Application of Benchun Decoction in preparation of drugs for treating ovarian cancer

Abstract

The present application relates to the technical field of medicine, and provides application of Ben Dung decoction in preparation of medicine for treating ovarian cancer, wherein the Ben Dung decoction is prepared from licorice, Szechuan lovage rhizome, Chinese angelica, pinellia, radix scrophulariae, radix puerariae, radix paeoniae, ginger and root white bark of prunus mira koehne with a mass ratio of (1.5-2.5):(1.5-2.5):(1.5-2.5):(3-5):(1-3):(6-10):(1.5-2.5):(3-5):(4-7). The Ben Dung decoction can significantly affect the morphology and cycle of ovarian cancer ES-2 and OV-1063 cells, effectively inhibit the malignant phenotypes such as proliferation, migration, infiltration and cloning of the ovarian cancer ES-2 and OV-1063 cells, and inhibit the expression level of CA125 and CEA proteins in the ovarian cancer OV-1063 cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical technology, and particularly relates to application of a Benchun decoction preparation in preparation of a medicine for treating ovarian cancer. BACKGROUND

[0002] Ovarian cancer is a malignant tumor of the ovary, and refers to a malignant tumor growing on the ovary. 90%-95% of ovarian cancers are primary cancers of the ovary, and 5%-10% are cancers transferred to the ovary from other parts. Since there are no symptoms in the early stage of ovarian cancer, and even if there are symptoms, they are not specific, and the screening effect is limited, early diagnosis is difficult. When diagnosed, 60%-70% of the cases are in the advanced stage, and the curative effect of the advanced cases is poor. Therefore, although the incidence of ovarian cancer is lower than that of cervical cancer and endometrial cancer, it ranks the third among gynecological malignant tumors, but the mortality rate is higher than that of cervical cancer and endometrial cancer, and ranks the first among gynecological cancers, which is the biggest disease that seriously threatens women's health.

[0003] Ovarian cancer has no specific name in traditional Chinese medicine, and is classified into the category of "cancer" and "accumulation" according to the symptoms of the disease. As early as in the "Water Swelling" of "Lingshu", it is pointed out that the stone accumulation is due to cold evil, qi stagnation and blood stasis, and the abdominal mass in the uterus is as large as a cup, and it is pointed out that the disease is located in the abdomen. It is also pointed out in "Complete Prescriptions for Women" that blood stasis, wind and cold, and phlegm retention are diseases; "Jingyue Quanshu" points out that qi deficiency and qi stagnation are diseases; "Medical Records of Chinese Medicine" describes blood stasis and phlegm accumulation as diseases; "Medical Source Theory" discusses that cancer is caused by the stagnation of women's uterus after childbirth; in traditional Chinese medicine, the cause of ovarian cancer is due to the deficiency of spleen and kidney, deficiency of qi and blood, and external evil such as phlegm, blood stasis, dampness and evil toxin flowing into the uterus. It is also pointed out in "Medical Records of Clinical Practice" that cancer should be treated by tonifying qi, promoting qi, tonifying blood, and removing blood stasis. It is believed in "Chinese Gynecology" that cancer is caused by the deficiency of internal qi, the invasion of external evil such as wind, cold and heat, emotional causes, damage to the bedroom, improper diet and other factors, leading to the dysfunction of five internal organs, qi stagnation, blood stasis, phlegm retention, dampness and other evil qi accumulating in the uterus, which is mainly divided into four types of qi stagnation, blood stasis, phlegm retention, dampness and blood stasis, and kidney deficiency.

[0004] At present, with the rapid development of molecular biology and information science, the research on ovarian cancer has also changed with the times, showing multi-stage, multi-factor and multi-target research. Migration and invasion are important biological characteristics of ovarian cancer, and inhibiting its development can effectively control the occurrence and development of ovarian cancer. Therefore, it is of great value to explore an effective traditional Chinese medicine preparation for the clinical treatment of ovarian cancer. SUMMARY

[0005] The application aims to provide application of Benchun Decoction in preparation of a drug for treating ovarian cancer.

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

[0007] The application provides application of Benchun Decoction in preparation of a drug for treating ovarian cancer, wherein the Benchun Decoction is prepared from licorice, Szechuan lovage rhizome, angelica sinensis, pinellia ternata, radix scrophulariae, pueraria lobata, paeonia lactiflora, ginger and prunus mira root white bark with a mass ratio of (1.5-2.5):(1.5-2.5):(1.5-2.5):(3-5):(1-3):(6-10):(1.5-2.5):(3-5):(4-7).

[0008] Preferably, the preparation process is as follows: water with a total mass of 12-14 times that of the above-mentioned components is added to the components, and then soaked, boiled, filtered, and obtained.

[0009] Preferably, the soaking time is 55-65 min, and the boiling time is 50-70 min.

