CN115991649A - Method for preparing acephating and application of acephating as anti-tumor metastasis medicine - Google Patents

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a method for preparing high-purity acetylalcaine from Arnebia euchroma (Royle) Johnst; meanwhile, the invention provides the application of the acephataine as the anti-melanoma transfer medicine for the first time. In vivo pharmacodynamic experiments prove that the composition has a certain degree of inhibition effect on nude mouse human melanoma A375 transplanted tumor; in vitro experiments prove that the acephataine has obvious inhibition effect on the growth and migration of human melanoma cells A375, and the acephataine provided by the invention has the advantages of obvious curative effect, small toxic and side effects and the like when being used for the transfer of melanoma, so that the acephataine has wide application prospect.

Description

Method for preparing acephating and application of acephating as anti-tumor metastasis medicine

Technical Field

The invention relates to the technical field of medicines. Relates to a preparation method of naphthoquinone compound acetylacarnine extracted and separated from arnebia root and application thereof in treating melanoma metastasis.

Background

Cancer is well known to be one of the major diseases that endanger human life and health. With recent studies on the pathogenesis and progress of tumors and methods of tumor treatment, the therapeutic effect of patients can be greatly improved. Tumor death is most often due to the occurrence of metastasis. Tumor metastasis means that the tumor metastasizes to distant organs, meaning that the tumor enters a late stage, medically known as the fourth stage, at which time death occurs quickly without treatment. While advanced tumors, most of which require chemotherapy for participation. Although chemotherapy can reduce cancer mortality, the existing antitumor drugs often kill tumor cells and have higher killing property on normal cells, so that toxic and side effects are higher. Thus, against such serious practical problems, governmental departments, research institutions and pharmaceutical enterprises in various countries have been highly concerned about the treatment of malignant tumors and the research and development of antitumor drugs for a long time, and have not been seriously invested. The search and discovery of compounds with remarkable anti-tumor effect from natural products is always one of the important methods for research and development of new drugs.

Sinkiang radix Arnebiae (Arnebiaeuchroma (Royle) Johnst) is perennial herb of Boraginaceae. The dried root of Sinkiang radix Arnebiae has effects of clearing heat and cooling blood, promoting blood circulation and removing toxic substances, promoting eruption and removing speckle, and can be used for treating blood heat toxin syndrome, burn, macula and purple black, measles, heat disease and macula, eczema, blood stranguria, bloody dysentery, pyocutaneous disease, erysipelas, etc. Along with the development of modern separation technology and the continuous and deep pharmacological and pharmacodynamic research, the antitumor value of the hydroxy naphthoquinone compounds contained in the arnebia euchroma (Royle) Johnst is recognized by the medical community.

Hydroxy naphthoquinone compounds in arnebia euchroma are mostly chiral compounds, and are called shikonins (R) with right optical activity due to R configuration, such as violaxanthin (Shikonin), acetylshikonin (acetylshikonin), beta-hydroxyisovaleryl Shikonin (beta-hydroxyisovaleryl Shikonin) and the like; s configuration and left optical activity are called as levo shikonins or acarnines (Akannin, S), such as beta-hydroxyisovaleryl acarnins (beta-hydroxyisovalerylalkinnin), beta-dimethyl shikonin (beta, beta-dimethyl shikonin) and beta, beta-dimethyl shikonin (beta, beta-dimethyl shikonin), and the like, and modern pharmacological research shows that the Xinjiang shikonin has the activities of resisting tumors, resisting influenza, resisting oxidization, resisting inflammation, regulating immunity and the like.

The research report shows that shikonin purified from arnebia root can completely inhibit the growth of ascites type sarcoma 180 cells at 50mg/kg and can prolong the life of tumor-bearing mice by 92.5% at 10 mg/kg. In addition, shikonin can enhance the radiation sensitivity and the drug resistance of glioblastoma in mice liver cancer H22 and Lewis lung cancer; the mechanism research shows that alkannin can induce apoptosis of melanoma cells by inhibiting autophagy to induce death of non-small cell lung cancer.

