Application of a natural product Oxirapentyn A in the preparation of anti-glioma drugs

Abstract

The present invention belongs to the field of biomedicine technology and specifically relates to the use of a natural product, Oxirapentyn A, in the preparation of anti-glioma drugs. The natural product, Oxirapentyn A, has significant anti-glioma activity and can inhibit the proliferation, migration, and invasion of glioma cells by promoting early and late apoptosis of glioma cells, showing excellent potential as an anti-glioma drug. Furthermore, the natural product, Oxirapentyn A, can be directly prepared through large-scale fermentation of marine fungi or obtained by simple derivatization of the fermentation product, Oxirapentyn B. It has a wide source, a simple production process, a short cycle, and low product cost, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology. More specifically, it relates to the use of a natural product, Oxirapentyn A, in the preparation of anti-glioma drugs. Background Art

[0002] Glioma originates from glial cells and is the most common primary malignant heterogeneous tumor of the central nervous system with high morbidity and mortality. Since most gliomas grow in an infiltrative manner and have unclear boundaries with surrounding brain tissue, treatment is very difficult, and drugs that can treat gliomas (mainly temozolomide and bevacizumab, etc.) are even rarer. Currently available drugs for treating gliomas, such as temozolomide chemotherapy drugs, have drug resistance, and bevacizumab is not significantly effective in improving the survival of glioma patients, all of which have different problems. In order to provide more drugs for treating gliomas, Chinese patent application CN102614209A discloses the application of compound echinoside A in the preparation of drugs for treating gliomas. This compound is a triterpenoid saponin compound extracted from sea cucumbers. Animal experiments have shown that echinoside A has a significant inhibitory effect on gliomas. However, the drug selection that can be used for research or treatment of gliomas is still limited, and more active ingredients still need to be provided for subsequent research or treatment of anti-gliomas. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the limited defects and deficiencies of existing anti-glioma drugs and provide an application of a natural product Oxirapentyn A in the preparation of anti-glioma drugs.

[0004] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0005] A natural product, Oxirapentyn A, is used in the preparation of an anti-glioma drug. The natural product, Oxirapentyn A, has the following structure:

[0006]

[0007] Marine fungal natural products are a major source of marine natural products. The search for compounds with anti-glioma activity from the abundant secondary metabolites of marine fungi is of great research significance. The inventors have isolated Oxirapentyn A, a terpene from the fermentation products of the ascidian fungus Beauveria felina SYSU-MS7908, which exhibits significant anti-glioma activity. A series of studies have demonstrated that Oxirapentyn A has a potent inhibitory effect on the proliferation, migration, and invasion of glioma cells and can promote both early and late apoptosis of glioma cells, suggesting promising application as an anti-glioma drug.

[0008] Furthermore, the natural product Oxirapentyn A may also be a pharmaceutically acceptable salt or solvate thereof.

[0009] Furthermore, the natural product Oxirapentyn A promotes apoptosis of early and late stages of brain glioma cells.

[0010] Furthermore, the natural product Oxirapentyn A inhibits the proliferation, migration and invasion of brain glioma cells.

[0011] Furthermore, the brain glioma cells are U251, T98G or U87-MG.

[0012] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the dosage form of the drug is oral, injection or microneedle.

[0014] Furthermore, the natural product Oxirapentyn A is isolated and purified from the fungal strain Beauveria felina SYSU-MS7908 derived from the marine ascidian with the deposit number GDMCC 61059.

[0015] Preferably, the separation and purification specifically comprises the following steps:

[0016] The ascidian fungus Beauveria felina SYSU-MS7908 was fermented at room temperature for 28 days in 10 kg of solid rice culture medium (100 parts of rice, 100 parts of 3% sea salt water, and 3 parts of peptone) sterilized at 121°C for 20 minutes; the fermentation product was extracted by immersion in an organic solvent (methanol or acetone) and extracted with ethyl acetate / water; the ethyl acetate extract was chromatographed on a silica gel column using a petroleum ether / ethyl acetate mobile phase system to collect the fraction containing 20% ​​by volume of ethyl acetate, and the natural product Oxirapentyn B (II) raw material was obtained in the gram range by ODS-C18 reverse phase chromatography; the remaining fraction containing 20% ​​by volume of ethyl acetate was purified by RP-HPLC (C18 column, 4.6×250 mm, 5 μm, MeOH / H2O, 70 / 30, 3 mL / min, T R =23-25 ​​min) can be separated and prepared to obtain the target heteroterpenoid compound Oxirapentyn A (I).

