Anthraquinone dimer compound as well as extraction method and application thereof

Abstract

The invention provides an anthraquinone dimer compound which is extracted from fermentation liquor of plant endophytic fungi and has an antibacterial effect.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to an anthraquinone dimer compound. Background Art

[0002] With the large-scale and unreasonable long-term use of antibiotics, the number of drug-resistant bacteria and their drug resistance have increased. Multidrug-resistant bacterial infections have undoubtedly become a global human health problem that urgently needs to be solved this century. Conservatively estimated, more than 700,000 people lose their lives every year worldwide due to severe diseases caused by infections with multidrug-resistant bacteria. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the common pathogenic drug-resistant bacteria causing hospital-acquired and community infections. Therefore, it is urgent to develop drugs for treating drug-resistant bacterial infectious diseases.

[0003] Natural products are an important source for the research and development of new drugs. Among the 1394 small molecule drugs for disease diagnosis and treatment launched globally between 1981 and 2019, 52.1% of the small molecule drugs are directly or indirectly related to natural products. According to literature reports, among the known 500,000 natural products, about 70,000 are derived from microorganisms, and about 33,500 of them have good biological activities. Among these 33,500 active natural products, 45.5% of the active natural products are derived from fungi, and 36.4% of the active natural products are derived from actinomycetes. Thus, it can be seen that fungal-derived active natural products play an important role among microbial natural products.

[0004] Therefore, exploring more drugs against drug-resistant bacteria with novel chemical structures, new action mechanisms or new action targets from fungi can effectively alleviate the current problem of bacterial drug resistance and has great potential. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides an anthraquinone dimer compound, which is extracted from the fermentation broth of an endophytic fungus of a plant and has an antibacterial effect.

[0006] To achieve the object of the present invention, the technical solution adopted by the present invention is:

[0007] An anthraquinone dimer compound, characterized in that the structure is as follows:

[0008]

[0009] The extraction method of the anthraquinone dimer compound of the present invention includes the following steps:

[0010] A. Fermentation: Ferment the fungus Diaporthe sp. CB10100 in rice or PDB medium to obtain a fermented product for standby;

[0011] B. Separation: The fermented product was extracted three times with ethyl acetate and then concentrated to obtain a crude extract paste. The aqueous suspension of the crude extract was extracted with petroleum ether, ethyl acetate, and n-butanol respectively to obtain the corresponding concentrates; the ethyl acetate fraction was separated by normal-phase silica gel column chromatography, ODS column chromatography gradient elution, and high-performance liquid chromatography to obtain Compound Cytoskyrin D.

[0012] Preferably, the fungus Diaporthe sp. CB10100 was cultured first and then fermented.

[0013] More preferably, before fermentation, the fungus Diaporthe sp. CB10100 was cultured in a 1000 mL Erlenmeyer flask containing 200 mL of PBD solution at 30 °C with shaking at 200 rpm for 3 days to obtain a fungal seed fermentation broth, and the fungal seed fermentation broth was inoculated at a volume of 10% of the large fermentation medium volume.

[0014] More preferably, each liter of the fermentation medium contains: 200 mL of a 35 g PDB / L solution or 200 g of rice and 200 mL of water added to a 1000 mL conical flask, which was sterilized and used for large-scale fermentation.

[0015] Preferably, in step B, the ethyl acetate fraction was separated by normal-phase silica gel column chromatography with a mobile phase of petroleum ether / ethyl acetate, and 9 fractions Fr.1-9 were collected. Fraction Fr.2 was eluted with a methanol / water ODS column chromatography gradient and then separated by high-performance liquid chromatography to obtain Compound Cytoskyrin D.

[0016] More preferably, the volume ratio change of the petroleum ether / ethyl acetate gradient elution was 20:1, 10:1, 5:1, 1:1, 1:5, 1:20;

[0017] The volume ratio change of the methanol / water ODS gradient elution was: 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8;

[0018] The chromatographic conditions for high-performance liquid chromatography were:

[0019] Chromatographic column: Yuexu AQ-C18, with a specification of 250×10 mm, 5 μm

[0020] Mobile phase: acetonitrile (A): 0.2% formic acid in water (B); gradient elution was used. From 0 to 11 min, the mobile phase ratio A:B was increased from 10:90 to 75:25. From 11 to 12 min, the mobile phase ratio was maintained at 75:25. From 12 to 19 min, the mobile phase ratio was changed from 75:25 to 10:90. From 19 to 22 min, the mobile phase ratio was maintained at 10:90;

[0021] Flow rate: 3 mL / min;

[0022] Column temperature: 35 °C.

