A new antibacterial compound and its preparation method and application

Abstract

The application discloses a new antibacterial compound and a preparation method and application thereof, and the new antibacterial compound is obtained by fermenting endophytic fungi (Epicoccum latusicollum) under stress of pathogenic fungi (Fusarium oxysporum), filtering, discarding mycelia, enriching the fermentation liquor by using a macroporous adsorption resin, and adopting high performance liquid preparation separation, and the chemical name of the new compound is (6E,8E)-13-acetoxy-3-hydroxy-8,10,12,14-tetramethylhexadeca-6,8-dienoic acid. The new compound obtained by fermenting endophytic fungi under stress of pathogenic fungi has significant antibacterial activity, and the application provides a scientific basis for mining endophytic fungi antibacterial natural compounds based on stress effects.

Description

Technical Field

[0001] This invention relates to the application of microorganisms, and more particularly to a method and application for isolating novel antimicrobial compounds by using pathogenic bacteria to stress endophytic bacteria. Background Technology

[0002] Endophytic fungi are a class of fungi that live within plant cells or, at a certain stage of their life cycle, within plant tissues, without causing significant disease. They maintain a long-term symbiotic relationship with their hosts, producing a series of secondary metabolites to protect the host from various stresses. Stress may induce endophytic fungi to produce specific secondary metabolites. In nature, fungi are frequently challenged by various stressors, and as a physiological response, they may activate adaptive metabolic reprogramming to survive these stresses. Therefore, stress may activate certain silent genes in plant endophytic fungi, inducing unique metabolic pathways and thus producing specific secondary metabolites. Using pathogenic fungi to stress endophytic fungi to find natural products with potential antibacterial activity provides a new method for discovering novel antibacterial compounds. Summary of the Invention

[0003] The purpose of this invention is to provide a novel antibacterial compound, its preparation method, and its application.

[0004] A new antibacterial compound, named (6E,8E)-13-acetoxy-3-hydroxy-8,10,12,14-tetramethylhexadeca-6,8-dienoic acid, has the following structural formula: compound (1).

[0005]

[0006] The aforementioned novel antimicrobial compounds or their pharmaceutical salts are used in the preparation of antimicrobial drugs and are pharmaceutically acceptable carriers.

[0007] The novel antibacterial compound or its pharmaceutical salt is used as an antifungal agent against pathogenic fungi such as *Botryosphaeria dothidea*, *Botrytis cinerea*, *Colletotrichum capsici*, *Fusarium oxysporum*, *Gloeosporium musarum*, *Lasiodiplodia theobromae*, *Nigrospora oryzae*, *Rhizoctonia solani*, and *Sclerotinia sclerotiorum*; the novel antibacterial compound or its pharmaceutical salt is used as an antibacterial agent against pathogenic bacteria such as *Bacillus subtilis* and *Staphylococcus aureus*.

[0008] The method for preparing the new antimicrobial compound involves simultaneously inoculating the pathogenic fungus *Fusarium oxysporum* and the endophytic fungus *Epicoccum latusicollum* into a culture medium for fermentation to obtain a culture broth. The culture broth is then separated into fermentation broth and mycelium by filtration or high-speed centrifugation. The mycelium is discarded, and the fermentation broth is enriched by macroporous adsorption resin and then separated by high-performance liquid chromatography to obtain the new antimicrobial compound.

[0009] The aforementioned method for preparing the new antibacterial compound is as follows: the culture medium is potato glucose broth (PDB), and the culture parameters are 18–28℃, 160–220 r / min for 5–30 days, and the high-speed centrifugation speed is 10,000–12,000 r / min.

[0010] The aforementioned method for preparing the new antibacterial compound involves activating and culturing the pathogenic fungus Fusarium oxysporum and the endophytic fungus Epicoccum latusicollum before fermentation, and then storing them at 4°C for 5–30 days or directly inoculating them into the fermentation medium.

[0011] The aforementioned method for preparing the new antibacterial compound involves inoculating Fusarium oxysporum and Epicoccum latusicollum at a ratio of 1:1 to 1:5; and activating the culture by inoculating Fusarium oxysporum and Epicoccum latusicollum into potato dextrose agar (PDA) medium and culturing at 28±1℃ for 3–7 days.

