Taxus chinensis extract as well as preparation method, application and pharmaceutical composition thereof

Abstract

The invention provides a taxus chinensis extract, a preparation method and application thereof and a pharmaceutical composition, and belongs to the technical field of extraction and separation. According to the invention, carbon dioxide is adopted as an extraction agent for supercritical extraction, and the ratio of the taxus chinensis extract to carbon dioxide is controlled, so that the extraction effect of effective components (such as paclitaxel, taxine, saruginine, flavone, polysaccharide and tannin) in taxus chinensis is improved, the mass percentage content of paclitaxel in the prepared extract is as high as 0.097-0.13%, and the mass percentage content of tannin in the prepared extract is as high as 0.097-0.13%. The content is far higher than the mass percentage content of paclitaxel in an extract (liquid) obtained by an extraction method in the prior art. The taxus chinensis extract provided by the invention contains various anti-cancer effective components, has a broad-spectrum and excellent anti-cancer effect on various cancer cells such as liver cancer, cervical cancer, lung cancer, rectal cancer and breast cancer, and is of great significance to research on preparation of anti-cancer drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of extraction and separation, and particularly relates to a Taxus extract, a preparation method and application thereof, and a pharmaceutical composition. Background Art

[0002] Taxus is a rare anti-cancer plant, which contains a variety of active ingredients. In addition to the anti-cancer drug ingredient - paclitaxel, it also contains a large number of other active ingredients including taxinine, cephalotaxine, flavonoids, polysaccharides, tannins, etc., and has significant anti-tumor effects. The existing extraction methods for the active ingredients in Taxus mainly include solvent extraction method, solid-phase extraction method and supercritical extraction method. Among them, the supercritical extraction method is currently the best extraction method for extracting active ingredients from Taxus. For example, Chinese Patent CN110151800A discloses a method for extracting a Taxus extract: adding pretreated Taxus raw materials into a supercritical extraction kettle, the pretreatment includes operations such as sorting, cleaning, cutting, drying, pulverizing, etc., and then loading the Taxus powder into the extraction kettle of the supercritical device, and introducing carbon dioxide and entrainer to extract the active ingredients in the Taxus raw materials. However, the mass percentage content of paclitaxel in the Taxus extract obtained by the above extraction method is only 0.002 - 0.010%, and the content of paclitaxel is low. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a Taxus extract, a preparation method and application thereof, and a pharmaceutical composition. The mass percentage content of paclitaxel in the Taxus extract prepared by the preparation method provided by the present invention is as high as 0.097 - 0.113%, and the content of paclitaxel is high.

[0004] To achieve the above purpose, the present invention adopts the following technical solutions:

[0005] The present invention provides a preparation method of a Taxus extract, comprising the following steps:

[0006] Mixing Taxus powder and an entrainer, and performing carbon dioxide supercritical extraction to obtain a Taxus extract;

[0007] The mass ratio of the dry weight of the Taxus powder to the mass of carbon dioxide is 1:3 - 4.

[0008] Preferably, the entrainer includes ethanol; the volume ratio of the dry weight of the Taxus powder to the entrainer is 6g:0.8 - 1.5mL.

[0009] Preferably, the carbon dioxide supercritical extraction is carried out in a supercritical extraction kettle;

[0010] The supercritical extraction kettle includes an extraction kettle and a separation kettle;

[0011] The temperature of the extraction kettle is 45 - 50 °C, and the pressure is 25 - 35 MPa.

[0012] Preferably, the temperature of the separation kettle is 35 - 45 °C, and the pressure is 7 - 9 MPa.

[0013] Preferably, the temperature of the extraction kettle is 48 - 50 °C, and the pressure is 28 - 30 MPa.

[0014] Preferably, the time of the supercritical carbon dioxide extraction is 3 - 4.5 h.

[0015] Preferably, the Taxus powder is obtained by drying and pulverizing Taxus.

[0016] The temperature of the drying is ≤ 70 °C, and the time is 20 - 24 h.

[0017] The present invention provides a Taxus extract prepared by the preparation method described in the above technical solution, and the Taxus extract contains paclitaxel with a mass percentage content of ≥ 0.097%.

[0018] The present invention provides the application of the Taxus extract described in the above technical solution in the preparation of anticancer drugs.

[0019] The present invention provides a pharmaceutical composition, which comprises the Taxus extract described in the above technical solution and a pharmaceutically acceptable excipient.

