Chaetomium sp., method for isolating secondary metabolites thereof and application

Abstract

The application provides a chaetomium globosum, a method for separating secondary metabolites of the chaetomium globosum and application, and belongs to the technical field of biological medicines. Arcopilus cupreus DGR-7 has been preserved in Guangdong Microbial Culture Collection Center on October 10, 2025, with a preservation number of GDMCC No: 67068; the application first utilizes rice medium to ferment A. cupreus strains, and 8 compounds are separated and purified from the strains, wherein 6 compounds are anthraquinones. In-vitro antibacterial experiments show that the chaetomium globosum Arcopilus cupreus The secondary metabolites of DGR-7, anthraquinones, have good bioactivity in inhibiting the growth of staphylococcus aureus, methicillin-resistant staphylococcus aureus, streptococcus salivarius, klebsiella pneumoniae, bauman acinetobacter, pseudomonas aeruginosa and escherichia coli.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a Chaetoceros bacterium, a method for isolating its secondary metabolites, and their applications. Background Technology

[0002] Chaetoceros ( Arcopilus cupreus Original name Chaetomium cupreum Ascomycota, Chaetomaceae ( Chaetomiaceae ), Arcopilus It belongs to the filamentous fungi family and is an effective biological control agent against some common plant pathogens, such as Fusarium oxysporum (Prunella vulgaris). Fusarium roseum Apple black spot fungus ( Venturia inequalis ), Inari spores ( Pyricularia oryzae ), Botrytis cinerea ( Botrytis cinerea These have excellent prevention and control effects.

[0003] Filamentous fungi have long been a focus of attention due to their role as an important source of secondary metabolites. Combining the number of biosynthetic gene clusters (BGCs) in the genomes of filamentous fungi with the number of genomes sequenced historically suggests that the secondary metabolites of filamentous fungi remain largely untapped.

[0004] Anthraquinones are the most abundant class of natural quinones. Anthraquinone components in traditional Chinese medicine possess a wide range of pharmacological effects. In experimental and clinical studies, their pharmacological effects have been demonstrated in numerous ways, including anti-inflammatory, antithrombotic, antitumor, antibacterial, and laxative / diuretic effects. Furthermore, the pharmacological applications of anthraquinone components are becoming increasingly widespread.

[0005] Currently regarding Arcopilus cupreus Research on the production of anthraquinone compounds, especially the application of high-yield, antibacterial "cage-like" bianthraquinone compounds, still lacks in-depth exploration, and existing research has not fully revealed the role of fungi. Arcopilus cupreus Potential uses. Summary of the Invention

[0006] The purpose of this invention is to provide a method for isolating Chaetoceros horn fungus and its secondary metabolites, and the application thereof. Arcopilus cupreus The anthraquinone compounds, secondary metabolites of DGR-7, exhibit good biological activity in inhibiting the growth of Staphylococcus aureus, methoxy-resistant Staphylococcus aureus, Streptococcus salivarius, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a type of chamomile. Arcopilus cupreusDGR-7 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 10, 2025, with accession number GDMCC No:67068.

[0009] The present invention also provides a method for isolating the aforementioned Chaetoceros hornfels. Arcopilus cupreus The method for processing secondary metabolites of DGR-7 includes the following steps:

[0010] The aforementioned Chaetoceros horn Arcopilus cupreus DGR-7 was inoculated into the culture medium for fermentation, extracted with ethyl acetate, concentrated, separated and purified to obtain the secondary metabolites;

[0011] The separation and purification methods include silica gel column chromatography and / or preparative high-performance liquid chromatography.

[0012] Preferably, the secondary metabolites include one or more of 1H-Indole-3-carbaldehyde, Isorhodoptilometrin, rugulosin D, Chrysophanol, Rugulosin A, Emodin, Endocrocin, and 1,3-diacyl glycerol.

[0013] Preferably, the culture medium is a rice solid culture medium, which is prepared by mixing rice and water at a mass-volume ratio of 1~2g:1.5~3mL.

[0014] Preferably, the fermentation temperature is 20~30℃ and the fermentation time is 28~32 days.

[0015] Preferably, the volume ratio of ethyl acetate to the fermentation product obtained by culture is 2-4:1; the number of extractions is 3-5; and the ethyl acetate layer is collected after extraction and concentrated under reduced pressure.

[0016] Preferably, the concentration method is vacuum concentration; the concentration temperature is 40~45℃, and the concentration vacuum degree is -0.05~-0.10MPa.

[0017] Preferably, the mobile phase system of the preparative liquid chromatography is 0.1% formic acid water-methanol; the mobile phase of the high performance liquid chromatography is 0.1% formic acid water-methanol, the column temperature is 30℃, the flow rate is 1mL / min, the injection volume is 10µL, and the sample concentration is 10mg / ml.

[0018] The present invention also provides the aforementioned Chaetoceros horn fungus. Arcopilus cupreusApplication of Rugulosin A, a secondary metabolite of DGR-7, in inhibiting the growth of Staphylococcus aureus, methoxy-resistant Staphylococcus aureus, Streptococcus salivarius, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, or Escherichia coli.