[0012] The aforementioned method for preparing the novel antibacterial compound involves using a 50×500mm macroporous adsorption resin-packed chromatography column for enrichment. The resin is washed with anhydrous ethanol and then eluted with pure water to achieve equilibrium. The collected supernatant is loaded onto a sample and then washed with pure water until the eluent is nearly colorless. The eluent is then eluted with 80% methanol / water (v / v) until the eluent is nearly colorless. This portion of the eluent is collected and concentrated under reduced pressure to constant weight using a rotary evaporator at 50°C to obtain the crude product.

[0013] The aforementioned method for preparing the novel antibacterial compound involves the following steps: The crude product is dissolved in 10–50 mL of an aqueous solution containing 50% acetonitrile / 0.1% acetic acid, filtered through a 0.45 μm filter membrane, and then separated by high-performance liquid chromatography (HPLC). The mobile phase in the HPLC separation is an acetonitrile-0.1% acetic acid aqueous solution, with gradient elution for 50–90 min at a flow rate of 50–100 mL / min. The detection wavelengths are 210 nm and 254 nm. The separated liquid is detected and collected by HPLC. The target compounds are then combined sequentially, concentrated to a small volume by rotary evaporation, and repeatedly purified to obtain the pure compound.

[0014] Compared with the prior art, the present invention prepares and isolates compound (1) from the fermentation products of Epicoccum latusicollum under the stress of pathogenic fungus Fusarium oxysporum. Compound (1) has significant broad-spectrum antibacterial activity and can be used to prepare antibacterial drugs. It provides candidate compounds for the research and development of new antibacterial drugs and provides a scientific basis and new ideas for the development and utilization of natural active substances of plant endophytic fungi. Attached Figure Description

[0015] Figure 1 The structural formula of compound (1) is shown below;

[0016] Figure 2 For compound (1) 1 H-NMR spectrum;

[0017] Figure 3 For compound (1) 13 C-NMR spectrum and DEPT spectrum;

[0018] Figure 4 For compound (1) 1 H- 1 H COSY spectrum;

[0019] Figure 5 The HSQC spectrum of compound (1);

[0020] Figure 6 The HMBC spectrum of compound (1);

[0021] Figure 7 The NOESY spectrum of compound (1);

[0022] Figure 8 The HR-ESI-MS spectrum of compound (1);

[0023] Figure 9 The antibacterial activity of compound (1) is shown.

[0024] In the diagram, A represents inhibition of *Botryosphaeria dothidea*, B represents inhibition of *Botrytis cinerea*, C represents inhibition of *Colletotrichum capsici*, D represents inhibition of *Fusarium oxysporum*, E represents inhibition of *Gloeosporium musarum*, F represents inhibition of *Lasiodiplodia theobromae*, G represents inhibition of *Nigrospora oryzae*, H represents inhibition of *Rhizoctonia solani*, I represents inhibition of *Sclerotinia sclerotiorum*, J represents inhibition of *Bacillus subtilis*, and K represents inhibition of *Staphylococcus aureus*. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0026] Example 1:

[0027] The technical solution of the present invention is that the compound is named (6E,8E)-13-acetoxy-3-hydroxy-8,10,12,14-tetramethylhexadeca-6,8-dienoic acid, and its specific structural formula is as follows: compound (1).

[0028]

[0029] Furthermore, compound (1) was isolated from the metabolites of co-fermentation of the pathogen Fusarium oxysporum and the endophytic fungus Epicoccum latusicollum.

[0030] The present invention also relates to a method for isolating the compound (1), comprising the following steps:

[0031] a. The pathogenic fungus *Fusarium oxysporum* was inoculated into potato dextrose agar (PDA) medium for activation culture at 28±1℃ for 3–7 days. The PDA medium consisted of 200g potato, 20g glucose, 15–20g agar, and 1000mL distilled water at a natural pH. Seed blocks were obtained and stored at 4℃ for 5–30 days or used directly. The selected seed blocks were inoculated into potato dextrose liquid (PDB) medium to obtain the seed culture of *Fusarium oxysporum*.

