Preparation method and application of compound Epiroridine acid B with anti-tumor effect
By isolating the new compound Epiroridin acid B from the fungus Myrothecium roridum, the problem of difficulty in finding effective anti-tumor small molecule compounds in the prior art is solved, and effective inhibition of malignant tumors such as melanoma is achieved.
Patent Information
- Application Number
- CN202510207088.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-25
- Publication Date
- 2025-05-30
AI Technical Summary
It is difficult to find effective low-toxic and efficient small molecule compounds to treat malignant tumor diseases such as melanoma.
A new compound, Epiroridin acid B, was isolated from the secondary metabolites of the fungus Myrothecium roridum, and the compound was prepared by steps such as fermentation and extraction.
Epiroridin acid B shows significant anti-tumor effects, can effectively inhibit the growth of melanoma cells, and the preparation process is simple, environmentally friendly and low-cost.
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Figure CN120058731A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of microbial application, and particularly relates to a compound with anti-tumor efficacy, its preparation method and application. Background Art
[0002] Malignant tumor diseases have a high degree of malignancy and an increasing incidence year by year, bringing a heavy burden to families and society and seriously affecting the health level of the people in our country. For example, melanoma can be caused by mutations in pigment cells in the eyes, gastrointestinal tract, genitals, sinuses, and meninges, and most commonly appears in the skin melanoma in an environment damaged by ultraviolet rays. Melanoma is the fifth most common cancer, and actively searching for low-toxic and highly effective small molecule compounds that can treat melanoma is of great significance for the current clinical application of melanoma.
[0003] Natural products have always been an important source of potential lead drugs. Natural products and their structural analogs have made significant contributions to disease treatment in history, especially for the treatment of diseases such as cancer. Compared with traditional chemically synthesized molecules, natural products have rich skeletal diversity and structural complexity, showing great advantages in new drug discovery. And fungal secondary metabolites are an important source of new structure natural products and are of great significance in exploring anti-tumor efficacy molecules. Therefore, it is a very feasible method to isolate and discover highly effective natural drugs with anti-tumor efficacy from fungal metabolites. Summary of the Invention
[0004] The purpose of the present invention is to provide a compound with anti-tumor efficacy, which is a new compound isolated from the secondary metabolites of the fungus Myrothecium roridum.
[0005] Another purpose of the present invention is to provide a preparation method of the compound.
[0006] Another purpose of the present invention is to provide the application of the compound in the preparation of drugs with anti-tumor efficacy.
[0007] Technical Solution: To achieve the above purposes, the technical solution adopted by the present invention is as follows:
[0008] A compound with anti-tumor efficacy, and such compounds have the following structure:
[0009]
[0010] The preparation method of the compound with anti-tumor efficacy described in the present invention includes the following steps:
[0011] Take the solid fermented mycelium of Myrothecium roridum on rice, extract it with an organic solvent, obtain an organic phase, and obtain an extract after recovering the solvent.
[0012] The above-mentioned extract is separated by chromatography to obtain the target product.
[0013] As an optimized scheme, for the preparation method of the compound with anti-tumor efficacy described above, the organic solvent used for extraction in step a is one of petroleum ether and ethyl acetate or their mixed solvent; the chromatographic separation material is silica gel, alumina or cyano-containing silane-bonded silica gel.
[0014] As an optimized scheme, the preparation method of the compound with anti-tumor efficacy described above includes the following steps:
[0015] (1) Inoculate the strain Myrothecium roridum on a PDA plate, culture it at 28 °C for 2 - 3 days, then inoculate it into a malt liquid medium, and culture it with shaking at 120 - 200 rpm for 2 - 5 days; then suck 40 mL of the seed liquid after 3 days of culture and inoculate it in batches into the rice medium, and culture it at a constant temperature of 28 °C for 28 days.
[0016] (2) After fermentation, soak the rice-fermented mycelium with an equal volume of ethyl acetate, crush the rice-fermented mycelium and perform ultrasonic extraction twice, and recover the solvent by rotary evaporation at 28 °C for the extract to obtain a crude paste extract;
[0017] (3) Perform column chromatography separation on the extract with normal-phase silica gel, elute it with petroleum ether - ethyl acetate with different volume ratios and ethyl acetate - methanol gradient elution, collect the fractions Fr.G1 - 15, and select the fractions Fr.G8 - 9 among them for semi-preparative high-performance liquid separation and purification to obtain the compound Epiroridin acid B. The preparation conditions are: 40% acetonitrile: 60% acid water (1‰ formic acid water) isocratic elution. Stationary phase: Fisher Wharton XBridge 5-μm C18 column (10×250 mm); flow rate: 2 mL / min; detection wavelength: 254 nm.