[0017] Furthermore, the natural product Oxirapentyn A is obtained by oxidizing the natural product Oxirapenty B:

[0018]

[0019] Oxirapentyn A can also be obtained in large quantities by derivatizing the natural product Oxirapentyn B, thus solving the drug source problem.

[0020] Furthermore, the oxidizing oxidant is selected from one or more of pyridinium chlorochromate (PCC), bromine, manganese dioxide, potassium permanganate, hydrogen peroxide, Jones reagent, and Dess-Martin oxidant.

[0021] Preferably, the derivatization specifically comprises the following steps:

[0022] In anhydrous dichloromethane (DCM), 1 eq of Oxirapentyn B (II, 20 mg) and 1.5 eq of pyridinium chlorochromate were stirred at 50°C for 2 h. The reaction solution was filtered, concentrated, and purified by flash silica gel column chromatography (petroleum ether / ethyl acetate system) to obtain the target compound Oxirapentyn A (I).

[0023] The present invention has the following beneficial effects:

[0024] The present invention provides the use of the natural product Oxirapentyn A in the preparation of an anti-glioma drug. Oxirapentyn A exhibits significant anti-glioma activity and can inhibit the proliferation, migration, and invasion of glioma cells by promoting early and late apoptosis, demonstrating its potential as an anti-glioma drug. Furthermore, Oxirapentyn A can be directly produced through large-scale fermentation of marine fungi or by simple derivatization of the fermentation product Oxirapentyn B. Oxirapentyn A boasts a wide range of sources, a simple production process, a short production cycle, and low product cost, offering broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figures are statistical graphs showing the proliferation inhibition rates of different cell lines U87-MG (a), U251 (b), and T98G (c) at different concentrations of Oxirapentyn A in Example 2 of the present invention; among them, compared with the NC negative control group (0.1% DMSO) and the drug-treated group, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[0026] Figure 2 The results of the effects of temozolomide, paclitaxel, and different concentration gradients of Oxirapentyn A on the migration of human glioma cells in Example 3 of the present invention are statistical graphs, (a) to (c) are micrographs (50×) of cell lines U87-MG, U251, and T98G, respectively, and (d) to (f) are statistical graphs of the migration rates of cell lines U87-MG, U251, and T98G, respectively; wherein, NC is a negative control group (0.1% DMSO), TMZ is a 450 μM temozolomide-treated group, and Taxol is a 1 μM paclitaxel-treated group. Compared with the drug-treated group, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001 in the NC group.

[0027] Figure 3 The results of the effects of temozolomide, paclitaxel, and different concentration gradients of Oxirapentyn A on the invasion of human glioma cells in Example 4 of the present invention are statistical graphs, (a) is a micrograph of U87-MG, U251, and T98G cells (200×), and (b) to (d) are statistical graphs of the results of counting the number of cells passing through the chamber in the cell lines U87-MG, U251, and T98G, respectively; NC is the negative control group (0.1% DMSO), TMZ is the 450 μM temozolomide-treated group, and Taxol is the 1 μM paclitaxel-treated group. Compared with the drug-treated group, * represents p < 0.05 in the NC group and *** represents p < 0.001.

[0028] Figure 4The figures are flow cytometric analyses of the effects of temozolomide, paclitaxel, and different concentration gradients of Oxirapentyn A on apoptosis in human glioma cells U87-MG (a), U251 (b), and T98G (c) in Example 5 of the present invention. Normal represents normal cells, Early Apoptosis represents early apoptosis, Later Apoptosis represents late apoptosis, Nercrosis represents necrotic cells, NC represents the negative control group (0.1% DMSO), TMZ represents the 450 μM temozolomide-treated group, and Taxol represents the 1 μM paclitaxel-treated group.

