75:1, 50:1, 30:1, 15:1, 5:1, 1:1, 0:1) as the eluent for gradient elution. Similar fractions were combined based on the thin layer chromatography color to obtain 8 components A1-A8;

[0041] 2) Fraction A3 was separated by ODS medium-pressure column chromatography using a gradient elution solution of acetonitrile-water (10% → 100%, 180 min). Similar fractions were combined based on TLC color development to obtain 12 fractions A3A-A3L.

[0042] 3) Fraction A3F was separated by reverse-phase HPLC (Wasters 1525 pump / 2998 photodiode array detector) with a gradient elution of acetonitrile-water (10% → 100%, 20 min) on a YMC-Pack Pro C18 RS column (10 × 250 mm, 5 μm) at a flow rate of 2.0 mL / min, detection wavelengths at 227 and 261 nm, and a retention time of 17 minutes to obtain the compound of Formula I of the present invention.

[0043] Step 3: Structural Identification

[0044] The molecular formula of the compound represented by formula I of the present invention is C 21 H 24 O6, 1D NMR, 2D NMR, HRESIMS, DP4 + probability analysis, calculation of ECD and other methods confirmed that the compound C of formula I of the present invention 21 H 24 The chemical structure of O6 is shown in Formula I:

[0045]

[0046] Compound C represented by formula I of the present invention 21 H 24 The physical and chemical properties and NMR data of O6 are as follows: white powder; [α]2D0-18.0 (c 0.10, MeOH); UV (MeOH)λmax (logε) 222 (2.38), 264 (2.15), 300 (1.88) nm; IRν max 2972,2931,1718,1682,1636,1617,1580,1454,1404,1377,1282,1241,1196,1176,1088,1032,985,908,856,800,773,735,693,544,453cm -1 ;ECD(MeOH)λmax(Δε)227(-2.22),261(-2.37),305(+1.60)nm; HRESIMS m / z 373.1661[M+H] + (calcdforC 21 H 25 O6,373.1651). The nuclear magnetic resonance spectrum data are shown in Table 1:

[0047] Table 1 Compound C of Formula I of the present invention 21 H 24 O6 NMR spectrum data (CDCl3, 700MHz)

[0048]

[0049]

[0050] The relative configuration of the compound was determined by analyzing NOESY spectra and NMR calculations combined with DP4+ probability analysis. H3-14 and H-12 have NOESY correlation, suggesting that Δ 12,13The NOESY correlation between 3-Me and H-2 indicates that 2-Me and 3-Me are on opposite sides of the ring, thus confirming the relative configuration of C-2 and C-3 to be 2R*3S*. The relative configuration of C-10 remains undetermined. To further determine the structure of the compound, the two possible relative configurations of 2R*, 3S*, 10R*-1 (Isomer 1) and 2R*, 3S*, 10S*-1 (Isomer 2) were analyzed. 1 H and 13 The chemical shifts of C-NMR were calculated and the measured spectrum data of compound 1 were compared with the calculated data of two possible structures. The DP4+ probability analysis results are as follows: Figure 1 As shown, Figure 1 This is a schematic diagram of the DP4+ probability analysis of the monoterpene naphthoquinone compound of the present invention, showing that the data of the structure 2R*, 3S*, 10R*-1 are in good agreement with the experimental data, so the relative configuration of compound 1 is 2R*, 3S*, 10R*. The absolute configuration of the compound was determined by using the calculated ECD, and the results are as follows: Figure 2 As shown, Figure 2 The calculated and measured ECD diagrams for the monoterpene naphthoquinone compounds of the present invention show good agreement between the calculated 2R, 3S, 10R-1 ECD curves and the measured spectra. Based on the calculated ECD results, the absolute configuration of compound 1 is 2R, 3S, 10R.

[0051] Example 2

[0052] In vitro antibacterial activity test of the compound prepared in Example 1 of the present invention

[0053] Methicillin-resistant Staphylococcus aureus was activated with LA solid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 18 g / L, pH 7.0). A single colony was picked from the activated plate and shaken and cultured for 1 day in MH liquid medium (MH broth 21 g / L, pH 7.0). The test strains were grouped and loaded onto 96-well plates, divided into a sample group (190 μL MH medium, 2 μL bacterial solution, 10 μL test compound), a positive control group (190 μL MH medium, 2 μL bacterial solution, 10 μL vancomycin), a negative control group (190 μL MH medium, 2 μL bacterial solution, 10 μL DMSO), and a blank control group (200 μL MH medium). Three parallel experiments were designed. After loading, the plates were stored in a 37°C constant temperature incubator and observed after 24 hours. The results showed that the compound prepared in Example 1 of the present invention had an inhibitory effect on methicillin-resistant Staphylococcus aureus, with an MIC value of 256 μg / mL. Therefore, the compound of the present invention can be used to prepare antibacterial drugs.

