A secondary metabolite, a preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a secondary metabolite, a preparation method and application thereof. The metabolite is named (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester, and is separated from fermentation liquor of endophytic fungus Fusarium oxysporum LZC03. The metabolite has the effects of anti-tumor and anti-oxidation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to a new secondary metabolite extracted and separated from endophytic fungus Fusarium oxysporum LZC03 isolated from Salvia plebeia R.Br., a preparation method and application thereof. BACKGROUND

[0002] Salvia plebeia R.Br. is an annual or biennial herbaceous plant of Labiatae and Salvia, and is distributed almost all over the country except for some areas in the northwest, and is rich in resources. The whole plant of Salvia plebeia R.Br. can be used as medicine, and has the effects of clearing heat and resolving toxins, diuresis and detumescence, and cooling blood and stopping bleeding.

[0003] Plant endophyte is a kind of microorganism existing in the interior of host plants, and does not cause the host plants to show obvious infection symptoms, and there is a strong interaction theory between the endophyte and the host. The endophyte exists in different plants, such as algae, moss, angiosperms, and gymnosperms. Moreover, the endophyte can be distributed in different tissue cells of plants, such as roots, stems, leaves, and fruits. Different endophytes are rich and diverse, and the metabolites produced by different endophytes are also inestimable.

[0004] At present, there is no report about (2-(4-(isopentenyl)oxy)phenyl)acetyl glycine methyl ester and its activity. SUMMARY

[0005] The purpose of the present application is to provide a secondary metabolite, a preparation method and application thereof. The metabolite is named (2-(4-(isopentenyl)oxy)phenyl)acetyl glycine methyl ester, and is separated from the fermentation broth of endophytic fungus Fusarium oxysporum LZC03. The metabolite has the effects of anti-tumor and anti-oxidation.

[0006] The technical scheme for solving the above technical problems is as follows:

[0007] The present application provides a new secondary metabolite, which is named (2-(4-(isopentenyl)oxy)phenyl)acetyl glycine methyl ester, and the structural formula is shown as formula 1a:

[0008]

[0009] The above compound is extracted and separated from endophytic fungus Fusarium oxysporum LZC03 isolated from the root part of Salvia plebeia R.Br., and is first discovered in the present application. Through biological activity determination, it is found that the compound has the effects of anti-tumor and anti-oxidation.

[0010] The application provides a preparation method of the secondary metabolite, and comprises the following steps: extracting and separating from a fermentation liquor of Fusarium oxysporum.

[0011] Further, the strain name of the Fusarium oxysporum is Fusarium oxysporum LZC03, the strain preservation number is CGMCC No.41251, the preservation unit name is China General Microbiological Culture Collection Center, the preservation address is No.3, Beichenxi Road, Chaoyang District, Beijing, China, and the preservation date is May 31, 2024.

[0012] The beneficial effects of the above scheme include that the new compound (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester extracted and separated from the secondary metabolite of the endophytic fungus Fusarium oxysporum LZC03 separated from the root part of the plant Artocarpus heterophyllus can be used for researching and developing new antitumor or antioxidant products, such as food, medicine and health care products.

[0013] Further, the preparation method of the fermentation liquor of the Fusarium oxysporum comprises the following steps: inoculating the Fusarium oxysporum into a culture medium containing rice and carrying out fermentation culture.

[0014] Further, the extracting and separating comprise the following steps:

[0015] (1) the fermentation liquor of the Fusarium oxysporum is extracted by using methanol, then extracted by using n-butanol, separated by using a silica gel column chromatography, the fraction eluted by using dichloromethane and methanol with a volume ratio of 100:2 is collected, and the fraction is detected by using thin layer chromatography to obtain a product Fr.2;

[0016] (2) the product Fr.2 is separated by using an ODS octadecyl bonded phase high-pressure chromatographic column, the fraction eluted by using methanol and water with a volume ratio of 30:70 is collected, and the fraction is detected by using thin layer chromatography to obtain a product Fr.2-1;

[0017] (3) the product Fr.2-1 is purified by using a semi-preparative HPLC to obtain (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester.

[0018] Further, in step (1), the silica gel column chromatography separation conditions include gradient elution by using a dichloromethane-methanol system with a volume ratio of 100:0-100:50.

