Secondary metabolite of fennel endophytic fungus fusarium oxysporum as well as preparation method and application of secondary metabolite

Abstract

The invention belongs to the field of plant endophytic fungus secondary metabolites, particularly relates to a secondary metabolite of fennel endophytic fungus fusarium oxysporum as well as a preparation method and application thereof, and provides a fennel endophytic fungus secondary metabolite with structures as shown in compounds 1-8. Wherein the compound 2 and the compound 7 have inhibitory activity on staphylococcus aureus, escherichia coli, bacillus cereus, bacillus subtilis, candida albicans and shigella, and have a certain inhibitory effect on cervical cancer Hela cell strains.

Description

Technical Field

[0001] The present invention belongs to the field of secondary metabolites of plant endophytic fungi, and particularly relates to secondary metabolites of anise endophytic fungus Fusarium oxysporum and a preparation method and application thereof. Background Art

[0002] Plant endophytic fungi are fungi that live inside plant tissues or organs but do not cause obvious pathological changes in the host plant. They are a type of specialized habitat fungi. Plants provide the environment and nutrients for endophytic fungi to survive; endophytic fungi inherit certain genetic characteristics of plants and produce secondary metabolites to help plants resist diseases and pests. Due to the specificity, complexity, and diversity of specialized habitat fungi, the structural types and biological activities of their secondary metabolites are also rich and diverse. To date, researchers have isolated compounds with structural types such as terpenes, alkaloids, glycosides, fatty acids, quinones, and flavonoids from cultures of specialized habitat fungi, and have discovered many compounds with significant activity against hypertension, hyperlipidemia, diabetes, and other diseases. Therefore, the search for active substances with novel structures in fungi is of great significance for drug discovery and the discovery of lead compounds. Summary of the Invention

[0003] The present invention provides secondary metabolites derived from the fennel endophytic fungus Fusarium oxysporum, including four polyketide compounds and four alkaloids or pharmaceutically acceptable salts thereof, and use of the secondary metabolites in the preparation of broad-spectrum antibacterial agents and anti-tumor drugs.

[0004] The technical solutions of the present invention are as follows:

[0005] The secondary metabolites of the fennel endophytic fungus Fusarium oxysporum include four polyketides and four alkaloids. The structures of the eight secondary metabolites are shown in compounds 1-8:

[0006]

[0007] The fennel endophytic fungus was identified as Fusarium oxysporum through morphological observation and molecular biological identification (ITS-rDNA). Its ITS sequence has been submitted to the NCBI platform with the accession number: PQ345338, and is currently preserved in the School of Life Sciences and Technology of Kunming University of Science and Technology. The colony of the fennel endophytic fungus Fusarium oxysporum is white, flocculent, and round with pink pigment deposition.

[0008] The present invention also provides a method for preparing eight secondary metabolites of the fennel endophytic fungus Fusarium oxysporum, and the specific steps are as follows:

[0009] (1) Cultivating the fennel endophytic fungus Fusarium oxysporum in PDB medium to prepare seed solution;

[0010] (2) inoculating the seed solution prepared in step (1) into a solid culture medium for fermentation and culturing at room temperature;

[0011] (3) soaking and extracting the solid fermentation product obtained in step (2) with a mixed solvent to obtain an extract;

[0012] (4) extracting the extract obtained in step (3) with ethyl acetate, and concentrating the extract under reduced pressure to obtain a fermentation extract;

[0013] (5) The extract prepared in step (4) is separated and purified to obtain compound 1-8, a secondary metabolite of the fennel endophytic fungus Fusarium oxysporum.

[0014] The PDB culture medium in step (1) is a potato liquid culture medium, which is composed of 6g potato extract powder and 20g glucose per 1L of water, and the culture conditions are 160r / min, 28°C, and culture for 3 days.

[0015] The solid culture medium described in step (2) is composed of 100 mL of 2% glucose solution by mass per 100 g of rice, and the culture conditions are: static culture at 28° C. and 50% humidity for 30 days.

