Application of alkaloid compounds in Erythrina variegata
By extracting and isolating the alkaloid compound Hecubine from *Cynodon dactylon*, an antitumor drug was prepared, which solved the problem of insufficient treatment for papillary thyroid carcinoma and clear cell renal carcinoma in existing technologies, and achieved significant antitumor effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- KUNMING MEDICAL UNIVERSITY
- Filing Date
- 2023-11-09
- Publication Date
- 2026-06-26
Smart Images
Figure CN117547532B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of antitumor technology, specifically the application of alkaloid compounds from *Cymbidium goeringii*. Background Technology
[0002] Malignant tumors are a major public health problem that seriously endangers people's health and social development. In recent decades, the incidence of cancer-related deaths has increased, and the number of patients diagnosed with papillary thyroid carcinoma and clear cell renal cell carcinoma has been rising. Therefore, the research and development of safe and effective anti-tumor drugs is urgently needed.
[0003] *Tabernaemontanabovina* Loureiro, a plant belonging to the genus *Tabernaemontanabovina* in the family Apocynaceae, is mainly produced in Guangdong, Hainan, and Yunnan provinces. Its roots are used medicinally. Plants in the *Tabernaemontanabovina* genus contain a large number of monoterpenoid indole alkaloids, which exhibit broad and significant biological activities. However, current research on the antitumor effects of *Tabernaemontanabovina* is limited. Summary of the Invention
[0004] The purpose of this invention is to provide an application of alkaloid compounds from *Cynodon dactylon* to solve the problems mentioned in the background art.
[0005] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:
[0006] The application of an alkaloid compound from *Cynodon dactylon* in the preparation of antitumor drugs, wherein the structural formula of the alkaloid compound is:
[0007] Preferably, the antitumor drug is an antitumor drug for human clear cell renal carcinoma and / or papillary thyroid carcinoma.
[0008] Preferably, the alkaloid compound is used to induce tumor cell apoptosis by affecting cell viability and cell proliferation.
[0009] Preferably, the extraction and separation method for the alkaloid compounds includes the following steps:
[0010] After drying, the branches and leaves of medicinal dog tooth flower are pulverized and then extracted with methanol under reflux. The extracts are combined and concentrated under reduced pressure to obtain the methanol extract of medicinal dog tooth flower.
[0011] The methanol extract of medicinal dog tooth flower was adjusted to pH 2-3 with hydrochloric acid aqueous solution, and then the solution was dissolved and filtered. The filtrate was extracted with petroleum ether and ethyl acetate respectively. The pH of the acidic aqueous layer was adjusted to 9-10 with ammonia water, and then extracted with ethyl acetate to obtain the alkaline aqueous layer.
[0012] The alkaline aqueous layer was adsorbed using macroporous adsorption resin, washed with water until colorless, then eluted with methanol, concentrated under reduced pressure to obtain an extract, and then subjected to column chromatography with silica gel, eluted with chloroform and methanol gradient to obtain the target component.
[0013] The target fraction was mixed with silica gel and subjected to column chromatography, followed by gradient elution with petroleum ether and acetone to obtain the target fraction.
[0014] After purification of the target fraction, the alkaloid compounds were obtained by separation.
[0015] Preferably, the temperature for methanol reflux extraction is controlled at 60°C.
[0016] Preferably, the macroporous adsorption resin is macroporous adsorption resin D101.
[0017] Preferably, the method for purifying the target fraction is to use gel Sepheedex LH-20 column chromatography and semi-preparative HPLC for purification.
[0018] The alkaloid compound provided in this invention can be extracted and isolated from *Cynodon dactylon*, a plant of the genus *Cynodon* in the family Apocynaceae. It can induce tumor cell apoptosis by affecting cell viability and cell proliferation, and has certain medicinal uses in the treatment of tumors such as human clear cell renal carcinoma and papillary thyroid carcinoma. Attached Figure Description
[0019] Figure 1 Hecubine is an alkaloid compound. 1 H-NMR and 13 C-NMR data graph.
[0020] Figure 2 This is a graph showing the cell viability of the alkaloid compound Hecubine on five types of human tumor cells.
[0021] Figure 3 This is a flow cytometry result of the alkaloid compound Hecubine on Caki-1 and IHH4 cells.
[0022] Figure 4 The figure shows the results of the clone formation experiment of the alkaloid compound Hecubine on Caki-1 and IHH4 cells. Detailed Implementation
[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0024] To address the technical problems raised in the background, this invention presents an embodiment of the invention that studies the antitumor activity of Hecubine, an alkaloid compound isolated from medicinal dog tooth flower, on five types of human tumor cells, providing a foundation for further development of antitumor drugs.
