CN110623963B

CN110623963B - Pharmaceutical composition for treating ovarian cancer and application thereof

Info

Publication number: CN110623963B
Application number: CN201911023749.7A
Authority: CN
Inventors: 常聪; 郑国华; 刘祖浩
Original assignee: Hubei College of Chinese Medicine
Current assignee: Hubei College of Chinese Medicine
Priority date: 2019-10-25
Filing date: 2019-10-25
Publication date: 2022-09-30
Anticipated expiration: 2039-10-25
Also published as: CN110623963A

Abstract

The invention relates to the technical field of ovarian cancer treatment, and discloses a pharmaceutical composition for treating ovarian cancer and application thereof. The pharmaceutical composition contains kaempferol and tripterine as active ingredients. In the invention, by combining kaempferol and tripterine, kaempferol can remarkably improve the anti-tumor curative effect of tripterine by blocking cell cycle, inducing apoptosis, inhibiting cell proliferation and migration and the like, and shows remarkable synergistic effect in inhibiting the growth of ovarian cancer cells.

Description

Pharmaceutical composition for treating ovarian cancer and application thereof

Technical Field

The invention relates to the technical field of ovarian cancer treatment, and particularly relates to a pharmaceutical composition for treating ovarian cancer and application thereof.

Background

Ovarian cancer is the most common malignancy in women over the age of 40, except for breast cancer. In recent years, the incidence of ovarian cancer has increased year by year. Mortality rates continue to rise as most patients are diagnosed at a middle or late stage. At present, the main treatment method of ovarian cancer clinically is to combine surgery and chemotherapy. The combination of platinum and taxol is a standard chemotherapy scheme for ovarian cancer, and although the survival time of a patient can be prolonged, the recurrence rate is high, the drug resistance problem is serious, and the 5-year survival rate is about 30%. Therefore, it is urgent to find a more effective treatment method for reversing the drug resistance of ovarian cancer.

Celastrol (CEL) is a pentacyclic triterpene monomer compound extracted from root bark of plant of Celastraceae, such as Tripterygium wilfordii hook F.H. and Celastrus. CEL has wide pharmacological activity, and can play a role in resisting inflammation, oxidation, rheumatism and tumors by regulating various molecular targets such as TNF-alpha, NF-kappa B, COX-2, VEGF, Akt and the like. As a natural antitumor drug, tripterine can inhibit the growth of multiple tumor cells such as ovarian cancer, prostatic cancer, lung cancer, liver cancer, leukemia, glioma, etc. However, studies have shown that celastrol may produce various toxic and side effects, such as heart toxicity, liver toxicity, neurotoxicity and blood toxicity, when used for a long time. The combination of the medicines with different action mechanisms can reduce the dosage, enhance the curative effect and relieve the toxic and side effect, thereby providing a more effective scheme for treating the ovarian cancer.

Kaempferol (Kaempferol, KAE) is a flavonoid, mainly derived from the rhizome of kaempferia galanga of the family zingiberaceae, and is also found in a wide variety of fruits and vegetables. The research shows that kaempferol can inhibit the proliferation and differentiation of tumor cell, regulate programmed apoptosis of tumor cell, block tumor cell period, and inhibit angiogenesis and cancer cell metastasis in tumor part.

At present, the research on the combined medication scheme of tripterine and kaempferol as the ovarian cancer resistance has not been reported.

Disclosure of Invention

The invention aims to provide kaempferol combined with tripterine for treating ovarian cancer, and particularly provides a pharmaceutical composition for treating ovarian cancer and application thereof.

The inventor of the invention discovers through research that by combining kaempferol and tripterine, kaempferol can remarkably improve the anti-tumor curative effect of the tripterine through the effects of blocking cell cycle, inducing apoptosis, inhibiting cell proliferation and migration and the like, and shows remarkable synergistic effect in inhibiting the growth of ovarian cancer cells, thereby completing the invention.

The invention provides a pharmaceutical composition for treating ovarian cancer, which contains kaempferol and tripterine as active components.

Optionally, the weight ratio of kaempferol to tripterine is 1-50: 1, preferably 8 to 40: 1.

optionally, the pharmaceutical composition is in the form of powder, tablet, pill, capsule, syrup, emulsion, suspension or injection.

The invention also provides application of the pharmaceutical composition in preparing a medicament for preventing and treating ovarian cancer.

Optionally, the kaempferol and tripterine have synergistic inhibitory effect on tumor cells.

Optionally, when the concentration of kaempferol is 5-20 μ g/mL, the tumor cells are synergistically increased gradually with the increase of the concentration of tripterine.