Through searching, no patent and report on the aspect of anti-melanoma metastasis of acetylalcaine in arnebia euchroma (Royle) Johnst has been found so far. Meanwhile, the process for extracting, separating and purifying the monomer compound acetoshikonin from the Sinkiang arnebia root has been reported to be complex, and organic reagents such as chloroform, methanol and the like which are harmful to human bodies are adopted as extraction solvents, so that the method is not suitable for industrial production.

Disclosure of Invention

The invention aims to provide a method for preparing acetylalcaine from arnebia euchroma (Royle) Johnst and application of acetylalcaine as an anti-melanoma transfer medicine. Overcomes the defects of the prior art, and can effectively solve the problems that the prior preparation process for preparing the acephate is complex and the extraction solvent is chloroform which is not suitable for industrial production; meanwhile, the invention provides the application of preparing the acetyl acarnine monomer from the arnebia euchroma (Royle) Johnst as the preparation of the anti-melanoma transfer medicine for the first time.

The first aspect of the invention provides a preparation method of the acetenin from the arnebia euchroma (Royle) Johnst, and the application of the acetenin prepared in the second aspect in preparing the anti-melanoma transfer medicine has the functions shown in the following (1) or/and (2); (1) inhibiting proliferation of cancer cells; (2) treating and/or preventing metastasis of melanoma.

One of the technical schemes of the invention is realized by the following measures: the method for preparing the acephatherin from the arnebia euchroma (Royle) Johnst comprises the following steps: in the first step, crushing, taking a proper amount of sinkiang arnebia root medicinal material, and crushing to obtain sinkiang arnebia root medicinal material coarse powder. And secondly, extracting, namely adding petroleum ether (60-90 ℃) into coarse powder of the sinkiang arnebia root medicinal material, performing cold leaching extraction for 2-3 times at room temperature, filtering to obtain filtrate, and combining the filtrate to obtain an extracting solution. And thirdly, concentrating, and concentrating the extracting solution to obtain pasty concentrated solution of the extracted part. Fourth, roughly separating, filling the column with silica gel to obtain a silica gel chromatographic column, dissolving the concentrated solution of the extraction part, adsorbing with silica gel, drying, and filling the column; sequentially eluting with petroleum ether, eluting with petroleum ether-ethyl acetate, analyzing with TLC, and taking acetylacarnine as reference, (developing solvent is that petroleum ether and ethyl acetate are combined, and formic acid=19:1:0.1), and concentrating under reduced pressure to obtain coarse fraction of acetylacarnine; fifthly, purifying, namely filling a column with silica gel H to obtain a silica gel H chromatographic column, dissolving a concentrated solution of a crude part of the acephating by using petroleum ether, and performing silica gel H chromatography by using petroleum ether: eluting with ethyl acetate; TLC analysis is adopted, the acephating is used as a reference, the same eluent with the main spot of the acephating is combined, and the acephating crude product is obtained after decompression and concentration; and sixthly, recrystallizing, namely taking an acephating crude product, dissolving and filtering the acephating crude product by using ethyl acetate, and adding petroleum ether for recrystallization. The red short needle-like crystal acetylacarnine is obtained, and the purity is more than 97 percent.

The preparation method for separating and purifying the acephataine from the sinkiang arnebia root has the advantages of simple preparation steps, high extraction rate and high purity in the preparation process, thereby greatly reducing the production cost.

The second technical scheme of the invention is realized by the following measures: the acetyl acarnine separated and purified from arnebia root is used for preparing anti-melanoma medicine.

Further, a method for detecting and preparing effective dosage of melanoma medicine by using an immunodeficiency mouse model is adopted, and is characterized in that the obtained melanoma sample is inoculated into an immunodeficiency mouse body, a melanoma immunodeficiency mouse model is obtained after 2-5 weeks, and an anti-melanoma medicine is given to the immunodeficiency mouse model by using an intraperitoneal injection method, so that the growth condition of melanoma in the mouse body is detected.

