Chromone compound as well as preparation method and application thereof

Abstract

The invention provides a chromone compound as well as a preparation method and application thereof, and belongs to the technical field of biomedicine. The structural formula of the chromone compound is shown as a formula (I). The compound provided by the invention is separated from a metabolite of Aspergillus sp. L06 derived from the root of Euphorbia tianschanica, is novel in structure, has remarkable clinical pathogenic bacterium resisting activity, has the minimum inhibitory concentration MIC of 4.0 mu g / mL to pseudomonas aeruginosa, and can become an active lead compound for developing novel clinical antibacterial medicines, a preparation method of the compound and a preparation method of the compound, and the compound can be applied to preparation of medicines for treating clinical pathogenic bacteria. Aspergillus fungi are used for fermentation culture, and then through extraction and separation, the chromone compound is rapidly, accurately and efficiently prepared from a fermentation product. The preparation method is low in cost, relatively simple in separation process, good in repeatability and easy to prepare the chromone compound on a large scale.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a chromogen ketone compound, its preparation method and application, and particularly to a novel chromogen ketone compound with anti-clinically pathogenic bacteria activity isolated from a strain of Aspergillus sp. L06, along with its preparation method and application. Background Technology

[0002] The evolution of clinical pathogens and drug resistance poses a major global public health challenge. World Health Organization data shows that infections caused by "superbugs" such as methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa are increasing by more than 15% annually, with severe cases resulting in a mortality rate 3-5 times higher than that of ordinary infections. In medical settings, these bacteria can spread through medical devices or colonize the respiratory tract and wounds of patients, prolonging treatment cycles by 3-4 times, and significantly increasing the risk of death, especially among immunocompromised individuals and the elderly and young children.

[0003] The development of existing antibacterial drugs lags behind the mutation of pathogenic bacteria. Due to overuse, the targets of traditional antibiotics have been broken down by resistance genes. For example, *Pseudomonas aeruginosa* can hydrolyze penicillin-type drugs and efflux quinolone drugs, causing the MIC value of ciprofloxacin to increase 8-10 times in ten years. The development of novel antibiotics is time-consuming (10-15 years), costly (over $1 billion), and prone to cross-resistance. Clinically, there is an urgent need for antibacterial lead compounds with novel structures and unique mechanisms.

[0004] Natural products are an important source of antibacterial drugs. Fungi, as "natural synthetic factories," can produce active molecules, such as penicillin and vancomycin. Fungi that live in symbiosis with medicinal plants have formed unique metabolic networks through co-evolution, and their products possess both stability and targeting properties.

[0005] In the extreme environments of Xinjiang's pastoral areas, symbiotic fungi on the roots of *Euphorbia tianshanensis* may produce unique antibacterial components under adverse conditions. *Aspergillus* fungi can synthesize compounds such as chromogenones. Chromogenones, due to their conjugated pyranone core, possess antibacterial properties and are less prone to cross-resistance. Therefore, screening for novel chromogenones from this fungus could provide new frameworks and candidate molecules for the development of drugs against drug-resistant pathogens. Summary of the Invention

[0006] The embodiments of the present invention provide a chromogen ketone compound, its preparation method and application, aiming to solve the problems raised in the background art.

[0007] This invention provides a chromogen ketone compound, the structural formula of which is shown in formula (Ⅰ);

[0008]

[0009] A method for preparing the chromogen ketone compounds described above includes the following steps:

[0010] Step (1): A strain of Aspergillus sp. L06 was isolated and purified from the root of the medicinal plant *Aspergillus tianshanensis*, which is native to pastoral areas of Xinjiang.

[0011] Step (2): Select rice culture medium to culture the fungus Aspergillus sp. L06 to obtain the fermentation product;

[0012] Step (3): Extract the fermentation product obtained in step (2) with anhydrous ethanol, and concentrate the obtained extract under reduced pressure to obtain crude extract;

[0013] Step (4): Dissolve the crude extract obtained in step (3) in water, extract with ethyl acetate, and concentrate the ethyl acetate phase under reduced pressure to obtain the crude extract.