[0025] Specifically, in one embodiment of the present invention, an application of an alkaloid compound from *Helicobacter pectinata* in the preparation of an antitumor drug is provided, wherein the alkaloid compound is named Hecubine, and its structural formula is:
[0026] In particular, the alkaloid compound Hecubine can be used to treat tumors such as human clear cell renal cell carcinoma and / or papillary thyroid carcinoma.
[0027] The mechanism of action of this alkaloid compound, Hecubine, is to induce tumor cell apoptosis by affecting cell viability and cell proliferation.
[0028] In a preferred embodiment of the present invention, the extraction and separation method of the above-mentioned alkaloid compounds includes the following steps:
[0029] S1. After drying the branches and leaves of medicinal dog tooth flower, pulverize them and then extract them by methanol reflux. Combine the extracts and concentrate them under reduced pressure to obtain the methanol extract of medicinal dog tooth flower.
[0030] S2. The methanol extract of medicinal dog tooth flower was adjusted to pH 2-3 with hydrochloric acid aqueous solution, and then the solution was dissolved and filtered. The filtrate was extracted with petroleum ether and ethyl acetate respectively. The acidic water layer was adjusted to pH 9-10 with ammonia water and then extracted with ethyl acetate to obtain the alkaline water layer.
[0031] S3. The alkaline aqueous layer was adsorbed with macroporous adsorption resin, washed with water until colorless, then eluted with methanol, concentrated under reduced pressure to obtain an extract, and then subjected to column chromatography with silica gel, eluted with chloroform and methanol gradient to obtain the target component.
[0032] S4. Mix the target component with silica gel and perform column chromatography, then elute with a gradient of petroleum ether and acetone to obtain the target fraction.
[0033] S5. After purifying the target fraction, the alkaloid compounds are obtained by separation.
[0034] In a preferred embodiment of the present invention, the temperature of methanol reflux extraction is controlled at 60°C, and the methanol reflux extraction can be performed multiple times, each time for 2-4 hours.
[0035] In a preferred embodiment of the present invention, the macroporous adsorption resin is macroporous adsorption resin D101.
[0036] In a preferred embodiment of the present invention, the method for purifying the target fraction is to use gel Sepheedex LH-20 column chromatography and semi-preparative HPLC for purification.
[0037] The following embodiments are implementation examples and related experimental examples of the technical solution of the present invention in practical applications, but are not limited thereto.
[0038] Example 1: This example provides a method for the extraction and separation of the alkaloid compound Hecubine, specifically including the following steps:
[0039] S1. Take 5 kg of medicinal dog tooth flower branches and leaves, dry and crush them, and extract them with methanol under reflux at 60℃ for 3 hours each time. Then combine the extracts and concentrate them under reduced pressure to obtain medicinal dog tooth flower methanol extract.
[0040] S2. Adjust the pH of the above-mentioned medicinal canine tooth flower methanol extract to 2-3 with 0.5% hydrochloric acid aqueous solution. After dissolving and filtering, extract the filtrate twice each with petroleum ether and ethyl acetate. Adjust the pH of the acidic aqueous layer to 9-10 with ammonia water, and then extract it twice with an equal volume of ethyl acetate to obtain the alkaline aqueous layer.
[0041] S3. The above alkaline water layer was adsorbed with macroporous adsorption resin D101, then washed with water until colorless, and then eluted with methanol. After concentration under reduced pressure, 30g of extract was obtained. Then, the sample was mixed with 40g of silica gel (100-200 mesh) for column chromatography and eluted with chloroform-methanol gradient (1:0-0:1, v / v) to obtain four components (AD).
[0042] S4. Take the above target component D (1.3g), mix it with 1.3g of silica gel, and then perform column chromatography with a 40g silica gel column. Then, perform gradient elution with petroleum ether-acetone (20:1-0:1, v / v) to obtain two fractions (D1-D2).
[0043] S5. The above low polarity component D1 was purified by gel Sepheedex LH-20 column chromatography (methanol-water gradient elution) and semi-preparative HPLC (acetonitrile / methanol-water) to obtain a white amorphous powder, which is the alkaloid compound Hecubine (10mg).
[0044] Example 2: The structure of the alkaloid compounds isolated in Example 1 was identified using modern spectroscopic analysis techniques. 1 H and 13 The C NMR data attribution is shown in Table 1 and... Figure 1 .