Further, the inhibition of the prepared acetylalcaine on the growth of melanoma A375 cells is detected by a CCK-8 method, and the inhibition of the cell migration and invasion capacity of the prepared acetylalcaine are observed after the prepared acetylalcaine is administered by a cell trace analysis method and a recombinant basement membrane invasion experiment.

The acephataine is extracted from lithospermum root, and related reports of the medicine for clinically treating tumors are not seen. Therefore, the invention is to prove that the natural compound acetylalcaine can effectively inhibit the growth and migration of melanoma through related experiments, thereby finally realizing that the compound can be used for clinical treatment of melanoma patients. The tumor treatment concentration in the body of the mice is 4-8 mg/kg body weight, and the medicine can be prepared into injection for patients at the dosage of 36-72 mg/kg. The compound has potential application value for clinically preventing and treating melanoma. Further, in vitro experiments show that the acetyl shikonin prepared in the arnebia euchroma (Royle) Johnst has an inhibition effect on the invasion and migration of melanoma A375 cells.

Further, the acephating is combined with a suitable pharmaceutically acceptable carrier. Such pharmaceutical compositions comprise a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The acephatidine according to the invention can be prepared in the form of injection, for example by adding co-solvents, solubilisers, emulsifiers and the like, such as ethanol, tween-80, polyoxyethylated castor oil physiological saline or aqueous solutions containing glucose and other adjuvants, by conventional methods. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. The amount of active ingredient administered is a therapeutically effective amount, for example, about 5.4 to 10.8mg/kg body weight per day. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc. In addition, the acephating of the present invention may also be used with other therapeutic agents.

There is no report on the use of acamprosate as a drug for clinical treatment of tumors. The related experiments of the invention prove that the preparation of the acephatic acid from the arnebia root can be applied to the clinical treatment of melanoma and melanoma metastasis.

Drawings

FIG. 1 is an HPLC chromatogram of acephating;

FIG. 2 is a nuclear magnetic resonance 1H-NMR chart of acamprosate;

FIG. 3 is a nuclear magnetic resonance 13C-NMR chart of acamprosate;

FIG. 4 is a graph showing the change in body weight of mice in a mouse model of transplantation melanoma immunodeficiencies with acamprin;

FIG. 5 shows tumor growth inhibition of acamprosate on a mouse model with transplanted melanoma;

FIG. 6 effect of different concentrations of acamprosate on melanoma A375 cell morphology;

FIG. 7 effects of acamprosate on inhibition of proliferation of melanoma A375 cells at various times and IC50 values;

FIG. 8 effect of different concentrations of acamprosate on migration of A375 cells;

FIG. 9 transplantation of different concentrations of acetylacarnine to A375 cell invasion.

Detailed Description

Acetylacarnine is a natural compound, and no research shows that the Acetylacarnine plays a role in the process of treating melanoma metastasis, and no medicine for treating tumors exists in clinic. The following describes the technical scheme of the present invention in further detail with reference to examples, but the scope of the present invention is not limited thereto.

The invention mainly prepares the acephatherin in the arnebia euchroma (Royle) Johnst, and detects the influence of the acephatherin on the growth and invasion migration of human melanoma by establishing an in-vivo model of a humanized xenograft model and selecting CCK-8, a cell trace analysis method and an experimental method of recombinant basement membrane invasion in vitro. The humanized xenograft model retains inherent genes and phenotypes, is a model which is closer to the human tumorigenesis and development process, and is very significant for personalized treatment. The invention shows that the acephataine can treat or prevent the growth and metastasis of tumors on a human melanoma and xenogenic tumor transplantation model mouse.