[0014] Step (5): The crude extract obtained in step (4) is loaded onto a normal-phase silica gel column by dry method, and then gradient elution is performed. The gradient eluents of each gradient are collected and concentrated. The same parts are combined by TLC monitoring to obtain 5 components A–E.

[0015] Step (6): The component E obtained in step (7) is wet-loaded onto a Sephadex LH-20 column, the eluent is collected and concentrated, and the same parts are combined by TLC monitoring to obtain 8 subcomponents E1–E8.

[0016] Step (7) involves separating and purifying the subfraction E4 obtained in step (6) to obtain the chromogen ketone compound shown in formula (Ⅰ).

[0017] In one embodiment of the present invention, in step (2), the culture conditions are 28°C and static culture for 35 days.

[0018] In one embodiment of the present invention, in step (3), the extraction method is to add an equal volume of anhydrous ethanol, sonicate at room temperature for 0.5 h, then let stand at room temperature for 2 h, filter, and concentrate the filtrate under reduced pressure.

[0019] In one embodiment of the present invention, in step (4), the volume of water and ethyl acetate used for extraction is the same, and the extraction is performed 3 times.

[0020] In one embodiment of the present invention, in step (5), the mobile phase used for gradient elution is a mixed solvent of petroleum ether and ethyl acetate, with volume ratios of 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1, respectively. After each gradient elution reaches the point where there are no spots on the TLC plate, the next gradient elution is performed. The TLC is then used to track the elution, and the same portions are combined and concentrated to obtain five components, A–E.

[0021] In one embodiment of the present invention, in step (6), the mobile phase used for gradient elution is methanol: dichloromethane = 1:1. One tube of eluent is collected every 2 minutes, tracked by TLC, and the same portions are combined and concentrated to obtain 8 sub-components E1–E8.

[0022] In one embodiment of the present invention, in step (7), the separation and purification are first performed by HPLC purification; the HPLC chromatographic conditions are as follows: the mobile phase is a mixed solvent of methanol and water with a volume ratio of 75:25, the flow rate is 2.0 mL / min, the retention time is 26.0 min, a C18 column is used with specifications of 5 μm and 10 × 250 mm, the column temperature is 30 °C, the injection volume is 50 μL, and a full-wavelength array ultraviolet detector is used with detection wavelengths of 210, 254, 280, and 365 nm.

[0023] The application of the chromogens described above in the preparation of agents against clinically pathogenic bacteria.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] 1) The chromone compound [ethyl7-(5,7-dimethoxy-4-oxo-4H-chromen-2-yl)heptanoate] in this invention is a newly discovered compound;

[0026] 2) The compound of the present invention was isolated from the metabolites of a fungus of the genus Aspergillus sp. L06 derived from the roots of *Pseudomonas aeruginosa*. It has a novel structure and significant activity against clinically pathogenic bacteria. The minimum inhibitory concentration (MIC) against *Pseudomonas aeruginosa* is 4.0 μg / mL. It can serve as an active lead compound for the development of novel antibacterial drugs. Therefore, the chromogens mentioned above have great application potential against clinically pathogenic bacteria.

[0027] 3) This invention also provides a method for preparing the compound, which involves fermentation culture using Aspergillus fungi, followed by extraction and separation to rapidly, accurately, and efficiently prepare the chromogenone compound from the fermentation product. This preparation method is low-cost, the separation process is relatively simple, and it has good reproducibility, making it easy to prepare the chromogenone compound on a large scale. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0029] Figure 1 The structural formula of the chromogen ketone compounds of this invention is shown below;

[0030] Figure 2 This is a high-resolution mass spectrum of the chromogen ketone compounds of the present invention;

[0031] Figure 3 The chromogen ketone compounds of this invention 1 H NMR spectrum;

[0032] Figure 4 The chromogen ketone compounds of this invention 13 C NMR and DEPT spectra;

[0033] Figure 5 The HSQC spectra of the chromogen ketone compounds of this invention are shown below.

