Application of iso-pasteuretin in preparation of medicine for treating diseases caused by methicillin-resistant staphylococcus aureus

Abstract

The invention discloses application of iso-pasteuretin in preparation of a medicine for treating diseases caused by methicillin-resistant staphylococcus aureus, and belongs to the technical field of biological medicine. According to the present invention, it is identified that the snowfield tea endophytic fungus 202109-Ts-F016 is the new Coprinopsis thamnolicola under coprinus pseudoratus, three monomeric compounds with anti-MRSA activity are separated from the secondary metabolite of the Coprinopsis thamnolicola, the three compounds can be adopted as the candidate inhibitors of the novel antibacterial target FtsZ, and the toxicity of the pasteurestin C and the iso-pasteurestin on the Vero cell is low. The invention provides an important basis and a material basis for the development of coprinus fungus metabolites and anti-MRSA new target drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of iso-pasteurestin in the preparation of a drug for treating diseases caused by methicillin-resistant Staphylococcus aureus. BACKGROUND

[0002] Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main causes of death due to bacterial infection, and is listed by WHO as one of the main pathogens threatening human health, with a mortality rate of up to 63.1%. Finding new anti-MRSA lead compounds is an urgent and long-term task to cope with the threat of drug-resistant bacteria. SUMMARY

[0003] The purpose of the present application is to provide the application of iso-pasteurestin in the preparation of a drug for treating diseases caused by methicillin-resistant Staphylococcus aureus, in order to solve the problems existing in the prior art.

[0004] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0005] One of the technical solutions of the present application is a compound iso-pasteurestin, which has the following structural formula:

[0006]

[0007] The second technical solution of the present application is the application of the compound iso-pasteurestin in the preparation of a drug for treating diseases caused by bacterial infection, including Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Listeria seeligeri, Listeria ivanovii, Listeria innocua, Listeria monocytogenes, Escherichia coli, Salmonella paratyphi A, Salmonella paratyphi B, Shigella flexneri, Klebsiella pneumoniae and drug-resistant Candida albicans.

[0008] The third technical solution of the present application is a snow tea endophytic fungus (Coprinopsis thamnoliicola) 202109-Ts-F016, which was deposited on July 11, 2025 at the China Center for Type Culture Collection, located in Wuhan, Wuhan University, China, with the accession number CCTCC NO:M 20251567.

[0009] The fourth technical solution of the present application is a microbial inoculant, which comprises the snow tea endophytic fungus 202109-Ts-F016.

[0010] The application discloses a snow tea endophytic fungus 202109-Ts-F016 or a microbial agent in the preparation of a drug for treating diseases caused by bacterial infection.

[0011] The application discloses a snow tea endophytic fungus 202109-Ts-F016 or a microbial agent in the preparation of the compound iso-pasteurestin.

[0012] The application discloses a snow tea endophytic fungus 202109-Ts-F016 or a microbial agent in the preparation of the compound iso-pasteurestin.

[0013] The application discloses a snow tea endophytic fungus 202109-Ts-F016 or a microbial agent in the preparation of the compound iso-pasteurestin.

[0014] Based on the above technical scheme, the application has the following technical effects:

[0015] The application identifies the snow tea endophytic fungus 202109-Ts-F016 as a new species of Coprinopsis thammoliicola, and three monomer compounds with anti-MRSA activity are separated from the secondary metabolites of Coprinopsis thammoliicola, including two known compounds pasteurestin C and illudin I and one new skeleton compound iso-pasteurestin, the three compounds can be used as candidate inhibitors of a new type of antibacterial target FtsZ, and pasteurestin C and iso-pasteurestin have low toxicity to Vero cells. The application provides an important basis and material basis for the development of metabolites of Coprinopsis fungi and new target drugs for anti-MRSA. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1Fig. 1 is a colony and microscopic morphology of the 202109-Ts-F016 strain; the scale size is 20 pm. Among them, a is the PDA plate culture of the 202109-Ts-F016 strain, b is the unstained hyphae diagram of the 202109-Ts-F016 strain, and c is the hyphae diagram of the 202109-Ts-F016 strain after cotton blue staining.

[0017] Figure 2 Fig. 5 is a phylogenetic tree of the 202109-Ts-F016 strain constructed based on ITS sequences.

[0018] Figure 3 Fig. 6 is the effect of PC, II and Iso-P at different action concentrations on the survival rates of HepG2 cells and Vero cells, respectively.

[0019] Figure 4 Fig. 7 is the survival rates of HepG2 cells and Vero cells treated by PC, II and Iso-P at MIC, respectively.

[0020] Figure 5 Fig. 8 is the survival rates of HepG2 cells and Vero cells treated by PC, II and Iso-P at MIC, respectively.

[0021] Figure 6 Fig. 9 is a GO enrichment analysis of differential genes.

[0022] Figure 7 Fig. 10 is a KEGG enrichment analysis of differential genes.

[0023] Figure 8 Fig. 11 is a PCA (a) and PLS-DA (b) analysis of MRSA metabolites in the active component treatment group and the control group.

[0024] Figure 9 Fig. 12 is a KEGG enrichment analysis of differential metabolites.

[0025] Figure 10 Fig. 13 is a Sankey diagram of the intersection path of the KEGG enrichment of the transcriptome and the metabolome. DETAILED DESCRIPTION

[0026] As used herein, "comprise", "include", "have", "contain", and the like, are open-ended terms, that is, meaning "including but not limited to".

[0027] The technical solutions described in the present application are conventional solutions in the art if not specifically stated, and the reagents or raw materials used are purchased from commercial channels or publicly disclosed if not specifically stated.

[0028] The present application provides a compound iso-pasteurestin, and the structural formula is as follows:

[0029]

[0030] The application also provides application of the compound iso-pasteurestin in preparation of a medicine for treating diseases caused by bacterial infection, wherein the bacteria include Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Listeria seeligeri, Listeria ivanovii, Listeria innocua, Listeria monocytogenes, Escherichia coli, Salmonella paratyphi A, Salmonella paratyphi B, Shigella flexneri, Klebsiella pneumoniae and drug-resistant Candida albicans.

[0031] The application also provides a Coprinopsis thamnoliicola 202109-Ts-F016, which is preserved in the China Center for Type Culture Collection on July 11, 2025, and the address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20251567.

[0032] The application also provides a microbial agent, which comprises the Coprinopsis thamnoliicola 202109-Ts-F016.

