CN111297943A

CN111297943A - Supercritical CO2Application of fluid extraction of salvia miltiorrhiza extract

Info

Publication number: CN111297943A
Application number: CN201911367055.5A
Authority: CN
Inventors: 贾力; 李飞杨; 卢余盛; 林国兴
Original assignee: Nanjing Licheng Biomedical Technology Co ltd; Fuzhou University; Minjiang University
Current assignee: Nanjing Licheng Biomedical Technology Co ltd; Fuzhou University; Minjiang University
Priority date: 2019-12-26
Filing date: 2019-12-26
Publication date: 2020-06-19

Abstract

The invention provides supercritical CO₂An application of a fluid extraction salvia miltiorrhiza extract relates to the technical field of medicines, and the salvia miltiorrhiza extract is applied to prevent the metastasis of tumor cells after tumor resection; the tumor cells are circulating tumor cells. The invention promotes the anoikis of the trace CTC in blood by specifically inhibiting the adhesion of the trace circulating tumor cells and vascular endothelial cells, effectively prevents the circulating tumor cells from transferring after the tumor operation and solves the drug resistance problem of the tumor cells.

Description

Supercritical CO2Application of fluid extraction of salvia miltiorrhiza extract

Technical Field

The invention relates to the technical field of medicines, in particular to supercritical CO₂Use of fluid extraction of Salvia miltiorrhiza Bunge extract is provided.

Background

Tumor metastasis is the leading cause of death in tumor patients, and clinically more than 90% of tumor patients die of tumor metastasis rather than primary tumors: (Hanahan and Weinberg 2011,Sethi and Kang 2011). At present, the survival rate after Chinese tumor surgery is estimated to be at least more than 2000 ten thousand, and the survival rate after Chinese tumor surgery is estimated to be at risk of tumor recurrence and metastasis, while the traditional 'anticancer drugs' cannot effectively prevent tumor metastasis due to serious toxic and side effects. Analysis of 22 clinical trials in 1990-2004 by Morgan et al revealed that "anticancer drugs" only increased the 5-year survival rate of tumor patients by 2.1%, (Morgan,Ward et al.2004). Early use of chemotherapeutic drugs does not prevent tumor metastasis, but rather accelerates tumor growth. Clinical studies of Sharouni and the like find that drug chemotherapy can accelerate the re-proliferation of non-small cell lung cancer, and the tumor volume doubling time of patients in a chemotherapy group is obviously shortened compared with patients who do not receive chemotherapy (El Sharouni,Kal et al.2003). Rustin et al, published in Lancet 2010 (Rustin,van der Burg et al.2010) The median survival time values of the early chemotherapy group and the delayed chemotherapy group of the ovarian cancer patients are not obviously different, and the median survival time value of the early chemotherapy group patients is shortened by 2 months instead, which indicates that the 'anticancer drug' can not inhibit tumor metastasis and has no obvious toxic or side effect on human. However, no drug that can prevent tumor metastasis is currently used clinically.

Tumor metastasis is a complex process involving multiple steps and multiple factors, and involves rearrangement and deformation of tumor cytoskeleton, and tumor cells shed from extracellular matrix are normally Anoikis (Anoikis), but a few of tumor cells are mutated, and cell surface integrin is changed, so that the tumor cells adapt to the environment in which the tumor cells are located, epithelial-mesenchymal transition (EMT) is generated, and Anoikis is resisted. The mutated Tumor Cells may break through the basement membrane, invade the capillary or lymphatic capillaries, form Circulating Tumor Cells (CTCs), and then may adhere to vascular endothelial Cells, interact with each other, and then penetrate out of the vascular system, and stay in specific tissues or organs, where the Tumor Cells proliferate and vascularize in new tissues or organs to form new metastases. The altered tumor cells may enter the blood or lymph vessels, and whether tumor cells entering the circulatory system adhere to the vascular endothelium becomes a critical step in tumor metastasis, depending on the expression of specific adhesion molecules on the surface of tumor cells and endothelial cells, including intercellular adhesion molecule (ICAM-1), Integrin (Integrin) on the surface of tumor cells, and a range of survival signals, oncogenes, growth factor receptors, and Integrin and growth factor receptor-associated enzymes.

The existing biological anti-cancer technology generally discusses the prevention of tumor metastasis from the perspective of tumor cells, but in practical application, the actual effect of preventing tumor metastasis again is limited aiming at the treatment of tumor cells, particularly after tumor resection, and the drug resistance is easily generated by the tumor cells aiming at the fact that the tumor cells are often single-target, so that the true prevention of tumor metastasis again cannot be achieved.

On the other hand, in the prior art, the tumor metastasis is not prevented from the viewpoint of the microenvironment of the tumor cells, so that the expressions of intercellular adhesion molecules (ICAM-1), tumor cell surface integrins (Integrin) and other related genes are simultaneously inhibited, and the tumor postoperative metastasis is effectively and comprehensively inhibited from the viewpoint of the microenvironment of the tumor cells.

Disclosure of Invention

In view of the above problems, the present invention provides supercritical CO₂The application of the fluid extraction of the salvia miltiorrhiza bunge extract can prevent tumor metastasis from the angle of a microenvironment where tumor cells are located, so as to inhibit the expression of intercellular adhesion molecules (ICAM-1), tumor cell surface integrins (Integrin) and other related genes, solve the problem of drug resistance of the tumor cells, and safely, effectively and comprehensively inhibit tumor postoperative metastasis at multiple target points. The specific invention content is as follows:

supercritical CO₂The application of the fluid extraction of the salvia miltiorrhiza extract, which is applied to prevent the metastasis of tumor cells after tumor resection operation;

the tumor cells are circulating tumor cells.