[0034] Figure 6 This is the HMBC spectrum of the chromogen ketone compounds of this invention.

[0035] Figure 7 The chromogen ketone compounds of this invention 1 H- 1 H COSY spectrum. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0038] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0039] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," and "counterclockwise," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0040] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0041] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0042] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can include direct contact between the first and second features, or contact between the first and second features through another feature between them. Furthermore, "above," "over," and "on top" of the second feature includes the first feature directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature includes the first feature directly below or diagonally below the second feature, or simply indicates that the first feature is at a lower horizontal level than the second feature.

[0043] Example

[0044] Please see Figure 1-7A chromogen ketone compound, the structural formula of which is shown in formula (Ⅰ);

[0045]

[0046] Example 2

[0047] Step (1): A strain of Aspergillus sp. L06 was isolated and purified from the root of the medicinal plant *Aspergillus tianshanensis*, which is native to pastoral areas of Xinjiang.

[0048] Step (2): Select rice culture medium to culture Aspergillus sp. L06 to obtain fermentation products;

[0049] Step (3): Extract the fermentation product obtained in step (2) with anhydrous ethanol, and concentrate the obtained extract under reduced pressure to obtain crude extract;

[0050] Step (4): Dissolve the crude extract obtained in step (3) in water, extract with ethyl acetate, and concentrate the ethyl acetate phase under reduced pressure to obtain the crude extract.

[0051] Step (5): The crude extract obtained in step (4) is loaded onto a normal-phase silica gel column by dry method, and then gradient elution is performed. The gradient eluents of each gradient are collected and concentrated. The same parts are combined by TLC monitoring to obtain 5 components A–E.

[0052] Step (6): The component E obtained in step (7) is wet-loaded onto a Sephadex LH-20 column, the eluent is collected and concentrated, and the same parts are combined by TLC monitoring to obtain 8 subcomponents E1–E8.

[0053] Step (7) involves separating and purifying the subfraction E4 obtained in step (6) to obtain the chromogen ketone compound shown in formula (Ⅰ).

[0054] Example 3

[0055] Step (1): A strain of Aspergillus sp. L06 was isolated and purified from the root of the medicinal plant *Aspergillus tianshanensis*, which is native to pastoral areas of Xinjiang.

[0056] Step (2): Select rice culture medium to culture Aspergillus sp. L06 to obtain fermentation products;

[0057] Step (3): Extract the fermentation product obtained in step (2) with anhydrous ethanol, and concentrate the obtained extract under reduced pressure to obtain crude extract;

[0058] Step (4): Dissolve the crude extract obtained in step (3) in water, extract with ethyl acetate, and concentrate the ethyl acetate phase under reduced pressure to obtain the crude extract.

[0059] Step (5): The crude extract obtained in step (4) is loaded onto a normal-phase silica gel column by dry method, and then gradient elution is performed. The gradient eluents of each gradient are collected and concentrated. The same parts are combined by TLC monitoring to obtain 5 components A–E.

[0060] Step (6): The component E obtained in step (7) is wet-loaded onto a Sephadex LH-20 column, the eluent is collected and concentrated, and the same parts are combined by TLC monitoring to obtain 8 subcomponents E1–E8.

[0061] Step (7) involves separating and purifying the subfraction E4 obtained in step (6) to obtain the chromogen ketone compound shown in formula (Ⅰ).

[0062] In step (1), the strain was isolated from the roots of *Pseudohypophys tianshanensis*.

[0063] In step (2), the culture conditions are static culture at 28℃ for 35 days.

[0064] In step (3), the extraction method is to add an equal volume of anhydrous ethanol, sonicate at room temperature for 0.5 h, then let stand at room temperature for 2 h, filter, and concentrate the filtrate under reduced pressure.