[0019] Further, in step (2), the ODS octadecyl bonded phase high-pressure chromatographic column separation conditions include gradient elution by using a methanol-distilled water system with a volume ratio of 30:70-80:20 at a flow rate of 21.00 mL / min.

[0020] Further, the purification condition of the semi-preparative HPLC in step (3) includes: CH3CN and H2O in a volume ratio of 30:70, Agela semi-preparative chromatographic column XB-C18-L 5 μm, flow rate of 3 mL / min, wavelength of 210 nm.

[0021] Specifically, the preparation method of the (2-(4-(isopentenyl)oxy)phenyl)acetyl glycine methyl ester can include the following steps:

[0022] Fusarium oxysporum LZC03 was inoculated into PDB medium and continuously cultured at 28°C in a 150 rpm shaker for 7 days; the fermentation seed liquid was inoculated into sterilized medium containing rice, the medium containing rice included rice and water, the ratio of rice and water was 100 g:120 mL, and when inoculation, the ratio could be 100 mL of fermentation seed liquid poured into each bottle of rice medium (each bottle containing 100 g of rice and 120 mL of water), and the medium was cultured at room temperature for 50 days; the fermentation product was soaked in methanol and ultrasonicated for 30 min, filtered, the filtrate was taken, the above steps were repeated, the filtrates were combined, suction filtered, and rotary evaporated to obtain a extract; 500 g of the extract was dispersed in water, extracted with n-butanol, concentrated to obtain an n-butanol layer extract; the n-butanol layer extract was separated by silica gel column chromatography, gradient eluted with a dichloromethane-methanol system in a volume ratio of 100:0-100:50, the fractions were collected, and the collected fractions were detected by thin layer chromatography (dichloromethane:methanol=9:1, volume ratio, Rf=0.75) to obtain Fr.2 (CH2Cl2:CH3OH=100:2); Fr.2 was gradient eluted by ODS octadecyl-bonded phase high-pressure chromatography column with a methanol-distilled water system in a volume ratio of 30:70-80:20 (flow rate 21.00 mL / min), the fractions were collected, and the collected fractions were detected by thin layer chromatography (dichloromethane:methanol=12:1, volume ratio, Rf=0.8) to obtain Fr.2-1 (CH3OH:H2O=30:70); Fr.2-1 was purified by semi-preparative HPLC, the semi-preparative condition including: CH3CN and H2O in a volume ratio of 30:70, Agela semi-preparative chromatographic column XB-C18-L 5 μm, flow rate of 3 mL / min, wavelength of 210 nm, to obtain compound 1a (retention time 28.00 min).

[0023] The application provides application of the new compound (2-(4-(isopentenyl)oxy)phenyl)acetyl glycine methyl ester in preparation of an anti-tumor product.

[0024] The application provides application of the new compound (2-(4-(isopentenyl)oxy)phenyl)acetyl glycine methyl ester in preparation of an anti-oxidation product.

[0025] The product in the above includes, but is not limited to, one or several of drugs, food, health products.

[0026] The application provides a fusarium oxysporum, the strain name of the fusarium oxysporum is LZC03, the strain preservation number is CGMCC No.41251, the name of the preservation unit is China General Microbiological Culture Collection Center, the preservation address is No.3, Beichen West Road, Chaoyang District, Beijing, China, and the preservation date is May 31, 2024.

[0027] The application provides application of the above fusarium oxysporum in preparation of a new compound (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester.

[0028] The above fusarium oxysporum can be used to prepare a new compound (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester, and the compound has the effects of antioxidation and antitumor. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a phylogenetic tree of the strain Fusarium oxysporum LZC03;

[0030] Figure 2 It is a 1H NMR spectrum of the compound 1a 1 1H NMR spectrum

[0031] Figure 3 It is a 13C NMR spectrum of the compound 1a 13 13C NMR spectrum

[0032] Figure 4 It is a HR-ESI-MS spectrum of the compound 1a

[0033] Figure 5 It is a HSQC spectrum of the compound 1a

[0034] Figure 6 It is a HMBC spectrum of the compound 1a. DETAILED DESCRIPTION

[0035] The application will be further described in detail below with reference to examples, so that those skilled in the art can implement the application according to the description.

