An anthraquinone compound, a preparation method thereof and application thereof in anti-mrsa

Abstract

The application discloses an anthraquinone compound, a preparation method of the anthraquinone compound and application of the anthraquinone compound in resisting MRSA, and the anthraquinone compound is obtained from fermentation products of Aspergillus carneus GXIMD00519 which is derived from Beibuwan coral. The active screening result shows that the anthraquinone compound 1 can significantly inhibit MRSA, and the minimum inhibitory concentration is 32 mu g / mL. The scanning electron microscope result shows that the anthraquinone compound 1 can cause the surface of MRSA to shrink and deform, the surface to dissolve, and the integrity of the bacteria to be destroyed, and the anthraquinone compound 1 has the potential to be developed into an anti-MRSA drug.

Description

Technical Field

[0001] This invention relates to the field of chemical technology, and in particular to an anthraquinone compound, its preparation method, and its application in anti-MRSA. Background Technology

[0002] Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important pathogens causing nosocomial infections worldwide. Mild cases can cause minor skin and soft tissue infections, while severe infections can lead to life-threatening endocarditis, chronic osteomyelitis, pneumonia, and bacteremia. MRSA is resistant to almost all penicillin antibiotics. Some international scholars have grouped MRSA infection, hepatitis B, and AIDS with the three most serious infectious diseases currently prevalent. Studies in the United States showed that in 2000-2001, patients infected with MRSA had an average hospital stay three times longer and a mortality rate five times higher than other hospitalized patients. Currently, vancomycin, linezolid, daptomycin, and tigecycline are commonly used antibiotics to treat MRSA. However, the use of these drugs has not significantly reduced the infection rate and mortality of MRSA; on the contrary, the selective pressure of antibiotics has led to more widespread drug resistance in the pathogen. There have been reports of vancomycin exhibiting a minimum inhibitory concentration (MIC) drift against MRSA, resulting in vancomycin-sensitive Staphylococcus aureus. Therefore, there is an urgent need to develop new antibiotics. Summary of the Invention

[0003] The purpose of this invention is to address the problem that MRSA in the prior art easily leads to resistance to existing antibiotics, and to provide an anthraquinone compound.

[0004] Another object of the present invention is to provide a method for preparing the anthraquinone compounds.

[0005] Another object of the present invention is to provide the use of the anthraquinone compounds in the preparation of anti-MRSA drugs.

[0006] The technical solution adopted to achieve the purpose of this invention is:

[0007] An anthraquinone compound, the structural formula of which is shown below:

[0008]

[0009] In the above technical solution, it was isolated from the fermentation product of Aspergillus carneus GXIMD00519, which is derived from corals in the Beibu Gulf.

[0010] In the above technical solution, the anthraquinone compounds are prepared through the following steps: Activation of *Aspergillus carneus* GXIMD00519 (from corals in the Beibu Gulf), followed by culture to obtain fermentation products; extraction of the fermentation products with an organic solvent to obtain an extract; concentration of the extract under reduced pressure to obtain a fermentation extract; mixing the fermentation extract with normal-phase silica gel using a dry method; loading the extract into a normal-phase silica gel column at a predetermined weight ratio; eluting the column with the mobile phase under ambient temperature and pressure; concentration of the eluent under reduced pressure; detection and merging of similar components by thin-layer chromatography to obtain 12 components F1 to F12; separation of component F9 by reversed-phase silica gel column chromatography to obtain 21 sub-components SF9-1 to SF9-21; and separation of SF9-11 by high-performance liquid chromatography to obtain the anthraquinone compounds.

[0011] In the above technical solution, Aspergillus carneus GXIMD00519, derived from corals in the Beibu Gulf, is activated on a plate culture medium. The activated mycelium is picked up with a sterile bamboo stick and placed into the culture medium. The culture is then statically incubated at a constant temperature of 22–28℃ for 25–35 days to obtain the fermentation product.

[0012] In the above technical solution, 80 grams of rice, 0.3-0.5 grams of yeast extract, 0.3-0.5 grams of glucose, 3-4 grams of sea salt, and 80-140 ml of water are mixed and placed in a container, and sterilized in an autoclave at 121°C for 20-30 minutes to obtain the culture medium.

[0013] In the above technical solution, the organic solvent is methanol, ethanol, dichloromethane, chloroform or ethyl acetate. The normal-phase silica gel used for dry mixing is 100-200 mesh. During dry mixing, the mass ratio of the fermentation extract to the normal-phase silica gel is 1:(1-2). The normal-phase silica gel used in the normal-phase silica gel chromatography column is 200-300 mesh. The mass ratio of the total mass of the dry mixing to the mass of the silica gel in the normal-phase silica gel chromatography column is 1:(5-30). When eluting the chromatography column, dichloromethane-methanol in ratios of 100:0, 90:10, and 80:20 are used as the mobile phase for elution.

