A glycosylated anthraquinone compound and its preparation method and use

Abstract

The invention discloses a glycosylated anthraquinone compound and a preparation method and use thereof. The compound is characterized in that the structural formula is shown in I. The preparation method comprises the steps of obtaining anthraquinone terpene fermentation product by fermenting and culturing actinomycetes with a preservation number of CCTCC NO: M2020953, then soaking the fermentation product in ethyl acetate to extract a crude extract, and then subjecting the crude extract to normal phase silica gel column chromatography, reverse medium pressure column chromatography and reverse phase semi-preparative high performance liquid chromatography separation and purification to obtain the anthraquinone compound. The anthraquinone compound has the use of Staphylococcus aureus and Escherichia coli inhibitors, and has the advantage that the anthraquinone compound has the effects of resisting Staphylococcus aureus, Escherichia coli and agricultural pathogenic fungi, and can be used to develop drugs for preventing and treating diseases caused by Staphylococcus aureus and Escherichia coli and drugs for fruit and vegetable diseases caused by agricultural pathogenic fungi.

Description

Technical Field

[0001] The invention relates to a glycosylated anthraquinone compound, in particular to a glycosylated anthraquinone compound extracted from marine actinomycetes and a preparation method and application thereof. Background Art

[0002] Staphylococcus aureus and Escherichia coli are the most common human bacterial pathogens, which can cause food spoilage and superficial infections in the human intestine, as well as systemic infections, seriously affecting the quality of life of patients and even endangering their lives. Bacterial infections mainly caused by Staphylococcus aureus and Escherichia coli are an increasingly serious problem in my country's clinical practice. They are opportunistic infections with the highest incidence in immunocompromised patients and infants. In the bodies of infected people, Staphylococcus aureus and Escherichia coli mostly exist in the form of biofilms. The formation of biofilms can not only enhance their drug resistance, but also protect them from attacks by the host immune system. Existing antibacterial drugs have a single mechanism of action, and long-term use causes a surge in drug resistance. The clinical demand for multi-channel treatment of Staphylococcus aureus and Escherichia coli is increasing.

[0003] Fruits and vegetables are rich in nutrients such as vitamins, trace elements and antioxidants, and are an important part of the human diet. However, most fruits and vegetables have a high water content and are rich in nutrients. They are easily infected by pathogenic fungi and rot during storage and processing. At the same time, fungi also accumulate a large amount of mycotoxins in the rotten parts of fruits and vegetables and the surrounding healthy tissues. Many mycotoxins are stable in nature during the storage and processing of fruits and vegetables after harvest. It is difficult to completely remove them under conventional storage and processing conditions, which will pose a potential threat to human and animal health. At present, the main types of mycotoxins detected in fresh fruits and vegetables and their products are aflatoxins, trichothecenes, ochratoxins, fumonisins, penicillins, and alternaria toxins. Not only does it cause serious losses to the global economy, but it is also highly toxic and can cause metabolic disorders, nerve paralysis, organ failure, fertility, carcinogenesis, and immune damage, making it a food safety and public health issue of global concern. Therefore, finding efficient, safe and easy-to-operate prevention and control technologies to reduce fungal toxin contamination in fruit and vegetable products and their products is of great significance to the safe production, circulation and people's dietary health of fresh fruits and vegetables and their products. It is urgent to research and develop new, safe and more effective biological preservatives to inhibit fungal contamination.

[0004] Anthraquinone is a class of naturally occurring molecules, widely found in actinomycetes, fungi, seaweed, sponges and mollusks. The skeleton type and number of anthraquinone are relatively large among terpenoid compounds, which are mainly manifested in effective antibacterial, anticancer and anti-inflammatory activities, and are important raw materials in the direction of medicine. The inventors discovered a new anthraquinone natural product in a chemical investigation of the ethyl acetate extract of marine actinomycete Steptomyces sp.4-7 fermented in a culture medium. At present, there is no report on the chemical structure and antibacterial activity of the compound, so there is no related drug on the market. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a glycosylated anthraquinone compound having an inhibitory effect on Staphylococcus aureus, Escherichia coli and agricultural pathogenic fungi, and a preparation method and use thereof.