Further, the tumor cell includes any circulating tumor cell of lung cancer, breast cancer or colon cancer.

Furthermore, the salvia miltiorrhiza extract is applied to a product for preventing tumor cell metastasis after a tumor resection operation or a product for preventing tumor cell metastasis after a tumor resection operation through a pharmaceutically acceptable carrier or auxiliary material.

Furthermore, the components of the salvia miltiorrhiza extract regulate the integrin protein on the surface of the circulating tumor cells from a plurality of channels, hinder the adhesion of the circulating tumor cells and platelets, reduce the adhesion capacity of the circulating tumor cells and vascular endothelium, and promote the anoikis of the circulating tumor cells, thereby inhibiting the migration of the circulating tumor cells and preventing the tumor from metastasizing again.

Furthermore, the salvia miltiorrhiza extract blocks the adhesion of tumor cells to endothelial cells by inhibiting the expression of adhesion proteins ICAM and E-Selectin on the endothelial cells.

Further, the salvia miltiorrhiza extract blocks the adhesion of tumor cells to endothelial cells by inhibiting the expression of adhesion protein CD29integrin β 1 on the tumor cells.

Further, the preparation method of the salvia miltiorrhiza extract comprises the following steps: adding 60ml of absolute ethanol into 200g of salvia miltiorrhiza powder, placing the mixture into a supercritical extraction kettle, and fixing the pressure of the separation kettle at 10MPa and the temperature of 50 ℃; the extraction pressure is 20MPa, the extraction temperature is 50 ℃, and CO is added₂The flow rate is 15kg/h, and the extraction time is 120 min.

Further, the components of the salvia miltiorrhiza extract comprise tanshinone IIA, tanshinone IIB, tanshinone I, cryptotanshinone, hydroxy tanshinone, tanshinone methyl ester, tanshinone, przewaquinone, tanshinol A, tanshinol I, tanshinol II, iso-tanshinone, tanshinol, salvianolic acid, tanshinol A, tanshinol B, tanshinolactone and protocatechualdehyde.

The invention mainly aims at the microenvironment where the tumor cells are located, particularly at circulating tumor cells entering blood vessels, and regulates integrin protein on the surface of the circulating tumor cells and the microenvironment where the tumor cells are located through the salvia miltiorrhiza extract so as to reduce the activity of platelets in the microenvironment, so that the effect of the platelets as a bridge between the tumor cells and endothelial cells is reduced. In addition, the Salvia miltiorrhiza extracts regulate adhesion proteins on the surface of endothelial cells. The approaches are combined to reduce the effect of the adhesion of the tumor cells to the endothelial cells, and prevent the subsequent tumor cells of the tumor resection operation from penetrating the endothelial cells to generate tissue metastasis.

The action way of the salvia miltiorrhiza extract for preventing the metastasis after the tumor operation mainly comprises the following aspects:

inhibiting the migration and invasion of tumor cells.

Promoting the anoikis of the tumor cells after the tumor cells are separated from the extracellular matrix.

Regulate the expression of circulating tumor cell surface integrins and vascular endothelial cell surface adhesion proteins into the vasculature, reducing cell adhesion.

Reducing the adhesion of CTC to blood platelet, preventing the adhesion of CTC to vascular endothelial cell, and promoting the anoikis of circulating tumor cell.

One aspect of the invention utilizes supercritical CO₂The salvia miltiorrhiza extract extracted by the fluid effectively regulates a plurality of passages and a plurality of proteins in a safe concentration range, inhibits the EMT process of tumor cells, regulates the expression of a plurality of enzymes and integrins related to tumor metastasis, regulates a plurality of proteins (Bcl-2, Bax, Bim, Bad and the like) of a Bcl-2 family, inhibits adhesion class kinase (FAK), inhibits integrin related kinase (ILK), inhibits PI3K/Akt passage, inhibits MAPK passage, reduces the activation of a plurality of growth factor receptors { Epithelial Growth Factor Receptor (EGFR), Platelet Derived Growth Factor Receptor (PDGFR), Hepatocyte Growth Factor Receptor (HGFR) and Vascular Endothelial Growth Factor Receptor (VEGFR) }, regulates the expression level of a plurality of integrins related to tumor metastasis, regulates the regulation of a plurality of miRNAs on the tumor cell metastasis process, increases intracellular activity oxygen and the like. CTC entering the circulatory system via these pathways reduce adhesion to platelets and fail to adhere toAttached vascular endothelium leads to CTC anoikis, and effectively prevents circulating tumor cells from generating metastasis.

On the other hand, the drug for multi-target therapy effectively solves the problem of drug resistance of tumor cells by searching multi-channel regulation. The components of the salvia miltiorrhiza extract can be mutually cooperated and matched, and the integrin protein on the surface of the circulating tumor cell is regulated from a plurality of channels, so that the adhesion of CTC and blood platelets is reduced, the adhesion capability of CTC and vascular endothelium is reduced, the anoikis of the circulating tumor cell is promoted, and the purpose of preventing the CTC from retransferring is achieved.

