Secondary metabolite, preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a secondary metabolite as well as a preparation method and application thereof. The name of the metabolite is (2-(4-(isopentenyl) oxy) phenyl) acetyl glycine methyl ester, and the metabolite is obtained by separation from a fermentation broth of an endophytic fungus Fusarium oxysporum LZC03. The metabolite has anti-tumor and anti-oxidation effects.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to a new secondary metabolite extracted and separated from an endophytic fungus Fusariumoxysporum LZC03 isolated from the plant Litchi grass, a preparation method and an application thereof. Background Art

[0002] Salvia plebeia R.Br. is an annual or biennial herbaceous plant of the Lamiaceae family and the genus Salvia. It is distributed almost all over the country except some areas in the northwest, and its resources are abundant. The whole plant of Salvia plebeia can be used as medicine, which has the effects of clearing away heat and detoxifying, diuresis and swelling, cooling blood and stopping bleeding.

[0003] Plant endophytes are a type of microorganisms that exist inside the host plant but do not cause the host plant to show obvious symptoms of infection, and there is a strong interaction theory between endophytes and the host. Endophytes exist in different plants, such as algae, mosses, angiosperms, gymnosperms, etc. In addition, endophytes can be distributed in different tissue cells of plants, such as roots, stems, leaves, fruits, etc. The endophytes of different plants are rich and diverse, and the metabolites that different endophytes can produce are also inestimable.

[0004] At present, there is no report on (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester and its activity. Summary of the invention

[0005] The object of the present invention is to provide a secondary metabolite, a preparation method and an application thereof. The metabolite is named (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester, which is isolated from the fermentation broth of the endophytic fungus Fusarium oxysporumLZC03. The metabolite has anti-tumor and antioxidant effects.

[0006] The technical solution to solve the above technical problems is as follows:

[0007] The present invention provides a new secondary metabolite, named (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester, and its structural formula is shown in Formula 1a:

[0008]

[0009] The above compound is extracted and separated from the endophytic fungus Fusarium oxysporum LZC03 separated from the root of the plant lychee grass, and is discovered for the first time by the present invention. Through biological activity assay, it is found that it has anti-tumor activity and antioxidant activity.

[0010] The present invention provides a method for preparing the secondary metabolites, comprising the following steps: extracting and separating from the fermentation liquid of Fusarium oxysporum.

[0011] Furthermore, the strain name of Fusarium oxysporum is Fusarium oxysporum LZC03, the strain preservation number is CGMCC No.41251, the name of the preservation unit is the General Microbiology Center of China Culture Collection Administration, the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China, and the preservation date is May 31, 2024.

[0012] The beneficial effects of adopting the above scheme include: the new compound (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester) extracted and separated from the secondary metabolites of the endophytic fungus Fusarium oxysporum LZC03 separated from the root of the plant lychee grass has anti-tumor and antioxidant activities, and can be used to research and develop new anti-tumor or antioxidant products, such as: food, medicine, health care products, etc.

[0013] Furthermore, the method for preparing the fermentation liquid of Fusarium oxysporum comprises the following steps: inoculating Fusarium oxysporum into a culture medium containing rice and fermenting and culturing the culture medium.

[0014] Further, the extraction and separation comprises the following steps:

[0015] (1) extracting the fermentation broth of Fusarium oxysporum with methanol, then extracting with n-butanol, separating by silica gel column chromatography, collecting the fractions eluted with a volume ratio of dichloromethane to methanol of 100:2, and detecting the fractions by thin layer chromatography to obtain the product Fr.2;

