Cynanchum atratum extract as well as preparation method and application thereof

Abstract

The invention discloses a cynanchum atratum extract as well as a preparation method and application thereof. The cynanchum atratum extract is an extract obtained by extracting cynanchum atratum with 50-80% ethanol. The cynanchum atratum is a root and a rhizome of a Tylophora yunnanensis Schlechter, which is a plant of the Schlechter family, and the root and the rhizome of the Tylophora yunnanensis Schlechter. Cell experiments and animal experiments prove that the extract obtained by extracting cynanchum atratum with 75% ethanol has the effects of inhibiting breast cancer and hepatoma carcinoma cell proliferation, inducing breast cancer and hepatoma carcinoma cell death and reducing tumor volume and tumor quality, has a remarkable effect on treating liver cancer and breast cancer, and has a practical application value.

Description

Technical Field

[0001] The present invention specifically relates to an extract of Cynanchum versicolor, a preparation method thereof, and uses thereof. Background Art

[0002] Malignant tumors, also known as cancers, are tumors composed of abnormal cells that have the ability to proliferate uncontrollably and may invade surrounding tissues or spread to other parts of the body through the blood and lymphatic systems. The causes of malignant tumors are diverse, including genetic factors, environmental factors, lifestyle, etc. The incidence and mortality of malignant tumors are on the rise globally and are the second leading cause of death after cardiovascular diseases. In China, the incidence and mortality of malignant tumors have also increased significantly, becoming one of the major diseases seriously threatening the health of residents. According to statistical data, liver cancer and breast cancer are common types of malignant tumors.

[0003] Cynanchum versicolor is the dried root and rhizome of Tylophora yunnanensis Schltr. Tylophora yunnanensis It is a commonly used medicinal material of the Yi ethnic group, with a bitter and pungent taste, slightly warm in nature, and belongs to the liver and spleen meridians. The "Yunnan Chinese Medicinal Materials Standard" (2005 edition) records that it is slightly bitter, cool in nature; belongs to the liver, kidney, and lung meridians; functions to clear heat, activate blood circulation, and disperse stasis and relieve pain; is used for traumatic injuries, swelling and pain due to blood stasis, and yin deficiency fever. "Yunnan Chinese Herbal Medicine" states that it can soothe tendons and activate blood circulation, regulate menstruation and relieve pain; treats traumatic injuries, rheumatic bone pain, hepatitis, gastric ulcer, consumptive disease, and malignant malaria. Research has found that Cynanchum versicolor has obvious anti-inflammatory effects, can also improve non-alcoholic fatty hepatitis, reduce body weight, regulate lipid levels, and improve intestinal microbiota imbalance and metabolic disorders.

[0004] Currently, there is no report on the effective treatment of malignant tumors with Cynanchum versicolor. Summary of the Invention

[0005] To solve the above problems, the present invention provides an extract of Cynanchum versicolor, which is an extract obtained by extracting Cynanchum versicolor with 50 - 80% ethanol; The Cynanchum versicolor is the root and rhizome of Tylophora yunnanensis Schltr. Tylophora yunnanensis rhizome.

[0006] Further, it is an extract obtained by extracting Cynanchum versicolor with 75% ethanol.

[0007] The present invention also provides a preparation method of the aforementioned extract of Cynanchum versicolor, including the following steps: Take Cynanchum versicolor, extract with 50 - 80% ethanol, concentrate and dry the extract, or dry the extract after removing impurities and purifying it, then the product is obtained.

[0008] Further, the extraction is reflux extraction or ultrasonic extraction; the power of the ultrasonic extraction is 600-800 W, the time is 20-30 min, and the solid-liquid ratio is 1 g:15-20 mL; the number of times of the reflux extraction is 3 times, the time for each time is 1-2 h, and the solid-liquid ratio is 1 g:8-10 mL.