[0016] The mixed solvent in step (3) is a mixture of dichloromethane and methanol in a volume ratio of 1:1 (v:v, 1:1); the soaking extraction is performed by extracting three times using a static extraction method, and the extract is obtained by combining and concentrating under reduced pressure.

[0017] The volume ratio of ethyl acetate to extract in step (4) is 1:1, extraction is carried out three times, the three ethyl acetate phase extracts are combined, and the fermentation extract is concentrated under reduced pressure.

[0018] The separation and purification method in step (5) includes one or a mixture of normal phase silica gel column chromatography, neutral / alkaline Al2O3 column chromatography, ODS column chromatography, semi-preparative HPLC, preparative plate PLC, semi-preparative high performance liquid chromatography, and Sephadex LH-20 dextran gel.

[0019] The present invention also provides the use of secondary metabolites of the fennel endophytic fungus Fusarium oxysporum in broad-spectrum antibacterial agents and anti-tumor drugs. The broad-spectrum antibacterial agents correspond to antibacterial agents such as Escherichia coli (CMCC(B)44102), Staphylococcus aureus (Staphylococcus aureusCMCC(B)26003), Bacillus cereus (Bacillus cereusBNCC336744), Bacillus subtilis (BNCC109047), Candida albicans (Candida albicansCMCC(F)98001) and Shigella Castellani; and the broad-spectrum antibacterial agents also have a certain inhibitory effect on cervical cancer Hela cell lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a colony morphology diagram of the fennel endophytic fungus Fusarium oxysporum;

[0021] Figure 2 This is the phylogenetic tree of the fennel endophytic fungus Fusarium oxysporum;

[0022] Figure 3 This is the inhibition zone diagram of the initial screening of antibacterial compounds. DETAILED DESCRIPTION

[0023] In order to better understand the present invention, the following will be described in detail with reference to specific embodiments and examples. It should be emphasized that these examples are for illustrative purposes only and do not constitute a limitation on the scope of the present invention.

[0024] The experimental methods in the examples are conventional methods unless otherwise specified; the materials, reagents, etc. used in the examples are commercially available unless otherwise specified.

[0025] Instruments and materials used in the examples:

[0026] UPLC-IT-TOF mass spectrometer (Shimadzu Corporation, Japan); Bruker AV 600 nuclear magnetic resonance instrument (TMS as internal standard, Bruker, Germany); medium-pressure column chromatography (Jiangsu Hanbang Instrument Co., Ltd.); ultra-clean workbench (SW-CJ-2FD, Suzhou Purification Equipment Co., Ltd.); constant temperature incubation oscillator (WYC-280AF, Shanghai Dute Scientific Instrument Co., Ltd.); vertical pressure steam autoclave (BKQ-B120Ⅱ, Shanghai Jiesheng Scientific Instrument Co., Ltd.); rotary evaporator (RE-5205, Shanghai Yuanhuai Intelligent Technology Co., Ltd.).

[0027] Sephadex (25-100 μm; Amersham Biosciences, Sweden) column chromatography silica gel (100-200 mesh), thin layer chromatography silica gel plate (5×10 cm, GF 254 ) (Qingdao Ocean Chemical Co., Ltd.); anhydrous glucose (Xilong Science Co., Ltd.); potato extract powder (Shanghai Yuanye Biotechnology Co., Ltd.); ciprofloxacin (Solarbio); methanol, ethyl acetate, dichloromethane, petroleum ether, etc. used in extraction and separation were of analytical grade (Yunnan Liyan Technology Co., Ltd.).

[0028] The strains in the examples are derived from:

[0029] The endophytic fungus Fusarium oxysporum was isolated from the roots of fennel samples in Kunming, Yunnan. The specific isolation method can be found in the literature Natural Product Research and Development, 2018, vol. 30, pp. 1928-1932; it was deposited in the College of Life Science and Technology of Kunming University of Science and Technology. Through morphological observation, such as Figure 1 As shown, the colony is white and flocculent, the center gradually turns pink, the hyphae are dense, and the edges are radial. Its molecular biological identification, such as Figure 2 As shown, the target strain has a sequence similarity of >90% with the Fusarium oxysporum cluster, and its species is determined to be Fusarium oxysporum. Its ITS sequence has been submitted to the NCBI platform with the accession number: PQ345338.