[0006] The technical solution adopted by the present invention to solve the above technical problems is:

[0007] 1. A glycosylated anthraquinone compound, the structural formula of which is shown in (I);

[0008]

[0009] 2. The preparation method of the above-mentioned glycosylated anthraquinone compound comprises the following steps:

[0010] (1) Fermentation production

[0011] The marine actinomycetes (Steptomyces sp. 4-7) with the accession number of CCTCC NO: M2020953 were streaked on a plate of Gao's solid medium No. 1, incubated upside down in an incubator at 28°C for 5 days, and then a single colony was picked and inoculated into liquid medium A, and incubated on a shaker at 28°C and 220 rpm / min for 11 days, and then the fermentation liquid was collected;

[0012] (2) Extraction

[0013] Adding an equal volume of ethyl acetate to the fermented product obtained in step (1), extracting repeatedly for 3 times, and then vacuum evaporating the ethyl acetate extract to obtain a crude extract;

[0014] (3) Isolation and preparation of compounds

[0015] The crude extract obtained in step (2) is first dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and then 200-300 mesh silica gel is added to mix the sample, and normal phase medium pressure column chromatography is performed, and gradient elution is performed using a petroleum ether-ethyl acetate solution in a volume ratio of 3:7 as an eluent, and the eluate is collected; the collected eluate is subjected to reverse phase medium pressure column chromatography, and linear gradient elution is performed using methanol-water with a methanol volume percentage of 30-100% as an eluent, and the eluted fractions are collected, and the fractions are arranged from large to small according to polarity, and 20 components are obtained by merging; the 13th component obtained is separated and purified by semi-preparative reverse phase high performance liquid chromatography using a mixed solution composed of acetonitrile and water in a volume ratio of 45:55 as a mobile phase to obtain a compound, whose structure is shown in (I):

[0016]

[0017] Furthermore, the Gao's No. 1 solid culture medium described in step (1) is prepared as follows: 20 g of soluble starch, 1 g of KNO3, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.01 g of FeSO4·7H2O and 20 g of agar, 1000 mL of distilled water, and the pH is adjusted to 7.4-7.6.

[0018] Furthermore, the preparation method of the liquid culture medium A described in step (1) is as follows: 20g starch, 2g yeast extract, 6g peptone, 1g calcium carbonate, 100mg potassium bromide, 40mg ferrous sulfate tetrahydrate and 30g sea salt are dissolved in 1000mL water.

[0019] Furthermore, in the linear gradient elution of the reversed-phase medium-pressure column chromatography in step (3), the volume percentage of methanol ranges from 30 to 100%, and the elution time is 120 min.

[0020] Furthermore, the flow rate of the compound separation preparation by semi-preparative reverse-phase HPLC described in step (3) is 2.0 mL / min.

[0021] 3. Use of the above-mentioned glycosylated anthraquinone compounds in the preparation of Staphylococcus aureus and / or Escherichia coli inhibitors.

[0022] 4. Use of the above-mentioned glycosylated anthraquinone compounds in the preparation of agricultural pathogenic fungi activity inhibitors.

[0023] Compared with the prior art, the advantages of the present invention are as follows: the present invention provides a glycosylated anthraquinone compound and a preparation method and use thereof, wherein a fermentation product is obtained by microbial fermentation culture, and then the fermentation product is soaked and extracted with ethyl acetate to obtain a crude extract, and then the crude extract is separated and purified by medium-pressure forward column chromatography, medium-pressure reverse column chromatography, and semi-preparative high-performance liquid chromatography, and the compound has significant anti-Staphylococcus aureus, anti-Escherichia coli and anti-agricultural fungi activities, and can be used for the development of drugs for inhibiting related diseases caused by Staphylococcus aureus, Escherichia coli and agricultural pathogenic fungi.

[0024] The above-mentioned marine actinomycetes (Steptomyces sp.4-7), with the deposit number CCTCC NO: M2020953, were deposited in the China Center for Type Culture Collection on December 21, 2020, and the deposit address is Wuhan University, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the compound of the present invention;

[0026] Figure 2 The carbon NMR spectrum of the compound of the present invention is

[0027] Figure 3 is the nuclear magnetic resonance DEPT-135 spectrum of the compound of the present invention;

[0028] Figure 4 is the COSY nuclear magnetic resonance spectrum of the compound of the present invention;

[0029] Figure 5 is the nuclear magnetic resonance HSQC spectrum of the compound of the present invention;

[0030] Figure 6 is the nuclear magnetic resonance HSQC spectrum of the compound of the present invention;

[0031] Figure 7 It is the nuclear magnetic resonance NOESY spectrum of the compound of the present invention. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below with reference to the accompanying drawings.