[0020] The present invention provides a preparation method of a Taxus extract. In the present invention, under a carbon dioxide atmosphere, pretreated Taxus and an entrainer are mixed, and supercritical extraction is carried out to obtain a Taxus extract. The present invention uses carbon dioxide as the extraction agent for supercritical extraction, and by controlling the dosage ratio of the Taxus extract to carbon dioxide, the extraction rate of effective components (such as paclitaxel, taxinine, cephalotaxine, flavonoids, polysaccharides, tannins) in Taxus is improved. The mass percentage content of paclitaxel in the prepared extract is above 0.097%, and the content of paclitaxel is much higher than that in the extract (liquid) obtained by the extraction method in the prior art. Moreover, the preparation method provided by the present invention is simple in operation. During the continuous production process, carbon dioxide can be recycled, the cost is low, and it is suitable for industrial production.

[0021] The present invention also provides a Taxus extract. The Taxus extract provided by the present invention contains a variety of anticancer effective components, has a broad-spectrum and excellent anticancer effect on various cancer cells such as liver cancer, cervical cancer, lung cancer, rectal cancer and breast cancer, and is of great significance for the research on the preparation of anticancer drugs. Description of the Drawings

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0023] In all the drawings of the present invention, H008 is the Taxus chinensis extract prepared by the present invention, and ZS is paclitaxel.

[0024] Figure 1 It is a comparison chart of the inhibitory effects of Taxus chinensis extract and paclitaxel on the growth of human hepatocellular carcinoma HepG2 cells;

[0025] Figure 2 It is a comparison chart of the inhibitory effects of Taxus chinensis extract and paclitaxel on the growth of human cervical cancer Hela cells;

[0026] Figure 3 It is a comparison chart of the inhibitory effects of Taxus chinensis extract and paclitaxel on human lung cancer A549 cells;

[0027] Figure 4 It is a comparison chart of the inhibitory effects of Taxus chinensis extract and paclitaxel on human rectal cancer HCT116 cells;

[0028] Figure 5 It is a comparison chart of the inhibitory effects of Taxus chinensis extract and paclitaxel on the growth of human breast cancer MDA-MB-231 cells;

[0029] Figure 6 It is a comparison chart of the inhibitory effects of Taxus chinensis extract and paclitaxel on the growth of human breast cancer MCF-7 cells;

[0030] Figure 7 It is a diagram of the effect of Taxus chinensis extract on the morphology of human hepatocellular carcinoma HepG2 cells;

[0031] Figure 8 It is a diagram of the effect of Taxus chinensis extract on the morphology of human cervical cancer Hela cells;

[0032] Figure 9 It is a diagram of the effect of Taxus chinensis extract on the morphology of human rectal cancer HCT116 cells;

[0033] Figure 10 It is a cell apoptosis loss diagram of human hepatocellular carcinoma HepG2 cells - normal control group;

[0034] Figure 11 It is a cell apoptosis loss diagram of human hepatocellular carcinoma HepG2 cells - 10 μg / mL Taxus chinensis extract treatment group;

[0035] Figure 12Apoptosis loss cell diagram of human liver cancer HepG2 cells - 20 μg / mL Taxus chinensis extract treatment group;

[0036] Figure 13 Apoptosis loss cell diagram of human liver cancer HepG2 cells - 20 ng / mL paclitaxel positive control group;

[0037] Figure 14 Bar chart of the effects of Taxus chinensis extract and paclitaxel on apoptosis of liver cancer HepG2 cells;

[0038] Figure 15 Apoptosis loss cell diagram of human cervical cancer Hela cells - normal control group;

[0039] Figure 16 Apoptosis loss cell diagram of human cervical cancer Hela cells - 10 μg / mL Taxus chinensis extract treatment group;

[0040] Figure 17 Apoptosis loss cell diagram of human cervical cancer Hela cells - 20 μg / mL Taxus chinensis extract treatment group;

[0041] Figure 18 Apoptosis loss cell diagram of human cervical cancer Hela cells - 20 ng / mL paclitaxel positive control group;

[0042] Figure 19 Bar chart of the effects of Taxus chinensis extract and paclitaxel on apoptosis of cervical cancer Hela cells;

[0043] Figure 20 Flow cytometry diagram of human liver cancer HepG2 cell cycle experiment - normal control group;

[0044] Figure 21 Flow cytometry diagram of human liver cancer HepG2 cell cycle experiment - 10 μg / mL Taxus chinensis extract treatment group;

[0045] Figure 22 Flow cytometry diagram of human liver cancer HepG2 cell cycle experiment - 20 μg / mL Taxus chinensis extract treatment group;

[0046] Figure 23 Flow cytometry diagram of human liver cancer HepG2 cell cycle experiment - 20 ng / mL paclitaxel positive control group;

