Anti-tumor compound, strain for preparing anti-tumor compound and preparation method of strain

Abstract

The invention discloses an anti-tumor compound, a strain for preparing the anti-tumor compound and a preparation method of the strain, relates to the technical field of medical biology, and provides a novel anti-tumor compound Phomeroid C which can effectively inhibit and kill tumor cells. Meanwhile, the invention provides a strain Penicillium sp.152 capable of preparing an anti-tumor compound Eupenifeldin and a new anti-tumor compound Phomeroid C. The strain has the characteristics of single product and high yield, and can be well applied to industrial preparation of the anti-tumor compounds.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical biotechnology, and in particular to an anti-tumor compound, a strain for preparing the anti-tumor compound and a preparation method thereof. Background Art

[0002] Commonly used drugs for treating tumors include: cytotoxic drugs (alkylating agents, antimetabolites, topoisomerase inhibitors, etc.); hormone drugs (adrenocortical hormones, androgens, estrogens, etc.); immune drugs (cytokines, monoclonal antibodies, etc.); targeted drugs (small molecule compounds, monoclonal antibodies, etc.). However, the rapid increase in tumor resistance has forced researchers to continuously search for new anti-tumor drug lead compounds.

[0003] The anti-tumor compound Eupenifeldin has excellent anti-tumor activity. It can cause G1 phase cell cycle arrest and cell apoptosis, reduce the levels of cell cycle proteins and survival proteins, and induce the production of reactive oxygen species (ROS), thereby affecting the growth and death of tumor cells. Eupenifeldin has always attracted much attention as an excellent anti-tumor drug lead compound due to its good anti-tumor activity. In the prior art, only application number: CN201510702627.6 discloses how to prepare the anti-tumor compound Eupenifeldin. How to expand the preparation channels of the anti-tumor compound Eupenifeldin and find new anti-tumor compounds are issues that need to be considered urgently in this field. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides an anti-tumor compound with good anti-tumor activity, a strain for preparing the anti-tumor compound and a preparation method thereof.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] Provided is an anti-tumor compound named Phomeroid C, comprising the following structure:

[0007]

[0008] An application of the anti-tumor compound is used for inhibiting liver cancer cell HepG2 and human non-small cell lung cancer A549 cell lines.

[0009] Furthermore, the antitumor compound is used to prepare antitumor drugs.

[0010] A Penicillium, named Penicillium sp.152, was registered and deposited with the Guangdong Provincial Microbiological Culture Collection Center on May 8, 2024, with the deposit number GDMCC 64483.

[0011] An application of the above strain is used for preparing anti-tumor compounds Phomeroid C and Eupenifeldin.

[0012] A method for preparing an antitumor compound using Penicillium sp.152 comprises the following steps:

[0013] S1: fermenting Penicillium sp.152 in a solid rice culture medium to obtain a fermentation product;

[0014] S2: adding an equal amount of ethyl acetate to the fermentation product for extraction, and gradient eluting the extract through a dichloromethane-methanol forward system; the gradient of the dichloromethane-methanol forward system is 100:0, 99:1, 98:2, 97:3, 96:4, 95:5, 93:7, 9:1; 8 fractions are obtained by elution, which are numbered Fr.A1-Fr.A8 in sequence;

[0015] S3: fractions Fr.A5 and Fr.A6 were taken, mixed and then gradient eluted with a normal silica gel petroleum ether-ethyl acetate system; the gradient of the normal silica gel petroleum ether-ethyl acetate system was 100%, 90%, 80%, 70%, 60%, 50%, 30%, 10%; 8 fractions were obtained by elution, which were numbered Fr.B1-Fr.B8 in sequence;

[0016] S4: Fraction Fr.B4 is taken and prepared using high performance liquid chromatography to obtain the anti-tumor compound Eupenifeldin.

[0017] Furthermore, the method also includes the preparation of the anti-tumor compound Phomeroid C. The specific method is: taking fraction Fr.B7 and using high performance liquid chromatography to prepare the anti-tumor compound Phomeroid C.

[0018] Furthermore, the solid rice culture medium is prepared by high-pressure sterilization at 121° C. for 30 minutes using 10% rice, 0.2% yeast powder, 0.2% peptone and 0.05% glucose.