[0014] In the above technical solution, the elution system used for reversed-phase silica column chromatography is acetonitrile-water at a ratio of 30:70 to 60:40.

[0015] In the above technical solution, the detection wavelength of high performance liquid chromatography is 254 nm, the flow rate is 3 mL / min, the chromatographic column is YMC 250×10 mm, 5 μm, the mobile phase is acetonitrile-water at a ratio of 45:55, and the elution time is 28.1 min.

[0016] In another aspect of the present invention, the anthraquinone compounds are used in the preparation of anti-MRSA drugs.

[0017] Another aspect of the present invention provides a method for preparing anthraquinone compounds, comprising the following steps: activating *Aspergillus carneus* GXIMD00519 (from Beibu Gulf coral) on a plate culture medium; picking the activated mycelium into the culture medium with a sterile bamboo stick; statically culturing at a constant temperature of 22-28℃ for 25-35 days to obtain a fermentation product; extracting the fermentation product with methanol, ethanol, dichloromethane, chloroform, or ethyl acetate to obtain an extract; concentrating the extract under reduced pressure to obtain a fermentation extract; dry mixing the fermentation extract with 100-200 mesh normal-phase silica gel; loading the extract into a normal-phase silica gel chromatography column at a weight ratio of 1:(5-30), wherein the normal-phase silica gel used in the column is 200-300 mesh; and sequentially mixing with 100:0. The elution column was a dichloromethane-methanol column with a ratio of 90:10 and 80:20. After the eluent was concentrated under reduced pressure, similar components were detected by thin-layer chromatography, yielding 12 components F1 to F12. Component F9 was separated by reversed-phase silica gel column chromatography with an acetonitrile-water elution system of 30:70 to 60:40, yielding 21 sub-components SF9-1 to SF9-21. SF9-11 was separated by high-performance liquid chromatography (HPLC) to obtain the anthraquinone compounds. The HPLC detection wavelength was 254 nm, the flow rate was 3 mL / min, the column was a YMC 250 × 10 mm, 5 μm, and the mobile phase was acetonitrile-water (45:55). The elution time was 28.1 min. The structural formula of the anthraquinone compounds is:

[0018]

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] 1. The anthraquinone compounds of the present invention have low production costs and simple production processes. They are found in the fermentation products of Aspergillus carneus GXIMD00519, which is derived from corals in the Beibu Gulf. They are easy to isolate and obtain.

[0021] 2. The anthraquinone compounds of the present invention have anti-MRSA activity. Attached Figure Description

[0022] Figure 1 This is a diagram showing the main NMR correlation signals of the novel anthraquinone compounds of this invention;

[0023] Figure 2 The 1H NMR spectrum (600MHz, DMSO-d6) of the novel anthraquinone compounds of this invention is shown.

[0024] Figure 3The carbon spectrum (150 MHz, DMSO-d6) of the novel anthraquinone compound of the present invention is shown.

[0025] Figure 4 The HSQC spectrum (DMSO-d6) of the novel anthraquinone compound of the present invention is shown.

[0026] Figure 5 The HMBC spectrum (DMSO-d6) of the novel anthraquinone compound of the present invention is shown.

[0027] Figure 6 The anthraquinone compounds of the present invention 1 H- 1 H COSY spectrum (DMSO-d6);

[0028] Figure 7 The NOESY spectrum (DMSO-d6) of the novel anthraquinone compounds of this invention is shown.

[0029] Figure 8 The HR-ESI-MS spectrum of the novel anthraquinone compounds of this invention;

[0030] Figure 9 Comparison of the calculated ECD spectrum (gray discontinuous line) of (1'S,4'S,5'R)-1 with the measured CD (black solid line) of the anthraquinone novel compound of the present invention (UV correction value: 25nm);

[0031] Figure 10 This is a comparison image of the MRSA scanning electron microscope morphology (right) of the anthraquinone compounds of the present invention after treatment and that of the untreated compounds (left).

[0032] The strain Aspergillus carneus GXIMD00519, derived from corals in the Beibu Gulf, was deposited on March 23, 2022, at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No:62314. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0034] Example 1

[0035] A method for preparing an anthraquinone compound includes the following steps:

[0036] Place 80 grams of rice, 0.4 grams of yeast extract, 0.4 grams of glucose, 3.6 grams of sea salt, and 120 ml of water into a 1-liter Erlenmeyer flask and sterilize in an autoclave at 121°C for 20 minutes to prepare the culture medium.