[0030] Example 1

[0031] (1) The endophytic fungus Fusarium oxysporum was removed from the slant and inoculated onto a PDA potato agar medium (1 L of deionized water, 6 g of potato extract powder, 15 g of agar powder, 20 g of glucose, sterilized at 121°C for 15 min) and then rejuvenated in a 28°C incubator. After the fungus recovered, it was inoculated into a PDB potato liquid medium (1 L of deionized water, 6 g of potato extract powder, 20 g of glucose, sterilized at 121°C for 15 min) and incubated at 28°C on a constant temperature shaker (160 rpm) for 3 days to prepare a seed solution.

[0032] (2) The prepared seed liquid was inoculated into a glucose rice solid medium (100 g of rice, 100 mL of a 2% glucose solution by mass, sterilized by autoclave at 121° C. for 15 min); the inoculated medium was placed in a constant temperature and humidity incubator and subjected to static fermentation for 30 days at 28° C. and 50% humidity;

[0033] (3) soaking the solid fermentation product obtained in step (2) in a mixed solvent of dichloromethane:methanol (v:v, 1:1) and allowing it to stand for extraction three times, combining and concentrating under reduced pressure to obtain an extract;

[0034] (4) extracting the extract obtained in step (3) three times with 1:1 volume of ethyl acetate, combining the three ethyl acetate extracts and concentrating under reduced pressure until there is no solvent to obtain a fermentation extract;

[0035] (5) The fermentation extract obtained in step (4) was subjected to column chromatography using a normal phase silica gel column to perform crude fractionation, and gradient elution was performed using an ethyl acetate-petroleum ether (v / v, 50:1→0:1) system to obtain six fractions (A–F):

[0036] Fraction E (806.0 g) was further separated by medium pressure liquid chromatography (MPLC) coupled with a C-18 reverse phase column using a methanol-water (40-100%) gradient elution to obtain subfractions E1–E 10 ;

[0037] Subsequently, subfraction E3 was purified by silica gel column chromatography and dextran gel (methanol) column chromatography to remove impurities, and compounds 1 (2.4 mg), 2 (1.0 mg), and 3 (1.0 mg) were isolated;

[0038] Subcomponent E7 is divided into 7 components, namely E 7a-7g , by silica gel column chromatography, eluted with ethyl acetate-petroleum ether (v / v, 2:1), and finally from component E 7b Compound 4 (4.9 mg) was obtained;

[0039] First, a preparative thin layer preparation (PLC) was performed using a developing solvent of petroleum ether-acetone (v / v, 2:1), followed by silica gel column chromatography and further purification using petroleum ether-acetone (v / v, 3:1), and finally from E 7e Compound 5 (3.0 mg) was obtained.

[0040] Component C was repeatedly subjected to silica gel column chromatography using a gradient elution system of ethyl acetate-petroleum ether (v / v, 50:1→0:1) to obtain three subcomponents (C 1-3 );

[0041] Compound 6 (0.7 mg) was obtained from C1 by Sephadex column chromatography eluting with methanol;

[0042] C2 was prepared by PLC thin layer chromatography using petroleum ether-acetone (v / v, 4:1) as the developing solvent, and compound 7 (0.9 mg) was finally purified from this fraction;

[0043] C3 was eluted with ethyl acetate-petroleum ether (v / v, 8:1), and the fraction was purified by repeated silica gel column chromatography to obtain compound 8 (6.2 mg).