[0047] Figure 24 Bar chart of the effects of Taxus chinensis extract and paclitaxel on the cell cycle of liver cancer HepG2 cells;

[0048] Figure 25 Flow cytometry diagram of human cervical cancer Hela cell cycle experiment - normal control group;

[0049] Figure 26 Flow cytometry graph of the cell cycle of human cervical cancer Hela cells - treatment group with 10 μg / mL Taxus chinensis extract;

[0050] Figure 27 Flow cytometry graph of the cell cycle of human cervical cancer Hela cells - treatment group with 20 μg / mL Taxus chinensis extract;

[0051] Figure 28 Flow cytometry graph of the cell cycle of human cervical cancer Hela cells - positive control group with 20 ng / mL paclitaxel;

[0052] Figure 29 Bar graph showing the effects of Taxus chinensis extract and paclitaxel on the cell cycle of cervical cancer Hela cells. Detailed implementation method

[0053] The present invention provides a preparation method of Taxus chinensis extract, comprising the following steps:

[0054] Mix Taxus chinensis powder and entrainer, and perform supercritical carbon dioxide extraction to obtain Taxus chinensis extract;

[0055] The mass ratio of the dry weight of Taxus chinensis powder to the mass of carbon dioxide is 1:3 - 4.

[0056] In the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art.

[0057] In the present invention, the Taxus chinensis powder is preferably obtained by drying and pulverizing Taxus chinensis. In the present invention, the Taxus chinensis is preferably Taxus media; the origin of the Taxus chinensis is preferably Beichuan Qiang Autonomous County, Mianyang City, Sichuan Province. In the present invention, the Taxus chinensis is preferably the branches and leaves of Taxus chinensis. In the present invention, the particle size of the Taxus chinensis powder is preferably 20 - 40 mesh, more preferably 20 - 30 mesh, and most preferably 25 - 30 mesh.

[0058] In the present invention, before drying, it is preferably further included to sort, wash and cut the Taxus chinensis. In the present invention, the sorting is preferably to remove foreign matters (obvious solids) other than Taxus chinensis branches. The present invention has no special limitation on the methods of washing and cutting, and conventional washing and cutting methods in the art can be used. In the present invention, the diameter of the cut Taxus chinensis segments is preferably ≤15 mm, and the length is preferably ≤10 mm.

[0059] In the present invention, the drying temperature is preferably ≤70°C, more preferably 40 - 60°C, and most preferably 47 - 50°C; the drying time is preferably 20 - 24 h, more preferably 21 - 23 h, and most preferably 21 - 22 h. The present invention has no special limitation on the pulverizing parameters, and it is only necessary to make the particle size of the Taxus chinensis powder reach 20 - 40 mesh.

[0060] In the present invention, the mass percentage content of paclitaxel in the Taxus powder is preferably ≥ 0.015%. In the present invention, the mass percentage content of paclitaxel in the Taxus powder is preferably obtained by liquid chromatography detection. In the present invention, the method of liquid chromatography detection preferably includes: dissolving the Taxus powder in acetonitrile to obtain a Taxus sample solution to be detected; dissolving the paclitaxel reference substance in acetonitrile to obtain a paclitaxel reference substance solution; respectively performing liquid chromatography detection on the Taxus sample solution to be detected and the paclitaxel reference substance solution, and comparing the obtained detection results to obtain the mass percentage content of paclitaxel in the Taxus powder.

[0061] In the present invention, the conditions of liquid chromatography detection preferably include: the chromatographic column is an octadecylsilane-bonded silica column; the mobile phase system includes mobile phase A, mobile phase B, and mobile phase C. Mobile phase A is methanol, mobile phase B is water, and mobile phase C is acetonitrile; the elution method is isocratic elution, and during the isocratic elution process, the volume ratio of mobile phase A, mobile phase B, and mobile phase C is 23:41:36; the detection wavelength is 227 nm, and the injection volume is 10 μL.

[0062] In the present invention, the entrainer preferably includes ethanol. In the present invention, the ratio of the dry weight of the Taxus powder to the volume of the entrainer is preferably 6 g: 0.8 - 1.5 mL, more preferably 6 g: 1.0 - 1.3 mL, and most preferably 6 g: 1.1 - 1.2 mL. In the present invention, ethanol is used as the entrainer, and no other organic solvents, chemical raw materials, and process water are added during the supercritical carbon dioxide extraction process, avoiding the residue of harmful substances, and the extraction process has no obvious adverse impact on the environment.

[0063] In the present invention, the supercritical carbon dioxide extraction is preferably carried out in a supercritical extraction autoclave; the supercritical extraction autoclave preferably includes an extraction autoclave and a separation autoclave.