[0010] Preferably, the filtering specification is 0.20-0.25 μm.

[0011] Preferably, the final concentration of the Benchun Decoction is 600-1800 μg / mL.

[0012] Preferably, the Benchun Decoction can inhibit the growth and proliferation of ovarian cancer ES-2 and OV-1063 cells, and change the cell morphology.

[0013] By using the above-mentioned technical solutions, the application has the following beneficial effects:

[0014] The Benchun Decoction can significantly affect the morphology and cycle of ovarian cancer ES-2 and OV-1063 cells, mainly arrest the ovarian cancer ES-2 cells in S phase to inhibit the cell growth, arrest the ovarian cancer OV-1063 cells in S and G2 phases to inhibit the cell growth, effectively inhibit the proliferation, migration, infiltration and cloning of ovarian cancer ES-2 and OV-1063 cells, and inhibit the expression level of CA125 and CEA proteins in the ovarian cancer OV-1063 cells. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1The quantity-effect relationship of the Benchun Decoction preparation on the growth inhibition of ovarian cancer ES-2 and OV-1063 cells Figure 1 A represents the influence on ovarian cancer ES-2 cells, and B represents the influence on ovarian cancer OV-1063 cells) ;

[0016] Figure 2 The time-effect relationship of the Benchun Decoction preparation on the growth inhibition of ovarian cancer ES-2 and OV-1063 cells Figure 2 A represents the influence on ovarian cancer ES-2 cells, and B represents the influence on ovarian cancer OV-1063 cells) ;

[0017] Figure 3 The influence of different groups on the growth morphology of ovarian cancer ES-2 and OV-1063 cells Figure 3 A represents the ovarian cancer ES-2 cell control group, B represents the ovarian cancer ES-2 cell Benchun Decoction preparation group, C represents the ovarian cancer OV-1063 cell control group, and D represents the ovarian cancer OV-1063 cell Benchun Decoction preparation group) ;

[0018] Figure 4 The influence of different groups on the cell cycle of ovarian cancer ES-2 and OV-1063 cells Figure 4 A represents the influence on ovarian cancer ES-2 cells, and B represents the influence on ovarian cancer OV-1063 cells) ;

[0019] Figure 5 The influence of different groups on the cell cycle of ovarian cancer ES-2 and OV-1063 cells Figure 5 A represents the ovarian cancer ES-2 cell control group, B represents the ovarian cancer ES-2 cell Benchun Decoction preparation group, C represents the ovarian cancer OV-1063 cell control group, and D represents the ovarian cancer OV-1063 cell Benchun Decoction preparation group) ;

[0020] Figure 6 The influence of different groups on the cell infiltration of ovarian cancer ES-2 and OV-1063 cells Figure 6 A represents the influence on ovarian cancer ES-2 cells, and B represents the influence on ovarian cancer OV-1063 cells) ;

[0021] Figure 7 The influence of different groups on the cell migration of ovarian cancer ES-2 and OV-1063 cells Figure 7 A represents the influence on ovarian cancer ES-2 cells, and B represents the influence on ovarian cancer OV-1063 cells) ;

[0022] Figure 8 The influence of different groups on the cell proliferation of ovarian cancer ES-2 and OV-1063 cells Figure 8 A represents the influence on ovarian cancer ES-2 cells, and B represents the influence on ovarian cancer OV-1063 cells) ;

[0023] Figure 9 Effects of different groups on the expression of CA125, HE4 and CEA proteins of ovarian cancer OV-1063 cells Figure 9 A represents the control group of ovarian cancer ES-2 cells, B represents the preparation group of the Benchun Decoction, C represents the control group of ovarian cancer OV-1063 cells, and D represents the preparation group of the Benchun Decoction for ovarian cancer OV-1063 cells;

[0024] Figure 10 Effects of different groups on the expression of CA125, HE4 and CEA proteins of ovarian cancer OV-1063 cells DETAILED DESCRIPTION

[0025] The application provides application of a Benchun Decoction preparation in preparation of a drug for treating ovarian cancer.

[0026] In the application, the Benchun Decoction preparation is prepared from licorice, Szechuan lovage rhizome, Chinese angelica, pinellia, radix scrophulariae, radix puerariae, paeony, ginger and white root bark of prunus mume, and the mass ratio of the licorice, Szechuan lovage rhizome, Chinese angelica, pinellia, radix scrophulariae, radix puerariae, paeony, ginger and white root bark of prunus mume is preferably (1.5-2.5):(1.5-2.5):(1.5-2.5):(3-5):(1-3):(6-10):(1.5-2.5):(3-5):(4-7), further preferably (1.8-2.3):(1.8-2.2):(1.7-2.1):(3.5-4.5):(1.5-2.5):(6.5-9.5):(1.7-2.2):(3.5-4.5):(5-6.5), and more further preferably 2:2:2:4:2:8:2:4:6.