[0065] In step (4), the volume of water and ethyl acetate used for extraction is the same, and the extraction is performed 3 times.

[0066] In step (5), the mobile phase used for gradient elution is a mixture of petroleum ether and ethyl acetate, with volume ratios of 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1, respectively. After each gradient elution reaches the point where there are no spots on the TLC plate, the next gradient elution is performed. The TLC is used to track the elution, and the same portions are combined and concentrated to obtain five components, A–E.

[0067] In step (6), the mobile phase used for gradient elution is methanol: dichloromethane = 1:1. One tube of eluent is collected every 2 minutes, and the eluent is tracked by TLC. The same portions are combined and concentrated to obtain 8 sub-components E1–E8.

[0068] In step (7), the separation and purification are performed using high performance liquid chromatography, and the detector is a full-wavelength diode array detector.

[0069] Application examples:

[0070] A novel chromogen ketone compound disclosed in this invention was isolated from rice fermentation products of the fungus Aspergillus sp. L06. The specific preparation steps are as follows:

[0071] 1. Strains Isolation and Purification

[0072] Clean the roots of *Euphorbia tirucalli*, disinfect them with alcohol, cut them into small pieces about 3 mm in length, place them on PDA medium, and incubate them at 30°C for 2 days. Pick a single colony and transfer it to another PDA medium for further culture and purification. Then, perform 16S rRNA gene sequencing to identify the isolated strain.

[0073] 2. Fermentation culture of microbial strains

[0074] The preserved bacterial culture was taken out and activated on PDA (potato dextrose agar) plates for 5 days. After spores grew, it was transferred to sterilized rice culture medium (100g rice + 80mL distilled water), for a total of 200 bottles, and incubated at 28℃ for 35 days.

[0075] 3. Extraction and extraction of fermentation products

[0076] After fermentation, anhydrous ethanol (40L) was added and extracted three times. The extract was concentrated under reduced pressure to obtain 0.5Kg of crude extract. Water was added to disperse the extract, and it was extracted three times with an equal volume of ethyl acetate. The ethyl acetate layer was collected and concentrated under reduced pressure to obtain 100g of crude extract.

[0077] 4. Isolation and purification of compounds

[0078] The crude extract was dissolved in 10 times its volume of ethyl acetate, and mixed with 1.5 times its mass of 80-100 mesh silica gel. The mixture was then packed into a 200-300 mesh silica gel column and loaded dry. A gradient elution was performed using a mixture of petroleum ether and ethyl acetate in ratios of 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1 (six gradients in the claim). After each gradient eluted until no spots were observed on the TLC plate, the next gradient was used. TLC was monitored, and identical fractions were combined and concentrated to obtain five components, A–E.

[0079] Component E (7g) was separated using an ODS reversed-phase column with a mixed solvent of methanol and water as the eluent. The eluent gradients were 20:80, 40:60, 80:20, and 100:0, respectively. After each gradient eluent reached the TLC plate with no spots, the next gradient was used for elution. The TLC was monitored, and identical fractions were combined and concentrated to obtain six subcomponents, E1–E6.

[0080] Component E5 (90 mg) was purified by gel chromatography using Sephadex LH-20 as the packing material and a 1:1 mixture of dichloromethane and methanol as the mobile phase. Thin-layer chromatography (TLC) was used for monitoring, and identical fractions were combined and concentrated to obtain three components E5.1–E5.5.

[0081] Component E5.2 (60 mg) was purified by semi-preparative HPLC. The mobile phase was a mixture of methanol and water (75:25 v / v), the flow rate was 2.0 mL / min, and the retention time was 26.0 min. A C18 column (5 μm, 10 × 250 mm) from Yuexu Technology (Shanghai) Co., Ltd. was used. The column temperature was 30℃, and the injection volume was 50 μL. A full-wavelength array UV detector was used, with detection wavelengths of 210, 254, 280, and 365 nm. The chromatographic peak with a retention time of 26 min was collected, yielding compound 1 [ethyl7-(5,7-dimethoxy-4-oxo-4H-chromen-2-yl)heptanoate] (10.9 mg).

