Application of active metabolite of marine actinomycetes HN2-56 in quorum sensing resistance

Abstract

The invention discloses an application of an active metabolite of marine actinomycetes HN2-56 in quorum sensing resistance. The active metabolites of the marine actinomycetes comprise propanediol tetraisopropyl alcohol ether, bisphenol A and 5-(2-methylphenyl)-4-pentenoic acid, the bisphenol A and the 5-(2-methylphenyl)-4-pentenoic acid have remarkable quorum sensing resisting activity, and the 5-(2-methylphenyl)-4-pentenoic acid has remarkable quorum sensing resisting activity on indicator bacterium chromobacterium violaceum. Compared with the prior art, the compound has no obvious growth inhibition on indicator bacteria and has anti-biofilm activity on the indicator bacteria staphylococcus aureus, and the propanediol tetraisopropyl alcohol ether is a prodrug of bisphenol A, so that the compound is expected to be applied to development of novel antibacterial substances taking a quorum sensing system as a target spot.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to application of marine actinomycete metabolites in anti-quorum sensing. Background Art

[0002] Quorum sensing is a phenomenon in which bacteria communicate with each other and coordinate group behavior by secreting and sensing one or several small molecular weight chemical signal molecules. This phenomenon indicates that the life activities between single-cell microorganisms are collective, and this group effect not only exists between cells of the same microorganism, but can also regulate the relationship between different microorganisms. Studies have found that important bacterial physiological functions such as pigment production, bioluminescence, virulence factor production and release, antibiotic synthesis, biofilm formation and maturation are all regulated by the quorum sensing system.

[0003] Due to the widespread and disorderly use of antibiotics, the drug resistance of pathogens has increased, and the types of drug-resistant strains have continued to increase. Many literature reports that strains that lack the quorum sensing system or whose system is inhibited have greatly reduced pathogenicity. Therefore, inhibiting the quorum sensing effect of pathogens may reduce the pathogenicity of pathogens. Quorum sensing inhibitors do not interfere with important life processes of bacteria, have less pressure on bacterial survival, and are not likely to cause bacterial mutations and produce drug resistance. They are a new target for the development of antibacterial substances.

[0004] Marine actinomycetes can produce metabolites with multiple biological activities and unique chemical structures such as antibacterial, antitumor, antiviral, anti-inflammatory, and enzyme inhibition, and are one of the important sources of natural products. These active natural products provide a wide range of valuable candidate drugs for the treatment of various diseases in plants, animals, and humans. Mangroves are located on tropical and subtropical coastal mudflats, with a special ecological environment (high temperature, high salt, low oxygen, and rich organic matter in the bottom mud). They contain extremely rich microbial resources, and their physiological characteristics are different from other habitats. There are some reports on active substances from marine actinomycetes in mangrove sediments, indicating that mangrove sediment actinomycetes are also an excellent source of natural active products. However, compared with the research on natural products of plants and terrestrial microorganisms in the same field, there are fewer studies on mangrove actinomycetes, which are in urgent need of development. It is of high theoretical significance and application value to explore more mangrove actinomycetes and their active natural product resources. Summary of the invention

[0005] Objective: To overcome the deficiencies in the prior art, the present invention provides an application of active metabolites of marine actinomycetes HN2-56 in anti-quorum sensing, with the intention of being applied to the development of new antibacterial substances targeting the quorum sensing system.

[0006] Technical solution: To solve the above technical problems, the present invention provides the application of active metabolites of marine actinomycetes HN2-56 in anti-quorum sensing, and the technical solution adopted is:

[0007] In the first aspect, the present invention provides the use of active metabolites of marine actinomycetes HN2-56 in anti-quorum sensing. The marine actinomycetes (Streptomyces chumphonensis) HN2-56 are deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101; the deposit date is November 4, 2024, and the deposit number is CGMCC No.32495.

