Marine fungus-derived bisindole derivative and application thereof

Abstract

The invention relates to a marine fungus-derived bisindole derivative and application thereof. Bisindole derivatives having structures represented by compounds 1, 2, 3, 4: # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of secondary metabolites of marine fungi, and particularly relates to a bis-indole derivative derived from a marine fungus and its applications. Background Art

[0002] Marine microorganisms are recognized as a rich source of bioactive secondary metabolites. More and more researchers have turned their attention to marine microorganisms, especially marine fungi. The unique living environment in the ocean endows marine-derived fungi with unique metabolic pathways, enabling them to produce many secondary metabolites with novel structures and remarkable activities. In recent years, the discovered secondary metabolites from marine sources include various structural types such as polyketides, alkaloids, terpenoids, and lipids, many of which have biological activities such as cytotoxicity, anti-inflammation, and antibacterial properties, and there are also active compounds with agricultural antibacterial and insecticidal activities. The bioactive secondary metabolites derived from marine fungi are not only hot resources for the development of the pharmaceutical industry but also important resources for the research and development of new natural agricultural fungicides. The present invention provides a marine fungus Aspergillus terreus N4-9, its crude extract, secondary metabolites, and their applications. Summary of the Invention

[0003] The present invention provides a marine fungus N4-9, which is isolated from the rhizosphere soil of the mangrove plant Sonneratia caseolaris. The mangrove plant Sonneratia caseolaris comes from the Dongzhai Harbor Mangrove Nature Reserve in Haikou, Hainan, China. The marine fungus N4-9 of the present invention is characterized by the following strain preservation information: Preservation unit name: China General Microbiological Culture Collection Center; Preservation unit address: No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Preservation date: April 6, 2023; Preservation number: CGMCC No. 40555; Taxonomic name: Aspergillus terreus.

[0004] Another embodiment of the present invention provides a crude extract of the marine fungus N4-9, which is characterized by the preparation method of the crude extract, including the following steps:

[0005] (1) Culturing the marine fungus N4-9 in a strain culture medium to obtain a seed liquid;

[0006] (2) Inoculating the seed liquid obtained in step (1) into a fermentation medium for fermentation culture to obtain a fermented product;

[0007] (3) Separating the fermentation broth and the thalli in the fermented product obtained in step (2). The fermentation broth is extracted with organic solvent A 2-4 times, and after combining the extraction liquids, it is concentrated under reduced pressure to obtain a fermentation broth extract; the thalli are extracted with organic solvent B 2-4 times, and after combining the extraction liquids, it is concentrated under reduced pressure to obtain a thallus extract; the fermentation broth extract and the thallus extract are combined to obtain the crude extract.

[0008] In the above preparation method of the present invention, the culture conditions and fermentation conditions of the strain (such as parameters such as medium selection, temperature, time, etc.) are all conventional experimental operations in the art, and those skilled in the art can make reasonable selections according to the experimental situation. For the convenience of understanding the present invention, only one of the conventional operation methods is listed below: The strain medium in step (1) is a commonly used strain medium in the art, preferably PDB medium, and the formula (per liter of water) is preferably: glucose 20 g / L, potato 200 g / L, sea salt 30 g / L; The strain culture in step (1) is preferably shake flask culture, the temperature is preferably 35-38 °C, the shake flask rotation speed is preferably 120-150 r / min, and the culture time is preferably 2-4 d. The fermentation medium in step (2) is a commonly used fermentation medium in the art, which can be the same as or different from the strain medium in step (1), preferably PDB medium, and the formula (per liter of water) is preferably: glucose 20 g / L, potato 150-300 g / L, sea salt 30 g / L; The inoculum amount in step (2) is preferably 1-5%, the fermentation culture temperature is from room temperature to 38 °C, and the culture time is 28-30 d. The organic solvent A in step (3) is preferably one or more of ethyl acetate, dichloromethane, chloroform or ether; The organic solvent B is preferably one or more of methanol, ethanol, THF or acetone. After the fermentation broth extract and the cell extract are combined, they are optionally dried to obtain a crude extract.