[0009] Further, the concentration method includes concentration using a rotary evaporator; and / or: the impurity removal method includes centrifugal separation, the rotation speed of the centrifugal separation is 5000-30000 r / min, and the time is 5-20 min; and / or: the purification method includes separation and purification using macroporous resin and / or organic membrane filtration purification; the macroporous resin includes D101 macroporous resin, AB-8 macroporous resin or X-5 macroporous resin, the elution reagent for separation and purification is an ethanol solution, the volume fraction of the ethanol solution is 10-80%, and the dosage is 2-20 times the column volume; the pore size of the organic membrane is 20000 Dalton; and / or: the drying method includes vacuum freeze-drying, the temperature of the vacuum freeze-drying is -120 to -40 °C, preferably -80 °C.

[0010] The present invention also provides the use of the foregoing Cynanchum versicolor extract in the preparation of a drug for treating tumors.

[0011] Further, the drug is a drug for treating breast cancer and / or liver cancer.

[0012] Furthermore, the drug is a drug for treating triple-negative breast cancer and / or hepatocellular carcinoma.

[0013] Furthermore, the drug has the effect of inhibiting cancer cell proliferation and / or inducing cancer cell death.

[0014] Furthermore, the drug has the effect of reducing tumor volume and / or tumor weight.

[0015] Further, the drug is a preparation prepared from the Cynanchum versicolor extract as an active ingredient and pharmaceutically acceptable excipients; the preparation is an oral preparation; the oral preparation includes solution, powder, granule, tablet, capsule.

[0016] The extract obtained by extracting Cynanchum versicolor with 75% ethanol in the present invention has been proven by cell experiments and animal experiments to have the effects of inhibiting the proliferation of BT549 and 4T1 triple-negative breast cancer cell lines, Huh7 and HepG2 liver cancer cells, inducing the death of triple-negative breast cancer cells and liver cancer cells, and reducing the tumor volume and tumor mass of triple-negative breast cancer, showing significant effects in the treatment of liver cancer and breast cancer. Using the ethanol extract of Cynanchum versicolor in the present invention for the treatment of liver cancer or breast cancer has practical popularization and application value.

[0017] Obviously, based on the above content of the present invention, according to the common general technical knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modification, substitution or change can be made.

[0018] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples, but this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Brief Description of the Drawings

[0019] Figure 1 TYE positive and negative total ion chromatogram; Figure 2 Effects of TYE on the viability of liver cancer cells and normal liver cells; A-D. Effects of TYE treatment on the viability of Huh7 and HepG2 liver cancer cells after 24 h and 48 h, respectively; E. Effect of TYE treatment on the morphology of normal liver cells of AML12 mice after 48 h; F-G. Effects of TYE treatment on the viability of AML12 cells after 24 h and 48 h; Figure 3 Effects of TYE on the viability of breast cancer cells; Figure 4 Effects of TYE on the clonal proliferation of liver cancer cells; Figure 5 Effects of TYE on the clonal proliferation of breast cancer cells; Figure 6 TYE promotes the death of liver cancer cells; Figure 7 TYE promotes the death of breast cancer cells; Figure 8 Effects of TYE on the cell cycle of liver cancer cells; Figure 9 Effects of TYE on the cell cycle of breast cancer cells; Figure 10 TYE inhibits the growth of breast cancer.

[0020] In the figure, compared with the control group (0 μg / mL), * p<0.05, ** p<0.01, *** p<0.001. Detailed implementation mode

[0021] The raw materials and equipment used in the detailed implementation mode of the present invention are all obtained by purchasing on the market.

[0022] Example 1 Preparation of the drug for treating tumors of the present invention Take 100 g of dried Cynanchum versicolor Bunge. herbs, crush them, and reflux extract with 10 times the amount of 75% ethanol for 3 times, 1.5 h each time. Combine the extraction solutions, filter them under vacuum, concentrate them with a rotary evaporator, and freeze-dry them under vacuum at -80 °C to obtain the extract powder of Cynanchum versicolor Bunge. ethanol extract. Add pharmaceutically acceptable excipients to make tablets.

[0023] Example 2 Preparation of the drug for treating tumors of the present invention Take 50 g of dried Cynanchum versicolor Bunge. herbs, crush them, and reflux extract with 8 times the amount of 75% ethanol for 3 times, 1 h each time. Combine the extraction solutions, filter them under vacuum, concentrate them with a rotary evaporator, and freeze-dry them under vacuum at -80 °C to obtain the extract powder of Cynanchum versicolor Bunge. ethanol extract. Add pharmaceutically acceptable excipients to make granules.