Drawings

FIG. 1A, FIG. 1B and FIG. 1C are high performance liquid chromatograms of the components of the Salvia miltiorrhiza Bunge extract of the present invention;

FIG. 2 shows the MTT cytotoxicity test results and analysis chart of the Salvia miltiorrhiza Bunge extract of the present invention;

FIG. 3 is a graph showing the comparison of the results of the inventive Salvia miltiorrhiza extract and paclitaxel AIR values;

FIG. 4 is a photomicrograph and results analysis of a scratch test of Salvia miltiorrhiza Bunge extract cells of the present invention;

FIG. 5 is a diagram of the test of the invasion of cells by Transwell and the analysis of the result of the present invention;

FIG. 6 shows the results and analysis chart of the effect of the Salvia miltiorrhiza Bunge extract on human umbilical vein endothelial cell surface proteins ICAM and E-Selectin;

FIG. 7 shows the result and analysis chart of the effect of Danshen extract on the CD29 protein on the surface of 231 human breast cancer cells;

FIG. 8 shows the experimental results and analysis chart of the adhesion of matrix of Salvia miltiorrhiza Bunge extract of the present invention;

FIG. 9 shows the results and analysis chart of the effect of the extract of Salvia miltiorrhiza Bunge on the adhesion of tumor cells to endothelial cells;

FIG. 10 shows the results and analysis chart of the effect of the extract of Salvia miltiorrhiza Bunge on the adhesion of platelets to tumor cells;

FIG. 11 is a diagram showing the analysis of the result of the adhesion of the platelets of the Salvia miltiorrhiza Bunge extract to the human lung cancer cells A549;

FIG. 12 is a graph showing the results of the adhesion of tumor cells to endothelial cells in the presence of platelets and the analysis thereof of an extract of Salvia miltiorrhiza Bunge according to the present invention;

FIG. 13 is a graph showing the survival and tumor metastasis results of mice with the extract of Salvia miltiorrhiza Bunge of the present invention.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further explained below.

Supercritical CO₂The application of the fluid extraction of the salvia miltiorrhiza extract, and the application of the salvia miltiorrhiza extract in products for preventing tumor cell metastasis after tumor resection operations or the preparation of products for preventing tumor cell metastasis after tumor resection operations.

If other methods are adopted for extraction, the extraction process and the extraction solvent are different, so that the extraction components are not complete, the content of effective components is insufficient, and the same effect of the invention cannot be achieved.

Further, the tumor cells include micro-circulating tumor cells of lung cancer, breast cancer or colon cancer.

Furthermore, the product for preventing tumor cell metastasis after tumor resection operation is prepared from the salvia miltiorrhiza extract and pharmaceutically acceptable carriers or auxiliary materials, is prepared into a conventional preparation used clinically, and can be applied to patients needing the treatment in modes of oral administration or parenteral administration and the like. Such as tablet, granule, capsule, powder, injection, dripping pill, inhalant, sublingual preparation, syrup, gel, ointment, suppository, preferably tablet, capsule, dripping pill and pill. These preparations can be prepared by adding pharmaceutical carriers such as excipient, binder, moisturizer, disintegrator, thickener, etc. by conventional methods. The medicinal carrier is selected from microcrystalline cellulose, powdered cellulose, mannitol, starch, lactose, gelatin, methylcellulose, dextrin, pregelatinized starch, superfine silica powder, hydroxypropyl methylcellulose, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, polyethylene glycol, xylitol, lactitol, glucose, sucrose, steviosin, potassium sorbate, glycine, mannitol, tartaric acid, silicon dioxide, calcium stearate, magnesium stearate, talcum powder and the like.

Furthermore, the salvia miltiorrhiza extract promotes the anoikis of the trace circulating tumor cells in blood by specifically inhibiting the adhesion of the trace circulating tumor cells and vascular endothelial cells, thereby inhibiting the migration of the tumor cells and preventing the tumor metastasis again.

Furthermore, the salvia miltiorrhiza extract blocks the adhesion of tumor cells to endothelial cells by inhibiting the expression of adhesion proteins ICAM and E-Selectin on the endothelial cells so as to prevent tumor metastasis again.

Furthermore, the salvia miltiorrhiza extract blocks the adhesion of tumor cells to endothelial cells by inhibiting the expression of adhesion protein CD29integrin β 1 on the tumor cells so as to prevent tumor metastasis again.

Furthermore, the salvia miltiorrhiza extract prevents the tumor metastasis again by inhibiting the adhesion of platelets to tumor cells and hindering the resistance of the platelets to the anoikis of the tumor cells.

The invention discovers the supercritical CO₂The fluid-extracted salvia miltiorrhiza extract can effectively regulate a plurality of channels and a plurality of proteins in a safe concentration range, inhibit the EMT process of tumor cells, regulate the expression of a plurality of enzymes and integrins related to tumor metastasis, regulate a plurality of proteins (Bcl-2, Bax, Bim, Bad and the like) of a Bcl-2 family, inhibit adhesion class kinase (FAK), inhibit integrin-related kinase (ILK), inhibit PI3K/Akt channel, inhibit MAPK channel and reduce the activation of a plurality of growth factor receptors { Epithelial Growth Factor Receptor (EGFR), platelet-derived growth factor receptor (PDGFR), Hepatocyte Growth Factor Receptor (HGFR) and vascular endothelial growth factor receptor (VEGF)The long factor receptor (VEGFR) regulates the expression level of a plurality of integrins related to tumor metastasis, regulates the regulation and control of a plurality of miRNA on the tumor cell metastasis process, and increases the oxygen content of active oxygen in cells. Through the approaches, the CTC entering the circulatory system is reduced in adhesion with platelets and cannot adhere to vascular endothelium, so that the CTC is anoikis and apoptotic, and the metastasis of circulating tumor cells is effectively prevented. The invention prevents tumor metastasis after operation, is different from an anticancer drug which causes serious toxic and side effects by directly killing tumor cells but cannot inhibit tumor metastasis, has low toxic and side effects, can be safely and effectively taken for a long time, and is obviously different from the anticancer drug which has serious toxic and side effects and cannot be taken for a long time (see table 1). The medicine for preventing tumor metastasis acts on the microenvironment where the circulating tumor cells are located, and adjusts the adhesion among the circulating tumor cells, extracellular matrix, vascular endothelial cells in a vascular system and platelets, so as to achieve the purpose of blocking the tumor cells from reaching a far-end metastatic tissue.