[0019] Furthermore, the elution procedure for preparing the anti-tumor compound Eupenifeldin or Phomeroid C by the high performance liquid chromatography method is: 0-20 min gradient elution from 60% to 100% acetonitrile: water according to the volume fraction ratio (the aqueous phase and acetonitrile contain 0.1% glacial acetic acid respectively). When preparing the anti-tumor compound Eupenifeldin, the retention time is 17.5 min; when preparing the anti-tumor compound Phomeroid C, the retention time is 12 min.

[0020] The beneficial effects of the present invention are:

[0021] The present invention provides a new anti-tumor compound Phomeroid C, which can effectively inhibit and kill tumor cells.

[0022] At the same time, a strain Penicillium sp.152 capable of preparing an antitumor compound Eupenifeldin and a new antitumor compound Phomeroid C is provided. The strain has the characteristics of a single product and high yield, and can be well applied to the industrial preparation of antitumor compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a real photo of the strain morphology of Penicillium sp.152;

[0024] Figure 2 This is the HPLC detection chart of the fermentation product obtained in Example 2;

[0025] Figure 3 This is a high-resolution mass spectrometry detection diagram of compound Ⅰ prepared in Example 2;

[0026] Figure 4 The compound I obtained in Example 2 1 H NMR detection image;

[0027] Figure 5 The compound I obtained in Example 2 13 C Nuclear magnetic resonance imaging;

[0028] Figure 6 The compound I obtained in Example 2 1 H- 1 H COSY NMR detection image;

[0029] Figure 7 This is the HSQC NMR detection image of compound Ⅰ prepared in Example 2;

[0030] Figure 8 This is the HMBC nuclear magnetic resonance detection image of compound Ⅰ prepared in Example 2;

[0031] Fig. 9 The compound I obtained in Example 2 1 H- 1 H NOESY NMR detection image;

[0032] Fig.10 This is a high-resolution mass spectrometry detection diagram of compound II prepared in Example 3;

[0033] Fig.11The compound II obtained in Example 3 1 H NMR detection image;

[0034] Fig.12 The compound II obtained in Example 3 13 C Nuclear magnetic resonance imaging;

[0035] Fig.13 The compound II obtained in Example 3 1 H- 1 H COSY NMR detection image;

[0036] Fig.14 This is the HSQC NMR detection image of compound II prepared in Example 3;

[0037] Fig.15 This is the HMBC nuclear magnetic resonance detection image of compound II prepared in Example 3;

[0038] Fig.16 The compound II obtained in Example 3 1 H- 1 H NOESY NMR detection image;

[0039] Fig.17 is the circular dichroism spectrum of compound II obtained in Example 3;

[0040] Fig.18 Schematic diagram of the structure of compound II prepared in Example 3;

[0041] Fig.19 This is a graph showing the anti-tumor activity test results of Phomeroid C. DETAILED DESCRIPTION

[0042] The specific implementation modes of the present invention are described below to facilitate those skilled in the art to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0043] Example 1 Identification of strain Penicillium sp.152

[0044] The Penicillium sp.152 strain of the present invention is isolated from the mud of the Yangtze River near Jiulong Temple in Jiulongpo District, Chongqing (water depth 0.3m). The isolation medium is a modified ISP2 medium, 2g / L yeast powder, 2.0g / L malt extract powder, 1g / L beef extract, 15.0g / L agar, 500mL tap water, the isolated strain is transferred to a PDA (300g / L peeled potatoes, cut into small pieces, boiled with water for 30min, filtered with gauze, the filtrate is taken, 20g / L glucose, 2% agar powder, 115℃ autoclaved for 30min) plate, and cultured at 28℃ for 5 days. Figure 1 As shown, the obtained colonies were off-white.

[0045] Take a small amount of mycelium and place it in a 1.5mL centrifuge tube, add 50μL 10×Buffer, shake to separate the mycelium, seal the centrifuge tube with sealing glue, place it in a 700w microwave oven and heat it on high heat for 2 minutes, then immediately take it out and ice bath for 2 minutes. After the ice bath, place it in a centrifuge and centrifuge it at 12000r / min for 1 minute, and aspirate the supernatant as a DNA template for later use.

