Application of secondary metabolite in preparation of anti-tumor product

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a secondary metabolite in preparation of an anti-tumor product. The name of the metabolite is (2-(4-(isopentenyl) oxy) phenyl) acetyl glycine methyl ester, the metabolite is separated from fermentation liquor of endophytic fungi Fusarium oxysporum LZC03, and the metabolite has the anti-tumor and anti-oxidation effects and can be used for preparing products with the anti-tumor and anti-oxidation effects.

Description

[0001] This invention is a divisional application. The original Chinese invention patent application number was 202510079936.6, the application date was January 18, 2025, and the patent title at the time of application was: A secondary metabolite, preparation method and application thereof. Technical Field

[0002] This invention belongs to the pharmaceutical field, specifically relating to the application of secondary metabolites in the preparation of antitumor products. Background Technology

[0003] Lychee grass ( Salvia plebeia R. Br. is an annual or biennial herb belonging to the Lamiaceae family and the Sage genus. It is distributed almost throughout China, except for parts of Northwest China, and is abundant in resources. The whole plant of R. Br. can be used medicinally, possessing properties such as clearing heat and detoxifying, promoting diuresis and reducing swelling, cooling the blood and stopping bleeding.

[0004] Plant endophytes are a class of microorganisms that exist within the host plant but do not cause obvious infection symptoms in the host plant, and there is a strong theory of interaction between endophytes and the host. Endophytes exist in various plants, such as algae, mosses, angiosperms, and gymnosperms. Furthermore, endophytes can be distributed in different plant tissues and cells, such as roots, stems, leaves, and fruits. The endophytes of different plants are rich and diverse, and the metabolic products produced by different endophytes are incalculable.

[0005] Currently, there are no reports on (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester and its activity. Summary of the Invention

[0006] The purpose of this invention is to provide a secondary metabolite, its preparation method, and its application. The metabolite is named (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester, and is obtained from endophytic fungi. Fusarium oxysporum The metabolite was isolated from the fermentation broth of LZCO3. This metabolite possesses antitumor and antioxidant properties.

[0007] The technical solution to the above-mentioned technical problem to be solved by the present invention is as follows: This invention provides a novel secondary metabolite, named (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester, with the structural formula shown in Formula 1a: .

[0008] The above-mentioned compounds are endophytic fungi isolated from the roots of the plant *Lychee rubra*. Fusarium oxysporum This substance was extracted and isolated from LZCO3, a discovery made for the first time in this invention. Bioactivity assays revealed that it possesses both antitumor and antioxidant activities.

[0009] This invention provides a method for preparing the above-mentioned secondary metabolites, comprising the following steps: from Fusarium oxysporum (… Fusarium oxysporum Extracted and separated from the fermentation broth of ).

[0010] Furthermore, the strain name of Fusarium oxysporum is Fusarium oxysporum (… Fusarium oxysporum LZC03, strain accession number CGMCC No. 41251, deposited at China General Microbiological Culture Collection Center, address No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, on May 31, 2024.

[0011] The beneficial effects of adopting the above-mentioned approach include: the endophytic fungi isolated from the roots of the plant *Lychee rubra* in this invention. Fusarium oxysporum The novel compound (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester) extracted and isolated from the secondary metabolites of LZC03 has antitumor and antioxidant activities and can be used to study and develop new antitumor or antioxidant products, such as food, drugs, and health products.

[0012] Furthermore, the preparation method of Fusarium oxysporum fermentation broth includes the following steps: inoculating Fusarium oxysporum into a culture medium containing rice and fermenting.

[0013] Furthermore, the extraction and separation process includes the following steps: (1) The fermentation broth of Fusarium oxysporum was extracted with methanol, then extracted with n-butanol, separated by silica gel column chromatography, and the fraction obtained by elution with dichloromethane and methanol at a volume ratio of 100:2 was collected. The fraction was detected by thin-layer chromatography to obtain product Fr.2. (2) Fr.2 was separated by high-pressure chromatography in ODS octadecyl bonded phase, and the fraction obtained by elution with methanol and water at a volume ratio of 30:70 was collected. The fraction was then subjected to thin-layer chromatography to obtain product Fr.2-1. (3) The product Fr.2-1 was purified by semi-preparative HPLC to obtain (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester.