[0029] Figure 5 Statistical graphs of the effects of temozolomide, paclitaxel, and different concentration gradients of Oxirapentyn A on apoptosis of human glioma cells U87-MG (a), U251 (b), and T98G (c) in Example 5 of the present invention. Normal represents normal cells, Early Apoptosis represents early apoptosis, Later Apoptosis represents late apoptosis, Nercrosis represents necrotic cells, NC represents the negative control group (0.1% DMSO), TMZ represents the 450 μM temozolomide-treated group, and Taxol represents the 1 μM paclitaxel-treated group. Compared with the drug-treated group, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001 in the NC group. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0031] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0032] Example 1 Preparation method of natural product Oxirapentyn A

[0033] 1. Method 1 (preparation by bacterial fermentation)

[0034] The ascidian symbiotic fungal strain Beauveria felina SYSU-MS7908 (deposited in Guangdong Provincial Microbiological Culture Collection Center on July 22, 2020, with the deposit number GDMCC No: 61059, the deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, disclosed and protected in Chinese patent application CN112646729A, and available to the public) was used for fermentation, and the resulting fermentation product was separated and purified to obtain the natural product Oxirapenty B, which was used as a raw material for an oxidation reaction to obtain the compound Oxirapenty A.

[0035] The preparation process of natural product Oxirapentyn B and natural product Oxirapentyn A is as follows:

[0036] The ascidian fungus Beauveria felina SYSU-MS7908 was fermented at room temperature for 28 days in 10 kg of solid rice culture medium (100 parts of rice, 100 parts of 3% sea salt water, and 3 parts of peptone) sterilized at 121°C for 20 minutes; the fermentation product was extracted by immersion in an organic solvent (methanol or acetone) and extracted with ethyl acetate / water; the ethyl acetate extract was chromatographed on a silica gel column using a petroleum ether / ethyl acetate mobile phase system to collect the fraction containing 20% ​​by volume of ethyl acetate, and the natural product Oxirapentyn B (II) raw material was obtained in the gram range by ODS-C18 reverse phase chromatography; the remaining fraction containing 20% ​​by volume of ethyl acetate was purified by RP-HPLC (C18 column, 4.6×250 mm, 5 μm, MeOH / H2O, 70 / 30, 3 mL / min, T R =23-25 ​​min) can be separated and prepared to obtain the target heteroterpenoid compound Oxirapentyn A (I).

[0037] 2. Method 2 (Synthesized using Oxirapentyn B as raw material)

[0038] The natural product Oxirapentyn B(II) undergoes 6-OH oxidation reaction with an oxidant (such as pyridinium chlorochromate) to form the terpene compound Oxirapentyn A(I).

[0039] The synthesis route of compound Oxirapentyn A using Oxirapentyn B as raw material is as follows:

[0040]

[0041] The specific steps include:

[0042] In anhydrous dichloromethane (DCM), 1 eq of Oxirapentyn B (II, 20 mg) and 1.5 eq of pyridinium chlorochromate were stirred at 50°C for 2 h. The reaction solution was filtered, concentrated, and purified by flash silica gel column chromatography (petroleum ether / ethyl acetate system) to obtain the target compound Oxirapentyn A (I).