[0024] Example 3 Preparation of the drug for treating tumors of the present invention Take 150 g of dried Cynanchum versicolor Bunge. herbs, crush them, and reflux extract with 8 times the amount of 70% ethanol for 3 times, 2 h each time. Combine the extraction solutions, filter them under vacuum, concentrate them with a rotary evaporator, and freeze-dry them under vacuum at -80 °C to obtain the extract powder of Cynanchum versicolor Bunge. ethanol extract. Add pharmaceutically acceptable excipients to make capsules.

[0025] Example 4 Preparation of the drug for treating tumors of the present invention Take 150 g of dried Cynanchum versicolor Bunge. herbs, crush them, and reflux extract with 10 times the amount of 70% ethanol for 3 times, 2 h each time. Combine the extraction solutions, filter them under vacuum, concentrate them with a rotary evaporator, and freeze-dry them under vacuum at -80 °C to obtain the extract powder of Cynanchum versicolor Bunge. ethanol extract. Add pharmaceutically acceptable excipients to make capsules.

[0026] Example 5 Preparation of the drug for treating tumors of the present invention Take 10 g of dried Cynanchum versicolor Bunge. herbs, crush them, add 150 mL of 50% ethanol aqueous solution, and ultrasonically extract for 20 min under the condition of 800 W. Centrifuge the extraction solution to remove impurities for 5 min under the condition of a rotation speed of 30000 r / min. Then load it onto a macroporous resin column (D101 macroporous resin), elute with 10 times the column volume of 70% ethanol, collect the eluate, filter it through an organic membrane with a pore size of 20000, collect the filtrate, and dry it to obtain the total extract of Cynanchum versicolor Bunge. Add pharmaceutically acceptable excipients to make powder.

[0027] Example 6 Preparation of the drug for treating tumors of the present invention Take 10 g of dried medicinal materials of Xiaobaiwei, crush them, add 200 mL of 80% ethanol aqueous solution, and ultrasonically extract them at 600 W for 30 min. The extract is centrifuged at 5000 r / min for 20 min to remove impurities, then loaded on a macroporous resin column (B-8 macroporous resin), eluted with 75% ethanol in 20 times the column volume, collected the eluate, passed through an organic membrane with a pore size of 20000, collected the filtrate, and dried to obtain the total extract of Xiaobaiwei, which was then added with pharmaceutically acceptable excipients to prepare granules.

[0028] Example 7 Preparation of the drug for treating tumors of the present invention Take 10 g of dried medicinal materials of Xiaobaiwei, crush them, add 200 mL of 50% ethanol aqueous solution, and ultrasonically extract at 600 W for 30 min. The extract is centrifuged at 5000 r / min for 20 min to remove impurities, then loaded on a macroporous resin column (X-5 macroporous resin), eluted with 10% ethanol in a volume of 2 times the column, collected the eluate, passed through an organic membrane with a pore size of 20000, collected the filtrate, and dried to obtain the total extract of Xiaobaiwei.

[0029] The beneficial effects of the present invention are further illustrated by test examples below. Experimental Example 1 Study on the inhibition of tumor growth by extract of Astragalus membranaceus 1. Methods 1 Materials The herbal medicine of Xiaobaiwei was purchased from Changzhentang Chinese Herbal Medicine Business Department in Guandu District, Kunming, Yunnan Province. It was identified as Yunnan Wawaertia, a plant of the Asclepiadaceae family, by Professor Xin Hailiang of the Pharmacognosy Teaching and Research Section of the School of Pharmacy of the Naval Medical University. Tylophora yunnanensis Schlechter's roots and rhizomes.

[0030] 1.1 Extract of Psoralea corylifolia Take 100 g of dried medicinal materials of Xiaobaiwei, crush them, and extract them with 10 times the amount of 75% ethanol for 3 times, each time for 1.5 h. Combine the extracts, vacuum filter, and concentrate them on a rotary evaporator. Further vacuum freeze-dry at -80℃ to obtain a total of 19.57 g of Xiaobaiwei ethanol extract powder (TYE), with a yield of 19.57%.