[0019] The present invention also provides the aforementioned Chaetoceros horn fungus. Arcopilus cupreus The use of Rugulosin A, a secondary metabolite of DGR-7, in the preparation of formulations that inhibit the growth of Staphylococcus aureus, methoxy-resistant Staphylococcus aureus, Streptococcus salivarius, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, or Escherichia coli.

[0020] The beneficial effects of this invention compared to the prior art are as follows:

[0021] This invention is the first to utilize rice culture medium for fermentation. A.cupreus The strain was identified, and eight compounds were isolated and purified from it, including six anthraquinone compounds. Content assays confirmed that the strain fermented on rice culture medium... A. cupreus The secondary metabolites are mainly anthraquinone compounds, and there is a high yield of compounds with a "cage-like" bianthraquinone dimer structure.

[0022] This invention, through in vitro antibacterial experiments, determined that *Chaetoceros*... Arcopilus cupreus The anthraquinone compounds, secondary metabolites of DGR-7, exhibit good biological activity in inhibiting the growth of Staphylococcus aureus, methoxysyl-resistant Staphylococcus aureus, Streptococcus salivarius, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli. In particular, compound 5 (Rugulosin A), with its cage-like bianthraquinone dimer structure, demonstrates broad-spectrum and potent antibacterial activity in in vitro antibacterial experiments, showing promising application prospects in the preparation of broad-spectrum antibacterial drugs.

[0023] Biological Preservation Instructions

[0024] The hornwort Arcopilus cupreus DGR-7 was deposited on October 10, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCCNo:67068. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 For HPLC analysis of Chaetoceros Arcopilus cupreus HPLC comparison of fermentation products obtained by DGR-7 on different culture media, where a is the Czapek's medium CK group, b is Czapek's medium, c is rice solid medium CK group, d is rice solid medium, e is Gao's No. 1 medium CK group, f is Gao's No. 1 medium, g is potato glucose broth medium CK group, and h is potato glucose broth medium.

[0027] Figure 2 This is a flowchart for the separation of compounds (secondary metabolites), where "Fr." represents "fraction", the number after Fr. represents the fraction number, PE represents petroleum ether, EA represents ethyl acetate, D represents dichloromethane, and M represents methanol.

[0028] Figure 3 The inhibition of fraction Fr.7 on Pseudomonas aeruginosa is shown, where W1 is vancomycin, L1 is chloramphenicol, and DM is fraction Fr.7.

[0029] Figure 4 The liquid phase diagram for the preparation of compound 3 is shown below.

[0030] Figure 5 The liquid phase diagram for the preparation of compound 5 is shown below.

[0031] Figure 6 The liquid phase diagram for the preparation of compound 7;

[0032] Figure 7 For compound 1 1 H NMR spectrum;

[0033] Figure 8 For compound 1 13 C NMR spectrum;

[0034] Figure 9 For compound 2 1 H NMR spectrum;

[0035] Figure 10 For compound 2 13 C NMR spectrum;

[0036] Figure 11 For compound 3 1 H NMR spectrum;

[0037] Figure 12 For compound 3 13 C NMR spectrum;

[0038] Figure 13 For compound 5 1 H NMR spectrum;

[0039] Figure 14 For compound 5 13 C NMR spectrum;

[0040] Figure 15 For compound 7 1 H NMR spectrum;

[0041] Figure 16 For compound 7 13 C NMR spectrum;

[0042] Figure 17 This is the high-resolution mass spectrum of compound 3;

[0043] Figure 18 This is the high-resolution mass spectrum of compound 5;

[0044] Figure 19 This is the high-resolution mass spectrum of compound 7;

[0045] Figure 20 The structural formulas of anthraquinone compounds 1-8 are shown.

[0046] Figure 21 strain A.cupreus HPLC chromatograms of fermentation products and mixed reference standards, where the black line represents the test sample, the red line represents the reference standard, 2 is Isorhodoptilometrin, 3 is Rugulosin D, 4 is Chrysophanol, 5 is Rugulosin A, 6 is Emodin, and 7 is Endocrocin.

[0047] Figure 22 The inhibitory effect of compound 5 on the growth of Escherichia coli, Staphylococcus aureus, and Streptococcus salivarius was determined by the filter paper disc agar diffusion method. From left to right, the bacteria are Escherichia coli, Staphylococcus aureus, and Streptococcus salivarius. In this diagram, HSL-5 represents compound 5, methanol (a) represents the negative control, and kanamycin and chloramphenicol (a) represent the positive controls, respectively.

[0048] Figure 23 This is a photograph of compound 5 inhibiting the growth of Staphylococcus aureus. Detailed Implementation

[0049] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0050] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0051] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0052] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0053] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0054] This invention provides a type of chamomile. Arcopilus cupreus DGR-7 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 10, 2025, with accession number GDMCC No:67068.

[0055] The present invention also provides a method for isolating the aforementioned Chaetoceros hornfels. Arcopilus cupreus The method for processing secondary metabolites of DGR-7 includes the following steps:

[0056] Chaetoceros Arcopilus cupreus DGR-7 was inoculated into the culture medium for fermentation, extracted with ethyl acetate, concentrated, separated and purified to obtain the secondary metabolites;

[0057] The separation and purification methods include silica gel column chromatography and / or preparative high-performance liquid chromatography.