[0033] The application also provides application of the Coprinopsis thamnoliicola 202109-Ts-F016 or the microbial agent in preparation of a medicine for treating diseases caused by bacterial infection, wherein the bacteria include Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Listeria seeligeri, Listeria ivanovii, Listeria innocua, Listeria monocytogenes, Escherichia coli, Salmonella paratyphi A, Salmonella paratyphi B, Shigella flexneri, Klebsiella pneumoniae and drug-resistant Candida albicans.

[0034] The application also provides application of the Coprinopsis thamnoliicola 202109-Ts-F016 or the microbial agent in preparation of the compound iso-pasteurestin.

[0035] The application also provides a preparation method of the compound iso-pasteurestin, which comprises inoculating the Coprinopsis thamnoliicola 202109-Ts-F016 or the microbial agent into a rice medium, and fermenting to prepare the compound iso-pasteurestin.

[0036] In some specific embodiments, the preparation method of the rice medium is that rice and water are mixed at a mass ratio of 2:3, and high-pressure sterilization is performed; the fermentation condition is that 15 ml of seed liquid is inoculated into 200 ml of rice medium, and static culture is performed at room temperature for 45 days.

[0037] The application also provides a medicine for treating diseases caused by bacterial infection, comprising the compound iso-pasteurestin, the endophytic fungus 202109-Ts-F016 of Ledum palustre or the microbial inoculant.

[0038] The endophytic fungus 202109-Ts-F016 strain used in the embodiments of the application was preserved in the China Center for Type Culture Collection on July 11, 2025, at an address of Wuhan University, Wuhan, China, and a preservation number of CCTCC NO: M20251567.

[0039] Embodiment 1

[0040] 1. Test materials

[0041] The names of the pathogenic bacteria used in the embodiments of the application are shown in Table 1.

[0042] Table 1. Names of test pathogenic bacteria

[0043]

[0044]

[0045] 2. Strain identification

[0046] (1) Morphological study

[0047] Colony observation: the 202109-Ts-F016 strain preserved in a slope was transferred to PDA solid culture medium, and the colony characteristics were observed after incubation at 28°C for 7-10 days; microscopic observation: a small amount of mycelium was inoculated on a fresh PDA plate, a sterile cover glass was inserted into the PDA medium at an angle of 0.5 cm from the edge of the mycelium, and the cover glass covered with mycelium was harvested after 3-4 days, fixed on a carrier glass soaked in anhydrous ethanol, uniformly dyed with cotton blue dye, and observed under a microscope to record and measure the structural shape, size, attachments and color characteristics; fruit body induction culture: after initial identification as Coprinopsis by ITS sequence sequencing, the strain was preliminarily identified as Coprinopsis.

[0048] (2) Molecular biology

[0049] The 202109-Ts-F016 strain was transferred to PDA solid medium and incubated at 28°C for 7-10 days. The ITS and LSU sequences were amplified by ITS1 / ITS4 and LR0R / LR7, respectively. The DNA was electrophoresed on agarose gel to check the purity. After passing the quality control, the DNA was sequenced by Shanghai Sunway Biotech Co., Ltd. The sequencing results were compared with the sequences in the NCBI database to find the most similar strain sequences. The most similar sequences were downloaded as reference sequences. The MEGA7.0 software was used for sequence alignment and maximum likelihood method to construct the phylogenetic tree of the 202109-Ts-F016 strain.

[0050] 3 In vitro antibacterial activity screening of the strain

[0051] (1) Pathogenic bacteria activity screening

[0052] In table 1, a small amount of bacteria was scraped in physiological saline, and 0.5 McFarland turbidity was applied. The bacterial solution was inoculated in LB broth (2%), and incubated at 37°C for 8-12 hours. Endophytic fungi were activated by transferring the endophytic fungi to fresh PDA plates and incubating at 28°C for 7 days.

[0053] After subculture of the 202109-Ts-F016 endophytic fungus, 5 fungal cakes were punched with a 6mm puncher under sterile conditions, inoculated in 200mL PDB fermentation medium, and incubated at 28°C and 150r / min for 7 days. After fermentation, an equal volume of ethyl acetate was added for repeated extraction three times. The combined extract was rotary evaporated at 35°C to obtain the crude extract, which was stored at 4°C for standby.

[0054] The punch method was used to determine the antibacterial activity of the endophytic fungus fermentation broth crude extract on pathogenic bacteria and drug-resistant Candida albicans. Sterile physiological saline was used to prepare a bacterial suspension of 1×10 5 CFU / mL for standby. Nutrient agar and bacterial solution were mixed at a ratio of 20:1 and poured onto the plate and cooled for standby. The endophytic fungus fermentation broth crude extract was prepared into a solution with a concentration of 100mg / mL using dimethyl sulfoxide (DMSO) as the solvent. The agar plate was punched (6mm in diameter), 20μL of endophytic fungus fermentation broth crude extract was added to each hole, and each strain was repeated three times. The same amount of DMSO solvent was added as a blank control. Levofloxacin was used as a positive control for pathogenic bacteria, and the concentration was set at 0.5mg / mL. Flucytosine tablets were used as a positive control for drug-resistant Candida albicans, and the concentration was 5mg / mL. After incubation at a suitable temperature for 18-24 hours or 48 hours, the diameter of the inhibition zone was observed.

[0055] The minimum inhibitory concentration and the minimum bactericidal concentration of MRSA were determined by using the constant broth method. The crude extract of 202109-1-F016 endophytic fungus fermentation broth was re-dissolved with 3% dimethyl sulfoxide (DMSO), and the sample was diluted with sterile MH broth to a certain concentration gradient (2.000, 1.750, 1.500, 1.250, 1.000, 0.750, 0.500, 0.250 mg / mL). Different concentrations of samples were inoculated with 2% (V / V) of activated bacteria solution (10 5 CFU / mL), and placed in a 37°C constant temperature incubator for 24h. An equal volume of 3% DMSO was added instead of the extract as a blank control.

[0056] (2) Plate antagonism experiment of test strain and plant pathogenic fungi

[0057] Activation of pathogenic fungi: A small amount of pathogenic fungal mycelium was inoculated with a needle to potato sucrose agar medium, and incubated at 28°C for 48h.