[0036] Potato dextrose agar medium (PDA) (article number: 02-023) is purchased from Beijing Aoboxing Biotechnology Co., Ltd., and is prepared according to the following proportion: potato 200 g, glucose 20 g, agar 20 g, and distilled water 1000 mL.

[0037] PDB medium was prepared according to the following proportions: mannitol 2%, glucose 2%, yeast extract 0.5%, peptone 1%, potato 20%, prepared with water, all in terms of mass percentage of water. Mannitol (item number: 01-063), peptone (item number: 01-001), yeast extract (item number: 01-014) and glucose (item number: 01-076) were all purchased from Beijing Aobosan Biotechnology Co., Ltd.

[0038] IMDM medium (item number: MA0231) and DMEM medium (item number: MA0212) were both purchased from Dalian Meilunbio Technology Co., Ltd.; RPMI-1640 medium (item number: R2405), fetal bovine serum (item number: F8687) and penicillin-streptomycin solution (item number: TMS-AB2) were all purchased from Merck.

[0039] 75% ethanol was purchased from Texas Sterilization Technology Co., Ltd.; sodium hypochlorite (NaClO) was purchased from Tianjin Tianda Chemical Reagent Factory; methanol (CH3OH) (batch number: 20211121) and dichloromethane (CH2Cl2) (batch number: 20211103) were both purchased from Tianjin Tianli Chemical Reagents Co., Ltd.; acetonitrile (CH3CN) (batch number: R142278) was purchased from Beijing Dima Technology Co., Ltd.; column chromatography silica gel was purchased from Qingdao Haoyang Chemical Factory Branch; thin layer chromatography silica gel plate (model: GF254) was purchased from Yantai Jiangyou Silica Gel Development Co., Ltd.; thiazolyl blue (MTT) (batch number: A4586) was purchased from Tianjin Alpha Biological Technology Co., Ltd.; dimethyl sulfoxide (DMSO) (item number: D6370) was purchased from Beijing Biotopped Co., Ltd.; 1,1-diphenyl-2-picrylhydrazyl (DPPH) (item number: 300267) was purchased from Merck; 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid diamine salt) (ABTS) (item number: A800764), potassium persulfate (item number: P816371) and PBS buffer (item number: P917808) were all purchased from Shanghai Mcllvane Biotech Co., Ltd.; L-ascorbic acid (item number: ST1434-25g) was purchased from Biyun Tian Biotechnology Co., Ltd.

[0040] Test cells: human leukemia cell line (HL-60) (item number: CL-0110), human colon cancer cell line (HCT-116) (item number: CL-0096), prostate cancer cell line (PC-3) (item number: CL-0185), pancreatic cancer cell line (ASPC-1) (item number: CL-0027) were all purchased from Wuhan Punsun Life Science Co., Ltd.

[0041] In the present application, all are conventional methods in the art unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, which can be obtained by commercial channels or prepared by conventional methods in the art.

[0042] The following will be introduced through specific examples.

[0043] Example 1

[0044] Fresh samples of Vitex negundo var. cannabifolia collected from Huangshan, Anhui were washed, and the surface of the samples was treated with 75% ethanol and 5% NaClO solution in a clean bench, and then washed with sterile water for 4 times to disinfect the surface of the Vitex negundo var. cannabifolia. The roots of the Vitex negundo var. cannabifolia with qualified surface disinfection were cut into 2 mm thin slices with a sterilized knife and inoculated on PDA culture medium plates, and placed in a 28°C constant temperature incubator for culture for 3-7 days, and the colonies around the root tissues were observed during the period.

[0045] Strain purification: the colony state was observed, and when the growth was good, the mycelium tip was picked and moved to a PDA culture medium plate, and cultured in a 28°C constant temperature incubator for 3-7 days for separation and purification, and the above steps were repeated until a single colony was obtained, which was named as LZC03.

[0046] Strain morphological characteristics: when the strain was cultured in a 28°C constant temperature incubator for 3 days, the colony on the PDA culture medium plate presented a dark white color on the front side and a red-brown flocculent colony on the back side, and after 7 days of culture, it presented a dark color.