[0058] In this invention, the secondary metabolites preferably include one or more of 1H-Indole-3-carbaldehyde, Isorhodoptilometrin, rugulosin D, Chrysophanol, Rugulosin A, Emodin, Endocrocin, and 1,3-diacyl glycerol; the culture medium is preferably rice solid culture medium, and the preparation method of the rice solid culture medium is preferably: mixing rice and water at a mass-volume ratio of 1~2g:1.5~3mL, more preferably 1.4~1.8g:2~2.5mL, and even more preferably 1.5g:2.4mL; the mixing is preferably carried out in a 1L Erlenmeyer flask, and the mixture is preferably autoclaved after mixing; the fermentation temperature is preferably 20~30℃, more preferably 24~28℃, and even more preferably 25℃; the fermentation time is preferably 28~32d, more preferably 30d; the ethyl acetate... The volume ratio of ethyl acetate to the fermentation product obtained from the culture is preferably 2-4:1, more preferably 3:1; the number of extractions is preferably 3-5 times, more preferably 4 times; after extraction, the ethyl acetate layer is preferably collected and concentrated under reduced pressure; the concentration method is preferably reduced pressure concentration; the concentration temperature is preferably 40-45℃, more preferably 42℃; the vacuum degree of the concentration is -0.05 to -0.10 MPa; the silica gel used in the silica gel column chromatography is preferably 300-400 mesh silica gel; the mobile phase system of the preparative liquid chromatography is preferably 0.1% formic acid water-methanol; the chromatographic column used in the preparative liquid chromatography is preferably 20 ID×250mm, 5C18-AR-Ⅱ; the mobile phase of the high performance liquid chromatography is preferably 0.1% formic acid water-methanol, column temperature 30℃, flow rate 1mL / min, injection volume 10µL, sample concentration 10mg / ml; the chromatographic column used in the high performance liquid chromatography is preferably a C18 column.

[0059] The present invention also provides the aforementioned Chaetoceros horn fungus. Arcopilus cupreus Application of Rugulosin A, a secondary metabolite of DGR-7, in inhibiting the growth of Staphylococcus aureus, methoxy-resistant Staphylococcus aureus, Streptococcus salivarius, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, or Escherichia coli.

[0060] The present invention also provides the aforementioned Chaetoceros horn fungus. Arcopilus cupreus The use of Rugulosin A, a secondary metabolite of DGR-7, in the preparation of formulations that inhibit the growth of Staphylococcus aureus, methoxy-resistant Staphylococcus aureus, Streptococcus salivarius, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, or Escherichia coli.

[0061] Example 1

[0062] Chaetoceros Arcopilus cupreus The DGR-7 strain was isolated from 20-year-old cinnamon plants collected on November 9, 2020, from a cinnamon planting base in Tanbin Town, Luoding City, Yunfu City, Guangdong Province (E: 111°22′54.74″, N: 22°49′27.66″, altitude 151 m). It was accurately identified by molecular biology (18S rDNA sequencing, ITS identification, whole genome sequencing) and morphology. It is preserved in the -80℃ freezer of Laboratory 110 of the School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, and the Guangdong Provincial Microbial Culture Collection Center.

[0063] Chaetoceros Arcopilus cupreus The DGR-7 strain was inoculated onto PDA plates (PDA medium preparation method: 39g of oxoid cm0139b potato dextrose agar (purchased from Thermo Fisher) was dissolved in pure water, and the volume was adjusted to 1L. After autoclaving at 121℃ for 20min, 10mL was dispensed into each plate). The plates were then incubated in a 28℃ incubator. When the strain had grown to 2 / 3 of the plate, a portion of the mycelium was picked using the mycelial tip purification method and inoculated into a new PDA plate. The culture was continued until the entire plate was covered.

[0064] Example 2

[0065] Suitable culture media were selected for fermentation of the strain. Potato glucose broth (PDB, purchased from Guangdong Huankai Microbial Technology Co., Ltd.), Gao's No. 1 medium, Czapek's medium, and rice solid medium were used for fermentation. The fermentation conditions for the first three liquid media were 130 rpm and 28℃ for 30 days, while the fermentation conditions for the rice solid medium were static incubation at 28℃ for 30 days. The obtained fermentation products were analyzed by HPLC. The specific methods are as follows:

[0066] Rice and pure water were mixed at a mass-to-volume ratio of 1g:1.5ml and placed in an 800mL Erlenmeyer flask. The mixture was then sterilized in an autoclave at 121℃ for 30 minutes. After the culture medium cooled to room temperature, it was placed in a clean bench and sterilized under ultraviolet light for 20 minutes to obtain sterilized rice solid culture medium.

