Application of Feilike in the preparation of products for preventing and treating liver cancer

Abstract

The present invention provides the use of Feilike in the preparation of products for the prevention and treatment of liver cancer, belonging to the technical field of liver cancer prevention and treatment. This invention proposes for the first time the use of Feilike in the preparation of products for the prevention and treatment of liver cancer, specifically inhibiting the vitality and proliferation of liver cancer cells, promoting apoptosis of liver cancer cells, and altering the morphology of liver cancer cells, thereby achieving the effective treatment of liver cancer. Furthermore, Feilike is a traditional Chinese medicine already in clinical use that can be directly applied to the human body without the need for clinical safety assessments. It has excellent application prospects and can rapidly increase the market share of this product.

Description

Technical Field

[0001] The invention belongs to the technical field of liver cancer prevention and treatment, and in particular relates to the application of Feilike in preparing products for preventing and treating liver cancer. Background Art

[0002] Feilike is a traditional Chinese medicine whose main ingredients include scutellaria baicalensis, peucedanum chinense, stemona root, gentiana scabra, sycamore root, hedyotis diffusa, and red tube wormwood. Currently, Feilike is available on the market in two dosage forms: Feilike mixture and Feilike capsules. Feilike capsules contain yellowish-brown to brown powder and granules and have a bitter taste. Feilike capsules are effective in relieving cough and asthma, clearing away heat and detoxifying, and promoting qi circulation and expectoration. They are used for cough, asthma, and excessive sputum, as well as for chronic bronchitis with the aforementioned symptoms. Existing technology does not document the technical content related to the treatment or prevention of liver cancer. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a use of Feili Ke in preparing products for preventing and treating liver cancer.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides the use of Feilike in preparing products for preventing and treating liver cancer.

[0006] The present invention also provides the use of Feilike in preparing a product for inhibiting the activity of liver cancer cells.

[0007] The present invention also provides the use of Feilike in preparing a product for inhibiting the proliferation of liver cancer cells.

[0008] The present invention also provides the use of Feilike in preparing a product for promoting apoptosis of liver cancer cells.

[0009] Preferably, the types of products include medicines.

[0010] Preferably, the Feilike includes Feilike mixture and Feilike capsules.

[0011] Preferably, the medicine includes injection, powder, tablet, granule, solution, emulsion, spray, ointment, plaster, patch, suppository or pill.

[0012] The present invention also provides a medicine for preventing and / or treating liver cancer, which comprises Feilike or an effective component of Feilike.

[0013] The present invention also provides a method for preparing the above-mentioned medicine, comprising the following steps: directly preparing the Feilike into a pharmaceutical preparation for preventing and treating liver cancer, or compounding the Feilike with other drugs for preventing and treating liver cancer to prepare a compound pharmaceutical preparation, or combining the active ingredients of the Feilike with a pharmaceutically acceptable carrier to prepare a pharmaceutical preparation for preventing and treating liver cancer.

[0014] Beneficial effects of the present invention:

[0015] This invention proposes for the first time the use of Feilike in the preparation of products for the prevention and treatment of liver cancer. Specifically, it inhibits the vitality and proliferation of liver cancer cells, promotes apoptosis, and alters their morphology, thereby effectively treating liver cancer. Furthermore, Feilike is a clinically approved traditional Chinese medicine that can be directly applied to the human body without the need for clinical safety assessments. This product has excellent application prospects and could rapidly increase its market share. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The effect of different concentrations of Feilike capsule solution on the cell morphology of human liver cancer cells HepG2 and HuH-7;

[0017] Figure 2 Effects of different concentrations of Feilike capsule solution on the cell viability of human liver cancer cells HepG2 and HuH-7. The left figure shows the results of human liver cancer cells HepG2, and the right figure shows the results of human liver cancer cells HuH-7.

[0018] Figure 3 The effect of different concentrations of Feilike capsule solution on the growth of human liver cancer cells HepG2 and HuH-7. The left picture shows the results of human liver cancer cells HepG2, and the right picture shows the results of human liver cancer cells HuH-7.