[0047] Strain identification: ITS sequencing analysis of the strain was performed by Shanghai Bioengineering Technology Service Co., Ltd., the measured sequence was subjected to Blast comparison in the NCBI database, the strain sequence with similar homology was downloaded, the phylogenetic tree was constructed by using software MEGA 11.0, and combined with morphology and molecular biology, it was identified as Fusarium oxysporum (red dot strain in China), the sequence similarity was 100.00%, and it was named as Fusarium oxysporum LZC03. Figure 1

[0048] On May 31, 2024, it was preserved in the China General Microbiological Culture Collection Center, the address is No. 1, Beichen West Road, Chaoyang District, Beijing, China, the strain name is Fusarium oxysporum LZC03, and the strain preservation number is CGMCC No. 41251.

[0049] Example 2 Preparation of compound 1a

[0050] ​Fusarium oxysporum LZC03 was inoculated into 500 mL conical flask containing 200 mL PDB medium, and continuously cultured at 28°C, 150 rpm for 7 days in a shaker. The fermentation seed liquid was prepared by the above method. 100 g of rice and 120 mL of water were placed in each of 80 500 mL conical flasks, which were autoclaved at 121°C for 30 min to obtain sterilized rice medium. Then the rice medium was taken out and cooled to room temperature, and 100 mL of fermentation seed liquid was poured into each flask, which was incubated at room temperature (20-25°C) for 50 days. After fermentation, 150 mL of methanol was added to each flask, and ultrasonic treatment was performed for 30 min. Then the mixture was filtered with 4 layers of sterile medical gauze, and the filtrate was collected. The residue was placed in a conical flask and soaked with 150 mL of methanol, and the above steps were repeated for 3 times. The extraction liquid was combined and filtered under reduced pressure. The rotary evaporation was performed at 75°C and 80 r / min to obtain 500 g of extract.

[0051] The 500 g of extract was dispersed in water, and extracted with n-butanol for 3 times. The n-butanol extract was concentrated under reduced pressure at 90°C and 80 r / min to obtain 98.0 g of extract (i.e. n-butanol layer extract). The n-butanol layer extract (98.0 g) was mixed with 147 g of silica gel (100-200 mesh), and the column silica gel was 588 g of silica gel (200-300 mesh), the protective silica gel was 65 g of silica gel (80-100 mesh), and the retention volume was 2 L. The silica gel column chromatography was performed with a gradient elution system of dichloromethane-methanol (100:0-100:50, by volume) to separate the extract. The fractions obtained by elution with different volume ratios of dichloromethane-methanol were collected, and the collected fractions were detected by thin layer chromatography (volume ratio of dichloromethane:methanol=9:1, Rf=0.75). The following 8 products were obtained, which were Fr.1 to Fr.8, respectively.

[0052] Fr.1 was the product (3.1 g) obtained by thin layer chromatography detection of the fraction separated by silica gel column chromatography with a volume ratio of CH2Cl2:CH3OH=100:1.

[0053] Fr.2 was the product (20.2 g) obtained by thin layer chromatography detection of the fraction separated by silica gel column chromatography with a volume ratio of CH2Cl2:CH3OH=100:2.

[0054] Fr.3 was the product (8.9 g) obtained by thin layer chromatography detection of the fraction separated by silica gel column chromatography with a volume ratio of CH2Cl2:CH3OH=100:3.

[0055] Fr.4 was the product (10.1 g) obtained by thin layer chromatography detection of the fraction separated by silica gel column chromatography with a volume ratio of CH2Cl2:CH3OH=100:5.

[0056] Fr.5 was the product (3.2 g) obtained by thin layer chromatography detection of the fraction obtained by silica gel column chromatography separation with the volume ratio of CH2Cl2:CH3OH = 100:10.

[0057] Fr.6 was the product (9.5 g) obtained by thin layer chromatography detection of the fraction obtained by silica gel column chromatography separation with the volume ratio of CH2Cl2:CH3OH = 100:15.

[0058] Fr.7 was the product (2.5 g) obtained by thin layer chromatography detection of the fraction obtained by silica gel column chromatography separation with the volume ratio of CH2Cl2:CH3OH = 100:20.

[0059] Fr.8 was the product (10.5 g) obtained by thin layer chromatography detection of the fraction obtained by silica gel column chromatography separation with the volume ratio of CH2Cl2:CH3OH = 100:50.