[0067] Pour the prepared PDB medium into 250mL Erlenmeyer flasks, 150mL per flask, and autoclave at 121℃ for 30min. After the medium has cooled to room temperature, place it in a laminar flow hood and sterilize under UV light for 20min. Pick the activated Chaetoceros horn fungi. Arcopilus cupreus DGR-7 strain mycelium blocks were placed in PDB medium and incubated in a shaker at 130 rpm and 28°C for 3 days to obtain seed culture. The 3-day cultured Chaetoceros horn was then... Arcopilus cupreusDGR-7 strain seed culture was inoculated at a volume ratio of 20% into sterilized rice solid medium, potato glucose broth medium, Gao's No. 1 medium, and Czapek's medium, respectively, and fermented indoors for 30 days to obtain fermentation products. Meanwhile, rice solid medium, potato glucose broth medium, Gao's No. 1 medium, and Czapek's medium without strain seed culture were used as control media (CK group).

[0068] The fermentation product was mixed with ethyl acetate at a volume ratio of 2:1, and ultrasonically extracted three times. The extract was concentrated under reduced pressure at 60℃ to obtain a paste, which was then evaporated to dryness to obtain the crude product. The dry weight of the crude product was weighed, yielding a total of 31.97 g of crude product. The crude product was redissolved in methanol to obtain an analyte with a concentration of 10 mg / mL. HPLC analysis was performed using a 10 µL injection volume, a column temperature of 30℃, a mobile phase of methanol-0.1% formic acid-water, and a flow rate of 1 mL / min. The results are as follows: Figure 1 As shown.

[0069] The effects of different culture media on the fermentation of the strain were compared and analyzed by HPLC. The results showed that a main peak could be observed when using rice solid culture medium for fermentation. Therefore, rice culture medium was selected for subsequent scale-up fermentation.

[0070] Example 3

[0071] The crude product (rice extract) obtained in Example 2 was separated using silica gel column chromatography. The procedure was as follows: the crude product was completely dissolved in ethyl acetate, and the solution was mixed with diatomaceous earth at a mass ratio of 1:2 and stirred evenly. A wet-packing column (solvent: petroleum ether) was used with a sample:300-mesh silica gel (1:35, m / m) and a column volume of 1.8 L. The mobile phase was eluted sequentially with petroleum ether:ethyl acetate = 50:1, 30:1, 20:1, 10:1, 5:1, 1:1, and methanol for three column volumes. Identical components were combined by HPLC or TLC analysis to obtain Fr.1~Fr.10 (e.g., ...). Figure 2 As shown, Figure 2 The document only shows the method flow for processing the substances involved in this invention; the other substances, which are only numbered, are unrelated to this invention and do not require detailed explanation.

[0072] The combined fractions were screened using the filter paper method, with the fraction exhibiting good antibacterial effects as the target for rapid separation of compounds. The results are as follows: Figure 3 As shown.

[0073] The results showed that among the fractions with good antibacterial activity screened by the filter paper disc method, fraction Fr.7 had good antibacterial activity against Pseudomonas aeruginosa, with an inhibition zone diameter of 8.99 cm.

[0074] Fr.2 (895.7 mg) was further subdivided using a smaller positive silica column and eluted with V (petroleum ether):V (ethyl acetate) ratios of 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, and 1:1 to give compound 4 (3.1 mg).

[0075] Fr.3 (238.8 mg) was further subdivided using a smaller positive silica column and eluted with V(dichloromethane):V(methanol) = 50:1, 30:1, 25:1, 20:1, 10:1 to give compound 6 (2.4 mg).

[0076] Fr.4 (265.4 mg) was purified by preparative liquid chromatography with gradient elution [V(methanol):V(water)=5%:95%→95%:5%, 30 min] at a flow rate of 10 mL / min to obtain compound 1 (λ=254 nm, RT=23.27 min, 6.3 mg) and compound 2 (λ=254 nm, RT=33.41 min, 7.6 mg).

[0077] Fr.7 (4.3859 g) was further subdivided using a smaller positive silica gel column. Dry loading was employed; 4.3859 g of the crude product was dissolved in methanol and then mixed thoroughly with diatomaceous earth at a mass ratio of 1:2. After complete grinding, the solvent was evaporated completely. Wet packing was used with 300-mesh silica gel. A certain amount of silica gel was weighed at a mass ratio of 1:35, fully swollen with petroleum ether, and then packed into a glass column with a column volume of 570 mL. Gradient elution was performed using dichloromethane and methanol at volume ratios of 100:0, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, and 50:50. Thin-layer chromatography was used for analysis, and fractions with similar characteristics were combined to obtain six components (Fr.7-1 to Fr.7-6).

[0078] Fr.7-3 (1.55 g) was subjected to ODS medium-pressure column chromatography [V(methanol):V(water) = 10%:90%, 30%:70%, 49%:51%, 68%:42%, 80%:20%, 100%, flow rate 25 mL / min] eluting 800 mL, and thin-layer chromatography was used for analysis. Fractions with the same composition were combined to finally obtain 5 fractions (Fr.7-3-1~Fr.7-3-5).

[0079] The fraction Fr.7-3-4 (136.95 mg) was further purified by preparative high performance liquid chromatography [V(methanol):V(0.1% formic acid water)=60:40, flow rate 10 mL / min] to prepare compound 3 (λ=210 nm, λ=254 nm, RT=32.6 min, 79.53 mg).

