A sesquiterpene lactone compound and its preparation and application

Abstract

The present invention discloses a sesquiterpene lactone compound, its preparation, and application. The compound is isolated from the plant Lepidoptera of the genus Lepidoptera in the Asteraceae family and obtained through solvent extraction, silica gel column chromatography, ODS column chromatography, and liquid chromatography separation. Experimental results demonstrate that the sesquiterpene lactone compound provided by the present invention can significantly inhibit the proliferation of glioma cells, induce apoptosis in glioma cells, and alter mitochondrial membrane potential in cells. Therefore, the compound is a candidate anti-glioma drug.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to a sesquiterpene lactone compound separated from Cyperus rotundus, an extraction method thereof, and an application of the sesquiterpene lactone compound in preparing a drug for treating glioma. Background Art

[0002] Cancer incidence has been increasing significantly in recent years. Glioma, a malignant tumor that develops in the brain, poses a serious threat to human health. Current drugs used to treat gliomas are often resistant to treatment and have significant side effects, necessitating the urgent need for new therapeutic agents.

[0003] Natural products are a source of innovative drug development. From 1981 to 2019, a total of 1,881 new drugs were launched globally, 46% of which were directly or indirectly derived from natural products. Anti-tumor drugs account for a significant number of these, demonstrating the importance of natural products in oncology drug development. my country boasts an extremely rich resource of traditional Chinese medicine (TCM), which plays a vital role in the treatment of diseases. TCM components are complex in structure and possess a wide range of biological activities, offering great potential for discovering active ingredients from TCM and developing them into anti-glioma drugs.

[0004] Rabbit umbrella ( Syneilesis aconitifolia Bunge (Bunge) Maxim. is a perennial herbaceous plant of the genus Bunge in the Asteraceae family. Its roots and whole plant are used as medicine, with a slightly warm nature and a pungent flavor. It has the effects of dispelling rheumatism, relaxing muscles and promoting blood circulation, and relieving pain. It can treat symptoms such as low back pain, traumatic injuries, rheumatic numbness, carbuncles, and swellings. Currently, the compounds isolated from Bunge primarily consist of active ingredients such as alkaloids, terpenes, and flavonoids. For example, Li Jun et al. analyzed the chemical composition of the hexane-extracted portion of the methanol extract of the dried Bunge whole plant, identifying nine compounds, five of which showed inhibitory effects on HeLa cells with uterine cancer. Summary of the Invention

[0005] In view of this, the present invention aims to separate new chemical substances from the wormwood in order to develop anti-tumor (especially anti-glioma) drugs.

[0006] The technical solutions of the present invention are as follows:

[0007] The first aspect of the present invention provides a sesquiterpene lactone compound isolated from Saconitifolia (named Saconitifolia B), the chemical formula of which is C 26 H 36 O6, and the structural formula is as follows:

[0008] .

[0009] The second aspect of the present invention provides the use of the above-mentioned sesquiterpene lactone compound in the preparation of a drug for treating glioma, and cell experiments show that the sesquiterpene lactone compound has at least the following functions:

[0010] a). Inhibit glioma cell proliferation;

[0011] b). Induce apoptosis of glioma cells and change mitochondrial membrane potential in glioma cells.

[0012] The sesquiterpene lactone compounds provided by the present invention belong to the eudesmane-type sesquiterpene. In addition to Saconitifolia B provided by the present invention, the prior art also discloses a eudesmane-type sesquiterpene lactone isolated from Saconitifolia serrata (i.e., Saconitifolia lactone A). However, its structure differs significantly from that of Saconitifolia B at positions C-3, C-6, C-8, and C-14. In addition, although Saconitifolia lactone A has an inhibitory effect on HeLa cells (human cervical cancer cells), its half inhibitory concentration (IC50) value after 72 hours of action on HeLa cells is 250 μg / mL. In contrast, the Saconitifolia B isolated and identified by the present invention has an IC50 value of 16.39 μM after 24 hours of action on GL261 cells (mouse glioma cells). This shows that the Saconitifolia B isolated by the present invention has obvious advantages in the treatment of gliomas.