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

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

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

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

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

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

[0022] Fusarium oxysporum LZC03 was inoculated into a PDB culture medium and continuously cultured in a shaking table at 28°C and 150 rpm for 7 days; the fermented seed liquid was inoculated into a sterilized culture medium containing rice, the culture medium containing rice includes rice and water, the ratio of rice to water is 100 g: 120 mL, and the ratio during inoculation can be 100 mL of fermented seed liquid poured into each bottle of rice culture medium (each bottle contains 100 g of rice and 120 mL of water), and cultured at room temperature for 50 days; the fermented product was soaked in methanol and ultrasonicated for 30 minutes, filtered, and the filtrate was taken, the above steps were repeated, the filtrate was combined, suction filtered, and rotary evaporated to obtain an extract; 500 g of the extract was dispersed in water, extracted with n-butanol, and concentrated to obtain an n-butanol layer extract; the n-butanol layer extract was separated by silica gel column chromatography, and gradient eluted with a dichloromethane-methanol system with a volume ratio of 100:0-100:50 The product was removed by elution, and the fractions were collected and subjected to thin layer chromatography (dichloromethane: methanol = 9: 1, volume ratio, Rf = 0.75) to obtain Fr.2 (CH2C12: CH3OH = 100: 2); Fr.2 was subjected to gradient elution using an ODS octadecyl bonded phase medium and high pressure chromatographic column with a methanol-distilled water system of a volume ratio of 30: 70-80: 20 (flow rate 21.00 mL / min), and the fractions were collected and subjected to 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 subjected to semi-preparative HPLC, and the semi-preparative conditions included: a volume ratio of CH3CN to H2O of 30: 70, an Agela semi-preparative chromatographic column XB-C18-L 5 μm, flow rate 3 mL / min, wavelength 210 nm, and compound 1a was purified (retention time 28.00 min).

[0023] The invention provides application of a novel compound (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester in preparing an anti-tumor product.

[0024] The invention provides application of a novel compound (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester in preparing an antioxidant product.

[0025] The above products include but are not limited to one or more of medicines, foods, and health products.

[0026] The present invention provides a strain of Fusarium oxysporum, the strain name is Fusarium oxysporum (Fusarium oxysporum) LZC03, the strain preservation number is CGMCC No.41251, the preservation unit name is General Microbiology Center of China Microorganism Culture Collection Administration, the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China, and the preservation date is May 31, 2024.

[0027] The present invention provides application of the above-mentioned Fusarium oxysporum in preparing a new compound (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester).

[0028] The novel compound (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester) can be prepared by using the above-mentioned Fusarium oxysporum. The compound has the effects of anti-oxidation and anti-tumor. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0030] Figure 2 For compound 1a 1 H NMR spectrum

[0031] Figure 3 For compound 1a 13 C NMR spectrum;

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

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

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

[0035] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0036] Potato dextrose agar medium (PDA) (Cat. No. 02-023) was purchased from Beijing Aoboxing Biotechnology Co., Ltd. and prepared in the following proportions: 200 g potato, 20 g glucose, 20 g agar, and 1000 mL distilled water.

[0037] PDB medium was prepared in the following proportions: 2% mannitol, 2% glucose, 0.5% yeast extract, 1% peptone, and 20% potato, prepared with water, all of which are relative to the mass percentage of water. Mannitol (Cat. No.: 01-063), peptone (Cat. No.: 01-001), yeast extract (Cat. No.: 01-014), and glucose (Cat. No.: 01-076) were purchased from Beijing Aoboxing Biotechnology Co., Ltd.

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

[0039] 75% ethanol was purchased from Dezhou Chengze Disinfection 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 purchased from Tianjin Tianli Chemical Reagent Co., Ltd.; acetonitrile (CH3CN) (batch number: R142278) was purchased from Beijing Dima Technology Co., Ltd.; column chromatography silica gel was purchased from Qingdao Ocean Chemical Plant 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 Biosciences Co., Ltd. Technology Co., Ltd.; dimethyl sulfoxide (DMSO) (Cat. No.: D6370) was purchased from Beijing Biotopped Co., Ltd.; 1,1-diphenyl-2-picrylphenylhydrazine (DPPH) (Cat. No.: 300267) was purchased from Merck; 2,2-diazo-bis-(3-ethylbenzothiazole-6-sulfonic acid diamine salt) (ABTS) (Cat. No.: A800764), potassium persulfate (Cat. No.: P816371) and PBS buffer (Cat. No.: P917808) were all purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; L-ascorbic acid (Cat. No.: ST1434-25g) was purchased from Beyotime Biotechnology Co., Ltd.