[0064] In the present invention, the temperature of the extraction autoclave is preferably 45 - 50 °C, more preferably 46 - 50 °C, and most preferably 48 - 50 °C; the pressure of the extraction autoclave is preferably 25 - 35 MPa, more preferably 27 - 32 MPa, and most preferably 28 - 30 MPa.

[0065] In the present invention, the temperature of the separation autoclave is preferably 35 - 45 °C, more preferably 37 - 42 °C, and most preferably 38 - 40 °C; the pressure of the separation autoclave is preferably 7 - 9 MPa, more preferably 7 - 8 MPa. In the extraction autoclave, carbon dioxide is in a supercritical state (fluid state), and together with the entrainer, it extracts the Taxus powder. The extracted extract enters the separation autoclave, the pressure decreases, carbon dioxide leaves the supercritical state and becomes a gas and escapes, and returns to the extraction autoclave through a pipeline, and the Taxus extract precipitates, completing one extraction.

[0066] In the present invention, the time of supercritical carbon dioxide extraction is preferably 3 to 4.5 h, more preferably 3 to 4 h, and most preferably 3.5 to 4 h. In the present invention, carbon dioxide is cycled and transformed between the fluid-gas state multiple times in the separation kettle and the extraction kettle to perform multiple extractions on Taxus powder, resulting in a high yield of Taxus extract and a high content of paclitaxel. Moreover, during the preparation process, carbon dioxide can be recycled multiple times, with low costs, and is more suitable for industrial production.

[0067] In the present invention, the Taxus extract is preferably used directly without post-treatment.

[0068] The present invention also provides a Taxus extract prepared by the above preparation method. The Taxus extract contains paclitaxel with a mass percentage content of ≥ 0.097%, preferably 0.097 to 0.15%, and more preferably 0.097 to 0.113%. In the present invention, the Taxus extract is preferably a paste. In the present invention, the mass percentage content of the entrainer in the Taxus extract is preferably 1.5 to 5%, more preferably 2 to 4%, and most preferably 3 to 3.33%.

[0069] The present invention provides the use of the Taxus extract according to the above technical solution in the preparation of anticancer drugs.

[0070] The present invention also provides a pharmaceutical composition, comprising the Taxus extract according to the above technical solution and a pharmaceutically acceptable excipient.

[0071] In order to further illustrate the present invention, the following describes in detail a Taxus extract, its preparation method, use, and pharmaceutical composition provided by the present invention with reference to the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0072] In all the test examples of the present invention, the specifications of the instruments and reagents used are as follows:

[0073] The reagents are as follows: (1) Biochemical reagents: Sodium chloride (Sangon Biotech, Shanghai, A610476-0001); Potassium chloride (Sangon Biotech, Shanghai, A100395-0100); Monosodium phosphate (Xilong Chemical Factory, Shantou, Guangdong); Disodium phosphate (Sangon Biotech, Shanghai, SB0878-500); Dimethyl sulfoxide (DMSO, Sangon Biotech, Shanghai, A610163-0250); Tris(hydroxymethyl)aminomethane (Tris, Sangon Biotech, Shanghai, A100826-0100); Sodium dodecyl sulfate (SDS, Sangon Biotech, Shanghai, A600485); Tween-20 (Sangon Biotech, Shanghai, A600560); Urethane (BSF171112, Hefei BASF Biotechnology Co., Ltd.); Glycine (Sangon Biotech, Shanghai, A502065-0500); Bovine serum albumin (BSA, Solarbio, Beijing, A8010); Matrix-Gel basement membrane matrix (standard type, without phenol red, C0372, Beyotime, Shanghai); Olive oil was purchased from Walmart (Huiyi premium grade, batch number: 203251081).

[0074] (2) Cell detection reagents: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit (Coolaber, Beijing, CM7461); Cell Cycle and Apoptosis Detection Kit (Yeasen Biotech Co., Ltd., Shanghai, C1208971); Annexin V-FITC / PI Apoptosis Detection Kit (Yeasen Biotech Co., Ltd., Shanghai, A5311020).

[0075] (3) Cells and cell culture reagents: HepG2 cells (hepatocarcinoma cells), Hela cells (cervical cancer cells), A549 cells (lung cancer cells), MDA-MB-231 cells (breast cancer cells), MCF-7 cells (breast cancer cells) and HCT-116 cells (rectal cancer cells) were all from the Cell Bank of the Chinese Academy of Sciences; Trypsin (Beyotime, Shanghai, C0201); Penicillin-Streptomycin solution (Gibco, USA, 15140-122); Fetal bovine serum (Gibco, USA, ST30-3302).

