A traditional Chinese medicine composition for treating esophageal cancer and a preparation method and application thereof

Abstract

The present application relates to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for treating esophageal cancer and a preparation method and application thereof. The present application provides a traditional Chinese medicine composition for treating esophageal cancer, which comprises the following components: zerumbet extract, kadsura extract and trillium extract; the concentration of the zerumbet extract is 61.23-102.16 μg / mL; the concentration of the kadsura extract is 13.41-45.20 μg / mL; and the concentration of the trillium extract is 6.42-15.06 μg / mL. The traditional Chinese medicine composition can inhibit the proliferation, migration and clone formation of esophageal cancer cells, induce cell cycle arrest, and down-regulate the expression of epidermal growth factor receptor, protein kinase B and mammalian target of rapamycin.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for treating esophageal cancer and a preparation method and application thereof. BACKGROUND

[0002] Esophageal cancer (EC) is one of the most common malignant tumors worldwide and the sixth leading cause of cancer-related death. Among them, esophageal squamous cell carcinoma (ESCC) is the most common histological type of esophageal cancer worldwide.

[0003] With the continuous development of molecular biology, omics, genetics and other fields, the occurrence and development mechanism of esophageal cancer at the gene and molecular level has been revealed, including the development of drugs targeting signaling pathways (such as EGFR pathway, HGF / c-Met pathway), epigenetics (such as DNA methyltransferase, histone modification, non-coding RNA), immune checkpoints (such as programmed death receptor 1 / programmed death ligand 1 (PD-1 / PD-L1), cytotoxic T lymphocyte-associated protein 4 (CTLA4)) into clinical. Epidermal growth factor receptor (EGFR) belongs to the ErbB receptor tyrosine kinase family. When combined with ligands, it dimerizes, and then EGFR autophosphorylation activates downstream PI3K / AKT / mTOR, RAS / MAPK and STAT3 signaling pathways, which are involved in tumorigenesis. EGFR is significantly up-regulated in ESCC tissues and is associated with clinical stage, tumor invasion and poor prognosis. The downstream PI3K / AKT pathway is abnormally activated in various tumors including esophageal cancer, and AKT is a target of PI3K downstream. After activation, it can phosphorylate and activate mTOR, which is an important regulator of mammalian cell growth, metabolism, aging and other life activities. Its abnormal regulation is closely related to the proliferation, apoptosis, migration, metabolism and drug resistance of tumor cells.

[0004] Conventional Western medicine treatment regimens have side effects, easy recurrence, drug resistance, and high cost, among others. Surgical resection, radiotherapy and chemotherapy are still the main clinical treatment methods for esophageal cancer. However, due to the limited efficacy of conventional treatment and severe adverse reactions, the results are not satisfactory. The comprehensive treatment method of traditional Chinese medicine combined with radiotherapy and chemotherapy is the advantage of treating esophageal cancer in China, which plays a significant role in synergistic effect and toxicity reduction. A large number of studies have shown that extracts and monomer compounds obtained from traditional Chinese medicine show potential anticancer activity against esophageal cancer. Exploring effective anticancer drugs from traditional Chinese medicine and clarifying their mechanisms of action have become a hot spot in the research and development of antitumor drugs, and are also a problem to be solved. SUMMARY

[0005] The present application aims to provide a traditional Chinese medicine composition for treating esophageal cancer, which can inhibit esophageal cancer cell proliferation, migration, clonogenicity, induce cell cycle arrest, and down-regulate the protein expression of epidermal growth factor receptor, protein kinase B and mammalian target of rapamycin.

[0006] To achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0007] The present application provides a traditional Chinese medicine composition for treating esophageal cancer, which comprises the following components: Zerumbet extract, Radix et Rhizoma Baphlantis extract and Radix et Rhizoma Polygonatum extract.

[0008] The concentration of the Zerumbet extract is 61.23-102.16 μg / mL; the concentration of the Radix et Rhizoma Baphlantis extract is 13.41-45.20 μg / mL; and the concentration of the Radix et Rhizoma Polygonatum extract is 6.42-15.06 μg / mL.

[0009] Preferably, the Zerumbet extract is prepared by mixing Zerumbet with ethanol, dark-soaking, suction-filtering, concentrating, drying and dissolving.

[0010] The initial concentration of the ethanol is 90-98 wt.%.

[0011] The mass-volume ratio of the Zerumbet and ethanol mixture is 1-3 g:4-6 mL.

[0012] The dark-soaking time is 6-8 days.

[0013] The suction-filtering is negative pressure suction-filtering, the pressure is-0.03 to-0.05 MPa, and the time is 1-2 min.

[0014] The concentration method is water bath rotary evaporation concentration, the temperature is 50-60℃, the rotation speed is 60-65 rmp, and the time is 10-20 min.

[0015] The drying method is vacuum freeze-drying, the vacuum degree is 2-5 Pa, the cold trap temperature is-60 to-70℃, and the time is 48-72 h.

[0016] Preferably, the Radix et Rhizoma Baphlantis extract is prepared by mixing Radix et Rhizoma Baphlantis with ethanol, dark-soaking, suction-filtering, concentrating, drying and dissolving.

[0017] The initial concentration of the ethanol is 90-98 wt.%.

[0018] The mass-volume ratio of the Radix et Rhizoma Baphlantis and ethanol mixture is 1-3 g:4-6 mL.

[0019] The light-avoiding soaking time is 6-8 days;

[0020] The suction filtration is negative pressure suction filtration; the pressure of the suction filtration is -0.03 to -0.05 MPa; and the time of the suction filtration is 1-2 min.