[0014] Furthermore, in step (1), the conditions for silica gel column chromatography separation include gradient elution using a dichloromethane-methanol system with a volume ratio of 100:0-100:50.

[0015] Furthermore, in step (2), the conditions for high-pressure column separation in the octadecyl bonded phase of ODS include: gradient elution with a methanol-distilled water system at a volume ratio of 30:70-80:20 and a flow rate of 21.00 mL / min.

[0016] 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 chromatographic column XB-C18-L 5 μm, a flow rate of 3 mL / min, and a wavelength of 210 nm.

[0017] Specifically, the preparation method of (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester may include the following steps: Will Fusarium oxysporum LZC03 was inoculated into PDB medium and cultured continuously for 7 days at 28℃ and 150 rpm in a shaker. The fermentation seed liquid was then inoculated into a sterilized rice-containing medium (rice and water in a 100 g:120 mL ratio). The inoculation ratio was 100 mL of fermentation seed liquid added to each bottle of rice medium (containing 100 g of rice and 120 mL of water). The mixture was then incubated statically at room temperature for 50 days. The fermentation product was then soaked in methanol and sonicated for 30 minutes. After filtration, the filtrate was collected, and the above steps were repeated. The filtrates were combined, filtered under vacuum, and evaporated by rotary evaporation to obtain an extract. 500g of the extract was dispersed in water, extracted with n-butanol, and concentrated to obtain the n-butanol layer extract. The n-butanol layer extract was separated by silica gel column chromatography using a gradient elution system of dichloromethane-methanol with a volume ratio of 100:0-100:50. The fractions were collected and analyzed by thin-layer chromatography (dichloromethane:methanol = 9:1, volume ratio, R...). f =0.75), yielding Fr.2 (CH2Cl2:CH3OH =100:2); Fr.2 was subjected to gradient elution on an ODS octadecyl-bonded phase high-pressure column with a methanol-distilled water system at a volume ratio of 30:70-80:20 (flow rate 21.00 mL / min), and the fractions were collected. The collected fractions were then analyzed by thin-layer chromatography (dichloromethane:methanol = 12:1, volume ratio, R f =0.8), to obtain Fr.2-1 (CH3OH:H2O = 30:70); Fr.2-1 was purified by semi-preparative HPLC under the following conditions: CH3CN to H2O volume ratio 30:70, Agela semi-preparative column XB-C18-L 5μm, flow rate 3 mL / min, wavelength 210nm, to obtain compound 1a (retention time 28.00 min).

[0018] This invention provides the application of the above-mentioned secondary metabolite (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester) in the preparation of antitumor products.

[0019] Furthermore, the secondary metabolites are derived from Fusarium oxysporum (… Fusarium oxysporum It was obtained by extraction and separation of the fermentation broth.

[0020] Furthermore, the strain name of the *Fusarium oxysporum* is *Fusarium oxysporum* (…). Fusarium oxysporum LZC03, strain preservation number is CGMCC No. 41251.

[0021] The aforementioned secondary metabolites can be used to treat one or more of the following: leukemia, colon cancer, pancreatic cancer, and prostate cancer.

[0022] The aforementioned secondary metabolites can be used to inhibit the proliferation of tumor cells.

[0023] This invention provides the application of the novel compound (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester) in the preparation of antioxidant products.

[0024] The products mentioned above include, but are not limited to, one or more of the following: pharmaceuticals, food, and health products.

[0025] This invention provides a strain of Fusarium oxysporum, named Fusarium oxysporum (… Fusarium oxysporum LZC03, strain accession number CGMCC No. 41251, deposited at China General Microbiological Culture Collection Center, address No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, on May 31, 2024.