[0008] Furthermore, the active metabolites of the marine actinomycetes include bisphenol tetraisopropyl ether (compound 1), bisphenol A (compound 2) and 5-(2-methylphenyl)-4-pentenoic acid (compound 3):

[0009]

[0010]

[0011] In a second aspect, the preparation method of the active metabolite of marine actinomycete HN2-56 comprises:

[0012] Step S1, fermenting the marine actinomycete HN2-56 strain using an improved Gao's medium No. 1 to obtain a fermentation liquid;

[0013] Step S2, filtering the fermentation liquid with gauze, and then extracting with ethyl acetate to obtain a crude extract;

[0014] Step S3, loading the crude extract solid, separating with a normal phase silica gel vacuum column, using petroleum ether, petroleum ether: ethyl acetate volume ratio of 30:1, petroleum ether: ethyl acetate volume ratio of 20:1, petroleum ether: ethyl acetate volume ratio of 10:1, petroleum ether: ethyl acetate volume ratio of 1:1, dichloromethane: methanol volume ratio of 20:1, dichloromethane: methanol volume ratio of 10:1, dichloromethane: methanol volume ratio of 1:1, methanol for gradient elution, collecting the eluate, analyzing by TLC, combining similar components, and obtaining a total of 12 components A, B, C, D, E, F, G, H, I, G, K, L; performing anti-quorum sensing activity assay on the 12 collected components, and screening out two active components, which are component I separated by elution with dichloromethane: methanol volume ratio of 20:1 and component G eluted with petroleum ether: ethyl acetate volume ratio of 1:1;

[0015] Step S4, subjecting the component I to reverse phase ODS pressure column separation, using 20V / V% methanol water, 40V / V% methanol water, 60V / V% methanol water, 80V / V% methanol water, and 100V / V% methanol gradient elution in sequence; and then subjecting the component eluted with 80V / V% methanol water to high performance liquid chromatography separation and purification to obtain compound 1;

[0016] Step S5, performing reverse phase ODS vacuum column separation on the component G, using 5V / V% methanol water, 20V / V% methanol water, 40V / V% methanol water, 60V / V% methanol water, 80V / V% methanol water, and 100V / V% methanol for gradient elution in sequence; then performing high performance liquid chromatography separation and purification on the components eluted with 20V / V% methanol water to obtain compounds 2 and compound 3 with different retention times.

[0017] In some embodiments, in step S1, the composition of the improved Gao's No. 1 medium is: soluble starch 40.0 g / L, KNO3 4.0 g / L, NaCl 0.5 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O0.5 g / L, FeSO4·7H2O0.01 g / L, pH 6.5.

[0018] In some embodiments, in step S1, the fermentation conditions are shaking culture at 28-32°C for 10-20 days; preferably shaking culture at 30°C and 140 rpm for 15 days.

[0019] In some embodiments, in step S2, extraction is performed with an equal volume of ethyl acetate.

[0020] In some embodiments, in step S4, during separation and purification by high performance liquid chromatography, the mobile phase is 51 V / V% chromatographic methanol water, and the flow rate is 2 mL / min.

[0021] In some embodiments, in step S5, during separation and purification by high performance liquid chromatography, the mobile phase is 60 V / V% chromatographic methanol water, and the flow rate is 2 mL / min.

[0022] In some embodiments, after step S5, the method further includes: performing anti-quorum sensing activity assay on compound 1, compound 2, and compound 3.

[0023] In some embodiments, the anti-quorum sensing activity assay is selected from at least one of filter paper agar diffusion method, pigment inhibition method, and biofilm method test.