[0009] Another embodiment of the present invention provides a bis-indole derivative, its tautomer or its pharmaceutically acceptable salt, characterized in that the bis-indole derivative has the structures shown in Compounds 1, 2, 3, and 4:

[0010]

[0011] Another embodiment of the present invention provides a preparation method of the above-mentioned bis-indole derivatives 1, 2, 3, and 4, characterized in that it comprises the following steps:

[0012] The crude extract of the marine fungus N4-9 is chromatographically separated to obtain bis-indole derivatives 1, 2, 3, and 4. The chromatographic separation is preferably one or a combination of several of reduced-pressure silica gel column chromatography, normal-phase silica gel column chromatography, reverse-phase silica gel column chromatography, gel column chromatography, HPLC, etc. The following preferred scheme is adopted: The crude extract of the marine fungus N4-9 is first subjected to reduced-pressure silica gel column chromatography, and petroleum ether-ethyl acetate is used as the eluent for gradient elution. The elution gradients are respectively 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 0:100. Two column volumes are collected for each gradient, and they are divided into 7 components according to the polarity. After the eluents obtained from the gradients 50:50 and 40:60 are combined and concentrated, they are subjected to Sephadex LH-20 gel column chromatography. The eluent is a mixed solvent of CH2Cl2 / MeOH, v / v, 1:1. Elution is carried out for 4-5 column volumes, and after reduced-pressure concentration, it is then subjected to reverse-phase column chromatography (ODS). The mobile phase is MeOH:H2O = 70:30 to obtain bis-indole derivatives 1, 2, 3, and 4. The obtained compounds 1-4 are optionally purified by semi-preparative HPLC. The ratios of the eluents or mobile phases described in the present invention are all volume ratios.

[0013] Another embodiment of the present invention provides a pharmaceutical composition, which is characterized in that the pharmaceutical composition uses the above-mentioned crude extract of the marine fungus N4-9, bis-indole derivatives 1, 2, 3, 4, their tautomers or their pharmaceutically acceptable salts as active ingredients. The pharmaceutical composition also includes pharmaceutically acceptable carriers, diluents or excipients. The dosage form of the pharmaceutical composition is selected from solid preparations, liquid preparations, semi-solid preparations, etc. The pharmaceutical composition is used for preventing and treating agricultural pathogenic bacteria, human pathogenic bacteria, and tumors.

[0014] Another embodiment of the present invention provides the application of the above-mentioned crude extract of the marine fungus N4-9, bis-indole derivatives 1, 2, 3, 4, their tautomers or their pharmaceutically acceptable salts in plant protection, especially in the prevention and treatment of agricultural pathogenic bacteria.

[0015] Another embodiment of the present invention provides the application of the above-mentioned marine fungus N4-9 in plant protection, especially in the prevention and treatment of agricultural pathogenic bacteria.

[0016] Another embodiment of the present invention provides the use of the above-mentioned marine fungus N4-9 or its crude extract, bisindole derivatives 1, 2, 3, 4, their tautomers or their pharmaceutically acceptable salts in the preparation of drugs for preventing and treating agricultural pathogenic bacteria, human pathogenic bacteria, and tumors. The agricultural pathogenic bacteria are selected from one or more of the pathogenic bacteria of strawberry gray mold, grape anthracnose, peach brown rot, tobacco brown spot, rice bakanae disease, rice blast, wheat take-all, cotton wilt, cabbage black rot, and potato blackleg. The human pathogenic bacteria are selected from one or more of Staphylococcus aureus, Bacillus subtilis, and Vibrio parahaemolyticus. The tumor is preferably the human gastric cancer cell line SGC-7901.

[0017] Another embodiment of the present invention provides the use of the above-mentioned marine fungus N4-9 in the preparation of bisindole derivatives 1, 2, 3, 4, their tautomers or their pharmaceutically acceptable salts.

[0018] Another embodiment of the present invention provides the use of the above-mentioned marine fungus N4-9 in the preparation of the crude extract of the above-mentioned marine fungus N4-9. Description of the Drawings

[0019] Figure 1 is the HMBC of compound 1 and 1 H- 1 H COSY main correlation signal diagram.

[0020] Figure 2 is the key ROESY correlation signal diagram of compound 1.