[0021] The concentration method is water bath rotary evaporation concentration; the concentration temperature is 50-60 DEG C; the concentration rotating speed is 60-65 rmp; and the concentration time is 10-20 min.

[0022] The drying method is vacuum freeze drying; the vacuum degree of the drying is 2-5 Pa; the cold trap temperature of the drying is -60 to -70 DEG C; and the drying time is 48-72 h.

[0023] As preferred, the preparation method of the Trillium tschonoskii Maxim extract is as follows: Trillium tschonoskii Maxim and ethanol are mixed, light-avoiding soaking is carried out, suction filtration, concentration, drying and dissolution are carried out to obtain the Trillium tschonoskii Maxim extract.

[0024] The initial concentration of the ethanol is 90-98 wt%;

[0025] The mass-volume ratio of the Trillium tschonoskii Maxim and ethanol mixing is 1-3 g:4-6 mL;

[0026] The light-avoiding soaking time is 6-8 days;

[0027] The suction filtration is negative pressure suction filtration; the pressure of the suction filtration is -0.03 to -0.05 MPa; and the time of the suction filtration is 1-2 min.

[0028] The concentration method is water bath rotary evaporation concentration; the concentration temperature is 50-60 DEG C; the concentration rotating speed is 60-65 rmp; and the concentration time is 10-20 min.

[0029] The drying method is vacuum freeze drying; the vacuum degree of the drying is 2-5 Pa; the cold trap temperature of the drying is -60 to -70 DEG C; and the drying time is 48-72 h.

[0030] The application further provides a preparation method of the traditional Chinese medicine composition, comprising the following steps: mixing Trillium tschonoskii Maxim extract, Anisomeles indica extract and Trillium tschonoskii Maxim extract to obtain the traditional Chinese medicine composition.

[0031] The application further provides application of the traditional Chinese medicine composition in preparation of a medicine for treating esophageal cancer.

[0032] The application further provides application of the traditional Chinese medicine composition prepared by the preparation method in preparation of a medicine for treating esophageal cancer.

[0033] The beneficial effects of the present application are as follows:

[0034] The present application takes EC-1, Eca109, TE-1, EC9706 four kinds of esophageal cancer cells as objects, and screens out effective traditional Chinese medicines against esophageal cancer from traditional Chinese medicines, i.e., Lingxiaozero, Dibutong and Delaying Grass; the traditional Chinese medicine composition can inhibit esophageal cancer cell proliferation, migration and clone formation, induce cell cycle arrest, and down-regulate the expression of epidermal growth factor receptor, protein kinase B and mammalian target of rapamycin, thereby providing a reference for basic research and clinical application of traditional Chinese medicine against esophageal cancer. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is the influence of each liquid on the morphology of EC-1 cells;

[0036] Figure 2 is the influence of each liquid on the morphology of Eca109 cells;

[0037] Figure 3 is the influence of each liquid on the morphology of EC9706 cells;

[0038] Figure 4 is the influence of each liquid on the morphology of TE-1 cells;

[0039] Figure 5 is the influence of each liquid on the clone formation rate (%) and morphology of four kinds of esophageal cancer cells;

[0040] Figure 6 is the influence of each liquid on the migration of four kinds of esophageal cancer cells;

[0041] Figure 7 is the influence of each liquid on the EC-1 cell cycle of four kinds of esophageal cancer cells;

[0042] Figure 8 is the influence of each liquid on the Eca109 cell cycle of four kinds of esophageal cancer cells;

[0043] Figure 9 is the influence of each liquid on the EC9706 cell cycle of four kinds of esophageal cancer cells;

[0044] Figure 10 is the influence of each liquid on the TE-1 cell cycle of four kinds of esophageal cancer cells;

[0045] Figure 11 is the influence of the heat-clearing and qi-moving and blood-activating prescription on the expression of EGFR / AKT pathway proteins of four kinds of esophageal cancer cells. DETAILED DESCRIPTION

[0046] The present application provides a traditional Chinese medicine composition for treating esophageal cancer, which comprises the following components: Lingxiaozero extract, Dibutong extract and Delaying Grass extract.

[0047] the concentration of the Zilingxiang extract is 61.23-102.16 μg / mL; the concentration of the Dikong extract is 13.41-45.20 μg / mL; and the concentration of the Yanlingcao extract is 6.42-15.06 μg / mL;

[0048] In the present application, the concentration of the Zilingxiang extract is preferably 71.23-92.16 μg / mL, further preferably 81.70 μg / mL; the concentration of the Dikong extract is preferably 23.41-35.20 μg / mL, further preferably 29.31 μg / mL; and the concentration of the Yanlingcao extract is preferably 10.42-11.06 μg / mL, further preferably 10.74 μg / mL.