[0033] Example 1

[0034] The structural formula of a glycosylated anthraquinone compound is shown in (I):

[0035]

[0036] Example 2

[0037] The method for preparing the glycosylated anthraquinone compound shown in structural formula (I) of Example 1 specifically comprises the following steps:

[0038] (1) Fermentation production

[0039] The marine actinomycetes (Steptomyces sp.4-7) with the accession number of CCTCC NO: M2020953 were cultured in Gao's solid medium No. 1 (prepared as follows: 20 g soluble starch, 1 g KNO3, 0.5 g K2HPO4, 0.5 g MgSO4·7H2O, 0.5 g NaCl, 0.5 g FeSO4·7H2O 0.01g of the culture medium was added with 20g of agar and 1000mL of distilled water, and the pH value was adjusted to 7.4-7.6 (0.01g). After inverting and culturing in an incubator at 28°C for 5 days, a single colony was picked and inoculated into liquid culture medium A. After culturing on a shaker at 28°C and a speed of 220rpm / min for 11 days, the fermentation broth was collected. The preparation method of liquid culture medium A is as follows: 20g of starch, 2g of yeast extract, 6g of peptone, 1g of calcium carbonate, 100mg of potassium bromide, 40mg of ferrous sulfate tetrahydrate, and 30g of sea salt were dissolved in 1000mL of water;

[0040] (2) Extraction

[0041] Adding an equal volume of ethyl acetate to the fermented product obtained in step (1), extracting repeatedly for 3 times, and then vacuum evaporating the ethyl acetate extract to obtain a crude extract;

[0042] (3) Isolation and preparation of compounds

[0043] The crude extract obtained in step (2) is first dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and then 200-300 mesh silica gel is added to mix the sample, and normal phase medium pressure column chromatography is performed, and gradient elution is performed using a petroleum ether-ethyl acetate solution in a volume ratio of 3:7 as an eluent, and the eluent is collected; the collected eluent is subjected to reverse phase medium pressure column chromatography, and linear gradient elution is performed using methanol-water with a methanol volume percentage of 30-100% as an eluent, and the elution time is 120min, and the eluted fractions are collected, arranged in descending order according to the polarity of the fractions, and 20 components are obtained by merging; the 13th component obtained is separated and purified by semi-preparative reverse phase high performance liquid chromatography using a mixed solution composed of acetonitrile and water in a volume ratio of 45:55 as a mobile phase at a flow rate of 2.0mL / min to obtain a compound, whose structure is shown in (I):

[0044]

