CN108578464B

CN108578464B - Application of caulis spatholobi extract in preparation of antitumor drugs

Info

Publication number: CN108578464B
Application number: CN201810538736.2A
Authority: CN
Inventors: 朱晓新; 李琦; 孙立东; 王娅杰; 杨庆; 陈颖; 李玉洁; 翁小刚; 蔡维艳
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2018-05-30
Filing date: 2018-05-30
Publication date: 2021-06-11
Anticipated expiration: 2038-05-30
Also published as: CN108578464A

Abstract

The invention relates to an application of a caulis spatholobi extract in preparing an anti-tumor medicament, wherein the caulis spatholobi extract is selected from the following components: water extract of caulis Spatholobi, ethyl acetate extract of caulis Spatholobi, and n-butanol extract of caulis Spatholobi. The preparation method of the caulis spatholobi water extract comprises the following steps: extracting caulis Spatholobi 70kg with 5 times of distilled water under reflux for 2 times each time for 2 hr, mixing, concentrating under reduced pressure, and vacuum drying with 75 deg.C steam for 7 hr. Obtaining 6kg of water extract of caulis Spatholobi to obtain water extract.

Description

Application of caulis spatholobi extract in preparation of antitumor drugs

The technical field is as follows:

the invention relates to a new application of a traditional Chinese medicine, in particular to an application of a spatholobus stem extract in preparing an anti-tumor medicine.

Background

Caulis Spatholobi is dried stem of Spatholobus suberectus Dunn of Leguminosae. Has the functions of promoting blood circulation, enriching blood, regulating menstruation, relieving pain, relaxing muscles and tendons and activating collaterals. Can be used for treating menoxenia, dysmenorrhea, amenorrhea, rheumatalgia, numbness, paralysis, and sallow complexion due to blood deficiency.

Platelets, one of the constituents in mammalian blood, are small, biologically active cytoplasm that is cleaved from the mature bone marrow megakaryocyte cytoplasm and is small in size and free of nuclei. Platelets play a vital role in hemostasis and in the pathophysiological processes of thrombosis. Platelets are the smallest blood cells, but the number is about 100 times the number of leukocytes. About 100-300 x10 ^9 platelets participate in the repair of damaged blood vessels in the blood circulation of an adult. Platelets aggregate with each other and can promote thrombin generation during clot formation.

The surface receptors of platelets, such as the GpIb-IX-V complex, are capable of binding to the fibronectin (GP) by binding Von Willebrand Factor (VWF) supporting the adhesion of platelets. Particularly, under high shear force of blood, platelet-specific α IIb β 3 integrin receptors induce platelet aggregation by binding to collagen receptors such as fibrinogen, GpIa-IIa collagen receptor, and platelet collagen receptor (GpVI). After platelets are abnormally activated, the cytoskeleton of the platelets undergoes structural reorganization, resulting in the release of alpha granules, dense bodies and lysosomes. Activated platelets cause conformational changes in the GpIIb-IIIa receptor, leading to binding to high affinity ligands and platelet aggregation [2 ]. Negatively charged phospholipids on the platelet surface are exposed, resulting in the production of Platelet Microparticles (PMPs), which produce procoagulant activity.

In addition to the blood clotting and hemostatic functions, increasing experimental evidence indicates that platelets are able to balance disease and health in more and more important aspects, on the one hand, they are able to promote wound healing, tissue regeneration, and on the other hand, they are also closely involved in the pathogenesis of certain major diseases, such as cancer.

Experimental and

test medicine

2016, 8, 34, 4 discloses an experimental study on the effects of different components of caulis Spatholobi on AA-induced platelet aggregation, but does not describe the relationship between platelet aggregation and tumor.

The close protection of platelets and tumors has been attracting attention and attention, and clinicians have observed the occurrence of thrombocytosis in patients with malignant tumors as early as over 100 years ago. In 1872, Riess first reported that the malignant tumor has thrombocytosis, and abnormal increase of thrombocytosis can cause thrombocytosis, which is often regarded as a passive and paraneoplastic disease. In the 19 th century, people found that tumors and thrombi are directly closely related. Thrombophlebitis is considered to be an early warning signal for malignant tumor. Patients with venous thromboembolism are at seven times more risk of having malignant tumors than other patients. In which platelets play a non-negligible role.