[0026] This invention provides the use of the above-mentioned Fusarium oxysporum in the preparation of a new compound (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester.

[0027] A new compound, (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester, can be prepared using the above-mentioned Fusarium oxysporum. This compound has antioxidant and antitumor effects. Attached Figure Description

[0028] Figure 1 strain Fusarium oxysporum Phylogenetic tree of LZC03; Figure 2 For compound 1a 1 H NMR spectrum Figure 3 For compound 1a 13 C NMR spectrum; Figure 4 The HR-ESI-MS spectrum of compound 1a; Figure 5 The HSQC spectrum of compound 1a; Figure 6 The image shows the HMBC spectrum of compound 1a. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0030] Potato glucose agar medium (PDA) (product number: 02-023) was purchased from Beijing Aoboxing Biotechnology Co., Ltd., and was prepared according to the following ratio: 200g potato, 20g glucose, 20g agar, and 1000mL distilled water.

[0031] PDB culture medium was prepared in the following proportions: mannitol 2%, glucose 2%, yeast extract 0.5%, peptone 1%, and potato 20%, diluted with water. All percentages are relative to water by mass. Mannitol (catalog number: 01-063), peptone (catalog number: 01-001), yeast extract (catalog number: 01-014), and glucose (catalog number: 01-076) were all purchased from Beijing Aoboxing Biotechnology Co., Ltd.

[0032] 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.

[0033] 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 both purchased from Tianjin Tianli Chemical Reagent Co., Ltd.; acetonitrile (CH3CN) (batch number: R142278) was purchased from Beijing Dima Technology Co., Ltd.; silica gel for column chromatography was purchased from Qingdao Ocean Chemical Plant Branch; thin-layer chromatography silica gel plates (model: GF254) were purchased from Yantai Jiangyou Silica Gel Development Co., Ltd.; thiazolyl blue (MTT) (batch number: A4586) was purchased from Tianjin Alpha Biotechnology Co., Ltd. The following products were purchased from Beijing Biotopped Co., Ltd.: Dimethyl sulfoxide (DMSO) (Catalog No.: D6370); 1,1-diphenyl-2-picrylhydrazine (DPPH) (Catalog No.: 300267) from Merck; 2,2-diazo-bis-(3-ethylbenzothiazol-6-sulfonic acid diamine salt) (ABTS) (Catalog No.: A800764), potassium persulfate (Catalog No.: P816371) and PBS buffer (Catalog No.: P917808) were all purchased from Shanghai Maclean Biotechnology Co., Ltd.; L-ascorbic acid (Catalog No.: ST1434-25g) was purchased from Beyotime Biotechnology Co., Ltd.

[0034] The test cells were all purchased from Wuhan Pronosai Life Science Technology Co., Ltd.: human leukemia cell line (HL-60) (catalog number: CL-0110), human colon cancer cell line (HCT-116) (catalog number: CL-0096), prostate cancer cell line (PC-3) (catalog number: CL-0185), and pancreatic cancer cell line (ASPC-1) (catalog number: CL-0027).

[0035] Unless otherwise specified, all methods used in this invention are conventional in the art. Unless otherwise specified, all materials, reagents, methods, and instruments used are conventional in the art, and those skilled in the art can obtain them commercially or prepare the solutions using conventional methods in the art.

[0036] The following is a description through specific embodiments.

[0037] Example 1 Fresh litchi grass samples collected from Huangshan, Anhui Province, were washed and treated with 75% ethanol and 5% NaClO solution in a clean bench. The samples were then rinsed four times with sterile water to disinfect the surface. The disinfected litchi grass roots were then sliced ​​into 2 mm thin slices using a sterile knife and inoculated onto PDA agar plates. These plates were then incubated at 28°C for 3-7 days, during which time the microbial community around the root tissue was observed.

[0038] Strain purification: Observe the colony status. When the growth is good, pick the tip of the hyphae and transfer it to a PDA medium plate. Incubate in a constant temperature incubator at 28℃ for 3-7 days for isolation and purification. Repeat the above steps until a single colony is obtained and name it LZC03.

