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

Abstract

The present invention provides the use of Jin Gu Lian in preparing products for the prevention and treatment of liver cancer, belonging to the technical field of liver cancer prevention and treatment. This invention, for the first time, proposes the use of Jin Gu Lian in preparing 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 changing the morphology of liver cancer cells, thereby achieving the effect of effectively treating liver cancer. Furthermore, Jin Gu Lian is a traditional Chinese medicine already used in clinical practice and can be directly applied to the human body without the need for clinical safety assessment. 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 golden bone lotus in preparing products for preventing and treating liver cancer. Background Art

[0002] The current main functions of Jin Gu Lian are to dispel wind and dampness, reduce swelling and relieve pain. It is used for joint swelling and pain caused by rheumatic arthritis, and difficulty flexing and extending joints. Jin Gu Lian is composed of radix polygoni multiflori, tangerine peel, safflower vine, maple, and cyperus rotundus, with corn starch as a supplement. In recent years, Traditional Chinese Medicine has garnered widespread attention in the treatment of liver cancer, but existing technology does not document the technical application of Jin Gu Lian in 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 the use of Jingulian 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 golden bristlecone lotus in preparing products for preventing and treating liver cancer.

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

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

[0008] The present invention also provides the use of Jingulian in preparing products that promote apoptosis of liver cancer cells.

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

[0010] Preferably, the golden bone lotus includes golden bone lotus capsules and golden bone lotus tablets.

[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 Herba Lycopodii or its effective ingredients.

[0013] The present invention also provides a method for preparing the above-mentioned medicine, comprising the following steps: directly preparing the golden bristle lotus into a pharmaceutical preparation for preventing and treating liver cancer, or compounding the golden bristle lotus with other drugs for preventing and treating liver cancer to prepare a compound pharmaceutical preparation, or combining the effective ingredients of the golden bristle lotus 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 Jin Gu Lian 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 of liver cancer cells, and alters their morphology, thereby effectively treating liver cancer. Furthermore, Jin Gu Lian is a clinically used 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 Jingulian capsule solution on the cell morphology of human liver cancer cells HepG2 and HuH-7;

[0017] Figure 2 The effect of different concentrations of Jingulian capsule solution on the cell viability of human liver cancer cells HepG2 and HuH-7. The upper figure shows the results of human liver cancer cells HepG2, and the lower figure shows the results of human liver cancer cells HuH-7.

[0018] Figure 3 The effect of different concentrations of Jingulian capsule solution on the growth of human liver cancer cells HepG2 and HuH-7. The upper figure shows the results of human liver cancer cells HepG2, and the lower figure shows the results of human liver cancer cells HuH-7.

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

[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 Jin Gu Lian in preparing products for preventing and treating liver cancer. The present invention also provides the use of Jin Gu Lian 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 Jin Gu Lian, and conventional commercially available products in this field can be used. In the present invention, the Jin Gu Lian preferably includes Jin Gu Lian capsules and Jin Gu Lian tablets. 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 Herba Lycopodii or its effective ingredients.

[0028] The present invention has no special restrictions on the source of Jin Gu Lian and the dosage form, excipients, etc. of the pharmaceutical preparation. In a specific embodiment of the present invention, the Jin Gu Lian is composed of Radix Tou Gu Xiang, Peach Leaves, Caulis Sargentodoxae, Acer octagonalis, and Gynostemma pentaphyllum.

[0029] The present invention also provides a method for preparing the above-mentioned medicine, comprising the following steps: directly preparing the golden bristle lotus into a pharmaceutical preparation for preventing and treating liver cancer, or compounding the golden bristle lotus with other drugs for preventing and treating liver cancer to prepare a compound pharmaceutical preparation, or combining the effective ingredients of the golden bristle lotus 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 solution: The contents of Jingulian capsule (Jingulian capsule was purchased from Guizhou Yibai Pharmaceutical Co., Ltd., and the contents refer to the contents obtained after removing the capsule shell) were prepared with normal saline to prepare Jingulian capsule solutions with concentrations of 1 mg / mL, 2 mg / mL, and 4 mg / mL, respectively.

[0036] (2) Preparation of MTT solution: Weigh 0.2 g of MTT powder, add 40 mL of sterilized double-distilled water, dissolve at 60°C to a concentration of 5 mg / mL, filter sterilize 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 rate of 100 cells / well in a 60 mm round dish and cultured. After the cells attached to the wall, different concentrations of sample solution (1 mg / mL, 2 mg / mL, and 4 mg / mL of Jin Gu Lian capsule solution) were added for 24 hours. Normal saline was used as the control group. Cell changes were observed under an inverted microscope.

[0042] The results are as follows Figure 1 As shown in the figure, 24 hours after treatment, the cell morphology was observed under a microscope. As the drug concentration increased, the cell number decreased significantly. Cell morphology changed, especially at high concentrations, from a long spindle to a round shape. The cells shrank and even fragmented.

[0043] Example 2

[0044] Cell viability test

[0045] Logarithmic phase cells 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 to the wall, different concentrations of sample solutions (1 mg / mL, 2 mg / mL, and 4 mg / mL of Jin Gu Lian capsule solution) were added for treatment. Normal saline was used as the control group. 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 the blue-purple crystalline formazan was completely dissolved, the absorbance value (OD) (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 figure, after treating HepG2 cells and HuH-7 cells with 1 mg / mL, 2 mg / mL, and 4 mg / mL sample solutions (Jin Gu Lian Capsule Solution) for 72 hours, the MTT assay was used to detect the inhibition rates of HepG2 cells and HuH-7 cells with different concentrations of sample solutions. Sample solutions of different concentrations could inhibit the viability of HepG2 cells and HuH-7 cells in a concentration-dependent manner. The IC 50 They were 1.36±0.04 mg / mL and 1.78±0.02 mg / mL respectively.