[0046] PCR amplification using universal primers for fungi:

[0047] The PCR reaction system (100 μL) was: 10 μL DNA template, 3 μL dimethyl sulfoxide DMSO (Adamas, catalog number 75927AG), 50 μL high-fidelity premix 2×Taqmix (Shanghai Jingnuo, catalog number 63000K-10ML), 10 μL primer ITS1primer with a concentration of 10 μmol / L, 10 μL primer ITS4 primer with a concentration of 10 μmol / L, and 17 μL ultrapure water ddH2O.

[0048] After amplification, the cells were placed in a PCR instrument for reaction. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 1 min, repeated for 35 cycles; extension at 72°C for 2 min; cooling and insulation at 4°C.

[0049] The DNA molecules were separated by gel electrophoresis, the target gel was cut, and the DNA molecules were recovered using the OMEGA gel recovery kit (OMEGA, catalog number Omega#R6376-01). The obtained 18S rDNA sequence was determined by Beijing Qingke Biotechnology Co., Ltd.

[0050] Example 2 Preparation of the antitumor compound Eupenifeldin using strain Penicillium sp.152

[0051] S1: Ferment Penicillium sp. 152 in solid rice culture medium to obtain a fermentation product (HPLC test results of the fermentation product are as follows Figure 2 As shown), the fermentation and further separation and purification method of Eupenifeldin are specifically as follows:

[0052] The Penicillium sp.152 strain grown on a PDA plate in a 28°C constant temperature incubator was taken from a 1 cm 2 The bacterial cells were inoculated into a 250 mL conical flask containing 80 mL TSBY liquid medium, which was prepared by high-pressure sterilization at 115 °C for 30 min, with 103 g / L sucrose, 30 g / L tryptic soytone, and 5 g / L yeast extract.

[0053] The conical flask was shaken and cultured at 28°C and 200 rpm for 2 days to serve as the fermentation seed solution.

[0054] The fermented seed liquid was added to a 500 mL conical flask containing 50 g of rice solid culture medium at a ratio of 5%, and then placed at 28° C. for pure culture for 7 days. Then 2.5% of Acinetobacter baumannii in the logarithmic growth phase was added and the silent fermentation was continued for 8 days, and a total of 2.5 kg of rice was fermented.

[0055] S2: The fermentation product was ultrasonically extracted three times with an equal volume of ethyl acetate, and then concentrated under reduced pressure on a rotary evaporator to obtain a total extract, which was then gradient eluted with a dichloromethane-methanol system forward system. The gradient of the dichloromethane-methanol system forward system was 100:0, 99:1, 98:2, 97:3, 96:4, 95:5, 93:7, and 9:1. Eight fractions were obtained, which were numbered Fr.A1-Fr.A8 in sequence.

[0056] S3: fractions Fr.A5 and Fr.A6 were taken, mixed and then gradient eluted with a normal silica gel petroleum ether-ethyl acetate system; the gradient of the normal silica gel petroleum ether-ethyl acetate system was 100%, 90%, 80%, 70%, 60%, 50%, 30%, 10%; 8 fractions were obtained by elution, which were numbered Fr.B1-Fr.B8 in sequence;

[0057] S4: Fraction Fr.B4 was used to prepare 1.5 g of compound Ⅰ by high performance liquid chromatography. R =17.5min.

[0058] The liquid chromatography column used was ODS-A, 250×10 mm, 5 μm, YMC, and the flow rate was 3 mL / min.

[0059] The specific elution procedure is: gradient elution from 60% to 100% acetonitrile: water (acetonitrile and water each contain 0.1% glacial acetic acid) from 0 to 20 min, and finally 1.5 g of Eupenifeldin is prepared at a retention time of 17.5 min.

[0060] Compound I was tested by high resolution mass spectrometry (HRESIMS). Figure 3 As shown, and nuclear magnetic resonance testing was performed. The results of nuclear magnetic resonance testing are as follows Figure 4-9 As shown in Table 1, compared with the NMR data in the literature (Mayerl F, Gao Q, Huang S, Klohr SE, Matson JA, Gustavson DR, Pirnik DM, Berry RL, Fairchild C, Rose WC. Eupenifeldin, a novel cytotoxic bistropolone from Eupenicillium brefeldianum. J Antibiot (Tokyo). 1993 Jul; 46 (7): 1082-1088.), it was determined that compound I was Eupenifeldin.

