Separation and purification method of emodin and helminthosporin metabolized by aspergillus cristatus of Fuzhuan tea and application of emodin and helminthosporin as antibacterial agent
By isolating Aspergillus coronatum strains from Fu brick tea, and using combined extraction and chromatography techniques, the antibacterial activity of eoldin and persorin were successfully isolated and purified, solving the problem of difficulty in isolating and purifying Aspergillus coronatum secondary metabolites in the prior art, and achieving its potential application in the food or biomedicine field.
Patent Information
- Application Number
- CN202510088154.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-20
- Publication Date
- 2025-06-13
AI Technical Summary
The prior art is difficult to systematically isolate and purify Aspergillus coronatal secondary metabolites, especially anthraquinone compounds with potential antibacterial activity, resulting in limited application in the food or biomedical field.
By isolating Aspergillus coronatum strain from Fu brick tea, using a combined extraction method of methanol and anhydrous ether, combined with chromatography and gradient elution technology, anthraquinone monomer compounds such as elevated and persorbentine were successfully isolated and purified.
The efficient separation and purification of emodin and streptosporin has been achieved, which significantly improves the identification and application potential of its antibacterial activity, has broad-spectrum antibacterial activity and a low minimum inhibitory concentration and minimum bactericidal concentration.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of separation, purification, development and utilization of natural fungal secondary metabolites, and particularly relates to a method for separating and purifying emodin and catenarin, the main antibacterial active components metabolized by Aspergillus cristatus derived from Fuzhuan tea, antibacterial activity evaluation, and their application as antibacterial agents in the fields of food or biomedicine. Background Art
[0002] The unique "golden flower fungus" in Fuzhuan tea is a golden granular fungus growing on the surface and inside of the tea brick. As a dominant fungus during the fermentation process of Fuzhuan tea, it has been identified as Aspergillus cristatus, which has an important impact on the sensory quality and health care effects of Fuzhuan tea. All along, the quantity and quality of the "golden flower fungus" have been used as important indicators for evaluating the quality of Fuzhuan tea. In recent years, due to the huge potential of fungal secondary metabolites, the natural products derived from fungi have novel structures and good biological activities, which have attracted wide attention. As a safe and non-toxic dominant fungus, people have increasingly paid attention to the development of its own value and functional research.
[0003] Research has found that during the "flowering" process of Fuzhuan tea, a large amount of Aspergillus cristatus grows and produces metabolites that are antagonistic to the growth and reproduction of other strains, so it can effectively prevent the growth of harmful miscellaneous bacteria. The antibacterial activity of the Fuzhuan tea soup is positively correlated with the fermentation time of Aspergillus cristatus, and it is speculated that it is closely related to the production of secondary metabolites of Aspergillus cristatus. Aspergillus cristatus contains a rich biosynthetic gene cluster of secondary metabolites and can produce a large number of secondary metabolites with various biological activities. According to their chemical structures, they can be mainly divided into anthraquinone derivatives, benzaldehyde derivatives, indole derivatives and other compounds. Due to the difficulty in separating and purifying the secondary metabolites of Aspergillus cristatus, it is difficult to systematically explore its secondary metabolites with potential antibacterial activity. Among them, anthraquinone compounds are also widely present in traditional Chinese medicines such as rhubarb, polygonum cuspidatum, cassia seed, and aloe. Anthraquinone monomers such as emodin and alizarin have been widely proven to have broad-spectrum antibacterial activity in vitro. Therefore, anthraquinone compounds may be the potential material basis for the antibacterial activity of the secondary metabolites of Aspergillus cristatus. Establishing a systematic separation and purification method for anthraquinone compounds metabolized by Aspergillus cristatus and screening anthraquinone monomer compounds with main antibacterial activity are of great significance for the development of efficient, safe and broad-spectrum new antibacterial agents, and can be used as food preservatives or antibacterial drugs, and have potential application prospects in the fields of food and biomedicine. Summary of the Invention
[0004] The present invention uses Aspergillus cristatus (MK346334, NCBI) isolated from Fuzhuan tea as the test strain, and develops an extraction, separation and purification method for emodin and desmorphine with antibacterial activity metabolized by it, aiming to prepare and purify anthraquinone monomer compounds with broad-spectrum antibacterial activity from the secondary metabolites of Aspergillus cristatus for use as antibacterial agents in industries such as food or biomedicine.