[0060] Fr.2 was subjected to rapid gradient elution by ODS octadecyl bonded phase high pressure column (20-35 μm, Tianjin Bonnal Jeger Technology Co., Ltd.) with the methanol:distilled water system (flow rate 21.00 mL / min) in the volume ratio of 30:70-80:20, and the fractions eluted by methanol:distilled water in different volume ratios were collected. The collected fractions were subjected to thin layer chromatography detection (volume ratio, dichloromethane:methanol = 12:1, Rf = 0.8) to obtain four fraction samples, Fr.2-1 to Fr.2-4.

[0061] Fr.2-1 was the product (5.1 g) obtained by thin layer chromatography detection of the fraction obtained by ODS octadecyl bonded phase high pressure column separation with the volume ratio of CH3OH:H2O = 30:70.

[0062] Fr.2-2 was the product (4.9 g) obtained by thin layer chromatography detection of the fraction obtained by ODS octadecyl bonded phase high pressure column separation with the volume ratio of CH3OH:H2O = 45:55.

[0063] Fr.2-3 was the product (3.5 g) obtained by thin layer chromatography detection of the fraction obtained by ODS octadecyl bonded phase high pressure column separation with the volume ratio of CH3OH:H2O = 60:40.

[0064] Fr.2-4 was the product (5.5 g) obtained by thin layer chromatography detection of the fraction obtained by ODS octadecyl bonded phase high pressure column separation with the volume ratio of CH3OH:H2O = 80:20.

[0065] Compound 1a (4 mg, retention time 28.00 min) was obtained by purifying Fr. 2-1 (5.1 g) by semi-preparative HPLC. The preparation conditions included: CH3CN-H2O (volume ratio 30:70), Agela semi-preparative chromatographic column XB-C18-L 5 μm, flow rate 3 mL / min, wavelength 210 nm.

[0066] Example 3

[0067] Compound 1a prepared by the method of Example 2 was a yellow powder (methanol), and its specific1H-NMR and 13 The C-NMR data are shown in Table 1, and this compound was first discovered in the present application.

[0068] Table 1.1H-NMR and C-NMR data of compound 1a 1 H and 13 C nuclear magnetic resonance data

[0069]

[0070] Figures 2 to 6 The1H-NMR, C-NMR, HR-ESI-MS, HSQC and HMBC spectra of the novel compound 1a of the present application were determined to confirm the structure of the compound. Specifically, compound 1a was a yellow powder (methanol), 1 H-NMR, 13 C-NMR, HR-ESI-MS, HSQC and HMBC spectra. Specifically, compound 1a was a yellow powder (methanol), 1 The1H-NMR (600 MHz, DMSO-d6) showed an amide proton signal at δ H 8.41 (1H, s). The signals at δ H 7.15 (2H, d, J = 8.4 Hz), 6.84 (2H, d, J = 8.4 Hz) indicated the presence of a para-substituted benzene ring in the structure. The proton signals at δ H 5.40 (1H, t, J = 6.6 Hz), 4.48 (2H, d, J = 7.2 Hz), 1.73 (3H, s), 1.69 (3H, s) indicated the presence of an oxygen-containing isopentenyl group in the structure. The signal at δ H 3.82 (2H, d, J = 6.0 Hz) indicated the hydrogen signal of a methylene group. The signal at δ H 3.61 (3H, s) indicated the presence of a methoxy group in the structure. The signal at δ H 3.38 (3H, s) indicated the presence of a methylene group connected to nitrogen in the structure.

[0071] 13 The C-NMR (150 MHz, DMSO-d6) gave 16 carbons, including two carbonyl signals at δ c 171.0, 170.3, and δC 157.0, 129.9, 129.9, 127.8, 114.3 and 114.3 are 6 carbon signals of the benzene ring, δ C 136.8, 120.0, 64.1, 25.3, 17.9 are 5 carbon signals of the oxygen-containing isopentenyl group, δ C 51.6 is 1 carbon signal of the methoxy group, δ C 41.0 is the methylene with nitrogen signal, δ C 40.6 is 1 carbon signal of the methylene.

[0072] The mass spectrometry of compound 1a was completed by Dalian Mondi Technology Co., Ltd. The mass-to-charge ratio m / z 314.1447 [M+Na] of HR-ESI-MS was measured + , indicating that the molecular formula of compound 1a is C 16 H 21 NO4.

[0073] The information of direct connection between all hydrogen and carbon in the structure was given by HSQC spectrum data, as shown in Table 1.