[0076] (4) Test drugs: The Taxus chinensis extract prepared in the present invention; Paclitaxel pure product (CAS: 33069-62-4, purity exceeding 99%, batch number 20221108) was provided by Shanghai HanXiang Biotechnology Co., Ltd.

[0077] The instruments are shown in Table 1.

[0078] Table 1 Instruments used in the test examples of the present invention

[0079]

[0080]

[0081] Example 1

[0082] Select Taxus branches and leaves, remove foreign substances other than Taxus branches and leaves by visual inspection, wash, cut the selected branches and leaves, dry them at 50 °C for 23 h, pulverize and sieve the dried Taxus branches and leaves to obtain Taxus powder with a particle size of 20 - 40 mesh, and store it in the warehouse after packaging and inspection.

[0083] The detection method for the mass percentage content of paclitaxel in Taxus powder is as follows:

[0084] Take paclitaxel reference standard and dissolve it in acetonitrile to obtain a paclitaxel reference standard solution with a concentration of 0.12 mg / mL; take 3 g of Taxus powder in a 150 mL stoppered conical flask, accurately add 50 mL of acetonitrile, weigh it, ultrasonically treat it for 30 min (ultrasonic power is 300 W, frequency is 40 kHz), take it out and let it cool, weigh it again, make up the lost weight with 50 v / v% methanol aqueous solution, shake well, filter, and take the subsequent filtrate to obtain the Taxus sample solution to be measured.

[0085] Accurately pipette 10 μL each of the Taxus sample solution to be measured and the paclitaxel reference standard solution, perform liquid chromatography detection to obtain the content of Taxus powder, and take Taxus powder with a theoretical plate number not less than 3000 calculated by paclitaxel for subsequent preparation. Among them, the conditions for liquid chromatography detection are: the chromatographic column is an octadecylsilane bonded silica column; the mobile phase system includes mobile phase A, mobile phase B, and mobile phase C, mobile phase A is methanol, mobile phase B is water, mobile phase C is acetonitrile; the elution method is isocratic elution, and during the isocratic elution process, the volume ratio of mobile phase A, mobile phase B, and mobile phase C is 23:41:36; the detection wavelength is 227 nm.

[0086] Add Taxus powder and ethanol to the extraction kettle of the supercritical extraction kettle, introduce carbon dioxide, and perform carbon dioxide supercritical extraction. Among them, the temperature of the extraction kettle is 50 °C, the pressure is 30 MPa, the temperature of the separation kettle is 40 °C, the pressure is 7 MPa, and the extraction time is 3.5 h. After the extraction is completed, collect the paste in the separation kettle to obtain Taxus extract. The solid-liquid ratio of Taxus powder (dry weight) to ethanol is 1 kg:0.2 L, and the mass ratio of Taxus powder (dry weight) to the introduced carbon dioxide is 1:3. Table 2 shows the detection results of each component in the Taxus extract (hereinafter referred to as "Batch A") prepared in this example.

[0087] Table 2 Detection Results of Each Component in Taxus Extract

[0088]

[0089] As can be seen from Table 2, the mass percentage content of paclitaxel in the Taxus chinensis extract prepared by the present invention is 0.097 - 0.113%, with a high content. At the same time, the Taxus chinensis extract also contains various anti-cancer and antibacterial active ingredients (such as taxinine, cephalotaxine, flavonoids, polysaccharides, tannins), which is of great significance for the research on the preparation of anti-cancer drugs.

[0090] Test Example 1

[0091] In order to prove the anti-cancer effect of the Taxus chinensis extract prepared by the present invention, a cell detection experiment was carried out on the Taxus chinensis extract prepared in Batch A of the present invention:

[0092] (1) Cell culture

[0093] MCF-7 and HCT-116 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin + streptomycin, and HepG2 cells, Hela cells, MDA-MB-231 cells and A549 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin + streptomycin. The culture environment was an incubator at 37°C with 5% CO2. When the cells grew to 80 - 90% confluence, at this time the cells were arranged more densely and the intercellular spaces were smaller, and subculture operations could be carried out. When subculturing the cells, aspirate the medium, slowly add an appropriate amount of PBS, wash the culture dish, wash away the floating dead cells, aspirate the PBS, then add fresh RPMI 1640 or DMEM medium to the culture dish. After digestion with trypsin, aspirate the trypsin, wash with PBS, aspirate the PBS, add medium and pipette to obtain a cell suspension, transfer it to a new culture dish and add an appropriate amount of medium to continue culturing.