[0040] Test cells: human leukemia cell line (HL-60) (Cat. No.: CL-0110), human colon cancer cell line (HCT-116) (Cat. No.: CL-0096), prostate cancer cell line (PC-3) (Cat. No.: CL-0185), and pancreatic cancer cell line (ASPC-1) (Cat. No.: CL-0027) were all purchased from Wuhan Pronocell Life Science Technology Co., Ltd.

[0041] In the present invention, unless otherwise specified, all methods are conventional in the art. The materials, reagents, methods and instruments used are conventional in the art unless otherwise specified, and those skilled in the art can obtain them through commercial channels or prepare the solutions used in the experiment using conventional methods in the art.

[0042] The following is an introduction through specific embodiments.

[0043] Example 1

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

[0045] Strain purification: Observe the status of the colony. When it grows well, pick the tip of the hyphae and transfer it to a PDA medium plate. Culture it in a 28°C constant temperature incubator for 3-7 days for separation and purification. Repeat the above steps until a single colony is obtained, which is named LZC03.

[0046] Morphological characteristics of the strain: When the strain was cultured in a constant temperature incubator at 28°C for 3 days, the front of the colonies on the PDA culture medium plate appeared dark white, and the back of the colonies appeared reddish brown flocculent colonies, which turned dark after 7 days of culture.

[0047] Identification of the strain: The strain was sequenced and analyzed by Shanghai Bioengineering Technology Service Co., Ltd. The measured sequence was compared with the NCBI database by Blast, and the strain sequence with similar homology to the strain sequence was downloaded. The phylogenetic tree was constructed using the software MEGA 11.0. Combining morphology and molecular biology, it was identified as Fusarium oxysporum ( Figure 1 The strain marked with a red dot) had a sequence similarity of 100.00% and was named Fusarium oxysporum LZC03.

[0048] On May 31, 2024, it was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. The strain name is Fusarium oxysporum LZC03, and the strain deposit number is CGMCC No.41251.

[0049] Example 2 Preparation of Compound 1a

[0050] Fusarium oxysporum LZC03 was inoculated into a 500mL conical flask containing 200mL PDB culture medium, and cultured continuously for 7 days at 28°C and 150rpm in a shaking table, and the fermentation seed liquid was prepared by the above method. 100g rice and 120mL water were placed in each bottle of a 500mL conical flask, totaling 80 bottles, and sterilized at 121°C for 30min to obtain a sterilized rice culture medium. After that, the rice culture medium was taken out and cooled to room temperature, and 100mL fermentation seed liquid was poured into each bottle, and cultured at room temperature (20-25°C) for 50 days. After the fermentation was completed, 150mL methanol was added to each bottle for soaking and ultrasonication for 30min, and then filtered with 4 layers of sterile medical gauze, the filtrate was squeezed out, the filtrate was collected, and the residue was placed in a conical culture bottle and continued to be soaked in 150mL methanol, and ultrasonication was repeated for 3 times. The extract was combined for suction filtration, and rotary evaporation was performed to dryness at 75°C and a speed of 80r / min to obtain 500g of extract.

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

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

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

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

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

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

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

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

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

[0060] Fr.2 was rapidly eluted with an ODS octadecyl bonded phase medium and high pressure chromatography column (20-35 μm, Tianjin Bona Aiger Technology Co., Ltd.) using a methanol: distilled water system with a volume ratio of 30:70-80:20 (flow rate 21.00 mL / min). The fractions obtained by elution with different volume ratios of methanol: distilled water were collected and detected by thin layer chromatography (volume ratio, dichloromethane: methanol = 12:1, Rf = 0.8) to obtain 4 fraction samples, namely Fr.2-1 to Fr.2-4.