[0074] The structure in compound 1a was determined by multiplicity and correlation shown in HMBC spectrum. In the HMBC spectrum, 5.40 (H-12) was correlated with 25.3 (C-14) and 17.9 (C-15); 4.48 (H-11) was correlated with 157.0 (C-8), 136.8 (C-13) and 120.0 (C-12); 1.73 (H-14) was correlated with 136.8 (C-13), 120.0 (C-12), 17.9 (C-15); 1.69 (H-15) was correlated with 136.8 (C-13), 120.0 (C-12), 25.3 (C-14) confirmed the presence of oxygen-containing isopentenyl group. Another group of HMBC 7.15 (H-6) was correlated with 157.0 (C-8), 114.3 (H-7), 40.6 (C-4). 6.84 (H-7) was correlated with 157.0 (C-8), 127.8 (C-5) confirmed the structure of benzene ring. 8.41 (NH), 3.82 (H-4), 3.38 (H-2) were correlated with 170.3 (C-3); 3.61 (H-16) and 3.38 (H-2) were correlated with 171.0 (C-1) confirmed the presence of glycine methyl ester structure fragment. By 4.48 (H-11) correlated with 157.0 (C-8), 7.15 (H-6) correlated with 40.6 (C-4), 3.82 (H-4) correlated with 170.3 (C-3), the oxygen-containing isopentenyl group was connected with glycine methyl ester and benzene ring.

[0075] Based on the data assignment of high resolution mass spectrometry and nuclear magnetic resonance spectroscopy, compound 1a is a new compound which has not been reported in the literature, named as (2-(4-(isopentenyl)oxy)phenyl)acetyl glycine methyl ester.

[0076] Example 4 Anti-tumor experiment

[0077] The MTT method was used to evaluate the inhibitory effect of compound 1a on the proliferation of human leukemia cell line (HL-60), human colon cancer cell line (HCT-116), pancreatic cancer cell line (ASPC-1) and prostate cancer cell line (PC-3).

[0078] The human leukemia cell line (HL-60) was cultured in IMDM medium containing 20% fetal bovine serum; the human colon cancer cell line (HCT-116) was cultured in RPMI-1640 medium containing 10% fetal bovine serum; the pancreatic cancer cell line (ASPC-1) and the prostate cancer cell line (PC-3) were cultured in DMEM medium containing 10% fetal bovine serum, and penicillin-streptomycin double antibody (the final concentration of penicillin was 1%, and the final concentration of streptomycin was 1%) was added to the medium, and the culture was carried out in a 37°C, 5% CO2 incubator.

[0079] DMSO was used to dissolve sample compound 1a and 5-fluorouracil (the final concentration of DMSO was <0.1%). Before the test, the prepared medium was diluted to the required concentration for the test.

[0080] The logarithmic growth phase of the test cells was taken, and 5-7) x 10 4 / mL of the cells were inoculated in a 96-well plate, 100 μL per well, and cultured in a 37°C, 5% CO2 incubator for 24 h. The original culture medium was then aspirated, and 100 μL of culture medium containing compound 1a was added to each well as the drug administration group. The concentration gradient of compound 1a was 1.25, 2.5, 5, 10, 20, 40, and 80 μM. The final concentration of 5-fluorouracil in the positive control group was the same as that in the sample group, i.e. 1.25, 2.5, 5, 10, 20, 40, and 80 μM, and the other conditions were the same as those in the sample group. The blank control group was added with 100 μL of culture medium. Each group had 6 replicate wells. After 48 h of continuous culture, 10 μL of MTT solution (the concentration of MTT was 5 mg / mL) was added to each well for incubation for 4 h. The supernatant was then discarded, and then 150 μL of DMSO was added to each well. After shaking at room temperature for 10 min, the absorbance value (OD) of each well was measured at 570 nm on an enzyme marker. The half growth inhibition concentration (IC 50 ) was obtained by regression analysis of the concentration-reaction data. The calculation formula for the inhibition rate of compound 1a on tumor cell proliferation was:

[0081] Inhibition rate (%) = (1-OD 给药组 / OD 空白对照组* 100%

[0082] The experimental results are shown in Table 2. Compound 1a has different degrees of inhibitory effects on the four tumor cells, and the inhibitory effect on human colon cancer HCT-116 cells is the most significant (IC 50 = 10.15 μM), which is better than that of the positive drug 5-fluorouracil (IC 50 = 15.00 μM).

