Application of isofelianol in the preparation of drugs for treating breast cancer

Abstract

The present invention discloses the use of isoflavone glycoside in the preparation of a drug for treating breast cancer. Isoflavone glycoside significantly inhibits the metastasis of MDA-MB-231 breast cancer cells. Wound repair experiments demonstrate the drug's efficiency in inhibiting tumor cell migration, and Transwell chamber invasion experiments demonstrate its effectiveness in inhibiting tumor cell invasion. Therefore, isoflavone glycoside can be used to prepare a drug for treating breast cancer metastasis.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to an application of isofelianol in the preparation of a drug for treating breast cancer. Background Art

[0002] Forsythia suspensa is one of the commonly used medicinal materials. It was first recorded in Shennong's Herbal Classic and is also included in the 2020 edition of the Chinese Pharmacopoeia. It is a plant of the Oleaceae family. Forsythia suspensa The dried fruit of Thunb. Vahl is harvested in autumn when it is still green and ripe, then decontaminated, steamed, and sun-dried. It is commonly known as "Qingqiao." The fruit is harvested when fully ripe, sun-dried, and decontaminated. It is commonly known as "Laoqiao." It has the effects of clearing heat and detoxifying, reducing swelling and dispersing stagnation, and dispersing wind-heat. It contains chemical components such as lignans, phenylethanoid glycosides, flavonoids, terpenes, volatile oils, and phenolic acids, and possesses anti-inflammatory, antibacterial, antiviral, and antioxidant activities.

[0003] Forsythia has a long history of use as a medicine. Phenylethanoid glycosides are its main active ingredients. Isoforsythiaside (IFA) is a phenylethanoid glycoside reported in 2012. Its structural formula is as follows:

[0004]

[0005] Isoforsythiaside has significant antioxidant and antibacterial effects and can be used to treat Alzheimer's disease. However, its anti-tumor activity has not yet been reported. Summary of the Invention

[0006] Purpose of the Invention: This invention addresses the shortcomings of existing technologies and provides a method for preparing a drug for treating breast cancer. Experiments have shown that isofysiaside significantly inhibits the metastasis of MDA-MB-231 breast cancer cells and, therefore, can be used to prepare a drug for treating breast cancer metastasis.

[0007] The experimental study of the present invention found that: Forsythiaside can significantly inhibit the metastasis of breast cancer. The scratch repair experiment proved the efficiency of the drug in inhibiting the migration of tumor cells. Compared with the solvent group, as the concentration of Forsythiaside increased, the scratch healing ability of MDA-MB-231 cells decreased significantly. This shows that Forsythiaside can significantly inhibit the migration ability of breast cancer MDA-MB-231 cells and is dose-dependent. The Transwell chamber invasion experiment proved the efficiency of the drug in inhibiting the invasion of tumor cells. As the concentration of Forsythiaside increased, the ability of MDA-MB-231 cells to pass through the polycarbonate bottom membrane of the Transwell chamber gradually decreased, weakening the ability of cells to migrate; in the Transwell invasion experiment, Matrigel, which simulates the extracellular matrix, was added to the chamber. The experimental results showed that the ability of MDA-MB-231 cells to degrade the Matrigel basement membrane decreased with the increase of Forsythiaside dose, reducing the ability of cells to invade. A lung metastasis model was established in BALB / c nude mice inoculated with MDA-MB-231 cells via the tail vein. Compared with the model group, the number of metastatic lesions in the isofysiaside-treated group was significantly reduced, and the diameter of most nodules was reduced. This suggests that isofysiaside has an inhibitory effect on breast cancer metastasis.

[0008] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0009] The present invention provides an application of isofysiaside in the preparation of a drug for treating breast cancer.

[0010] The isofysiaside can be used as the sole active ingredient to prepare drugs for treating breast cancer.

[0011] The isofysiaside can be used in combination with other drugs to prepare drugs for treating breast cancer.

[0012] The breast cancer therapeutic drug comprises isoflavone glycoside and a pharmaceutically acceptable carrier or excipient.

[0013] The excipients include one or more of a wetting agent, a preservative, an antioxidant, an emulsifier, a solubilizer, a disintegrant, a binder or a diluent.

[0014] The wetting agent is selected from at least one of water and ethanol.

[0015] The preservative is selected from at least one of benzoic acid and its salts, sorbic acid and its salts, or parabens.

[0016] The antioxidant is selected from at least one of sulfite, ascorbic acid, bisulfite, gallic acid and lipids thereof.

[0017] The emulsifier is selected from at least one of Tweens, Spans, pectin, agar, glycerol fatty acid esters, sodium alginate or silicon dioxide.

[0018] The solubilizing agent is selected from one of Tweens, polyoxyethylene fatty alcohol ethers, sulfates or sulfonates.

[0019] The disintegrant is selected from at least one of starch, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose or cross-linked polyvinyl pyrrolidone.