[0061] Table 1

[0062] No. <![CDATA[δ C ]]> <![CDATA[δ H ]]> 1 112.9 6.92(s) 2 163 3 173.4 4 125.7 7.16(s) 5 151.5 6 118.7 7 34.2 2.85(dd,J=17.2,5.2Hz) 2.37(s) 8 32.2 1.81(s) 9 46.7 0.79(dd,J=14.0,4.0Hz) 1.80(m) 10 34.8 11 144.1 5.80 (dd, J = 16.4, 1.6 Hz) 12 126 5.70 (ddd, J = 16.4, 10.4, 4.4 Hz) 13 46.4 2.53 (dd, J = 13.6, 10.4 Hz) 2.74(s) 14 80.6 16 160.4 17 113.6 6.95(s) 18 163.3 19 172.7 20 124.8 7.13(s) 21 150.6 22 122.5 23 33.1 2.41(m) 3.38(m) 24 41.7 2.20(m) 25 30.3 1.54(t,J=11.9Hz) 2.21(s) 26 70.9 4.21(d,J=11.2Hz) 27 82.1 29 159.6 30 27.5 2.38(s) 31 29.8 1.08(s) 32 27.2 1.11(s) 33 19.4 1.41(s) 34 27.6 2.41(s) 35 16.2 1.15(s)

[0063] Table 1 shows the NMR data of compound I (400 MHz, CDCl3), where δ C is the C spectrum value, δ H is the H spectrum value. It proves that the strain Penicillium sp.152 can successfully prepare the anti-tumor compound Eupenifeldin.

[0064] Example 3 Preparation of antitumor compound Phomeroid C using strain Penicillium sp.152

[0065] S1: Fermenting Penicillium sp.152 in a solid rice culture medium to obtain a fermentation product; specifically:

[0066] The Penicillium sp.152 strain grown on a PDA plate in a 28°C constant temperature incubator was taken from a 1 cm 2 The bacterial cells were inoculated into a 250 mL conical flask containing 80 mL TSBY liquid medium, which was prepared by high-pressure sterilization at 115 °C for 30 min, with 103 g / L sucrose, 30 g / L tryptic soytone, and 5 g / L yeast extract.

[0067] The conical flask was shaken and cultured at 28°C and 200 rpm for 2 days to serve as the fermentation seed solution.

[0068] The fermented seed liquid was added to a 500 mL conical flask containing 50 g of rice solid culture medium at a ratio of 5%, and then placed at 28° C. for pure culture for 7 days. Then 2.5% of Acinetobacter baumannii in the logarithmic growth phase was added and the silent fermentation was continued for 8 days, and a total of 2.5 kg of rice was fermented.

[0069] S2: The fermentation product was ultrasonically extracted three times with an equal volume of ethyl acetate, and then concentrated under reduced pressure on a rotary evaporator to obtain a total extract, which was then gradient eluted with a dichloromethane-methanol system in a forward system with a gradient of 100:0, 99:1, 98:2, 97:3, 96:4, 95:5, 93:7, and 9:1; 8 fractions were obtained, which were numbered Fr.A1-Fr.A8 in sequence;

[0070] S3: fractions Fr.A5 and Fr.A6 were taken, mixed and then gradient eluted with a normal silica gel petroleum ether-ethyl acetate system; the gradient of the normal silica gel petroleum ether-ethyl acetate system was 100%, 90%, 80%, 70%, 60%, 50%, 30%, 10%; 8 fractions were obtained by elution, which were numbered Fr.B1-Fr.B8 in sequence;

[0071] S4: Fraction Fr.B7 was used to prepare 38 mg of compound II by high performance liquid chromatography. R =12min.

[0072] The liquid chromatography column used was ODS-A, 250×10 mm, 5 μm, YMC, and the flow rate was 3 mL / min.

[0073] The specific elution procedure is: 0-20min gradient elution with acetonitrile:water from 60% to 100% by volume (the aqueous phase and acetonitrile each contain 0.1% glacial acetic acid), and Phomeroid C is prepared at a retention time of 12min.