[0094] (2) MTT assay (cytotoxicity assay)

[0095] Collect MCF-7 cells, HCT-116 cells, HepG2 cells, Hela cells, MDA-MB-231 cells and A549 cells in the logarithmic growth phase, adjust the cell suspension concentration, add cells to a 96-well plate, and finally add 200 μL of culture medium to each well. Plate the cells to adjust the density to 1000 - 10000 cells / well, and fill the edge wells with sterile PBS. Incubate in an incubator at 37 °C and 5% CO2 for 12 h. Dissolve the Taxus chinensis extract and paclitaxel prepared in the present invention with DMSO, and add the Taxus chinensis extract at each concentration gradient to adherent cells. A total of 6 gradients are set, which are 1, 5, 10, 20, 40, and 80 μg / mL respectively. The positive control drug is paclitaxel, and a total of 6 gradients are set, which are 1, 5, 10, 20, 40, and 80 ng / mL respectively. The negative control adds an equal dose of DMSO solution. Set 5 replicates for each concentration, and incubate in an incubator at 37 °C and 5% CO2 for 24 h, 48 h, and 72 h respectively. Measure the cytotoxicity at each time point using an MTT kit. During the MTT test, add 20 μL of MTT (5 mg / mL) solution to each well of cells, and culture the cells in an incubator at 37 °C and 5% CO2 for 2 h. Discard the cell culture medium, add 100 μL of dimethyl sulfoxide solution to completely dissolve the blue-violet crystal formazan on the cell plate. Analyze the optical density value at 490 nm (OD490) using an Epoch microplate spectrophotometer.

[0096] Figure 1 Figure for comparing the inhibitory effects of Taxus chinensis extract and paclitaxel on the growth of human liver cancer HepG2 cells; Figure 2 Figure for comparing the inhibitory effects of Taxus chinensis extract and paclitaxel on the growth of human cervical cancer Hela cells; Figure 3 Figure for comparing the inhibitory effects of Taxus chinensis extract and paclitaxel on human lung cancer A549 cells; Figure 4 Figure for comparing the inhibitory effects of Taxus chinensis extract and paclitaxel on human rectal cancer HCT116 cells; Figure 5 Figure for comparing the inhibitory effects of Taxus chinensis extract and paclitaxel on the growth of human breast cancer MDA-MB-231 cells; Figure 6 Figure for comparing the inhibitory effects of Taxus chinensis extract and paclitaxel on the growth of human breast cancer MCF-7 cells. It can be Figures 1 to 6 seen that the Taxus chinensis extract prepared in the present invention can significantly inhibit the growth of human liver cancer, cervical cancer, lung cancer, rectal cancer and breast cancer cells, has a dose-dependent effect, and the effect is better than that of paclitaxel calculated by equal dose conversion.

[0097] (3) Cell apoptosis experiment

[0098] HepG2 and Hela cells were respectively seeded in a 96-well plate and cultured in an incubator at 37°C with 5% CO2. After cell attachment, the culture medium was aspirated, and the cells were divided into 4 groups. Culture media containing an equal amount of DMSO solution (control group), 10 μg / mL Taxus chinensis extract, 20 μg / mL Taxus chinensis extract, and 20 ng / mL paclitaxel positive drug were added respectively, and the cells were cultured for 24 h.

[0099] The cell apoptosis experiment was detected using a cell apoptosis detection kit. The specific steps were as follows: First, the cells were digested with trypsin, centrifuged to collect the cells, washed twice with PBS, and centrifuged again to collect the cells. Then the cells were resuspended with Binding Buffer, and AnnexinV-FITC and PI Staining Solution were added to label the cells. The reaction was carried out in the dark at room temperature for 10 - 15 min. Finally, Binding Buffer was added, and the prepared sample was placed on ice and detected using a flow cytometer. Detection was performed at an excitation wavelength of 488 nm, and the light scattering was also detected simultaneously. Quantification was carried out using FlowJo_v10.8.1 software.

[0100] Figure 7 Figure showing the effect of Taxus chinensis extract on the morphology of human hepatocarcinoma HepG2 cells; Figure 8 Figure showing the effect of Taxus chinensis extract on the morphology of human cervical cancer Hela cells; Figure 9 Figure showing the effect of Taxus chinensis extract on the morphology of human rectal cancer HCT116 cells. It can be seen from Figures 7 to 9 that in terms of cell morphology, it was observed that Taxus chinensis extract could induce apoptosis and necrosis of hepatocarcinoma, cervical cancer, and rectal cancer cells.