[0032] b. Inoculate the endophytic fungus (Epicoccum latusicollum) into potato dextrose agar (PDA) medium for activation culture at 28±1℃ for 3–7 days to obtain seed culture blocks. Store the seed culture blocks at 4℃ for 5–30 days or use them directly. Select the seed culture blocks and inoculate them into potato dextrose liquid agar (PDB) medium to obtain the seed culture of the endophytic fungus (Epicoccum latusicollum).

[0033] c. Inoculate the seed culture or seed blocks of Fusarium oxysporum and endophytic fungus Epicoccumlatusicollum obtained in steps a and b into potato dextrose liquid medium (PDB) at an inoculation ratio of 1:1 to 1:5, place on a shaker, and incubate at 18–28°C and 160–220 r / min for 5–30 days to obtain the fermentation product.

[0034] d. Separate the fermentation product from step c into fermentation broth and mycelium by vacuum filtration or high-speed centrifugation at 10,000–12,000 r / min. Discard the mycelium and collect the supernatant for enrichment with macroporous adsorption resin. Specifically: First, pack a 50×500 mm chromatography column with macroporous adsorption resin, then wash the resin with anhydrous ethanol, followed by elution with pure water to achieve equilibrium; load the collected supernatant onto a sample, then wash with pure water until the eluent is nearly colorless; then elute with 80% methanol / water (v / v) until the eluent is nearly colorless, collect this eluent, and concentrate it to constant weight under reduced pressure using a rotary evaporator at 50°C to obtain the crude product.

[0035] e. The crude product obtained in step d is subjected to preparative high-performance liquid chromatography (HPLC) to obtain the pure compound. Specifically, the crude product is dissolved in 10–50 mL of an aqueous solution containing 50% acetonitrile / 0.1% acetic acid, filtered through a 0.45 μm filter membrane, and then preparatively separated by HPLC (mobile phase: acetonitrile-0.1% acetic acid aqueous solution, gradient elution for 50–90 min, flow rate: 50–100 mL / min, detection wavelengths: 210 nm and 254 nm). The separated liquid is detected and collected by HPLC, and the target analytes are combined sequentially and concentrated to a small volume by rotary evaporation. This purification process is repeated until the pure compound is obtained.

[0036] The potato glucose liquid culture medium (PDB) is composed of 200g peeled potatoes, 20g glucose, and 1000mL distilled water, with a natural pH.

[0037] The endophytic fungus *Epicoccum latusicollum* described in this invention was deposited on March 8, 2022, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC NO.40110, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The depositor requested the name, strain number, or symbol to be designated for its culture: *Epicoccum latusicollum* HGUP191049. Its ITS sequence has a GenBank accession number of MZ541971.1. Any strain with an ITS sequence similarity ≥98% is considered to be of the same species.

[0038] The pathogenic fungus *Fusarium oxysporum* described in this invention was isolated from the root rot disease of *Pseudostellaria heterophylla*, from the Plant Pathology Laboratory of Guizhou University, strain number: FO44. Its ITS sequence GenBank accession number is: MN538904.1. Strains with ITS sequence similarity ≥98% are considered to be of the same species.

[0039] Through experiments, this invention found that compound (1) had a minimum inhibitory concentration (MIC) of 15.6 μg / mL against *Botryosphaeria dothidea*, *Botrytis cinerea*, *Lasiodiplodia theobromae*, *Nigrospora oryzae*, and *Rhizoctonia solani*; and a MIC of 62.5 μg / mL against *Colletotrichum capsici*, *Sclerotinias clerotiorum*, *Gloeosporium musarum*, and *Fusarium oxysporum*. Furthermore, it also exhibited inhibitory activity against *Bacillus subtilis* and *Staphylococcus aureus*. These results indicate that compound (1) of this invention possesses significant broad-spectrum antibacterial activity.