[0074] Compound II is in the form of white powder. High resolution mass spectrometry HRESI-MS is performed on compound II. The test results are as follows: Fig.10 As shown, [M+H] + The test result is 581.2742, indicating that the molecular formula of compound II is C 33 H 40 O9 has 14 unsaturated degrees. The compound II was tested by NMR, and the test results are as follows Figure 11-16 As shown in Table 2, Figure 11-16 It can be seen that 1H spectrum and HSQC spectrum showed that compound II contained 6 methyl groups (including two benzyl groups), 5 methylene groups, 9 methine groups, and 13 quaternary C atoms. 1 H- 1 The HCOZY spectrum showed that compound II contained three independent coupling systems: H2-2 / H-3 / H-4; H2-6 / H-7 / H2-25; H-9 / H2-10 / H-11 / H2-12. Combined with the related signals of HMBC spectrum H3-20 / C-1, C-2, C-11; H3-21 / C-4, C-5, C-6; H3-22 / C-4, C-5, C-6; H3-22 / C-4, C-5, C-6; H2-6 / CC-4, C-5, C-7, C-8, C-21, C-22; H3-23 / C-7, C-8, C-9, it was suggested that there was an 11-membered sesquiterpene structural unit (C) in the structure. The related signals of HMBC spectrum H-29 / C-27, C-28, C-30, C-31; H-32 / C-27, C-30, C-31, C-33, C-34; H3-34 / C-27, C-32, C-33 indicate the existence of a seven-membered aromatic ring - tropolone (A). The HMBC spectrum of H2-26 / C-6, C-7, C-8, C-27, C-28, C33 and C8 (δ C 83.4), C-27(δ C 162.4) chemical shifts indicate that C-8 and C-27 are connected to the same oxygen atom and form a tetrahydropyran ring unit, which connects the two structural units A and B through C-6 / 7 and C-27 / 28. C 86.9), C-28(δ C 162.7) chemical shifts suggest that C-1 and C-14 are both connected to the same oxygen atom and form another tetrahydropyran ring structural unit, while the correlation of H-15 / C-14 and C-5 in the HMBC spectrum suggests that there is an unsaturated acetyl substitution at the C-14 position. H-18 is correlated with the HMBC of C-13, C-17, C-19, and C-25, and H3-25 is correlated with the HMBC of C-13, C-17, and C-18, suggesting that there is another structural unit connected to the 13th position of the E structural unit. By comparing the molecular weight of the high-resolution mass spectrum, it is suggested that there is still an O atom and an unsaturation that has not been assigned, indicating that C-19 may be connected to C-13 through an ester bond to form a ring, which is also consistent with C-13 (δ C 89.4).

[0075] In terms of the relative configuration of compound II, the NMR data showed that the coupling constant of H-3 and H-4 was 16.0Hz, indicating that the C-3 / C-4 olefin was in the trans E configuration. The NOESY correlation spectrum showed that H-9 was correlated with H3-20, suggesting that they were on the same plane. The larger coupling constants of H-9 and H-10β (1.32) suggested that they might be in the trans configuration, while in the NOESY correlation spectrum, H-9 was correlated with H-10α (2.43), and H-10β (1.32) was correlated with H3-23 and H-11, suggesting that H-11, H-10β, H3-23, and H-7 were on the same plane and in the opposite plane to H-9. The configuration of C-13 was obtained by ECD calculation. The specific determination method was to first dissolve the compound II to be tested in methanol and measure the absorbance under a UV spectrophotometer so that its absorbance was within the range of 0.6-0.8A. Turn on the circular dichroism spectrometer, preheat for 20 minutes, set the scanning wavelength to 200-600nm, perform blank calibration, put the configured sample to be tested into the sample cell for measurement, and export the raw data after the measurement to obtain the following: Fig.17 Shown is the circular dichroism (CD) spectrum.