[0021] Figure 3 is the ECD diagram of compound 1.

[0022] Figure 4 is the HMBC of compound 2 and 1 H- 1 H COSY main correlation signal diagram.

[0023] Figure 5 is the key NOESY correlation signal diagram of compound 2.

[0024] Figure 6 is the HMBC of compound 3 and 1 H- 1 H COSY main correlation signal diagram.

[0025] Figure 7 is the key NOESY correlation signal diagram of compound 3.

[0026] Figure 8 is the HMBC of compound 4 and 1 H- 1The main relevant signal diagram of H COSY.

[0027] Figure 9 It is the key NOESY relevant signal diagram of Compound 4.

[0028] Figure 10 It is of Compound 1 1 1H NMR (600 MHz, DMSO) diagram.

[0029] Figure 11 It is of Compound 1 13 13C NMR (150 MHz, DMSO) diagram.

[0030] Figure 12 It is of Compound 2 1 1H NMR (600 MHz, CDCl3) diagram.

[0031] Figure 13 It is of Compound 2 13 13C NMR (150 MHz, CDCl3) diagram.

[0032] Figure 14 It is of Compound 3 1 1H NMR (600 MHz, CDCl3) diagram.

[0033] Figure 15 It is of Compound 3 13 13C NMR (150 MHz, CDCl3) diagram.

[0034] Figure 16 It is of Compound 4 1 1H NMR (600 MHz, CDCl3) diagram.

[0035] Figure 17 It is of Compound 4 13 13C NMR (150 MHz, CDCl3) diagram.

[0036] Figure 18 It is the anti - phytopathogenic bacteria activity diagram of Compound 1 (upper figure) and 3 (lower figure) (a: 25 μg / p, b: 50 μg / p, c: 100 μg / p, d: Penicilin 5 μg / p).

[0037] Figure 19 It is the result diagram of the cell migration experiment of Compound 2. Detailed implementation mode

[0038] Example 1

[0039] (1) Cultivation of marine fungus N4 - 9 strain

[0040] The culture medium for culturing the marine fungus N4-9 is prepared by adding to every 1000 mL of water: 200 g of potatoes boiled to obtain juice, 20 g of glucose, and 30 g of crude sea salt; it is dispensed into conical flasks during use. Cultured on a shaker at a temperature of 36 - 38 °C, a shaker rotation speed of 120 r / min, and a culture time of 3 days to obtain a seed solution.

[0041] (2) Fermentation of the marine fungus N4-9

[0042] The fermentation medium for the fermentation culture of the marine fungus N4-9 is prepared by adding to every 1000 mL of water: 200 g of potatoes boiled to obtain juice, 20 g of glucose, and 30 g of crude sea salt; it is dispensed into conical flasks during use (100 1-L conical flasks, each containing 400 mL of PDB medium). The seed solution is inoculated into the medium in the conical flasks, with an inoculation amount of 1.0%, and the fermentation culture temperature is at room temperature. The static culture time is 30 days to obtain a fermentation product.

[0043] (3) Preparation of the crude extract of the marine fungus N4-9

[0044] Take the fermentation product obtained after the fermentation culture in step (2), separate the fermentation broth and the thalli. The fermentation broth is extracted 3 times with ethyl acetate, and the extract is concentrated under reduced pressure to obtain a fermentation broth extract (about 26 g); the thalli are extracted 3 times with methanol and concentrated under reduced pressure to obtain a thalli extract (about 35 g); the fermentation broth extract and the thalli extract are combined (optionally with a further drying operation) to obtain the crude extract.

[0045] Example 2

[0046] The crude extract of the marine fungus N4-9 obtained in Example 1 is first subjected to silica gel column chromatography under reduced pressure (preferably 100 - 200 mesh silica gel), and petroleum ether - ethyl acetate is used as the eluent for gradient elution. The elution gradients are 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 0:100. Two column volumes are collected for each gradient, and they are divided into 7 components according to the polarity. Among them, the eluates obtained from the gradients 50:50 and 40:60 are combined and concentrated, and then subjected to Sephadex LH-20 gel column chromatography. The eluent is a mixed solvent of CH2Cl2 / MeOH, v / v, 1:1, and eluted for 4 - 5 column volumes. After concentration under reduced pressure, it is further subjected to reverse-phase column chromatography (ODS), and the mobile phase is MeOH:H2O = 70:30 to obtain bisindole derivatives 1 (19.1 mg), 2 (5.1 mg), 3 (29.2 mg), and 4 (24.3 mg).

