Pharmaceutical composition for preventing and treating lung cancer

Abstract

The invention relates to a pharmaceutical composition for preventing and treating lung cancer, and belongs to the technical field of natural pharmaceutical compositions. In order to solve the problem that the risk of drug resistance exists when a single component of icariin is used for preventing and treating cancers, the invention provides a pharmaceutical composition for preventing and treating lung cancers, which comprises a component icariin derived from herba epimedii and a component daphnetin derived from stellera chamaejasme. The icariin and daphnoretin are compounded for preventing and treating lung cancer, and the pharmaceutical composition has the advantages of multiple components, multiple targets, small adverse reaction and synergistic interaction after compatibility. In-vitro experiments prove that through compatibility of icariin and daphnetin, proliferation, migration and invasion of lung cancer cell lines A549 and Lewis cells can be inhibited, apoptosis of the two cells is promoted, and the effect is better than that of a single icariin component. The possibility is provided for application of the icariin and daphnetin composition in lung cancer treatment, and clinical application of the icariin and daphnetin composition to lung cancer can be further developed on the basis of the anticancer effect of the composition.

Description

Technical Field

[0001] The invention belongs to the technical field of natural medicine compositions, and in particular relates to a medicine composition for preventing and treating lung cancer. Background Art

[0002] Epimedium is an important medicine for warming yang and tonifying the kidney. It is warm in nature, pungent and sweet in taste, and enters the kidney and liver meridians. By warming and tonifying kidney yang, epimedium can significantly improve symptoms such as soreness of waist and knees, fear of cold limbs, frequent urination at night, impotence and premature ejaculation caused by kidney deficiency. In recent years, significant progress has been made in the development of effective ingredients of epimedium, and its effective ingredients mainly include flavonoids, polysaccharides, lignans, alkaloids, etc. Among them, flavonoids are the most abundant and widely used effective ingredients in epimedium, among which icariin (ICA), icariin, and total flavonoids of epimedium are natural medicinal ingredients with anti-cancer potential. As a first-line treatment drug, icariin has shown significant efficacy in the treatment of advanced hepatocellular carcinoma. Clinical trial results show that icariin can significantly improve the overall survival rate of patients with good safety. Studies have shown that icariin can inhibit neutrophil infiltration and the synthesis of neutrophil extracellular traps (NETs), thereby significantly reducing the spread and progression of urothelial carcinoma.

[0003] Although icariin has certain efficacy and safety when used as a single ingredient for anticancer treatment, it still has some potential disadvantages and limitations. Long-term use of icariin as a single anticancer drug may induce drug resistance in tumor cells. Tumor cells may bypass the anticancer mechanism of icariin through gene mutation or signal pathway rearrangement, resulting in drug failure. Moreover, the occurrence and development of cancer often involve multiple complex signal pathways and abnormal gene expression. Therefore, drugs with a single target may not be able to fully inhibit the growth and spread of tumors. Therefore, the combination of icariin with other natural active ingredients to improve the anti-cancer effect is a potential anti-cancer strategy.

[0004] Since there are complex interactions between different natural ingredients, which may lead to changes in drug effects or even adverse reactions, there are still many technical difficulties and challenges in screening out natural active ingredients that have synergistic effects with icariin and determining the optimal ratio between them. Summary of the invention

[0005] In order to solve the problem of drug resistance risk when a single ingredient of icariin is used for preventing and treating cancer, the present invention provides a pharmaceutical composition for preventing and treating lung cancer.

[0006] The technical solution of the present invention: A pharmaceutical composition for preventing and treating lung cancer, comprising components derived from Epimedium and components derived from Stellera chamaejasme.

[0007] Furthermore, the component derived from Epimedium is icariin, and the component derived from Stellera chamaejasme is daphnetin.

[0008] Furthermore, the molar ratio of icariin to daphnetoside is 1:3~3:1.

[0009] Furthermore, the molar ratio of icariin to daphnetoside is 1:1.

[0010] A use of a pharmaceutical composition for preventing and treating lung cancer in preparing a medicine for preventing and treating lung cancer, wherein the pharmaceutical composition for preventing and treating lung cancer comprises a component derived from epimedium and a component derived from stellera chamaejasme.

[0011] Furthermore, the component derived from Epimedium is icariin, and the component derived from Stellera chamaejasme is daphnetin.

[0012] Furthermore, the molar ratio of icariin to daphnetoside is 1:3~3:1.

[0013] Furthermore, the molar ratio of icariin to daphnetoside is 1:1.