[0005] The present invention is achieved through the following technical solutions:
[0006] A method for separating and purifying emodin and desmorphine metabolized by Aspergillus cristatus from Fuzhuan tea comprises the following steps:
[0007] (1) Inoculate the Aspergillus cristatus strain isolated from Fuzhuan tea into a PDA medium containing 10% sucrose, culture at 28 °C for 30 days, and then freeze-dry the culture product to constant weight.
[0008] (2) Initially extract the dried culture product in step (1) with methanol. Under the conditions of a solid-liquid ratio of 1:50 (w / v), an extraction temperature of 35 °C, and an extraction time of 3 h, perform ultrasonic extraction 3 times, centrifuge (4000 rpm, 15 min) to discard the culture medium residue, combine the methanol extracts, concentrate under reduced pressure at 35 °C, and freeze-dry to obtain the methanol extract.
[0009] (3) Secondarily extract the methanol extract in step (2) with anhydrous ether. Extract 3 times at a solid-liquid ratio of 1:50 (w / v), mix the ether extract with an equal volume of deionized water in a separating funnel for extraction, let it stand for 3 h, separate the ether layer, and concentrate under reduced pressure at 35 °C to a suitable volume.
[0010] (4) Mix the ether concentrate in step (3) with 10% Na₂CO₃ 3 at a solid-liquid ratio of 1:2 and mix well. Collect the alkaline solution layer, add an appropriate amount of HCl to acidify the alkaline solution to pH = 2, and the anthraquinone components will precipitate. Centrifuge (4000 rpm, 15 min) to discard the supernatant, collect the precipitate, add an appropriate volume of water, and vacuum freeze-dry for 2 days to obtain the anthraquinone extract.
[0011] (5) The anthraquinone extracts in steps (4) and (5) were separated and purified by chromatography using a C18 chromatographic column. A gradient elution mode was adopted, with formic acid water (0.1%, v / v) as mobile phase A and acetonitrile containing 0.1% formic acid as mobile phase B. The gradient elution conditions for the anthraquinone extract F1 were as follows: 0 - 40 min, 35% - 45% phase B; 40 - 80 min, 45% - 95% phase B; 80 - 85 min, isocratic elution with 95% phase B; 85 - 90 min, 95% - 100% phase B. The chromatographic peaks of Fr9 and Fr10 were collected, concentrated under reduced pressure at 35°C, and vacuum freeze-dried to obtain the purified anthraquinone monomers: emodin and chaetocin.
[0012] In the present invention, HPLC-Q-TOF-MS analysis, MALDI-TOF-MS analysis, HPLC qualitative analysis, and NMR analysis were used to identify the structures of the purified emodin and chaetocin.
[0013] Specifically, in the present invention, Escherichia coli (G - ) and Staphylococcus aureus (G + ) were used as the test bacteria to evaluate the antibacterial activity of the purified anthraquinone monomers. The results showed that both emodin and chaetocin had high broad-spectrum antibacterial activity, low MIC and MBC, and significantly inhibited their growth, reproduction, and biofilm formation ability. The emodin and chaetocin metabolized by Aspergillus cristatus have the potential to be used as broad-spectrum antibacterial agents and can be applied in the food or biopharmaceutical fields. Description of the Drawings
[0014] Figure 1 It is the high-performance liquid chromatogram for extracting the anthraquinone compounds metabolized by Aspergillus cristatus in the present invention.
[0015] Figure 2 It is the semi-preparative liquid chromatogram and high-performance liquid chromatogram for separating and purifying emodin and chaetocin in the present invention.
[0016] Figure 3 It is the first-order mass spectrum and second-order mass spectrum of the separated emodin and chaetocin in the positive and negative ion modes in the present invention.
[0017] Figure 4 It is the mass spectrum of the purified emodin and chaetocin in the present invention and the HPLC qualitative analysis of emodin and the standard product.
[0018] Figure 5 It is for the purified chaetocin in the present invention 1 H NMR and 13 C NMR spectra.
[0019] Figure 6Antibacterial activities of purified emodin and monorden of the present invention against Escherichia coli and Staphylococcus aureus.
[0020] Figure 7 Dynamic antibacterial curves of purified emodin and monorden of the present invention against Escherichia coli and Staphylococcus aureus.
[0021] Figure 8 Inhibitory ability of purified emodin and monorden of the present invention against bacterial biofilm formation. Detailed implementation manners
[0022] To better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. The described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0023] Example 1: Extraction method of anthraquinone compounds metabolized by Aspergillus cristatus from Fuzhuan tea, including the following technical steps:
[0024] (1) Inoculate the Aspergillus cristatus strain isolated from Fuzhuan tea into a PDA medium containing 10% sucrose, and culture it at 28 °C for 30 days. Then freeze-dry the culture product to constant weight.