Optionally, when the concentration of tripterine is 1-4 μ g/mL, the inhibition rate of tumor cell growth gradually increases with the increase of kaempferol concentration.

The invention has the advantages and effects that: in-vitro MTT (methyl thiazolyl tetrazolium) method experiments prove that the pharmaceutical composition can obviously inhibit the growth of A2780 ovarian cancer cells, and almost all combined CDI (critical dimension) values are equal to or less than 0.7, which shows that the synergistic effect of the two drugs is very obvious. Cell cycle experimental results show that when the concentration of the tripterine is 0.6 mu G/mL, the concentration range of kaempferol is 5-20 mu G/mL, G can be reduced ₀ /G ₁ Proportion of cells in phase, arrest of cells at G ₂ the/M phase, which is the inhibition of cell proliferation by interfering with the cell cycle. The apoptosis experiment result shows that when the concentration of the tripterine is 0.6 mug/mL, the kaempferol can induce apoptosis in the concentration range of 5-20 mug/mL and is dose-dependent. Migration experiment results show that when the concentration of the tripterine is 0.2 mug/mL, the kaempferol can inhibit the migration of cells within the concentration range of 1-8 mug/mL and is dose-dependent, and the transfer capacity of tumor cells is reduced. The results show that the combination of kaempferol and tripterine can improve the curative effect of the tripterine on ovarian cancer cells and reduce the dose of the tripterine, thereby reducing the toxic and side effects of the tripterine and having wide application prospect.

Drawings

FIG. 1 is a graph showing the effect of kaempferol and tripterine combination on the survival rate of A2780 ovarian cancer cells;

FIG. 2 is a graph showing the effect of kaempferol and tripterine composition on cell cycle distribution of A2780 ovarian cancer cells;

FIG. 3 is a graph showing the effect of kaempferol and tripterine combination on apoptosis of A2780 ovarian cancer cells;

FIG. 4 is a graph showing the effect of kaempferol and tripterine combination on migration of A2780 ovarian cancer cells.

Detailed Description

The following describes the embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For numerical ranges, each range between its endpoints and individual point values, and each individual point value can be combined with each other to give one or more new numerical ranges, and such numerical ranges should be construed as specifically disclosed herein.

The pharmaceutical composition for treating ovarian cancer of the present invention contains kaempferol and tripterine as active ingredients.

In the pharmaceutical composition, the weight ratio of kaempferol to celastrol may be 1-50: 1, preferably 8 to 40: 1.

the pharmaceutical composition can also contain pharmaceutically acceptable auxiliary materials. The pharmaceutically acceptable excipients may be of conventional choice in the art. In a specific embodiment, the pharmaceutically acceptable excipient is one or more selected from diluents, disintegrants, binders and lubricants. The diluent may be, for example, microcrystalline cellulose. The disintegrant may be, for example, at least one of sodium starch croscarmellose, sodium starch glycolate and sodium croscarmellose. The binder may be, for example, polyvinylpyrrolidone having a molecular weight of 5000 to 50000 (the molecular weight herein refers to a number average molecular weight). The lubricant may be, for example, at least one of magnesium stearate, stearic acid, colloidal silica, and talc.

In the pharmaceutical composition, the content of the active ingredient in the pharmaceutical composition may be 1 to 100. mu.g/mL, preferably 5 to 25. mu.g/mL.

The pharmaceutical composition according to the present invention may be in various forms conventional in the art, and specifically, may be, for example, a powder, a tablet, a pill, a capsule, a syrup, an emulsion, a suspension or an injection.

The present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited thereto.

Example 1

The preparation of the anti-ovarian pharmaceutical composition comprises the following steps:

the active components of the pharmaceutical composition are kaempferol and tripterine, wherein the weight ratio of kaempferol to tripterine is 10: 1, preparing the two medicaments into solutions respectively, and then mixing the solutions fully and uniformly.

Example 2

The application of the anti-ovarian cancer pharmaceutical composition comprises the following steps:

(1) cytotoxicity assays and drug interaction index assessment

Human ovarian cancer cells A2780 at 1 × 10 ⁵ The density of each cell/well is inoculated into a 96-well plate, incubated for 24 hours, and then the cells are exposed to kaempferol (5-40 mug/mL) and tripterine (0.625-10 mug/mL) at different concentrations for 24 hours. The cells were then incubated with 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) for 4 hours, the formazan crystals formed were dissolved in 150 μ L DMSO, mixed well with a micro-shaker, and the absorbance (OD) was measured at 492nm using a microplate reader and the experiment was repeated three times and averaged. The cell survival rate is equal to the ratio of the OD value of the administered group to the OD value of the blank group, i.e., the cell survival rate is equal to (OD) _{Sample set} /OD _{Control group} ) X 100%. Drawing cell growth chart with the drug concentration as abscissa and the cell survival rate as ordinate, and calculating IC ₅₀ (median inhibition rate), IC ₅₀ The results are shown in table 1, which indicates the drug concentration at which the inhibition of cell growth is 50%.