[0083] Table 2 Inhibitory effect of compound 1a on tumor cell proliferation (IC 50 , μM)

[0084]

[0085] Example 5 Test of antioxidant activity

[0086] The DPPH method was used to detect the antioxidant activity, including the following steps: 3.94 mg of DPPH was accurately weighed and dissolved with anhydrous ethanol, and then diluted to 10 mL in a brown volumetric flask, diluted 5 times, and prepared into a DPPH working solution with a concentration of 0.2 mM. The concentration of compound 1a was prepared as 100, 50, 25, 10, 5, and 1 μg / mL, respectively. 100 μL of sample solution was taken in a 96-well plate with a pipette, 100 μL of DPPH working solution was added as a sample group, 100 μL of anhydrous ethanol was added instead of DPPH working solution, and 100 μL of sample solution with different concentrations was added as a control group, and 100 μL of anhydrous ethanol was added to 100 μL of DPPH working solution as a blank group. After mixing, the reaction was carried out in the dark for 30 min, and the absorbance of each well was measured at 517 nm wavelength with an enzyme marker. L-ascorbic acid was used as a positive control to determine the DPPH free radical scavenging rate.

[0087] The scavenging rate was calculated by the following formula: DPPH free radical scavenging rate (%) = [1-(A1-A2) / A0] x 100%, wherein the absorbance value of the blank group is A0, the absorbance value of the sample group is A1, and the absorbance value of the control group is A2.

[0088] ABTS method was used to detect the antioxidant activity, including the following steps: 7.0 mmol / L of ABTS solution and 2.45 mmol / L of potassium persulfate solution were prepared respectively, and the above two solutions were mixed in equal volume, and stored at room temperature for 12-16 hours in the dark, and then a certain amount of prepared solution was diluted with PBS buffer (0.01 mol / L, pH 7.4) to make the absorbance value measured at 734 nm be 0.70±0.02, and the ABTS working solution was obtained. The concentration of compound 1a was prepared as 100, 50, 25, 10, 5, 1 μg / mL respectively. 150 μL of ABTS working solution was placed in a 96-well plate, 100 μL of sample solution of different concentrations was added to 150 μL of ABTS working solution in the sample group, and ethanol was used instead of ABTS working solution in the control group, that is, 100 μL of sample solution of different concentrations was added to 150 μL of ethanol in the control group, and 100 μL of ethanol was added to 150 μL of ABTS working solution in the blank group. Then, mix well, avoid light and stand for 20 min, and then measure the absorbance of each well at 734 nm with an enzyme-labeled instrument. L-ascorbic acid was used as a positive control to determine the ABTS free radical scavenging rate

[0089] The scavenging rate was calculated by the following formula: ABTS free radical scavenging rate (%) = [1-(A1-A2) / A0]x100%, wherein the absorbance value of the blank group is A0, the absorbance value of the sample group is A1, and the absorbance value of the control group is A2.

[0090] The experimental results are shown in Table 3. In the DPPH free radical scavenging ability experiment, compared with the positive drug L-ascorbic acid, the antioxidant capacity of compound 1a was more significant, and compound 1a had good anti-DPPH free radical ability. In the ABTS free radical scavenging ability experiment, compound 1a also had antioxidant capacity.

[0091] Table 3 antioxidant activity (IC 50 , μg / mL)

[0092]

[0093] Although the embodiments of the present application have been disclosed as above, they are not limited only to the application listed in the specification and embodiments, and can be fully applied to various fields suitable for the present application, and additional modifications can be easily realized by those skilled in the art, and therefore the present application is not limited to specific details, without departing from the general concept defined by the claims and equivalent scope.

Claims

1. A secondary metabolite, characterized in that, Methyl (2-(4-(isopentenyl)oxy)phenyl)acetate, having the structure shown in 1a:

2. Use of the secondary metabolite according to claim 1 for the preparation of an antioxidant product.

3. A strain of Fusarium oxysporum, characterized in that, The strain is named Fusarium oxysporum LZC03, and the strain preservation number is CGMCC No. 41251.

4. Use of the Fusarium oxysporum according to claim 3 for the preparation of the secondary metabolite according to claim 1.

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