CN105726726A - Combined application of extract of traditional Chinese medicaments coptischinensis and evodia rutaecarpa and 5-fluorouracil to preparation of medicament for treating intestinal cancer - Google Patents

Abstract

The invention provides the use of extracts of Coptis chinensis and Evodia rutaecarpa (Zuojinfang) combined with 5?FU in preparing a medicine for treating intestinal cancer. The combination of Zuojinfang and 5?FU significantly increased the survival rate of cells, greatly reduced the toxic and side effects of 5?FU, and promoted the high expression of Bax protein and the low expression of Bcl?2 and Survivin proteins in cells, and promoted intestinal The apoptosis of cancer cell HT?29 is better than Zuojinfang or 5?FU alone in the treatment of intestinal cancer.

Description

Application of extracts of Coptidis Rhizoma and Evodia rutaecarpa combined with 5-fluorouracil in the preparation of drugs for treating intestinal cancer

technical field

The invention relates to a new application of combined treatment of traditional Chinese medicine compound Zuojin Fang and chemotherapeutic drugs.

Background technique

Colorectal cancer is one of the common malignant tumors in the digestive system, including colon cancer and rectal cancer. Every year, about 1.2 million patients are diagnosed with colorectal cancer worldwide, and more than 600,000 patients die directly or indirectly from colorectal cancer , its morbidity and mortality rank third and fourth respectively. The incidence of colorectal cancer in men is higher than that in women, and the incidence of colorectal cancer is lower in the age group below 40 years old, but it will increase with age. Colorectal cancer is one of the most common and currently most concerned malignant tumors in China. Since the 1990s, the incidence of colorectal cancer in developed countries has been declining year by year, but it is still rising year by year in developing countries such as China. This epidemiological feature is more obvious in male patients, which seriously threatens human health.

Surgery is considered to be the main method for the treatment of colon cancer. However, due to the concealment of colon cancer and the limitations of diagnosis and treatment methods, most colon cancers have entered the middle and late stages of colon cancer when they are discovered, and are not suitable for surgical treatment. Currently, colorectal cancer is generally treated with chemotherapy, supplemented by radiotherapy and biological immunotherapy. However, chemotherapy has huge adverse reactions, leading to acute liver injury and kidney injury, which greatly limits the amount of chemotherapy drugs and hinders the efficacy of drugs. In addition, radiotherapy can easily lead to radiation brain and spinal cord injuries, radiation cranial nerve damage, and radiation salivary gland damage. And radiation-induced complications such as cancer.

Therefore, traditional Chinese medicine combined with chemotherapy is more and more advantageous in the treatment of advanced colorectal cancer. The research on the treatment of colorectal cancer by traditional Chinese medicine has involved the fields of tumor occurrence, development, death, aging, etc., and has played an active role in the treatment of colorectal cancer. A large number of medical practices have proved that: traditional Chinese medicine combined with adjuvant therapy (chemical, targeted, gene, radiation, endocrine, biological therapy, etc.) The quality has obvious advantages. In addition, the cost of traditional Chinese medicine is relatively low and the practicability is wide. However, traditional Chinese medicine anti-intestinal cancer preparations are monotonous and have uncertain curative effects. At present, there are no clinical preparations with extensive therapeutic effects. Most of the traditional Chinese medicine preparations are used as adjuvant therapy. Cancer traditional Chinese medicine and its compound are widely used and recognized by the international community.

Zoujin Perscription (Zoujin Perscription) comes from "Danxi Xinfa". It is composed of the monarch drug Coptischinensis Fr and the adjuvant drug Evodiarutaecarp (Juss.) benth. in a weight ratio of 6:1. It is recorded in various pharmacopoeias. Its medicinal properties Slightly cold, mainly treats liver stagnation into fire, stomach loss and subsidence, hypochondriac pain, abdominal distension, vomiting and acid regurgitation, noisy like hunger, stringy and rapid pulse, bitter mouth and red tongue, etc. It is more prone to stomach fire. Modern medical research shows that Zuojin Fang has the functions of protecting gastric mucosa, inhibiting gastric acid secretion, anti-ulcer, and affecting gastrointestinal motility. Currently clinically used for acute and chronic gastritis and peptic ulcer. Recently, studies have shown that Zuojin Fang can induce the apoptosis of colon cancer cells HT-29 cultured in vitro. It significantly inhibits the expression of Bax protein in tumor tissue and reduces the expression of Bcl-2 and Survivin proteins, thereby significantly inhibiting the apoptosis of human colon cancer cells. Growth of xenograft tumors in mice.