[0019] Figure 4 The results of the clone formation assay of different concentrations of Feilike capsule solution on human liver cancer cells HepG2 and HuH-7 are shown;

[0020] Figure 5 The results of apoptosis assay of different concentrations of Feilike capsule solution on human liver cancer cells HepG2 and HuH-7 are shown;

[0021] Figure 6 The results of Hoechst 33258 fluorescence detection;

[0022] Figure 7 Results of tumor volume in different groups;

[0023] Figure 8 The figures are the tumor weights of different groups. DETAILED DESCRIPTION

[0024] The present invention provides the use of Feilike in preparing products for preventing and treating liver cancer. The present invention also provides the use of Feilike in preparing products for inhibiting the activity of liver cancer cells, inhibiting the proliferation of liver cancer cells and promoting the apoptosis of liver cancer cells.

[0025] In the present invention, the types of products preferably include medicines. The present invention has no special restrictions on the specific source of Feilike, and conventional commercially available products in this field can be used. In the present invention, the Feilike preferably includes Feilike mixture and Feilike capsules. In the present invention, the liver cancer cells preferably include human liver cancer cells HepG2 and HuH-7. The present invention has no special restrictions on the sources of the human liver cancer cells HepG2 and HuH-7, which can be purchased commercially or frozen in the laboratory. The human liver cancer cells HepG2 and HuH-7 described in the embodiments of the present invention are all retained by the Guizhou Natural Products Research Center.

[0026] In the present invention, the pharmaceutical preparation preferably includes injection, powder, tablet, granule, solution, emulsion, spray, ointment, plaster, patch, suppository or pill. The present invention has no special requirements for the excipients of each dosage form, and any conventional excipient used in the art for preparing the dosage form can be used.

[0027] The present invention also provides a medicine for preventing and / or treating liver cancer, which comprises Feilike or an effective component of Feilike.

[0028] The present invention has no particular limitation on the source of Feili Ke, the dosage form of the pharmaceutical preparation, the excipients, etc. In a specific embodiment of the present invention, the main active ingredients of Feili Ke preferably include Scutellaria baicalensis, Peucedanum chinense, Stemona tuberosum, Gentiana scabra, Phyllanthus chinensis root, Hedyotis diffusa and Herba Lycopodii.

[0029] The present invention also provides a method for preparing the above-mentioned medicine, comprising the following steps: directly preparing the Feilike into a pharmaceutical preparation for preventing and treating liver cancer, or compounding the Feilike with other drugs for preventing and treating liver cancer to prepare a compound pharmaceutical preparation, or combining the active ingredients of the Feilike with a pharmaceutically acceptable carrier to prepare a pharmaceutical preparation for preventing and treating liver cancer.

[0030] In the preparation method of the drug of the present invention, matters not described in detail, such as excipients and how to prepare the drug, are all conventional choices in the art.

[0031] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] In the following examples, unless otherwise specified, all methods are conventional.

[0033] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0034] The experimental materials required in the following examples are prepared as follows

[0035] (1) Preparation of sample solutions: The contents of Feilike capsules (Felike capsules were purchased from Guizhou Jianxing Pharmaceutical Co., Ltd., and the contents refer to the contents obtained after removing the capsule shell) were prepared with normal saline to prepare Feilike capsule solutions with concentrations of 1 mg / mL, 2 mg / mL, and 4 mg / mL, respectively.

[0036] (2) Preparation of MTT solution: Weigh 0.25 g of MTT powder, add 50 mL of sterilized double-distilled water, and dissolve by ultrasonication to a concentration of 5 mg / mL. Sterilize by filtration with a 0.2 μm filter membrane and store at 4°C.

[0037] (3) Cell lines: Human hepatoma cells HepG2 and HuH-7, both of which were stored at the Guizhou Natural Products Research Center. Cells were cultured in DMEM medium containing 10% FBS in a 37°C, 5% CO2 incubator.

[0038] In the following examples, each experiment was repeated three times independently. All data are expressed as mean ± SD. Student's t-test was performed using GraphPad Prism 7. P < 0.05 was considered statistically significant, *P < 0.05, **P < 0.01.

[0039] Example 1

[0040] Cell morphology test

[0041] Cells in logarithmic growth phase were collected, and HepG2 cells and HuH-7 cells were collected at a rate of 3×10 5 Cells were seeded at a density of 100 cells / well in a 6-well plate and cultured overnight. After the cells attached, they were treated with different concentrations of sample solution (1 mg / mL, 2 mg / mL, and 4 mg / mL of Feilike capsule solution). A normal saline solution control group served as the control. After 24 hours of sample treatment, the cells were observed and photographed under an inverted microscope (200×).