[0024] Beneficial effects: The marine actinomycete Streptomyces chumphonensis HN2-56 of the present invention has high-efficiency and low-toxic anti-quorum sensing activity, and can metabolize to produce the compound 5-(2-methylphenyl)-4-pentenoic acid with anti-quorum sensing activity and the important chemical raw material bisphenol A and its derivative propanediol tetraisopropanol ether, and the yield is relatively large. The present invention separates, purifies and analyzes the active metabolites of the marine actinomycete HN2-56 by means of column chromatography, high-performance liquid chromatography, thin-layer chromatography and the like, and obtains propanediol tetraisopropanol ether, bisphenol A and 5-(2-methylphenyl)-4-pentenoic acid. Among them, bisphenol A and 5-(2-methylphenyl)-4-pentenoic acid have significant anti-quorum sensing activity. 5-(2-methylphenyl)-4-pentenoic acid has significant anti-quorum sensing activity against the indicator bacteria Chromobacterium violaceum, and has no obvious growth inhibition on the indicator bacteria. It has anti-biofilm activity against the indicator bacteria Staphylococcus aureus. Bisphenol A tetraisopropanol ether is a prodrug of bisphenol A. Therefore, they are expected to be used in the development of new antibacterial substances targeting the quorum sensing system. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of TLC analysis results of compound 2 (GB-1) and bisphenol A standard (S) in the example of the present invention;

[0026] Figure 2 Schematic diagram of TLC analysis results of the fermentation broth extract of marine actinomycetes HN2-56 at different days (5th, 7th, 9th, 12th, and 15th days) in the fermentation culture of the present invention;

[0027] Figure 3 This is a schematic diagram of the HPLC purification results of compound 1 (ID1) in the example of the present invention;

[0028] Figure 4 This is a schematic diagram of the TLC analysis results of compound 1 (ID1) in the example of the present invention;

[0029] Figure 5 Schematic diagram of HPLC separation results of compound 2 (GB-1) and compound 3 (GB-2) in the examples of the present invention;

[0030] Figure 6 Schematic diagram of TLC analysis results of compound 2 (GB-1) and compound 3 (GB-2) in the examples of the present invention.

[0031] Figure 7 Schematic diagram of the separation process of active metabolites of marine actinomycete strain HN2-56 in an embodiment of the present invention.

[0032] Figure 8Schematic diagram of the anti-quorum sensing activity test results of the compounds in the examples of the present invention: a filter paper agar diffusion test results (sample amount 250 μg / piece), b pigment inhibition test results (test concentration 250 μg / mL, negative control is an equal volume of solvent DMSO), c biofilm test results (test concentration 150 μg / mL, negative control is an equal volume of solvent DMSO). DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present application and its application or use. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0034] Unless otherwise specifically stated, the relative arrangement, numerical expressions and numerical values ​​of the parts and steps set forth in these embodiments do not limit the scope of the present application. At the same time, it should be understood that, for ease of description, the sizes of the various parts shown in the accompanying drawings are not drawn according to the actual proportional relationship. The technology, method and equipment known to those of ordinary skill in the relevant field may not be discussed in detail, but in appropriate cases, the technology, method and equipment should be considered as a part of the authorization specification. In all examples shown and discussed here, any specific value should be interpreted as being merely exemplary, rather than as a limitation. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar reference numerals and letters represent similar items in the following drawings, so that once a certain item is defined in an accompanying drawing, it does not need to be further discussed in subsequent drawings.

[0035] This embodiment provides the application of the active metabolites of marine actinomycetes HN2-56 in anti-quorum sensing. The marine actinomycetes (Streptomyces chumphonensis) HN2-56 are deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101; the deposit date is November 4, 2024, and the deposit number is CGMCC No. 32495.

[0036] Furthermore, the active metabolites of the marine actinomycetes include bisphenol A tetraisopropyl ether (compound 1), bisphenol A (compound 2) and 5-(2-methylphenyl)-4-pentenoic acid (compound 3), and the compound structures are shown below.

[0037]

[0038] In this embodiment, the Chromobacterium violaceum ATCC 12472 involved was provided by the laboratory of Professor Qian Peiyuan of the Hong Kong University of Science and Technology in 2010 and has been used and preserved for a long time in the laboratory of the inventor of the present application; the marine actinomycete strain Streptomyces chumphonensis HN2-56 (deposit number CGMCC No.32495) was isolated from the bottom mud of mangroves in the South China Sea.