[0058] The antibacterial activity experiment of plant pathogenic fungi was carried out by using the plate confrontation method. The fungus cake was prepared by punching the edge of the endophytic fungus colony with a sterile puncher (Φ = 6mm). The preparation of plant pathogenic fungus cake was the same as that of endophytic fungus. The plant pathogenic fungus was inoculated in the center of the PDA plate, and the endophytic fungus cake was inoculated at the edge of the plate for confrontation culture. The culture was carried out at 28°C for 7d, and the results were observed. The plate inoculated with only pathogenic fungus was used as a control (CK), and each treatment was set with 3 replicates. The radius (R) of the control group colony and the radius (r) of the pathogenic fungus colony growing towards the endophytic fungus were measured, and the inhibition rate ((R-r) / R×100%) was calculated. The average value of three replicates was taken.

[0059] 4 Fermentation extraction, activity-oriented separation and identification of strains

[0060] The mycelium of the endophytic fungus 202109-Ts-F016 was inoculated on a PDA plate and cultured at 28°C for 7 days. Ten fungal cakes were punched out with a 9 mm puncher and inoculated into 400 mL of PDB culture solution, which was shaken and cultured for 7 days to obtain a seed solution. 15 mL of the seed solution was inoculated into 200 mL of rice medium (rice and water were mixed at a mass ratio of 2:3 and autoclaved) and incubated at room temperature for 45 days. The fermented material was stirred with a glass rod, extracted with ethyl acetate three times and distilled water three times, and rotary evaporated to obtain 35 g of a crude extract. The 35 g of the crude extract was separated by normal phase silica gel column chromatography, eluted with dichloromethane:methanol, and the elution gradient was 100:1, 50:1, 20:1 and methanol in turn, and 21 fractions (Fr.1-Fr.21) were obtained. The 21 fractions were tested for antibacterial activity by the microbroth method using MRSA as the indicator bacteria, and Fr.8, Fr.9 and Fr.10 were screened out as having antibacterial activity, and the others had no antibacterial activity. The active fraction Fr.8 (3 g) was eluted with petroleum ether: ethyl acetate, and the elution gradient was 50:1, 25:1, 20:1, 10:1, 8:1, 6:1, 4:1, 3:1, 1:1 and ethyl acetate in turn, and 11 fractions (Fr.8.1-Fr.8.11) were obtained. Fr.8.1-Fr.8.11 were tested for antibacterial activity by the TLC-bioautography technique, and Fr.8.2-Fr.8.7 had antibacterial activity. The active fraction Fr.8.2 (900 mg) was eluted with petroleum ether: ethyl acetate, and the elution gradient was 15:1, 10:1, 8:1, 6:1 and ethyl acetate in turn, and 20 fractions (Fr.8.2.1-Fr.8.2.20) were obtained. Fr.8.2.1-Fr.8.2.20 were tested for antibacterial activity by the TLC-bioautography technique, and Fr.8.2.1-Fr.8.2.9 had antibacterial activity. The active fraction Fr.8.2.6 (20 mg) was subjected to gel column chromatography to obtain compound 1 (5 mg), which was verified for antibacterial activity by the TLC-bioautography technique and determined to be an antibacterial monomer compound. The active fraction Fr.8.2.12 (43 mg) was isocratically eluted with petroleum ether: ethyl acetate 6:1 to obtain compound 2 (5 mg), which was verified for antibacterial activity by the TLC-bioautography technique and determined to be an antibacterial monomer compound.Fr.9 (826 mg) was separated by normal phase silica gel column chromatography, eluted with petroleum ether: ethyl acetate, elution gradient was 10:1, 6:1, 4:1, 3:1, 1:1 and ethyl acetate in turn, 10 fractions (Fr.9.1~Fr.9.10) were obtained, Fr.9.1~Fr.9.10 were tested for antibacterial activity by TLC-bioautography technology, fractions Fr.9.3, Fr.9.4, Fr.9.5 had antibacterial activity, Fr.9.3 (20 mg) was subjected to preparative thin layer chromatography to obtain compound 3 (5 mg), compound 3 was verified for antibacterial activity by TLC-bioautography technology, and it was determined to be an antibacterial monomer compound. The total amount of Fr.10 fraction was small, so it was not further separated. The monomer compounds obtained by separation were determined by one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, mass spectrometry analysis and combined with literature to determine the chemical structure; the structure novelty of the compounds was determined by searching the Dictionary of Natural Products (DNP) and SciFinder Scholar databases.

[0061] 4Molecular docking to explore the potential of monomer compounds to bind to FtsZ

[0062] PC190723 is currently an FtsZ inhibitor verified by in vitro and in vivo experiments, and it is essential to identify compounds that can mimic the interaction mode of PC190723 to discover efficient FtsZ inhibitors. Secondly, considering that pasteurestin C, illudin I and iso-pasteurestin are all terpenoids, the FtsZ inhibitor totarol of terpenoids was selected to increase the comparability of the results. PC190723 and totarol were used as references in the molecular docking study to analyze the affinity and interaction mode of pasteurestin C, illudin I and iso-pasteurestin to FtsZ.

[0063] 5Effect of monomer compounds on the survival rate of HepG2 and Vero cells

[0064] CCK-8 method was used to test the toxicity of the compounds. Cells were seeded at a density of 1.5×10 4 in 96-well plates, and placed in an incubator until complete adhesion; the compound stock solution was diluted with DMEM high glucose medium to 4MIC, 2MIC, MIC, 0.5MIC, and slowly added to the cells at a volume of 100 μL per well, with 4 replicate wells in each group. After 24 h of culture, the supernatant was aspirated, and the prepared CCK-8 mixed solution was added to each well. After 1.5 h of incubation, the culture medium turned orange yellow, and the optical absorbance value (OD value) of each well was immediately measured at 450 nm wavelength on a microplate reader. The data was recorded and the results were calculated.

[0065] The calculation formula is: cell survival rate (%) = [(OD experimental group-OD blank group) / (OD control group-OD blank group)] x 100%.

[0066] 6The test data is expressed as mean ± standard deviation (Mean ± SD), and the data processing and analysis are carried out by using Graphpad prism and SPSS software. The statistical method is one-way analysis of variance. P<0.05 indicates significant difference, and P<0.01 indicates extremely significant difference.