[0027] In the application, the preparation process is as follows: water with a mass of 12-14 times that of the total mass of the above components is added to the components, and then soaked, boiled, filtered, and obtained. In the application, the water is preferably ultrapure water, and the water amount is further preferably 12.5-13.5 times that of the total mass of the above components, and more further preferably 13 times. The soaking time is preferably 55-65 min, further preferably 58-62 min, and more further preferably 60 min. After soaking, boiling is performed, and the boiling is performed by first boiling with a strong fire and then boiling with a weak fire. The weak fire boiling time is preferably 50-70 min, further preferably 57-64 min, and more further preferably 60 min. After boiling, filtering is performed, and medical gauze filtering is preferably adopted.

[0028] In the present application, the filtering is followed by centrifugation, and the supernatant is taken and filtered again to obtain the Benchun Decoction preparation. The rotation speed of the centrifugation is preferably 2500-3500 r / min, further preferably 2800-3200 r / min, and more preferably 3000 r / min. The centrifugation time is preferably 30-40 min, further preferably 32-37 min, and more preferably 35 min. The specification of the re-filtering is preferably 0.20-25 μm, 0.21-0.23 μm, and more preferably 0.22 μm.

[0029] In the present application, the final concentration of the Benchun Decoction preparation is preferably 600-1800 μg / mL, further preferably 800-1700 μg / mL, and more preferably 1600 μg / mL.

[0030] In the present application, the Benchun Decoction preparation can inhibit the growth and proliferation of ovarian cancer ES-2 and OV-1063 cells, and change the cell morphology.

[0031] The technical solutions provided by the present application are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0032] The traditional Chinese medicinal materials used in the present application are purchased from the Third Affiliated Hospital of Henan University of Chinese Medicine.

[0033] Example 1

[0034] The application of the Benchun Decoction preparation in the preparation of a drug for treating ovarian cancer is as follows:

[0035] Glycyrrhiza, Chuanxiong, Danggui, Banxia, Huangqi, Gegen, Shaoyao, Shengjiang, and Ganli Genbaipi with a mass ratio of 2:2:2:4:2:8:2:4:6 are added with 13 times the total mass of ultrapure water, and soaked for 60 min. Then, the mixture is boiled with a high flame of an electric stove, and then boiled with a low flame for 60 min. The mixture is filtered through eight layers of medical gauze into a 50 mL centrifuge tube, and centrifuged at 3000 r / min for 35 min. The supernatant is taken and filtered through a 0.22 μm microporous filter into a sterile centrifuge tube. The mixture is dried and concentrated, and then divided into 2 mL sterile EP tubes. The mixture is stored in a -20℃ freezer. The final concentration of the Benchun Decoction preparation is 1600 μg / mL.

[0036] Example 2

[0037] The application of the Benchun Decoction preparation in the preparation of a drug for treating ovarian cancer is as follows:

[0038] Glycyrrhiza, Chuanxiong, Danggui, Banxia, Huangqi, Gegen, Shaoyao, Shengjiang and Ganli root white skin in a mass ratio of 1.5:2.5:1.5:3:1:6:1.5:3:4 were added with 12 times of the total mass of ultrapure water, soaked for 55 min; then boiled with a stove, first with a strong fire, and then with a weak fire for 70 min, filtered with eight layers of medical gauze into a 50 mL centrifuge tube, centrifuged at 2500 r / min for 40 min, and then the supernatant was taken, filtered with a 0.22 μm microporous filter into a sterile centrifuge tube, dried, and concentrated and determined, and then divided into 2 mL sterile EP tubes, and stored in a -20℃ refrigerator. The final concentration of the Benxun decoction preparation for use was 800 μg / mL.

[0039] Example 3

[0040] The application of the Benxun decoction preparation in the preparation of a drug for treating ovarian cancer, the steps are as follows:

[0041] Glycyrrhiza, Chuanxiong, Danggui, Banxia, Huangqi, Gegen, Shaoyao, Shengjiang and Ganli root white skin in a mass ratio of 2.5:1.5:2.5:5:3:10:2.5:5:7 were added with 14 times of the total mass of ultrapure water, soaked for 65 min; then boiled with a stove, first with a strong fire, and then with a weak fire for 50 min, filtered with eight layers of medical gauze into a 50 mL centrifuge tube, centrifuged at 3500 r / min for 30 min, and then the supernatant was taken, filtered with a 0.22 μm microporous filter into a sterile centrifuge tube, dried, and concentrated and determined, and then divided into 2 mL sterile EP tubes, and stored in a -20℃ refrigerator. The final concentration of the Benxun decoction preparation for use was 1800 μg / mL.

[0042] Test Example 1

[0043] (I) Cell culture

[0044] Human ovarian cancer cells ES-2 and OV-1063 were purchased from the Cell Resource Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences.