[0020] The binder is selected from at least one of starch slurry, sodium carboxymethyl cellulose, povidone, hydroxypropyl cellulose, methyl cellulose or ethyl cellulose.

[0021] The diluent is selected from at least one of starches, sugars, celluloses or inorganic salts.

[0022] The dosage form of the breast cancer therapeutic drug is tablets, capsules, granules, suspensions, oral solutions, injections or infusions.

[0023] The medicaments of the present invention can be administered in various known ways, such as oral administration, injection, and the like. The medicaments of the present invention can be administered alone or in combination with other drugs. Oral compositions can be in any orally acceptable dosage form, including but not limited to tablets, capsules, granules, suspensions, and oral solutions.

[0024] Sterile injectable compositions can be formulated using suitable dispersing agents or wetting agents and suspending agents according to techniques known in the art. Pharmaceutically acceptable carriers and solvents that can be used include water, sodium chloride solution, and the like.

[0025] The drug of the present invention can be prepared into common preparations, and can also be prepared into sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

[0026] The actual dosage level of the active ingredient in the medicament of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends on a variety of factors, including the route of administration, time of administration, rate of excretion, duration of treatment, other drugs, compounds, and / or materials used in combination with isofelianidin, the age, sex, weight, general health, and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0027] The isofysiaside significantly inhibits the metastatic ability of breast cancer MDA-MB-231 cells.

[0028] The isofysiaside significantly inhibits the migration and invasion ability of breast cancer cells MDA-MB-231. Beneficial effects

[0029] The isofusin of the present invention can be used as an active ingredient in drugs that inhibit breast cancer cell metastasis, providing a new option for the preparation of drugs that inhibit breast cancer metastasis. The isofusin of the present invention can significantly inhibit the metastasis of MDA-MB-231 breast cancer cells. Therefore, isofusin can be used to prepare drugs for the treatment of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The results of the scratch inhibition experiment of isofusin (IFA) on MDA-MB-231 cells are shown in Figure 2. Figure 1 A: IFA scratch test on MDA-MB-231 cells, scale bar = 100 μm; Figure 1 B is the quantitative results of the scratch test; n = 3, x ± SD. Compared with the solvent group, ** p <0.01 indicates a significant difference. *** p <0.001 was considered to be extremely significant.

[0031] Figure 2 The results of the metastasis and invasion inhibition experiment of isofusin (IFA) on MDA-MB-231 cells are shown in Figure 2. Figure 2 A: IFA Transwell assay on MDA-MB-231 cells, scale bar = 100 μm; Figure 2 B is the quantitative results of migration experiment; Figure 2 C is the quantitative results of invasion assay; n=3, x±SD. Compared with the solvent group, ** p <0.01 indicates a significant difference. *** p <0.001 was considered to be extremely significant.

[0032] Figure 3 The results of the experiment on the inhibition of MDA-MB-231 cell lung metastasis by isofelian glycoside (IFA) in mice; Figure 3 A. Figure 3 B is the lung results and nodule quantification of mice in each group; Figure 3 C is the graph showing the changes in body weight of mice in each group during the treatment period; Figure 3 D is the H&E staining of the lungs of mice in each group, scale bar = 100 μm; n = 6, x ± SD; compared with the Model, * p <0.5 indicates significant difference. ** p <0.01 indicates a significant difference. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0034] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0036] Example 1 Scratch repair experiment

[0037] MDA-MB-231 breast cancer cells (Cell Bank of the Chinese Academy of Sciences, Shanghai) in the logarithmic growth phase were seeded in 6-well plates. The next day, when the cells adhered to the wall and the density reached 90%, a 200 μL sterile pipette tip was used to streak the cells on the surface of the single cell layer.

[0038] After scratching, the cells were washed three times with sterile PBS. MDA-MB-231 cells were treated with 4, 16, and 32 μM isoflavone glycosides (Shanghai Yuanye Biotechnology Co., Ltd.) and an equal volume of PBS. Serum-free medium (Glass Life Science Technology Co., Ltd., Shanghai) was used for this experiment. The cells were incubated in a 37°C, 5% CO2 incubator for 24 h.

[0039] The wound healing of cells was observed and photographed under an inverted microscope at 0 and 24 hours. Image J software was used to calculate the distance and area between the cell scratches and the wound healing rate. The wound healing rate was calculated as (scratch width at 0 hours - scratch width at 24 hours) / scratch width at 0 hours × 100%.

[0040] The experimental results are as follows Figure 1 As shown in the figure, compared with the solvent group, the scratch healing ability of MDA-MB-231 cells decreased significantly with the increase of the concentration of forsythiaside. This indicates that forsythiaside can significantly inhibit the migration ability of breast cancer MDA-MB-231 cells in a dose-dependent manner.