TABLE 1 differentiation of anticancer drugs and tumor metastasis preventive drugs

Example 1

A Saviae Miltiorrhizae radix extract is prepared by supercritical CO₂Fluid extraction is carried out by taking salvia miltiorrhiza, drying, sieving with 40 mesh sieve, precisely weighing salvia miltiorrhiza powder 200g, adding into 60ml absolute ethyl alcohol, mixing, placing into supercritical extraction kettle, fixing separation kettle pressure at 10MPa and temperature at 50 ℃. The optimal extraction pressure is 20MPa, the extraction temperature is 50 ℃, and CO is used₂The flow rate is 15kg/h, and the extraction time is 120 min. Preheating the extraction kettle, the separation kettle and the storage tank to a selected temperature, pressurizing the system to 20MPa for circular extraction, keeping constant temperature and constant pressure, and obtaining the salvia miltiorrhiza extract when the selected time is reached. And identifying by High Performance Liquid Chromatography (HPLC) to obtain three groups of maps shown in figure 1A, figure 1B and figure 1C. The component in fig. 1A is tanshinone IIA, and other components cryptotanshinone and tanshinone I can be distinguished according to the polarity. The component in FIG. 1B is salvianolic acid B. FIG. 1C shows a schematic diagram of aIt is classified into protocatechualdehyde.

To verify the effect of the present invention, the applicant set the following tests.

Test 1

Taking the salvia miltiorrhiza extract obtained in the example 1 to carry out MTT cytotoxicity test, the details are as follows:

using a medium containing 10% fetal bovine and 1% double antibody 1640, 5% CO at 37 deg.C₂Culturing human lung cancer cell A549 in a normal temperature incubator, changing the solution once a day, and digesting the cell with pancreatin every other day. Cells were counted using a cell counting plate, and cells in the log phase of growth were plated in 96-well plates at 0.8 ten thousand/100. mu.l/well, and dosed overnight. The concentrations of active cells of the radix Salviae Miltiorrhizae extract prepared by the culture medium are 0, 5, 10, and 20 μ g/ml respectively. One day later, the culture medium is sucked off, 100 mul of MTT solution is added into each well to act for 4h, the MTT solution is sucked off, DMSO solution is added to act on a shaking table for 10min, the light absorption value at 570nm is measured to obtain the light absorption value shown in figure 2, the light absorption value of a blank control group is taken as a reference, and the light absorption value of an administration group is compared with the blank group to obtain the percentage histogram shown in figure 2.

As shown in fig. 2, the concentration of the salvia miltiorrhiza extract is in relatively low concentration ranges of 0, 5, 10 and 20 mug/ml, and the MTT result shows that the activity of the tumor cell a549 is not significantly different. The results indicate that the salvia miltiorrhiza extract has little lethality to tumor cells at a certain concentration level. The safety of the salvia miltiorrhiza extract on cells is verified.

Test 2

Taking the salvia miltiorrhiza extract obtained in the embodiment 1, testing and calculating the AIR values of the paclitaxel and the salvia miltiorrhiza extract, which are specifically as follows:

in order to prove that the tumor metastasis preventing medicament is different from the traditional anti-cancer medicament (the anti-cancer medicament kills tumor cells through cytotoxicity action, and one mode of the action of the tumor metastasis preventing medicament is to intervene the adhesion of tumor cells to vascular endothelium instead of killing the tumor cells), a new tumor metastasis preventing medicament curative effect evaluation standard is defined, and the tumor metastasis preventing efficiency of the medicament is evaluated by calculating the adhesion inhibition rate of the tumor metastasis preventing medicament. Adhesion inhibition ratio (Adhesion inhibi)Ion ratio, AIR) is the ratio of the drug IC10 (meaning the concentration of the drug that causes 10% inhibition of proliferation of the test cells) to the EC50 (meaning the concentration of the drug that causes 50% decrease in the efficiency of adhesion between tumor cells and endothelial cells), i.e., AIR ═ IC₁₀/EC₅₀. The larger the AIR value, the smaller the toxic side effect of the drug cell, which indicates that the tested drug mainly acts by specifically inhibiting the adhesion between the tumor cell and the vascular endothelial cell, but does not inhibit the adhesion between the tumor cell and the vascular endothelial cell by killing the tumor cell.

The results of figure 3, which tests and calculates AIR values, show that the red sage root extract value is about 1.6 and the paclitaxel value is about 0.5. According to the data obtained in FIGS. 2 and 9 of the present application, IC10 was 36. mu.g/ml, EC50 was 10. mu.g/ml, and AIR value of the extract of Salvia miltiorrhiza was 4. According to the results of FIGS. 3A and 3B of the present application, IC10 was 10. mu.M, EC50 was 20. mu.M, and thus the AIR value of paclitaxel was 0.5. Experiments prove that compared with the traditional anticancer medicine taxol, the AIR value of the salvia miltiorrhiza extract is obviously increased, the toxicity is obviously reduced, and the effect of preventing tumor metastasis is obviously enhanced, which shows that the cell toxicity and the side effect of the salvia miltiorrhiza extract are small, and the salvia miltiorrhiza extract can be used for preventing tumor cell metastasis by specifically inhibiting the adhesion between tumor cells and vascular endothelial cells.