[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0019] The new structure compound Epiroridin acid B produced by the fermented fungus Myrothecium roridum in the present invention has a significant anti-tumor effect and can be used for preparing drugs with anti-tumor efficacy. In addition, the present invention uses the fungus Myrothecium roridum to prepare this new compound, which has simple operation, is environmentally friendly and has low cost. Description of the Drawings
[0020] Figure 11H-NMR spectrum of epiroridin acid B in DMSO-d6 (500 Hz).
[0021] Figure 2 13C-NMR spectrum of epiroridin acid B in DMSO-d6 (125 Hz).
[0022] Figure 3 DEPT spectrum of epiroridin acid B.
[0023] Figure 4 1H-1H COSY spectrum of epiroridin acid B.
[0024] Figure 5 HSQC spectrum of epiroridin acid B.
[0025] Figure 6 HMBC spectrum of epiroridin acid B.
[0026] Figure 7 NOESY spectrum of epiroridin acid B.
[0027] Figure 8 HR-ESI-MS spectrum of epiroridin acid B.
[0028] Figure 9 UV spectrum of epiroridin acid B (MeOH).
[0029] Figure 10 IR spectrum of epiroridin acid B.
[0030] Figure 11 Key 1H–1H COSY, HMBC spectra (A) and NOESY spectrum (B) correlation diagrams of epiroridin acid B.
[0031] Figure 12 Single crystal X-ray diffraction pattern of epiroridin acid B. Specific implementation mode
[0032] The present invention will be further described in detail below in conjunction with specific embodiments and with reference to data. It should be understood that these embodiments are only for illustrative purposes of the present invention and do not limit the scope of the invention in any way.
[0033] Myrothecium roridum (ATCC 16297) is derived from a standard strain publicly sold by the American Type Culture Collection.
[0034] Example 1
[0035] The preparation of compound Epiroridin acid B comprises the following steps:
[0036] (1) Fermentation of Myrothecium roridum
[0037] Seed liquid fermentation: An appropriate amount of mycelium was picked from the PDA plate of the strain Myrothecium roridum (ATCC 16297) with an inoculation loop and inoculated into a 1 L conical flask containing 400 mL of ME culture medium. A total of 12 flasks were fermented and cultured on a constant temperature shaker at 28 °C and 180 rpm for 3 days.
[0038] Preparation of rice culture medium: 40 mL of seed liquid was aspirated and inoculated into the rice culture medium. A total of 100 flasks were fermented and cultured in a constant temperature environment at 28 °C for 28 days.
[0039] (2) Extraction of secondary metabolites
[0040] After fermentation, it was soaked with an equal volume of ethyl acetate. The rice was mashed and ultrasonically extracted twice. The extract was concentrated under reduced pressure to obtain a total of 146.61 g of extract.
[0041] (3) Isolation and purification of secondary metabolites of Myrothecium roridum
[0042] Take 24.14 g of the paste and mix it with 25 g of silica gel for wet sample mixing. The sample was added to a silica gel column containing 250 g of silica gel by dry loading method. First, a gradient elution was carried out with petroleum ether - ethyl acetate as the elution system, and the elution ratios were: 1:0 (4 L), 50:1 (4 L), 25:1 (4 L), 10:1 (4 L), 5:1 (4 L), 2:1 (4 L), 1:1 (4 L), 0:1 (4 L); then a gradient elution was carried out with ethyl acetate - methanol as the elution volume, and the elution ratios were: 50:1 (2.4 L), 20:1 (2.4 L), 10:1 (2.4 L), 5:1 (2.4 L), 2:1 (4 L), 1:1 (2.4 L), 0:1 (4.5 L). A total of 15 fractions (Fr.G1 - 15) were obtained in sequence.
[0043] Take the fraction of Fr:G8 - Fr:G9 (462.67 mg), and separate and purify it by semi-preparative high performance liquid chromatography to obtain compound Epiroridin acid B. The preparation conditions are as follows: isocratic elution with 40% acetonitrile: 60% acidified water (1‰ formic acid water). Stationary phase: Fisher Wharton XBridge 5-μm C18 column (10×250 mm); flow rate: 2 mL / min; detection wavelength: 254 nm.