In light of the above pathological phenomena, modern oncology studies have increasingly recognized that the progression of tumorigenesis is not a parallel concomitant event with the pathological alteration of platelets, and that there is a high causal link between the two.

Tumor cells have the pathological potential of inducing the numerical and functional changes of platelets, and further promote the formation of thrombus. The change has good prompting effect on clinical diagnosis of malignant tumor and thrombus. After that, a plurality of research reports and clinical examples show that the thrombocythemia phenomenon exists in a plurality of solid tumors such as lung cancer, esophageal cancer, gastric cancer, breast cancer, liver cancer, colorectal cancer, pancreatic cancer and the like, and the prognosis of a patient with the tumor suffering from the thrombocythemia is poor.

The facts indicate that the thrombocytosis can cause the high coagulation state of blood and the occurrence of thrombus in a tumor patient, and the thrombocytosis indicates that the thrombocytosis is closely related to the proliferation of tumor cells, hematogenous metastasis and angiogenesis in the tumor and plays an important role in the disease progression and metastasis of the tumor, wherein TCIPA is the most direct embodiment and the most central regulation process of the interaction between the thrombocytosis and the tumor.

Tumor cells are capable of inducing platelet activation, which activates and aggregates platelets around tumor cells to form a platelet-tumor cell complex, a process known as TCIPA. This phenomenon was first discovered in 1865 and gradually systematically and scientifically explained and verified. On the one hand, tumor cells can induce platelet number increase and function activation by actively enhancing the expression of procoagulant molecules such as ADP, Thrombin and the like, and form and maintain the hypercoagulable state of blood of patients. On the other hand, the abnormally activated blood platelets can reversely act on the tumor cells through the effects of physical combination, biological shielding and the like on the tumor cells, protect the tumor cells from being damaged by physical shearing force and killing and removing of an immune system in a circulating system, and promote the adhesion and colonization of the tumor cells on a target organ.

TCIPA is taken as a target spot, and aspirin is used for medicine intervention, so that the transfer resistant effect is high. However, the successful use of aspirin does not represent a widely applicable or sustainable use. Behind the effective use, there are also limitations in use, such as aspirin resistance, which is prevalent in drug-treated patients, with a maximum incidence of up to 60%. And in some patients with the medicine, gastric ulcer, gastrorrhagia, tinnitus and hemorrhagic stroke are accompanied, and after aspirin is used in teenagers, the risk of occurrence of Raynaud's syndrome is caused, and liver and kidney failure, brain injury and even death can be caused quickly. The generation of a plurality of side effects greatly limits the application of the treatment strategy in clinic, thereby promoting the development and emergence of other non-aspirin anti-TCIPA medicines.

Under the theoretical guidance that anti-TCIPA is the core event of anti-tumor metastasis, under the condition that aspirin can effectively prevent tumor metastasis and under the condition that aspirin has a plurality of defects, the traditional Chinese medicine related knowledge is applied, and from the traditional Chinese medicine theory, the search for a new medicine taking anti-TCIPA as a target point becomes the primary problem of the inventor.

The invention content is as follows:

the invention researches the Chinese medicinal caulis spatholobi and obtains the following technical solution.

The invention provides an application of a caulis spatholobi extract in preparing an anti-tumor medicament, wherein the caulis spatholobi extract is selected from the following components: water extract of caulis Spatholobi, ethyl acetate extract of caulis Spatholobi, and n-butanol extract of caulis Spatholobi.

The application of the invention is that the caulis spatholobi extract is prepared by the following steps: extracting caulis Spatholobi 70kg with 5 times of distilled water under reflux for 2 times each for 2 hr, mixing, concentrating under reduced pressure, and vacuum drying with 75 deg.C steam for 7 hr. Obtaining 6kg of water extract of the caulis spatholobi to obtain the water extract.

The application of the invention is that the spatholobus stem ethyl acetate extract is prepared by the following steps: extracting caulis Spatholobi 70kg with 5 times of distilled water under reflux for 2 times each for 2 hr, mixing, concentrating under reduced pressure, and vacuum drying with 75 deg.C steam for 7 hr. Obtaining 6kg of caulis Spatholobi water extract, dissolving the water extract in 7.2L of distilled water, extracting with ethyl acetate 10.8L each time for 8 times; mixing the ethyl acetate layer extractive solutions, evaporating solvent, and drying to obtain ethyl acetate extract.