[0044] Compounds 1-8 prepared in Example 1 were 1 H-NMR and 13 C-NMR analysis and comparison of the data with the literature showed the following results:

[0045] Compound 1 is a yellow oil (CHCl3); ESI-MS: m / z 305 [M+H]+; molecular formula: C 15 H 12 O7; 1 H-NMR (600MHz, CDCl3) δ: 8.09 (1H, s, H-3), 6.68 (1H, s, H-6), 5.19 (1H, q, J = 6.9Hz, H-10), 4.01 (3H, s, -OCH3), 1.66 (3H, d, J = 6.8Hz, H-11); 13 C-NMR (150 MHz, CDCl3) δ: 166.7 (s, C-1), 143.2 (d, C-3), 123.6 (s, C-3a), 182.3 (s, C-4), 107.3 (s, C-4a), 161.1 (s, C-5), 107.7 (d, C-6), 157.5 (s, C-7), 150.6 (s, C-8), 114.2 (s, C-8a), 186.1 (s, C-9), 117.1 (s, C-9a), 64.4 (d, C-10), 21.2 (q, C-11), 56.9 (q, -OCH3). Compare the NMR data with those in the literature for nectriafurone.

[0046] Compound 2 is a yellow oil (CHCl3); ESI-MS: m / z 319 [M+H]+; molecular formula: C 17 H 18 O6; 1H-NMR (600MHz, CDCl3) δ: 7.45 (1H, s, H-4), 6.86 (1H, s, H-2), 4.74 (1H, dd, J=19.2, 2.6Hz, H-11a), 4.39 (1H, dt, J=19.2, 2.6Hz, H-11b), 4.04 (3H, s , 3-OCH3), 4.02 (3H, s, 1-OCH3), 3.27 (3H, s, 9-OCH3), 2.81 (1H, dd, J=18. 7, 3.1Hz, H-12a), 2.53 (1H, dt, J=18.6, 3.3Hz, H-12b), 1.53 (3H, s, H-14); 13 C-NMR (150MHz, CDCl3) δ: 162.7 (s, C-1), 103.9 (d, C-2), 165.2 (s, C-3), 105.7 (d, C-4), 136.7 (s, C-5), 183.8 (s, C-6), 180.4 (s, C-7), 137.2 (s, C-8), 97.3 (s, C-9) , 114.6(s, C-10), 58.7(t, C-11), 32.3(t, C-12), 145.1(s, C-13), 22.7(q, C-14), 56.7(q, 1-OCH3), 56.9(q, 3-OCH3), 48.7(q, 9-OCH3). The NMR data are basically consistent with those in the literature for colelomycerone A.

[0047] Compound 3 is a brown oil (CHCl3); ESI-MS: m / z 357 [M+H]+; molecular formula: C 19 H 16 O7; 1 H-NMR (600MHz, CDCl3) δ: 10.95 (1H, s, 8-OH), 6.49 (1H, s, Hz, H-7), 6.41 (1H, s, H-4`), 6.34 (1H, s, H-6`), 6.27(1H,s,H-5), 5.98(1H,s,H-4), 4.08(2H,s,H-9), 3.85(3H,s,H-10), 2.62(3H,s,H-12); 13C-NMR (150MHz, CDCl3) δ: 165.7 (s, C-1), 155.9 (s, C-3), 105.7 (d, C-4), 138.6 (s, C-4a) ,101.7(d,C-5),167.2(s,C-6),101.3(d,C-7),163.6(s,C-8),99.9(s,C-8a),39.5(t,C -9), 55.7(q, C-10), 203.6(s, C-11), 32.5(q, C-12), 138.7(s, C-1'), 115.5(s, C-2'), 166.9(s, C-3'), 103.9(d, C-4'), 161.3(s, C-5'), 112.9(d, C-6'). The NMR data are basically consistent with those of karimunone B in the literature.