[0039] Embodiment 1:

[0040] The activation culture and fermentation method of strain HN2-56 includes:

[0041] The marine actinomycete HN2-56 strain in the cryopreserved tube was placed at room temperature for natural thawing, and all of them were inoculated into 50mL Gao's No. 1 liquid culture medium (soluble starch 20.0g / L, KNO3 1g / L, NaCl 0.5g / L, K2HPO4 0.5g / L, MgSO4·7H2O0.5g / L, FeSO4·7H2O 0.01g / L, sea salt 33g / L, pH7.4-7.6), and cultured at 30°C, 140rpm shaking. After 5 days of culture, the bacterial liquid was transferred to 100mL Gao's No. 1 liquid culture medium at a 5% inoculation rate, and cultured at 30°C, 140rpm shaking for 7 days. The bacteria were centrifuged to remove the cells, and the fermentation broth was extracted twice with an equal volume of ethyl acetate. The organic phase was concentrated by rotary evaporator to obtain a crude strain extract sample, which was weighed and tested.

[0042] Comparison of thin layer chromatography (TLC) of crude extract of HN2-56 with compound 2 and standard bisphenol A: HN2-56 was cultured according to the above fermentation method, and the fermentation broth was collected on the 5th, 7th, 9th, 12th and 15th days, respectively. The crude extract was extracted with ethyl acetate, and the crude extract was dissolved in a small amount of ethyl acetate and spotted on a silica gel thin layer plate for TLC analysis. The developing solvent was dichloromethane CH2Cl2: methanol MeOH = 20:1. Figure 1 and Figure 2 As shown, all samples were f The value of 0.47 has a yellow center and a light purple outer circle spot. This color spot has the same color characteristics as the pure compound 2 and the standard bisphenol A. f This type of spot was not seen in other strains that were fermented and extracted during the same period, indicating that the isolated compound 2 (bisphenol A) was not derived from experimental material contamination, but was a secondary metabolite synthesized by HN2-56.

[0043] Example 2: Anti-quorum sensing activity assay method

[0044] Filter paper agar diffusion method: With Chromobacterium.violaceum 12472 (the quorum sensing effect regulates the pigment synthesis of this strain) as the indicator bacteria, 100 μL of CV12472 bacterial solution after overnight culture is inoculated on the solid LB medium, 250 μg of the sample to be tested (crude strain extract or isolated pure compound) is added to the sterile filter paper (diameter 6 mm), and after the solvent evaporates, the filter paper is placed upside down on the above solid culture medium, cultured at 30°C for 1-2 days, and the morphology of the pigment inhibition zone formed around the filter paper is observed. If it is a transparent inhibition zone, it indicates that the test bacteria inhibits the growth of the indicator bacteria. If it is a fuzzy inhibition zone, it indicates that the test bacteria can inhibit the production of pigments controlled by the quorum sensing effect of the indicator bacteria, but does not affect the growth of the indicator bacteria. The test showed that the crude extract of the fermentation broth of the strain HN2-56 after activation culture and fermentation had a significant inhibitory effect on the production of purple pigment by the indicator bacteria at 250 μg / tablet. The diameter of the pigment inhibition zone was 11 mm, which was a fuzzy circle. It had no obvious inhibitory effect on the growth of the indicator bacteria.

[0045] Pigment inhibition method: Add 948 μL of LB medium and 50 μL of activated cultured Chromobacterium violaceum to a short test tube, and add 2 μL of pure compound dissolved in DMSO at the same time, so that the final concentration of the compound in the system is 250 μg / mL. The negative control is a negative control with only the corresponding volume of DMSO added without adding a sample. After culturing at 30°C and 140 rpm for 24 to 48 hours (the culturing time depends on the growth of indicator bacteria and pigment production in the negative control), transfer the culture medium to a finger-shaped tube, centrifuge at 10,000 rpm for 20 minutes, remove the supernatant, add 0.5 mL of dimethyl sulfoxide (DMSO), vortex to fully dissolve the pigment in the bacteria, and centrifuge at 10,000 rpm for 10 minutes to precipitate the bacteria and debris. Determine the supernatant (OD 样品 OD 阴性对照 ) and solvent DMSO (OD 空白对照 ) at a wavelength of 570 nm. The inhibition rate was calculated according to the formula.