Currently, preoperative fluorouracil (5-FU)-based chemoradiotherapy is the standard treatment for locally advanced rectal cancer. 5-FU is a radiosensitizer, but the treatment effect on patients with distant metastases is not good, it will cause some problems such as anal and sexual function damage, and it cannot further prolong the survival time. Therefore, 5-FU has the disadvantages of low bioavailability, low selectivity for tumor cells, high toxicity and side effects, and the therapeutic dose is close to the toxic dose, which greatly limits its clinical application.

Chinese patent application 101785807A discloses the application of extracts of Coptis chinensis and Evodia rutaecarpa with a mass ratio of 6:1 in the preparation of drugs for treating gastric cancer, and the application of the traditional Chinese medicine compound Zuojin Fang in the preparation of drugs for treating gastric cancer.

Contents of the invention

The object of the present invention is to provide the use of extracts of Coptidis Rhizome and Evodia rutaecarpa in combination with 5-FU (5-fluorouracil) in the preparation of medicaments for treating intestinal cancer.

According to one aspect of the present invention, Chinese medicine Coptidis and Evodia extracts are combined with 5-FU (5-fluorouracil) for the treatment of intestinal cancer, preferably Chinese medicine Coptidis and Evodia extracts with a mass ratio of 6:1 Combined with the chemotherapeutic agent 5-fluorouracil, it is more preferred to prepare the extract by the preparation method of the traditional Chinese medicine compound Zuojin Fang, so the compound Zuojin Fang and Zuojin Pills prepared according to the compound are included in the scope of the present invention In addition, those skilled in the art can understand that the compound medicine prepared by taking Zuojinfang as the basic prescription and adding other auxiliary medicinal materials is also included in the present invention. In the treatment of intestinal cancer, the combination refers to simultaneous administration or sequential administration, that is, administering 5-FU before, simultaneously or after administering the extract to patients.

The present invention proves through cell in vitro tests that the combination of the extracts of the Chinese medicine Coptidis Rhizoma and Evodia rutaecarpa and 5-FU has better therapeutic effect on intestinal cancer than that of the extracts alone or 5-FU alone. The extracts of Coptis chinensis and Evodia rutaecarpa combined with 5-FU significantly improved the survival rate of normal colorectal mucosal epithelial cells of FHC human beings, greatly reduced the cytotoxic effect of 5-FU, and promoted the expression of Bax protein in HT-29 cells. High expression and low expression of Bcl-2 and Survivin proteins can promote the apoptosis of human intestinal cancer cells. The research results of the present invention have great significance for reducing the toxic and side effects of clinical anti-intestinal cancer chemotherapeutic drugs and improving the therapeutic effect.

Description of drawings

Fig. 1 is the CCK-8 test result of Zuojinfang of the present invention acting on FHC human colorectal mucosal epithelial cells, showing the relationship between extract concentration, action time and FHC survival rate;

Fig. 2 is the CCK-8 test result of 5-FU of the present invention acting on FHC human colorectal mucosal epithelial cells, showing the relationship between 5-FU concentration, action time and FHC survival rate;

Fig. 3 is the flow cytometric detection result of Zuojin Fang of the present invention acting on colon cancer cell HT-29, showing the relationship between extract concentration, action time and HT-29 apoptosis rate;

Fig. 4 is the flow cytometry detection result of 5-FU of the present invention acting on colon cancer cell HT-29, shows the relation of 5-FU concentration, action time and HT-29 apoptosis rate;

Figure 5 is the blank control, 1.00μg/mL Zuojinfang, 50.0μg/mL5-FU and 1.00μg/mL Zuojinfang combined with 50.0μg/mL5-FU respectively acted on the CCK-8 test results of FHC cells for 24 hours, showing the specific concentration The relationship between the extract, 5-FU, extract combined with 5-FU and FHC survival rate;

Figure 6 is the blank control, 1.00μg/mL Zuojinfang, 100.0μg/mL5-FU, and 1.00μg/mL Zuojinfang combined with 100.0μg/mL5-FU respectively acted on FHC cells for 24 hours. The CCK-8 test results show specific concentrations The relationship between the extract, 5-FU, extract combined with 5-FU and FHC survival rate;