[0045] Table 1: Alkaloids1 H-NMR and 13 C-NMR data attribution information
[0046]
[0047]
[0048] Example 3: The in vitro antitumor activity of the alkaloid compound Hecubine obtained in Example 1 against five types of human tumor cells, RKO, Caki-1, IHH4, MDA-MB-231, and HepG2, was studied, as follows:
[0049] Experimental reagents: DMEM High Glucose medium (Servicebio, G4510), 0.25% trypsin (Servicebio, G4001), Fetal Bovine Serum (Ozfan, FBSRY0901), penicillin / streptomycin P / S (Servicebio, G4003), cell-grade DMSO (Servicebio, GC203005), CCK-8 assay kit (Servicebio, G4103), Annexin V-FITC apoptosis assay kit (Beyotime, C1062).
[0050] Experimental equipment: 96-well plate (Servicebio CCP-96H), 6-well plate (Servicebio CCP-6H), culture flask (Servicebio CCF-25), 1.5ml centrifuge tube (EP-150-M), pipette (Eppendorf), full-wavelength microplate reader (Thermo Fisher), carbon dioxide incubator (Thermo Fisher), cell counter (Countstar Mira FL).
[0051] Experimental Procedure: Human tumor cells RKO, Caki-1, IHH4, MDA-MB-231, and HepG2 were cultured in 89% DMEM High Glucose medium, 10% heat-inactivated FBS (fetal bovine serum), and 1% penicillin / streptomycin P / S, and placed in a 37°C, 5% CO2 incubator. Tumor cells in the logarithmic growth phase were cultured at 5 × 10⁶ cells / well. 3Cells were seeded at 100 μL / 100 μL in 96-well plates and incubated for 24 h. After cell attachment, the cells were divided into three groups: blank group, control group, and experimental group (100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, and 6.25 μg / mL). Different concentrations of drug solution were added to the experimental groups, while the blank group was treated with an equal volume of cell-free culture medium. The control group was not treated with drugs but was seeded with cells. Each group had three replicates. A positive control was also included, containing oxaliplatin. After drug administration, the cells were incubated for 48 h. Then, 10 μL of CCK-8 solution was added to each well (in the dark), and the cells were incubated for 2 h. The absorbance (OD) was measured at 450 nm using a microplate reader. Cell viability and the IC50 (half-maximal inhibitory concentration) of the compound were calculated. The experiment was repeated three times.
[0052] Cell viability (%) = [OD(experimental group) - OD(blank group)] / [OD(control group) - OD(blank group)] × 100%.
[0053] The calculation method for the half-inhibitory concentration (IC50) was as follows: Cell viability data were entered into GraphPad Prism 9.0.0 software to generate cell inhibition rate curves and IC50 values. Then, IBM SPSS Statistics 24 statistical software was used to perform statistical analysis on all experimental data. express.
[0054] The results of the above experiments are shown in Table 2 and Figure 2 As shown in Table 2, the alkaloid compound Hecubine and the positive control drug oxaliplatin inhibited the growth of human liver cancer cells (HepG2), colorectal cancer cells (RKO), renal clear cell carcinoma skin metastases (Caki-1), thyroid papillary carcinoma cells (IHH4), and breast cancer MDA-MB-231 cells. Their IC50 values are detailed in Table 2.
[0055] Table 2
[0056]
[0057]
[0058] Experimental results showed that the alkaloid compound Hecubine had a certain inhibitory effect on the growth of human hepatocellular carcinoma (HepG2), colorectal carcinoma (RKO), renal clear cell carcinoma skin metastases (Caki-1), papillary thyroid carcinoma (IHH4), and breast cancer MDA-MB-231 cells. The IC50 value of Hecubine against IHH4 cells was basically consistent with that of oxaliplatin. Furthermore, the IC50 value of Hecubine against Caki cells was not significantly different from that of oxaliplatin. When the concentration of Hecubine reached 25 μg / mL or higher, its inhibitory effect on Caki-1 cells was stronger than that of oxaliplatin. When the concentration of Hecubine reached 50 μg / mL or higher, its inhibitory effect on RKO cells was stronger than that of oxaliplatin. To further verify the antitumor activity of the alkaloid compound Hecubine, apoptosis and cell cloning experiments were conducted on Caki-1 and IHH4 cells in the embodiments of this invention, as detailed in Examples 4 and 5 below.