[0048] Compound 4 colorless crystals (MeOH); ESI-MS: m / z 289 [M+H] +; molecular formula: C 16 H 16 O5; 1 H-NMR (600MHz, CD3OD) δ: 7.12 (1H, d, J = 1.2, H-10), 6.27 (1H, d, J = 2.3Hz, H-5) , 6.28 (1H, d, J = 2.3Hz, H-7), 3.86 (3H, s, 8-OCH3), 1.07 (3H, d, J = 7.2, 3-CH3); 13 C-NMR (150 MHz, CD3OD) δ: 206.6 (s, C-1), 43.3 (t, C-2), 37.7 (d, C-3), 71.8 (d, C-4), 139.8 (s, C-4a), 102.8 (d, C-5), 160.6 (s, C-6), 99.7 (d, C-7), 161.9 (s, C-8), 109.4 (s, C-8a), 164.7 (s, C-9), 109.3 (s, C-9a), 114.2 (d, C-10), 141.2 (s, C-10a), 56.3 (q, 8-OCH3), 18.3 (q, 3-CH3). The NMR data are basically consistent with the isoasperflavin in the literature.

[0049] Compound 5 is a yellow oil (CHCl3); ESI-MS: m / z 284 [M+H]+; molecular formula: C 16 H 13 NO4; 1H-NMR (600MHz, CDCl3) δ: 9.42 (1H, s, H-1), 7.83 (1H, s, H-4), 7.45 (1H, d, J=2.5Hz, H-5 ), 6.88 (1H, d, J = 2.5Hz, H-7), 4.03 (3H, s, H-16), 4.01 (3H, s, H-17), 2.76 (3H, s, H-15); 13 C-NMR (150 MHz, CDCl3) δ: 149.9 (d, C-1), 164.3 (s, C-3), 117.8 (d, C-4), 103.8 (d, C-5), 165.3 (s, C-6), 105.5 (d, C-7), 162.8 (s, C-8), 180.8 (s, C-9), 183.8 (s, C-10), 137.8 (s, C-11), 115.8 (s, C-12), 125.7 (s, C-13), 137.2 (s, C-14), 25.2 (q, C-15), 56.3 (q, C-16), 56.9 (q, C-17). The NMR data are basically consistent with the scorpinone in the literature.

[0050] Compound 6 is a yellow oil (CHCl3); ESI-MS: m / z 438 [M+H]+; molecular formula: C 28 H 39 NO3; 1 H-NMR (600MHz, CDCl3) δ: 9.98 (1H, s, 4`-OH), 7.42 (2H, m, H-3`and H-5`), 7.38 (2H, m, H-2`and H-6`), 7.31 (1H, m, H-4`), 5.18 (1H, d, J = 9.4Hz, H-13), 5.02 (1H, d, J = 10.2Hz, H-7), 3.52 (1H, overlapped, H-11), 3.49 (3H, s, H-23), 1.61 (3H, s, H-21), 0.89 (3H, d, J = 6.5Hz, H-20), 0.83 (3H, t, J = 6.5Hz, H-18), 0.81 (3H, d, J = 6.5Hz, H-19), 0.73 (3H, d, J = 6.5Hz, H-22); 13C-NMR (150MHz, CDCl3) δ: 161.4 (s, C-2), 110.3 (s, C-3), 161.8 (s, C-4), 115.1 (s, C-5), 136.3 (d, C-6), 78.1 (d, C-7), 30.7 (t, C-8), 32.2 (t, C-9), 32.4 (d, C-10), 92.6 (d, C-11), 130.3 (s, C-12), 138.0 (d, C-13), 29.6 (d, C-14), 44.7 (t, C-15), 32. 0(d, C-16), 28.9(t, C-17), 11.3(q, C-18), 19.6(q, C-19), 20.7(q, C-20), 11.6(q, C-21), 17.7(q, C-22), 37.1(q, C-23), 134.1(s, C-1`), 129.2(d, C-2`), 128.3(d, C-3`), 127.4(d, C-4`), 128.3(d, C-5`), 129.2(d, C-6`). The NMR data are basically consistent with those of sambutoxin A in the literature.

