CN115919845A - Application of brucea javanica element A as medicine for treating liver cancer - Google Patents

Abstract

The brucine A is used as a medicine for treating liver cancer; the molecular formula of the brucine A is as follows: C26H34O11 molecular weight: 522.5416 the application specifically is: brucine A inhibits the growth of human hepatoma cells. The application specifically comprises the following steps: brucine A inhibits the invasive ability of human hepatoma cells. The application specifically comprises the following steps: brucine A inhibits the proliferative capacity of human hepatoma cells.

Description

The Application of Bactanin A as a Drug for the Treatment of Liver Cancer

technical field

The invention relates to the new application field of brucetine A, in particular to the application of brucetine A as a medicine for treating liver cancer.

Background technique

Liver cancer is a malignant tumor of the liver, which can be divided into two categories: primary and secondary. Primary liver malignant tumors originate from the epithelial or mesenchymal tissues of the liver. The former is called primary liver cancer, which is one of the most common and extremely harmful malignant tumors in my country; according to the 2015 my country's malignant tumors released by the National Cancer Center Data show that there are 370,000 new cases of liver cancer, ranking fourth among malignant tumors, and 326,000 deaths, ranking second among malignant tumors. Liver cancer is not sensitive to chemotherapy and radiotherapy, and the commonly used treatment methods include surgical resection, liver transplantation, vascular intervention, and radiofrequency ablation. Some patients with early liver cancer have a better prognosis because they can undergo radical surgery, such as resection or liver transplantation. The survival time of patients with advanced liver cancer is usually only half a year to one and a half years. In addition, the recurrence rate of liver cancer patients is relatively high, and the tumor recurrence and metastasis rate is as high as 40%-70% within 5 years after surgical resection. Therefore, it is particularly important to find new therapeutic drugs for liver cancer.

Bruceine A (bruceineA, BA), also known as bruceine A, is a bitterwood lactone compound extracted from the fruit of the plant Bruceine javanica (Linnaeus) Merrill. Traditional Chinese medicine believes that Brucea javanica is bitter in taste, cold in nature, and slightly poisonous, and it belongs to the large intestine and liver meridian, and has the effects of clearing away heat, detoxifying, stopping malaria, stopping dysentery and corroding superfluous skin [7]. In southern my country, Brucea javanica is mostly used as an antimalarial drug, and Brucea javanica oil emulsion is often used clinically for adjuvant treatment of lung cancer and lung cancer brain metastases, liver cancer and digestive tract tumors, but whether the monomer component BA in Brucea javanica has anti-malarial Tumor effect, there is no experimental data.

The molecular formula of brucein A: C26H34O11 Molecular weight: 522.5416 The structural formula is as follows:

There are many applications of brucein, most of which are patents related to brucein D, and brucein A is used for diabetic nephropathy and bone metabolic diseases. There is no report about the use of Bruceatin A in tumor therapy.

Contents of the invention

In order to solve the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a new application of Brucea Brucea for the treatment of liver cancer, confirm the inhibitory effect of Brucea Brucea on the proliferation of liver cancer cells from in vivo experiments, and confirm the effect of Brucea Brucea on the proliferation of liver cancer cells from in vitro experiments. The inhibitory effect of brucein A on the proliferation, invasion and metastasis of liver cancer cells proves that brucein A can be used in the treatment of liver cancer. The brucein A involved in the present invention can be used for the preparation of antitumor drugs for treating liver cancer.

To achieve the above object, the technical solution of the present invention is:

The application of brucein A as a drug for treating liver cancer; the molecular formula of said brucein A: C26H34O11 molecular weight: 522.5416 structural formula is as follows:

Further, the application is specifically: Brucella Bacterin A inhibits the growth of human liver cancer cells.

Further, the application is specifically: Bruceavenin A inhibits the invasion ability of human liver cancer cells.

Further, the application is specifically: Brucella Bacterin A inhibits the proliferation ability of human liver cancer cells.

Compared with the prior art, the beneficial effects of the present invention are:

The application of Bruceavenin A as a drug for the treatment of liver cancer, the new use of Bruceavenin A in the treatment of liver cancer, the in vivo experiment confirmed the inhibitory effect of Bruceabrunic A on the proliferation of liver cancer cells, and the in vitro experiment confirmed the effect of Bruceavenin A on liver cancer Inhibition of cell proliferation, invasion, and metastasis, thus proving that brucein A can be used for the treatment of liver cancer. The brucein A involved in the present invention can be used for the preparation of antitumor drugs for treating liver cancer.