[0080] The fraction Fr.7-3-5 (1.2153g) was further purified by preparative high performance liquid chromatography (V(methanol):V(0.1% formic acid water)=70:30, flow rate 10mL / min) to prepare compound 5 (λ=210nm, λ=254nm, RT=21.4min, 23.5mg).

[0081] Fr.7-4 (1.33 g) was subjected to ODS medium-pressure column chromatography [V(methanol):V(water) = 20%:80%, 50%:50%, 65%:45%, 80%:20%, 100%, flow rate 25 mL / min], eluting 800 mL. Thin-layer chromatography was used for analysis, and fractions with the same composition were combined to finally obtain 4 fractions (Fr.7.4.1~Fr.7.4.4).

[0082] The fraction Fr.7-4-2 (90.58 mg) was further purified by preparative high performance liquid chromatography (V(methanol):V(0.1% formic acid water)=60:40, flow rate 10 mL / min) to prepare compound 7 (λ=210 nm, λ=254 nm, RT=24.4 min, 9.5 mg).

[0083] Meanwhile, 9g of crude product (rice extract) was subjected to ODS medium-pressure column chromatography [V(methanol):V(water) = 50%:50%, 60%:40%, 70%:30%, 80%:20%, 100%, flow rate 20mL / min], eluting 800mL to obtain 28 fractions (ZY.1~ZY.28). ZY.28 (1.15g) was subjected to silica gel column chromatography (300~400 mesh). The mixture was eluted with ethyl acetate at a ratio of V(petroleum ether):V(ethyl acetate) = (1:0, 40:1, 10:1, 1:1) and V(dichloromethane):V(methanol) = 40:1, yielding 8 fractions (ZY28-1~ZY28-8). ZY.28-1 (75 mg) was separated by preparative thin-layer chromatography with a ratio of V(petroleum ether):V(ethyl acetate) = 25:1 to obtain compound 8 (30 mg).

[0084] Experimental Example 1

[0085] The following experiments were conducted using an AVANCEIII 500MHz nuclear magnetic resonance spectrometer (purchased from Bruker Beyspin GmbH, Switzerland), an Orbitrap Exploris 120 ultra-high performance liquid chromatography-mass spectrometry system (purchased from Thermo Fisher Scientific), and a Q-Tof Micro high-resolution mass spectrometer (purchased from Waters, USA):

[0086] Compounds 1 and 2 obtained in Example 3 were dissolved in deuterated methanol (purchased from CIL, USA) and analyzed by 1H and 1C NMR spectra in NMR tubes. Compound 5 was dissolved in deuterated methanol and analyzed by 1H and 1C NMR spectra in NMR tubes, as well as by Q-Tof Micro high-resolution mass spectrometry. Compounds 3 and 7 were dissolved in deuterated dimethyl sulfoxide (purchased from CIL, USA) and analyzed by 1H and 1C NMR spectra in NMR tubes, as well as by Q-Tof Micro high-resolution mass spectrometry. The results are as follows: Figures 4 to 19 As shown.

[0087] The results showed that compound 1 was a white powder with the molecular formula C9H9NO. 1 HNMR (500MHz, Methanol-d4)δ9.85(1H,s,-CHO),8.07(1H,s),8.12(1H,dd,J=8.15,7.15Hz),7.45(1H,dd,J=7.75,7.9Hz),7.19–7.26(2H,m,H-5,H-6). 13 CNMR (126MHz, methanol-d4) δ 187.44 (-CHO), 139.73 (C-2), 120.11 (C-3), 125.71 (C-3'), 123.62 (C-4), 122.39 (C-5), 125.01 (C-6), 113.14 (C-7), 138.94 (C-7'). Based on C-10, 1H-10, and literature searches, compound 1 was confirmed as 1H-Indole-3-carbaldehyde.

[0088] Compound 2 is an orange-yellow powder, according to HRESIMS m / z: 313.0715 [MH]. - The molecular formula is C 17 H 14 O6 has 11 degrees of unsaturation in its molecule. 1 HNMR (500 MHz, Methanol-d4)δ7.62 (1H,d,J =1.6Hz), 7.17(1H,d,J = 2.4Hz), 7.15 (1H,d,J =1.6Hz), 6.54 (1H,d,J =2.3Hz), 4.05 (1H,m),2.81 (1H,d,J = 2.2Hz), 2.80 (1H,d,J =3.6Hz), 1.23 (3H,d,J=6.15Hz). 13CNMR (126MHz, methanol-d4) δ 163.32 (C-1), 125.94 (C-2), 150.75 (C-3), 122.10 (C-4), 134.56 (C-4a), 110.38 (C-5), 167.74 (C-6), 109.10 (C-7), 166.52 (C-8), 191.80 (C-9), 115.33 (C-9a), 183.31 (C-10), 136.86 (C-10a), 46.57 (C-1'), 68.96 (C-2'), 23.34 (C-3'). Based on the C-10, 1H-10, and literature search, compound 2 was confirmed as isorhodoptilometrin.