[0040] Structural identification of compound (1)

[0041] Compound (1): White powder, with a quasi-molecular ion peak at m / z 381.2640 [MH] according to HR-ESI-MS data. - (C 22 H 37 The calculated value of O5 is 381.2746, and its molecular formula is determined to be C. 22 H 38 O5, with an unsaturation degree of 4. Compound (1) 1 The H-NMR spectrum showed three olefinic proton signals δ. H 5.66 (d, J = 15.6 Hz, 1H, H⁻⁷), 5.53 (dt, J = 15.6, 6.9 Hz, 1H, H⁻⁶), 5.19 (d, J = 9.2 Hz, 1H, H⁻⁹), two δ-proton signals of the oxymethyl group. H 4.56 (dd, J = 7.2, 5.1 Hz, 1H, H⁻¹³), 3.77 (tt, J = 8.0, 4.7 Hz, 1H, H⁻³), 5 aliphatic methylene hydrogen protons δ H2.26(dd, J = 14.8, 4.8 Hz, 1H, H⁻²) & 2.17(dd, J = 14.8, 8.2 Hz, 1H, H⁻²'), 2.17(m, 1H, H⁻⁵) & 2.06(m, 1H, H⁻⁵'), 1.43(m, 2H, H⁻⁴), 1.40(m, 1H, H⁻¹⁵) & 0.99(m, 1H, H⁻¹⁵'), 1.28(ddd, J = 13.1, 9.3, 3.3 Hz, 1H, H⁻¹¹) & 0.98(m, 1H, H⁻¹¹'), 3 aliphatic methine hydrogen protons δ H 2.48 (m, 1H, H-10), 1.77 (m, 1H, H-12), 1.63 (m, 1H, H-14), 6 methyl protons δ H 1.66(s,3H,H-20), 0.87(d,J=7.2Hz,3H,H-19), 0.83(t,J=7.2Hz,3H,H-16), 0.83 (t,J=7.2Hz,3H,H-22), 0.82(d,J=7.2Hz,3H,H-18), 0.81(d,J=7.2Hz,3H,H-17).

[0042] Compound (1) 13 C-NMR spectroscopy combined with DEPT spectroscopy revealed two carbonyl carbon signals δc 173.6 (C-1) and 170.4 (C-21), and one sp. 2 The hybrid quaternary carbon signal δc is 130.9 (C-8), with 3 sps. 2 Hybridized methylene carbon signals δc 137.1 (C-9), 134.6 (C-7), 127.4 (C-6); two oxygen-bound methylene carbon signals δc 81.0 (C-13), 66.7 (C-3); five aliphatic methylene carbon signals δc 42.9 (C-2), 38.0 (C-11), 36.9 (C-4), 28.4 (C-5), 23.6 (C-15); three aliphatic methylene carbon signals δc 35.0 (C-14), 31.3 (C-12), 29.4 (C-10); and six methyl carbon signals δc 19.7 (C-19), 16.9 (C-18), 15.3 (C-17), 12.4 (C-20), 11.0 (C-16), 11.0 (C-22).

[0043] According to J 6,7 (15.6Hz) The double bond between C-6 and C-7 was determined to be inverse (E) form. Based on the correlation signal between H-9 and H-7 in the NOESY spectrum, the double bond between C-9 and C-8 was determined to be inverse (E) form. 1 H- 1 The correlation signal in the H COSY spectrum is obtained as follows: Figure 4 The two structural segments are shown by the thick solid lines. In the HMBC spectrum, H-13 and H-22 show correlated signals to C-21, H-20 to C-9, C-8, and C-7, and H-3 and H-2 to C-1. Based on these findings, the planar structure of compound (1) is inferred as follows: Figure 1 As shown. A search by SciFinder Scholar did not find any literature reports on this compound. It was named (6E,8E)-13-acetoxy-3-hydroxy-8,10,12,14-tetramethylhexadeca-6,8-dienoic acid. This compound (1) is a new compound.

[0044] Table 1. Compound (1) 1 H-NMR and 13 C-NMR data (600 / 150MHz, DMSO-d6)

[0045]

[0046]

[0047] The antibacterial activity of compound (1) was tested using the micro-dilution method.

[0048] 1. Test reagents: The prepared compound (1) was dissolved in dimethyl sulfoxide (DMSO) to obtain a concentration of 0.5 mg / mL.

[0049] 2. The pathogenic fungi used in this experiment were *Botryosphaeria dothidea*, *Botrytis cinerea*, *Colletotrichum capsici*, *Fusarium oxysporum*, *Gloeosporium musarum*, *Lasiodiplodia theobromae*, *Nigrospora oryzae*, *Rhizoctonia solani*, and *Sclerotinia sclerotiorum*; the pathogenic bacteria were *Bacillus subtilis* and *Staphylococcus aureus*.