[0013] The third aspect of the present invention provides an anti-glioma drug, which uses the sesquiterpene lactone compound provided by the present invention as one of the active ingredients; it is understandable that the drug may further contain pharmaceutically acceptable excipients.

[0014] A fourth aspect of the present invention provides a method for preparing the above-mentioned sesquiterpene lactone compound, comprising the following steps:

[0015] S1, using an extractant to extract the cyperus rotundus, collecting the extract and concentrating it to obtain a concentrate;

[0016] S2. Subjecting the concentrated solution obtained in S1 to silica gel column chromatography, eluting with a first eluent, and collecting a crude fraction rich in the sesquiterpene lactone compound, wherein the first eluent is any one of petroleum ether, petroleum ether-ethyl acetate, dichloromethane-methanol, ethyl acetate, and n-butanol;

[0017] S3, subjecting the crude fraction obtained in S2 to ODS column chromatography, performing gradient elution using methanol-water as the second eluent, and collecting the target fraction rich in the sesquiterpene lactone;

[0018] S4. Separate the target fraction obtained in S3 by high performance liquid chromatography (HPLC) to obtain the sesquiterpene lactone compound.

[0019] Preferably, in the above method, the extractant is water and / or alcohol, wherein the alcohol includes methanol and ethanol; more preferably, the extractant is a mixture of water and alcohol, and the alcohol concentration is 10-95% (volume ratio), wherein the ethanol concentration is 60-90% is the best.

[0020] In step S1 of the above method, the amount of the extractant added is preferably 8-15 times the amount of the Myrica ovata (mass ratio). The Myrica ovata can be dried and chopped before soaking. The extraction temperature can be room temperature (15-30°C, and the extraction time at room temperature is preferably 1-7 days), or the extraction can be performed under heating conditions and the temperature is preferably 60°C to the boiling point of the extractant.

[0021] In step S2 of the above method, when the first eluent used is a mixed phase (such as petroleum ether-ethyl acetate), the volume ratio can be 100:0, 95:5, 9:1, 8:2, 7:3, 6:4, 1:1, or 0:100.

[0022] In step S3 of the above method, the volume ratio of the second eluent, i.e., methanol-water (methanol:water), can be 0:100, 5:95, 1:9, 2:8, 3:7, 4:6, 1:1, 6:4, 7:3, 8:2, 9:1, or 100:0, and gradient elution can be performed in ascending order of polarity of the second eluent.

[0023] Preferably, in step S4 of the above method, the HPLC separation conditions include: YMC ODS column (5 μm, 250×20 mm), 80% (v / v) MeOH / H 2 O, 3.0 mL / min.

[0024] The beneficial effects of the present invention are as follows: the present invention extracts and separates components from the whole plant of the cyperus rotundus, and identifies a new eucalyptol-type sesquiterpene lactone compound therefrom. The compound can significantly inhibit the proliferation of glioma cells, induce glioma cell apoptosis, and change the mitochondrial membrane potential in glioma cells. Therefore, the sesquiterpene lactone compound provided by the present invention has anti-glioma activity and can be used as a candidate anti-glioma drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a high-resolution mass spectrum of the sesquiterpene lactone compound of the present invention.

[0026] Figure 2 The nuclear magnetic resonance of the sesquiterpene lactone compound of the present invention is 1 H NMR spectra ( 1 H NMR, 600 MHz, CDCl3)

[0027] Figure 3 The nuclear magnetic resonance of the sesquiterpene lactone compound of the present invention is13 C NMR ( 13 C NMR, 150 MHz, CDCl3).

[0028] Figure 4 The figure is the nuclear magnetic resonance HSQC spectrum of the sesquiterpene lactone compound of the present invention.

[0029] Figure 5 The figure is the HMBC nuclear magnetic resonance spectrum of the sesquiterpene lactone compound of the present invention.

[0030] Figure 6 The nuclear magnetic resonance of the sesquiterpene lactone compound of the present invention is 1 H- 1 H COSY spectrum.

[0031] Figure 7 The figure is the nuclear magnetic resonance NOESY spectrum of the sesquiterpene lactone compound of the present invention.