[0025] (2) Initially extract the dried culture product in step (1) with methanol. Under the conditions of a solid-liquid ratio of 1:50 (w / v), an extraction temperature of 35 °C, and an extraction time of 3 h, perform ultrasonic extraction 3 times, centrifuge (4000 rpm, 15 min) to discard the culture medium residue, combine the methanol extracts, and concentrate them under reduced pressure at 35 °C, and then freeze-dry to obtain a methanol extract.
[0026] (3) Perform a second extraction on the methanol extract in step (2) with anhydrous ether. Extract 3 times according to a solid-liquid ratio of 1:50 (w / v). Mix the ether extract with an equal volume of deionized water in a separating funnel for extraction. After standing for 3 h, separate the ether layer and concentrate it under reduced pressure at 35 °C to an appropriate volume.
[0027] (4) Mix the ether concentrate in step (3) with 10% NaCO 3 Mix them thoroughly according to a solid-liquid ratio of 1:2, collect the alkaline solution layer, add an appropriate amount of HCl to acidify the alkaline solution to pH = 2, and the anthraquinone components will precipitate. Centrifuge (4000 rpm, 15 min) to discard the supernatant, collect the precipitate, add an appropriate volume of water, and vacuum freeze-dry for 2 days to obtain an anthraquinone extract.
[0028] Example 2: Separation and purification method of emodin and helminthosporin metabolized by Aspergillus cristatus, comprising the following technical steps:
[0029] (1) Analyze the extracted anthraquinone compounds by high performance liquid chromatography. Chromatographic conditions: Agilent technologies 1200 series high performance liquid chromatograph; Inertsil ODS-3 chromatographic column (4.6 mm × 250 mm, 5 μm); Mobile phase: A: ultrapure water containing 0.1% formic acid; B: acetonitrile containing 0.1% formic acid; Gradient elution conditions: 0 - 35 min, 35% - 95% phase B; 35 - 45 min, 95% phase B; Elution flow rate is 0.8 mL / min; Column temperature 35°C; Injection volume: 20 μL; PDA detector: 280 nm and 440 nm. As shown in the appendix Figure 1 , the secondary metabolites of Aspergillus cristatus are well separated (compounds 1 - 23); By using the extraction method of anthraquinone compounds in Example 1, most of the impurity peaks can be removed, so as to obtain the main anthraquinone extract (containing anthraquinone monomer compounds 3, 4, 6, 7, 9, 10, 13, 14, 15, 16).
[0030] (2) Separate and purify the anthraquinone extract by chromatography in combination with a C18 chromatographic column. Use a gradient elution mode, and use formic acid water (0.1%, v / v) as mobile phase A and acetonitrile containing 0.1% formic acid as mobile phase B. The gradient elution conditions are: 0 - 40 min, 35% - 45% phase B; 40 - 80 min, 45% - 95% phase B; 80 - 85 min, 95% isocratic elution of phase B; 85 - 90 min, 95% - 100% phase B; Set the elution flow rate to 6 mL / min, the signal collection wavelength to 440 nm, collect the chromatographic peaks of Fr9 and Fr10, concentrate under reduced pressure at 35°C, and vacuum freeze-dry to obtain purified anthraquinone monomers: emodin (Fr9) and helminthosporin (Fr10). The results are as shown in the appendix Figure 2 A. Identify the sample purity by high performance liquid chromatography in step (1). The results are as shown in the appendix Figure 2 B - C. The purified Fr9 (emodin) and Fr10 (helminthosporin) components are both composed of single peaks, and the purities are 97.2% and 99.3% respectively.
[0031] Example 3: Structure identification of anthraquinone monomers Fr9 and Fr10 metabolized by Aspergillus cristatus
[0032] (1) High performance liquid chromatography - quadrupole - time of flight mass spectrometry (HPLC - Q - TOF - MS) analysis
[0033] Use The 5600+ type liquid chromatography-mass spectrometry (AB SCIEX) is equipped with a Kinetex C18 chromatographic column (2.1×100mm., 2.6um, 100A) for the separation of anthraquinone compounds. Mobile phase: A: water containing 0.1% (v / v) formic acid, B: acetonitrile; Gradient elution conditions: 0 min, 5% B phase; 35 min, 95% B phase; 38 min, 95% B phase; 40 min, 5% B phase, and the elution flow rate is 0.3 mL / min.