[0031] 1.2 Analysis of chemical components of Psoralea corylifolia extract Accurately weigh 0.2 g of TYE, add 10 mL of methanol, weigh accurately, ultrasonicate for 30 min, cool to room temperature, weigh accurately again, make up the weight with methanol, filter through a 0.22 μm organic membrane and set aside. HPLC conditions: Mobile phase: A: 0.1% formic acid aqueous solution; B: 0.1% formic acid acetonitrile; Flow rate: 0.2 mL / min; Detection wavelength: 280 nm; Column: Agilent ZORBAX-SB C18 (100 mm × 4.6 mm i.d., 1.8 µm); Injection volume: 2 μL; Column oven temperature: 30 °C; Gradient elution program: 0 - 5 min, 5%B - 20%B; 5 - 10 min, 20%B - 55%B; 10 - 25 min, 55%B - 75%B; 25 - 40 min, 75%B - 95%B. MS conditions: Negative ion scanning mode; Scanning range: m / z 100 - 1500; Nebulizing gas (GS1): 50 psi; Nebulizing gas (GS2): 50 psi; Curtain gas (CUR): 35 psi; Ion source temperature (TEM): 550 °C; Ion source voltage (IS): -4500 V; First stage scan: Declustering potential (DP): 100 V; Collision energy (CE): 10 V; Second stage scan: Use TOF MS~Product Ion~IDA mode to acquire MS data, CID energy is 20, 40 and 60 V. Before injection, use a CDS pump to perform mass axis calibration to make the mass axis error less than 2 ppm.

[0032] 2 Effect test of Cynanchum versicolor Bge. var. incanum (Maxim.) Hsiao on tumors 2.1 Cell viability detection Take logarithmic growth phase Huh7 and HepG2 liver cancer cells, BT549 and 4T1 breast cancer cells, and AML12 normal liver cells respectively, and inoculate them into 96-well plates at a density of 5×10 3 cells per well. Treat the cells with basal medium containing 0 (control group), 1, 3, 5, 10, 20, 40, 50, 100 μg / mL Cynanchum versicolor Bge. var. incanum (Maxim.) Hsiao extract (TYE) for 24 h and 48 h, and set up a blank group without cells, with 6 replicates in each group. After incubation, add 10 μL of CCK-8 to each well and incubate for 1 h. Measure the absorbance (A) at 450 nm with an enzyme-labeled instrument, and calculate the cell survival rate. Cell survival rate (%) = (OD value of experimental well - OD value of blank well) / (OD value of control group - OD value of blank well) × 100%.

[0033] 2.2 Colony formation assay Huh7 and HepG2 liver cancer cells in the logarithmic growth phase, as well as BT549 and 4T1 breast cancer cells, were respectively seeded in 12-well plates at a density of 500 cells / well and cultured for 24 h (breast cancer) or 48 h (liver cancer). After that, the cells were treated with different concentrations of TYE (0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL) for 24 h or 48 h. Then, the normal medium was replaced and the cells were continuously cultured for 8 days. After removing the medium, the cells were washed once with PBS and fixed with 4% paraformaldehyde at room temperature for 20 min. The cells were stained with crystal violet for 15 min, washed, dried, and photographed for analysis.

[0034] 2.3 Live / Dead Cell Staining Huh7 and HepG2 liver cancer cells in the logarithmic growth phase, as well as BT549 and 4T1 breast cancer cells, were respectively seeded in 96-well plates at a density of 5×10 3 cells / well. After culturing for 24 h until the cells adhered, the cells were treated with different concentrations of TYE for 24 h (breast cancer) or 48 h (liver cancer), with 3 replicates in each group. The medium was aspirated, and the cells were washed once with PBS. 100 μL of staining working solution (prepared with PBS, containing 2 μM Calcein AM and 8 μM PI) was added to each well, and the cells were incubated in the dark at 37°C for 30 min. Images were collected with an inverted fluorescence microscope.