[0051] Compound 7 is a white amorphous powder (MeOH); ESI-MS: m / z 181 [M+H] +; molecular formula: C9H7NO2; 1 H-NMR (600MHz, CD3OD) δ: 8.08 (1H, d, J=7.8Hz, H-4), 7.95 (1H, s, H-2), 7.44 (1H, d, J=7.9Hz, H-7), 7.19 (2H, m, H-5and H-6); 13 C-NMR (150 MHz, CD3OD) δ: 133.4 (d, C-2), 113.0 (s, C-3), 127.7 (s, C-3a), 122.1 (d, C-4), 122.4 (d, C-5), 123.6 (d, C-6), 113.0 (d, C-7), 138.3 (s, C-7a), 169.5 (s, C-8). The NMR data are basically consistent with those in the literature for 1H-indole-3-carboxylic acid.

[0052] Compound 8 is a white amorphous powder (CHCl3); ESI-MS: m / z 262[M+H]+; molecular formula: C 15 H 19 NO3; 1H-NMR (600MHz, CDCl3) δ: 7.26–7.20 (4H, m, H-12, 13, 15, 16), 7.16 (1H, m, H-14), 5.51 (1H, d, J = 7.1Hz, H-3 ), 4.88 (1H, d, J = 8.7Hz, H-6), 3.01 (3H, s, 4-CH3), 0.78 (3H, d, J = 6.7Hz, H-9), 0.38 (3H, d, J = 6.7Hz, H-8); 13 C-NMR (150 MHz, CDCl3) δ: 170.0 (s, C-2), 57.2 (d, C-3), 169.6 (s, C-5), 75.6 (d, C-6), 29.7 (d, C-7), 17.4 (q, C-8), 18.4 (q, C-9), 34.8 (t, C-10), 136.6 (s, C-11), 129.1 (d, C-12, 16), 128.6 (d, C-13, 15), 126.8 (d, C-14), 32.5 (q, 4-CH3). The NMR data are basically consistent with those in the literature (3R, 6R)-4-Methyl-6-(1-methylethyl)-3-phenylmethylperhy-dro-1,4-oxazine-2,5-dione.

[0053] From the above, we can see that the structural formulas of the eight compounds 1-8 are shown below:

[0054]

[0055] Example 2

[0056] The filter paper method was used to preliminarily screen the antibacterial activity of compounds 1-8, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values ​​of the active compounds were determined in combination with the two-fold dilution method.

[0057] 1. Filter paper method for measuring inhibition zone

[0058] Use a 6mm hole punch to make the filter paper into circular filter paper pieces. Sterilize the prepared culture medium, tweezers, filter paper pieces, pipette tips, etc. under high pressure for later use.

[0059] The strain stored in the glycerol tube was inoculated onto the LB plate, and the test strain was rejuvenated at 37°C. The recovered test strain was inoculated into LB broth and cultured in a constant temperature shaker at 37°C and 160 r / min for 24 hours to prepare the test bacterial solution.

[0060] The sample was dissolved in DMSO to prepare a 10 μg / μL sample solution, and the sample solution was added to a dried sterile filter paper, 5 μL was added to each filter paper, and a drug-loaded filter paper with a drug-load of 50 μg / piece was prepared for use; the test bacterial solution was diluted 100 times, and the diluted bacterial solution was added to the LB plate, 1 mL was added to each plate, and the entire plate was evenly coated with a coating rod. The drug-loaded filter paper was evenly distributed and placed on the coated plate. Each plate was set with a positive control (levofloxacin) and a negative control (DMSO) group. Each plate can hold up to 8 filter papers, and each group was set up with three parallels (repeated 3 times). The LB plate with the filter paper was placed upright in a 37°C incubator. After 24 hours, the strains on each plate were observed in three situations. An obvious inhibition zone appeared, which preliminarily determined that the compound had antibacterial activity.

[0061] 2. Determination of minimum inhibitory concentration (MIC value) by two-fold dilution method

[0062] The strain stored in the glycerol tube was inoculated into LB broth and cultured at 37°C and 160 rpm for 24 h for activation.

[0063] After mixing the activated bacterial solution, take 100 μL and add it to 100 mL of clean LB broth for a 1000-fold dilution. Use a 12-channel gun to add 198 μL of the diluted bacterial solution to the first row of a 96-well plate. Add 100 μL of bacterial solution to each of the remaining rows. Add 2 μL of each test compound solution (10 mg / mL) in DMSO as the solvent to the first row. Set up 3 replicate wells for each compound and dilute the bacterial solution in each well using the two-fold dilution method.