[0049] In the present application, the Zilingxiang extract is prepared by mixing Zilingxiang and ethanol, dark-soaking, suction filtration, concentration, drying, and dissolution;

[0050] The initial concentration of the ethanol is 90-98 wt%, preferably 94%;

[0051] The mass-volume ratio of the mixture of Zilingxiang and ethanol is 1-3 g:4-6 mL, preferably 1 g:5 mL;

[0052] The dark-soaking time is 6-8 d, preferably 7 d;

[0053] The suction filtration is negative pressure suction filtration; the pressure of the suction filtration is -0.03 to -0.05 MPa, preferably -0.04 MPa; and the suction filtration time is 1-2 min, preferably 1.5 min;

[0054] The concentration method is water bath rotary evaporation concentration; the concentration temperature is 50-60 °C, preferably 55 °C; the concentration rotation speed is 60-65 rmp, preferably 62.5 rmp; and the concentration time is 10-20 min, preferably 15 min;

[0055] The drying method is vacuum freeze drying; the vacuum degree of the drying is 2-5 Pa, preferably 3.5 Pa; the cold trap temperature of the drying is -60 to -70 °C, preferably -65 °C; and the drying time is 48-72 h, preferably 60 h;

[0056] The solvent used for the dissolution is one or several of dimethyl sulfoxide, water, a fetal bovine serum-free medium, and a fetal bovine serum-containing medium.

[0057] In the present application, the preparation method of the extract of Radix Trichosanthis is as follows: mixing Radix Trichosanthis and ethanol, light-proof soaking, suction filtration, concentration, drying, and dissolution to obtain the extract of Radix Trichosanthis.

[0058] The initial concentration of the ethanol is 90-98 wt%, preferably 94%.

[0059] The mass-volume ratio of the mixing of Radix Trichosanthis and ethanol is 1-3 g:4-6 mL, preferably 1 g:5 mL.

[0060] The light-proof soaking time is 6-8 d, preferably 7 d.

[0061] The suction filtration is negative pressure suction filtration; the pressure of the suction filtration is -0.03 to -0.05 MPa, preferably -0.04 MPa; the suction filtration time is 1-2 min, preferably 1.5 min.

[0062] The concentration method is water bath rotary evaporation concentration; the concentration temperature is 50-60℃, preferably 55℃; the concentration rotation speed is 60-65 rmp, preferably 62.5 rmp; the concentration time is 10-20 min, preferably 15 min.

[0063] The drying method is vacuum freeze drying; the vacuum degree of the drying is 2-5 Pa, preferably 3.5 Pa; the cold trap temperature of the drying is -60 to -70℃, preferably -65℃; the drying time is 48-72 h, preferably 60 h.

[0064] The solvent used for the dissolution is one or more of dimethyl sulfoxide, water, fetal bovine serum-free medium, and fetal bovine serum-containing medium.

[0065] In the present application, the preparation method of the extract of Radix Trichosanthis is as follows: mixing Radix Trichosanthis and ethanol, light-proof soaking, suction filtration, concentration, drying, and dissolution to obtain the extract of Radix Trichosanthis.

[0066] The initial concentration of the ethanol is 90-98 wt%, preferably 94%.

[0067] The mass-volume ratio of the mixing of Radix Trichosanthis and ethanol is 1-3 g:4-6 mL, preferably 1 g:5 mL.

[0068] The light-proof soaking time is 6-8 d, preferably 7 d.

[0069] The suction filtration is negative pressure suction filtration; the pressure of the suction filtration is -0.03 to -0.05 MPa, preferably -0.04 MPa; the suction filtration time is 1-2 min, preferably 1.5 min.

[0070] The concentration method is water bath rotary evaporation concentration; the concentration temperature is 50-60 DEG C, preferably 55 DEG C; the concentration rotation speed is 60-65 rmp, preferably 62.5 rmp; the concentration time is 10-20 min, preferably 15 min;

[0071] The drying method is vacuum freeze drying; the drying vacuum degree is 2-5 Pa, preferably 3.5 Pa; the drying cold trap temperature is -60 to -70 DEG C, preferably -65 DEG C; the drying time is 48-72 h, preferably 60 h;

[0072] The solvent used for dissolving is one or several of dimethyl sulfoxide, water, fetal bovine serum-free medium, fetal bovine serum-containing medium.

[0073] The application further provides a preparation method of the traditional Chinese medicine composition, comprising the following steps: mixing Lingxian extract, Dinei Rong extract and Yanchengao extract to obtain the traditional Chinese medicine composition.

[0074] The application further provides application of the traditional Chinese medicine composition in preparation of a medicament for treating esophageal cancer.

[0075] The application further provides application of the traditional Chinese medicine composition prepared by the preparation method in preparation of a medicament for treating esophageal cancer.

[0076] The technical solutions provided by the application will be described in detail below in combination with examples, but they should not be understood as limitations to the protection scope of the application.

[0077] The human esophageal cancer cell lines Eca109, EC-1, TE-1 and EC9706 used in the experimental examples are from the Key Laboratory of Signal Transduction of Traditional Chinese Medicine Prescriptions in Henan Province of Henan University of Chinese Medicine.

[0078] The cell cycle detection kit used in the experimental examples is purchased from Hangzhou Lianke Biotechnology Co., Ltd., the BCA protein quantification kit and the ECL hypersensitive luminescence kit are purchased from Kangwei Century Biotechnology Co., Ltd., the low-melting-point agarose dry powder is purchased from Beijing Solabio Technology Co., Ltd., the CIM-Plate16 detection plate is purchased from American Eisen Bioscience Co., Ltd., the GAPDH rabbit anti-human polyclonal antibody is purchased from Beijing Boao Sun Biotechnology Co., Ltd., the goat anti-rabbit immunoglobulin G is purchased from Wuhan Dr. Deki Biotechnology Co., Ltd., the AKT rabbit anti-human polyclonal antibody is purchased from GeneTex Co., Ltd., the skimmed milk powder is purchased from Wandashan Dairy Co., Ltd., and the mTOR rabbit anti-human monoclonal antibody and the EGFR rabbit anti-human monoclonal antibody are purchased from American Cell Signaling Technology.