[0082] 5. Physicochemical properties and structural analysis of compounds

[0083] Pale yellow oil; UV(MeOH)λmax: 208, 233, 288 nm; HRESIMS m / z [M+Na]+385.1627 (calcd for C 20 H 26 O6Na + 385.1627), from which we can deduce that its molecular formula is C 20 H 26 O6. Regarding it. 1 H NMR data analysis revealed three methyl proton signals (δ¹⁸O₂). H 3.92, 3.90, 1.24), two of which are oxymethyl protons (δ). H 3.92, 3.90); seven methylene proton signals (δ H 4.11, 2.63, 1.75, 1.64, 1.43, 1.43); three methylene proton signals (δ H (6.05, 6.51, 6.64). Further research on... 13 Analysis of C10 NMR, DEPT, and HSQC spectral data confirmed that compound 1 contains 20 carbons, including one methyl group (δ¹⁰). C 14.5), 2 methoxy groups (δ C 56.5, 56.5), 7 methylene groups (δ C25.8, 27.6, 29.6, 29.7, 34.3, 35.0, 61.4), 3 sps 2 Methionine (δ) C 94.0, 97.2, 111.3), 7 sps 2 Quaternary carbon (δ C 109.2, 161.8, 162.1, 166.4, 169.7, 175.5, 180.1). The 1D NMR data of compound 1 were compared with those of penithochromone F reported in the literature.

[11] The data from both compounds were found to be largely consistent, suggesting they share the same parent nucleus; the difference lies in compound 1 having an additional hydroxymethylene signal (δ). H 4.11,δ C 61.4). To determine the complete planar structure of compound 1, its 2D NMR data ( Figure 2 ) to conduct analysis. 1 H– 1 The H COSY spectrum contains two spin systems: H2-9 / H2-10 / H2-11 / H2-12 / H2-13 / H2-14 and H2-17 / H3-18. The HMBC spectrum shows H-14 (δ... H 2.32) and H-17 (δ) H 4.11) are both similar to C-15 (δ C 175.5) is related, thus C-14(δ) C 35.0) and C-17 (δ C 61.4) linked by ester bonds; H-8 (δ H 6.51) and C-8a(δ C 161.8), C-6(δ) C 94.0), C-4a(δ C 109.2) related, H-6(δ) H 6.64) and C-8(δ) C 97.2) and C-4a(δ C 109.2) related, H-3(δ) H 6.05) and C-4a(δ C 109.2), C-4(δ) C 180.1), C-2(δ) C 169.7) is related, therefore it can be inferred that compound 1 is benzo[a]γ - pyranone skeleton; H2-9(δ) H 2.63) and C-2(δ C 169.7) and C-3(δ CBased on the correlation with 111.3), it can be determined that the side chain is connected to the parent core via C-2. Combining the above data, it can be determined that the difference between compound 1 and the known compound penithochromone F is that the ketone carbonyl group at position 15 is replaced by an ethoxy group instead of a methoxy group. Therefore, the structure of the compound is determined. For the NMR data required for compound structure analysis, please refer to (…). Figures 3-6 Table 1);

[0084] Table 1 Compound 1 1 H NMR and 13 C NMR data (400MHz and 100MHz, CD3OD)

[0085]

[0086]

[0087]

[0088] 6. Experiments on the activity of compounds against clinically resistant bacteria:

[0089] 5.1 The target compound and ciprofloxacin (positive control) were dissolved in physiological saline containing 0.20% DMSO to prepare a solution with an initial concentration of 6.4 mg / mL.

[0090] 5.2 Clinically pathogenic bacteria, namely Staphylococcus aureus (SAU), Acinetobacter baumannii (ABA), Enterobacter cloacae (ECL), Escherichia coli (ECO), and Pseudomonas aeruginosa (PAE), were activated in LB (lysate broth) medium for 8 hours. After activation, 50 μL of the bacterial solution was added to 50 mL of LB medium to obtain a diluted bacterial solution.