[0044] Structure analysis of Epiroridin acid B
[0045] Epiroridin acid B is a white powder. HR-ESI-MS m / z: 559.2200 [M-H] - (C 29 H 35 O 11 Calculated value 559.2185), and the molecular formula is C 29 H 36 O 11 , and the degree of unsaturation is 12. By analyzing 1 H-NMR (Table 1) data, it can be seen that this compound contains 3 methyl signals (δ H 0.73, s, 14-H3; 1.46, s; 12'-H3; 0.95, d, J = 6.2 Hz, 14'-H3), 2 characteristic hydrogen signals of exocyclic epoxides (δ H 2.79, d, J = 3.8 Hz; 3.05, d, J = 3.8 Hz, 13-H2), 5 olefinic proton signals (δ H 6.55, d, J = 4.8 Hz, 10-H; 6.18, dd, J = 15.7, 2.4 Hz, 7'-H; 7.20, dd, J = 15.7, 11.4 Hz, 8'-H; 6.75, t, J = 11.4 Hz, 9'-H; 5.77, d, J = 11.4 Hz, 10'-H;), and from these signal information, it can be speculated that this compound is a macrocyclic trichothecene compound. Combining the analysis of 13 C-NMR (Table 1) data, it can be determined that this compound contains 29 carbon units, and further determined that this compound contains 3 methyl signals (δ C 6.7, C-14; 16.9, C-12'; 17.9, C-14'), 3 carbonyl carbon signals (δ C 167.6, C-16; 168.0, C-1'; 166.1, C-11'), and there is 1 less carbonyl signal compared with verrucarin Z. Also combining the DEPT spectrum, it can be seen that there are 7 methylene groups (δ C34.4, C-3; 19.3, C-7; 21.8, C-8; 46.9, C-13; 63.0, C-15; 39.2, C-4'; 67.9, C-5'). Combined with 1 the H-NMR data and 13 the C-NMR data, it can be determined that the basic skeleton of this compound is basically the same as that of verrucarin Z. Figures 1 - 10 Analysis of the NOESY of the compound shows that there is a correlation between H-11 and H-4, and a correlation between H-4 and H-15. Thus, it is judged that H-4, H-11, and H-15 are in the same orientation, designated as β. Since there is no obvious NOESY correlation among H-2, H-13, and H-14, nor is there an obvious correlation with H-4, H-11, and H-15, referring to the literature, it is speculated that H-2, H-13, and H-14 are in the same orientation, which is α. Also, because there is a correlation between H-14 and H-12', it is judged that H-14 and H-12' are in the same orientation, which is α; there is a correlation between H-15 and H-6', so it is judged that H-15 and H-6' are in the same orientation, which is β. In addition, according to the chemical shift values of H-2' (δ H 3.50) and 13'-CH 3 (δ H 1.46), it shows that the epoxide is trans-substituted, that is, 12'-CH 3 and 2'-H are in opposite orientations, which is β. There is no obvious correlation between H-13' and other hydrogen signals. Finally, the absolute configuration of the compound is determined to be 2R, 4R, 5S, 6R, 11R, 12S, 2'S, 3'R, 6'R, 13'R by single crystal X-ray diffraction (the data is stored in the Cambridge Crystallographic Data Centre, no. CCDC - 2357112) ( Figure 12 ).
[0046] Table 1. 1 H NMR and 13 C-NMR Data of Epiroridin acid B (δ in ppm, J in Hz)
[0047]
[0048] Example 2 Anti-tumor Efficacy Evaluation
[0049] The following method was used to resuscitate A375 (melanoma cells), HCT-116 (human colon cancer cells), and H23 (human non-small cell lung cancer cells): 1) Prepare a DMEM medium containing 10% (volume percentage concentration) fetal bovine serum and 1% double antibody (penicillin and streptomycin), and incubate it at 37 °C and 5% CO 2Place for 2 hours under the following conditions. 2) Take out the cryopreserved cells and dissolve them in a 37°C water bath. 3) After dissolution, aspirate the cells into an EP tube containing 2 ml of culture medium, pipette to mix well, and centrifuge at 3000 r / min for 3 min. 4) Discard the supernatant, add the culture medium obtained in step 1), gently pipette the cells to mix well, transfer them to a culture dish, and culture at 37°C and 5% CO 2 Culture for 24 hours under the conditions to resuscitate the cells.
[0050] Cell passage: 1) Inoculate the resuscitated cells in a culture dish and culture them in DMEM medium containing 10% fetal bovine serum and 1% double antibody. 2) When the cell confluence reaches 90%, aspirate the culture medium, add 500 μL of trypsin for digestion, and then add 2 mL of DMEM medium containing 10% fetal bovine serum and 1% double antibody, pipette to detach the cells. 3) Transfer the cells to a 15 mL EP tube and centrifuge at 3000 r / min for 3 min. 4) Discard the supernatant and resuspend in a new DMEM medium containing 10% fetal bovine serum and 1% double antibody to obtain a suspension of approximately 5×10 5 cells / ml.