[0039] Morphological characteristics of the strain: When the strain was cultured in a constant temperature incubator at 28℃ for 3 days, the colonies on the PDA medium plate appeared dark white on the front and reddish-brown flocculent colonies on the back. After 7 days of culture, the colonies turned dark.

[0040] Strain identification: ITS sequencing analysis of the strain was performed by Shanghai Bioengineering Technology Service Co., Ltd. The obtained sequences were compared with those in the NCBI database using BLAST. Sequences of strains with similar homology to the strain were downloaded, and a phylogenetic tree was constructed using MEGA 11.0 software. Combining morphological and molecular biological analysis, the strain was identified as… Fusarium oxysporum ( Figure 1 The strain marked with a red dot (with a sequence similarity of 100.00%) was named... Fusarium oxysporum LZC03.

[0041] On May 31, 2024, it was deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. The strain name is *Fusarium oxysporum*.Fusarium oxysporum LZC03, strain preservation number CGMCC No. 41251.

[0042] Example 2 Preparation of compound 1a Will Fusarium oxysporum LZC03 was inoculated into 500 mL Erlenmeyer flasks containing 200 mL of PDB medium and cultured continuously for 7 days at 28℃ and 150 rpm in a shaker to prepare the fermentation seed liquid. 80 Erlenmeyer flasks, each containing 100 g of rice and 120 mL of water, were autoclaved at 121℃ for 30 min to obtain sterilized rice culture medium. The rice culture medium was then removed and cooled to room temperature. 100 mL of the fermentation seed liquid was added to each flask, and the mixture was incubated statically at room temperature (20-25℃) for 50 days. After fermentation, each flask was soaked in 150 mL of methanol and sonicated for 30 min. The mixture was then filtered through four layers of sterile medical gauze, the filtrate was squeezed out, and the residue was collected. The residue was placed in an Erlenmeyer flask and soaked in 150 mL of methanol again. This process was repeated three times. The extracts were combined and filtered, then evaporated to dryness at 75℃ and 80 rpm to obtain 500 g of extract.

[0043] 500 g of the extract was dispersed in water and extracted three times with n-butanol. The extract was then concentrated under reduced pressure at 90℃ and 80 r / min to obtain 98.0 g of extract (i.e., the n-butanol layer extract). The n-butanol layer extract (98.0 g) was mixed with 147 g of silica gel (100-200 mesh). The column silica gel consisted of 588 g of 200-300 mesh silica gel and 65 g of 80-100 mesh silica gel as a protectant, with a retention volume of 2 L. Separation was performed by silica gel column chromatography using a gradient elution system of dichloromethane-methanol at a volume ratio of 100:0-100:50. Fractions obtained from different volume ratios of dichloromethane-methanol were collected, and the collected fractions were analyzed by thin-layer chromatography (volume ratio, dichloromethane:methanol = 9:1, R...). f =0.75), yielding the following 8 products, namely Fr.1 to Fr.8.

[0044] Fr.1 is the product (3.1 g) obtained by thin-layer chromatography analysis of the fraction separated by silica gel column chromatography with a volume ratio of CH2Cl2:CH3OH=100:1.

[0045] Fr.2 is the product (20.2 g) obtained by thin-layer chromatography analysis of the fraction separated by silica gel column chromatography with a volume ratio of CH2Cl2:CH3OH=100:2.

[0046] Fr.3 is the product (8.9 g) obtained by thin-layer chromatography analysis of the fraction separated by silica gel column chromatography with a volume ratio of CH2Cl2:CH3OH=100:3.

[0047] Fr.4 is the product (10.1 g) obtained by thin-layer chromatography analysis of the fraction separated by silica gel column chromatography with a volume ratio of CH2Cl2:CH3OH=100:5.

[0048] Fr.5 is the product (3.2 g) obtained by thin-layer chromatography analysis of the fraction separated by silica gel column chromatography with a volume ratio of CH2Cl2:CH3OH=100:10.