Test 3

The salvia miltiorrhiza extract of the invention obtained in example 1 was used for a cell scratch test, which was as follows:

the cell culture method was the same as in test 1. Cell counting was performed using a cell counting plate, and a549 cells in logarithmic growth phase were plated in a 24-well plate at 20 ten thousand/500 μ l/well and subjected to a scratching experiment overnight. A straight line was gently drawn at the center of a 24-well plate using a 100. mu.l pipette tip, gently washed three times with physiological saline, and 1ml of a medium containing 2% fetal calf was used to prepare the cells affected by the extract of Salvia miltiorrhiza Bunge at concentrations of 0, 1, 5, and 10. mu.g/ml, respectively. Taking a picture by using a fluorescence microscope in a bright field, taking the picture once again after 24 hours, and comparing and analyzing the two-time taking result to obtain a microscope picture and a result analysis chart shown in figure 4. Fig. 4 is a histogram of percentage obtained by using the experimental migration distance of the control group as a reference distance and the ratio of the migration distance of the administration group to the migration distance of the control group.

As shown in fig. 4, the relative mobility of the salvia miltiorrhiza extract at concentrations of 0, 1, 5 and 10 μ g/ml was 100%, 77%, 55% and 28%, respectively, and the scoring results showed that the tumor cell a549 migrated gradually decreased at higher concentrations. The results show that the salvia miltiorrhiza extract effectively inhibits the migration of tumor cells.

Test 4

The salvia miltiorrhiza extract obtained in example 1 is used for a Transwell cell invasion test, which comprises the following specific steps:

the cell culture method was the same as in test 1. Cell counting was performed using a cell counting plate, 250. mu.l of 0.1% BSA containing 20 ten thousand A549 cells was added to the upper chamber of a Transwell dish, and the concentrations of the extracts of Salvia miltiorrhiza were set to 0, 1, 5, and 10. mu.g/ml, respectively, and 750. mu.l of 20% fetal bovine-containing medium was added to the lower chamber. In the presence of 5% CO₂Culturing for 24h in a normal-temperature incubator, discarding upper and lower chamber liquid, washing with physiological saline for three times, adding 800 μ l paraformaldehyde into the lower chamber, fixing for 30min, discarding paraformaldehyde, adding 800 μ l crystal violet solution diluted by 1:1 with physiological saline, acting for 30min, taking a picture under an upright microscope, taking the number of invasion of cells of a control group as a reference number, and taking the result of the ratio of the number of invasion of an administration group and the number of invasion of the control group as the result of a percentage histogram, thereby obtaining a graph 5.

As shown in FIG. 5, the relative invasion rates of the salvia miltiorrhiza extract at concentrations of 0, 1, 5 and 10 mug/ml are respectively 100%, 88%, 35% and 20%, and the Transwell results show that the higher the concentration is, the tumor cell invasion is obviously reduced. The results show that the salvia miltiorrhiza extract effectively inhibits the invasion of tumor cells.

Test 5

The salvia miltiorrhiza extract obtained in the embodiment 1 is taken to carry out flow detection on the influence of the salvia miltiorrhiza extract on the expression of endothelial cell adhesion protein ICAM (intercellular adhesion factor) and E-Selectin, and the specific steps are as follows:

using a medium containing 10% fetal bovine, 1% double-resistant ECM, 5% CO at 37 deg.C₂Human umbilical vein endothelial cells were cultured in a room temperature incubator, cells in logarithmic growth phase were plated in 6-well plates, 40 ten thousand/ml, 2ml per well, overnight, and then TNF- α (tumor necrosis factor, an inflammatory factor) was used)10ng/ml, adding 0, 5 and 10 mug/ml of radix salviae miltiorrhizae extract simultaneously, wherein one hole is not added with TNF- α as a control group, acting together in an incubator for 4h, sucking out the culture medium, digesting the cells by using pancreatin without EDTA, after digesting, gently blowing and beating the cells, centrifugally collecting the cells, gently blowing and beating the cells uniformly by using 1ml of PBS solution, adding an antibody ICAM with fluorescence, incubating for 30min, gently washing once by using PBS, centrifuging, suspending the cells combined with the antibody by using a proper amount of PBS, finally measuring ICAM (intercellular adhesion factor) of endothelial cells by using a flow cytometer, and measuring the expression quantity of E-Selectin, respectively measuring the protein quantity of ICAM and the protein quantity of E-Selectin in figure 6, and taking the protein expression quantity of a blank group as a reference group, and taking the result of the ratio of the protein expression quantity of the experimental group and the protein expression quantity of the blank group as the result of a percentage histogram in figure 6.

As shown in FIG. 6, the results of the measurement show that the concentration of the Salvia miltiorrhiza Bunge extract is in the range of 0, 5 and 10 μ g/ml, the relative expression amounts of ICAM protein are 100%, 60% and 50%, the control group is 4%, the relative expression amounts of E-Selectin protein are 100%, 85% and 65%, and the control group is 7%, and the results show that the expression levels of endothelial cells and adhesion-related proteins ICAM and E-Selectin are gradually reduced as the concentration is higher. The results show that the salvia miltiorrhiza extract can inhibit the expression of adhesion-related proteins ICAM and E-Selectin on endothelial cells, and provide basis for blocking the adhesion of tumor cells to the endothelial cells.