Experimental example 1, acetyl acarnine in Sinkiang arnebia root is prepared by the following preparation method: firstly, taking dried roots of sinkiang arnebia root, and crushing to obtain crude powder of sinkiang arnebia root; secondly, extracting, namely 1kg of coarse powder of the sinkiang arnebia root medicinal material in the first step, adding 6L of petroleum ether (60-90 ℃), soaking for 12 hours at room temperature, filtering, repeating the steps for 3 times, and combining and filtering to obtain a filtrate to obtain an extracting solution; concentrating the extractive solution at 45deg.C under reduced pressure to 0.25L to obtain dark red crude extract; fourth, roughly separating, filling the column with 1kg of silica gel to obtain a silica gel chromatographic column, dissolving the concentrated solution of the extraction part, adsorbing with silica gel, drying, and filling the column; eluting with 5L petroleum ether sequentially, eluting with 5L petroleum ether-ethyl acetate (50:1), performing TLC analysis with acephataine as reference, (developing solvent is petroleum ether: ethyl acetate: formic acid=19:1:0.1), mixing eluents with main spots as the same components of acephataine, concentrating under reduced pressure at 45deg.C to obtain coarse fraction of acephataine; fifthly, purifying, namely filling a column with silica gel H to obtain a silica gel H chromatographic column, dissolving a concentrated solution of a crude part of the acephating by using petroleum ether, and performing silica gel H chromatography by using 5L petroleum ether: eluting with ethyl acetate (19:1); TLC analysis is adopted, the acephating is used as a reference, the same eluent with the main spot of the acephating is combined, and the acephating crude product is obtained after decompression concentration at 45 ℃; and sixthly, recrystallizing, namely taking an acephating crude product, dissolving the acephating crude product with 1 time of ethyl acetate, filtering, and adding 5 times of petroleum ether for recrystallization. Thus obtaining the red short needle-like crystal acetylacarnine. The acetoacarnine obtained was analyzed by HPLC as shown in FIG. 1. Further characterization was confirmed by 1H-NMR and 13C-NMR using deuterated chloroform as shown in FIGS. 2 and 3, 1H-NMR (CDCl 3) δH=1.58 (S, 3H), 1.70 (S, 3H), 2.15 (S, 3H), 2.43-2.64 (m, 2H), 5.12 (t, 1H, J=6.8 Hz), 6.02 (dd, lH, J=7.2, 4.8 nz), 6.99 (S, 1H), 7.18 (S, 1H), 7.18 (S, IH), 12.42 (S, lH), 12.57 (S, 1H). 13C-NMR (CDCl 3), δc=17.9q, 20.9q,25.7q,32.8t,69.5d, 111.5S,111.8S,117.6d,131.4d,132.7d,132.9d,136.1s,148.2 s,166.9S,167.5S,169.8s.176.8s,178.2s.

Experimental example 2 tumor-inhibiting effect of Sinkiang arnebia root acetyl acarnine on melanoma immunodeficiency mice prepared by the invention

2.1 materials

2.1.1 animals: immunodeficient mice, females, weighing 16+ -2 kg, were supplied by Peking Vitrelli laboratory animal technologies Inc., were placed in an SPF-grade environment and then placed in an IVC system for feeding. The experimental animals were kept under a 12:12 hour light/dark cycle, free access to food and water, and room temperature was controlled at 21 ℃.

2.1.2 cell lines: human melanoma a375 cells, supplied by Qiao Xin boat biotechnology limited in the Shanghai.

2.1.3 reagents DMEM medium, FBS, green streptomycin diabody and EDTA-pancreatin were purchased from BI company and dimethyl sulfoxide (DMSO) was purchased from Sigma company.

2.1.4 reagent configuration: taking 10mg of acephatherin prepared from a scheme of arnebia euchroma (Royle) Johnst, dissolving in 500 mu L of DMSO; then 50 mu L of the solution is taken, 4.95ml of physiological saline is added to prepare 0.2mg/ml of acephate solution; taking 2.0ml of 0.2mg/ml of acephate solution, adding 2.0ml of physiological saline, and preparing 0.1mg/ml of acephate solution; 2.0ml of 0.1mg/ml of an acephate solution is taken, and 2.0ml of physiological saline is added to prepare 0.05mg/ml of the acephate solution.