[0101] Figure 10 Flow cytometry plot of apoptotic cells of human hepatocarcinoma HepG2 cells - normal control group, Figure 11 Flow cytometry plot of apoptotic cells of human hepatocarcinoma HepG2 cells - 10 μg / mL Taxus chinensis extract treatment group, Figure 12 Flow cytometry plot of apoptotic cells of human hepatocarcinoma HepG2 cells - 20 μg / mL Taxus chinensis extract treatment group, Figure 13 Flow cytometry plot of apoptotic cells of human hepatocarcinoma HepG2 cells - 20 ng / mL paclitaxel positive control group. It can be seen from Figures 10 to 13 that there was no obvious apoptosis and necrosis in the hepatocarcinoma cells of the normal control group; obvious apoptosis and necrosis occurred in the hepatocarcinoma cells treated with 10 μg / mL Taxus chinensis extract; more significant apoptosis and necrosis occurred in the hepatocarcinoma cells treated with 20 μg / mL Taxus chinensis extract; apoptosis and necrosis occurred in the hepatocarcinoma cells treated with 20 ng / mL paclitaxel, but it was weaker than that of Taxus chinensis extract with equivalent dose conversion.

[0102] Figure 14Bar chart showing the effects of Taxus chinensis extract and paclitaxel on the apoptosis of HepG2 liver cancer cells. As can be seen from Figure 14 it, the Taxus chinensis extract can significantly increase the proportion of liver cancer cell death and apoptosis, showing a dose-dependent effect, and the effect is better than that of paclitaxel calculated by equivalent dose conversion.

[0103] Figure 15 Flow cytometry plot of apoptosis of human cervical cancer Hela cells - normal control group, Figure 16 Flow cytometry plot of apoptosis of human cervical cancer Hela cells - 10 μg / mL Taxus chinensis extract treatment group, Figure 17 Flow cytometry plot of apoptosis of human cervical cancer Hela cells - 20 μg / mL Taxus chinensis extract treatment group, Figure 18 Flow cytometry plot of apoptosis of human cervical cancer Hela cells - 20 ng / mL paclitaxel positive control group. As can be seen from Figures 15 to 18 it, there was no obvious apoptosis and necrosis in the cervical cancer cells of the normal control group; obvious apoptosis and necrosis occurred in the cervical cancer cells treated with 10 μg / mL Taxus chinensis extract; more significant apoptosis and necrosis occurred in the cervical cancer cells treated with 20 μg / mL Taxus chinensis extract, and apoptosis and necrosis occurred in the cervical cancer cells treated with 20 ng / mL paclitaxel, but it was weaker than that of the Taxus chinensis extract calculated by equivalent dose conversion.

[0104] Figure 19 Bar chart showing the effects of Taxus chinensis extract and paclitaxel on the apoptosis of Hela cervical cancer cells. As can be seen from Figure 19 it, the Taxus chinensis extract can significantly increase the proportion of cervical cancer cell death and apoptosis, showing a dose-dependent effect, and the effect is better than that of paclitaxel calculated by equivalent dose conversion.

[0105] (4) Cell cycle experiment

[0106] HepG2 and Hela cells were respectively seeded in well plates and cultured in an incubator at 37°C and 5% CO2. After attachment, the culture medium was aspirated, and the cells were divided into 4 groups, and the culture medium containing an equal amount of DMSO solution (control group), 10 μg / mL Taxus chinensis extract, 20 μg / mL Taxus chinensis extract, and 20 ng / mL paclitaxel positive drug was added respectively, and cultured for 24 h. The cell cycle experiment was carried out using a cell cycle and apoptosis detection kit. HepG2 and Hela cells were respectively treated. First, the cells were digested with trypsin to prepare a single-cell suspension, the cells were collected by centrifugation, fixed with 70 v / v% ethanol, centrifuged and resuspended with PBS, centrifuged again, and then resuspended with the prepared propidium iodide staining solution, incubated at 37°C in the dark for 30 min, and filtered through a 400-mesh sieve. Finally, detection was carried out using a flow cytometer, detection was carried out at an excitation wavelength of 488 nm, and at the same time the light scattering was detected, and quantification was carried out using FlowJo_v10.8.1 software.