Test 6

The salvia miltiorrhiza extract obtained in the embodiment 1 is taken for flow detection of the adhesion protein CD29(integrin β) of the salvia miltiorrhiza extract to tumor cells₁) The effect of expression was tested as follows:

using L-15 medium containing 10% fetal bovine and 1% double antibody at 37 deg.C and 5% CO₂Culturing the human breast cancer cell MDA-MB-231 in a normal temperature incubator. Cells in logarithmic growth phase were plated in 6-well plates at 40 ten thousand/mL, 2mL per well. Standing overnight for cell attachment, adding Saviae Miltiorrhizae radix extract with concentration of 0, 1, 5, 10 μ g/ml, acting for 24 hr, sucking off culture medium, digesting the cells with pancreatin without EDTA, gently blowing and beating the cells, and separatingCells were collected from the heart. After gently washing the cells with PBS, the cells were blown evenly with PBS, and the cells were incubated for 30min with the fluorescent antibody CD 29. The cells bound to the antibody were suspended with an appropriate amount of PBS solution after gentle washing once with PBS solution, centrifugation. Finally, the expression level of CD29 (intercellular adhesion factor) on the surface of the tumor cells MDA-MB-231 was measured by flow cytometry, and the ratio of the protein expression level of the experimental group to that of the blank group was calculated as a percentage histogram, using the protein expression level of the blank group as a reference group, and the results are shown in FIG. 7.

As shown in FIG. 7, the relative protein expression amounts of the Saviae Miltiorrhizae radix extract at 0, 1, 5, and 10 μ g/ml were respectively 100%, 97%, 95%, and 91%, and the results showed that the higher the concentration, the protein CD29(integrin β) associated with tumor cell MDA-MB-231 surface adhesion₁) The result shows that the salvia miltiorrhiza extract can inhibit the adhesion-related protein CD29(integrin β) on the tumor cells₁) Provides basis for blocking the adhesion of tumor cells to endothelial cells.

Test 7

Taking the salvia miltiorrhiza extract obtained in the embodiment 1 to perform an intervention test of the salvia miltiorrhiza extract on the adhesion of tumor cells to extracellular matrix, which comprises the following specific steps:

the cell culture method was the same as in test 1. Each well of a 96-well plate was coated with 1% Matrigel at 37 ℃ with 5% CO₂After overnight at room temperature in the incubator, the Matrigel solution was aspirated. Cell counting was performed using a cell counting plate, and A549 cells in the logarithmic growth phase were plated in a 96-well plate at 8X 10³One dose per 100. mu.l/well, and the drug is added after overnight. The concentrations of active cells of the radix Salviae Miltiorrhizae extract prepared by using the culture medium are 0, 1, 5, and 10 μ g/ml respectively. After one day the medium was aspirated, the 96-well plate was gently washed three times with PBS buffer, and dead and non-adherent cells were washed away. Adding 100 μ l MTT solution into each well, allowing the solution to act for 4h, removing the MTT solution by suction, adding DMSO solution, allowing the solution to act on a shaker for 10min, and measuring absorbance at 570 nm. Matrigel coated 96-well plates, mimicking the extracellular matrix environment, where cells normally adhere to the extracellular matrix, but are detached from the extracellular matrix upon drug stimulationAnd then fall down. The blank group without drug was used as an experimental control group, and the MTT results showed cells that normally adhered to the matrix. The cells in the administration group are partially detached from the matrix relative to the blank group, so that the cells adhered to the matrix in the administration group are reduced. The ratio of the administered group to the control group was obtained as a result of the percentage histogram, i.e., fig. 8.

As shown in FIG. 8, the relative compliance rates of the Saviae Miltiorrhizae radix extract concentrations at 0, 1, 5, 10 μ g/ml matrix are respectively: 100%, 95%, 80%, 65%, the results showed a gradual decrease in tumor cell adhesion matrix at higher concentrations. The results show that the salvia miltiorrhiza extract can inhibit the adhesion of tumor cells to extracellular matrix, thereby promoting the anoikis of the desquamated tumor cells.

Test 8

The salvia miltiorrhiza extract of the invention obtained in example 1 is used for tumor cell adhesion endothelial cell tests, which are as follows:

the cell culture method is the same as test 5. human umbilical vein endothelial cells are paved on a 6-well plate, 30 thousand cells and 1ml of culture medium are added in each well, after overnight, 10ng TNF- α stimulating factor is added for acting for 4h to activate the endothelial cells, then the digested A549 cells are evenly mixed and placed in 1ml of PBS buffer solution, 10 mu l/ml rhodamine is used for shading and staining the tumor cells, after 20 minutes, the PBS is used for washing the tumor cells twice, the tumor cells are counted to be 20 ten thousand/ml by 1640 without phenol red, meanwhile, 0, 1, 5 and 10 mu g/ml of salvia miltiorrhiza extract is mixed in, the endothelial cell culture medium is sucked off, 1ml of 1640 suspension tumor cells without phenol red and the endothelial cells are incubated for 1h, after the culture medium is sucked off in each well, the PBS is used for washing twice, the non-adhered tumor cells are removed, then 1ml of 1640 culture medium without phenol red is continuously added, finally, 5-6 pictures are taken under a fluorescence microscope, the number of the tumor cells adhered on the blank group endothelial cells is used as a control group, and the percentage of the tumor cells adhered on the group is used as a contrast group, and a contrast group, namely, and a histogram is 9.