[0047] The structure characterization data of Compounds 1 - 4 are as follows. According to the data of one-dimensional and two-dimensional NMR, MS, ECD, etc., the structures of Compounds 1 - 4 can be determined as follows:

[0048]

[0049] Compound 1: [α] 20 D +24 (c, 0.2, MeOH); UV (MeOH) λ max (logε) 301 (2.2), 276 (2.2), 334 (3.4) nm; ECD (0.1 mM, MeOH) λ max (Δε) 316 (+11.49), 260 (+7.47), 234 (-42.77), 212 (+31.95) nm; IR (KBr) ν max 2923, 2863, 1731, 1610, 1463, 1263, 1174, 1048, 743 cm –1 ; 1 1H NMR (DMSO, 600 MHz) and 13 13C NMR (DMSO, 150 MHz), see Table 1; HRESIMS m / z 755.3208 (calcd for C 44 H 48 N2NaO8, 755.3303).

[0050] Table 1 1H NMR data (600 MHz, DMSO) and 1 13C NMR data (150 MHz, DMSO) of Compound 1 13

[0051]

[0052]

[0053]

[0054] Compound 2: [α] 20 D +23 (c, 0.1, MeOH); UV (MeOH) λ max (logε) 291 (1.4), 274 (1.6), 224 (2.3), 206 (2.0) nm; IR (KBr) ν max 3390, 2928, 1736, 1628, 1564, 1454, 1259, 1027, 922, 747, 665, 514 cm –1 ; 1 1H NMR (CDCl3, 400 MHz) and 13 ​13C NMR (CDCl3, 150 MHz), see Table 2; HRESIMS m / z 737.3177 (calcd for C 44 H 46 N2NaO7, 737.3197).

[0055] Table 2 1H NMR data (600 MHz, CDCl3) and 1 13C NMR data (150 MHz, CDCl3) of Compound 2 13

[0056]

[0057]

[0058]

[0059] Compound 3: [α] 20 D +58 (c, 0.1, MeOH); UV (MeOH) λ max (log ε) 301 (0.8), 275 (0.8), 218 (1.4), 204 (1.4) nm; IR (KBr) ν max 3305, 2912, 1739, 1564, 1458, 1261, 1017, 921, 746, 666 cm –1 ; 1 1H NMR (DMSO, 600 MHz) and 13 13C NMR (DMSO, 150 MHz), see Table 3; HRESIMS m / z 669.2593 (calcd for C 39 H 38 N2NaO7, 669.2571).

[0060] Table 3 1H NMR data (600 MHz, CDCl3) and 1 13C NMR data (150 MHz, CDCl3) of Compound 3 13

[0061]

[0062]

[0063] Compound 4: [α] 20 D +38 (c, 0.1, MeOH); UV (MeOH) λ max ​​(logε) 269(0.4), 221(0.7), 202(0.5) nm; IR(KBr) ν max 2914, 2849, 1730, 1649, 1585, 1451, 1293, 1182, 1038, 744 cm –1 ; 1 H NMR(CDCl3, 400 MHz) and 13 C NMR(CDCl3, 150 MHz), see Table 4; HRESIMS m / z 669.2556 (calcd for C 39 H 38 N2NaO7, 669.2571).