[0045] The water bath was set to 37℃, and the human ovarian cancer cells ES-2 and OV-1063 stored in liquid nitrogen were quickly thawed in the water bath, and the thawed cell suspension was transferred to a 15 mL centrifuge tube with a pipette, 5 mL of culture medium was added, and then mixed uniformly by blowing, and then centrifuged at 1000 r / min for 10 min. After centrifugation, the supernatant was removed, 2 mL of culture medium was added and mixed thoroughly, and then transferred to a 25 cm2 culture flask. Add 1 mL of serum and 7 mL of culture medium to a cell culture dish. Mix well by pipetting and shaking in a cross-hatching motion. Incubate at 37°C with 5% CO2 and saturated humidity. Observe cell growth daily. After 2-3 days of culture, observe cell adhesion. When the cell confluence reaches 70%-80%, remove the original culture medium from the dish. Wash the cells twice with 5 mL of RPMI 1640 medium (GIBCO Company). Add 3 mL of 0.25% EDTA-containing trypsin and digest for 5-6 minutes in an incubator. Observe under an inverted microscope. When more than half of the cells have detached from the cell wall, add 0.5 mL of FBS (AusGeneX Company) to stop digestion. Mix the cells thoroughly by pipetting and transfer them to a 50 mL centrifuge tube. Centrifuge at 1000 rpm for 10 minutes. After centrifugation, aspirate the liquid from the top, add 5 mL of serum-free culture medium to the tube, and gently pipette to ensure homogeneity. Perform cell counting at a concentration of 1 × 10⁻⁶ cells / mL. 6 Cells were seeded at a density of 1 mL in a cell culture dish, 1 mL of serum was added, and the remaining volume was adjusted to 10 mL with culture medium. The cells were then cultured in a cell culture incubator.

[0046] (II) The dose- and time-dependent relationship between the Ben Tun Tang preparation and the inhibition of human ovarian cancer ES-2 and OV-1063 cell growth was determined by MTT assay.

[0047] Based on the Ben Tun Tang preparation obtained in Example 1, a concentration gradient of 1600 μg / mL, 800 μg / mL, 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL and 25 μg / mL was prepared by serial dilution for later use.

[0048] Based on the observed cell state, once the confluence rate reaches 70%-80% or higher, the cells are seeded using a medium containing 10% FBS at a concentration of 1×10⁻⁶. 4The cells are seeded in 96-well plates at a density of 200 μL per well, and are placed in an incubator for 24 hours after seeding. After 24 hours, the supernatant in each well is removed, and 200 μL of 1640 culture medium containing 10% FBS is added to the first three columns of the 96-well plate as a control group. 200 μL of the prepared drug gradient from low to high is added to the next nine columns as three different drug groups, and the cells are cultured for another 48 hours after drug addition. After 48 hours, MTT (Sigma Company) is added, the supernatant in the well is removed, 100 μL of MTT stock solution is added to each well at a ratio of 1:9 with the culture medium, and the cells are cultured for 4 hours. The supernatant in the well is removed by suction, 150 μL of DMSO (Amresco Company) is added to each well, and the cells are allowed to stand for 20 minutes. The cells are gently shaken until they are uniformly dissolved, and the optical density (OD value) of the cells is measured by an enzyme-labeled instrument at a wavelength of 570 nm. The inhibition rate of human ovarian cancer ES-2 and OV-1063 cells is calculated. The experimental results are analyzed by using IBM SPSS 21.0 software, and the mean ± standard deviation is expressed, and regression analysis is used for analysis.

[0049] Cell inhibition rate (%) = (1-drug group OD value / control group OD value) x 100%

[0050] 1. Dose-effect relationship

[0051] Table 1 Dose-effect relationship of the preparation of Benchun decoction on the growth inhibition of ovarian cancer ES-2 and OV-1063 cells

[0052]

[0053]

[0054] It can be seen from Table 1 and Figure 1 that after the preparation of Benchun decoction acts on human ovarian cancer ES-2 and OV-1063 cells, the inhibition rate gradually increases with the increase of the drug concentration, showing a dose-effect dependent relationship.

[0055] 2. Time-effect relationship

[0056] Table 2 Time-effect relationship of the preparation of Benchun decoction on the growth inhibition of ovarian cancer ES-2 and OV-1063 cells

[0057]

[0058] It can be seen that after the preparation of Benchun decoction acts on human ovarian cancer ES-2 and OV-1063 cells, the inhibition rate gradually increases with the increase of the drug concentration, showing a dose-effect dependent relationship. Figure 2 ​

[0059] 3. The half-maximal inhibitory concentration (IC50) of the formulation on cell proliferation was calculated using SPSS 21.0 software. 50 ).