The application of the invention is that the suberect spatholobus stem n-butanol extract is prepared by the following steps: extracting caulis Spatholobi 70kg with 5 times of distilled water under reflux for 2 times each for 2 hr, mixing, concentrating under reduced pressure, and vacuum drying with 75 deg.C steam for 7 hr. Obtaining caulis Spatholobi water extract 6kg, obtaining water extract, dissolving water extract in 7.2L distilled water, extracting with n-butanol 14.4L each time for 9 times, mixing n-butanol layer extractive solutions, evaporating solvent, and drying to obtain n-butanol extract.

The application of the invention, wherein the inhibition of tumor metastasis is realized by platelet aggregation induced by suberect spatholobus stem anti-extract tumor cells.

The application of the invention, wherein the inhibition of tumor metastasis is realized by the inhibition of platelet activation aggregation and anticoagulation of the spatholobus stem extract.

The use of the invention, wherein the tumor is selected from the group consisting of: colon cancer, breast cancer, lung cancer, pancreatic cancer or liver cancer.

Preferably, the tumor is colon cancer.

The invention further provides an extract of the spatholobus stem with ethyl acetate, and the preparation method comprises the following steps: extracting caulis Spatholobi 70kg with 5 times of distilled water under reflux for 2 times each for 2 hr, mixing, concentrating under reduced pressure, and vacuum drying with 75 deg.C steam for 7 hr. Obtaining 6kg of caulis Spatholobi water extract, dissolving the water extract in 7.2L of distilled water, extracting with ethyl acetate 10.8L each time for 8 times; mixing the ethyl acetate layer extractive solutions, evaporating solvent, and drying to obtain ethyl acetate extract.

The invention also comprises a pharmaceutical composition of the spatholobus stem ethyl acetate extract.

The most preferred of the present invention is the ethyl acetate extract of spatholobus stem.

The medicinal composition containing the caulis spatholobi extract comprises any preparation form suitable for oral administration, such as tablets, capsules, oral liquid, granules, pills, dripping pills, powder and the like.

The pharmaceutical composition can be prepared by the conventional preparation method.

In application, the dosage is adjusted according to patient condition, and can be administered 1-3 times per day. The beneficial effects of the present invention are further illustrated by experimental data as follows:

in the following experiments, the adopted medicine is caulis spatholobi extract, named as SET, and the preparation method of the extract can refer to any technical scheme in the prior art, can also be purchased from the market, or can be prepared by the preparation method of the invention.

If purchased, the following products may be purchased:

caulis Spatholobi extract from Siennaesen Biotech Ltd

Extracting parts: the rattan extraction solvent: water property: fineness of brown yellow powder: the specification of 100 percent passing through a 80-mesh sieve is as follows: 5: 1 or 10: 1

The following experiments were carried out according to the specific conditions of the present experiment.

Experiment 1, study of drug effect of SET in inhibiting platelet aggregation and blood coagulation

The study procedure is shown in FIG. 1

Experiment, 2, SET in vitro anti-TCIPA pharmacodynamic study

The study procedure is shown in FIG. 2

Experiment 3, SET in vivo anti-tumor metastasis drug effect research

The study procedure is shown in FIG. 3

The research results are as follows:

SET can remarkably prolong the calcium ion plasma recalcification time, and the SET with anticoagulation activity increased in dose dependence by 40 mug/mL has better platelet aggregation inhibiting activity of 20, 40 and 80 mug/mL, can remarkably inhibit ADP-induced platelet aggregation and inhibit the increase in dose dependence of the activity.

Experiment 2, SET in vitro anti-TCIPA pharmacodynamic study

The SET can effectively inhibit platelet adhesion induced by 4T1 cells and remarkably reduce the aggregation rate of platelets. SET is capable of specifically inhibiting TCIPA process.

Experiment 3, SET in vivo anti-tumor metastasis drug effect research

In an in vivo experiment, SET can obviously inhibit a lung metastasis model constructed in a tail vein injection mode and reduce the strength of a lung metastasis focus. The daily continuous administration can improve the lung tissue structure, obviously relieve the actual degree, finally prolong the life cycle of the mice with pulmonary metastasis and reduce the death rate.