[0059] Example 4: Apoptosis detection experiment: Caki-1 and IHH4 cells were divided into two groups of 5 × 10⁶ cells per well. 5 Cells were seeded at 1000 cells / mL in 6-well plates and incubated at 37°C with 5% CO2 for 24 hours. Then, the cells were treated with the alkaloid compound Hecubine at IC50 value. After 48 hours of drug treatment, the cells were collected and stained with Annexin V-FITC and PI staining solution for apoptosis. After incubation at 20°C in the dark for 10 minutes, the cells were immediately detected by flow cytometry.
[0060] The above experimental results are as follows Figure 3 As shown, Figure 3 The results are from flow cytometry analysis of the alkaloid compound Hecubine on Caki-1 and IHH4 cells. The results show that after 48 hours of treatment with the alkaloid compound Hecubine, the number of Caki-1 and IHH4 cells decreased significantly, and the cell necrosis rate increased significantly, indicating that the alkaloid compound Hecubine induces apoptosis in Caki-1 and IHH4 cells (P < 0.001).
[0061] Example 5: Cell colony formation assay to detect cell proliferation capacity. Specifically, cells in the logarithmic growth phase were seeded at 100 cells / well in a 6-well plate. When visible colonies appeared in the culture plate, the cells were maintained by administering the IC50 of the alkaloid compound Hecubine. The cells were cultured at 37°C and 5% CO2 for 2 weeks. The culture was then terminated, the supernatant was discarded, and the cells were washed with PBS and fixed with 4% paraformaldehyde for 20 minutes. After washing with PBS, the cells were stained with crystal violet for 30 minutes. The staining solution was washed away with running water, and the cells were dried and photographed.
[0062] The above experimental results are as follows Figure 4 As shown, Figure 4 The results of the colony formation experiment of the alkaloid compound Hecubine on Caki-1 and IH H4 cells showed that, compared with the normal group of cells, the alkaloid compound Hecubine significantly reduced the colony formation of IHH4 and Caki-1 cells (P < 0.01) and had a significant inhibitory effect on the proliferation of IHH4 and Caki-1 cells.
[0063] In summary, the alkaloid compound Hecubine obtained in the embodiments of the present invention has a certain degree of inhibitory effect on skin metastatic cells of clear cell renal cell carcinoma (Caki-1) and papillary thyroid carcinoma cells (IHH4). It can significantly inhibit the proliferation of Caki-1 cells and IHH4 cells, leading to necrosis of Caki-1 cells and IHH4 cells. Its anti-tumor effect on Caki-1 cells is comparable to that of the positive control drug oxaliplatin, and its effect on IHH4 cells is better than that of the positive control drug oxaliplatin when the concentration is higher than 25 μg / mL.
[0064] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.
Claims
1. The application of an alkaloid compound from *Cymbidium goeringii* in the preparation of antitumor drugs, characterized in that, The structural formula of the alkaloid compound is: The antitumor drug is an antitumor drug for clear cell carcinoma of the kidney and / or papillary thyroid carcinoma.
2. The application according to claim 1, characterized in that, The alkaloid compounds are used to induce tumor cell apoptosis by inhibiting cell viability and cell proliferation.
3. The application according to claim 1, characterized in that, The extraction and separation method for the alkaloid compounds includes the following steps: After drying, the branches and leaves of medicinal dog tooth flower are pulverized and then extracted with methanol under reflux. The extracts are combined and concentrated under reduced pressure to obtain the methanol extract of medicinal dog tooth flower. The methanol extract of medicinal dog tooth flower was adjusted to pH 2-3 with hydrochloric acid aqueous solution, and then the solution was dissolved and filtered. The filtrate was extracted with petroleum ether and ethyl acetate respectively. The pH of the acidic aqueous layer was adjusted to 9-10 with ammonia water, and then extracted with ethyl acetate to obtain the alkaline aqueous layer. The alkaline aqueous layer was adsorbed using macroporous adsorption resin, washed with water until colorless, then eluted with methanol, concentrated under reduced pressure to obtain an extract, and then subjected to column chromatography with silica gel, eluted with chloroform and methanol gradient to obtain the target component. The target fraction was mixed with silica gel and subjected to column chromatography, followed by gradient elution with petroleum ether and acetone to obtain the target fraction. After purification of the target fraction, the alkaloid compounds were obtained by separation.
4. The application according to claim 3, characterized in that, The temperature for methanol reflux extraction was controlled at 60℃.
5. The application according to claim 3, characterized in that, The macroporous adsorption resin is macroporous adsorption resin D101.
6. The application according to claim 3, characterized in that, The target fraction was purified using gel Sepheedex LH-20 column chromatography and semi-preparative HPLC.