[0037] Aspergillus carneus GXIMD00519 (strain deposited at Guangdong Provincial Microbial Culture Collection Center on March 23, 2022, accession number: GDMCC No: 62314) from corals in the Beibu Gulf was activated on a suitable agar plate. The activated mycelia were picked up with a sterile bamboo stick and transferred to approximately 100 bottles of the prepared culture medium. The mixture was incubated statically at 25°C for 30 days to obtain the fermentation product. The fermentation product was extracted three times with ethyl acetate, and the extract was concentrated under reduced pressure to obtain the fermentation extract (the fermentation product can also be extracted with methanol, ethanol, dichloromethane, or chloroform).

[0038] The fermentation extract was dry-mixed with normal-phase silica gel (100-200 mesh) and then packed into a normal-phase silica gel column (200-300 mesh) at a weight ratio of 1:20. Under ambient temperature and pressure, the column was eluted sequentially with dichloromethane-methanol at ratios of 100:0, 90:10, and 80:20. After concentration under reduced pressure, similar components were analyzed by thin-layer chromatography, yielding 12 fractions (F1–F12). Fraction F9 was separated by reversed-phase silica gel column chromatography. The elution system was acetonitrile-water 30%–60%, yielding 21 subfractions (SF9-1–SF9-21). SF9-11 was prepared by high performance liquid chromatography (detection wavelength 254 nm, flow rate 3 mL / min, column YMC 250×10 mm, 5 μm, mobile phase 45:55 acetonitrile-water), yielding an anthraquinone compound 1 (53 mg) with a peak time of 28.1 min.

[0039] Example 2

[0040] The anthraquinone compounds obtained in Example 1 were analyzed.

[0041] Anthraquinone compound 1 is a reddish-brown powder. High-resolution mass spectrometry at 383.0760 [MH] was used to analyze it. - The NMR data confirmed that the molecular formula of compound 1 is C16. 20 H 16 O8 (calculated value 383.0767). The NMR data and related signals of anthraquinone compound 1 are shown in Table 1.

[0042] Table 1: NMR data and related signals of anthraquinone compound 1

[0043]

[0044]

[0045] 1 (125MHz, DMSO-d6)

[0046] 2 (600MHz, DMSO-d6)

[0047] NMR data indicate that anthraquinone compound 1 contains one methyl group, two methylene groups, five methine groups (including three methine groups on the benzene ring and two methine groups with oxygen ions), and twelve quaternary carbons (including two carbonyl quaternary carbons, nine quaternary carbons on the benzene ring, and one quaternary carbon with an oxygen ion).

[0048] NMR data and UV absorption UV(MeOH)λ max The NMR values ​​(logε) 224(3.11), 239(2.66), 294(3.03), 322(2.57), 352(2.13), 441(2.61) nm indicate that compound 1 is an anthraquinone compound. Literature review revealed that the NMR data of anthraquinone compound 1 is extremely similar to those of compound averufin (Yamazaki M, et al., Chemical and Pharmaceutical Bulletin, 1988, 36, 670-675). The most significant difference lies in the E ring of the compound. 1 H- 1 The H COSY spectrum showed correlations between H-3' and H-4' and H-2', while the HMBC spectrum showed correlations between H-4' and C-2', 3', and 5', and CH3-6' and C-4', 5', indicating an additional hydroxyl group at C-4' compared to compound averufin. The relative configuration of anthraquinone compound 1 was determined using the NOESY spectrum. NOESY correlation signals were present at H-1', H-4', and CH3-6', indicating they are on the same side of the E ring. The absolute configuration of anthraquinone compound 1 was calculated using Gaussian16 software. By comparing with the measured CD spectrum, the absolute configuration of the compound was determined to be (1'S, 4'S, 5'S), as shown in the following structural formula:

[0049]

[0050] Example 3

[0051] Activity screening of anthraquinone compound 1 showed that it significantly inhibited MRSA, with a minimum inhibitory concentration (MIC) of 32 μg / mL. Scanning electron microscopy results indicated that anthraquinone compound 1 caused MRSA cell surface shrinkage and dissolution, disrupting bacterial integrity and demonstrating its potential for research and development as an anti-MRSA drug.