Figure 7 is the blank control, 1.00 μg/mL extract, 50.0 μg/mL5-FU, 1.00 μg/mL extract combined with 50.0 μg/mL5-FU acted on the flow cytometry results of HT-29 cells respectively, showing specific concentrations The relationship between the extract, 5-FU, extract combined with 5-FU and HT-29 apoptosis rate;

Figure 8 is the blank control, 1.00 μg/mL extract, 100.0 μg/mL5-FU, 1.00 μg/mL extract combined with 100.0 μg/mL5-FU acted on HT-29 cells by flow cytometry, showing the specific concentration The relationship between the extract, 5-FU, extract combined with 5-FU and HT-29 apoptosis rate;

Figure 9 is the blank control, 1.00μg/mL Zuojinfang, 100.0μg/mL5-FU, 1.00μg/mL Zuojinfang combined with 100.0μg/mL5-FU respectively acted on HT-29 cells for 24 hours to regulate BAX, Bcl- 2 and Western Blotting gel electrophoresis of Survivin protein expression (n=6);

Figure 10 is the blank control, 1.00μg/mL extract, 50.0μg/mL5-FU, 100.00μg/mL5-FU, 1.00μg/mL extract combined with 50.0μg/mL5-FU and 1.00μg/mL extract combined with 100.0μg /mL5-FU acted on HT-29 cells respectively under the microscope of cell morphology (×200).

detailed description

1. Test materials

Cell lines: FHC cells (normal human colorectal epithelial cells), HT-29 cells (human colon cancer cells) were purchased from the Cell Center of the Animal Experiment Department of Sun Yat-sen University. Use DMEM complete medium containing 10% FBS by volume fraction, culture at 37°C, 5% CO2 incubator for 3 days for one passage, and the passage ratio is 1:3. Cells in growth phase were taken for experiments.

Extract of Coptis chinensis and Evodia rutaecarp (hereinafter referred to as extract or Zuojinfang): the traditional Chinese medicine Coptis chinensis Fr and Evodiarutaecarp (Juss.) benth. Concentrate and freeze-dry to make an aqueous extract. After the original medicinal material is extracted with water, the water extract 260 (mg)/crude drug (g) is obtained.

Zuojinfang traditional decoction freeze-dried powder: provided by our laboratory, take 30g Coptis rhizome and 5g Evodia rutaecarpa, add 8 times of distilled water to soak for 30min, add water to decoct 3 times, each time for 30min, filter, combine 3 times of filtrate, freeze-dry, each The dry powder contains 3.98g of crude drug; as determined by HPLC, Zuojinfang freeze-dried powder contains 39.85% berberine, 9.19% palmatine, 4.86% jatrorrhizine, 0.17% evodiamine, and 0.19% evodiamine.

The method of preparing Zuojinfang at the dosage concentration in the test: Dissolve Zuojinfang freeze-dried powder in deionized water to make a 1mg/mL mother solution, and dilute it with cell culture medium to the required concentration during the experiment.

5-FU: purchased from Tianjin Jinyao Amino Acid Co., Ltd., specification: 10ml: 0.25g, batch number: 1412011.

The method for preparing 5-FU at the administration concentration in the test: take an appropriate amount of 5-fluorouracil injection stock solution and add cell culture solution to prepare a 1mg/ml mother solution, and dilute it with cell culture solution to the required concentration during the experiment.

2. Detection method and data statistics method

1. CCK-8 detection of cell viability

CCK-8 detection kit was used to observe the influence of Zuojinfang or 5-FU or Zuojinfang combined with 5-FU on the survival rate of cultured FHC cells within a specified time, in which the survival rate of untreated control group cells was set as Set at 100%.