[0064] Table 4 1 1H NMR data (600 MHz, CDCl3) and 13 13C NMR data (150 MHz, CDCl3) of Compound 4

[0065]

[0066]

[0067] Example 3 Activity Screening of Crude Extract from Fungus N4-9

[0068] (1) Evaluation of Anti-Plant Pathogenic Fungal Activity

[0069] The anti-plant pathogenic fungal activity of the crude extract of fungus N4-9 obtained in Example 1 was evaluated by the method of using a poisoned culture medium. The 8 test strains of agricultural pathogenic bacteria included the pathogen of strawberry gray mold (Botrytis cinerea), the pathogen of grape anthracnose (Colletotrichum gloeosporioides), the pathogen of peach brown rot (Monilinia fructicola), the pathogen of tobacco brown spot (Alternaria alternate), the pathogen of rice bakanae disease (Fusarium fujikuroi), the pathogen of rice blast (Magnaporthe grisea), the pathogen of wheat take-all (Gaeumannomyces graminis), and the pathogen of cotton wilt (Fusarium oxysporum); Pretreatment of the crude extract: The crude extract was dissolved with DMSO to prepare a solution of 1 mg / mL. Appropriate amounts of the sample solution were respectively added to the PDA solid medium that had been sterilized at high temperature and shaken well, so that the concentration of the crude extract in the medium in each conical flask was 100 μg / mL, 50 μg / mL, and 25 μg / mL respectively. The medium containing the crude extract was poured into a petri dish for standby. DMSO was added to the blank PDA medium and poured into a petri dish as a control. Activity test: Discs of 8 strains of agricultural pathogenic bacteria with a diameter of 10 mm were respectively taken to the center of the solid petri dish containing the crude extract prepared in advance. Three parallels were set for each concentration of each fungus, and the blank PDA petri dish added with DMSO was used as a control. It was cultured in an incubator at 37°C. When the control group was about to cover the petri dish, the diameter of the colony was measured to calculate the inhibition rate of the sample on the growth of the hyphae of the pathogenic bacteria at a specific concentration.

[0070] Inhibition rate of hyphal growth (%) = [(diameter of colony growth in control - diameter of colony growth in treatment) / diameter of colony growth in control] × 100%

[0071] The test results are shown in Table 5. The results show that the crude extract showed antibacterial activity against most plant pathogenic fungi. Among them, the antibacterial activity against the pathogen of peach brown rot was the most obvious. When the concentration was 25 μg / mL, the inhibition rate still reached 53.85%. This indicates that the crude extract of fungus N4-9 and its active ingredients have the activity of inhibiting agricultural pathogenic bacteria (especially agricultural pathogenic fungi) and have the function of plant protection.

[0072] Table 5 Anti-plant pathogenic fungal activity of the crude extract of fungus N4-9

[0073]

[0074]

[0075] (2) Anti-human pathogenic bacteria activity

[0076] The filter paper method was used to preliminarily screen the antifungal activity of the crude extract of fungus N4-9 obtained in Example 1 against human pathogenic bacteria. The indicator bacteria tested included 4 strains of Staphylococcus aureus ATCC (43300, 29213, 33591, 25923). Filter paper method activity test: Use a pipette to suck 200 μL of the bacterial solution into the LB solid plate, and use a sterile cotton swab to evenly spread the bacterial solution on the entire surface of the plate. The sample to be tested was dissolved into a methanol solution with a concentration of 50 μg / μL. Use a pipette to accurately suck 0.5, 1, 2 μL (i.e., the test concentrations are 25, 50, 100 μg per piece) of the solution and drop them onto the filter paper respectively. Wait until the filter paper is completely dry and stick it to the plate coated with the indicator bacteria. Penicillin was used as a positive control drug, and the amount of drug on each filter paper was 5 μg. Three parallels were made for each group and placed in an incubator at 28 °C for incubation, usually for 18-24 h. Pay attention to observing the formation of the inhibition zone, and it is also necessary to measure the diameter of the inhibition zone as an experimental record (inhibition zone diameter = blank circle diameter - filter paper diameter, unit: mm).

[0077] The results showed that the crude extract showed significant antibacterial activity against all 4 strains of Staphylococcus aureus, and the diameter of the inhibition zone was greater than 20 mm.

[0078] Activity test of Example 4 against plant pathogenic bacteria

[0079] The filter paper method (the test method is the same as that in Example 3) was used to test the antifungal activity of Compounds 1 and 3 against plant pathogenic bacteria. The test strains included: Xanthomonas campestris and Erwinia carotovora. The test results are shown in the following table.