[0034] Mass spectrometry conditions: Electrospray ionization source (ESI) is used, with positive and negative ion scanning modes. In the positive ion mode, the electrospray voltage is 5500V, the vaporization temperature is 550°C, the curtain gas (CUR) is 40 psi, the declustering voltage (DP) is 80V, and the collision energy (CE) is 45V; in the negative ion mode, the electrospray voltage is 4500V, the vaporization temperature is 550°C, CUR is 40 psi, DP is -80V, and CE is -45V. The scanning mass number is m / z 50 - 1000 Da.
[0035] As shown in Figure 3 Appendix 15 H 10 O 5 , the theoretical mass number is 270.05282 [M], and the actually measured m / z 271.0603 [M+H] in the positive ion mode + ; the actually measured m / z 269.0457 [M-H] in the negative ion mode - , and the ion fragments m / z 241.0567 [M-H-CO] 2 in the MS - spectrum, 225.0613 [M-H-CO-O] - , 197.0650 [M-H-CO-O-CO] - , which is consistent with Emodin, and its structure can be preliminarily identified as Emodin.
[0036] As shown in Figure 3 Appendix 15 H 10 O 6 , the theoretical mass number is 286.04774 [M], and the actually measured m / z 287.0552 [M+H] in the positive ion mode + ; the actually measured m / z 285.0407 [M-H] in the negative ion mode - , and the ion fragments m / z 257.0521 [M-H-CO] 2 in the MS - spectrum, 241.0565 [M-H-CO-O]- ,229.0569 [M-H-CO-O-C] - ,213.0607 [M-H-CO-O-CO] - ,201.0610 [M-H-CO-O-CO-C] - ,Its structure can be preliminarily identified as catenarin.
[0037] (2) MALDI-TOF-MS mass spectrometry analysis
[0038] Mix the purified anthraquinone monomer Fr9 or Fr10 (5 mg / mL) with the DHB matrix (20 mg / mL) evenly in a ratio of 1:1. Take 1 μL and spot it on the target plate, and let it dry naturally. Then use the Bruker Autoflex MALDI-TOF mass spectrometry system to analyze Fr9 and Fr10. The mass spectrometry conditions are set as follows: positive ion mode, ion acceleration voltage 20 kV, mass-to-charge ratio (m / z) scanning range 100 - 1000. As shown in Figure 4 Figure A-B, the mass spectrometry results show that the [M+H] + of Fr9 is 271.1, which is consistent with the molecular weight of the standard emodin; the [M+H] + of Fr10 is 287.3, which is consistent with the molecular weight of catenarin.
[0039] (3) Qualitative analysis of anthraquinone monomer Fr9 by high performance liquid chromatography
[0040] Analyze the anthraquinone monomer Fr9 and the standard emodin by high performance liquid chromatography. Chromatographic conditions: Agilent technologies 1200series high performance liquid chromatograph; Inertsil ODS-3 chromatographic column (4.6 mm × 250 mm, 5 μm); mobile phase: A: ultrapure water containing 0.1% formic acid; B: acetonitrile containing 0.1% formic acid; gradient elution conditions: 0 - 35 min, 35% - 95% phase B; 35 - 45 min, 95% phase B; elution flow rate 0.8 mL / min; column temperature 35°C; injection volume: 20 μL; PDA detector: 280 nm and 440 nm. As shown in Figure 4 Figure C, there is no significant difference in the retention time of the anthraquinone monomer Fr9 and the standard emodin. The purified anthraquinone monomer Fr9 (compound 15) is identified as emodin.
[0041] (4) NMR identification of anthraquinone monomer Fr10
[0042] The anthraquinone monomer Fr10 was identified using a Bruker Avance DRX500 low-field nuclear magnetic resonance. Using deuterated chloroform and deuterated dimethyl sulfoxide as solvents, at a temperature of 298 K, a working frequency of 500 MHz, the hydrogen spectrum ( 1 H) and carbon spectrum ( 13 C) were collected for analysis. As shown in the appendix Figure 5 , in Table 1 1 the chemical shifts of the 13 H NMR and
[0043] 13 C NMR spectra indicated that the anthraquinone monomer Fr10 (compound 16) was identified as catenarin.