[0051] MTT drug concentration screening: Inoculate the cell suspensions of A375, HCT-116, and H23 into 96-well plates at 100 μl / well respectively, transfer them to an incubator, and culture at 37°C and 5% CO 2 Culture under the conditions. When the culture time is 24 h and 80% of the cells are adherent, in the drug treatment group, aspirate the old culture medium, add 100 μl of Epiroridin acid B solution with a final concentration of 10 μM to each well, and culture for 48 h; in the control group, replace the Solanapyrone A solution with culture medium, and the others are the same as the drug treatment group; in the blank group, there are no cells and no drug solution is added. Aspirate the old culture medium, add 10 μl of MTT solution (5 mg / ml, i.e., 0.5% MTT, prepared with serum-free culture medium, and can be accelerated to dissolve in a water bath at 37°C or 60°C after vortex oscillation, pay attention to light protection) to each well, continue to culture for 3 h, and then aspirate the supernatant. Add 150 μl of DMSO to each well, shake gently on a shaker at low speed for 10 min to fully dissolve the crystals. Calculate the cell survival rate according to the following formula: Cell survival rate = (drug treatment group - control group) / (blank group - control group) × 100%.
[0052] The results showed that the inhibitory activities of Epiroridin acid B against A549, HCT-116 and H23 cells reached 99.59%, 98.43% and 94.42% respectively at a concentration of 10 μM. Then, the MTT drug concentration screening method was used to investigate the half-maximal inhibitory concentration (IC50) of Epiroridin acid B and the positive drug control Sorafenib against A549, HCT-116 and H23 cells. The results are shown in Table 2. The IC50 values of Epiroridin acid against the above-mentioned cells were 0.097 μM, 0.052 μM and 0.023 μM, which were significantly lower than those of the positive drug.
[0053] Table 2. IC 50 values (μM)
[0054] Compound A375 HCT-116 H23 Epiroridinacid B 0.097 0.052 0.023 Sorafenib 1.8 2.0 2.4 。
[0055] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.
Claims
1. A compound having anti-tumor efficacy, Epiroridin acid B, characterized in that: The structural formula of the compound is as follows:
2. The method for preparing the compound according to claim 1, characterized in that: The following steps are involved: The solid fermentation mycelium of rice of Myrothecium roridum (ATCC 16297) is extracted with an organic solvent to obtain an organic phase, and the solvent is recovered to obtain an extract. The extract is separated by chromatography to obtain the desired product.
3. The method for preparing the compound according to claim 2, characterized in that: The organic solvent used for extraction is one of petroleum ether and ethyl acetate or a mixed solvent thereof; the chromatographic separation material is silica gel, alumina or silane bonded silica gel containing cyano groups.
4. The method for preparing the compound according to claim 1, characterized in that: The following steps are involved: (1) The strain Myrothecium roridum (ATCC 16297) was activated with a PDA plate and inoculated into a conical flask containing malt culture medium, and then cultured on a constant temperature shaker. The seed liquid after culture was then aspirated and inoculated into a rice culture medium in batches, and then cultured in a constant temperature environment; (2) After the fermentation is completed, the rice fermentation mycelium is soaked in ethyl acetate, crushed and then ultrasonically extracted, the extract is concentrated under reduced pressure, and separated using normal phase silica gel and reverse phase ODS columns in turn; When normal phase silica gel is used for separation, petroleum ether-ethyl acetate is used as the elution system for gradient elution first; then ethyl acetate-methanol is used as the elution volume for gradient elution to obtain 15 fractions Fr.G1-15; (3) Fractions Fr.G8-9 were selected and purified by semi-preparative HPLC to obtain compound Epiroridin acid B.
5. The method for preparing the compound according to claim 4, characterized in that: The following steps are involved: The preparation conditions of step (3) are: acetonitrile-1‰ formic acid water in a volume ratio of 40:60, isocratic elution, stationary phase: Fisher Wharton XBridge 5-μm C18 column, specification: 10×250mm; flow rate: 2mL / min; detection wavelength: 254nm.
6. Use of the compound according to claim 1 and its salt or derivative in the preparation of anti-tumor drugs.
7. Use of the compound according to claim 1 and its salt or derivative in the preparation of anti-melanoma, colon cancer or lung cancer drugs.