[0042] The results are as follows Figure 1 As shown, after HepG2 cells and HuH-7 cells were treated with 1 mg / mL, 2 mg / mL, and 4 mg / mL Feilike capsule solutions for 24 hours, observation under an inverted microscope showed that the number of cells decreased and their morphology changed significantly.

[0043] Example 2

[0044] Cell viability test

[0045] Cells in logarithmic growth phase were collected, and HepG2 cells and HuH-7 cells were collected at a rate of 6×10 3Cells were seeded at a density of 100 μL / well in a 96-well plate and cultured overnight. After the cells attached, different concentrations of sample solution (1 mg / mL, 2 mg / mL, and 4 mg / mL of Feili Ke capsule solution) were added. A normal saline control group was used. After 72 hours of drug treatment, 10 μL of 5 mg / mL MTT solution was added to each well and incubated at 37°C for 4 hours. The supernatant was discarded, and 160 μL of DMSO was added to each well to dissolve the blue-purple crystalline formazan. After complete dissolution of the blue-purple crystalline formazan, the absorbance (OD) at 490 nm was measured with a microplate reader.

[0046] The calculation method of inhibition rate: inhibition rate = (OD value of control group - OD value of treatment group) / OD value of control group. 50 ) calculation method: Use the forecast function to obtain the corresponding IC 50 .

[0047] The results are as follows Figure 2 As shown in the results, after treating cells with different concentrations of sample solutions (1 mg / mL, 2 mg / mL, 4 mg / mL of Feili Ke capsule solution) for 72 h, each concentration could inhibit the viability of HepG2 cells and HuH-7 cells in a concentration-dependent manner, with IC 50 They were 1.16±0.17 mg / mL and 1.85±0.13 mg / mL respectively.

[0048] Example 3

[0049] Cell growth curve assay

[0050] Cells in logarithmic growth phase were collected, and HepG2 cells and HuH-7 cells were collected at a rate of 6×10 3 Cells were seeded at a density of 100 cells / well in a 96-well plate and cultured overnight. After the cells attached to the wall, sample solutions of different concentrations (1 mg / mL, 2 mg / mL, and 4 mg / mL of Feili Ke capsule solution) were added for treatment. Normal saline was used as the control group. OD values (490 nm) were measured at 0 h, 24 h, 48 h, 72 h, and 96 h, and growth curves were drawn.

[0051] The results are as follows Figure 3 As shown in the data, after treating cells with sample solutions of different concentrations (1 mg / mL, 2 mg / mL, 4 mg / mL of Feilike capsule solution) for different time periods (0 h, 24 h, 48 h, 72 h, 96 h), the growth rates of HepG2 cells and HuH-7 cells gradually slowed down, indicating that Feilike capsule solution can inhibit the proliferation of HepG2 cells and HuH-7 cells in a concentration- and time-dependent manner.

[0052] Example 4

[0053] Cell clone formation assay

[0054] Cells in logarithmic growth phase were collected, and HepG2 cells and HuH-7 cells were collected at a rate of 3×10 5 The cells were seeded at a density of 1000 cells / well in a 6-well plate and cultured overnight. After the cells adhered to the wall, sample solutions of different concentrations (1 mg / mL, 2 mg / mL, and 4 mg / mL of Feili Ke capsule solution) were added. Normal saline was used as the control group. The cells were treated for 24 hours and then cultured in the medium for 14 days. After 14 days, the cells were fixed with methanol for 30 minutes and stained with crystal violet solution (1%) for 15 minutes. The cells were washed with PBS three times, and finally the clones in each group were counted.

[0055] The results are as follows Figure 4 As shown, compared with the control group, the number of cell clones in the groups treated with different concentrations of sample solution (1 mg / mL, 2 mg / mL, and 4 mg / mL of Feilike capsule solution) was less than that of the control group in a concentration-dependent manner. This indicates that the sample solution (Felike capsule solution) reduced the proliferation capacity of HepG2 cells and HuH-7 cells after treatment.