[0076] The molecular Merck force field (MMFF) was used to perform a random conformational search on Spartan'14. Subsequently, the IEFPCM model was used to further perform ECD calculations at the B3LYP / 6-31G(d) level in methanol, and the Gaussion09 software was used to optimize the conformations with a Boltzmann distribution greater than 5%. At the B3LYP / 6-311G(d, p) level, the optimized stable conformations were selected for further ECD calculations. The Boltzmann distribution was used to weight the overall ECD data, and the ECD curves and enantiomeric ECD curves were drawn using SpecDis1.70.1 software with a half-bandwidth of 0.2eV based on the Boltzmann calculation contribution of each conformation after UV correction. The corrected calculated spectrum was compared with the measured spectrum to determine the absolute configuration of compound II. Fig.17 The comparison of the calculated and experimental spectra is shown, where expt is obtained from the experiment, and calcd.2a and calcd.2b are calculated. Therefore, based on the previous relative configuration results, the absolute configuration of compound II can be determined as follows: 1S, 7R, 8S, 9R, 11R, 13S. The structure of compound II is shown in Fig.18 As shown, it was named Phomeroid C.

[0077] Table 2

[0078] No. <![CDATA[δ C ]]> <![CDATA[δ H ]]> 1 86.9 2 47.4 2.69(m) 3 126.4 5.72 (ddd, J = 16.0, 10.0, 4.8 Hz) 4 146.1 5.94(d,J=16.0Hz) 5 36 6 47.8 1.81(d,J=14.4Hz) 0.75(dd,J=14.4,4.4Hz) 7 33.1 1.98(s) 8 83.4 9 71.6 4.16(d,J=11.6Hz) 10 33 1.32(t,J=13.6Hz) 2.43(m) 11 41.9 2.73 (dd, J = 13.2, 4.8 Hz) 12 38.7 1.53 (dd, J = 15.2, 2.8 Hz) 3.08(t,J=14.4Hz) 13 89.4 14 162.7 15 104.1 4.97(s) 16 168.5 17 170.8 18 120.2 6.05(s) 19 173.4 20 21.1 1.57(s) 21 30.1 1.04(s) 22 27.5 1.12(s) 23 16.3 1.08(s) 25 14.4 2.21(s) 26 35.5 2.45(m) 2.96 (dd, J = 17.6, 5.2 Hz) 27 121.7 28 162.4 29 115.5 7.08(s) 30 165.2 31 174.3 32 126.1 7.12(s) 33 153.9 34 27.5 2.41(s)

[0079] Example 4 Antitumor Activity Detection of Compound Phomeroid C

[0080] The compound Phomeroid C prepared in Example 3 was used to perform a tumor cell cytotoxicity test on tumor cells HepG2 and A549. The specific method is as follows:

[0081] (1) Tumor cell recovery and plating: Take the tumor cells out of liquid nitrogen and immediately thaw them in 37°C warm water. After thawing, add culture medium (DMEM 89%, fetal bovine serum 10%, double antibody 1%) to the suspension for tumor cell recovery. After the tumor cells are passaged twice, collect the logarithmic phase tumor cells, wash the dead tumor cells with 2mL PBS and then aspirate them. Add 2mL trypsin to digest for 30-180s. Observe the state of the tumor cells in real time under a microscope. When the tumor cells shrink, become brighter, and no longer connect under a microscope, add 2mL culture medium to terminate the digestion. Centrifuge for 5min, pour out the culture medium, and add 3mL culture medium to resuspend. Add 100μL of mixed tumor cell suspension (about 10,000 tumor cells per well) to each well of the 96-well plate to avoid tumor cell precipitation, which will lead to uneven number of tumor cells in each well. Set up 8 concentration gradients and a blank group and control group, and culture them in a 37°C incubator with 5% CO2 for 12h to wait for tumor cells to adhere to the wall.

[0082] (2) CCK-8 (Cell Counting Kit-8) experiment: Compound Phomeroid C and positive drug Adriamycin were prepared in advance into solutions of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, and 0.78125 μM, respectively. The tumor cells were taken out of the incubator and observed under a microscope to see if they were attached to the wall. The culture medium of the tumor cells that were attached to the wall was aspirated, and the tumor cells were washed with 100 μL PBS per well, and 100 μL of the prepared drug was added. The cells were cultured in a 5% CO2, 37°C incubator for 24 h. Prepare CCK-8 reagent at a ratio of CCK-8: culture medium = 10:1. Protect from light throughout the process and use it immediately after preparation. Aspirate the original culture medium and add 100 μL of CCK-8 mixed solution to each well at an angle against the wall of the plate. Be careful to prevent the formation of bubbles. Incubate for 30 minutes. After the mixed solution turns yellow, use a microplate reader to measure the OD value at 450 nm.