Description of drawings

Figure 1: Inhibitory effect of brucetin A on the proliferation of human liver cancer MHCC97-H, HepG2 and HCCLM3 cells;

Figure 2: Transwell cell invasion after treatment of HCCLM3 cells in various dose groups of Brucella A;

Figure 3: Each dose group of Brucella Bacterin A inhibited the proliferation potential of liver cancer cells HCCLM3;

Figure 4. Tumor growth in each group in the human liver cancer HCCLM3 subcutaneously transplanted tumor model;

Figure 5 shows HCCLM3 transplanted tumors in nude mice after the administration of Brucella Bacterin A;

Figure 6: Tumor weight of HCCLM3 transplanted tumors in nude mice after administration of Bructusin A.

Detailed ways

The technical solution of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments:

As shown in Figure 1, the experimental materials used in the following examples are as follows: cell strains: human liver cancer cell lines MHCC97-H, HepG2 and HCCLM3 are provided by Henan Provincial Industrial Microbial Strain Engineering Technology Research Center; 96 orifice plates are purchased from Costar company (Cat. No.: 3599); DMEM high glucose medium was purchased from Gibco (Cat. No.: C11995500BT); fetal bovine serum was purchased from Gibco (Cat. No.: 10270-106); CCK-8 kit was purchased from Meilun Bio ( Catalog number: MA0218); DMSO was purchased from Beijing Suolaibao Technology Co., Ltd. (catalog number: D8370); Bruceine A (BruceineA, BRA) was purchased from Hubei Yongkuo Technology Co., Ltd. (catalogue number JOT-11226, batch 22061309 ), HPLC purity 98.73%, sealed at -20°C.

The experimental apparatus used in the following examples is as follows:

Experimental equipment: ultra-clean bench (Suzhou purification model BHC-1000IIA2), CO2 incubator (Thermo model: 3111), microplate reader (Thermo MultiscanMK3)

Example 1: Effect of Brucedin A on the growth of human liver cancer cells

Experimental method: Weigh 2mg, add 0.38mL DMSO to dissolve, and prepare a 10mM/L solution. Using the CCK-8 method to evaluate the growth inhibitory effect of brucein A on human hepatoma cells: Human hepatoma cells (MHCC97-H, HepG2 and HCCLM3) were digested with trypsin, counted, and treated with complete medium (10% fetal bovine serum) +90% DMEM high-glucose medium) and mix well to make a cell suspension with a concentration of 2×104 cells/mL. Add 100 μL of cell suspension (2×103 cells per well) to each well of a 96-well plate, and then place it in a 5% CO2 incubator at 37°C for 16 hours; dilute the drug to the required gradient concentration with complete medium (3.9nM/L-4000nM/L), add 100μL of the corresponding drug-containing medium to each well, and set up a vehicle (DMSO) control group and a cell-free culture medium control group at the same time, and then place the 96-well plate at 37 ° C 5% CO2 culture Incubate for 72 hours. Then add 20μLCCK-8 solution to each well, continue to cultivate for 2 hours, detect the absorbance or OD value of each well with a microplate reader at a wavelength of λ=450nm, and subtract the average value of the cell-free culture medium from the average value of each multiple well as the value. The OD value of the cells in the group was compared with the vehicle control, and the cell survival rate after drug treatment was calculated.

Results: As shown in Figure 1, brucein A has a significant growth inhibitory effect on human liver cancer MHCC97-H, HepG2 and HCCLM3 cells, and its half inhibitory concentration (IC50) is less than 300nM/L, respectively 148.60nM/L and 216.50nM /L and 51.86nM/L.

Figure 1: Inhibitory effect of brucetin A on the proliferation of human liver cancer MHCC97-H, HepG2 and HCCLM3 cells;

a. The data are represented by "average inhibition rate ± standard deviation of each concentration".

Example 2: Bactanin A inhibits the invasion ability of human liver cancer cells

Need testing product: described brucein A solution is prepared by the method described in embodiment one.