[0060] Based on the different concentration gradients and corresponding inhibition rates in Table 1, the half-maximal inhibitory concentrations (IC50) of Ben Tun Tang against ES-2 and OV-1063 were calculated using SPSS 21.0 software.

[0061] Table 3. IC50 of Bentun Decoction preparation on human ovarian cancer cells. 50 value

[0062]

[0063] It can be seen that the Ben Tun Tang preparation has a high and stable inhibition rate on human ovarian cancer ES-2 and OV-1063 cells.

[0064] Effect of Experiment Example 2 on the malignant phenotype of human ovarian cancer cells

[0065] (a) Observing changes in cell growth morphology

[0066] ES-2 cells are poorly differentiated clear cell carcinoma of the ovary, and are fibroblast-like cells that grow adherently; OV-1063 cells are epithelial cell carcinoma cells, and are epithelial cell-like cells that grow adherently.

[0067] Human ovarian cancer ES-2 and OV-1063 cells were used with 1×10 6 Cells / dish density seeding Two cell culture dishes were set up: a control group and a Bentun Decoction preparation group. The cells were cultured in a cell culture incubator at 37℃ and 5% CO2. After 24 hours, the supernatant was aspirated. The control group was given 200 μL of medium containing 10% FBS, while the Bentun Decoction preparation group was given 200 μL of complete medium calculated based on the IC50 values ​​of the Bentun Decoction preparation (90.500 μg / mL for ES-2 cells and 114.000 μg / mL for OV-1063 cells). The cells were then cultured in the incubator for an additional 48 hours. After 48 hours, the morphological changes of the cells in each group were observed under an inverted microscope, and photographs were taken at 200× magnification and stored.

[0068] Depend on Figure 3It can be seen that for ovarian cancer ES-2 cells, the control group cells are uniformly distributed, the adhesion state is good, the shape is fibrous, and the nuclear division phase is obvious; the cell gap of the Benxun decoction preparation group is larger, and the number of cells is significantly reduced. For ovarian cancer OV-1063 cells, the growth state of the control group cells is good, the cells grow closely and evenly, the shape is polygonal, and the cell boundary is clear; the cell gap of the Benxun decoction preparation group becomes larger, the cell surface is rough, the boundary is not clear, and there are dead cells. That is, the IC 50 The Benxun decoction preparation with IC50 value concentration respectively acts on ovarian cancer ES-2 and OV-1063 cells, and has different effects on the morphology and number of ovarian cancer cells, and has obvious inhibitory effect.

[0069] (ii) Cell cycle determination

[0070] The cell cycle is mainly divided into interphase and mitosis, and the whole cycle can be represented as G1 phase (pre-DNA synthesis phase) → S phase (DNA synthesis phase) → G2 phase (post-DNA synthesis phase) → M phase (resting phase).

[0071] Human ovarian cancer ES-2 and OV-1063 cells were inoculated in 6-well plates at a density of 0.4x10 6 cells / well, and control group and Benxun decoction preparation group were set up, and cultured in a cell special incubator at 37°C and 5% CO2. After 24h, the supernatant was aspirated with a suction device, the control group was added with 2mL of RPMI1640 culture medium containing 10% FBS (RPMI1640 culture medium was purchased from GIBCO Company), and the Benxun decoction preparation group was added with 2mL of complete culture medium with IC50 value of Benxun decoction preparation as the calculated concentration, and continued to culture for 48h. After 48h, the supernatant in each well of the plate was aspirated, the cells were washed once with 2mL PBS per well, and then 1.2mL trypsin without EDTA was added per well, and the cells were digested for 5min. After observing that the cells were round and detached under a microscope, 0.2mL FBS (AusGeneX Company) was added per well to stop the digestion, and the cells were gently blown into a 15mL centrifuge tube, and each sample was labeled. After centrifugation at 2000r / min for 5min, the supernatant was aspirated, 2mL PBS was added and blown to make the cells uniform, and then centrifuged at 2000r / min for 5min. The supernatant was aspirated with a suction device, 2mL PBS was added and blown to make the cells uniform, and then counted, and 1x10 6 cells / tube were transferred into a 15mL centrifuge tube and centrifuged at 2000r / min for 5min. The old supernatant was aspirated with a suction device, 1mL PBS was added to suspend the cells, 2.4mL pre-cooled absolute ethanol was added, and the cells were blown evenly. The cells were fixed in a 4°C refrigerator for 2h to overnight.

[0072] After cell fixation, centrifuge at 2000 rpm for 5 min, discard the fixative, wash cells twice with 1 mL PBS, centrifuge at 2000 rpm for 5 min, aspirate the supernatant, add 50 μL RNase per tube and incubate at 37°C for 30 min, then add 450 μL LPI per tube, gently tap to mix, and stain at 4°C in the dark for 30-60 min. Filter through a 300-mesh nylon filter into a dedicated flow cytometry tube, turn on the FCM, and analyze the cell cycle after 10 min. The results are shown in Tables 4-5. Figures 4-5 .