As shown in FIG. 9, the relative adhesion rates of the Saviae Miltiorrhizae radix extract at 0, 1, 5, 10 μ g/ml are 100%, 88%, 85%, 50%, and the results show that the tumor cells A549 with the higher concentration gradually decrease the adhesion of endothelial cells. The result shows that the salvia miltiorrhiza extract can inhibit the adhesion of tumor cells to endothelial cells and promote the anoikis of the tumor cells.

Test 9

The salvia miltiorrhiza bunge extract obtained in example 1 is taken to carry out an adhesion test of platelets and tumor cells by confocal microscope shooting, which comprises the following specific steps:

the cell culture method was the same as in test 1. Tumor cells A549 were plated in confocal dishes at 30 ten thousand/ml/dish overnight. The cell membrane and nucleus of tumor cells were stained with PKH26 dye and DAPI dye, respectively, for 20 min. Suspending 10 μ l blood with 1ml PBS solution, adding 20 μ M ADP, and adding Saviae Miltiorrhizae radix extract with concentration of 0, 10, 20 μ g/ml to blood, wherein one group without ADP is used as negative control group. Platelets in blood were stained with CFSE dye for 15 min. The stained tumor cells and platelets were incubated for 45min with 1640 that does not contain phenol red, and the adhesion of the tumor cells and platelets was photographed under a confocal laser microscope, and the percentage histogram of the ratio of the number of platelets adhered to the tumor cells in the group to which ADP was added to the control group to the number of platelets adhered to the tumor cells in the group to which ADP was not added to the control group was taken as the result of the percentage histogram, which is shown in FIG. 10.

As shown in FIG. 10, the relative adhesion rates of the Saviae Miltiorrhizae radix extract at concentrations of 0, 10 and 20 μ g/ml are respectively: 100%, 70%, 65%, the results demonstrate a reduction in the number of tumor cells adhering to platelets at higher concentrations. The result shows that the salvia miltiorrhiza extract can inhibit the adhesion of the blood platelets to the tumor cells and prevent the resistance of the blood platelets to the anoikis of the tumor cells.

Test 10

The salvia miltiorrhiza extract obtained in the example 1 is taken to carry out an influence test of the salvia miltiorrhiza extract on platelets carried by tumor cells, and the specific steps are as follows:

collecting digested A549 cells with count of 80 ten thousand/ml, suspending with 500 μ l PBS, adding Saviae Miltiorrhizae radix extract with concentration of 0, 10, 20 μ g/ml, adding fresh blood 1.5 ml, adding 20 μ M ADP to stimulate platelet, incubating antibodies CD61, CD29, and acting for 20 min. And finally, acting the erythrocyte lysate on the blood for 20min to break the erythrocytes. Tumor cells were collected by centrifugation. The platelet-carrying status of tumor cells was determined by flow cytometry. The assay used flow analysis results using CD29 labeled breast cancer cells a549, CD61 labeled platelets. The number of platelets on the tumor cells of the blank non-drug-added group was used as the control group, and the ratio of the number of platelets on the tumor cells of the drug-added group to the number of platelets on the control group was used as the result of the percentage histogram. The fluorescence intensity of CD61 detected here using a flow cytometer represents the number of platelets, and the results are shown in FIG. 11.

As shown in FIG. 11, the relative adhesion rates of the Saviae Miltiorrhizae radix extract at concentrations of 0, 10 and 20 μ g/ml were 100%, 85% and 80%, respectively, and the results demonstrated that the number of adhered platelets on tumor cells decreased at higher concentrations. The results indicate that the salvia miltiorrhiza extract can inhibit the adhesion of platelets on tumor cells.

Test 11

The salvia miltiorrhiza extract of the invention obtained in example 1 is used for a tumor cell adhesion endothelial cell test co-incubated with platelets, which comprises the following steps:

the cell culture method is the same as test 5, a 6-well plate is paved by using human umbilical vein cells, 30 ten thousand cells and 1ml of culture medium are added in each well, after overnight, 10ng TNF- α stimulating factor is added for acting for 4h to activate endothelial cells, then the digested A549 cells are uniformly mixed and placed in 1ml of PBS buffer solution, 10 mu l/ml rhodamine is used for shading and staining tumor cells, after 20 minutes, the PBS is used for washing the tumor cells twice, the tumor cells are counted into 20 ten thousand/ml by using 1640 without phenol red, 10 mu l of fresh blood is prepared in each well, ADP is added to activate platelets, 0, 1, 5 and 10 mu g/ml of red sage root extract is added, the counted tumor cells and the fresh blood are uniformly mixed by using 1ml1640 without phenol red, the endothelial cell culture medium is sucked, the tumor cells and the blood are incubated together for 1h, after the culture medium is sucked in each well, the PBS is used for washing twice, the tumor cells without adhesion are removed, 1ml of culture medium without phenol red is further added, finally, the number of the PBS and the contrast ratio of the number of the endothelial cells in each well is taken as a control group, and the number of the contrast of the group is 12.

As shown in FIG. 12, the relative adhesion rates of the Saviae Miltiorrhizae radix extract at concentrations of 0, 1, 5 and 10. mu.g/ml were 100%, 78%, 72% and 67%, respectively, and the results showed that the tumor cells A549 co-incubated with blood at higher concentrations gradually decreased the adhesion of endothelial cells. The results show that the salvia miltiorrhiza extract can inhibit the adhesion of tumor cells to endothelial cells under the condition of co-incubation with blood and promote the anoikis of the tumor cells.