[0050] 3. Antifungal tests are as follows:

[0051] Activation of test fungi: Plant pathogenic fungi such as *Botryosphaeria dothidea*, *Botrytis cinerea*, *Colletotrichum capsici*, *Fusarium oxysporum*, *Gloeosporium musarum*, *Lasiodiplodia theobromae*, *Nigrospora oryzae*, *Rhizoctonia solani*, and *Sclerotinia sclerotiorum* were inoculated onto PDA medium in a clean bench and cultured in a constant temperature incubator at 28±1℃ for 3–7 days to serve as test fungi.

[0052] Take 1-3 mL of compound (1) reagent from step 1, filter it through a microporous membrane (0.22 μm) and set it aside. The antifungal test was conducted using the filter paper diffusion method: 6 mm diameter circular filter paper discs were made using a perforator, sterilized at 121℃ for 30 min, and then set aside. On a clean bench, the mycelial cake (diameter: 6 mm) of the test bacteria was inoculated onto a PDA plate (diameter: 9 cm). Sterile filter paper discs were placed at equal intervals along the other edge of the plate, and then the discs were soaked in 10 μL of compound (1) reagent (0.5 mg / mL). DMSO was used as a negative control. All plates were incubated at 28℃ for 2-7 days. The radial growth radius (R1) of the mycelium on the negative control plate and the radial growth radius (R2) of the mycelium on the experimental plate containing compound (1) reagent were measured. The inhibition rate (%) was calculated as (R1-R2) / R1×100%. A higher inhibition rate indicates a better antifungal effect on the test bacteria. The compound was serially diluted using a 2-fold dilution method and then subjected to an antifungal test. If the inhibition rate was close to 0, it indicated no antibacterial effect; this was the minimum inhibitory concentration (MIC). This process was repeated three times to determine the MIC value of the compound.

[0053] PDA medium composition: 200g potato (peeled), 20g glucose, 15-20g agar, 1000mL distilled water, natural pH.

[0054] 4. Antibacterial tests are as follows:

[0055] Activation of test bacteria: In a clean bench, Bacillus subtilis and Staphylococcus aureus were streaked onto nutrient agar (NA) medium and incubated at 37°C for 12–24 h. Single colonies were then picked and inoculated onto LB medium and incubated at 220 rpm at 37°C for 12–24 h to serve as test bacteria.

[0056] Take 1-3 mL of compound (1) reagent from step 1, filter it through a microporous membrane (0.22 μm) and set it aside. The antibacterial test was conducted using the filter paper diffusion method: 6 mm diameter circular filter paper discs were made using a perforator, sterilized at 121°C for 30 min, and then set aside. On a clean bench, the bacterial suspension was quickly added to the still-unsolidified NA medium (45-65°C). 10 mL of the bacterial suspension was added to every 100 mL of NA medium, shaken well, and quickly poured into a plate. After cooling, different culture media for the tested bacteria were obtained. A sterile filter paper disc (diameter: 6 mm) was placed in the center of the NA plate containing the tested bacteria, and then the disc was soaked in 10 μL of compound (1) reagent (0.5 mg / mL). DMSO was used as a negative control. The plates were placed in a 37°C incubator and incubated for 12-24 h. The inhibitory effect of compound (1) on the tested bacteria was then observed. When the diameter of the inhibition zone is greater than 7 mm, it indicates that the compound has antibacterial activity. The larger the diameter of the inhibition zone, the better the antibacterial effect on the tested bacteria. Compound (1) was diluted sequentially using a 2-fold serial dilution method and then subjected to antibacterial tests. If the diameter of the inhibition zone is less than or equal to 7 mm, it indicates that there is no antibacterial effect. The test was repeated three times to determine the MIC value of the compound.

[0057] NA medium composition: 10.0g peptone, 3.0g beef meal, 5.0g sodium chloride, 15.0g agar, 1000mL distilled water, pH 7.3±0.1.

[0058] LB medium composition: 10.0g peptone, 5.0g yeast extract, 10.0g sodium chloride, pH 7.0.