[0079] Example 1

[0080] Zieria fructus extract: 50 g of Zieria fructus was placed in a 500 mL brown bottle, 250 mL of 95% ethanol was added for cold soaking, and it was shaken once a day for 5 minutes each time. After 7 days of soaking, negative pressure filtration was first performed at -0.04 MPa, then water bath rotary evaporation was performed at a temperature of 56°C and a speed of 65 rpm, and then vacuum freeze drying was performed at 4 Pa and -65°C. The alcohol-extracted dried Zieria fructus was dissolved with dimethyl sulfoxide to prepare a Zieria fructus extract with a concentration of 300 mg / mL.

[0081] Radix et rhizoma bupleuri extract: 50 g of Radix et rhizoma bupleuri was placed in a 500 mL brown bottle, 250 mL of 95% ethanol was added for cold soaking, and it was shaken once a day for 5 minutes each time. After 7 days of soaking, negative pressure filtration was first performed at -0.05 MPa, then water bath rotary evaporation was performed at a temperature of 56°C and a speed of 60 rpm, and then vacuum freeze drying was performed at 3 Pa and -65°C. The alcohol-extracted dried Radix et rhizoma bupleuri was dissolved with dimethyl sulfoxide to prepare a Radix et rhizoma bupleuri extract with a concentration of 300 mg / mL.

[0082] Radix et rhizoma trillii extract: 50 g of Radix et rhizoma trillii was placed in a 500 mL brown bottle, 250 mL of 95% ethanol was added for cold soaking, and it was shaken once a day for 5 minutes each time. After 7 days of soaking, negative pressure filtration was first performed at -0.05 MPa, then water bath rotary evaporation was performed at a temperature of 56°C and a speed of 63 rpm, and then vacuum freeze drying was performed at 5 Pa and -65°C. The alcohol-extracted dried Radix et rhizoma trillii was dissolved with dimethyl sulfoxide to prepare a Radix et rhizoma trillii extract with a concentration of 300 mg / mL.

[0083] The Zieria fructus extract, Radix et rhizoma trillii extract, and Radix et rhizoma bupleuri extract with a concentration of 300 mg / mL were diluted with a fetal bovine serum-free medium to prepare Zieria fructus extract, Radix et rhizoma trillii extract, and Radix et rhizoma bupleuri extract with a concentration of 4 mg / mL. After filtration with a 0.22 μm microporous filter, the concentrations of the extracts were adjusted with a fetal bovine serum-containing medium, and then the extracts were mixed. The concentration of Zieria fructus extract in the prepared mixture was 67.77 μg / mL, the concentration of Radix et rhizoma bupleuri extract was 19.59 μg / mL, and the concentration of Radix et rhizoma trillii extract was 8.84 μg / mL. The prescription was a heat-clearing, qi-moving, and blood-activating prescription.

[0084] The medium was an RPMI-1640 medium.

[0085] Example 2

[0086] The zero-ginger extract, the ground not contain extract, and the trillium tschonoskii extract having a concentration of 300 mg / mL described in Example 1 were further adjusted in concentration with distilled water and mixed, and the prepared mixture had a concentration of 102.16 μg / mL of the zero-ginger extract, a concentration of 45.20 μg / mL of the ground not contain extract, and a concentration of 15.06 μg / mL of the trillium tschonoskii extract.

[0087] Example 3

[0088] The zero-ginger extract, the ground not contain extract, and the trillium tschonoskii extract having a concentration of 300 mg / mL described in Example 1 were further adjusted in concentration with distilled water and mixed, and the prepared mixture had a concentration of 61.23 μg / mL of the zero-ginger extract, a concentration of 13.41 μg / mL of the ground not contain extract, and a concentration of 7.81 μg / mL of the trillium tschonoskii extract.

[0089] Example 4

[0090] The zero-ginger extract, the ground not contain extract, and the trillium tschonoskii extract having a concentration of 300 mg / mL described in Example 1 were further adjusted in concentration with distilled water and mixed, and the prepared mixture had a concentration of 64.77 μg / mL of the zero-ginger extract, a concentration of 18.82 μg / mL of the ground not contain extract, and a concentration of 6.42 μg / mL of the trillium tschonoskii extract.

[0091] Comparative Example 1

[0092] The zero-ginger extract having a concentration of 4 mg / mL of Example 1 was filtered through a 0.22 μm microfiltration head, and the concentration of the extract was further adjusted with a 10% fetal bovine serum-containing medium to obtain a zero-ginger extract (zero-ginger) having a concentration of 67.77 μg / mL.

[0093] Comparative Example 2

[0094] The ground not contain extract having a concentration of 4 mg / mL of Example 1 was filtered through a 0.22 μm microfiltration head, and the concentration of the extract was further adjusted with a 10% fetal bovine serum-containing medium to obtain a ground not contain extract (ground not contain) having a concentration of 19.59 μg / mL.

[0095] Comparative Example 3

[0096] The trillium tschonoskii extract having a concentration of 4 mg / mL of Example 1 was filtered through a 0.22 μm microfiltration head, and the concentration of the extract was further adjusted with a 10% fetal bovine serum-containing medium to obtain a trillium tschonoskii extract (trillium tschonoskii) having a concentration of 8.84 μg / mL.