[0049] Fr.6 is the product (9.5 g) obtained by thin-layer chromatography analysis of the fraction separated by silica gel column chromatography with a volume ratio of CH2Cl2:CH3OH=100:15.

[0050] Fr.7 is the product (2.5 g) obtained by thin-layer chromatography analysis of the fraction separated by silica gel column chromatography with a volume ratio of CH2Cl2:CH3OH=100:20.

[0051] Fr.8 is the product (10.5 g) obtained by thin-layer chromatography analysis of the fraction separated by silica gel column chromatography with a volume ratio of CH2Cl2:CH3OH=100:50.

[0052] Fr.2 was subjected to rapid gradient elution using a high-pressure column (20-35 μm, Tianjin Bona Ager Technology Co., Ltd.) in an ODS octadecyl-bonded phase with a methanol:distilled water system at a volume ratio of 30:70-80:20 (flow rate 21.00 mL / min). Fractions obtained from elution with different volume ratios of methanol:distilled water were collected, and the collected fractions were analyzed by thin-layer chromatography (volume ratio, dichloromethane:methanol = 12:1, R...). f =0.8), resulting in four fraction samples, Fr.2-1 to Fr.2-4.

[0053] Fr.2-1 is the product (5.1 g) obtained by thin-layer chromatography analysis of the fraction separated by high-pressure chromatography column in ODS octadecyl bonded phase with a volume ratio of CH3OH:H2O=30:70.

[0054] Fr.2-2 is the product (4.9 g) obtained by thin-layer chromatography analysis of the fraction separated by high-pressure chromatography in an ODS octadecyl bonded phase with a volume ratio of CH3OH:H2O = 45:55.

[0055] Fr.2-3 is the product (3.5 g) obtained by thin-layer chromatography analysis of the fraction obtained by high-pressure chromatography in an ODS octadecyl bonded phase with a volume ratio of CH3OH:H2O=60:40.

[0056] Fr.2-4 is the product (5.5 g) obtained by thin-layer chromatography analysis of the fraction separated by high-pressure chromatography in an ODS octadecyl bonded phase with a volume ratio of CH3OH:H2O=80:20.

[0057] 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 5 μm, flow rate 3 mL / min, wavelength 210 nm.

[0058] Example 3 Compound 1a, prepared using the method of Example 2, is a yellow powder (methanol). 1 H-NMR and 13 The C-NMR data are shown in Table 1. This compound is the first discovery of its kind in this invention.

[0059] Table 1 Compound 1a 1 H and 13 C NMR data

[0060] Figures 2 to 6 For the new compound 1a of the present invention 1 H-NMR, 13 The structures of the compounds were determined by C-NMR, HR-ESI-MS, HSQC, and HMBC spectra. Specifically, compound 1a is a yellow powder (methanol). 1 H-NMR (600 MHz, DMSO- d 6 ) displayed δ H An amide proton signal is present at 8.41 (1H, s). δ H 7.15 (2H, d, J =8.4 Hz), 6.84 (2H, d, J The signal at 8.4 Hz indicates the presence of a para-substituted benzene ring in the structure. δ H 5.40 (1H, t, J =6.6 Hz), 4.48 (2H, d, JThe proton signals at 7.2 Hz, 1.73 (3H, s), and 1.69 (3H, s) indicate the presence of an oxyisoprene group in the structure. δ H 3.82 (2H, d, J The signal at 6.0 Hz indicates that it is a hydrogen signal of the methylene group. δ H The signal at 3.61 (3H, s) indicates the presence of a methoxy group in the structure. δ H The signal at 3.38 (3H, s) indicates the presence of a nitrogen-containing methylene group in the structure.

[0061] 13 C-NMR (150MHz, DMSO-) d 6 ) gives 16 carbons, including δ C The two carbonyl signals at 171.0 and 170.3, δ C 157.0, 129.9, 129.9, 127.8, 114.3, and 114.3 represent the signals of six carbons on the benzene ring. δ C 136.8, 120.0, 64.1, 25.3, and 17.9 represent the carbon signals on five oxygen-containing isopentenyl groups. δ C 51.6 represents the carbon signal on a single methoxy group. δ C The methylene group with a nitrogen signal at position 41.0 δ C 40.6 represents the carbon signal on a single methylene group.