[0043] Among them, the physical and chemical properties of compounds Oxirapenty A and Oxirapenty B are as follows:

[0044] Oxirapenty A: 1 H NMR (400MHz, CDCl3) δH 5.45 (brs, 1H, H-4a'), 5.35 (d, J=

[0045] 1.06Hz,1H,H-4b'),4.95(t,J=2.5Hz,1H,H-2),4.62(s,1H,H-9),3.82(s,1H,H-8),3.23(brs,1H,H-5),2.60(dd,J=15.0,2.6Hz, 1H,H-3a),2.09(s,3H,H-2”),1.91(dd(1.0,1.5)s,3H,H-5’),1.46(d,J=2.6Hz,1H,H-3b),1.44(s,3H,H-11),1.22(s,3H,H-10). 13 C NMR(101MHz,CDCl3)δC 194.60,170.38,125.39,124.96,88.75,79.52,75.08,73.03,68.33,64.20,63.9 9,61.76,54.43,32.77,25.29,23.02,21.69,21.01.HR-ESIMSm / z333.1340[M+H] + (calcd.for C 18 H 21 O6,333.1338).

[0046] Oxirapenty B: 1 H NMR (400 MHz, CDCl3) δ H5.39(s,1H,H-4a'),5.32(m,1H,H-4b'),4.89(t,J=3.0Hz,1H,H-2),4.47(d, J=6.0Hz,1H,H-6),4.47(s,1H,H-9),3.40(s,1H,H-8),3.01(s,1H,H-5),2.5 0(dd,J=14.7,2.8Hz,1H,H-3a),2.13(s,3H,H-2”),1.89(d,J=1.0Hz,3H,H-5 '),1.50(dd,J=14.7,3.2Hz,1H,H-3b),1.43(s,3H,H-11),1.26(s,3H,H-10); 13 C NMR (101 MHz, CDCl3) δ C 170.44,125.43,124.25,86.37,83.99,74.58,73.66,63.81,63.75,60.15,60. 14,52.98,51.69,32.48,25.36,23.03,21.54,20.86.HREIMSm / z335.1498[M+H] + (calcd.for C 18 H 23 O6,335.1495).

[0047] The heteroterpene compound Oxirapenty A (I) prepared in Example 1 was used as the research object to test its effects on the proliferation, migration, invasion and apoptosis of brain glioma cells (U251, T98G, U87-MG).

[0048] Experimental materials: paclitaxel (positive control), temozolomide (positive control), U251 (human glioma cells), T98G (human glioblastoma multiforme cells), U87-MG (human astrocytic glioblastoma cells), dimethyl sulfoxide (DMSO), MTT (5 mg / mL), Matrigel (Solarbio), Transwell cell culture chamber (corning), BSA (Solarbio), cell apoptosis detection kit (Beyotime Company).

[0049] The compound was dissolved in DMSO to prepare a 10 mM stock solution, which was diluted with DMEM medium to the required concentration (DMSO content was less than 0.5%) before use.

[0050] Example 2 Effect of Oxirapentyn A on the Inhibition of Glioma Cell Proliferation

[0051] 1. Experimental methods

[0052] U251, T98G, and U87-MG glioma cells were removed from liquid nitrogen and revived for culture and passage. Cells in the logarithmic growth phase were diluted using DMEM complete medium to a cell concentration of approximately 2.5 × 10 4 / mL, add 200μL to each well of a 96-well plate and place it in a CO2 incubator for 24 hours. Use DMEM complete medium to dilute Oxirapentyn A to 50μM, 25μM, 12.5μM, 6.25μM, 3.125μM, and 1.5625μM, respectively. Remove the incubated 96-well plate, carefully remove the old culture medium, and add 200μL of culture medium containing different concentrations of the test sample. DMSO is used as a negative control and temozolomide solution is used as a positive control. Set up 3 parallel test wells for each test sample, and set up a set of blank wells (no cells) on each plate. Place the set 96-well plate in a CO2 incubator and culture for 12h, 24h, and 48h.