[0089] Compound 3 is a yellow powder. According to HRESIMS m / z: 581.1074 [M+Na]+, its molecular formula is C2. 30 H 22 O 11 The molecule contains 20 degrees of unsaturation. 1 HNMR(500MHz,DMSO-d6)δ11.81 (brs,9-OH),11.06(s,9-OH'),2.75(1H,d,J=5.4Hz,2-H),2.92(1H,d,J=5.1Hz,2'-H),4.28(1H,brs,3-H),4.57(1H,brs, 3'-H),3.48(1H,brs,4-H),3.75(1H,brs,4'-H),7.45(1H,s,6-H),7.42(1H,s,6'-H),7.22(1H,s,8-H),7.21(1H,s,8'-H); 13CNMR(126MHz,DMSO-d 6)δ178.1 / 198.9(C-1 / C-1'),55.8 / 63.5(C-2 / C-2'),70.2 / 69.0(C-3 / C-3'),48.1 / 44.1(C-4 / C -4'),53.7 / 64.0(C-5 / C-5'),120.5 / 120.1(C-6 / C-6'),148.5(C-7),124.0 / 123.8(C-8 / C-8'),1 61.0 / 161.1 (C-9 / C-9'), 114.5 / 113.4 (C-10 / C-10'), 184.7 / 192.3 (C-11 / C-11'), 106.8 / 74.6 (C-12 / C-12'), 193.1 / 192.9 (C-13 / C-13'), 132.3 / 133.5 (C-14 / C-14'), 21.6 / 21.6 (C-15 / C-15'). Based on carbon and hydrogen spectroscopy and literature search, compound 3 was confirmed as Rugulosin D.

[0090] Compound 4 is a yellow powder, and according to HRESIMS m / z: 252.8715 [MH]-, its molecular formula is C4. 15 H 10 O4 has 11 degrees of unsaturation in its molecule. 1 HNMR(500MHz,DMSO-d6)δ11.01(s,1H),10.93(s,1H),7.06(dd,J=10.5,9.5Hz,1H),6.98(d ,J=9.5Hz,1H),6.83(d,J=1Hz,1H),6.53(d,J=2.8Hz,1H),6.49(d,J=1Hz,1H),1.98(s,3H); 13 CNMR (126MHz, DMSO-d6) δ 191.0 (C-9), 180.8 (C-10), 161.4 (C-8), 161.0 (C-1), 148.8 (C-3), 137.0 (C-6), 133.2 (C-10a), 132.1 (C-4a), 124.0 (C-2), 123.5 (C-7), 120.2 (C-4), 118.8 (C-5), 115.3 (C-8a), 113.6 (C-9a), 21.3 (-CH3). Based on the C-10, 1H-10, and literature searches, compound 4 was confirmed as Chrysophanol.

[0091] Compound 5 is a yellow powder, and according to HRESIMS m / z: 565.1109 [M+Na]+, its molecular formula is C. 30 H 22 O 10The molecule contains 20 degrees of unsaturation. 1 HNMR(500MHz,Methanol-d4)δ7.10(2H,s,H-2 / H-2'),7.49(2H,s,H-4 / H-4'),3.28(2H,m,H-5 / H-5 '),4.53(2H,d,J=5.55Hz,H-6 / H-6'),2.85(2H,d,J=5.1Hz,H-7 / H-7'),2.43(6H,s,3-CH3 / 3'CH3). 13 CNMR(126MHz,Methanol-d4)δ162.86(C-1 / C-1'),124.95(C-2 / C-2'),149.39(C- 3 / C-3'),121.65(C-4 / C-4'),133.79(C-4a / C-4a'),49.25(C-5 / C-5'),70.05(C- The carbon and hydrogen spectra of compound 5 were 6 / C-6'), 59.44 (C-7 / C-7'), 107.71 (C-8a / C-8a'), 186.27 (C-9 / C-9'), 115.94 (C-9a / C-9'), 195.31 (C-10 / C-10'), 58.10 (C-10a / C-10a'), and 22.03 (3-CH3 / 3'-CH3). Based on carbon and hydrogen spectra and literature search, compound 5 was confirmed as Rugulosin A.

[0092] Compound 6 is a yellow powder, HRESIMS m / z: 268.5732 [MH], with the molecular formula C. 15 H 10 O5 has 11 degrees of unsaturation in its molecule. 1 HNMR(500MHz,DMSO-d6)δ12.01(s,1H),11.54(s,1H),11.21(brs,1H),7.38(s ,1H),7.10(s,1H),7.03(d,J=2.6Hz,1H),6.42(d,J=2.6Hz,1H),2.21(s,3H). 13CNMR (126MHz, DMSO-d6) δ 189.5 (C-9), 181.0 (C-10), 164.8 (C-3), 164.1 (C-1), 160.6 (C-8), 147.6 (C-6), 134.4 (C-4a), 131.9 (C-10a), 124.0 (C-7), 120.1 (C-5), 112.8 (C-8a), 108.6 (C-4), 108.5 (C-9a), 107.3 (C-2), 20.7 (CH3). Based on the C-10, 1H-10, and literature searches, compound 6 was confirmed as Emodin.