[0062] Mass spectrometry analysis of compound 1a was performed by Dalian Mengdi Technology Co., Ltd. The mass-to-charge ratio (m / z) measured by HR-ESI-MS was 314.1447 [M+Na]. + This indicates that its molecular formula is C 16 H 21 NO4.

[0063] The information on all direct hydrogen-carbon connections in the structure is given by HSQC spectral data, as shown in Table 1.

[0064] The structure of compound 1a was determined by the multiplicity and correlations shown in the HMBC spectra. In the HMBC spectra, 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), and 17.9 (C-15); and 1.69 (H-15) correlated with 136.8 (C-13), 120.0 (C-12), and 25.3 (C-14), confirming the presence of oxyisopentene. Another set of HMBCs, 7.15 (H-6), is associated with 157.0 (C-8), 114.3 (H-7), and 40.6 (C-4). 6.84 (H-7) is associated with 157.0 (C-8) and 127.8 (C-5), confirming the structure of the benzene ring. 8.41 (NH), 3.82 (H-4), and 3.38 (H-2) are associated with 170.3 (C-3); 3.61 (H-16) and 3.38 (H-2) are associated with 171.0 (C-1), confirming the presence of a glycine methyl ester structural fragment. The oxyisoprene and glycine methyl ester were linked to the benzene ring by correlations of 4.48 (H-11) with 157.0 (C-8), 7.15 (H-6) with 40.6 (C-4), and 3.82 (H-4) with 170.3 (C-3).

[0065] In summary, based on the data from high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy, compound 1a is a novel compound not previously reported in the literature, and is named (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester.

[0066] Example 4 Anti-tumor experiment 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) was evaluated using the MTT assay.

[0067] Human leukemia cell line (HL-60) was cultured in IMDM medium containing 20% ​​fetal bovine serum; human colon cancer cell line (HCT-116) was cultured in RPMI-1640 medium containing 10% fetal bovine serum; pancreatic cancer cell line (ASPC-1) and prostate cancer cell line (PC-3) were cultured in DMEM medium containing 10% fetal bovine serum, with penicillin-streptomycin antibiotics added to the medium (final concentration of penicillin was 1%, and final concentration of streptomycin was 1%), and cultured in an incubator at 37°C and 5% CO2.

[0068] Compound 1a and 5-fluorouracil were dissolved in DMSO (final DMSO concentration <0.1%). The solution was then diluted to the required concentration using prepared culture medium before the experiment.

[0069] Take test cells in the logarithmic growth phase and use (5-7) × 10 4 The culture medium was seeded at a density of 100 μL / mL in 96-well plates, and incubated at 37°C in a 5% CO2 incubator for 24 h. After incubation, the original culture medium was discarded, and 100 μL of culture medium containing compound 1a was added to each well as the drug treatment group. The concentration gradient of compound 1a was set at 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 all other conditions were the same as those in the sample group. 100 μL of culture medium was added to the blank control group. Each group was divided into 6 replicates. After culturing for another 48 h, 10 μL of MTT solution (MTT concentration of 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 shaking at room temperature for 10 min, the absorbance (OD) of each well was measured at 570 nm using a microplate reader. The half-maximal inhibitory concentration (IC50) was determined by regression analysis of the concentration-reaction data. 50 The formula for calculating the inhibition rate of tumor cell proliferation by compound 1a is as follows: Inhibition rate (%) = (1-OD) 给药组 / OD 空白对照组 )*100% The experimental results are shown in Table 2. Compound 1a showed varying degrees of inhibitory effects on four types of tumor cells, with the most significant inhibitory effect on human colon cancer HCT-116 cells (IC50). 50 =10.15μM), superior to the positive control drug 5-fluorouracil (IC50). 50 =15.00 μM).