[0014] Furthermore, the drug for preventing and treating lung cancer is in an oral dosage form or a parenteral dosage form, and also includes a pharmaceutically acceptable carrier or excipient.

[0015] Furthermore, the oral dosage form is tablets, capsules, soft capsules, oral liquids, syrups, granules, pills, orodisintegrating tablets, sustained-release tablets, sustained-release capsules, controlled-release tablets or controlled-release capsules; the parenteral dosage form is water injection, lyophilized powder injection, sterile powder injection or infusion.

[0016] Beneficial effects of the present invention: The present invention uses icariin and daphnetin in combination for the prevention and treatment of lung cancer, which has the advantages of multiple components, multiple targets, few adverse reactions, and synergistic effects after combination. In vitro experiments have shown that the combination of icariin and daphnetin can inhibit the proliferation, migration and invasion of lung cancer cell lines A549 and Lewis cells, promote apoptosis of the two cells, and the effect is better than that of a single component of icariin. This provides a possibility for the application of the combination of icariin and daphnetin in the treatment of lung cancer. Based on the anti-cancer effect of the combination, its clinical application for lung cancer can be further developed. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a comparison chart of the OD values ​​of A549 cells in each group in the CCK8 experiment of Example 6; Figure 2 This is a comparison chart of the OD values ​​of Lewis cells in each group in the CCK8 experiment in Example 6; Figure 3These are crystal violet staining photos of A549 cells in each group in the cloning experiment of Example 6; Figure 4 This is a comparison chart of the number of clones of A549 cells in each group in the cloning experiment of Example 6; Figure 5 These are crystal violet staining photos of Lewis cells in each group in the cloning experiment of Example 6; Figure 6 This is a comparison chart of the number of Lewis cell clones in each group in the cloning experiment of Example 6; Figure 7 The scatter plots of A549 cells in each group at 24h and 48h in flow cytometry of Example 6 are shown; Figure 8 This is a comparison chart of the apoptosis rates of A549 cells in each group at 24h and 48h in flow cytometry in Example 6; Fig. 9 The scatter plots of Lewis cells in each group at 24h and 48h in flow cytometry of Example 6; Fig.10 This is a comparison chart of the apoptosis rates of Lewis cells in each group at 24h and 48h in flow cytometry in Example 6; Fig.11 The photos of the wound healing of A549 cells in each group in the scratch experiment of Example 6; Fig.12 This is a comparison chart of the cell migration rates of A549 cells in each group in the scratch experiment of Example 6; Fig.13 The photos of the Lewis cell scratch healing in each group in the scratch experiment of Example 6; Fig.14 This is a comparison chart of the cell migration rates of Lewis cells in each group in the scratch experiment of Example 6; Fig.15 These are the staining micrographs of A549 cells in each group in the Transwell experiment of Example 6; Fig.16 This is a comparison chart of the number of cell invasion of A549 cells in each group in the Transwell experiment of Example 6; Fig.17 These are the staining micrographs of Lewis cells in each group in the Transwell experiment of Example 6; Fig.18 This is a comparison chart of the cell invasion numbers of Lewis cells in each group in the Transwell experiment of Example 6; Fig.19 This is a flow cytometry comparison diagram of A549 cells in each group in the flow cytometry cycle experiment of Example 6; Fig. 20This is a comparison diagram of the cell cycle distribution of A549 cells in each group in the flow cell cycle experiment of Example 6; Fig.21 This is a flow cytometry cycle comparison diagram of Lewis cells in each group in the flow cytometry cycle experiment of Example 6; Fig. 22 This is a comparison diagram of the cell cycle distribution of each group of Lewis cells in the flow cytometry cycle experiment in Example 6. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described below in conjunction with the embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the protection scope of the present invention. The process equipment or devices not specifically noted in the following embodiments are all conventional equipment or devices in the art. If not specifically specified, the raw materials used in the embodiments of the present invention can be obtained commercially; if not specifically specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0019] Example 1 This embodiment provides a pharmaceutical composition for preventing and treating lung cancer, which is composed of icariin and daphnetin in a molar ratio of 1:1.

[0020] Example 2 This embodiment provides a pharmaceutical composition for preventing and treating lung cancer, which is composed of icariin and daphnetin in a molar ratio of 1:2.

[0021] Example 3 This embodiment provides a pharmaceutical composition for preventing and treating lung cancer, which is composed of icariin and daphnetin in a molar ratio of 2:1.

[0022] Example 4 This embodiment provides a pharmaceutical composition for preventing and treating lung cancer, which is composed of icariin and daphnetin in a molar ratio of 1:3.