[0091] 5.3 Using ciprofloxacin as a positive control, 2 μL of the target compound was added to 198 μL of the target bacterial culture, and the antagonistic activity of the target compound against the above eight clinically resistant bacteria was tested using the two-fold dilution method. (Activity results are shown in Table 2).

[0092]

[0093] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A chromogen ketone compound, characterized in that, The structural formula of the chromogen ketone compounds is shown in formula (Ⅰ); 2. A method for preparing the chromogen ketone compound of claim 1, characterized in that, Includes the following steps: Step (1): A strain of Aspergillus sp. L06 was isolated and purified from the root of the medicinal plant *Aspergillus tianshanensis*, which is native to pastoral areas of Xinjiang. Step (2): Select rice culture medium to culture the fungus Aspergillus sp. L06 to obtain the fermentation product; Step (3): Extract the fermentation product obtained in step (2) with anhydrous ethanol, and concentrate the obtained extract under reduced pressure to obtain crude extract; Step (4): Dissolve the crude extract obtained in step (3) in water, extract with ethyl acetate, and concentrate the ethyl acetate under reduced pressure to obtain the crude extract. Step (5): The crude extract obtained in step (4) is loaded onto a normal-phase silica gel column by dry method, and then gradient elution is performed. The gradient eluents of each gradient are collected and concentrated. The same parts are combined by TLC monitoring to obtain 5 components A–E. Step (6): The component E obtained in step (7) is wet-loaded onto a Sephadex LH-20 column, the eluent is collected and concentrated, and the same parts are combined by TLC monitoring to obtain 8 subcomponents E1–E8. Step (7) involves separating and purifying the subfraction E4 obtained in step (6) to obtain the chromogen ketone compound shown in formula (Ⅰ).

3. The method for preparing chromogen ketone compounds according to claim 2, characterized in that, In step (2), the culture conditions are 28°C and static culture for 35 days.

4. The method for preparing chromogen ketone compounds according to claim 2, characterized in that, In step (3), the extraction method is to add an equal volume of anhydrous ethanol, sonicate at room temperature for 0.5 h, then let stand at room temperature for 2 h, filter, and concentrate the filtrate under reduced pressure.

5. The method for preparing chromogen ketone compounds according to claim 2, characterized in that, In step (4), the volume of water and ethyl acetate used for extraction is the same, and the extraction is performed 3 times.

6. The method for preparing chromogen ketone compounds according to claim 2, characterized in that, In step (5), the mobile phase used for gradient elution is a mixture of petroleum ether and ethyl acetate, with volume ratios of 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1, respectively. After each gradient elution reaches the point where there are no spots on the TLC plate, the next gradient elution is performed. The TLC is used to track the elution, and the same portions are combined and concentrated to obtain five components, A–E.

7. The method for preparing chromogen ketone compounds according to claim 2, characterized in that, In step (6), the mobile phase used for gradient elution is methanol: dichloromethane = 1:

1. One tube of eluent is collected every 2 minutes, and the eluent is tracked by TLC. The same parts are combined and concentrated to obtain 8 sub-components E1–E8.

8. The method for preparing chromogen ketone compounds according to claim 2, characterized in that, In step (7), the separation and purification are first carried out by HPLC purification; the HPLC chromatographic conditions are as follows: the mobile phase is a mixed solvent of methanol and water with a volume ratio of 75:25, the flow rate is 2.0 mL / min, the retention time is 26.0 min, and a C18 column with specifications of 5 μm and 10 × 250 mm is used. Column temperature: 30℃, injection volume per injection: 50μL, using a full-wavelength array UV detector with detection wavelengths of 210, 254, 280, and 365nm.

9. The use of the chromogens of claim 1 in the preparation of agents against clinically pathogenic bacteria.

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