[0067] 7Results

[0068] 7.1Strain identification

[0069] 202109-Ts-F016 strain was cultured on PDA medium at 28℃ constant temperature and dark for 7d, the mycelium was milky white, grew straight up, the central mycelium was relatively loose, the peripheral mycelium was gathered, and the colony was thick (Fig. 1a). Figure 1 Under the light microscope, the mycelium was bifurcated and branched, with septum (Fig. 1b-c). Figure 1

[0070] 7.2Molecular biology

[0071] In the BLASTn alignment of ITS sequence, the ITS sequence of strain 202109-Ts-F016 (GenBank: PV053245) and Coprinopsis alnivora F-296 (GenBank: PQ641272) had the highest sequence homology (94.48%), with 36 base differences; in the BLASTn alignment of LSU sequence, the LSU sequence of strain 202109-Ts-F016 (GenBank: PV055854) and Coprinus alcobae SZMC-NL-0767 (GenBank: HQ847122) had the highest sequence homology (99.22%), with 10 base differences. The phylogenetic tree analysis based on ITS sequence showed that strain 202109-Ts-F016 was clustered with Coprinopsis alnivora F-296 and Coprinus alcobae SZMC-NL-0767 (Fig. 2). Figure 2 ​), 202109-Ts-F016 formed a monophyletic branch with other members of Coprinopsis known before, with obvious evolutionary differences. Combined with the macro- and micro-morphological characteristics of 202109-Ts-F016, it was suggested that 202109-Ts-F016 was a new species unknown before. 202109-Ts-F016 was an endophytic fungus isolated from Thamnolia subuliformis, and thus was named as Coprinopsis thamnoliicola according to its host of origin. The new species has been registered in MycoBank, and the holotype was deposited in HMAS (HMAS 353933). ITS and LSU gene sequences have been submitted to GenBank database with accession numbers PV053245 and PV055854, respectively.

[0072] 7.3 In vitro antimicrobial activity of the strain

[0073] 202109-Ts-F016 showed a broad spectrum of antimicrobial activity against Gram-positive pathogenic bacteria, especially against S. aureus, MRSA and S. epidermidis, with extremely sensitive diameters of the inhibitory zone of 25.5 ± 0.7 mm, 22.4 ± 0.6 mm and 23.0 ± 0.6 mm, respectively. In addition, 202109-Ts-F016 showed moderate inhibition against the tested Listeria, and showed certain growth inhibition against drug-resistant C. albicans, and plant pathogenic fungi, Rhizoctonia solani and Botrytis cinerea. These indicated that endophytic fungus 202109-Ts-F016 had potential application prospects in the development of new types of antibacterial and antifungal drugs.

[0074] Table 2 Antimicrobial activity screening of the crude extract of the fermentation broth of endophytic fungus 202109-Ts-F016 from Thamnolia subuliformis

[0075]

[0076] Note: The diameter of the inhibitory zone (DIZ) ≤ 6 mm was judged as no antimicrobial activity; 6 < DIZ ≤ 8 mm, low sensitivity; 8 < DIZ ≤ 14 mm, moderate sensitivity; 14 < DIZ < 20 mm, high sensitivity; DIZ ≥ 20 mm, extremely sensitive.

[0077] Table 3 Results of plate antagonism test of the test strains with plant pathogenic fungi

[0078]

[0079] 7.4 Structure identification and antimicrobial activity of the compound

[0080] Two known compounds and one new compound were obtained and identified from the fermentation extract of strain 202109-Ts-F016, which were pasteurestin C (1), illudin I (2) and iso-pasteurestin (3), respectively. The minimum inhibitory concentrations against MRSA were 125 μg / mL, 125 μg / mL and 62.5 μg / mL, respectively.

[0081] The structural formulas of pasteurestin C, illudin I and iso-pasteurestin are as follows:

[0082]

[0083] Compound 3, iso-pasteurestin, white oil, easily soluble in dichloromethane, developed on silica gel thin layer plate with petroleum ether-ethyl acetate (1:1), the Rf value was 0.156, no obvious dark spots were observed under UV lamp 254 nm, and pink spots were presented after heating with 10% H2SO4 in EtOH solution. ESI-MS m / z 250.33 [M+H]+, the molecular formula was determined as C 15 H 22 O3, unsaturation degree was 5. According to the hydrogen spectrum results, two hydroxyl proton signals [δH 5.20 (1H, d, J = 5.0 Hz, H-17), 4.83 (1H, s, H-16)], three singlet methyl proton signals [δH 1.42 (3H, s, H-12), 1.11 (3H, s, H-10), 0.97 (3H, s, H-11)], five methylene proton signals [δH 2.00 (2H, m, H-7), 2.00 (1H, m, H-14), 1.79 (1H, m, H-14), 2.00 (1H, m, H-13), 1.79 (1H, m, H-13), 3.69 (1H, m, H-15), 3.41 (1H, m, H-15), 1.88 (1H, d, J = 13.7 Hz, H-9), 1.51 (1H, d, J = 13.7 Hz, H-9)], and one singlet methine proton signal [δH 3.47 (1H, s, H-3)] were obtained. According to the carbon spectrum results, the compound had 15 carbon signals, including a group of double bond carbon signals (δC 144.2, 128.9), two oxygen-containing carbon signals (δC 87.6, 64.6), three methyl groups (δC 29.3, 28.4, 11.7), five methylene groups (δC 64.6, 45.1, 43.5, 31.4, 18.1), and four quaternary carbon signals (δC 144.2, 128.9, 56.3, 37.0).

[0084] One double bond carbon is one unsaturation, and the remaining 4 unsaturations suggest 4 rings, 1 H- 1 HCOSY showed one relevant fragment, two magnetically non-equivalent H on C-9. In the HMBC spectrum, H-3 was correlated with C-15, C-2, C-4, confirming C-3 was connected with C-2, C-4, H2-15 was correlated with C-2, C-4, C-5, C-3, confirming C-15 was connected with C-4, and C-2, C-15 were oxygenated carbons, so C-3, C-2, C-4, C-15 together formed a 5-membered oxygen ring; H a -13 was correlated with C-14, C-4, C-5, C-6, H a -14 was correlated with C-13, C-4, C-5, C-3, confirming C-13 was connected with C-14, C-5, and C-14 was connected with C-13, C-4, so C-13, C-14, C-4, C-5 together formed a 4-membered ring; H2-7 was correlated with C-1, C-2, C-8, C-9, confirming C-7 was connected with C-1 and C-8; H a -9 was correlated with C-2, C-8, C11, C-1, C-3, confirming C-9 was connected with C-8, C-2, i.e. C-1, C-2, C-8, C-9, C-7 together formed a five-membered ring, so there was one remaining saturation, C-10, C-11, C-12 were primary carbons and did not participate in ring formation, C-1, C-2, C-4, C-5, C-6 were quaternary carbons, and C3 was connected with C-2, C-4, so C-1, C-2, C-3, C-4, C-5, C-6 together formed a cyclohexene. H3-10 was correlated with C-8, C-7, H3-11 was correlated with C-8, C-9, confirming C-10, C-11 were connected with C-8, H3-12 was correlated with C-1, C-6, C-5, C-12 was connected with C-6. The relative configuration of compound 3 was further determined according to the ROESY spectrum, and its chemical structure was deduced as (2aS,8aR,9R)-6,6,8-trimethyl-1,2,6,7-tetrahydro-3H-2a,4a-methanocyclobuta[e]cyclopenta[b]oxepine-8a,9(5H)-diol, which was named iso-pasteurestin, and the nuclear magnetic data is shown in Table 4.