[0023] Example 5 This embodiment provides a pharmaceutical composition for preventing and treating lung cancer, which is composed of icariin and daphnetin in a molar ratio of 3:1.

[0024] Example 6 This example verifies the effect of the pharmaceutical composition for preventing and treating lung cancer provided in Example 1 on inhibiting the proliferation of lung cancer cells through in vitro experiments.

[0025] 1. Research subjects: A549 and Lewis lung cancer cell lines 2. Experimental Materials Icariin and daphnetin were purchased from Shanghai Yuanye Biotechnology Co., Ltd., CCK8 reagent, crystal violet dye, and 1640 culture medium were purchased from White Shark Biotechnology Co., Ltd., and cell apoptosis kit and cell cycle kit were purchased from Aibixin Biotechnology Co., Ltd.

[0026] 3. Instruments Microplate reader (Diatek, DR-200Bs), flow cytometer (Luminex, Guava easyCyte), microscope (Thermo Fisher Scientific, EVOS), centrifuge (Shanghai Anting Scientific Instrument Factory, TGL-16c).

[0027] 4. Cell culture methods A549 and Lewis cells were cultured in 1640 medium (10% fetal bovine serum, 1% double antibody), 5% CO2, and 37°C cell culture incubator.

[0028] (V) Detection of lung cancer cell proliferation using CCK8 method The experiment was divided into a blank group (CON), an icariin group (ICR), and an icariin plus daphnetin group (ICR+RXS). A549 and Lewis cells were cultured at 3×10 3 Each well was inoculated into a 96-well plate and administered in suspension. The concentration of the ICR and ICR+RXS groups was 50 μM. The molar ratio of icariin to daphnetin in the ICR+RXS group was 1:1. CCK8 reagent was added to each well 24 h, 48 h, and 72 h after administration. After incubation for 1.5 h, the absorbance at 450 nm was detected using an enzyme reader. The results are shown in Figure 1 and Figure 2 shown.

[0029] Figure 1 and Figure 2 The results showed that compared with the blank control group, the OD values ​​of the ICR group and the ICR+RXS group were reduced, which indicated that icariin and the combination of icariin and daphnetin could inhibit the proliferation of A549 and Lewis lung cancer cells, and at 48 h, the inhibitory effect of the combination of icariin and daphnetin was better than that of the single component of icariin.

[0030] (VI) Cloning experiment to detect lung cancer cell proliferation A549 and Lewis cells were seeded into 6-well plates at 500 cells / well and divided into ICR group and ICR+RXS group. The drugs were suspended and administered in the same way as in (V). The medium was changed every two days. After significant clones appeared in both groups, the cells were fixed with 4% paraformaldehyde for 15 minutes and stained with crystal violet solution for 30 minutes. After taking pictures, the images were analyzed using ImageJ software. The results are shown in the figure. Figure 3-Figure 6 shown.

[0031] Figure 3-Figure 6 The results showed that compared with the administration of icariin alone, the number of clones was significantly reduced after the combined administration of icariin and daphnetin, indicating that the combined administration of icariin and daphnetin had a significant proliferation inhibitory effect on A549 and Lewis lung cancer cells, and the effect was better than that of icariin alone.

[0032] (VII) Detection of apoptosis of lung cancer cells by flow cytometry A549 and Lewis cells were cultured at 1×10 5 The cells were inoculated into 6-well plates and divided into ICR group and ICR+RXS group. The drugs were administered in the same way as in (V). After 48 h of administration, the cells were digested and centrifuged, and stained with Annexin V-FITC / PI double staining method. Flow cytometry was used to detect cell apoptosis in the two groups. The results are shown in Table 5. Figure 7-Figure 10 shown.

[0033] Figure 7-Figure 10 The results showed that compared with the administration of icariin alone, the proportions of the lower right quadrant (early apoptosis) and upper right quadrant (late apoptosis / necrosis) in the group administered with the combination of icariin and daphnetin increased significantly, while the proportion of the lower left quadrant decreased, indicating that the combination of icariin and daphnetin promoted the apoptosis of A549 and Lewis cells, and the effect was better than that of icariin alone.

[0034] 8. Scratch assay to detect the migration of lung cancer cells A549 and Lewis cells were cultured at 5×10 4 Cells were seeded into 6-well plates and divided into ICR group and ICR+RXS group. The cells covered the bottom of the well and then scratched. Each group was given drugs at the same time. The drug administration method was the same as (V). The migration of cells was observed under a microscope 24h, 48h, and 72h after drug administration. The results are shown in Figure 5. Figure 11-Figure 14 shown.