[0032] Figure 8 This is the effect of the sesquiterpene lactone compounds of the present invention on the activity of GL261 cells.

[0033] Figure 9 The figure shows the effect of the sesquiterpene lactone compounds of the present invention on the apoptosis of GL261 cells at different concentrations (0, 5, 10, 20, 30 μM).

[0034] Figure 10 The figure shows the effect of the sesquiterpene lactone compounds of the present invention on the mitochondrial membrane potential of GL261 cells at different concentrations (0, 5, 10, 20, 30 μM). DETAILED DESCRIPTION

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The term "include," "comprising," and any variations thereof, in the specification and claims of the present invention are intended to cover non-exclusive inclusions. The terms "first," "second," and the like in the specification and claims of the present invention are used solely to distinguish between different objects and should not be construed as indicating or implying relative importance or implicitly indicating a specific order or primary-secondary relationship of the indicated technical features.

[0036] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0037] If no specific techniques or conditions are specified in the following examples, the procedures were carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product instructions; if no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0038] Example 1

[0039] This study isolated and identified the sesquiterpene lactone compound Saconitifolia B from the genus Saconitifolia, specifically by the following steps:

[0040] (1) Chop the dried whole herb of Cyperus rotundus into small pieces, add it to 10 times the amount of 60% ethanol solvent and extract it twice, each extraction time is 3 days, combine the extracts, filter, and concentrate under reduced pressure to obtain the concentrated solution.

[0041] (2) The concentrate was separated by silica gel column chromatography and eluted with a mixed solvent of petroleum ether-ethyl acetate (100:0, 95:5, 9:1, 8:2, 7:3, 6:4, 1:1, 0:100) to obtain four fractions.

[0042] (3) The third fraction obtained in step (2) was subjected to ODS column chromatography and gradient eluted with methanol-water (100:0, 95:5, 9:1, 8:2, 7:3, 6:4, 1:1, 0:100) to obtain 12 fractions.

[0043] (4) The seventh fraction obtained in step (3) was separated by HPLC to obtain sesquiterpene lactones, wherein the HPLC conditions were: YMC ODS column (5 μm, 250×20 mm), 80% MeOH / H2O, 3.0 mL / min.

[0044] The obtained sesquiterpene lactone was structurally identified, and the identification results were as follows: Figure 1-7 As shown, the specific data of mass spectrum, nuclear magnetic hydrogen spectrum and carbon spectrum are as follows:

[0045] HRESIMS m / z 445.2628 [M+H] in positive ion mode + (C 26 H 37 Calculated data for O6: 445.2585).

[0046] 1 H-NMR (600 MHz, CDCl3): δ H1.73 (2H, m,H-1a, H-1b), 1.70 (1H, m, H-2a), 1.89 (1H, m, H-2b), 5.14 (1H, m, H-3), 2.19 (1H, m, H-4), 6.17 (1H, H-6), 4.90 (1H, dd, J = 12.6, 7.8 Hz, H-8), 1.59 (1H, m, H-9a), 2.20 (1H, m, H-9b), 1.97 (1H, m, H-10), 1.81 (3H, s, H-13), 0.94 (3H, s, H-14), 0.98 (3H, d, J = 9.0 Hz, H-15), 2.30 (1H, s, H-2′), 2.24 (2H, m, H-4′), 1.11 (3H, t, J = 9.0 Hz, H-5′), 2.22 (3H, s, H-6′), 6.02 (1H, qd, J = 8.4, 1.8 Hz, H-3′′), 1.94 (3H, J = 1.8 Hz, H-4′′), 1.85 (3H,m, H-5′′).

[0047] 13 C-NMR (150 MHz, CDCl3): δ C 27.0 (C-1), 25.5 (C-2), 71.0 (C-3), 35.7 (C-4), 45.4 (C-5), 70.4 (C-6), 159.0 (C-7), 77.2 (C-8), 34.5 (C-9), 35.4 (C-10), 122.5 (C-11), 174.4 (C-12), 8.5 (C-13), 20.2 (C-14), 8.7 (C-15), 167.0 (C-1′), 113.0 (C-2′), 165.8 (C-3′), 34.1 (C-4′), 12.0 (C-5′), 19.0 (C-6′), 165.2 (C-1′′), 128.2 (C-2′′), 137.8 (C-3′′), 15.8 (C-4′′), 20.8 (C-5′′).