[0080]

[0081] Penicilin: 5 μg / p

[0082] Compound 1 showed moderate antibacterial activity against Erwinia carotovora and Xanthomonas campestris. When the concentration of the compound was 25 μg per piece, the sizes of the inhibition zones were 11.27 ± 0.25 and 10.33 ± 0.25 mm respectively. Compound 3 had weak antibacterial activity against Erwinia carotovora and Xanthomonas campestris. When the concentration of the compound was 25 μg per piece, the sizes of the inhibition zones were 4.23 ± 0.15 and 4.27 ± 0.21 mm respectively.

[0083] Activity test of Example 5 against human pathogenic bacteria

[0084] The minimum concentration dilution method was selected to conduct experimental tests on the antibacterial activity of the compound. The test strains included Staphylococcus aureus ATCC (43300, 33591, 25923, 29213), Bacillus subtilis (Bacillus subtilis ATCC 19659), and Vibrio Parahemolvticus (Vibrio Parahemolvticus ATCC17802). The positive control drugs selected were vancomycin hydrochloride and oxacillin sodium, and the bacterial medium was used as the blank control.

[0085] The prepared LB liquid medium needs to be sterilized at high temperature in the sterilizer and set aside for later use. The test strains are taken out from the -80°C refrigerator for activation. An appropriate amount of bacterial liquid is aspirated and placed into the blank LB liquid medium, and cultured on a shaker at 18 r / min, 37°C for 24 hours to obtain the seed liquid. The sample is dissolved in DMSO at a concentration of 100 μg / mL, and the concentration of the positive control drug is also dissolved to 25 μg / mL.

[0086] Before the activity test, the seed liquid needs to be aspirated into the blank LB medium and shaken well for dilution. Then, a 96-well plate is taken out, and the sample wells and control wells are marked for distinction. 198 μL of the diluted blank bacterial liquid is aspirated and added to the first row of sample wells, and 100 μL of the blank bacterial liquid is dropped into the remaining seven rows. Then, 2 μL of the sample and vancomycin hydrochloride are aspirated and added to the corresponding first row of sample wells. Subsequently, 100 μL of the bacterial liquid containing the sample to be tested is added to the next sample well, and dilution is gradually achieved until 100 μL of the mixed liquid is taken out from the last sample well. Then, all the wells are filled with 200 μL of the bacterial liquid. Each sample needs to be done in 3 parallels and diluted in 8 gradients. After 24 hours, the OD 600 value is read by an enzyme-linked immunosorbent assay (ELISA) reader and the growth inhibition rate is calculated, and the IC 50 value is calculated using the software GraphPad Prism 8.

[0087] The experimental results are shown in the following table.

[0088]

[0089] Example 6 Cytotoxic Activity Test

[0090] The MTT method was used to screen the in vitro cytotoxic activity of Compound 2. The screening cells were human gastric cancer cell line SGC-7901, and the positive control drug was cisplatin. The absorbance (OD) value of the sample was obtained at a wavelength of 490 nm, and then the growth inhibition rate of each compound against the indicated cancer cells was calculated respectively. The specific operation is as follows:

[0091] Preparation of solutions. The sample was dissolved in DMSO to form a stock solution with a concentration of 1 mg / mL for later use in the experiment. Preparation of PBS: 0.2 g of KCl, 0.2 g of KH2PO4, 8 g of NaCl, and 1.54 g of Na2HPO4·12H2O were added to 1 L of ultrapure water and stirred until dissolved. The prepared solvent was sterilized and then filtered through a microporous membrane to remove impurities, and stored in a refrigerator at room temperature. Preparation of MTT: 0.25 g of MTT powder was dissolved in the prepared PBS solution, and the amount of solvent should be as small as possible and diluted to 50 mL. After filtering through the membrane, it was stored in the dark at -20 °C.