[0048] Example 3

[0049] Cell growth curve assay

[0050] HepG2 cells and HuH-7 cells in the logarithmic phase were collected and the cells were divided into 6×10 3 Cells were seeded at a density of 1000 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 Jingulian 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, compared with the control group, different concentrations of sample solutions (1 mg / mL, 2 mg / mL, and 4 mg / mL of Jingulian capsule solution) can inhibit the proliferation of HepG2 cells and HuH-7 cells, and with the increase of concentration, the growth rate of HepG2 cells and HuH-7 cells gradually slowed down, indicating that the sample 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 amount of cells / well was inoculated into a 60mm round culture dish for cultivation. After the cells adhered to the wall, different concentrations of sample solution (1mg / mL, 2mg / mL, 4mg / mL of Jin Gu Lian capsule solution) were added for 24h. With normal saline as the control group, the cells were digested and collected and counted. According to 1000 cells / well, they were planted in a 6-well plate and continuously cultured in an incubator with DMEM culture medium containing 10% FBS, and fresh culture medium was replaced every three days. After 14 days, the culture medium was discarded, washed 2-3 times with PBS, fixed with methanol for 15min, stained with 0.1% crystal violet for 15min, washed 5-8 times with PBS, air-dried, photographed, and the number of clones containing more than 50 cells was counted under a light microscope.

[0055] The results are as follows Figure 4 As shown in the figure, compared with the control group, the number of clones and the diameter of the clones decreased significantly with the increase of the concentration of the sample solution, and had a certain concentration dependence, which indicated that the sample solution could inhibit the proliferation of HepG2 cells and HuH-7 cells in vitro.

[0056] Example 5

[0057] Cell apoptosis detection

[0058] HepG2 cells and HuH-7 cells were cultured at a rate of 3 × 10 5 Cells were seeded at a concentration of 100 μL / well in a 60 mm round dish for culture. After the cells adhered to the wall, different concentrations of sample solution (1 mg / mL, 2 mg / mL, and 4 mg / mL of Jin Gu Lian capsule solution) were added and the cells were collected after 48 hours. Normal saline was used as the control group. The cells were washed three times with PBS, resuspended in 100 μL 1× Binding Buffer, and incubated in the dark for 15 minutes at room temperature. The apoptosis rate was calculated by flow cytometry.

[0059] The results are as follows Figure 5 As shown, compared with the control group, sample solutions of different concentrations can induce apoptosis in human liver cancer cells HepG2 and HuH-7 in a concentration-dependent manner.

[0060] Example 6

[0061] Hoechst 33258 staining

[0062] Take a clean cover glass and soak it in 70% ethanol for 5 minutes or longer, wash it three times with sterilized PBS in a biosafety cabinet, and then wash it once with cell culture medium. 5 Cells were planted in 60mm round culture dishes and cultured overnight. After the cells adhered to the wall the next day, the supernatant was discarded and sample solutions of different concentrations (1mg / mL, 2mg / mL, and 4mg / mL of Jin Gu Lian capsule solution) were added, with normal saline as the control group. After 48 hours of action, the culture medium was aspirated, 0.5mL of fixative was added, and fixed for 10 minutes. Wash twice with PBS, shaking manually during washing for 3 minutes each time, and aspirate all the liquid. Add 0.5mL of Hoechst 33258 staining solution and stain for 5 minutes. Shake manually several times during the period. After staining, wash twice with PBS for 3 minutes each time and aspirate all the liquid. Drop a drop of anti-fluorescence quenching sealing solution on the slide, cover with a coverslip with cells, let the cells contact the sealing solution, and try to avoid bubbles. Observe and take pictures with an inverted fluorescence microscope.

[0063] The results are as follows Figure 6 As shown in the figure, compared with the control group, sample solutions of different concentrations can induce cell apoptosis, and the nuclei of apoptotic cells are densely stained or fragmented and densely stained.

[0064] Example 7

[0065] Establishment of animal tumor models

[0066] After BALB / c nude mice were adapted for one week, HuH-7 cells in the logarithmic growth phase were collected and cultured at a rate of 2×10 6 The cells were inoculated subcutaneously at the dorsal level of the neck of BALB / c nude mice at a density of 10 cells per mouse. Two weeks later, the mice were randomly divided into four groups, six mice per group: a negative control group (administered with an equal volume of normal saline); a low-dose Jingulian Capsule group (Jingulian Capsule-L) administered with 195 mg / kg body weight by oral gavage; a medium-dose Jingulian Capsule group (Jingulian Capsule-M) administered with 390 mg / kg body weight by oral gavage; and a high-dose Jingulian Capsule group (Jingulian Capsule-H) administered with 780 mg / kg body weight by oral gavage, once daily for 14 days. Tumor volume was measured every other day with 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, Jingulian Capsule can significantly inhibit the growth of subcutaneous transplanted tumors in nude mice and reduce tumor weight.

[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 Golden Bone Lotus in the preparation of products for preventing and treating liver cancer.

2. The application of golden bone lotus in the preparation of products that inhibit the vitality of liver cancer cells.

3. Application of golden bone lotus in the preparation of products that inhibit the proliferation of liver cancer cells.

4. Application of golden bone lotus 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 is a drug.

6. The use according to any one of claims 1 to 4, characterized in that The golden bone lotus is a golden bone lotus capsule or a golden bone lotus tablet.

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.

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