[0046] Pigment inhibition rate (%) = (OD 阴性对照 -OD 样品 ) / (OD 阴性对照 -OD 空白 )×100%

[0047] Biofilm method: The sample to be tested is dissolved in DMSO to a 50 mg / mL sample solution. Add 947 μL of LB medium, 50 μL of overnight cultured indicator bacteria Staphylococcus aureus SA43300 and 3 μL of sample solution to a 24-well plate, so that the final concentration of the sample solution in the test system is 150 μg / mL. Make four parallels for each sample, and the negative control is the same volume of solvent DMSO. Incubate in a 30°C constant temperature incubator for 24 hours. Use a pipette to completely remove the bacterial solution, and rinse the 24-well plate with pure water to remove the residual bacteria that have not formed a film. Add 200 μL of 0.4% crystal violet solution to the 24-well plate to completely cover the biofilm formed in the well, stain for 30 minutes, then rinse the crystal violet stain with pure water, air dry, add 200 μL of DMSO solution to the well to fully dissolve the stained biofilm, then transfer it to a 96-well plate, and measure its OD value at 570nm. The size of the OD value is positively correlated with the number of bacteria in the indicator bacteria biofilm, that is, the growth situation. The inhibition rate is calculated using the same formula in the pigment inhibition method to quantitatively evaluate the inhibitory effect of the test sample on the indicator bacteria biofilm.

[0048] Embodiment 3:

[0049] like Figure 7 As shown, the extraction and separation method of the metabolites of strain HN2-56 includes:

[0050] Step S1, using improved Gao's No. 1 medium (soluble starch 40.0 g / L, KNO3 4.0 g / L, NaCl 0.5 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 0.01 g / L, pH 6.5) to ferment the marine actinomycete strain HN2-56 in large quantities, at 30°C, 140 rpm shaking culture for 15 days, a total of 80 L.

[0051] In the present application, orthogonal experiments were performed to optimize the soluble starch, KNO3 content and pH in the culture medium, and it was found that when HN2-56 was cultured using the above-mentioned improved Gao's No. 1 culture medium, the yield and anti-quorum sensing activity of the crude extract of the strain fermentation broth were increased by 177.9% and 41.8% respectively compared with the original Gao's No. 1 culture medium.

[0052] Step S2, the fermentation broth was filtered through 8 layers of gauze and then extracted with an equal volume of ethyl acetate to obtain 10.23 g of crude extract;

[0053] Step S3, loading the crude extract solid, separating it with a normal phase silica gel vacuum column, gradient eluting with petroleum ether PE: ethyl acetate EtOAc (volume ratio 1:0, 30:1, 20:1, 10:1, 1:1), dichloromethane CH2Cl2: methanol MeOH (volume ratio 20:1, 10:1, 1:1, 0:1), collecting the eluate, analyzing it by TLC, combining similar components, and obtaining a total of 12 components A, B, C, D, E, F, G, H, I, G, K, L; performing anti-quorum sensing activity assay on the 12 collected components, and screening out two active components, which are component I separated by elution with dichloromethane: methanol in a volume ratio of 10:1 and component G eluted with petroleum ether: ethyl acetate in a volume ratio of 1:1;

[0054] Step S4, purifying the active components (component I and component G) again through an ODS column, specifically comprising:

[0055] The component I separated by elution with dichloromethane: methanol volume ratio of 20:1 on the normal phase vacuum column was subjected to reverse phase ODS pressure column separation, and 20V / V% methanol water, 40V / V% methanol water, 60V / V% methanol water, 80V / V% methanol water, 100V / V% methanol gradient elution were used in sequence, and then the components eluted with 80V / V% methanol water were separated and purified by high performance liquid chromatography, the mobile phase was 51V / V% chromatographic methanol water, the flow rate was 2mL / min, as Figure 3 , Figure 4 As shown, compound 1 (ID-1, 6.9 mg) was obtained at a retention time of 82 min;