[0023] The application of the anthraquinone dimer compound described in the present invention in the preparation of antibacterial drugs, and the bacteria inhibited include methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, and methicillin-sensitive Staphylococcus aureus. Description of the Drawings

[0024] Figure 1 is the 1 1H NMR of compound Cytoskyrin D;

[0025] Figure 2 is the 13 13C NMR of compound Cytoskyrin D;

[0026] Figure 3 is the DEPT90 of compound Cytoskyrin D;

[0027] Figure 4 is the DEPT135 of compound Cytoskyrin D;

[0028] Figure 5 is the two-dimensional nuclear magnetic resonance spectrum HSQC of compound Cytoskyrin D;

[0029] Figure 6 is the two-dimensional nuclear magnetic resonance spectrum HMBC of compound Cytoskyrin D;

[0030] Figure 7 is the two-dimensional nuclear magnetic resonance spectrum 1 1H- 1 1H COSY of compound Cytoskyrin D;

[0031] Figure 8 is the two-dimensional nuclear magnetic resonance spectrum NOESY of compound Cytoskyrin D;

[0032] Figure 9 is the high-resolution mass spectrum of compound Cytoskyrin D;

[0033] Figure 10 is the ultraviolet absorption spectrum of compound Cytoskyrin D. Detailed Embodiments

[0034] In order to more clearly and detailedly illustrate the technical solutions of the present invention, the present invention will be further described below through relevant embodiments. The following embodiments are only for specifically illustrating the implementation methods of the present invention and do not limit the protection scope of the present invention.

[0035] Example 1

[0036] An anthraquinone dimer compound, the structure of which is as follows:

[0037]

[0038] Example 2

[0039] The method for extracting anthraquinone dimer compounds of the present invention comprises the following steps:

[0040] A. Fermentation: fermenting the fungus Diaporthe sp. CB10100 in rice to obtain a fermented product for later use;

[0041] B. Separation: The fermented product was extracted three times with ethyl acetate and then concentrated to obtain a crude extract. The aqueous suspension of the crude extract was extracted with petroleum ether, ethyl acetate and n-butanol, respectively, to obtain a corresponding concentrate. The ethyl acetate portion was subjected to normal phase silica gel column chromatography with petroleum ether / ethyl acetate as the mobile phase to obtain 9 fractions Fr.1-9. Fraction Fr.2 was eluted with methanol / water ODS column chromatography gradient and then separated by high performance liquid chromatography to obtain compound Cytoskyrin D.

[0042] The Diaporthe sp.CB10100 strain was collected from the rhizome of the medicinal plant Caulis Sinensis from the Xuefeng Mountains in Huaihua City, Hunan Province. The strain was stored on the growth medium PDA slant (recorded in Frontiers in Chemistry, 2021, Volume 9).

[0043] The genome sequence of the fungus Diaporthe sp. CB10100 described in the present invention is included in the NCBI database.

[0044] Example 3

[0045] The method for extracting anthraquinone dimer compounds of the present invention comprises the following steps:

[0046] A. Fermentation: fermenting the fungus Diaporthe sp. CB10100 in PDB medium to obtain a fermentation product for later use;

[0047] Each liter of culture medium for fermentation contains: 200 mL of a solution containing 35 g PDB / liter is added to a 1000 mL conical flask and sterilized for large-scale fermentation.

[0048] B. Isolation: The fermented product was extracted three times with ethyl acetate and then concentrated to obtain a crude extract paste. The aqueous suspension of the crude extract was extracted with petroleum ether, ethyl acetate, and n-butanol respectively to obtain the corresponding concentrates. The ethyl acetate fraction was subjected to normal-phase silica gel column chromatography with a mobile phase of petroleum ether / ethyl acetate, and 9 fractions Fr.1 - 9 were collected. Fraction Fr.2 was eluted with a methanol / water ODS column chromatography gradient and then separated by high-performance liquid chromatography to obtain compound Cytoskyrin D.

[0049] Example 4

[0050] The extraction method of the anthraquinone dimer compound of the present invention includes the following steps:

[0051] A. Fermentation: First, the fungus Diaporthe sp. CB10100 was cultured in a 1000 mL flat-bottom conical flask containing 200 mL of PBD solution, shaken at 30 °C and 200 rpm for 3 days to obtain a fungal seed fermentation broth. The fungal seed fermentation broth was inoculated at a volume of 10% of the large fermentation medium volume.

[0052] Each liter of the fermentation medium contains: 200 g of rice and 200 mL of water were added to a 1000 mL conical flask, sterilized and used for large-scale fermentation.

[0053] B. Isolation: The fermented product was extracted three times with ethyl acetate and then concentrated to obtain a crude extract paste. The aqueous suspension of the crude extract was extracted with petroleum ether, ethyl acetate, and n-butanol respectively to obtain the corresponding concentrates. The ethyl acetate fraction was subjected to normal-phase silica gel column chromatography with a mobile phase of petroleum ether / ethyl acetate, and 9 fractions Fr.1 - 9 were collected. Fraction Fr.2 was eluted with a methanol / water ODS column chromatography gradient and then separated by high-performance liquid chromatography to obtain compound Cytoskyrin D.