2.1.5 preparation of complete Medium: 10ml of FBS, 49.5ml of DMEM and 0.5ml of neomycin diabody are added.

2.2 method

2.2.1 cell culture and Collection

After resuscitating the human melanoma A375 cells, they were grown in DMEM medium (100U/ml penicillin, 100. Mu.g/ml streptomycin) containing 10% fetal bovine serum, incubated in an incubator with 5% CO2 at 37℃saturated humidity to 80% confluency, and passaged with 0.25% trypsin and 0.02% EDTA. A375 cells in logarithmic growth phase were taken, after pancreatin digestion, washed 2 times with PBS, and after cell counting, diluted to 1X 107 cell sap.

2.2.2 establishment and grouping of the transplantation tumor models

30 immunodeficient mice were inoculated with A375 cell sap at the underarm of the right forelimb, 0.2 ml/mouse.

2.2.3 therapeutic Studies of laboratory animals

One week after inoculation, the mice began to group when the tumor node at the back became about 200 cubic millimeters, i.e., the mice were evenly distributed to each group according to tumor volume size, with 6 mice per group. The interventions were given at the following doses:

blank control group: physiological saline containing 1% dmso;

model control group: physiological saline containing 1% dmso;

60mg/kg dacarbazine group: as a positive drug control, 12mg of dacarbazine was dissolved in 4ml of physiological saline containing 1% DMSO.

4mg/kg of acalcet group: 10mg of acephatherin was dissolved in 500. Mu.L of DMSO; then 50 mu L of the solution is taken, 4.95ml of physiological saline is added to prepare 0.2mg/ml of acephate solution;

2mg/kg of acalcet group: taking 2.0ml of 0.2mg/ml of acephate solution, adding 2.0ml of physiological saline, and preparing 0.1mg/ml of acephate solution;

1mg/kg of acalcet group: 2.0ml of 0.1mg/ml of an acephate solution is taken, and 2.0ml of physiological saline is added to prepare 0.05mg/ml of the acephate solution.

Mice were intraperitoneally injected with acalcet at a rate of 20 μl/g based on the weight of the mice every 1 day, and the weight of the mice and tumor volume were recorded every 1 day. When the tumor volume of the control group mice is about 1500 cubic millimeters (about 12 days), the experiment is terminated, the tumor tissue is taken out, the tumor weight is weighed, and the tumor inhibition rate is calculated: tumor growth inhibition (%) = (average tumor weight of transplanted tumor model group-average tumor weight of administration group)/average tumor weight of transplanted tumor model group×100%. The specific results are shown in FIGS. 4 and 5.

EXAMPLE 3 inhibition of melanoma A375 cell growth and migration invasion by Acetylacarnine

3.1 materials

3.1.1 cell lines: human melanoma a375 cells, supplied by Qiao Xin boat biotechnology limited in the Shanghai.

3.1.2 reagent DMEM Medium, FBS, green streptomycin diabody and EDTA-pancreatin were purchased from BI, CCK-8 was purchased from Borson reagent, transwell cell and Matrigel Matrix were purchased from corning, and Crystal Violet was purchased from Soilebao.

3.1.3 configuration of reagents: the test sample was sinkiang arnebia root acetylalcaine prepared in example 1. Accurately weighing a proper amount of sample, and preparing a 0.1M storage solution by using DMSO for pharmacological activity test.

3.2 method

3.2.1 Effect of Acetylacarnine on melanoma A375 cell morphology

A375 cells in the logarithmic growth phase were inoculated into 6-well plates at a cell density of 1X 105 cells/mL, and the cells were allowed to adhere to the walls in a 5% CO2 incubator at 37℃overnight. Experiments were divided into 0 (blank), 400, 800, 1600nM acetoshikonin, added to the corresponding wells, placed in an incubator for 48h, and after incubation, the cell morphology was observed and recorded using an inverted microscope.