[0053] The 96-well plates that had been treated for 12h, 24h, and 48h were removed and the culture medium was slowly removed with a pipette. The MTT reagent was diluted 10-fold with serum-free DMEM medium, and the diluted MTT solution was added to the 96-well plate (100μL / well). The plate was incubated in a CO2 incubator for 4h to allow blue crystals to fully form. The liquid in the plate was carefully removed with a pipette, and 100μL DMSO (Formans solution) was added to fully dissolve the blue precipitate. The absorbance (OD value) of each well was read at a wavelength of 570nm on a microplate reader. The glioma cell inhibition rate of Oxirapentyn A was calculated according to the following formula:

[0054] Inhibition rate = (OD 实验组 -OD 阴性对照组 ) / OD 阴性对照组 ×100%

[0055] 2. Experimental results

[0056] Results see Figure 1 As shown in the figure, after 48 hours of treatment, Oxirapentyn A has good cell proliferation inhibitory activity against human glioma cells U87-MG, U251, and T98G (IC 50 were 6.84±0.67 μM, 20.14±0.93 μM, and 12.48±0.33 μM, respectively), and showed concentration dependence.

[0057] Example 3 Effect of Oxirapentyn A on Migration of Brain Glioma Cells

[0058] 1. Experimental methods

[0059] U251, T98G, and U87-MG glioma cells in good condition were trypsinized and seeded into 6-well plates, with 5×10 cells per well. 5 The cells were then cultured at 37°C, 5% CO2 and 95% relative humidity. When the cell density reached about 90%, a 200μL pipette tip was used to make vertical scratches from top to bottom in a clean bench. The scratched cells were washed away with PBS buffer, and photos were taken to record the distance of the initial scratch. The old culture medium was removed, and different concentrations (10μM, 5μM, 2μM) of Oxirapentyn A were added for 48 hours. In the experiment, 0.5% DMSO was added as the negative control group, and the common anti-tumor drug paclitaxel (1μM) and the anti-glioma drug temozolomide (450μM) were added as the positive control. An inverted microscope was used to observe and obtain single-layer images, and the cell front edge movement distance and scratch width were measured to analyze the cell migration ability.

[0060] 2. Experimental results

[0061] Results see Figure 2 As can be seen from the figure, Oxirapentyn A can significantly inhibit the migration of human glioma cells. The inhibitory effect is concentration-dependent and stronger than that of the positive control temozolomide.

[0062] Example 4 Effect of Oxirapentyn A on the Invasion of Brain Glioma Cells

[0063] 1. Experimental methods

[0064] The night before the experiment, Matrigel was placed in a 4°C refrigerator overnight from -20°C to allow the Matrigel to melt from a solid state to a liquid state. Dilute the Matrigel at a ratio of 1:3 using high-glucose DMEM medium on ice and add it to the upper chamber surface of the bottom membrane of the Transwell chamber. Place it in an incubator to air-dry for 3 hours. Carefully aspirate the residual liquid in the chamber, add 50 μL of 10 g / L BSA serum-free culture medium to each chamber, and culture at 37°C for 30 minutes. After digesting the cells with trypsin, wash them with PBS 1 to 2 times to remove the influence of the serum in the original culture medium, and then resuspend the cells with serum-free culture medium to a cell density of 5×10 5Cells were cultured at a concentration of 10 μM, 5 μM, or 2 μM in the upper chamber of each Transwell chamber. Add 200 μL of cell suspension to the upper chamber of the Transwell chamber, and add 600 μL of complete culture medium containing 10% FBS to the lower chamber of the 24-well culture plate. Oxirapentyn A was added to the upper chamber of each Transwell chamber at final concentrations of 10 μM, 5 μM, and 2 μM as experimental groups. A negative control group was treated with 0.5% DMSO, and a positive control group was treated with paclitaxel at a final concentration of 1 μM and temozolomide at a final concentration of 450 μM. Care was taken to prevent air bubbles from forming between the chamber and the lower chamber. The plate was incubated at 37°C in a CO2 incubator for 48 hours. The chamber was removed, rinsed twice with PBS, and fixed in 4% paraformaldehyde for 20 minutes in the 24-well plate. The plate was then rinsed twice with PBS, and stained in 0.1% crystal violet solution for 15 minutes in the 24-well plate. After staining, the stain was discarded, and cells on the upper layer of the chamber bottom membrane were carefully removed with a cotton swab. An inverted microscope was used to take pictures, and 5 fields of view were randomly counted for each chamber sample. The average value was taken and the effect of the drug on the invasion ability of tumor cells was statistically analyzed.