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

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

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

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

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

[0066] Example 3

[0067] Compound 1a prepared by the method of Example 2 is a yellow powder (methanol). The specific 1H-NMR and 13 C-NMR data are shown in Table 1. This compound is discovered for the first time by the present invention.

[0068] Table 1 Compound 1a 1 H and 13 C NMR data

[0069]

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

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

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

[0073] The HSQC spectrum data provides information on all hydrogen-carbon direct connections in the structure, as shown in Table 1.

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

[0075] In summary, based on the data attribution of high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy, compound 1a is a new compound that has not been reported in the literature and is named (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester.

[0076] Example 4 Antitumor Experiment

[0077] The MTT assay 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 was added to the culture medium (the final concentration of penicillin was 1%, and the final concentration of streptomycin was 1%), and cultured in a 37°C, 5% CO2 incubator.

[0079] The sample compound 1a and 5-fluorouracil were dissolved in DMSO (final DMSO concentration < 0.1%) and diluted to the required concentration in the prepared culture medium before the experiment.

[0080] Take the test cells in the logarithmic growth phase and use (5-7)×10 4 / mL were inoculated in a 96-well plate, with 100 μL in each well. After culturing in a 37°C, 5% CO2 incubator for 24 hours, the original culture medium was aspirated and discarded, and 100 μL of culture medium containing compound 1a was added to each well as the dosing group, and the concentration gradient of compound 1a was set to 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 other conditions were the same as those in the sample group; 100 μL of culture medium was added to the blank control group; 6 replicate wells were set for each group. After 48 h of continuous culture, 10 μL of MTT solution (MTT concentration was 5 mg / mL) was added to each well and incubated for 4 h. The supernatant was discarded, and then 150 μL of DMSO was added to each well. After oscillation at room temperature for 10 min, the absorbance value (OD) of each well was measured at 570 nm on a microplate reader. The half growth inhibition concentration (IC) was obtained by regression analysis of the concentration-response data. 50 ). The calculation formula of the inhibition rate of compound 1a on tumor cell proliferation is:

[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, among which the inhibitory effect on human colon cancer HCT-116 cells is the most significant (IC 50 =10.15μM), which is better than 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 Antioxidant Activity Test

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

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

[0088] The antioxidant activity was detected by the ABTS method, including the following steps: preparing an ABTS solution with a final concentration of 7.0 mmol / L and a potassium persulfate solution with a final concentration of 2.45 mmol / L, respectively, mixing the two solutions in equal volumes, storing them at room temperature in the dark for 12-16 hours, taking an appropriate amount of the prepared solution, diluting it with PBS buffer (0.01 mol / L, pH 7.4) so ​​that the absorbance measured at 734 nm is 0.70±0.02, and obtaining the ABTS working solution. The concentrations of compound 1a were prepared at 100, 50, 25, 10, 5, and 1 μg / mL, respectively. 150 μL ABTS working solution was placed in a 96-well plate. The sample group added 100 μL sample solutions of different concentrations to 150 μL ABTS working solution. The control group replaced the ABTS working solution with anhydrous ethanol, that is, 100 μL sample solutions of different concentrations were added to 150 μL anhydrous ethanol. The blank group added 100 μL anhydrous ethanol to 150 μL ABTS working solution. After that, mix well, keep it away from light for 20 minutes, and measure the absorbance of each well at a wavelength of 734 nm using an enzyme reader. 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] × 100%, wherein the absorbance value of the blank group was A0, the absorbance value of the sample group was A1, and the absorbance value of the control group was 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, compound 1a has a more significant antioxidant ability, and compound 1a has a better ability to resist DPPH free radicals. In the ABTS free radical scavenging ability experiment, compound 1a also has antioxidant ability.

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

[0092]

[0093] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A secondary metabolite, characterized in that The name is (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester, and the structural formula is shown in 1a:

2. Use of the secondary metabolite according to claim 1 in the preparation of antioxidant products.

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

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

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