[0073] Table 4. Effects of different groups on the cell cycle of ES-2 ovarian cancer cells.

[0074] Group G1 phase (%) S phase (%) G2 phase (%) Control group 56.28 35.59 8.13 Ben-tun decoction preparation 50.79 41.32 7.89

[0075] Table 5. Effects of different groups on the cell cycle of ovarian cancer OV-1063 cells.

[0076] Group G1 phase (%) S phase (%) G2 phase (%) Control group 77.69 13.67 8.64 Ben-tun decoction preparation 75.2 14.78 10.02

[0077] It can be seen that, regarding ovarian cancer ES-2 cells, compared with the control group, the proportion of cells in the Bentun Decoction preparation group decreased in the G1 phase, increased in the S phase, and decreased in the G2 phase. Regarding ovarian cancer OV-1063 cells, compared with the control group, the proportion of cells in the Bentun Decoction preparation group decreased in the G1 phase, while the proportions of cells in both the S and G2 phases increased. These results indicate that the Bentun Decoction preparation described in this invention inhibits the growth of ovarian cancer ES-2 cells by arresting them in the S phase, and inhibits the growth of ovarian cancer cells by arresting OV-1063 cells in the S and G2 phases.

[0078] (III) Cell proliferation, migration, and invasion experiments

[0079] 1. Cell infiltration and migration assays

[0080] For the wetting experiment, Matrigel (Corning Company) needs to be laid in advance; for the migration experiment, no matrigel is required. Melt the Matrigel in a 4°C freezer and dilute it with FBS-free culture medium at a ratio of 1:40. The entire process should be performed on ice to prevent the Matrigel from solidifying.

[0081] Remove the upper and lower chambers of the CIM-Plate VIEW16 plate for the infiltration experiment from the operating table. Place the upper chamber in the CIM fixture. Add 50 μL of pre-diluted Matrigel to each well of the upper chamber, then gently aspirate 30 μL from each well, ensuring that each well contains 20 μL of Matrigel coating the upper chamber of the CIM-Plate VIEW16 plate. Avoid air bubbles throughout the process, and keep the Matrigel on ice. Place the upper chamber and fixture with the added Matrigel in an incubator until solidification (estimated process time 4 hours). After the Matrigel has solidified, remove it from the incubator. Simultaneously, remove the lower chamber of the CIM-Plate VIEW16 from its packaging on the operating table and place both on the CIM fixture, ensuring the blue marker is positioned at the upper left of the fixture. Add 165 μL of complete culture medium to each well of the control group (for migration and infiltration experiments) and 165 μL of IC50-containing medium to each well of the Ben Tun Tang preparation group. 50 Add the drug solution (avoiding air bubbles during addition) to create an upward-convex meniscus in the lower chamber. Rotate the clamp and lower chamber 90° to the front position. Place the sensor side of the upper chamber of the Matrigel-covered wetting plate (excluding the migration plate) into the corresponding lower chamber, ensuring all wells in both chambers are aligned (avoiding air bubbles during placement). Once aligned, press down by hand until a click is heard. Then, add 30 μL / well of FBS-free culture medium to the upper chambers of the wetting and migration plates and equilibrate in an incubator for 1 hour.

[0082] After equilibration, the CIM-Plate 16 plate was placed on the RTCADP Analyzer, and the system automatically scanned to check for good instrument contact. After the check, baseline detection was initiated to ensure that the Cell Index of all wells was below 0.063. Cells and IC preparation were performed normally during equilibration. 50 Drug solution. Adjust the cell concentration to (2-5) × 10⁻⁵ using FBS-free culture medium. 5 Prepare cells / mL (different concentrations are set according to different cell types) for later use, and prepare IC50-containing medium using FBS-free 1640 medium. 50 Prepare the drug solution. Remove the infiltration and migration plates from the RTCA instrument after baseline detection. Seed cells in the upper chamber at a concentration of (2-5)×10⁻⁵ cells / well at 100 μL / well. 5 Cells / mL of cell solution were added to each well of the drug-treated group, and 5 μL of pre-prepared FBS-free drug solution was added. The cells were left to stand on the operating table for 30 minutes to allow them to settle before being placed on the RTCA. The system automatically scanned the cells, measuring once every 15 minutes, and each channel was set to a detection time of 48 hours.

[0083] Depend on Figures 6-7It can be seen that, relative to the control group, the Ben Dung decoction preparation group has a certain inhibitory effect on the infiltration and migration of ovarian cancer ES-2 cells and ovarian cancer OV-1063 cells.