2. Detection of cell apoptosis rate by flow cytometry

Adjust the density of HT-29 cells to 2×10 ⁵ /mL, and seed them into 6-well plates at 1.5 mL/well. Each group has 6 replicate wells. After 24 hours of drug action, scrape the cells with a cell brush, centrifuge at 800r·min-1 for 5 minutes, remove the supernatant, add PBS to resuspend, and adjust the cell concentration to 1×10 ⁶ /mL. Take an appropriate amount into a flow tube, add 500uL of working solution, then add fluorescent dye, filter through a 350-mesh nylon mesh filter to remove cell clumps, and after staining at room temperature in the dark for 15 minutes, use a flow cytometer to detect the cell apoptosis rate (excitation light wavelength At 488 nm, FITC fluorescence was detected with a bandpass filter with a wavelength of 515 nm, and PI was detected with a filter with a wavelength greater than 560 nm). The measured data is processed according to the software configured by the flow cytometer, and the sum of the early apoptosis rate represented by the lower right plus the middle and late apoptosis rate represented by the upper right represents the apoptosis rate.

3. Observation of cell morphology

HT-29 cells (2×10 ⁵ /mL) were inoculated on the coverslips in 6-well plates, and each experimental group was given different treatment factors as required, and then freshly prepared 4% paraformaldehyde (pH 7.4) was added Fix cells at 37°C for 30 min, wash twice with PBS, add 5 mg/L Hochest33258 staining solution for staining for 30 min, wash with PBS, and use mounting solution (20 mmol/L citric acid, 50 mmol/L disodium hydrogen phosphate, 50% glycerol, pH 5.5) After mounting the slides, observe and take pictures with a fluorescence microscope.

4. Statistical processing

All the above data were statistically analyzed using SPSS 10.0 statistical software, and the data were expressed as mean ± standard deviation One-way ANOVA (One-way ANOVA) was used for comparison between groups.

[Example 1] Effect of Zuojin Fang or 5-FU alone on the survival rate of FHC cells

1.1 The effect of Zuojin Fang on the survival rate of FHC cells

Take human normal colorectal mucosal epithelial cells FHC cells in logarithmic growth phase, use DMEM medium to make cell suspension (5×104/mL) into 96-well plate, 100 μL/well, and administer after 12 hours of culture, the experimental group was added Different concentrations of test drugs were cultured for 24 and 48 hours respectively, 10 μL of CCK-8 solution was added to each well, the culture plate was incubated in the incubator for 4 hours, and the absorbance (OD) at 450 nm was measured with a microplate reader. Cell inhibition rate=(1-average OD value of experimental group/average OD value of control group)×100%.

The results of the CCK-8 experiment are shown in Table 1. Table 1 lists the survival rate of FHC cells after different concentrations of Zuojin Fang acted on FHC cells for 24 hours and 48 hours (n=6).

Table 1 The effect of different concentrations of Zuojinfang on the viability of FHC cells for different times (n=6)

*: p<0.05 compared with the control group; **: p<0.01 compared with the control group;

Figure 1 is the graph of Table 1, from which the relationship between the left golden square and the survival rate of FHC cells can be seen more intuitively.

The results of CCK-8 experiment showed that: 1) Zuojinfang had little effect on the viability of FHC cells, that is, when the administration concentration increased to a certain level, the cell viability was slightly weakened with the increase of administration concentration and the prolongation of the action time ; 2) A small dose of Zuojin Fang can promote the proliferation and division of human normal colorectal mucosal epithelial cells.

Effect of 1.25-FU on the survival rate of FHC cells

Take human normal colorectal mucosal epithelial cells FHC logarithmic growth phase cells, use DMEM medium to make cell suspension (5×104/mL) into 96-well plate, 100 μL/well, and administer after 12 hours of incubation, the experimental group was added Different concentrations of test drugs were cultured for 24 and 48 hours respectively, 10 μL of CCK-8 solution was added to each well, the culture plate was incubated in the incubator for 4 hours, and the absorbance (OD) at 450 nm was measured with a microplate reader. The survival rate of the untreated control group cells was set as 100%. Cell inhibition rate=(1-average OD value of experimental group/average OD value of control group)×100%.

The results of the CCK-8 experiment are shown in Table 2. Table 2 lists the survival rate of FHC cells (n=6) after different concentrations of 5-FU acted on the FHC cells for 24 hours and 48 hours.

Table 2 The effect of different concentrations of 5-FU on the survival rate of FHC cells for different times (n=6).

*: p<0.05 compared with the control group; **: p<0.01 compared with the control group

Fig. 2 is a graph of Table 2. From Fig. 2, the relationship between 5-FU and the survival rate of FHC cells can be seen more intuitively (n=6).