Test 12

The salvia miltiorrhiza extract of the invention obtained in example 1 was used for animal experiments, and the details are as follows:

after the purchased nude mice were bred in a mouse room for 1 week, 31 BALB/C nude mice were divided into four groups, 10 control groups, 7 salvia miltiorrhiza extract high dose groups, 7 salvia miltiorrhiza extract low dose groups and 7 paclitaxel groups. The Saviae Miltiorrhizae radix extract is prepared from 0.5% sodium carboxymethylcellulose (CMC-Na) into high dose group of 50mg/kg/d, low dose group of 5mg/kg/d, and paclitaxel group of 10mg/kg/d, and the control group is administered with 100 μ l solvent three days earlier. Then, 100 ten thousand human melanoma a375 cells suspended in 100 μ l of pbs solution were injected into the tail vein to obtain survival curves of four groups of mice. After death, the mice were dissected and counted for lung tumor nodules. FIG. 13 is obtained.

As shown in FIG. 13, the mean number of tumor nodules of the high-dose group, the mean number of tumor nodules of the low-dose group and the mean number of tumor nodules of the paclitaxel group were 4, 5 and 12, respectively, and the mean number of tumor nodules of the control group was 13, which indicates that the survival period of the nude mice was significantly prolonged and the number of nodules of the pulmonary metastasis of the nude mice was reduced. The result shows that the salvia miltiorrhiza extract has the effect of preventing tumor metastasis in an animal model of tumor cell metastasis and has more advantages compared with the traditional anticancer drug taxol.

At present, the clinical medicine for treating tumor metastasis has a great blank, and many medicines are failed in preclinical experiments, and the most main reason is the drug resistance of tumor cells. Therefore, the problem of drug resistance of tumor cells can be effectively solved by the troublesome search of multi-channel regulation and multi-target treatment drugs. The components of the salvia miltiorrhiza extract can be mutually cooperated and matched, and the integrin protein on the surface of the circulating tumor cell is regulated from a plurality of channels, so that the adhesion of CTC and blood platelets is reduced, the adhesion capability of CTC and vascular endothelium is reduced, the anoikis of the circulating tumor cell is promoted, and the purpose of preventing the CTC from retransferring is achieved. The medicine for preventing tumor metastasis acts on the microenvironment where the circulating tumor cells are located, and the adhesion among the circulating tumor cells, extracellular matrix, vascular endothelial cells in a vascular system and platelets is adjusted, so that the aim of safely and effectively blocking the tumor cells from reaching a far-end metastatic tissue is fulfilled.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection of the claims.

Claims

1. Supercritical CO₂The application of the fluid extraction of the salvia miltiorrhiza extract is characterized in that: the salvia miltiorrhiza extract is applied to preventing the metastasis of tumor cells after tumor resection operation;

the tumor cells are circulating tumor cells.

2. The supercritical CO of claim 1₂The application of the fluid extraction of the salvia miltiorrhiza extract is characterized in that: the tumor cell comprises any circulating tumor cell of lung cancer, breast cancer or colon cancer.

3. The supercritical CO of claim 1₂The application of the fluid extraction of the salvia miltiorrhiza extract is characterized in that: the salvia miltiorrhiza extract is applied to a product for preventing tumor cell metastasis after a tumor resection operation or a product for preventing tumor cell metastasis after a tumor resection operation through a pharmaceutically acceptable carrier or auxiliary material.

4. The supercritical CO of claim 1₂The application of the fluid extraction of the salvia miltiorrhiza extract is characterized in that: the components of the Saviae Miltiorrhizae radix extract can regulate the integrin protein on the surface of circulating tumor cell from multiple channels to blockThe adhesion of the circulating tumor cells and the blood platelets reduces the adhesion capacity of the circulating tumor cells and the vascular endothelium, promotes the anoikis of the circulating tumor cells, and thus inhibits the migration of the circulating tumor cells and prevents the tumor from metastasizing again.

5. The supercritical CO of claim 4₂The application of the fluid extraction of the salvia miltiorrhiza extract is characterized in that: the salvia miltiorrhiza extract blocks the adhesion of tumor cells to endothelial cells by inhibiting the expression of adhesion proteins ICAM and E-Selectin on the endothelial cells.

6. The supercritical CO of claim 4₂The application of the fluid extraction of the salvia miltiorrhiza extract is characterized in that the salvia miltiorrhiza extract blocks the adhesion of tumor cells to endothelial cells by inhibiting the expression of adhesion protein CD29integrin β 1 on the tumor cells.

7. The supercritical CO of claim 1₂The application of the fluid extraction of the salvia miltiorrhiza extract is characterized in that: the preparation method of the salvia miltiorrhiza extract comprises the following steps: adding 60ml of absolute ethanol into 200g of salvia miltiorrhiza powder, placing the mixture into a supercritical extraction kettle, and fixing the pressure of the separation kettle at 10MPa and the temperature of 50 ℃; the extraction pressure is 20MPa, the extraction temperature is 50 ℃, and CO is added₂The flow rate is 15kg/h, and the extraction time is 120 min.

8. The supercritical CO of claim 8₂The application of the fluid extraction of the salvia miltiorrhiza extract is characterized in that the components of the salvia miltiorrhiza extract comprise tanshinone IIA, tanshinone IIB, tanshinone I, cryptotanshinone, hydroxy tanshinone, tanshinone methyl ester, danshenxinone, przewaquinone A, tanshinol I, tanshinol II, iso-tanshinone, tanshinol, salvianolic acid, tanshinol A, tanshinol B, tanshinol lactone and protocatechualdehyde.