[0084] 2. Cell proliferation experiment

[0085] The E-Plate VIEW16 detection plate was taken out at the operating table, 50 μL / well of RPMI1640 culture medium containing 10% was added, and it was placed on the RTCA. The system automatically scanned to ensure that the baseline was below 0.063, and the cell concentration was adjusted to (2-5) x 10 5 cells / mL, 100 μL / well was added to the E-Plate16 detection plate, and it was placed in the clean bench for 30 min. The RTCA was detected. After 24 h, the RTCA was paused, the E-Plate16 detection plate was slowly taken out from the incubator, 5 μL / well of the Ben Dung decoction preparation group was added, and RPMI1640 culture medium without FBS was used to add the IC 50 concentration of the liquid medicine, and the detection was continued for 48 h.

[0086] From Figure 8 It can be seen that, relative to the control group, the Ben Dung decoction preparation group has a certain inhibitory effect on the proliferation of ovarian cancer ES-2 cells and ovarian cancer OV-1063 cells.

[0087] (IV) Soft agar colony formation experiment

[0088] 3 mL / well of 0.6% lower agar liquid was poured into a 6-well plate, and it was placed at room temperature to solidify. After solidification was observed, the ovarian cancer cells were taken out from the incubator, the cells were treated according to the normal cell treatment method, the cell concentration was adjusted to 1 x 10 4 cells / mL, 2 mL / well of upper agar liquid and 100 μL (1000 cells) of cell suspension were added, 10 μL of the Ben Dung decoction preparation group was added, and the IC 50 concentration of the liquid medicine was added. It was placed at room temperature to solidify, and it was continued to be cultured in the 37°C incubator. The change of cell clone formation was observed under the microscope. After 14 days, the cell clone number was counted under the microscope, and the cell clone formation rate was calculated.

[0089] Cell clone colony formation rate (percentage) = (average number of clones / number of cells inoculated per well) x 100%.

[0090] Table 6 Effect of different groups on ovarian cancer ES-2 and OV-1063 cell clone formation

[0091]

[0092] From Table 6 and Figure 9It can be seen that, compared with the control group, the size and morphology of ovarian cancer ES-2 and OV-1063 cell clones in the Ben Tun Tang preparation group changed, with the cell clones becoming irregular in shape and smaller in size.

[0093] Effects of Experiment 3 on the expression of CA125, HE4, and CEA proteins in human ovarian cancer OV-1063 cells

[0094] The CA125 mouse monoclonal antibody, CEA mouse monoclonal antibody, and HE4 mouse monoclonal antibody used in this invention were all purchased from Roche Diagnostics GmbH.

[0095] 1. Extraction of cell proteins

[0096] Ovarian cancer OV-1063 cells were divided into groups of 1.25 × 10⁻⁶. 6 Inoculation density of 1 per dish In culture dishes, each cell type was divided into a control group and a Bentun Decoction preparation group. Cells were cultured normally for 24 hours in an incubator at 37℃ and 5% CO2. The old culture medium was then removed using a suction device. The control group was given RPMI 1640 medium containing 10% FBS, while the Bentun Decoction preparation group was given medium at an IC50 concentration according to the Bentun Decoction preparation. 50 The calculated concentration of the complete culture medium was used for further culture for 48 hours. The supernatant was discarded, and the cells were washed 3 times by shaking the culture dish 6-8 times using the cross-shaking method with 5 mL of 0.01M PBS at 4℃. After washing, the pre-prepared lysis buffer was added, and the cells were lysed for 30 minutes (shaking once every 6 minutes, repeating 5 times). After lysis, the cells were scraped 3 times with a cell scraper, and the cells were collected in 1.5 mL of EP. The cells were centrifuged at 14000 rpm / min at 4℃ for 30 minutes. After centrifugation, the supernatant was transferred to a 1.5 mL EP tube and stored at -20℃.

[0097] 2. Determine protein concentration

[0098] Prepare 1 5mL EP tube, prepare BCA (BCA protein concentration determination kit purchased from Solarbio Company) working solution in advance (BCA reagent and Cu reagent are added according to the ratio of 50:1). First dilute the BSA standard with PBS to a concentration of 500 μg / μL, and then add 0 μL, 2 μL, 4 μL, 6 μL, 8 μL, 12 μL, 16 μL, 20 μL of BSA with a concentration of 500 μg / μL to 18 test tubes in turn, and the rest is supplemented with PBS to 20 μL. After dilution of the sample 2 / 1 times, 4 / 1 times, 8 / 1 times, 20 μL / well is added to the 96-well plate, and then 200 μL of BCA working solution prepared in advance is added to each well, and it is placed at 37°C for 30 min. Set the wavelength of the enzyme label to 562 nm, analyze the OD value, and draw the standard curve of the protein. According to the standard curve determined, the protein concentration is detected.