[0041] Example 2 Transwell chamber invasion assay

[0042] Matrigel (Shanghai Nova Pharmaceutical Technology Co., Ltd., Shanghai) was placed on the microporous filter membrane of a Transwell chamber (Thermo Fisher Scientific, Shanghai) and placed in an incubator at 37°C for 1 hour. MDA-MB-231 cells in the logarithmic growth phase were taken and diluted to a concentration of 5×10 6Activated chambers were placed in a 24-well plate, and 400 μL of the cell suspension containing forsythiaside (4, 16, and 32 μM) was slowly and evenly added dropwise to the upper chamber of the Transwell. 600 μL of culture medium containing 30% serum (Glass Life Science Technology Co., Ltd., Shanghai) was added to the lower chamber and incubated in a 37°C incubator for 24 hours. After 24 hours, the chambers were removed, and unmigrated cells in the upper chamber were gently wiped away with a cotton swab. The cells were fixed with 4% paraformaldehyde for 20 minutes and stained with 0.5% crystal violet for 30 minutes. The cells were then rinsed with PBS to remove any excess color and observed under a microscope. Five fields of view (top, bottom, left, right, and center) were randomly selected, and the average number of migrated cells was calculated.

[0043] like Figure 2 As shown, as the concentration of forsythiaside A increases, the ability of MDA-MB-231 cells to penetrate the polycarbonate basement membrane of the Transwell chamber gradually decreases, weakening the cell migration ability. In the Transwell invasion assay, Matrigel, a simulated extracellular matrix, was added to the chamber. The experimental results showed that the ability of MDA-MB-231 cells to degrade the Matrigel basement membrane decreased with increasing doses of forsythiaside A, thereby reducing the cell invasion ability. These results indicate that forsythiaside A inhibits the migration and invasion ability of breast cancer MDA-MB-231 cells in a dose-dependent manner.

[0044] Example 3 Construction of a lung metastasis model in Balb / c nude mice by intravenous inoculation of MDA-MB-231 cells

[0045] MDA-MB-231 cells in the logarithmic growth phase were digested with digestive enzymes and washed twice with PBS to remove residual serum. Cells were counted using a cell counting plate and diluted with pre-cooled PBS to a cell concentration of 1×10 7 The cell suspension was placed on ice. A breast cancer lung metastasis model was established by tail vein injection of breast cancer cells. Female Balb / c nude mice (Nanjing Qinglongshan Animal Breeding Farm, 5-6 weeks old, 18 g) were immobilized using a tail vein clamp. Each mouse was inoculated with 100 µL of MDA-MB-231 cell suspension via the tail vein. The inoculated mice were randomly divided into six groups, each consisting of six mice: blank control group; model group; model group + positive drug group (Taxol, Shanghai Yuanye Biotechnology Co., Ltd., 10 mg / kg); model group + isofelianside (12 mg / kg); model group + isofelianside (25 mg / kg); and model group + isofelianside (50 mg / kg). The blank control and model groups were injected with an equal volume of PBS buffer. Following tail vein inoculation, drugs were administered intraperitoneally every three days. Body weights of the mice were measured and recorded for a total of 33 days. Breast cancer cell metastasis was observed in vivo, and lung metastases were removed for further pathological examination.

[0046] like Figure 3 As shown in Figures A and 3B, images of breast cancer metastatic nodules in the lungs of mice were collected. Compared with the model group, the number of metastatic lesions in the various forsythiaside-treated groups was significantly reduced, and the diameter of most nodules was reduced. During treatment, changes in mouse weight were observed. Compared with the normal group, there was no significant change in mouse weight after forsythiaside-treated mice ( Figure 3 C). Mouse lung tissue was collected and H&E staining was performed for pathological index detection. Results ( Figure 3 D) shows that lung tissue from mice in the control group showed clear alveolar structure, no degeneration or shedding of bronchial epithelial cells, and no clear tumor tissue. In the model group, tumor cells were distributed diffusely in the lung tissue, with unclear boundaries between tumor cells. In the lung tissues of mice treated with various forsythiasides, nuclear atypicality and slightly vacuolated chromatin were observed, and tumor nodules were visible, which were fewer than in the model group. These results demonstrate that various forsythiasides significantly inhibit the lung metastasis of MDA-MB-231 breast cancer cells in vivo.

[0047] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. The use of isofysiaside in the preparation of an anti-triple-negative breast cancer metastasis drug, characterized in that: The structural formula of the isofysiaside is: 。 2. The use according to claim 1, characterized in that The isofysiaside is used as the sole active ingredient in preparing a drug for resisting triple-negative breast cancer metastasis.

3. The use according to claim 1, characterized in that The isofysiaside is used in combination with other drugs to prepare drugs for resisting triple-negative breast cancer metastasis.

4. The use according to any one of claims 1 to 3, characterized in that The anti-triple-negative breast cancer metastasis drug comprises isoflavone glycoside and pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that The excipients include one or more of a wetting agent, a preservative, an antioxidant, an emulsifier, a solubilizer, a disintegrant, a binder or a diluent.

6. The use according to claim 4, characterized in that The dosage forms of the anti-triple-negative breast cancer metastasis drug are tablets, capsules, granules, suspensions, oral solutions and injections.

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