[0097] Inhibition of esophageal cancer cells by the traditional Chinese medicine composition of Experimental Example 1

[0098] Cell culture: the culture method of Eca109, EC-1, TE-1, EC9706 is as follows: the four kinds of esophageal cancer cells of Eca109, EC-1, TE-1, EC9706 are placed in a 37℃, 5% CO2, saturated humidity incubator for culture, the culture solution uses RPMI-1640 culture medium containing 10% fetal bovine serum, 100 U / mL penicillin / streptomycin, is subcultured once every 3 days, is subcultured 2 times, and esophageal cancer cells are obtained, and a cell suspension is prepared with a culture medium containing 10% fetal bovine serum.

[0099] According to the cell subculture step, the cell suspension is poured into a sample groove, and the esophageal cancer cells are inoculated in a 96-well culture plate at a density of 1×10 4 cells / well with a 12-hole gun, 200 μl per hole, and are placed in a cell culture box for culture.

[0100] After the cells are cultured for 24 h, the original culture solution is removed, 200 μl of RPMI-1640 culture medium containing 10% fetal bovine serum is added to the blank group, and 200 μl of the heat-clearing and qi-moving blood-activating formula, zerumbet, gendarussa, and trillium tschonoskii described in Example 1 and Comparative Examples 1-3 is added to the drug groups, respectively, and the inhibition rate of the esophageal cancer cells to the drugs is detected at 12 h, 24 h, 36 h, 48 h, and 60 h after the drugs are added, and the detection results are shown in Tables 1-4.

[0101] Table 1 Inhibition of EC-1 cells by each drug solution

[0102]

[0103]

[0104] Table 2 Inhibition of EC9706 cells by each drug solution

[0105]

[0106] Table 3 Inhibition of Eca109 cells by each drug solution

[0107]

[0108] Table 4 Inhibition of TE-1 cells by each drug solution

[0109]

[0110]

[0111] As can be seen from Tables 1-4, the inhibition rates of the single herbs and the formula on the four kinds of esophageal cancer cells increase with the increase of the drug concentration and the extension of the action time, showing a good dose-time dependent relationship, and the heat-clearing and qi-moving blood-activating formula has the strongest inhibition effect on the four kinds of cells when the drug acts for 48 hours.

[0112] Effect of the composition of the experimental example 2 on cell morphology

[0113] According to the cell passage procedure, count the cells, and inoculate the cell suspension described in the experimental example 1 into a 24-well plate at a number of 5x10 4 cells / well, 0.5 mL / well, and place in an incubator for 24 h; wash with phosphate buffer solution for 3 times; set up a blank group and a drug group, each with 3 replicate wells; add 0.5 mL of culture medium containing 10% fetal bovine serum to the blank group (control), and add 0.5 mL of the Qingre Xingqi Huoxue Formula, Zhongjingxiang, Diburong, and Yianlingcao described in the experimental example 1 and the comparative examples 1-3 to the drug group, respectively; continue to incubate for 24 h; discard the old culture medium, wash with phosphate buffer solution for 3 times, and leave 200 μl of phosphate buffer solution in each well; observe the cell morphology under an inverted microscope (100X) and take photos. The results are shown in Figures 1-4 .

[0114] As can be seen from Figures 1-4 , the cells in the blank group (control) of EC-1, EC9706, Eca109, and TE-1 grow adherently, have clear cell boundaries, good refractive index, and dense and uniform distribution, have an oval or fusiform shape, and have obvious nucleoli. After the cells in each group are treated with the drugs, the number of cells is obviously reduced, and the cell morphology in each group changes similarly, except that the cell morphology in the group treated with Zhongjingxiang does not change obviously. The changes include that part of the cells become shrunken and round, have vacuoles, have widened intercellular spaces, have blurred or disappeared boundaries, have poor adhesion, and there are floating dead cells and cell debris in the culture dish.

[0115] Effect of the composition of the experimental example 3 on esophageal cancer cell clones

[0116] (1) Pave the lower layer of gel: mix 1.2% agar with an equal volume of 2X culture medium containing 20% fetal bovine serum to prepare 0.6% lower layer gel, and pave 0.8 mL / well in a 24-well plate, and stand at room temperature until the agar is solidified.

[0117] (2) Pave the upper layer of gel: according to the cell passage procedure described in the experimental example 1, count the cells, and adjust the cell suspension described in the experimental example 1 to a density of 1x10 4 cells / mL; mix 0.6% agar with an equal volume of 2X culture medium containing 20% fetal bovine serum to prepare 0.35% upper layer agar, and add 0.8 mL of upper layer agar and 40 μl (400 cells) of single cell suspension to each well.

[0118] (3) Drug addition: 10 μl of the heat-clearing and qi-moving blood-activating formula, Zerumbet, Disporum, and Trillium described in Example 1 and Comparative Examples 1 to 3 was added to the drug addition groups, and an equal volume of a 10% fetal bovine serum-containing medium was added to the control group; the medium was allowed to solidify at 25°C, and the medium was incubated in a cell incubator at 37°C and 5% CO2for 15 days.

[0119] (4) Counting: the formation of the clone spheres was observed under an inverted microscope, the clone formation rate was calculated, and a photograph was taken; the clone formation rate = average number of cell clones / number of cells inoculated per well x 100%; the results are shown in Table 5 and Figure 5 .