[0054] The present invention provides a new monoterpene naphthoquinone compound and a preparation method thereof for marine drug research, and provides a scientific basis for the development and utilization of marine antibacterial drugs in my country.

[0055] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with this patent can make slight changes or modifications to equivalent embodiments using the above technical content without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

1. A monoterpene naphthoquinone compound derived from Streptomyces symbiotically attached to a sponge, characterized in that: The structure is shown in Formula I:

2. The monoterpene naphthoquinone compound derived from sponge-associated Streptomyces according to claim 1, characterized in that: The monoterpene naphthoquinone compound derived from sponge-associated Streptomyces diacarni is derived from the fermentation product of sponge-associated Streptomyces diacarni LHW51701.

3. A method for preparing a monoterpene naphthoquinone compound derived from sponge-associated Streptomyces according to claim 1 or 2, characterized in that: The following steps are involved: The first step is to prepare the total extract Streptomyces diacarni LHW51701, a sponge-associated bacterium, was inoculated into TSBY medium and then into MYGS medium for further culture. The extract was extracted with an equal volume of ethyl acetate to obtain an ethyl acetate extract, which was then dissolved in 95% methanol-water, defatted with n-hexane, and concentrated under reduced pressure to obtain a methanol total extract. The second step is separation and purification 1) The methanol total extract was separated by normal phase silica gel vacuum column chromatography using a dichloromethane-methanol solution as the eluent for gradient elution. Similar fractions were combined according to the thin layer chromatography color to obtain 8 components A1-A8; 2) Fraction A3 was separated by ODS medium-pressure column chromatography using an acetonitrile-water gradient elution. Similar fractions were combined based on TLC color development to obtain 12 fractions A3A-A3L. 3) Component A3F is separated by reverse-phase high performance liquid chromatography to obtain the monoterpene naphthoquinone compounds derived from the sponge-associated Streptomyces.

4. The method for preparing monoterpene naphthoquinone compounds derived from sponge-associated Streptomyces according to claim 3, characterized in that: The conditions for culturing in TSBY medium in the first step are: 220 rpm, temperature 30°C, and culturing for six days; The conditions for inoculating into MYGS medium and continuing to culture in the first step are: 220 rpm, temperature 30° C., and culture for one week.

5. The method for preparing monoterpene naphthoquinone compounds derived from sponge-associated Streptomyces according to claim 3, characterized in that: In the first step, TSBY medium: sucrose 100 g / L, tryptone soy broth 30 g / L, yeast extract 5 g / L, defoamer 1 mL / L; In the first step, MYGS culture medium: 30 g / L sea salt, 10 g / L malt extract, 4 g / L glucose, 4 g / L yeast extract, and 1 mL / L defoaming agent.

6. The method for preparing monoterpene naphthoquinone compounds derived from sponge-associated Streptomyces according to claim 3, characterized in that: In the second step, in 1), the conditions for gradient elution using dichloromethane-methanol solution as the eluent are: elution with dichloromethane-methanol solution with a volume ratio of 100:1, 75:1, 50:1, 30:1, 15:1, 5:1, 1:1, and 0:1 in sequence.

7. The method for preparing monoterpene naphthoquinone compounds derived from sponge-associated Streptomyces according to claim 3, characterized in that: In the second step, in 2), the volume ratio of acetonitrile-water solution in gradient elution is 10%→100%, and the time is 180 min.

8. The method for preparing monoterpene naphthoquinone compounds derived from sponge-associated Streptomyces according to claim 3, characterized in that: In the second step, in 3), the conditions for reversed-phase high-performance liquid separation are: the eluent is acetonitrile-water gradient elution, the chromatographic column is YMC-Pack Pro C18 RS, the flow rate is 2.0 mL / min, the detection wavelengths are 227 and 261 nm, and the retention time is 17 minutes.

9. Use of the monoterpene naphthoquinone compound derived from sponge-associated Streptomyces according to claim 1 or 2 in the preparation of antibacterial drugs.

10. The use according to claim 9, characterized in that The bacteria is methicillin-resistant Staphylococcus aureus.

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