[0035] Figure 11-Figure 14 As shown, compared with the administration of icariin alone, the migration speed of A549 and Lewis lung cancer cells in the combination administration group of icariin and daphnetin was slower and the cell migration rate was lower, indicating that the inhibitory effect of the combination of icariin and daphnetin on the migration of A549 and Lewis lung cancer cells is better than that of icariin alone.

[0036] 9. Transwell assay to detect lung cancer cell invasion A549 and Lewis cells were cultured at 5×10 4Cells were inoculated into the Transwell chamber containing matrix gel and divided into ICR group and ICR+RXS group. The drugs were suspended and administered in the same way as (V). After 48 hours of action, they were fixed with 4% paraformaldehyde and stained with crystal violet dye. After washing with clean water, the cells were photographed under a microscope to observe the invasion of the two groups of cells. The results are shown in Figure 15-18 shown.

[0037] Figure 15-18 The results showed that compared with the administration of icariin alone, the number of cells passing through the Transwell membrane in the group administered with the combination of icariin and daphneline was less, indicating that the inhibitory effect of the combination of icariin and daphneline on the invasion of A549 and Lewis lung cancer cells was better than that of icariin alone.

[0038] 10. Flow cytometry detection of lung cancer cell cycle A549 and Lewis cells were cultured at 1×10 5 Cells were inoculated into 6-well plates and divided into ICR group and ICR+RXS group. The drugs were administered by suspension. The administration method was the same as (V). 48 hours after administration, the cells were digested and centrifuged and fixed with 75% alcohol at 4°C overnight. The cells were stained with PI the next day. The cell cycle of the two groups was detected by flow cytometry. The results are shown in Figure 19-22 shown.

[0039] Figure 19-22 The results showed that compared with the administration of icariin alone, the proportion of G1 phase cells increased, and the proportion of S phase and G2 / M phase cells decreased in the group administered with the combination of icariin and daphneline. This indicates that the combined administration of icariin and daphneline can make A549 and Lewis lung cancer cells stay in the G1 phase and unable to enter the next proliferation stage, and has a better regulatory effect on the cell proliferation rate.

Claims

1. A pharmaceutical composition for preventing and treating lung cancer, characterized in that: The pharmaceutical composition comprises components derived from epimedium and components derived from stellera chamaejasme.

2. A pharmaceutical composition for preventing and treating lung cancer according to claim 1, characterized in that: The component derived from Epimedium is icariin, and the component derived from Stellera chamaejasme is daphnetin.

3. A pharmaceutical composition for preventing and treating lung cancer according to claim 2, characterized in that: The molar ratio of icariin to daphnetin is 1:3-3:

1.

4. A pharmaceutical composition for preventing and treating lung cancer according to claim 3, characterized in that: The molar ratio of icariin to daphnetoside is 1:

1.

5. Use of a pharmaceutical composition for preventing and treating lung cancer in the preparation of a drug for preventing and treating lung cancer, characterized in that: The pharmaceutical composition for preventing and treating lung cancer comprises components derived from epimedium and components derived from stellera chamaejasme.

6. Use of a pharmaceutical composition for preventing and treating lung cancer according to claim 5 in preparing a drug for preventing and treating lung cancer, characterized in that: The component derived from Epimedium is icariin, and the component derived from Stellera chamaejasme is daphnetin.

7. Use of a pharmaceutical composition for preventing and treating lung cancer according to claim 6 in preparing a drug for preventing and treating lung cancer, characterized in that: The molar ratio of icariin to daphnetin is 1:3-3:

1.

8. Use of a pharmaceutical composition for preventing and treating lung cancer according to claim 7 in preparing a drug for preventing and treating lung cancer, characterized in that: The molar ratio of icariin to daphnetin is 1:

1.

9. Use of a pharmaceutical composition for preventing and treating lung cancer according to claim 8 in preparing a drug for preventing and treating lung cancer, characterized in that: The medicine for preventing and treating lung cancer is in an oral dosage form or a parenteral dosage form, and also includes a pharmaceutically acceptable carrier or excipient.

10. Use of a pharmaceutical composition for preventing and treating lung cancer according to claim 9 in preparing a drug for preventing and treating lung cancer, characterized in that: The oral dosage form is tablets, capsules, soft capsules, oral liquids, syrups, granules, dripping pills, orodisintegrating tablets, sustained-release tablets, sustained-release capsules, controlled-release tablets or controlled-release capsules; the parenteral dosage form is water injection, lyophilized powder injection, sterile powder injection or infusion.

Images