[0048] Example 2

[0049] This example verifies the inhibitory effect of the sesquiterpene lactone compound Saconitifolia B of the present invention on glioma cell proliferation, which specifically includes the following steps:

[0050] (1) Cell recovery.

[0051] Thaw frozen GL261 cells (mouse glioma cells) quickly in a 37°C water bath and centrifuge at 1000 rpm for 5 minutes. After centrifugation, discard the supernatant and resuspend the cells in fresh DMEM medium. Plate the cells in a 10 cm dish and mix thoroughly. Incubate the cells in a 37°C, 5% CO2, and constant humidity incubator. Change the medium after 24 hours.

[0052] (2) Propagation.

[0053] When the cell density reaches approximately 70%, discard the culture medium. Wash the cells twice with sterile PBS and add 1.0 mL of trypsin solution. Digest the cells for approximately 3 minutes. Once the cells become round, add 2.0 mL of culture medium to terminate the digestion. Use a pipette to repeatedly pipette the cells until they are completely detached, then transfer them to a 5 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. After centrifugation, discard the supernatant, add fresh DMEM medium, resuspend the cells evenly, and seed them into a new culture dish at a 1:3 ratio. Continue culturing in a cell culture incubator.

[0054] (3) Laying the boards.

[0055] After passage 2-3, cells in the logarithmic phase were collected and adjusted to an appropriate cell density. The cells were then plated in triplicate in a 96-well cell culture plate, with 100 μL per well, containing approximately 10,000 cells. The cells were cultured in a cell culture incubator.

[0056] (4) Add medicine.

[0057] After the cells were stably attached, the old culture medium was removed. 100 μL of fresh DMEM culture medium was added to each well of the blank control group; 100 μL of DMEM culture medium containing the sesquiterpene lactone compound of the present invention was added to each well of the drug-treated group to achieve final drug concentrations of 5, 10, 20, and 30 μM, respectively. The cells were then cultured in a cell culture incubator.

[0058] (5) Add CCK8.

[0059] After 24 hours of incubation, remove the culture medium from the 96-well plates and add 100 μL of a mixture of CCK8 and DMEM (1:9 ratio) to each well. Continue incubating in a cell culture incubator for 1 hour. Finally, measure the absorbance (OD) of each well at 450 nm using a microplate reader.

[0060] The experimental results of CCK8 assay for cell activity are as follows Figure 8As shown, the sesquiterpene lactone compound provided by the present invention can significantly inhibit the proliferation of GL261 cells, and its IC50 value is 16.39 μM.

[0061] Example 3

[0062] The sesquiterpene lactone compound Saconitifolia B of the present invention has the effect of inducing apoptosis of glioma cells. This example also uses GL261 cells as an example and is demonstrated through the following experiments:

[0063] (1) Cell culture,

[0064] Same as steps (1) and (2) of Example 2.

[0065] (2) Dosing treatment.

[0066] GL261 cells in the logarithmic growth phase were evenly plated in a 12-well plate with 1 mL of culture medium per well and 2 × 10 cells per well. 5 Cells were cultured at 37°C in a cell culture incubator containing 5% CO2 and constant humidity. When the cell density reached approximately 70%, the culture medium was aspirated and 1 mL of sesquiterpene lactone compounds (5, 10, 20, and 30 μM) prepared in the culture medium was added. An equal volume of DMSO was added to the culture medium of the control group, and the cells were cultured in the incubator for 24 h.

[0067] (3) Determination of apoptotic cell staining.