From the test results, it can be known that: 1) the effect of 5-FU on the viability of FHC cells presents a "concentration-time-dependent" relationship, that is, with the increase of the administration concentration and the prolongation of the action time, the cell viability gradually weakens; 2) and Zuo Jin Fang is different, a small dose of 5-FU can cause cytotoxicity, and its toxicity is also enhanced as the concentration of the drug increases.

[Example 2] Effect of Zuojinfang or 5-FU alone on the apoptosis rate of HT-29 cells

2.1 Effect of Zuojin Fang on the apoptosis rate of HT-29 cells

Take HT-29 cells in the logarithmic growth phase, adjust the cell density to 2×10 ⁵ /mL with DMEM medium, and seed them into 6-well plates at 1.5 mL/well. Each group had 6 replicate wells, cultured for 12 hours, then added the corresponding drugs for 24 hours, scraped the cells with a cell brush, centrifuged at 800r·min-1 for 5 minutes, removed the supernatant, added PBS to resuspend, and adjusted the cell concentration to 1×10 ⁶ / mL. Take an appropriate amount into a flow tube, add 500uL of working solution, and then add fluorescent dye, filter through a 350-mesh nylon mesh filter to remove cell clumps, stain at room temperature in the dark for 15 minutes, and then run a flow cytometer to detect the cell apoptosis rate. The measured data is processed according to the software configured by the flow cytometer, and the sum of the early apoptosis rate represented by the lower right plus the middle and late apoptosis rate represented by the upper right represents the apoptosis rate.

The test results are shown in Table 3. Table 3 lists the apoptosis rate of HT-29 cells after different concentrations of Zuojin Fang acted on HT-29 cells for 24 hours (n=6).

Table 3 Effects of different concentrations of Zuojin Fang on the apoptosis rate of HT-29 cells for 24 hours (n=6)

*: p<0.05 compared with the control group; **: p<0.01 compared with the control group

Fig. 3 is a graph of Table 3, from which the relationship between 5-FU and the apoptosis rate of HT-29 cells can be seen more intuitively (n=6).

From the test results, it can be seen that: 1) The effect of Zuojinfang on the apoptosis rate of HT-29 presents a "concentration-time-dependent" relationship, that is, with the increase of the administration concentration and the prolongation of the action time, the apoptosis rate gradually increases ; 2) A small dose of Zuojinfang can promote the apoptosis of HT-29 cells.

2. Effect of 25-FU on apoptosis rate of HT-29 cells

Take the cells in the logarithmic growth phase, adjust the cell density to 2×10 ⁵ /mL with DMEM medium, and seed them into 6-well plates at 1.5 mL/well. Each group had 6 replicate wells, cultured for 12 hours, then added the corresponding drugs for 24 hours, scraped the cells with a cell brush, centrifuged at 800r·min-1 for 5 minutes, removed the supernatant, added PBS to resuspend, and adjusted the cell concentration to 1×10 ⁶ / mL. Take an appropriate amount into a flow tube, add 500uL of working solution, and then add fluorescent dye, filter through a 350-mesh nylon mesh filter to remove cell clumps, stain at room temperature in the dark for 15 minutes, and then run a flow cytometer to detect the cell apoptosis rate. The measured data is processed according to the software configured by the flow cytometer, and the sum of the early apoptosis rate represented by the lower right plus the middle and late apoptosis rate represented by the upper right represents the apoptosis rate.

The test results are shown in Table 4. Table 4 lists the apoptosis rate of HT-29 cells after different concentrations of 5-FU acted on HT-29 cells for 24 hours (n=6).

Table 4 Effects of different concentrations of 5-FU on the apoptosis rate of HT-29 cells for 24h (n=6)

*: p<0.05 compared with the control group; **: p<0.01 compared with the control group

Figure 4 is the graph of Table 4, from which the relationship between 5-FU and the apoptosis rate of HT-29 cells can be seen more intuitively.

From the test results, it can be known that: 1) The effect of 5-FU on the apoptosis rate of HT-29 presents a "concentration-time-dependent" relationship, that is, with the increase of the administration concentration and the prolongation of the action time, the apoptosis rate gradually increases 2) The apoptotic rates of HT-29 cells treated with 50.00ug·mL ^-1 and 100.00ug·mL ^-1 5-FU for 24h were (50.40±1.45)% and (65.30±1.27)%, respectively.