[0120] Table 5 Effects of each drug solution on the clone formation rate (%) of four esophageal cancer cells

[0121] EC-1 Eca109 EC9706 TE-1 control 35.75±2.65 28.42±4.73 34.25±2.65 41.33±3.51 Linglingxiang 20.83±2.31** 19.08±3.21** 21.42±4.16** 25.25±4.00** Dibutong 9.17±1.53** 11.83±2.52** 10.83±2.52** 13.17±4.16** Yanlingcao 11.58±3.06** 12.83±2.08** 12.75±4.58** 11.17±2.08** Qingre Xingqi Huoxue Fang 10.83±3.79** 11.67±3.51** 13.08±3.21** 15.08±4.04**

[0122] Note: compared with the control group, **P < 0.001.

[0123] As can be seen from Table 5 and Figure 5 . Morphologically, the clone spheres of the control group were observed under a microscope to be spherical and clear in outline; compared with the control group, the clone spheres of each drug addition group were significantly smaller and irregular in shape. Quantitatively, the clone formation rate of the control group was between 28% and 42%, and the clone formation rate of Eca109 was the lowest at 28.42%. After the addition of the drugs, the number of clone spheres was significantly reduced, and the clone formation rate was between 9% and 26%.

[0124] Effects of the Chinese medicinal composition on the migration of esophageal cancer cells in Experimental Example 4

[0125] (1) Drug addition to the upper and lower chambers: the upper and lower chambers of the CIM-Plate plate were prepared, 165 μl of a 10% fetal bovine serum-containing medium was added to each well of the lower chamber of the control group, and 165 μl of the heat-clearing and qi-moving blood-activating formula, Zerumbet, Disporum, and Trillium described in Example 1 and Comparative Examples 1 to 3 was added to each well of the drug addition groups, and three replicates were set. The upper and lower chambers were fastened, 30 μl of a fetal bovine serum-free medium was added to the upper chamber, and the upper and lower chamber plate was placed in a cell incubator for 1 h.

[0126] (2) Preparation of a cell suspension: the cells were counted according to the cell subculturing procedure, the cell concentration was adjusted to 5 x 10 5 cells / mL with a fetal bovine serum-free medium, and the mixture was uniformly mixed to obtain a 5 x 10 5 cell fetal bovine serum-free cell suspension.

[0127] (3)Detect baseline: Put the CIM-Plate on the RTCA, the system automatically scans "Scan Plate", and displays "Connection OK" on the Message page, indicating that it has been connected. At this time, the baseline is detected to ensure that the CellIndex of each well is below 0.063.

[0128] (4)Seed plate: Take the CIM-Plate with the detected baseline from the RTCA, and add 100 μl of 5 x 10 4 cells fetal bovine serum cell suspension to each well of the upper chamber, and add 5 μl of the heat-clearing and qi-moving blood-activating formula, Zhongjingxiang, Dibutong, and Yanyingcao of Example 1 and Comparative Examples 1-3 to the drug group. Let stand for 30 min, and then put back on the RTCA after the cells are settled.

[0129] (5) After the system automatically scans "Scan Plate", set interval 15 min, Duration 48 h, and start real-time dynamic detection; the detection results are shown in Figs. 7-10. Figure 6

[0130] As can be seen from Figure 6 , each group of prescriptions can significantly inhibit the migration of the four esophageal cancer cells. The migration effect of each group of prescriptions in the four esophageal cancer cells is heat-clearing and qi-moving blood-activating formula > Dibutong > Yanyingcao > Yanyingcao.

[0131] Effect of the traditional Chinese medicine composition on the cell cycle in Experimental Example 5

[0132] Inoculate 5 x 10 5 cells / well of esophageal cancer cells in a 6-well plate, and incubate in a cell incubator; 24 h later, aspirate the supernatant, add 2 mL of 10% fetal bovine serum medium to the blank group (control), and add 2 mL of the heat-clearing and qi-moving blood-activating formula, Zhongjingxiang, Dibutong, and Yanyingcao of Example 1 and Comparative Examples 1-3 to the drug group, and continue to incubate in the incubator for 48 h; wash the cells with pre-cooled phosphate buffer solution twice, digest and centrifuge at 2000 rpm for 4 min, and then perform counting on 1 x 10 6 cells, add RNAase and PI staining agent according to the cell cycle detection kit instructions, react in the dark at 25°C for 30 min, filter with a 200-mesh nylon filter, and then detect by flow cytometry. The experiment is repeated three times, and the detection results are shown in Figs. 7-10, wherein Figure 7 is the effect on the cell cycle of EC-1, Figure 8 is the effect on the cell cycle of EC9706, Figure 9 is the effect on the cell cycle of Eca109, and Figure 10 is the effect on the cell cycle of TE-1.

[0133] From​Figure 7 As can be seen from Table 2, after adding drugs, the Lingxiang group had less effect on the cell cycle; the Dibutong group had a significant decrease in G1 phase cells (P<0.01) and an increase in G2 phase cells (P<0.01); the Yanchang group had a decrease in G1 phase cells (P<0.05) compared with the control group; the Qingre Xingqi Huoxue group had an increase in G2 phase cells (P<0.01) compared with the control group.

[0134] As can be seen from Table 3, after adding drugs, the Lingxiang group and the Yanchang group had less effect on the cell cycle; the Dibutong group had a significant decrease in S phase cells (P<0.01) and an increase in G2 phase cells (P<0.01); the Qingre Xingqi Huoxue group had an increase in G2 phase cells (P<0.01) and a decrease in S phase cells (P<0.01) compared with the control group. Figure 8 As can be seen from Table 4, after adding drugs, the Qingre Xingqi Huoxue group had less effect on the cell cycle; the Lingxiang group had a significant decrease in G1 phase cells (P<0.01) and an increase in G2 phase cells (P<0.01); the Dibutong group had a significant decrease in G1 phase cells (P<0.01) and an increase in G2 phase cells (P<0.01); the Yanchang group had a decrease in G1 phase cells (P<0.05) and an increase in S phase cells (P<0.05).