The results of the drug effect study of SET for inhibiting platelet aggregation and blood coagulation are shown in FIGS. 4-6, the results of the drug effect study of SET for in vitro anti-TCIPA are shown in FIGS. 7-8, and the results of the drug effect study of SET for in vivo anti-tumor metastasis are shown in FIGS. 9-13.

Description of the drawings:

FIG. 1, experiment 1, study of the drug effect of SET in inhibiting platelet aggregation and blood coagulation

The study procedure is shown in FIG. 1

In the experiment, an ADP induced platelet aggregation model is constructed in vitro through an in vitro anti-platelet aggregation experiment, the induction effect of tumor cells in the blood internal environment is simulated, and the optimal drug effect dosage of the SET for resisting platelet aggregation is preliminarily determined. Meanwhile, through a calcium recovery experiment, the influence of SET on the blood coagulation time is verified, and the anticoagulation effect of the caulis spatholobi extract is more intuitively reflected. Finally, the optimal drug concentration and action time of SET for resisting platelet aggregation are determined.

FIG. 2, experiment, 2, SET study of in vitro anti-TCIPA drug effect

The study procedure is shown in FIG. 2

In the experiment, CFDA-SE is used for carrying out fluorescence labeling on platelets, mutual adhesion and aggregation of tumors and the platelets are judged visually and definitely through a laser confocal microscope, then the number of the platelets aggregated on the surfaces of the tumor cells is quantitatively identified through a flow cytometer, and meanwhile, the drug effect of SET on platelet adhesion resistance is evaluated.

FIG. 3, experiment 3, SET in vivo drug effect study on anti-tumor metastasis

The study procedure is shown in FIG. 3

In the experiment, tumor cells stably expressing fluorescence are injected into a blood circulation system of a mouse in a tail vein injection mode, the growth process of tumor cell metastasis foci is observed in real time through a living small animal imaging system, the size of the metastasis foci is finally evaluated according to fluorescence intensity, and the morphological change in the tumor is visually judged through immunohistochemical HE (high-intensity immuno sorbent assay) staining.

FIGS. 4 to 6, the results of the study of the drug effect of SET on the inhibition of platelet aggregation and blood coagulation

FIGS. 7-8, the results of the in vitro anti-TCIPA efficacy study of SET

FIGS. 9-13, the results of the in vivo efficacy study of SET for antitumor metastasis

FIG. 14 Effect of caulis Spatholobi extracts on platelet LDH Release

FIG. 15 the effect of the caulis Spatholobi series extract on proliferation of colon cancer cells MC38

FIG. 16 the effect of the caulis Spatholobi series extract on proliferation of colon cancer cell HCT116

FIG. 17 the efficacy of a caulis Spatholobi extract against ADP-induced platelet aggregation

FIG. 1820 h morphological changes of cells in each group

FIG. 19 Effect of caulis Spatholobi extract on MC38 migration

FIG. 20 Effect of caulis Spatholobi extract on MC38 migration

The results of fig. 19 and 20 show that: in the blank group, the cells partially migrated and had a certain migration ability. Compared with the blank group, the model group has an increased cell migration rate after platelet co-culture; in the aspirin group, the n-butanol group, and the water group, the cell migration number was significantly reduced compared to the model group (P < 0.05). Compared with the model group, the ethyl acetate extract can obviously reduce the increase of cell migration number caused by platelets (P <0.01), and the effect is obviously better than that of the positive group and other groups (P < 0.01). The above experimental results show that the platelet can induce and enhance the migration ability of MC38 cells, and the addition of the ethyl acetate extract of spatholobus stem can effectively reduce the migration ability of MC38 cells.

FIG. 21 Effect of Millettia dielsiana extracts on E-cadherin protein expression in HCT116 cells

The results in FIG. 21 show that: in the blank group, the expression level of E-cadherin was normal in HCT116 cells. Compared with the blank group, the model group can cause the expression level of the E-cadherin protein of HCT116 cells to be remarkably reduced (P <0.01) after the platelet co-culture; when the suberect spatholobus stem series extracts are added, the expression level of the E-cadherin protein can be obviously improved (P is less than 0.01), and the protein is superior to the positive aspirin. The above experimental results demonstrate that: the platelets can induce the reduction of the expression of E-cadherin of HCT116 cells, and the three suberect spatholobus stem extracts can obviously increase the expression of E-cadherin, and the effect of the ethyl acetate group is most obvious.