[0059] 5. Antifungal test results: The MIC values ​​of compound (1) against Botryosphaeria dothidea, Botrytis cinerea, Lasiodiplodia theobromae, Nigrospora oryzae, and Rhizoctonia solani were all 15.6 μg / mL; the MIC values ​​against Colletotrichum capsici, Sclerotinia sclerotiorum, Gloeosporium musarum, and Fusarium oxysporum were all 62.5 μg / mL.

[0060] 6. Antibacterial test results: Compound (1) has inhibitory activity against Bacillus subtilis and Staphylococcus aureus, with MIC values ​​of 125-250 μg / mL.

[0061] The above embodiments are merely preferred technical solutions of the present invention and should not be considered as limitations on the present invention. The embodiments and features described in these embodiments can be arbitrarily combined without conflict. The scope of protection of the present invention should be limited to the technical solutions described in the claims, including equivalent substitutions of the technical features described in the claims. That is, equivalent substitutions and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. A novel antibacterial compound, characterized in that: The compound was named (6E,8E)-13-acetoxy-3-hydroxy-8,10,12,14-tetramethylhexadeca-6,8-dienoic acid, and its specific structural formula is shown in compound (1). ； Compound (1).

2. The application of the novel antibacterial compound as described in claim 1, characterized in that: The novel antibacterial compound or its pharmaceutical salt is used as an antifungal agent for the preparation of pathogenic fungi such as *Botryosphaeria dothidea*, *Botrytis cinerea*, *Colletotrichum capsici*, *Fusarium oxysporum*, *Gloeosporium musarum*, *Lasiodiplodia theobromae*, *Nigrospora oryzae*, *Rhizoctonia solani*, and *Sclerotinias clerotiorum*; the novel antibacterial compound or its pharmaceutical salt is used as an antibacterial agent for the preparation of pathogenic bacteria such as *Bacillus subtilis* and *Staphylococcus aureus*.

3. The method for preparing the novel antibacterial compound as described in claim 1, characterized in that: The pathogen Fusarium oxysporum and the endophytic fungus Epicoccum latusicollum were simultaneously inoculated into a culture medium and fermented to obtain a culture broth. The culture broth was separated into fermentation broth and mycelium by vacuum filtration or high-speed centrifugation. The mycelium was discarded, and the fermentation broth was enriched by macroporous adsorption resin and then separated by high-performance liquid chromatography to obtain a new antibacterial compound. The culture medium is potato glucose broth (PDB), and the culture parameters are 18-28℃, 160-220 r / min for 5-30 days, and high-speed centrifugation at 10,000-12,000 r / min. The pathogen *Fusarium oxysporum* and the endophytic fungus *Epicoccum latusicollum* were activated and cultured before fermentation, and then stored at 4°C for 5–30 days or directly inoculated into the fermentation medium. The inoculation ratio of the pathogen *Fusarium oxysporum* and the endophytic fungus *Epicoccum latusicollum* is 1:1 to 1:5; the activation culture is carried out by inoculating the pathogen *Fusarium oxysporum* and the endophytic fungus *Epicoccum latusicollum* into potato dextrose agar (PDA) medium and culturing at 28±1℃ for 3 to 7 days. The macroporous adsorption resin enrichment was carried out by packing a 50×500 mm chromatography column with macroporous adsorption resin, washing the resin with anhydrous ethanol, and then eluting with pure water to equilibrate; the collected supernatant was loaded onto the sample, and then washed with pure water until the eluent was nearly colorless; then eluted with 80% methanol / water (v / v) until the eluent was nearly colorless, the eluent was collected, and concentrated under reduced pressure to constant weight at 50°C using a rotary evaporator to obtain the crude product; The high-performance liquid chromatography (HPLC) preparation and separation method involves dissolving the crude product in 10–50 mL of an aqueous solution containing 50% acetonitrile / 0.1% acetic acid, filtering through a 0.45 μm filter membrane, and then performing HPLC preparation and separation. The mobile phase in the HPLC preparation and separation method is an acetonitrile-0.1% acetic acid aqueous solution, with gradient elution for 50–90 min at a flow rate of 50–100 mL / min. The detection wavelengths are 210 nm and 254 nm. The separated liquid is detected and collected by HPLC, and the target compounds are combined sequentially. The mixture is then concentrated to a small volume by rotary evaporation and repeatedly purified to obtain the pure compound.

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