[Example 3] Combination of Zuojinfang and 5-FU

3.1 Selection of combination drug concentration

When the concentration of Zuojin Fang is 1.00ug·mL-1, the cell survival rate is (106.68±1.65)% when the FHC cells are treated for 24 hours, the cell survival rate is (100.16±1.23)% after 48 hours, and the apoptosis rate is 24 hours when the HT-29 cells are treated (37.90±1.06)%, 1.00ug·mL ^-1 was selected as the concentration of Zuojinfang combined medication.

3. Selection of concentration of 25-FU combined drug

When the concentration of 5-FU was 50.00ug·mL-1, the cell survival rate was (47.76±1.48)% when the FHC cells were treated for 24 hours, and the cell survival rate was (37.69±1.62)% after 48 hours. (50.40±1.45)%.

When the concentration of 5-FU was 100.00ug·mL-1, the cell survival rate was (42.42±1.74)% when the FHC cells were treated for 24 hours, and the cell survival rate was (32.79±1.95)% after 48 hours. (65.30±1.27)%, select 50.00, 100.00ug·mL-1 as the concentration of 5-FU combined drug.

3.3 The effect of Zuojinfang combined with 5-FU on the survival rate of FHC cells

Take human normal colorectal mucosal epithelial cells FHC logarithmic growth phase cells, use DMEM medium to make cell suspension (5×104/mL) into 96-well plate, 100 μL/well, and administer after 12 hours of incubation, the experimental group was added Different concentrations of test drugs were cultured for 24 and 48 hours respectively, 10 μL of CCK-8 solution was added to each well, the culture plate was incubated in the incubator for 4 hours, and the absorbance (OD) at 450 nm was measured with a microplate reader. Cell inhibition rate=(1-average OD value of experimental group/average OD value of control group)×100%.

The test results are shown in Table 5 and Table 6. Table 5 and Table 6 list the survival rate of the control group, 1.00ug·mL ^-1 Zuojin Fang, 50.00ug·mL ^-1 5-FU and the combination of the two drugs on FHC cells for 24h and 48h respectively ( n=6).

Table 5 Effects of Zuojinfang and 5-FU combined drug action on the survival rate of FHC cells at different times (n=6)

*: p<0.05 between the combined drug group and the Zuojinfang 1.00ug·mL ^-1 group; #: p<0.05 between the combined drug group and the 5-FU50.00ug·mL ^-1 group

Figure 5 is the graph in Table 5. From Figure 5, we can more intuitively see the relationship between Zuojinfang and 50.00ug·mL ^-1 5-FU combined with 24h and the survival rate of FHC cells.

Table 6 lists the control group, 1.00ug·mL-1Zuojinfang, 100.00ug·mL-15-FU and the combination of the two respectively acted on the survival rate of FHC cells after 24h and 48h (n=6) .

Table 6 The effect of Zuojinfang and 5-FU combined drug action on the survival rate of FHC cells at different times (n=6)

*: p<0.05 between the combined drug group and the Zuojin Fang 1.00ug·mL ^-1 group; #: p<0.05 between the combined drug group and the 5-FU100.00ug·mL ^-1 group

Figure 6 is the graph in Table 6. From Figure 6, it can be seen more intuitively the relationship between Zuojinfang and 100.00ug·mL ^-1 5-FU combined for 24 hours and the survival rate of FHC cells.

From the test results, it can be seen that the combination of Zuojinfang and 5-FU can greatly improve the cell survival rate of FHC in human normal colorectal mucosal epithelial cells. Compared with Zuojinfang and 5-FU alone, the combined drug group had p<0.05, and there was a significant statistical difference, that is, the cytoprotective effect of the combined drug group was better than that of each single drug group.

3.4 Effects of Zuojinfang combined with 5-FU on the apoptosis rate of HT-29 cells

Take the cells in the logarithmic growth phase, adjust the cell density to 2×10 ⁵ /mL with DMEM medium, and seed them into 6-well plates at 1.5 mL/well. Each group had 6 replicate wells, cultured for 12 hours, then added the corresponding drugs for 24 hours, scraped the cells with a cell brush, centrifuged at 800r·min-1 for 5 minutes, removed the supernatant, added PBS to resuspend, and adjusted the cell concentration to 1×10 ⁶ / mL. Take an appropriate amount into a flow tube, add 500uL of working solution, and then add fluorescent dye, filter through a 350-mesh nylon mesh filter to remove cell clumps, stain at room temperature in the dark for 15 minutes, and then run a flow cytometer to detect the cell apoptosis rate. The measured data is processed according to the software configured by the flow cytometer, and the sum of the early apoptosis rate represented by the lower right plus the middle and late apoptosis rate represented by the upper right represents the apoptosis rate.