3.2.2 effects of Acetylacarnine on proliferation of melanoma A375 cells

Taking A375 cells in logarithmic phase, preparing single cell suspension after digestion by pancreatin, inoculating 2×103 cells/well into 96-well plate, and placing in 37 deg.C 5% CO2 incubator overnight to make cells adhere to wall. The experiment is carried out by setting a model group, an acetyl shikonin different concentration group and a blank control group, and 5 compound holes are arranged in each group. Acetylshikonin stock solution was diluted with medium and added to corresponding wells to final concentrations of 2400, 1200, 600, 300, 150, 75, 37.5, 18.75nM, and medium with corresponding concentrations of DMSO was added to each of the model and blank. After culturing in an incubator for 24, 48 and 72 hours, 10. Mu.L of CCK-8 reagent was added to each well, and the mixture was incubated in the incubator for 2 hours, and absorbance (A) was measured at 450nm using an ELISA reader. The mean value of absorbance (A) of the cells of the model group is taken as 100% of the number of living cells, namely the relative growth inhibition rate of the model group is zero. According to the formula: cell relative growth inhibition (%) = (a model group-a experimental group)/(a model group-a blank control group) ×100%.3.2.3 Effect of Acetylacarnine on melanoma A375 cell migration

Taking A375 cells in logarithmic phase, preparing single cell suspension after digestion by pancreatin, regulating the cell density to 5X 105 cells/mL, inoculating the cell suspension into 6-well plates, 2mL each well, culturing at 37 ℃ under 5% CO2, and after the cells are completely adhered and the fusion rate reaches 100%, performing cell scratching by using a 200 mu L sterile gun head. After the scratch was completed, 2mL of complete medium with acetoacarnine concentration of 400, 800, 1600nM was added to each of the drug administration groups, scratch widths were recorded for 0h and 48h under a microscope and photographed. The experimental results were analyzed using ImageJ software, the scratch area was measured and the scratch healing rate was calculated. Healing rate (%) = (0 h scratch area-48 h scratch area)/0 h scratch area x 100%.3.2.4 Effect of Acetaminophen on melanoma A375 cell invasion

After dilution with 50. Mu.l matrigel in medium 1:8, 100. Mu.l of the upper surface of the bottom membrane of the coated transwell chamber was used to form a membrane at the bottom. A375 cells in logarithmic growth phase were inoculated into cells at a density of 100. Mu.L per mL of 1X 105 cells, after cell adhesion, 0.1mL of medium containing N-acetylacarnine at a concentration of 400, 800 and 1600nM was added to each of the drug administration groups, medium containing 15% FBS was added to the lower chamber, after culturing for 24 hours, cells were fixed with 4% multi-step formaldehyde, stained with 0.5% crystal violet, matrigel and cells in the upper chamber were wiped off with a cotton swab, and the cells adhered to the lower side of the basement membrane of the cell were counted under a microscope.

3.3 results

3.3.1 Effect of Acetylacarnine on melanoma A375 cell morphology

Morphological observations showed that cell morphology changed after a375 cells were treated with different concentrations (0, 400, 800, 1600, nM) of acamprosate for 48 h: the blank group cells have better growth condition, firm adhesion and tight connection among cells, are in a regular long shuttle shape, have rich cytoplasm, and have a few of polygonal cells; as the concentration of acephating increases, the number of adherent cells is obviously reduced, the change from long shuttle to irregular shape with different sizes, the suspension cells are gradually increased, the cells become round and fall off, shrinkage and cavitation appear, and obvious apoptosis characteristics are presented, as shown in fig. 6.