[0064] Each plate was equipped with a positive control (levofloxacin) and a negative control (DMSO). The plate was placed in a 37°C constant temperature environment for incubation and observed after 12 hours. The concentration of the compound at which no bacterial growth occurred was the minimum inhibitory concentration (MIC).

[0065] 3. Subculture method to measure the minimum bactericidal concentration (MBC value)

[0066] The minimum bactericidal concentration (MBC) was determined by the subculture method, that is, the liquid in the MIC well and the well with the next concentration below the MIC was transferred to a fresh LB agar plate and the growth of colonies was observed after culture. No growth was the MBC.

[0067] 4. Results Analysis

[0068] The inhibition zone experiment showed (attached Figure 3), there were obvious inhibition zones around the filter paper pieces of compounds 2 and 7, indicating that compounds 2 and 7 had antibacterial effects. The minimum inhibitory concentration and minimum bactericidal concentration of compounds 2 and 7 were then determined by the two-fold dilution method. The results are shown in Table 1. Compound 7 had a good inhibitory effect on all six pathogens, and it was speculated that its activity might be related to the nitrogen atom in its molecule.

[0069] Table 1 Minimum inhibitory concentration and minimum bactericidal concentration (MIC, μg / mL) of compounds 2 and 7

[0070]

[0071] Example 3

[0072] Evaluation of anti-cervical cancer Hela cell activity:

[0073] 1. Cell recovery

[0074] The cell cryopreservation tube stored in liquid nitrogen was taken out, placed in a sterile ziplock bag, and quickly placed in a 37°C water bath. When there were still small pieces of ice in the cell cryopreservation tube, it was quickly taken out. 1 mL of DMEM complete medium (hereinafter referred to as DMEM complete medium) was slowly added to the cryopreservation tube with a pipette on the operating table, and then pipetted 30 times to fully suspend the cells; centrifuged at 800 rpm for 4 minutes; discarded the medium in the clean bench, added 1 mL of new complete medium to the centrifuge tube with a pipette, and repeatedly pipetted 30 times to fully suspend the cells, and then transferred to T 25 Tighten the cap in the bottle and shake it in a cross-shaped manner. 25 The cell suspension was evenly suspended in the culture medium. Subsequently, the culture flask was labeled according to the standard, and the surface was disinfected with 75% ethanol, and finally transferred to a CO2 incubator for culture.

[0075] 2. CCK-8 method to measure IC of Hela cells 50 value

[0076] When the cell density reaches 80-90%, discard the culture medium in the bottle under sterile environment, rinse with PBS 3 times, add 2mL trypsin, and incubate in the incubator for 2min. Digest until the adherent cells become quicksand-like when the bottle is gently tapped (use a pipette to take 1mL of complete culture medium and add it to the culture bottle to terminate digestion). Use a Pasteur pipette to gently blow the adherent cells to fully suspend them, and try to avoid bubbles. Use a pipette to transfer the cell suspension to a centrifuge tube containing 2mL of complete culture medium, centrifuge, discard the culture medium in the centrifuge tube, and leave the cell sediment at the bottom. Use a pipette to add 1mL of new complete culture medium to the centrifuge tube, blow and mix the cells several times, count the cell suspension with a hemocytometer, and finally calculate the total number of cells in the cell suspension before dilution. According to the experimental requirements, a suitable single-cell suspension is prepared, and finally the cells are inoculated into a 96-well plate (each well contains 100μL of single-cell suspension, 2×10 4 cells) and were incubated in a culture incubator for 24 h before administration.

[0077] After 24 h, the sample was prepared into 6 concentration gradients (3.2, 6.25, 12.5, 25, 50, 100 μM / L) using complete culture medium. The culture medium in each well of the 96-well plate was aspirated and discarded with a pipette, and the culture medium containing the sample of the corresponding concentration was added to each well, 100 μL per well. The 96-well plate after administration was placed in a cell culture incubator and cultured for 24 h.