[0068] Collect the cell culture medium obtained in step (2) into a 2 mL EP tube. Then, add 0.2 mL of 0.25% trypsin without EDTA to the 6-well plate and digest at room temperature for 2 minutes. Then, add an equal amount of culture medium to terminate the digestion. Then, use a pipette to gently blow down the cells and transfer them to the above 2 mL EP tube. Centrifuge at 1000 rpm for 5 minutes. After aspirating the supernatant, add 1 mL of pre-cooled PBS to resuspend and then transfer them to a 1.5 mL EP tube. Repeat the washing twice, centrifuge at 1000 rpm for 5 minutes, discard the above PBS, add a mixture of 100 µL 1× Binding Buffer, 5 µL Annexin V-FITC and 5 µL PI Staining Solution to the EP tube, and react at room temperature in the dark for 10 minutes. Then, add 400 µL 1× Binding Buffer, mix gently and filter with nylon cloth to obtain the filtrate. The obtained filtrate is detected by flow cytometry within 1 hour.

[0069] (4) Determination of mitochondrial membrane potential cell staining.

[0070] Aspirate the cell culture medium into a 2mL EP tube and add 0.2mL of trypsin digestion buffer to digest the cells. Incubate at room temperature until adherent cells flow down with gentle shaking. Stop digestion by adding collected cell culture medium. Gently pipette the cells down and transfer them to the same 2mL EP tube. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and collect the cells. Resuspend the cells in 0.5mL of cell culture medium and add 0.5mL of JC-1 working staining solution. Mix by inverting several times. Incubate in a cell culture incubator at 37°C for 20 minutes, mixing every few minutes. After the 37°C incubation, centrifuge at 600g at 4°C for 4 minutes to pellet the cells. Discard the supernatant, wash twice with JC-1 staining buffer, pellet the cells, and discard the supernatant. Resuspend the tube in 200μL of JC-1 staining buffer, store on ice, and analyze by flow cytometry.

[0071] The above test results are as follows Figure 9 and 10 As shown, the results showed that after GL261 cells were treated with different concentrations of the sesquiterpene lactone compounds of the present invention for 24 hours, the proportion of apoptotic cells increased significantly and the mitochondrial membrane potential changed significantly with the increase of the compound concentration.

[0072] In summary, the present invention isolated and identified the sesquiterpene lactone compound Saconitifolia B from the umbelliferae for the first time. This new compound can effectively inhibit the proliferation of glioma cells, induce apoptosis of glioma cells, and change the mitochondrial membrane potential in cells. Therefore, it can be used as a candidate drug for anti-glioma.

[0073] It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solutions of the present invention rather than to limit them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A sesquiterpene lactone compound, characterized in that: It has the following structural formula: 。 2. Use of the sesquiterpene lactone compound according to claim 1 in the preparation of a drug for treating glioma.

3. The use according to claim 2, characterized in that The sesquiterpene lactone compound has at least the following functions: a) inhibiting glioma cell proliferation; b) Induce apoptosis of glioma cells and change mitochondrial membrane potential in glioma cells.

4. An anti-glioma drug, characterized in that: The sesquiterpene lactone compound according to claim 1 is one of the active ingredients.

5. The drug according to claim 4, characterized in that Contains pharmaceutically acceptable excipients.

6. A method for preparing the sesquiterpene lactone compound according to claim 1, characterized in that: The following steps are involved: S1, using an extractant to extract the cyperus rotundus, collecting the extract and concentrating it to obtain a concentrate; S2. Subjecting the concentrated solution obtained in S1 to silica gel column chromatography, eluting with a first eluent, and collecting a crude fraction rich in the sesquiterpene lactone compound, wherein the first eluent is any one of petroleum ether, petroleum ether-ethyl acetate, dichloromethane-methanol, ethyl acetate, and n-butanol; S3, subjecting the crude fraction obtained in S2 to ODS column chromatography, performing gradient elution using methanol-water as the second eluent, and collecting the target fraction rich in the sesquiterpene lactone compound; S4. Separate the target fraction obtained in S3 by high performance liquid chromatography to obtain the sesquiterpene lactone compound.

7. The method according to claim 6, characterized in that The extractant is water and / or alcohol, and the alcohol includes methanol and ethanol.

8. The method according to claim 7, characterized in that The extractant is a mixture of water and alcohol, and the alcohol concentration is 10-95%.

9. The method according to claim 6, characterized in that The first eluent is petroleum ether-ethyl acetate.

10. The method according to claim 6, characterized in that The separation conditions in step S4 are: YMC ODS column, 80% MeOH / H2O, 3.0 mL / min.

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