3.3.2 inhibition of proliferation of melanoma A375 cells by Acetylacarnine

The CCK-8 method detection results show that after different concentrations of the acephating (2400, 1200, 600, 300, 150, 75, 37.5 and 18.75 nM) are respectively acted on the A375 cells for 24, 48 and 72 hours, the acephating is found to have a certain inhibition effect on the proliferation of the A375 cells at 20-2400 nM, and the inhibition effect is obviously enhanced along with the increase of the concentration and the action time of the acephating, and the time-dose dependency is realized (see figure 40). The IC50 s of acalcet effect a375 cells 24, 48 and 72h were (837.94 ±17.26), (340.07 ±39.63), (156.47 ±24.72) nM, respectively, and the differences were statistically significant (P < 0.001), see fig. 7.

3.3.3 inhibition of melanoma A375 cell migration by Acetylacarnine

And (3) analyzing the influence of the acephate on the migration capacity of the A375 cells by adopting a scratch experiment, wherein after the scratch is performed for 48 hours, the scratch distance and the scratch area of a control group are smaller than those of an acephate administration group, and the higher the concentration of the acephate, the larger the scratch width and the scratch area. The scratch healing rate of the acalcet group was significantly reduced with increasing concentration and cell mobility was significantly reduced (400 nM, P <0.05, 800nM, P <0.01, 1600nM, P < 0.005) compared to the control group, see fig. 8.

3.3.4 inhibition of melanoma A375 cell invasion by Acetylacarnine

The effect of acamprosate on the invasive capacity of a375 cells was analyzed using transwell experiments, and 24h after drug intervention, the number of cells across the basement membrane was greater in the control group than in the acamprosate-dosed group, and significantly decreased as the concentration of acamprosate increased, the number of cells across the pores was smaller (400 nM, 800nM and 1600nM P < 0.005), see fig. 9.

With the above-described preferred embodiments according to the present invention as an illustration, the above-described descriptions can be used by persons skilled in the relevant art to make various changes and modifications without departing from the scope of the technical idea of the present invention. The technical scope of the present invention is not limited to the description, but must be determined according to the scope of the claims.

Claims (10)

1. A method for preparing acephatherin and application thereof in preparing medicines for preventing and treating tumors, wherein the acephatherin is prepared from arnebia euchroma (Roxb.) kuntze, and any one or at least two of acephatherin and racemate thereof contained in a medicinal composition.

2. Use according to claim 1, characterized in that said acephating and its racemate inhibit the invasion and metastasis of melanoma.

3. The acephating and racemates thereof according to claim 1, characterized in that they are prepared according to the following method:

3-1, crushing, namely crushing a proper amount of sinkiang arnebia root medicinal material to obtain sinkiang arnebia root medicinal material coarse powder;

3-2, extracting, namely adding petroleum ether (60-90 ℃) into coarse powder of the sinkiang arnebia root medicinal material, performing cold leaching extraction for 2-3 times at room temperature, filtering to obtain filtrate, and combining the filtrate to obtain extract;

3-3, concentrating the extracting solution to obtain a pasty concentrated solution of the extracted part;

step 3-4, coarsely separating, filling the column with silica gel to obtain a silica gel chromatographic column, dissolving the concentrated solution of the extraction part, adsorbing with silica gel, drying, and filling the column; sequentially eluting with petroleum ether, eluting with petroleum ether-ethyl acetate, analyzing with TLC, and taking acetylacarnine as reference, (developing solvent is that petroleum ether and ethyl acetate are combined, and formic acid=19:1:0.1), and concentrating under reduced pressure to obtain coarse fraction of acetylacarnine;

3-5, purifying, filling a column with silica gel H to obtain a silica gel H chromatographic column, dissolving a concentrated solution of a crude part of the acephataine with petroleum ether, and performing silica gel H column chromatography by using petroleum ether: eluting with ethyl acetate; TLC analysis is adopted, the acephating is used as a reference, the same eluent with the main spot of the acephating is combined, and the acephating crude product is obtained after decompression and concentration;

and 3-6, recrystallizing, taking an acephate crude product, dissolving and filtering the acephate crude product with ethyl acetate, adding petroleum ether for recrystallization, and obtaining red short needle crystals, and acephate.