[0056] The component G obtained by eluting with petroleum ether: ethyl acetate volume ratio of 1:1 is active, and is further separated by reverse phase ODS vacuum column, and gradient elution is performed using 5V / V% methanol water, 20V / V% methanol water, 40V / V% methanol water, 60V / V% methanol water, 80V / V% methanol water, and 100V / V% methanol in sequence. After the activity of each collected component is determined, the active component eluted with 20V / V% methanol water is separated and purified by high performance liquid chromatography, and the mobile phase is 60V / V% chromatographic methanol water, and the flow rate is 2mL / min. Figure 5 , Figure 6 As shown, compound 2 (GB-1, 31.5 mg) and compound 3 (GB-2, 25.8 mg) were obtained at retention times of 27 min and 48 min, respectively.

[0057] Example 4: Structural Identification of Compound 1, Compound 2 and Compound 3

[0058] Compound 1 (ID-1) is a yellow oil that is easily soluble in dichloromethane or methanol;

[0059] TLC analysis, when the mobile phase was dichloromethane:methanol volume ratio of 10:1, at 254nm, R f There are dark spots at 0.5, which become light pink spots after anisaldehyde coloration;

[0060] 1 H NMR (Table 1) showed that compound 1 had a high affinity for the aromatic protons [δ H 6.82 (2H, d, J = 8.8 Hz, H-2 / 6) and 7.08 (2H, d, J = 8.8 Hz, H-3 / 5)], indicating the presence of one para-substituted benzene ring in the structure. 1 H- 1 H COSY showed a CH2-CH-CH2 spin coupling fragment [δ H 3.93,3.81(H2-9) / 3.76(H-10) / 3.42(H2-11)]. Since all the methylene and methine groups in this fragment move to the low field due to the deshielding effect, it is speculated that each carbon atom is connected to an oxygen atom, which is a glycerol fragment. HMBC shows that H2-9[δ H 3.93 (1H, dd, J = 9.6, 4.3 Hz) and 3.81 (1H, dd, J = 9.6, 6.1 Hz)] and C-1 (δ C 156.6), proving that the C-1 position of the aromatic ring was replaced by glycerol. C 41.1), H3-8 and C-7 and C-4 (δ C 142.6), indicating that the para position of glycerol on the aromatic ring is replaced by ethyl. 3 Hybridized quaternary carbon, and the chemical shift is relatively high field (δ C 41.1), therefore, it is inferred that the compound has two completely symmetrical fragments connected to each other through C-8. Further comparison with the literature confirmed that compound 1 (ID-1) is bisphenol tetraisopropyl ether 2,2-[bis-4-(2,3-dihydroxypropoxy)phenyl]propane.

[0061] Compound 2 (GB-1) is a light yellow amorphous powder, which is easily soluble in methanol or dichloromethane;

[0062] When the mobile phase was dichloromethane:methanol in a volume ratio of 30:1, at 254 nm, R f There is a dark spot at 0.3, and after anisaldehyde coloration, it becomes a light purple spot with a yellow center and an outer circle;

[0063] 1The H NMR spectrum is similar to that of compound 1, except that the glycerol in compound 1 is missing and the C-1 position is replaced by a hydroxyl group. By comparing the data, compound 2 (GB-1) was determined to be bisphenol A (BPA).

[0064] Compound 3 (GB-2) is a colorless amorphous powder, which is easily soluble in methanol or dichloromethane;

[0065] When the mobile phase was dichloromethane:methanol in a volume ratio of 30:1, at 254 nm, R f There are dark spots at a value of 0.5, which become light yellow spots after anisaldehyde coloration;