[0093] Compound 7 is an orange-red powder, and according to HRESIMS m / z: 313.03538[MH]-, its molecular formula is C7. 16 H 10 O7 has 12 degrees of unsaturation in its molecule. 1 HNMR(500MHz,DMSO-d6)δ6.59(1H,d,J=2.35Hz,H-2),7.09(1H,d,J=2.4Hz,H-4),7.43(1H,s,H-5),12.45(1H,s,8-OH),2.46(3H,s,H-11). 13 CNMR (126MHz, DMSO-d6) δ 164.48 (C-1), 108.08 (C-2), 165.11 (C-3), 108.24 (C-4), 134.89 (C-4a), 119.81 (C-5), 144.80 (C-6), 129.27 (C-7), 114.47 (8a), 188.73 (C-9), 109.45 (C-9a), 181.57 (C-10), 132.89 (C-10a), 20.81 (C-11), 167.75 (C-12). Based on the C-10, 1H-10, and literature searches, compound 7 was confirmed as Endocrocin.

[0094] Compound 8 is a colorless oil with the molecular formula C. 47 H 76 O5. 1 H NMR(500MHz,Methanol-d4) δ0.95(3H,brs,H-1)1.97(1H,t,J=7.45Hz,H-11),1.56(1H,s,H-13),2.43(2H,t,J= 6.55Hz,H-28), 1.26(2H,m,H-30,H-31), 1.21(2H,s,H-32,H-11), 5.01(1H,m,H-36).13 CNMR(126MHz,Methanol-d4)δ14.48(C-1), 14.51(C-2), 14.50(C-3), 23.62( C-4), 23.73(C-5), 24.18(C-6), 26.01(C-7), 26.01(C-8), 26.02(C-9), 26.5 6(C-10), 28.11(C-11), 28.15(C-12), 28.17(C-13), 30.15(C-14), 30.17(C- 15), 30.20(C-16), 30.25(C-17), 30.35(C-18), 30.38(C-19), 30.45(C-20), 3 0.47(C-21), 30.58(C-22), 30.61(C-23), 30.67(C-24), 30.70(C-25), 30.76 (C-26), 30.78(C-27), 30.80(C-28), 30.84(C-29), 32.66(C-30), 32.66(C-3 1), 33.06 (C-32), 33.07 (C-33), 34.82 (C-34), 77.02 (C-35), 128.71 (C-36), 129.01 (C-37), 129.09 (C-38), 130.82 (C-39), 130.90 (C-40), 175.85 (C-41). Based on carbon and hydrogen spectroscopy and literature search, compound 8 was confirmed as 1,3-diacyl glycerol.

[0095] The structural formulas of compounds 1 to 8 are as follows: Figure 20 As shown.

[0096] Experimental Example 2

[0097] The content of anthraquinone components in the crude ethyl acetate extract of the fungal fermentation product obtained in Example 2 was determined using a Shimadzu LC-2030C high-performance liquid chromatograph, with compounds 2, 3, 4, 5, 6, and 7 purified in Example 3 used as reference standards. A COSMOSIL analytical column (4.6 ID × 250 mm, 5C) was used. 18 -AR-Ⅱ), column temperature 30℃, HPLC conditions: 0.1% formic acid aqueous solution (A)-methanol (B) as mobile phase, gradient elution (0~5min, 40%~55%B; 5.01~30min, 62%B; 30.01~40min, 75%B; 40~55min, 75%~98%B), flow rate 1mL / min, detection wavelength 254nm, injection volume 10μL.

[0098] (1) Instrument precision experiment

[0099] The instrument accurately weighs 1 mg of the reference standard and completely dissolves it to 1 mg / mL. The solution is then injected six times consecutively using a high-performance liquid chromatograph. The RSD values ​​of the peak areas of the reference standard are calculated for each of the six measurements to confirm the instrument's precision.

[0100] (2) Plotting the standard curve

[0101] Six reference standards were precisely prepared into six solutions of different concentrations for each of the six reference standards. These solutions were then sequentially injected into a high-performance liquid chromatograph (HPLC) to detect the peak area of ​​the reference standard chromatographic peaks. The mass concentration (mg / mL) of the reference standard was set as the x-axis (X), and the corresponding peak area as the y-axis (Y), thus deriving the standard curve equation.

[0102] (3) Stability test

[0103] Six portions of the crude product from Example 3 were accurately weighed and prepared to a concentration of 1 mg / mL. These were then injected repeatedly at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h. The RSD values ​​of the peak areas from these six measurements were calculated to confirm the stability of the crude product solution within 24 h.

[0104] (4) Repeatability experiment

[0105] Six portions of the crude product from Example 3 were weighed and prepared to a concentration of 1 mg / mL. The six test samples were then sequentially analyzed by HPLC. The RSD values ​​of the peak areas from the six measurements were calculated to determine whether the assay method had good reproducibility.

[0106] (5) Spiking recovery experiment

[0107] Six portions of the test sample with known anthraquinone content, each weighing 5 mg accurately, were precisely added to the mixed reference standard. The six crude mixed products were then subjected to HPLC analysis sequentially, and the recoveries of the added reference standard were calculated.

[0108] (6) Calculate the content of anthraquinone compounds in the crude extract solution.