[0045] Example 3

[0046] Compound I of the present invention is a yellow solid powder. The cation HR-ESI-MS gives a quasi-molecular ion peak m / z 817.3483 [M+Na] + , its molecular formula is determined to be C 43 H 54 O 14 (The calculated molecular weight is 817.3514), indicating that its unsaturation is 17. 1 H and 13 C NMR spectra and 2D data are shown in Figure 1-7 and Table 1: Figure 3-7 is the main two-dimensional correlation of the compounds of the present invention; Figure 2 and Figure 3 It can be seen that the compound contains 43 carbon atoms, of which 12 are quaternary carbon atoms (C-1, C-3, C-4a, C-6a, C-7, C-7a, C-8, C-9, C-11a, C-12, C-12a, C-12b), 9 are methylene atoms (C-2, C-4, C-2A, C-2B, C-3B, C-2C, C-3C, C-2D, C-3D), and 17 are methine atoms (C-5, C-6, C-10, C-11, C-1A, C-3A, C-4A, C-5A, C-1B, C-4B, C-5B, C-1C, C-4C, C-5C, C-1D, C-4D, C-5D). Figure 1 The 1H NMR high field signal analysis revealed five methyl carbon signals (C-13, C-6A, C-6B, C-6C, C-6D); Figure 4 The COSY spectrum shows that H-5 and H-6, H-10 and H-11 are aromatic hydrogen protons with coupling; Figure 5 The HSQC spectrum shows that the four aromatic hydrogen protons correspond to the four aromatic carbon atoms C-5, C-6, C-10, and C-11. Figure 2 The three carbon spectrum low-field chemical shifts C-1, C-7, and C-12 show that the compound has a carbonyl structure, and the presence of two pairs of coupled aromatic hydrogen protons indicates the presence of an anthraquinone skeleton in the compound. By consulting the literature and comparing the NMR spectra of the known compound marangucycline A, it can be found that both have the spectral characteristics of a disaccharide connected by β-D-olive sugar (olivose sugar ring A) and α-L-amino sugar (amicetose sugar ring B). Figure 4 The COSY spectra show that H-1A / H-2A / H-3A / H-4A / H-5A / H-6A, H-1B / H-2B / H-3B / H-4B / H-5B / H-6B have COSY correlation and simultaneously bind Figure 5The HSQC spectrum shows the chemical shift correspondence of the sugar ring. The NOESY correlation of H-10 (δH 7.89) to H-1A (δH 4.90) shows that the connection between sugar ring A and the anthraquinone skeleton is C-9-C-1A. The NOESY correlation of 3A-OH to H-4A shows that sugar ring A is β-D-olive sugar. The HMBC correlation of H-1B to C-3A shows that the connection between sugar ring A and B is the same as that of the known compound marangucycline A. Combined with the 1D and 2D NMR spectra, it can be determined that sugar ring B is α-L-amino sugar. From the NOESY correlation of C-13 to H-1C and the low-field chemical shift of C-13 and C-1C, it can be inferred that the sugar ring C is connected to the C-3 position of the six-membered ring through COC, and from the NOESY correlation and chemical shift of H-1D to C-4C, it can be inferred that the connection between sugar ring D and C is C-4C-OC-1D. The structure of the sugar CD part of the compound was finally determined to be a disaccharide side chain composed of two α-L-rhodinoses connected by C-4C-OC-1D through the comparison of 1D and 2D NMR spectra, and the compound was finally determined to be a new compound.

[0047] Table 1. Compound I 1 H and 13 C NMR data (CDCl3-d6)

[0048]

[0049]

[0050] Note 1: s - singlet, d - doublet, m - multiplet.

[0051] Note 2: 1 H was obtained at 600 MHz NMR; 13 C was obtained at 150 MHz NMR.

[0052] Example 4

[0053] 1. Anti-Staphylococcus aureus and Escherichia coli activity of glycosylated anthraquinone compounds

[0054] (1) Experimental samples

[0055] Preparation of the sample solution: The test sample is the pure compound I separated and purified in Example 1 above. Accurately weigh an appropriate amount of the sample and use DMSO to prepare a solution of the required concentration for testing antibacterial activity. The indicator bacteria used in this experiment are Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922).

[0056] (2) Experimental methods

[0057] 96-well plate antifungal test: Pick single colonies of Staphylococcus aureus and Escherichia coli and inoculate them into LB liquid culture medium, add 4μL 12.8mg / mL sample and 96μL MH culture medium solution to each well. Through gradient dilution, the final concentrations were 128μg / mL, 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, and 0.5μg / mL. DMSO was used as a negative control, and tetracycline at the same concentration was used as a positive control. Bacterial growth was observed after culturing at 28°C for 8 hours, and the entire experiment was repeated 3 times.

[0058] (3) Experimental results

[0059] In the 96-well plate antibacterial test, the MIC of compound I against Staphylococcus aureus and Escherichia coli was determined to be 32 μg / ml.

[0060] 2. Anti-agricultural pathogenic fungi activity of glycosylated anthraquinone compounds

[0061] (1) Experimental samples

[0062] Preparation of the test sample solution: The test sample is the pure compound I separated and purified in Example 1 above. An appropriate amount of the sample is accurately weighed and prepared into a solution of the required concentration with DMSO for testing the antifungal activity. The indicator bacteria used in this experiment are Penicillium expansum ATCC5761, Aspergillus niger ATCC16404 and Botrytis cinereaCGMCC3.3579.