[0083] (3) Data processing: Calculate the survival rate of tumor cells, that is, the inhibition rate of the compound on tumor cells:

[0084]

[0085] OD in the formula 实验孔 OD 空白孔 OD 对照孔The drug concentration was used as the horizontal axis, and the calculated tumor cell inhibition rate was used as the vertical axis. The GraphPadPrism8 software was used for graphical analysis. The results are shown in the figure. Fig.19 As shown, Fig.19 The left side is a schematic diagram comparing the inhibition rates of Phomeroid C and the positive control group Adriamycin on HepG2 liver cancer cells. Fig.19 The right side is a schematic diagram comparing the inhibition rates of Phomeroid C and the positive control group Adriamycin on A549 human non-small cell lung cancer cells.

[0086] Depend on Fig.19 It can be seen that Phomeroid C has a good inhibition rate on both HepG2 liver cancer cells and A549 human non-small cell lung cancer cells, which proves that Phomeroid C can be used to prepare drugs that inhibit tumor cells.

Claims

1. An antitumor compound, characterized in that: Named Phomeroid C, it includes the following structure:

2. Use of the antitumor compound according to claim 1, characterized in that: Used to inhibit liver cancer cell HepG2 and human non-small cell lung cancer A549 cell lines.

3. The use according to claim 2, characterized in that: The antitumor compound is used for preparing antitumor drugs.

4. A Penicillium, characterized in that Named Penicillium sp.152, it was registered and deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 8, 2024, with the deposit number GDMCC 64483.

5. A use of the strain according to claim 4, characterized in that: Used to prepare anti-tumor compounds PhomeroidC and Eupenifeldin.

6. A method for preparing an antitumor compound using Penicillium sp.152, characterized in that: The steps include: S1: fermenting Penicillium sp.152 in a solid rice culture medium to obtain a fermentation product; S2: adding an equal amount of ethyl acetate to the fermentation product for extraction, and gradient eluting the extract through a dichloromethane-methanol forward system; the gradient of the dichloromethane-methanol forward system is 100:0, 99:1, 98:2, 97:3, 96:4, 95:5, 93:7, 9:1; 8 fractions are obtained by elution, which are numbered Fr.A1-Fr.A8 in sequence; S3: fractions Fr.A5 and Fr.A6 were taken, mixed and then gradient eluted with a normal silica gel petroleum ether-ethyl acetate system; the gradient of the normal silica gel petroleum ether-ethyl acetate system was 100%, 90%, 80%, 70%, 60%, 50%, 30%, 10%; 8 fractions were obtained by elution, which were numbered Fr.B1-Fr.B8 in sequence; S4: Fraction Fr.B4 is taken and prepared using high performance liquid chromatography to obtain the anti-tumor compound Eupenifeldin.

7. The method for preparing the antitumor compound according to claim 6, characterized in that: The invention also includes the preparation of the anti-tumor compound Phomeroid C. The specific method is: taking the fraction Fr.B7 and using high performance liquid chromatography to prepare the anti-tumor compound Phomeroid C.

8. The method for preparing the antitumor compound according to claim 6, characterized in that: The solid rice culture medium is prepared by using 10% rice, 0.2% yeast powder, 0.2% peptone and 0.05% glucose and sterilizing by high pressure at 121° C. for 30 minutes.

9. The method for preparing an antitumor compound according to any one of claims 6-7, characterized in that: The elution procedure for preparing the antitumor compound Eupenifeldin or Phomeroid C by the high performance liquid chromatography method is: 0-20min gradient elution with acetonitrile:water from 60% to 100% by volume fraction (the aqueous phase and acetonitrile each contain 0.1% glacial acetic acid), when preparing the antitumor compound Eupenifeldin, the retention time is 17.5min; when preparing the antitumor compound Phomeroid C, the retention time is 12min.

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