[0052] Minimum inhibitory concentration (MIC) against MRSA 90 Assay method: The MIC of the test sample was determined using the 2:1 dilution method. Indicator bacteria MRSA, activated on LBA medium, were inoculated onto LB medium and cultured with shaking (30℃, 180 rpm) until the logarithmic growth phase. The bacterial suspension concentration was then adjusted to 10⁻⁶. 6 CFU / mL. Pipettes 195 μL of bacterial suspension into a 96-well plate, then add 5 μL of sample solution (anthraquinone compound 1 solution) at gradient concentrations to final concentrations of 64, 32, 16, 8, and 4 μg / mL. After mixing and sealing, incubate at 30°C for 24 h. Bacterial growth is then measured using a microplate reader at 600 nm. Penicillin G is used as a positive control; negative controls and blank controls are also included, with three replicates for each sample. The antibacterial activity of the samples is calculated as: inhibition rate = (blank value - sample value) / blank value × 100%. MIC 90 The minimum inhibitory concentration (MIC) for achieving an inhibition rate of 90% has been verified to be 32 μg / mL.

[0053] MRSA scanning electron microscopy experimental method: The concentration of MRSA bacterial suspension was adjusted to 10⁶ CFU / mL, and a treatment group (anthraquinone compound 1 concentration of 4 MIC) and a control group (DMSO) were set up. After incubation at 37℃ and shaking at 180 rpm for 24 h, sample preparation was performed. The sample was washed three times with PBS (pH 7.0), fixed in PBS buffer with 2.5% glutaraldehyde for about 2 h, and then washed three times with PBS. Gradual dehydration was performed using different concentrations (30%, 40%, 50%, 60%, 70%, 80%, 90%, and anhydrous ethanol) for 15 minutes each time. Tert-butanol was used to replace anhydrous ethanol. After freeze-drying, gold was sputtered onto the cells, and the micromorphology at 1 μm, 400 nm, and 200 nm was observed using a scanning electron microscope (Zeiss Sigma 300). The micromorphology at 400 nm was the most obvious. Anthraquinone compound 1 caused the surface of MRSA bacteria to shrink and deform, and the surface dissolved, thus destroying the integrity of the bacteria. It has the potential to be studied and developed into an anti-MRSA drug.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing an anthraquinone compound, characterized by, The anthraquinone compound has a structural formula as shown in the following formula: The preparation method comprises the following steps: activating the Aspergillus carneus GXIMD00519 from the Beibu Gulf corals The activated GXIMD00519 is transferred to a culture medium for culture to obtain a fermentation product, the fermentation product is extracted with an organic solvent to obtain an extract, the extract is concentrated under reduced pressure to obtain a fermentation extract, the fermentation extract is dry-mixed with normal-phase silica gel, the normal-phase silica gel is filled into a normal-phase silica gel chromatographic column according to a predetermined weight ratio, the chromatographic column is eluted under normal temperature and pressure by using a mobile phase, the eluate is concentrated under reduced pressure, and then thin-layer chromatography is used to detect and combine similar components, so that 12 components F1-F12 are obtained, the component F9 is separated by reverse-phase silica gel column chromatography to obtain 21 sub-components SF9-1-SF9-21, and the sub-component SF9-11 is separated by high-performance liquid chromatography to obtain the anthraquinone compound.

2. The method for preparing anthraquinone compounds as described in claim 1, characterized in that, The Aspergillus carneus GXIMD00519 from the Beibu Gulf corals is activated on a plate culture medium, and the activated mycelium is picked up with a sterile bamboo stick and placed in a culture medium, and then the culture medium is cultured at a constant temperature of 22-28 DEG C for 25-35 days to obtain a fermentation product.

3. The method for preparing anthraquinone compounds as described in claim 1, characterized in that, 80-140ml of water are mixed in a container, and then the mixture is sterilized in a high-pressure sterilization pot at 121 DEG C for 20-30 minutes to obtain the culture medium.

4. The method for preparing anthraquinone compounds as described in claim 1, characterized in that, The organic solvent is methanol, ethanol, dichloromethane, chloroform or ethyl acetate, the normal-phase silica gel used for dry mixing is 100-200 mesh, the mass ratio of the fermentation extract to the normal-phase silica gel is 1:(1-2) during dry mixing, the normal-phase silica gel used in the normal-phase silica gel chromatographic column is 200-300 mesh, and the mass ratio of the total mass of the dry-mixed normal-phase silica gel to the normal-phase silica gel in the chromatographic column is 1:(5-30), and the mobile phase used for eluting the chromatographic column is dichloromethane-methanol with a proportion of 100:0, 90:10 and 80:20 in sequence.

5. The method for preparing anthraquinone compounds as described in claim 1, characterized in that, The elution system used in the reverse-phase silica gel column chromatography is acetonitrile-water with a proportion of 30:70-60:

40.

6. The method for preparing anthraquinone compounds as described in claim 1, characterized in that, The detection wavelength of the high-performance liquid chromatography is 254nm, the flow rate is 3ml / min, the chromatographic column is YMC 250x10mm, 5um, the mobile phase is acetonitrile-water with a proportion of 45:55, and the peak time is 28.1min.

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