[0085] Table 4 Nuclear magnetic data of compound 3

[0086]

[0087] 7.5 Molecular docking to explore the potential of monomeric compounds to bind to FtsZ

[0088] The molecular docking results show that PC190723 forms multiple interactions with FtsZ, including hydrogen bonds with key residues Thr159, Asn192 and Gly47; halogen interactions with Leu131 and Gly130. In addition, PC190723 also forms a π-σ interaction with Ala48. Totarol forms hydrophobic interactions with Leu33, Ala38 and Leu95, and a hydrogen bond with Val234. These interactions provide important reference benchmarks for evaluating the binding potential of natural compounds. Among the evaluated compounds, iso-pasteurestin forms a hydrogen bond with Gly47 and a hydrophobic interaction with Ala48, showing the same interactions as PC190723, indicating that iso-pasteurestin can be a candidate compound for FtsZ inhibitors. Pasteurestin C forms hydrophobic interactions with Leu33, Ala38 and Leu95, which is the same as totarol, indicating that pasteurestin C also has the potential to inhibit FtsZ. Illudin I forms a hydrophobic interaction with Ala38, which is the same as totarol. In addition, it also forms hydrogen bonds with Ala38, Ile226 and Asn233, indicating that illudin I has the potential to inhibit FtsZ. In addition, among the evaluated compounds, the docking score is greater than -5 kcal / mol, proving good docking binding.

[0089] 7.6 Effect of monomer compounds on HepG2 and Vero cell survival rate

[0090] In the present application, at the minimum inhibitory concentration, the survival rates of HepG2 cells of pasteurestin C, illudin I and iso-pasteurestin are 86.76%, 68.02% and 76.7% respectively, and the survival rates of Vero cells are 82.39%, 88.05% and 83.38% respectively, indicating that pasteurestin C and iso-pasteurestin have lower toxicity to HepG2 cells and Vero cells, while illudin I has higher toxicity to HepG2 cells. In addition, with the increase of the concentration multiples of pasteurestin C and illudin I, the survival rates of HepG2 cells and Vero cells of the two respectively show a downward trend, but they do not decrease step by step with the increase of the concentration multiples. This is similar to the effect of iso-pasteurestin on the survival rate of Vero cells Figure 3pasteurestin C, illudin I and iso-pasteurestin, respectively, PC, II and Iso-P, hereinafter). Secondly, the difference of iso-pasteurestin at different concentrations on the survival rate of HepG2 cells was not statistically significant (P>0.05).

[0091] By comparing the difference of pasteurestin C, illudin I and iso-pasteurestin at the minimum inhibitory concentration on the toxicity of HepG2 cells and Vero cells, it was found that the compound pasteurestin C had the lowest toxicity on HepG2 cells (P<0.05), and the difference of the toxicity of the three compounds on Vero cells was not statistically significant (P>0.05). Figure 4 By comparing the difference of illudin I on the toxicity of HepG2 cells and Vero cells, it was found that illudin I had lower toxicity on HepG2 cells (P<0.05), and the difference of the toxicity of pasteurestin C and iso-pasteurestin between HepG2 cells and Vero cells was not statistically significant (P>0.05).

[0092] Figure 5

[0093] In summary, the fermentation broth extract of Coprinopsis thamnoliicola showed a wide range of antibacterial effect. For gram-positive pathogenic bacteria, especially for Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and epidermal Staphylococcus aureus, it reached extremely sensitive. In addition, it showed certain growth inhibition effect on drug-resistant Candida albicans, plant pathogenic fungi Rhizoctonia solani and Botrytis cinerea. It is shown that the extract of the endophytic fungus has potential application prospect in the development of new antibacterial and antifungal drugs, especially in dealing with MRSA infection, which may have important significance, which indicates that 202109-Ts-F016 can be used as a research object for further screening of anti-MRSA lead compounds.

[0094] Through bioactivity-guided separation and purification of the secondary metabolites of Coprinopsis thamnoliicola, three anti-MRSA active compounds were obtained, including two known compounds pasteurestin C, illudin I and one new skeleton compound iso-pasteurestin.

[0095] ​​In the molecular docking experiment, PC190723 is an FtsZ inhibitor verified by in vivo and in vitro experiments, and totarol is a promising terpenoid FtsZ inhibitor. The molecular docking results show the amino acid residues involved in the hydrogen bonds and hydrophobic interactions of their interaction with FtsZ, while pasteurestin C, illudin I and iso-pasteurestin also exhibit partial amino acid residues with the same mode of action, which indicates that they can mimic the interaction mode of PC190723 or totarol. Therefore, it is speculated that pasteurestin C, illudin I and iso-pasteurestin can be candidate molecules of FtsZ inhibitors. Therefore, compounds 1-3 have strong drug development potential.

[0096] In the cytotoxicity test, pasteurestin C and iso-pasteurestin have relatively low toxicity to HepG2 cells and Vero cells, while illudin I has high toxicity to HepG2 cells. In addition, the effects of pasteurestin C, illudin I on the survival rate of HepG2 cells and Vero cells and the effect of iso-pasteurestin on the survival rate of Vero cells show a trend of concentration-dependent decrease, and the decrease is not strictly stepwise. It is speculated that at a certain concentration, the effect of the compound on the cell may tend to be saturated, resulting in a decrease in the survival rate, and the cell may adapt to the compound, which will cause the survival rate to decrease at a slower rate at high concentrations. In addition, pasteurestin C, illudin I and iso-pasteurestin may also affect cell survival through multiple mechanisms, and since the dominant mechanism at different concentrations may be different, the cell survival rate may not decrease step by step with the multiplication of the concentration of the compound. Through the evaluation of in vitro cytotoxicity experiment, it is proved that pasteurestin C and iso-pasteurestin have low toxicity to HepG2 cells and Vero cells.