The specific implementation mode is as follows:

the experimental procedures and experimental results of the present invention are further illustrated by the following examples.

Example 1

Cell culture

Cell passage is carried out when the cell growth confluency reaches 70-80%. The old medium in the original flask was carefully aspirated for future use, avoiding pipette contact with the cells, and the cells in the flask were washed 2 times with PBS buffer. 1mL of 0.05% trypsin (containing EDTA) was added to allow the cells to infiltrate sufficiently, digestion was carried out at 37 ℃ and the digestion time was controlled under an inverted microscope. When the cell spacing increases and the refractive index increases, the digestion reaction is terminated by adding old medium. The cell suspension in the culture bottle is sucked by using an elbow suction pipe, and the cells attached to the wall of the culture bottle are slightly and repeatedly blown and beaten, so that the cells are completely separated. The cell suspension was transferred to a sterile centrifuge tube and centrifuged at 1000rpm for 5 min. Centrifuging, removing supernatant, adding new late-stage culture medium into a centrifuge tube, re-suspending cells, blowing uniformly, carrying out passage according to a required proportion or carrying out other related experiments, and culturing the cell culture bottle in a cell culture box at 37 ℃ containing 5% CO2 with the relative humidity of 95%. Platelet extraction

The total volume of blood taken from the orbit of the mouse is 5mL, and all mice do not take any anticoagulant drugs and drugs which have influence on the platelet function within two weeks. Placing in 3.8% sodium citrate anticoagulation tube, inverting, and mixing. The blood samples were stored at room temperature and tested in the next experiment within two hours.

Preparation of PRP

The sodium citrate anticoagulated blood 280g was centrifuged for 10min and the supernatant plasma and middle buffy coat were carefully transferred to a new EP tube. Centrifugation at 280g was repeated for 10 minutes, and the supernatant plasma was aspirated to obtain Platelet Rich Plasma (PRP).

Drug activity screening using platelet activation as an indicator

And opening the platelet aggregation instrument, the computer and the computer display in sequence. The siliconizing reaction cup with the magnetic bar is inserted into a pre-warming tank and pre-warmed for 10 minutes to 37 ℃. After 1mL of the solution in the siliconized tube obtained in the above step was placed in a reaction cup, the reaction cup was pre-warmed for 5 minutes, an electrode was inserted, and the other end was connected to an aggregometer. The stirring speed of the magnetic bar was set at 1200 rpm. And selecting a Run Test button in an aggregoment menu through computer operation, and inputting the grouping name of the experimental objects. Clicking OK after selecting the resistance method, and observing an operation curve on a screen. The baseline of the curve was set at 0% using the Impense Zero Knob, the calibre button was pressed, the Gain button was adjusted to position the aggregation curve at 50%, ADP was chosen as the inducer, run for 6 minutes, and Stop test was clicked. And determining Set start time and stop time, and automatically calculating the resistance value. The electrode was taken out, washed with tap water and physiological saline, and wiped clean with dust-free absorbent paper. And after the experiment is finished, the temperature switch of the instrument, the power supply and the computer are turned off for one time.

Tail vein injection model construction

The growth and the vascular process of the primary focus crossing the tumor cell metastasis focus the target point of the drug effect on the survival process of the tumor cell in the blood, eliminate the influence of the tumor in situ on the lung metastasis, and provide a more specific action zone for the drug effect detection of SET.

Example 2

Method

2.1 cell lines and cell cultures

Mouse colon cancer cell mc38 and human colon cancer cell HCT116 were purchased from beijing synergetics cell bank. The cell culture conditions were 1640 medium containing 10% calf serum and 1% diabase (penicillin 100U. mL-1 and streptomycin 100. mu.g. mL-1), and subcultured in an incubator at 37 ℃ and 5% CO 2.