[0054] The volume ratio change of the petroleum ether / ethyl acetate gradient elution is 20:1, 10:1, 5:1, 1:1, 1:5, 1:20;

[0055] The volume ratio change of the methanol / water ODS gradient elution is: 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8;

[0056] The chromatographic conditions of high-performance liquid chromatography are:

[0057] Chromatographic column: Yuexu AQ-C18, with a specification of 250×10 mm, 5 μm

[0058] Mobile phase: Acetonitrile (A): 0.2% formic acid in water (B); Gradient elution was used, with the mobile phase ratio of A:B increasing from 10:90 to 75:25 from 0 - 11 min, remaining 75:25 from 11 - 12 min, changing from 75:25 to 10:90 from 12 - 19 min, and remaining 10:90 from 19 - 22 min;

[0059] Flow rate: 3 mL / min;

[0060] Column temperature: 35 °C.

[0061] Structural analysis of Cytoskyrin D:

[0062] Cytoskyrin D was fully structurally characterized through comprehensive structural analysis.

[0063] (1) High-resolution mass spectrometry (HRESIMS) analysis yielded the [M + H] molecular ion peak (m / z) of Cytoskyrin D as 561.1029, which is consistent with the calculated value of 561.1028 for its standard molecular formula C + H 29 H 20 O 12 [C 29 H 21 O 12 +H] + ( Figure 9 ).

[0064] (2) Ultraviolet spectrum analysis demonstrated that Cytoskyrin D contains 5 absorption peaks at 193.6 nm, 256.1 nm, 292.9 nm, 333.5 nm, and 389.7 nm ( Figure 10 ).

[0065] Cytoskyrin D is yellow in color; UV(MeOH) max 193.6 nm, 256.1 nm, 292.9 nm, 333.5 nm, 389.7 nm see Figure 10 ; HRESIMS: Calculated value for the molecular formula C 29 H 20 O 12 : 561.1028, measured value: 561.1029, see Figure 9 ;

[0066] (3) NMR data: See Table 1. Figures 1-4 is a one-dimensional nuclear magnetic resonance spectrum, Figures 5-8 is a two-dimensional nuclear magnetic resonance spectrum, where Figure 1 is the 1 H NMR of Cytoskyrin D, DMSO-d6 (500 MHz); Figure 2 is of Cytoskyrin D 13 13C NMR, pyridine-d 5 (125 MHz); Figure 3 is the DEPT90 of Cytoskyrin D, Figure 4 is the DEPT135 of Cytoskyrin D and Figures 5-7 is a two-dimensional nuclear magnetic resonance spectrum, where Figure 5 is the HSQC of Cytoskyrin D, Figure 6 is the HMBC of Cytoskyrin D; Figure 7 is of Cytoskyrin D 1 1H- 1 1H COSY; Figure 8 is the two-dimensional nuclear magnetic resonance spectrum NOESY of compound Cytoskyrin D; Figure 9 is the high-resolution mass spectrum of compound Cytoskyrin D; Figure 10 is the ultraviolet absorption spectrum of compound Cytoskyrin D.

[0067]

[0068] Table 1

[0069]

[0070]

[0071] Antibacterial Activity Test of Compound Cytoskyrin D

[0072] The minimum inhibitory concentration of compound Cytoskyrin D against methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus (Staphylococcus aureus ATCC 29213), methicillin-sensitive Staphylococcus aureus (MSSA), Escherichia coli (Escherichia coli ATCC 25922), Klebsiella pneumoniae (Klebsiella pneumoniae ATCC 113), and Pseudomonas aeruginosa was determined by the plate dilution method. First, the bacteria were cultured overnight for 13 hours at 37 °C and 220 rpm in LB medium, and the OD 600 = 1.0, and the bacterial solution concentration was diluted to OD 600After dilution by 10,000 times after reaching 0.2 - 0.3, the diluted bacterial solution was obtained. Then, 2 μL of the diluted bacterial solution was dropped onto the plates containing compounds with different concentrations, and cultured in an incubator at 37 °C for 16 hours. The lowest compound concentration at which bacteria did not grow at all on the plate was the minimum inhibitory concentration of the compound.

[0073] Table 2 Minimum inhibitory concentration MIC (μg / mL) of compound Cytoskyrin D against bacteria

[0074]

[0075] As shown in Table 2, the minimum inhibitory concentrations of compound Cytoskyrin D against Gram-positive bacteria Methicillin-resistant Staphylococcus aureus and Staphylococcus aureus were both 0.25 μg / mL, the minimum inhibitory concentration against Methicillin-sensitive Staphylococcus aureus was 0.5 μg / mL, and the minimum inhibitory concentration against Gram-negative bacteria Escherichia coli was 32 μg / mL, all better than the positive drug Ampicillin.