[0097] Example 2

[0098] Mechanism of action of active substances of snow tea endophytic fungus Coprinopsis thamnoliicola against MRSA

[0099] 1. Differential gene expression analysis after treatment of MRSA with active ingredients

[0100] (1) MRSA treatment and RNA extraction: 2% bacterial liquid (1x10 5CFU / mL and 3% active component (1 / 2 MIC) were added to 50 mL MHB broth and incubated at 37°C with shaking until the logarithmic growth phase. The bacterial pellet was collected by centrifugation, flash-frozen in liquid nitrogen for 10 s, thawed on ice, and then PBS buffer was added. After freezing and centrifugation, the bacterial pellet was collected and stored at -80°C for later use. The control group contained no active component. Three biological replicates were prepared for each group. RNA was extracted using TRIzol reagent. The concentration of the prepared RNA samples was determined by agarose gel electrophoresis, and purity and integrity were assessed using an Agilent 2100 analyzer. The test results are categorized as follows: Category A: Sample quality meets the requirements for library construction and sequencing, and the total quantity meets the requirements for one or more library constructions; Category B: Sample quality meets the requirements for library construction and sequencing, and the total quantity meets the requirements for one but less than two library constructions; Category C: Sample quality does not fully meet the requirements for library construction and sequencing, but library construction can be attempted; Category D: Sample quality does not meet the requirements for library construction and sequencing at all, and is not recommended for use. Samples classified as A or B have a library construction success rate of over 95%; samples classified as C or D are not recommended for use. After meeting the standards and completing the test, RNA samples should be stored at -80℃ for later use.

[0101] (2) Library preparation: The library was constructed using the VAHTS Universal V6 RNA-seq Library Prep (NRM604-01) kit. The library construction steps included: mRNA purification and fragmentation, first-strand cDNA synthesis, second-strand cDNA synthesis and purification of the second-strand products, end repair and 3'-terminal A addition, adapter ligation, and mRNA enrichment. After library construction, quality control was performed using the Qsep-400 method. Even if the peak patterns fully met the sequencing standards or had minor issues that did not affect sequencing, the libraries were acceptable for sequencing. The constructed libraries were sequenced using the Illumina HiSeq PE150 high-throughput sequencing platform.

[0102] (3) Transcriptome data analysis: After filtering, trimming, and base correction of the raw sequencing data, high-quality clean data was obtained. Reads containing rRNA were removed using the rRNA alignment method. The rRNA-filtered reads were then aligned with the Staphylococcus aureus ATCC 43300 genome using Bowtie2 software to obtain Mapped Data. Simultaneously, the quality of the transcriptome alignment results was assessed, including sequencing saturation, gene coverage, read distribution in different regions of the reference genome, and read distribution across different chromosomes. After passing the quality assessment, the expression levels of genes and transcripts were quantitatively analyzed using RSEM software. After obtaining the read counts, DESeq was used. 2The software analyzes the inter-sample gene differential expression, identifies the differentially expressed genes, and screens the differentially expressed genes between the treatment group and the control group according to the criteria of |log2FC|>1 (i.e., the fold change (FC) is more than 2 times) and FDR<0.05, and performs bioinformatics analysis such as GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) on the differential genes.

[0103] 2. Analysis of differential metabolites after treatment of MRSA with active ingredients

[0104] (1) MRSA treatment and extraction of intracellular metabolites: The MRSA bacteria were treated in the same manner as described above. After treatment, 25 mg of the sample was weighed into an EP tube at low temperature, homogenization beads were added, 500 μL of extraction solution (methanol: acetonitrile: water = 2:2:1) (V / V) containing an isotopically labeled internal standard mixture was added, vortexed for 30 s, and then transferred to a homogenizer for homogenization (35 Hz, 4 min). The sample was then transferred to ice water and ultrasonicated for 5 min, and this step was repeated 3 times. The sample was then placed at -40°C for 1 h, centrifuged at 4°C at 12000 rpm (centrifugal force 13800 (×g), R = 8.6 cm) for 15 min, and the supernatant was transferred to a sample vial for detection.

[0105] (2) LC-MS detection: A Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph was used to separate the target compounds by chromatography using a Waters ACQUITY UPLC BEH Amide (2.1 mm x 50 mm, 1.7 μm) liquid chromatography column. The liquid chromatography A phase was an aqueous phase containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia water, and the B phase was acetonitrile. The sample tray temperature was 4°C, and the injection volume was 2 μL. The Orbitrap Exploris 120 mass spectrometer was capable of collecting primary and secondary mass spectrometry data under the control of the control software (Xcalibur, version: 4.4, Thermo). The detailed parameters are as follows: sheath gas flow rate 50 Arb, Aux gas flow rate 15 Arb, capillary temperature 320°C, full MS resolution 60000, MS / MS resolution 15000, collision energy: SNCE 20 / 30 / 40, and spray voltage 3.8 kV (positive) or -3.4 kV (negative), respectively.

[0106] 3. Screening of potential antibacterial protein targets associated with the metabolome and transcriptome and molecular docking

[0107] In the transcriptomic results, the screened differential genes can be directly used as the target points of the active component against MRSA. Secondly, by comparing the differential metabolites and the metabolic pathways enriched by the differential genes in each omics result, the KEGG pathways significantly enriched by both of them can be the main pathways of the antibacterial activity of the active component. The differential genes in these pathways can be important antibacterial target points. Therefore, using the antibacterial compound isolated in this example as a ligand and the above protein target points as receptors, molecular docking was performed using ChemOffice, AutoDock software and Uniprot database (https: / / www.uniprot.org / ), to further analyze the potential target points and pathways.

[0108] 4Differential gene expression analysis after treatment of MRSA by the active component

[0109] (1) Quality inspection results of total RNA of MRSA bacteria

[0110] After the extraction of total RNA of MRSA bacteria in the control group (D) and the treatment group (S) was completed, agarose gel electrophoresis was used for concentration quantitative detection, and the purity and integrity of the RNA were detected using Agilent 2100. The results showed that the average concentration of the RNA was 644.01 ng / μL; the average total amount of the sample was 22.54 ug; and the RIN value was 10. According to the results, each group was determined to be A, and subsequent library construction and sequencing could be performed.

[0111] (2) Sequencing data quality and reference genome alignment results

[0112] The CleanReads were aligned with the specified reference genome using Bowtie2 software, and the results showed that the alignment rate was more than 90%, and the repeatability between samples was good, so that further differential gene screening and annotation analysis could be performed.