Treatment method: Human liver cancer cells HCCLM3 were digested with trypsin, counted, and mixed with DMEM high-glucose medium to make a cell suspension with a concentration of 1×105 cells/mL. Add 600 μL of complete medium to the lower chamber of the transwell of the 24-well plate, set up the vehicle DMSO control group and the Brucevenin A treatment group (50 nM/L and 200 nM/L), and each group has 4 parallel wells. Place the Transwell chamber in a 24-well plate with tweezers, take 100 μL of the cell suspension and add it to the upper chamber of the transwell pre-coated with Matrigel, and place it in an incubator for 24 hours. Remove the small chamber, aspirate the medium, and gently wipe off the Matrigel and the cells in the upper chamber with a cotton swab. Take a new 24-well plate and add 600 μL of 4% paraformaldehyde, put the chamber in and fix it for 20-30 minutes, discard the fixative solution, stain with 0.1% crystal violet staining solution for 10 minutes, wash with deionized water 3 times, and dry it properly , photographed under a high-power microscope. After staining, add 600 μL of 33% acetic acid to each Transwell chamber to completely elute the crystal violet. After fully shaking, take 100 μL of the eluate and measure the light absorbance (OD570) at 570 nm with a microplate reader.

Results: See Figure 2. The results of the Transwell cell invasion assay showed that, compared with the vehicle (DMSO) control group, each dose group (50nM/L and 200nM/L) of Brucevenin A significantly inhibited the transwell invasion ability of HCCLM3 cells (BRA( 50nM/L) vs. vehicle, P<0.0001; BRA (200nM/L) vs. vehicle, P<0.0001).

Figure 2: Transwell cell invasion after treatment of HCCLM3 cells in various dose groups of Brucella A;

Note: a. The data are expressed as "mean ± standard deviation".

b. Use one-way ANOVA and Dunnett's multiple comparisons test to compare the significant difference in the apoptosis rate between the control group and each dose group. A P value of less than 0.05 was considered to have a significant difference.

Example 3: Bactanin A inhibits the proliferation ability of human liver cancer cells (EdU experiment)

Need testing product: described brucein A solution is prepared by the method described in embodiment one.

Treatment method: Human liver cancer cells HCCLM3 were digested with trypsin, counted, and mixed with complete medium (10% fetal bovine serum + 90% DMEM high-glucose medium) to make a cell suspension with a concentration of 5×104 cells/mL. solution, added to a 24-well culture plate pre-covered, and 0.5 mL of cell suspension was added to each well, and then placed in a 5% CO2 incubator at 37 ° C for 16 hours. Set solvent control group (DMSO), BRA high-dose group (200nM/L) and BRA low-dose group (50nM/L). After drug treatment for 48 hours, 10 μM/L EdU solution was added to label proliferating cells. Wash once with PBS, add 4% paraformaldehyde to fix cell slides, then add EdU detection solution and incubate in the dark for 30 minutes, use anti-fluorescence quencher containing DAPI to seal the slides, observe EdU labeling under a fluorescence microscope, each cell The EdU-labeled cells in 4 fields of view were randomly counted under the 20× objective field of view. EdU labeling rate=(Number of EdU-labeled cells/Number of DAPI-stained cells)×100%. Result: see Fig. 3, the EdU experiment result shows, compares with vehicle (DMSO) control group, each dose group (50nM/L and 200nM/L) of Bruceavenin A all significantly inhibits the proliferative potential of HCCLM3 cell (BRA (50nM/L ) vs. vehicle, P<0.0001; BRA (200nM/L) vs. vehicle, P<0.0001).

Figure 3: Each dose group of Brucella Bacterin A inhibited the proliferation potential of liver cancer cells HCCLM3;

Note: a. The data are expressed as "mean ± standard deviation".

b. Use one-way ANOVA and Dunnett's multiple comparisons test to compare the significant difference of EdU labeling rate between the control group and each dose group. A P value of less than 0.05 was considered to have a significant difference.

Example 4: Drug efficacy experiment of nude mice subcutaneously transplanted with HCCLM3 tumor model

Experimental animals: BALB/c nude mice, male and female, 4 weeks old, weighing about 17g, purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., production license number: S C X K (Xiang) 2019-0004, animal Certificate number: 430727221102657814. Breeding environment: SPF grade.