[0066] 1 H NMR (Table 2) showed δ H 7.13-7.42 has 4 aromatic protons, suggesting the presence of 1,2-disubstituted benzene rings in the structure; δ H 6.66 (1H, d, J = 15.3 Hz, H-8) and 6.08 (1H, dt, J = 15.3, 6.4 Hz, H-9) suggest that there is a pair of trans-olefinic double bonds in the structure; a single peak methyl group (δ H 2.33, H3-7), indicating the presence of an aromatic methyl group in the structure; in addition, there are two methylene groups [δ H 2.57 (4H, m, H2-9 and H2-10)]. 13 C NMR showed 12 carbon atoms and 1 carbonyl carbon (δ C 179.6), 8 SP 2 Hybridized aromatic and olefinic carbons (δ C 125.7-136.6), 2 SP 3 Hybridized methylene (δ C 34.1,28.4) and 1 SP 3 Hybridized methyl (δ C 19.9). By comparing the microspectral 13 C NMR data showed that the compound was completely consistent with 5-(2-Methylphenyl)-4-pentenoic acid reported by Mukku et al. in 2002, so the compound was identified as 5-(2-methylphenyl)-4-pentenoic acid.

[0067] Table 1 Compound 1 and Compound 2 1 H NMR (600 MHz) and 13 C NMR data (DMSO-d6)

[0068]

[0069]

[0070] Table 2 Compound 3 1 H NMR (600 MHz) and 13 C NMR (150MHz) data (CDCl3)

[0071] Position <![CDATA[δ H ]]> <![CDATA[δ C ]]> 1 136.6,C 2 135.2,C 3 7.41,m 125.7,CH 4 7.15,m 126.2,CH 5 7.15,m 127.3,CH 6 7.15,m 130.3,CH 7 2.33,s <![CDATA[19.9,CH3 <!-- 7 -->]]> 8 6.66,d(15.3) 129.5,CH 9 6.08,dt(15.3,6.4) 129.2,CH 10 2.57,m <![CDATA[34.1,CH2]]> 11 2.57,m <![CDATA[28.4,CH2]]> 12 179.6,C

[0072] Example 5: Testing of anti-quorum sensing activity of pure compounds

[0073] The anti-quorum sensing activity of the three pure compounds 1-3 obtained was tested by the filter paper method and the pigment inhibition method. The results showed that in the filter paper method, bisphenol A (compound 2) and 5-(2-methylphenyl)-4-pentenoic acid (compound 3) had significant quorum sensing inhibition at 250 μg / piece. The diameters of the purple pigment inhibition zone for CV12472 were 27.8 mm and 28.5 mm, respectively. The pigment inhibition zone was a fuzzy circle, and there was no inhibitory effect on bacterial growth ( Figure 8 a); In the pigment inhibition test, at a concentration of 250 μg / mL, the two had no significant inhibition on the growth of Chromobacterium violaceum, the bacterial solution was turbid but basically no purple pigment was produced, and the pigment inhibition rates were 73.1% and 82.6% respectively ( Figure 8 b). The anti-biofilm activity of the two active compounds was tested and it was found that the inhibition rates of bisphenol A and 5-(2-methylphenyl)-4-pentenoic acid on the biofilm of the drug-resistant Staphylococcus aureus strain SA43300 at 150 μg / mL were 56.8% and 26.7%, respectively. Figure 8 c). Although no activity was detected in the activity test for bisphenol A tetraisopropyl ether (Compound 1), it is a prodrug of Compound 2 bisphenol A, and can be converted into active Compound 2 during bacterial metabolism and released in vivo through enzymatic or non-enzymatic conversion; therefore, it can also be used in the preparation of antibacterial drugs targeting the quorum sensing system.

[0074] The technical means disclosed in the present application are not limited to the technical means disclosed in the above implementation mode, but also include technical solutions composed of any combination of the above technical features. The present application has been disclosed in the above preferred embodiments, but it is not used to limit the present application. Any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present application.

Claims

1. An active metabolite of marine actinomycete HN2-56, characterized in that: The marine actinomycetes ( Streptomyces chumphonensis ) HN2-56, the depository is the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, zip code 100101; the deposit date is November 4, 2024, and the deposit number is CGMCC No.32495; The active metabolites of the marine actinomycetes include at least one of bisphenol A tetraisopropyl ether, bisphenol A and 5-(2-methylphenyl)-4-pentenoic acid, and the chemical structural formulas are as follows: ； ； 。 2. Use of the active metabolite of marine actinomycete HN2-56 as claimed in claim 1 in the preparation of antibacterial drugs targeting the quorum sensing system.