[0109] Content determination experiments revealed that six anthraquinone compounds... A. cupreus The content percentages in rice fermentation products are shown in Table 1 and... Figure 21 As shown in Table 2, the results of the methodological investigation are presented.

[0110] Table 1 A. cupreus Content of anthraquinone compounds in secondary metabolites of rice fermentation

[0111]

[0112] Table 2. Results of Methodological Investigation

[0113]

[0114] Experimental Example 3

[0115] Antimicrobial susceptibility testing strips were affixed to PDA plates. Each plate contained one crude product from Test Example 2, one negative control, and one positive control, with three parallel experiments performed. The test plates containing fungi were incubated at 28°C for 12 hours, and the size of the inhibition zone was measured using calipers and recorded. The average value was calculated. Results are as follows: Figure 22 As shown.

[0116] Simultaneously, the minimum inhibitory concentrations (MICs) of compounds 1-8 against Staphylococcus aureus, methoxyresistant Staphylococcus aureus, Streptococcus salivarius, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli were determined using the microbroth dilution method. Chloramphenicol and kanamycin were used as positive controls.

[0117] The test samples and positive control stock solutions were prepared using methanol or dimethyl sulfoxide to a concentration of 200 μg / mL, and the pathogenic bacteria were activated using LB broth solid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.). The cultures were incubated overnight at 37°C for 12 h. Aliquots of the pathogenic bacterial suspension were prepared in 96-well plates, divided into groups: sample groups diluted to various concentrations (containing culture medium, bacterial suspension, and test sample); positive control group (containing culture medium, bacterial suspension, and positive control, but no test sample); negative control group (containing culture medium and bacterial suspension, but no positive control or test sample); and blank control (containing only culture medium, without bacterial suspension, positive control, or test sample). Each sample was tested in triplicate and incubated at 37°C for 18 h. Bacterial growth was observed, and the absorbance at VU=600 nm was measured using a microplate reader. If the culture medium remained clear and the absorbance showed no significant change, the compound at that concentration inhibited bacterial growth. Conversely, it did not inhibit bacterial growth. Results are as follows: Figure 23 As shown.

[0118] The results showed that compound 5 inhibited the growth of Escherichia coli, Staphylococcus aureus, and Streptococcus salivarius, with inhibition zone diameters of 8.41 mm, 17.56 mm, and 13.57 mm, respectively.

[0119] The results showed that compound 5 could effectively inhibit the growth of Staphylococcus aureus, with a MIC value of 1.56 μg / mL; the positive control drug had a MIC value of 50 μg / mL.

[0120] In summary, this invention, guided by the principle of inhibiting the bioactivity of human pathogens, isolated and purified a series of anthraquinone components, among which compound 5 exhibits the best bioactivity. Compound 5 not only inhibits both Gram-positive and Gram-negative bacteria, but also, when fermented on rice culture medium, inhibits the activity of these bacteria. A. cupreus Compound 5 can be obtained in large quantities. Its stable and abundant availability, along with its broad-spectrum and strong growth-inhibiting effects against human pathogenic bacteria, demonstrates the great potential of compound 5 for development into a broad-spectrum antibiotic.

[0121] As can be seen from the above embodiments, the present invention provides a method for isolating Chaetoceros hornella and its secondary metabolites, as well as its applications. Arcopilus cupreus The anthraquinone compounds, secondary metabolites of DGR-7, exhibit good biological activity in inhibiting the growth of Staphylococcus aureus, methoxy-resistant Staphylococcus aureus, Streptococcus salivarius, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli.

[0122] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An isolated Chaetomium sp. Arcopilus cupreus DGR-7, characterized in that, the chaetomium Arcopilus cupreus DGR-7 was deposited with Guangdong Microbial Culture Collection Center on October 10, 2025, and the deposit number is GDMCC No: 67068; The method includes the following steps: [The text abruptly ends here, likely due to an incomplete sentence or a missing section.] Arcopilus cupreus DGR-7 was inoculated into a culture medium for fermentation, followed by ethyl acetate extraction, concentration, separation, and purification to obtain the secondary metabolites. The separation and purification methods included silica gel column chromatography and / or preparative high-performance liquid chromatography. The secondary metabolites included one or more of indole-3-carboxaldehyde, isothiazolinone, rhein D, chrysophanol, rhein A, emodin, endorphin, and 1,3-diacid-glycerol esters. The culture medium was a rice solid medium, prepared by mixing rice and water at a mass-to-volume ratio of 1–2 g: 1.5–3 mL. The fermentation temperature is 20–30℃, and the fermentation time is 28–32 days; the volume ratio of ethyl acetate to the fermentation product obtained from the culture is 2–4:1; the extraction is performed 3–5 times; the ethyl acetate layer is collected after extraction and concentrated under reduced pressure; the concentration method is reduced pressure concentration; the concentration temperature is 40–45℃, and the concentration vacuum degree is -0.05 to -0.10 MPa; the mobile phase of the preparative high-performance liquid chromatography is 0.1% formic acid-water-methanol, the column temperature is 30℃, the flow rate is 1 mL / min, the injection volume is 10 µL, and the sample concentration is 10 mg / mL.

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