4. The preparation method according to claim 3-2, wherein 1kg of sinkiang arnebia root crude fraction is added with 6 to 12 liters of petroleum ether solution, and the mixture is soaked and extracted for 2 to 3 times at room temperature for 8 to 12 hours each time; concentrating the extractive solution at 35-45deg.C, and recovering petroleum ether under reduced pressure to 1/36-1/72 of the total volume of petroleum ether added during reflux extraction to obtain pasty concentrate.

5. The method according to claim 3-4, wherein the column chromatography silica gel is 200-300 mesh silica gel, the mass of the silica gel is 8-12 times of the mass of the extract, and the concentrated solution of the extracted part is prepared by petroleum ether: dissolving ethyl acetate (1:1), adsorbing with silica gel, drying, and loading into column; sequentially eluting with 3-6 times of petroleum ether, eluting with 3-6 times of petroleum ether-ethyl acetate (volume ratio of 50:1), and eluting at a rate of 1-5 column volumes per hour; TLC analysis was performed (developing solvent condition: petroleum ether: ethyl acetate: formic acid=19:1:0.1), and the same eluent was combined, and concentrated under reduced pressure at 35℃to 45℃to obtain a crude fraction of acamprosate.

6. The method according to claim 3-5, wherein the column is packed with silica gel H of 30 to 60 mesh to obtain a silica gel H column, the crude fraction concentrate of acamprosate is dissolved with 1 to 3 times of petroleum ether in the volume of the crude fraction concentrate, and the column chromatography is performed with 3 to 6 times of petroleum ether in the volume of the column: ethyl acetate (volume ratio 19:1) at a rate of 1 to 5 column volumes per hour; TLC analysis is adopted, the same eluent with main spots of the acephating is combined, and the acephating crude product is obtained after decompression concentration at 35 ℃ to 45 ℃.

7. The process for preparing the pure product of acetylacarnine with purity more than 95% is obtained by dissolving acetylacarnine Ning Cupin with ethyl acetate in 1 to 3 times of mass volume, adding petroleum ether in 1 to 5 times of volume and ethyl acetate, filtering with qualitative filter paper at normal pressure, standing at 4 ℃ for 2 to 8 hours, and filtering with short needle-like crystals in red in the solution with qualitative filter paper at normal pressure.

8. The method for preparing acephatherin according to claim 3, wherein (1) the acephatherin is dissolved in DMSO and physiological saline to prepare 0.2g/ml acephatherin solution, and a method for preparing an effective dose of a melanoma drug is adopted by using an immunodeficient mouse model, wherein the obtained melanoma sample is inoculated into an immunodeficient mouse, and after 2-5 weeks, a melanoma immunodeficient mouse model is obtained, and an immunodeficient mouse anti-melanoma drug is administered to the immunodeficient mouse model by using an intraperitoneal injection method, and the growth of melanoma in the tumor in the mouse is detected: the mice with melanoma immunity deficiency were intraperitoneally injected with 4mg/kg, 2mg/kg and 1mg/kg lavage, and the growth of melanoma in the mice after administration of acacetin was examined. (2) The method for preparing acephatherin according to claim 3, wherein the cell migration and invasion capacity of melanoma A375 cell lines are observed after administration of acephatherin by cell trace analysis and recombinant basement membrane invasion assay.

9. Use according to claims 1 and 2, characterized in that it is a pharmaceutically acceptable salt, ester, solvate, acceptable carrier or excipient; the carrier comprises liposome, micelle, dendrimer, microsphere or microcapsule.

10. The use according to claims 1, 2, 8 and 9, said adjuvant comprising any one or a combination of at least two of excipients, diluents, carriers, flavouring agents, binders or fillers; the dosage forms of the medicine comprise tablets, capsules, granules, powder, injection, spray, film, suppositories, nasal drops or drop pills.

Images