[0035] 2.4 Flow Cytometry Cell Cycle Analysis Huh7 and HepG2 liver cancer cells in the logarithmic growth phase, as well as BT549 and 4T1 breast cancer cells, were respectively seeded in 6-well plates at a density of 4×10 4 cells / well. After culturing for 24 h until the cells adhered, the breast cancer cells were treated with different concentrations of TYE (0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL) for 24 h or the liver cancer cells were treated for 48 h, with 3 replicates in each group. The cells were collected, washed, centrifuged, and the supernatant was discarded. 50 μL of H 2 O was added to resuspend the cell pellet, and then 1 mL of pre-cooled 70% ethanol was added to resuspend the cells, and the cells were fixed at 4°C overnight. The cell suspension was centrifuged, the supernatant was aspirated, washed, and centrifuged again to collect the cell pellet. 100 μL of staining working solution (staining buffer: propidium iodide PI staining solution (20X): RNase A (50X) = 100:5:2) was added to each tube of the sample, and the cell pellet was gently pipetted to fully resuspend the cells. After incubating at 37°C in the dark for 30 min, the cells were detected by flow cytometry.

[0036] 2.5 Animal Experiments To construct an orthotopic breast cancer tumor model, 20 4-week-old SPF-grade BALB / c female mice were purchased and fed adaptively for one week. 4T1 cells were digested and resuspended in pre-cooled PBS to a cell density of 1×10 7 / mL, then transferred to a centrifuge tube and stored on ice, and the tumor was completed within 2 hours. 100 μL of cells were injected into the second breast of the right side of the mouse. When the tumor grew to 60-80 cubic millimeters, the mice were randomly divided into four groups, namely the control group, the positive group, and the TYE low-dose and high-dose groups, with 5 mice in each group. The mice in the control group were gavaged with 1% CMC-Na every day, and the mice in the positive group were intraperitoneally injected with 5 mg / kg of oxaliplatin every two days; the mice in the treatment group were gavaged with 25 mg / kg and 50 mg / kg of TYE every day. Tumor volume and body weight were measured every 2 days. Tumor volume was calculated according to the following formula: volume = 1 / 2 × (length × width × width). After 21 days of TYE treatment, the mice were killed, and the tumor tissues were removed and weighed.

[0037] 3 Results 3.1 Analysis results of chemical components of Rhizoma Cibotii The results of LC-MS test of ethanol extract of Psoralea corylifolia are shown in Figure 1 . We consulted the relevant literature on the chemical components of Rhizoma Cibotii at home and abroad, and collected and sorted out the components in Rhizoma Cibotii with the help of Scifinder and Reaxys databases. Figure 1 The compounds represented by each peak in the TYE positive and negative total ion current graph were compared with the compounds in the compound library, and no known compounds were found.

[0038] 3.2 Results of the test on the inhibition of liver cancer and breast cancer cell viability by the extract of Asparagus cochinchinensis After incubating Huh7 and HepG2 human liver cancer cell lines with different doses of TYE for different time periods, the CCK8 method was used to detect cell viability. The results showed that TYE significantly inhibited Huh7 cell viability after incubation for 24 h at a concentration of 20-100 μg / mL, with a significant dose-effect relationship; the effect was more significant after incubation for 48 h, and 10 μg / mL showed a very significant inhibitory effect ( Figure 2 AB). TYE at a concentration of 1-100 μg / mL incubated for 24 h did not inhibit the viability of HepG2 cells ( Figure 2 C), but the 3-100 μg / mL dose significantly inhibited the viability of HepG2 cells after 48 h of incubation ( Figure 2 D). Subsequently, the effect of TYE on normal hepatocytes was further observed. The results showed that after TYE incubated normal hepatocytes of AML12 mice for 48 hours, the cell morphology did not change significantly compared with the control group, even at a dose of 100 μg / mL ( Figure 2E). CCK8 analysis revealed that incubation with TYE for 24 h and 48 h did not affect the viability of AML12 cells ( Figure 2 F-G), indicating that TYE has good safety and selectivity. Further tests on the viability of BT549 and 4T1 breast cancer cells found that treatment with extremely low doses of TYE for 24 h showed significant inhibitory effects, which were significantly stronger than those on Huh7 and HepG2 human liver cancer cells ( Figure 3 ).