[0135] Figure 9 As can be seen from Table 5, after adding drugs, the Lingxiang group had a decrease in G1 phase cells (P<0.01); the Yanchang group had a decrease in G1 phase cells (P<0.01) and an increase in S phase cells (P<0.05); the Dibutong group and the Qingre Xingqi Huoxue group had a significant decrease in G1 phase cells (P<0.01) and an increase in G2 phase cells (P<0.01).

[0136] As can be seen from Table 6, after adding drugs, the Lingxiang group had a decrease in G1 phase cells (P<0.01); the Yanchang group had a decrease in G1 phase cells (P<0.01) and an increase in S phase cells (P<0.05); the Dibutong group and the Qingre Xingqi Huoxue group had a significant decrease in G1 phase cells (P<0.01) and an increase in G2 phase cells (P<0.01). Figure 10 Effect of the traditional Chinese medicine composition in Experimental Example 6 on the expression of epidermal growth factor receptor / protein kinase B (EGFR / AKT) pathway-related proteins

[0137] (1) Protein extraction and quantification: 24 hours after inoculation, the control group was subjected to routine cell replacement, and the drug groups were added with 2 mL of the Qingre Xingqi Huoxue formula described in Example 1; then, after 48 hours of drug incubation, the cells were scraped into 10 mL EP tubes with a spatula; centrifuged at 1000 rpm for 10 min; resuspended in a phosphate buffer solution and transferred to 1.5 mL EP tubes; centrifuged again at 1000 rpm for 10 min; ultrasonicated at 20 kHz with an interval of 4 s for 12 s; ice-bath lysed for 1 h; low-temperature high-speed centrifuged at 4°C and 15000 rpm for 15 min; and the supernatant was collected. According to the instructions of the BCA kit (purchased from Kangwei Century Biotechnology Co., Ltd.), the OD values of each well at 562 nm were measured, and the protein liquid concentration was determined by data analysis. The results are shown in Table 6.

[0138]

[0139] ​​(2) Protein quantification and denaturation: according to the configuration of BCA working solution, the BCA standard was diluted with phosphate buffer solution to a concentration of 2000 μg / mL, 1500 μg / mL, 1000 μg / mL, 750 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 25 μg / mL, 0 μg / mL, a total of 9 concentrations, the sample to be tested was diluted 5 times with phosphate buffer solution, 25 μl of the prepared standard or sample was added to each well, and 200 μl of BCA working solution was added to each well, and two duplicate wells were set for each group. After incubation in a 37°C incubator for 30 min, cool to room temperature, measure the OD value of each well at 562 nm wavelength, then calculate the protein concentration.

[0140] The protein concentration of each group was diluted with lysis solution to a concentration of 2000 μg / mL, 4 times the volume of protein sample was added to 1 volume of 5X loading buffer, mixed well, and denatured at 95°C for 10 min.

[0141] (3) Gel preparation and electrophoresis: prepare 8% separation gel, slowly pour from the upper right corner of the glass plate, and press the gel for about 20 min with ultrapure water; after the gel is solidified, pour out the ultrapure water, absorb the excess water with filter paper, and pour in enough 5% concentrated gel and vertically insert the comb; after the separation gel is gelled, place the gel on a fixed device filled with electrophoresis liquid, remove the comb, add the previously thawed marker and the sample to be tested, and open the electrophoresis instrument to run the concentrated gel at 60V and the separation gel at 90V to the appropriate position.

[0142] (4) Transfer and blocking: remove the gel, cut off the excess part, cut the nitrocellulose membrane to the appropriate size, place it in the transfer slot according to the sandwich stacking method, add enough transfer solution, and transfer at 200mA and 4°C constant current. After the end, cut the nitrocellulose membrane (NC membrane) according to the molecular weight of the target protein, and block with 5% skimmed milk powder.

[0143] (5) Hybridization and development: prepare a hybridization bag of appropriate size, wrap the NC membrane, add the primary antibody, seal the opening tightly, and incubate overnight at 4°C; rinse with 1xTBST for about 30 min, shake on the shaker, add the secondary antibody (1:5000), incubate at room temperature for 1-2h; remove the membrane, rinse for about 20 min, according to the ECL luminescent kit instructions, add the developing agent, avoid light incubation for about 2 min, and develop with the gel imaging system. The results are shown in Figure 11

[0144] Table 6 Effect of Qingre Xingqi Huoxue Decoction on EGFR / AKT pathway protein expression in esophageal cancer cells

[0145] Group AKT EGFR MTOR EC-1 control (control) 0.468±0.118 0.476±0.050 0.368±0.152 EC-1 Qingre Xingqi Huoxue Fang 0.173±0.049* 0.274±0.042 0.196±0.066 Eca109 control (control) 0.498±0.126 0.512±0.062 0.456±0.099 Eca109 Qingre Xingqi Huoxue Fang 0.232±0.018 0.325±0.056* 0.251±0.034 EC9706 control (control) 0.497±0.034 0.663±0.141 0.543±0.032 EC9706 Qingre Xingqi Huoxue Fang 0.149±0.146 0.198±0.095 0.234±0.061 TE-1 control (control) 0.777±0.788 2.381±0.228 0.760±0.007 TE-1 Qingre Xingqi Huoxue Fang 0.182±0.203 1.039±0.514 0.297±0.116

[0146] From Table 6 and Figure 11 ​As can be seen, the heat-clearing and qi-moving blood-activating prescription described in Embodiment 1 can down-regulate the expression of epidermal growth factor receptor, protein kinase B and mammalian target of rapamycin of esophageal cancer cells, indicating that the Chinese medicinal composition of the present application has an inhibitory effect on the proliferation and migration of esophageal cancer cells, which is related to the inhibition of the epidermal growth factor receptor / protein kinase B pathway.