[0099] 3. Preparation of gel

[0100] Fix the clean glass plate on the support, carefully pour the prepared 6% SDS-PAGE separation gel into the glass plate, and pour to 1.2 cm away from the glass plate. Press the gel with ultrapure water, and let the gel solidify for 20-60 min (depending on the specific situation). After the gel solidifies, pour out the ultrapure water, and carefully pour the 4% SDS-PAGE concentrated gel prepared in advance into the glass plate. Insert the green comb, and let it solidify for 20-60 min.

[0101] 4. Sample loading and electrophoresis

[0102] Load 20 μg / group as the total protein amount, calculate the protein loading volume according to the protein concentration measured in advance, and add 5x loading buffer to make the final concentration 1x. Turn on the constant temperature test tube heater in advance, and denature at 99°C for 10 min. Fix the gel in the electrophoresis tank filled with 1x electrophoresis buffer, slowly load the sample, and add 5 μL Marker and 20 μg of the loaded protein in turn. Electrophorese at a voltage of 60V for 3h, and stop when the sample containing blue bromophenol blue in the electrophoresis tank is close to the lower edge of the glass plate.

[0103] 5. Membrane transfer and blocking

[0104] Take out the gel, remove the excess gel, and transfer it to the transfer clamp in the order of transfer clamp negative electrode-sponge-filter paper-gel-nitrocellulose membrane (NC membrane)-filter paper-sponge-transfer clamp positive electrode from bottom to top. Clamp the transfer clamp, place it in the transfer tank, and note that the positive and negative electrodes of the transfer clamp correspond to the positive and negative electrodes of the transfer tank, respectively. Transfer the membrane at a constant voltage of 60V for 2h (the transfer time may be adjusted according to the molecular weight). After the transfer is completed, mark the molecular weight according to the color of the Marker, and block the NC membrane with skimmed milk powder at room temperature for 3h.

[0105] 6. Antibody incubation

[0106] After blocking with skim milk powder, place the NC membrane into a hybridization bag, add the corresponding primary antibody, and incubate on a shaker at room temperature for 2 hours. Wash with 1×TTBS for 30 minutes, repeating TTBS for 5 minutes per wash, for a total of 6 washes. After washing, transfer the membrane to a hybridization bag, add the corresponding secondary antibody, and incubate on a shaker at room temperature for 2 hours. Wash with 1×TTBS for 60 minutes, repeating TTBS for 10 minutes per wash, for a total of 6 washes. After washing, prepare for development.

[0107] 7. Development

[0108] Take luminescent agent A and solution B (ECL luminescence kit purchased from Santa Cruz Company), mix them evenly at a ratio of 1:1, and use the mixture to develop the bands using a chemiluminescence imaging system.

[0109] The expression of CA125, HE4, and CEA proteins in ovarian cancer OV-1063 cells was analyzed, and the results (e.g.) Figure 10 The results showed that, compared with the control group, the Bentun Decoction preparation group had significant differences in the expression of CA125 and CEA proteins in human ovarian cancer OV-1063 cells, and the expression of each protein was significantly downregulated after drug treatment; it had no significant effect on the expression level of HE4 protein in human ovarian cancer OV-1063 cells.

[0110] As can be seen from the above examples and experimental cases, the Ben Tun Tang preparation described in this invention can significantly affect the morphology and cell cycle of ovarian cancer ES-2 and OV-1063 cells. It mainly arrests ovarian cancer ES-2 cells in the S phase to inhibit cell growth, and arrests ovarian cancer OV-1063 cells in the S and G2 phases to inhibit the growth of ovarian cancer cells. It can also effectively inhibit the proliferation, migration, invasion and cloning of ovarian cancer ES-2 and OV-1063 cells and inhibit the expression levels of CA125 and CEA proteins in ovarian cancer OV-1063 cells.

[0111] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of a preparation of the deer antler velvet in the manufacture of a medicament for the treatment of ovarian cancer, characterized in that, The Benchun decoction preparation is prepared from licorice, Szechuan lovage rhizome, angelica sinensis, pinellia ternata, scrophularia ningpoensis, pueraria lobata, paeonia lactiflora, ginger and root white skin of prunus mira koehne with a mass ratio of (1.5-2.5):(1.5-2.5):(1.5-2.5):(3-5):(1-3):(6-10):(1.5-2.5):(3-5):(4-7).

2. Use according to claim 1, characterized in that, The preparation process is as follows: adding water with a total mass of 12-14 times of the above components, soaking, then decocting and filtering to obtain the Benchun decoction preparation.

3. Use according to claim 2, characterized in that, The soaking time is 55-65 min, and the decocting time is 50-70 min.

4. Use according to claim 2, characterized in that, The filtering specification is 0.20-0.25 μm.

5. The use according to claim 1, characterized in that, The final concentration of the Benchun decoction preparation is 600-1800 μg / mL.

6. Use according to claim 1, characterized in that, The Benchun decoction preparation can inhibit the growth and proliferation of ovarian cancer ES-2 and OV-1063 cells and change the cell morphology.

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