TABLE 1

(2) Drug interaction index assessment

And (3) analyzing the interaction between kaempferol and tripterine: when kaempferol is used alone, cell viability is shown as a, and when celastrol is used alone, cell viability is shown as B. The two drugs were used in combination, and the cell viability was indicated as AB. The formula for the drug interaction Coefficient (CDI) is CDI ═ AB/(a × B). If CDI >1, the combination of the two drugs is considered to have antagonistic effect; if CDI <1, the combination of the two drugs is considered to have synergistic effect; when the CDI is <0.7, the two drugs are considered to have significant synergy when combined.

In order to evaluate whether the composition of kaempferol and tripterine has synergistic effect on ovarian cancer cells, the MTT method is adopted to detect the cell survival rate of different administration groups. As shown in FIG. 1, tripterine and kaempferol can inhibit the proliferation of A2780 cells with IC ₅₀ 2.3. mu.g/mL and 23.6. mu.g/mL, respectively. When the kaempferol and tripterine composition is used, the cell survival rate of different administration groups is lower than that of one drug used alone. By calculating the CDI values of the drug interaction coefficients, it was found that almost all of the compositions had CDI values<0.7, the kaempferol and tripterine composition can inhibit the proliferation of A2780 cells and has obvious synergistic effect.

(3) Cell cycle experiments

A2780 cells at 1X 10 ⁶ The density of each cell/well is inoculated in a 6-well plate, after 24 hours of culture, a culture medium containing different proportions of kaempferol and tripterine compositions is added, and the culture is carried out in an incubator for 24 hours. The cells were then washed 3 times with PBS, trypsinized for a few minutes, harvested by centrifugation, removed of PBS and fixed in 70% cold ethanol for 24 hours. The fixed cells were washed several times with PBS to remove ethanol, and then stained with RNase A enzyme at a concentration of 10. mu.g/mL and Propidium Iodide (PI) staining solution at 37 ℃ for 30min in the absence of light. After staining was completed, washing with PBS, trypsinizing, and cell cycle distribution was measured with flow cytometry.

As shown in fig. 2, G in the blank group ₀ /G ₁ Period and G ₂ The percentages of the/M phase are 54.05% and 12.38%, respectively, G in the CEL 0.6. mu.g/mL group, CEL 0.6. mu.g/mL + KAE 5. mu.g/mL group, CEL 0.6. mu.g/mL + KAE 10. mu.g/mL group, CEL 0.6. mu.g/mL + KAE 20. mu.g/mL group ₀ /G ₁ The phase percentages are 54.95%, 53.17%, 45.03% and 31.26%, respectively; g ₂ The percentage of the/M phase was 13.48%, 13.52%, 27.16% t and 38.79%, respectively. Compared with the administration of only one medicine of tripterine, the two medicinesThe pharmaceutical composition can reduce G after treatment ₀ /G ₁ Proportion of cells in phase, increase G ₂ The ratio of the phases/M. With increasing kaempferol dosage, G ₂ The ratio of/M phase increased, indicating that kaempferol can interfere with tumor cell cycle distribution, inhibit cell proliferation, and is dose dependent. The inventors speculate that the reason is that kaempferol up-regulates P21 protein which is an important cell cycle regulator and activates Chk2 which is a cycle regulatory point kinase, and down-regulates Cdc25C and Cdc2 protein, thereby blocking the cell cycle at G ₂ And a/M period.

(4) Apoptosis assay

A2780 cells at 1X 10 ⁶ The density of each cell/well is inoculated in a 6-well plate, after 24 hours of culture, a culture medium containing different proportions of kaempferol and tripterine compositions is added, and the culture is continued for 24 hours in an incubator. The cells were then washed 3 times with PBS, trypsinized for a few minutes, harvested by centrifugation, redispersed by the addition of 500. mu.LPBS, and stained with 5. mu.LannexinV-FITC and 5. mu.LPI in the dark for 10 minutes. After staining, the cells were washed with PBS and the mean fluorescence intensity inside the cells was measured by flow cytometry.