[0113] (3) Differential expression gene screening

[0114] Based on the screening criteria of FDR <0.05 and |log2FC| >1, a total of 88 genes differentially expressed under the treatment of the active component at the sub-inhibitory concentration (0.5 MIC) were screened, of which 35 differentially expressed genes (DEGs) were significantly up-regulated, and 53 DEGs were significantly down-regulated.

[0115] (4) GO enrichment analysis of differential genes

[0116] Among the 88 differentially expressed genes screened, 65 had annotation information in the GO database, accounting for 73.9%. Among them, 53 were biological processes (BP), involving cell lysis, ion transport, and nucleic acid metabolism and synthesis, etc. 6 were cell components (CC), located in the extracellular region, membrane raft, membrane microdomain, cell anatomy entity, and cell component. 30 were metabolic functions (MF), involving tryptophan synthetase activity, endopeptidase activity, peptidase activity, molecular entity transmembrane transporter activity, catalytic activity, and action on a protein, etc. Each functional classification was arranged in descending order of Gene ratio, and the top 5 were selected for visualization. Figure 6

[0117] (5) KEGG enrichment analysis of differential genes

[0118] The KEGG enrichment analysis of differential genes showed that among the 88 differential genes, 56 were annotated in the KEGG database, and 33 differential genes were annotated in the map pathway information, participating in 20 metabolic pathways, involving Staphylococcus aureus infection, pyrimidine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, ABC transporter, two-component system, tuberculosis, biofilm formation-Pseudomonas aeruginosa, phenazine biosynthesis, bacterial invasion of epithelial cells, alanine, aspartate and glutamate metabolism, nucleotide excision repair, sulfur metabolism, glycine, serine and threonine metabolism, arginine biosynthesis, nitrogen metabolism, glycerolipid metabolism, protein export, homologous recombination purine metabolism, quorum sensing. Among them, Staphylococcus aureus infection enriched the most genes, with 9 genes, accounting for 16.07% of the total annotated genes. Figure 7

[0119] 5 Differential metabolite analysis of MRSA treated with active components

[0120] (1) Total ion current plot of metabolites

[0121] After treating MRSA with active components, the ion peak spectrum of metabolites under positive ion mode (POS) and negative ion mode (NEG) can be obtained by LC-MS detection. Direct comparison shows that there are obvious differences in ion peaks between the treatment group and the control group under the two detection modes. Further peak detection, extraction, alignment, integration using R development internal program based on XCMS, and analysis combined with Biotree DB (V3.0) software, a total of 80 metabolites were identified.

[0122] (2) PCA and OPLS-DA analysis​​

[0123] Based on the intracellular metabolite composition and content in each sample, PCA and OPLS-DA analysis were performed. In the PCA analysis diagram, each scatter point represented a sample, and the color and shape of the scatter point represented different groups. The closer the scatter points were distributed, the more similar the types and contents of metabolites in the samples were. Conversely, the greater the overall metabolic level difference was. From Fig. 2a, it can be seen that the 3 samples in each group were well clustered, indicating that the homogeneity within the group was good, and the metabolite composition was similar. There was no overlap and cross between the active component treatment group and the control group, and they were effectively distinguished, which indicated that there were differences between the two groups. Compared with PCA, OPLS-DA obtained better grouping results, as shown in Fig. 2b. The parallel samples of the active component treatment group and the control group were clustered in a certain area, and the treatment groups of different active components were significantly distinguished, indicating that there were significant differences in metabolic composition between the active component group and the control group. Figure 8 Figure 8

[0124] (3) Screening of differential metabolites

[0125] Taking VIP>1 and P-value≤0.05 as the screening criteria, 41 differential metabolites were significantly changed after the active component acted on the MRSA bacteria, mainly involving amino acids, fatty acids and energy metabolism. Compared with the control group, 34 differential metabolites were down-regulated, which were acetylpiclinic acid, uridine diphosphate-N-acetylglucosamine, gamma-glutamylmethionine, N6-acetyl-L-lysine, gamma-glutamyl lysine, N6, N6, N6-trimethyl-L-lysine, lysine, pantothenic acid, glycolic acid, pyridoxol, pyridoxal, biotin, thiamine monophosphate, orotic acid (vitamin B13), 4-hydroxy-1-L-prolyl-L-proline, betaine aldehyde, methionine, valine, leucine, isoleucine, glutamic acid, arginine, phosphothreonine, orthophosphohomoserine, pentylphenylalanine, and phenylalanine. There were 7 differential metabolites up-regulated, which were uridine 5'-monophosphate, acetylglycine, cysteine, proline, glutamine, 2-oxoadipic acid, and indoleacetic acid.

[0126] (4) KEGG enrichment analysis of differential metabolites

[0127] ​​KEGG enrichment analysis of differentially metabolites revealed that 16 out of 41 differentially metabolites were annotated in the KEGG database, participating in 40 metabolic pathways. These pathways involved metabolic pathways, microbial metabolism in different environments, cofactor biosynthesis, secondary metabolite biosynthesis, lysine degradation, amino acid biosynthesis, pyrimidine metabolism, carbon metabolism, 2-oxocarboxylic acid metabolism, interconversion of pentose and glucuronic acid, glyoxylic acid and dicarboxylic acid metabolism, C5-branched dicarboxylic acid metabolism, glycerophospholipid metabolism, glycine, serine and threonine metabolism, lysine biosynthesis, vitamin B6 metabolism, biotin metabolism, ABC transporters, and nucleotide metabolism. A bubble diagram was created by selecting the top 15 metabolic pathways (see [link to diagram]). Figure 9 ).

[0128] 6. Screening and molecular docking of potential antibacterial targets associated with metabolomics and transcriptomics

[0129] (1) The intersection of differentially metabolites and differentially expressed genes in the KEGG pathway and related proteins in the pathway

[0130] Twenty pathways were enriched in transcriptomics analysis, and 40 pathways were enriched in metabolomics analysis. Five KEGG pathways were jointly enriched: pyrimidine metabolism, glycine, serine, and threonine metabolism, ABC transporters, two-component systems, and quorum sensing, involving 15 differentially expressed genes: orotidine decarboxylase (pyrF), dihydroorotase (pyrC), aspartate carbamoyltransferase catalytic subunit (pyrB), carbamoyl phosphate synthase small chain (carA), thioredoxin reductase (pstS), and phosphate transport system permease protein (pstB). The presence of proteins involved in phosphate transport system permease (pstA), heme transport system ATP-binding protein (hrtA), teichoic acid transport permease (tagG), histidine protein kinase (saeS), response regulator (saeR), potassium ion transport ATPase KdpC subunit (kdpC), potassium ion transport ATPase ATP-binding subunit 1 (kdpB), tryptophan synthase α chain (trpA), tryptophan synthase β chain (trpB), and tryptophan synthase component II (trpG) suggests that these genes may be potential antibacterial targets for the active components against MRSA. Pathways and related genes co-regulated by metabolomics and transcriptomics, such as... Figure 10 As shown.