[0056] Example 5

[0057] Cell apoptosis detection

[0058] Cells in logarithmic growth phase were collected, and HepG2 cells and HuH-7 cells were collected at a rate of 3×10 5 Cells were seeded at a density of 100 μL / well in a 6-well plate and cultured overnight. After cell attachment, different concentrations of sample solution (1 mg / mL, 2 mg / mL, and 4 mg / mL of Feilike capsule solution) were added. Normal saline was used as the control group. Cells were collected after 48 hours. They were washed three times with PBS and resuspended in 100 μL of 1× Binding Buffer. 5 μL of Annexin V-FITC and 5 μL of propidium iodide were added and incubated at room temperature in the dark for 15 minutes. The apoptotic rate was calculated by flow cytometry.

[0059] The results are as follows Figure 5 As shown, after treating cells with different concentrations of sample solutions (1 mg / mL, 2 mg / mL, and 4 mg / mL of Feilike capsule solution) for 48 hours, the apoptosis rate of cells in each sample solution treatment group was higher than that in the control group in a concentration-dependent manner. This indicates that the sample solution (Felike capsule solution) can significantly affect the apoptosis of HepG2 and HuH-7 cells.

[0060] Example 6

[0061] Hoechst 33258 fluorescence detection

[0062] Cells in logarithmic growth phase were collected, and HepG2 cells and HuH-7 cells were collected at a rate of 3×10 5Cells were seeded at a density of 100 cells / well in a 6-well plate and cultured overnight. After the cells adhered, sample solutions of different concentrations (1 mg / mL, 2 mg / mL, and 4 mg / mL of Feili Ke capsule solution) were added for treatment. Normal saline was used as the control group. After 48 hours of treatment, 1 mL of fixative was added to each well and the cells were placed at room temperature for 10 minutes. The cells were washed twice with 1× PBS solution for 3 minutes each time. 0.5 mL of Hoechst dye was added to each well and the cells were placed in the dark for 5 minutes. The dye was removed and the cells were washed twice with 1× PBS solution again and observed under an inverted fluorescence microscope.

[0063] The results are as follows Figure 6 As shown, different concentrations of the sample solution (1 mg / mL, 2 mg / mL, and 4 mg / mL of Feili Ke capsule solution) significantly damaged the nuclei of HepG2 and HuH-7 cells. Compared with the control group, the nuclei of the treated cells became lobed and fragmented. This phenomenon was concentration-dependent, further demonstrating that the sample solution can significantly affect the apoptosis of HepG2 and HuH-7 cells.

[0064] Example 7

[0065] Establishment of animal tumor models

[0066] HuH-7 cells in the logarithmic growth phase were collected and the cell concentration was adjusted to 2×10 10 Cells / L, take 100 μL of cell suspension (2×10 6 Two weeks later, the mice were randomly divided into four groups, each consisting of six mice: a low-dose Feilike capsule group (Felike capsule-L) that received 468 mg / kg body weight by gavage; a medium-dose Feilike capsule group (Felike capsule-M) that received 936 mg / kg body weight by gavage; a high-dose Feilike capsule group (Felike capsule-H) that received 1872 mg / kg body weight by gavage; and a negative control group (a negative control group) that received an equal volume of normal saline once daily for 14 days. Tumor volume was measured every other day using a vernier caliper. After the final dose, the mice were dissected and the tumor weights were measured.

[0067] The results are as follows Figure 7 and Figure 8 As shown in the results, Feilike capsule can significantly inhibit the growth of tumor volume and reduce tumor weight in tumor-bearing mice.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of Feili Ke in the preparation of products for preventing and treating liver cancer.

2. Application of Feilike in the preparation of products that inhibit the activity of liver cancer cells.

3. Application of Feilike in the preparation of products that inhibit the proliferation of liver cancer cells.

4. Application of Feilike in the preparation of products that promote apoptosis of liver cancer cells.

5. The use according to any one of claims 1 to 4, characterized in that The product described is a drug.

6. The use according to any one of claims 1 to 4, characterized in that The Feilike is a Feilike mixture or a Feilike capsule.

7. The use according to claim 5, characterized in that The medicine is an injection, powder, tablet, granule, solution, emulsion, spray, ointment, plaster, patch, suppository or pill.

Images