2.2 preparation of the reagent

The caulis spatholobi extract is extracted from laboratory of Yangxuwei teacher of Beijing university medical department: reflux-extracting caulis Spatholobi 70kg with 5 times of distilled water for 2 times each for 2 hr, concentrating under reduced pressure, vacuum drying with steam at 75 deg.C for 7 hr. Obtaining 6kg of water extract of caulis Spatholobi, dissolving in 7.2L of distilled water, extracting with 10.8L of ethyl acetate for 8 times; then extracted with 14.4L of n-butanol, 9 times. Finally, 300g of ethyl acetate extract, 581g of n-butanol extract and aqueous layer extract were obtained.

The preparation of the medicine comprises the following steps: the ethyl acetate extract, n-butanol extract and aqueous layer extract powders were weighed up to 35mg each, respectively, dissolved in 175 μ L DMSO, sufficiently dissolved, and mixed until no precipitate was formed. Then respectively taking 30 mu L of the mixture in a mortar, adding 1470 mu L of normal saline while stirring until the solution is clear and transparent, no precipitate or crystal is separated out, and the final concentration is 4 mg/mL. Subpackaging with new sterile EP tube, and storing at-20 deg.C.

2.3 preparation of animal Whole blood sample and preparation of platelet-rich plasma PRP

Blood is taken from the orbit of the mouse and is put into an anticoagulant tube containing 20U/mL sodium citrate, and the mixture is slightly mixed to obtain a whole blood sample. The sodium citrate anticoagulated blood vessel was centrifuged at 280g for 10min and the supernatant plasma and middle buffy coat were carefully transferred to a new EP tube. Centrifugation at 280g was repeated for 10 minutes, and the supernatant plasma was aspirated to obtain Platelet Rich Plasma (PRP).

2.4 Lactate Dehydrogenase (LDH) detection

LDH kit detection (manufactured by Nanjing technology Co., Ltd.) was used, and sample treatment and enzyme activity measurement were carried out according to the kit instructions. After the reaction, the OD value was measured at 450nm using a microplate reader.

2.5 MTT colorimetric method

Preparing single cell suspension from MC38 and HCT116 tumor cells in logarithmic growth phase, adjusting cell concentration to 2 × 107. L-1, inoculating into 96-well plate, culturing at 37 deg.C in 5% CO2 constant temperature incubator for 24 hr, adding 50 μ L medicinal or non-medicinal culture solution into each well, setting blank control group, medicinal group with each concentration, and positive control group, and setting 3 multiple wells in each group. After further culturing for 72 hours, 10. mu.L of MTT (5 g.L-1) was added to each well, and further culturing was carried out under the same conditions for 4 hours. Discarding the culture solution, adding 150 μ L DMSO into each well, shaking thoroughly, mixing well to dissolve the crystal, measuring the absorbance (A value) at 470nm on an enzyme-linked immunosorbent assay, and calculating the cell proliferation Inhibition Rate (IR). Cell growth inhibition (%) × (1-experimental OD/control OD) × 100%. The experiment was repeated 3 times.

2.6 resistance method for detecting ADP-induced whole blood platelet aggregation resistance experiment

Respectively arranging a blank group, a modeling group, a water extraction group, an ethyl acetate extract group, a normal butanol extract group and a positive medicine group. The concentration of the drug groups is 100 mug/mL, and the concentration of the positive drug aspirin is 300 mug M. In addition to the blank, 10. mu.L of an ADP inducer at a concentration of 10mM was added to each group. The method comprises the following specific steps: 0.5mL of blood per tube was aliquoted into each set of siliconized tubes and an equal volume of test drug was added separately. The siliconized test tubes with magnetic rods were inserted into the pre-warming bath and pre-warmed for 5 minutes to 37 ℃. The siliconized tube was then transferred into a reaction cuvette, the electrode was inserted, and the other end was connected to an aggregometer platelet aggregation instrument. The stirring speed of the magnetic bar was set at 1200rpm, and the operation was carried out after selecting the resistance method, and the operation curve was observed on the screen. ADP is selected as an inducer, the experiment is stopped after running for 5 minutes, and the resistance value is automatically calculated.

2.7 morphological Observation of cells

Mouse mc38 cells were plated 1 × 106 per well in six-well plates containing 1mL1640 medium per well.