[0065] 2. Experimental results

[0066] Results see Figure 3 As can be seen from the figure, both 5μM and 10μM Oxirapentyn A can significantly reduce the number of three types of glioma cells passing through the chamber (p<0.001), indicating that Oxirapentyn A can inhibit the invasion of human glioma cells and has a significant effect on inhibiting the metastasis of glioma in the brain.

[0067] Example 5 Effect of Oxirapentyn A on the Apoptosis Rate of Brain Glioma Cells

[0068] 1. Experimental methods

[0069] Dilute the cells in logarithmic growth phase to a cell density of approximately 5 × 10 5Cells were seeded into 6-well plates at 100 μg / mL and incubated in an incubator for 24 hours. The old culture medium was removed and various concentrations of Oxirapentyn A (10 μM, 5 μM, and 2 μM) were added for 48 hours. 0.5% DMSO was added as a negative control, while paclitaxel (1 μM) and temozolomide (450 μM) were added as positive controls. After 48 hours, the original culture medium was aspirated from the plate and stored in a test tube. Each group of cells was washed with PBS and digested with an appropriate amount of trypsin. Digestion was terminated by adding the original culture medium, and the cells were pipetted. The washed PBS solution and trypsin-digested cells were collected in a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was carefully aspirated from the tube, and the cells were resuspended in 1.5 mL of pre-chilled PBS. The cells were centrifuged at 1000 rpm for 5 minutes to remove cell debris from the PBS and cell suspension. Add 100 μL of Annexin-V FITC / PI conjugate, 5 μL of Annexin-V FITC, and 10 μL of PI, and incubate at room temperature in the dark for 30 minutes. Use unstained cells to adjust the cell gain, use cells stained with Annexin-V FITC and PI to adjust the compensation, and use cells stained with Annexin-V FITC and PI to divide the cells into four quadrants. After adjustment, analyze the sample for apoptosis rate.

[0070] 2. Experimental Results

[0071] Results see Figures 4-5 As can be seen from the figure, 10μM Oxirapentyn A can significantly promote the apoptosis of human glioma cells in the early and late stages, among which the pro-apoptotic effect on U87-MG cells is the strongest.

[0072] The above results indicate that the natural product Oxirapenty A has good anti-glioma activity and can inhibit the proliferation, migration and invasion of glioma cells by promoting early and late apoptosis of glioma cells. Therefore, the natural product Oxirapenty A has good potential as an anti-glioma drug.

[0073] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A use of a natural product Oxirapentyn A in the preparation of an anti-glioma drug, characterized in that: The natural product Oxirapentyn A has the following structure:

2. The application according to claim 1, characterized in that The natural product Oxirapentyn A may also be in the form of a pharmaceutically acceptable salt thereof.

3. The application according to claim 1, characterized in that The natural product Oxirapentyn A promotes the apoptosis of early and late stage brain glioma cells.

4. The application according to claim 1, characterized in that The natural product Oxirapentyn A inhibits the proliferation, migration and invasion of brain glioma cells.

5. The use according to claim 3 or 4, characterized in that: The brain glioma cells are U251, T98G or U87-MG.

6. The application according to claim 1, characterized in that: The drug also includes pharmaceutically acceptable excipients.

7. The use according to claim 1, characterized in that The dosage form of the drug is oral preparation, injection or micro-injection.

8. The application according to claim 1, characterized in that: The natural product Oxirapentyn A is isolated and purified from the fungus strain Beauveria felina SYSU-MS7908 derived from the marine ascidian with the preservation number GDMCC61059.

9. The use according to claim 1, characterized in that: The natural product Oxirapentyn A is obtained by oxidation of the natural product Oxirapenty B:

10. The use according to claim 9, characterized in that: The oxidizing agent is selected from one or more of pyridinium chlorochromate, bromine, manganese dioxide, potassium permanganate, hydrogen peroxide, Jones reagent, and Dess-Martin oxidant.

Images