[0131] (2) Molecular docking

[0132] The transcriptomic analysis results show that 53 differential genes are down-regulated, combined with differential gene function annotation, involving 5 differential genes related to virulence factors: gamma-hemolysin component A (hlgA), gamma-hemolysin component B (hlgB), gamma-hemolysin component C (hlgC), histidine protein kinase (saeS), and response regulator (saeR); 4 differential genes related to biofilm formation: phosphate transport system permease protein (pstB), phosphate transport system permease protein (pstA), phosphate transport system permease protein (pstC), and phosphate-specific transport system auxiliary protein PhoU (phoU); 1 differential gene related to cell wall synthesis: tryptophan synthetase component II (tagG); 1 differential gene related to multidrug efflux pump (norB), 1 differential gene related to arginine metabolism regulation: Arginine repressor (argR), and 1 differential gene related to iron metabolism: iron-regulated surface determinant protein B (isdB). The above genes all belong to target points closely related to antibiosis. At the same time, the transcriptomic combined with metabolomic results reveal that the differential genes enriched in the intersection pathways of the two may be important potential target points of the active components against MRSA. Therefore, the anti-MRSA monomeric compounds isolated and identified in the present embodiment are used as ligands, and the potential protein target points are used as receptors to perform molecular docking. The results show that the above protein target points are all well combined with the three compounds (the binding energy is greater than 5 kcal, which is well combined). Among them, the catalytic subunit of aspartate carbamoyltransferase (pyrB) has the highest binding energy with compounds pasteurestin C and illudin I, and the phosphate-specific transport system auxiliary protein PhoU (phoU) has the highest binding energy with compound iso-pasteurestin.

[0133] In summary, the differential metabolites and differential genes are involved in the antibacterial mechanism of biofilm formation and cell wall synthesis, so it is speculated that the two antibacterial mechanisms may play a dominant role in the antibacterial process of the active components. In addition, the intersection analysis of the pathways enriched by the differential substances of the transcriptome and the metabolome shows that there are 5 intersection pathways, namely pyrimidine metabolism, glycine, serine and threonine metabolism, ABC transporter and two-component system and quorum sensing, indicating that these pathways may be important pathways for the active components to exert antibacterial effect on MRSA. The target points involved in these pathways are important target points for the antibacterial effect of the active components, including orotidine decarboxylase (pyrF), dihydroorotate dehydrogenase (pyrC), aspartate carbamoyltransferase catalytic subunit (pyrB), small chain of carbamoyl phosphate synthase (carA), thioredoxin reductase (pstS), phosphate transport system permease protein (pstB), phosphate transport system permease protein (pstA), heme transport system ATP binding protein (hrtA), teichoic acid transport permease protein (tagG), histidine protein kinase (saeS), response regulator (saeR), potassium ion transport ATPase KdpC subunit (kdpC), potassium ion transport ATPase ATP binding subunit 1 (kdpB), tryptophan synthetase alpha chain (trpA), tryptophan synthetase beta chain (trpB) and tryptophan synthetase component II (trpG). Molecular docking is performed on the potential and important antibacterial target points obtained by the present application, and the results show that each potential protein target point is well combined with the three compounds. In general, the mechanism of the active components to resist MRSA involves cell wall synthesis, biofilm formation, multidrug efflux pump, energy metabolism and iron metabolism, and the combined analysis of omics reveals that the active substances of Coprinopsis thamnoliicola mainly act on five important target points, namely phosphate transport system related proteins (pstB, pstA, pstC), regulatory proteins (phoU) and tryptophan synthetase (tagG), inhibit the cell wall synthesis of MRSA, and interfere with the biofilm formation to exert the antibacterial effect on MRSA. Phosphate uptake (Pst system) and tryptophan synthesis are essential pathways for bacterial survival, which exist widely in various pathogenic bacteria (such as Mycobacterium tuberculosis and Staphylococcus aureus), and few existing antibiotics target such pathways, and the clinical drug resistance rate is less than 1%, which indicates that the drug resistance mutation risk of compounds 1-3 is low.

[0134] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. For ordinary skilled users in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments are not required to be exhausted. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A compound iso-pasteurestin, characterized in that, The structural formula is as follows:

2. Use of the compound iso-pasteurestin according to claim 1 for the manufacture of a medicament for the treatment of diseases caused by bacterial infections, characterized in that, The bacteria include Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Listeria ivanovii, Listeria monocytogenes, Listeria innocua, Listeria seeligeri, Escherichia coli, Salmonella paratyphi A, Salmonella paratyphi B, Shigella flexneri, Klebsiella pneumoniae and drug-resistant Candida albicans.

3. An endophytic fungus (Coprinopsis thamnoliicola) 202109-Ts-F016, which is characterized in that, The strain was preserved in the China Center for Type Culture Collection on July 11, 2025, and the address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20251567.

4. A microbial inoculant, characterized in that, The endophytic fungus 202109-Ts-F016 of Leontice sibiricum L. in claim 3 is included.

5. The use of the snow tea endophytic fungus 202109-Ts-F016 of claim 3 or the microbial agent of claim 4 in the preparation of a drug for treating diseases caused by bacterial infection. The bacteria include Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Listeria ivanovii, Listeria monocytogenes, Listeria innocua, Listeria seeligeri, Escherichia coli, Salmonella paratyphi A, Salmonella paratyphi B, Shigella flexneri, Klebsiella pneumoniae and drug-resistant Candida albicans.

6. The use of the endophytic fungus 202109-Ts-F016 of Leontice sibiricum L. in claim 3 or the microbial agent in claim 4 in the preparation of the compound iso-pasteurestin in claim 1.

7. The method for preparing the compound iso-pasteurestin as described in claim 1, characterized in that, The endophytic fungus 202109-Ts-F016 of Leontice sibiricum L. in claim 3 or the microbial agent in claim 4 is inoculated into a rice culture medium to ferment and prepare the compound iso-pasteurestin.

8. A medicament for treating a disease caused by a bacterial infection, characterized by, The compound iso-pasteurestin in claim 1, the endophytic fungus 202109-Ts-F016 of Leontice sibiricum L. in claim 3 or the microbial agent in claim 4 is included.

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