The test results are shown in Table 7 and Table 8. Table 7 and Table 8 list the control group, Zuojinfang at selected concentrations, 5-FU at selected concentrations, and Zuojinfang at selected concentrations combined with 5-FU at selected concentrations acting on HT-29 cells for 24 hours After that, the apoptosis rate of HT-29 cells (n=6).

Table 7 Effects of Zuojinfang and 5-FU in combination for 24 hours on the apoptosis rate of HT-29 cells (n=6)

*: p<0.05 between the combined drug group and the Zuojinfang 1.00ug·mL ^-1 group; #: p<0.05 between the combined drug group and the 5-FU50.00ug·mL ^-1 group

Fig. 7 is the graph of Table 7. From Fig. 7, it can be seen more intuitively the relationship between Zuojinfang and 5-FU combined medication and the apoptosis rate of HT-29 cells.

Table 8 Effects of Zuojinfang and 5-FU in combination for 24 hours on the apoptosis rate of HT-29 cells (n=6)

*: p<0.05 between the combined drug group and the Zuojin Fang 1.00ug·mL ^-1 group; #: p<0.05 between the combined drug group and the 5-FU100.00ug·mL ^-1 group

Figure 8 is the graph of Table 8. From Figure 8, it can be seen more intuitively the relationship between Zuojinfang and 5-FU combined medication and the apoptosis rate of HT-29 cells.

It can be seen from the test results that 1.00ug·mL ^-1 Zuojin Fang was combined with 50.00ug·mL ^-1 and 100.00ug·mL ^-1 5-FU to act on HT-29 cells for 24 hours, and the cell apoptosis rate was greatly increased. Compared with Zuojin Fang and 5-FU alone, the combined drug group has a significant statistical difference, p<0.05, that is, the effect of the combined drug group on inducing apoptosis is better than that of the individual drug groups.

3.5 Effects of Zuojinfang combined with 5-FU on Bcl-2, Bax and Survivin proteins

Protein extraction: Add 10 μL of PMSF to 1 mL of cell lysate RIPA, take about 2.4×10 ⁶ cells that have been treated with drugs and add to the RIPA, lyse on ice for 30 min, centrifuge at 10,000 r·min ^-1 for 10 min, and take the supernatant.

The protein concentration was determined by BCA, and the wavelength of the microplate reader was A562, and the protein concentration was calculated according to the standard curve. Cast 12% separating gel and 4% stacking gel for SDS-PAGE gel. Aspirate 80μg total protein and electrophoresis at a constant voltage of 100V until bromophenol blue reaches the bottom of the separating gel. Wet electroporation with constant voltage of 100V, 120min, transfer protein from SDS-PAGE gel to PVDF membrane. 3% skimmed milk powder/PBS-T was blocked, diluted Rabbitanti-Bcl-2/-Bax/-Survivin at 1:500, and incubated overnight at 4°C. 1:3000 diluted secondary antibody Goatanti-RabbitIgG, incubated for 1h, enhanced chemiluminescence (enhancedchemiluminescence, ECL) development. UVI gel imaging system camera.

Figure 9 lists the blank control, 1.00μg/mL Zuojinfang, 100.0μg/mL5-FU, and 1.00μg/mL Zuojinfang combined with 100.0μg/mL5-FU respectively acted on HT-2924h to regulate BAX and Bcl in cells -2 and Survivin protein expression of Western Blotting gel electrophoresis (n = 6).

It can be seen from the test results that after 1.00ug·mL ^-1 Zuojinfang and 100.00ug·mL ^-1 5-FU acted on HT-29 cells for 24 hours, the expression of Bax increased, while the expression of Bcl-2 and Survivin proteins decreased ; Zuojinfang combined with 5-FU (1.00+100.00) combined treatment for 24 hours, the expression of Bax further increased, while the expression of Bcl-2 and Survivin protein further decreased, compared with each single treatment group, P<0.05, the difference was significant.