[0076] Cytotoxic activity screening

[0077] The cytotoxicity of compound Cytoskyrin D against non-small cell lung cancer cell line (A549), breast cancer cell line (SKBR-3) and human normal colon cells (NCM-460) was evaluated by CCK8 method, with Mitomycin as the positive drug control. A single cell suspension was prepared with a culture medium (DMEM) containing 10% fetal bovine serum, and inoculated into a 96-well plate at 5000 - 10000 cells per well, with a volume of 100 μL per well. Adherent cells were inoculated and cultured 12 h in advance; the test compound solution was added, and screening was carried out at a compound concentration of 50 μM, and 3 replicates were set for each treatment; after incubation at 37 °C for 48 h, the supernatant in the 96-well plate was aspirated, 20 μL of CCK8 solution and 100 μL of culture medium were added to each well, and incubation was continued for 4 h to make the reaction proceed fully; at a wavelength of 490 nm, the optical absorbance values of each well were read with an enzyme-linked immunosorbent detector (Bio-Rad 680), and the results were recorded to obtain the survival rates of the 3 cell lines at a compound concentration of 50 μM.

[0078] Table 3 Cell survival rates of Cytoskyrin D at a concentration of 50 μM

[0079]

[0080] At a concentration of 50 μM, the cell survival rates of compound Cytoskyrin D all exceeded 50%, indicating that the cytotoxic activity of this compound was very poor.

[0081] The DMEM medium was purchased from Thermo Fisher Scientific (gibico).

[0082] The above-described embodiments merely represent specific implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention.

Claims

1. An anthraquinone dimer compound, characterized in that: The structure is as follows:

2. The method for extracting anthraquinone dimer compounds according to claim 1, characterized in that: The following steps are involved: A. Fermentation: fermenting the fungus Diaporthe sp. CB10100 in rice or PDB medium to obtain a fermented product for later use; B. Separation: The fermented product was extracted three times with ethyl acetate and then concentrated to obtain a crude extract. The aqueous suspension of the crude extract was extracted with petroleum ether, ethyl acetate and n-butanol, respectively, to obtain a corresponding concentrate. The ethyl acetate portion was separated by normal phase silica gel column chromatography, ODS column chromatography gradient elution and high performance liquid chromatography to obtain compound Cytoskyrin D.

3. The method for extracting anthraquinone dimer compounds according to claim 2, characterized in that: The fungus Diaporthe sp. CB10100 is first cultured and then fermented.

4. The method for extracting anthraquinone dimer compounds according to claim 3, characterized in that: Before fermentation, the fungus Diaporthe sp.CB10100 was cultured in a 1000 mL flat-bottom conical flask containing 200 mL of PBD solution at 30°C and 200 rpm with shaking for 3 days to obtain a fungal seed fermentation liquid, which was inoculated with a volume of 10% of the volume of the large fermentation medium.

5. The method for extracting anthraquinone dimer compounds according to claim 3, characterized in that: Each liter of culture medium for fermentation contained: 200 mL of a solution of 35 g PDB / liter, or 200 g rice and 200 mL water in a 1000 mL conical flask, which was sterilized for large-scale fermentation.

6. The method for extracting anthraquinone dimer compounds according to claim 2, characterized in that: In the step B, the ethyl acetate portion is subjected to normal phase silica gel column chromatography with petroleum ether / ethyl acetate as the mobile phase to collect 9 fractions Fr.1-9, and the fraction Fr.2 is subjected to gradient elution by methanol / water ODS column chromatography and then separated by high performance liquid chromatography to obtain compound Cytoskyrin D.

7. The method for extracting anthraquinone dimer compounds according to claim 6, characterized in that: The volume ratio of petroleum ether / ethyl acetate gradient elution was changed to 20:1, 10:1, 5:1, 1:1, 1:5, and 1:20; The volume ratio of methanol / water ODS gradient elution varied as follows: 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8; The chromatographic conditions of HPLC were: Chromatographic column: Yuexu AQ-C18, specification: 250×10mm, 5μm Mobile phase: acetonitrile (A): 0.2% formic acid water (B); gradient elution was used, the mobile phase ratio A:B increased from 10:90 to 75:25 during 0-11 min, the mobile phase ratio was maintained at 75:25 during 11-12 min, the mobile phase ratio was changed from 75:25 to 10:90 during 12-19 min, and the mobile phase ratio was maintained at 10:90 during 19-22 min; Flow rate: 3 mL / min; Column temperature: 35°C.

8. Use of the anthraquinone dimer compound according to claim 1 in the preparation of antibacterial drugs.

9. Use of the anthraquinone dimer compound according to claim 8 in the preparation of antibacterial drugs, characterized in that: The inhibited bacteria included methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, and methicillin-sensitive Staphylococcus aureus.

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