[0043] Table 1 Chemical shifts of anthraquinone monomer Fr10 in 1 H NMR and 13 C NMR spectra
[0044]
[0045] Example 4: Antibacterial activities of emodin and catenarin metabolized by Aspergillus cristatus against Escherichia coli and Staphylococcus aureus
[0046] Experiment 1: Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of emodin and catenarin against Escherichia coli and Staphylococcus aureus
[0047] (1) Bacterial suspension preparation: Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) stored at -20 °C were respectively inoculated on LB agar medium, and then single colonies were picked and inoculated into 25 mL of sterile LB broth. They were shaken at 37 °C and 180 r / min for 12 h until the logarithmic growth phase, and the bacterial suspension concentration was diluted to 1×10 5 CFU / mL with sterile LB broth.
[0048] (2) Sample solution preparation: The test samples emodin (Fr9) and catenarin (Fr10) and the positive drug ceftriaxone sodium (CRO) were dissolved in DMSO to a final concentration of 20 mg / mL.
[0049] (3) Sampling in 96-well plates: Add 190 μL of the above-diluted bacterial solution to each well in the first column of the 96-well plate, and add 100 μL of the diluted bacterial solution to the remaining wells; add 10 μL of the test sample to each well in the first column, and pipette and mix well to make the final concentration 1000 μg / mL. Add DMSO as the negative control group (Control) and add CRO as the positive control group; then pipette 100 μL of the bacterial solution from the first column and add it to the second column, mix well, and successively dilute it to the 12th column according to the two-fold dilution method, and pipette and discard 100 μL of the liquid. Incubate the 96-well plate in a 37 °C constant temperature incubator for 24 h and then read the results.
[0050] (4) Investigation of MIC and MBC: The lowest sample concentration at which the well is clear and no bacteria grow as observed with the naked eye is defined as the MIC of the test sample; Pipette 10 μL of the 96-well plate culture onto the LB solid medium respectively, and incubate it at 37 °C for 12 h. The lowest sample concentration at which no colonies grow in the LB solid medium is the MBC of the test sample.
[0051] As shown in Table 2 and the appendix Figure 6 : Fr9 (emodin) and Fr10 (monorden) have high in vitro antibacterial activities against Escherichia coli and Staphylococcus aureus, can inhibit both Gram-positive bacteria and Gram-negative bacteria simultaneously, and have broad-spectrum antibacterial properties. Among them, the MIC and MBC of Fr9 (emodin) against Escherichia coli are 15.63 μg / mL and 62.5 μg / mL respectively; the MIC and MBC of Fr9 against Staphylococcus aureus are 7.81 μg / mL and 31.25 μg / mL respectively; the MIC and MBC of Fr10 (monorden) against Escherichia coli are 7.81 μg / mL and 15.63 μg / mL respectively; the MIC and MBC of Fr10 against Staphylococcus aureus are 15.63 μg / mL and 62.5 μg / mL respectively.
[0052] Table 2 MIC and MBC of emodin and monorden metabolized by Aspergillus cristatus against Escherichia coli and Staphylococcus aureus
[0053]
[0054] Experiment 2: Dynamic antibacterial curves of emodin and monorden against Escherichia coli and Staphylococcus aureus
[0055] (1) Bacterial solution preparation: Inoculate the activated Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) into sterile LB broth at a ratio of 2% respectively, shake and culture at 37 °C and 180 r / min for 12 h until the logarithmic growth phase, and dilute the bacterial solution concentration to 1×10 5CFU / mL.
[0056] (2) Preparation of sample solution: Dissolve the test samples emodin (Fr9), helminthosporin (Fr10), and the positive drug ceftriaxone sodium (CRO) in DMSO to a final concentration of 781, 391, and 195 μg / mL.
[0057] (3) Treatment of test samples: Mix 10 μL of test samples at different concentrations (781, 391, 195 μg / mL) with 990 μL of diluted bacterial solution to a final concentration of 7.81, 3.91, and 1.95 μg / mL; add DMSO as the negative control group (Control), and add CRO as the positive control group. Then incubate at 37 °C and 180 r / min for 24 h, take samples every 2 h to measure OD600nm, and plot the time-dynamic antibacterial curve.
[0058] As shown in the Figure 7 attachment: Emodin (Fr9) and helminthosporin (Fr10) can both delay Escherichia coli and Staphylococcus aureus from reaching the logarithmic phase of bacterial growth at concentrations of 7.81, 3.91, and 1.95 μg / mL. At a concentration of 7.81 μg / mL, they significantly inhibit the growth of Escherichia coli and Staphylococcus aureus, making the bacterial turbidity increase very slowly within 24 h and the number of bacteria decrease significantly, indicating that emodin and helminthosporin have an obvious inhibitory effect on the growth and reproduction of Escherichia coli and Staphylococcus aureus, and the strength of the inhibitory effect is positively correlated with the concentration.