The experimental animals are all kept in an independent ventilated box with constant temperature and humidity. The temperature of the breeding room is 20-26°C, the humidity is 40-70%, the ventilation is 10-20 times per hour, and the alternating time of day and night is 12h/12h; continuous supply of cobalt 60 radiation Sterilized full-price pellet feed for rats, unlimited free intake, drinking tap water (used after high-pressure steam sterilization), continuous water supply from drinking bottles, free intake. The rat box is a polysulfone mouse box, which is used after autoclaving, and the size is 325mm×210mm×180mm; the bedding material is autoclaved corncob, and there are 5 animals in each box; the experimental animals are marked with ear tags.

Experimental materials: Bruceine A (Bruceine A, BRA) was purchased from Hubei Yongkuo Technology Co., Ltd. (catalogue number JOT-11226, batch 22061309), HPLC purity 98.73%, sealed at -20°C. PEG300 was purchased from Haian Petrochemical Plant in Jiangsu Province; PBS was purchased from Tianjin Haoyang Huake Biotechnology Co., Ltd.; DMSO was purchased from Beijing Suolaibao Technology Co., Ltd.

Drug preparation: weigh Brucea Bacterin A powder and dissolve it in a solvent solution (10% DMSO+30% PEG300+60% PBS).

Experimental method: Human liver cancer HCCLM3 cells were cultured in RPMI-1640 culture medium (purchased from Gibco, USA) containing 10% fetal bovine serum. HCCLM3 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration (2×107/mL) for subcutaneous tumor inoculation in nude mice. 100 μL of cell suspension (containing 2×106 HCCLM3 cells) was inoculated in the right armpit of nude mice. When the tumor grows to an average volume of about 50 mm3, they are randomly divided into male and female groups according to the size of the tumor, and administered on the day of grouping, and the day of administration is defined as day 0. Nude mice were administered orally via hepatic administration, and the administration volume was 100 μL/nude mouse. Measure the long diameter a and short diameter b of the tumor with a vernier caliper every other day

Table 1. Dosing regimen instructions

Experimental results: No nude mice died during the administration, and the administration could be tolerated. On the 24th day of group administration, the volume of the tumor in the vehicle control group was 639.05±111.58 mm3, and the test was terminated, and the tumor was taken and weighed. Tumor volume calculation formula: tumor volume (mm3) = 1/2 x (a x b2) (wherein a represents the long diameter and b represents the short diameter). At the end of the test, the average tumor volume of the brucein A200 mg/kg administration group was 252.05 ± 50.04mm3, which was statistically significantly different from that of the control group (P=0.0001) (see Figure 4, Figure 5);

Figure 4. Tumor growth in each group in the human liver cancer HCCLM3 subcutaneously transplanted tumor model;

Note: a. The data are expressed as "mean ± standard deviation";

b. Unpaired t-test (Welch's correction) was used to compare the significant difference in tumor weight between the vehicle control group and the Brucevenin A administration group. P value less than 0.05 is considered to have significant difference;

Figure 5 shows HCCLM3 transplanted tumors in nude mice after the administration of Brucella Bacterin A;

Tumor samples were collected at the end of the experiment and the tumor weight (g) was measured, and the results were consistent with the tumor volume measurements. See Figure 6 for the tumor weights of the administration group and the control group. Compared with the solvent control tumor weight (0.534±0.080g), the tumor weight after administration of brucein A200 mg/kg was 0.147±0.062g, and the difference was significant (P<0.0001). The results showed that brucein A had good in vivo antitumor activity in liver cancer xenograft tumor model.

Figure 6: Tumor weight of HCCLM3 transplanted tumors in nude mice after administration of Bructusin A;

Note: a. The data are expressed as "mean ± standard deviation";

b. Use the unpaired t test (Welch's correction) to compare the significant difference in tumor weight between the vehicle control group and the administration group. A P value of less than 0.05 was considered to have a significant difference.

The above is only a specific implementation of the present invention, but the scope of protection of the present invention is not limited thereto, and any changes or replacements that do not come to mind through creative work shall be covered within the scope of protection of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope defined in the claims.

Claims (4)

1. The brucine A is used as the medicine for treating liver cancer; the molecular formula of the brucine A is as follows: C26H34O11 molecular weight: 522.5416 is of the formula:

2. use according to claim 1, characterized in that: the application specifically comprises the following steps: brucine A inhibits the growth of human hepatoma cells.

3. Use according to claim 1, characterized in that: the application specifically comprises the following steps: brucine A inhibits the invasive ability of human hepatoma cells.

4. Use according to claim 1, characterized in that: the application specifically comprises the following steps: brucine A inhibits the proliferative capacity of human hepatoma cells.

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