[0039] 3.3 Inhibitory effect of Cynanchum versicolor extract on the clonal proliferation of breast and liver cancer cells After incubating Huh7 and HepG2 liver cancer cells with TYE for 48 h respectively, the cells were cultured in normal medium continuously for 8 days. After staining, it was found that compared with the control group (0 μg / mL), the clonal proliferation areas of both liver cancer cells were significantly reduced. Statistical analysis showed significant differences and an obvious dose-effect relationship, with the inhibitory effect on Huh7 cells being particularly obvious ( Figure 4 ). This indicates that TYE significantly inhibited the growth of both liver cancer cells. The results of the plate clonal proliferation test on BT549 and 4T1 breast cancer cells showed that the inhibitory effect of TYE on breast cancer cell proliferation was more significant than that on liver cancer cells ( Figure 5 ).

[0040] 3.4 Promotion of the death of breast and liver cancer cells by Cynanchum versicolor extract After treating Huh7 and HepG2 cells with different doses of TYE for 48 h, Calcein / PI staining (green represents live cells, red represents dead cells) was used for analysis. The results showed that with the increase in the dose of TYE, the number of live cells in both liver cancer cells decreased significantly, and the number of dead cells increased significantly. After statistical analysis, the ratio of dead cells to live cells in both liver cancer cell lines was significantly different from that of the control group ( Figure 6 ). In promoting the death of breast cancer BT549 and 4T1 cells, TYE showed a similar trend and effect to that of liver cancer cell lines ( Figure 7 ).

[0041] 3.5 Induction of cell cycle arrest in breast and liver cancer cells by Cynanchum versicolor extract Flow cytometry detection found that after treating two liver cancer cells with different doses of TYE for 48 h, the cell cycle was arrested at the G2 / M phase. The proportion of Huh7 cells in the G2 / M phase increased from 9.50 ± 3.44% in the control group to 22.41 ± 1.10% in the 40 μg / mL dose group, and the proportion of HepG2 cells in the G2 / M phase increased from 8.97 ± 1.66% in the control group to 29.55 ± 1.11% in the 40 μg / mL dose group. Except for the 5 μg / mL dose group of HepG2 cells, the other groups were significantly different from the control group (0 μg / mL) ( Figure 8). These results indicate that TYE induces cell cycle arrest in liver cancer cells, thereby increasing cell death. TYE also significantly induces cell cycle arrest in breast cancer cells, except that the arrest is in the G1 phase ( Figure 9 ).

[0042] 3.6 Animal experiments TYE was continuously administered to mice by gavage at a dose of 25 mg / kg.d -1 and 50 mg / kg.d -1 for 21 days. It significantly inhibited the growth of triple-negative breast cancer tissues. Compared with the control group, both the tumor volume and weight were significantly reduced; moreover, compared with the oxaliplatin positive group, there was no significant difference. The results are shown in Figure 10 .

[0043] 4. Discussion There are very few literature reports on the chemical constituents of Tylophora yunnanensis, also known as Cynanchum atratum var. albidum, and its related plants. It has been reported that 11 compounds were isolated and identified from the 90% ethanol extract of the rhizome of Tylophora yunnanensis, including p-hydroxyacetophenone, 2,4-dihydroxyacetophenone, 3,4-dihydroxyacetophenone, 3-hydroxy-4-methoxyacetophenone, neotylophorin, tylophorone, β-amyrin, tylophorine B, stearic acid, β-sitosterol, and daucosterol; 18 compounds were identified from the petroleum ether extract of the stems and leaves of Tylophora yunnanensis, including sterols, flavonoids, alkaloids, esters, carboxylic acids, etc. However, in this invention, liquid chromatography-mass spectrometry technology was used to detect the chemical constituents of TYE, and the mass spectrometry data of TYE was compared and analyzed in detail with the compounds reported in the literature of Cynanchum atratum var. albidum, and no known chemical constituents were identified. This indicates that the extract obtained by extracting Cynanchum atratum var. albidum with 75% ethanol contains new components, and their efficacy is still unclear.