3.6 Effects of Zuojinfang combined with 5-FU on the morphology of HT-29 cells

HT-29 cells (2×10 ⁵ ·mL ^-1 ) were inoculated on the coverslips in 6-well plates, each experimental group was given corresponding drugs as required, and freshly prepared 4% paraformaldehyde (pH 7.4) was added , fix the cells at 37°C for 30 min, wash twice with PBS, add 5 mg/L Hochest33258 staining solution for staining for 30 min, wash with PBS, wash with mounting solution (20mmol·L ^-1 citric acid, 50mmol·L ^-1 disodium hydrogen phosphate , 50% glycerol, pH 5.5) after mounting the slides, observed and photographed with a fluorescence microscope.

The test results are shown in Figure 10. Figure 10 is the blank control, 1.00 μg/mL Zuojinfang, 50.0 μg/mL5-FU, 100.00 μg/mL5-FU, 1.00 μg/mL Zuojinfang combined with 50.0 μg/mL5-FU And 1.00μg/mL Zuojin Fang combined with 100.0μg/mL5-FU acted on HT-2924h cell morphology microscope observation picture (×200).

It can be seen from the test results that after Hoehest33258 fluorescent staining, under the fluorescence microscope, the cell membranes of HT-29 cells in the blank group were complete, the nucleus was plump, the nucleoplasm was lightly stained, and the density was uniform, as shown in Figure 10A; 1.00ug·mL ^-1 left After being treated with gold square, 50.00ug·mL ^-1 5-FU, and 100.00ug·mL ^-1 5-FU for 24 hours, the cells showed typical apoptotic morphological features: cell membrane shrinkage, relatively smaller nuclei, significantly enhanced fluorescence, and became highly blocky , see Figures 10B, 10C, and 10D; Zuojinfang+5-FU (1.00+50.00) and Zuojinfang+5-FU (1.00+100.00) were combined for 24 hours. In addition to the above characteristics, the cells showed apoptotic bodies The number is obviously more than that of each single medication group, see Fig. 10E, 10F.

In summary, the combination of Zuojinfang and 5-FU has a better therapeutic effect on intestinal cancer than Zuojinfang, or 5-FU alone. The cell survival rate of FHC in rectal mucosal epithelial cells greatly reduced the cytotoxic effect of 5-FU, and promoted colon cancer by promoting the high expression of Bax protein and the low expression of Bcl-2 and Survivin proteins in intestinal cancer cell HT-29 cells. Apoptosis of cells. The research results of the present invention have great significance for reducing the toxic and side effects of clinical anti-intestinal cancer chemotherapeutic drugs and improving the therapeutic effect.

Claims (7)

1. Chinese Drug Rhizomes of Coptis is combined in the medicine of preparation treatment intestinal cancer with 5-fluorouracil with the extract of Fructus Evodiae Purposes.

2. purposes as claimed in claim 1, wherein said Chinese Drug Rhizomes of Coptis is mass ratio with the extract of Fructus Evodiae Chinese Drug Rhizomes of Coptis and the extract of Fructus Evodiae for 6:1.

3. purposes as claimed in claim 2, wherein said extract is water extract.

4. purposes as claimed in claim 1, wherein said combine for by 5-fluorouracil Chinese Drug Rhizomes of Coptis and Wu Before the extract of the Fructus Evodiae, give patient simultaneously or after.

5. purposes as claimed in claim 1, the extract of wherein said Chinese Drug Rhizomes of Coptis and Fructus Evodiae is according to Chinese medicine Prepared by the method for compound Zuojin prescription.

6. purposes as claimed in claim 5, the preparation method of wherein said Zuojin prescription includes: take 30g Rhizoma Coptidis With 5g Fructus Evodiae, add 8 times of distilled water immersions 30 minutes, boiling 3 times, each 30 minutes, filter, Merge 3 filtrates, lyophilization, every gram of dry powder 3.98g Han crude drug；Measure through HPLC method, Zuojin prescription lyophilizing Containing berberine 39.85% in powder, palmatine 9.19%, jateorhizine 4.86%, rutaecarpin 0.17%, Fructus Evodiae Alkali 0.19%.

7. purposes as claimed in claim 1, wherein said intestinal cancer is colon cancer.