A blank group, a modeling group, a water extraction group, an ethyl acetate extract group, a normal butanol extract group and a positive medicine group are respectively arranged. The remaining groups, except the blank, were plated 24h before the addition of 200. mu.LPRP. The concentration of the drug group is 100 mug/mL, and the concentration of the positive drug aspirin is 300 mug M. The images were taken for comparison after 20h of co-incubation.

2.8 Transwell method for detecting migration capability of MC38

MC38 cells in the logarithmic growth phase were digested to a concentration of 106/ml, and 0.1ml (1X 105 cells) was added to each chamber. The cells were divided into a blank control group, a model control group (platelet co-culture in the upper chamber) and each administration group according to the treatment method. The lower chamber was filled with 600. mu.L of 1640 medium containing 10% FBS. After 18h the chamber was removed and the cells on the chamber filter were gently wiped off with a cotton swab. Inhibition of cell migration was also calculated: the inhibition of cell migration was (number of control-blank-control migrated cells)/number of control migrated cells × 100%.

2.9 Western blot method for detecting protein expression levels of MC38 and HCT116 cells CDH1, taking MC38 and HCT116 cells in logarithmic growth phase, inoculating the cells in a 12-well plate at a cell density of 5 × 104/well, culturing for 24h, adding PRP and caulis Spatholobi extracts to each component except a blank group for treatment, discarding supernatant after 4h and 20h, washing the cells for 3 times by using precooled PBS, adding RIPA ice to each well for cracking for 30min, completely cracking the cells, transferring the cells to a centrifugal tube, centrifuging for 5min at 13000 × g, sucking the supernatant, and quantitatively and uniformly diluting the BCA kit to the same concentration. Loading 12 μ L of protein per well, separating protein by SDS-PAGE, transferring to PVDF membrane by wet transfer method, blocking with 5% BSA for 1h, adding primary antibody, washing membrane with TBST 3 times at 4 deg.C overnight, adding secondary antibody, incubating at room temperature for 2h, washing membrane with TBST 3 times, and emitting light. The experiment was repeated 3 times and the grey values were counted.

3 statistical methods and treatments

The experimental data are analyzed by adopting IBM SPSS 23.0 software for data statistics and adopting a single-factor variance method, the result is expressed by x +/-s, the difference P <0.05 has statistical significance, and the difference P <0.01 has very significant statistical significance.

4 results of the experiment

4.1 selection of the concentration of the caulis Spatholobi extract

In order to eliminate the direct effect of the drug on tumor cells or platelets, the LDH experiment and the MTT experiment are firstly adopted to evaluate the effect of the drug on the platelets and the tumor cells respectively, so that the survival and the function of the platelet or tumor are not greatly influenced.

The results in FIG. 14 show that: the LDH activity of the platelets is not significantly different from that of a negative control group under the action of five drug concentrations (25 mu g/mL, 50 mu g/mL, 100 mu g/mL, 200 mu g/mL and 400 mu g/mL) of the caulis spatholobi series extract. The experimental results show that: the caulis Spatholobi series extract has no influence on platelet LDH release.

Figure 15 results show that: when the three extracts are under the action of low concentration (<100 mu g/mL) and short time (24H), the inhibition rate of the three extracts on MC38 cells is lower than 20%. When the time exceeds 24H and the medicine concentration is more than 200 mug/mL, each extract has stronger inhibition effect on MC38 cells.

The results in FIG. 16 show that: the ethyl acetate extract and the n-butanol extract have weak proliferation inhibition effect on HCT116 cells under 5 concentration doses. When the action time of the water layer group is 24H, the HCT116 cells are inhibited at the concentration of only 800 mu g/mL; when the water layer group action time is 48H, each concentration has obvious inhibition effect on HCT116 cell proliferation.

In combination with the above experimental results, we selected 100 μ g/mL as the appropriate dosing concentration for each subsequent drug group in vitro experiment.

4.2 resistance method for detecting platelet aggregation of each extract of caulis Spatholobi for resisting ADP induction

The most direct pathological phenomenon of TCIPA is abnormal activation of platelet function. Furthermore, an ADP-induced platelet aggregation resistant experiment is selected to quickly and effectively evaluate and screen the pharmacodynamic activity of the suberect spatholobus stem series extract for resisting platelet aggregation in vitro.