[0070] Table 2. Inhibitory effect of compound 1a on tumor cell proliferation (IC50) 50 (μM)

[0071] Example 5 Antioxidant Activity Test The antioxidant activity was detected using the DPPH method, including the following steps: Accurately weigh 3.94 mg of DPPH, dissolve it in anhydrous ethanol, and dilute to a 10 mL amber volumetric flask. Dilute 5-fold to prepare a 0.2 mM DPPH working solution. Compound 1a was prepared at concentrations of 100, 50, 25, 10, 5, and 1 μg / mL. 100 μL of sample solution was pipetted into a 96-well plate. 100 μL of DPPH working solution was added to each well as a sample group. 100 μL of anhydrous ethanol was added to each well instead of the DPPH working solution as a control group. The blank group consisted of 100 μL of DPPH working solution mixed with 100 μL of anhydrous ethanol. After mixing, the mixture was reacted in the dark for 30 min. The absorbance of each well was measured at 517 nm using a microplate reader. L-ascorbic acid was used as a positive control to determine the DPPH free radical scavenging rate.

[0072] The clearance rate is calculated using the following formula: DPPH free clearance rate (%) = [1 - (A1 - A2) / A0] × 100%, where 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.

[0073] The antioxidant activity was detected using the ABTS method, which included the following steps: 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 were prepared. Equal volumes of the two solutions were mixed and stored at room temperature in the dark for 12–16 h. An appropriate amount of the prepared solution was diluted with PBS buffer (0.01 mol / L, pH 7.4) until the absorbance at 734 nm was 0.70 ± 0.02, thus obtaining the ABTS working solution. Compound 1a was prepared at concentrations of 100, 50, 25, 10, 5, and 1 μg / mL. 150 μL of ABTS working solution was placed in a 96-well plate. For the sample group, 100 μL of sample solution of different concentrations was added to 150 μL of ABTS working solution. The control group used anhydrous ethanol instead of ABTS working solution, i.e., 100 μL of sample solution of different concentrations was added to 150 μL of anhydrous ethanol. The blank group used 100 μL of anhydrous ethanol to add to 150 μL of ABTS working solution. After mixing, the plates were incubated in the dark for 20 min, and the absorbance of each well was measured at 734 nm using a microplate reader. L-ascorbic acid was used as a positive control to determine the ABTS free radical scavenging rate. The scavenging rate is calculated using the following formula: ABTS free radical scavenging rate (%) = [1 - (A1 - A2) / A0] × 100%, where 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.

[0074] The experimental results are shown in Table 3. In the DPPH free radical scavenging test, compound 1a showed significantly greater antioxidant capacity compared to the positive control drug L-ascorbic acid, indicating that compound 1a possesses better ability to scavenge DPPH free radicals. Compound 1a also demonstrated antioxidant capacity in the ABTS free radical scavenging test.

[0075] Table 3 Antioxidant activity of compound 1a (IC50) 50 (μg / mL)

[0076] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. The application of secondary metabolites in the preparation of antitumor products, characterized in that, The secondary metabolite is named (2-(4-(isopentenyl)oxy)phenyl)acetylglycine methyl ester, with the structural formula shown in 1a: 。 2. The application according to claim 1, characterized in that, The secondary metabolites are from Fusarium oxysporum (… Fusarium oxysporum It was obtained by extraction and separation of the fermentation broth.

3. The application according to claim 2, characterized in that, The strain of Fusarium oxysporum is named Fusarium oxysporum (…). Fusarium oxysporum LZC03, strain preservation number CGMCC No. 41251.

4. The application according to any one of claims 1-3, characterized in that, The product in question is a medicine or health supplement.

5. The application according to any one of claims 1-3, characterized in that, It has one or more of the following properties: anti-leukemia, anti-colon cancer, anti-pancreatic cancer, and anti-prostate cancer.

6. The application according to any one of claims 1-3, characterized in that, It is used to inhibit the proliferation of tumor cells.

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