The proliferation and differentiation of tumor cells are different from those of normal cells, and the defect or the blockage of apoptosis is often shown, so that the promotion of the apoptosis of the tumor cells is an important strategy for treating tumors. As shown in FIG. 3, the early apoptosis rates in the blank group, CEL 0.6. mu.g/mL + KAE 5. mu.g/mL group, CEL 0.6. mu.g/mL + KAE 10. mu.g/mL group, CEL 0.6. mu.g/mL + KAE 20. mu.g/mL group were 0.01%, 1.85%, 5.06%, 6.31%, and 9.98%, respectively; the late apoptosis rates were 0%, 0.04%, 1.84%, 1.89% and 3.1%, respectively; the total apoptosis rates were 0.01%, 1.89%, 6.90%, 8.20% and 13.08%, respectively. The pharmaceutical composition can increase the rate of early apoptosis, the rate of late apoptosis and the total rate of apoptosis after treatment compared to administration of tripterine alone. With the increase of the kaempferol dosage, the three apoptosis rates are increased, which indicates that the kaempferol can induce the tumor cell to be apoptotic and is in dosage dependence. When kaempferol is 20 mug/mL, the total apoptosis rate after the action of the composition is 6.92 times of that when the tripterine is used alone. The inventors speculate that the reason for this is that kaempferol binds to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), inducing apoptosis in ovarian cancer cells.

(5) Cell scratch test

A2780 cells at 4X 10 ⁶ Inoculating the cells/well in a 6-well plate, culturing until the cell fusion degree reaches 90%, drawing a straight line at the bottom of the plate by using a sterile 200-mu L pipette tip, adding a serum-free culture medium containing kaempferol and tripterine composition in different proportions, and continuously culturing for 48 hours. The scratch distance was measured by taking photographs with an inverted fluorescence microscope at 0, 24h, and 48 h. Uniformly selecting 30 points on each side edge of the scratch, taking the central line of the points to represent the edge of the scratch, measuring the scratch distance, and calculating the cell mobility by using the following formula:

cell mobility (scratch spacing) _0h -scratch spacing _{24h or 48h} ) X 100%/scratch spacing _0h

To assess the effect of kaempferol and tripterine combinations on the migration capacity of ovarian cancer cells, we used the scratch method to determine the rate of migration of a2780 cells from the edge of the scratch. As shown in FIG. 4, the cell mobilities at 24h for the blank group, CEL 0.2. mu.g/mL + KAE 1. mu.g/mL group, CEL 0.2. mu.g/mL + KAE 2. mu.g/mL group, CEL 0.2. mu.g/mL + KAE 8. mu.g/mL group were 14.74%, 10.37%, 9.02%, 5.20%, 1.18%, and 1.03%, respectively; cell mobilities at 48h were 22.16%, 19.74%, 12.31%, 7.56%, 2.35% and 1.19%, respectively. The migration capacity of the cells is reduced after the treatment with the pharmaceutical composition compared with the administration of only one drug, and the reduction range is increased along with the increase of the kaempferol dosage. When the kaempferol concentration is 8 mu g/mL, the cell migration of 24h and 48h is 9.93 percent and 6.03 percent of that of the tripterine alone respectively, which shows that the kaempferol can inhibit the migration capability of tumor cells and is dose-dependent. The inventors speculate that the reason for this is that kaempferol reduces tumor cell migration by inhibiting the activity of Matrix Metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA).

In conclusion, the composition of kaempferol and tripterine shows a remarkable synergistic effect in inhibiting the growth of ovarian cancer cells, and the kaempferol can remarkably improve the curative effect of the tripterine on ovarian cancer cells and reduce the administration dosage of the tripterine by blocking the cell cycle, inducing apoptosis, inhibiting cell proliferation and migration and the like, thereby relieving the toxic and side effects caused by long-term use of the tripterine.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims

1. The application of a pharmaceutical composition in preparing a medicament for treating ovarian cancer is characterized in that the pharmaceutical composition contains kaempferol and tripterine as active components;

the kaempferol and the tripterine have a synergistic inhibition effect on tumor cells;

when the concentration of kaempferol is 5-20 mug/mL, the synergy of the kaempferol and tripterine on tumor cells is gradually increased along with the increase of the concentration of tripterine;

when the concentration of the tripterine is 1-4 mug/mL, the inhibition rate on the growth of tumor cells is gradually increased along with the increase of the kaempferol concentration;

the weight ratio of kaempferol to tripterine is 1-50: 1;

the tumor cells are A2780 ovarian cancer cells.

2. The use of claim 1, wherein the weight ratio of kaempferol to celastrol is 8-40: 1.

3. the use according to claim 1, wherein the pharmaceutical composition is in the form of a powder, tablet, pill, capsule, syrup, emulsion, suspension or injection.