[0044] The CCK8 experiment results of this invention show that the extract of Cynanchum atratum var. albidum (TYE) significantly inhibits the viability of Huh7 liver cancer cells, especially after 48 h of treatment. Although TYE cannot inhibit the viability of HepG2 cells after 24 h of incubation, it shows a significant inhibitory effect after 48 h. To determine whether TYE is toxic to normal liver cells, the effects on the morphology and viability of the normal mouse liver cell line AML12 were observed. As a result, TYE did not significantly change the cell morphology even when treated at a concentration as high as 100 μg / mL for 48 h, and there was no significant effect on the viability of normal liver cells after incubation for 24 h and 48 h, with no significant difference compared with the control group. These results suggest that Cynanchum atratum var. albidum may not exert its effect through direct cytotoxicity and has good safety and selectivity. The plate cloning assay and PI / Calcein AM staining analysis further confirmed the effect of TYE in inhibiting the proliferation and inducing cell death of liver cancer cells.

[0045] Flow cytometry analysis was used in this invention and it was found that after 48 h of treatment with TYE, the cell cycles of Huh7 and HepG2 liver cancer cells were significantly arrested at the G2 / M phase, and after 24 h of treatment, the cell cycles of BT549 and 4T1 breast cancer cells were significantly arrested at the G1 phase. At the same time, animal experiments confirmed that TYE had a significant therapeutic effect on breast cancer.

[0046] In summary, the ethanol extract of Cynanchum versicolor Bunge ex Decne. in this invention has been proven by cell experiments and animal experiments to have the effects of inhibiting the proliferation of triple-negative breast cancer cells and liver cancer cells, inducing the death of triple-negative breast cancer cells and liver cancer cells, and reducing the tumor volume and tumor mass of triple-negative breast cancer, showing significant effects in the treatment of liver cancer and breast cancer.

Claims

1. A Herba Microcarpaeidae extract, characterized in that: It is an extract obtained by extracting the small white violet with 50-80% ethanol; The small white violet is a plant of the Asclepiadaceae family. Tylophora yunnanensis Schlechter's roots and rhizomes.

2. The Herba Microphyllae extract according to claim 1, characterized in that: It is an extract obtained by extracting small white mulberry with 75% ethanol.

3. A method for preparing the Herba Microphyllae extract according to claim 1, characterized in that: The steps include: Take Xiaobaiwei, add 50~80% ethanol to extract, concentrate and dry the extract, or remove impurities and purify the extract and then dry it.

4. The preparation method according to claim 3, characterized in that: The extraction is reflux extraction or ultrasonic extraction; the power of the ultrasonic extraction is 600-800 W, the time is 20-30 min, and the solid-liquid ratio is 1 g:15-20 mL; the number of reflux extractions is 3 times, each time lasts 1-2 h, and the solid-liquid ratio is 1 g:8-10 mL.

5. The preparation method according to claim 3, characterized in that: The concentration method includes using a rotary evaporator to concentrate; And / or: the impurity removal method includes centrifugal separation, the speed of centrifugal separation is 5000-30000 r / min, and the time is 5-20 min; And / or: the purification method includes separation and purification by macroporous resin and / or purification by organic membrane filtration; the macroporous resin includes D101 macroporous resin, AB-8 macroporous resin or X-5 macroporous resin, the elution reagent for separation and purification is ethanol solution, the volume fraction of ethanol solution is 10-80%, and the amount is 2-20 times the column volume; the pore size of the organic membrane is 20000 Daltons; And / or: the drying method includes vacuum freeze drying, and the vacuum freeze drying temperature is -120~-40°C, preferably -80°C.

6. Use of the Herba Microphyllae extract according to claim 1 in the preparation of a drug for treating tumors.

7. The use according to claim 6, characterized in that: The medicine is a medicine for treating breast cancer and / or liver cancer.

8. The use according to claim 6, characterized in that: The drug is a drug for treating triple-negative breast cancer and / or hepatocellular carcinoma.

9. The use according to claim 6, characterized in that: The drug has the effects of inhibiting cancer cell proliferation, inducing cancer cell death, reducing tumor volume and / or reducing tumor weight.

10. The use according to claim 6, characterized in that: The medicine is a preparation prepared by using the extract of Astragalus membranaceus as the active ingredient and adding pharmaceutically acceptable auxiliary materials; the preparation is an oral preparation; the oral preparation includes solution, powder, granule, tablet and capsule.

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