The results in fig. 17 show that, compared with the model group, the positive drugs aspirin, ethyl acetate extract and n-butanol extract can significantly inhibit ADP-induced platelet aggregation (P <0.01), and the efficacy of the ethyl acetate extract of spatholobus stem is due to the aspirin group and the n-butanol group. In addition, the water layer group has no significant difference compared with the model group. The experimental results show that the ethyl acetate extract and the n-butanol extract may contain the active ingredient for resisting platelet aggregation, while the water layer component has lower active ingredient.

4.3 Effect and detection of caulis Spatholobi extract on morphology in tumor cells

After initially determining the active site of the drug, we first evaluated the effect of the spatholobus stem series extract on MC38 cells from morphological index.

As can be seen in fig. 18 (lower right corner enlarged partial view): in the blank, the cells were oval and tightly connected. Compared with the blank group, after the model group is co-cultured by the platelets, the cells are in a long fusiform shape, the aspect ratio is increased, the pseudopodia is increased, the connection among the cells is loose, and the cells are easy to shed. In the aspirin group, the aspect ratio of cells is reduced, the pseudopodia is reduced, and the cells are not easy to fall off. In the spatholobus stem ethyl acetate extract group, the aspect ratio of cells is recovered to be normal, and the false feet are disappeared. The n-butanol group did not change significantly compared to the water-extracted group compared to the model group. The experimental phenomenon shows that the platelets induce the mc38 cells to generate obvious mesenchymal-like changes; the addition of the ethyl acetate extract of the caulis Spatholobi can reverse the mesenchyma-like change.

4.4 Effect of caulis Spatholobi extract on MC38 migration

Once the tumor cells have EMT change, the migration capacity of the tumor cells is greatly improved, and the tumor cells are invaded into blood vessels, so that the distant organ metastasis is carried out through blood circulation. Therefore, based on the change of morphological experiments, the influence of the caulis Spatholobi series extract on the migration capability of tumor cells was tested.

4.5 Effect of Millettia dielsiana extract on E-cadherin protein expression in HCT116 cells

At the molecular level, we detected the most central molecule in the changes in EMT, E-cadherin. The results in FIG. 21 show that: in the blank group, the expression level of E-cadherin was normal in HCT116 cells. Compared with the blank group, the model group can cause the expression level of the E-cadherin protein of HCT116 cells to be remarkably reduced (P <0.01) after the platelet co-culture; when the suberect spatholobus stem series extracts are added, the expression level of the E-cadherin protein can be obviously improved (P is less than 0.01), and the protein is superior to the positive aspirin. The above experimental results demonstrate that: the platelets can induce the reduction of the expression of E-cadherin of HCT116 cells, and the three suberect spatholobus stem extracts can obviously increase the expression of E-cadherin, and the effect of the ethyl acetate group is most obvious.

Claims

1. The application of a caulis spatholobi extract in preparing a medicine for resisting tumor metastasis in blood circulation is disclosed, wherein the caulis spatholobi extract is selected from the following components: ethyl acetate extract of caulis Spatholobi or n-butanol extract of caulis Spatholobi;

the preparation method of the spatholobus stem ethyl acetate extract comprises the following steps:

reflux-extracting caulis Spatholobi 70kg with 5 times of distilled water for 2 times each time, 2 hr each time, mixing, concentrating under reduced pressure, vacuum drying with 75 deg.C steam for 7 hr to obtain caulis Spatholobi water extract 6kg, dissolving the water extract in 7.2L distilled water, extracting with ethyl acetate 10.8L each time for 8 times; mixing the ethyl acetate layer extractive solutions, evaporating solvent, and drying to obtain ethyl acetate extract;

the preparation method of the suberect spatholobus stem n-butyl alcohol extract comprises the following steps:

reflux-extracting caulis Spatholobi 70kg with 5 times of distilled water each time for 2 times and 2 hr each time, mixing, concentrating under reduced pressure, vacuum drying with 75 deg.C steam for 7 hr to obtain caulis Spatholobi water extract 6kg, dissolving the water extract in 7.2L distilled water, extracting with n-butanol 14.4L each time for 9 times, mixing n-butanol layer extractive solutions, distilling off solvent, and drying to obtain n-butanol extract;

wherein the tumor is: colon cancer;

wherein said inhibition of tumor metastasis is achieved by the anti-tumor cell-induced platelet aggregation of an extract of spatholobus stem.