[0078] After 24 hours, the liquid in the well plate was discarded, and 100 μL of new complete medium containing 10% CCK-8 by volume was added. The 96-well plate replaced with the complete medium containing CCK-8 was moved into the incubator and incubated for 2 hours. After the incubation, the OD (optical density) value of each well was measured at 450 nm.

[0079] The cell survival rate was calculated according to the formula: cell survival rate = [(OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group)] × 100%;

[0080] There were experimental group (containing cells, complete culture medium, CCK-8, sample, doxorubicin), control group (containing cells, complete culture medium, CCK-8, DMSO), and blank group (complete culture medium, CCK-8).

[0081] The calculated results were imported into Graphpad Prism software to determine the half-maximal inhibitory concentration (IC 50 The results are shown in Table 2. It can be seen from Table 2 that compounds 2 and 7 have inhibitory activity against cervical cancer Hela cells.

[0082] Table 2 IC of inhibition of Hela cell activity 50 value

[0083]

[0084] The above embodiments only show preferred implementation schemes of the present invention. Reasonable modifications and improvements that can be made by those skilled in the art without departing from the core concept of the present invention should be included in the scope of patent protection.

Claims

1. The structures of the secondary metabolites of the fennel endophytic fungus Fusarium oxysporum are shown in the following compounds 1-8:

2. The method for preparing the secondary metabolites of the fennel endophytic fungus Fusarium oxysporum according to claim 1, characterized in that: The specific steps are as follows: (1) Cultivating the fennel endophytic fungus Fusarium oxysporum in PDB medium to prepare seed solution; (2) inoculating the seed solution prepared in step (1) into a solid culture medium for fermentation and culturing at room temperature; (3) soaking and extracting the solid fermentation product obtained in step (2) with a mixed solvent to obtain an extract; (4) extracting the extract obtained in step (3) with ethyl acetate, and concentrating the extract under reduced pressure to obtain a fermentation extract; (5) The extract prepared in step (4) is separated and purified to obtain compound 1-8, a secondary metabolite of the fennel endophytic fungus Fusarium oxysporum.

3. The method for preparing the secondary metabolites of the fennel endophytic fungus Fusarium oxysporum according to claim 2, characterized in that: The PDB medium in step (1) is a potato liquid culture medium, which is composed of 6g potato extract powder and 20g glucose per 1L of water, and the culture conditions are 160r / min, 28°C, and culture for 3 days.

4. The method for preparing the secondary metabolites of the fennel endophytic fungus Fusarium oxysporum according to claim 2, characterized in that: In step (2), the solid culture medium is prepared by adding 100 mL of 2% glucose solution by mass to every 100 g of rice, and the culture is statically cultured at 28° C. and 50% humidity for 30 days.

5. The method for preparing the secondary metabolites of the fennel endophytic fungus Fusarium oxysporum according to claim 2, characterized in that: The mixed solvent in step (3) is a mixture of dichloromethane and methanol in a volume ratio of 1:1; the soaking extraction is performed by extracting three times using a static extraction method, and the extracts are combined and concentrated under reduced pressure to obtain an extract.

6. The method for preparing the secondary metabolites of the fennel endophytic fungus Fusarium oxysporum according to claim 2, characterized in that: In step (4), the volume ratio of ethyl acetate to the extract is 1:1, extraction is carried out three times, the three ethyl acetate phase extracts are combined, and concentrated under reduced pressure to obtain a fermentation extract.

7. Use of the secondary metabolites of the fennel endophytic fungus Fusarium oxysporum according to claim 1 in broad-spectrum antibacterial agents and anti-tumor drugs.

8. The use according to claim 7, wherein the broad-spectrum antibacterial agent corresponds to Escherichia coli, Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Candida albicans, and Shigella.

9. The use according to claim 7, wherein the anti-tumor drug is a drug against cervical cancer Hela cell lines.

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