[0063] (2) Experimental methods

[0064] 96-well plate method for antifungal test: Agricultural fungi were inoculated into PDA plates and incubated at 28°C to prepare spore suspensions. The spore density of the test microorganisms was adjusted to 10 5 ~10 6 CFU / mL. The fungal cultures were inoculated into 96-well plates. The compounds and positive controls were dissolved in DMSO and added to the wells of the 96-well culture plates for serial dilution, with the highest and lowest concentrations ranging from 512 to 1 μg / mL. One column was left in each 96-well plate as a negative control, containing DMSO (0.5%), and the positive control was prochloraz. The 96-well plates were incubated at 28°C for approximately 48 hours and then visually analyzed. All tests were performed in triplicate.

[0065] (3) Experimental results

[0066] In the 96-well plate antifungal test, the MICs of compound I against Penicillium expansum (ATCC5761), Aspergillus niger (ATCC16404) and Botrytis cinerea (CGMCC3.3579) were determined to be 12, 4 and 8 μg / ml, respectively.

[0067] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A glycosylated anthraquinone compound, characterized in that The structural formula of the compound is shown in (I); 2. A method for preparing the glycosylated anthraquinone compound according to claim 1, characterized in that The steps include: (1) Fermentation production The marine actinomycetes (Steptomyces sp.4-7) with the accession number of CCTCC NO: M2020953 were streaked on a plate of Gao's solid medium No. 1, and incubated upside down in an incubator at 28°C for 5 days, and then a single colony was picked and inoculated into liquid medium A, and then cultured on a shaker at a temperature of 28°C and a speed of 220 rpm / min for 11 days, and then the fermentation liquid was collected, wherein the preparation method of the liquid medium A is as follows: 20g starch, 2g yeast extract, 6g peptone, 1g calcium carbonate, 100mg potassium bromide, 40mg ferrous sulfate tetrahydrate and 30g sea salt were dissolved in 1000mL water; (2) Extraction Adding an equal volume of ethyl acetate to the fermented product obtained in step (1), extracting repeatedly for 3 times, and then vacuum evaporating the ethyl acetate extract to obtain a crude extract; (3) Isolation and preparation of compounds The crude extract obtained in step (2) is first dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and then 200-300 mesh silica gel is added to mix the sample, and normal phase medium pressure column chromatography is performed, and gradient elution is performed using a petroleum ether-ethyl acetate solution in a volume ratio of 3:7 as an eluent, and the eluate is collected; the collected eluate is subjected to reverse phase medium pressure column chromatography, and linear gradient elution is performed using methanol-water with a methanol volume percentage of 30-100% as an eluent, and the eluted fractions are collected, and the fractions are arranged from large to small according to polarity, and 20 components are obtained by merging; the 13th component obtained is separated and purified by semi-preparative reverse phase high performance liquid chromatography using a mixed solution composed of acetonitrile and water in a volume ratio of 45:55 as a mobile phase to obtain a compound, whose structure is shown in (I):

3. The method for preparing a glycosylated anthraquinone compound according to claim 2, characterized in that The preparation method of Gao's No. 1 solid culture medium described in step (1) is as follows: 20g of soluble starch, 1g of KNO3, 0.5g of K2HPO4, 0.5g of MgSO4·7H2O, 0.5g of NaCl, 0.01g of FeSO4·7H2O and 20g of agar, 1000mL of distilled water, and the pH is adjusted to 7.4-7.

6.

4. The method for preparing a glycosylated anthraquinone compound according to claim 2, characterized in that: In the linear gradient elution of the reverse phase medium pressure column chromatography in step (3), the volume percentage of methanol ranges from 30 to 100%, and the elution time is 120 min.

5. The method for preparing a glycosylated anthraquinone compound according to claim 2, characterized in that: The flow rate of the semi-preparative reversed-phase HPLC compound separation preparation described in step (3) is 2.0 mL / min.

6. Use of the glycosylated anthraquinone compound according to claim 1 in the preparation of Staphylococcus aureus and / or Escherichia coli inhibitors.

7. Use of the glycosylated anthraquinone compound according to claim 1 in the preparation of an agricultural pathogenic fungus activity inhibitor.

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