[0092] Adjust the cell concentration to 1.0×10 5 cells / mL, aspirate the cells and inoculate them into the well plate, 100 μL per well, and culture in an incubator for one day; specific concentrations of the test sample and cisplatin should also be added to each well, and 100 mL of culture medium should also be added to the negative and blank control groups (5 in parallel); after a period of time, 20 μL of MTT solution was added to each well and cultured for another 4 h; then the solution in the well was aspirated, and 100 μL of DMSO was added for dissolution. The ultrasonic oscillation time was about 10 min, and the absorbance (OD) value required was read with an enzyme-linked immunosorbent assay (ELISA) reader. The calculation formula for the proliferation inhibition rate of tumor cells is as follows:

[0093]

[0094] The cell scratch assay was used to observe whether the test sample affected the migration ability of cells. First, a cross was drawn on the bottom of the well plate with a water pen. The concentration of cancer cells was adjusted to 1.0×106 cells / mL, inoculated into the well plate and cultured statically. After the formation of a single cell layer could be observed, it was rinsed 2-3 times with PBS solvent, and then the prepared sample solution was added for intervention. After 48 h, it was necessary to take pictures to record the migration of cancer cells after sample treatment.

[0095] The results showed that compound 2 had a strong inhibitory effect on cell proliferation, and the IC 50 value of the compound was 4.457 μg / mL. The in vitro cell scratch method was used to test the effect of compound 2 on the migration ability of tumor cells. After 48 h of observation, compared with the scratch at 0 h, it was found that the scratch width of the NC control group (negative control) was significantly narrowed, the cell density at the scratch increased, and the scratch healed to a certain extent. However, for the scratches at two concentrations of compound 2, the scratch width did not change significantly, but the cell density of the scratch decreased significantly, especially when the compound concentration was 10 μg / mL, the above phenomenon was more significant, indicating that compound 2 had an anti-migration effect on SGC-7901 gastric cancer cells.

Claims

1. A marine fungus N4-9, characterized in that The name of the strain preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration; the preservation number is CGMCC No.40555.

2. A crude extract of the marine fungus N4-9 according to claim 1, characterized in that The method for preparing the crude extract comprises the following steps: (1) Cultivating the marine fungus N4-9 in a bacterial culture medium to obtain a seed solution; (2) inoculating the seed liquid obtained in step (1) into a fermentation medium for fermentation to obtain a fermentation product; (3) Separating the fermentation broth and bacterial cells in the fermented product obtained in step (2), extracting the fermentation broth with organic solvent A for 2 to 4 times, combining the extracts and concentrating under reduced pressure to obtain a fermentation broth extract; extracting the bacterial cells with organic solvent B for 2 to 4 times, combining the extracts and concentrating under reduced pressure to obtain a bacterial cell extract; and combining the fermentation broth extract and the bacterial cell extract to obtain the crude extract.

3. A bisindole derivative, a tautomer thereof or a pharmaceutically acceptable salt thereof, characterized in that The bisindole derivatives have the structures shown in compounds 1, 2, 3, and 4:

4. The method for preparing the bisindole derivatives 1, 2, 3 and / or 4 according to claim 3, characterized in that The steps include: The crude extract of the marine fungus N4-9 described in claim 2 is separated by chromatography to obtain the bisindole derivatives 1, 2, 3 and / or 4.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the crude extract of claim 2, the bisindole derivatives 1, 2, 3 and / or 4 of claim 3, their tautomers or pharmaceutically acceptable salts thereof as active ingredients. The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient. The dosage form of the pharmaceutical composition is selected from solid preparations, liquid preparations, semisolid preparations, etc.

6. Use of the crude extract according to claim 2, the bisindole derivatives 1, 2, 3 and / or 4 according to claim 3, their tautomers or pharmaceutically acceptable salts thereof in plant protection, especially in controlling agricultural pathogens.

7. Use of the marine fungus N4-9 according to claim 1 in plant protection, especially in controlling agricultural pathogens.

8. Use of the marine fungus N4-9 according to claim 1 or the crude extract according to claim 2, the bisindole derivatives 1, 2, 3 and / or 4 according to claim 3, their tautomers or pharmaceutically acceptable salts thereof in the preparation of drugs for preventing and controlling agricultural pathogens, human pathogens and tumors.

9. Use of the marine fungus N4-9 according to claim 1 in the preparation of bisindole derivatives 1, 2, 3 and / or 4, their tautomers or pharmaceutically acceptable salts thereof.

10. Use of the marine fungus N4-9 according to claim 1 in preparing the crude extract according to claim 2.

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