3. The method for preparing the active metabolite of marine actinomycete HN2-56 according to claim 1, characterized in that: include: Step S1, fermenting the marine actinomycete HN2-56 strain using an improved Gao's medium No. 1 to obtain a fermentation liquid; Step S2, filtering the fermentation liquid with gauze, and then extracting with ethyl acetate to obtain a crude extract; Step S3, loading the crude extract solid, separating with a normal phase silica gel vacuum column, using petroleum ether, petroleum ether: ethyl acetate volume ratio of 30:1, petroleum ether: ethyl acetate volume ratio of 20:1, petroleum ether: ethyl acetate volume ratio of 10:1, petroleum ether: ethyl acetate volume ratio of 1:1, dichloromethane: methanol volume ratio of 20:1, dichloromethane: methanol volume ratio of 10:1, dichloromethane: methanol volume ratio of 1:1, methanol for gradient elution, collecting the eluate, analyzing by TLC, combining similar components, and obtaining a total of 12 components A, B, C, D, E, F, G, H, I, G, K, L; performing anti-quorum sensing activity assay on the 12 collected components, and screening out two active components, which are component I separated by elution with dichloromethane: methanol volume ratio of 20:1 and component G eluted with petroleum ether: ethyl acetate volume ratio of 1:1; Step S4, subjecting the component I to reverse phase ODS pressure column separation, using 20 V / V% methanol water, 40 V / V% methanol water, 60 V / V% methanol water, 80 V / V% methanol water, and 100 V / V% methanol gradient elution in sequence; and then subjecting the component eluted with 80 V / V% methanol water to high performance liquid chromatography separation and purification to obtain compound 1; Step S5, subjecting the component G to reverse-phase ODS vacuum column separation, using 5 V / V% methanol water, 20 V / V% methanol water, 40 V / V% methanol water, 60 V / V% methanol water, 80 V / V% methanol water, and 100 V / V% methanol for gradient elution in sequence; and then subjecting the components eluted with 20 V / V% methanol water to high performance liquid chromatography separation and purification to obtain compounds 2 and compound 3 with different retention times.

4. The method for preparing the active metabolite of marine actinomycetes HN2-56 according to claim 3, characterized in that: In step S1, the composition of the improved Gao's No. 1 medium is: soluble starch 40.0 g / L, KNO3 4.0 g / L, NaCl0.5 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 0.01 g / L, pH 6.

5.

5. The method for preparing the active metabolite of marine actinomycetes HN2-56 according to claim 3, characterized in that: In step S1, the fermentation conditions are shaking culture at 28-32° C. for 10-20 days; preferably, shaking culture at 30° C. and 140 rpm for 15 days.

6. The method for preparing the active metabolite of marine actinomycetes HN2-56 according to claim 3, characterized in that: In step S2, extraction is performed with an equal volume of ethyl acetate.

7. The method for preparing the active metabolite of marine actinomycetes HN2-56 according to claim 3, characterized in that: In step S4, during the separation and purification by high performance liquid chromatography, the mobile phase is 51 V / V% chromatographic methanol water, and the flow rate is 2 mL / min.

8. The method for preparing the active metabolite of marine actinomycetes HN2-56 according to claim 3, characterized in that: In step S5, during the separation and purification by high performance liquid chromatography, the mobile phase is 60 V / V% chromatographic methanol water, and the flow rate is 2 mL / min.

9. The method for preparing the active metabolite of marine actinomycetes HN2-56 according to claim 3, characterized in that: After step S5, the method further includes: performing anti-quorum sensing activity assay on compound 1, compound 2, and compound 3.

10. The method for preparing the active metabolite of marine actinomycete HN2-56 according to claim 3 or 9, characterized in that: The anti-quorum sensing activity assay is selected from at least one of a filter paper agar diffusion method, a pigment inhibition method, and a biofilm method test.

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