[0107] Figure 20 Flow cytometry diagram of the cell cycle of human hepatocarcinoma HepG2 cells - normal control group, Figure 21 Flow cytometry diagram of the cell cycle of human hepatocarcinoma HepG2 cells - 10 μg / mL taxus extract treatment group, Figure 22 Flow cytometry diagram of the cell cycle of human hepatocarcinoma HepG2 cells - 20 μg / mL taxus extract treatment group, Figure 23 Flow cytometry diagram of the cell cycle of human hepatocarcinoma HepG2 cells - 20 ng / mL paclitaxel positive control group. It can be Figures 20 to 23 seen that the hepatocarcinoma cells in the normal control group are mainly in the G1 phase, while there are fewer cells in the G2 phase; after treatment with 10 μg / mL taxus extract, the G1-phase hepatocarcinoma cells are significantly reduced, while the G2-phase cells are significantly increased; after treatment with 20 μg / mL taxus extract, the G1-phase hepatocarcinoma cells are further significantly reduced, while the G2-phase cells are further significantly increased. After treatment with 20 ng / mL paclitaxel, the G1-phase hepatocarcinoma cells are significantly reduced, while the G2-phase cells are significantly increased, but the effect is weaker than that of the taxus extract with the same dose conversion.

[0108] Figure 24 Bar chart of the effects of taxus extract and paclitaxel on the cell cycle of human hepatocarcinoma HepG2 cells. It can be Figure 24 seen that taxus extract can inhibit the proportion of G1-phase hepatocarcinoma cells, up-regulate the proportion of G2-phase cells, terminate the cell cycle at the G2 phase, and has a dose-dependent effect, and the effect is better than that of paclitaxel.

[0109] Figure 25 Flow cytometry diagram of the cell cycle of human cervical cancer Hela cells - normal control group, Figure 26 Flow cytometry diagram of the cell cycle of human cervical cancer Hela cells - 10 μg / mL taxus extract treatment group, Figure 27 Flow cytometry diagram of the cell cycle of human cervical cancer Hela cells - 20 μg / mL taxus extract treatment group, Figure 28 Flow cytometry diagram of the cell cycle of human cervical cancer Hela cells - 20 ng / mL paclitaxel positive control group. It can be Figures 25 to 26 seen that the cervical cancer cells in the normal control group are mainly in the G1 phase, while there are fewer cells in the G2 phase; after treatment with 10 μg / mL taxus extract, the G1-phase cervical cancer cells are significantly reduced, while the G2-phase cells are significantly increased; after treatment with 20 μg / mL taxus extract, the G1-phase cervical cancer cells are further significantly reduced, while the G2-phase cells are further significantly increased. After treatment with 20 ng / mL paclitaxel, the G1-phase cervical cancer cells are significantly reduced, while the G2-phase cells are significantly increased, but the effect is weaker than that of the taxus extract with the same dose conversion.

[0110] Figure 29Bar chart showing the effects of Taxus chinensis extract and paclitaxel on the cell cycle of cervical cancer Hela cells. It can be seen from Figure 29 that Taxus chinensis extract can inhibit the proportion of G1-phase cells in cervical cancer, up-regulate the proportion of G2-phase cells, cause the cell cycle to terminate in the G2 phase, and has a dose-dependent effect, with a better effect than paclitaxel.

[0111] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A preparation method of Taxus chinensis extract, characterized in that, It includes the following steps: Mix Taxus powder and entrainer, and perform supercritical carbon dioxide extraction to obtain Taxus extract; The mass ratio of the dry weight of the Taxus powder to the mass of carbon dioxide is 1:3 - 4.

2. The preparation method according to claim 1, wherein The entrainer includes ethanol; the volume ratio of the dry weight of the Taxus powder to the entrainer is 6 g:0.8 - 1.5 mL.

3. The preparation method according to claim 1, characterized in that, The supercritical carbon dioxide extraction is carried out in a supercritical extraction kettle; The supercritical extraction kettle includes an extraction kettle and a separation kettle; The temperature of the extraction kettle is 45 - 50 °C, and the pressure is 25 - 35 MPa.

4. The preparation method according to claim 3, wherein The temperature of the separation kettle is 35 - 45 °C, and the pressure is 7 - 9 MPa.

5. The preparation method according to claim 3, wherein The temperature of the extraction kettle is 48 - 50 °C, and the pressure is 28 - 30 MPa.

6. The preparation method according to any one of claims 1 to 5, characterized in that, The time of the supercritical carbon dioxide extraction is 3 - 4.5 h.

7. The preparation method according to claim 1 or 2, characterized in that, The Taxus powder is obtained by drying and pulverizing Taxus; The temperature of the drying is ≤70 °C, and the time is 20 - 24 h.

8. The Taxus chinensis extract prepared by the preparation method according to any one of claims 1 to 7, characterized in that, The Taxus extract contains paclitaxel with a mass percentage content ≥0.097%.

9. Use of the Taxus extract according to claim 8 in the preparation of an anticancer drug.

10. A pharmaceutical composition, characterized in that, It includes the Taxus extract according to claim 8 and pharmaceutically acceptable excipients.

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