[0059] Experiment 3: Determination of the ability of emodin and helminthosporin to inhibit the biofilm formation of Escherichia coli and Staphylococcus aureus
[0060] Inoculate the activated Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) into sterile LB broth at a ratio of 2% respectively, shake and culture at 37 °C and 180 r / min for 12 h until the logarithmic growth phase, and dilute the bacterial solution concentration to 1×10 5CFU / mL. The diluted bacterial solution was mixed with test samples emodin (Fr9) or helminthosporin (Fr10) at different concentrations to a final concentration of 15.63, 7.81, 3.91 μg / mL. Meanwhile, DMSO was set as the negative control group (Control), and they were cultured in a 24-well cell culture plate for 24 h. The planktonic bacteria were carefully removed, and then the same volume of 99% methanol was added for fixation at room temperature for 20 min. The methanol was discarded, and it was dried at room temperature for 10 min; 200 μL of 0.1% crystal violet staining solution was added for staining for 10 min, and then washed 3 times with the same volume of sterile PBS, dried at room temperature for 20 min, and the crystal violet was fully dissolved with 33% acetic acid. After decolorization at room temperature for 5 min, the absorbance value at OD570nm was detected by an enzyme-linked immunosorbent assay (ELISA) for quantification to test the inhibitory ability of the test sample on biofilm formation. As shown in Figure 8 A - D: Emodin and helminthosporin significantly inhibited the formation of biofilms of Escherichia coli and Staphylococcus aureus at a concentration of 15.63 μg / mL, and the strength of the inhibitory effect was positively correlated with the concentration.
[0061] In summary, emodin and helminthosporin have high antibacterial activity, low MIC and MBC against Escherichia coli and Staphylococcus aureus, and have an obvious inhibitory effect on their growth, reproduction and biofilm formation ability.
[0062] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. The separation and purification method of emodin and streptosporin metabolized by Aspergillus cristatus and their application as antibacterial agents.
2. The separation and purification method of emodin and streptosporin metabolized by Aspergillus cristatus as claimed in claim 1 and its use as an antibacterial agent, characterized in that: The emodin and streptosporin metabolized by Aspergillus cristatus are prepared by the following method: (1) The Aspergillus cristatus strain isolated from Fuzhuan tea was inoculated into a PDA medium containing 10% sucrose, and after culturing at 28° C. for 30 days, the culture product was freeze-dried to a constant weight; (2) The culture product dried in step (1) was preliminarily extracted with methanol, and ultrasonic extraction was performed three times at a solid-liquid ratio of 1:50 (w / v), an extraction temperature of 35° C., and an extraction time of 3 h. The culture medium residue was discarded by centrifugation (4000 rpm, 15 min), and the methanol extracts were combined, concentrated under reduced pressure at 35° C., and freeze-dried to obtain a methanol extract; (3) extracting the methanol extract in step (2) for a second time with anhydrous ether, extracting three times at a solid-liquid ratio of 1:50 (w / v), mixing the ether extract with an equal volume of deionized water in a separation funnel for extraction, standing for 3 hours, separating the ether layer, and concentrating under reduced pressure at 35° C. to an appropriate volume; (4) The ether concentrate in step (3) is fully mixed with 10% NaCO3 at a solid-liquid ratio of 1:2, the alkaline solution layer is collected, and an appropriate amount of HCl is added to acidify the alkaline solution to pH = 2, so that anthraquinone components can be precipitated. Centrifuge (4000 rpm, 15 min) and discard the supernatant. The precipitate is collected, an appropriate volume of water is added, and vacuum freeze-dried for 2 days to obtain an anthraquinone extract.
3. The purification of anthraquinone compounds according to claim 1 or 2, characterized in that: The anthraquinone extract is further separated and purified by chromatography with a C18 chromatographic column to obtain anthraquinone monomers: emodin and streptosporin.
4. The use of emodin and streptosporin according to claim 1, 2 or 3, characterized in that: The purified anthraquinone monomers are identified as emodin and catenarin, have low MIC and MBC and broad-spectrum antibacterial activity against Escherichia coli and Staphylococcus aureus, and have obvious inhibitory effects on their growth, reproduction and biofilm formation ability.
5. The use of emodin and streptosporin according to claim 1, 2, 3 or 4, characterized in that: Emodin and Catenarin metabolized by Aspergillus cristatus are used as antimicrobial agents in the food or biopharmaceutical industries.