[0147] As can be seen from the above embodiments, the present application provides a Chinese medicinal composition for treating esophageal cancer, which can inhibit the proliferation, migration and clonogenicity of esophageal cancer cells, induce cell cycle arrest, and down-regulate the expression of epidermal growth factor receptor, protein kinase B and mammalian target of rapamycin.

[0148] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the scope of protection of the present application.

Claims

1. A traditional Chinese medicine composition for treating esophageal cancer, characterized in that, Consist of: Radix Zanthoxyli extract, Radix Lysimachiae extract, Radix Trillium extract; The concentration of the Radix Zanthoxyli extract is 61.23-102.16 μg / mL; the concentration of the Radix Lysimachiae extract is 13.41-45.20 μg / mL; and the concentration of the Radix Trillium extract is 6.42-15.06 μg / mL; The preparation method of the Radix Zanthoxyli extract is as follows: mixing Radix Zanthoxyli and ethanol, dark-soaking, suction filtration, concentration, drying, and dissolution to obtain the Radix Zanthoxyli extract; The preparation method of the Radix Lysimachiae extract is as follows: mixing Radix Lysimachiae and ethanol, dark-soaking, suction filtration, concentration, drying, and dissolution to obtain the Radix Lysimachiae extract; The preparation method of the Radix Trillium extract is as follows: mixing Radix Trillium and ethanol, dark-soaking, suction filtration, concentration, drying, and dissolution to obtain the Radix Trillium extract.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The preparation method of the Radix Zanthoxyli extract is as follows: mixing Radix Zanthoxyli and ethanol, dark-soaking, suction filtration, concentration, drying, and dissolution to obtain the Radix Zanthoxyli extract; The initial concentration of the ethanol is 90-98 wt%; The mass-volume ratio of the Radix Zanthoxyli and ethanol is 1-3 g:4-6 mL; The dark-soaking time is 6-8 d; The suction filtration is negative pressure suction filtration; the pressure of the suction filtration is-0.03--0.05 MPa; and the suction filtration time is 1-2 min; The concentration method is water bath rotary evaporation concentration; the concentration temperature is 50-60 ℃; the concentration rotating speed is 60-65 rmp; and the concentration time is 10-20 min; The drying method is vacuum freeze drying; the vacuum degree of the drying is 2-5 Pa; the cold trap temperature of the drying is-60--70 ℃; and the drying time is 48-72 h.

3. The traditional Chinese medicine composition according to claim 2, characterized in that, The preparation method of the Radix Lysimachiae extract is as follows: mixing Radix Lysimachiae and ethanol, dark-soaking, suction filtration, concentration, drying, and dissolution to obtain the Radix Lysimachiae extract; The initial concentration of the ethanol is 90-98 wt%; The mass-volume ratio of the Radix Lysimachiae and ethanol is 1-3 g:4-6 mL; The dark-soaking time is 6-8 d; The suction filtration is negative pressure suction filtration; the pressure of the suction filtration is-0.03--0.05 MPa; and the suction filtration time is 1-2 min; The concentration method is water bath rotary evaporation concentration; the concentration temperature is 50-60 ℃; the concentration rotating speed is 60-65 rmp; and the concentration time is 10-20 min; The drying method is vacuum freeze drying; the vacuum degree of the drying is 2-5 Pa; the cold trap temperature of the drying is-60--70 ℃; and the drying time is 48-72 h.

4. The traditional Chinese medicine composition according to claim 3, characterized in that, The preparation method of the Radix Trillium extract is as follows: mixing Radix Trillium and ethanol, dark-soaking, suction filtration, concentration, drying, and dissolution to obtain the Radix Trillium extract. The initial concentration of the ethanol is 90-98 wt%; The mass-volume ratio of the Radix Trillium and ethanol is 1-3 g:4-6 mL; The dark-soaking time is 6-8 d; The suction filtration is negative pressure suction filtration; the pressure of the suction filtration is-0.03 to-0.05 MPa; the time of the suction filtration is 1 to 2 min; The method of concentration is water bath rotary evaporation concentration; the temperature of the concentration is 50 to 60 DEG C; the rotating speed of the concentration is 60 to 65 rmp; the time of the concentration is 10 to 20 min; The method of drying is vacuum freeze drying; the vacuum degree of the drying is 2 to 5 Pa; the cold trap temperature of the drying is-60 to-70 DEG C; the time of the drying is 48 to 72 h.

5. The preparation method of the traditional Chinese medicine composition according to any one of claims 1-4, characterized in that, The method comprises the following steps: mixing Linglingxiang extract, Dibuteng extract and Yanchengao extract to obtain a traditional Chinese medicine composition.

6. The use of the traditional Chinese medicine composition according to any one of claims 1 to 4 in the preparation of a medicament for treating esophageal cancer.

7. The use of the traditional Chinese medicine composition prepared by the preparation method according to any one of claim 5 in the preparation of a medicament for treating esophageal cancer.

Images